CA2395781C - Detection and treatment of polycystic kidney disease - Google Patents

Abstract

Compositions useful for examining the PKD1 gene are provided. In addition, methods for detecting mutations of the PKD1 gene, which can be associated with autosomal dominant polycystic kidney disease in humans, are provided. Methods for diagnosing a mutant PKD1 gene sequence in a subject also are provided, as are methods of treating a subject having a PKD1-associated disorder.

Description

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DETECTION AND TREATMENT OF POLYCYSTIC KIDNEY DISEASE

BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The present invention relates generally to the diagnosis and treatment of polycystic kidney disease and more specifically to probes and agents useful in diagnosing and treating polycystic kidney disease and related disorders.
BACKGROUND INFORMATION
Autosomal doininant polycystic kidney disease (ADPKD), also called adult-onset polycystic kidney disease, is one of the most common hereditary disorders in Izumans, affecting approximately one individual in a thousaiid. The prevalence in the United States is greater than 500,000, with 6,000 to 7,000 new cases detected yearly (Striker et al., Am. J. Nephrol. 6:161-164, 1986; Iglesias et al., Am. J. Kid.
Dis.

2:630-639, 1983). The disease is considered to be a systemic disorder, characterized by cyst formation in the ductal organs such as kidney, liver, and pancreas, as well as by gastrointestinal, cardiovascular, and musculoskeletal abnormalities, including colonic diverticulitis, berry aneurysms, hernias, and mitral valve prolapse (Gabow et al., Adv. Nephrol. 18:19-32, 1989; Gabow, New Eng. J. Med. 329:332-342, 1993).
The most prevalent and obvious symptom of ADPKD is the formation of kidney cysts, which result in grossly enlarged kidneys and a decrease in renal-concentrating ability. In approximately half of ADPKD patients, the disease progresses to end-stage renal disease, and ADPKD is responsible for 4-8% of the renal dialysis and transplantation cases in the United States and Europe (Proc. Eur.
Dialysis and Transplant Assn., Robinson and Hawlcins, eds., 17:20, 1981).

Few diagnostics are available for the identification and characterization of mutations of the PKD1 gene, which is located on human chromosome 16. A major factor contributing to the difficulty in identifying and characterizing mutations of the PKD1 gene is that greater than 70% of the length of the PKD1 gene is replicated on chromosome 16 and elsewhere, resulting in at least six PKD1 homologs.
Significantly, the PKD1 lloinologs share a very high sequence identity with the PKD1 gene, including sequences having greater than 95% identity with the PKD 1 gene. As such, oligonucleotides that have been examined for use as specific probes, or as primers for amplification, of PKD 1 gene sequences have been found to cross-llybridize with the PKD 1 homologs, and the inability to identify PKD 1 locus specific probes has prevented accurate analysis of PKD 1 geiie inutations.

The identification and characterization of PKD1 gene mutations have been further hindered, in part, because transcription of the PKD 1 gene results in production of a 14 kilobase (lcb) mRNA, which is highly GC-rich. In addition, unlike the remainder of the PKD 1 gene, which is extremely compact (approximately 13.51cb mRNA coded within approximately 30 kb genomic DNA), exon 1 is separated from the rest of the gene by an intron of approximately 19 lcb. Thus, previous investigators have simply placed the 5' anchor primer within the first intron and used it as a link to more 3' sequences. Exon 1 has several other features that have been major obstacles to its amplification, including an extremely high GC content (approximately 85%), and the ability to replicate with high fidelity in PKD 1 gene homologs.
Furthermore, no effective method for DNA based analysis of PKD 1 gene exon 22, which is flanked on both ends by introns that contain lengthy polypyrimidine tracts.
Accordingly, very few positions within the replicated segment and flanking exon 22 are suitable for the design of PKD 1 -specific primers.

A few oligonucleotides useful for examining regions of the human PKD 1 gene, have been described. For example, the primer set fortll below as SEQ ID NO: 11 has been described in U.S. Pat. No. 6,017,717, and the primer set forth as SEQ ID
NO: 18 has been described by Watnick et al. (Hum. Mol. Genet. 6:1473-1481, 1997).
Also, the primers set forth below as SEQ ID NOS:9, 10, 49 to 51, and 61 to 105 have been described by Watnick et al. (Am. J. Hum. Genet. 65:1561-1571, 1999). The primers set forth below as SEQ ID NOS: 9 and 10 and SEQ ID NOS: 11 and 12 also were more recently described by Phakdeekitcharoen et al. (Icdney International 58:1400-1412, 2000). In addition, a primer set forth as SEQ ID NO:13 in U.S. Pat. No.
6,071,717 has a nucleotide sequence that is substantially identical to that set forth below as SEQ ID
NO:10, and a primer designated TWR2 by Watniclc et al. (Mol. Cell 2:247-251,1998) has a nucleotide sequence that is substantially identical to that set forth below as SEQ ID
NO:12.

Despite the large number of fatnilies having diseases associated with PKDI
geile mutations, the potential clinical and scientific impact of mutation studies, and the availability of a genomic structure, the fact that only a relatively small niunber of PKD 1 mutations have been described demonstrates the relative paucity of data due to the complicated genomic structure of the PKD1 gene. Thus, there exists a need for diagnostic methods suitable for examining the PKD1 gene and for identifying disorders related to PKD 1 gene mutations. The present invention satisfies this need and provides additional advantages.

SUMMARY OF THE INVENTION
The present invention provides compositions and methods that allow for the selective examination of the human PKD 1 gene, including the detection and identification of PKD1 gene mutations. For example, the compositions of the invention include oligonucleotide primers that are useful for selectively amplifying a region of a PKDl gene, but not a corresponding region of a PKD1 homolog.
Accordingly, the present invention relates to a PKD 1 gene specific primer, which can be one of a primer pair. A primer of the invention includes a 5' region and adjacent PKDl-specific 3' region, wherein the 5' region has a nucleotide sequence that can hybridize to a PKD1 gene sequence and, optionally, to a PKD 1 homolog sequence, and the 3' region has a nucleotide sequence that selectively hybridizes only to a PKD 1 gene sequence, and particularly not to a PKDl gene homolog sequence, except that a primer of the invention does not have a sequence as set forth in SEQ ID NO:
11, SEQ
ID NO:18, SEQ ID NO:52, or SEQ ID NO:60. A 5' region of a primer of the .
invention generally contains at least about ten contiguous nucleotides, and the 3' region contains at least one 3' terininal nucleotide, wherein the at least one 3' terminal nucleotide is identical to a nucleotide that is 5' and adjacent to the nucleotide sequence of the PKD 1 gene to which the 5' region of the primer can hybridize, and is different fiom a nucleotide that is 5' and adjacent to a nucleotide sequence of the PKD

homolog to which the 5' region of the primer can hybridize. Generally, the primer includes a 5' region of about 14 to 18 nucleotides and a 3' region of about 2 to 6 nucleotides, particularly about 2 to 4 nucleotides. For example, a primer of the invention can have a sequence as set forth in any of SEQ ID NOS:3 to 10, 12 to 17, 19 to 51 and 61 to 113.

The present invention also relates to an isolated mutant PKDI polynucleotide, or an oligonucleotide portion thereof. The polynucleotides of the invention are exemplified by mutation of SEQ ID NO:1, which appear to be normal variants that are not associated with a PKD1-associated disorder, for example, a polynucleotide or oligonucleotide that includes nucleotide 474, wherein nucleotide 474 is a T;
nucleotide 487, wherein nucleotide 487 is an A; nucleotide 9367, wherein nucleotide 9367 is a T; nucleotide 10143, wlierein nucleotide 10143 is a G;
nucleotide 10234, wherein nucleotide 10234 is a C; nucleotide 10255, wherein nucleotide 10255 is a T; or a combination thereof; and by mutations of SEQ ID
NO: 1 that are associated with a PKD1-associated disorder, for exainple, a polynucleotide or oligonucleotide that includes nucleotide 3110 of SEQ ID NO:1, wherein nucleotide 3110 is a C; nucleotide 8298 of SEQ ID NO: 1, wherein nucleotide 8298 is a G; nucleotide 9164 of SEQ ID NO:1, wherein nucleotide 9164 is a G;
nucleotide 9213 of SEQ ID NO:1, wherein nucleotide 9213 is an A; nucleotide of SEQ ID NO:1, wherein nucleotide 9326 is a T; nucleotide 10064 of SEQ ID
NO:1, wherein nucleotide 10064 is an A; or a combination thereof. The invention also provides a vector containing such a polynucleotide, or an oligonucleotide portion thereof, and provides a host cell containing such a polynucleotide or oligonucleotide, or vector.

A PKD1-specific primer of the invention is exemplified by an oligonucleotide that can selectively hybridize to a nucleotide sequence that flanks and is within about fifty nucleotides of a nucleotide sequence selected from about nucleotides to 4209; nucleotides 17907 to 22489; nucleotides 22218 to 26363; nucleotides to 30615; nucleotides 30606 to 33957; nucleotides 36819 to 37140; nucleotides to 41258; and nucleotides 41508 to 47320 of SEQ ID NO:l . The primer, which can be one of a primer pair, can have a nucleotide sequence substantially identical to any of SEQ ID NOS: 3 to 18, provided that when the primer is not one of a primer pair, the primer does not have a sequence as set forth in SEQ ID NO: 11, SEQ ID NO:
18, 5 SEQ ID NO:52, or SEQ ID NO:60. Accordingly, the present invention further relates to a primer pair that can amplify a portion of a PKD1 gene, for example, the wild type PKD1 gene set fortli as SEQ ID NO: 1, wllerein the aiilplification product can include about nucleotides 2043 to 4209; nucleotides 17907 to 22489; nucleotides 22218 to 26363; nucleotides 26246 to 30615; nucleotides 30606 to 33957; nucleotides to 37140; nucleotides 37329 to 41258; nucleotides 41508 to 47320; or a combination thereof. A primer pair of the invention is useful for performing PKD 1-specific anlplification of a portion of a PKD 1 gene.

Primer pairs of the invention are exemplified by a pair including at least one forward primer and at least one reverse primer of the oligonucleotides sequences set forth in SEQ ID NOS:3 to 18 or a sequence substantially identical thereto. In one embodiment, the primer pair includes SEQ ID NOS:3 and 4; SEQ ID NOS:5 and 6;
SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID
NOS:13 and 14; SEQ ID NOS:15 and 16; SEQ ID NOS:17 and 18; or SEQ ID NOS:9 and 113. Also provided are primer pairs useful for performing nested amplification of a PKD 1 -specific amplification product of a PKD 1 gene, for example, the primer pairs set forth as SEQ ID NOS:19 and 20; SEQ ID NOS:21 and 22; SEQ ID NOS:23 and 24;
SEQ ID NOS:25 and 26; SEQ ID NOS:27 and 28; SEQ ID NOS:29 and 30; SEQ ID
NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ ID NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID NOS:41 and 42; SEQ ID NOS:43 and 44;
SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48; SEQ ID NOS:49 and 50; SEQ ID
NOS: 51 and 61; SEQ ID NOS:62 and 63; SEQ ID NOS:64 and 65; SEQ ID NOS:66 and 67; SEQ ID NOS:68 and 69; SEQ ID NOS:70 and 71; SEQ ID NOS:72 and 73;
SEQ ID NOS:74 and 75; SEQ ID NOS:76 and 77; SEQ ID NOS:78 and 79; SEQ ID
NOS:80 and 81; SEQ ID NOS:82 and 83; SEQ ID NOS:84 and 85; SEQ ID NOS:86 and 87; SEQ ID NOS:88 and 89; SEQ ID NOS:90 and 91; SEQ ID NOS:92 and 93;
SEQ ID NOS:94 and 95; SEQ ID NOS:96 and 113; SEQ ID NOS:97 and 98; SEQ ID

NOS:99 and 100; SEQ ID NOS:101 and 102; SEQ ID NOS:103 and 104; SEQ ID
NOS: 105 and 106; SEQ ID NOS:107 and 108; SEQ ID NOS:109 and 110; or SEQ ID
NOS:111 and 112. In another embodiment, the invention relates to a plurality of primer pairs, which can include two or more primer pairs that are useful for generating two or more PKD 1-specific amplification products of a PKD 1 gene;
or can include two or more primer pairs that are useful for generating a PKD 1-specific amplification product of a PKD 1 gene and for generating a nested amplification product of the PKD1-specifc amplification product.

The present invention also relates to a purified mutant PKD 1 polypeptide, or a peptide portion thereof, comprising an anlino acid sequence of a mutant of SEQ
ID
NO:2. A mutant PKD 1 polypeptide, or peptide portion thereof can be substantially identical to a sequence of SEQ ID NO:2 and, for example, include amino acid residue 88 of SEQ ID NO:2, wherein residue 88 is a V; residue 967 of SEQ ID
NO:2, wherein residue 967 is an R; residue 2696 of SEQ ID NO:2, wherein residue 2696 is an R; residue 2985 of SEQ ID NO:2, wherein residue 2985 is a G; residue 3039 of SEQ ID NO:2, wherein residue 3039 is a C; residue 3285 of SEQ ID NO:2, wherein residue 3285 is an I; or residue 3311 of SEQ ID NO:2, wherein residue 3311 is an R;
or can include residue 3000 of a truncated mutant PKD 1 polypeptide ending at amino acid residue 3000 with respect to SEQ ID NO:2, wherein residue 3001 is absent (and the mutant PKD1 polypeptide is truncated) due to the presence of a STOP codon in the encoding mutant PKD1 polynucleotide; or a combination of such mutations.
Also provided is a purified antibody that specifically binds to a mutant PKD 1 polypeptide, or to a peptide thereof.
The present invention further relates to a primer or an oligonucleotide of the invention immobilized to a solid support. In addition, the primer or oligonucleotide can be one of a plurality of primers, oligonucleotides, or a combination thereof, each of which is immobilized to a solid support. The solid support can be any support, including, for exainple, a microchip, in which case, the primers, oligonucleotides, or combination thereof can be arranged in array, particularly an addressable array. The primers, oligonucleotides, or combination thereof also can be degenerate with respect to each other, and specific for a wild type PKD 1 polyiiucleotide, a mutant polynucleotide, including a variant, or combinations thereof, and, therefore, provide a means for multiplex analysis. Accordingly, the present invention provides compositions coinprising one or a plurality of iiumobilized primers or oligonucleotides of the invention, or combinations thereof.

The present invention also relates to a method of detecting a PKD 1 polynucleotide in a sample, wherein the PKD1 polynucleotide is a wild type PKDl polynucleotide having a sequence as set forth in SEQ ID NO:1, or a mutant PKD

polynucleotide, which can be a variant PKD1 polynucleotide that has a sequence different from SEQ ID NO:1 but is not associated with a PKD1-associated disorder or can be a mutant PKDl polynucleotide that is associated with a PKD 1 -associated disorder. A method of the invention can be performed, for example, by contacting nucleic acid molecules in a sample suspected of containing a PKD 1 polynucleotide with at least one primer pair under conditions suitable for amplification of a PKDl polynucleotide by the primer pair; and generating a PKD1-specific amplification product under said conditions, thereby detecting a PKD1 polynucleotide in the sample. The primer pair can be any primer pair as disclosed herein, for example, a primer pair such as SEQ ID NOS:3 and 4; SEQ ID NOS:5 and 6; SEQ ID NOS:7 and 8;
SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID NOS:13 and 14; SEQ ID
NOS:15 and 16; SEQ ID NOS:17 and 18; or SEQ ID NOS:9 and 113; or can be a combination of such primer pairs.

A method of detecting a PKD 1 polynucleotide can further include, upon generating a PKD 1-specific amplification product, contacting the amplification product with at least a second primer pair, under conditions suitable for nested amplification of the PKD1-specific amplification product by the second primer pair, and generating a nested amplification product. The second primer pair can be any primer pair that can produce a nested amplification product of the PKD 1-specific amplification product, for example, a second primer pair sucli as SEQ ID
NOS:19 and 20; SEQ ID NOS:21 and 22; SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26;
SEQ ID NOS:27 and 28; SEQ ID NOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID

NOS:33 and 34; SEQ ID NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID NOS:41 and 42; SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46;
SEQ ID NOS:47 and 48; SEQ ID NOS:49 and 50; SEQ ID NOS:51 and 61; primer pairs foznled using consecutive primers set forth in Table 2 as SEQ ID NOS:62 to 96, 113, and 97 to 112; or a coinbination thereof.

Upon detecting a PKD 1 polynucleotide in a sample according to a method of the invention, an additional step of detecting the presence or absence of a mutation in an aniplification product of the PKD 1 polynucleotide in the sample as compared to a corresponding nucleotide sequence in SEQ ID NO:1. As such, a method of the invention provides a ineans to identify a PKD 1 polynucleotide in a sample as a mutant PKD 1 polynucleotide or a wild type PKD 1 polynucleotide, wherein detecting the absence of a inutation in the amplification product identifies the PKD1 polynucleotide in the sample as a wild type PKD1 polynucleotide, and wherein detecting the presence of a mutation in the amplification product identifies the PKD 1 polynucleotide in the sample as a mutant PKD1 polynucleotide, which can be a variant PKDl polynucleotide, or can be mutant PKD 1 polynucleotide associated with a PKD 1-associated disorder, the latter of which are exemplified by a polynucleotide that is substantially identical to SEQ ID NO: 1, and wherein at least nucleotide 474 is a T;
nucleotide 487 is an A; nucleotide 3110 is a C; nucleotide 8298 is a G;
nucleotide 9164 is a G; nucleotide 9213 is an A; nucleotide 9326 is a T; nucleotide 9367 is a T;
nucleotide 10064 is an A; nucleotide 10143 is a G; nucleotide 10234 is a C; or nucleotide 10255 is a T.

The presence or absence of a mutation in an amplification product generated according to a method of the invention can be detected any method useful for detecting a mutation. For example, the nucleotide sequence of the amplification product can be determined, and can be compared to the corresponding nucleotide sequence of SEQ ID NO:1. The melting temperature of the amplification product also can be determined, and can be compared to the melting temperature of a corresponding double stranded nucleotide sequence of SEQ ID NO:1. The melting teinperature can be deterinined using a method such as denaturing high performance liquid chromatography.

An advantage of a method of the invention is that a large number of samples can be examined serially or in parallel. Accordingly, a method of the invention can be performed with respect to a plurality of samples, and can be performed using a high throughput format, for example, by organizing the samples of a plurality of samples in an array suc11 as in an array is on a microchip. The method can further include detecting the presence or absence of a mutation in an amplification product of the samples of the plurality of samples, for example, by determining the melting temperature of the amplification product and comparing it to the melting temperature of a corresponding nucleotide sequence of SEQ ID NO:1 using a method such as denaturing high performance liquid chromatography, or the presence or absence of a mutation can be performed using any method useful for such a purpose, for example, matrix-assisted laser desorption time of flight mass spectrometry or high throughput conformation-sensitive gel electrophoresis, each of which is readily adaptable to a high throughput analysis format.

In another einbodiment, the presence or absence of a mutation in an amplification product can be detected by contacting the amplification product with the oligonucleotide of the invention, under condition suitable for selective hybridization of the oligonucleotide to an identical nucleotide sequence; and detecting the presence or absence of selective hybridization of the oligonucleotide to the amplification product. Using such a method detecting the presence of selective hybridization identifies the PKD 1 polynucleotide in the sample as a mutant PKD 1 polynucleotide, and detecting the absence of selective hybridization identifies the PKD1 polynucleotide as a wild type PKD1 polynucleotide. Where an absence of a mutation is detected, the PKDl polynucleotide in the sample is identified as a wild type PKD 1 polynucleotide. In comparison, where the presence of a mutation is identified, the mutant PKD1 polynucleotide so identified can be further examined to determine whether the inutant PKD1 polynucleotide is a variant PKD1 polynucleotide, which is associated witll a normal phenotype with respect to PKD1, for example, where the amplification product has a nucleotide sequence substantially identical to SEQ
ID
NO:l, and including C474T, G487A, G4885A; C6058T; G6195A; T7376C; C7696T;
G8021A; C9367T, A10143G, T10234C, or a combination thereof, or is a mutant polynucleotide associated with a PKD1-associated disorder, for example, where the 5 amplification product has a nucleotide sequence substantially identical to SEQ ID
NO:l, and 'ulcluding T3110C, G3707A; T6078A; C7433T; T8298G; A9164G; G9213A, C9326T; G10064A; an insertion of GCG between nucleotides G7535 and A7536; or a combination thereof, each of which is associated with ADPKD (see Example 2;
see, also, Phakdeekitcharoen et al., ICdney International 58:1400-1412, 2000, which is 10 incorporated herein by reference).

The present invention further relates to a metllod of detecting the presence of a inutant PKD 1 polynucleotide in a sample. In one embodiment, a method of the invention is performed by aniplifying a nucleic acid sequence in a sample suspected of containing a mutant PKD 1 polynucleotide using a primer pair of the invention, for example, a primer pair selected from SEQ ID NOS:3 and 4; SEQ ID NOS:5 and 6;
SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID
NOS:13 and 14; SEQ ID NOS:15 and 16; SEQ ID NOS:17 and 18; or SEQ ID NOS:9 and 113, thereby obtaining a PKD 1-specific amplification product of a PKD 1 gene sequence; and detecting a mutant PKD1 polynucleotide in the amplification product.
The mutant PKDI nucleotide in the amplification product can be detected using any method useful for detecting a mutation in a polynucleotide, for example, using denaturing high performance liquid cliromatograph. In anotller embodiment, a method of the invention is performed by contacting a sample suspected of containing a mutant PKD 1 polynucleotide with a probe comprising an isolated polynucleotide of the invention, or an oligonucleotide portion thereof, under conditions such that the probe selectively hybridizes to a mutant PKD 1 polynucleotide, and detecting specific hybridization of the probe and a PKD 1 polynucleotide, thereby detecting the presence of a mutant PKD 1 polynucleotide sequence in the sample.
The present invention further relates to a method of identifying a subject having or is at risk of having a PKD1-associated disorder. Such a method can be performed, for example, by contacting nucleic acid molecules in a sample from a subject with at least one primer pair of the invention under coiiditions suitable for amplification of a PK D 1 polynucleotide by the primer pair, tllereby generating an amplification product; and testing an amplification product for the presence or absence of a mutation indicative of a PKD1-associated disorder. As disclosed herein, the absence of such a mutation identifies the subject as not having or at risk of the having a PKD 1-associated disorder, wherein the presence of such a mutation identifies the subject as having or is at risk of having a PKD1-associated disorder, for example, ADPKD or acquired cystic disease.
A primer pair useful in a diagnostic method of the invention can include at least one primer pair selected from SEQ ID NO:3 and 4; SEQ ID NO:5 and 6; SEQ ID
NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID NOS:13 and 14; SEQ
ID NOS:15 and 16; SEQ ID NOS:17 and 18; and SEQ ID NOS:9 and 113. The subject can be any subject having a PKD 1 gene and susceptible to a PKD1-associated disorder, including a vertebrate subject, and particularly a mammalian subject such as a cat or a human. In addition, the diagnostic method can be performed in a high throughput format, thereby allowing the examination of a large number samples in a cost-effective manner.
The diagnostic method can further include contacting the amplification product generated as described above with at least a second primer pair, under conditions suitable for nested amplification of the amplification product by a second primer pair, thereby generating a nested amplification product. The second primer pair can be, for example, a primer pair selected from SEQ ID NOS:19 and 20;
SEQ ID
NOS:21 and 22; SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID NOS:27 and 28; SEQ ID NOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID NOS:33 and 34;
SEQ ID NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID
NOS:41 and 42; SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48; SEQ ID NOS:49 and 50; SEQ ID NOS:51 and 61; a primer pair formed using two consecutive primers set forth in Table 2 as SEQ ID NOS:62 to 96, 113, and to 112 (i.e., SEQ ID NOS: 62 and 63, SEQ ID NOS:64 and 65, and so on); and a combination tliereof, in which case, the step of testing the ainplification product for the presence or absence of a mutation comprises testing the nested amplification product.
It should be recognized that the selection of a primer pair for nested anlplification is based, in part, on the sequence of the PKD 1 -specific amplification product that is to be used as a template for the nested amplification, i.e., nested primer pairs are selected such that they can hybridize to a target PKD 1 -specific amplification product and can a.inplify the target sequence.

An amplification product can be tested for the presence or absence of the mutation, for example, by determining the nucleotide sequence of the amplification product, and coinparing it to a corresponding nucleotide sequence of SEQ ID
NO: 1;
by determining the melting temperature of the amplification product, and comparing it to the melting temperature of a corresponding nucleotide sequence of SEQ ID
NO: 1, for example, using a niethod such as denaturing high performance liquid chromatography; or by contacting the amplification product with an oligonucleotide probe contaiiiing nticleotide 474 of SEQ ID NO: 1, wherein nucleotide 474 is a T;
nucleotide 487 of SEQ ID NO:1, wherein nucleotide 487 is an A; nucleotide 3110 of SEQ ID NO: 1, wherein nucleotide 3110 is a C; nucleotide 8298 of SEQ ID NO: 1, wherein nucleotide 8298 is a G; nucleotide 9164 of SEQ ID NO:1, wherein nucleotide 9164 is a G; nucleotide 9213 of SEQ ID NO:1, wherein nucleotide 9213 is an A;
nucleotide 9326 of SEQ ID NO:1, wherein nucleotide 9326 is a T; nucleotide 9367 of SEQ ID NO:1, wherein nucleotide 9367 is a T; nucleotide 10064 of SEQ ID NO:l, wherein nucleotide 10064 is an A; nucleotide 10143 of SEQ ID NO:1, wherein nucleotide 10143 is a G; nucleotide 10234 of SEQ ID NO: 1, wherein nucleotide 10234 is a C; and nucleotide 10255 of SEQ ID NO:1, wherein nucleotide 10255 is a T, under conditions suitable for selective hybridization of the probe to a mutant PKD1 polypeptide, which can be a normal variant or can be a mutant PKD1 polynucleotide associated with a PKD1-associated disorder.

The present invention also relates to a method of diagnosing a PKD1-associated disorder in a subject suspected of having a PKDl-associated disorder.
Sucll a method is performed by amplifying a nucleic acid sequence in a sample obtained from the subject using a primer pair suitable for PKD1-specific amplification of a PKD 1 gene sequence, for example, a primer pair such as SEQ ID NO:3 and 4;
SEQ ID NOS:5 and 6; SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID NOS:13 and 14; SEQ ID NOS:15 and 16; SEQ ID NOS:17 and 18, or SEQ ID NOS:9 and 113, thereby obtaining a PKD1-specific first amplification product;
and detecting a mutation of a PKDl gene sequence in the PKD1-specific first amplification product, wherein the mutation is indicative of a PKD1-associated disorder, thereby diagnosing a PKD 1-associated disorder in the subject.

In one embodiment, the diagnostic method includes a step of further amplifying the first amplification product using a second set of primer pairs to obtain a nested ainplification product; and detecting a PKD1 gene mutation in the nested amplification product. The second set of primer pairs can be any primer pairs useful for amplifying the PKD1-specific first a.niplification product, including, for example, the primer pairs exemplified by SEQ ID NOS:19 and 20; SEQ ID NOS:21 and 22;
SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID NOS:27 and 28; SEQ ID
NOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ ID NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID NOS:41 and 42;
SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48; SEQ ID
NOS:49 and 50; SEQ ID NOS:51 and 61; or any of the primer pairs formed using consecutive primers set forth in Table 2 as SEQ ID NOS:62 to 96, 113, and 97 to 112.
In another method, the diagnostic method includes a step of contacting the PKD 1-specific first amplification product or second amplification product with a probe comprising an isolated polynucleotide, or an oligonucleotide portion thereof, comprising a mutant of SEQ ID NO:1, under conditions such that the probe can selectively hybridize to a mutant PKD 1 polynucleotide; and detecting selective hybridization of the probe to the first amplification product, thereby diagnosing a PKD1-associated disorder in the subject. The probe can be, for example, an oligonucleotide portion of SEQ ID NO:1 that includes one or more of nucleotide is a T; nueleotide 487 is an A; nucleotide 3110 is a C; ilucleotide 8298 is a G;
nucleotide 9164 is a G; nucleotide 9213 is an A; nucleotide 9326 is a T;
nucleotide 9367 is a T; nucleotide 10064 is an A; nucleotide 10143 is a G; nucleotide 10234 is a C; or nucleotide 10255 is a T.

The present invention also relates to a method of detecting the presence of a mutant PKD1 polypeptide in a sample. Such a metllod can be performed, for example, by contacting a sample suspected of containing a mutant PKD1 polypeptide with an antibody that specifically binds to a mutant PKD1 polypeptide, under conditions which allow the antibody to bind to the mutant PKD1 polypeptide and detecting specific binding of the antibody and the mutant PKD1 polypeptide in the sample. The detection of an immunocomplex of the antibody and a mutant PKDI
polypeptide, for example, indicates the presence of a mutant PKD1 polypeptide in the sample. In one embodiment, the method is performed by contacting a tissue sample from a subject suspected of containing a PKD1 polypeptide with the antibody that specifically binds a mutant PKD1 polypeptide under conditions that allow the antibody interact with a PKD 1 polypeptide and detecting specific binding of the antibody and the PKDI polypeptide in the tissue.

The present invention further relates to a kit for detecting a mutant PKD1 polynucleotide, which can be a variant PKDI polynucleotide or a mutant PKD1 polynucleotide associated with a PKD 1 -associated disorder. The lcit can contain, for example, a carrier means containing therein one or more containers wherein a first container contains a nucleotide sequence useful for detecting a wild type or mutant PKD1 polynucleotide. As such, a nucleotide sequence useful in a kit of the invention can be an oligonucleotide coinprising at least ten contiguous nucleotides of SEQ ID
NO:1, including at least one of nucleotide 474, wherein nucleotide 474 is a T;
nucleotide 487, wherein nucleotide 487 is an A; nucleotide 3110, wherein nucleotide 3110 is a C; a position corresponding to nucleotide 3336, wherein nucleotide 3336 is deleted; nucleotide 3707, wherein nucleotide 3707 is an A;
nucleotide 4168, wherein nucleotide 4168 is a T; nucleotide 4885, wherein nucleotide 4885 is an A; nucleotide 5168, wherein nucleotide 5168 is a T;
nucleotide 6058, wherein nucleotide 6058 is a T; nucleotide 6078, wherein nucleotide 6078 is an A; nucleotide 6089, wherein nucleotide 6089 is a T;

nucleotide 6195, wherein nucleotide 6195 is an A; nucleotide 6326, wherein nucleotide 6326 is a T; a position corresponding to nucleotides 7205 to 7211, wherein nucleotides 7205 to 7211 are deleted; nucleotide 7376, wherein nucleotide 7376 is a C; a nucleotide sequence corresponding to nucleotides 7535 to 7536, wherein a GCG
5 nucleotide sequence is inserted between nucleotides 7535 and 7536;
nucleotide 7415, wherein nucleotide 7415 is a T; nucleotide 7433, whereiua nucleotide 7433 is a T;
nucleotide 7696, wherein nucleotide 7696 is a T; nucleotide 7883, wherein nucleotide7883 is a T; nucleotide 8021, wherein nucleotide 8021 is an A; a nucleotide sequence corresponding to nucleotide 8159 to 8160, wherein nucleotides 8159 to 10 are deleted; nucleotide 8298, wherein nucleotide 8298 is a G; nucleotide 9164, wherein nucleotide 9164 is a G; nucleotide 9213, wherein nucleotide 9213 is an A;
nucleotide 9326, wherein nucleotide 9326 is a T; nucleotide 9367, wherein nucleotide 9367 is a T; nucleotide 10064, wllerein nucleotide 10064 is an A;
nucleotide 10143, wherein nucleotide 10143 is a G; nucleotide 10234, wherein 15 nucleotide 10234 is a C; or nucleotide 10255, wherein nucleotide 10255 is a T. A
nucleotide sequence useful in a kit of the invention also can comprise one or both primers of a pr.iuner pair, particularly at least a forward primer and a reverse primer as set fortli in SEQ ID NOS: 3 to 18; and tlie lcit can further include at least a second primer pair, including a forward and reverse primer as set forth in SEQ ID NOS: 19 to and 61 to 113. In another aspect, the present invention relates to a kit containing an antibody that specifically binds to a mutant PKD1 polypeptide or peptide portion thereof.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a schematic showing the genomic structure of the PKD 1 gene (SEQ ID NO: 1) and the relative position of locus-specific templates and primers.
Figure 2 shows the relative position of the BPF6-BPR6 long-range PCR
template and the much shorter PKD1-specific exon 28 product, 28F-BPR6. The dashed line below exon 28 identified the long range PCR amplification product that resulted when BPF6, the sequence of which is common to the PKD1 gene and to the homologs, was used in combination with the homolog-specific primer, BPR6HG.

DETAILED DESCRIPTION OF THE INVENTION
The present invention provides conlpositions and methods for identifying polycystic kidney disease-associated protein-1 (PK.D1) gene variants and mutants, and for diagnosing PKD1-associated disorders in a subject. Prior to the present disclostire, the ability to selectively examine the entire PKD 1 gene for mutations was precluded due to the high sequence homology of the PKD 1 gene and the PKD 1 gene homologs, including those present with the PKD1 gene on human chromosome 16.
As disclosed herein, polynucleotide sequences have now been developed that are useful as probes and primers for examining the entire PKD 1 gene. Accordingly, the present invention provides polynucleotides, and oligonucleotide portions thereof, of a PKD 1 gene and of PKD 1 gene mutants that are useful for detecting PKD 1 mutations, and that can be diagnostic of a PKD 1 -associated disorder.

Autosomal dominant polycystic kidney disease (ADPKD) exhibits a transmission pattern typical of autosomal dominant inheritance, where typically each offspring of an affected individual has a 50% chance of inheriting the causative gene.
Linkage studies indicated that a causative gene is present on the short arm of chromosome 16, near the a globin cluster; this locus was designated PKD1 (Reeders et al., Nature, 3 2 7:542, 1985.) Though otller PKD-associated genes exist (for example, PKD2), defects in PKD1 appear to cause ADPKD in about 85-90% of affected families (Parfrey et al., New Eng. J. Med. 323:1085-1090, 1990;
Peters et al., Contrib. Nephrol. 97:128-139, 1992).

The PKD1 gene has been localized to chromosomal position 16p13.3, specifically to an interval of approximately 6001cb betweeii the markers ATPL
and CMM65 (D16S84). This region is rich in CpG islands that often flank transcribed sequences; it has been estimated that this interval contains at least 20 genes. The precise location of the PKD 1 gene was pinpointed by the finding of an ADPKD
fainily whose affected members carry a translocation that disrupts a 141cb RNA
transcript associated with this region (European PKD Consortium, Cell, 77:881, 1994).

The genomic structure of the PKD 1 gene, whicli is illustrated in Figure 1(SEQ
ID NO:1; see Appendix A; see, also, GenBank Accession No. L39891), extends over approximately 50 kb, contains 46 exons, aiid is bisected by two large polypyiimidine tracts of approximately 2.5 kb and 0.5 kb, respectively, in introns -2 1 and 22 (indicated by "...CCTCCTCCT..." in Figure 1). The replicated portion of the gene, wliicll begins prior to the 5'UTR and is believed to end in exon 34 (Figtu-e 1; stippled region), covers approximately two thirds of the 5' end of the gene and is duplicated several tinies in a highly siinilar, transcribed fashion elsewhere in the htunan genome (Germino et al., Genomics 13:144-151, 1992; European Chroniosome 16 Tuberous Sclerosis CoiLsortium, 1993, Cell 75:1305-1315). The encodeci PKDI polypepl:ide is shown as SEQ ID NO:2 (see Appendix A; see, also, GenBank Accession Noõ P98161). It should be recognized that SEQ ID NO:2 is not the same amino"acid sequence as that shown to be encoded by GenBai-ilc Accession No. L39891 (see, also, GenBank AAB59488), presumably due to errors in predicting the encoded PKD 1 polypeptide from the PKD I
gene sequence. Instead, the wild type PKD1 polypeptide sequence is shown in SEQ ID
NO:2 (GenBank Accession No. P98161).

The present invention provides a PKD1 gene specific primer, which can be one of a primer pair. A primer of the invention includes a 5' region and adjacent PKD 1-specific 3' region, wherein the 5' region has a nucleotide sequence that can hybridize to a PKD I gene sequence or to a PKD I gene sequence and a PKD I
gene homolog sequence, and the 3' region has a nucleotide sequence that selectively hybridizes only to a PKD1 gene sequence, and particularly not to a PKD1 gene homolog sequence, except that a primer of the invention does not have a sequence as set forth in SEQ ID NO:11, SEQ ID NO:18, SEQ ID NO:52, or SEQ ID NO:60.
Thus, a primer of the invention can have a seqtzence as set forth in any of SEQ ID
NOS:3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 9-2, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 1.12 and 113, as well as a sequence that is substantially identical ls to any of SEQ ID NOS:3 to 51 and 61 to 113, provided the sequence comprises a 5' region that can hybridize to a PKD1 gene sequence or to a PKD 1 gene sequence and a PKDl gene homolog sequence, and a 3' region that selectively hybridizes to a PKDl gene sequence, but not to a PKD1 gene homolog sequence; and provided the sequence is not otherwise specifically excluded herein.

As disclosed herein, a primer of the invention can be prepared by aligning SEQ ID NO:1 with the PKDI gene homologs contained in GenBank Accession Nos. AC002039, AC010488, AC040158, AF320593 and AF320594;
(see, also, Bogdanova et al., Genomics 74:333-34I, 2001), and identifying regions having potential sequence differences, then selecting as PKD1-specific primers those sequences that match over at least about ten nucleotides and that have a mismatcli at or adjacent to the 3' terminus of the matched regions (see Example 1; see, also, Phakdeekitcharoen et a;l., supra, 2000). Such primers are referred to as "PKDl-specific prilners" because, while they can hybridize to a PKD 1 gene and a PKD
1 gene homologue, an extension product only can be generated upon hybridization to a PKD 1 gene due to the r,nismatch of one or more nucleotides in the 3' region when the primer hybridizes to a PKD1 gene homologue. Confirmation that a selected oligonucleotide is a PK:D1-specific primer can be made using methods as disclosed herein (Example 1) or otherwise kno`vn in the art. For example, a simple and straightforward method for determining that a primer is a PKD 1-specific primer of the invention is to perform a primer extension or an amplification reaction using the putative PKD1-specific primer and templates including a PKD1 gene sequence and PKDI gene homolog sequences, and detecting a single extension product or amplification product generated from the PKDI gene template, but not the PKD1 gene homolog templates. Sequences identified as PKD 1-specific primers using this or a.nother method can be confirnied by performing various control experiments as described by Watnick et: al. (supra, 1999), for example, by comparing an amplification product obtained in a cell having a PKD 1 gene with the products, if any, produced using the radiation hybrid cell line, 145.19, which lacks the PKD1 gene but contains PKD1 gene homologs.

A nucleotide sequence suspected of being useful as a PKD1-specific primer also cai be compared against a human genomic DNA database using, for example, a BLAST search or otlier algorithin, to confirm that the nucleotide sequence meets the requirements of a PKD 1-specific primer as defined herein. For example, a putative PKD 1 -specific primer can be examined at the National Center for Biotechnology Information (NCBI), which can be accessed on the world wide web, by selecting the "Blast" option, thereafter selecting the "Search for short nearly exact matches", entering in the sequence to be examined, and, using the default search algorithms (word size 7), searching the "nr" database, which include all non-redundant GenBank+EMBL+DDBJ+PDB sequences, but no EST, SST, GSS or HTGS
sequences; output can be restricted to showing only the top ten matclles.

In a PKD 1-specific primer of the invention, the 5' region contains at least about ten contiguous nucleotides, generally at least about 12 nucleotides, and usually about 14 to 18 nucleotides. In addition, the 3' region of the primer contains at least one 3' terminal nucleotide, and can include a sequence of at least about 2 to nucleotides, particularly about 2 to 4 nucleotides. Where the 3' region consists of a single 3' terminal nucleotide, the primer is selected such that the 3' terminal nucleotide is identical to a nucleotide that is 5' and adjacent to the nucleotide sequence of the PKD 1 gene to which the 5' region of the primer can hybridize, and is different from a nucleotide that is 5' and adjacent to a nucleotide sequence of the PKD 1 homolog to which the 5' region of the primer can hybridize, i.e., provides a mismatched nucleotide. Where the 3' region of the PKD1-specific primer contains two or more nucleotides, one or more of the nucleotides can be mismatched, and the mismatched nucleotide can, but need not include the 3' terminal nucleotide, provided that when the inismatched nucleotide or nucleotides do not include the 3' terminal nucleotide, the primer cannot be extended when hybridized to a PKDl gene homolog.

PKD1-specific primers of the invention are exemplified by primers that can selectively hybridize to a nucleotide sequence that flanks and is within about fifty nucleotides of a nucleotide sequence of SEQ ID NO:1 selected from about nucleotides 2043 to 4209; nucleotides 17907 to 22489; nucleotides 22218 to 26363;
nucleotides 26246 to 30615; nucleotides 30606 to 33957; nucleotides 36819 to 37140;
nucleotides 37329 to 41258; and nucleotides 41508 to 47320. A primer of the invention is exemplified by any of SEQ ID NOS: 3 to 10, 12 to 17, 19 to 51, and 61 5 to 113, and can have a sequence substantially identical to any of SEQ ID
NOS:3 to 51 and 61 to 113, provided the sequence meets the requirements of a PKD1-specific primer as disclosed 1lerein, and provided the sequence is not a sequence as set forth in any of SEQ ID NO:11, SEQ ID NO:18, SEQ ID NO:52, and SEQ ID NO:60.

10 A primer is considered to be "substantially identical" to any of SEQ ID
NOS:3 to 51 and 61 to 113 if the primer has at least about 80% or 85%, generally at least about 90%, usually at least about 95%, and particularly at least about 99%
sequence identity with one of SEQ ID NOS:3 to 51 and 61 to 113, and has a 5' region and adj acent PKD 1-specific 3' region, wherein the 5' region has a nucleotide sequence that 15 can hybridize to a PKD1 gene sequence or to a PKD1 gene sequence and a PKDl gene hoinolog sequence, and the 3' region has a nucleotide sequence that selectively hybridizes only to a PKD 1 gene sequence, and particularly not to a PKD 1 gene homolog sequence, as defined herein, except that a primer of the invention does not have a sequence as set forth in SEQ ID NO:11, SEQ ID NO:18, SEQ ID NO:52, or 20 SEQ ID NO:60. As such, a primer of the invention can include one or a few, but no more than about four or five, more or fewer nucleotide than a primer as set forth in SEQ ID NOS:3 to 51 and 61 to 113, provided the primer meets the functional requirements as defined herein.

The present invention also provides primer pairs. In one embodiinent, a primer pair of the invention comprising a forward and reverse PKD1-specific primer as disclosed 1lerein. As such, a primer pair of the invention can amplify a portion of SEQ ID NO:1 including about nucleotides 2043 to 4209; nucleotides 17907 to 22489;
nucleotides 22218 to 26363; nucleotides 26246 to 30615; nucleotides 30606 to 33957;
nucleotides 36819 to 37140; nucleotides 37329 to 41258; nucleotides 41508 to 47320;
or a combination thereof. In general, a primer pair of the invention can produce an amplification product of about ten kilobases or shorter, generally about 7500 bases or shorter, and particularly about six kilobases or shorter. Primer pairs of the invention are exemplified by a forward primer and a reverse primer selected from SEQ ID
NOS:3 to 18, for example, by any of SEQ ID NOS:3 and 4; SEQ ID NOS:5 and 6;
SEQ
ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID NOS:13 and 14; SEQ ID NOS:15 and 16; SEQ ID NOS:17 and 18; and SEQ ID NOS:9 and 113, which can be used to produce PKD1-specific amplification products of about 0.31cilobases to about 5.8 kilobases.

As disclosed herein, a set of eight polymerase chain reaction (PCR) primer pairs can be used to prepare PKD 1 -specific amplification products that encompass all of the exons and their flanlcing introns within the replicated region of the PKD 1 gene. In view of the disclosed nucleotide sequences of the primers and of SEQ ID NO:1, it will be recognized that additional PCR priiner pairs useful for a preparing PKD1-specific first amplification product can be based on the exemplified primers and primer pairs, but can include one or few additional nucleotides (based on SEQ ID NO:1) at one or both ends of the exemplified priuners, or can have one or a few nucleotides of an exemplified primer deleted, and their usefulness can be determined by comparing an amplification product generated using the derived or modified primer with a PKD1-specific ainplification product as disclosed herein. As such, a primer pair based, for example, on SEQ ID NOS: 3 and 4 can be used to generate a PKD-1 specific amplification product containing about nucleotides 2043 to 4209 of SEQ ID NO:2, where in reference to "about" nucleotides 2043 to 4209 of SEQ ID NO:2 accounts for the disclosure that a primer pair used for amplification can be identical or substantially identical to SEQ
ID NOS: 3 and 4.
Accordingly, the present invention provides primer pairs comprising a forward primer and a reverse primer having nucleotide sequences as set forth in SEQ ID
NOS:3 to 18; prinier pairs exemplified by SEQ ID NOS:3 and 4; SEQ ID NOS:5 and 6;
SEQ ID
NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID NOS:13 and 14; SEQ ID NOS:15 and 16; SEQ ID NOS:17 and 18; and SEQ ID NOS:9 and 113;
and substantially identical priuner pairs that comprise primers based on or derived from the exemplified primers, such primer pairs being useful for preparing a PKD 1 -specific amplification product. The primer pairs shown as SEQ ID NOS: 9 and 10 and SEQ
ID
NOS: 11 and 12 have been described by Phalcdeekitcharoen et al. (supra, 2000), as have the PKD 1 specific amplification products generated using these primers.

It should be recognized that certain primers and certain primer pairs exemplified herein are not considered to be encompassed w:ihin the present invention. For exainple, the primer set forth in SEQ ID NO:11 has been described in U.S. Pat. No.
6,017,717 (column 24, SEQ ID NO:15); and the primer set forth in SEQ ID NO: 18 has been described by Watnick et al. (Hum. Mol.
Genet.
6:1473-1481,1997; see page 1479; KG8R25), and, therefore, neither of these primers is considered to be a primer of the invention.
Nevertheless, the primers set forth as SEQ ID NOS: 11 and 18 can be encompassed witlun the primer pairs of the invention, including within various disclosed and exemplified primer pairs, for example, the primer pairs set forth as SEQ ID
NOS:11 and 12 and as SEQ ID NOS:17 and 18, as well as within combinations of two or more primer pairs, for example, a combination comprising SEQ ID NOS:l 1 and 12 and SEQ
ID NOS:13 and 14.

The primers set forth in SEQ ID NO:9 and SEQ ID NO: 10 have been described by Watnick etal. (Azn. J. Hum. Genet. 65:1561-1571, 1999) and, therefore, can be specifically excluded from certain embodiinents of the invention, as desired, for example, as encompassed within the primers of the invention. It should be recognized, however, that the combination of SEQ
ID NOS:9 and 10 as a primer pair is not described by Watnick et al. (supra, 1999). SEQ
ID NOS:49 to 51 and 61 to 105 also have been described by Watnick et al.
(supra, 1999) and, therefore, can be specifically excluded from certain embodiments of the invention, as desired.

Except as provided herein, a primer of the invention is exemplified by any of SEQ ID NOS:3 to 51 and 61 to 113, as well as substantially identical oligonucleotide primers that are based on or derived from SEQ ID NOS:3 to 51 and 61 to 113. It should be recognized, however, that the primer set forth as SEQ ID NO:12 is substantially similar to the primer designated TWR2 by Watnick et al. (Mol. Cell 2:247-251, 1998, page 250;
5'-GCAGGGTGAGCAGGTGGGGCCATCCTA-3'; SEQ ID NO:60), and that the primer set forth as SEQ ID NO:10 is substantially identical to SEQ ID NO:13 in U.S.
S Pat. No. 6,071,717 (5'-AGGTCAACGTGGGCCTCCAAGTAGT-3'; SEQ ID NO:52).
As such, a primer having the nucleotide sequence of SEQ ID NO:52 or of SEQ ID
NO:60 is specifically excluded from the priiners that otherwise would be encompassed within the scope of primers that have a sequence substantially identical to the sequence of the primer set forth as SEQ ID NO: 12 or SEQ ID NO: 10, respectively.

The present invention also provides an isolated mutant PKD1 polynucleotide, or an oligonucleotide portion thereof comprising a mutation as disclosed herein. As used herein, the tenn "isolated" or "purified," when used in reference to a polynucleotide, oligonucleotide, or polypeptide, nleans that the material is in a form other than that in which it normally is found in nature. Thus, where a polynucleotide or polypeptide occurs in a cell in nature, an isolated polvnucleotide or purified polypeptide can be one that separated, at least in part, from the materials with which it is normally associated. In general, an isolated polynucleotide or a purified polypeptide is present in a form in which it constitutes at least about 5 to 10% of a composition, usually 20% to 50% of a composition, particularly about 50% to 75% of a composition, and preferably about 90% to 95% or more of a composition.
Methods for isolating a polynucleotide or polypeptide are well known and routine in the art.

As part of or following isolation, a polynucleotide can be joined to other polynucleotides, such as DNA molecules, for example, for mutagenesis studies, to form fiision protei.ns, or for propagation or expression of the polynucleotide in a host.
The isolated polynucleotides, alone or joined to otlier polynucleotides, such as vectors, can be introduced into host cells, in culture or in whole organisms.
Such polynucleotides, when introduced into host cells in culture or in whole organisms, nevertheless are considered "isolated" because they are not in a form in which they exist in nature. Similarly, the polynucleotides, oligonucleotides, and polypeptides can be present in a composition such as a media formulation (solutions for introduction of polynucleotides, oligonucleotides, or polypeptides, for example, into cells or compositions or solutioiis for chemical or enzymatic reactions which are not naturally occurring compositions) and, therein remain isolated polynucleotides, oligonucleotides, or polypeptides within the meaning of that term as it is employed herein. An isolated polynucleotide can be a polynucleotide that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous in a genome or other naturally occurring cellular DNA molecule in nature. Thus, a recombinant polynucleotide, which can comprise a polynucleotide incorporated into a vector, an autonomously replicating plasinid, or a virus; or into the genomic DNA of a prokaryote or eulcaryote, which does not normally express a PKD 1 polypeptide.

As used herein, the term "polynucleotide" or "oligonucleotide" or "nucleotide sequence" or the like refers to a polymer of two or more nucleotides or nucleotide analogs. The polynucleotide can be a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule, and can be single stranded or double stranded DNA or RNA, or a double stranded DNA:RNA hybrid. A polynucleotide or oligonucleotide can contain one or more modified bases, for example, inosine or a tritylated base.
The bonds linking the nucleotides in a polymer generally are phosphodiester bonds, but can be other bonds routinely used to link nucleotides including, for example, phosphorothioate bonds, thioester bonds, and the lilce. A polynucleotide also can be a chemically, enzyinatically or metabolically modified form.

As used herein, the terin "mutant PKD1 polynucleotide" ineans a nucleotide sequence that has one or a few nucleotide changes as compared to the nucleotide sequence set forth as SEQ ID NO: 1. The nucleotide change can be a deletion, insertion or substitution, and can be silent such that there is no change in the reading frame of a polypeptide encoded by the PKD1 polynucleotide, or can be a change that results in an amino acid change or in the introduction of a STOP codon into the polynucleotide, or a change in a nucleotide sequence involved in traiiscription or translation of the PKD 1 polynucleotide, for example, a change that results in altered splicing of a PKD1 gene transcript into an mRNA (see Example 2). As disclosed herein, a mutant PKD 1 polynucleotide can be a polyinorphic variant, which, other than one or a few nucleotide changes with respect to SEQ ID NO: 1, encodes a PKD 1 polypeptide and does not correlate with a PKDl associated disorder, particularly ADPKD, or can be a mutant PKD 1 polynucleotide that contains one or more mutations that correlate with a associated disorder such as ADPKD (see Example 2).

For convenience of discussion and for use as a fiame of reference, the PKD1 nucleotide sequence set forth in SEQ ID NO:1 is referred to as a "wild type polynucleotide" or a "wild type PKD 1 gene" sequence, and, similarly, the polypeptide set forth as SEQ ID NO:2 is referred to as a "wild type PKIDl polypeptide."
However, 10 while the presence of the wild type PKD 1 gene sequence (i.e., SEQ ID NO:1) in an individual correlates to the absence of ADPKD in the individual, it should be recognized that polyinorphic varialts of SEQ ID NO:1 also are found in individuals that do not exhibit ADPKD or other PKD1-associated disorder. The term "variants" or "polymoiphic variants" is used 1lerein to refer to inutant PKD 1 polynucleotide sequences 15 (with respect to SEQ ID NO:1) that do not correlate with the signs or symptoms characteristic of a PKD1 associated disorder such as ADPKD. Variant PKD1 polynucleotides include, for example, nucleotide substitutions that do not result in a change in the encoded amino acid, i.e., silent mutations, such as G4885A, in which the wild type and mutant codons both encode a threonine (T1558T), and C6058T, in which 20 the wild type and mutant codons both encode a serine (S 1949S; see Example 2; see, also, Phalfdeekitcharoen et al., supra, 2000); those that do not segregate with the disease, or those that are found in a panel of unaffected individuals. As such, it should be recognized that the tei7n "mutant PKD 1 polynucleotide" broadly encompasses PKD I
variants, which do not correlate with a PKD 1 associated disorder, as well as mutant 25 PKD1 polynucleotides that correlate or are associated with a PKD1 associated disorder.
Examples of mutant PKD 1 polynucleotide sequences, including variant PKD 1 polynucleotide sequence, include sequences substantially as set forth in SEQ
ID NO: 1, but having a mutation at nucleotide 474, wherein nucleotide 474 is a T;
nucleotide 487, wherein nucleotide 487 is an A; nucleotide 3110, wherein nucleotide 3110 is a C; a position corresponding to nucleotide 3336, wherein nucleotide 3336 is deleted;
nucleotide 3707, wherein nucleotide 3707 is an A; nucleotide 4168, wherein nucleotide 4168 is a T; nucleotide 4885, wherein nucleotide 4885 is an A;
nucleotide 5168, whereiii nucleotide 5168 is a T; nucleotide 6058, wherein nucleotide 6058 is a T; nucleotide 6078, wherein nucleotide 6078 is an A;
nucleotide 6089, wherein iiucleotide 6089 is a T; nucleotide 6195, wherein nucleotide 6195 is an A; nucleotide 6326, wherein nucleotide 6326 is a T; a position corresponding to nucleotides 7205 to 7211, wherein nucleotides 7205 to 7211 are deleted; nucleotide 7376, wherein nucleotide 7376 is a C; a nucleotide sequence corresponding to nucleotides 7535 to 7536, wherein a GCG nucleotide sequence is inserted between nucleotides 7535 and 7536; nucleotide 7415, wherein nucleotide 7415 is a T; nucleotide 7433, wherein nucleotide 7433 is a T; nucleotide 7696, wherein nucleotide 7696 is a T; nucleotide 7883, wherein nucleotide 7883 is a T;
nucleotide 8021, wherein nucleotide 8021 is an A; a nucleotide sequence corresponding to nucleotide 8159 to 8160, wherein nucleotides 8159 to 8160 are deleted;
nucleotide 8298, wliereiii nucleotide 8298 is a G; nucleotide 9164, wherein nucleotide 9164 is a G; nucleotide 9213, wherein nucleotide 9213 is an A;
nucleotide 9326, wherein nucleotide 9326 is a T; nucleotide 9367, wherein nucleotide 9367 is a T; nucleotide 10064, wllerein nucleotide 10064 is an A;
nucleotide 10143, wherein nucleotide 10143 is a G; nucleotide 10234, wherein nucleotide 10234 is a C; or nucleotide 10255, wherein nucleotide 10255 is a T;
or a combiuiation thereof (see Example 2; see, also, Tables 3 and 4). Examples of a mutant PKD1 polynucleotide of the invention also include a polynucleotide that encodes a PKD 1 polypeptide having substantially as set forth in SEQ ID NO:2, but having an A88V, W967R, G1166S; V1956E; R1995H; R2408C; D2604N; L2696R, R2985G, R3039C, V32851, H3311R inutation, or a combination tliereof, as well as polypeptides that have, for example, an addition of a Gly residue between amino acid residues 2441 and 2442 of SEQ ID NO:2 due to an insertion, or that terminate with amino acid 3000 of SEQ ID NO:2 due to the presence of a STOP codon at the position in SEQ ID NO:1 that would otlierwise encode amino acid 3001 (see, also, Table 4; Example 2).

Additional examples of mutant PKD1 polynucleotides of the invention include polynucleotide sequences that selectively hybridize to the coinplements of the polynucleotide sequences, or oligonucleotide portions thereof, as disclosed herein, under highly stringent hybridization conditions, e.g., hybridization to filter-bound DNA in 0.5M NaHPO4i 7% soditun dodecyl sulfate (SDS), I mM EDTA at 65 C, and washing in 0.1 x SSC/0.1 % SDS at 6S C (Ausubel et al., Cui7-ent Protocols in Molecular Biology, (Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York 1989), and supplements; see p. 2.10.3; Sambrook et aL, Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratoiy Press, 1989), as well as polynucleotides that encode a PKD I polypeptide substantially as set for-th in SEQ ID NO:2, but having one or more mutations; or an RNA
corresponding to such a polynucleotide.

A polynucleotide or polypeptide sequence that is "substantially identical" to a PKD I polynucleotide of SEQ ID NO:1 or a polypeptide sequence of SEQ ID NO:2 generally is at least 80% or 85%, usually at least about 90%, and particularly at least about 95%, and preferably at least about 99% identical to the nucleotide sequence or amino acid sequence as set forth in SEQ ID NO: I or SEQ ID NO:2, respectively.
It should be recognized, however, that a mutation in a PKD 1 gene sequence can result in the expression of a h-uncated PKD 1 polypeptide, or even a complete loss of expression of the PKDl polypeptide. As such, while a mutant PKDI
polynucleotide is identified as being substantially identical to SEQ ID NO: 1, it may not always be possible to make the sarne comparison with respect to the encoded polypeptides. In one aspect of the inventrton, a polynucleotide or polypeptide sequence that is substantially identical to SEQ ID NO:1 or 2 will vary at one or more sites having a mutation, for example, a mutation present in a mutant PKD I polynucleotide as set forth in the preceding paragraph. Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison WI 53705).

A polynucleotide or oligonucleotide portion thereof of the invention can be useful, for example, as a probe or as a primer for an amplification reaction.
Reference to an "oligonucleotide portion" of a mutant PKD 1 polynucleotide means a nucleotide sequence of the inutant PKD 1 polynucleotide that is less than the full length polynucleotide. Generally, a polynucleotide useful as a probe or a primer coiitains at least about 10 nucleotides, a.nd usually contains about 15 to 30 nucleotides or more (see, for example, Tables 1 and 2). Polynucleotides can be prepared by any suitable method, including, for example, by restriction enzyme digestion of an appropriate polynucleotide, by direct chemical synthesis using a method such as the phosphotriester method (Narang et al., 1979, Meth. Enzyinol., 68:90-99); the phosphodiester method (Brown et al., 1979, Meth. Enzymol., 68:109-151); the diethylphosphoramidite method (Beaucage et al., 1981, Tetrahedron Lett., 22:1859-1862); the triester method (Matteucci et al., 1981, J. Am. Chem. Soc., 103:3185-3191), including by automated synthesis methods; or by a solid support method (see, for example, U.S. Pat. No. 4,458,066). In addition, a polynucleotide or oligonucleotide can be prepared using recoinbinant DNA methods as disclosed herein or otherwise known in the art.
An oligonucleotide of the invention can include a portion of a mutant PKD 1 polynucleotide, including, for example, a sequence substantially identical to that of SEQ ID NO: 1, except wherein nucleotide 474 is a T; or wherein nucleotide 487 is an A;
or wherein nucleotide 3110 is a C; or wherein nucleotide 8298 is a G; or wherein nucleotide 9164 is a G; or wherein nucleotide 9213 is an A; or wherein nucleotide 9326 is a T; or wherein nucleotide 9367 is a T; or wherein nucleotide 10064 is an A; or wherein nucleotide 10143 is a G; or wherein nucleotide 10234 is a C; or wherein nucleotide 10255 is a T; or wherein the oligonucleotide contains a combination of such substitutions with respect to SEQ ID NO: 1. Thus, as disclosed herein, the oligoilucleotide can be any length and can encompass one or more of the above mutations.

An oligonucleotide of the invention can selectively hybridize to a mutant PKD

polynucleotide sequence as disclosed herein. As such, the oligonucleotide does not hybridize substantially, if at all, to a wild type PKDl polynucleotide (i.e., to SEQ ID
NO: 1). As used herein, the tenn "selectively hybridize" refers to the ability of an oligonucleotide (or polynucleotide) probe to hybridize to a selected sequence, but not to a higlily related nucleotide sequence. For example, a oligonucleotide of the invention selectively 1lybridizes to a mutant PK-D 1 polynucleotide, but not substantially to a corresponding sequence of SEQ ID NO:1. As such, hybridization of the oligonucleotide to SEQ ID NO:1 generally is not above baclcground, or, if sonie hybridization occurs, is at least about ten-fold less than the amount of hybridization that occurs with respect to the mutant PKD1 polynucleotide.

In addition, the tenn "hybridize" is used herein to have its commonly understood meaning of two nucleotide sequences that can associate due to shared complenlentarity.
As disclosed herein, a primer of the invention can hybridize to PDK1 gene and may also hybridize to a PDK1 gene homolog, but generally does not substantially hybridize to a nucleotide sequence other than a PKD 1 gene or PKD 1 gene homolog. Desired hybridization conditions, including those that allow for selective hybridization, can be obtained by varying the stringency of the hybridization conditions, based, in part, on the length of the sequences involved, the relative G:C content, the salt concentration, and the lilce (see Sambrook et al., supra, 1989). Hybridization conditions that are highly stringent conditions are used for selective hybridization ai7d can be used for hybridization of a primer or primer pair of the invention to a PKD1 gene or PKD1 gene homolog, and include, for example, washing in 6 x SSC/0.05% sodium pyrophosphate at about 37 C (for 14 nucleotide DNA probe), about 48 C (for 17 nucleotide probe), about 55 C (for a 20 nucleotide probe), and about 60 C (for a 23 nucleotide probe).
As disclosed lierein, polynucleotides that selectively hybridize to a mutant PKD

polynucleotide provide a means to distinguish the mutant PKD1 polynucleotide from a wild type PKD 1 polyiiucleotide.
A polynucleotide or oligonucleotide of the invention can be used as a probe to screen for a particular PKD 1 variant or mutant of interest. In addition, the oligonucleotides of the invention include a PK.D 1 antisense molecule, which can be useful, for example, in PKD1 polynucleotide regulation an.d amplification reactions of PKDl polynucleotide sequences, including mutant PKD1 polynucleotide sequences.
Further, such oligonucleotides can be used as part of ribozyme or triple helix sequence for PKD1 gene regulation. Still further, such oligonucleotides can be used as a component of diagnostic method, whereby the level of PKD I transcript can be determined or the presence of an ADPKD-causing allele can be detected.
Further, such oligonucleotides can be used, for example, to screen for and identify PKD 1 homologs from other species.

The term "primer" or "PCR primer" refers to an isolated natural or synthetic oligonucleotide that can act as a point of initiation of DNA synthesis wlzen placed under conditions suitable for primer extension. Synthesis of a primer extension product is initiated in the presence of nucleoside triphosphates and a polymerase in an 10 appropriate buffer at a suitable teinperature. A primer can comprise a plurality of primers, for exaniple, where there is some ambiguity in the information regarding one or both ends of the target region to be synthesized. For instance, if a nucleic acid sequence is determined from a protein sequence, a primer generated to synthesize nucleic acid sequence encoding the protein sequence can comprise a collection of 15 primers that contains sequences representing all possible codon variations based on the degeneracy of the genetic code. One or more of the primers in this collection will be hoinologous with the end of the target sequence or a sequence flanlcing a target sequence. Likewise, if a conserved region shows significant levels of polymorphism in a population, mixtures of primers can be prepared that will amplify adjacent 20 sequences.

During PCR ainplification, primer pairs flanlcing a target sequence of interest are used to amplify the target sequence. A primer pair typically comprises a forward primer, which hybridizes to the 5' end of the target sequence, and a reverse primer, 25 wlzich hybridizes to the 3' end of the target sequence. Except as otherwise provided herein, primers of the present inveiition are exemplified by those having the sequences set forth as SEQ ID NOS:3 to 51 and 61 to 113 (see Tables 1 and 2).
Forward primers are exemplified by SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49; and reverse primers are 30 exemplified by SEQ ID NOS:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50. A primer pair of the invention includes at least one forward primer and at least one reverse primer that allows for generation of an amplification product, which can be a long range PKD1-specific amplification product or a nested amplification product of such an amplification product, including a forward and reverse primer as set forth in SEQ ID NOS:3 to 18 and of SEQ ID
NOS:19 to 51 and 61 to 113, provided that the forward primer is 5' (or upstream) of the reverse primer witll reference to a target polynucleotide sequence, and that the primers are in sufficient proximity such that an amplification product can be generated.

Nucleic acid sequences that encode a fusion protein can be produced and can be operatively linlced to expression control sequences. Such fusion proteins and compositions are useful in the development of antibodies or to generate and purify peptides and polypeptides of interest. As used herein, the term "operatively linked"
refers to a juxtaposition, wherein the components so described are in a relationship permitting them to function in their intended manner. For exainple, an expression control sequence operatively linlced to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences, whereas two operatively linked coding sequences can be ligated such that they are in the saine reading frame and, therefore, encode a fusion protein.
As used herein, the term "expression control sequences" refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linlced. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
Thus, expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signals for introns, maintenance of the correct reading frame of that gene to permit proper translation of the mRNA, and STOP codons. Control sequences include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
Expression control sequences can include a promoter.

A polynucleotide of the invention can comprise a portion of a recombinant nucleic acid molecule, which, for exanple, can encode a fusion protein. The polynucleotide, or recoinbinant nucleic acid molecule, can be inserted into a vector, which can be an expression vector, and can be derived from a plasmid, a virus or the like. The expression vector generally contains an origin of replication, a promoter, and one or more genes that allow phenotypic selection of transformed cells containing the vector. Vectors suitable for use in the present invention include, but are not liinited to the T7-based expression vector for expression in bacteria (Rosenberg et al., Gene 56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988); baculovirus-derived vectors for expression in insect cells; and the like.
The choice of a vector will also depend on the size of the polynucleotide sequence and the host cell to be employed in the methods of the invention.
Thus, the vector used in the invention can be plasmids, phages, cosmids, phagemids, viruses (e.g., retroviruses, parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and the like), or selected portions thereof (e.g., coat protein, spike glycoprotein, capsid protein).
For example, cosmids and phagemids are typically used where the specific nucleic acid sequence to be analyzed or modified is large because these vectors are able to stably propagate large polynucleotides. Cosmids and phagemids are particularly suited for the expression or manipulation of the PKD 1 polynucleotide of SEQ ID NO: 1 or a mutant PKD 1 polynucleotide.

In yeast, a number of vectors containing constitutive or inducible promoters can be used (see Ausubel et al., supra, 1989; Grant et al., Meth. Enzymol.
153:516-544, 1987; Glover, DNA Cloning, Vol. II, IRL Press, Washington D.C., Ch. 3, 1986;
and Bitter, Meth. Enzymol. 152:673-684, 1987; and The Molecular Biology of the Yeast Saccharomyces, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I
and II, 1982). A constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL can be used ("Cloning in Yeast," Ch. 3, Rothstein, In "DNA
Cloning"
Vol. 11, A Practical Approach, ed. Glover, IRL Press, 1986). Alternatively, vectors can be used which promote integration of foreign DNA sequences into the yeast chromosome. The construction of expression vectors and the expression of genes in transfected cells involves the use of molecular cloning tecliniques also well known in the art (see Sanibrook et al., supra, 1989; Ausubel et al., supra, 1989).
These methods include in vitro recombinant DNA techniques, synthetic techniques aiid in vivo recombination/genetic recombination.

A polynucleotide or oligonucleotide can be contained in a vector and can be introduced into a cell by transformation or transfection of the cell. By "transformation" or "transfection" is meant a permanent (stable) or transient genetic change induced in a cell following incorporation of new DNA (i.e., DNA
exogenous to the cell). Where the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell.

A transformed cell or host cell can be any prokaryotic or eulcaryotic cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a polynucleotide sequence of the invention or fragment thereof.
Transformation of a host cell can be carried out by conventional tecluiiques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, conlpetent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaC12 method by procedures well known in the art, or using MgC12 or RbCl.
Transformation can also be performed after forming a protoplast of the host cell or by electroporation.

When the host is a eulcaryote, such methods of transfection include the use of calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or the use of virus vectors, or other metlzods known in the art. One method uses a eulcaryotic viral vector, such as simian virus 40 (SV40) or bovine papillomavirus, to transiently infect or transform eulcaryotic cells and express the protein.
(Eulcaryotic Viral Vectors, Cold Spring Harbor Laboratoiy, Gluznlan ed., 1982). Preferably, a eulcaryotic host is utilized as the host cell as described herein. The eulcaryotic cell can be a yeast cell (e.g., Saccharonzyces cerevisiae), or can be a mammalian cell, including a human cell.

A variety of host-expression vector systems can be utilized to express a PKD I
polynucleotide sequence such as SEQ ID NO:1, a coding sequence of SEQ ID NO:1 or a mutant PKD1 polynucleotide. Such host-expression systems represent vehicles by wliich the nucleotide sequences of interest can be produced and subsequently purified, and also represent cells that, wllen transformed or transfected with the appropriate nucleotide coding sequences, can express a PKD 1 protein, including a PKD 1 variant or inutant polypeptide or peptide portion thereof in situ. Such cells include, but are not limited to, inicroorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recoinbinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a PKD1 polyiiucleotide, or oligonucleotide portion thereof (wild type, variant or other mutant); yeast (e.g., Saccharoinyces, Pichia) transformed with recombinant yeast expression vectors containing a PKD1 polynucleotide, or oligonucleotide portions thereof (wild type, variant or other PKD1 mutant); insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing a PKDI
polynucleotide, or oligonucleotide portion thereof (wild type, PKD 1 variant or other mutant); plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmi.d expression vectors (e.g., Ti plasinid) containing a mutant PKD1 polynucleotide, or oligonucleotide portion thereof; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recoinbinant expression constructs containing promoters derived from the genome of manunalian cells (e.g., metallothionein promoter) or from marmnalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K
promoter).

In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the PKD1 protein (wild type, variant or other PKD1 inutaiit) being expressed. For example, when a large quatitity of sucli a protein is to be produced, for the generation of antibodies, which can be used to identify or diagnose PKD I -associated diseases or disorders, or to screen peptide libraries, vectors that direct the expression of high levels of fusion protein products that are readily 5 purified can be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J: 2:1791), in which a polynucleotide, or oligonucleotide portion thereof (wild type, variant or other mutant) can be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye and Inouye, Nucl. Acids Res.
13:3101-10 3109,1985; Van Heeke and Schuster, J. Biol. Chem. 264:5503-5509, 1989); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed 15 to include thrombin or factor Xa protease cleavage sites so that the cloned PKD 1 protein, variant or mutant can be released from the GST moiety.

In an insect system, Autographa califoNnica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera f ugiperda 20 cells. A PKD 1 polynucleotide, or oligonucleotide portion thereof can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
Successful insertion of a PKD 1 polynucleotide, or oligonucleotide portion thereof will result in inactivation of the polyhedrin gene and production of non-occluded 25 recombinant virus (i.e., virus laclcing the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera fi ugiperda cells in wliich the inserted gene is expressed (see Sinith et al., 1983, J. Virol.
46:584; U.S. Pat.
No. 4,215,051).

30 In manvnalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenoviru.s is used as an expression vector, a polynucleotide, or oligonucleotide portion thereof, can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chiineric gene can then be inserted in the adenovirus genonle by in vitro or in vivo recombination. hisertion in a non-essential region of the viral genome such as the El or E3 region results in a recombinant virus that is viable and capable of expressing a PKD1 protein (e.g., wild-type, variants or mutants tllereof) in infected hosts (Logan aizd Shenk, Proc. Natl. Acad. Sci., USA 81:3655-3659, 1984). Specific initiation signals can also be required for efficient translation of an inserted PKD 1 sequence.
These signals include the ATG initiation codon and adjacent sequences. Where an entire PKD1 polynucleotide, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals can be ileeded. However, where only a portion of a PKDI sequence is inserted, exogenous translational control signals, including, for example, an ATG
initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and syntlietic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, and the like (see Bittner et al., Meth. Enzymol. 153:516-544,1987).

In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the expressed polypeptide in a specific fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein being expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the polypeptide can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and the like.

For long tenn, higll yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express a PKD 1 protein, includ'uig wild-type, variants or mutants of PKD 1, can be engiileered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter and/or enhancer sequences, transcription tenninators, polyadenylation sites, and the like), and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be grown for 1-2 days in an enriched media, then switched to selective media.
The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines that express a PKD 1 variant or mutant polypeptide. Such engineered cell lines can be particularly useful in screenuig and evaluation of compounds that affect the endogenous activity of a variant or mutant PKD 1 polypeptide.
Such engineered cell lines also can be useful to discriuninate between factors that have specific vs. non-specific effects. In particular, mutant cell lines should lack key functions, and various mutations can be used to identify lcey functional domains using in vivo assays.

A nulnber of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalslci, Proc. Natl. Acad.
Sci.
USA 48:2026, 1962), and adenine phosphoribosyltransferase (Lowy et al., Cel122:817, 1980) genes can be employed in tl{ , hgprt' or aprf cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA 77:3567, 1980;
O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527, 1981); gpt, which confers resistance to mycophenolic acid Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072, 1981);
neo, which confers resistance to the aininoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol. 150:1, 1981); and /zygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147, 1984) genes. Accordingly, the invention provides a vector that contains a mutant PKD 1 polynucleotide, or oligonucleotide portion thereof, or one or more primers or their complements, including an expression vector that contains any of the foregoing sequences operatively associated with a regulatory element that directs the expression of a coding sequence or primer; and also provides a host cell that contains any of the foregoing sequences, alone or operatively associated with a regulatory element, which can directs expression of a polypeptide encoded the polynucleotide, as appropriate.

In addition to mutant PKDI polynucleotide sequences disclosed herein, homologs of mutant PKD I polynucleotide of the invention, including a non-human species, can be identified and isolated by molecular biological tecluiiques well known in the art. Further, mutant PKD 1 alleles and additional normal alleles of the huinan PKD 1 polynucleotide, can be identified using the methods of the invention.
Still filrther, there can exist genes at other genetic loci within the human genome that encode proteins having; extensive homology to one or more domains of the PKD 1 polypeptide (SEQ ID NO:2). Such genes can also be identified including associated variants and mutants by the methods of the invention.

A homolog of a. mutant PKD1 polynucleotide sequence can be isolated by performing a polymerase chain reaction (PCR; see U.S. Pat. No. 4,683,202) using two oligonucleotide primers, which can be selected, for example, from among SEQ ID NOS:3 to 51, preferably from among SEQ
ID NOS: 3 to 18, or can be degenerate primer pools designed on the basis of the amino acid sequences af a PKD 1 polypeptide such as that set forth in SEQ ID
NO:2 or a mutant thereof as disclosed herein. The template for the reaction can be eDNA

obtained by reverse transcription of mRNA prepared from human or non-human cell lines or tissue lcnown to express a PKD 1 allele or PKD 1 homologue. The PCR
product can be subcloned and sequenced or manipulated in any number of ways (e.g., further manipulated by nested PCR) to insure that the amplified sequences represent the sequences of a PKD 1 or a PKD mutant polynucleotide sequence. The PCR

fragment can then be used to isolate a full length PKD 1 cDNA clone (including clones containing a mutant PKD1 polynucleotide sequence) by labeling the amplified fragment and screening a nucleic acid library (e.g., a bacteriophage cDNA
library).

Alternatively, the labeled fragment can be used to screen a genomic library (for review of cloning strategies, see, for example, Sambrook et al., supra, 1989;
Ausubel et al., supra, 1989).

The present invention also provides a purified mutant PKD 1 polypeptide, or a peptide portion thereof. As disclosed herein, a mutant PKD1 polypeptide has an amino acid sequence substantially identical to SEQ ID NO:2, and includes a mutation resulting in the deletion, addition (insertion), or substitution of an amino acid of SEQ
ID NO:2, or is tz-uncated with respect to SEQ ID NO:2. Examples of such mutations include, with respect to SEQ ID NO:2, an A88V, W967R, G1166S; V1956E;
R1995H; R2408C; D2604N; L2696R, R2985G, R3039C, V32851, or H3311R
mutation, an addition of a Gly residue between amino acid residues 2441 and 2442 of SEQ ID NO:2 due to an insertion, or a truncated PKD 1 polypeptide terminates with amino acid 3000 of SEQ ID NO:2 due to the presence of a STOP codon at the position in SEQ ID NO:1 that would otherwise encode amino acid 3001; as well as mutant PKDI polypeptides having a combination of such mutations (see Table 4).
A mutant PKD1 polypeptide or peptide portion thereof can contain one or more of the exemplified mutations. As used herein, reference to a peptide portion of SEQ ID NO:2 or of a mutant PKD 1 polypeptide refers to a contiguous amino acid sequence of SEQ ID NO:2 or of SEQ ID NO:2 including a mutation as disclosed herein, respectively, that contains fewer amino acids than full length wild type PKD 1 polypeptide. Generally, a peptide portion of a PKD 1 polypeptide or a mutant polypeptide contains at least about five amino acids (or amino acid derivatives or modified amino acids), each linked by a peptide bond or a modified form thereof, usually contains at least about eight amino acids, particularly contains about ten amino acids, and can contain twenty or thirty or more amino acids of SEQ ID
NO:2.
In particular, where the peptide is a peptide portion of a mutant PKD 1 polypeptide, the peptide includes a mutant ainino acid with respect to SEQ ID NO:2.

The mutant PKD 1 polypeptides and peptide fragments thereof of the invention include a PKDI polypeptide or peptide having a sequence substantially identical to that set forth in SEQ ID NO:2, and having one or a combination of the following mutations:
A88V, W967R, L2696R, R2985G, R3039C, V32851, or H331 1R, or a mutation resulting in termiiiation of the mutant PKDI polypeptide at amino acid 3000 (with respect to SEQ ID NO:2) due to the presence of a STOP codon at the position that 5 otherwise would encode amino acid 3001. The wild type PKDl polypeptide (SEQ
ID
NO:2) contains 4303 amino acid residues and has a predicted molecular mass of approximately 467 kilodaltons (kDa). Further encompassed by the present invention are inutant PKD1 polypeptides that are truncated with respect to SEQ ID NO:2, for example, a niutation of SEQ ID NO: 1 resulting in a G9213A, which results in premature 10 tennination of the encoded PKD 1 polypeptide (see Example 2). Such truncated products can be associated witli PKD 1 -associated disorders such as ADPKD (see, also, Table 4).
PKDI polypeptides that are fi.uictionally equivalent to a wild type P.KDl polypeptide, including variant PKD1 polypeptides, which can contain a deletion, 15 insertion or substitution of one or more amino acid residues with respect to SEQ ID
NO:2, but that nevertheless result in a phenotype that is indistingiiishable from that conferred by SEQ ID NO:2, are encompassed within the present invention. Such amino acid substitutions, for example, generally result in similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, amphipatic nature or the lilce of the residues 20 involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids hlclude lysine and arginine;
amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleuchle, valine, glycine, alanine, asparagine, glutamine, serine, tlireonine, pllenylalanine and tyrosine. In many cases, however, a nucleotide substitution 25 can be silent, resulting in no change in the encoded PKD 1 polypeptide (see Exainple 2).
Such variant PKD1 polynucleotides are exemplified by those encoded by the variant PKD 1 polynucleotide sequences substantially identical to SEQ ID NO: 1 (SEQ ID
NO:2), but containing (encoding) G487A (A92A), C9367T (G3052G), T10234C
(L3341L), and G10255T (R3348R) as shown in Table 3 (see, also, Example 2), and by 30 C9494T (L3095L).

Mutant PKD 1 polypeptides and peptide portions thereof that are substantially identical to the PKD1 polypeptide SEQ ID NO:2 or peptide portions thereof, which cause ADPKD symptoins, are encompassed within the scope of the invention. Such mutant PKD 1 polypeptides and peptide portions thereof can include dominant mutant PKD 1 polypeptides, or PKD 1 related polypeptides functionally equivalent to such mutant PKD1 polypeptides. Examples of mutant PKD1 polypeptide sequences include a polypeptide sequences substantially identical to SEQ ID NO:2 having one or more amino acid substitutions such as A88V, W967R, L2696R, R2985G, R3039C, V32851, or H3311R, or truncated after amino acid 3000. A peptide portion of a mutant PKDI
polypeptide can be 3, 6, 9, 12, 20, 50, 100 or more amino acid residues in length, and includes at least one of the mutations identified above.

A PKD 1 wild type or inutant polypeptide, or peptide portions thereof, can be purified fiom natural sources, as discussed below; can be chemically synthesized; or can be recombinantly expressed. For example, one skilled in the art can synthesize peptide fragments correspoiiding to a mutated portion of the PKD1 polypeptide as set forth in SEQ ID NO:2 (e.g., including residue 3110) and use the synthesized peptide fiagment to generate polyclonal and monoclonal antibodies. Synthetic polypeptides or peptides can be prepared by chemical synthesis, for example, solid-phase chemical peptide synthesis methods, which are well lcnown (see, for example, Merrifield, J. Am. Chem.
Soc., 85:2149-2154, 1963; Stewart and Young, Solid Phase Peptide Synthesis, Second ed., Pierce Chemical Co., Rockford, I11., pp. 11-12), and have been employed in commercially available laboratory peptide design and synthesis lcits (Cambridge Research Biochemicals). Such commercially available laboratory kits have generally utilized the teachings of Geysen et al., Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide for synthesizing peptides upon the tips of a multitude of rods or pins, each of which is connected to a single plate. When such a system is utilized, a plate of rods or pins is inverted and inserted into a second plate of corresponding wells or reservoirs, which contain solutions for attaching or anchoring an appropriate amino acid to the tips of the pins or rods. By repeating such a process step, i.e., inverting and inserting the tips of the rods or pins into appropriate solutions, amino acids are built into desired peptides.

A number of available FMOC peptide synthesis systems are available. For example, asseinbly of a polypeptide or fraginent can be carried out on a solid support using an Applied Biosystems, Inc., Model 431A automated peptide synthesizer.
Such equipment provides ready access to the peptides of the invention, either by direct synthesis or by synthesis of a series of fragments that can be coupled using other known techniques. Accordingly, methods for the cheinical synthesis of polypeptides and peptides are well-known to those of ordinary skill in the art, e.g., peptides can be synthesized by solid phase techniques, cleaved from the resin and purified by preparative high perfoi7nance liquid cllromatography (see, e.g., Creighton, 1983, Proteins: Sti-uctures and Molecular Principles, W. H. Freeman & Co., N.Y., pp. 50-60). The composition of the synthetic peptides can be confirmed by amino acid analysis or sequencing;
e.g., using the Edman degradation procedure (see e.g., Creigllton,1983, supra at pp. 34-49). Thus, fragments of the PKD 1 polypeptide, variant, or mutant can be ch.emically synthesized.
Peptides can then be used, for example, to generate antibodies useful in the detection of PKD 1 variants and inutants, as well as the diagnosis of PKD 1-associated disorder (e.g., ADPKD).

A PKD1 polypeptide or peptide, including variants or mutants of the invention, can be substantially purified fiom natural sources (e.g., purified from cells) using protein separation techniques, well known in the art. Such methods can separate the polypeptide away from at least about 90% (on a weight basis), and from at least about 99% of other proteins, glycoproteins, and other macromolecules normally found in such natural sources. Such purification techniques can include, but are not limited to ammonium sulfate precipitation, inolecular sieve chromatography, and/or ion exchange chromatography. Alternatively, or additionally, the PKD 1 polypeptide, variant, or mutant can be purified by immunoaffinity chromatography using an immunoabsorbent column to which an antibody is immobilized that is capable of specifically binding the PKDl polypeptide, variant, or mutant. Such aii antibody can be monoclonal or polyclonal in origin. For example, an antibody that specifically binds to a mutant PKD 1 polypeptide does not bind to a wild-type PKDl polypeptide or peptide thereof.
If the PKDI polypeptide is glycosylated, the glycosylation pattern can be utilized as part of a purification scheme via, for example, lectin chromatography.

The cellular sources from which the PKD1 polypeptide, variant, or mutants thereof can be purified 'Hlclude, for example, those cells that are shown by northern and/or westenl blot aiialysis to express a PKD 1 polynucleotide, variant, or nlutant sequence. Preferably, such cellular sources are renal cells including, for exaniple, renal tubular epithelial cells, as well as biliaty duct cells, skeletal muscle cells, lung alveolar epithelial cell, placental cells, fibroblasts, lymphoblasts, intestinal epithelial cells, and endothelial cells. Other sources include biological fluids, fractionated cells such as organelle preparations, or tissues obtained from a subject. Examples of biological fluids of use with the invention are blood, sei-um, plasma, urine, mucous, and saliva.
Tissue or cell samples can also be used with the invention. The samples can be obtained by many methods such as cellular aspiration, or by surgical renloval of a biopsy sample.

PKD I polypeptides, vaiiants, or mutants of the invention can be secreted out of the cell. Such extracellular forms of the PKD1 polypeptide or mutants thereof can preferably be purified from whole tissue rather than cells, utilizing any of the techniques described above. PKDl expressing cells such as those described above also can be grown in cell culture, tuider conditions well known to those of skill in the art. PKD 1 polypeptide or mutants tliereof can then be purified from the cell media using any of the techniques discussed above.

A PKD1 polypeptide, variant, or nnitant can additionally be produced by recombinant DNA technology using the PKD I nucleotide sequences, variants and mutants described above coupled with techniques well known in the art.
Alternatively, RNA capable of encoding PKDI polypeptides, or peptide fragments thereof, can be chemically synthesized using, for exainple, autonlated or sen-u-autoinated synthesizers (see, for exarnple, "Oligonucleotide Syntliesis", 1984, Gait, ed., IRL Press, Oxford).

When used as a component in the assay systems described herein, the mutant PKD1 polypeptide or peptide can be labeled, either directly or indirectly, to facilitate detection of a complex foimed between the PKDI polypeptide and an antibody or nucleic acid sequence, for example. Any of a variety of suitable labeling systems can be used including, but not l:imited to, radioisotopes such as !`SI, enzyme labeling systenls such as biotin-avidin or ]aorseradish peroxidase, which generates a detectable colorimetr-ic signal or light when exposed to substrate, and fluorescent labels.

The present invention also provides antibodies that specifically bind a PKD1 mutant or PKD I variant, except that, if desired, an antibody of the invention can exclude an antibody as described in U.S. Pat. No. 5,891,628, or an antibody that that specifically binds a PKD 1 mutant as described in U.S.
Pat. No. 5,891,628. Antibodies that specifically bind a mutant PKD 1 polypeptide are useful as diagnostic or therapeutic reagents and, therefore, can be used, for example, in a diagnostic assay for identifying a subject having or at risk of having ADPKD, and are particularly convenient when provided as a kit.
As used herein, the term "specifically binds," when used in reference to an antibody and an antigen or epitopic portion thereof, means that the antibody and the antigen (or epitope) have a dissociation constant of at least about 1 x 10-7, generally at least about 1 x 10-8, usually at least about 1 x10-9, and particularly at least about 1 x 10"10or less. Methods for identifying and selecting an antibody having a desired specificity are well known and routine in the art (see, for example, Harlow and Lane, "Antibodies: A Laboratoiy Manual" (Cold Spring Harbor Pub. 1988), which is incorporated herein by reference.

Methods for proclucing antibodies that can specifically bind one or more PKD 1 polypeptide epitopes, particularly epitopes unique to a mutant PKD1 polypeptide, are disclosed herein or otherwise well lalown and routine in the art. Such antibodies can be polyclonal antibodies or monoclonal antibodies (mAbs), and can be humanized or chimeric antibodies, single chain antibodies, anti-idiotypic antibodies, and epitope-binding fragments of any of the above, including, for example, Fab fragments, F(ab')2 fragments or fragments produced by a Fab expression library. Such antibodies can be used, for example, in the detection of PKDl polypeptides, or mutant PKDl polypeptides, including variant PKD1 polypeptides, wluch can be in a biological sample, or can be used for the inhibition of abnorznal PKDl activity. Thus, the antibodies can be utilized as part of ADPIM treatment methods, as well as ui diagnostic methods, for example, to detect the presence or amount of a PKDI polypeptide.

For the production of antibodies that bind to PKD 1, including a PKD 1 variant or PKD1 mutant, various tiost animals can be immunized by injection with a PKD1 polypeptide, mutant polypeptide, vaiiant, or a portion thcreof. Such host animals can include but are not liunited to, rabbits, mice, and rats. Various adjuvants can be used to 10 increase the inuntuiological response, depending on the host species, including, but not limited to, Freund's (complete and ulcomplete), inineral gels such as aluminum hydroxide, suiface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole lunpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calnlette-Guerin) or Colynebacterium 15 parvuriz.

Antibodies that bind to a mutant PKD 1 polypeptide, or peptide portion thereof, of the invention can be prepared usnlg an intact polypeptide or fragments containing small peptides of interest as the iunmunizing antigen. The polypeptide or a peptide used 20 to inununize an animal can be derived fronl ti-anslated eDNA or chemical synthesis, and can be conjugated to a carrier protein, if desired. Such commonly used carriers that can be chemically coupled to the peptide include keyhole limpet heinocyanin, thyroglobulin, bovine sen.un albumin, tetanus toxoid and otllers as described above or otherwise known in the art. The coupled polypeptide or peptide is then used to immunize the animal and 25 antiseruni can be collected. If desired, polyclonal or monoclonal antibodies can be purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is botuld. Any of various tecluliques commonly used in imn-iunology for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies, can be used (see for example, Coligan, et 30 aL, Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991).

Anti-idiotype technology can be used to produce monoclonal antibodies that mimic an epitope. For example, an atiti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region that is the unage of the epitope bound by the first monoclonal antibody. Antibodies of the invention include polyclonal antibodies, monoclonal antibodies, and fragments of polyclonal and monoclonal antibodies that specifically bind to a mutant PKD 1 polypeptide or peptide portion thereof.

The preparation of polyclonal antibodies is well I:nown to those skilled in the art (see, for example, Green et aL, Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992); Coligan et aL, Production of Polyclonal Antisera. ui Rabbits, Rats, Mice and Hamsters, in Current Protocols in Immunology, section 2.4.1 (1992), which are incorporated herein by reference).
The preparation of monoclor.tal antibodies likewise is conventional (see, for example, Kohler and Milstein, Nature, 256:495, 1975; see, also Coligan et al., supra, sections 2.5.1-2.6.7; and Harlow et al., supra, 1988).
Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a ser-um sample, removing the spleen to obtain B lymphocytes, fusing tlie B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A SepharoseTM , size-exclusion chromatography, and ion-exchange chromatography (see Coligan et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al., Purification of Imrnunoglobulin G (IgG), in Methods in Molecular Biology, Vol. 10, pages 79-104 (Hmnana Press 1992)). Methods of in vitro and in vivo multiplication of hybridoma cells expressing monoclonal antibodies is well-known to those skilled in the art. Multiplication in vitl=o can be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally repleiushed by a inanunalian serum such as fetal calf senun or trace elements and growth-sustaining supplements such as noimal mouse peritoneal exudate cells, spleen cells, bone mai7-ow macrophages. Production in viti-o provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies. Large scale lrybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture.
Multiplication in vivo caui be carried out by injecting cell clones into mammals histocompatible with the parent cells, e.g., syngeneic mice, to cause growth of antibody-producing twnors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane tetranethylpentadecane prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.
Therapeutic applications for antibodies disclosed herein are also part of the present invention. For example, antibodies of the present invention can be derived from subhuman priniate antibodies. General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in Goldenberg el al., International Application Publication No. WO 91/11465, 1991; Losman et al., Int. J. Cancer, 46:310, 1990.

An anti-PKD 1 arttibody also can be derived from a'"humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse iznmunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counteiparts. The use of antibody components derived fi-om hunlanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine inuntmoglobulin variable domains are described, for example, by Orlandi et al., Proc. Nati. Acad. Sci. USA 86:3833, 1989.

Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature, 321:522, 1986; Riechmann et al., Nature 332:323, 1988;
Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Nati. Acad.
Sci. USA, 89:4285, 1992; Sandliu, Crit. Rev. Biotech. i 2:437, 1992; and Singer el al., J. Immunol.
150:2844, 1993.

Antibodies of the invention also can be derived from human antibody fragments isolated fi-om a combinat:orial imrnunoglobulin library (see, for example, Barbas et al., Methods: A Companion to Methods in Enzymology, Vol. 2, page 119, 1991; Winter et al., Ann. Rev. Immunol. 12:433, 1994).
Cloning and expression vectors that are useful for producing a human inirnunoglobulin phage library can be obtained, for example, from Stratagene (La Jolla CA).
In addition, antibodies of the present invention can be derived fi-om a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific lnunan antibodies in response to antigenic challenge.
In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived fi-om embryonic stem cell lines that contain targeted disruptions of the endogenous heavy aiid light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the nlice can be used to produce human antibody-secreting hybridomas. Methods for obtaining hunlan antibodies from transgenic nlice are described by Green et al., Nature Genet., 7:13 (1994);
Lonberg et al., Nature, 368:856 (1994); and Taylor et al., Int. Inullunol., 6:579 (1994).

Antibody fragments of the invention can be prepared by proteolytic hydrolysis of an antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For exaniple, antibody fi-agments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragnlent denoted F(ab')2.
This fragment can be fiu-ther cleaved using a thiol redticing agent, and optionally a blocking group for the sulfhydryl groups resulting fi-om cleavage of distflfide linlcages, to produce 3.5S Fab' monovalent fi-agments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, (see, also, Nisonhoff et al., Arch. Biochem. Biophys,. 89:230, 1960; Porter, Biochem. J.
73:119, 1959; Edelnlan et al., Meth. Enzylnol. 1:422, 1967; and Coligan et al., at sections 2.8.1-2.8.10 and 2.10.1-2.10.4). Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques can also be used, provided the fragn7ents bind to the antigen that is recogliized by the intact antibody.

Fv fragments coniprise an association of VH and VL chains, for exanlple, which can be noncovalent (see Inbar et al., Proc. Natl. Acad. Sci. USA 69:2659, 1972). The variable chains also can be linked by an intei-molecular disulfide bond, can be crosslinked by a chemical such as glutaraldehyde (Sandhu, supra, 1992), or F, fra.gments comprisulg Vn and VL chains can be connected by a peptide linker. These single chain antigen bind'uig proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encodirig the VI{ and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E: coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for exaniple, by Whitlow et al., Methods: A
Companion to Metli. Enzymol., 2:97, 1991; Bird et al., Science 242:423, 1988; Ladner et al., U.S.
Patent No. 4,946,778; Pack et al., BioTecluiology t 1:1271, 1993; and Sandhu, supra, 1992).

Another form of an antibody fragment is a peptide coding for a single complementarity detelrnining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes eneoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, for example, Larrick et al., Methods: A Companion to Meth. Enzymol., 2:106, 1991).

A variety of inethods can be employed utilizing reagents such as a mutant PKD1 polynucleotide, or oligonucleotide portion tliereof and aitibodies directed against a mutant PKD1 polypeptide or peptide. Specifically, such reagents can be used for the detection of the presence of PKD 1 mutations, e. g. , molecules present in diseased tissue 5 but absent from, or present in greatly reduced levels coinpared or relative to the corresponding non-diseased tissue.

The methods described herein can be perfoi7iied, for example, by utilizing pre-packaged kits, wllich can be diagnostic kits, comprising at least one specific 10 oligonucleotide portion of a PKD 1 gene or mutant PKD 1 polynucleotide, a primer pair, or an anti-PKD1 antibody reagent as disclosed herein, which can be conveniently used, for example, in a clinical setting to diagnose subjects exhibiting PKDI
abnormalities or to detect PKDl-associated disorders, including ADPKD. Any tissue in which a polynucleotide is expressed can be utilized in a diagnostic method of the invention.

Nucleic acids fiom a tissue to be analyzed can be isolated using procedures that are well known in the art, or a diagnostic procedures can be performed directly on a, tissue section (fixed or frozen), which can be obtained from a subject by biopsy or resection, without furtller purification. Oligonucleotide sequences of the invention can be used as probes or primers for such in situ procedures (Nuovo, 1992, PCR in situ hybridization: protocols and applications, Raven Press, N.Y.). For example, oligonucleotide probes useful in the diagnostic methods of the invention include nucleotide sequences having at least 10 contiguous nucleotides and having a sequence substantially identical to a portion of SEQ ID NO: 1, and including nucleotide 474, wherein nucleotide 474 is a T; nucleotide 487, wherein nucleotide 487 is an A;
nucleotide 3110, wherein nucleotide 3110 is a C; nucleotide 8298, wherein nucleotide 8298 is a G; nucleotide 9164, wherein nucleotide 9164 is a G;
nucleotide 9213, wherein nucleotide 9213 is aii A; nucleotide 9326, wherein nucleotide 9326 is a T; nucleotide 9367, wherein nucleotide 9367 is a T;
nucleotide 10064, wlierein nucleotide 10064 is an A; nucleotide 10143, wherein nucleotide 10143 is a G; nucleotide 10234, wherein nucleotide 10234 is a C;
nucleotide 10255, wllerein nucleotide 10255 is a T, or a combination thereof.
Primers useful in the present invention include those set forth in SEQ ID NOS:3 to 18 and SEQ
ID NOS: 19 to 51 and 61 to 112. Sucli prnners flaiAc and can be used to amplify sequences containing one or more mutated nucleotides of a mutant PKD1 polynucleotide.

PKD I polynucleotide seq uences, either RNA or DNA, can be used in hybridization or aniplification assays of biological sainples to detect abnormalities of PKD1 expression; e.g., Southem or noithern blot analysis, single stranded confoimational polymorphism (SSCP) analysis including in situ hybridization assays, or polymerase chain reaction analyses, including detecting abnormalities by a methods such as denaturing high performance liqtiid chromatography (DHPLC; also referred to as temperature-modulated heteroduplex chromatography) or confoimation sensitive gel electrophoresis (CSGE)., both of which are readily adaptable to high tlvroughput analysis (see, for example, Kiistensen et al., BioTechniques 30:318-332, 2001; Leung et al., BioTechniques 30:334-340, 2001). Such analyses can reveal quantitative abnoi-inalities in the expression patteni of the PKD I
polynucleotide, and, if the PKD I mutation is, for example, an extensive deletion, or the result of a chromosomal rearrangement, can reveal more qualitative aspects of the PKD 1 abnormaIity.

Diagnostic methods for detecting a mutant PKD I polynucleotide can involve, for example, contacting and incubating nucleic acids derived fi=om a tissue sample being analyzed, with one or more labeled oligonucleotide probes of the invention or with a primer or primer pair of the invention, under conditions favorable for the specific annealing of these reagents to their complementary sequences within the target molecule.
After incubation, non-annealed oligonucleotides are removed, and hybridization of the probe or pruner, if any, to a nucleic acid from the target tissue is detected.
Using such a detection scheme, the target tissue nucleic acid can be inunobilized, for example, to a solid support such as a rrlembrane, or a plastic surface such as that on a microtiter plate or polystyrene beads. In this case, after incubation, non-annealed, labeled nucleic acid reagents are easily removed. Detection of the remaining, annealed, labeled nucleic acid ieagents is accomplished using standard techniques well known to those in the art.

Oligonucleotide probes or primers of the invention also can be associated with a solid matrix such as a cl-iip in an an ay, thus providing a means for high throughput methods of analysis. Microfabricated ailays of large numbers of oligonucleotide probes (DNA chips) are useful :for a wide variety of applications. Accordingly, methods of diagnosing or detecting a PKD1 variant or mutant can be implemented using a DNA
chip for analysis of a PK:D 1 polynucleotide and detection of mutations therein. A
methodology for large scale analysis on DNA chips is described by Hacia et al.
(Nature Genet. 14:441-447, 199(5; U.S. Pat. No. 6,027,880;
see, also, Kristensen et al., supra, 2001). As described in Hacia et aL, high density arrays of over 96000 oligonucleotides, each about 20 nucleotides in length, are immobilized to a single glass or silicon chip using light directed chemical synthesis.
Contingent on the nun7ber and design of the oligonucleotide probe, potentially every base in a sequence can be exaniuned for altei-ations.

Polynucleotides or oligonucleotides applied to a cliip can contain sequence variations, which can be used to identify mutations that are not yet known to occur in the population, or they can only those mutations that are known to occur, including those disclosed herein (see Example 2). Exaniples of oligonucleotides that can be applied to the chip include oligonucleotides containing at least 10 contiguous nucleotides and having a sequence substantially identical to a portion of SEQ ID NO:l, and including nucleotide 474, wherein nucleotide 474 is a T; nucleotide 487, wherein nucleotide 487 is an A; nucleotide 3110, wherein nucleotide 3110 is a C; a position corresponding to nucleotide 3336, wherein nucleotide 3336 is deleted; nucleotide 3707, wherein nucleotide 3707 is an A; nucleotide 4168, wherein nucleotide 4168 is a T;
nucleotide 4885, wherein nucleotide 4885 is an A; nucleotide 5168, wherein nucleotide 5168 is a T; nucleotide 6058, wherein nucleotide 6058 is a T;
nucleotide 6078, wherein nucleotide 6078 is an A; nucleotide 6089, vvherein nucleotide 6089 is a T; nucleotide 6195, whereui nucleotide 6195 is an A.

nucleotide 6326, wherein nucleotide 6326 is a T; a position corresponding to nucleotides 7205 to 7211, wherein nucleotides 7205 to 7211 are deleted;
nucleotide 7376, wherein nucleotide 7376 is a C; a nucleotide sequence corresponding to nucleotides 7535 to 7536, wherein a GCG nucleotide sequence is inserted between nucleotides 7535 and 7536; nucleotide 7415, wherein nucleotide 7415 is a T;
nucleotide 7433, wherein nucleotide 7433 is a T; nucleotide 7696, wherein nucleotide 7696 is a T; nucleotide 7883, wherein nucleotide 7883 is a T;
nucleotide 8021, wherein nucleotide 8021 is an A; a nucleotide sequence corresponding to nucleotide 8159 to 8160, wherein nucleotides 8159 to 8160 are deleted;
nucleotide 8298, wherein nucleotide 8298 is a G; nucleotide 9164, wherein nucleotide 9164 is a G; nucleotide 9213, wlzerein nucleotide 9213 is an A;
nucleotide 9326, wherein nucleotide 9326 is a T; nucleotide 9367, wherein nucleotide 9367 is a T; nucleotide 10064, wherein nucleotide 10064 is an A;
nucleotide 10143, wherein nucleotide 10143 is a G; nucleotide 10234, wherein nucleotide 10234 is a C; nucleotide 10255, wherein nucleotide 10255 is a T; or a combination thereof.

Prior to hybridization with oligonucleotide probes on the chip, the test sample is isolated, amplified and labeled (e.g. fluorescent markers). The test polynucleotide sample is then hybridized to the iminobilized oligonucleotides. The intensity of sequence-based techniques of the target polynucleotide to the iminobilized probe is quantitated and compared to a reference sequence. The resulting genetic information can be used in molecular diagnosis. A common utility of the DNA chip in molecular diagnosis is screening for lcnown inutations.

In addition to DNA chip methodology, methods using machinery adapted to DNA analysis can allow for commercialization of the disclosed methods of detection of PKD 1 mutations and diagnosis of ADPKD. For example, genotyping by mass spectrometry can be used, or matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry can be used for mass genotyping of single-base pair and short tandem repeat mutant and variant sequences. For example, PCR
amplification of the region of the inutation with biotin attached to one of the primers can be conducted, followed by immobilization of the amplified DNA to streptavidin beads.
Hybridization of a primer adjacent to the variant or mutant site is performed, then extension with DNA
polymerase past the variant or mutant site in the presence of dNTPs and ddNTPs is performed. When suitably designed according to the sequence, this results in the addition of or-dy a few additional bases (Braun, Little, Koster, 1997). The DNA is then processed to reniove unused nucleotides and salts, and tl2e short primer plus mutant site is removed by denaturation and trausferTed to silicon wafers using a piezoelectric pipette.
The mass of the primer+variant or niutant site is then determined by delayed extraction MALDI-TOF inass spectrometry. Single base pair and tandem repeat variations in sequence are easily deteimined by their rnass. This final step is very rapid, reqturing only 5 sec per assay, and all of these steps can be automated, providing the potential of performing up to 20,000 genotypings per day. This technology is rapid, extremely accurate, and adaptable to any variant or mutation, can identify both single base pair and short tandem repeat variants, and adding or removing variant or mutant sequences to be tested can be done in a few seconds at trivial cost.

Another diagnostic methods for the detection of mutant PKD1 polynucleotides involves amplification, for exarnple, by PCR (see U.S. Patent No. 4,683,202), ligase chain reaction (Barany, :Proc. Natl. Acad. Sci. USA 88:189-193, 1991 a), self sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990), transcriptional amplification system (Kwoh et al., Proc. Natl. Acad. Sci. USA
86:1173-1177, 1989), Q-Beta ReplicaseT"' (Lizardi et al., Bio/Technology 6:1197, 1988), or any other RNA aniplificatiori method, followed by the detection of the aniplification products. The present invention provides reagents, niethods and compositions that can be used to overcome prior difficulties with diagnosing ADPKI).

Using the primer pairs and methods described herein, the entire replicated segment of the PKD 1 gene, including exons 1 and 22, can be amplified from genomic DNA to generate a set of eight long range amplification products, which range in size from about 0.3 kb to 5.8 kb (Table 1; see, also, Figure 1). The availability of widely scattered PKD1-specific primers provides a means to anchor PKD1-specific amplification, and the ability to use various primer combinations provides a means to produce longer or shorter amplification products as desired. For example, the largest PKDI fragment, which is amplified by primers BPF13 and KG8R25 (see Table 1;
SEQ ID NOS: 17 and 18, respectively), can be divided into two shorter segments by using the PKD 1-specif c primer, KG85R25 (SEQ ID NO:18), with forward nested primer F32 (5'-GCCTTGCGCAGCTTGGACT-3'; SEQ ID NO:53), and using BPF13 (SEQ ID NO:17) and a second specific primer, 31R
(51-ACAGTGTCTTGAGTCCAAGC-3'; SEQ ID NO:54).

It should be recognized that, while many of the primers disclosed herein are positioned wit11 intronic sequences of the PKD 1 gene, others such as SEQ ID
NO: 16 are positioned in coding sequences. As such, a cDNA molecule can obtained from a target RNA molecule, for example, by reverse transcription of the RNA molecule using a 10 primer such as SEQ ID NO: 16 and an appropriate second primer positioned 5' or 3' to SEQ ID NO: 16. In this embodiment, a PKD 1 RNA can be isolated fiom any tissue in which wild type PKD1 is known to be expressed, including, for example, kidney tissue, nucleated peripheral blood cells, and fibroblasts. A target sequence witliin the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR
15 amplification reaction, or the like. An amplification product can be detected, for example, using radioactively or fluorescently labeled nucleotides or the like and an appropriate detection system, or by generating a sufficient amount of the amplification product such that it can be visualized by ethidium bromide staining and gel electrophoresis.
Genomic DNA from a subject, including from a cell or tissue sample, can be used as the template for generating a long range PKD1-specific amplification product.
Methods of isolating genomic DNA are well known and routine (see Sambrook et al., supra, 1989). Amplification of the genomic PKD1 DNA has advantages over the cDNA amplification process, including, for example, allowing for analysis of exons and introns of the PKD 1 gene. As such, a target sequence of interest associated witli either an intron or exon sequence of a PKD1 gene can be amplified and characterized.
A target sequence of interest is any sequence or locus of a PKD 1 gene that contains or is thought to contain a inutation, including those inutations that correlate to a PKD1-associated disorder or disease.

Using primers flanking the target sequence, a sufficient number of PCR cycles is performed to provide a PKD 1-specific amplification product coiresponding to the target sequence. If desired, additional amplification can be perfonned, for exainple, by performing a nested PCR reaction. Examples of primers usefuI for generating a PKDI-specific fu-st amplification product from genomic DNA include the primer pairs having sequences as exemplified in SEQ ID NO:3 and 4; SEQ ID NOS:5 and 6;
SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID
NOS: 13 and 14; SEQ ID NOS: 15 and 16; and SEQ ID NOS: 17 and I S. The PKD1-specific first amplification product can be further amplified using nested primers specific for a target sequence, including the primer pairs exemplified as SEQ ID NOS:
19 and 20;
SEQ ID NOS:21 and 22; SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID
NOS:27 and 28; SEQ 11) NOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ ID NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and 40;
SEQ ID NOS:4I and 42; SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID
NOS:47 aiid 48; SEQ II) NOS:49 and 50; SEQ ID NOS:51 and 61; and the primer pairs fonned using consecutive primers set forth in Table 2 as SEQ ID NOS:62 to 96, 113, and 97 to 112.

The anlplified target sequences can be examined for changes (i.e., mutations) with respect to SEQ ID NO:1 using any of various well known methods as disclosed herein or otherwise known in the art. For example, the amplification products can simply be sequenced using routine DNA sequencing methods, particularly where only one or few amplification products are to be examined. However, DNA sequencing will be more valuable as a inethod of detecting mutations according to a method of the invention as sequeneing technology iinproves and becomes more adaptable to high throughput screening assays. In addition, methods that are useful for detecting the presence of a mutation in a DNA sequence include, for example, DHPLC (Huber et al., NucI. Acids Res. 21:106I-10666, 1993; Liu et al., Nuc1. Acids Res. 26:1396-1400, 1998;
Choy et al., Ann. HLUn. Genet. 63:383-391, 1999; Ellis et al., Hum. Mutat.
15:556-564, 2000; see, also, Kristensen et al., supra, 2001); CSGE (Leung et al., supra, 2001); single-stranded conformation analysis (SSCA;
Orita et al., Proc. Nati. Acad. Sci., USA 86:2766-2770, 1989); denaturing gradient gel electrophoresis (DGGE; Sheffield et al., Proc. Natl. Acad. Sci., USA 86:232-23 6, 1989);
RNase protection assays; allele-specific oligonucleotides (ASOs; Handelin and Shuber, Current Protocols in Hunian Genetics, Suppl. 16 (John Wiley & Sons, Inc.
1998), 9:9.4.1-9.4.8); the use of proteins that recognize nucleotide mismatches, such as the E. coli mutS protein; and allele-specific PCR.

For allele-specific PCR, primers are used that hybridize at their 3' ends to a particular mutations. Examples of primers that can be used for allele specific PCR
include an oligonucleotide of at least 10 nucleotide of SEQ ID NO:1 and that has at its 3' end nucleotide 474, wherein nucleotide 474 is a T; nucleotide 487, wherein nucleotide 487 is an A; nucleotide 3110, wherein nucleotide 3110 is a C;
nucleotide 8298, wherein nucleotide 8298 is a G; nucleotide 9164, wherein nucleotide 9164 is a G; nucleotide 9213, wherein nucleotide 9213 is an A;
nucleotide 9326, wherein nucleotide 9326 is a T; nucleotide 9367, wherein nucleotide 9367 is a T; nucleotide 10064, wherein nucleotide 10064 is an A;
nucleotide 10143, wherein nucleotide 10143 is a G; nucleotide 10234, wherein nucleotide 10234 is a C; or nucleotide 10255, wherein nucleotide 10255 is a T.
If the particular mutation is not present, an amplification product is not observed.
Amplification Refractory Mutation System (ARMS) can also be used (see European Patent Application Publ. No. 0332435; Newton et aZ., Nucl. Acids. Res. 17:2503-2516, 1989).

In the SSCA, DGGE and RNase protection methods, a distinctive electrophoretic band appears. SSCA detects a band that migrates differentially because the sequence change causes a difference in single-strand, intramolecular base pairing.
RNase protection involves cleavage of the mutant polynucleotide into two or more smaller fiagments. DGGE detects differences in migration rates of mutant sequences compared to wild-type sequences, using a denaturing gradient gel. In an allele-specific oligonucleotide assay, an oligonucleotide is designed that detects a specific sequence, and the assay is performed by detecting the presence or absence of a hybridization signal. In the mutS assay, the protein binds only to sequences that contain a nucleotide mismatch in a heteroduplex between mutant and wild-type sequences.

Denaturing gradient gel electrophoresis is based on the inelting behavior of the DNA fiagments and the use of denaturing gradient gel electrophoresis as shown by Fischer and Lerman, Proc. Natl. Acad. Sci. USA 80:1579-83,1983; Myers et al.;
Nucl.
Acids Res. 13:3111-3129, 1985; Lerman et al., in Molecular Biol. of Homo Sapiens, Cold Spring Harbor Lab. (1986) pp. 285-297. DNA fragments differing by single base substitutions can be separated from each other by electrophoresis in polyacrylamide gels containing an ascending gradient of the DNA denaturants urea and formamide.
Two identical DNA fragments differing by only one single base pair, will initially move through the polyacrylamide gel at a constant rate. As they migrate into a critical concentration of denaturant, specific domains within the fragments melt to produce partially denatured DNA. Melting of a domain is accompaiiied by an abrupt decrease in mobility. The position in the denaturant gradient gel at which the decrease in mobility is observed corresponds to the melting temperature of that domain. Since a single base substitution within the melting domain results in a melting temperature difference, partial denaturation of the two DNA fragments will occur at different positions in the gel. DNA molecules can therefore be separated on the basis of very small differences in the melting temperature. Additional improvements to this DGGE have been made as disclosed by Borresen in US Patent No. 5,190,856. In addition, after a first DGGE
analysis, an identified product can be cloned, purified and analyzed a second time by 'DGGE.

Denaturing high performance liquid chromatography (DHPLC; Kristensen et al., supra, 2001) and high tliroughput conformation sensitive gel electroplioresis (HTCSGD;
Leung et al., supra, 2001) are particularly useful methods for detecting a mutant PKD1 polynucleotide sequence because the methods are readily adaptable to high throughput analysis. In addition, these methods are suitable for detecting known mutations as well as identifying previously unknown mutations. As such, these methods of detection can be adopted for use in clinical diagnostic settings. DHPLC, for example, can be used to rapidly screen a large number of samples, for example, 96 samples prepared using a 96 well microtiter plate format, to identify those showing a change in the denaturation properties. Where such a change is identified, confirmation that the PKD 1 polynucleotide in the sample showing the altered denaturation property is a mutant PKD 1 polynucleotide can be confirmed by DNA sequence analysis, if desired.

An oligonucleotide probe specific for a mutant PKD 1 polynucleotide also can be used to detect a inutant PKD I polynucleotide in a biological sample, including in a biological fluid, in cells or tissues obtained from a subject, or in a cellular fraction suclz as an organelle preparation. Cellular sources useful as samples for identifying a inutant PKDI polynucleotide include, for example, renal cells including renal tubular epithelial cells, bile duct cells, skeletal muscle cells, lung alveolar epithelial cells, placental cells, fibroblasts and lyinphocytes. Biological fluids useful as samples for identifying a znutant PKDl polynucleotide include, for example, whole blood or serum or plasma fractions, urine, mucous, and saliva. A biological sample such as a tissue or cell sample can be obtained by any method routinely used in a clinical setting, including, for example, by cellular aspiration, biopsy or other surgical procedure.

The oligonucleotide probe can be labeled with a compound that allows detection of binding to a mutant PKD 1 polynucleotide in the sample. A
detectable compound can be, for example, a radioactive label, which provides a highly sensitive means for detection, or a non-radioactive label such as a fluorescent, luminescent, chemiluminescent, or enzymatically detectable label or the lilce (see, for exaniple, Matthews et al., Anal. Biochem. 169:1-25, 1988).

The method of detection can be a direct or indirect method. An indirect detection process can involve, for exainple, the use of an oligonucleotide probe that is labeled with a hapten or ligand such as digoxigenin or biotin. Following hybridization, the target-probe duplex is detected by the formation of an antibody or streptavidin complex, which can further include an enzyme such as horseradish peroxidase, alkaline phosphatase, or the like. Such detection systems can be prepared using routine methods, or can be obtained from a commercial source. For example, the GENIUS detection system (Boehringer Mannheim) is useful for mutational analysis of DNA, and provides an indirect method using digoxigenin as a tag for the oligonucleotide probe and an anti-digoxigenin-antibody-alkaline phosphatase conjugate as the reagent for identifying the presence of tagged probe.

Direct detection methods can utilize, for example, fluorescent labeled 5 oligonucleotides, lanthanide chelate labeled oligonucleotides or oligonucleotide-enzyme conjugates. Examples of fluorescent labels include fluorescein, rhodamine aiid phthalocyanine dyes. Examples of lanthanide chelates include complexes of europium (Eu3+) or terbium (Tb3}). Oligonucleotide-enzyme conjugates are particularly useful for detecting point mutations when using target-specific 10 oligonucleotides, as they provide vezy high sensitivities of detection.
Oligonucleotide-enzyme conjugates can be prepared by a number of methods (Jablonslci et aL, Nucl. Acids Res. 14:6115-6128, 1986; Li et al., Nucl.
Acids. Res.
15:5275-5287, 1987; Ghosh et al., Bioconjugate Chem. 1:71-76, 1990). The detection of target nucleic acids using these conjugates can be carried out by filter 15 hybridization methods or by bead-based sandwich hybridization (Ishii et al., Bioconjugate Chem. 4:34-41, 1993).

Methods for detecting a labeled oligonucleotide probe are well known in the art and will depend on the partictllar label. For radioisotopes, detection is by 20 autoradiography, scintillation counting or phosphor imaging. For hapten or biotin labels, detection is withi antibody or streptavidin bound to a reporter enzyme such as horseradish peroxidase or allcaline phosphatase, which is then detected by enzymatic means. For fluorophor or lanthanide chelate labels, fluorescent signals can be measured with spectrofluorimeters, with or without time-resolved mode or using 25 automated microtiter plate readers. For enzyme labels, detection is by color or dye deposition, for example, p-nitrophenyl phosphate or 5-bromo-4-chloro-3-indolyl phosphate/nitroblue teti-azoliuin for alkaline phosphatase, and 3,3'-dianunobenzidine-NiCI-) for horseradish peroxidase, fluorescence by 4-methyl umbelliferyl phosphate for alkaline phosphatase, or chemiluminescence by the alkaline phosphatase dioxetane 30 substrates LumiPhos 530 (Lumigen Inc., Detroit MI) or AMPPD and CSPD
(Tropix, Inc.). Chemiluminescent detection can be carried out with X-ray or PolaroidTM
film, or by using single photon counting luminoineters, which also is a useful detection fonnat for alkaline phosphatase labeled probes.

Mutational analysis can also be carried out by methods based on ligation of oligonucleotide sequences that anneal immediately adjacent to each other on a target DNA or RNA molecule (Wu and Wallace, Genomics 4:560-569, 1989; Landren et al., Scienee 241:1077-1080, 1988; Nickerson et al., Proc. NatI. Acad. Sci. USA
87:8923-8927, 1990; Barany, supra, 1991a). Ligase-inediated covalent attaclunent occurs only when the oligonucleotides are coirectly base-paired. The ligase chain reaction (LCR) and the oligonucleotide ligation assay (OLA), which utilize the thernlostable Taq ligase for target amplification, are particularly useful for interrogating mutation loci.
The elevated reaction temperatures permit the ligation reaction to be conducted with high stringency (Barany, PCR Methods and Applications 1:5-16, 1991b; Grossman et al., NucI. Acids. Res. 22:4527-4534, 1994).

Analysis of point mutations in DNA can also be carried out by using PCR and variations thereof Mismatches can be detected by competitive oligonucleotide priming under hybridization conditions where binding of the perfectly matched primer is favored (Gibbs et al.., Nucl. Acids. Res. 17:2437-2448, 1989). In the amplification refractoiy mutation system technique (ARMS), primers can be designed to have perfect matches or misrnatches witli target sequences either internal or at the 3' residue (Newton et al_, supra, 1989). Under appropriate conditions, only the perfectly annealed oligonucleotide can ftinction as a primer for the PCR
reaction, thus providing a method of (liscrimination between iionnal and mutant sequences.

Detection of single base mutations in target nucleic acids can be conveniently accomplished by differential hybridization techniques using sequence-specific oligonucleotides (Suggs et al., Proc. Natl. Acad. Sci. USA 78:6613-6617, 1981;
Conner et al., Proc. Natl. Acad. Sci. USA 80:278-282, 1983; Saiki et al., Proc. Natl.
Acad. Sci. USA 86:6230-6234, 1989). Mutations can be diagnosed on the basis of the higher themial stability of the perfectly matched probes as compared to the mismatched probes. The hybridization reactions can be carried out in a filter-based format, in which the target nucleic acids are immobilized on nitrocellulose or nylon meinbranes and probed witli oligonucleotide probes. Any of the lenown hybridization formats can be used, including Southern blots, slot blots, reverse dot blots, solution hybridization, solid suppor-t based sandwich hybridization, bead-based, silicon chip-based and microtiter well-based hybridization formats.

An alternative strategy involves detection of the niutant sequences by sandwich hybridization methods. In this strategy, the mutant and wild type target nucleic acids are separated from non-homologous DNA/RNA using a common capture oligonucleotide iinmobilized on a solid support and detected by specific oligonucleotide probes tagged with reporter labels. The capture oligonucleotides can be immobilized on microtiter plate wells or on beads (Gingeras et al., J.
Infect. Dis.
164:1066-1074, 1991; Richen et al., Proc. Natl. Acad. Sci. USA 88:11241-11245, 1991).

Another method for analysis of a biological sample for specific mutations in a PKDl polynucleotide sequence (e.g., mutant PKD1 polynucleotides, or oligonucleotide portions thereof) is a multiplexed primer extension metllod.
In this method primer is hybridized to a nucleic acid suspected of containing a mutation such that the primer is hybridized 3' to the suspected mutation. The primer is extended in the presence of a mixture of one to three deoxynucleoside triphosphates and one of three chain terminating deoxynucleoside triphosphates selected such that the wild-type extension product, the inutant DNA-derived extension product and the primer each are of different lengths. These steps can be repeated, such as by PCR or RT-PCR, and the resulting primer extended products and primer are then separated on the basis of molecular weight to thereby enable identification of mutant DNA-derived extension product.

In one aspect of the invention, the OLA is applied for quantitative inutational analysis of PKD1 polynucleotide sequences (Grossman, et al., supra, 1994). In this einbodiment of the invention, a thermostable ligase-catalyzed reaction is used to link a fluorescently labeled coinmon probe with allele-specific probes. The latter probes are sequence-coded with non-nucleotide mobility modifiers that confer unique electrophoretic mobilities to the ligation products.

Oligonucleotides specific for wild type or mutant PKD 1 sequences can be synthesized with different oligomeric nucleotide or non-nucleotide modifier tails at their 5' termini. Examples of nucleotide modifiers are inosine or thymidine residues, whereas examples of non-nucleotide modifiers include pentaethyleneoxide (PEO) and hexaethyleneoxide (HEO) monomeric units. The non-nucleotide modifiers are preferred and most preferably PEO is used to label the probes. When a DNA
template is present, a thermostable DNA ligase catalyzes the ligation of norinal and mutant probes to a common probe bearing a fluorescent label. The PEO tails modify the mobilities of the ligation products in electrophoretic gels. The combination of PEO
tails and fluorophor labels (TET and FAM (5-carboxy-fluorescein derivatives)), HEX
and JOE (6-carboxy-fluorescein derivatives), ROX (6-carboxy-x-rhodamine), or TAMRA (N, N, N', N'-tetramethyl-6-carboxy-rhodamine; Perlcin-Elmer, ABI
Division, Foster City CA) allow multiplex analysis based on size and color by providing unique electrophoretic signatures to the ligation products. The products are separated by electrophoresis, and fluorescence intensities associated with wild type and mutant products are used to quantitate heteroplasmy. Tlzus, wild type and mutant, including variant, sequences are detected and quantitated on the basis of size and fluorescence intensities of the ligation products. This method further can be configured for quantitative detection of multiple PKD 1 polynucleotide mutations in a single ligation reaction.
Mismatch detection or mutation analysis can also be performed using mismatch specific DNA intercalating agents. Such agents intercalate at a site having a mismatch followed by visualization on a polyacrylamide or agarose gel or by electrocatalysis. Accordingly, PKD I polynucleotide sequences can be contacted with probes specific for a PK D1 mutation or probes that are wild type for an area having a specific mutation under conditions such that the PKD 1 polynucleotide and probe hybridize. The hybridized sequences are then contacted with a mismatch intercalating agent and, for example, separated on a gel. Visualized bands on the gel correspond to a sequence having a mismatch. If the probes are wild-type probes mismatches will occur if the target PKD 1 sequence contains a mismatch. If the probes are specific for a mutated sequence misinatches will be present where the target PKDI sequence is wild type, but the hybridized or duplex sequeilces will not contain mismatches where the probe sequence hybridizes to a PKD1 sequence containing the same mutation.
For quantitative analysis of PKD 1 mutations using OLA, oligonucleotide probes are preferably labeled with fluorophor labels that provide spectrally distinguishable characteristics. In one embodiment, oligonucleotides are labeled with 5' oligomeric PEO tails. Synthesis of such 5' labeled oligonucleotides can be carried out, for example, using an automated syntllesizer using standard phosphoramidite cliemistry. Following cleavage from resin and deprotection with ammonium hydroxide, the (PEO)õ -oligonucleotides can be purified by reverse phase HPLC.
Oligonucleotides with 3'-FAM or TET dyes (Perkin Elmer) and 5'-phosphates can be synthesized and purified by the procedure of Grossman et al., supra, 1994. The 5'-PEO-labeled probes can be synthesized to have 5'-PEO-tails of differing lengths to facilitate distinguishing the ligated probe products both electrophoretically by size and by spectral characteristics of the fluorophor labels.

The oligonucleotide probes are used for identifying inutant PKD1 polynucleotides, which can be indicative of a PKD 1 -associated disorder such as ADPKD. Preferably, the probes are specific for one or more PKD 1 nucleotide positions of SEQ ID NO:1 selected from nucleotide 474, wherein nucleotide 474 is a T;
nucleotide 487, wherein nucleotide 487 is an A; nucleotide 3110, wllerein nucleotide 3110 is a C; nucleotide 8298, wherein nucleotide 8298 is a G;
nucleotide 9164, wherein nucleotide 9164 is a G; nucleotide 9213, wherein nucleotide 9213 is an A; nucleotide 9326, wherein nucleotide 9326 is a T;
nucleotide 9367, wherein nucleotide 9367 is a T; nucleotide 10064, wherein nucleotide 10064 is an A; nucleotide 10143, wherein nucleotide 10143 is a G;
nucleotide 10234, wherein nucleotide 10234 is a C; or nucleotide 10255, wherein nucleotide 10255 is a T. The oligonucleotide probes for the OLA assay are typically designed to have calculated melting temperatures of about 40 C to 50 C, generally about 48 C, by the nearest neighbor metliod (Breslaur et al., Proc. Natl.
Acad. Sci.
USA 83:9373-9377, 1986) so that the ligation reaction can be performed at a temperature range of about 40 C to 60 C, typically from about 45 C to about 55 C.
5 The wild type and mutant, including variant, oligonucleotide probes can be synthesized with various coinbinations of PEO oligomeric tails and fluorescein dyes such as TET and FAM. These combinations of mobility modifiers and fluorophor labels furnish electrophoretically unique ligation products that can enable the monitoring of two or more PKD1 nucleotide sites in a single ligation reaction.
In one embodiment, a method of diagnosing a PKD1-associated disorder in a subject is performed by amplifying a portion of a PKDI polynucleotide in a nucleic acid sainple from a subject suspected of having a PKD I -associated disorder witll at least a first primer pair to obtain a first amplification product, wherein said first primer pair is a primer pair of claim 3; amplifying the first amplification product with at least a second primer pair to obtain a nested amplification product, wherein the second primer pair is suitable for performing nested amplification of the first amplification product; and determining whether the nested amplification product has a inutation associated with a PKD1-associated disorder, wherein the presence of a inutation associated wit11 a PKD 1 -associated disorder is indicative of a associated disorder, thereby diagnosing a PKD1-associated disorder in the subject.
The method can be performed using a first primer pair selected from SEQ ID
NOS:3 and 4; SEQ ID NOS:5 and 6; SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID
NOS:11 and 12; SEQ ID NOS:13 and 14; SEQ ID NOS:15 and 16; SEQ ID NOS:17 and 18; and a combination tllereof, and a second primer pair selected from SEQ
ID
NOS:19 and 20; SEQ ID NOS:21 and 22; SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID NOS:27 and 28; SEQ ID NOS:29 and 30; SEQ ID NOS:31 and 32;
SEQ ID NOS:33 and 34; SEQ ID NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID
NOS:39 and 40; SEQ ID NOS:41 and 42; SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48; SEQ ID NOS:49 and 50; SEQ ID NOS:51 and 61;
SEQ ID NOS:62 and 63; SEQ ID NOS:64 a.nd 65; SEQ ID NOS:66 and 67; SEQ ID
NOS:68 and 69; SEQ ID NOS:70 and 71; SEQ ID NOS:72 and 73; SEQ ID NOS:74 and 75; SEQ ID NOS:76 and 77; SEQ ID NOS:78 and 79; SEQ ID NOS:80 and 81;
SEQ ID NOS:82 and 83; SEQ-ID NOS:84 and 85; SEQ ID NOS:86 and 87; SEQ ID
NOS:88 and 89; SEQ ID NOS:90 and 91; SEQ ID NOS:92 and 93; SEQ ID NOS:94 and 95; SEQ ID NOS:96 and 113; SEQ ID NOS:97 and 98; SEQ ID NOS:99 and 100;
SEQ ID NOS:101 and 102; SEQ ID NOS:103 and 104; SEQ ID NOS: 105 and 106;
SEQ ID NOS:107 and 108; SEQ ID NOS:109 and 110; or SEQ ID NOS:111 and 112;
and a combination thereof.

In another einbodiment, a method of diagnosing a PKD1-associated disorder in a subject is performed by amplifying a portion of PKD1 polynucleotide in a nucleic acid sample from a subject suspected of having a PKD1-associated disorder with a first primer pair to obtain a first amplification product; amplifying the first amplification product using a second primer pair to obtain a second ainplification product; and detecting a mutation in the second amplification product, wherein the mutation comprises SEQ ID NO:1 wherein nucleotide 3110 is a C; nucleotide 3336 is deleted; nucleotide 3707 is an A; nucleotide 5168 is a T; nucleotide 6078 is an A;
nucleotide 6089 is a T; nucleotide 6326 is a T; nucleotides 7205 to 7211 are deleted;
nucleotide 7415 is a T; nucleotide 7433 is a T; nucleotide 7883 is a T; or nucleotides 8159 to 8160 are deleted; nucleotide 8298 is a G; nucleotide 9164 is a G;
nucleotide 9213 is an A; or nucleotide 9326 is a T; nucleotide 10064 is an A;
or wherein a GCG nucleotide sequence is inserted between nucleotides 7535 and 7536; or a combination thereof, thereby diagnosing a PKD1-associated disorder in the subject.

The present invention also provides a method of identifying a subject having or at risk of having a PKD1-associated disorder. Such a method can be performed, for example, by comprising contacting nucleic acid molecules in a sample from a subject with at least one primer pair of the invention under conditions suitable for amplification of a PKD1 polynucleotide by the primer pair, thereby generating a PKD1-specific amplification product; and testing an ainplification product for the presence or absence of a mutation indicative of a PKD 1 -associated disorder, wherein the absence of the mutation identifies the subject a not having or at risk of the having a PKD 1 -associated disorder, and wherein the presence of the mutation identifies the subject as having or is at risk of having a PKD1-associated disorder. The primer pair can be, for example, selected from SEQ ID NO:3 and 4; SEQ ID NO:5 and 6; SEQ
ID
NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID NOS:13 and 14; SEQ ID NOS:15 and 16; or SEQ ID NOS:17 and 18. The PKD1-associated disorder can be autosomal dominant polycystic kidney disease, acquired cystic disease, or any other PKD-1 associated disorder, and the subject can be, for example, a vertebrate, particularly a huinan subject.

Such a method is particularly adaptable to a high throughput format, and, if desired, can include a step of coritacting the PKD 1-specific amplification product with at least a second primer pair, under conditions suitable for nested amplification of the PKDl-specific amplification product by a second primer pair, thereby generating a nested amplification product, then testing the nested amplification product for the presence or absence of a mutation indicative of a PKD1-associated disorder.
The second primer pair can be any primer pair suitable for nested amplification of the PKD 1-specif c ainplification product, for example, a primer pair selected from SEQ
ID NOS:19 and 20; SEQ ID NOS:21 and 22; SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID NOS:27 and 28; SEQ ID NOS:29 and 30; SEQ ID NOS:31 and 32;
SEQ ID NOS:33 and 34; SEQ ID NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID
NOS:39 and 40; SEQ ID NOS:41 and 42; SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48; SEQ ID NOS:49 and 50; SEQ ID NOS:51 and 61;
SEQ ID NOS:62 and 63; SEQ ID NOS:64 and 65; SEQ ID NOS:66 and 67; SEQ ID
NOS:68 and 69; SEQ ID NOS:70 and 71; SEQ ID NOS:72 and 73; SEQ ID NOS:74 and 75; SEQ ID NOS:76 and 77; SEQ ID NOS:78 and 79; SEQ ID NOS:80 and 81;
SEQ ID NOS:82 and 83; SEQ ID NOS:84 and 85; SEQ ID NOS:86 and 87; SEQ ID
NOS:88 and 89; SEQ ID NOS:90 and 91; SEQ ID NOS:92 and 93; SEQ ID NOS:94 and 95; SEQ ID NOS:96 and 113; SEQ ID NOS:97 and 98; SEQ ID NOS:99 and 100;
SEQ ID NOS:101 and 102; SEQ ID NOS:103 and 104; SEQ ID NOS: 105 and 106;
SEQ ID NOS:107 a.nd 108; SEQ ID NOS:109 and 110; or SEQ ID NOS:111 and 112;
and a combination thereof.

Testing an ainplification product for the presence or absence of the inutation can be performed using any of various well lalown methods for examining a nucleic acid molecule. For exainple, nucleotide sequence of the amplification product can be determined, and compared with the nucleotide sequence of a corresponding nucleotide sequence of SEQ ID NO:1. The amplification product also can be tested by determining the melting temperature of the amplification product, and comparing the melting temperature to the melting temperature of a corresponding nucleotide sequence of SEQ ID NO: 1. The melting temperature can be determined, for example, using denaturing high performance liquid chromatography.

Where a nested ainplification is to be performed, the method can include a step directed to reducing contamination of the PKD1-specific amplification product by genomic DNA prior to contacting the PKD1-specific amplification product with the at least second set of primer pairs. For example, contamination of the PKDl-specific amplification product can be reduced by diluting the PKIDl-specific amplification product.

The mutation indicative of a of PKD 1 associated disorder can be, for example, a nucleotide sequence substantially identical to SEQ ID NO: 1, wherein nucleotide 3110 is a C; nucleotide 8298 is a G; nucleotide 9164 is a G;
nucleotide 9213 is an A; nucleotide 9326 is a T; or nucleotide 10064 is an A; or can be a nucleotide sequence substantially identical to SEQ ID NO: 1, wherein nucleotide 3336 is deleted;
nucleotide 3707 is an A; nucleotide 5168 is a T; nucleotide 6078 is an A;
nucleotide 6089 is a T; nucleotide 6326 is a T; nucleotides 7205 to 7211 are deleted;
nucleotide 7415 is a T; nucleotide 7433 is a T; nucleotide 7883 is a T; or nucleotides 8159 to 8160 are deleted; or wherein a GCG nucleotide sequence is inserted between nucleotides 7535 and 7536.

Data that is collected pursuant to a step of detecting the presence or absence of a mutation indicative of a PKD1-associated disorder in an amplification product, which can be an ainplification product generated according to a method of the invention, inch.iding, for example, a PKD 1-specific amplification product or a nested amplification product, can be accuinttlated, and can be formatted into a form that facilitates determining, for example, whetller a subject is at risk of a PKD1-associated disorder. As such, the data can be formatted into a report that indicates whether a subject is at risk of a PK_D1-associate disorder. The report can be in any of various forms, including, for example, contained in a computer random access or read-only memory, or stored on a diskette, CD, DVD, magnetic tape; presented on a visual display such as a computer monitor or other cathode ray tube or liquid crystal display;
or printed on paper. Furtherinore, the data, which can be formatted into a report, can be transmitted to a user interested in or privy to the information. The data or report can be transmitted using any convenient medium, for example, via the internet, by facsimile or by mail, depending on the form of the data or report.

Also provided is a method of detecting the presence of a mutant PKD1 polynucleotide in a sainple by contacting a sample suspected of containing a mutant PKD1 polynucleotide with an oligonucleotide of the invention under conditions that allow the oligonucleotide to selectively hybridize with a mutant PKD I
polynucleotide; and detecting selective hybridization of the oligonucleotide and a mutant PKD 1 polynucleotide, thereby detecting the presence of a mutant PKD 1 polynucleotide sequence in the sample. In another embodiment, a inethod of detecting the presence of a mutant PKD 1 polypeptide in a sample is provided, for example, by contacting a sample suspected of containing a mutant PKD 1 polypeptide witll an antibody of the iiivention under conditions that allow the antibody to specifically bind a mutant PKD1 polypeptide; and detecting specific binding of the antibody and the mutant PKD 1 polypeptide in the sample, thereby detecting the presence of a mutant PKD 1 polypeptide in a sample. The mutant PKD 1 polypeptide can have a sequence, for example, substantially as set forth in SEQ ID NO:2, and having a mutation of A88V, W967R, L2696R, R2985G, W3001X, R3039C, V32851, H3311R, or a combination thereof (see, also, Table 4).

Antibodies tliat can specifically bind wild type or mutant PKD1 polypeptides, or peptide portions thereof, can also be used as ADPKD diagnostic reagents. Such reagents provide a diagnostic inetllod that can detect the expression of abnormal PKD1 proteins or of abnormal levels of PKD1 protein expression, including the detection of mutant PKDl polypeptides or aberrant cellular localization of a PK:D1 protein. For example, differences in the size, electronegativity, or antigenicity of the mutant PKD
1 protein relative to a wild type PKD 1 protein can be detected.

Diagnostic methods for the detection of mutant PKD1 polypeptides or peptide portions thereof can involve, for example, immunoassays wherein epitopes of a mutant PKDI polypeptide are detected by their interaction with an anti-PKDl specific antibody (e.g., an anti-mutant PKD 1 specific antibody). For exainple, an antibody that 10 specifically binds to a mutant PKDl polypeptide does not bind to a wild-type PKDl polypeptide or peptide thereof. Particular epitopes of PKDl to which antibodies can be developed include peptides that are substantially identical to SEQ ID NO:2, and having at least five ainino acids, including amino acid residue 88, wherein residue 88 is a V; residue 967, wherein residue 967 is an R; residue 2696, wherein residue 2696 15 is an R; residue 2985, wherein residue 2985 is a G; residue 3039, wherein residue 3039 is a C; residue 3285, wherein residue 3285 is an I; or residue 3311, wherein residue 3311 is an R; or a C-terminal peptide including amino acid residue 3000, where residue 3001 is absent and the mutant PKD1 polypeptide is truncated due to the presence of a STOP codon in the encoding inutant PKD 1 20 polynucleotide.

Antibodies, or fragments of antibodies, such as those described, above, are useful in the present invention and can be used to quantitatively or qualitatively detect the presen.ce of wild type or mutant PKD 1 polypeptides or peptide portions thereof, for 25 example. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorimetric detection.

The antibodies (or fraginents thereof) useful in the present invention can, 30 additionally, be employed histologically, as in irnmunofluorescence or immunoelectron microscopy, for in situ detection of PKD1 polypeptide, peptides, variants or mutants thereof. Detection can be accomplished by removing a histological specimen from a subject, and applying thereto a labeled antibody of the present invention. The histological sa.inple can be taken from a tissue suspected of exliibiting ADPKD. The antibody (or fiagment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to deteimine not only the presence of PKD1 polypeptides, but also their distribution in the examined tissue. Using the present invention, those of ordinary slcill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to acliieve such in situ detection.

Immunoassays for wild type or niutant PKD1 polypeptide or peptide portions thereof typically comprise incubating a biological satnple, such as a biological fluid, a tissue extract, freshly harvested cells, or cells that have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying a PKD

polypeptide, mutant PKD 1 polypeptide and peptide portions thereof, and detecting the bound antibody by any of a number of techniques well-lalown in the art. The biological sample can be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support that is capable of in~unobilizing cells, cell particles or soluble proteins. The support can then be washed with suitable buffers followed by treatment with the detectably labeled mutant PKD1 specific antibody, preferably an antibody that recognizes a developed include peptides that are substantially identical to SEQ ID NO:2, and having at least five amino acids, including aniino acid residue 88, wherein residue 88 is a V; residue 967, wherein residue 967 is an R; residue 2696, wherein residue 2696 is an R; residue 2985, wherein residue 2985 is a G; residue 3039, wherein residue 3039 is a C;
residue 3285, wherein residue 3285 is an I; or residue 3311, wherein residue 3311 is an R;
or a C-terminal peptide including amino acid residue 3000, where residue 3001 is absent and the mutant PKD1 polypeptide is truncated due to the presence of a STOP
codon in the encoding mutant PKD 1 polynucleotide (see, also, Table 4). The solid phase support can then be washed with the buffer a second time to remove unbound antibody, and the amount of bound label on solid support can be detected by conventional means specific for the label.

A "solid phase support" or "carrier" can be any support capable of binding an antigen or a.n antibody. Well-kiiown supports or catriers include glass, polystyrene, polypropylene, polyetllylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Tllus, the support configuration can be spl7erical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface can be flat such as a sheet, test strip, or the lilce. Those skilled in the art will lrnow many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

The binding activity of a given lot of an anti-mutant PKD1 antibody can be determined according to well known methods. Those skilled in the art will be able to determine operative and optinial assay conditioiis for each determination by employing routine experimentation. One of the ways in which the mutant PKD1-specific antibody can be detectably labeled is by linking the antibody to an enzyme and use the enzyme labeled antibody in an enzyme immunoassay (EIA; Voller, "The Enzyme Linked Immunosorbent Assay (ELISA):, Diagnostic Horizons 2:1-7, 1978; Microbiological Associates Quarterly Publication, Wallcersville, Md.); Voller et al., J. Clin.
Pathol.
31:507-520, 1978; Butler, Meth. Enzymol. 73:482-523, 1981; Maggio (ed.), "Enzyme Immunoassay", CRC Press, Boca Raton FL, 1980; Ishilcawa et al., (eds.), "Enzyme linmunoassay", Kgalcu Shoin, Tolcyo, 1981). The enzyme that is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluoriinetric or by visual means.

Enzyines that can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, a-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribo;nuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetyleholinesterase. The detection can be accomplished by colorimetric methods that employ a cluomogenic substrate for the enzyme.
Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in conlparison witll siiniIarly prepared standards. In addition, detection can be accomplished using any of a variety of other immunoassays, including, for example, by radioactively labeling the antibodies or ailtibody fragments and detecting PKD
1Nuild type or mutant peptides using a radioinununoassay (RIA; see, for exanlple, Weintraub, Principles of Radioirrnnunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endociine Society, March, 1986).
T he radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

The antibody also can be labeled with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most comrnonly used fluorescent labeling compounds are fluorescein isothiocyanate, i-hodaniine, phycoerythrin, pllycocyanin, allophycocyanin, o-phthaldehyde and fluorescanune. The antibody can also be detectably labeled using fluorescence emitting metals such as 1S2Eu, or others of the lanthanide seiies. These metals cazn be attaclied to the antibody using such metal chelating groups as dietlrylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence, of the chemiluminescent-tagged antibody is then determined by detecting the presence of lui-ninescence that arises during the course of a chemical reaction. Examples of parficularly useful chemiluminescent labeling compounds are luminol, isoluminol, theroinatic acridinium ester, imidazole, acridinium salt and oxalate ester. Lilcewise, a biolun-iinescent conlpound can be used to label the antibody of the present invention. Biolurninescence is a type of chenuluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a biolununescent protein is determined by detecting the presence of luminescence. Important bioluminescent coinpounds for purposes of labeling are luciferin, luciferase and aequorin.

In vitro systems can be designed to identify compounds capable of binding a mutatlt PKD1 polynucleotide of the invention (e.g., a polynucleotide having a sequence substantially identical to SEQ ID NO:1 and having a mutation such as C474T;
G487A;
T311OC; T8298G; A9164G; G9213A; C9326T; C9367T; G10064A; A10143G;
T10234C; or G10255T). Such coinpounds can include, but are not limited to, peptides made of D-and/or L-configuration ainino acids in, for exainple, the form of random peptide libraries (see, e.g., Lain et al., Nature 354:82-84, 1981), phosphopeptides in, for example, the form of random or partially degenerate, directed phosphopeptide libraries (see, e.g., Songyang et al., Cell 72:767-778,1993), antibodies, and small or large organic or inorganic molecules. Coinpounds identified can be useful, for exainple, in modulating the activity of PKD 1 protei.uis, variants or inutailts. For example, mutant PKD 1 polypeptides of tlie invention can be useful in elaborating the biological function of the PKD 1 protein. Such mutants can be utilized in screens for identifying compounds that disrupt normal PKD1 interactions, or can in themselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to a mutant PK-D1 protein uzvolves preparing a reaction mixture of the PKD1 protein, which can be a mutant, including a variant, and the test compound under conditions and for a time sufficient to allow the two components to interact, then isolating the interaction product (complex) or detecting the complex in the reaction mixture. Sucli assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring PKD 1 or the test substance onto a solid phase and detecting PKD 1 test substance complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid pliase. In either approach, the order of addition of reactants can be varied to obtain different information about the coinpounds being tested.

In addition, metllods suitable for detecting protein-protein interactions can be employed for identifying novel PKD1 cellular or extracellular protein interactions based upon the inutailt or variant PKD1 polypeptides of the invention. For example, some traditional inethods that can be einployed are co-immunoprecipitation, crosslinking and copurification through gradients or chromatographic columns. Additionally, methods that result in the sinlultaneous identification of the genes coding for the protein 5 interacting with a target protein can be employed. These metliods include, for example, probing expression libraries with labeled target protein, using this protein in a manner similar to antibody probing of 2~gt libraries. One such method for detecting protein interactions in vivo is the yeast two hybrid system. One version of this system has been described (Cliien et al., Proc. Natl. Acad. Sci. USA $8:9578-9582, 1991) and can be 10 performed using commercially available reagents (Clontech; Palo Alto CA).

A PKD1 polypeptide (e.g., a variant or inutant) of the invention can interact with one or more cellular or extracellular proteins in vivo. Such cellular proteins are referred to herein as "binding partners". Compounds that disrupt such interactions can be useful 15 in regulating the activity of a PKDl polypeptide, especially mutant PKD1 polypeptides.
Such coinpounds include, for exainple, molecules such as antibodies, peptides, peptidomimetics and the like.

In instances whereby ADPKD symptoms are associated with a mutation within 20 the PKD 1 polyiiucleotide (e.g., SEQ ID NO:1 having a mutation at T3110C;
T8298G;
A9164G; G9213A; C9326T; G10064A or the like; see Exanple 2), which produces PKD1 polypeptides having aberrant activity, compounds identified that disrupt suc11 activity can therefore inhibit the aberrant PKD 1 activity and reduce or treat associated symptoms or ADPKD disease, respectively (see Table 4). For example, 25 compounds can be identified that disrupt the interaction of mutant PKD I
polypeptides with cellular or extracellular proteins, for example, the PKD2 gene product, but do not substantially effect the interactions of the norinal PKD 1 protein. Such compounds can be identified by comparing the effectiveness of a coinpound to disrupt interactions in an assay containing normal PKD1 protein to tliat of an assay containing mutant 30 polypeptide, for exatnple, a two hybrid assay.

The basic principle of the assay systems used to identify compounds that interfere with the interaction between the PKD 1 protein, preferably a mutant protein, and its cellular or extracellular protein binding partner or partners involves prepariuig a reaction mixture containing the PKDl protein and the binding partner under conditions and for a tiine sufficient to allow the two proteins to interact or bind, thus forming a complex. In order to test a compound for inhibitory activity, reactions are conducted in the presence or absence of the test compound, i.e., the test compound can be iiiitially included in the reaction mixture, or added at a time subsequent to the addition of PKDl and its cellular or extracellular binding partner; controls are incubated without the test compound or with a placebo. The foimation of any complexes between the PKD1 protein and the cellular or extracellular binding partner is then detected. The formation of a complex or interaction in the control reaction, but not in the reaction mixture containing the test compound indicates that the compound interferes with the interaction of the PKD 1 protein and the binding partner. As noted above, complex formation or component interaction within reaction mixtures containing the test compound and normal PKDl protein can also be compared to complex formation or component interaction within reaction mixtures containing the test compound and mutant PKD1 protein. This comparison can be important in those cases wherein it is desirable to identify coinpounds that disrupt interactions of mutant but not normal PKD1 proteins.

Any of the binding compoluids, including but not limited to, compounds such as those identified in the foregoing assay systems can be tested for anti-ADPKD
activity.
ADPKD, an autosomal dominant disorder, can involve underexpression of a wild-type PKD 1 allele, or expression of a PKD 1 polypeptide that exhibits little or no activity. In such an instance, even though the PKDI polypeptide is present, the overall level of normal PKD 1 polypeptide present is insufficient and leads to ADPKD
symptoms. As such increase in the level of expression of the normal PKD1 polypeptide, to levels wherein ADPKD symptoms are anzeliorated would be useful.
Additionally, the tenn can refer to an increase in the level of normal PKD1 activity in the cell, to levels wherein. ADPKD symptoms are ameliorated.

The identified coinpounds that inhibit PKD1 expression, synthesis and/or activity can be administered to a patient at tllerapeutically effective doses to treat polycystic kidney disease. A therapeutically effective dose refers to that atnount of the compound sufficient to result in amelioration of syinptoms of polycystic lcidney disease. Toxicity and therapeutic efficacy of such coinpotuzds can be determined by standard phannaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit large therapeutic indices are preferred.
While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of adininistration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell cult-Lire assays. A dose can be foimulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in huinans. Levels in plasma can be measured, for example, by high performance liquid chromatography. Additional factors that can be utilized to optimize dosage can include, for example, such factors as the severity of the ADPKD symptoms as well as the age, weight and possible additional disorders that the patient can also exhibit.
Those skilled in the art will be able to determine the appropriate dose based on the above factors.
Pharlnaceutical compositions for use in accordance witli the present invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates can be fonnulated for administration by inhalation (either tlhrough the mouth or the nose) or oral, buccal, parenteral or rectal administration.

For oral administration, the pharmaceutical coinpositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl inethylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calciwn hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodiusn starch glycollate); or wetting agents (e.g:, sodium lawyl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for exanlple, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral admiiustration can be suitably formulated to give controlled release of the active compound. For buccal administration the compositions can take the foi7n of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the for-m of an aerosol spray presentation from pressurized paclcs or a nebuliser, with the use of a suitable propellant such as dichlorodifluoromefihane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin, for use in an inhaler can be foimulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds can be formulated for parenteral adinirustration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and call contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder foim for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g, containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds can also be formulated as a depot preparation. Such long acting fonnulations can be administered by iunplantation (for example subcutaneously or intratnuscularly) or by intramuscular injection. Thus, for exainple, the compounds can be fornmulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The compositions can, if desired, be presented in a pack or dispenser device that can contain one or inore unit dosage forms containing the active ingredient.
The pack can for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration.
Alternatively, ADPKD can be caused by the production of an aberrant mutant form of the PKD 1 protein, that either interferes with the normal allele product or introduces a novel function into the cell, which then leads to the mutant phenotype. For example, a mutant PKD 1 protein can compete with the wild type protein for the binding of a substance required to relay a signal inside or outside of a cell.

Cell based aiid animal model based assays for the identification of compounds exliibiting anti-ADPKD activity are also encompassed within the present invention.
Cells that contain and express mutant PKD1 polynucleotide sequences (e.g., a sequeiice 5 substantially identical to the sequence as set forth in SEQ ID NO:1 and having one or more mutations of a C474T; G487A; T3110C; T8298G; A9164G; G9213A; C9326T;
C9367T; G10064A; A10143G; T10234C; G10255T or the like; see Example 2), which encode a inutant PKDI polypeptide, and thus exhibit cellular phenotypes associated with ADPKD, can be utilized to identify compoluzds that possess anti-ADPKD
activity. Such 10 cells can include cell lines consisting of naturally occuiring or engineered cells that express mutant or express both normal and inutant PKD1 polypeptides. Such cells include, but are not limited to renal epithelial cells, including primary and immortalized human renal tubular cells, MDCK cells, LLPCK1 cells, and human renal carcinoina cells. Methods of transfoin-iing cell with PKD 1 polynucleotide sequences encoding 15 wild-type or mutant proteins are described above.

Cells that exhibit ADPKD-like cellular phenotypes, can be exposed to a compound suspected of exhibiting anti-ADPKD activity at a sufficient concentration and for a time sufficient to elicit an anti-ADPKD 1 activity in the exposed cells.
After 20 exposure, the cells are examined to determine whether one or more of the ADPKD-like cellular phenotypes has been altered to resemble a more wild type, non-ADPKD
phenotype.

Among the cellular phenotypes that can be followed in the above assays are 25 differences in the apical/basolateral distribution of membrane proteins.
For example, normal (i.e., non-ADPKD) renal tubular cells in sitaa and in culture under defined conditions have a characteristic pattern of apical/basolateral distribution of cell surface markers. ADPKD renal cells, by contrast, exhibit a distribution pattern that reflects a partially reversed apical/basolateral polarity relative to the normal 30 distribution. For example, sodium-potassium ATPase generally is found on the basolateral membranes of renal epithelial cells, but also can be found on the apical surface of ADPKD epithelial cells, both in cystic epithelia in vivo and in ADPKD

cells in culture (Wilson etal., An1. J. Plrysiol. 260:F420-F430, 1991).
Another marlcer that exhibits an alteration in polarity in normal versus ADPKD
affected cells is the EGF receptor, which is normally located basolaterally, but in ADPKD
cells is mislocated to the apical surface. Such a apical/basolateral niarlcer distribution phenotype can be followed, for example, by standard immunohistology techniques using antibodies specific to a markers of interest.

Assays for the function of PKDI also ean include a nieasui-e of the rate of cell gromrth or apoptosis, since dysregulation of epithelial cell growth can be a key step in cyst formation. The cysts are fluid filled structures lined by epithelial cells that are both hyper-proliferative and hyper-apoptotic (Evan et al., Kidney Intemational 16:743-750, 1979; Kovacs and Gomba, Kidney Blood Press. Res. 21:325-328, 1998;
Lanoix et al., Oncogene 13: 1153-1160, 1996; Woo, New Engl. J. Med. 333:18-25, 1995). The cystic epithelium has a high mitotic rate in vivo as measured by PCNA staining (Nadasdy et al., J. Am.
Soc.
Nephrol. 5:1462-1468;, 1995), and increased levels of expression of other markers of proliferation (Klingel et al., Ainer.
J. Kidney Dis. 19:22-30, 1992). In addition, cultured cells from ADPKD cystic lddneys have increased growth rates in vitro (Wilson et al., Kidney Int. 30:371-380, 1986;.Wilson, Amer. J.
Kidne), Dis.
17:634-63 7, 1991).

Further, in studies of rodent models of polycystic kidney disease, the epithelial cells that line cysts of a.ninials with naturally occurring forms of PKD
showed abnormalities similar to those reported in hLUnan ADPKD (Harding et al., 1992;
Ramasubbu et trl., J. Am. Soc. Nephrol. 9:937-945, 1998; Rankin et al., J.
Cell Physiol. 132:578-586, 1992; Rankin et al., In Vitro Cell Devel. Biol. Anim.
32:100-106, 1996 ). Moreover, mice that have transgenic over-expression of either c-myc or SV40-large T antigen developed PKD (Kelley et al. J. Am. Soc. Nephrol. 2:84-97, 1991; Trudel et al., Kidney Int.
39:665-671 1991). Also, expression of recombinant full length PKD1 in epithelial cells reduced their rate of growth and induced resistance to apoptosis when challenged with stimuli such as sei-tim starvatioii or exposure to UV light, which are known to stimulate apoptosis (Boletta et al., Mol. Ce116:1267-1273, 2000).
As such, biochemical pathways that are activated by PKD I expression, including, for example, JAK2, STAT1/3, P13 kinase, p2l, and AKT, can provide surrogate niarkers foi- PKD I activity.

The propensity of an epithelial cell to form tubules provides still another assay for the function of PKD 1. Isz vivo, PKD is characterized by cystic transformation of renal tubules and pancreatic and biliary ductules. Iiz vitro, expression of full length PKD1 induces spontaneous tubulogenesis in MDCK cells (Boletta et al., supra, 2000). In this model system, control MDCK cells, which did not express recombinant wild type fiill length PK:D1, formed cystic structures unless treated with hepatocyte growth factor or with fibroblast conditioned medium when cultured suspended in collagen. In contrast, MDCK cells that expressed the full length wild type recombinant form of Pli;Dt spontaneously fonned tubules in the absence of exogenous factors wheri cultured in this manner. As such, this model system can be used to identify ligands that bind to and activate the PKDI protein, to determine pathways that are targeted for activation by therapeutic agents, and as an assay system to evaluate the effect of sequence variants on PKD 1 function.

Additionally, assays for the fiinction of a PKD 1 polypeptide can, for example, include a measure of extracellular matrix (ECM) components, such as proteoglycans, laminin, fibronectin and. the like, in that studies in both ADPKD and in rat models of acquired cystic disease (Carone et al., Kidney International 35:1034-1040, 1989) have shown alterations in such components. Thus, any compound that serves to create an extracellular matrix envirotunent that more fully mimics the normal ECM should be considered as a candidate for testing for an ability to ameliorate ADPKD
symptoms.
In addition, it is contemplated that the present invention can be used to measure the ability of a compound, such as those identified in the foregoing binding assays, to prevent or inhibit disease in animal models for ADPKD. Several naturally-occuTing mutations for renal cystic disease have been found in animals, and are accepted in the art as models of ADPKD and provide test systems for assaying the effects of compounds that interact with PKD I proteins. Of these models, the Han:SPRD rat model, provides an autosomal dominant model system (see, for example, Kaspareit-Rittinghausen et al., Vet. Path. 26:195, 1989), and several recessive models also are available (Reeders, Nature Genetics 1:235, 1992). In addition, knock-out mice, in which the PKD1 or PKD2 gene has been disrupted, are available and provide a relevant model system for genetic forms of ADPKD. As such, the PKD1 and PKD2 knock-out mice can be useful for confirming the effectiveness in vivo of compounds that interact with PKD1 proteins in vitro (see, for exaniple, Wu et al., Nat. Genet. 24:75-78, 2000; Kim et al., Proc. Natl. Acad.
Sci., USA 97:1731-1736, 2000; Lu et al., Nat. Genet.21:160-161, 1999; Wu et al., Cell 93:177-188, 1998; Lu et al., Nat. Genet. 17:179-181, 1997).

Aiumal models eAhibiting ADPKD-like symptoms associated with one or more of the mutant PKD1 polynucleotide sequences as disclosed herein can also be engineered by utilizing the PKDI polynucleotide sequences such in conjunction with well known methods for producing transgenic animals. Animals of any species, ineluding, but not liinited to, mice, rats, rabbits, guinea pigs, pigs, mini-pigs, goats, and non-hunian primates, e.g., baboons, squiirels, monkeys, and chimpanzees can be used to generate such ADPKD anunal models or transgenic ani.mals. In instances where the PKDI mutation leading to ADPKD symptoms causes a drop in the level of PKD1 protein or causes an ineffective PKDI protein to be made (e.g., the PKDI
mutation is a dominant loss-of-function mutation, such as a W3001X, i.e., truncated after amino acid residue 3000, or a T3 l lOC mutation; see, also, Table 4) various strategies can be utilized to generate animal models exhibiting ADPKD-like symptoms.

The present invention also provides transgenic non-human organisms, including invertebrates, vertebrates and mammals. For purposes of the subject invention, these animals are referred to as "transgenic" when such animal has had a heterologous DNA
sequence, or one or more additional DNA sequences normally endogenous to the anunal (collectively referred to herein as "transgenes") chromosomally integrated into the germ cells of the animal. The transgenic aiiimal (including its progeny) will also have the transgene integrated into the chromosomes of somatic cells.

Various methods to make the transgenic animals of the subject invention can be employed. Generally speaking, three such methods can be employed. In one such method, an embryo at the pronuclear stage (a "one cell embryo") is harvested from a female and the transgene is microinjected into the embryo, in which case the transgene will be chromosomally integrated into both the germ cells and somatic cells of the resulting mature animal. In another such method, embryonic stem cells are isolated and the transgene incoiporated therein by electroporation, plasmid transfection or microinjection, followed by reintroduction of the stem cells into the embryo where they colonize alid contribute to the germ line. Methods for microinjection of mammalian species is described in U.S. Pat. No. 4,873,191.
In yet another such metliod, embryonic cells are infected with a retrovirus containing the transgene whereby the germ cells of the embryo have the transgene chromosomally integrated therein. When the animals to be made transgenic are avian, because avian fertilized ova generally go through cell division for the first twenty hours in the oviduct, microinjection into the pronucleus of the fertilized egg is probleinatic due to the inaccessibility of the pronucleus. Therefore, of the methods to make transgenic animals described generally above, retrovirus infection is preferred for avian species, for example as described 'ui U.S. Pat. No. 5,162,215. If microinjection is to be used with avian species, however, the method of Love et al., (Biotechnology, 12, 1994) can be utilized whereby the embryo is obtained from a sacrificed hen approximately two and one-half hours after the laying of the previous laid egg, the transgene is microinjected into the cytoplasm of the germinal disc and the embryo is cultured in a host shell until maturity. When the animals to be inade transgenic are bovine or porcine, microinjection can be hampered by the opacity of the ova thereby making the nuclei difficult to identify by traditional differential interference-contrast microscopy. To overcome this problem, the ova can first be centrifuged to segregate the pronuclei for better visualization.

The non-human traiisgenic animals of the invention include, for example, bovine, porcine, ovine alid avian animals (e.g., cow, pig, sheep, chiclcen, turkey). Such transgenic non-human animals are produced by introducing a transgene into the germline of the non-huinan animal. Embryonal target cells at various developmental stages can 5 be used to introduce transgenes. Different methods are used depending on the stage of development of the embiyonal target cell. The zygote is the best target for microinjection. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incoiporated into the host gene before the first cleavage (Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985). As a 10 consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This w411 in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.

15 The terin "transgenic" is used to describe an animal that includes exogenous genetic material within all of its cells. A transgenic animal can be produced by cross-breeding two chimeric animals that include exogenous genetic material within cells used in reproduction. Twenty-five percent of the resulting offspring will be transgenic i.e., animals that include the exogenous genetic material within all of their cells in both 20 alleles. Fifty percent of the resulting animals will include the exogenous genetic material within one allele and 25% will include no exogenous genetic material.

In the microinjection method usefid in the practice of the invention, the transgene is digested and purified fiee from any vector DNA e.g. by gel electrophoresis.
It is 25 preferred that the trailsgene include an operatively associated promoter that interacts with cellular proteins involved in transcription, ultimately resulting in constitutive expression. Promoters usefiil in this regard include those from cytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpes vu2is, as well as those from the genes encoding metallothionein, slceletal actin, P-enolpyruvate carboxylase (PEPCK), 30 pliosphoglycerate (PGK), DHFR, and thymidine kinase. Promoters for viral long terminal repeats (LTRs) such as Rous Sarcoma Virus can also be employed. When the animals to be made transgenic are avian, preferred promoters include those for the chiclcen (3-globin gene, chiclcen lysozyme gene, and avian leukosis virus.
Constructs useful in plasmid transfection of embryonic stem cells will employ additional regulatory elements well lcnown in the art sucli as enhancer elements to stimulate transcription, splice acceptors, termination and polyadenylation signals, and ribosome binding sites to permit translation.

Retroviral infection can also be used to introduce transgene into a non-huznan animal, as described above. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retro viral infection (Jaenich, Proc. Natl. Acad. Sci. USA 73:1260-1264, 1976). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan et al., In "Manipulating the Mouse Embryo" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY 1986)). The viral vector system used to introduce the transgene is typically a replication-defective retro virus carrying the transgene (Jahner et al., Proc. Natl. Acad. Sci. USA 82:6927-6931, 1985; Van der Putten et al., Proc. Natl.
Acad. Sci USA 82:6148-6152, 1985). Transfection is easily and efficiently obtained by culttuing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart, et al., EMBO J. 6:383-388, 1987). Alternatively, infection can be perforined at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al., Nature 298:623-628, 1982). Most of the founders will be mosaic for the transgene suice incorporation occurs only in a subset of the cells that formed the transgenic nonliuinan animal. Further, the founder can contain various retroviral 'ulsertions of the transgene at different positions in the genome that generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line, albeit with low efficiency, by intrauterine retroviral infection of the midgestation embryo (Jahner et al., supra, 1982).

A third type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells are obtained from pre-implantation embiyos cultured in vitro and fused with embryos (Evans et al. Nature 292:154-156, 1981; Bradley et al., Nature 309:255-258, 1984; Gossler et al., Proc. Natl. Acad. Sci. USA 83:9065-9069, 1986; and Robertson et al., Nature 322:445-448, 1986). Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retro virus-mediated transduction.
Such transformed ES cells can tlzereafter be combined with blastocysts from a nonhutnan animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (for review see Jaenisch, Science 240:1468-1474, 1988).
The transgene can be any piece of DNA that is inserted by artifice into a cell, and becomes part of the genome of the organism (i.e., either stably integrated or as a stable extrachromosomal element) that develops from that cell. Such a transgene can include a gene that is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or can represent a gene homologous to an endogenous gene of the organism.
Included within this definition is a transgene created by the providing of an RNA
sequence that is transcribed into DNA, then incorporated into the genome. The transgenes of the invention include DNA sequences that encode a mutant PKD1 polypeptide, for example, a polypeptide having an amino acid sequence substantially identical to SEQ ID
NO:2 and having a mutation of a A88V, a W967R, a L2696R, an R2985G, an R3039C, a V32851, a H331 1R, or any combination thereof; or encoding a truncated PKD1 polypeptide ending at amino acid 3000 (also referred to herein as "W3001X", where "X" indicates STOP codon; see, also, Table 4) and include sense, antisense, and dominant negative encoding polyiiucleotides, which can be expressed in a transgenic non-human animal. The term "transgenic" as used herein also includes any organism whose genome has been altered by in vitro manipulation of the early embryo or fertilized egg or by any transgenic technology to induce a specific gene lcnoclcout. The term "gene lcnockout" as used herein, refers to the targeted disruption of a gene in vivo with complete or partial loss of function that has been achieved by any transgenic technology familiar to those in the art. In one embodiment, transgenic animals having a gene knockout are those in which the target gene has been rendered nonfunctional by an insertion targeted to the gene to be rendered non-functional by homologous recombination.

The invention also includes animals having heterozygous mutations in or partial inhibition of function or expression of a PKD1 polypeptide. One of skill in the art would readily be able to determine if a particular mutation or if an antisense molecule was able to partially inhibit PKD 1 expression. For example, in vitro testing can be desirable initially by conlparison witli wild-type (e.g., coinparison of northern blots to examine a decrease in expression). After an embryo has been microinjected, colonized with transfected embryonic stenl cells or infected with a retrovir-us containing the transgene (except for practice of the subject invention in avian species, which is addressed elsewhere herein), the einbryo is implanted into the oviduct of a pseudopregnant female.
The progeny are tested for incorporation of the transgene by Southern blot analysis of blood samples using transgene specific probes. PCR is particularly useful in this regard.
Positive progeny (Po) are crossbred to produce offspring (P1) that are analyzed for transgene expression by northern blot analysis of tissue samples.

In order to distinguish expression of like species transgenes from expression of an endogenous PKD 1-related gene, a marker gene fragment can be included in the construct in the 3' untranslated region of the transgene and the northern blot probe designed to probe for the marker gene fragment. The serum levels of a PKD1 polypeptide can also be measured in the transgenic animal to detennine the level of PKD 1 expression. A method of creating a transgenic organism also can include methods of inserting a transgene into, for example, an embryo of an already created transgenic organism, the organism being transgenic for a different unrelated gene or polypeptide.
Transgenic organisms of the invention are highly useful in the production of organisms for study of, for example, polycystic kidney disease or PKD1-related diseases or disorders and in identifying agents or drugs that inhibit or modulate polycystic kidney disease, PKD 1 associated disorders and inheritance.
Expression of a mutant huinan PKD1 polynucleotide can be assayed, for example, by standard northern blot analysis, and the production of the mutant htunan PKD 1 polypeptide can be assayed, for example, by detecting its presence using an antibody directed against the mutant huznan PKD 1 polypeptide. Those animals found to express the mutant human PKD1 polypeptide can then be observed for the development of ADPKD-like symptoms.

As discussed above, animal models of ADPKD can be produced by engineering animals containing mutations in a copy of an endogenous PKD 1 gene that correspond to inutations within the huinan PKD 1 polynucleotide. Utilizing such a strategy, a PKD 1 homologue can be identified and cloned from the animal of interest, usin.g techniques such as those described herein. One or more mutations can be engineered into such a PKD1 homologue that correspond to mutations within the huinan PKD1 polynucleotide, as discussed above (e.g., resulting in a mutation of the amino acid sequence as set forth in SEQ ID NO:2 and having a mutation of a A88V, a W967R, a L2696R, an R2985G, a W3001X, an R3039C, a V3285I, a H3311R, or any combination thereof; see, also, Table 4). As disclosed herein, a mutant polypeptide produced by such an engineered corresponding PKD1 homologue can exhibit an aberrant PKD1 activity that is substantially siinilar to that exhibited by a mutant human PKD 1 protein. The engineered PKD1 homologue can then be introduced into the genome of the animal of interest, using techniques such as those described, above. Accordingly, any of the ADPKD
animal models described herein can be used to test compounds for an ability to ameliorate ADPKD symptoms, including those associated with the expression of a mutant PKDl polypeptide substantially identical to SEQ ID NO:2 and having the mutation A88V, W967R, L2696R, R2985G, W3001X, R3039C, V32851, H3311R, or a combination thereof (see Example 2 and Table 4).
As discussed above, mutations in the PKDI polynucleotide that cause ADPKD
can produce a form of the PKD 1 protein that exhibits an aberrant activity that leads to the formation of ADPKD symptoms. A variety of techniques can be utilized to inhibit the expression, synthesis, or activity of such mutant PKDl polynucleotides and polypeptides. For example, compounds such as those identified through assays described, above, which exhibit inhibitory activity, can be used in accordance with the invention to ameliorate ADPKD symptoins. Such molecules can include, but are not limited, to small and large organic molecules, peptides, and antibodies.
Furtlier, antisense and ribozyme molecules that inhibit expression of a PK-Dl polynucleotide, (e.g., a mutant PKD1 polynucleotide), can also be used to inhibit the aberraiit PKD1 activity. Such techniques are described, below. In yet another embodiment, triple helix molecules can be utilized in inhibiting aberrant PKD1 activity.

Among the coinpounds that can exhibit anti-ADPKD activity are antisense, ribozyme, and triple helix inolecules. Such molecules can be designed to reduce or inhibit mutant PKD1 activity by inodulating the expression or synthesis of 5 polypeptides. Techniques for the production and use of such molecules are well known to those of slcill in the art.

Double stranded interfering RNA molecules are especially useful to inhibit expression of a target gene. For exainple, double stranded RNA molecules can be 10 injected into a target cell or organisni to inhibit expression of a gene and the resultant polypeptide's activity. It has been found that such double stranded RNA
molecules are more effective at inhibiting expression than either RNA strand alone (Fire et al., Nature, 19:391(6669):806-11, 1998).

15 When a disorder is associated with abnormal expression of a PKD1 polypeptide (e.g., overexpression, or expression of a mutated form of the protein), a therapeutic approach that directly interferes with the translation of a PKD1 polypeptide (e.g., a wild type, variant or mutant PKD1 polypeptide) is possible. Alteinatively, similar methodology can be used to study gene activity. For example, antisense nucleic acid, 20 double stranded interfering RNA or ribozymes could be used to bind to a PKD1 mRNA
sequence or to cleave it. Antisense RNA or DNA molecules bind specifically with a targeted gene's RNA message, interrupting the expression of that gene's protein product.
The antisense binds to the messenger RNA forming a double stranded molecule that cannot be translated by the cell. Antisense oligonucleotides of about 15 to 25 25 nucleotides are preferred since they are easily synthesized and have an inhibitory effect just lilce antisense RNA lnolecules. In addition, chemically reactive groups, such as iron-linlced ethylenediaminetetraacetic acid (EDTA-Fe) can be attached to an antisense oligonucleotide, causing cleavage of the RNA at the site of hybridization.
Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of 30 a specific mRNA molecule (Weintraub, Scientific American, 262:40, 1990). In the cell, the antisense nucleic acids hybridize to the co2-responding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate a mRNA that is double-stranded.
Antisense oligomers of at least about 15 nucleotides also are preferred because they are less lilcely to cause problems wheii iiitroduced into the target PKD1 polypeptide producing cell.
The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus-Salcura, Anal. Biochem.,172:289, 1988).

Use of an oligonucleotide to stall transcription is Icnown as the triplex strategy since the oligomer winds around double-helical DNA, forming a three-strand helix.
Therefore, these triplex compounds caii be designed to recognize a unique site on a chosen gene (Maher et al., Antisense Res. and Devel., 1:227, 1991; Helene, Anticancer Drug Design, 6:569, 1991).

Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases.
Through the modification of nucleotide sequences that encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA
molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
There are two basic types of ribozymes nainely, tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) asid "hammerhead"-type. Tetrahymena-type ribozymes recognize sequences that are four bases in length, while "hammerhead"-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA
species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-base recognition sequences are preferable to shorter recognition sequences. These and other uses of antisense and ribozymes methods to inhibit the in vivo translation of genes are lcnown in the art (e.g., De Mesmaeker et al., Curr. Opin. Struct. Biol., 5:343, 1995; Gewirtz et al., Proc. Natl.
Acad. Sci. USA, 93:3161,1996b; Stein, Chem. and Biol. 3:319, 1996).

Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target inolecule for ribozyme cleavage sites, which include the following sequence: GUA, GUU and GUC. Once identified, short RNA sequences of about 15 to 30 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of caiididate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.

It is possible that the antisense, ribozyme, or triple helix molecules described herein can reduce or inhibit the translation of mRNA produced by inutant PKD 1 alleles of the invention. In order to ensure that substantial nornial levels of PKD1 activity are maintained in the cell, nucleic acid inolecules that encode and express PKD1 polypeptides exhibiting normal PKD 1 activity can be introduced into cells that do not contain sequences susceptible to whatever antisense, ribozyme, or triple llelix treatments.
Such sequences can be introduced via gene therapy methods such as those described, below. Alternatively, it can be preferable to coadminister normal PKD1 protein into the cell or tissue in order to maintain the requisite level of cellular or tissue PKD1 activity.

Antisense RNA and DNA molecules, ribozyme molecules and triple helix molecules of the invention can be prepared by any method lrnown in the art for the sy7lthesis of DNA and RNA molecules. These include techniques for cheniically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well luiown in the art such as for example solid phase phosphoramidite chemical synthesis.
Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA
sequences encoding the antisense RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polyinerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Various well luiown modifications to the DNA molecules can be introduced as a means of increasing intracellular stability aiid half-life. Possible modifications include, but are not liinited to, the addition of flatil{ing sequences of ribonucleotide or deoxyribonucleotides to the 5' or 3' end or both of the molecule or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide baclcbone.

As discussed above, mutations in the PKD I polynucleotide that cause ADPKD
can lower the level of expression of the PKD 1 polynucleotide or;
alternatively, can cause inactive or substantially inactive PKDl proteins to be produced. In either instance, the result is an overall lower level of normal PKD1 activity in the tissues or cells in which PKD 1 is normally expressed. This lower level of PKD 1 activity, then, leads to ADPKD
symptoms. Thus, such PKD1 mutations represent dominant loss-of-function mutations.
For example, a polynucleotide having a sequence as set forth in SEQ ID NO:1 and having a mutation of a G9213A results in early termination of PKD1.

For exanZple, normal PKD1 protein, at a level sufficient to ameliorate ADPKD
symptoms can be adininistered to a patient exhibiting such symptoms or having a mutant PKD1 polynucleotide. Additionally, DNA sequences encoding normal PKDl protein can be directly administered to a patient exhibiting ADPKD symptoms or administered to prevent or reduce ADPKD symptoms where they have been diagnosed as having a PKD 1 inutation identified herein but have not yet demonstrated symptoms. Such administration can be at a concentration sufficient to produce a level of PKDI
protein such that ADPKD syinptoms are ameliorated.
Further, subjects with these types of mutations can be treated by gene replacement therapy. A copy of the normal PKD 1 polynucleotide can be inserted into cells, renal cells, for example, using viral or non-viral vectors that include, but are not limited to vectors derived from, for example, retroviiuses, vaccinia virus, adeno-associated virus, herpes vin.ises, bovine papilloma virus or non-viral vectors, such as plasmids. In addition, techniques fiequently employed by those skilled in the art for introducing DNA into mammalian cells can be utilized. For example, methods including but not limited to electroporation, DEAE-dextran mediated DNA transfer, DNA
guns, liposomes, direct injection, and the like can be utilized to transfer recombinant vectors iilto host cells. Alternatively, the DNA can be transferred into cells through conjugation to proteins that are normally targeted to the inside of a cell. For example, the DNA can be conjugated to viral proteins that nonnally target viral particles into the targeted host cell.

Adininistering the whole gene or polypeptide is not necessary to avoid the appearance of ADPKD symptoms. The use of a"minigene" therapy approach also can serve to ameliorate such ADPKD symptoms (see Ragot et al., Nature 3:647, 1993;
Duncldey et al., Hum. Mol. Genet. 2:717-723, 1993). A nzinigene system uses a portion of the PKD 1 coding region that encodes a partial, yet active or substantially active PKD 1 polypeptide. As used herein, "substantially active" means that the polypeptide serves to ameliorate ADPKD symptoms. Thus, the minigene system utilizes only that portion of the normal PKDl polynucleotide that encodes a portion of the PKD1 polypeptide capable of ameliorating ADPKD symptoms, and can, therefore represent an effective and even more efficient ADPKD therapy than full-length gene therapy approaches.
Such a minigene can be inserted into cells and utilized via the procedures described, above, for fiill-length gene replacement. The cells into which the PKD1 minigene are to be introduced are, preferably, those cells, such as renal cells, wlv.ch are affected by ADPKD. Alternatively, any suitable cell can be transfected with a PKD1 minigene so long as the minigene is expressed in a sustained, stable fashion and produces a polypeptide that aineliorates ADPKD symptoms.

A therapeutic minigene for the amelioration of ADPKD symptoms can comprise a nucleotide sequence that encodes at least one PKD1 polypeptide peptide domain, particularly a domain having an amino acid sequence substantially identical to a peptide portion SEQ ID NO:2 and having a mutation as shown in Table 4, for example, an A88V, W967R, L2696R, R2985G, W3001X, R3039C, V32851, or H3311R mutation. Miiiigenes that encode such PKD1 polypeptides can be synthesized and/or engineered using the PKD 1 polynucleotide sequence (SEQ ID NO:1).

The materials for use in the assay of the invention are ideally suited for the preparation of a kit. Such a kit can comprise a carrier means containing one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. One of the 5 container means can coinprise a probe that is or can be detectably labeled.
Such probe can be an oligonucleotide comprising at least 10 contiguous nucleotides and having a sequence of a fiagment of SEQ ID NO:l including: nucleotide 474, wherein nucleotide 474 is a T; nucleotide 487, wherein nucleotide 487 is an A;
nucleotide 3110, wherein nucleotide 3110 is a C; nucleotide 8298, wherein nucleotide 8298 is a G;
10 nucleotide 9164, wherein nucleotide 9164 is a G; nucleotide 9213, wherein nucleotide 9213 is an A; nucleotide 9326, wherein nucleotide 9326 is a T;
nucleotide 9367, wherein nucleotide 9367 is a T; nucleotide 10064, wherein nucleotide 10064 is aii A; nucleotide 10143, wherein nucleotide 10143 is a G;
nucleotide 10234, wllerein nucleotide 10234 is a C; or nucleotide 10255, wherein 15 nucleotide 10255 is a T (see, also, Exainple 2).

A kit containing one or more oligonucleotide probes of the invention can be useful, for exaiuple, for qualitatively identifying the presence of mutant polynucleotide sequences in a sample, as well as for quantifying the degree of binding 20 of the probe for determining the occurrence of specific strongly binding (hybridizing) sequences, thus indicating the lilcelihood for a subject having or predisposed to a disorder associated with PKD 1. Where the lcit utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing reagents for amplification of the target nucleic acid sequence. When it is desirable to amplify the 25 mutant target sequence, this can be accomplished using oligonucleotide primers, which are based upon identification of the flanking regions contiguous with the target nucleotide sequence. For example, primers such as those listed below in Tables and 2 can be included in the kits of the invention. The kit can also contain a container comprising a reporter means such as an enzymatic, fluorescent, or radionuclide label, 30 which can be bound to or incorporated into the oligonucleotide and can facilitate identification of the oligonucleotide.

The following examples are intended to illustrate but not limit the invention.
EXAMPLES
The present invention is based upon the use of widely spaced PKD1-specific anchor primers in long range PCR to generate 5 kb to 10 kb PKD 1 polynucleotide segments. After appropriate dilution, the PCR products can be used as a template for mutation screening using any one of a variety of methods. Accordingly, a number of mutants have been identified in families with PKD 1 -associated disorders.

Using a nuinber of PKD 1-specific primers, eight templates ranging in size from about 0.3 to 5.8 kb were generated that span from the 5' untranslated region to intron 34 and cover all exons in the replicated region including exon 1 and exon 22 (Example 1). These reagents were used to evaluate 47 Asian PKD 1 families (Example 2). Variant nucleotide sequences were found throughout the PKD1 polynucleotide sequence.

Forty-one Thai and 6 Korean ADPKD fatnilies were studied. Samples from 50 healthy Thai blood donors collected in blood banks served as normal controls.
Genomic DNA was extracted from either fresh or frozen whole blood that had been stored for up to five years using commercially available kits (PuregeneTM, GentraTm) or standard phenol-chlorofoim methods. For the N23HA and 145.19 cell lines (Cell 77:881-894, 1994; Germino et al., Ani J. Hum. Genet. 46:925-933, 1990;
Ceccherini et al., Proc. Natl. Acad. Sci. USA 89:104-108, 1992, each of which is incorporated herein by reference; see, also, Watnick et al., siapra, 1997), genomic DNA was isolated using the Pluegene DNA isolation kit.

LONG RANGE SPECIFIC TEMPLATES

A two-part strategy was used to generate and validate PKD 1-specific primers that could be used to amplify the replicated portion of PKD 1. The sequence of (SEQ ID NO: 1) was aligned with that of two homologues present in GenBank (Accession Number AC002039) and identified potential sequence differences.

Candidate primers were designed such that the mismatches were positioned at or adjacent to the 3' end of the oligonucleotide so as to maximize their specificity for PKD 1.

The primers were tested for specificity using rodent-human somatic cell hybrids that either contained only human 16p 13.3 and tlierefore, human PKD 1 (145.19, a radiation hybrid), or that lacked 16pl3.3 and contained only the human PKD1-homologues (N23HA). Figure 2 presents a representative example of this approach using the primer pair, BPF6 and the PKD1-specific primer BPR6. This primer pair amplified a product of the correct length (4.5 kb) under the stated conditions only w11en total human genomic DNA or 145.19 DNA is used as template.
Similar results were obtained when BPR6 was used in combination with the non-specific primer 28F to generate a much shorter product.

As a final control, the absence of amplified product was verified using N23HA
as template to confirm that the results obtained using total human genomic DNA
and145.19 DNA were due to the specificity of the primer and not the result of other causes (i.e., difference in quality of DNA or ratio of human/rodent template).
A
primer specific for the homologues (BPR6HG) was designed that was positioned the same distance from BPF6 as BPR6 and used to amplify a specific band of the same size as the corresponding PKD 1-long range product. As predicted, a product of the correct size was amplified from both N23HA and total genomic DNA, but not from 145.19.

A total of eight primer pairs can be used to generate a series of templates that range in size from about 0.3kb to 5.81cb and include all exons and their flanking intron sequences in the replicated portion of PKD 1(exons 1 to 34). Table 1 summarizes the details for each prodtict and includes the sequence of each primer, its respective position within the gene, its expected size, and the optimal annealing temperature and extension time for its amplification. Figure 1 illustrates the relative position of each product with respect to the overall gene structure. It should be noted that exon 1 and its flanking sequences were particularly problematic to evaluate. Primer design was Table 1 Oligonucleotide primers for Long-range sPecific templates from exon 1-34 of PKDl gene Template Priiners Sequence 5'-3' Position Size Tm ET SEQ
(5') (kb) ( C) (Min) ID
NO:
Tl BPF14* CCATCCACCTGCTGTGTGAC 2043 2.2 69 7 3 CTGGTAAAT

AAGCAT
T2-7 BPF9* ATTTTTTGAGATGGAGCTTC 17907 4.6 68 7 5 ACTCTTGCAGG

T8-12 BPF12 CCGCCCCCAGGAGCCTAGAC 22218 4.2 68 7 7 G
BPR5* CATCCTGTTCATCCGCTCCA 26363 8 CGGTTAC
T13-15 F13 TGGAGGGAGGGACGCCAAT 26246 4.4 68 7 9 C
R27* GTCAACGTGGGCCTCCAAGT 30612 10 T15-21 F26* AGCGCAACTACTTGGAGGCC 30603 3.4 70 4.5 11 C

CCATCCTAC
T22 BPF15 GAGGCTGTGGGGGTCCAGTC 36815 0.3 72 1 13 AAGTGG
BPR12* AGGGAGGCAGAGGAAAGGG 37136 14 CCGAAC
T23-28 BPF6 CCCCGTCCTCCCCGTCCTTTT 37325 4.2 69 7 15 GTC
BPR6* AAGCGCAAAAGGGCTGCGT 41524 16 CG
T29-34 BPF13* GGCCCTCCCTGCCTTCTAGG 41504 5.8 68 8 17 CG
KG8R25* GTTGCAGCCAAGCCCATGTT 47316 18 A
Tm - annealing teinperature; ET - extension time; *- PKD 1-specific primer.
Bold type in BPR12 primer sequence identifies intentional replacement of C by A to enliance discrimination of PKD1 from homologs.

greatly limited by the high degree of homology and extreme GC bias in the region. A
combination of widely space primers (to generate a fragment considerably larger than the segment of interest) and the GC melt system were used to circunivent these obstacles.

Specific details concerning the primer sequences, annealing temperatures and extension times used for each long-range (LR) template are provided in Table 1(all sequences in Tables 1 and 2 are shown in 5' to 3' orientation from left to right). Three hundred to 400 ng of genomic DNA was used as template for each LR product, except for exon 1(see below). The long range PCR amplification was performed as follows in a Perkin Elmer 9600 thernial cycler: denaturation at 95 C for 3 min followed by 35 cycles of a two-step protocol that included denaturation at 95 C for 20 sec followed by annealing and extension at a temperature and for a time specific for each primer pair (Table 1). A final extension at 72 C for 10 min was included in each program. The total PCR volume was 50 l using 4 U of rTth DNA polymerase XL
(CetusT"', Perkin Elmer) and a final MgOAC2 concentration of 0.9 mM. A hot start protocol as recommended by the manufacturer was used for the first cycle of amplification. For the exon 1 LR product (TI), the LR was generated using 500 ng of genomic DNA. The lorig range PCR amplification was modified as follows:
denaturation 95 C for 1 min followed by 35 two-step cycles of denatiu-ation at for 30 sec followed by aruiealing and extension at 69 C for 7 min. The total PCR
volume was 50 l using I l of AdvantageTm-GC genomic polymerase (Clontech), GC melt of 1.5 M and final MgOAC2 concentration of 1.1 mM.

The long-range templates were serially diluted (1:104 or 1:105 ) to remove genomic contaminationõ then used as templates for nested PCR of 200-400 bp exonic fragments. A total of 17 new primer pairs were developed for exons 1-12 and exon 22. The sequences and PCR conditions for each new pair are summarized in Table 2. Primer sequenees and PCR conditions for exons 13-21 and 23-34 are described in Watnick et al., Am. J. Hum. Genet. 65:1561-1571, 1999; and Watnick et al., Hum. Mol. Genet. 6:1473-1481, 1997.

Intron based primers were positioned approximately 30-50 bp away from consensus splice sites. Exons larger than approximately 400 bp were split into overlapping fragments of less than or equal to 350 bp. Two l of diluted long range (LR) product was used as template for amplification of each exon. Single strand conformation analysis was performed using standard protocols. SSCA analysis was performed by use of 8% polyaciylamide gels with 5% glycerol added. The Table 2 Nested Primers Used for Mutation Detection Exons Prinzer Priiner Sequence 5' 3' Fragment T. ( C) SEQ
size (bp) ID
NO:
Tl 1F1 GGTCGCGCTGTGGCGAAGG 328 67 19 Tl 1F2 ACGGCGGGGCCATGCG 348 67 21 Tl 1R2 GCGTCCTGGCCCGCGTCC 22 C

GG

C

A

T8-12 11midF GCTTGCAGCCACGGAAC 386 65 47 T8-12 11midR GCAGTGCTACCACTGAGAAC 48 TAC

AG

TABLE 2 (cont.) Exons Primer Sequence 5' 3' Fragment T,õ ( C) SEQ ID
size (bp) NO:

13 13F: TGGAGGGAGGGACGCCAATC 308 67 62 13R: GAGGCTGGGGCTGGGACAA 63 14 14F: CCCGGTTCACTCACTGCG 220 64 64 14R: CCGTGCTCAGAGCCTGAAAG 65 15 15F16: CGGGTGGGGAGCAGGTGG 280 67 66 15R16: GCTCTGGGTCAGGACAGGGG 67 A
15 15F15: CGCCTGGGGGTGTTCTTT 270 64 68 15R15: ACGTGATGTTGTCGCCCG 69 15 15F14: GCCCCCGTGGTGGTCAGC 250 67 70 15R14: CAGGCTGCGTGGGGATGC 71 15 15F13: CTGGAGGTGCTGCGCGTT 256 67 72 15R13: CTGGCTCCACGCAGATGC 73 15 15F12: CGTGAACAGGGCGCATTA 270 65 74 15R12: GCAGCAGAGATGTTGTTGGA 75 C
15 15F11: CCAGGCTCCTATCTTGTGACA 259 60 76 15R11: TGAAGTCACCTGTGCTGTTGT 77 15 15F10: CTACCTGTGGGATCTGGGG 217 67 78 15R10: TGCTGAAGCTCACGCTCC 79 15 15F9: GGGCTCGTCGTCAATGCAAG .267 67 80 15R9: CACCACCTGCAGCCCCTCTA 81 15 15F8: 5CCGCCCAGGACAGCATCTTC 261 64 82 15R8: CGCTGCCCAGCATGTTGG 83 15 15F7: CGGCAAAGGCTTCTCGCTC 288 64 84 15R7: CCGGGTGTGGGGAAGCTATG 85 15 15F6: CGAGCCATTTACCACCCATA 231 65 86 G
15R6: GCCCAGCACCAGCTCACAT 87 15 15F5: CCACGGGCACCAATGTGAG 251 64 88 15R5: GGCAGCCAGCAGGATCTGAA 89 15 15F4: CAGCAGCAAGGTGGTGGC 333 67 90 15R4: GCGTAGGCGACCCGAGAG 91 15 15F3: ACGGGCACTGAGAGGAACTT 206 64 92 C
15R3: ACCAGCGTGCGGTTCTCACT 93 15 15F2: GCCGCGACGTCACCTACAC 265 67 94 15R2: TCGGCCCTGGGCTCATCT 95 15 15F1: GTCGCCAGGGCAGGACACAG 228 68 96 R27': AGGTCAACGTGGGCCTCCAA 113 15 15F1-1: ACTTGGAGGCCCACGTTGAC 276 69 97 C
15R1-1: TGATGGGCACCAGGCGCTC 98 15 15F1-2: CATCCAGGCCAATGTGACGG 266 64 99 T
15R1-2: CCTGGTGGCAAGCTGGGTGT 100 T
16 16F: TAAAACTGGATGGGGCTCTC 294 56 101 16R: GGCCTCCACCAGCACTAA 102 TABLE 2 (cont.) Exons Primers Primer Sequence 5'-3' Fragment Tm ( C) SEQ ID
size (bp) NO:
17 17F: GGGTCCCCCAGTCCTTCCAG 244 67 103 17R: TCCCCAGCCCGCCCACA 104 18 18F: J GCCCCCTCACCACCCCTTCT 342 67 105 18R: TCCCGCTGCTCCCCCCAC 106 19 19F: IGATGCCGTGGGGACCGTC 285 67 107 19R: GTGAGCAGGTGGCAGTCTCG 108 20 20F: CCACCCCCTCTGCTCGTAGGT 232 64 109 20R: GGTCCCAAGCACGCATGCA 110 21 21F: TGCCGGCCTCCTGCGCTGCTG 232 67 111 A
TWR2-1: GTAGGATGGCCCCACCTGCT 112 CACCCTGC

radiolabeled PCR products were diluted with loading buffer, were denatured by heating at 95 C for 5 min, then were placed on ice prior to being loaded and run on the gel at room temperature. Gels were run at 400 V overnight, dried, and placed on X-Omat rM XAR film (Kodak) at room temperature. Aberrantly migrating bands detected by SSCA were cut from the gel and eluted into 100 l of sterile water overnight. The eluted products were re-amplified using the same set of primers, purified using CentriconTm -100 columns (Amicon) and then sequences.
Variants that we:re predicted to alter a restriction site were confirmed by restriction enzyme digestion analysis of re-amplified products. In cases where the change did not alter a restriction site, primers were designed with mismatches that create a new restriction site when combined with the point mutation in question. The following prinier colnbinations were utilized:
ASP 1+26R (AS P 1; 5'-CTGGTGACCTACATGGTCATGGCC GAGATC-3';
SEQ ID NO:55);
ASP2+30R (AS P2; 5'-GGTTGTCTATCCCGTCTACCTGGCCCTCCT-3';
SEQ ID NO:56);
ASP3 + 30F (ASP3; 5'-GTCCCCAGCCCCAGCCCACCTGGCC-3'; SEQ ID
NO:57).

When possible, segregation of the variant with the disease phenotype was tested. In cases wllere a missense change was unable to be determined on the riormal haplotype (and tlius be a normal variant) the mutation was tested for in a panel of 50 normal controls.

MUTATION SCREENING
The new PKD1-specific products were generated from one affected member of each of the 47 Asian families and then used as template for mutation detection of exons 1-12 and 22-34. Table 2 lists the sequence and PCR condition for primer pairs that were used for nested amplification of individual exons and their adjacent intronic sequence. Overlapping pairs were designed for segments >400 base pairs in length.
A total of 13 novel variants were detected by SSCA using the conditions described above. Two are higllly likely to be pathogenic mutations, four are predicted to encode missense substitutions not found in normals and seven are normal variants (see Table 3).

The first pathogenic mutation is a G to A transition at position 9213 in exon 25 that is predicted to result in a nonsense codon (W3001X). Its presence was confirmed by restriction analysis using the enzyme Mae I and it was found to segregate with disease. This variant is predicted to truncate the protein near the carboxyl end of the Receptor for Egg Jelly (REJ) domain. The W3001X mutation results in a greatly truncated product missing all of the membrane spanning elements, intervening loops and carboxy terminus. The second mutation (T3110C) is predicted to result in a non-conservative amino acid substitution (W967R) at a critical position of one of the PKD repeats. The mutation is unique to the family in which it was found and was not observed in a screen of over 100 normal Thai chromosomes.
The W967R missense mutation is predicted to disrupt the secondary structure of PKD
domain 3. The WDFGDGS (SEQ ID NO:58) motif within the CC' loop region is the most conserved sequence of the PKD domains. The tryptophan is replaced is the first residue of the turn at the end of the C strand and is conserved in 14 out of domains. Moreover, it is evolutionarily conserved in mouse and Fugu polycystin-1.

Table 3 Mutations Identified in the PKD1 Gene in a Thai Population Patient 7 Exon Nucleic Acid Codon Change Consequence Confirmation Change Enzyme Pathogenic RAMA28-01 12 T3110C W967R Missense BsaW 1 (disrupt PKD (cut NC) domain3) RAMA59-02* 25 G9213A W3001X Nonsense (early Mae I
termination) Variants not found in 100 cliromosomes R4MA3-02* 22 T8298G L2696R Missense HinP1I
RAMA87-01* 25 A9164G R2985G Missense BsrB 1 R4MA87-01* 25 C9326T R3039C Missense Fau I(cut NC) RAMA45-03* 29 G10064A V32851 Missense Bsm I
Probable normal variants RAMA7-06 2 C474T A88V Missense Hph I
RAMA 107-01 2 G487A A92A Silent change TspR I
RAMA94-01 25 C9367T G3052G Silent change Sfo I (cut NC) RAMA66-01 30 A10143G H3311R Missense Nsp I (cut NC) RAM466-01 30 T10234C L3341L Silent change ASP1 + BseR
I
RAM451-01 30 G10255T R3348R Silent change ASP2 + MSC
I
*- Segregation with disease; 0- cannot test for segregation; NC - Normal control;
HG - Present in one copy of the homologues; ASP - Allele-specific primer.

These pathogenic inutations add to previously identified pathogenic mutations, including a deletion of G3336 (AG3336) in exon 13, resulting in a frame shift after amino acid 1041 (FS1041); C4168T (Q1653)X), C6089T (Q1960X) and C6326T
(Q2039X) mutations in exon 15, each resulting in a nonsense termination;
OG7205-G7211 in exon 16, resulting in a FS2331; a C7415T (R2402X) mutation in exon 18, resulting in a nonsense termination; a C7883T (Q2558X) mutation in exon 19, resulting in a ilonsense termination; and a OC8159-T8160 mutation in exon 21, resulting in a FS2649 (Phalcdeelciteharoen et al., supra, 2000). In addition, probable pathogenic inutations including G3707A (G1166S) and T6078A (V1956E) missense mutations in exon 15, and a C7433T (R2408C) missense mutation and an insertion of a GCG trinucleotide between G7535 and G7536 (extra G1y2422) in exon 18 have been identified (Phalcdeelciteharoen et al., supra, 2000).

Four additional mutations unique to one of the families also were identified (see Table 3). The mutants segregate with disease, and were not observed in a screen of over 100 normal Thai chromosomes. Three of the four variants are predicted to result in non-conservative amino acid substitutions. Two of them (A9164G, C9326T) are present in the same allele of a single family (RAMA87). As such, these mutations meet several criteria expected of disease-producing mutations, including they are not found in normal, ethiiically matched chromosomes, they segregate with the disease, and they result in non-conservative substitutions.
In one case a heteroduplex pattern was discovered for the exon 22 product of the proband by standard agarose electrophoresis. The heteroduplex pattern was confirmed to segregate with disease and subsequently determined that the novel variant was the result of a T to G transversion at position 8298. This mutation is predicted to substitute arginine for leucine at position 2696 of the protein sequence.
This non-conservative substitution is within the REJ domain. Interestingly, the R3039C substitution occurs near a newly described putative proteolytic cleavage site of polycystin-1, His(3047)-Leu-Thr-Ala(3050) (SEQ ID NO:59). In the corresponding position of Fugu and murine polycystin-1, glutainic acid and arginine, respectively, are present, suggesting a non-critical role for a non-polar residue at this location.

Seven nucleotide substitutions that are likely normal variants were also identified. Two are missense variants that do not segregate with disease in the family in which they were discovered. The C474T substitution results in the conservative replacement of valine by alanine at position 88 in the first leucine ricll (LRR) repeat.
The amino acid is not conserved between species and is not predicted to disrupt the LRR structure. The second missense variant, A10143G, substitutes arginine for histidine at position 3311 witllin the first extracellular loop between TM2 and TM3.
It too, is a conservative change involving a residue whose identity is not evolutionarily conserved at this position. The otlier five variants were silent nucleotide substitutions that were unique to the pedigree in which they were found and not found in more than 100 normal chromosomes. It is possible that these variants can be pathogenic by affecting gene splicing in the region. Two of the normal variants of exon 30, A10143G (H3311R) and T10234C (L3341L), were clustered together in a single PKD1 haplotype. Interestingly, both variants also are present in at least one of the homologues, suggestiiig a previous gene conversion event as the original of these PKD 1 variants. Additional PKD 1 variants, which do not appear to be associated with a PKD1-associated disorder, include two silent mutations, G4885A (T1558T) and C6058T (S1949S), and a missense mutation, G6195A (R1995H), in exon 15; a silent T7376C (L2389L) mutation in exon 17; a silent C7696T (C2495C) mutation in exon 18; and a missense G8021A (D2604N) mutation in exon 20 (Phakdeelcitcharoen et al., supra, 2000).

Table 4 summarizes the clinical findings for the probands of 17 Thai families.
The genotypes and phenotypes for patients with ADPKD are shown. It has been estimated on the basis of studies of Caucasian populations that approximately 15% of mutations are localized to the nonreplicated portion of the PKD 1 gene. If the same frequency is true for the Thai population (the patients were not screened for mutations in the nonreiterated portion), then the present studies have identified approximately 45% to 54 percent of all mutations present in the nonreplicated region. This detection rate likely can be increased by using more sensitive detection methods such as DHPLC (Kristensen et al., supra, 2001), HTCSGD (Leung et al., supra, 2001), or the lilce.

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Although the invention has been described witli reference to the above examples, it will be Lulderstood that modifications and variations are encompassed within the spirit and scope of the iuvention. Accordingly, the invention is limited only by the following claims.

SEQUENCE LISTING
<110> THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE
GERMINO, Gregory G.
WATNICK, Terry J.
PHAKDEEKITCHAROEN, Bunyong <120> DETECTION AND TREATMENT OF POLYCYSTIC KIDNEY DISEASE
<130> 581-259 <140> CA 2,395,781 <141> 2001-07-13 <150> US 60/283,691 <151> 2001-04-13 <150> US 60/218,261 <151> 2000-07-13 <160> 113 <170> PatentIn ver'sion 3.0 <210> 1 <211> 53522 <212> DNA
<213> Homo sapiens <400> 1 tgtaaacttt ttgagacagc atctcaccc:t gttccccagg ctggagtgca gtggtgtgat 60 catggctcac tgcagcgtca acctcctggg tctacttgat ctgtaaactt cgagggaagg 120 tgtaataaac cctcctgcaa tgtctttgt.t tttcaaaatc tttgtatttc acagtttagc :180 ttcgtgggtt gatgttctat tttgtttttg tgtgtgtgtg tgtgtgtttt gtgttttttt :240 ttgagacaca gtcttgctct tgttgcccag gctggagtgc aatggtgtga tcttggctca :300 ctgcaacttc cacctcttgg gttcaagaga ttctcctgcc tcagccttcc gagtagctag :360 gattacaggc gccgccacca caccccgcta attttgtatt tttagtagag atggggtttc 420 tccatattgg tcaggctggt ctcaaactc.c cgacctcagg tgatccgccc acctcagcct 480 cccaaaatgc tgggattaca ggcgtgagtc accgcacctg gccaatgttc tatttttgag 540 aacacaacag ttcataatat attctacata gaccatacct gttatgtgta gataaacaga 600 ctcttttccc atttaacacc ttttgcctta ggtttatttt tctggtatca atactggcac 660 acttactttg tttgcagttt cctgtctttt tttttttttt tttttttttt gagacagagt '720 ctcactctgt cacccaggct ggagtgaagt ggcgggatct cggctcactg caacctctac '780 ctcctgggtt catgcgattc tcctgccr_ca gcttcccgaa tagctgagac cacaactgtg E340 tgccaccatg cccagccaat ttttgtattt ttagtagaca cggggtttca ccatactggc 900 caggatggct caatctcttg acctcgtgat ccacctgcct ccgcctccca aagtgctggg 960 attacaggca tgagccactg tgcctggcct ttttttttct ttttgagatg gagtctcact 1020 ctgtcaccca ggctggagtg cagtggggta acctcaggtc actgcgacct ccgcctcccg 1080 ggttccagtg attctcctgc ctcagcctcc cgagtagctg ggattacagg cacccaccac 1140 catgcctggc taatttttgt atttttagta gagacggggt tttgccacgt tggccaggtt 1200 ggtctcgaac tcttggcctc atgtgacccg cctgccttgg cctcccaaag tgctgggatt 1260 acaggtgtga gccactgtgc ctggcctggc tttcttgttt cttttctcct cttctagttt 1320 ccccctttta ggctaacaat tattcactgt taataaaaac cctcaggtct gtattttatc 1380 aagaaacatt tccctcacgt cttcttccct gaaccaaaca agatctctgg cacattttat 1440 ttgctctgtc tcaccacatg gattttgttt ttttgtttct ttgttttttg agatggagtc 1500 tcactcttgt tgcccaggct ggagtgccat ggcacaatct cagctcactg caacctccac 1560 ctcctgggtt caagcgattc tcctgtctca gcctcctgag tagctgggat tacaggcgcg 1620 tggcaccacc cccagctaat ttttgtattt ttagtagaga cggggtttca ccatgttggt 1680 caggctggtc tcgaactcct gaccttgtga tctgcccacc ttggcctccc aaagtgctgg 1740 gattacaggc atgagccacc acgcccggcc cccatggttt ttcaaatagt ttagaatttc 1800 atttccaggt aactaatttg cttctttaaa catatgtctt ttctatttaa gaaatccttt 1860 ctaaacaatt gcattttatt ccacaaccgc cttcaaacaa tcattgagac ttggttaatc 1920 tgttttgctc atttggcagc agtttcttgt ggctgtttct tccctccact ggagtccttg 1980 aatcttaagt ctgtcatttg actgcaatta aaagctgggt ttggaataca atcgcagcct 2040 taccatccac ctgctgtgtg acctggtaaa tttctttttt tttttttgag acggagtctt 2100 gctctgttgc ccaggctgga gtgcagtggc acaacctctg cctcccaggt tcaagcgatt 2160 ctactgcctc aggctcccta gtagctggga ttataggtgc ctgccaccat gcccagctga 2220 tttttgtatt tttagtagag atgaggtttc accatgttgg ctaggctggt ctcgaacttc 2280 tgatcttgtg atctgcccgc ctcggcctcc caaagtgctg ggattacagg catgagccac 2340 cactcccagc cagttctttt tttctttttt ccattttttt ttttttcgag acaggatctt 2400 actcttttgc ccaggcggga gtgcagtggc acaatcacgg ctcagcgcag ccactgccta 2460 ctgggctcac acgctcctcc ggcctcagcc tctcgagtac ctgggactac aagcgtgagc 2520 cagtttggct aattttggct aatttttgta gaaacggggt ctcgccatgt tggccaggct 2580 ggtctccaac tcctggactc aagggatcca ccttCCtCCc cctctcaaag ttctgggatt 2640 accggagtga gccactgtgc cctgctggca aatttcttaa actgtctgtg cctcagtgac 2700 ctcatttaat aaagggaata attgtagcac actttttcta gagctgtgaa gattcaatgg 2760 aataaataag gcaataaatg aatggatggg gaatgaagga tgtgggtttc ctccctcttg 2820 tctttcaata agctctcacc atcaacctcc cattgcctgt tctctctctt CCCCCtctCt 2880 ccctctgtct ctctctcagc caggaaacct ggggtaggga ggcttggagc cagcgggtgc 2940 gtcgggaggc tgcgggtact gactcgggcc gcgcacggag atcgcgggag aaggatccac 3000 aaccgcggaa gaaggatcag ggtggagcct gtggctgctg caggaggagg aacccgccgc 3060 ctggcccaca ccacaggaga agggcggagc agatggcacc ctgcccaccg cttcccgccc 3120 acgcacttta gcctgcagcg gggcggagcg tgaaaaatag ctcgtgctcc tcggccgact 3180 ctgcagtgcg acggcggtgc ttccagacgc tccgccccac gtcgcatgcg ccccgggaac 3240 gcgtggggcg gagcttccgg aggccccgcc ctgctgccga ccctgtggag cggagggtga 3300 agcctccgga tgccagtccc tcatcgctgg cccggtcgcg ctgtggcgaa gggggcggag 3360 cctgcacccg ccccgccccc cctcgccccg tccgccccgc gccgcgcggg gaggaggagg 3420 aggagccgcg gcggggcccg cactgcagcg ccagcgtccg agcgggcggc cgagctcccg 3480 gagcggcctg gccccgagcc ccgagcgggc gtcgctcagc agcaggtcgc ggccgcagcc 3540 ccatccagcc cgcgcccgcc atgccgtccg cgggccccgc ctgagctgcg gcctccgcgc 3600 gcgggcgggc ctggggacgg cggggccatg cgcgcgctgc cctaacgatg ccgcccgccg 3660 cgcccgcccg cctggcgctg gccctgggcc tgggcctgtg gctcggggcg ctggcggggg 3720 gccccgggcg cggctgcggg ccctgcgagc ccccctgcct ctgcggccca gcgcccggcg 3780 ccgcctgccg cgtcaactgc tcgggccgcg ggctgcggac gctcggtccc gcgctgcgca 3840 tccccgcgga cgccacagcg ctgtgagtag cgggcccagc ggcacccggg agaggccgcg 3900 ggacgggcgg gcgtgggcgg gttccctggc ccgggacggg aagcaggacg cgggccagga 3960 cgctcccagg ggcgaggctc cggcgcggca cggcgggccc tgctaaataa ggaacgcctg 4020 gagccgcggt tggcacggcc ccggggagcc gaaaaacccc gggtctggag acagacgtcc 4080 cacccggggg ctctgcagac gccagcgggg gcggggcgcg gaggccgcgc tcagctggga 4140 ggacaaacag tcgctaattg gagaggaatt gggatgcggc ctggggctgc ggggtacccg 4200 gagaggtggg gatggctgta gggggcggca gggaagagtt ccaggaggtg tctggaaaag 4260 gatttgatgg atgtgcaaga attgggctga tgcttaggaa ggggcgatga ggtgggtcca 4320 gaagaagggg ggtgaacggt gtgagcaaag accgtgaggc tggaggctgg ccacgggagg 4380 tgtgaggggt aggggcaggg tgggaggtgg gctcgcgggt gggctggggt catgaagggc 4440 ctcaggcgct ctgctattgg gttccaaggc tatcctgaga acaggggtga ggggggattg 4500 ccgtgggggg ttaaagcctt gtcatgttcg ctttcgggag ataaaaacaa caggtggcct 4560 ttatggagac gctgcccaga gccaggtctg tgccaggctc ctgttggggg tcgtcatgcg 4620 gaatcctgac tctgaccatc cgaggcatag ggaccgtgga gatttgcatt tcacagatga 4680 ggaaacaggt ttggagaggt gacacgacct gtcccaggca tcacagccgg gatgtgcata 4740 gcaggggttt ggaactatga ggtgcccagg acccagggtt ggattgaaaa gggcggaggg 4800 gactaagata agcagacagt tgtccccagc gctggggaga gtcttgggac cagtctgatg 4860 ccttgtattt cccaggctcc aggctcctcg ccgggacagt gtctccttgg gtgcgtgctg 4920 gatccctggg ggacgtggca catccccagg cttgctaaac attgggtggg ttctggcatt 4980 tggttttgta acgtttctgg gtcactcccg cctgtggcca cccttcctta ggggagccgt 5040 gtgtccttgg ggctttgctg ggtggtctcg agggtgggag aagaatgggt tctcctggac 5100 caatggagcc cgtgcccctc ggggccacat tgctcctgcg ctccctgact gcggacgcgt 5160 gtgtctcgcg gctgtctctg tggagatggc ctcctcctgc ctggcaacag cacccacaga 5220 attgcatcag acctacccca cccgttgttt gtgatgctgt agctgagggc tcctctgtct 5280 gccaggccgg tcactgggga ctctgtccag ggcctggtgg ttcctgcttc ccagcacctg 5340 atggtgtcca tgagagcagc ccctcaggag ctgtccggga gagaagggcg ctggtggctg 5400 ctgagcggag agcaaggccc gtgttctcca ggcccttggc acagcagtgg agcccccgcc 5460 cctgccttgt gttgtcctct taggctctgg tcctggggtt tggaggaggg ggaccctggg 5520 agttggtggc ctgtcccagc ctgagctggc aagattccga atgccaggcc ccccaagtgt 5580 gcaacagggc acagggtgac ctcatgtggg caggtgggtg ctgttctgta cacacctggg, 5640 gccgccgctg ggagagttct ggaaggtggg gtgaggggac ccatggcaaa ctagggcctt 5700 aggaaggatg tgaaggccct ggctggcccc ccaggccacc ctctgtgctg tggggcagcc 5760 cagccatttt gctgtctacc ctgcaaactc ctcctcgggg agacggctgg gttttcccca 5820 gggaagaggg gtcaagctgg gagaggtgaa ggacacagat cacagctgct ggcaggtgtt 5880 caagggtcca agagcgttgc tgtctgggtg tcaccagtag ccttcctggg gggctcacgc 5940 aggtgcctct ccacttgtgg ctccctggct gctgaagctc agcagggaca gctgtgtcca 6000 gttccaggtg gaggacagcc ggggcttctg aggccacagc ctgccttggg ttaatgatgc 6060 tgccgagagg tggtggcttt tggaaaagat ggcgtactgc aaaacgtgct gctctgcgtg 6120 gctcgaagct tcgtggggag acgtgggcag agccgtggct gactcacaga ccccccaccc 6180 cagagcctgc cctgccctcc ctgccccgac ccttctccct cctgacccat gtgttttttt 6240 tttttttttt tttttttgag acagagttca ctcttgttgc caaggctgga gtgcaatggc 6300 acgatctcgg ctcatggcaa cctccgcctc ctgggttcaa gcgctttttc ctgcctcagc 6360 ctcccgagta gctgggatta caggcgtgca ccaccatgcc tggctaattt tgtattttta 6420 gtagagacag ggtttctcca tattggtcag gctggtcttg aactcctgac ctcagatgat 6480 ccgcccgcct cggcctccca aagtgctggg attacaggca tgagccacca cgcccagccc 6540 tgacccatgt tttgaaccaa attccagcca cccttttatc tgcaagcatt ttggagggca 6600 tcgcaatact gcagacccac ctaacacaac agacagttcc ttcatgccac cgaaggcctg 6660 gtgtgttcac atttttggtt taatagtttg aattaagagc caaataaggt ccacacactg 6720 caattagttg atgtcttttt ttttttcttt tttttttttt ttttgagacg gagtcttgct 6780 cttgtctcca ggccgcagtg cagtggcatg atctcagctc accgcaacct ccgactccct 6840 ggttcaagcg attctcctgc ctcagcctcc cgagtacctg gtagctgggt ttacaggcat 6900 gcaccaccgt gcccagctaa tttttgtatt tttagtagag acggggtttt actgtgttgg 6960 ccaggatggt ctcgatctcc tgacctcgtg atctgcccac ctcggcctcc caaagtgctg 7020 ggattacagg cgtgagccac cgcacccggc caatgtcttt taaaaatata tacttttttt 7080 ttttttttga gacggagttt cgctcttgtt gcccaggctg gagtgcagtg gcgcgatctc 7140 acctcacggc aacctccgcc tcccgggttc aagtgattct cctgcctcag cctctccagt 7200 agctgggatt acaggcatgt gccaccatgc ctggctaatt ttgtattttt aggagagacg 7260 gggtttctcc acgttggtca ggctggtctc aaactcctga cctcaggtga tccgcctgcc 7320 ttggcctccc aaagtgttgg gattacaggt gtgagccaac gcgcccagac aaaaatatat 7380 gtgtgtcttt aaggctggtc aagcaaagca gtaggactgg agaaagaatg aagaattcta 7440 cctggctgtg atcaattcgt tgtgaacacc actgtgcttg gaccagctag ctgatgtctt 7500 ttgttttgtt ttgtttgaga cggagtctgg ctctgtcacc caggctggag gacaatggtg 7560 tgatctcggc tcactgcagc ctccatctcc cgggttcaag cgattctcct gcctcagcct 7620 cctgagtagc tgggattaga ggcgcgcgcc accacgcccg gctaattttt aaaaatattt 7680 ttagtagaga tggggtttca ccatgttggt caggctggtc ttgaactctt ggccttaggt 7740 gatctgcttg cctcggcctc ccaaagtgct gggattacag gtgtgagtga tgtattttat 7800 ttatttattt atttatttat ttttattatt tgagatggag tctcactctg ttgcccaggc 7860 tggagtgcag cagtgccatc tcagctcact gcaagctccg cctcctgggt tcacgccatt 7920 ctcctgcctc agcctcctga gtagcctgga ctggtgcccg ccaccatgcc cagctaattt 7980 tttgtatttt tagtagagac ggggtttcac cgtgttagcc aggatggtct ggatctcctg 8040 acctcgtgat cctcccgcct cagcctccca aagtgctggg attacaggct tgagccaccg 8100 cctgtctttt aaatgtccga tgatgtctag gagcttccct tcctctcttt ttccttgtgc 8160 aatttgttga agaaactggc tcctgcagcc tggatttctc gctgtgtctt gggggtgcca 8220 cctccatggt gtcacctccg tggtgctgtg agtgtgtgct ttgtgtttct tgtaaattgg 8280 tcgttggagc cgacatccca ttgtcccaga ggttgtcctg gctggcactg gcctaggtgt 8340 agatgtcatc agctcagggc cccctgctct aaaggccact tctggtgctg gttgccactc 8400 accctggctg ggggtcacct gggtctgctg ctgtctcgca aatgctgggg tccaggactg 8460 ggcacatcga gggacttggt aggtgcttgg ttcactgatg taaaatatag gagcacccgg 8520 ggccttgccc tttcccacct gcatccctga atgacaggag agtgtgggag agtgtaggga 8580 cagcaggcgc agaccccggg gcccctgcct gggattggcg tcggggaaga caggcattct 8640 ggagcgaccc ctaggcctga tgccttagag cgcaactgcc agagacacag cttccttggg 8700 gggctggcca ggccacggag gggccctggc tcccatttct ggtccctgga tcctgagagc 8760 gaggactagg gattgtcacc aaggcctcca tgagccctca gcagaaggag ggccaccctc 8820 gagggctccg ttatcactgg agcccgcgtt caaccaacac gcagatgatt ctccaaggac 8880 agagatggat gatggggagg gggctggcct ggaaggaccc ccagtgcagg tgacattgaa 8940 gccaggtttc aaagctccca cagggagctg cccagagaga gtccccaagg ggcaaggtga 9000 ctcgggggca ggggtagggc ctctgtcagg agagcctagg agaggcctgt gtcttctagg 9060 aagagccctg gcagccgagc ggaggcagtg gtgaggacct gcatcctgca tgtccagctg 9120 gcctcacccg gggtccctga gccgggtctt acgtggctcc cgcactcggg cgttcagaac 9180 gtgcctgcgt gagaaacggt agtttcttta ttagacgcgg atgcaaactc gccaaacttg 9240 tggacaaaaa tgtggacaag aagtcacacg ctcactcctg tacgcgattg ccggcagggg 9300 tgggggaagg gatggggagg ctttggttgt gtctgcagca gttgggaatg tggggcaccc 9360 gagctcccac tgcagaggcg actgtggaga cagagagcac ctgcaggtca tccatgcagt 9420 atcggcttgc atccagatca tacagggaac actatgattc aacaacagac agggaccccg 9480 tttaaacatg gacaaggggt cactcacgcc tggaatccca gcagtttggg aggccagggt 9540 gggtggatcg cttgagccca ggagtttgac accagcctgg gcaacagggt gagaccccgg 9600 tctctaaaaa ataaaagaac attggccggg cgtggtggta tgcatctgtg gtcccagcta 9660 ttcaggagac tgaggtggga catcacttga gccgaggagg tcaaggctgc agtgagctgt 9720 gatcacacca ctgcactcca ggctgggtca cagagcaaga ccctgtctca aaaaaaaaaa 9780 aaaaaaaaaa aaaaaatcac aggatctgaa cagagatttc tccaaagaag acgcacagat 9840 ggccaacagc gtgtgagaag atggtcggcc tcattagtca tgagggaaac gtaaatcaaa 9900 accactgtcc agccgggcgc ggtgcctcac gcctgtaatc ccagcacttt aggagagcag 9960 atggcttgag gccaggagtt tgaggccagc ctgggcaaca tagcgagacc aataaataga 10020 tattagtggt ggcgcctgta gtcccagcta gttgggaggc tgagggggga ggattccctg 10080 agtctatgag gttgagactg cagttagctg tgatggtgcc actgcactcc agcctgggcg 10140 actaggaaac ggtctttaaa aaaaaaaaaa aaaaacaggg tgggcgcggt ggttcacgcc 10200 tgtaatctca gcactttggg aggccaaggt ggggggatca caaggtcagg agtttgtgac 10260 cagcctgacc aacatggtga aaccccgttc tactaaaaat acaaaaatta gcgaggtgtg 10320 gtcgtgggcg cctgtaatcc cagctaatta ggaggctgag gcaggagaat cacttgaacc 10380 cgggaggcgg aggttgcagt gagccaatat cacaccactg cactctagcc tggtcaacag 10440 agcgagactc tgtctcaaaa aaaaaaaatg ctgagcgtgg tggcgcatgc ctgtagtctc 10500 agctactttg ggggctgagg caggagaatc gcttgaacct gggaggcaga ggtcgcagtg 10560 aggcaagatt gcaccattgc actccagcct gggagacaga gtgaaactct gtctcaaaaa 10620 gaaaaggtct aggaagagtc cgcaccctct ccccgcggtg gccacgccgg gctccgcgct 10680 gagccctctg tgttcttgtc tctccatacc tcatcacggc accgcagggt tgcagccact 10740 cctggtctca ttttacacac caggaaattg aggctctttg agaagccgtg gtgatgattt 10800 catcagcatg ctctggggca gacccctgca gccgcacagg gtgcctgggg cccacactag 10860 tgccctggtt tatagacaga cagaggtggc agtggcgctt ccgagtcggg ctgcgatgtg 10920 cttgcactcc ccgaggggct gaggggccct gcgcccaggt gcagctgctt gggtgctgcc 10980 agcccctccc acctctccct ccctgccagc ccctcccacc tctccctccc tgCCagCCCc 11040 tcccacctct ccctccctgc cagcccctcc cacctctccc tccctgccag cccctcccac 11100 ctctccctcc ctgccagccc ctcccacctc tccctccctg ccagcccctc ccacctctcc 11160 ctcCctgCCa gcccctccca cctctccctc cctccagccc ctcccacctc tcCCtcCCtg 1122.0 CcagcCCCtc ccacctctcc ctccctgcca gcccctccca cctctccctc cctgccagcc 11280 cctcccacct ctccctccct gccagcccct cccacctCtc cctccctgcc agcccctccc 11340 acctCtCCCt cCctgCCagc ccctcccacc tctccctccc tggctcatcc CtgCtgtgtC 11400 ccttctctct agtttcctgt tcagtttcag gaaggaggct gggaacccag atgtagggaa 11460 tttgcgccct ggagtcagac ctgggttcac gtcccagcgc ctccacctct ggtgtgacct 11520 tggtccagtc tctcagcctc agtttcctca cctgtaaagt gggctccatg attagatgca 11580 ccctgcaggg cagtgtagca gtgacctggc tcagccactg gcagccccaa caatcatacc 11640 ttgttaaagt agctctgtcg gttccctcag gggttccggg ggcccattcc cctgtcctcc 11700 atgcactgtg agacctgccc tgccacagag cagagtgtaa cagcctgagg gtgagagcca 11760 gacactgtgc ctgtgcttag accagacact ggacgacggg agccagtgca gcctgggcgg 11820 gtggactcct atggacccct cagcacccag cctcggtgcc ttcagcgcag ggccgcgtgg 11880 ctgtgggggc tcacaagacc cggcccactc ctgcttgtgc ctacatctgg gtgtttgccc 11940 attggtgcct tttgacgcgt tctggtgtgt gtgagacgtg cggggctggg aagtgttggc 12000 agagccgcga gtaccgtcct cactcctttt gttcttttga cgtaagctgg cgagtggcac 12060 tgcctgagtt ccgctcagtg cccgccctga tgtgcggacc ccgctgcatt cttgctgtta 12120 ggtggtggcg gtgtgcgctg tcgctggtgg gcaccgagag tctttgggag ctttggggag 12180 gttgtgccaa gcctgagcct cgacgtcccc cttcccggct ttctgttggc tcttctgagg 12240 ccagggcatc tctatgaggg cctcctgctg gagccgtctc tgtggatctc ctctgccatc 12300 ctggcccatg agtgggtgat gcgctggcca ccatctggtg acagtggccg ggcaccgctg 12360 ccaaatgtgg gtcccgcatc tgcaagcccc tccctgggtc ccctagggta tggggtggtt 12420 ctgccactgc cctcgctccc ccaccttggg gtgcctctcc ccctgctcgt gggggagacc 12480 ctgcctggga tctgctttcc agcaaggaat atactttgga gggagacaca catgttcttt 12540 tctggagctc tgcagtggcc acggcagccc agcccgccaa gcaccctgga atgaaaacat 12600 cccgctgctg tctgggcctg gcctgcactc tgctgcctgc gctccagctg gctgaggccg 12660 ggcacgtctg cgggcacagc agcgggggcg ccacagtctc cctgcagagt gagcgcagct 12720 ggaaaatgca gctcacgccc tttcccagaa cacctcgctc ttcatggctt ggcagctgtc 12780 cttgcctagg ggccagggtg cccaggcact ggtggcagga gaagggctac atctggggct 12840 gaggcgggct gggtcctttt ctccctgcag ctcccgaggc ccagccctgg cccagcctgg 12900 cattcctgac cttagcagcg ccatgatctg aagacaggct ggcttctgtg aggccacctc 12960 agaaagggct ttgtgcccag gcagaggcgg aagccagctc ttccttctgg ttgaggcagg 13020 aatgaggcca gcgctgggca agcccatgcc cagggaacgt cacagctgtg ggagtacagg 13080 ggctccgggt tctgagcccg tccactgtgc atcgtggccc tggcctcagg atggctcgta 13140 ccatcattgg ctgtgcccac agccgagtgg gtgatgggat tccggctgcc ccgctggatc 13200 tgtgctgctg ccctctccag ggcactgctg tgcccgcaca gccgggcgca gatggccagt 13260 ttgcttgccc ccccccccac catcctcttc ctaccttggc ttcctccatt gacacactgg 13320 accctgctgg ctgcccgggg aggtgtttgg gggatggtgt tgggggagga ggagggcccc 13380 ttgagcctca gtgtgcccat caggagcgta aggtcagtgc agcacctgcc cacacaggct 13440 gtgaagggtg ggagtggaga gggatgcaag ggggtcacaa cgcctggctc catgtcagct 13500 gcgtgcaggg gcaccaggag ccggccctca ttctcccctt gaactggaag ggtggccccg 13560 accccagcgg caggtagcat acgtatgaag cgctctcctt cctacacccc acaggtgggc 13620 tcgtctccag acggcccttt ttgagctggc tgtgtttttc catctgtgta ggcaaggaca 13680 tcgcagactc ccctttctca tctccctcgt tcagcctccg aggccggagt ctccatccct 13740 gtgcctgcct gtgggtcccg ggaggacctg aggctgccca tgtcaccccc ggcatctcat 13800 cctggggaca gttcagccgt gggagggatc tgtaaggaca gaatgccgct gagcctgggg 13860 ctccccagct agtctcacac cccgtgtctg ggacccagag accctcgtgc agggctctgt 13920 tgcttggggc ctggcagcct cgtcctgtat cagaggctgc cacccccacc cctcgtgggg 13980 ccagggttgt ggccggcctc cctggccctc cccatggaag tggtaggcgg agccagcagc 14040 catctgccca gcccggggct gcactgtttt ttttcaaatg agcaccgtcc caaactgcag 14100 cccgttaatt taaacaggat catttccggc cctggaagcc gcctcactct ccttaaatag 14160 aaaggagcac agcgcagagg gaaacagatg aggtcatggc tcggctggcc cagcgaggaa 14220 ggggccgcag tgggggtggc actgccgcct gtcCCCtgtc ctctccagcg cccacactgc 14280 agcccatttc ctcaccctgg gcctgctctc gggagggacg ggcctggggg tcctcttgct 14340 gggcggaggg gaaccagctc ctccaggaga ggacggggcc tggcaggggg catggggcct 14400 ccctgggtct ggcgtcctgt cctgcccctg ccgagggagg agcggttaca taagctccgc 14460 aggcggcccc tccgagccgg tccccccagc ccagtttcca gtgaggcggc cagcgcgggc 14520 gggggtgccg ggcctggcgc acacccgctg ctgaccacac gtgtctggaa tgtgcagatg 14580 tttctttggg ggctccgtcc ggcccccaga ccccactcag catctggtct ggggagtggg 14640 cgcctggggc actcagctct gagtgtgaga ctctgaggca ggtctggttt gtctggggcc 14700 attccctctg ctgtggattg ggagggcccc gggagctgcc ccacacccag ggaagttctc 14760 ctcagtccca ctgttgcatt ccccgacccc ggctcccccg gcccaggagc gcctgtgggg 14820 .cagaaggccc agccccaaga cttcccggcc ctgccagcct caggcttcac ccaccctcgc 14880 gccaactgtg ggcagagccc agggggaggg caggagagcc agcgcctggc tgggaacacc 14940 cctgaggggc cgaggctcca gggcgagggg gcccgacctg gggttcacac gcccgggtgg 15000 cgggcagacc cgctgcagca tgagacacgt gtcagctacc tcgggccggc aggctggccc 15060 tgctgcccac agccctggga cgtggcccca cctgtgacgg gtgtggaggg gcagcctcca 15120 ggcctggcca caccctctgc tgttgctgct cctgctccag gattggcaag ggtgctggga 15180 aggggtgaag acccgtactg tggccacaca cctgggactt ccttctccac ccagtggtgc 15240 cccagcagcc gctaaggagc ccgctgggtc ccacgctagg atggtcctaa ctcctCCcgc 15300 cttccagatc ggacgctcgg cgctggggac cccttgtgtc ccggggctgg ggcaccgtcc 15360 tgcccccatg ggggtgtact cctcccgaca agcttggctt cagcttccct gggagcacat 15420 cctggccctc gggcacccat caggctgtcc ctgtgcacct ggctcccacc cttccagctc 15480 atagcaggaa ctggggtgag gagtgcgtgg ggcagcaagg gcctgggacc ccagaggacc 15540 ctgcactctg ctctgtgctc ttgcctgggc ttagggccgc tcggtggtcc tgctgccaga 15600 tgcctgggcc ctgctgtgtc ccccatcctt gcagggaacc agaacgtggg ggcagggcat 15660 cagacagcgg cgatgatgtc acctggcggg tgcagaggaa gcccgagggg cggggtgggg 15720 gggctggcgc gaggctgcct ggctaggcct tggcgttccc ccagaacggc gatggcaaaa 15780 gcagatggag acgtgaaaaa gtacgggagc aagcgaggtg aggactccac ggggacccct 15840 gtgctgttcc ctgtccctga agcccacacc tgagtcctgc ccagggcaga tgcttccaca 15900 cccagggggc acctgagtcc tacccagggc agacgcttcc acaccctggg ggctggggga 15960 ctgcacctgg ctcctgtctg ggccccagct tcattccact gccctgggcc ctgggagctc 16020 ggccgagcgg ggtccccaag accttgctgc atttctgggc cttgggctgg ggtgagggcc 16080 gggagaagga gccagcctgg agcctggcac gcagggagtg catggccaga accggtgaca 16140 ggcagggctg cctgctggcg tggaagaagt gtccatggca cccccaggcc tggttcacag 16200 tgggatgggc ggggagccgg ggggctctgg ggtcctcggc tgacctgccc ccacccctgc 16260 cctggcttgt cagctcccag cagcagccac tcttgatgga ttttccagaa aatgaggtgt 16320 ggccaaacat cttcaggctt ttccttcttt cctttctccc gtggcctggg tgggagctgc 16380 tccccatgcc tgggggcagg tgcgagagcc tgtgcccctc cctggggcag tttcacagct 16440 gtgtcccttc cagggggcct gcctgtgttc accgtggcct ctgcagcacc tctcgcccct 16500 tagggctcct gcgcctcggg tcccggtgcc tcatttctcc ctaaagcatt ggttctgctg 16560 ccgccgcagc cgctggaaag tccctcctca ggtctaactg cagttcctca cggcacagtg 16620 ttccccctcg ggcatggtgc ttgggcagtg ggtgtgagtc cagctgcctc accctgtctc 16680 gagaatggcc tcttgctggt ctcccagcca ccaccctgtc ccaccccacg gcggggatgg 16740 tgtggatgcc tagcagcgcg gctgtgggcc cacccatcct tatgggcagt ggggagcacc 16800 tcagcccgtg tccctacctt ggtgtagagg aggggacggc agagaagcag ggttcagtta 16860 ggggggaagt ggtggccctg ccggaggggc cgttccctgt gtgcctggcc cccagatcct 16920 ctcccctccc ggagcccagg gcacaggcat aggctctctg agtgtcccac agcccctggg 16980 ggaagggaac tgcaccccca accgtgccct ccatccgcag atggaacgag aagctccggg 17040 agccagtgcc cagcgtctca tctgtctggg cacccagccc aggtgagggc ctggctccac 17100 cgtccgtggc tggtgctgct tcctggcacg gagaaggcct cggctgctct gtcccctcag 17160 ctggggtggc ctctggtccc cttctttgtt ggttcccttc tcaagctctt gccctggccc 17220 cgggccccac cgggcagcct gtgtgtgcgt ctctcctgcg ccgggtaggc tcctgtggga 17280 gcggagctcc ggtgggagga gcagggctgg aggctggcag gggctgggcg ggtgttcagg 17340 gatggaggcc gccccggctt ggggctggct gccgggtggt cattgctggg aagagcaagt 17400 ctaggcggag gcacctgctg ggtcactcgt ggggagggtg acacctgggg aagtagaggc 17460 ccgtggcagg aggtgaggcc tcggggtcct ggggagcagg ggggtggtgt gcagacctgc 17520 ggagccatag tcctgtgcca ggagcactac tgggagtgcg tgggaccagg aggggtgccc 17580 agggtgggcg gcagagtgac ccccgaggtg cttgaggccg aggggaggtg gagttctcgg 17640 tttgccccag ctctctgtct actcacctcc gcatcaccag ctccaggacc tggtttgtaa 17700 ctcgggcagc tctgaaaaga gagacatgct gccgccctgt ggtttctgtt gctttttctt 17760 cactgactac tgacatggga tgtttttcct acggctgtga ccaattgtgc ttcttctaat 17820 tgcctggttt ttcttttttt gtttttggag ttttctcttt ctttcctccc tccctctcac 17880 CCtCcatCCt tttttttttt atttttattt tttgagatgg agcttcactc ttgcaggatg 17940 gggtgctgga gtgcaggggt gcgatctcag ctcactgcaa cctctgcctc gcgggttcaa 18000 gtgattctcc tgcctaagcc tcctgagtag ctggaattac aggtgcttgc caccacgccc 18060 gactaattct gtagttttgg tagagacagg gtgtctccgt gttggtcggt ctggtcttga 18120 actcctgacc tcaggtgatg cgcccgcctc agcctcccaa agtgctggga ttacaggcag 18180 gagccattgc acccggctct ttccccttct CcttttCttC tctctctcct cCctttcttt 18240 cttttctttt cttttttttt tcttttgaga tggagtctcg ctctgtcacc aggctggatt 18300 gcagtggcgt gatcttggct cactgcaacc ttcgcctccc gggttcacgt gattctcctg 18360 cctcagcctc ctgagtggct ggcactacag gctcccgccg ccatgcccgg ctaatttttg 18420 catttttagt agagacaggg tttcaccctg ttggccagga tggtctcgat ctcttgatct 18480 catgatccac ccaccttggc ctcccaaagt tctggcatta caggagtgag ccaccgtgcc 18540 cggccatctt tctttccttg ctttctcttt gttttctttc gagaccgggt cttgctctgt 18600 cgcccaggct ggactgcagt ggcacaatca tagctcactg cagcctcgac ttccctggct 18660 caagcgatcc ttcctcctca gccccccgag tagctggaac tacagttaca cactaccatg 18720 cctggctgat tctttttttc cttgtagaga tggggtcttg ctatgctgtc catcctggtc 18780 tcaaactcct ggccttccca aagcactggg tttacaggca taagccacca cacccagttt 18840 ccttttcttc tttttaactg gaatagttga cgttttcttt attagctgtg tgtcaggagg 18900 gtatttttgg cctttagtat gtcgtgtaag ttgctagtgc ttttctgaga ttgtagtttg 18960 ttttctaatt ttatttatat tttgcgtaga agttgtgtat tttagatgga gttaggtcgg 19020 ctggtctttg atgttttatt tattaattat gtatgtattt atttattttt gaggtagagt 19080 ctcgccgttt cacccaggct ggagtacagt gatgcgatct cagctccctg tagccttgac 19140 ctctctgggc tcaagtgatt tttctctcct ctacctcccg agtacttggg accccaggcg 19200 catgccgcca tgcctggcta atgtgtattt tttgtagata cggggtctca ctgtgttgcc 19260 cagggtggtt tcaaaatcct gggcccaggc gatccttccg tctcagctcc cacggtgctg 19320 tgttaccggc gtgtgcccag tgcctggccg tcttggaggt cttgtttctc tgggtttatg 19380 cctcgaggtg gcgcctgctc ccctgtgctc cctggtagcc tggtagtgag cctgcttctc 19440 acacagtcat acctggttgt ggtcccacag tgggaccacc ctgttgggtt cagaacagga 19500 gatgggggcc cctcgagtct gtgtgggggc tgtggacagg gttgggagac cttggctctg 19560 tgggggactg tggacagggg atggggggcc ttggccctgc gtgggatggg ttgggggtcc 19620 gtgcccttcc tggccctggg tggacaggtc catgtggcac tcggcatagg gctgagatgg 19680 gtgcagaggg ctgaggcccc caggcctctc ctggcttggt ttccccagat gagtgttcat 19740 ttgggtcttc catcagaaag tcccctcctg acctctggga gtggggagct caagggtggg 19800 aggccatagc ttggggatgc tggcaatgtg tgggatgggc ccagggaagg cctctggcct 19860 actaggggct ctggccctga cccacggcca ctcactcctc agagacgtct cccacaacct 19920 gctccgggcg ctggacgttg ggctcctggc gaacctctcg gcgctggcag agctgtgagt 19980 gtcccccagt cgtgccagca tgcggggctc actccgggtg ggctggcggc accgcctctt 20040 gctgctcagc tgtgggggct tccatcagct ttgccgaatc ccCcgtctct tccagggata 20100 taagcaacaa caagatttct acgttagaag aaggaatatt tgctaattta tttaatttaa 20160 gtgaaatgta agttgtggtt ctttgggtgg ggtcctggct ggaccccagg cccccaatat 20220 cccttctgcc ctcccagttg gtccgtgtcc ccttccaggc ttgagaccag atcctggggg 20280 cagttcactg cctgcttgga gccccccagt gccggcttgg ttggggcagg ggaggcggtg 20340 ctgtcagggt ggctccaggg cctggttgcc agtggggggc tggcatagac ccttcccacc 20400 agacctggtc cccaacacct gcccctgccc tgcagaaacc tgagtgggaa cccgtttgag 20460 tgtgactgtg gcctggcgtg gctgccgcga tgggcggagg agcagcaggt gcgggtggtg 20520 cagcccgagg cagccacgtg tgctgggcct ggctccctgg ctggccagcc tctgcttggc 20580 atccccttgc tggacagtgg ctgtggtgag tgccggtggg tggggccagc tctgtccttc 20640 ccagccaggt gggacctggg ccctgcagac actgggcagg gctcaggaag gcctctctgg 20700 ggggggcctc cgggccaagg gaacagcatg ggagcctgtg agtgcggcgg gcggatgtgg 20760 gggcgtgggg tggagccagg aggagcagaa cccggggtcc agtggctgcc tcttctaggt 20820 gaggagtatg tcgcctgcct ccctgacaac agctcaggca ccgtggcagc agtgtccttt 20880 tcagctgccc acgaaggcct gcttcagcca gaggcctgca gcgccttctg cttctccacc 20940 ggccagggcc tcgcagccct ctcggagcag ggctggtgcc tgtgtggggc ggcccagccc 21000 tccagtgcct cctttgcctg cctgtccctc tgctccggcc ccccgccacc tCctgCCCCC 21060 acctgtaggg gccccaccct cctccagcac gtcttccctg cctccccagg ggccaccctg 21120 gtggggcccc acggacctct ggcctctggc cagctagcag ccttccacat cgctgccccg 21180 ctccctgtca ctgccacacg ctgggacttc ggagacggct ccgccgaggt ggatgccgct 21240 gggccggctg cctcgcatcg ctatgtgctg cctgggcgct atcacgtgac ggccgtgctg 21300 gccctggggg ccggctcagc cctgctgggg acagacgtgc aggtggaagc ggcacctgcc 21360 gccctggagc tcgtgtgccc gtcctcggtg cagagtgacg agagcctcga cctcagcatc 21420 cagaaccgcg gtggttcagg cctggaggcc gcctacagca tcgtggccct gggcgaggag 21480 ccggcccgag gtgagtgtct gctgcccact ccccttcctc cccagggcca tccagatggg 21540 gcagagcctg gtacccccgt cttgggccca cactgaccgt tgacaccctc gttcccaccg 21600 gtctccagcg gtgcacccgc tctgcccctc ggacacggag atcttccctg gcaacgggca 21660 ctgctaccgc ctggtggtgg agaaggcggc ctggctgcag gcgcaggagc agtgtcaggc 21720 ctgggccggg gccgccctgg caatggtgga cagtcccgcc gtgcagcgct tcctggtctc 21780 ccgggtcacc aggtgcctgc ccccaccccc cgaggggcca taggttggga gatctctgaa 21840 gcactggggc agagactgcg gctggggagt ctcaggagga aggaggtggg agctgggccg 21900 gccctggtga gcaggtggcg ccggccggtg gggccgttcc tgtcagctct gcagatgcag 21960 aggtggacat gagctggggg cagcctccgg acactcctgg gcacgccata cgggaggtgg 22020 cctgcacggg gatccctgcc ggtacccaca ggccccgtgg gtgggtgctg ctgtgagcct 22080 gggctggtgg gccctggtct ccgggctctg agcctcagtt tccccatctg gaaaggggga 22140 cagtgatggg gctcccagcg ggctgctgtg agggtgggag gatggaggag tgccctgagc 22200 cccctgccat cccacacccg cccccaggag cctagacgtg tggatcggct tctcgactgt 22260 gcagggggtg gaggtgggcc cagcgccgca gggcgaggcc ttcagcctgg agagctgcca 22320 gaactggctg cccggggagc cacacccagc cacagccgag cactgcgtcc ggctcgggcc 22380 caccgggtgg tgtaacaccg acctgtgctc agcgccgcac agctacgtct gcgagctgca 22440 gcccggaggt gtgcgggggg ccaggcaggg gcctgagacg ctggctgtgg ttaggggcct 22500 gccgagcgcc cgcggtggag cctgggctga ggaggagggg ctggtggggg ggttttcggg 22560 cggctcggtc cccagtctgt tcgtcctggt gtcctgggcc ctggcccggc gcctcactgt 22620 gcactcgcca ccccaggccc agtgcaggat gccgagaacc tcctcgtggg agcgcccagt 22680 ggggacctgc agggacccct gacgcctctg gcacagcagg acggcctctc agccccgcac 22740 gagcccgtgg aggtagtcgg ccccccacgt tctacaacct gccctcctgc ctgcccctgg 22800 aggccttgcc tgccctgccc actgtgggtc tcgccaaaaa acttgggggc cttaatgttg 22860 cttgtgccca gtgaagatgg ttgggaaaat ccagagtgca gagaggaaag cgtttactca 22920 cattacctcc aggccttttc tctgagcgtg tgtgagttat tcctgaaagg caggtcaggg 22980 gtcctgcccc ccatggacag tttccaccgg agtcttcctc tcgagcgaca ggagccaggc 23040 ctgtgggggt ctgatggctc gctctccttc cctcccctct tcctgggaag ttcgggtagg 23100 gggagtctgg gcttcaggct gggatggggt ctgtggagct gaggcggccc cctgcccacc 23160 aggtcatggt attcccgggc ctgcgtctga gccgtgaagc cttcctcacc acggccgaat 23220 ttgggaccca ggagctccgg cggcccgccc agctgcggct gcaggtgtac cggctcctca 23280 gcacagcagg tgggactctg ggtggtgggt ggtgggtggt gggcgccgca ggactcgggg 23340 tggcctctct gagctttcac gtctgctggt cctgtggcca ccagagtggt tcccagtctt 23400 aggtggacag agcaggggtt ccagagacac cagctcattc caggtgtcct gggggtggat 23460 tgggtggggc ctgcctgggg gccggcctgg gtcagtcggc tggccggaga cggacgcagc 23520 actgggctgg gagtgctgcc caggtgggga gacctgtcct cacagcaagg ccaggattgc 23580 tggtgcaggc agttgggcat ctctgacggt ggcctgtggg caaatcaggg ccccaacacc 23640 ctcccctcct cacagggacc ccggagaacg gcagcgagcc tgagagcagg tccccggaca 23700 acaggaccca gctggccccc gcgtgcatgc cagggggacg ctggtgccct ggagccaaca 23760 tctgcttgcc gctggacgcc tcctgccacc cccaggcctg cgccaatggc tgcacgtcag 23820 ggccagggct acccggggcc ccctatgcgc tatggagaga gttcctcttc tccgttcccg 23880 cggggccccc cgcgcagtac tcggtgtgtg gccctgacct gggtctgttc cctgcatctc 23940 ctcaggccac cttcctgtct gctgcccagg gtctgggtct gtgcaccaga cacacccagc 24000 CtgcaggCCC CtCCCacgtC CttgCCaCCt ctgacctccg acctctgcag tgCCCtCggC 24060 cctctcccag tgggagaagc tctcgcctgg gcccttggca cgagctgtgc ctcctcttcc 24120 tctctcccag cacagctgct ccttcctgtc tgccaggtct tggcctgtgt cctctccccg 24180 tgtgtccccc ggtctgcaac tgtcctgcct gtccttgtca cgagcactgt ggggaggctc 24240 cttgaggtgt ggctgacgaa gcggggagcc ctgcgtgtcc accctcatcc gtcgtgcggg 24300 ggtccacggg ccatgaccgt gaggacgtga tgcagccctg cctccctctc cacaggtcac 24360 cctccacggc caggatgtcc tcatgctccc tggtgacctc gttggcttgc agcacgacgc 24420 tggccctggc gccctcctgc actgctcgcc ggctcccggc caccctggtc cccgggcccc 24480 gtacctctcc gccaacgcct cgtcatggct gccccacttg ccagcccagc tggagggcac 24540 ttgggcctgc cctgcctgtg ccctgcggct gcttgcagcc acggaacagc tcaccgtgct 24600 gctgggcttg aggcccaacc ctggactgcg gctgcctggg cgctatgagg tccgggcaga 24660 ggtgggcaat ggcgtgtcca ggcacaacct ctcctgcagc tttgacgtgg tctccccagt 24720 ggctgggctg cgggtcatct accctgcccc ccgcgacggc cgcctctacg tgcccaccaa 24780 cggctcagcc ttggtgctcc aggtggactc tggtgccaac gccacggcca cggctcgctg 24840 gcctgggggc agtgtcagcg cccgctttga gaatgtctgc cctgccctgg tggccacctt 24900 cgtgcccggc tgcccctggg agaccaacga taccctgttc tcagtggtag cactgccgtg 24960 gctcagtgag ggggagcacg tggtggacgt ggtggtggaa aacagcgcca gccgggccaa 25020 cctcagcctg cgggtgacgg cggaggagcc catctgtggc ctccgcgcca cgcccagccc 25080 cgaggcccgt gtactgcagg gagtcctagt ggtgagtatg gccgaggctc caccaccagc 25140 ccccaggcag gtgcctgcag acagggtgct cacacagggc gtgaggcctg gcttcccagt 25200 gagggcagca gcccagttac tggggacgtc ggccccgggc aggtcctgct ggctggctcc 25260 tcgggctacc tggtgggctt taaattcctg gaaagtcacg gctctgacag tggctccgct 25320 aactcattcc actgtctcat ttcacaaaat gaatttaaaa CtCtgCtCCc tgacctcaca 25380 cgagcccccg tgagtctctc acgccctctg ctgtgttctc gcctggctaa agcgagtggc 25440 ttttgaggtg gagtctgaac ccctgatggg aaactgcggg ctgcccgcgg tgccaccatg 25500 ctgggtacat gggggacagg gctgtctcca tcttgcgggt acctgcctct tcaccagggg 25560 ccttgggagg ggccatcaga aatggcgtga cctgtgcagc ctgtcctggg ttctgtaagc 25620 cagtgtaggt gcctcccctc actgctccga gctctctggg tgaggagctg gggcaagagc 25680 gccgggaggg tctgagaaga ctcagagaga ggtggactct ttgtagctgg tactaggttt 25740 gctttacaga tggggaaact gaggcacaga gaggttgagg cattagtagt actacatggc 25800 tggctggaga gccggacagt gagtgtccca gcccgggctt ggctcccatg gcatgcagag 25860 ccccgggcac ctcctctcct ctgtgccccg cgtgggactc tccagcccga cgggaggtgt 25920 gtccaggagg cgacaggcta agggcagagt cctccacaga gcccaggctg acaccattcc 25980 ccccgcagag gtacagcccc gtggtggagg ccggctcgga catggtcttc cggtggacca 26040 tcaacgacaa gcagtccctg accttccaga acgtggtctt caatgtcatt tatcagagcg 26100 cggcggtctt caagctctca gtaggtgggc gggggtgggg aggggagggg atggggcggg 26160 gcagggcggg ggcgggctcc accttcacct ctgccttctg ctctgcttca tgctgcccga 26220 ggacgctgcc atggctgtgg gtgagtggag ggagggacgc caatcagggc caggcctctc 26280 acctgccacc tgggctcact gacgcctgtc cctgcagctg acggcctcca accacgtgag 26340 caacgtcacc gtgaactaca acgtaaccgt ggagcggatg aacaggatgc agggtctgca 26400 ggtctccaca gtgccggccg tgctgtcccc caatgccacg ctagcactga cggcgggcgt 26460 gctggtggac tcggccgtgg aggtggcctt cctgtgagtg actcgggggc cggtttgggg 26520 tgggcaccag gctcttgtcc cagccccagc ctcagccgag ggacccccac atcacggggt 26580 tgcttttctg agcctcggtt tccctgtctg ttgggaggta actgggtgca caggagccct 26640 gaggctgcac gggagccggg agaggcctca gcacagccgg gtgggccctg aatggaggcc 26700 cggggcgtga ctgcagagtg gagcctcggc tgggtcccaa gcaccccctg cCCCgcCaCC 26760 gcccacccct gtcccggttc actcactgcg tcccaccgcc ccggcaggtg gacctttggg 26820 gatggggagc aggccctcca ccagttccag cctccgtaca acgagtcctt cccgg.ttCca 26880 gacccctcgg tggcccaggt gctggtggag cacaatgtca tgcacaccta cgctgcccca 26940 ggtgagggat gagggggtga gggggccact gcctttcagg ctctgagcac gggtcccccc 27000 agctCCCCag tcaagctgcc CCCcttcCtc cccaacagcc ctcactgtga cctCacctgg 27060 gctgatggct taggccctac tggggtgagg gaggggccag gcgtgggggg agtggacagg 27120 gaagctgggc ccctgaactg cgccccccgc cctccccggg cctggctctt gctgctctgc 27180 tgccccgagt gcagctgcac ttggaggcgg tgcgtcctcg ccaggcagcc ctcagtgctg 27240 ctacacctgt gctccgtccc gcacgtggct tgggagcctg ggacccttaa ggctgggccg 27300 caggtgcagc cgttcacccc gggctcctca ggcggggggc ttctgccgag cgggtgggga 27360 gcaggtgggg gtgccgcggc tgccccactc gggcctgtcc ccacaggtga gtacctcctg 27420 accgtgctgg catctaatgc cttcgagaac cggacgcagc aggtgcctgt gagcgtgcgc 27480 gcctccctgc cctccgtggc tgtgggtgtg agtgacggcg tcctggtggc cggccggccc 27540 gtcaccttct acccgcaccc gctgccctcg cctgggggtg ttctttacac gtgggacttc 27600 ggggacggct cccctgtcct gacccagagc cagccggctg ccaaccacac ctatgcctcg 27660 aggggcacct accacgtgcg cctggaggtc aacaacacgg tgagcggtgc ggcggcccag 27720 gcggatgtgc gcgtctttga ggagctccgc ggactcagcg tggacatgag cctggccgtg 27780 gagcagggcg cccccgtggt ggtcagcgcc gcggtgcaga cgggcgacaa catcacgtgg 27840 accttcgaca tgggggacgg caccgtgctg tcgggcccgg aggcaacagt ggagcatgtg 27900 tacctgcggg cacagaactg cacagtgacc gtgggtgcgg ccagccccgc cggccacctg 27960 gcccggagcc tgcacgtgct ggtcttcgtc ctggaggtgc tgcgcgttga acccgccgcc 28020 tgcatcccca cgcagcctga cgcgcggctc acggcctacg tcaccgggaa cccggcccac 28080 tacctcttcg actggacctt cggggatggc tcctccaaca cgaccgtgcg ggggtgcccg 28140 acggtgacac acaacttcac gcggagcggc acgttccccc tggcgctggt gctgtccagc 28200 cgcgtgaaca gggcgcatta cttcaccagc atctgcgtgg agccagaggt gggcaacgtc 28260 accctgcagc cagagaggca gtttgtgcag ctcggggacg aggcctggct ggtggcatgt 28320 gcctggcccc cgttccccta CCgctacacC tgggactttg gcaccgagga agccgccccc 28380 acccgtgcca ggggccctga ggtgacgttc atctaccgag acccaggctc ctatcttgtg 28440 acagtcaccg cgtccaacaa catctctgct gccaatgact cagccctggt ggaggtgcag 28500 gagcccgtgc tggtcaccag catcaaggtc aatggctccc ttgggctgga gctgcagcag 28560 ccgtacctgt tctctgctgt gggccgtggg cgccccgcca gctacctgtg ggatctgggg 28620 gacggtgggt ggctcgaggg tccggaggtc acccacgctt acaacagcac aggtgacttc 28680 accgttaggt ggccggctgg aatgaggtga gccgcagcga ggcctggctc aatgtgacgg 28740 tgaagcggcg cgtgcggggg ctcgtcgtca atgcaagccc cacggtggtg cccctgaatg 28800 ggagcgtgag cttcagcacg tcgctggagg ccggcagtga tgtgcgctat tcctgggtgc 28860 tctgtgaccg CtgCaCgCCC atccctgggg gtcctaccat ctcttacacc ttCCgCtCCg 28920 tgggcacctt caatatcatc gtcacggctg agaacgaggt gggctccgcc caggacagca 28980 tcttcgtcta tgtcctgcag ctcatagagg ggctgcaggt ggtgg.gcggt ggccgctact 29040 tccccaccaa ccacacggta cagctgcagg ccgtggttag ggatggcacc aacgtctcct 29100 acagctggac tgcctggagg gacaggggcc cggccctggc cggcagcggc aaaggcttct 29160 cgctcaccgt ctcgaggccg gcacctacca tgtgcagctg cgggccacca acatgctggg 29220 cagcgcctgg gccgactgca ccatggactt cgtggagcct gtggggtggc tgatggtggc 29280 cgcctccccg aacccagctg ccgtcaacaa aagcgtcacc ctcagtgccg agctggctgg 29340 tggcagtggt gtcgtataca cttggtcctt ggaggagggg ctgagctggg agacctccga 29400 gccatttacc acccatagct tccccacacc cggcctgcac ttggtcacca tgacggcagg 29460 gaacccgctg ggctcagcca acgccaccgt ggaagtggat gtgcaggtgc ctgtgagtgg 29520 cctcagcatc agggccagcg agcccggagg cagcttcgtg gcggccgggt cctctgtgcc 29580 cttttggggg cagctggcca cgggcaccaa tgtgagctgg tgctgggctg tgcccggcgg 29640 cagcagcaag cgtggccctc atgtcaccat ggtcttcccg gatgctggca ccttctccat 29700 ccggctcaat gcctccaacg cagtcagctg ggtctcagcc acgtacaacc tcacggcgga 29760 ggagcccatc gtgggcctgg tgctgtgggc cagcagcaag gtggtggcgc ccgggcagct 29820 ggtccatttt cagatcctgc tggctgccgg ctcagctgtc accttccgcc tgcaggtcgg 29880 cggggccaac cccgaggtgc tccccgggcc ccgtttctcc cacagcttcc cccgcgtcgg 29940 agaccacgtg gtgagcgtgc ggggcaaaaa ccacgtgagc tgggcccagg cgcaggtgcg 30000 catcgtggtg ctggaggccg tgagtgggct gcaggtgccc aactgctgcg agcctggcat 30060 cgccacgggc actgagagga acttcacagc ccgcgtgcag cgcggctctc gggtcgccta 30120 cgcctggtac ttctcgctgc agaaggtcca gggcgactcg ctggtcatcc tgtcgggccg 30180 cgacgtcacc tacacgcccg tggccgcggg gctgttggag atccaggtgc gcgccttcaa 30240 cgccctgggc agtgagaacc gcacgctggt gctggaggtt caggacgccg tccagtatgt 30300 ggccctgcag agcggcccct gcttcaccaa ccgctcggcg cagtttgagg ccgccaccag 30360 ccccagcccc cggcgtgtgg cctaccactg ggactttggg gatgggtcgc cagggcagga 30420 cacagatgag cccagggccg agcactccta cctgaggcct ggggactacc gcgtgcaggt 30480 gaacgcctcc aacctggtga gcttcttcgt ggcgcaggcc acggtgaccg tccaggtgct 30540 ggcctgccgg gagccggagg tggacgtggt cctgcccctg caggtgctga tgcggcgatc 30600 acagcgcaac tacttggagg cccacgttga cctgcgcgac tgcgtcacct accagactga 30660 gtaccgctgg gaggtgtatc gcaccgccag ctgccagcgg ccggggcgcc cagcgcgtgt 30720 ggccctgccc ggcgtggacg tgagccggcc tcggctggtg ctgccgcggc tggcgctgcc 30780 tgtggggcac tactgctttg tgtttgtcgt gtcatttggg gacacgccac tgacacagag 30840 catccaggcc aatgtgacgg tggcccccga gcgcctggtg cccatcattg agggtggctc 30900 ataccgcgtg tggtcagaca cacgggacct ggtgctggat gggagcgagt cctacgaccc 30960 caacctggag gacggcgacc agacgccgct cagtttccac tgggcctgtg tggcttcgac 31020 acaggtcagt gcgtggcagg gccgtcctcc atgcccctca cccgtccaca cccatgagcc 31080 cagagaacac ccagcttgcc accagggctg gcccgtcctc agtgcctggt gggccccgtc 31140 ccagcatggg gagggggtct cccgcgctgt ctcctgggcc gggctctgct ttaaaactgg 31200 atggggctct caggccacgt cgccccttgt tctcggcctg cagagggagg ctggcgggtg 31260 tgcgctgaac tttgggcccc gcgggagcag cacggtcacc attccacggg agcggctggc 31320 ggctggcgtg gagtacacct tcagcctgac cgtgtggaag gccggccgca aggaggaggc 31380 caccaaccag acggtgggtg ccgcccgccc ctcggccact tgccttggac agcccagcct 31440 ccctggtcat ctactgtttt ccgtgtttta gtgctggtgg aggccgcacg ctctcccctc 31500 tctgtttctg atgcaaattc tatgtaacac gacagcctgc ttcagctttg cttccttcca 31560 aacctgccac agttccacgt acagtcttca agccacatat gctctagtgg caaaagctac 31620 acagtcccct agcaatacca acagtgagga agagcccctt cccaccccag aggtagccac 31680 tgtccccagc ccatgtccct gttgctggat gtggtgggcc ggttctcacc ctcacgctcc 31740 cctctctgga ccggccagga ggcttggtga ccctgagccc gtggtggctg ctcctgctgc 31800 tgtcaggcgg ggcctgctgg tgccccagag tgggcgtctg ttccccagtc cctgctttcc 31860 tcagctggcc tgattggggg tcttcccaga ggggtcgtct gaggggaggg tgtgggagca 31920 ggttccatcc cagctcagcc tcctgaccca ggccctggct aagggctgca ggagtctgtg 31980 agtcaggcct acgtggcagc tgcggtcctc acacccacac atacgtctct tctcacacgc 32040 atccccccag gggccctcag tgagcattgC ctgcctcctg ctagggtcca gctgggtcca 32100 gtacaccaga acgcacactc cagtgtcctc tgccctgtgt atgcccttcc gccgtccaag 32160 ttggaaggtg gcaaaccgga tgagtatcct gggagggagt gagctcaccg gcagtggcca 32220 ggcccctggg aaacctggag tttgggagca gcatcctcca tgggtccccc agtccttcca 32280 gcaggccaaa tagacctgtg ttggaggtaa ccccactccc acgccaggtg ctgatccgga 32340 gtggccgggt gcccattgtg tccttggagt gtgtgtcctg caaggcacag gccgtgtacg 32400 aagtgagccg cagctcctac gtgtacttgg agggccgctg cctcaattgc agcagcggct 32460 ccaagcgagg ggtgagtgtt gagcggggtg tgggcgggct ggggatgggt cccatggccg 32520 aggggacggg gcctgcaggc agaagtgggg ctgacagggc agagggttgc gccccctcac 32580 caccccttct gcctgcagcg gtgggctgca cgtacgttca gcaacaagac gctggtgctg 32640 gatgagacca ccacatccac gggcagtgca ggcatgcgac tggtgctgcg gcggggcgtg 32700 ctgcgggacg gcgagggata caccttcacg ctcacggtgc tgggccgctc tggcgaggag 32760 gagggctgcg cctccatccg cctgtccccc aaccgcccgc cgctgggggg ctcttgccgc 32820 ctcttcccac tgggcgctgt gcacgccctc accaccaagg tgcacttcga atgcacgggt 32880 gagtgcaggc ctgcgtgggg ggagcagcgg gatcccccga ctctgtgacg tcacggagcc 32940 ctcccgtgat gccgtgggga ccgtccctca ggctggcatg acgcggagga tgctggcgcc 33000 ccgctggtgt acgccctgct gctgcggcgc tgtcgccagg gccactgcga ggagttctgt 33060 gtctacaagg gcagcctctc cagctacgga gccgtgctgc ccccgggttt caggccacac 33120 ttcgaggtgg gcctggccgt ggtggtgcag gaccagctgg gagccgctgt ggtcgccctc 33180 aacaggtgag ccaggccgtg ggagggcgcc cccgagactg ccacctgctc accaccccct 33240 ctgctcgtag gtctttggcc atcaccctcc cagagcccaa cggcagcgca acggggctca 33300 cagtctggct gcacgggctc accgctagtg tgctcccagg gctgctgcgg caggccgatc 33360 cccagcacgt catcgagtac tcgttggccc tggtcaccgt gctgaacgag gtgagtgcag 33420 cctgggaggg gacgtcacat ctgctgcatg cgtgcttggg accaagacct gtacccctgc 33480 ctggagcttt gcagagggct catcccgggc cccagagata aatcccagtg accctgaagc 33540 agcaccccga ccttccgctc ccagcagcca cacccaccgg gccctctccg gcgtctgctt 33600 tccacaatgc agcccccgcc caggagggcc catgtgctta ccctgttttg cccatgaaga 33660 aacagctcag tgttgtgggt cagtgcccgc atcacacagc gtctagcacg taactgcacc 33720 ccgggagtcg tgggcatctg ctggcctcct gccggcctcc tgcgctgctg acagcttgct 33780 gtgccccctg cctgccccag tacgagcggg ccctggacgt ggcgcagagc ccaagcacga 33840 gcggcagcac cgagcccaga tacgcaagaa catcacggag actctggtgt ccctgagggt 33900 ccacactgtg gatgacatcc agcagatcgc tgctgcgctg gcccagtgca tggtaggatg 33960 gccccacctg ctcaccctgc cccgcatgcc tgccagggca ctgggttcag ccccccaggg 34020 cagacgggca gcttggccga ggagctgagc ctccagcctg ggctccttcc tgccatggcg 34080 ttcctcggtc tctgacctgc ttcagtagcc tcagccgttc tgtcctgtgt gaacgcaggg 34140 tgcctctcgg gggacccagg gtgtaaagag gggcccagat gtggggaggg actaagaaga 34200 tgctgctctg tgccctccac tctCCCCtcc cctcccctcc cccttccctc ccctagcccc 34260 tCCCctCCtC ccctccccta gcccttcccc tcctcccctc ccctagccct ttcccttctt 34320 CCCCCCCagC ccttcccctc ctCCCctCCC CtagCCCttC CCCtCCtCCC ctcccctacc 34380 CCttCCCCtC CtCCCCtCCC CtagacCttC CCCtCaCCtC CtCCCgctga gcccctccac 34440 tcgtcCCCca gCCCCtCCCt CCCctagCCC CtCCCCtCCC CCttCCtCCC CtCCtCCCCC 34500 tcccctcctc CCCCtCCCtC ttCCtcCCCC tCCCCtCCtc ccccttcctc CCCtCtCCtC 34560 CCCCtCCCCt CCtgtCCCCC CtCCtCCCCt CCtCCCtCCt CCCCtCCtCC CCCCtCCtCC 34620 tCCCCCtCCt CCCtCCtCCC tCCtCCCCCt CCtCCtCCtC CCCtCCtCCC tcctcccctc 34680 CtCCCCtCCC ctcctccccc tCCCCCCtCC CttcCtCCCc CtCCCCCCtC CCCtCCtCCC 34740 CCtCtcctCC tCCCatCCct cCtCCCatCc CtCCtCCCCg ttcccattct ctcccctccc 34800 CCttCCattt ctccctcctc cCCctgCCCt CCtCtCCtCC tcaCctCCCC ttctccgctc 34860 ctttcttctc ctccctccct ttctC'tcctc cctccccttc tCCCCttCtC CtCttCtCCC 34920 CttCtCctCt cttttcatcc ttcccttctt CCCtCCtttC CtCCtCtttt CCCtCttCtC 34980 ccccctcctc ccctccttcc tCCtCCCatt cCCCCtCCtC CCCCctCCca ttCCCCCtCC 35040 tcccctcctt CCtCCtCCCa ttacccctcc tctCCtCCCC tCCtCCCaCC CCCCtCtCCt 35100 cccggctcct CtCCtCCCCt CCtCatCCCC CtCCtctCCt tcCCtCCtaa CCCCCCtCCt 35160 ctcctcccct CCtCatCCCC ctcctctcct tCCCtCCtCC tatCCCCCct CCtCtCCtCC 35220 cctcctccta ttccccctcc tctcctcccc tccttcctcc tcctCtCCtC ccatgccccc 35280 tcctcccctc ctcccatccc cctcctcccc tcctCCCtcC tcccatccca tccccctcct 35340 CtCCtCCCCt tctctcccct CCtCtCCtCC CCtCCtCtCC tctcctcctc tCCtCCCCtC 35400 CtCCCatCCC ccctcctccc atcccccctc ctctcctccc CdCtCctCtC ctccccactc 35460 ctctcctccc ctcatccccc tCCtCtctCC tCCCCtCCCC CtCCtCtCCt tCCCtCCtCC 35520 tttCCtCCCC tCCCCCtCCt tccccctcct ccccctcctt CtCCCCatCC cccttcccct 35580 tctcctcctc tcccctcccc cttctctttt tccctcctcc tCCCttcCtc CtCCCCtCtt 35640 ctcccctttt cccttttctc ttcctctcct ccccttctcc cctcctgtcc tCCCtCCCtt 35700 tctctctttC tttCCtcCCt ttccttctcc cctgttctcc tCCcttCCCt tctccccttt 35760 tCttCCCtCC tcctttcctc ccctcctcct tttctctgtt tctcttcctt tcccctccac 35820 tttccccttc ctttcccctc tcctttctcc ttCCtttCCt CtCCCcttCt cttccttttc 35880 CtCtCtCCCC ttcttttccc tcttcccctc CCCtCCtCtt CCCCtCCCCt CCtCttCCCC 35940 tCCCCtcCtc ttCCCCtCCC CtCCtCttCC CCtCtCCtCC tcttcCCCtC ccctcctctt 36000 tCCCtCCCCt CttCtCCtCC CCtCCtCtCC cctcttCCCC tCCCCtCCtC ttccctcccc 36060 ttcccctccc ctcctcttcc CtCCCCttCC cctcccctcc tCttCCCtCC CCttCCCCtC 36120 CtCttCCttC ctctcttccc CtCCCCtCCt CttCCCtCCC CtCttCCCCt CCCCttCtCt 36180 tctCCtCCCc ttctCttCCC ctcccctttt CttCCCtCtC CttgtCttCC ctgccctcct 36240 cttccctccc CtCCtCttCc ctcccctctt cccctctcct CCtCttCCCt CCCCtCttCC 36300 tCtttCCtCt tCCCCtCCCC tCCtCCtCCC tCCCCtttCC CCtCttCCCC tCCCCtCCgc 36360 ttccctcccc tttCtCCCCC ttctCtCCCC tCCCCtctCC ccccttctct cCCCtCCCCt 36420 ctcccccttc tctCCCCtCC cctctccccc ttctctcccc tCtCCtCtCC CCCttCtCtC 36480 ccccttctct CCCCCttCtC tctCCCCttC tCtCCCCCtt CtCtCCCCtC CCCCCttCtC 36540 tCCCCtCCCC tctccccctt ctctcccctc CCCtCtCCCC tgtCCtCtCc tctCCaCCCt 36600 tctctcccct CCCCtCtCCt CtCCCCCttC CCtCtCCtCt CCCCCttCtC tCCCCtCCCC 36660 tctcctctcc CCCCttttct ccactcccct CtCCtCtCtC CCCtCCtCCt ccgctctcat 36720 gtgaagaggt gccttgtgtg gtcggtgggc tgcatcacgt ggtccccagg tggaggccct 36780 gggtcatgca gagccacaga aaatgcttag tgaggaggct gtgggggtcc agtcaagtgg 36840 gctctccagc tgcagggctg ggggtgggag ccaggtgagg acccgtgtag agaggagggc 36900 gtgtgcaagg agtggggcca ggagcggggc tggacactgc tggctccaca caggggccca 36960 gcagggagct cgtatgccgc tcgtgcctga agcagacgct gcacaagctg gaggccatga 37020 tgctcatcct gcaggcagag accaccgcgg gcaccgtgac gcccaccgcc atcggagaca 37080 gcatcctcaa catcacaggt gccgcggccc gtgccccatg ccacccgccc gccccgtgcg 37140 gCCCtttCct CtgCCtCCCt cctcccccca accgcgtcgc ctttgcccca tcccatcttc 37200 gtccccctcc cctcccccca attcccatcc tcatccccct cccccaattc CCattCtCCt 37260 CCCCCtCCCC cttccctatt accatccctt ttctCCatCt CtCtCCCCtt ttctccattt 37320 CCCCCCCCgt CCtCCCcgtC CttttgtCca ttCCCCtcat cttcctcatc CCCCtcatcC 37380 cccttCCCCt cccttatccc ccttcccctc cctttccccc tgctCCtCtt CttCtCCCtt 37440 ctcttttCtC tacccttttc cttccttttt cctccctctc cccatcatcc CCCtCatCtt 37500 cgtcctcatc cccatcacct tccccctccc ccctccacca ctctctctcc agcttccccc 37560 ttCCttCtgC CtgCaCCtCg ctctctgccc cctcaggttc cccctttctc ccagccccca 37620 cCCtCCggCt cccccttttt gcctgccccc aCCCtCCctC taCCtCCCtg tctctgcact 37680 gacctcacgc atgtctgcag gagacctcat ccacctggcc agctcggacg tgcgggcacc 37740 acagccctca gagctgggag ccgagtcacc atctcggatg gtggcgtccc aggcctacaa 37800 CCtgacctct gccctcatgc gcatcctCat gcgCtCCcgc gtgctcaacg aggagcccct 37860 gacgctggcg ggcgaggaga tcgtggccca gggcaagcgc tcggacccgc ggagcctgct 37920 gtgctatggc ggcgccccag ggcctggctg ccacttctcc atccccgagg ctttcagcgg 37980 ggccctggcc aacctcagtg acgtggtgca gctcatcttt ctggtggact ccaatccctt 38040 tccctttggc tatatcagca actacaccgt ctccaccaag gtggcctcga tggcattcca 38100 gacacaggcc ggcgcccaga tccccatcga gcggctggcc tcagagcgcg ccatcaccgt 38160 gaaggtgccc aacaactcgg actgggctgc ccggggccac cgcagctccg ccaactccgc 38220 caactccgtt gtggtccagc cccaggcctc cgtcggtgct gtggtcaccc tggacagcag 38280 caaccctgcg gccgggctgc atctgcagct caactatacg ctgctggacg gtgcgtgcag 38340 cgggtggggc acacgcggcc ccctggcctt gttcttgggg ggaaggcgtt tctcgtaggg 38400 cttccatggg tgtctctggt gaaatttgct ttctgtttca tgggctgctg ggggcctggc 38460 cagagaggag ctgggggcca cggagaagca ggtgccagct ctggtgcaga ggctcctatg 38520 ctttcaggcc cgtggcagag ggtgggctca ggagggccat cgtgggtgtc ccccgggtgg 38580 ttgagcttcc cggcaggcgt gtgacctgcg CgttCtgCCc caggccacta cctgtctgag 38640 gaacctgagc cctacctggc agtctaccta cactcggagc cccggcccaa tgagcacaac 38700 tgctcggcta gcaggaggat ccgcccagag tcactccagg gtgctgacca ccggccctac 38760 accttcttca tttccccggg gtgagctctg.cgggccagcc tggcagggca gggcagggca 38820 tcatgggtca gcattgcctg ggttactggc cccatgggga cggcaggcag cgaggggact 38880 ggaccgggta tgggctctga gactgcgaca tccaacctgg cggagcctgg gctcacgtcc 38940 gctacccctt ccctgcccag gagcagagac ccagcgggga gttaccatct gaacctctcc 39000 agccacttcc gctggtcggc gctgcaggtg tccgtgggcc tgtacacgtc cctgtgccag 39060 tacttcagcg aggaggacat ggtgtggcgg acagaggggc tgctgcccct ggaggagacc 39120 tcgcCCCgCC aggccgtctg cCtcaCCCgc cacctcaccg ccttcggcgc cagcctcttc 39180 gtgcccccaa gccatgtccg ctttgtgttt cctgtgagtg accctgtgct cctgggagcc 39240 tctgcagagt cgaggagggc ctgggtgggc tcggctctat cctgagaagg cacagcttgc 39300 acgtgacctc ctgggcccgg cggctgtgtc ctcacaggag ccgacagcgg atgtaaacta 39360 catcgtcatg ctgacatgtg ctgtgtgcct ggtgacctac atggtcatgg ccgccatcct 39420 gcacaagctg gaccagttgg atgccagccg gggccgcgcc atccctttct gtgggcagcg 39480 gggccgcttc aagtacgaga tcctcgtcaa gacaggctgg ggccggggct caggtgaggg 39540 gcgcagcggg gtggcagggc ctcccctgct ctcactggct gtgctggttg caccctctgg 39600 gagtgagtct cgtcgcaggc gtcagaacaa ggcagttttt gcagtgctgt gtgaagggct 39660 cgtgtgttca tcctgggaat gacctcgtga gcactcactg tccctgagga ctaggacagc 39720 tcctagctgg aagtaggtgc cagtcagtca gggtgggcag cccacgttct gcacagtagc 39780 gtggccccac aagtgacgtg agcatcgcta ccactgtggg agactgtgca tccacccgcg 39840 atcctgactg catagctcgt ctctcagacg gaggcgccag caccctcccc gtggctgttt 39900 cttcagtacc tccattttcc tttcattgga attgcccttc tggcattccc tttttgtttt 39960 cgtttttctt tttttagaga cggagtctca ctctgttgcc caggctggag tgcaatggca 40020 tgatcttggc tcacagcaac ttccagctcc cgggtttaag ccattcccct taagcgattc 40080 tcctgagtag ctgggagtac aggtgcacac caccacaccc agttaatttt tcaccatgtc 40140 agccaggcga actcctgacc tcaggtgatc cgcctgcctc ggcctgccag agtgctggga 40200 tgacaggtgt gagccaccac acctggctgt gttcccattt tttatctctg tgctgctttc 40260 ctcttcattg cccagttctt tcttttgatt acctactttt aaaaactgtc ggccgggcgc 40320 ggtggctcac acctgtaatc cgagcacttt gggaggccag gcaggcaaat cacggggtca 40380 ggagatcgag accatcctgg ctaacggtga aaccctgtct ctaataaaaa gtacaaaaaa 40440 attagcccgg cgtagtggca ggcgcctgta gtcccagctc cttgggagac tgaggcagga 40500 gaatggcgtg aacccgggag gcggagcttg cagtgagctg agattgcgcc actgcactcc 40560 agcctgggtg acacagcaag actccatctc aaaaaaaaaa gaaaaaaaat actgtcacct 40620 gggtctgtca ctgggagagg aggtgacaca gcttcacgct ttgcagtctg tgcatgaact 40680 gagggacggg tgtgtggtgc gggtcaccgg ttgtggcatg actgaggcgt ggacaggtgt 40740 gcagtgcggg tcactggttg tggtgtggac tgaggcgtgt gcagccatgt ttgcatgtca 40800 caagttacag ttctttccat gtaacttaat catgtccttg aggtcctgct gttaattgga 40860 caaattgcag taaccgcagc tccttgtgta tggcagagcc gtgcaaagcc gggactgcct 40920 gtgtggctcc ttgagtgcgc acaggccaaa gctgagatga cttgcctggg atgccacacg 40980 tgttgggcag cagaccgagc ctcccacccc tccctcttgc ctcccaggta ccacggccca 41040 cgtgggcatc atgctgtatg gggtggacag ccggagcggc caccggcacc tggacggcga 41100 cagagccttc caccgcaaca gcctggacat cttccggatc gccaccccgc acagcctggg 41160 tagcgtgtgg aagatccgag tgtggcacga caacaaaggt ttgtgcggac cctgccaagc 41220 tctgcccctc tgcccccgca ttggggcgcc ctgcgagcct gacctccctc ctgcgcctct 41280 gcagggctca gccctgcctg gttcctgcag cacgtcatcg tcagggacct gcagacggca 41340 cgcagcgcct tcttcctggt caatgactgg ctttcggtgg agacggaggc caacgggggc 41400 ctggtggaga aggaggtgct ggccgcgagt aaggcctcgt tccatggtcc cactccgtgg 41460 gaggttgggc agggtggtcc tgccccgtgg cctcctgcag tgcggccctc cctgccttct 41520 aggcgacgca gcccttttgc gcttccggcg cctgctggtg gctgagctgc agcgtggctt 41580 ctttgacaag cacatctggc tctccatatg ggaccggccg cctcgtagcc gtttcactcg 41640 catccagagg gccacctgct gcgttctcct catctgcctc ttcctgggcg ccaacgccgt 41700 gtggtacggg gctgttggcg actctgccta caggtgggtg ccgtaggggt cggggcagcc 41760 tcttcctgcc CagCCCttCC tgcccctcag cctcacctgt gtggCCtCCt CtcctCCaCa 41820 cagcacgggg catgtgtcca ggctgagccc gctgagcgtc gacacagtcg ctgttggcct 41880 ggtgtccagc gtggttgtct atcccgtcta cctggccatc ctttttctct tccggatgtc 41940 ccggagcaag gtgggctggg gctggggacc cgggagtact gggaatggag cctgggcctc 42000 ggcaccatgc ctagggccgc cactttccag tgctgcagcc agagggaaag gcgtccacca 42060 aaggctgctc gggaagggtc aacacacttg agcagcctta gctagactga ccagggagaa 42120 agagagaaga ctcagaagcc agaatggtga aagaacgagg gcactttgct aagcagacgc 42180 cacggacgac tgcacagcag cacgccagat aactcagaag aagcaagcac gcggctgtgc 42240 acgcttccga aatgcactcc agaagaaaat ctcagtacat ctataggaag tgaagaggct 42300 gagttagtcc cttagaaacg tcccagtggc cgggccgggt gtggtggctc acgcctgtaa 42360 tcccaacact tcaggtggcc gaggtgggcg gatctgagtc caggagtttg agaccagcct 42420 gggcaacata gcaagacccc atctatataa aacattaaaa agggccaggc gcggtggctc 42480 acgcctgtaa tcccagcact ttgggaggcc gaggcgggca gatcacttga ggtcaggagt 42540 tcgagaccag cctggccaac acaatgaaac cccgactcta ctacaaatac aaaaacttag 42600 ctgggcatgg tggcgggcgc ctgtagtccc agctactcga gaggctgagg caggagaatg 42660 gcatgaaccc aggaggcgga gcttgcagtg agccgagatt gcgccactgc actccatcct 42720 gggcaacgga gcaagactcc atctccaaaa aaaaaaaaaa aaaatcccac aaagaaaagc 42780 tcaggctcag agccttcacg atagaatttt tctaagcagt taaggaagaa ttaacaccaa 42840 tccttcacag actctttcca agaatacagc aggtgggaac gcttcccatt catacggaaa 42900 cgggaggccg caccccttag gaatgcacac gtggggtcct caagaggtta catgcaaact 42960 aaccccagca gcacacagag aaggcgcata agccgcgacc aggaggggtt gctcccgagt 43020 ccgtggcagg aaccagaggc cacatgtggc tgctcgtatt taagttaatt aaaatggaac 43080 gatggccggg tgtggtggct cacacctgta atcccagcac tttgggaggc ggaggcgggc 43140 agatcacttg aggtcaggag ttccaagacc agcctggcca acacagtgaa accccgtctc 43200 tactaaaaat acaaaaaatt agctgggcat ggtggcaggc acctgtaatc ccagctactc 43260 aggaggctga gccaggacaa tcgcctgaac gcgggaggtg gaggttgcag tgagctgaga 43320 ttgcgccatt gcactccagc ctgggtgaca gcgagactcc atctaaaaaa gaaaatatga 43380 aatttaaaac tctgttcctt agctgcacca gtctgctgtc aagtgttcag tggcacacgt 43440 cgcgaggggc tgccatcacg gacggtgcag atgtcccata tatccagcat tctaggacat 43500 tctgtcagat ggcaccgggc tctgtcctgt ctgctgagga ggtggcttct catccctgtc 43560 ctgagcaggt ctgagctgcc gcccgctgac cactgccctc gtcctgcagg tggctgggag 43620 cccgagcccc acacctgccg ggcagcaggt gctggacatc gacagctgcc tggactcgtc 43680 cgtgctggac agctccttcc tcacgttctc aggcctccac gctgaggtga ggactctact 43740 gggggtcctg ggctgggctg ggggtcctgc cgccttggcg cagcttggac tcaagacact 43800 gtgcacctct cagcaggcct ttgttggaca gatgaagagt gacttgtttc tggatgattc 43860 taagaggtgg gttccctaga gaaacctcga gccctggtgc aggtcactgt gtctggggtg 43920 ccgggggtgt gcgggctgcg tgtccttgct gggtgtctgt ggctccatgt ggtcacacca 43980 cccgggagca ggtttgctcg gaagcccagg gtgtccgtgc gtgactggac gggggtgggc 44040 tgtgtgtgtg acacatcccc tggtaccttg ctgacccgcg ccacctgcag tctggtgtgc 44100 tggccctccg gcgagggaac gctcagttgg ccggacctgc tcagtgaccc gtccattgtg 44160 ggtagcaatc tgcggcagct ggcacggggc caggcgggcc atgggctggg cccagaggag 44220 gacggcttct ccctggccag cccctactcg cctgccaaat ccttctcagc atcaggtgag 44280 ctggggtgag aggagggggc tctgaagctc acccttgcag ctgggcccac cctatgcctc 44340 ctgtacctct agatgaagac ctgatccagc aggtccttgc cgagggggtc agcagcccag 44400 cccctaccca agacacccac atggaaacgg acctgctcag cagcctgtga gtgtccggct 44460 ctcgggggag gggggattgc cagaggaggg gccgggactc aggccaggca gccgtggttc 44520 ccgcctgggg tagggtgggg tggggtgcca gggcagggct gtggctgcac cacttcactt 44580 ctctgaacct ctgttgtctg tggaaagagc ctcatgggat ccccagggcc ccagaacctt 44640 ccctctaggg agggagcagg ctcatggggc tttgtaggag cagaaaggct cctgtgtgag 44700 gctggccggg gccacgtttt tatcttggtc tcagagcagt gagaaattat gggcgggttt 44760 ttaaataccc catttttggc cgggcgcggt ggctcacacg tgtaatccca gcactttggg 44820 aggccgaggt gggcagatga cctgaggtca gcagttcgag accagcctgg ccaacatggc 44880 gaaaccccgt ctctactaaa aatacaaaaa attagccggg catgctggca ggcgcctgta 44940 gtcccagtta ctcgggagac tgaggtagga gaatcgattg aacctggtag gtgaaggttg 45000 tagtgagccg agatcgcgcc actgcactcc agcctgggca acaagagcga aactccgtct 45060 caaaaacaaa aaaattcctc aatttcttgg ttgttttgta acttatcaac aaatggtcat 45120 atagaggtta ccttgtatgt agtcacgcac atagtcacgc acatggcagc cggcggcgga 45180 gcgcacccac ggcgtgttcc cacgcgtgtg accccgggct ctgccatgcc ctcctatgct 45240 caggtgtgct gaggtccaca cggccctgcc gttgcactgc agctgcctgc aggattcagt 45300 gcagtggcat gcagtgcagg tgcggtgccc cggagccaca ggccacacca cagggcctgc 45360 atgcacaggg gctgcggtgt ctgggtttgg gtaactacgc cctgtgacat ttgcacagca 45420 acagaattac ctaatgacgc atttctcaga acacatccct ggcactaagt ggtgcgtgac 45480 tgctgctttt gcatccacat ctagtttgat ttgtgtgtta ttcctttgag tgcttctcat 45540 tgttaagcaa ccaagaacta aagaggtatg aactgcccct ggactcaaac aaaaaggaaa 45600 acttcctgat ttacaaaagg cagataacca tcacatgagg gcatctttat gaataaattg 45660 ctggttggtt ttaaaaatac agagtatggg gaaatccagg ggtagtcact acatgctgac 45720 cagccccagg tatctccggc ccaaagctct gtgaaatcca gattcagtgc ttccgcgggg 45780 atttctgacg gcagctcaga ctccgcatcc acacagagcg cgtggccctc accctcccgg 45840 cttcctcaac ccttggccgt cccttgctcg gacagtgctt cgggctgacc aggtcggagg 45900 cttgggtttg tcctggaccc ctctgcgtcc ttcctcactg cagcctccag cgcgtcccgt 45960 ggctcctttc ccaacgcaga gcacggcctt ccctgcgcct gagcctgcac cctccgtcct 46020 ggcggcgcct ctgccctggc attccctgcc actccatgcc tccctattgg ccattctccg 46080 tctctgccag cgagagcctg ctccctgagt cagaccctga gtcatttgtg ttgctataaa 46140 ggaatagttg aggctgggtt attttttatt tttatttatt tttttgagat ggagtctctg 46200 ttgcccagac tggagtgcag tcgcatgatc tcggctcact gcaaagtctg cctcccacgt 46260 tcaagcagtt atctgcctca gcctcccaag tagctaagat tacaggcgcc cgccgccaca 46320 gccggctaat tttttgtgtg tgtgttttag tagagaggag gtttcaccat cttagccagg 46380 ctggtcttga actcctgacc tcgtgatcca cccatctcag cctcccaaaa tgctgagatt 46440 acaggcgtga gccaccacgc ctgaccaagt tgaggctagg tcatttttta attttttgta 46500 aagacagggt ctcactgtct ccaactcctg agctcaagtg atcctcctgc ctcagcctcc 46560 tgaagtgctg ggattacagg cttgagacac tgcgcccagc caagagtgtc ttttatcctc 46620 cgagagacag caaaacagga agcattcagt gcagtgtgac cctgggtcag gccgttcttt 46680 cggtgatggg ctgacgaggg cgcaggtacg ggagagcgtc ctgagagccc gggactcggc 46740 gtctcgcagt tggtctcgtc ctccccctca acgtgtcttc gctgcctctg tacctcttct 46800 ctagcagctc tgggaccggg catatcagca tggtggcccg atgcagtggc acagcctcgg 46860 tggtcactgg ctcctggaga cacaagcaga tctctggcct cagggagccc tacacactgt 46920 tgggatttga aaggcattca tatgtttcct tgtccagaag ttaattttag gccataaacc 46980 tgcatgggac agacacactg gcgtctctag attgtagaga tgcttgttgg atggttgaga 47040 cccaatcata gtttgcaggg ttgaaggggg gctcattgca ccctgagaga ctgtgcactg 47100 ctgtaagggc agctggtcag gctgtgggcg atgggtttat cagcagcaag cgggcgggag 47160 agggacgcag gcggacgcct gacttcggtg cctggagtgg ctcttggttc cctggctccc 47220 agcaccactc ccactctcgt ttggggtagg gtcttccggc tttttgtcgg ggggaccctg 47280 tgacccaaga ggctcaagaa actgcccgcc caggttaaca tgggcttggc tgcaactgcc 47340 tcctggaggc cgggatgaat tcacagccta ccatgtccct caggtccagc actcctgggg 47400 agaagacaga gacgctggcg ctgcagaggc tgggggagct ggggccaccc agcccaggcc 47460 tgaactggga acagccccag gcagcgaggc tgtccaggac aggtgtgctt gcgtagcccc 47520 gggatgcccc tagcccctcc ctgtgagctg cctctcacag gtctgtctct gcttccccag 47580 gactggtgga gggtctgcgg aagcgcctgc tgccggcctg gtgtgcctcc ctggcccacg 47640 ggctcagcct gctcctggtg gctgtggctg tggctgtctc agggtgggtg ggtgcgagct 47700 tccccccggg cgtgagtgtt gcgtggctcc tgtccagcag cgccagcttc ctggcctcat 47760 tcctcggctg ggagccactg aaggtgaggg ggctgccagg ggtaggctac aggcctccat 47820 cacgggggac ccctctgaag ccaccccctc cccaggtctt gctggaagcc ctgtacttct 47880 cactggtggc caagcggctg cacccggatg aagatgacac cctggtagag agcccggctg 47940 tgacgcctgt gagcgcacgt gtgccccgcg tacggccacc ccacggcttt gcactcttcc 48000 tggccaagga agaagcccgc aaggtcaaga ggctacatgg catgctgcgg gtgagcctgg 48060 gtgcggcctg tgcccctgcc acctccgtct CttgtCtCCC aCCtCCCaCC Catgcacgca 48120 ggacactcct gtcccccttt cctcacctca gaaggccctt aggggttcaa tgctctgcag 48180 cctttgcccg gtctcCCtCc taccccacgc cccccacttg ctgccccagt ccctgccagg 48240 gcccagctcc aatgcccact cctgcctggc cctgaaggcc cctaagcacc actgcagtgg 48300 cctgtgtgtc tgcccccagg tggggttccg ggcagggtgt gtgctgccat taccctggcc 48360 aggtagagtc ttggggcgcc ccctgccagc tcaccttcct gcagccacac ctgccgcagc 48420 catggctcca gccgttgcca aagccctgct gtcactgtgg gctggggcca ggctgaccac 48480 agggcccccc cgtccaccag agcctcctgg tgtacatgct ttttctgctg gtgaccctgc 48540 tggccagcta tggggatgcc tcatgccatg ggcacgccta ccgtctgcaa agcgccatca 48600 agcaggagct gcacagccgg gccttcctgg ccatcacgcg gtacgggcat ccggtgcact 48660 ggtctgtctt ctgggcttta gttttgcctt tagtccagcc agaccctagg ggacatgtgg 48720 acatgtgtag atacctttgt ggctgctaga actggaggta ggtgctgctg gcatcagtag 48780 gcagagggga gggacacagg tccgtgtctt gcagtgcaca ggacgggccc atgacagaca 48840 actgtctgcc ccagaacatc cccaggataa ggctgagaag cccaggtcta gccgtggcca 48900 gcagggcagt gggagccatg ttccctgggt ctctggtggc cgctcactcg aggcgggcat 48960 ggggcagtag gggctggagc gtgtgactga tgctgtggca ggtctgagga gctctggcca 49020 tggatggccc acgtgctgct gccctacgtc cacgggaacc agtccagccc agagctgggg 49080 cccccacggc tgcggcaggt gcggctgcag gaaggtgagc tggcagggcg tgccccaaga 49140 cttaaatcgt tcctcttgtt gagagagcag cctttagcgg agctctggca tcagccctgc 49200 tccctagctg tgtgaccttt gccctcttaa caccgccgtt tccttctctg tatatgagag 49260 atggtaacgt tgtctaattg atggctgctg ggagggttcc ctggggtggc gccgaaccag 49320 agctcaggcg agctggccag caggaaacac tcctgttggg ttttgatgag gccctggccc 49380 cggcctgggg ctctgtgtgt ttcagcactc tacccagacc ctcccggccc cagggtccac 49440 acgtgctcgg ccgcaggagg cttcagcacc agcgattacg acgttggctg ggagagtcct 49500 cacaatggct cggggacgtg ggcctattca gcgccggatc tgctggggtg agcagagcga 49560 gggccccggg cgtctacgcc aaggacaagg gagtagttct ccaggagtgc cgcggcctcc 49620 tgaccagcct ggctccgggg tgccggaagg gctggggtgc ggcacccacg ccacccctct 49680 ccggcagggc atggtcctgg ggctcctgtg ccgtgtatga cagcgggggc tacgtgcagg 49740 agctgggcct gagcctggag gagagccgcg accggctgcg cttcctgcag ctgcacaact 49800 ggctggacaa caggtgggag CtCCCtCCCC tgccctctcc ggggtggccg cagtcaccag 49860 ccaggagccc accctcactc ctccggcccc cgctggccta ggcggcttcc acagcccctc 49920 agccacgcct gcactgcgcg gtccccgcag ctdccgccct gccacccgct cctactgacc 49980 cgcaccctct gcgcaggagc cgcgctgtgt tcctggagct cacgcgctac agcccggccg 50040 tggggctgca cgccgccgtc acgctgcgcc tcgagttccc ggcggccggc cgcgccctgg 50100 ccgccctcag cgtccgcccc tttgcgctgc gccgcctcag cgcgggcctc tcgctgCCtc 50160 tgctcacctc ggtacgcccg tccccggcca gaccccgcgc ctcccaccgg cagcgtcccg 50220 ccccctcgcg gggccccgcc cggcagcgtc tcacccctcg cagcgccccg ccccctcgca 50280 gcgtcccgcc ccctcgcagg gccccgcccc ggcagcgtcc cgccccctcg tagggccccg 50340 ccccggcagc gtcCcgCCCC ctcgcagggC cccgccccgg cagcgtccct CCcgcCCtCC 50400 tgaccgcgcc ccccacaggt gtgcctgctg ctgttcgccg tgcacttcgc cgtggccgag 50460 gcccgtactt ggcacaggga agggcgctgg cgcgtgctgc ggctcggagc ctgggcgcgg 50520 tggctgctgg tggcgctgac ggcggccacg gcactggtac gcctcgccca gctgggtgcc 50580 gctgaccgcc agtggacccg tttcgtgcgc ggccgcccgc gccgcttcac tagcttcgac 50640 caggtggcgc agctgagctc cgcagcccgt ggcctggcgg cctcgctgct cttcctgctt 50700 ttggtcaagg tgagggctgg gccggtgggc gcggggctgg gcgcacaccc cagggctgca 50760 agcagacaga tttctcgtcc gcaggctgcc cagcagctac gcttcgtgcg ccagtggtcc 50820 gtctttggca agacattatg ccgagctctg ccagagctcc tgggggtcac cttgggcctg 50880 gtggtgctcg gggtagccta cgcccagctg gccatcctgg taggtgactg cgcggccggg 50940 gagggcgtct tagctcagct cagctcagct gtacgccctc actggtgtcg ccttccccgc 51000 agctcgtgtc ttcctgtgtg gactccctct ggagcgtggc ccaggccctg ttggtgctgt 51060 gccctgggac tgggctctct accctgtgtc ctgccgagtc ctggcacctg tCaCCCctgc 51120 tgtgtgtggg gctctgggca ctgcggctgt ggggcgccct acggctgggg gctgttattc 51180 tccgctggcg ctaccacgcc ttgcgtggag agctgtaccg gccggcctgg gagccccagg 51240 actacgagat ggtggagttg ttcctgcgca ggctgcgcct ctggatgggc ctcagcaagg 51300 tcaaggaggt gggtacggcc cagtgggggg gagagggaca cgccctgggc tctgcccagg 51360 gtgcagccgg actgactgag CCCctgtgCC gcccccagtt ccgccacaaa gtccgctttg 51420 aagggatgga gccgctgccc tctcgctcct ccaggggctc caaggtatcc ccggatgtgc 51480 ccccacccag cgctggctcc gatgcctcgc acccctccac ctcctccagc cagctggatg 51540 ggctgagcgt gagcctgggc cggctgggga caaggtgtga gcctgagccc tcccgcctcc 51600 aagccgtgtt cgaggccctg ctcacccagt ttgaccgact caaccaggcc acagaggacg 51660 tctaccagct ggagcagcag ctgcacagcc tgcaaggccg caggagcagc cgggcgcccg 51720 ccggatcttc ccgtggccca tccccgggcc tgcggccagc actgcccagc cgccttgccc 51780 gggccagtcg gggtgtggac ctggccactg gccccagcag gacacccctt cgggccaaga 51840 acaaggtcca ccccagcagc acttagtcct ccttcctggc gggggtgggc cgtggagtcg 51900 gagtggacac cgctcagtat tactttctgc cgctgtcaag gccgagggcc aggcagaatg 51960 gctgcacgta ggttccccag agagcaggca ggggcatctg tctgtctgtg ggcttcagca 52020 ctttaaagag gctgtgtggc caaccaggac ccagggtccc ctccccagct cccttgggaa 52080 ggacacagca gtattggacg gtttctagcc tctgagatgc taatttattt ccccgagtcc 52140 tcaggtacag cgggctgtgc ccggccccac cccctgggca gatgtccccc actgctaagg 52200 ctgctggctt cagggagggt tagcctgcac cgccgccacc ctgcccctaa gttattacct 52260 ctccagttcc taccgtactc cctgcaccgt ctcactgtgt gtctcgtgtc agtaatttat 52320 atggtgttaa aatgtgtata tttttgtatg tcactatttt cactagggct gaggggcctg 52380 cgcccagagc tggcctcccc caacacctgc tgcgcttggt aggtgtggtg gcgttatggc 52440 agcccggctg ctgcttggat gcgagcttgg ccttgggccg gtgctggggg cacagctgtc 52500 tgccaggcac tctcatcacc ccagaggcct tgtcatcctc ccttgcccca ggccaggtag 52560 caagagagca gcgcccaggc ctgctggcat caggtctggg caagtagcag gactaggcat 52620 gtcagaggac cccagggtgg ttagaggaaa agactcctcc tgggggctgg ctcccagggt 52680 ggaggaaggt gactgtgtgt gtgtgtgtgt gcgcgcgcgc acgcgcgagt gtgctgtatg 52740 gcccaggcag cctcaaggcc ctcggagctg gctgtgcctg cttctgtgta ccacttctgt 52800 gggcatggcc gcttctagag cctcgacacc cccccaaccc ccgcaccaag cagacaaagt 52860 caataaaaga gctgtctgac tgcaatctgt gcctctatgt ctgtgcactg gggtcaggac 52920 tttatttatt tcactgacag gcaataccgt ccaaggccag tgcaggaggg agggccccgg 52980 cctcacacaa actcggtgaa gtcctccacc gaggagatga ggcgcttccg ctggcccacc 53040 tcatagccag gtgtgggctc ggctggagtc tgtgcagggg ctttgctatg ggacggaggg 53100 tgcaccagag gtaggctggg gttggagtag gcggcttcct cgcagatctg aaggcagagg 53160 cggcttgggc agtaagtctg ggaggcgtgg caaccgctct gcccacacac ccgccccaca 53220 gcttgggcag ccagcacacc ccgctgaggg agccccatat tCCctacccg ctggcggagc 53280 gcttgatgtg gcggagcggg caatccactt ggaggggtag atatcggtgg ggttggagcg 53340 gctatgatgc acctgtgagg ccatctgggg acgtaggcag ggggtgagct cactatcagg 53400 tggcacctgg gcctgtccca ccagctcacg cctggaccca cccccactca catttgcgtg 53460 cagggccatc tggcgggcca cgaagggcag gttgcggtca gacacgatct tggccacgct 53520 gg 53522 <210> 2 <211> 4303 <212> PRT
<213> Homo sapiens <400> 2 Met Pro Pro Ala Ala Pro Ala Arg Leu Ala Leu Ala Leu Gly Leu Gly Leu Trp Leu Gly Ala Leu Ala Gly Gly Pro Gly Arg Gly Cys Gly Pro Cys Glu Pro Pro Cys Leu Cys Gly Pro Ala Pro Gly Ala Ala Cys Arg Val Asn Cys Ser Gly Arg Gly Leu Arg Thr Leu Gly Pro Ala Leu Arg Ile Pro Ala Asp Ala Thr Glu Leu Asp Val Ser His Asn Leu Leu Arg Ala Leu Asp Val Gly Leu Leu Ala Asn Leu Ser Ala Leu Ala Glu Leu Asp Ile Ser Asn Asn Lys Ile Ser Thr Leu Glu Glu Gly Ile Phe Ala Asn Leu Phe Asn Leu Ser Glu Ile Asn Leu Ser Gly Asn Pro Phe Glu Cys Asp Cys Gly Leu Ala Trp Leu Pro Gln Trp Ala Glu Glu Gln Gln Val Arg Val Val Gln Pro Glu Ala Ala Thr Cys Ala Gly Pro Gly Ser Leu Ala Gly Gln Pro Leu Leu Gly Ile Pro Leu Leu Asp Ser Gly Cys Gly Glu Glu Tyr Val Ala Cys Leu Pro Asp Asn Ser Ser Gly Thr Val Ala Ala Val Ser Phe Ser Ala Ala His Glu Gly Leu Leu Gln Pro Glu Ala Cys Ser Ala Phe Cys Phe Ser Thr Gly Gln Gly Leu Ala Ala Leu Ser Glu Gin Gly Trp Cys Leu Cys Gly Ala Ala Gln Pro Ser Ser Ala Ser Phe Ala Cys Leu Ser Leu Cys Ser Gly Pro Pro Ala Pro Pro Ala Pro Thr Cys Arg Gly Pro Thr Leu Leu Gln His Val Phe Pro Ala Ser Pro Gly Ala Thr Leu Val Gly Pro His Gly Pro Leu Ala Ser Gly Gln Leu Ala Ala Phe His Ile Ala Ala Pro Leu Pro Val Thr Asp Thr Arg Trp Asp Phe Gly Asp Gly Ser Ala Glu Val Asp Ala Ala Gly Pro Ala Ala Ser His Arg Tyr Val Leu Pro Gly Arg Tyr His Val Thr Ala Val Leu Ala Leu Gly Ala Gly Ser Ala Leu Leu Gly Thr Asp Val Gln Val Glu Ala Ala Pro Ala Ala Leu Glu Leu Val Cys Pro Ser Ser Val Gln Ser Asp Glu Ser Leu Asp Leu Ser Ile Gln Asn Arg Gly Gly Ser Gly Leu Glu Ala Ala Tyr Ser Ile Val Ala Leu Gly Glu Glu Pro Ala Arg Ala Val His Pro Leu Cys Pro Ser Asp Thr Glu Ile Phe Pro Gly Asn Gly His Cys Tyr Arg Leu Val Val Glu Lys Ala Ala Trp Leu Gln Ala Gln G1u Gln Cys Gln Ala Trp Ala Gly Ala Ala Leu Ala Met Val Asp Ser Pro Ala Val Gln Arg Phe Leu Val Ser Arg Val Thr Arg Ser Leu Asp Val Trp Ile Gly Phe Ser Thr Val Gln Gly Val Glu Val Gly Pro Ala Pro Gln Gly Glu Ala Phe Ser Leu Glu Ser Cys Gln Asn Trp Leu Pro Gly Glu Pro His Pro Ala Thr Ala Glu His Cys Val Arg Leu Gly Pro Thr Gly Trp Cys Asn Thr Asp Leu Cys Ser Ala Pro His Ser Tyr Val Cys Glu Leu Gln Pro Gly Gly Pro Val Gln Asp Ala Glu Asn Leu Leu Val Gly Ala Pro Ser Gly Asp Leu Gln Gly Pro Leu Thr Pro Leu Ala Gln Gln Asp Gly Leu Ser Ala Pro His Glu Pro Val Glu Val Met Val Phe Pro Gly Leu Arg Leu Ser Arg Glu Ala Phe Leu Thr Thr Ala Glu Phe Gly Thr Gln Glu Leu Arg Arg Pro Ala Gln Leu Arg Leu Gln Val Tyr Arg Leu Leu Ser Thr Ala Gly Thr Pro Glu Asn Gly Ser Glu Pro Glu Ser Arg Ser Pro Asp Asn Arg Thr Gln Leu Ala Pro Ala Cys Met Pro Gly Gly Arg Trp Cys Pro Gly Ala Asn Ile Cys Leu Pro Leu Asp Ala Ser Cys His Pro Gln Ala Cys Ala Asn Gly Cys Thr Ser Gly Pro Gly Leu Pro Gly Ala Pro Tyr Ala Leu Trp Arg Glu Phe Leu Phe Ser Va1 Pro Ala Gly Pro Pro Ala Gln Tyr Ser Val Thr Leu His Gly Gln Asp Val Leu Met Leu Pro Gly Asp Leu Val Gly Leu Gln His Asp Ala Gly Pro Gly Ala Leu Leu His Cys Ser Pro Ala Pro Gly His Pro Gly Pro Arg Ala Pro Tyr Leu Ser Ala Asn Ala Ser Ser Trp Leu Pro His Leu Pro Ala Gln Leu Glu Gly Thr Trp Gly Cys Pro Ala Cys Ala Leu Arg Leu Leu Ala Gln Arg Glu Gln Leu Thr Val Leu Leu Gly Leu Arg Pro Asn Pro Gly Leu Arg Leu Pro Gly Arg Tyr Glu Val Arg Ala Glu Val Gly Asn Gly Val Ser Arg His Asn Leu Ser Cys Ser Phe Asp Val Val Ser Pro Val Ala Gly Leu Arg Val Ile Tyr Pro Ala Pro Arg Asp Gly Arg Leu Tyr Val Pro Thr Asn Gly Ser Ala Leu Val Leu Gln Val Asp Ser Gly Ala Asn Ala Thr Ala Thr Ala Arg Trp Pro Gly Gly Ser Leu Ser Ala Arg Phe Glu Asn Val Cys Pro Ala Leu Val Ala Thr Phe Val Pro Ala Cys Pro Trp Glu Thr Asn Asp Thr Leu Phe Ser Val Val Ala Leu Pro Trp Leu Ser Glu Gly Glu His Val Val Asp Val Val Val Glu Asn Ser Ala Ser Arg Ala Asn Leu Ser Leu Arg Val Thr Ala Glu Glu Pro Ile Cys Gly Leu Arg Ala Thr Pro Ser Pro Glu Ala Arg Val Leu Gln Gly Val Leu Val Arg Tyr Ser Pro Val Val Glu Ala Gly Ser Asp Met Val Phe Arg Trp Thr Ile Asn Asp Lys Gln Ser Leu Thr Phe Gln Asn Val Val Phe Asn Val Ile Tyr Gln Ser Ala Ala Val Phe Lys Leu Ser Leu Thr Ala Ser Asn His Val Ser Asn Val Thr Val Asn Tyr Asn Val Thr Val Glu Arg Met Asn Arg Met Gln Gly Leu Gln Val Ser Thr Val Pro Ala Val Leu Ser Pro Asn Ala Thr Leu Ala Leu Thr Ala Gly Val Leu Val Asp Ser Ala Val Glu Val Ala Phe Leu Trp Thr Phe Gly Asp Gly Glu Gln Ala Leu His Gln Phe Gln Pro Pro Tyr Asn Glu Ser Phe Pro Val Pro Asp Pro Ser Val Ala Gln Val Leu Val Glu His Asn Val Thr His Thr Tyr Ala Ala Pro Gly Glu Tyr Leu Leu Thr Val Leu Ala Ser Asn Ala Phe Glu Asn Leu Thr Gln Gln Val Pro Val Ser Val Arg Ala Ser Leu Pro Ser Val Ala Val Gly Val Ser Asp Gly Val Leu Val Ala Gly Arg Pro Val Thr Phe Tyr Pro His Pro Leu Pro Ser Pro Gly Gly Val Leu Tyr Thr Trp Asp Phe Gly Asp Gly Ser Pro Val Leu Thr Gln Ser Gln Pro Ala Ala Asn His Thr Tyr Ala Ser Arg Gly Thr Tyr His Val Arg Leu Glu Val Asn Asn Thr Val Ser Gly Ala Ala Ala Gln Ala Asp Val Arg Val Phe Glu Glu Leu Arg Gly Leu Ser Val Asp Met Ser Leu Ala Val Glu Gln Gly Ala Pro Val Val Val Ser Ala Ala Val Gln Thr Gly Asp Asn Ile Thr Trp Thr Phe Asp Met Gly Asp Gly Thr Val Leu Ser Gly Pro Glu Ala Thr Val Glu His Val Tyr Leu Arg Ala Gln Asn Cys Thr Val Thr Val Gly Ala Gly Ser Pro Ala Gly His Leu Ala Arg Ser Leu His Val Leu Val Phe Val Leu Glu Val Leu Arg Val Glu Pro Ala Ala Cys Ile Pro Thr Gln Pro Asp Ala Arg Leu Thr Ala Tyr Val Thr Gly Asn Pro Ala His Tyr Leu Phe Asp Trp Thr Phe Gly Asp Gly Ser Ser Asn Thr Thr Val Arg Gly Cys Pro Thr Val Thr His Asn Phe Thr Arg Ser Gly Thr Phe Pro Leu Ala Leu Val Leu Ser Ser Arg Val Asn Arg Ala His Tyr Phe Thr Ser Ile Cys Val Glu Pro Glu Val Gly Asn Val Thr Leu Gln Pro Glu Arg Gln Phe Val Gln Leu Gly Asp Glu Ala Trp Leu Val Ala Cys Ala Trp Pro Pro Phe Pro Tyr Arg Tyr Thr Trp Asp Phe Gly Thr Glu Glu Ala Ala Pro Thr Arg Ala Arg Gly Pro Glu Val Thr Phe Ile Tyr Arg Asp Pro Gly Ser Tyr Leu Val Thr Val Thr Ala Ser Asn Asn Ile Ser Ala Ala Asn Asp Ser Ala Leu Val Glu Val Gln Glu Pro Val Leu Val Thr Ser Ile Lys Val Asn Gly Ser Leu Gly Leu Glu Leu Gln Gln Pro Tyr Leu Phe Ser Ala Val Gly Arg Gly Arg Pro Ala Ser Tyr Leu Trp Asp Leu Gly Asp Gly Gly Trp Leu Glu Gly Pro Glu Val Thr His Ala Tyr Asn Ser Thr Gly Asp Phe Thr Val Arg Val Ala Gly Trp Asn Glu Val Ser Arg Ser Glu Ala Trp Leu Asn Val Thr Val Lys Arg Arg Val Arg Gly Leu Val Val Asn Ala Ser Arg Thr Val Val Pro Leu Asn Gly Ser Val Ser Phe Ser Thr Ser Leu Glu Ala Gly Ser Asp Val Arg Tyr Ser Trp Val Leu Cys Asp Arg Cys Thr Pro Ile Pro Gly Gly Pro Thr Ile Ser Tyr Thr Phe Arg Ser Val Gly Thr Phe Asn Ile Ile Val Thr Ala Glu Asn Glu Val Gly Ser Ala Gln Asp Ser Ile Phe Val Tyr Val Leu Gln Leu Ile Glu Gly Leu Gln Val Val Gly Gly Gly Arg Tyr Phe Pro Thr Asn His Thr Val Gln Leu G1n Ala Val Val Arg Asp Gly Thr Asn Val Ser Tyr Ser Trp Thr A1a Trp Arg Asp Arg Gly Pro Ala Leu Ala Gly Ser Gly Lys Gly Phe Ser Leu Thr Val Leu Glu Ala Gly Thr Tyr His Val Gln Leu Arg Ala Thr Asn Met Leu Gly Ser Ala Trp Ala Asp Cys Thr Met Asp Phe Val Glu Pro Val Gly Trp Leu Met Val Ala Ala Ser Pro Asn Pro Ala Ala Val Asn Thr Ser Val Thr Leu Ser Ala Glu Leu Ala Gly Gly Ser Gly Val Val Tyr Thr Trp Ser Leu Glu Glu Gly Leu Ser Trp Glu Thr Ser Glu Pro Phe Thr Thr His Ser Phe Pro Thr Pro Gly Leu His Leu Val Thr Met Thr Ala Gly Asn Pro Leu Gly Ser Ala Asn Ala Thr Val Glu Val Asp Val Gln Val Pro Val Ser Gly Leu Ser Ile Arg Ala Ser Glu Pro Gly Gly Ser Phe Val Ala Ala Gly Ser Ser Val Pro Phe Trp Gly Gln Leu Ala Thr Gly Thr Asn Val Ser Trp Cys Trp Ala Val Pro Gly Gly Ser Ser Lys Arg Gly Pro His Val Thr Met Val Phe Pro Asp Ala Gly Thr Phe Ser Ile Arg Leu Asn Ala Ser Asn Ala Val Ser Trp Val Ser Ala Thr Tyr Asn Leu Thr Ala Glu Glu Pro Ile Val Gly Leu Val Leu Trp Ala Ser Ser Lys Val Val Ala Pro Gly Gln Leu Val His Phe Gln Ile Leu Leu Ala Ala Gly Ser Ala Val Thr Phe Arg Leu Gln Val Gly Gly Ala Asn Pro Glu Val Leu Pro Gly Pro Arg Phe Ser His Ser Phe Pro Arg Val Gly Asp His Val Val Ser Val Arg Gly Lys Asn His Val Ser Trp Ala Gln Ala Gln Val Arg Ile Val Val Leu Glu Ala Val Ser Gly Leu Gln Val Pro Asn Cys Cys Glu Pro Gly Ile Ala Thr Gly Thr Glu Arg Asn Phe Thr Ala Arg Val Gln Arg Gly Ser Arg Val Ala Tyr Ala Trp Tyr Phe Ser Leu Gln Lys Val Gln Gly Asp Ser Leu Val Ile Leu Ser Gly Arg Asp Val Thr Tyr Thr Pro Val Ala Ala Gly Leu Leu Glu Ile Gln Val Arg Ala Phe Asn Ala Leu Gly Ser Glu Asn Arg Thr Leu Val Leu Glu Val Gln Asp Ala Val Gln Tyr Val Ala Leu Gln Ser Gly Pro Cys Phe Thr Asn Arg Ser Ala Gln Phe Glu Ala Ala Thr Ser Pro Ser Pro Arg Arg Val Ala Tyr His Trp Asp Phe Gly Asp Gly Ser Pro Gly Gln Asp Thr Asp Glu Pro Arg Ala Glu His Ser Tyr Leu Arg Pro Gly Asp Tyr Arg Val Gln Val Asn Ala Ser Asn Leu Val Ser Phe Phe Val Ala Gln Ala Thr Val Thr Val Gln Val Leu Ala Cys Arg Glu Pro Glu Val Asp Val Val Leu Pro Leu Gln Val Leu Met Arg Arg Ser Gln Arg Asn Tyr Leu Glu Ala His Val Asp Leu Arg Asp Cys Val Thr Tyr Gln Thr Glu Tyr Arg Trp Glu Val Tyr Arg Thr Ala Ser Cys Gln Arg Pro Gly Arg Pro Ala Arg Val Ala Leu Pro Gly Val Asp Val Ser Arg Pro Arg Leu Val Leu Pro Arg Leu Ala Leu Pro Val Gly His Tyr Cys Phe Val Phe Val Val Ser Phe Gly Asp Thr Pro Leu Thr Gln Ser Ile Gln Ala Asn Val Thr Val Ala Pro Glu Arg Leu Val Pro Ile Ile Glu Gly Gly Ser Tyr Arg Val Trp Ser Asp Thr Arg Asp Leu Val Leu Asp Gly Ser Glu Ser Tyr Asp Pro Asn Leu Glu Asp Gly Asp Gln Thr Pro Leu Ser Phe His Trp Ala Cys Val Ala Ser Thr Gln Arg Glu Ala Gly Gly Cys Ala Leu Asn Phe Gly Pro Arg Gly Ser Ser Thr Val Thr Ile Pro Arg Glu Arg Leu Ala Ala Gly Val Glu Tyr Thr Phe Ser Leu Thr Val Trp Lys Ala Gly Arg Lys Glu Glu Ala Thr Asn Gin Thr Val Leu Ile Arg Ser Gly Arg Val Pro Ile Val Ser Leu Glu Cys Val Ser Cys Lys Ala Gln Ala Val Tyr Glu Val Ser Arg Ser Ser Tyr Val Tyr Leu Glu Gly Arg Cys Leu Asn Cys Ser Ser Gly Ser Lys Arg Gly Arg Trp Ala Ala Arg Thr Phe Ser Asn Lys Thr Leu Val Leu Asp Glu Thr Thr Thr Ser Thr Gly Ser Ala Gly Met Arg Leu Val Leu Arg Arg Gly Val Leu Arg Asp Gly Glu Gly Tyr Thr Phe Thr Leu Thr Val Leu Gly Arg Ser Gly Glu Glu Glu Gly Cys Ala Ser Ile Arg Leu Ser Pro Asn Arg Pro Pro Leu Gly Gly Ser Cys Arg Leu Phe Pro Leu Gly Ala Val His Ala Leu Thr Thr Lys Val His Phe Glu Cys Thr Gly Trp His Asp Ala Glu Asp Ala Gly Ala Pro Leu Val Tyr Ala Leu Leu Leu Arg Arg Cys Arg Gln Gly His Cys Glu Glu Phe Cys Val Tyr Lys Gly Ser Leu Ser Ser Tyr Gly Ala Val Leu Pro Pro Gly Phe Arg Pro His Phe Glu Val Gly Leu Ala Val Val Val Gln Asp Gln Leu Gly Ala Ala Val Val Ala Leu Asn Arg Ser Leu Ala Ile Thr Leu Pro Glu Pro Asn Gly Ser Ala Thr Gly Leu Thr Val Trp Leu His Gly Leu Thr Ala Ser Val Leu Pro Gly Leu Leu Arg Gln Ala Asp Pro Gln His Val Ile Glu Tyr Ser Leu Ala Leu Val Thr Val Leu Asn Glu Tyr Glu Arg Ala Leu Asp Val Ala Ala Glu Pro Lys His Glu Arg Gln His Arg Ala Gln Ile Arg Lys Asn Ile Thr Glu Thr Leu Val Ser Leu Arg Val His Thr Val Asp Asp Ile Gln Gln Ile Ala Ala Ala Leu Ala Gln Cys Met Gly Pro Ser Arg Glu Leu Val Cys Arg Ser Cys Leu Lys Gln Thr Leu His Lys Leu Glu Ala Met Met Leu Ile Leu Gln Ala Giu Thr Thr Ala Gly Thr Val Thr Pro Thr Ala Ile Gly Asp Ser Ile Leu Asn Ile Thr Gly Asp Leu Ile His Leu Ala Ser Ser Asp Val Arg Ala Pro Gln Pro Ser Glu Leu Gly Ala Glu Ser Pro Ser Arg Met Val Ala Ser Gln Ala Tyr Asn Leu Thr Ser Ala Leu Met Arg Ile Leu Met Arg Ser Arg Val Leu Asn Glu Glu Pro Leu Thr Leu Ala Gly Glu Glu Ile Val Ala Gln Gly Lys Arg Ser Asp Pro Arg Ser Leu Leu Cys Tyr Gly Gly Ala Pro Gly Pro Gly Cys His Phe Ser Ile Pro Glu Ala Phe Ser Gly Ala Leu Ala Asn Leu Ser Asp Val Val Gln Leu Ile Phe Leu Val Asp Ser Asn Pro Phe Pro Phe Gly Tyr Ile Ser Asn Tyr Thr Val Ser Thr Lys Val Ala Ser Met Ala Phe Gln Thr Gln Ala Gly Ala Gln Ile Pro Ile Glu Arg Leu Ala Ser Glu Arg Ala Ile Thr Val Lys Val Pro Asn Asn Ser Asp Trp Ala Ala Arg Gly His Arg Ser Ser Ala Asn Ser Ala Asn Ser Val Val Val Gln Pro Gln Ala Ser Val Gly Ala Val Val Thr Leu Asp Ser Ser Asn Pro Ala Ala Gly Leu His Leu Gln Leu Asn Tyr Thr Leu Leu Asp Gly His Tyr Leu Ser Glu Glu Pro Glu Pro Tyr Leu Ala Val Tyr Leu His Ser Glu Pro Arg Pro Asn Glu His Asn Cys Ser Ala Ser Arg Arg Ile Arg Pro Glu Ser Leu Gln Gly Ala Asp His Arg Pro Tyr Thr Phe Phe Ile Ser Pro Gly Ser Arg Asp Pro Ala Gly Ser Tyr His Leu Asn Leu Ser Ser His Phe Arg Trp Ser Ala Leu Gln Val Ser Val Gly Leu Tyr Thr Ser Leu Cys Gln Tyr Phe Ser Glu Glu Asp Met Val Trp Arg Thr Glu Gly Leu Leu Pro Leu Glu Glu Thr Ser Pro Arg Gln Ala Val Cys Leu Thr Arg His Leu Thr Ala Phe Gly Ala Ser Leu Phe Val Pro Pro Ser His Val Arg Phe Val Phe Pro Glu Pro Thr Ala Asp Val Asn Tyr Ile Val Met Leu Thr Cys Ala Val Cys Leu Val Thr Tyr Met Val Met Ala Ala Ile Leu His Lys Leu Asp Gln Leu Asp Ala Ser Arg Gly Arg Ala Ile Pro Phe Cys Gly Gln Arg Gly Arg Phe Lys Tyr Glu Ile Leu Val Lys Thr Gly Trp Gly Arg Gly Ser Gly Thr Thr Ala His Val Gly Ile Met Leu Tyr Gly Val Asp Ser Arg Ser Gly His Arg His Leu Asp Gly Asp Arg Ala Phe His Arg Asn Ser Leu Asp Ile Phe Arg Ile Ala Thr Pro His Ser Leu Gly Ser Val Trp Lys Ile Arg Val Trp His Asp Asn Lys Gly Leu Ser Pro Ala Trp Phe Leu Gln His Val Ile Val Arg Asp Leu Gln Thr Ala Arg Ser Ala Phe Phe Leu Val Asn Asp Trp Leu Ser Val Glu Thr Glu Ala Asn Gly Gly Leu Val Glu Lys Glu Val Leu Ala Ala Ser Asp Ala Ala Leu Leu Arg Phe Arg Arg Leu Leu Val Ala Glu Leu Gln Arg Gly Phe Phe Asp Lys His Ile Trp Leu Ser Ile Trp Asp Arg Pro Pro Arg Ser Arg Phe Thr Arg Ile Gln Arg Ala Thr Cys Cys Val Leu Leu Ile Cys Leu Phe Leu Gly Ala Asn Ala Val Trp Tyr Gly Ala Val Gly Asp Ser Ala Tyr Ser Thr Gly His Val Ser Arg Leu Ser Pro Leu Ser Val Asp Thr Val Ala Val Gly Leu Val Ser Ser Val Val Val Tyr Pro Val Tyr Leu Ala Ile Leu Phe Leu Phe Arg Met Ser Arg Ser Lys Val Ala Gly Ser Pro Ser Pro Thr Pro Ala Gly Gln Gln Val Leu Asp Ile Asp Ser Cys Leu Asp Ser Ser Val Leu Asp Ser Ser Phe Leu Thr Phe Ser Gly Leu His Ala Glu Gln Ala Phe Val Gly Gin Met Lys Ser Asp Leu Phe Leu Asp Asp Ser Lys Ser Leu Val Cys Trp Pro Ser Gly Glu Gly Thr Leu Ser Trp Pro Asp Leu Leu Ser Asp Pro Ser Ile Val Gly Ser Asn Leu Arg Gln Leu Ala Arg Gly Gln Ala Gly His Gly Leu Gly Pro Glu Glu Asp Gly Phe Ser Leu Ala Ser Pro Tyr Ser Pro Ala Lys Ser Phe Ser Ala Ser Asp Glu Asp Leu Ile Gln Gln Val Leu Ala Glu Gly Val Ser Ser Pro Ala Pro Thr Gln Asp Thr His Met Glu Thr Asp Leu Leu Ser Ser Leu Ser Ser Thr Pro Gly Glu Lys Thr Glu Thr Leu Ala Leu Gln Arg Leu Gly Glu Leu Gly Pro Pro Ser Pro Gly Leu Asn Trp Glu Gln Pro Gln Ala Ala Arg Leu Ser Arg Thr Gly Leu Val Glu Gly Leu Arg Lys Arg Leu Leu Pro Ala Trp Cys Ala Ser Leu Ala His Gly Leu Ser Leu Leu Leu Val Ala Val Ala Val Ala Val Ser Gly Trp Val Gly Ala Ser Phe Pro Pro Gly Val Ser Val Ala Trp Leu Leu Ser Ser Ser Ala Ser Phe Leu Ala Ser Phe Leu Gly Trp Glu Pro Leu Lys Val Leu Leu Glu Ala Leu Tyr Phe Ser Leu Va1 Ala Lys Arg Leu His Pro Asp Glu Asp Asp Thr Leu Val Glu Ser Pro Ala Val Thr Pro Val Ser Ala Arg Val Pro Arg Val Arg Pro Pro His Gly Phe Ala Leu Phe Leu Ala Lys Glu Glu Ala Arg Lys Val Lys Arg Leu His Gly Met Leu Arg Ser Leu Leu Val Tyr Met Leu Phe Leu Leu Val Thr Leu Leu Ala Ser Tyr Gly Asp Ala Ser Cys His Gly His Ala Tyr Arg Leu Gln Ser Ala Ile Lys Gln Glu Leu His Ser Arg Ala Phe Leu Ala Ile Thr Arg Ser Glu Glu Leu Trp Pro Trp Met Ala His Val Leu Leu Pro Tyr Val His Gly Asn Gln Ser Ser Pro Glu Leu Gly Pro Pro Arg Leu Arg Gln Val Arg Leu Gln Glu Ala Leu Tyr Pro Asp Pro Pro Gly Pro Arg Val His Thr Cys Ser Ala Ala Gly Gly Phe Ser Thr Ser Asp Tyr Asp Val Gly Trp Glu Ser Pro His Asn Gly Ser Gly Thr Trp Ala Tyr Ser Ala Pro Asp Leu Leu Gly Ala Trp Ser Trp Gly Ser Cys Ala Val Tyr Asp Ser Gly Gly Tyr Val Gln Glu Leu Gly Leu Ser Leu Glu Glu Ser Arg Asp Arg Leu Arg Phe Leu Gln Leu His Asn Trp Leu Asp Asn Arg Ser Arg Ala Val Phe Leu Glu Leu Thr Arg Tyr Ser Pro Ala Val Gly Leu His Ala Ala Val Thr Leu Arg Leu Glu Phe Pro Ala Ala Gly Arg Ala Leu Ala Ala Leu Ser Val Arg Pro Phe Ala Leu Arg Arg Leu Ser Ala Gly Leu Ser Leu Pro Leu Leu Thr Ser Val Cys Leu Leu Leu Phe Ala Val His Phe Ala Val Ala Glu Ala Arg Thr Trp His Arg Glu Gly Arg Trp Arg Val Leu Arg Leu Gly Ala Trp Ala Arg Trp Leu Leu Val Ala Leu Thr Ala Ala Thr Ala Leu Val Arg Leu Ala Gln Leu Gly Ala Ala Asp Arg Gln Trp Thr Arg Phe Val Arg Gly Arg Pro Arg Arg Phe Thr Ser Phe Asp Gln Val Ala His Val Ser Ser Ala Ala Arg Gly Leu Ala Ala Ser Leu Leu Phe Leu Leu Leu Val Lys Ala Ala Gln His Val Arg Phe Val Arg Gln Trp Ser Val Phe Gly Lys Thr Leu Cys Arg Ala Leu Pro Glu Leu Leu Gly Val Thr Leu Gly Leu Val Val Leu Gly Val Ala Tyr Ala Gln Leu Ala Ile Leu Leu Val Ser Ser Cys Val Asp Ser Leu Trp Ser Val Ala Gln Ala Leu Leu Val Leu Cys Pro Gly Thr Gly Leu Ser Thr Leu Cys Pro Ala Glu Ser Trp His Leu Ser Pro Leu Leu Cys Val Gly Leu Trp Ala Leu Arg Leu Trp Gly Ala Leu Arg Leu Gly Ala Val Ile Leu Arg Trp Arg Tyr His Ala Leu Arg Gly Glu Leu Tyr Arg Pro Ala Trp Glu Pro Gln Asp Tyr Glu Met Val Glu Leu Phe Leu Arg Arg Leu Arg Leu Trp Met Gly Leu Ser Lys Val Lys Glu Phe Arg His Lys Val Arg Phe Glu Gly Met Glu Pro Leu Pro Ser Arg Ser Ser Arg Gly Ser Lys Val Ser Pro Asp Val Pro Pro Pro Ser Ala Gly Ser Asp Ala Ser His Pro Ser Thr Ser Ser Ser Gln Leu Asp Gly Leu Ser Val Ser Leu Gly Arg Leu Gly Thr Arg Cys Glu Pro Glu Pro Ser Arg Leu Gln Ala Val Phe Glu Ala Leu Leu Thr Gln Phe Asp Arg Leu Asn Gln Ala Thr Glu Asp Val Tyr Gln Leu Glu Gln Gln Leu His Ser Leu Gln Gly Arg Arg Ser Ser Arg Ala Pro Ala Gly Ser Ser Arg Gly Pro Ser Pro Gly Leu Arg Pro Ala Leu Pro Ser Arg Leu Ala Arg Ala Ser Arg Gly Val Asp Leu Ala Thr Gly Pro Ser Arg Thr Pro Leu Arg Ala Lys Asn Lys Val His Pro Ser Ser Thr <210> 3 <211> 29 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer BPF14 <400> 3 ccatccacct gctgtgtgac ctggtaaat 29 <210> 4 <211> 26 <212> DNA

<213> Artificial sequence <220>
<223> PCR primer BPR9 <400> 4 ccacctcatc gccccttcct aagcat 26 <210> 5 <211> 31 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer BPF9 <400> 5 attttttgag atggagcttc actcttgcag g 31 <210> 6 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer BPR4 <400> 6 cgctcggcag gcccctaacc 20 <210> 7 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer BPF12 <400> 7 ccgcccccag gagcctagac g 21 <210> 8 <211> 27 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer BPR5 <400> 8 catcctgttc atccgctcca cggttac 27 <210> 9 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer F13 <400> 9 tggagggagg gacgccaatc 20 <210> 10 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer R27 <400> 10 gtcaacgtgg gcctccaagt 20 <210> 11 <211> 2].
<212> DNA
<213> Artificial sequence <220>
<223> PCR primer F26 <400> 11 agcgcaacta cttggaggcc c 21 <210> 12 <211> 28 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer R2 <400> 12 gcagggtgag caggtggggc catcctac 28 <210> 13 <211> 26 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer BPF15 <400> 13 gaggctgtgg gggtccagtc aagtgg 26 <210> 14 <211> 25 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer BPR12 <400> 14 agggaggcag aggaaagggc cgaac 25 <210> 15 <211> 24 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer BPF6 <400> 15 ccccgtcctc cccgtccttt tgtc 24 <210> 16 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer BPR6 <400> 16 aagcgcaaaa gggctgcgtc g 21 <210> 17 <211> 22 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer BPF13 <400> 17 ggccctccct gccttctagg cg 22 <210> 18 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer KG8R25 <400> 18 gttgcagcca agcccatgtt a 21 <210> 19 <211> 19 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 1F1 <400> 19 ggtcgcgctg tggcgaagg 19 <210> 20 <211> 16 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 1R1 <400> 20 cggcgggcgg catcgt 16 <210> 21 <211> 16 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 1F2 <400> 21 acggcggggc catgcg 16 <210> 22 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 1R2 <400> 22 gcgtcctggc ccgcgtcc 18 <210> 23 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 2F
<400> 23 ttggggatgc tggcaatgtg 20 <210> 24 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 2R

<400> 24 gggattcggc aaagctgatg 20 <210> 25 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 3F
<400> 25 ccatcagctt tgccgaatcc 20 <210> 26 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 3R
<400> 26 agggcagaag ggatattggg 20 <210> 27 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 4F
<400> 27 agacccttcc caccagacct 20 <210> 28 <211> 19 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 4R
<400> 28 tgagccctgc ccagtgtct 19 <210> 29 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 5F1 <400> 29 gagccaggag gagcagaacc c 21 <210> 30 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 5R1 <400> 30 agagggacag gcaggcaaag g 21 <210> 31 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 5F2 <400> 31 cccagccctc cagtgcct 18 <210> 32 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 5R2 <400> 32 cccaggcagc acatagcgat 20 <210> 33 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 5F3 <400> 33 ccgaggtgga tgccgctg 18 <210> 34 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 5R3 <400> 34 gaaggggagt gggcagcaga c 21 <210> 35 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 6F
<400> 35 cactgaccgt tgacaccctc g 21 <210> 36 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 6R
<400> 36 tgccccagtg cttcagagat c 21 <210> 37 <211> 19 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 7F
<400> 37 ggagtgccct gagccccct 19 <210> 38 <211> 19 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 7R
<400> 38 cccctaacca cagccagcg 19 <210> 39 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 8F
<400> 39 tctgttcgtc ctggtgtcct g 21 <210> 40 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 8R
<400> 40 gcaggagggc aggttgtaga a 21 <210> 41 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 9F
<400> 41 ggtaggggga gtctgggctt 20 <210> 42 <211> 17 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 9R
<400> 42 gaggccaccc cgagtcc 17 <210> 43 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 1OF
<400> 43 gttgggcatc tctgacggtg 20 <210> 44 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer lOR
<400> 44 ggaaggtggc ctgaggagat 20 <210> 45 <211> 17 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 11F2 <400> 45 ggggtccacg ggccatg 17 <210> 46 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 11R2 <400> 46 aagcccagca gcacggtgag 20 <210> 47 <211> 17 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer llmidF
<400> 47 gcttgcagcc acggaac 17 <210> 48 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer llmidR
<400> 48 gcagtgctac cactgagaac 20 <210> 49 <211> 23 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 11F1 <400> 49 tgcccctggg agaccaacga tac 23 <210> 50 <211> 22 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 1.1R1 <400> 50 ggctgctgcc ctcactggga ag 22 <210> 51 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 12F
<400> 51 gaggcgacag gctaaggg 18 <210> 52 <211> 25 <212> DNA
<213> Artificial sequence <220>
<223> Primer for PCR
<400> 52 aggtcaacgt gggcctccaa gtagt 25 <210> 53 <211> 19 <212> DNA
<213> Artificial sequence <220>
<223> Forward nested primer F32 <400> 53 gccttgcgca gcttggact 19 <210> 54 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Second specific primer 31R
<400> 54 ,acagtgtctt gagtccaagc 20 <210> 55 <211> 30 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer <400> 55 ctggtgacct acatggtcat ggccgagatc 30 <210> 56 <211> 30 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer <400> 56 ggttgtctat cccgtctacc tggccctcct 30 <210> 57 <211> 25 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer <400> 57 gtccccagcc ccagcccacc tggcc 25 <210> 58 <211> 7 <212> PRT
<213> Homo,sapiens <400> 58 Trp Asp Phe Gly Asp Gly Ser <210> 59 <211> 4 <212> PRT
<213> Homo sapiens <400> 59 His Leu Thr Ala <210> 60 <211> 27 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer <400> 6o gcagggtgag caggtggggc catccta 27 <210> 61 <211> 19 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 12R-2 <400> 61 catgaagcag agcagaagg 19 <210> 62 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 13F
<400> 62 tggagggagg gacgccaatc 20 <210> 63 <211> 19 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 13R
<400> 63 gaggctgggg ctgggacaa 19 <210> 64 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 14F
<400> 64 cccggttcac tcactgcg 18 <210> 65 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 14R

<400> 65 ccgtgctcag agcctgaaag 20 <210> 66 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F16 <400> 66 cgggtgggga gcaggtgg 18 <210> 67 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R16 <400> 67 gctctgggtc aggacagggg a 21 <210> 68 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F15 <400> 68 cgcctggggg tgttcttt 18 <210> 69 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R15 <400> 69 acgtgatgtt gtcgcccg 18 <210> 70 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F14 <400> 70 gcccccgtgg tggtcagc 18 <210> 71 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R14 <400> 71 caggctgcgt ggggatgc 18 <210> 72 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F13 <400> 72 ctggaggtgc tgcgcgtt 18 <210> 73 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R13 <400> 73 ctggctccac gcagatgc 18 <210> 74 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F12 <400> 74 cgtgaacagg gcgcatta 18 <210> 75 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R12 <400> 75 gcagcagaga tgttgttgga c 21 <210> 76 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F11 <400> 76 ccaggctcct atcttgtgac a 21 <210> 77 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R11 <400> 77 tgaagtcacc tgtgctgttg t 21 <210> 78 <211> 19 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F10 <400> 78 ctacctgtgg gatctgggg 19 <210> 79 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R10 <400> 79 tgctgaagct cacgctcc 18 <210> 80 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F9 <400> 80 gggctcgtcg tcaatgcaag 20 <210> 81 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R9 <400> 81 caccacctgc agcccctcta 20 <210> 82 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F8 <400> 82 ccgcccagga cagcatcttc 20 <210> 83 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R8 <400> 83 cgctgcccag catgttgg 18 <210> 84 <211> 19 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F7 <400> 84 cggcaaaggc ttctcgctc 19 <210> 85 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R7 <400> 85 ccgggtgtgg ggaagctatg 20 <210> 86 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F6 <400> 86 cgagccattt accacccata g 21 <210> 87 <211> 19 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R6 <400> 87 gcccagcacc agctcacat 19 <210> 88 <211> 19 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F5 <400> 88 ccacgggcac caatgtgag 19 <210> 89 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R5 <400> 89 ggcagccagc aggatctgaa 20 <210> 90 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR pimer 15F4 <400> 90 cagcagcaag gtggtggc 18 <210> 91 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R4 <400> 91 gcgtaggcga cccgagag 18 <210> 92 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F3 <400> 92 acgggcactg agaggaactt c 21 <210> 93 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R3 <400> 93 accagcgtgc ggttctcact 20 <210> 94 <211> 19 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F2 <400> 94 gccgcgacgt cacctacac 19 <210> 95 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R2 <400> 95 tcggccctgg gctcatct 18 <210> 96 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F1 <400> 96 gtcgccaggg caggacacag 20 <210> 97 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15F1-1 <400> 97 acttggaggc ccacgttgac c 21 <210> 98 <211> 19 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R1-1 <400> 98 tgatgggcac caggcgctc 19 <210> 99 <211> 21 <212> DNA
<213> Artificialsequence <220>
<223> PCR primer 15F1-2 <400> 99 catccaggcc aatgtgacgg t 21 <210> 100 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 15R1-2 <400> 100 cctggtggca agctgggtgt t 21 <210> 101 <211> 20 <212> DNA

<213> Artificial sequence <220>
<223> PCR primer 16F
<400> 101 taaaactgga tggggctctc 20 <210> 102 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 16R
<400> 102 ggcctccacc agcactaa 18 <210> 103 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 17F
<400> 103 gggtccccca gtccttccag 20 <210> 104 <211> 17 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 17R
<400> 104 tccccagccc gcccaca 17 <210> 105 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 18F
<400> 105 gCCCCCtCac caccccttct 20 <210> 106 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 18R
<400> 106 tcccgctgct ccccccac 18 <210> 107 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 19F
<400> 107 gatgccgtgg ggaccgtc 18 <210> 108 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 19R
<400> 108 gtgagcaggt ggcagtctcg 20 <210> 109 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 20F
<400> 109 ccaccccctc tgctcgtagg t 21 <210> 110 <211> 19 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 20R
<400> 110 ggtcccaagc acgcatgca 19 <210> 111 <211> 22 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer 21F
<400> 111 tgccggcctc ctgcgctgct ga 22 <210> 112 <211> 28 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer TWR2-1 <400> 112 gtaggatggc cccacctgct caccctgc 28 <210> 113 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> PCR primer R27' <400> 113 aggtcaacgt gggcctccaa 20

Claims (35)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A set of primers for detecting the presence or absence of a mutation in a polynucleotide, said set of primers comprising at least a first and second set of primer pairs, each primer of said first set of primer pairs comprising a 5' region and adjacent 3' region, said 5' region comprising a nucleotide sequence that selectively hybridizes to a PKD1 gene sequence, or to a PKD1 gene sequence and a PKD1 gene homolog sequence, and said 3' region comprising a nucleotide sequence that hybridizes to a PKD1 gene sequence, wherein the set of primers can amplify the PKD1 gene sequence as set forth in:

nucleotides 22218 to 26363 of SEQ ID NO:1, nucleotides 36819 to 37140 of SEQ ID NO:1 or nucleotides 37329 to 41258 of SEQ ID NO: 1, or a nucleotide sequence complementary to the PKD1 gene sequence, wherein each primer of said first set of primer pairs hybridizes to a nucleotide sequence flanking and within fifty nucleotides of one of the PKD1 gene sequences, wherein the first set of primer pairs is for amplifying a first amplification product, and the second set of primer pairs is for amplifying the first amplification product to provide a second amplification product, wherein primers for the first set of primer pairs are selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8; SEQ ID NO:13, SEQ ID NO:14, SEQ
ID
NO: 15 and SEQ ID NO: 16, and primers for the second set of primer pairs are selected from the group consisting of SEQ ID NOS:39 to 51, and 61, and wherein the second amplification product is an authentic PKD1 gene.

2. The set of primers of claim 1, wherein the second set of primer pairs comprises SEQ ID
NOS:39 and 40; SEQ ID NOS:41 and 42; SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46;
SEQ ID NOS:47 and 48; SEQ ID NOS:49 and 50; or SEQ ID NOS:51 and 61.

3. The set of primers of claim 1, wherein at least one primer pair is selected from SEQ ID
NOS:7 and 8; SEQ ID NOS:13 and 14; and SEQ ID NOS:15 and 16.

4. The set of primers of claim 1, wherein the first set of primer pairs comprises SEQ ID NOS:7 and 8; SEQ ID NOS:13 and 14; or SEQ ID NOS:15 and 16.

5. The set of primers of claim 1, wherein at least one primer pair is selected from SEQ ID
NOS:39 and 40; SEQ ID NOS:41 and 42; SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46;
SEQ ID NOS:47 and 48; SEQ ID NOS:49 and 50; and SEQ ID NOS:51 and 61.

6. A solid matrix, comprising the set of primers of claim 1, wherein the set of primers is immobilized on the solid matrix.

7. The solid matrix of claim 6, which comprises a plurality of immobilized sets of primers.

8. The solid matrix of claim 6, wherein the solid matrix is a microchip.

9. A method of detecting the presence or absence of a mutation in a PKD1 polynucleotide in a sample from a subject, the method comprising:
(a) contacting nucleic acid molecules in a sample with a first primer pair under conditions suitable for amplification of a first PKD1 polynucleotide by the first primer pair, thereby generating a PKD1-specific amplification product under said conditions, wherein each primer of said first primer pair comprises a 5' region and adjacent 3' region, said 5' region comprising a nucleotide sequence that selectively hybridizes to a PKD1 gene sequence, or to a PKD1 gene sequence and a PKD1 gene homolog sequence, and said 3' region comprising a nucleotide sequence that hybridizes to a PKD1 gene sequence, wherein the first primer pair can amplify a PKD1 gene sequence as set forth in:
nucleotides 2043 to 4209 of SEQ ID NO: 1;
nucleotides 17907 to 22489 of SEQ ID NO:1;
nucleotides 22218 to 26363 of SEQ ID NO: 1;
nucleotides 26246 to 30615 of SEQ ID NO: 1;

nucleotides 30606 to 33957 of SEQ ID NO:1;
nucleotides 36819 to 37140 of SEQ ID NO:1;
nucleotides 37329 to 41258 of SEQ ID NO:1, or nucleotides 41508 to 47320 of SEQ ID NO: 1, and wherein each primer in the first primer pair hybridizes to a nucleotide sequence flanking and within fifty nucleotides of one of the PKD1 gene sequences, wherein primers from the first primer pair are selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10; SEQ
ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17 and SEQ ID NO:18;
(b) contacting the PKD 1-specific amplification product with a second primer pair under conditions suitable for nested amplification of the PKD 1-specific amplification product by the second primer pair, wherein primers from the second primer pair are selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID
NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID
NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ
ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID
NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID
NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ
ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID
NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID
NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ
ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID
NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NOS:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:113, SEQ ID NO:97, SEQ ID NO:98, SEQ ID
NOS:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID
NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID
NOS:109, SEQ ID NO:110, SEQ ID NO:111 and SEQ ID NO:112, thereby generating a nested amplification product; and (c) identifying the presence or absence of a mutation in the nested amplification product, by comparing the sequence of the nested amplification product to SEQ ID NO: 1, thereby detecting the presence or absence of a mutation in the PKD1 polynucleotide in the sample.

10. The method of claim 9, wherein the first primer pair comprises SEQ ID NO:3 and 4; SEQ
ID NOS:5 and 6; SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12;
SEQ
ID NOS:13 and 14; SEQ ID NOS:15 and 16; or SEQ ID NOS:17 and 18.

11. The method of claim 9, wherein the second primer pair comprises SEQ ID
NOS:19 and 20;
SEQ ID NOS:21 and 22; SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID
NOS:27 and 28; SEQ ID NOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ ID
NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID NOS:41 and 42; SEQ
ID
NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48; SEQ ID NOS:49 and 50;
SEQ ID NOS:51 and 61; SEQ ID NOS:62 and 63; SEQ ID NOS:64 and 65; SEQ ID
NOS:66 and 67; SEQ ID NOS:68 and 69; SEQ ID NOS:70 and 71; SEQ ID NOS:72 and 73; SEQ ID
NOS:74 and 75; SEQ ID NOS:76 and 77; SEQ ID NOS:78 and 79; SEQ ID NOS:80 and 81; SEQ
ID
NOS:82 and 83; SEQ ID NOS:84 and 85; SEQ ID NOS:86 and 87; SEQ ID NOS:88 and 89;
SEQ ID NOS:90 and 91; SEQ ID NOS:92 and 93; SEQ ID NOS:94 and 95; SEQ ID
NOS:96 and 113; SEQ ID NOS:97 and 98; SEQ ID NOS:99 and 100; SEQ ID NOS:101 and 102; SEQ
ID
NOS:103 and 104; SEQ ID NOS:105 and 106; SEQ ID NOS:107 and 108; SEQ ID
NOS:109 and 110; or SEQ ID NOS:111 and 112.

12. The method of claim 9, wherein amplification is performed by a polymerase chain reaction.

13. The method of claim 9, wherein the PKD1 polynucleotide is a variant PKD1 polynucleotide.

14. The method of claim 13, wherein the variant PKD1 polynucleotide comprises a nucleotide sequence substantially identical to SEQ ID NO:1, wherein nucleotide 474 is a T; nucleotide 487 is an A; nucleotide 4884 is an A; nucleotide 6058 is a T; nucleotide 6195 is an A; nucleotide 7376 is a C; nucleotide 7696 is a T; nucleotide 8021 is an A; nucleotide 9367 is a T; nucleotide 10143 is a G; nucleotide 10234 is a C; or nucleotide 10255 is a T.

15. The method of claim 9, wherein identifying the presence or absence of a mutation in the nested amplification product is performed using a primer extension reaction assay, wherein the primer extension reaction is performed using a detectably labeled primer and a mixture of deoxynucleotides and dideoxynucleotides, and wherein the primer and mixture are selected so as to enable differential extension of the detectably labeled primer in the presence of a wild type PKD1 polynucleotide as compared to a mutant PKD1 polynucleotide.

16. The method of claim 9, wherein the method is performed in a high throughput format using a plurality of samples.

17. The method of claim 16, wherein the plurality of samples are in an array.

18. The method of claim 17, wherein the array comprises a microtiter plate.

19. The method of claim 17, wherein the array is on a microchip.

20. A method of identifying a subject at risk for an autosomal dominant polycystic kidney disease (ADPKD), the method comprising:
(a) contacting nucleic acid molecules in a sample from a subject with a first primer pair under conditions suitable for amplification of a first PKD1 polynucleotide by the first primer pair, thereby generating a PKD1-specific amplification product under said conditions, wherein each primer of said first primer pair comprises a 5' region and adjacent 3' region, said 5' region comprising a nucleotide sequence that selectively hybridizes to a PKD1 gene sequence, or to a PKD1 gene sequence and a PKD1 gene homolog sequence, and said 3' region comprising a nucleotide sequence that hybridizes to a PKD1 gene sequence, wherein the first primer pair can amplify a PKD1 gene sequence as set forth in:
nucleotides 2043 to 4209 of SEQ ID NO:1;
nucleotides 17907 to 22489 of SEQ ID NO:1;
nucleotides 22218 to 26363 of SEQ ID NO:1;
nucleotides 26246 to 30615 of SEQ ID NO:1;
nucleotides 30606 to 33957 of SEQ ID NO:1;
nucleotides 36819 to 37140 of SEQ ID NO:1;
nucleotides 37329 to 41258 of SEQ ID NO:1, or nucleotides 41508 to 47320 of SEQ ID NO:1, and wherein each primer in the first primer pair hybridizes to a nucleotide sequence flanking and within fifty nucleotides of one of the PKD1 gene sequences, wherein primers for the first primer pair are selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10; SEQ
ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17 and SEQ ID NO:18;
(b) contacting the PKD1-specific amplification product with a second primer pair under conditions suitable for nested amplification of the PKD1-specific amplification product by the second primer pair, wherein primers for the second primer pair are selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID
NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID
NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ
ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID
NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID
NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ
ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID
NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID
NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ
ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID
NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NOS:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:113, SEQ ID NO:97, SEQ ID NO:98, SEQ ID
NOS:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID
NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID
NOS:109, SEQ ID NO:110, SEQ ID NO:111 and SEQ ID NO:112, thereby generating a nested amplification product; and (c) detecting the presence or absence of a mutation indicative of ADPKD in the nested amplification product, wherein the absence of the mutation identifies the subject as not at risk for ADPKD, and wherein the presence of the mutation identifies the subject as at risk for ADPKD.

21. The method of claim 20, wherein the first primer pair comprises SEQ ID
NO:3 and 4; SEQ
ID NO:5 and 6; SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12;
SEQ ID
NOS:13 and 14; SEQ ID NOS:15 and 16; or SEQ ID NOS:17 and 18.

22. The method of claim 20, wherein the method is performed in a high throughput format.

23. The method of claim 20, wherein detecting the presence or absence of a mutation indicative of ADPKD in the nested amplification product comprises comparing data from said amplification product with accumulated data representative of the presence or absence of the mutation.

24. The method of claim 23, further comprising formatting the data into a report indicating whether said mutation is present and said subject is at risk for ADPKD.

25. The method of claim 20, wherein the second primer pair comprises SEQ ID
NOS: 19 and 20; SEQ ID NOS:21 and 22; SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID
NOS:27 and 28; SEQ ID NOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ
ID
NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID NOS:41 and 42;
SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48; SEQ ID
NOS:49 and 50; SEQ ID NOS:51 and 61; SEQ ID NOS:62 and 63; SEQ ID NOS:64 and 65; SEQ ID
NOS:66 and 67; SEQ ID NOS:68 and 69; SEQ ID NOS:70 and 71; SEQ ID NOS:72 and 73; SEQ
ID
NOS:74 and 75; SEQ ID NOS:76 and 77; SEQ ID NOS:78 and 79; SEQ ID NOS:80 and 81;
SEQ ID NOS:82 and 83; SEQ ID NOS:84 and 85; SEQ ID NOS:86 and 87; SEQ ID
NOS:88 and 89; SEQ ID NOS:90 and 91; SEQ ID NOS:92 and 93; SEQ ID NOS:94 and 95; SEQ ID
NOS:96 and 113; SEQ ID NOS:97 and 98; SEQ ID NOS:99 and 100; SEQ ID NOS:101 and 102;
SEQ ID
NOS:103 and 104; SEQ ID NOS:105 and 106; SEQ ID NOS:107 and 108; SEQ ID
NOS:109 and 110; or SEQ ID NOS:111 and 112.

26. The method of claim 20, wherein detecting the presence or absence of a mutation comprises determining the nucleotide sequence of the nested amplification product, and comparing the nucleotide sequence to the corresponding nucleotide sequence of SEQ ID NO:1.

27. The method of claim 20, wherein detecting the presence or absence of a mutation comprises determining the melting temperature of the nested amplification product, and comparing the melting temperature to the melting temperature of the corresponding nucleotide sequence of SEQ
ID NO:1.

28. The method of claim 20, wherein detecting the presence or absence of a mutation is performed using denaturing high performance liquid chromatography.

29. The method of claim 20, wherein the mutation indicative of ADPKD comprises a nucleotide sequence substantially identical to SEQ ID NO:1, wherein nucleotide 3110 is a C; nucleotide 8298 is a G; nucleotide 9164 is a G; nucleotide 9213 is an A; nucleotide 9326 is a T; or nucleotide 10064 is an A.

30. The method of claim 20, wherein the mutation indicative of ADPKD comprises a nucleotide sequence substantially identical to SEQ ID NO:1, wherein nucleotide 3336 is deleted; nucleotide 3707 is an A; nucleotide 5168 is a T; nucleotide 6078 is an A; nucleotide 6089 is a T; nucleotide 6326 is a T; nucleotides 7205 to 7211 are deleted; nucleotide 7415 is a T;
nucleotide 7433 is a T;

nucleotide 7883 is a T; nucleotides 8159 to 8160 are deleted; or wherein a GCG
nucleotide sequence is inserted between nucleotides 7535 and 7536.

31. A method of diagnosing an autosomal dominant polycystic kidney disease (ADPKD) in a subject, the method comprising:
(a) amplifying a portion of a PKD1 polynucleotide in a nucleic acid sample from a subject with at least a first primer pair to obtain a first amplification product, wherein each primer of said first primer pair comprises a 5' region and adjacent 3' region, said 5' region comprising a nucleotide sequence that selectively hybridizes to a PKD1 gene sequence, or to a PKD1 gene sequence and a PKD1 gene homolog sequence, and said 3' region comprising a nucleotide sequence that hybridizes to a PKD1 gene sequence, wherein the first primer pair can amplify a PKD1 gene sequence as set forth in:
nucleotides 2043 to 4209 of SEQ ID NO:1;
nucleotides 17907 to 22489 of SEQ ID NO:1;
nucleotides 22218 to 26363 of SEQ ID NO:1;
nucleotides 26246 to 30615 of SEQ ID NO:1;
nucleotides 30606 to 33957 of SEQ ID NO:1;
nucleotides 36819 to 37140 of SEQ ID NO:1;
nucleotides 37329 to 41258 of SEQ ID NO:1, or nucleotides 41508 to 47320 of SEQ ID NO:1, and wherein each primer in the first primer pair hybridizes to a nucleotide sequence flanking and within fifty nucleotides of one of the PKD1 gene sequences, wherein primers for the first primer pair are selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10; SEQ
ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17 and SEQ ID NO:18;

(b) amplifying the first amplification product with at least a second primer pair to obtain a nested amplification product, wherein the second primer pair is suitable for performing nested amplification of the first amplification product, wherein primers for the second primer pair are selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID
NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ
ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID
NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ
ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID
NO:50, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID
NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ
ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID
NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID
NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ
ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID
NO:93, SEQ ID NOS:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:113, SEQ ID NO:97, SEQ ID
NO:98, SEQ ID NOS:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID
NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ
ID NOS:109, SEQ ID NO:110, SEQ ID NO:111 and SEQ ID NO:112; and (c) determining whether the nested amplification product has a mutation associated with ADPKD, wherein the presence of a mutation associated with ADPKD is indicative of ADPKD, thereby diagnosing ADPKD in the subject.

32. The method of claim 31, wherein the method is performed in a high throughput format using a plurality of nucleic acid samples.

33. The method of claim 31, wherein the first primer pair comprises SEQ ID
NO:3 and 4; SEQ
ID NO:5 and 6; SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12;
SEQ ID
NOS:13 and 14; SEQ ID NOS:15 and 16; or SEQ ID NOS:17 and 18.

34. The method of claim 31, wherein the second primer pair comprises SEQ ID
NOS:19 and 20; SEQ ID NOS:21 and 22; SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID
NOS:27 and 28; SEQ ID NOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ
ID
NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID NOS:41 and 42;
SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48; SEQ ID
NOS:49 and 50; SEQ ID NOS:51 and 61; SEQ ID NOS:62 and 63; SEQ ID NOS:64 and 65; SEQ ID
NOS:66 and 67; SEQ ID NOS:68 and 69; SEQ ID NOS:70 and 71; SEQ ID NOS:72 and 73; SEQ
ID
NOS:74 and 75; SEQ ID NOS:76 and 77; SEQ ID NOS:78 and 79; SEQ ID NOS:80 and 81;
SEQ ID NOS:82 and 83; SEQ ID NOS:84 and 85; SEQ ID NOS:86 and 87; SEQ ID
NOS:88 and 89; SEQ ID NOS:90 and 91; SEQ ID NOS:92 and 93; SEQ ID NOS:94 and 95; SEQ ID
NOS:96 and 113; SEQ ID NOS:97 and 98; SEQ ID NOS:99 and 100; SEQ ID NOS:101 and 102;
SEQ ID
NOS:103 and 104; SEQ ID NOS:105 and 106; SEQ ID NOS:107 and 108; SEQ ID
NOS:109 and 110; or SEQ ID NOS:111 and 112.

35. A kit for detecting the presence or absence of a mutation in a PKD1 gene, the kit comprising a set of primers, said set of primers comprising a first and second set of primer pairs, each primer of said first set of primer pairs comprising a 5' region and adjacent 3' region, said 5' region comprising a nucleotide sequence that selectively hybridizes to a PKD1 gene sequence, or to a PKD1 gene sequence and a PKD1 gene homolog sequence, and said 3' region comprising a nucleotide sequence that selectively hybridizes to a PKD1 gene sequence, wherein the set of primers can amplify the PKD1 gene sequence is as set forth in:
nucleotides 22218 to 26363 of SEQ ID NO:1, nucleotides 36819 to 37140 of SEQ ID NO:1 or nucleotides 37329 to 41258 of SEQ ID NO:1, or a nucleotide sequence complementary to the PKD1 gene sequence, wherein each primer of said first set of primer pairs hybridizes to a nucleotide sequence flanking and within fifty nucleotides of the PKD1 gene sequence, wherein the first set of primer pairs is for amplifying a first amplification product, and the second set of primer pairs is for amplifying the first amplification product to provide a second amplification product, wherein primers for the first set of primer pairs are selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14; SEQ
ID

NO:15 and SEQ ID NO:16, and primers for the second set of primer pairs are selected from the group consisting of SEQ ID NOS:39 to 51, and 61, and wherein the second amplification product is an authentic PKD1 gene.