WO1997032996A1 - Direct molecular diagnosis of friedreich ataxia - Google Patents

Abstract

The invention relates generally to methods for the diagnosis and therapeutic treatment of Friedreich Ataxia. Friedreich ataxia (FRDA) is an autosomal recessive, degenerative disease that involves the central and peripheral nervous system and the heart. A gene, X25, was identified in the critical region for the FRDA locus on chromosome 9q13. The gene encodes a 210 amino acid protein, frataxin, that has homologues in distant species such as C. elegans and yeast. A few FRDA patients have been found to have point mutations in X25, but the vast majority are homozygous for a variable, unstable GAA trinucleotide expansion in the first X25 intron. Mature X25 mRNA was severely reduced in abundance in individuals with FRDA. Carriers and individuals at risk for developing FRDA can be ascertained by the methods of the present invention. Further, the methods of the present invention provide treatment to those individuals having FRDA.

Description

DIRECT MOLECULAR DIAGNOSIS OF FRIEDREICH ATAXIA

This invention was supported in part by a grant from the United States Government through federal funds (NINDS NS34192) The U S government has certain rights to this invention

FIELD OF THE INVENTION

This invention relates generally to methods for the diagnosis screening and therapeutic treatment of Friedreich ataxia Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease that involves the central and peripheral nervous system and the heart A gene X25. was lαeπtified in the critical region for the FRDA locus on chromosome 9q 13 The X25 gene encodes a 210 ammo acid protein, frataxin that has nomologues in distant species such as C elegans and yeast A few FRDA patients have been found to have point mutations in X25, but the vast majority are homozygous for a variable unstable GAA tπnucieotide expansion in the first X25 intron Mature X25 mRNA was severely reduced in abundance in individuals with FRDA BACKGROUND OF THE INVENTION

Friedre_ich ataxia (FRDA) is the most common heredⁱtary ataxⁱa. with ₅ an est_imated prevalence of 1 in 50,000 and a deduced carrier frequency of 1 /120 in the European population FRDA is an autosomal recessive degenerat_ive disease characterized by progressive gait and limb ataxia, a lack of tendon reflexes in the legs, loss of position sense, dysarthria, and pyram_idal weakness of the legs. Hypertrophic cardiomyopathy is _in found in almost all patients. Diabetes mellitus is seen in about 10% of the cases_, carbohydrate intolerance in an additional 20%. and a reduced insul_in response to arginine stimulation in all cases. The age of onset ⁱs usually around puberty_, and almost always before age twenty-fⁱve^. Most pat_ients are wheelchair bound by their late twenties and currently there

1₅ is no treatment to slow progression of the disease.

The first pathologic changes are thought to occur in the dorsal root gangl_ia w_ith loss of large sensory neurons, followed by deterioration of the sensory posterior columns, spinocerebellar tracts and corticospinal motor tracts of the spinal cord, and atrophy of large sensory fibers ⁱn ₀ per_ipheral nerves_. Only occasional mild degenerative changes are seen in the cerebellum, pons and medulla. While most symptoms are a consequence of neuronai degeneration, cardiomyopathy and diabetes are thought to reflect independent sites of primary degeneration. Overall_, the pathology of FRDA is very different from that of other

2₅ hereditary ataxias. particularly the dominant forms and ataxia- telang_iectas_ia. where the cerebellum is the primary site of degeneration.

The mutated gene in FRDA has been mapped to chromosome 9q13- q21_.1. S. Chamberlain, et al., Nature, 334:248 (1988); and the FRDA cand_idate region has been narrowed to a 150 kb segment flanked by the

*,o ZO-2 gene (distal) and the marker F8101 (proximal), L Montermⁱni et al., Am J Hum Genet 57 1061 (1995) Previously proposed candidate genes are excluded the X104/CSFA1/Z0-2 gene on the basis of the absence of deleterious mutation in patients and the STM7 and PRKACG genes because they lie in entirety on the centromeric side of F8101 (Figure 1A)

SUMMARY OF THE INVENTION

It is a particular object of the present invention to provide a method of screening individuals for a mutation that leads to Friedreich's ataxia, comprising determining the number of GAA repeats in an introπ of the X25 gene

It is a further object of the present invention to provide a method of screening individuals for a mutation that leads to Friedreich's ataxia, comprising the steps of measuring expression of the X25 gene at the mRNA or protein levels

It is another object of the present invention to provide a method of screening individuals for a mutation that leads to Friedreich's ataxia, comprising the step of detecting a variation in a size of a (GAA)_n repeat in a first intron of a X25 gene by measuring the length of said repeat wherein n for normal individuals ranges from 1 -22 and n for affected individuals is more than about 120-900

It is another object of the present invention to provide a method of screening individuals for a mutation that leads to Friedreich's ataxia, comprising the steps of sequencing DNA from an individual, and comparing said sequence from said individual to SEQ ID NOS 1-12 to determine what differences, if any, there are between the two sequences

It is yet a further object of the present invention to provide a method of treating Friedreich's ataxia in an individual, comprising the step of 7 administering an effective pharmacologic dose of a protein having an ammo acid sequence substantially similar to SEQ ID NO 4 to said individual

It is an additional ob_ject of the present invention to provide a method of treat_ing Friedreich's ataxia in an individual, comprising adminⁱstration of a nucleic acid vector containing an X25 gene capable of expression in a pharmacologically acceptable carrier to said individual.

It _is a further ob_ject of the present invention to provide compositions of matter having SEQ ID NOS 1 -32.

Other and further ob_jects, features and advantages will be apparent and the invention more readily understood from a reading of the following specification and by reference to the accompanying drawings form_ing a part thereof_, wherein the examples of the presently preferred embodiments of the invention are given for the purposes of disclosure.

DESCRIPTION OF THE DRAWINGS

Figure 1(A) Transcription map of the FRDA critical interval. Distances are in kilobase pairs from the first Not I site upstream to the ZO-2 gene The critical FRDA region is between the F8101 marker and the ZO-2 gene M Miu I site, N, Not I site; E, Eag I site: S. Sac II site; B, BssH I site Figure 1 (B) Alignment of the exon 5a-contaιnιπg isoform of frataxin with translated ORFs contained within a C elegans cosmid (CELT59G1 ) and a S cerevisiae EST (T38910) Identical ammo acids are boxed The putative signal peptide is underlined Am o acids _involved by point mutation (L106X and II54F) are indicated by vertical arrows The exon 5b-containiπg isoform diverges at position 161 , and its 1 1 COOH-terminal ammo acids are RLTWLLWLFHP

Figure 2 Northern blot analysis of X25 transcripts. A ³ P-labeled 5^'- RACE product containing exons 1 -5b was hybridized to a multiple tissue Northern blot (Clontech), containing 2 μg of poly-A + RNA in each lane

The membrane was washed at 50° with 0 1 x SSC 0 1 % SDS then exposed to x-ray film at -70° for 7 days The lower panel shows a successive hybridization of the same blot with an actin probe (provided by the blot manufacturer)

Figure 3 Southern blot analysis showing FRDA-associated expanded restriction fragments Lanes 1 and 12, normal controls, lanes 2-7, individuals from a Saudi Arabian FRDA family lanes 8-11 , individuals from a Louisiana Acadian (Cajun) FRDA family Affected subjects are in lanes 3-5 and 9-10, heterozygous carriers in lanes 2, 6-8_, and 11 The position of molecular weight markers is indicated on the side The constant bands correspond to exons 2 and 3 (15 kb), and to a related sequence outside of the FRDA region (5 kb) Ten μg of genomic DNA from each individual were digested with Eco Rl, run in a 0.6% agarose gel, and blotted onto a nylon membrane (Hybond+) The blot was hybridized with a ³ P-labeled X25 cDNA probe After a highest stringency wash with 0 1 x SSC, 0 1 % SDS for 5' at 65°, the blot was exposed to x-ray film at -70° for two days

Figure 4 An automated sequence of the FRDA-associated expanded region from a cosmid subclone The CTT strand was sequenced

Figure 5: Automated sequence of the FRDA-associated expanded region containing the expanded repeat in a FA patient The CTT strand was sequenced It is interesting to note the presence of two imperfect repeats in the patient (the 7th and 8th in the sequenced strand) that are not present on the normal sequence and which could indicate a polymorphic variant present on the chromosome in which the original expansion occurred (>

Figure 6(A) Example of PCR analysis of normal alleles of the GAA repeat Lane 1 is the 1 kb ladder DAN size marker lanes 2-6 are normal controls previously identified to be heterozygous at the repeat The

GAA-F/GAA-R primers were used for amplification Fragments vary in size in the 480-520 bp range

Figure 6(B): PCR amplification of the expanded GAA repeat in a FRDA carrier (lane 3) and in a patient (lane 4) Lane 1 is the 1 kb ladder DNA marker lane 2 is a normal control The Bam/2500 primers were used for PCR Expanded alleles have a slightly fuzzy appearance Instability of the repeat is indicated by the presence of two distinct bands in the oatieπt lane although the patient is an offspring of consanguineous parents Also, the carrier in lane 3 is the patient's mother, but the corresponding expanded allele does not exactly match in size any of her offspring bands Figure 7 Segregation of the L106X mutation and of the GAA expansion in a FRDA family The SSCP pattern shown in A indicates the paternal origin of the point mutation while Southern blot analysis, shown in B, indicates the maternal origin of the expansion NR indicates an unrelated normal control Figure 8 RT-PCR analysis of X25 mRNA in FRDA subjects^, obligate carriers and normal controls Reactions were performed on total RNA extracted from lymphoblastoid cell Iines. The serine hydroxymethyltraπsferase (SHMT) transcript (encoded by a gene on chromosome 17) was used as a control for RNA amount Mock reactions without reverse transcπptase (-RT) were also performed as a negative control In the case of SHMT, the PCR following the -RT reactions generated a product of larger size than the product expected from the cDNA because a fragment of genomic DNA (contaminating the RNA preparation) containing a small intron was amplified In all three r panels the lane marked with r t is a negative control (water) lane 9 corresponds to a normal control individual lanes 1 and 4 to obligate carriers of FRDA lanes 2 3, and 5 to 8 to individuals with FRDA To generate cDNA from the X25 transcript, the RT reaction was primed with the oligonucleotide E2R (SEQ ID NO 13), then PCR was performed between this primer and the πF primer (SEQ ID NO 14)

DETAILED DESCRIPTION OF THE INVENTION

It will be apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope of the invention

As used herein "FRDA" refers to Friedreich ataxia an autosomal recessive degenerative disease that involves the central and peripheral nervous system as well as the heart As used herein, "GAA expansion" refers to multiple (GAA)_n repeats located 1 4 kb downstream from exon 1 in an intron of the X25 gene

As used herein, the "X25" gene refers to the gene identified on chromosome 9q13 that is in the critical region of the FRDA-determinative locus As used herein the term "polymerase chain reaction or "PCR" refers to the PCR procedure described in the patents to Mullis et al U S Patent Nos 4 683 195 and 4,683,202 The procedure basically involves ( 1 ) treating extracted DNA to form single-stranded complementary strands (2) adding a pair of oligonucleotide primers, wherein one primer of the pair is substantially complementary to part of the sequence in the sense strand and the other primer of each pair is substantially complementary to a different part of the same sequence in the complementary antisense strand, (3) annealing the paired primers to the complementary sequence, (4) simultaneously extending the annealed primers from a 3^' terminus of each primer to synthesize an O 97/32996 PCΪ7EP97/01070 s extension product complementary to the strands annealed to each primer wherein said extension products after separation from the complement serve as templates for the synthesis of an extension product for the other primer of each pair, (5) separating said extension products from said templates to produce single-stranded molecules and (6) amplifying said single-stranded molecules by repeating at least once said annealing, extending and separating steps

As used herein the term "pulsed field gel electrophoresis" or "PFGE" refers to a procedure described by Schwartz, et al., Cold Springs Harbor Symposium, Quantitative Biology, 47 189-195 (1982) The procedure basically comprises running a standard electrophoresis gel (agarose, polyacrylamide or other gel known to those skilled in the art) under pulsing conditions One skilled in the art recognizes that the strength of the field as well as the direction of the field is pulsed and rotated in order to separate megabase DNA molecules Current commercial systems are computer controlled and select the strength, direction and time of pulse depending on the molecular weight of DNA to be separated

As used herein the phrase "gene transcript" shall mean the RNA product that results from transcribing a genetic (DNA) template "Gene" shall mean a hereditary unit- in molecular terms, a sequence of chromosomal DNA that is required for production of a functional product. As used herein the phrase "messenger RNA" or "mRNA" shall mean an RNA transcribed from the DNA of a gene, that directs the sequence of ammo acids of the encoded polypeptide

As used herein the phrase "copy DNA" or "cDNA" shall mean DNA synthesized from a primer hybridized to a messenger RNA template

As used herein the phrase "oligonucleotide" shall mean a short nucleic acid molecule (usually 8 to 50 base pans), synthesized for use as a probe or primer J

As used herein, the phrase "primer" shall mean a short DNA or RNA molecule that is paired with a complementary DNA or RNA template, wherein the short DNA or RNA molecule provides a free 3'-OH terminus at which a DNA polymerase starts synthesis of a nucleotide chain. It is a particular object of the present invention to provide a method of screening individuals for a mutation that leads to Friedreich's ataxia, comprising the steps of digesting DNA from an individual to be tested with a restriction endonuclease: and measuring the length of a restriction fragment length polymorphism (RFLP) by hybridization to probes that recognize a region encompassing a GAA repeat in a first intron of an X25 gene and performing Southern Blot analysis, wherein an RFLP corresponding to a GAA repeat longer than a normal range of 7-22 triplets, usually more than about 120, is an indication of said mutation that leads to Friedreich's ataxia. It is a further object of the present invention to provide a method of screening individuals for a mutation that leads to Friedreich's ataxia, comprising the steps of measuring expression of an X25 gene by determining an amount of mRNA expressed from the X25 gene and from known controls, and comparing the amount of mRNA from the X25 gene to the amount of mRNA from the known controls, wherein a reduced amount of mRNA from the X25 gene indicates individuals having said mutation that leads to Friedreich's ataxia.

It is an additional object of the present invention to provide a method of screening individuals for a mutation that leads to Friedreich's ataxia, wherein the amounts of mRNA is determined by the steps of extracting mRNA from individuals to be tested; preparing cDNA from said mRNA, amplifying said cDNA to produce amplification products; and comparing relative amounts of X25 and control cDNA present, wherein a reduced amount of mRNA from the X25 gene indicates individuals having said mutation that leads to Friedreich's ataxia. It is an a_dditional ob_ject of the present invention to provide a method of screening individuals for a mutation that leads to Friedreich s ataxia by detecting tne amount of specific proteins encoded by X25 in cells from patients using antibodies specific for the X25 proteins It is another ob_ject of the present invention to provide a method of screening individuals for a mutation that leads to Friedreich's ataxia, comprising the step of detecting a variation in a size of a (GAA)_n repeat in a first intron of a X25 gene by measuring the length of said repeat, wherein n for normal individuals ranges from 1 -22 and n for affected individuals is more than about 120-900

It is an aαditional ob_ject of the present invention to provide a method for detecting a GAA polymorphism in a first intron of an X25 gene comprising the steps of performing a PCR assay to produce amplified products of said first intron of said X25 gene and measuring the length of said amplified products with molecular techniques known in the art

It is another ob_ject of the present invention to provide a method of screening individuals for a mutation that leads to Fnedreich's ataxia, comprising the steps of sequencing DNA from an individual, and comparing said sequence from said individual to SEQ ID NOS 1 -12 to determine wnat differences if any, there are between said sequence from said mαividual and said SEQ ID NOS 1-12

It is yet a further ob_ject of the present invention to provide a method of treating Fnedreich's ataxia in an individual, comprising the step of administering a pharmacologic effective dose of a protein having an ammo acid sequence substantially similar to SEQ ID NO 4 to said individual

It is an additional ob_ject of the present invention to provide a method of treating Fnedreich's ataxia in an individual, comprising administration of a nucleic acid vector containing an X25 gene capable of expression and a pharmacologically acceptable carrier to said individual //

It is a further object of the present invention to provide compositions of matter having SEQ ID NOS 1 -32

The therapeutic compositions of the present invention can be formulated according to known methods to prepare pharmacologically 5 useful compositions The compositions of the present invention or their functional derivatives are combined in admixture with a pharmacologically acceptable carrier vehicle Suitable vehicles and their formulations are well known in the art In order to form a pharmacologically acceptable composition suitable for effective lo therapeutic administration such compositions will contain an effective amount of the X25 gene or its equivalent or the functional derivative thereof or the frataxin protein or its equivalent or the functional derivative thereof together with the suitable amount of carrier vehicle The nucleic acid therapeutic composition of the present invention will 5 usually be formulated in a vector The frataxin protein therapeutic composition will usually be administered as a purified protein in a pharmacologically suitable carrier The compositions can be administered by a variety of methods including parenterally, by injection, rapid infusion nasopharyngeal absorption, dermal absorption or orally o The compositions may alternatively be administered intramuscularly or intravenously In addition the compositions for pareπteral administration can further include sterile aqueous or nonaqueous solutions, suspensions and emulsions Examples of known nonaqueous solvents include propylene glycol polyethylene glycol vegetable oils such as olive oil and mjectable organic esters, such as ethyl oleate Carriers, adjuncts or occlusive dressings can be used to increase tissue permeability and enhance absorption Liquid dosage forms for oral administration may generally comprise a liposome solution Suitable forms for suspension include emulsions, solutions syrups and elixirs containing inert diluents commonly used in the art such as purified water Besides the inert dilutants. such compositions can also include wetting agents emulsifying and suspending agents or sweetening, flavoring, coloring or perfuming agents.

Additionally_, pharmaceutical methods may be employed to control the duration of action These are well known in the art and include control release preparations and can include appropriate macromolecules, for example polymers, -polyesters, polyamino acids, polyvinyl, pyrolidone, ethyienevinylactate_, methylceliulose,carboxymethylcellulose,or protamine sulfate The concentration of macromolecules, as well as the methods of incorporation, can be adjusted in order to control release. Additionally_, the vector could be incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogells. poly (lactic acid) or ethylene v ylacetate copolymers In addition to being incorporated_, these agents can also be used to trap the vectors in microcapsules. These techniques are well known in the art.

A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in detectable change in the physiology of a recipient patient.

Generally_, the dosage needed to provide an effective amount of composition will vary depending on such factors as the recipient's age, condition_, sex and extent of disease, if any, and other variables which can be ad_justed by one of ordinary skill in the art.

One skilled in the art will appreciate readily that the present invention is well adapted to carrying out the ends and advantages mentioned as well as those inherent herein The probes, primers, methods, procedures and techniques described are presently representative of the preferred embodiments, are intended to be exemplary, and are not /3 intended as limitations on the scope of the invention Changes therein and other uses will occur to those skilled in the art. and are encompassed within the spirit of the invention or defined by the scope of the appended claims. All references specifically cited herein are incorporated by reference.

The following examples are offered by way of illustration and are not intended to limit the invention in any manner:

Example 1 : Localizing and Sequencing the FRDA Critical Region

Potential exons were identified in the FRDA critical region by direct cDNA selection exon amplification, and computer prediction from random sequences Twelve cosmids spanning 120 kb of the critical FRDA interval, plus 80 kb immediately proximal to the interval were subcloned individually as Bam HI - Bgl II fragments into pSPLI and pSPL3 exon-trapp g vectors and transfected into COS-7 (A6) cells for splicing of potential exons See D.M. Church et al., Nature Genet. 6:98 (1994) The same cosmids were used for hybridization-selection from uncloned cDNAs synthesized from human cerebellum and spinal cord poly-A+RNA. See J.G Morgan et al.. Nucl. Acids Res. 20:5173 (1992). Finally, seven of the cosmids were subcloned as Sau 3AI, Apo I, and Hae III fragments and about 1500 random single pass sequences were generated. These sequences were analyzed using the GRAILI a and GRAIL2 (E.C. Uberbacher and R.J. Mural, Proc. Natl. Acad. Sci. USA 88: 1 1261 (1991 ). and FEXH (V.V. Solovyev et al., Nucl. Acids Res. 22, 5156 (1994)) programs

These analyses yielded 19, 5, and 17 potential coding sequences, respectively, including two that matched known genes, namely the protein kinase A gamma catalytic subunit gene (obtained by cDNA selection and random sequencing), and a mitochondhal adenylate kinase 3 pseudogene (obtained by random sequencing) One exon, called d26, was identified independently by two approaches Nested primers based on the d26 sequence when used in a rapid amplification of cDNA 5 end (5'-RACE) experiment on a heart cDNA template yielded two independent but overlapping products The 5 -RACE was performed using the Clontech RACE-ready cDNA kit according to the manufacturer's instructions Sequence from these clones matched another amplified exon and an expressed sequence tag (EST) from a human liver+spleen cDNA library (Homosapiens cDNA clone 126314, 5' sequence (GeπBank accession number R06470)) This gene called X25 apparently had alternate transcripts because the sequences at the 3 end of the EST and RACE products were different

The gene structure of X25 (Figure 1 A) was resolved by obtaining introπic sequences flanking the identified exons by inverse PCR and by direct sequencing of cosmids The EST clone contained 4 exons, and the longer RACE product contained one additional 5' exon This exon mapped with the CpG island at position 100 on the genomic map A transcription start site was predicted 388 bp upstream of the exon I donor splice site and a TATA box was found 28 bp further upstream by the TSSG program Five exons ( 1 to 5a where exon 5a corresponds to the 3 end of the EST) were found to be spread over 40 kb They contain an open reading frame (ORF) encoding a 210 ammo-acid protein which was named frataxin (Figure 1 B) An alternative exon (5b), corresponding to d26, was localized at about 40 kb from exon 5a in the teiomeπc direction Exon 5b also has an in-frame stop codon, so that the alternative transcript encodes a shorter, 171 ammo acid protein, whose 1 1 COOH- terminal residues differ from the mam isoform Nucleotide sequences of the X25 exons have been deposited in the GenBank database under the accession numbers U43748 to U43753 The 3' end of the transcript encoding the alternative form was IS investigated by 3' RACE (see Froman and Martin Technique 1 165 (1989)) 2 μg total RNA from Heia cells was used with nested primers in exon 5b₃ and showed that, depending on the alternate usage of the 3' donor splice site in exon 5b either a transcript ending with this exon, or a longer transcript including an additional non-coding exon 6 could be generated This longer 3' RACE product ended with the poly-A tail of a downstream Alu sequence Genomic sequence of exon 6 showed that it contains 3 Alu sequences in tandem, followed by a polyadenylation signal 1050 bp away form the acceptor splice site Exon 6 was mapped 13 kb telomenc to exon 5b (Fig 1A) Splice sites of all 7 exons (1 to 4, 5a 5b and 6) conform to the canonical consensus

Example 2: Expression of the X25 Transcript

Poly A+ Northern blots of different human tissues revealed the highest expression of X25 in heart, intermediate levels in liver, skeletal muscle and pancreas, and minimal expression in other tissues, including whole brain (Figure 2) A 1 3 kb major transcript was identified in agreement with the predicted size of an exon 5a-contaιnιng mRNA Fainter bands of 1 05, 2 0 2 8, and 7 3 kb were also detected Further hybridizations of the northern blot with exon 5a- and 5b-specιfιc probes revealed that the 1 05 and 2 0 kb bands contained exon 5b, while sequences matching exon 5a were found in the 1 8 and 7 3 kb bands in addition to the major 1 3 kb band A northern blot of total RNA from selected parts of the central nervous system (CNS) revealed high expression of the 1 3 kb transcript in the spinal cord, with less expression in cerebellum, and very little in cerebral cortex (not shown) Overall expression of X25 appeared to be highest in the primary sites of degeneration in FRDA both within and outside the CNS IL

To investigate the nature of the larger transcripts, a fetal brain cDNA library was screened with the EST clone (exons 2-5a) Among nine positives four clones were isolated whose sequence extended beyond the limits of the previously identified X25 mRNAs Sequence analysis of these clones indicated that they originated from a related gene differing from X25 at several positions and with stop codons in the sequence corresponding to X25 exon 1 Three of the cDNAs, which are identical in the portion that has been sequenced, extend respectively for 0 5, 1 and

2 kb upstream of exon 1 Their sequence presents numerous divergences from X25 in the part corresponding to exon 1 , mostly CpG d ucleotides changed in TG or CA, then being almost identical in the part corresponding to exons 2 to 4

An additional 1 6 kb cDNA begins with a sequence closely matching exon 5a even in its UTR with only occasional single base changes and short insertions/deletions The X25 related gene was excluded from the critical FRDA region, and at least one mtronless copy exists in the genome, as indicated by Southern blot and PCR analysis Southern blot analysis with an X25 exon 1 -5a cDNA probe revealed a prominent 5 kb Eco Rl band in genomic DNA that did not correspond to any exon and was absent in YAC and cosmid DNA from the critical FRDA region Several additional bands also absent from cloned DNA from the FRDA region appeared when blots were washed at lower stringency (1 X SSC at room temperature) The primers nF2 (5'-

TCCCGCGGCCGGCAGAGTT-3') [SEQ ID NO 14] and E2R (5'- CCAAAGTTCCAGATTTCCTCA-3') [SEQ ID NO 13], which can amplify a 173 bp fragment spanning exons 1 and 2 of the X25 cDNA, generated a PCR product of corresponding size from genomic DNA, but not from cloned DNA from FRDA region, indicating the presence of sequences with high similarity to a processed X25 transcript elsewhere in the genome '7

Example 3: Computer Database Search.

A BLASTN DNA database search with the X25 DNA sequence and a BLASTP search with the translated sequence did not reveal any significant match However a TBLASTN search in which the protein sequence was compared to the six-frame translation of the DNA databases yielded highly significant matches with an ORF contained in a C elegans cosmid (P = 7 6 x 10^'13) and with a S cerevisiae EST (P=2 0 x 10^'10) (Fig 1 B) In both cases the closest match involved a 27-aa segment of the protein (positions 141 -167) encoded in exons 4 and 5a, showing 25/28 and 22/27 amiπ-oacid identity with the C elegans and S cerevisiae sequences respectively, and 65% identity at the DNA level Secondary structure predictions for the X25-encoded protein suggested an σ-helical structure for the NH₂-termιnal 30 ammo acids and the regions between residues 90-1 10 and 185-195, with possible interspersed β-sheet regions around residues 125-145 and 175-180 Secondary structure prediction was performed with the SSP and NNSSP programs which are designed to locate secondary structure elements (V V Solovyev and A A Salamov CABIOS 10 661 (1994)) The TMpred program was used to predict putative transmembrane domains (K Hoffmann and W Stoffel Biol Chem Hoppe-Seyler 374 166 (1993)) PSORT was used to predict possible protein sorting signals (K Nakai and M Kanehisa Proteins Structure, Function, and Genetics 1 1 95 (1991 )) No transmembrane domain was identified As computer analysis of the am o acid sequence suggests that the frataxin protein contains an N-terminal hydrophobic signal, it may be a precursor for a secreted protein with a growth factor or hormone-like action, making frataxin an ideal protein for expression in bacteria, yeast and mammalian cells Example 4: Determining the Nature of the Mutation Leading to FRDA.

All six coding exons of X25 in 184 FRDA patients were amplified with flanking primers and screened for mutations. The following intronic primers were used to amplify the X25 exons: exon 1 (240 bp), F: 5'- AGCACCCAGCGCTGGAGG-3'[SEQ ID N015], R: 5'-

CCGCGGCTGTTCCCGG-3' [SEQ ID NO 16]; exon 2 (168 bp), F: 5'- AGTAACGTACTTCTTAACTTTGGC-3' [SEQ ID NO 17]; R: 5'-

AGAGGAAGATACCTATCACGTG'-3' [SEQ ID NO 18], exon 3 (227 bp), F. 5 - AAAATGGAAGCATTTGGTAATCA-3' [SEQ ID NO 19], R: 5'- AGTGAACTAAAATTCTTAGAGGG-3' [SEQ ID NO 20]; exon 4 (250 bp), F: 5 -AAGCAATGATGACAAAGTGCTAAC-3' [SEQ ID NO 21]; R: 5'- TGGTCCACAATGTCACATTTCGG-3' [SEQ ID NO 22]; exon 5a (223 bp), F 5'- CTGAAGGGCTGTGCTGTGGA-3' [SEQ ID NO 23], R: 5'- TGTCCTTACAAACGGGGCT-3' [SEQ ID NO 24], exon 5b (224 bp), F: 5'-CCCATGCTCAAGACATACTCC-3' [SEQ ID NO 25], R: 5'- ACAGTAAGGAAAAAACAAACAGCC-3' [SEQ ID NO 26], Amplifications for exons 2 3. 4. 5a. and 5b consisted of 30 cycles using the following conditions: 1 mm. at 94°, 2 min. at 55°, 1 m . at 72°. To amplify the highly GC-rich exon 1 , the annealing temperature was raised to 68° and 10% DMSO was added to the reaction. The search for mutations was conducted using single-strand conformation polymorphism (SSCP) analysis (see M Orita et al., Genomics 5:874 (1989)) in 168 FRDA patients, and chemical clevage (see J. A Saleeba et al., Hum. Mutat. 1 :63 (1992)) in 16. Three point mutations that introduce changes in the X25 gene product were identified. Point Mutations The first change in a French family with two affected siblings consisted of a T→ G traπsversion in exon 3 that changed a leucine codon (TTA) into a stop codon (TGA)(L106X) The second case in a Spanish family with one affected member was an A→G transition that disrupted the acceptor splice site at the end of the third intron, changing the invariant AG into a GG Finally, a change from isoleucine to phenylalaniπe (I154F) was found in exon 4 in five patients from three Southern Italian families This conservative change of an hydrophobic ammo acid affects an invariant position within the highly conserved domain shared between human, worm and yeast In all three cases affected individuals were heterozygous for the point mutation The I154F mutation was also found in 1 out of 417 chromosomes from 210 control individuals from the same Southern Italian population, which is compatible with the possibility that this is a disease-causing mutation (Assuming a FRDA carrier frequency in Italy of 1/120 individuals and a frequency of II54F of 1 /40 FRDA chromosomes in Southern Italians, one individual in 3.300 in that population is expected to be a carrier of 1 154F Finding such an individual in a random sample of 210 subjects can occur with >6% probability ) Intron 1 Expansion. Southern blot analysis did not reveal any difference between FRDA patients and normal controls, when DNAs digested with Msp I Taq I, or Bst XI were hybridized with an X25 cDNA probe thereby excluding major rearrangements Hybridization of Eco Rl-digested DNAs from FRDA patients, however revealed that the fragment containing exon 1 was on average 2 5 kb larger than in normal controls, with no detectable normal band FRDA carriers were heterozygous for an enlarged- and a normal-sized fragment The size of the enlarged fragment was clearly variable even among FRDA carriers who were related (Figure 3) The enlarged region was localized further 2o to a 5 2 kb Eco Rl/Not I fragment within the first intron of X25 which was subcloned from a cosmid and sequenced

Oligonucleotide primers were designed to amplify this fragment using a long-range PCR technique and its increased in size in FRDA patients was confirmed The Perkin-Elmer XL long-PCR reagent kit was used to set up the reactions utilizing standard conditions as suggested by the manufacturer and primers 5200Eco (5'- GGGCTGGCAGATTCCTCCAG- 3') [SEQ ID NO 27] and 5200Not (5'-GTAAGTATCCGCGCCGGGAAC- 3') [SEQ ID NO 28] Amplifications were performed in a Perkin-Elmer 9600 machine and consisted of 20 cycles of the following steps 94° for 20 sec 68° for 8 mm followed by further 17 cycles in which the length of the 68° increased by 15 sec /cycle The generated amplification product is 5 kb from normal chromosomes and about 7 5 kb from FRDA chromosomes Cosmid sequence analysis revealed a (GAA)₉ repeat apparently derived from a poly-A expansion of the canonical A₅TACA₆ sequence linking the two halves of an Alu repeat (Figure 4 showing the reverse complementary sequence) The (GAA)₉ repeat is located 1 4 kb downstream from exon 1 and restriction analysis of long-range PCR fragments from FRDA patients located the abnormal size increase within 100 bp from this triplet repeat Digestion of the same fragments with Mbo II, whose recognition site is GAAGA, suppressed size difference between patients and controls, indicating that the GAA repeat may be involved Direct sequencing proved that the mutation consists of an almost pure GAA repeat expansion (Figure 5) PCR primers were then designed to evaluate the presence and size of the GAA expanded repeat FRDA patients, and any variability of the repeat in normal individuals

(Figure 6)

The pr_imers GAA-F (5 -GGGATTGGTTGCCAGTGCTTAAAAGTTAG- 3')[SEQ ID N029] and GAA-R (5'- ATCTAAGGACCATCATGGCCACACTTGCC-3') [SEQ ID NO 30] flank the GAA repeat and generate a PCR product of 457 + 3π bp (n = number of GAA triplets) With these primers, efficient amplification of normal alleles could be obtained by using the traditional PCR procedure with Taq polymerase. after 30 cycles consisting of the following steps. 94° for 45 se , 68° for 30 sec . 72° for 2 mm. Enlarged alleles were much less efficiently amplified, particularly when present together with a normal aliele, therefore, use of these primers is not indicated for FRDA carrier detection A more efficient amplification of expanded alleles. also in FRDA carriers is obtained using the primers Bam (5'- GGAGGGATCCGTCTGGGCAAAGG-3') [SEQ IDNO 31 ] and 2500F (5'- CAATCCAGGACAGTCAGGGCTTT-3') [SEQ ID NO 32]. These primers generated a -1 5 kb ( 1398 bp) normal fragment Amplification was conducted using the long PCR protocol, in 20 cycles composed of the following steps. 94° for 20 sec, 68° for 2 m . and 30 sec , followed by further 17 cycles in which the length of the 68° step was increased by 15 sec/cycle.

Seventy-nine unrelated FRDA patients with typical disease, including five patients known to carry X25 point mutations, were tested for the GAA expansion by Southern analysis and/or by PCR The patients previously known to carry point mutations were all heterozygous for the expansion Segregation analysis within families indicated that the point mutation and the GAA expansion had different parental origin (Figure 7), demonstrating that the point mutations-including the conservative missense mutation H54F-are disease causing. Homozygosity for expanded alleles was demonstrated in 71 of the 74 patients without previously-detected X25 point mutations, and heterozygosity was demonstrated in three.

Overall, according to these data the GAA expansion accounted for about 98% of the FRDA phenotype. The sizes of the enlarged alleles were found to vary between 200 and more than 900 GAA unⁱts, with most alleles containing 700-800 repeats Instability of expanded repeats during parent-offspring transmission was clearly demonstrated, both d_irectly by analysis of parent-offspring pairs and indirectly by the detection of two distinct alleles in affected children of consanguineous parents, who are expected to be homozygous-by-descent at the FRDA locus PCR products corresponding to expanded repeats appeared as slightly blurred bands suggesting the occurrence of only a limited degree of somatic mosaicism for different size repeats due to mitotic instability, at least in lymphocyte DNA (Figure 6B) Seventy-seven normal individuals who were tested by Southern analysis were homozygous for a normal allele PCR analysis of additional 98 normal controls also did not show any expansion, and revealed that the GAA repeat is polymorphic its length varying from 7 to 22 units (Figure 6A) Smaller alleles were more prevalent

GAA repeats, up to 30-40 units are common in many organisms and are sometimes polymorphic as in the 3' UTR of the rat polymeric Ig receptor, however they have not previously been associated with disease A recently proposed theoretical model suggests that ability to form a hairpin structure is crucial for the susceptibility of trinucleotide repeats to give rise to large expansions (See A M Gray et al . Cell 81 533 (1995)) According to this model, CAG/CTG or CGG/CCG repeats are predicted to be expansion prone, while the GAA/CCT repeat has lowest propensity to expand making the FRDA expansion an unexpected finding A striking linkage disequilibrium between FRDA and a polymorphism in a newly-identified exon of the ZO-2 gene (about 120 kb telomenc to the expanded triplet repeat) in French and Spanish families suggests a single origin for the FRDA expansion, but it is also compatible with a multistep or recurrent expansion on an allele at rⁱsk (See Imbert et al Nature Genet 4 72 (1993) where the absolute lⁱnkage disequilibrium in myotonic dystrophy is expanded by recurrent mutations on such a risk allele ) The fact that RDA is autosomal recessive makes the natural history of the mutation at the population level strikingly different from any other known disease due to trinucleotide expansions. In fragile X and myotonic dystrophy, where expansions of comparable size occur in non-coding sequences, carriers have severe early-onset disease and a strong reproductive disadvantage. Large expansions in these diseases are newly formed from unstable alleles of intermediate sizes, resulting in the phenomenon of anticipation. In FRDA, large expanded alleles are transmitted by asymptomatic carriers, and new expansion events in heterozygotes would go undetected at the pheπotypic level. Absence of negative selection against heterozygotes plays the key role in maintaining the frequency of large FRDA expanded alleles as high as 1 per 250 chromosomes, at least one order of magnitude higher than any other characterized trinucleotide expansion. Conversely, deletions of CTG repeats in myotonic dystrophy with reversion to normal size alleles have been observed (see Imbert et al., Nature Genet. 4:72 (1993)) wherein the absolute linkage disequilibrium in myotonic dystrophy is explained by recovered mutations in such a risk allele In the sample of FRDA families in the study of the present invention large expanded alleles were present in all tested symptomatic carriers, and, despite their size instability, neither new expansions deriving from an intermediate allele nor reversions to normality were detected. Although the occasional occurrence of such events cannot be excluded in the general population given the large number of heterozygous individuals, it appears that the frequency is low enough not to introduce detectable distortions in the pattern of FRDA inheritance, particularly inconsistencies in linkage results. Example 5: Quantification of the FRDA Transcript

When the X25 transcript was amplified with primers connecting exons 1 and 2 FRDA patients showed either undetectable or extremely low mRNA levels when compared to carriers and unrelated controls

RT-PCR. RT-PCR was done on lymphoblast RNA from two normal controls, two obligate carriers and six patients, using the exon 2 reverse primer E2R (5'- CCAAAGTTCCAGATTTCCTGA-3-) [SEQ ID NO 13] and the exon 1 forward primer nF (5^"-CAGGCCAGACCCTCAC-3-) [SEQ ID

NO 14] As a precaution to avoid amplification of X25-related sequences not deriving from the FRDA region transcript the nF primer was chosen to have no match with the non-9q13 related gene PCR reactions were carried out for 25 cycles in order to maintain linearity between starting and final concentrations of DNA fragments PCR products were blotted onto nylon membranes and hybridized with the ³²P end-labeled internal oligonucleotide nF2 ('5-

TCCCGCGGCCGGCAGAGTT-3') [SEQ ID NO 15] This observation suggests that either an abnormality in RNA processing, or an interference with the transcription machinery, occur as a consequence of the intronic GAA expansion

Patients with deleterious point mutations affecting X25 clearly demonstrate that no other gene in the region, which could in principle, be affected by a GAA expansion, is involved in the causation of FRDA The restricted expression of X25 in the sites of degeneration or malfunction distinguishes FRDA from the dominant ataxias and from ataxia telangiectasia, where expression of the causative gene is ubiquitous A severely reduced X25 mature mRNA is expected to result in a similarly low level of frataxin Reduced frataxin in spinal cord, heart 2 r and pancreas is likely the primary cause of neuronal degeneration, cardiomyopathy and increased risk of diabetes

RNase Protection In order to synthesize antisense πboprobes, two regions of the X25 cDNA were subcloned in a plasmid vector containing the T7 RNA polymerase promoter. Two separate segments of then X25 cDNA. one containing exons 1 and 2 (partial) and the other containing exons 4 (partial) and 5b were subcloned accordingly. 1 μg of linearized plasmid was used as a template for in vitro transcription (using the Ambion Maxiscπpt kit) in a reaction containing 3 μM - ³ P UTP. The reaction was carried out at 37°C for an hour, after which the DNA template was completely digested by RNase-free DNase treatment Full-length labeled transcripts were then purified following proparative denaturing polyacrylamide gel electrophoresis. A human GAPDH riborprobe (pTRI-GAPDH human Ambion) was also generated as a control.

The RNase protection assay was performed using the RPAII Riboπuclease protection assay kit from Ambion following the manufacturer's recommendations Briefly, 20 μg of total RNA extracted from patient and control lymphoblastoid cell Iines was mixed with 8 x 10⁴ cpm-labeled nboprobe in a 20 μl reaction, denatured and allowed to incubate at 45°C for 16 hours. 2 μg of RNA was used for the control GAPDH reaction For each riboprobe, yeast RNA control hybridizations were performed as well RNase (RNase A/RNase T1 mixture) treatment was carried out for 30 minutes at 37°C. The reaction products were ethanol precipitated and resuspended in formamide loading dye. These products were denatured and electrophoresed on a pre-heated 5% polyacrylamide/8 M urea gel in 1x Tris-borate buffer at 35 watts constant power. The gel was dried and exposed to an X-ray film for 6 days at -70°C using intensifying screens. The sizes of the protected fragments were estimated accurately using a sequence ladder that had been co- electrophoresed with the sample

Example 6: Therapeutics.

FRDA is caused by abnormalities in the X25 gene leading to a deficiency of its protein product frataxin, occasionally due to point mutations that generate a truncated protein but most commonly, to a GAA expansion in the first intron that causes supression of gene expression Therapeutic administration of frataxin to FRDA patients is therefore an aspect of the present invention Large amounts of recombinant frataxin is produced by cloning X25 cDNA ⁱnto an expression vector that is tranformed into a suitable organism Expression vectors that lead to production of high amounts of recombinant protein can be purified by several techniques and prepared for systemic or local administration to patients Computer analysis of the frataxin sequence suggests that frataxin is not a membrane protein, and is likely secreted Both characteristics make frataxin an ideal protein for administration

Another approach is examining the function of the frataxin protein and identifying compounds that can induce a cellular response or modification in the cell metabolism in cells that produce and/or respond to frataxin Such compounds overcome the consequences of the lack of frataxin protein in FRDA

Additionally_, one can inactivate the murine X25 homoiog via homologous recombination to provide an animal model for Fnedreich's ataxia in order to test various therapeutic strategies

Finally, the coding sequence for frataxin is inserted into a suitable expression vector that is administered to FRDA patients The coding sequence of frataxin is inserted in the genome of a modified RNA or DNA v_irus which is administered systemically or locally to patients, or used to transduce cultured cells from patients that are then re-implanted into the patient body Alternatively, non-viral vectors are utilized and administered directly to the patients or to patient's cultured cells that are re-implanted into the patient

One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein The nucleotides proteins peptides, methods, procedures and techniques described herein are presently representative of the preferred embodiments are intended to be exemplary and are not intended as limitations on the scope Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention or defined by the scope of attached claims

SEQUENCES

SEQUENCE ID (SEQ ID NO 1 )

1 GATCGAGAAT AGGGCCTGAG ACTTTGTATT TCTACCAAGT TTCCAGGTGA 51 TGCTGAGGCT GCTGGCCCAG CGACCACATT TGATAATCAT AGCCCTCTGA 101 TAAATCCTAT CAAAATATCC TAATGGCAGA GCAAGGGAAT TCTGGTGATA 151 TCCTCCCCTA CCCATAACCT GACAGCTATT AGGATCTGCC TACTTGAGGC 201 TAAAAGCAAC CAAGAGAGGA ACAGCTACAG TGTACCACAG AGTCCCTCAA

251 CATCTTTGCC CACGCCACGG TGCCCCAGCT TCTTACCAAG TGTGCCTGAT 301 TCCTCTTGAC TACCTCCAAG GAAGTGGAGA AGGACAAGTT CTTGCGAAGC 351 CTTCGTCTTC TCTGATATGC TATTCTATGT CTATTTCTTT GGCCAAAAAG 401 ATGGGGCAAT GATATCAACT TTGCAGGGAG CTGGAGCATT TGCTAGTGAC 451 CTTTCTATGC CAGAACTTGC TAAGCATGCT AGCTAATAAT GATGTAGCAC

501 AGGGTGCGGT GGCTCACGCC TGTAATCTCA GCACTTTGGG CGGCCGAGGC 551 GGGCGGATCA CCTGAGGTCA GGAGTTCGAG ACCAGCCTGG CCAACATGAT 601 GAAACCCCAT CTCTACTAAA AATACAAAAA TTAGCCAGGC GTGGTGGTGG 651 GCACCTGCAA TCCCAGCTAC TCTGGAGGCT GAGACAGAAT CTCTTGAACC 701 CAGGAGGTGG AGATTGCAGT GAGCAGAGAT GGCACCACTG CATACCAGCC

751 TGGGCAACAA AGCAAGACTC TGTCTCAAAT AATAATAATA ATAATAACTA 801 ATGATGCAGC TTTCTCTCTC TGAGTATATA ATGCAGTTCT GATGATGTGA 851 GGAAGGGCCT CACTGTTGGT GTGGCAGAGA CTGACACCAT TGCTTGCAAT 901 GAAAACACTG CCCTTCGGTG CCTATGGGCT CTCCCTTTAT GGTTTCAGGG 951 AGGGCTTCTC AACCTTGGGA GAATTTTGGA CTGGATAGTT CTTTGTTGCA 1001 CAGGTGGGGG GCTGTCCTGC ACATCACAGG ATGTTTCATC CCTGGCCTCT 1051 ACCTACTAGA TGCCAGTAGA ACATACCCAC CCCACAGCTG CCTGTTGTGA 1101 CAATCAAAAG CATCTCCAGA TACTTTGCAG GGGGAAAATG ATTTCTCCAG 1 151 GCCTGGCATA TACATAACAG TATTTAAGCA GCTGCCTAGA ATTAATTAAA 1201 CACAGAAGGA TGTCTCTCAT CCAGAATGCC CTGGACCACC TCTTTGATAG

1251 GCAATCAGAT CCCACCTCCT CCACCCTATT TTTGAAGGCC CTGTGCCAAC 1301 ACCACTTCTT CCATGAATAC TTCCTTGATT CCCCCATCCC TAGCTCTATA 1351 TAAATCTCCC ACTCAACACT CACACCTGTT AGTTTACATT CCTCTTGACA 1401 CTTGTCATTT AGCATCCTAA GTATGTAAAC ATGTCTCTCT TCACGATTCA 1451 CAAAGTGGCT TTGGAAGAAC TTTAGTACCT TCCCATCTTC TCTGCCATGG

1501 AAAGTGTACA CAACTGACAT TTTCTTTTTT TTTAAGACAG TATCTTGCTA 1551 TGATGGCCGG GCTGGAATGC TGTGGCTATT CACAGGCACA ATCATAGCTC 1601 ACTGCAGCCT TGAGCTCCCA GGCTCAAGTG ATCCTCCCGC CTCAGCCTCC 165¹ 'GAGTAGCTG AGATCACAGG CATGCACTAC CACACTCGGC TCACATTTGA 1701 ' ATCCTCTAA AGCATATATA AAATGTGGAG GAAAACTTTC ACAATTTGCA

1751 TCCCTTTGTA ATATGTAACA GAAATAAAAT TCTCTTTTAA AATCTATCAA 1801 CAATAGGCAA GGCACGGTGG CTCACGCCTG TCGTCTCAGC ACTTTGTGAG 1851 GCCCAGGCGG GCAGATCGTT TGAGCCTAGA AGTTCAAGAC CACCCTGGGC 1901 AACATAGCGA AACCCCCTTT CTACAAAAAA TACAAAAACT AGCTGGGTGT 1951 GGTGGTGCAC ACCTGTAGTC CCAGCTACTT GGAAGGCTGA AATGGGAAGA 2001 CTGCTTGAGC CCGGGAGGGG GAAGTTGCAG TAAGCCAGGA CCACACCACT 2051 GCACTCCAGC CTGGGCAACA GAGTGAGACT CTGTCTCAAA CAAACAAATA 2101 AATGAGGCGG GTGGATCACG AGGTCAGTAG ATCGAGACCA TCCTGGCTAA 2151 ACGGTGAAA CCCGTCTCTA CTAAAAAAAA AAAAAAAATA CAAAAAATTA 2201 CCAGGCATG GTGGCGGGCG CCTGTAGTCC CAGTTACTCG GGAGGCTGAG

2251 GCAGGAGAAT GGCGTGAAAC CGGGAGGCAG AGCTTGCAGT GAGCCGAGAT 2301 CGCACCACTG CCCTCCAGCC TGGGCGACAG AGCGAGACTC CGTCTCAATC 2351 AATCAATCAA TCAATAAAAT CTATTAACAA TATTTATTGT GCACTTAACA 2401 GGAACATGCC CTGTCCAAAA AAAACTTTAC AGGGCTTAAC TCATTTTATC 2451 CTTACCACAA TCCTATGAAG TAGGAACTTT TATAAAACGC ATTTTATAAA

2501 CAAGGCACAG AGAGGTTAAT TAACTTGCCC TCTGGTCACA CAGCTAGGAA 2551 GTGGGCAGAG TACAGATTTA CACAAGGCAT CCGTCTCCTG GCCCCACATA 2601 CCCAACTGCT GTAAACCCAT ACCGGCGGCC AAGCAGCCTC AATTTGTGCA 2651 TGCACCCACT TCCCAGCAAG ACAGCAGCTC CCAAGTTCCT CCTGTTTAGA 2701 ATTTTAGAAG CGGCGGGCCA CCAGGCTGCA GTCTCCCTTG GGTCAGGGGT

2751 CCTGGTTGCA CTCCGTGCTT TGCACAAAGC AGGCTCTCCA TTTTTGTTAA 2801 ATGCACGAAT AGTGCTAAGC TGGGAAGTTC TTCCTGAGGT CTAACCTCTA 2851 GCTGCTCCCC CACAGAAGAG TGCCTGCGGC CAGTGGCCAC CAGGGGTCGC 2901 CGCAGCACCC AGCGCTGGAG GGCGGAGCGG GCGGCAGACC CGGAGCAGCA 2951 TGTGGACTCT CGGGCGCCGC GCAGTAGCCG GCCTCCTGGC GTCACCCAGC

3001 CCGGCCCAGG CCCAGACCCT CACCCGGGTC CCGCGGCCGG CAGAGTTGGC 3051 CCCACTCTGC GGCCGCCGTG GCCTGCGCAC CGACATCGAT GCGACCTGCA 3101 CGCCCCGCCG CGCAGTAAGT ATCCGCGCCG GGAACAGCCG CGGGCCGCAC 3151 GCCGCGGGCC GCACGCCGCA CGCCTGCGCA GGGAGGCGCC GCGCACGCCG 3201 GGGTCGCTCC GGGTACGCGC GCTGGACTAG CTCACCCCGC TCCTTCTCAG

3251 GGTGGCCCGG CGGAAGCGGC CTTGCAACTC CCTTCTCTGG TTCTCCCGGT 3301 TGCATTTACA CTGGCTTCTG CTTTCCGAAG GAAAAGGGGA CATTTTGTCC 3351 TGCGGTGCGA CTGCGGGTCA AGGCACGGGC GAAGGCAGGG CAGGCTGGTG 3401 GAGGGGACCG G÷TCCGAGGG GTGTGCGGCT GTCTCCATGC TTGTCACTTC 3451 TCTGCGATAA CT7GTTTCAG TAATATTAAT AGATGGTATC TGCTAGTATA

3501 TACATACACA TAATGTGTGT GTCTGTGTGT ATCTGTATAT AGCGTGTGTG 3551 TTGTGTGTGT GTGTTTGCGC GCACGGGCGC GCGCACACCT AATATTTTCA 3601 AGGCTGGATT TTTTTGAACG AAATGCTTTC CTGGAACGAG GTGAAACTTT 3651 CAGAGCTGCA GAATAGCTAG AGCAGCAGGG GCCCTGGCTT TTGGAAACTG 3701 ACCCGACCTT TATTCCAGAT TCTGCCCCAC TCCGCAGAGC TGTGTGACCT

3751 TGGGGGATTC CCCTAACCTC TCTGAGACGT GGCTTTGTTT TCTGTAGGGA 3801 GAAGATAAAG GTGACGCCCA TTTTGCGGAC CTGGTGTGAG GATTAAATGG 3851 GAATAACATA GATAAAGTCT TCAGAACTTC AAATTAGTTC CCCTTTCTTC 3901 CTTTGGGGGG TACAAAGAAA TATCTGACCC AGTTACGCCA CGGCTTGAAA 3951 GGAGGAAACC CAAAGAATGG CTGTGGGGAT GAGGAAGATT CCTCAAGGGG

4001 AGGACATGGT ATTTAATGAG GGTCTTGAAG ATGCCAAGGA AGTGGTAGAG 4051 GGTGTTTCAC GAGGAGGGAA CCGTCTGGGC AAAGGCCAGG AAGGCGGAAG 4101 GGGATCCCTT CAGAGTGGCT GGTACGCCGC ATGTATTAGG GGAGATGAAA 4151 GAGGCAGGCC ACGTCCAAGC CATATTTGTG TTGCTCTCCG GAGTTTGTAC 4201 TTTAGGCTTA AACTTCCCAC ACGTGTTATT TGGCCCACAT TGTGTTTGAA Jo

4251 GAAACTTTGG GATTGGTTGC CAGTGCTTAA AAGTTAGGAC TTAGAAAATG 4301 GATTTCCTGG CAGGACGCGG TGGCTCATGC CCATAATCTC AGCACTTTGG 4351 GAGGCCTAGG AAGGTGGATC ACCTGAGGTC CGGAGTTCAA GACTAACCTG 4401 GCCAΔCATGG TGAAACCCAG TATCTACTAA AAAATACAAA AAAAAAAAAA 4451 AAAAAGAAGA AGAAGAAGAA GAAGAAGAAG AAAATAAAGA AAAGTTAGCC

4501 GGGCGTGGTG TCGCGCGCCT GTAATCCCAG CTACTCCAGA GGCTGCGGCA 4551 GGAGAATCGC TTGAGCCCGG GAGGCAGAGG TTGCATTAAG CCAAGATCGC 4601 CCAATGCACT CCGGCCTGGG CGACAGAGCA AGACTCCGTC TCAAAAAATA 4651 ATAATAATAA ATAAAAATAA AAAATAAAAT GGATTTCCCA GCATCTCTGG 4701 AAAAATAGGC AAGTGTGGCC ATGATGGTCC TTAGATCTCC TCTAGGAAAG

4751 CAGACATTTA TTACTTGGCT TCTGTGCACT ATCTGAGCTG CCACGTATTG 4801 GGCTTCCACC CCTGCCTGTG TGGACAGCAT GGGTTGTCAG CAGAGTTGTG 4851 TTTTGTTTTG TTTTTTTGAG ACAGAGTTTC CCTCTTGTTG CCCAGGCTGG 4901 AGTGCAGTGG CTCAGTCTCA GCTCACTGCA ACCTCTGCCT CCTGGGTTCA 4951 AGTGATTCTC CTGCCTCAGC CTCCCGAGTA GCTGGGATTA TCGGCTAATT

5001 TTGTATTTTT AGTAGAGACA GATTTCTCCA TGTTGGTCAG GCTGGTCTCG 5051 AACTCCCAAC CTCAGGTGAT CCGCCCACCT CGCCCTCCCA AAGTGCTGGA 5101 ATTACAGGCG TGAGCCACCG CGTCTGGCCA TCAGCAGAGT TTTTAATTTA 5151 GGAGAATGAC AAGAGGTGGT ACAGTTTTTT AGATGGTACC TGGTGGCTGT 5201 TAAGGGCTAT TGACTGACAA ACACACCCAA CTTGGCGCTG CCGCCCAGGA

5251 GGTGGACACT GGGTTTCTGG ATAGATGGTT AGCAACCTCT GTCACCAGCT 5301 GGGCCTCTTT TTTTCTATAC TGAATTAATC ACATTTGTTT AACCTGTCTG 5351 TTCCATAGTT CCCTTGCACA TCTTGGGTAT TTGAGGAGTT GGGTGGGTGG 5401 CAGTGGCAAC TGGGGCCACC ATCCTGTTTA ATTATTTTAA AGCCCTGACT 5451 GTCCTGGATT GACCCTAAGC TCCCCCTGGT CTCCAAAATT CATCAGAAAC

5501 TGAGTTCACT TGAAGGCCTC TTCCCCACCC TTTTCTCCAC CCCTTGCATC 5551 TACTTCTAAA GCAGCTGTTC AACAGAAACA GAATGGGAGC CACACACATA 5601 ATTCTACATT TTCTAGTTAA AAAGAAAAAA AAATCATTTT CAACAATATA 5651 TTTATTCAAC CTAGTACATA CAAAATATTA TCATTCCAAC ATGTAATCAG 5701 TATTTTAAAA ATCAGT ATG AGACCAGGCA CGGTGGCTCC CGACTGTAAT

5751 CCCAGGACTT TGGGAGGCCG AGGCGAGTGG ATCATCTGAG ATCAGGAGTT 5801 CAAGACCAGC CTGGCCAACA TGGTGAAACC CCATCTCTAC TAAACACTAG 5851 CTCAGCATGG TGGTGGGTGC CTGTAGTCCC AGCTACTCGG GAGGCTGAGG 5901 CATGAGAATC ACTTGAGCCC AGGAGGCAGA GGTTGCAGTG AGCCAAGATT 5951 TTGGGGGATT CTGTGACATA CAAAAAAAAT CAGTAATAAG ATATCTTGCA

6001 TACTCTTTTC GTACTCATAT ACTTCCAGCA TATCTCAATT CACAATTTCT 6051 AAGTAAATGC TCTATCTGTA TTTACTTTTA TAAAATTCAC AATTAAAAAT 6101 GAAGGTTCAC ATAGTCAAGT TGTTCCAAAC ACACTTAAAT GTCTCCTAGG 6151 CTGGGTGTGG TTGCTCACAC CTGTAATCCC AGCACTTTGG GAGGCTGAGA 6201 TGGGCGGATC ACCTGAGGTC AGGAGTTTGA GACCAGCCTG GCCAACATGG 6251 TGAAACCCCG TCTCTACTAA AAATACAAAA ATTAGCTGGA TGTGGTGGCA 6301 CTCACATGTA ATCCCAGCTA CTCAGGAGGC TGAGGCAGGA TAATTGCTTG 6351 AACCCGGGAG GTGGTGGAGG TTGCAGTGAG CCGAGATCGC ACCACTGCCT 6401 TCCAACCTGG GCGACAGAGC GAGACTCCGT CTCAAAAAAA AAAAAAAGGC 6451 TCCTAATAAC TTTATTACTT TATTATCACC TCAAATAATT AAAATTAAAT 6501 GAAGTTGAAA ATCCAGGTCC TCAGTCCCAT TAGCCACATT TCTAGTGCTC 6551 AGTAGCCACG GGGGCTGGTG ACCACCACAT GGGACAGCAT ATTTAGTACC 6601 TGATCATTGG TTCTCAGATC TGGCTACTCA GCAGAACCAA GAATCCACAG 6651 AAACGGCTTT TAAAAGCACA GCCCCACAGC CCCCAGCCCC AGCCTTACTA 6701 CCTGGAGGCT GGGAAGGACT CTGATTCCAC GAGGCAGCCT ATGTTTTTTG

6751 ATGGAGGGAT GTGACAGGGG CTGCATCTTT AACGTTTCCT CTTAAATACT 6801 GGAGACAGCT TCGAGGAGGA GATAACTGGA TGTGTCTTAG TCCATTTGAT 6851 GGAGGGATGT GACGGGGCTG CGTCTTTAAC GTTTCCTCTT AAATACCGGA 6901 GACAGCTTCG AGAAGGAGAT AACTGGATGT TTCTTAGTCC ACTTTCTGTT 6951 GCTTGTGACA GAATACCTGA AACTGGGCAA TTTATATGGT AAAAAATTTT

7001 CTTCTTACTG CTCTGGAGGC TGAGAAGTCC AAAGTCAAGT CCCTTCTTGC 7051 TGGTGGGGAC TTTGCAGAGT ATTGAGGCGG CACCGGGCGT CATATGGTAA 7101 GGGGCTGAGT GTGCTACCTC AGGTGTCTTT TTCTTTTCTT ATAAAGCCTA 7151 ACTAGTTTCA CTCCCATGAT AACCCATTAA TCTATGAATG GATTAATCCA 7201 TTATTGAGGG AAGAACCTTC ATGACCCAGT CACCGCTTAA AGGCCCCACC

7251 TCTCAATACT GCCACATCGG GAATTAAGTT TCAACATGAG TTTCGGAGGT 7301 GACAAACATT CAAACCATAG CATGCTGTCT CTTAAATGAC TCAATAAGCT 7351 CCTGTGGCAT CCACTTCTGC ATGCCTTGGG CAGCTTTTAG ACATCTGTCC 7401 ATTTTCCTAG AGGGACAAGA CCACCACCTG TGATCCTATG ACCTTTTGGC 7451 TTTAGGCCTA ACAAGCAGGT TATACCCTCA CTCACTTTCA AATCATTTTT

7501 ATTGTCTTGC AGACAATTTA CACAAGTTTA CACATAGAAA AGGATATGTA 7551 AATATTTATA CGCTGCCGGG CGCGGTGGCT CACGCCTGTA ATCCCAGCAC 7601 TTTGGGAGGC CGAGGCAGGT GGATCACGAG TTCAGGAGAT GGAGACCATC 7651 CTGGCTAATA CGATGAAACC CCATCTCTAC TAAAAATACA AAAAATTAGC 7701 CGGGCGTGGT GACGGGTGCC TGTAGTCCCC ACTACTCGGG ACGCTGAGGC

7751 AGGAGAATGG CGTGAACCCG GGAGGCAGAG CTTGCAGTGA TCCGAGATCG 7801 TGCCACTGCA CTCCAGCCTG GGTGACAGAG CGAGACTGCA TCTCAAAGAA 7851 AAAAATAAAT AAATAAATAA ATATTTATAC TGCTTATAAA CTAATAATAA 7901 ATGCTATGGT CTGCATGTTT GTGTCACCCC ACCATTCATA TGTTAAAACC 7951 TAATCACCAA AGTGATATTA GGAGGTGGGG CCCTTGGGAG GTGATGAGGT

8001 ATGAGGGTGG AGCCCATATG ATTGGGATTA GTGCCCTTCT AAAATAGCCC 8051 AACGGAGCCC AGTGACAAGG CATCATCTAT GAACCAGGAA ACTGGCCCTC 8101 ACCAGACACC AAAGCTGTTG GTGCATTGAT CTTGGATTTC CCACCCTCCA 8151 GGACTCTAAG AAACACATTT CTATTGTTTA TAAGCCACCC AGTGGCTGGT 3201 ATTTTGTTAT AACATCCCAG ACTAAGACAA ATAACAAATA CTTGTATCCC

8251 TGACACCAGG TTAAGAGATA GAATTTGTTT GTTCCTCTGG AGGCCCTTGT 8301 CTTCACCCCA TCACTGCCCT GTCCTCCCTG GAGGAATCTG CCAGCCCGAA 8351 TTC SE^QUENCE ID 2 fSEQ ID NO 2

1 TTTACAGGGC ATAACTCATT TTATCCTTAC CACAATCCTA TGAAGTAGGA 51 ACTTTTATAA AACGCATTTT ATATNCAAGG GCACAGAGAG GNTAATTAAC 101 TTGCCCTCTG GTCACACAGC TAGGAAGTGG GCAGAGTACA GATTTACACT

151 AGGCATCCGT CTCCTGNCCC CACATANCCA GCTGCTGTAA ACCCATACCG 201 GCGGCCAAGC AGCCTCAATT TGTGCATGCA CCCACTTCCC AGCAAGACAG 251 CAGCTCCCAA GTTCCTCCTG TTTAGAATTT TAGAAGCGGC GGGCCACCAG 301 GCTGCAGTCT CCCTTGGGTC AGGGGTCCTG GTTGCACTCC GTGCTTTGCA 351 CAAAGCAGGC TCTCCATTTT TGTTAAATGC ACGAATAGTG CTAAGCTGGG

401 AAGTTCTTCC TGAGGTCTAA CCTCTAGCTG CTCCCCCACA GAAGAGTGCC 451 TGCGGCCAGT GGCCACCAGG GGTCGCCGCA GCACCCAGCG CTGGAGGGCG 501 GAGCGGGCGG CAGACCCGGA GCAGCATGTG GACTCTCGGG CGCCGCGCAG 551 TAGCCGGCCT CCTGGCGTCA CCCAGCCCGG CCCAGGCCCA GACCCTCACC 501 CGGGTCCCGC GGCCGGCAGA GTTGGCCCCA CTCTGCGGCC GCCGTGGCCT

651 GCGCACCGAC ATCGATGCGA CCTGCACGCC CCGCCGCGCA AGTTCGAACC 701 AACGTGGCCT CAACCAGATT TGGAATGTCA AAAAGCAGAG TGTCTATTTG 751 ATGAATTTGA GGAAATCTGG AACTTTGGGC CACCCAGGCT CTCTAGATGA 301 GACCACCTAT GAAAGACTAG CAGAGGAAAC GCTGGACTCT TTAGCAGAGT 851 TTTTTGAAGA CCTTGCAGAC AAGCCATACA CGTTTGAGGA CTATGATGTC

901 TCCTTTGGGA GTGGTGTCTT AACTGTCAAA CTGGGTGGAG ATCTAGGAAC 951 CTATGTGATC AACAACAGAC GCCAAACAAG CAAATCTGGC TATCTTCTCC 1001 ATCCAGTGGA CCTAAGCGTT ATGACTGGAC TGGGAAAAAC TGGGTGTTCT 1051 CCCACGACGG CGTGTCCCTC CATGAGCTGC TGGCCGCAGA GCTCACTAAA 1 101 GCCTTAAAAA CCAAACTGGA CTTGTCTTGG TTGGCCTATT CCGGAAAAGA

1 151 TGCTTGATGC CCAGCCCCGT TTTAAGGACA TTAAAAGCTA TCAGGCCAAG 1201 ACCCCAGCTT CATTATGCAG CTGAGGTGTG TTTTTTGTTG TTGTTGTTGT 1251 TTATTTTTTT TATTCCTGCT TTTGAGGACA CTTGGGCTAT GTGTCACAGC 1301 TCTGTACAAA CAATGTGTTG CCTCCTACCT TGCCCCCAAG TTCTGATTTT ' 351 TAATTTCTAT GGAAGATTTT TTGGATTGTC GGATTTCCTC CCTCACATGA

1401 TACCCCTTAT CTTTTATAAT GTCTTATGCC TATACCTGAA TATAACAACC 1451 TTTAAAAAAG CAAAATAATA AGAAGGAAAA ATTCCAGGAG GGA

SEQUENCE ID NO 3 (SEQ ID NO 3)

1 ATGTGGACTC TCGGGCGCCG CGCAGTAGCC GGCCTCCTGG CGTCACCCAG 51 CCCGGCCCAG GCCCAGACCC TCACCCGGGT CCCGCGGCCG GCAGAGTTGG

151 CCCCACTCTG CGGCCGCCGT GGCCTGCGCA CCGACATCGA TGCGACCTGC 201 ACGCCCCGCC GNGCAAGTTC GAACCAACGT GGCCTCAACC AGATTTGGAA 251 TGTCAAAAAG CAGAGTGTCT ATTTGATGAA TTTGAGGAAA TCTGGAACTT 301 TGGGCCACCC AGGCTCTCTA GATGAGACCA CCTATGAAAG ACTAGCAGAG 351 GAAACGCTGG ACTCTTTAGC AGAGTTTTTT GAAGACCTTG CAGACAAGCC

401 ATACACGTTT GAGGACTATG ATGTCTCCTT TGGGAGTGGT GTCTTAACTG 451 TCAAACTGGG TGGAGATCTA GGAACCTATG TGATCAACAA GCAGACGCCA 501 AACAAGCAAA TCTGGCTATC TTCTCCATCC AGGTTAACGT GGCTCCTGTG 551 GCTGTTCCAT CCCTGAGGAA AAGTGAGGAC CATGCTCTCC AAACAGGCCA 601 TGTGCTGGAC TACCTCTGTT TCTGTCTCCT GGGATTCCAA TCAGCAAGTG

651 AGCAACGAAG CAACCCAGAC AGTGTGGTTC ATAGGATGGC TGG

SEQUENCE ID NO 4 (SEQ ID NO 4)

1 MWTLGRRAVA GLLASPSPAQ AQT TRVPRP AELAPLCGRR GLRTDIDATC 51 TPRRASSNQR GLNQIWNVKK QSVYL NLRK SGTLGHPGSL DETTYERIAE 101 ETLDSLAEFF EDLADKPYTF EDYDVSFGSG VLTVKLGGDL GTYVINKQTP

151 NKQIWLSSPS SGPKRYDWTG KNWVFSHDGV SLHELLAAEL TKALKTKLDL 201 SWLAYSGKDA

SE^QUENCE ID NO 5 (SEQ ID NO 5 ι

1 MWTLGRRAVA GLLASPSPAQ AQTLTRVPRP AELAPLCGRR GLRTDIDATC 51 TPRRASSNQR GLNQIWNVKK QSVYLMNLRK SGTLGHPGSL DETTYERLAE

101 ETLDSLAEFF EDLADKPYTF EDYDVSFGSG VLTVKLGGDL GTYVINKQTP 151 NKQIWLSSPS RLTWLLWLFH P

SEQUENCE ID NO 6 (SEQ ID NO 6)

1 CAAGCCTGGG CGACAGAGCG AGCTCCGTCN CAACCAATNA ACCAATCAAT AAAATCTANN 61 AACAATATTT ATTGTGCACT TAACAGGAAC ATGCCCTGTC CAAAAAAAAC TTTACAGGGC

121 TTAACTCATT TTATCCTTAC CACAATCCTA TGAAGTAGGA ACTTTTATAA AACGCATTTT 181 ATAAACAAGG CACAGAGAGG TTAATTAACT TGCCCTCTGG TCACACAGCT AGGAAGTGGG 241 CAGAGTACAG ATTTACACAA GGCATCCGTC TCCTGGCCCC ACATACCCAA CTGCTGTAAA 301 CCCATACCGG CGGCCAAGCA GCCTCAATTT GTGCATGCAC CCACTTCCCA GCAAGACAGC 361 AGCTCCCAAG TTCCTCCTGT TTAGAATTTT AGAAGCGGCG GGCCACCAGG CTGCAGTCTC

421 CCTTGGGTCA GGGGTCCTGG TTGCACTCCG TGCTTTGCAC AAAGCAGGCT CTCCATTTTT 481 GTTAAATGCA CGAATAGTGC TAAGCTGGGA AGTTCTTCCT GAGGTCTAAC CTCTAGCTGC 541 TCCCCCACAG AAGAGTGCCT GCGGCCAGTG GCCACCAGGG GTCGCCGCAG CACCCAGCGC 601 TGGAGGGCGG AGCGGGCGGC AGACCCGGAG CAGCATGTGG ACTCTCGGGC GCCGCGCAGT 561 AGCCGGCCTC CTGGCGTCAC CCAGCCCGGC CCAGGCCCAG ACCCTCACCC GGGTCCCGCG

721 GCCGGCAGAG TTGGCCCCAC TCTGCGGCCG CCGTGGCCTG CGCACCGACA TCGATGCGAC 781 CTGCACGCCC CGCCGNGCAG TAAGTATCCG CGCCGGGAAC AGCCGCGGGC CGCACGCCGY Θ41 GGGCCGCACG CCGCACGCCT GCGCAGGGAG GCGCCGCGCA CGCCGGGGTC GCTCCGGGTA 901 CGCGCGCTGG ACTAGCTCAC CCCGCTCCTT CTCAGGGTGG CCCGGCGGAA GCGGCCTTGC 961 AACTCCCTTC TCTGGTTCTC CCGGTTGCAT TTACACTGGC TTCTGCTTTC CGAAGGAAAA

1021 GGGGACATTT TGTCCTGCGG TGCGACTGCG GGTCAAGGCA CGGGCGAAGG CAGGGCAGGC 1081 TGGTGGAGGG GACCGGTTCC GAGGGGTGTG CGGCTGTCTC CATGCTTGTC ACTTCTCTGC 1141 GATAACTTGT TTCAGTAATA TTAATAGATG GTATCTGCTA GTATATACAT ACACATAATG 1201 TGTGTGTCTG TGTGTATCTG TATATAGCGT GTGTGTTGTG TGTGTGTGTT TGCGCGCACG 1261 GGCGCGCGCA CACCTAATAT TTTCAAGGCT GGATTTTTTT GAACGAAATG CTTTCCTGGA

1321 ACGAGGTGAA ACTTTCAGAG CTGCAGAATA GCTAGAGCAG CAGGGGCCCT GGCTTTTGGA 1381 AACTGACCCG ACCTTTATTC CAGATTCTGC CCCACTCCGC AGAGCTGTGT GACCTTGGGG 1441 GATTCCCCTA ACCTCTCTGA GACGTGGCTT TGTTTTCTGT AGGGAGAAGA TAAAGGTGAC 1501 GCCCATTTTG CGGACCTGGT GTGAGGATTA AATGGGAATA ACATAGATAA AGTCTTCAGA 1561 ACTTCAAATT AGTTCCCCTT TCTTCCTTTG GGGGGTACAA AGAAATATCT GACCCAGTTA

1621 CGCCACGGCT TGAAAGGAGG AAACCCAAAG AATGGCTGTG GGGATGAGGA AGATTCCTCA 1681 AGGGGAGGAC ATGGTATTTA ATGAGGGTCT TGAAGATGCC AAGGAAGTGG TAGAGGGTGT 1741 TTCACGAGGA GGGATCCGTC TGGGCAAAGG CCAGGAAGGC GGAAGGGGAT CCCTTCCGAG 1801 TGGCTGGTAC GCCGCCTGTA NTATGGGAGA GGATCCCTTC AGAGTGGCTG GTACGCCGCA 1361 TGTATTAGGG GAGATGAAAG AGGCAGGCCA CGTCCAAGCC ATATTTGTGT TGCTCTCCGG

1921 AGTTTGTACT TTAGGCTTAA ACTTCCCACA CGTGTTATTT GGCCCACATT GTGTTTGAAG 1981 AAACTTTGGG ATTGGTTGCC AGTGCTTAAA AGTTAGGACT TAGAAAATGG ATTTCCTGGC 2041 AGGACGCGGT GGCTCATGCC CATAATCTCA GCACTTTGGG AGGCCTAGGA AGGTGGATCA 2101 CCTGAGGTCC GGAGTTCAAG ACTAACCTGG CCAACATGGT GAAACCCAGT ATCTACTAAA 2161 AAATACAAAA AAAAAAAAAA AAAAGAAGAA GAAGAAGAAG AAGAAGAAGA AAATAAAGAA

2221 AAGTTAGCCG GGCGTGGTGT CGCGCGCCTG TAATCCCAGC TACTCCAGAG GCTGCGGCAG 2281 GAGAATCGCT TGAGCCCGGG AGGCAGAGGT TGCATTAAGC CAAGATCGCC CAATGCACTC 2341 CGGCCTGGGC GACAGAGCAA GCTCCGTCTC AAAAAATAAT AATAATAAAT AAAAATAAAA 2401 AATAAAATGG ATTTCCCAGC ATCTCTGGAA AAATAGGCAA GTGTGGCCAT GATGGTCCTT 2461 AGATC 3S-

SEQUENCE ID NO 7 (SEQ ID NO 7)

1 AATTTACTCC GAAACTAGCT TGGGTGAGGG GTACAAAGCA TCCTGCCTTT CTTTAAAAGT 61 GCTGCTTCCC CTTGGAAGTA GAAAGTGGAC ACTTTTATAA GGTAAGGGGG GAAGTGTGCA

121 AGGGCAAGTG GGGGGGTCCC TCTGCTAGTT CCGTGCATAC TCTACAGGAC AGTTGACTTG 181 GCACCTTCCT GGTTAGTAAT AAGCTGTAGC AGTGGCCAAG TGGGCATGCT TTCAGTATGC 241 CCTCCCAGTG AATGAAAGTC CTGAGGCAAC CCCCAAGGGT GGAAGTGCCA GGCCACCACC 301 CACTGGAGGT GAAAGTTCCG TGATGGGTTT GCTTTGGTCT GCGAATCTAC TGTCATGTGG 361 AGAGATCTGT GCTCTGGAAG AGCATACAGT TAGAAAAGCT TGCCCTGAAG GGAATGTATG

421 GTGAAGGGGA GGTGAAAGGT TATATTTGCA TTTCTGAAGG GCTAAGTAGG AAACCGGGAA 481 CCAGGGGAGA GGAGAAGAGA AGAGAGGATA ATTTTTTTTA AGAAAAGCAA CATATTCCCT 541 TTTTCTTAGA AAAAATGGAG CACTCGGTTA CAGGCACTCG AATGTAGAAG TAGCAATATA 601 TAAATTATGC ATTAATGGGT TATAATTCAC TGAAAAATAG TAACGTACTT CTTAACTTTG 661 GCTTTCAGAG TTCGAACCAA CGTGGCCTCA ACCAGATTTG GAATGTCAAA AAGCAGAGTG

721 TCTATTTGAT GAATTTGAGG AAATCTGGAA CTTTGGGCCA CCCAGGGTAA GATAAAGCAN 731 CTTNCACGTG ATAGGTATCT TCCTCTNTCC TTCCCTGCCT CTCCCATTAG AACCTGGTTT 841 TCTTTCTGAG CAGCAACAAT NTTAGGCATC TTTCCATGTG ACTGAGTATC CACCACATTA 901 TTTTTAATGA AATAGTATTA GATTGCATGG ATGTGACATA ATCCATTTAA CNGATCNCCT 961 ACTGTTGGAC ATTCAGGTTG TTTTCAGAGT TTNATATTAT TTTATTTAAT ACCCTAATAG

1021 TTAGAGCAGG CCATGCTTNT NTTACAAATA GGACCCAAAT ATTTAATAGC TCAAACCAAT 1081 AACGGTNTGT GTCCTCCTCT CTGGGCAGTA CAGGGTTGGC ATACCTCTGA AGTGATTAGG 1141 GNCTACACTC ATTCNAGCTT CCAGTTGGCC TTATCTGTCA GTGCCTACT

SEQUENCE ID NO 8 (SEQ ID NO 3)

1 AAAATGGAAG CATTTGGTAA TCATGTTTGG GTTTTGTGCT TCCTCTGCAG CTCTCTAGAT 61 GAGACCACCT ATGAAAGACT AGCAGAGGAA ACGCTGGACT CTTTAGCAGA GTTTTTTGAA

121 GACCTTGCAG ACAAGCCATA CACGTTTGAG GACTATGATG TCTCCTTTGG GGTACCTCTT 181 GACTTCTTTT ATTTTTCTGT TTCCCCCTCT AAGAATTTTA GTTCACT

SE^QUENCE ID NO 9 (SEQ ID NO 9)

1 AAGCAATGAT GACAAAGTGC TAACTTTTTC TTGTTTTAAT TTCTTTATGC TTTTTTTCCA 51 CCTAATCCCC TAGAGTGGTG TCTTAACTGT CAAACTGGGT GGAGATCTAG GAACCTATGT

121 GATCAACAAG CAGACGCCAA ACAAGCAAAT CTGGCTATCT TCTCCATCCA GGTATGTAGG 181 TATGTTCAGA AGTCAACATA TGTAATTCTT AAAGACTTCC GAAATGTGAC ATTGTGGACC 41 A 3t

SEQUENCE ID NO 10 (SEQ ID NO 10⁾

1 TCATCTGAAG GGCTGTGCTG TGGAATTACT ATGCATTTGT TTTGTCTTCC AGTGGACCTA 61 AGCGTTATGA CTGGACTGGG AAAAACTGGG TGTTCTCCCA CGACGGCGTG TCCCTCCATG 121 AGCTGCTGGC CGCAGAGCTC ACTAAAGCCT TAAAAACCAA ACTGGACTTG TCTTGGTTGG 181 CCTATTCCGG AAAAGATGCT TGATGCCCAG CCCCGTTTTA AGGACATTAA AAGCTATCAG 241 GCCAAGACCC CAGCTTCATT ATGCAGCTGA GGTGTGTTTT TTGTTGTTGT TGTTGTTTAT 301 TTTTTTTATT CCTGCTTTTG AGGACACTTG GGCTATGTGT CACAGCTCTG TACAAACAAT 361 GTGTTGCCTC CTACCTTGCC CCCAAGTTCT GATTTTTAAT TTCTATGGAA GATTTTTTGG 421 ATTGTCGGAT TTCCTCCCTC ACATGATACC CCTTATCTTT TATAATGTCT TATGCCTATA 481 CCTGAATATA ACAACCTTTA AAAAAGCAAA ATAATAAGAA GGAAAAATTC CAGGAGGG

SEQUENCE ID NO 1 1 (SEQ ID NO 1 1 )

1 CCTAGGAGGT GTAGCCTGGG AACCATAGGC AAGAATAATT AACTCAGCTC CTCGGTTAGT 61 GCCTCCTCAG TTCGAGATGG AATTTATTTG CAGGCATGGC TCCTTAATAT GCCAAACCCA

121 TGCTCAAGAC ATACTCCTTC TCCTGGAAGG TTAACGTGGC TCCTGTGGCT GTTCCATCCC 181 TGAGGAAAAG TGAGGACCAT GCTCTCCAAA CAGGCCATGT GCTGGACTAC CTCTGTTTCT 241 GTCTCCTGGG ATTCCAATCA GCAAGTGAGC AACGAAGCAA CCCAGACAGT GTGGTTCATA 301 GGATGGCTGG GTAAGTGGCT GTTTGTTTTT TCCTTACTGT GGATATGTAT CAGTGAAGGA 361 ATCTGTAGAA CATTCTTGAT GGGAACATTT AGTCATATCA AGTCAATAAA TTAATGTTTA

421 GGCTGGGAC

SEQUENCE ID NO 12 (SEQ ID NO 12)

1 TTTACAGGGC ATAACTCATT TTATCCTTAC CACAATCCTA TGAAGTAGGA ACTTTTATAA 61 AACGCATTTT ATATNCAAGG GCACAGAGAG GNTAATTAAC TTGCCCTCTG GTCACACAGC 121 TAGGAAGTGG GCAGAGTACA GATTTACACT AGGCATCCGT CTCCTGNCCC CACATANCCA

181 GCTGCTGTAA ACCCATACCG GCGGCCAAGC AGCCTCAATT TGTGCATGCA CCCACTTCCC 241 AGCAAGACAG CAGCTCCCAA GTTCCTCCTG TTTAGAATTT TAGAAGCGGC GGGCCACCAG 301 GCTGCAGTCT CCCTTGGGTC AGGGGTCCTG GTTGCACTCC GTGCTTTGCA CAAAGCAGGC 361 TCTCCATTTT TGTTAAATGC ACGAATAGTG CTAAGCTGGG AAGTTCTTCC TGAGGTCTAA 421 CCTCTAGCTG CTCCCCCACA GAAGAGTGCC TGCGGCCAGT GGCCACCAGG GGTCGCCGCA

481 GCACCCAGCG CTGGAGGGCG GAGCGGGCGG CAGACCCGGA GCAGCATGTG GACTCTCGGG 541 CGCCGCGCAG TAGCCGGCCT CCTGGCGTCA CCCAGCCCGG CCCAGGCCCA GACCCTCACC 601 CGGGTCCCGC GGCCGGCAGA GTTGGCCCCA CTCTGCGGCC GCCGTGGCCT GCGCACCGAC 661 ATCGATGCGA CCTGCACGCC CCGCCGCGCA AGTTCGAACC AACGTGGCCT CAACCAGATT 721 TGGAATGTCA AAAAGCAGAG TGTCTATTTG ATGAATTTGA GGAAATCTGG AACTTTGGGC

781 CACCCAGGCT CTCTAGATGA GACCACCTAT GAAAGACTAG CAGAGGAAAC GCTGGACTCT 841 TTAGCAGAGT TTTTTGAAGA CCTTGCAGAC AAGCCATACA CGTTTGAGGA CTATGATGTC 901 TCCTTTGGGA GTGGTGTCTT AACTGTCAAA CTGGGTGGAG ATCTAGGAAC CTATGTGATC 961 AACAAGCAGA CGCCAAACAA GCAAATCTGG CTATCTTCTC CATCCAGTGG ACCTAAGCGT 1021 TATGACTGGA CTGGGAAAAA CTGGGTGTTC TCCCACGACG GCGTGTCCCT CCATGAGCTG

1081 CTGGCCGCAG AGCTCACTAA AGCCTTAAAA ACCAAACTGG ACTTGTCTTG GTTGGCCTAT 1141 TCCGGAAAAG ATGCTTGATG CCCAGCCCCG TTTTAAGGAC ATTAAAAGCT ATCAGGCCAA 1201 GACCCCAGCT TCATTATGCA GCTGAGGTGT GTTTTTTGTT GTTGTTGTTG TTTATTTTTT 1261 TTATTCCTGC TTTTGAGGAC ACTTGGGCTA TGTGTCACAG CTCTGTACAA ACAATGTGTT 1321 GCCTCCTACC TTGCCCCCAA GTTCTGATTT TTAATTTCTA TGGAAGATTT TTTGGATTGT

1381 CGGATTTCCT CCCTCACATG ATACCCCTTA TCTTTTATAA TGTCTTATGC CTATACCTGA 1 41 ATATAACAAC CTTTAAAAAA GCAAAATAAT AAGAAGGAAA AATTCCAGGA GGGAAAAAAA 1501 AAAAA SEQUENCE ID NO 13 (SEQ ID NO 13)

1 CCAAAGTTCC AGATTTCCTC A

SEQUENCE ID NO 14 (SEQ ID NO 14)

1 TCCCGCGGCC GGCAGAGTT

SEQUENCE ID NO 15 (SEQ ID NO 15)

1 AGCACCCAGC GCTGGAGG

33

SEQUENCE ID NO 16 (SEQ ID NO 16)

1 CCGCGGCTGT TCCCGG

SEQUENCE ID NO 17 (SEQ ID NO 17)

1 AGTAACGTAC TTCTTAACTT TGGC

SEQUENCE ID NO 18 (SEQ ID NO 18)

1 AGAGGAAGAT ACCTATCACG TG

SEQUENCE ID NO 19 (SEQ ID NO 19)

1 AAAATGGAAG CATTTGGTAA TCA

SEQUENCE ID NO 20 (SEQ ID NO 20)

1 AGTGAACTAA AATTCTTAGA GGG

SEQUENCE ID NO 21 (SEQ ID NO 21 )

1 AAGCAATGAT GACAAAGTGC TAAC

SEQUENCE ID NO 22 (SEQ ID NO 22)

1 TGGTCCACAA TGTCACATTT CGG

SEQUENCE ID NO 23 (SEQ ID NO 23)

1 CTGAAGGGCT GTGCTGTGGA o

SEQUENC E ID NO 24 (SEQ ID NO 24)

1 TGTCCTTACA AACGGGGCT

SEQUENCE ID NO 25 (SEQ ID NO 25)

1 CCCATGCTCA AGACATACTC C

SEQUENCE ID NO 26 (SEQ ID NO 26)

1 ACAGTAAGGA AAAAΔCAAAC AGCC

SEQUENCE ID NO 27 (SEQ ID NO. 27)

1 GGGCTGGCAG ATTCCTCCAG

SEQUENCE ID NO 23 (SEQ ID NO. 28).

1 GTAAGTATCC GCGCCGGGAA C

SEQUENCE ID NO 29 (SEQ ID NO. 29)

1 GGGATTGGTT GCCAGTGCTT AAAAGTTAG

SEQUENCE ID NO 30 (SEQ ID NO. 30)

1 GATCTAAGGA CCATCATGGC CACACTTGCC

SEQUENCE ID MO 31 (SEQ ID NO 31 ) 1 GGAGGGATCC GTCTGGGCAA AGG

SEQUENCE ID NO 32 (SEQ ID NO 32)

1 CAATCCAGGA CAGTCAGGGC TTT

SEQUENCE ID NO 33 (SEQ ID NO 33)

1 TCCCGCGGCC GGCAGAGTT

Claims

What is claimed is.

1. A method of screening individuals for a mutation that leads to Friedreich's ataxia. comprising the steps of digesting DNA from an individual to be tested with a restriction endonuciease: and measuring the length of a restriction fragment length polymorphism (RFLP) by hybridization to probes that recognize a region encompassing a GAA repeat in the first intron of an X25 gene and performing Southern Blot analysis, wherein an RFLP having said GAA expansion of more than about 120 is an indication of said mutation that leads to Friedreich's ataxia.

2. The method of claim 1 . wherein the restriction endonuciease is EcoRI.

3. The method of claim 1 , wherein the probe used for performing said Southern Blot is SEQ ID NO 2.

4 The method of claim 1 . wherein the probe used for performing said Southern Blot is an amplification product obtained by performing PCR on said DNA with SEQ ID NO 16 and SEQ ID NO 17.

5. A method of screening individuals for a mutation that leads to Friedreich's ataxia. comprising the steps of measuring expression of an X25 gene by determining an amount of mRNA expressed from said X25 gene and from known controls, and comparing the amount of mRNA from said X25 gene to the amount of mRNA from the known controls. 13

6 The method of claim 5, wherein the mRNA is determined by the steps of extracting mRNA from individuals. to be tested; preparing cDNA from mRNA; amplifying said cDNA to produce amplification products; and comparing relative amounts of X25 and control amplification products present wherein a reduced amount of mRNA from the X25 gene indicates individuals having said mutation that leads to Friedreich's ataxia lo

7. The method of claim 6, wherein the comparing step includes electrophoresis of said amplification products; traπsfering said amplification products to a solid support; hybridizing said amplification products to a probe, and quantifying of X25 amplification products 5 versus control gene amplification products.

8. The method of claim 6, wherein said probe is SEQ ID NO 14

9. The method of claim 5, wherein said control gene is serine o hydroxymethyltraπsferase (SHMT)

10. A method of screening individuals for a mutation that leads to Friedreich's ataxia. comprising the step of detecting a variation in a size of a (GAA)_n repeat in a first intron of a X25 gene by measuring a length of saiα repeat, wherein n for normal individuals ranges from 1 -22 and n for a ected individuals is 120.

1 1. The method of claim 10, wherein said size of said repeat is measured by restriction endonuciease digestion of sample DNA and Southern Blot analysis.

12 The method of claim 10 wherein said size of said repeat is determined by pulsed field gel electrophoresis

13 The method of claim 10 wherein SEQ ID NO 29 and SEQ ID NO 30 are used in said detecting step

14 The method of claim 10 wherein SEQ ID NO 31 and SEQ ID NO 32 are used in said detecting steps

15 A method for detecting a GAA polymorphism in a first intron of an X25 gene comprising the steps of performing a PCR assay to produce amplified products of said first intron of said X25 gene and measuring the length of said amplified products

16 The method of claim 15, wherein SEQ ID NO 29 and SEQ ID NO 30 are used in said PCR assay

17 The method of claim 15 wherein SEQ ID NO 31 and SEQ ID NO 32 are used in said PCR assay

18 A method of screening individuals for a mutation that leads to Fnedreich's ataxia comprising the steps of sequencing DNA from an individual and comparing said sequence from said individual to SEQ ID NOS 1 -12 to determine what differences there are between said sequence from said individual and SEQ ID NOS 1 -12

19 A method of treating Fnedreich's ataxia, comprising the step of administering a pharmacologic dose of a protein having an ammo acid sequence substantially similar to SEQ ID NO 4 to an individual 4 r

20 A method of treating Friedreich's ataxia comprising administration to an individual of a nucleic acid vector containing an X25 gene capable of expression

21 As a composition of matter, the molecule having SEQ ID NO 1

22 As a composition of matter the molecule having SEQ ID NO 2

23 As a composition of matter, the molecule having SEQ ID NO 3

24 As a composition of matter the molecule having SEQ ID NO 4

25 As a composition of matter the molecule having SEQ ID NO 5

26 As a composition of matter the molecule having SEQ ID NO 6

27 As a composition of matter, the molecule having SEQ ID NO 7

28 As a composition of matter the molecule having SEQ ID NO 8

29 As a composition of matter, the molecule having SEQ ID NO 9

30 As a composition of matter the molecule having SEQ ID NO 10

31 As a composition of matter the molecule having SEQ ID NO 11

32 As a composition of matter, the molecule having SEQ ID NO 12

33 As a composition of matter, the molecule having SEQ ID NO 13

34. As a composition of matter, the molecule having SEQ ID NO 14.

35. As a composition of matter, the molecule having SEQ ID NO 15.

36. As a composition of matter, the molecule having SEQ ID NO 16.

37 As a composition of matter, the molecule having SEQ ID NO 17.

38. As a composition of matter, the molecule having SEQ ID NO 18.

39. As a composition of matter the molecule having SEQ ID NO 19.

40. As a composition of matter, the molecule having SEQ ID NO 20.

41. As a composition of matter, the molecule having SEQ ID NO 21.

42. As a composition of matter, the molecule having SEQ ID NO 22.

43. As a composition of matter, the molecule having SEQ ID NO 23.

44. As a composition of matter, the molecule having SEQ ID NO 24.

45. As a composition of matter, the molecule having SEQ ID NO 25.

46. Λs a composition of matter, the molecule having SEQ ID NO 26.

47. As a composition of matter, the molecule having SEQ ID NO 27.

48. As a composition of matter, the molecule having SEQ ID NO 28.

49 As a composition of matter, the molecule having SEQ ID NO 29

50 As a composition of matter the molecule having SEQ ID NO 30.

51 As a composition of matter, the molecule having SEQ ID NO 31.

52. As a composition of matter, the molecule having SEQ ID NO 32.