WO1997005234A2 - Gene for friedreich's ataxia - Google Patents

Abstract

Idenfification of a gene, STM7, encoding for a novel phosphatidylinositol isoform responsible for Friedreich's Ataxia is claimed as well as splice variants resulting from recombination events. Of particular importance is the area of STM7 gene spanning exons 1 to 17, inclusive. The nucleic and amino acid sequences, cDNA sequences, antibodies and DNA probes complementary to the nucleic and amino acid sequences are also claimed as part of the present invention. Transgenic animals and diagnostic protocols are also given.

Description

GENE FOR FRIEDREICH'S ATAXIA

This invention is based on the identification of a gene encoding Friedreich's Ataxia and relates to the application of that discovery for diagnostic, therapeutic and modelling purposes.

Friedreich's Ataxia (FRDA) is a progressive neurodegenerative disorder affecting the sensory afferents of both the central and peripheral nervous system. The disease, autosomal recessively inherited with an incidence of approximately 1 in 50,000, is the most common of the hereditary ataxias. Onset commonly occurs between the ages of 8 and 15 years, presenting as ataxia of gait accompanied by dysarthria, areflexia, extensor plantar responses and distal loss of joint position and vibration sense. Sensorineural deafness and optic atrophy occur in approximately 10% and 25% of cases respectively. Electrocardiographic abnormality is commonly detected leading to concentric ventricular or asymmetric septal hypertrophy, which is the most common cause of premature death. Diabetes mellitus or impaired glucose tolerance is detected in more than 30% of patients.

Pathologically, the disease is characterised by degeneration of the posterior columns and spinocerebellar tracts within the spinal cord, extensive loss of the larger cell bodies in the dorsal root ganglia and loss of the large myelinated axons in the peripheral nerves and marked degeneration of the pyramidal tracts, particularly in the lumbar region - consistent with a 'dying-back' process.

In 1988, we generally assigned the FRDA locus to chromosome 9ql3-21.1 by genetic linkage analysis and fluorescent in situ hybridisation studies (Chamberlain et al. , Nature 334248-249 (1988); Shaw etal. , Cytogenet. Cell Genet. 53221-224 (1990)). Following the demonstration of locus homogeneity (Chamberlain et al. , Am. J. Hum. Genet. 44 518-521 (1989); Richter et al. , Cytogenet. Cell Genet. 51 1066 (1989); Pandolfo et al , Am. J. Hum. Genet. 47 228-235 (1990)), the considerable phenotypic variation seen within the disorder was postulated to reflect the existence of a number of mutations within a single gene. Linkage analysis supported a location for FRDA within 1Mb of the two original linked loci, D9S15 and D9S5 (Chamberlain et al. , Nature 334248-249 (1988); Fujita et al. , Genomics

4 110-111 (1989)), with the most probable order being: qter - D9S15 - D9S5 - FRDA - cen (Chamberlain et al , Am. J. Hum. Genet. 52 99-109 (1993)). Subsequent strategies by those in the art to clone this gene therefore focused solely on the cloning of d e 1Mb region immediately proximal to D9S5 and to identifying new highly polymorphic microsatellite markers for the detection of rare recombination events which position the gene more precisely (Duclos et al. , Hum. Mol. Genet. 6 909-914 (1994); Rodius et al. , Am. J. Hum. Genet. 54 1050-1059 (1994)). These markers have also been investigated for linkage disequilibrium in a number of geographically distinct patient populations.

Strategies to clone the FRDA gene proved problematic based on its proximity to the variable heterochromatic region of chromosome 9 (Shaw et al. , Cytogenet. Cell Genet. 53 221-224 (1990)), hampering efforts to generate the centromeric boundary of the FRDA region. The paucity of recombination events to refine the location of the gene complicated matters further. However, it is the unexpected nature of the mutation itself which has provided the final sting in the tail and contributed most to the extended time lapse between mapping this gene in 1988 and its eventual isolation.

We disclose here the isolation of an 860kb interval immediately proximal to the

D9S5 locus as well as the construction of a 230kb cosmid contig widiin this interval. In this interval, we have for the first time isolated and cloned die gene associated widi Friedreich's Ataxia, is hereby designated generally as STM7. The contig traverses die majority of me putative CpG-islands detected in previous physical mapping studies (Wilkes et al , Genomics 9 90-95 (1991)). During our investigations, we detected strong allelic association with markers proximal to D9S202 and in particular, with the locus D9S888. Exon-trapping carried out in cosmids comprising the 230kb contig initiated from D9S888, led to our original identification of STM7. Investigation of the genomic organisation of the reported

2780bp sequence (STM7.I), including a 1620bp open reading frame, revealed that the sequence initially comprised 16 exons, with me putative in-frame initiation and stop codons located in exons 4 and 16 respectively.

The candidacy of the STM7 gene was strengd ened considerably following database comparation of the STM7.I sequence. The deduced amino acid sequence of STM7.I demonstrated homology with two S. cerevisiae gene products, Fablp and die multicopy suppressor of me STT4 mutation, Mss4p, with inferred roles in the phosphoinositide cycle and possibly in the localisation of the phospholipid metabolic enzymes or metabolites. The potential for the human homologue participating in neuronal protein trafficking offered an attractive explanation for the perturbation of these processes by mutation within STM7. Northern analysis using probes from the cloned sequence demonstrated a complex expression pattern, with multiple transcripts ranging from 1.3- > 6.5kb detected in a number of tissues, and evidence of alternative splicing and developmental regulation. Two major transcripts, approximately 3.9 and 1.9kb in size were detected in most tissues studied, with me longest transcripts observed in skeletal muscle (6.3kb) and placenta ( > 6.5kb).

STM7.I shows remarkable homology with the S. cerevisiae FAB1 (P34756), MSS4

(D 13716) genes as well as a high degree of overall homology with a newly isolated human placental phosphatidylinositol-4-phosphate 5-kinase type II (PtdInsP5K-II) cDNA ((U14957) (showing 29% identity and 50% similarity at the amino acid level)). Multiple sequence alignment generated using the PILEUP program (Genetics Computer Group, University of Wisconsin) of the predicted amino acid sequence for STM7.I (X92493) wim me deduced amino acid sequences for MSS4, PtdInsP5K-II and FAB1 is shown in Figure 5, with particular emphasis on residues common to all four sequences. The highest level of overall homology is seen between STM7.I and Mss4p. The level of conservation observed between Fablp,

Mss4p, STM7.I and me 300-amino acid region constituting me putative kinase domain within PtdInsP5K-II, seemingly infers that STM7.I is a novel member of the PtdInsP5K family. The clustering of highly conserved residues present in all four sequences possibly indicates critical elements of the catalytic core. The putative phosphate-binding loop is also highly conserved in STM7.I (residues

120-129). No significant similarity between FAB1 and MSS4 can be detected outside of tiiis catalytic domain, with the unique nature of the remaining sequences for each postulated to confer specificity of function. A schematic representation of the alignment of the four proteins is shown in Figure 13. In contrast to the location of the kinase domain at the C-terminal ends of the Fablp and Mss4p gene products, the catalytic domain present in STM7.I at least, is located towards the 5 '-end of the transcript.

Molecular investigations linking the STM7 gene wim the predominant mutation observed in individuals wim Friedreich's ataxia, provides major insight into the biology of this genetic defect. PtdInsP5K regulates the synthesis of phosphatidylinositol-4, 5-biphosphate (PtdIns(4,5)P2), a critical component of several intracellular signalling pathways modulating diverse functions including the regulation of secretion, cell proliferation, differentiation, motility and vesicle-mediated transport. PtdInsP5K exists as two isoforms, types I and II, distinguished by lack of immunocross-reactivity (Jenkins et al. , J. Biol. Chem. 269: 11547-11554 (1994)). Based on the diversity of the proposed sites for PtdIns(4,5)P2 metabolism, the existence of multiple isoforms for both types I and II has been predicted and confirmed by immunological investigation (Jenkins et al. , J. Biol Chem. 269: 11547-54 (1994) and Bazenet et al , J. Biol. Chem. 265: 18012-22 (1990)).

The high expression of the > 6.5kb placental transcript implies the general importance of STM7 in cellular processes. Based on the high degree of overall homology with Mss4p in particular, a defect in vesicular trafficking would quite possibly be consistent wim the degeneration of the axon from the periphery towards the central nervous system. Each neuronal type has a requirement for characteristic membrane proteins for the packaging and processing of important metabolites such as neurotransmitters, determining specificity for receptors or carrier molecules in the vesicular membrane. Reconciliation of the ubiquitous expression of STM7 with the specificity of the pathology may be resolved dierefore, by the extent of the alternative splicing in the gene. The absence of particular exons could effect major consequences, including me loss of modification sites, creating microheterogeneity and thereby affecting intermolecular recognition; the loss of binding sites for regulatory sequences, including Rho; and modification of ligand binding affinity.

The recent cloning and characterisation of the gene causing ataxia telangiectasia (ATM) provides clues to an alternative function of the protein product of die

Friedreich's Ataxia gene (Savitsky et al , Science 268: 1749-1753 (1995)). The ATM gene, witii C-terminal similarity to the catalytic domains of the PtdlnsP 3-kinases, is involved in die maintenance of genome stability with potential roles in the surveillance of DNA damage and d e regulation of cell cycle progression. Previous investigation of these properties in cell lines cultured from FRDA individuals has demonstrated that the effect of mutation in the Friedreich's Ataxia gene generates a similar, but less severe, cellular phenotype. In a first embodiment, die present invention provides an isolated and purified DNA sequence encoding for Friedreich's Ataxia. In particular, this would encode for exons 1-17 of the gene designated STM7 and as is seen in Figures 7 and 22. Further, the DNA sequences encoding for specific split variants, occurring due to recombination events and as described in Figure 10 and Figure 16 are also claimed herein.

The invention specifically incorporates the nucleotide sequences of STM7.I which includes exon 1-16, inclusive. STM7.IIIa which includes exons 1-13, 17, 19-22; STM7.IIIb which includes exons 1-15, 17, 19, 21-22; and STM7.IIIc which includes exons 1-15, 19-22.

According to the present invention, there is also provided a polypeptide encoded by the STM7 gene having activity comparable to that of phosphatidylinositol-4- phosphate 5-kinase (PtdInsP5K), which:

a) comprises a polypeptide protein having d e amino acid sequences shown in Figures 8 and 16; b) has one or more amino acid substitutions, deletions or insertions relative to PtkInsP5K; or c) is a fragment of a polypeptide as defined in a) or b) above, which is at least ten amino acids long.

The term "polypeptide" is used herein in a broad sense to indicate mat a particular substance has more than one peptide bond. It therefore includes within its scope substances which may sometimes be referred to in the literature as peptides, polypeptides or proteins. The skilled person is able to determine whether or not a particular polypeptide has the activity of PtdInsP5K by using techniques known in the art. For example, by characterisation ofthe molecular weight by SDS polyacrylamide gel electrophoresis at about 90 kDa and also by enzymatic analysis. The existence of PtdlnsP kinase activity can be readily assayed and the absence of activity using Ptdlns as a substrate acts as a control. The identity of the enzymatic product of PtdlnsP kinase which is Ptdlns 4,5-biphosphate (PtdInsP₂) can be confirmed by deacylation and HPLC.

Polypeptides of the present invention may be produced by techniques known to those skilled in the art. For example gene cloning techniques may be used to provide a nucleic acid sequence encoding such a polypeptide. By using an appropriate expression system (a eukaryotic, prokaryotic or cell free system) the polypeptide can then be produced. It can then be purified using standard purification techniques.

Alternatively, chemical synthesis techniques may be used to produce polypeptides of the present invention. Such techniques generally utilise solid phase synthesis. Chemical synthesis techniques which allow polypeptides having particular sequences to be produced have now been automated. Machines capable of chemically synthesising polypeptides are available, for example, from Applied Biosystems.

Preferred methods of producing polypeptides of d e present invention are discussed in greater detail later on.

A polypeptide of die present invention may be provided in substantially pure form. Thus it may be provided in a composition in which it is the predominant polypeptide component present (i.e. where it is present at a level of more man 50% of the total polypeptide present in the composition; preferably at a level of more than 75%, of more than 90%, or of more than 95% of the total polypeptide present in the composition; when determined on a weight/ weight basis).

In order to more fully appreciate the present invention, polypeptides widiin the scope of a), b) or c) above will now be discussed in greater detail.

Polypeptides within the scope of a)

A polypeptide widiin the scope of a) may consist of the particular amino acid sequence shown in Figure 8, or may have an additional N-terminal and/or an additional C-terminal amino acid sequence. Additional peptide sequences may also occur within the particular amino acid sequence shown in Figure 8.

Additional N-terminal or C-terminal sequences may be provided for various reasons. Techniques for providing such additional sequences are well known in the art. These include using gene cloning techniques to ligate nucleic acid molecules encoding polypeptides or parts thereof, followed by expressing a polypeptide encoded by the nucleic acid molecule produced by ligation.

Additional sequences may be provided in order to alter die characteristics of a particular polypeptide. This can be useful in improving expression or regulation of expression in particular expression systems. For example, an additional sequence may provide some protection against proteolytic cleavage. This has been done for the hormone Somatostatin by fusing it at its N-terminus to part of the β galactosidase enzyme (Itakwa et al, Science 198: 105-63 (1977)).

Additional sequences can also be useful in altering die properties of a polypeptide to aid in identification or purification. For example, a signal sequence may be present to direct the translocation of the polypeptide to a particular location within a cell or two export die polypeptide from the cell. Different signal sequences can be used for different expression systems. Another example of the provision of an additional sequence is where a polypeptide is linked to a moiety capable of being isolated by affinity chromatography. The moiety may be an antigen or an epitope and the affinity column may comprise immobilised antibodies or immobilised antibody fragments which bind to said antigen or epitope (desirably with a high degree of specificity). The fusion protein can usually be eluted from the column by addition of an appropriate buffer.

Additional N-terminal or C-terminal sequences may, however, be present simply as a result of a particular technique used to obtain a substance of die present invention and need not provide any particular advantageous characteristic.

Polypeptides within the scope of b) Turning now to the polypeptides defmed in b) above, it will be appreciated by the person skilled in the art that these are variants of the polypeptides given in a) above.

The skilled person will appreciate that various changes can sometimes be made to the amino acid sequence of a polypeptide which has a particular activity to produce variants (often known as "muteins") which still have said activity. Such variants of the polypeptides described in a) above are widiin the scope of the present invention and are discussed in greater detail below in sections (i) to (iii). They include allelic and non-allelic variants.

(i) Substitutions

An example of a variant of the present invention is a polypeptide as defined in a) above, apart from the substitution of one or more amino acids witii one or more other amino acids. The skilled person is aware tiiat various amino acids have similar properties. One or more such amino acids of a polypeptide can often be substituted by one or more other such amino acids widiout eliminating a desired activity of that polypeptide.

For example, the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred tiiat glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic).

Otiier amino acids which can often be substimted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains).

Substimtions of tiiis nature are often referred to as "conservative" or "semi- conservative" amino acid substimtions.

(ii) Deletions

Amino acid deletions can be advantageous since die overall length and the molecular weight of a polypeptide can be reduced whilst still retaining activity. This can enable the amount of polypeptide required for a particular purpose to be reduced. For example if the polypeptide is to be used in medicine, dosage levels can be reduced. (iii) Insertions

Amino acid insertions relative to a polypeptide as defined in a) above can also be made. This may be done to alter die properties of the polypeptide (e.g. to assist in identification, purification or expression, as explained above in relation to fusion proteins).

Polypeptides incorporating amino acid changes (whether substimtions, deletions or insertions) relative to the sequence of a polypeptide as defined in a) above can be provided using any suitable techniques. For example, a nucleic acid sequence incorporating a desired sequence change can be provided by site directed mutagenesis. This can then be used to allow die expression of a polypeptide having a corresponding change in its amino acid sequence.

Whatever amino acid changes may be made, preferred polypeptides of the present invention have at least 50% sequence identity with the polypeptide as shown in

Figure 1, more preferably the degree of sequence identity is at least 75%. Sequence identities of at least 90% or of at least 95% are most preferred.

The degree of amino acid sequence identity can be calculated, for example, using a program such as "bestfit" (Smith and Waterman, Advances in Applied

Mathematics, 482-489 (1981)) to find the best segment of similarity between any two sequences. The alignment is based on maximising the score achieved using a matrix of amino acid similarities, such as that described by Schwarz and Day hof (1979) Atlas of Protein Sequence and Strucmre, Dayhof, M.O., Ed pp 353-358.

Where high degrees of sequence identity are present there may be relatively few differences in amino acid sequence. Thus for example they may be less than 20,less than 10, or even less than 5 differences. Polypeptides within the scope of c)

As discussed supra, it is often advantageous to reduce the length of a polypeptide. Feamre c) of the present invention therefore covers fragments of the polypeptides a) or b) above which are at least 10 amino acids long.

Desirably these fragments are at least 20, at least 50 or at least 100 amino acids long.

Preferred Polypeptides of the Present Invention Prefeπed polypeptides within the scope of the present invention include the amino acid sequences for exons 1-16, inclusive and most preferably exons 7-11, inclusive.

In a second embodiment a purified DNA molecule comprising the cDNA associated witii FRDA is also claimed. In particular, the cDNA associated with exons 7-11 is of particular value as well as the cDNA sequences of Figures 18 to 21.

After further investigation, an additional sequence, designated X25, was also found by the inventors to constitute part of the STM7 gene for Friedreich's Ataxia.

These smdies which utilised RT-PCR and Northern analysis are discussed fully below (Example 2). The demonstration that exons comprising X25 are part of the STM7 gene and tiiat a newly identified exon 17 (as seen in Figure 9) which was derived from the 3 'UTR of PRKACG and is transcribed in die opposite direction to the testis-specific transcript of STM7 gene, was yet another unexpected twist in die characterisation of the Friedreich's Ataxia gene. Inteφretation of the combined physical mapping data indicates that the STM7 gene spans a genomic interval of approximately 450kb, which includes 95% of die original 300kb FRDA critical region. The ancestral recombination event which defines the centromeric boundary of the critical region, thus excluding STM7.I, must therefore be intragenic. Coding exons which have been identified range from 49-300bp in size, whilst intron size varies from 0.118-75kb, indicating die highly dispersed organisation of the exonic sequences within this genomic region. It is clear from our research that segments of six splice variants have been characterised. The presence of exon 16 in the STM7.I transcript, with the first three nucleotides constituting the first in-frame stop codon (TAA), highlights two important points - the existence of at least three alternative polyadenylation sites within STM7, and more importantly, the ability of the gene to generate transcripts which terminate upstream of a GAA triplet repeat motif located in intron 18 (originally codon 1 of

X25, see Figure 9).

A further embodiment of the present invention features substantially pure antibodies selected against segments of the STM7 gene.

By "substantially pure antibody" is meant antibody which is at least 60%, by weight, free from the proteins and naturally occurring organic molecules with which it is naturally selected. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, an STM7 antibody. A substantially pure antibody of tiiis type maybe obtained, for example, by affinity chromatography using recombinantly produced STM7 polypeptides and standard techniques. Specifically, computer prediction of potential beta mrns in the STM7-produced polypeptide, as these beta mrns are often reasonable predictors of β-cell determinants as well as hydrophilicity is considered. Antibodies generated from conserved regions of the kinase domain of the PtdInsP5K gene are prefeπed and contemplated under this invention and particularly antibodies raised against the following peptide sequences: AAPGKQNEEKTYKKTA; and LKEKEEETPQNVPDAK

Other prefeπed antibodies are generated from conserved regions of the X25 sequence which have the following peptide sequences:

CNKQTPNKQIWLSSPS; and CNLRKSGTLGHPGSLD

The antibodies described above may be monoclonal or polyclonal.

Polyclonal antibodies can be raised by stimulating their production in a suitable animal host (e.g. a mouse, rat ,guinea pig, rabbit, sheep, goat or monkey) when the substance of the present invention is injected into the animal. If necessary an adjuvant may be administered together with the substance of the present invention.

The antibodies can then be purified by virtue of their binding to a substance of the present invention.

Monoclonal antibodies can be produced from hybridomas. These can be formed by fusing myeloma cells and spleen cells which produce the desired antibody in order to form an immortal cell line. Thus the well known Kohler & Milstein technique (Nature 25652-55 (1975)) or variations upon this technique can be used.

Techniques for producing monoclonal and polyclonal antibodies which bind to a particular polypeptide are now well developed in d e art. They are discussed in standard immunology textbooks, for example in Roitt et al. , Immunology second edition (1989), Churchill Livingstone, London. In addition to whole antibodies, the present invention includes derivatives thereof which are capable of binding to polypeptides of the present invention.

Thus the present invention includes antibody fragments and synthetic constructs. Examples of antibody fragments and synthetic constructs are given by Dougall et al. in Tibtech 12 372-379 (September 1994).

Antibody fragments include, for example, Fab, F(ab')₂ and Fv fragments (these are discussed in Roitt et al. [supra], for example).

Fv fragments can be modified to produce a synthetic construct known as a single chain Fv (scFv) molecule. This includes a peptide linker covalently joining V_h and V_j regions which contribute to the stability of the molecule.

Other synthetic constructs which can be used include CDR peptides. These are synthetic peptides comprising antigen binding determinants. Peptide mimetics may also be used. These molecules are usually conformationally restricted organic rings which mimic the strucmre of a CDR loop and which include antigen- interactive side chains.

Synthetic constructs include chimaeric molecules. Thus, for example, humamsed (or primatised) antibodies or derivatives tiiereof are within the scope of the present invention. An example of a humanised antibody is an antibody having human framework regions, but rodent hypervariable regions.

Synthetic constructs also include molecules comprising an additional moiety which provides the molecule with some desirable property in addition to antigen binding. For example die moiety may be a label (e.g. a fluorescent or radioactive label). Alternatively, it may be a pharmaceutically active agent. The antibodies or derivatives thereof of the present invention have a wide variety of uses. They can be used in purification and/or identification of polypeptides of the present invention. Thus they may be used in diagnosis.

They can be provided in the form of a kit for screening for the polypeptides of the present invention.

Nucleic Acid Molecules of the Present Invention and Uses Thereof

In addition to tiie polypeptides of the present invention and the uses thereof discussed above, the present invention also provides nucleic acid molecules.

Such nucleic acid molecules: a) code for a polypeptide according to die present invention; or b) are complementary to molecules as defmed in a) above; or c) hybridise to molecules as defined in a) or b) above.

These nucleic acid molecules and their uses are now discussed in greater detail below:

The polypeptides of the present invention can be coded for by a large variety of nucleic acid molecules, taking into account the well known degeneracy of the genetic code. All of these molecules are within the scope of the present invention.

They can be inserted into vectors and can be cloned to provide large amounts of

DNA or RNA for further smdy. Suitable vectors may be introduced into host cells to enable the expression of polypeptides of the present inventions using techniques known to the person skilled in the art. Alternatively, cell free expression systems may be used. Techmques for cloning, expressing and purifying polypeptides are well known to the skilled person. Various techniques are disclosed in standard text books such as in Sambrook et al. [Molecular Cloning 2nd Edition, Cold Spring Harbor Laboratory Press (1989)]; in Old & Primrose [Principles of Gene Manipulation 5th Edition, Blackwell Scientific Publications (1994); and in Stryer [Biochemistry 4th

Edition, W H Freeman and Company (1995)].

By using appropriate expression systems different forms of polypeptides of the present invention may be expressed.

For example, the polypeptide may be provided in glycosylated or non-glycosylated form. Non-glycosylated forms can be produced by expression in prokaryotic hosts, such as E. coli, whereas glycosylated forms can be produced in eukaryotic hosts, such as S. cerevisiae.

Polypeptides comprising N-terminal methionine may be produced using certain expression systems, whilst in others the mature polypeptide will lack this residue.

Polypeptides may initially be expressed to include signal sequences. Different signal sequences may be provided for different expression systems. Alternatively signal sequences may be absent (e.g. where it is not desired to secrete a polypeptide from a host).

Any suitable expression system may be used as described above. Prefeπed vectors include, but are not limited to, pGEX 5X-1 and prefeπed hosts include, but are not limited to E. coli DH5.

In addition to nucleic acid molecules coding for substances according to the present invention (refeπed to herein as "coding" nucleic acid molecules), the present invention also includes nucleic acid molecules complementary thereto. Thus, for example, both strands of a double stranded nucleic acid molecule are included within the scope of the present invention (whether or not they are associated with one another). Also included are mRNA molecules and complementary DNA molecules (e.g. cDNA molecules). Prefeπed cDNA sequences of the invention are those in Figures 18 to 21.

Nucleic acid molecules which can hybridise to any of die nucleic acid molecules discussed above are also covered by die present invention. Such nucleic acid molecules are refeπed to herein as "hybridising" nucleic acid molecules.

Hybridising nucleic acid molecules can be useful as probes or primers, for example.

Desirably such hybridising molecules are at least 10 nucleotides in lengtii and preferably are at least 20 or at least 50 nucleotides in length. The hybridising nucleic acid molecules may specifically hybridise to nucleic acids widiin the scope of a) or b) above.

Prefeπed hybridising molecules hybridise to such molecules under stringent hybridisation conditions. One example of stringent hybridisation conditions is where attempted hybridisation is carried out at a temperamre of from about 35 °C to about 65°C using a salt solution which is about 0.9 molar. However, the skilled person will be able to vary such parameters as appropriate in order to take into account variables such as probe lengtii, base composition, type of ions present, etc.

In addition to being used as probes, hybridising nucleic acid molecules of the present invention can be used as antisense molecules to alter the expression of substances of the present invention by binding to complementary nucleic acid molecules. This technique can be used in antisense therapy. Such molecules may also be used to produce ribozymes. Ribozymes can be used to regulate expression by binding to and cleaving RNA molecules which include particular target sequences.

A hybridising nucleic acid molecule of the present invention may have a high degree of sequence identity along its length with a nucleic acid molecule within the scope of a) or b) above (e.g. at least 50%, at least 75% or at least 90% sequence identity). As will be appreciated by the skilled person, the higher the sequence identity a given single stranded nucleic acid molecule has with another nucleic acid molecule, the greater the likelihood that it will hybridise to a nucleic acid molecule which is complementary to that other nucleic acid molecule under appropriate conditions.

In view of the foregoing description die skilled person will appreciate that a large number of nucleic acids are within the scope of the present invention.

Unless the context indicates otherwise, nucleic acid molecules of the present invention may have one or more of the following characteristics:

1) they may be DNA or RNA;

2) they may be single or double stranded;

3) they may be provided in recombinant form i.e. covalently linked to a 5' and/or a 3' flanking sequence to provide a molecule which does not occur in nature;

4) they may be provided without 5' and/or 3' flanking sequences which normally occur in nature;

5) they may be provided in substantially pure form (thus they may be provided in a form which is substantially free from contaminating proteins and/or from other nucleic acids); 6) they may be provided with introns (e.g. as a full length gene) or without introns (e.g. as cDNA).

It is yet a further aspect of this invention to provide non-human animal models of

Friedreich's Ataxia which exhibit expression of STM7, through the preparation of transgenic animals having the STM7 gene incoφorated into its genome. Non¬ human animals having incoφorated into its genome either exons 1-16 of STM7.I or the exons combinations of splice variants STM7.IIIa, STM7.IIIb and STM7.IIIc are highly prefeπed under the scope of the present invention. As used herein, the phrase "incoφorated into its genome" is intended to refer to mice, rats or other mammals which have a selected transgene introduced into their germ cells and/or somatic cells such that it is stably incoφorated and is capable of carrying out a desired function. The term "genome" is intended to include the entire DNA complement of an organism, including the nuclear DNA component, chromosomal or extrachromosomal DNA, as well as the cytoplasmic domain (e.g. mitochondrial DNA). Thus, die present invention contemplates that one or more transgenes may be stably incoφorated into an organism's germ cells or somatic cells, in a functional form, and achieve a desired effect, e.g., such as confeπing a selected trait onto the transgenic animal.

While the invention is directed in prefeπed embodiments to the generation of transgenic mice or rats, and in particular to transgenic mice having the propensity to develop Friedreich's Ataxia, it is believed that an appropriate transgene (i.e. STM7.I gene) may be incoφorated as set forth herein. There is no reason why otiier non-human mammals, such as cows, pigs, rabbits, guinea pigs, sheep, hamsters, or even goats, could not also be employed in tiiis regard. In other aspects, the present invention also concerns the general methodology for preparation of transgenic mice or rat lines. These methods generally include first preparing a group of transgenic mice or rats having incoφorated into their genome at least one selected transgene, selecting at least one founder from said group of transgenic mice or rats and breeding the founder or founders to establish at least one line of transgenic mice or rats having the selected transgene incoφorated into their genome.

The initial group of transgenic mice or rats are generally prepared by introduction of DNA which includes the selected transgene into germ cells of the mouse or rat

(typically fertilised eggs), which germ cells are then employed to generate the complete animal. Introduction of the DNA into the germ cells is most conveniently achieved by a technique known as microinjection wherein a solution containing DNA is introduced through the aid of a microscope and a microinjector pipet which deposits intact DNA into one of the two pronuclei. However, the inventors contemplate that other technique may be employed for introduction ofthe DNA into the genome, including in vitro fertilisation using sperm as a carrier of exogenous DNA, electroporation or transfection into a rat embryonic stem cell line and introduction of these cells into a rat blastocyst.

Prefeπed forms of transgenic animals according to this aspect of the invention include, but are not limited to, animals where expression of the native gene has been deleted or otherwise prevented, animals where the predominant Friedreich's Ataxia mutations have been introduced and animals where other common mutations have been introduced.

A particularly prefeπed method for preparing transgenic mice or rats includes subjecting a female to hormonal conditions effective to promote superovulation, followed by fertilising eggs of the superovulated female, and introducing the selected transgene into the fertilised eggs. In this embodiment, the fertilised eggs having the selected transgene are transfeπed into a pseudopregnant female mouse or rat, and brought to term. In that rats have traditionally been found to be difficult to effect superovulation, one will typically desire to employ the technique developed by Armstrong et al , (1988), wherein immature rats are superovulated by a continuous infusion of follicle stimulating hormone, such as FoUtropin Registered TM (Vetrepharm Inc., London, Ontario, Canada).

After superovulation is effected and the females are bred, die resultant eggs are collected (by flushing from the oviduct). While the presently prefeπed method for fertilising the eggs is by breeding the female with a fertile male, superovulated eggs could be fertilised by artificial insemination of the female, or after removal, by in vitro fertilisation.

After fertilisation, the selected transgene is introduced into the fertilised eggs by a convenient method, for example, microinjection. (Note that it is even possible to introduce die DNA prior to fertilisation.)

The next step, wherein fertilised eggs having the selected transgene are transfeπed into a pseudopregnant female, can be accomplished using techniques known in the art, for example, the techniques which are typically employed in connection with transgenic mice (see e.g., Brinster et al , (1985)).

Once the fetuses in the pseudopregnant female have been brought to term, a founder animal is identified by standard techniques of hybridisation of transgene

DNA to genomic DNA from weaning offspring. The word "founder" is intended to refer to a transgenic animal which develops from the microinjected egg. The founders are tested for expression of a functional gene by any suitable assay of the gene product. Typically, cells obtained form the founders, such as white cells (leukocytes), are tested by immunofiuorescence and flow cytometry with a test antibody against the gene product, such as AAPGKQNEEKTYKKTA, LKEKEEETPQNVPDAKCNKQTPNKQIWLSSPS,orCNLRKSGTLGHPGSLD. Founders that express the gene, particularly those that express the gene at levels and with a tissue distribution that is comparable to that found for selected genes in general, are then bred to establish a line or lines of transgenic rats which have a selected transgene incoφorated into their genomes.

The inventors contemplate that the foregoing technique can successfully be employed to develop mouse and rat lines having any of a number of genes, DNA segments or transgenically derived traits introduced into their genomes. As used herein, the term "line" is intended to refer to animals that are direct descendants of one founder and bearing one transgene locus stably integrated into their germline. Furthermore, it is contemplated that inbred lines can be developed from such lines wherein the rats that are used for microinjection are members of established inbred strains. As used herein, the term "inbred line" is intended to refer to genetically identical at all endogenous loci.

Since Friedreich's Ataxia is an autosomal recessive disorder, it is an excellent candidate for somatic cell therapy, as it may be assumed that introduction of a single expressing copy of the normal gene into appropriate cells will coπect the pathophysiology.

The exact location of the primary lesion remains to be established. Degeneration of the spinal cord and cerebellum dominates the pathology, although other parts of the central nervous system may be involved. Loss of large myelinated axons in peripheral nerves occurs almost universally, leading to the suggestion that some form of 'dying-back' process is occuπing. Extensive loss of large myelinated cells in the dorsal root ganglia detected in cases of Friedreich's Ataxia at autopsy suggest that these cells may provide die focus for delivery of the exogenous gene. However, coπection to other groups of neurons, including Purkinje cells, is also contemplated by this invention. Finally, hypertrophic cardiomyopathy is commonly associated with Friedreich's Ataxia and represents the major cause of mortality in these patients. Consequently, future strategies will also need to facilitate coπection of the deficit in the heart, although it is not clear whether this pathology relates to an innervation or structural abnormality.

Having established the full genomic sequence of the FRDA gene and its regulatory elements (Fig. 7), coπection of the biochemical abnormality in vitro using neuronal and non-neuronal cell lines. These cell lines would include, for example,

PC 12 neuroblastoma. Transfection with constructs containing the sequences is contemplated under the scope of the present invention to STM7.I or the splice variants (STM7.IIIa, STM7.IIIb and STM7.IIIc), possibly introducing fragments of DNA up to 500kb in size as yeast artificial chromosome clones, may be carried out initially in neuroblastoma and fibroblastoid cell lines, then extended to include cell lines more pertinent to the nature of the degeneration, for instance in the dorsal root ganglia neurons and Purkinje cells. Confirmation of expression of the gene with the required specificity and at appropriate levels widiin these cells would then allow coπection of the defect in vivo.

Successful delivery of an exogenous gene to die nervous system depends on transport through the brain vascular endothelium, which constimtes the blood-brain barrier (BBB) in vivo. Strategies to achieve in vivo coπection of FRDA fall into four broad categories: A) physiologically based - receptor mediated transfer utilising chimeric peptides; B) pharmacologically based - delivery by encapsulation into liposomes; C) direct delivery of plasmid constructs, and D) the utilisation of viral vectors. These liposomes, plasmid constructs and viral vectors developed and then used in the treatment of Friedreich's Ataxia form yet another embodiment of the invention. A) Carrier-mediated transport. This system can be used to augment delivery to the brain due to the large number of carriers known to be present at the brian capillary endothelium. Agents which would not normally cross the barrier can be coupled to brain transport vectors including proteins such as cationised albumin (Kumagai et al. (1987)) or the murine OX26 monoclonal antibody directed against the rat transferrin receptor (Jeffries et al. , (1984)), the most efficient transporter known to date. Because of the differential expression of transport caπiers between tissues and in various disease states, it may also be possible to determine some degree of selectivity of delivery (Smith et al , (1993)).

The transferrin receptor is known to be highly expressed on brain capillary endothelium and facilitates receptor-mediated transcytosis of either circulating transferrin or the OX26 antibody through the BBB. High yield coupling to the brian-transport vector is achieved by the use of an avidin-biotin system (Yoshikawa et al. , (1992)), the DNA construct can be subsequently released by incoφoration of a disulphide linker which is rapidly cleaved in brain, but not in brain capillaries (Bickel et al , (1993)). This system may also be applicable for the delivery of anti-sense oligonucleotide-based therapies.

B) Encapsulation in liposomes. The utilisation of liposomes as a delivery system for providing the PtdInsP5K (STM7) gene seems particularly attractive as the technology has already been used extensively for the delivery of drugs to the CNS and appears to offer a more benign method of delivery, particularly important for coπection in non-proliferating cells such as neurons. Liposomes fuse to the cell membrane and the associated DNA is delivered to the cytoplasm, from which it will migrate to the nucleus if appropriate targeting signals are provided. Liposomes of a diameter of about approximately 100-500 nanometres would be a prefeπed size under this aspect of the invention. Considerable progress has been made in die encapsulation capacity by modification of formulation and specifically by the use of charged phospholipids (Puglisi et al. , (1992)). The technology has been successfully used to deliver agents to ameliorate brain dysfunction including anti-convulsants (Mori etal , (1992)), ischemia (Phelan et al , (1991)), anti-oxidant dependent recovery from brain injury by liposome entrapped CuZn-SOD (Chan (1992)) and die facilitation of behavioural recovery in a Parkinson mouse model using dopamine-containing liposomes (During et al. , (1992)). Recently, the technology has been adapted as a vehicle for die delivery of foreign recombinant genes, including the CFTR gene cuπently being used in clinical trials.

Smdies to address the issues associated with the long-term expression of such genes, including autoimmune damage and possible incoφoration into germ line cells (Nabel et al, (1992)) appear to yield favourable results with no immunopathological evidence of damage in several species. In addition, liposomes offer the potential of encapsulating large DNA constructs, possibly up to 500kb in size.

More recently, enhanced tissue uptake of liposomes has been demonstrated following intravenous introduction of die therapeutic agent by modification of the

BBB using intracarotid administration of etoposide (Gennuso et al , (1993)) and by direct delivery into the cerebrospinal fluid using multivesicular liposomes (Kim et al. , (1990)), although little is known about penetration from the CSF into brain parenchyma.

C) Direct introduction of plasmid DNA into neurons. Recent smdies have demonstrated d e feasibility of introducing plasmid DNA into neurons utilising either retrograde axonal transport following direct injection into sciatic nerve (Schenk et al. , (1993)) or by lipofection. Lipofection has been used to transfect reporter genes into fetal brain cells in culmre which are then transplanted into adult host brain demonstrating expression in glia and neurons for up to two months (Jiao et al , (1992)).

D) Viral vectors. Such vectors have been the prefeπed choice for neuronal gene delivery to date. In the case of Friedreich's Ataxia, where the primary lesion is thought to occur within the dorsal root ganglia cells, the latency of the heφes simplex virus within these cells normally, suggests that this viral vector would be eminently suitable to deliver coπection to this group of sensory neurons. We would also propose use of adeno-associated virus (AAV).

E) Other Routes. We also propose use of segments of tetanus toxoid for specific delivery to tiie dorsal root ganglia.

The efficacy of each system to deliver exogenous sequences to both neuronal and non-neuronal cells in culmre will be evaluated in terms of levels of expression, dose-dependence, tissue (or sub-population) specificity, long term persistence and for potentially toxic effects. Such evaluative steps would also be clearly within the capabilities of those skilled in the art. Modification of the strategy to establish the limit in construct size for effective uptake by all delivery systems will be undertaken and is also under the capabilities of one skilled in the art. Extension to animal smdies both in normal mice and the transgenic model will facilitate comparison of gene delivery in vivo and we propose to evaluate delivery via the circulation (receptor-mediated transfer and liposomes) or via injection into dorsal root ganglia, cerebellum, peripheral nerve and cerebrospinal fluid (naked DNA and liposomes). We propose to monitor uptake of the construct into nervous tissue, establish levels and patterns of expression for die exogenous sequence, investigate immunological response and evaluate methods for improving integration via modification of the blood brain barrier. Reference to the following Figures is made throughout the present invention.

FIGURE 1 shows the genomic map of the FRDA region on chromosome 9q showing alignment of the Y887A2 and Y761D3 clones and the position of the cosmid contig in relation to the FRDA critical region (Duclos et al. (1994)).

[NOTE: The YAC41 clone has been repositioned according to recent analysis. The clone was found to be inverted and now spans the interval + 120 - +480kb, rather than +240 - +600kb reported in Wilkes et al. (1991)]. The position of rare cutter restriction sites in genomic, YAC and cosmid DNA are summarised as follows: N=NσtI, B=5^HII, S=SαcII, E=Eagl. (R) and (L) represent the right and left ends of the YAC clones respectively.

FIGURE 2 shows a 230kb cosmid contig initiated from the D9S888 (FR8) locus and constructed using clones isolated from the chromosome 9-specific cosmid library [LLO9ΝC01]. The relative location of the left end of Y761D3 (D3L) and the presence of rare-cutter restriction sites within these clones are shown. [N=NøtI, Genomic organisation of the sixteen exons comprising STM7.I and location witii respect to the proximal boundary of the FRDA candidate region (D9S887/ D9888) is illustrated. Centromeric cosmids which contain exons 1 and 2 have not yet been identified. The five exons isolated following exon-trapping protocols are indicated (*).

FIGURE 3 shows exonic organisation within the 2780bp comprising STM7.I showing the location of the 1620bp open reading frame. The five exons captured by exon-trapping are shown as hatched boxes. Primers used for RT-PCR analysis and to generate (rt) probes for Northern hybridisation and cDNA library screening are indicated. Alignment of the two cerebellum cDNA clones is shown. The hatched region of the cDNA clone cer-5B2 indicates sequence divergence to intronic sequence which coincides witii a consensus splice-donor site within the clone. FIGURE 4 shows hybridisation of the n900 fragment from STM7.I to a commercially prepared poly(A)⁺ RNA blot showing the existence of multiple transcripts in a variety of tissues. The two major transcripts 3.9kb and 1.9kb are present in all tissues, with the exception of the 3.9kb transcript which is absent from liver.

FIGURE 5 depicts alignment generated by the PILEUP program (Genetics Computer Group, University of Wisconsin) of the predicted amino acid sequence for STM7.I with the deduced amino acid sequence of the S. cerevisiae MSS4 [D13716], the S. cerevisiae FAB1 gene [P34756] and the human homologue of the phosphatidylinositol-4-phosphate 5-kinase type II gene [U14957] . Exon boundaries for STM7.I are indicated. Residues conserved in all four sequences, constituting elements of the kinase domain, are denoted by black boxes. Grey boxes denote conserved residues between two or more of the amino acid sequences. Residues 120-129 constitute the putative phosphate-binding loop. Dots indicate gaps introduced into the sequence.

FIGURE 6 shows schematic representation of two alternative splicing events detected using RT-PCR amplification. STM7.1 shows the exonic organisation of the transcript characterised by exon-trapping and RT-PCR analysis. STM7.IIA illustrates the loss of exons 3-6 detected in 14-week fetal brain RNA and STM7.IIB the loss of exon 15 in poly(A)⁺ RNA in a variety of tissues. Primers used for first strand syntiiesis and RT-PCR amplification are described in text. For STM7.IIB, the position of the first in-frame stop codon is located 3bp further downstream.

FIGURE 7 shows the nucleotide sequence for STM7.I.

FIGURE 8 shows the complete amino acid sequence for STM7.I. FIGURE 9 depicts sequence analysis of STM7.IIIb showing die transition (a) from exon 15 of STM7.I (X92493) to a newly identified exon 17 derived from the 3'UTR of PRKACG (M34182), and (b) from exon 17 to exon 19 (X25; exon 2) (R06470). The presence of exon 15 and exon 19 as contiguous sequence, as determined by analysis of STM7.IIIc, is shown in (c).

FIGURE 10 depicts schematic representation of the three splice variants generated from RT-PCR amplification of placental poly(A)+ RNA using nested primers compared to the linear aπay of exons 7-24. Numbers in parentheses indicate the original designation of exons comprising X25. Open and closed boxes represent coding and non-coding sequences respectively. The alternatively spliced exons are identical to d e published sequences and utilise die reported splice boundaries. Conventional splice donor/acceptor sites are utilised for exon 17.

FIGURE 11 depicts genomic organisation of the exons constituting the reported

STM7.I and X25 transcripts with respect to the 300kb and 150kb FRDA critical regions. Exons have been numbered in accordance witii their order in STM7. The previous numbering system for X25 is indicated in parentheses. Exon 17, derived from the 3'UTR of the PRKACG gene, is located distal to exon 15, as determined from mapping smdies caπied out in the high resolution cosmid contig spanning the region. The transcriptional directions of the ZO-2 and PRKACG genes are shown.

FIGURE 12 depicts a sequential hybridisation of (a) the RT400 fragment generated from STM7.I and (b) STM7.IIIb to a commercially prepared poly(A)⁺ RNA blot derived from multiple tissues [Lane 1: heart; 2: brain; 3: placenta; 4: lung; 5: liver; 6: skeletal muscle; 7: kidney; 8: pancreas]. An additional 1.3kb transcript, similar in size to that reported for X25, is detected by RT400 at a comparatively low level of expression in heart and skeletal muscle, when compared witii the published hybridisation pattern for RT900⁴. STM7.IIIb detects the major 3.9kb and 1.3kb transcripts reported for STM7.I (RT900) and X25 respectively. Hybridisation of STM7.IIIb to a human brain MTN blot (c) indicates that the 3.9kb transcript is more highly expressed in most tissues, with the exception of spinal cord, when compared to the 1.3kb transcript. [Lane 9: cerebellum; 10: cerebral cortex; 11: medulla; 12: spinal cord; 13: occipital pole; 14: frontal lobe; 15: temporal lobe; 16: putamenj.

FIGURE 13 depicts schematic alignment of the amino acid sequences of STM7.I, PtdInsP5K-II, Mss4p and Fablp showing the relative location of the putative kinase domain in each sequence. The regions of high overall homology are indicated by shading.

FIGURE 14 depicts a sequence composition of the primers used for RT-PCR amplification of placental poly(A)⁺ mRNA.

FIGURE 15 depicts a thin layer chromatographic separation of a PtdInsP₂ standard (lane 1) and the products from PtdlnsP kinase assays of GST (lane 2) and GST-STM7 (lane 3). The position of PtdInsP₂ is indicated by an aπow. The minor product in tiiese assays is the breakdown product, lysoPtdInsP₂.

FIGURE 16 depicts nucleotide sequences of the STM7 exons. the variants are composed of:

STM7.I of exons 1-16, inclusive STM7.IIa (partial) of exons 2 and 7

STM7.IIb (partial) of exons 14, 16

STM7.IIIa (partial) of exons 9-13, 17, 19-22

STM7.IIIb (partial) of exons 13-15, 17, 19, 21, 22

STM7.IIIC (partial) of exons 14, 15, 19-22 FIGURE 17 depicts a map of cDNA positions with respect to STM7.

FIGURE 18 depicts the cDNA sequence of 'cer5B2' of STM7 (exons 1-8).

FIGURE 19 depicts the cDNA sequence of 'cerRT400' of STM7 (partial exon 13 -

Exon 16).

FIGURE 20 depicts the cDNA sequence of 'NFBA' of STM7 (partial exon 14 through exon 16).

FIGURE 21 depicts the cDNA sequence of 'ORF' (Open Reading Frame) of STM7.

FIGURE 22 depicts pairs of intronic primer sequences which facilitated PCR amplification of all STM7 exons and exon-intron junctions.

EXAMPLE 1

For ease of following the procedures used in isolating the STM7 gene to Friedreich's Ataxia, each stage will be addressed under separate headings.

Extension of the YAC contig

We previously reported a yeast artificial chromosome clone contig spanning approximately 475kb including die D9S15 and D9S5 loci (Wilkes et al. , Genomics 9 90-95 (1991)). This original contig extended approximately 250kb centromeric to D9S5. In order to complete the cloning of the 1Mb interval immediately proximal to this locus, extension of the contig was achieved by PCR screening of the GENETHON mega- YAC library (Albertsen et al. , Proc. Natl. Acad. Sci. 87 4256-4260 (1990)) with primers amplifying the loci D9S15, D9S5 and D9S202 (Pandolfo et al. , Am. J. Hum. Genet. 47 228-235 (1990)). Screening of the GENETHON mega- YAC library with primers amplifying the D9S15 and D9S202 loci resulted in the isolation of two YAC clones, Y761D3 and Y887A2, approximately 1.2Mb and 1Mb respectively. The fidelity of each clone for the corresponding genomic region was confirmed by fluorescent in situ hybridisation analysis. Orientation of Y761D3 with respect to the centromere was achieved by dual labelling smdies using cosmid probes isolated from, or in close proximity to, the ends of this clone. These smdies also confirmed orientation of the linkage group (Hillermann et al , Cytogenet. Cell Genet, (in press) (1995)), previously based on the inteφretation of a single recombination event (Chamberlain et al. , Am. J. Hum. Genet. 52 99-109 (1993)).

Positioning of these clones within the genomic region was initially achieved by determining the presence or absence of twelve 9ql3 region sequence tagged sites (STSs) within the clones by PCR amplification. The clone Y761D3 was found to contain the loci D9Slll, D9S15, D9S110, D9S327, D9S411E, FR1, D9S202, FD2 and D9S886, whereas Y887A2 contained D9S11E, FR1, D9S202, FD2, D9S886, D9S887, D9S888 and D9S889 (Figure 1). A more precise location was then achieved by comparative genomic and YAC pulsed field restriction mapping of the two end fragments generated from each clone. This demonstrated tiiat Y761D3 approximated to 1.16Mb in size, spanning the interval -250kb to +91 Okb and that Y887A2 was approximately 990kb in size, with the left end located at + 1 lOkb and the right end located at + 1100kb (Figure 1). These clones provided the basis for the subsequent strategies to isolate potential coding sequences from the region.

Isolation of chromosome 9 region-specific cosmids

Region-specific cosmids were isolated by screening a chromosome-9 gridded cosmid library (LL09NC01) with isolated human inserts (Baxendale et al. , Nucleic Acids Res. 19 6651 (1991)) and end-probes generated from the YAC clones Y761D3 and Y887A2 (Hermanson et al. , Nucleic Acid Res. 19 4943-4948 (1991)) and oligonucleotides for known loci in the region. Probes were labelled by random priming with [α-³²P] dCTP (800Ci/mmol), with the exception of oligonucleotide probes which were end-labelled witii [γ-³ P] ATP ( >5000 Ci/mmol). YAC inserts were competed with lmg human placental DNA (Sigma) and pYAC4 vector for 6 hours at 65 °C and YAC-end clones with lmg human placental DNA for 1 hour at 65 °C. Random-primed probes were hybridised at 65 °C overnight and washed to 0.1 x SSC/0.1 %SDS. End-labelled oligonucleotides were hybridised at 55 °C overnight and washed to 2 x SSC/0.1%SDS at room temperamre.

Positive cosmid clones detected following screening of the library with YAC inserts were grown in microtitre plates and gridded onto filters for subsequent screening procedures. Having identified cosmids which contained anchor loci within the region (microsatellite markers, YAC-ends), the contig was constructed by cosmid walking using end-clones generated using vectorette technology (Riley et al , Nucleic acids Res. 18 2887-2890 (1990)).

Construction of cosmid contigs

Cosmid contigs were initiated at each of the anchor loci - D9S5, D9S411E, FR1, D9S202, FD2, D9S886, D9S888 and D9S889. Fluorescent in situ hybridisation smdies were caπied out on all clones to confirm their fidelity for the FRDA region. Particular emphasis was placed on die extension of die contig anchored by the D9S888 locus in view of the strong linkage disequilibrium observed between FRDA and tiiis marker. Genetic evidence in support of this location has been reported by Duclos et al. (1994), where extensive haplotype analysis led to the detection of recombination events which define the FRDA critical region to an interval spanning approximately 300kb, flanked by the loci D9S886 and D9S887/888. The location of the contig initiated from the D9S888 locus in relation to the FRDA critical region is shown in Figures 1 and 2. The contig spans approximately 230kb extending 160kb distal to the D9S887/888 loci into the FRDA critical region and also includes D9S889. Details ofthe twelve overlapping cosmid clones comprising the contig are reported in Figure 2.

As part of the strategy to isolate potential coding sequences, all clones were initially screened for the presence of rare-cutter restriction sites including NotI, SacII and BssΗII to facilitate detection of putative CpG- islands within the contig. The restriction data is summarised in Figures 1 and 2. Three SacII sites were detected - in the overlapping regions of cl8C8 and c44D2, the overlapping regions of cosmids cl8F10 and cl68D2 and the unique region of cl68D2. Association of the SαcII sites with other rare-cutter restriction sites suggested the presence of two putative CpG-islands located approximately 35kb and 115kb distal to D9S887/888 (Figures 1 and 2). A single NotI site was detected in the overlapping regions of the clones c214G3 and c292F4. This site is located 35kb proximal to D9S887/888, previously identified as d e centromeric bracket for the FRDA candidate interval (Duclos et al, Hum. Mol. Genet. 6 909-914 (1994)).

Exon trapping

Exon trapping to isolate coding sequence from the cosmid contig was carried out according to Buckler et al. (1991), using either individual clones or pools of five cosmids. Cosmid DΝA was double-digested with Bglll and BamHl and ligated into the .BαmHI cloning site of either of the exon-splicing vectors pSPLl (Buckler et al , Proc. Natl. Acad. Sci. 88 4005-4009 (1991)) or pSPL3 (Gibco, BRL).

Following transformation using XLl-Blue competent cells (Stratagene), DΝA was isolated and electroporated into COS-7 cells. Cytoplasmic RΝA was isolated after 72 hours incubation. cDΝA synthesis was followed by two rounds of PCR amplification using nested primers for d e pSPL vectors. Products were cloned into pAMPIO (Gibco-BRL) for sequencing using the dideoxy chain termination method (Sanger et al , Proc. Natl. Acad. Sci. 74 5463-5467 (1977)).

Exon trapping carried out in cosmid clones positioned at or distal to D9S887/888 (c22G2-c3Hl) as shown in Figure 2, resulted in die isolation of five exons ranging from 78bp-128bp in size. Two of these exons, subsequently designated exons 5 and 6 (Figure 2), were captured from the cosmid clone c22G2, previously shown to contain the D9S887/888/889 loci by hybridisation analysis. Exons 7 and 12 were isolated from clone cl68D2 distal to 22G2 and exon 15 was trapped from the clone c3Hl, located approximately 140kb distal to D9S888.

Confirmation of a single gene by reverse transcriptase amplification of fetal spinal cord mRNA

The transcriptional direction of the trapped exons is determined from the relative positions of the splice donor and acceptor sites in the pSPL vectors. Primers derived from each exon were synthesised in both the 5 '-3' (pl) and 3 '-5' (p2) direction (Figure 3) and utilised for RT-PCR amplification of fetal spinal cord total

RNA to investigate whether the sequences constitute a single gene and to establish die direction of transcription in relation to the centromere. Amplifications were caπied out using combinations of primers from each trapped exon, including those from exon 15, despite its genomic location. Primer pairs e5pl/e6p2, e5pl/e7p2, e7pl/el2ρ2 and el2pl/el5p2 generated fragments of 173, 347, 753 and 411bρ respectively, confirming that all exons are part of a single gene and indicating that exons 5, 6 and 7 are present as contiguous sequence. Inteφretation of the PCR product size generated between e7pl-el2p2 and el2pl-el5p2 indicated the presence of additional coding sequence (650 and 300bp respectively) between these exons. The total length of the transcript as generated by RT-PCR amplification is approximately 1.5kb in length. Further analysis revealed that the entire transcript constimtes open reading frame. Since the relative physical locations of the trapped exons had been established previously and primers in the reverse orientation failed to amplify, the transcriptional direction from centromere to telomere could be established.

First strand synthesis was performed using between 0.5-lug of poly(A)⁺ RNA isolated from fetal tissue, 20pmol of primer, 0.5mM dNTPs, 200U Superscript II reverse transcriptase (Gibco-BRL) and RT-reaction buffer [final concentrations:

50mM Tris-HCl (pH 8.3), 75mM KCl, 3mM MgCl₂] to a total volume of 20ul. After treatment with RNase H, RT-PCR was performed using 2ul of the cDNA as template, 20pmol of each RT-PCR primer, 0.5mM dNTPs, IU of Taq polymerase (Flowgen) and RT-reaction buffer to a total volume of 50ul.

cDNA library screening

Isolation of clones coπesponding to the candidate gene was achieved by screening 10⁶ pfu from a cDNA library prepared from the cerebellum of a 2-year old child (Stratagene) using: a) 200ng of a probe generated by PCR amplification of the trapped partial exon 7 (80bp) labelled with [α-³²P] dCTP (800Ci/mmol); and b)

50ng of a RT-PCR fragment (rt400), generated by amplification between primers el2pl and el5p2, using random-primed labelling (Feinberg et al. , Anal. Biochem. 132 6-13 (1983)). Following overnight hybridisation at 55°C, filters were washed to 1 x SSC/0.1% SDS and exposed to autoradiography.

Cloning of the 5' and 3' regions

Extension of the 5' end was achieved by screening a cerebellum cDNA library with the cryptically-spliced exon 7 (80bp). This resulted in the isolation of a 1.5kb cDNA clone, designated cer-5B2 (Figure 3). Sequence analysis confirmed that the clone contained the exons 5, 6 and 7 as contiguous sequence and included additional coding sequence both 5' and 3' to these exons.

The open reading frame was extended by an additional 93bp in the 5' direction with a putative in-frame initiation codon located 69bp upstream of the boundary with exon 5. This additional sequence was later determined to constitute exon 4 and part of exon 3. An additional 396bp 5' to the first upstream stop codon were characterised. Analysis of die sequence immediately 3' to exon 7 revealed an additional 300bp of coding sequence, followed by approximately 400bp of sequence with multiple stop codons in all frames indicating probable genomic contamination of the cDNA clone. Comparison of the cDNA sequence with that obtained by RT-PCR, revealed a divergence which coincided with the transition from coding to non-coding sequence at a consensus splice-donor site within the clone.

Extension of the sequence in the 3' region was achieved by a combination of two methodologies - 3' RACE using a set of nested primers derived from exons 12 and 15 and screening of the above cDNA library using a 400bp fragment (rt400) generated by RT-PCR using primers derived from exon 12 (el2pl) and exon 15 (el5p2). A 900bp fragment was isolated using the 3' RACE methodology, whilst a cDNA clone, cer-rt400, proved to be 1125bp in length. Analysis and alignment of these two sequences revealed that the 900bp 3 '-RACE product is completely contained within cer-rt400 and that both include a polyadenylation signal and a polyA tract. Comparison of the combined sequences with that derived from RT- PCR analysis demonstrated that the alignment of the cDNA imtiated at nucleotide position (nt) 1646, with exons 13, 14 and 15 present as contiguous sequence. The sequence extended 738bp distal to exon 15, with the first three bases following the junction with 15 constituting the first in-frame stop codon followed by the 3'UTR. Characterisation of the gene

Alignment of sequence data generated from both RT-PCR and cDNA analysis resulted in a total sequence 2780bp in length, of which 1620bp constimtes open reading frame. This sequence was subsequently designated STM7. The putative in-frame initiation codon is located within exon 4 at nt 420. The first three bases of exon 16 constitute the first in-frame stop codon (TAA) at position nt 2040, with the polyadenylation signal (AAT AAA) located at nt 2749, 19 bases upstream of the polyA tract.

Exon-intron boundary analysis demonstrated that this sequence comprises 16 exons, with coding region present in exons 4 through 15. All exon-intron junctions comply with gt/ag consensus donor/acceptor splice site sequences (Padgett et al , Ann. Rev. Biochem. 55 1119-1150 (1986)). Exon 16 includes the first in-frame stop codon, 729bp of the 3 'UTR region and the polyA tract.

From the span of the cosmid contig, the gene is located in an interval extending at least 220kb, including the D9S887/888/889 loci (Figure 1). Exons 7-12 are clustered within 35kb as defined by die cosmid cl68D2.

Although the gene extends approximately 150kb distal to D9S887/888 into die

FRDA critical region, exons 1-5 (including the putative initiation site) are located centromeric to this locus. The gene also traverses the three putative CpG-islands detected in the cosmid contig, although neither the respective restriction sites or alternative CpG-rich regions were detected in the cloned sequence. An API site was detected at position -56 and an AP2 site at position -50. Other regulatory elements were absent from the proposed 5 'UTR.

3'-RACE

First-strand synthesis was performed on lug poly (A) ⁺ RNA isolated from fetal spinal cord using the 3 '-RACE kit (Gibco-BRL) according to manufacmrer's recommendations. After treatment with RNase H, one round of RT-PCR was performed using AP-oligo-(dT) and a forward primer from exon 9 (e9pl). Following electrophoretic separation on 2% agarose, products were excised, subjected to a second round of PCR using the AP primer and the nested primer el2pl and cloned into pCRII TA- vector (Invitrogen) for sequencing.

Northern analysis

Hybridisation of a RT-PCR fragment (rt900; Figure 3) generated using primers eόpl and el2p2, to a commercially prepared poly(A)⁺ RNA blot (MTN, Clontech) is shown in Figure 4. The probe was denamred for 5 minutes in the presence of lOug yeast tRNA (Boehringer) and lOug of salmon sperm DNA (Promega) and quenched on ice for 1 minute. Hybridisation was carried out at 42 °C for 18 hours and the filter washed to 0.1 x SSC, 0.1 % SDS. Autoradiography was carried out between 1-7 days depending on signal intensity. A complicated hybridisation pattern was detected even at high stringency, including probable weak hybridisation to residual 28S and 18S ribosomal bands. Two major transcripts, approximately 3.9kb and 1.9kb in size were detected in all tissues studied with the exception of the 3.9kb band being absent from liver. Additional weaker hybridisation signals were also observed for heart, placenta and skeletal muscle (3.3kb), lung (2.8kb), liver (6.0kb), skeletal muscle (6.3kb). Finally, a strong hybridisation signal approximately 6.5kb in size was also detected in placenta poly(A)⁺ RNA.

Subsequent hybridisation of die Northern filter with an rt400 fragment generated between el2pl and el5p2 resulted in the detection of identical transcripts with variable intensity, with the exception of an additional 2.7kb transcript in liver, only detected using this second probe (data not shown). Sequence homology

Database searches using nucleotide and amino acid sequences of STM7 were carried out using the SWISSPROT database. This resulted in the detection of strong homology with the Saccharomyces cerevisiae FAB1 gene (P34756) - amino acids 118-161 (47% identity), 248-273 (38% identity) and 340-389 (30% identity)

[P= 1.2e-06].

Comparison of the amino acid sequence with the translated version of the EMBL DNA database generated even stronger homology with S. cerevisiae DNA for multicopy suppressor of the STT4 mutation (MSS4) (D 13716) - 33.9% identity with 55.7% similarity [P=3.7e-32]. Alignment of the deduced amino acid sequence for STM7.I with the deduced MSS4 FAB1 gene and phosphatidylinositol- 4-phosphate 5-kinase type II gene (U14957) sequence is shown in Figure 5.

Exon-intron boundaries

Sequence flanking each exon was obtained by digesting the appropriate cosmid with Alul, EcoRV, Pvuϊl or Rsάl, ligating on a blunt-ended vectorette cassette (Riley et al. , Nucleic Acids Res. 18 2887-2890 (1990)) and amplifying between exon-specific and vectorette primers. Amplified products were resolved on a 2% agarose gel, excised and gel purified (Geneclean, BiolOl), cloned into a pCRII

T A- vector (Invitrogen) and double strand sequenced. Unique intronic primer pairs flanking each exon were then designed to facilitate genomic mutation screening procedures.

Mutation screening

Systematic screening for mutation was caπied out in a panel of patients, heterozygotes and normal unrelated control individuals using heteroduplex analysis following a) RT-PCR analysis caπied out in total RNA isolated from blood leucocytes and b) genomic amplification of exons using flanking intronic primers. For the RT-PCR analysis, first strand synthesis was performed using the primers e8p2 and elόpό as described previously, and pairs of nested primers designed to generate fragments ranging from 90-400bp which span the sequence. Genomic amplification of exons was carried out by amplification of 250ng DNA in a total volume of 50ul containing lOmM Tris-HCl (pH 8.8), 50mM KCl, 1.5mM

MgCl₂, 0.1 % Triton X-100, O.lmM each of dATP, dCTP, dGTP and dTTP, IU Taq polymerase (Dynazyme, Flowgen) and 5pmol of each intronic primer. 35 cycles of amplification were performed; 94°C for 30 seconds, 55 °C for 1 min and 72°C for 1 min followed by 1 cycle at 72°C for 10 min. lOul of the PCR product was denamred at 95 °C for 5 minutes, followed by slow cooling to room temperamre. Samples were mixed with 2ul of MDE loading buffer, resolved on 0.5 x MDE (AT Biochem), O.όxTBE, 350V at room temperamre for 12-16 hours and fragments visualised with EtBr.

No mutations were detected in the sequence isolated. However, systematic screening of exons for mutation in the panel of patients, heterozygotes and normal controls led to the detection of an insertion (T) in the flanking intronic sequence, 71-78bp downstream of the donor splice-site of exon 7. This insertion proved to be polymoφhic, present in both normal and affected individuals witii a frequency of 25%.

Evidence for alternative splicing

Northern analysis showed variation in signal intensity depending on the region of STM7 used for hybridisation, suggesting tiiat alternative transcription may be a feamre of this gene. In order to detect exon-splicing events, RT-PCR was carried out using primer pair combinations covering the existing transcript and RNA derived from a variety of fetal tissues including liver, heart, placenta and fetal brain (11 and 14 weeks). The expected products were generated in all cases. However, smaller products were also observed in two instances: a) for the 14-week fetal brain RNA, using the primer e8p2 for first strand synthesis and the primers e2p3 and e7p2 for amplification (sequence analysis revealed that this product lacked exons 3-6 (Figure 6:IIA). The absence of expression of this smaller transcript in fetal brain of 11 weeks gestational age suggests that alternative transcription is also subject to developmental control); and b) for all tissues using the primer el6p2 for first strand synthesis and tiie primers el4pl and el6p6 for amplification (sequence analysis revealed that this product lacked exon 15 (Figure 6:IIB)). We are therefore extending these smdies to include fetal and neonatal tissue sampled at various intervals during die development and maturation of the nervous system.

The namral history of Friedreich's Ataxia indicates tiiat the nervous system develops normally since most patients experience several years of normal neurological function. However, it is highly likely that neurodegradation is occurring at least from the time of maturation of the nervous system, with the deficit only manifesting once the number of sensory neurons necessary for normal function falls below an arbitrary threshold number. It is also highly probable that the level of gene expression of Friedreich's Ataxia at maturation is being affected in a manner similar to that of nerve growth factor, where high levels of neurotrophin are required for development, however the expression is down- regulated to levels essential for neuronal maintenance. Our present invention will be of assistance to those skilled in the art in understanding the normal function of the gene as well as providing great insight into how mutation within this sequence may give rise to the neurodegenerative mechamsm causing Friedreich's Ataxia. The production of antibodies, which forms a part of this invention will be necessary to facilitate the quantification of gene expression in both affected and normal control tissues and for use in more specific in situ hybridisation smdies. The location of the FRDA gene is highly consistent with the disequilibrium profile detected in our smdies. Spanning a genomic interval of at least 220kb, of which 150kb extends distal to D9S887/888, die location is consistent with an intragenic recombination event in the pedigree used to define die centromeric boundary for die critical region. However, with more than 70kb of the genomic sequence of this gene extending proximal to D9S887/888, including at least two coding exons (designated as exons 4 and 5) and the 5 'UTR, this location explains the observation of rare recombination events which position FRDA centromeric to D9S887/888.

Without wishing to be bound by theory, the predominant neuronal degeneration observed in Friedreich's Ataxia is likely explained by die generation of neuron- specific transcript(s) by either the presence of tissue-specific promoters or alternative splicing as seen for the regulation of neuronal versus non-neuronal expression of aromatic L-amino acid decarboxylase (Albert et al , Proc. Natl. Acad. Sci. 89 12053-12057 (1992)), choline acetyltransferase (Misawa et al , J.

Biol. Chem. 267 20392-20399 (1992)), and the transcription of the tropomyosin genes (Had et al , Brain Res. Mol. Brain Res. 18 77-86 (1993)). Selectivity for specific neuronal sub-populations may also be a consequence of alternative splicing with regional differences in expression within the brain related to transcript lengtii. In the rat, for example, the selective neuronal expression of the inositol (1,4,5)- triphosphate receptor is determined by alternatively spliced forms of the gene (Schnell et al , Mol. Brain Res. 17 212-216 (1993)).

Although Northern analysis revealed the presence of multiple transcripts, the evidence for alternative splicing appears stronger than for the existence of a gene family. Hybridisation of segments of the sequence to genomic DNA from a variety of mammalian species as detailed infra argues in favour of a single gene, highly conserved throughout evolution. In addition, all cDNA clones screened proved to be directly related to the reported sequence, supporting our findings. It is interesting to note the proximity of a putative CpG-island to the cluster of exons present in cl68D2. Again, without wishing to be bound by theory, this raises the possibility of an alternative promoter in this region, which could give rise to a transcript approximating in size to one of tiie major transcripts (1.9kb).

Receptor-mediated activation of inositol phospholipid metabolism is one of the major signal transduction pathways in mammalian cells. The MSS4 gene has been identified in the course of smdies designed to elucidate this pathway in yeast. Essential for cell growth, its ability to suppress mutation in a yeast gene encoding a phosphatidylinositol 4-kinase, has implicated its role in the phosphoinositide cycle, although probably not in the conventional PKC1 pathway. Biochemical analysis would suggest that MSS4 is involved witii the localisation of the phospholipid metabolic enzyme or metabolites (Yoshida et al. , Mol. Gen. Genet. 242 631-640 (1994)). The homology detected between the STM7 gene of the present invention and the yeast gene FAB1, also implicated in the endocytic- vacuolar pathway, would support this functional role (Yamamoto and Koshland; [accession number: U01017]). By analogy, mutation in the human gene likely results in the perturbation of neuronal protein trafficking or even synaptic transmission, leading to the pattern of degeneration seen in FRDA.

Another aspect of this invention will therefore be to characterise the number and incidence of mutations in families afflicted with FRDA. This will facilitate making a coπelation between a FRDA genotype and its coπesponding phenotype to explain the considerable clinical variability observed within the disorder. Anotiier aspect of this invention will therefore be the production of diagnostic kits utilising the nucleic acid or amino acid sequence of the STM7 gene.

Experiments using a mixmre of oligonucleotides derived from the STM7 sequence was found to hybridise to a variety of autopsy and surgically-derived tissues. This reveals that STM7 is expressed in all tissues affected by this disorder, including heart, muscle, pancreas, nervous tissue and specifically, Purkinje cells of the cerebellum.

EXAMPLE 2

RT-PCR analysis demonstrates that X25 constitutes part of the STM7 gene

Following first strand synthesis, primary amplification was undertaken using the primer e7pl (STM7.I; exon 7) against the T3B primer located within the 3 'UTR associated with exon 5a of X25 gene. Secondary amplification using nested primers located within either STM7.I exon 9 (e9p3), 13 (el3p3) or 14 (el4pl) against T3B resulted in the generation of three fragments - STM7.HIa (1142bp), STM7.JJIb (914bp) and STM7.IIIc (791bp) respectively.

Sequence analysis of the three products revealed that each contained elements of both STM7.I and X25. Direct sequencing of STM7.IIIa detected die loss of exons 14 and 15 from STM7.I and the incoφoration of 62bp of novel sequence, designated exon 17. Database comparison of the novel sequence revealed that the new exon constimtes part of the 3' -UTR of another gene located within the excluded region, the catalytic subunit Cg of the cAMP-dependent protein kinase

(PRKACG). This testis-specific gene is located distal to exon 15 of die STM7.I transcript and is transcribed in the opposite direction (from the telomere towards the centromere) to both STM7.I and X25. Sequence coπesponding to exons 19-22 (exons 2-5a of X25) was contiguous with exon 17, indicating die loss of exon 18 (X25; exon 1). For this variant, the open reading frame extends from at least exon

9 to the reported first in-frame stop codon in exon 22 (X25; exon 5a), indicating the potential for a transcript longer than the 2.7kb variant originally described. Deviation from the predicted size of STM7.IIIb results from the loss of exons 18 and 20 (in X25 this would be equivalent to exons 1 and 3). Sequence transitions from exon 15 to 17 and from exon 17 to 19 (X25; exon 2) are shown in Figures 9a, 9b, 10, 11 and 16. The inclusion of the novel exon repositions the first in-frame stop codon within exon 17, located 13nt downstream of the acceptor splice site. Finally, sequence analysis of STM7.IIIc revealed the presence of exons 14 and 15 of the STM7.I transcript and exons 19-22 (X25; exons 2-5a) as contiguous sequence (Figure 9c). Once again, the splice variant utilises the reported stop codon in exon 22. Schematic representation of the splice variants detected are shown in Figure 10. The reassignment of exonic sequences coπesponding to X25 with respect to their revised location within the STM7 gene is shown in Figure 11.

Northern analysis Figure 12 shows a comparison of the signals obtained following sequential hybridisation of a commercially prepared multiple tissue poly(A)+ RNA blot (MTN; Clontech) with RT-PCR fragments coπesponding to (a) RT400 (generated using primers el2pl and el5p24) (data not published previously); and (b) STM7.IIIb. Comparison of the hybridisation pattern obtained for RT400 with our data for RT900 4 [the two fragments representing contiguous sequence extending from exon 6 to 15] (Carvajal et al , Human Mol. Gen. 4: 1411-19 (1995)), demonstrated that both probes detect almost identical transcripts witii variable intensity. Of particular note however, is the detection of an additional 1.3kb transcript by RT400 at a comparatively low level of expression in heart and skeletal muscle. A strong hybridisation signal was obtained for this transcript following sequential hybridisation of the filter with individual 30mer oligonucleotide probes designed to coπespond to exons 5, 12 and 15. However, confirmation that this transcript is almost certainly identical to that detected following hybridisation with STM7.IIIb questions the direct relationship reported between the X25 sequence and the 1.3kb transcript; the apparent contribution of sequence derived from exons 5-22 to the 1.3kb transcript implying that extensive alternative splicing has occuπed. In addition to the 3.9kb transcript seen in several tissues as reported previously, STM7.IIIb also detects a 3.7kb transcript in heart, placenta, skeletal muscle and pancreas and low intensity signals were observed for the 5.0kb (heart) and 6.3kb (heart and skeletal muscle) transcripts previously reported for RT900. Notably, die 1.9kb major transcript and the >6.5kb placental transcript detected by RT900 were absent, indicating tiiat exons 13-22 do not make a major contribution to these variants, suggesting additional alternative splicing involving the 5' end of the gene. Hybridisation of STM7.IUb to a human brain MTN blot (Clontech) is shown in Figure 12c. Whilst the level of expression for the 1.3kb transcript was extremely low, as reflected by a barely discernable signal, the 3.9kb transcript appeared to be highly expressed in most tissues with the exception of spinal cord. These data demonstrate the expression of at least two splice variants of STM7 in adult human neurological tissue.

EXAMPLE 3

Expression and enzymatic activity of recombinant STM7 protein The full length STM7 cDNA coπesponding to the reported 2.7kb transcript was subcloned into pGEX 5X-1 (Pharmacia), expressed as a glutathione S-transferase (GST) fusion protein in E. coli DH5, and purified from cell lysates using glutathione Sepharose. GST-STM7 was eluted from glutathione sepharose (Pharmacia) using elution buffer containing lOmM glutathione, 200mM Tris.HCl (pH 7.5) and checked for purity and quantity by SDS-PAGE. Eluates containing approximately lOOng of GST-STM7 or GST alone from control samples were assayed for phosphatidylinositol 4-monophosphate (PtdlnsP) kinase activity. Assays were performed in 50mM Tris-HCl (pH 7.5), lOOmM KCl, lOmM MgC12, 50μM PtdlnsP (Sigma), 20μM ATP and 5μCi of g[32P]ATP (Amersham). Assays were started by addition of ATP, incubated at 37 °C for 20min, and terminated by the addition of 50μl of IM HCl. Phospholipids were extracted into 500μl chloroform : methanol : H₂O (15:5:5), vortexed briefly and the phases separated by centrifugation. The organic phase was re-extracted with 500μl of methanol : IM HCl (1:1) and dried under a stream of nitrogen, resuspended in chloroform and analysed by thin layer chromatography according to Morgan et al. The identity of the phospholipid products was confirmed by deacylation and ion exchange hplc as described by Serunian et al , Methods Enzymol. 198 78-87 (1991).

The absence of TATA and CAAT regulatory elements is regarded to be indicative of a housekeeping or growth control gene and is consistent with the widespread expression of STM7 in a variety of tissues. Ubiquitous expression is also observed for other genes giving rise to neurological disorders such as Huntington's disease (Strong et al. , Nature Genet. 5 259-265 (1993)) and spinocerebellar ataxia 1 (On et al , Nature Genet. 4 221-226 (1993)). Although Friedreich's Ataxia is commonly considered in terms of its neurological phenotype, the clinical spectrum includes hypertrophic cardiomyopathy, skeletal abnormalities and an increased incidence of both clinical and chemical diabetes. More recently clinical investigations, including MRI, have demonstrated the involvement of the cerebral cortex, indicating that the degeneration is not as limited as previously predicted. Widespread expression does not argue therefore, against the viability of this gene as the one encoding FRDA.

EXAMPLE 4

Antibodies were raised against peptides synthesised using Abacus system and Fmoc chemistry. Peptides were analysed by HPLC to ensure "immunological grade" purity, prior to conjugation. Each immunisation requires 50-100μg of antigen, approximately 5mg of custom peptides is originally synthesised. the peptide is then conjugated to BSA or keyhole limpet haemocyanin (KLH) and mixed with Complete or Incomplete Freund's Adjuvant for immunisation. Peptide sequences from conserved regions of the PtdInsP5K gene and the X25 sequence were used having the sequences:

AAPGKQNEEKTYKKTA; and

LKEKEEETPQNVPDAK and

CNKQTPNKQIWLSSPS; and CNLRKSGTLGHPGSLD

respectively.

Using a rabbit as host, the conjugated peptides were injected subcutaneously at multiple sites. A "two-week" immunisation schedule was applied, with bleeds performed in alternate weeks. The initial immunisation was given in Complete Freund's with all subsequent immunisation boosts given in Incomplete Freund's Adjuvant.

Prefeπed features of each aspect of the invention are as for each other aspect, mutatis mutandis.

Claims

1. An isolated and purified DNA molecule encoding the STM7.I gene associated with FRDA wherein said gene is characterised by the nucleotide sequences as set forth in either of Figures 7 or 16.

2. An isolated and purified DNA molecule encoding the STM7.I gene associated with FRDA wherein said gene is characterised by the amino acid sequences as set forth in Figure 8 or a functionally active variant or derivative thereof.

3. A purified gene sequence comprising the DNA sequence of claim 1.

4. The isolated and purified DNA molecule of claims 1 and 2 wherein said DNA molecule encodes for phosphatidylinositol-4-phosphate 5-kinase (PtdInsP5K) or an analogue or fragment of such DNA molecule which encodes for an isoform of PtdInsp5K.

5. An expression vector for the expression of PtdInsP5K in a recombinant host cell wherein said expression vector contains the DNA molecule of claim 1.

6. A host cell which expresses a recombinant PtdInsp5K peptide wherein said host cell contains the expression vector of claim 5.

7. A process for the expression of PtdInsP5K in a recombinant host cell, comprising:

(a) transfecting the expression vector of claim 5 into a suitable host cell; and

(b) culturing the host cells under conditions which allow expression of PtdinsP5K from the expression vectors.

8. A fusion protein comprising the amino acid sequence of claim 2.

9. A DNA probe comprising a DNA sequence of at least about 10 nucleotides, preferably 20 and most preferably 50 nucleotides, selected from the sequence of claim 1.

10. A transgenic host comprising a nucleic acid segment encoding at least exons 1 to 16 inclusive of STM7.I encoding PtdInsP5K or an PtdInsP5K isoform.

11. The nucleic acid segment of claim 10 which includes exons 7 to 11, inclusive.

12. A host of claim 10 or 11 which is a primary or immortalised eukaryotic cell line.

13. A host of claim 10 or 11 which is a bacterium.

14. A host of claim 10 or 11, wherein the nucleic acid segment is integrated into the host genome.

15. A host of claim 10 or 11, which is a non-human animal having the nucleic acid segment of claim 10 incoφorated into its germline and which is capable of expressing PtdInsP5k.

16. A host of claim 15, wherein the recombinant PtdinsP5K protein is the sole

PtdInsP5K protein produced by the animal.

17. A transgenic non-human animal with germ cells or somatic cells comprising a heterologous STM7 gene encoding PtdInsP5K, which gene upon expression promotes the neuropathological characteristics of Friedreich' s Ataxia in the animal .

18. A diagnostic method for determining an inherited predisposition to Friedreich's Ataxia in a subject comprising detecting in the subject the presence of an allele of PtdInsP5K, an isoform or fragment thereof, wherein said allele has the sequence of claim 1 or claim 2.

19. A method according to claim 18, wherein the detecting step comprises (i) mixing a nucleic acid sample from the subject with one or more polynucleotide probes capable of hybridising selectively to a PtdInsP5K gene allele in a reaction and (ii) monitoring the reaction to determine the presence of the gene allele in the sample, thereby indicating whether the subject is at risk for FRDA.

20. A method according to claim 19, wherein one probe is a polynucleotide comprising a sequence of at least about 10 nucleotides spanning exons 1 to 16, inclusive of STM7.

21. The method of claim 20 wherein the polynucleotide comprises a sequence spanning exons 7 to 11, inclusive.

22. An antibody to PtdInsP5k wherein said antibody is raised against a peptide with an amino acid sequence of AAPGKQNEEKTYKKTA or

LKEKEEETPQNVPDAK.

23. An antibody to a conserved region of the X25 sequence wherein said antibody is raised against a peptide with an amino acid sequence of CNKQTPNKQIWLSSPS or CNLRKSGTLGHPGSLD .