WO2002027022A2

WO2002027022A2 - Methods for diagnosing and treating autosomal dominant optic atrophy

Info

Publication number: WO2002027022A2
Application number: PCT/GB2001/004284
Authority: WO
Inventors: Shomi Bhattacharya; Bernd Wissinger; Christiana Alexander; Marcela Votruba
Original assignee: University College London; University Eye Hospital
Priority date: 2000-09-26
Filing date: 2001-09-26
Publication date: 2002-04-04
Also published as: WO2002027022A3; GB0023555D0; AU2001290103A1

Abstract

The invention relates generally to the OPA1 gene, including variants thereof, as well as their transcripts and gene products. OPA1 is involved in the disease process of autosomal dominant optic atrophy. The present invention also relates to methods of screening for and detection of carriers of a defective OPA1 gene, diagnosis of a defective OPA1 gene, prenatal OPA1 gene defect screening and detection, gene therapy utilising recombinant technologies and drug therapy using the information derived from the gene, protein, and the metabolic function of the protein.

Description

Improvements in and relating to Treatments for Eye Disease

Field of the invention The present invention relates generally to the diagnosis and treatment of autosomal dominant optic atrophy. More particularly, the invention relates to the identification of a gene involved in causing the disease. The invention relates to the gene, including variants thereof, as well as their transcripts and gene products. The present invention also relates to methods of screening for and detection of carriers of a defective gene, diagnosis of a defective gene, prenatal gene defect screening and detection, gene therapy utilising recombinant technologies and drug therapy using the information derived from the gene, protein, and the metabolic function of the protein.

Background of the invention

Autosomal dominant optic atrophy (ADOA) is the most prevalent hereditary optic neuropathy resulting in progressive loss of visual acuity, centrocoecal scotoma, bilateral temporal atrophy of the optic nerve with an onset within the first two decades of life. (Hoyt, C.S. Autosomal dominant optic atrophy. A spectrum of disability. Ophthalmology. 87,245-251 (1980) and Votruba, M. et al .

Clinical features in affected individuals from 21 pedigrees with dominant optic atrophy. Arch . Ophthalmol . 116,351-358 (1998) .)

ADOA occurs with an estimated disease prevalence of between 1:12,000 and 1:50,000. (Kivlin, J.D., Lovrien, E.W., Bishop, D.T. & Maumenee, I.H. Linkage analysis in dominant optic atrophy. Am . J. Hum. Genet . 35, 1190-1195 (1983), Kjer, B., Eiberg, H. , Kjer, P. & Rosenberg, T. Dominant optic atrophy mapped to chromosome 3q region. II. Clinical and epidemiological aspects. Acta Ophthalmol . Scand. 1996 74,3-7 (1996) and Lyle, W.M. Genetic risks. Waterloo, Ontario, University of Waterloo Press. (1990).) The disease is highly variable in expression and shows incomplete penetrance in some families (Hoyt, C.S. Autosomal dominant optic atrophy. A spectrum of disability. Ophthalmology. 87,245-251 (1980), Votruba, M. et al . Clinical features in affected individuals from 21 pedigrees with dominant optic atrophy. Arch . Ophthalmol . 116,351-358 (1998) and Johnston, R.L., Seller, M.J., Behnam, J.T., Burdon, M.A. & Spalton, D.J. Dominant optic atrophy. Refining the clinical diagnostic criteria in light of genetic linkage studies. Ophthalmology. 106,123-128 (1999).) Histopathological post-mortem examination of donor eyes suggests that the fundamental pathology of ADOA is a primary degeneration of retinal ganglion cells followed by ascending atrophy of the optic nerve. (Johnston, P.B.,

Gaster, R.N. , Smith, V.C. & Tripathi, R.C . A clinicopathologic study of autosomal dominant optic atrophy. Am. J. Ophthalmol . 88,868-875 (1979) and Kjer, P., Jensen, O.A. & Klinken, L. Histopathology of eye, optic nerve and brain in a case of dominant optic atrophy. Acta Ophthalmol . (Copenh) .61, 300-312 (1983) .)

The predominant locus for ADOA ( OPAl , OMIM#165500) has been mapped to a 1.4 cM interval on chromosome 3q28-q29 (Eiberg, H., Kjer, B., Kjer, P. & Rosenberg, T. Dominant optic atrophy (OPAl) mapped to chromosome 3q region. I. Linkage analysis. Hum. Mol . Genet . 3, 977-980 (1994), Jonasdottir, A., Eiberg, H., Kjer, B., Kjer, P. & Rosenberg, T. Refinement of the dominant optic atrophy locus (OPAl) to a 1.4-cM interval on chromosome 3q28-3q29, within a 3-Mb YAC contig. Hum. Genet . 99, 115-120 (1997) and Brown, J. et al . Clinical and genetic analysis of a family affected with dominant optic atrophy. Arch . Ophthalmol . 115,95-99 (1997)) flanked by markers D3S3669 and D3S3562. Linkage in a single family defined a second locus on 18ql2.2. -ql2.3 ( OPA4) . (Kerrison, J.B. et al . Genetic heterogeneity of dominant optic atrophy, Kjer type: Identification of a second locus on chromosome 18ql2.2-12.3. Arch . Ophthalmol . 117,805-810 (1999) .) The precise chromosomal location and the sequence of the OPAl gene has remained unknown until now.

Glossary of terms

"Gene", as used herein, includes the coding sequence, non-coding introns, and upstream and downstream control elements of a gene.

"OPAl gene", as used herein encompasses, except where otherwise specified, any OPAl gene of any species, i.e. of any animal, especially human, including a normal OPAl gene, its functional equivalents and any mutant form of the gene. "Normal OPAl gene", as used herein encompasses an OPAl gene which, upon transcription and translation, gives rise to a normal OPAl polypeptide, for example a form of the gene found in subjects who do not have clinically diagnosed autosomal dominant optic atrophy. "OPAl polypeptide" and "OPAl gene product" are used herein interchangeably and, as used herein, encompass, except where otherwise specified, a polypeptide encoded by the coding sequence of any OPAl gene, including a normal OPAl gene and any mutant form of the gene and including any fragment of less than full length and including any immature polypeptide .

"OPAl protein" as used herein encompasses, except where otherwise specified, a protein corresponding to the sequence of any OPAl gene, including a normal OPAl gene and any mutant form of the gene and including any intermediate immature protein.

"Defective OPAl gene" is taken herein to mean an OPAl gene comprising one or more mutations, which may be in the coding sequence or in a control sequence, which cause the gene product of the gene not to carry out its normal function and/or cause the gene product to be produced at so low a level that it does not carry out its function effectively.

Summary of the invention

A gene, defects in which are involved in the autosomal dominant optic atrophy disease process, hereinafter called the OPAl gene, has been identified, isolated, its cDNA cloned, and its transcripts and gene products identified and sequenced. Mutations of the gene were identified in seven independent human families, members of which families were diagnosed clinically as having ADOA. The mutations identified include mis-sense and non-sense alterations, deletions and insertions, which segregate with disease in these families. With the identification and sequencing of the gene, its transcripts and its gene products, nucleic acid probes and primers derived from the OPAl gene and antibodies capable of binding to a product of the OPAl gene can be used in a variety of hybridisation and immunological assays to screen for, and to detect the presence of, either a normal or a defective OPAl gene or gene product. Assay kits for such screening and diagnosis are provided.

It has been found that the OPAl polypeptide is required in the body on a regular basis. Gene therapy is therefore a particularly convenient way to treat autosomal dominant optic atrophy as it enables the provision of a constant supply of polypeptide or correction of the defective gene, for example as discussed below.

Gene therapy may be carried out by means of supplementation of cells lacking a functional OPAl polypeptide with a normal OPAl gene product, or fragment or analogue thereof. Production of a suitable gene product, fragment or analogue thereof may be achieved using recombinant techniques. For example, a suitable vector may be inserted into a host cell and expressed in that cell. Synthesis of the gene product may take place in vi tro by culture of suitable cells followed by isolation of the product. Suitable cells may be obtained by treatment with a vector including a DNA fragment encoding the gene product arranged to integrate into the host genome. Optionally the integration may occur by homologous recombination. Various ' recombination vectors are known. Alternatively, synthesis may take place in vivo in the body by administering to the body a nucleic acid or vector including the gene, or exogenous cells engineered to synthesise the gene product. Such cells may optionally be enclosed in a suitable scaffold.

A defective gene may be corrected or modified. Such gene therapy may take several forms . The gene may be corrected ex vivo in cells obtained from a sufferer of the disease in question. In the laboratory, the cells may be manipulated to correct the gene defect using recombinant techniques to alter the genomic sequence, or the defect may be corrected by introduction into the cells of one or more autonomous DNA molecules capable of expressing a normal gene product. After manipulation, the cells may be returned to the patient.

Alternatively, correction or modification may take place in si tu in the patient. The patient may be treated with a suitable, optionally targeted, vector which comprises DNA of the normal gene sequence. DNA may be a DNA fragment and may be associated with portions which facilitate integration into the host genome. Optionally, the integration may occur by homologous recombination. Various recombination vectors are known, for example RNA/DNA chimeric oligonucleotide based vectors. The vector may, alternatively, be constructed so as to correct the defect by expression of a normal gene product from an autonomous DNA molecule within the cell. Advantageously, gene therapy is targeted to the optic nerve or retina. This may be achieved by targeting a gene therapy vehicle to optic nerve or retinal cells. Alternatively or additionally, it may be achieved by placing the gene under the control of a specific expression control element that directs expression in nerve cells, for example optic nerve cells or retinal cells.

Detailed description of the invention According to a first aspect, the invention provides a method of detecting an OPAl gene in a sample obtained from a subject which method comprises contacting the sample with at least one oligonucleotide and determining whether binding occurs, wherein the at least one oligonucleotide comprises at least one of the following: a) the DNA sequence of Figure 2 (SEQ. ID.NO: 1) from nucleotide residue position 1 to position 2935, b) a DNA sequence encoding an OPAl polypeptide having the sequence according to Figure 3 (SEQ . ID.NO : 2 ) from amino acid residue position 1 to position 978, c) a DNA sequence which consists of at least 16 sequential nucleotides of the sequence of a) , d) a DNA sequence which comprises at least 18 nucleotides and encodes a fragment of the amino acid sequence of Figure 3 (SEQ . ID. O: 2) , e) a DNA sequence encoding an epitope encoded by at least 18 sequential nucleotides in the sequence of Figure 2 (SEQ. ID.NO: 1) from nucleotide residue position 1 to position 2935, f) a DNA sequence encoding a mutant of a sequence according to a) , b) , c) , d) or e) , g) a DNA sequence complementary to a sequence according to a) , b) , c) , d) , e) or f) , and h) an RNA sequence corresponding to a sequence according to a) , b) , c) , d) , e) , f) or g) .

An oligonucleotide c) is, for example, a DNA sequence which consists of at least 18 sequential nucleotides selected from the DNA sequence of Figure 2 (SEQ . ID.NO: 1) from nucleotide residue position 1 to position 2935, preferably, at least 21 sequential nucleotides from nucleotide residue position 1 to position 2935.

An oligonucleotide d) is, for example, a DNA sequence which comprises at least 21 nucleotides and encodes a fragment of the amino acid sequence of Figure 3 (SEQ . ID .NO : 2 ) .

An oligonucleotide e) is, for example, a DNA sequence encoding an epitope encoded by at least 21 sequential nucleotides in the sequence of Figure 2 (SEQ. ID.NO: 1) .

In a preferred embodiment, the oligonucleotide is directed to a mutant OPAl gene. Preferably, the oligonucleotide for a mutant OPAl gene is an oligonucleotide according to the first aspect of the invention and includes one or more mutations selected from the group consisting of: a) 869 G>A; b) 1016 del C; c) 1096 OT; d) Del 1296 CAT; e) 1354 del G; f) 1644 ins T; g) Deletion of exon 20.

The symbol ">" indicates that the base indicated to the left of the symbol is replaced by the base to the right of the symbol. The symbol "del" indicates that the base or bases named at or starting from the position indicated is/are deleted from the sequence. The symbol "ins" indicates that the named base is inserted into the sequence at the base position named. Preferably, the oligonucleotide is (i) a mutant DNA sequence;

(ii) a DNA sequence complementary to a mutant DNA sequence; or (iii) an RNA sequence corresponding to a sequence according to (i) or (ii) The open reading frame of the OPAl gene extends from position 56 to position 2935 of the nucleotide sequence shown in Figure 2 (corresponding to amino acid position 19 to 978 in Figure 3) . Accordingly, position 1 in the coding sequence corresponds to position 56 in Figure 2 (SEQ . ID.NO: 1) . The nucleotide positions described for the mutations are counted from the translation start position. The ATG "start" codon of the OPAl gene at positions 56 to 58 is indicated in bold in Figure 2 and the starting amino acid methionine is indicated in bold in Figure 3. In the nucleotide and amino acid designations referred to herein (except in reference to the cDNA sequence in Figure 2 (SEQ . ID.NO: 1) or the amino acid sequence in Figure 3 (SEQ. ID.NO: 2) ) position 1 corresponds to position 56 in the nucleotide sequence and position 1 corresponds to position 19 in the amino acid sequence respectively. Furthermore, position 2935 of the nucleotide sequence of Figure 2 (SEQ. ID.NO: 1) corresponds nucleotide position 2880 of the coding sequence and amino acid position 978 corresponds to amino acid position 960 of the expressed polypeptide.

Position 2935 of the nucleotide sequence of Figure 2 (SEQ. ID.NO: 1) forms the third position of the codon corresponding to amino acid position 978 of the amino acid sequence of Figure 3 (SEQ . ID.NO: 2 ) . In view of the fact that the ORF extends from position 56 of the nucleotide sequence shown in Figure 2 it is the sequence from nucleotide residue position 56 to position 2935 that is of particular interest. Likewise, with regard to the amino acid sequence of Figure 3 (SEQ . ID.NO: 2) , it is the sequence from nucleotide residue position 19 to position 978 that is of particular interest.

According to a second aspect of the invention, there is provided a method of detecting a mutant OPAl gene in a sample obtained from a subject which method comprises contacting the sample with an oligonucleotide and determining whether binding occurs, wherein the at least one oligonucleotide comprises a mutant of at least one of the following: a) the DNA sequence which corresponds to the DNA sequence of Figure 2 (SEQ . ID.NO : 1) from nucleotide residue position 1 to position 2935, b) a DNA sequence encoding an OPAl polypeptide having the sequence according to Figure 3 (SEQ . ID.NO: 2 ) from amino acid residue position 1 to position 978, c) a DNA sequence which consists of at least 16 sequential nucleotides of the sequence of a) , d) a DNA sequence which comprises at least 18 nucleotides and encodes a fragment of the amino acid sequence of Figure 3 (SEQ . ID.NO: 2) , e) a DNA sequence encoding an epitope encoded by at least 18 sequential nucleotides in the sequence of Figure 2

(SEQ. ID.NO: 1) from nucleotide residue position 1 to position

2935, or a DNA sequence complementary to such a mutant DNA sequence or an RNA sequence corresponding to such a mutant DNA sequence or its complement.

In a preferred embodiment, the mutation is selected from the group consisting of: a) 869 G>A; b) 1016 del C; c) 1096 OT; d) Del 1296 CAT; e) 1354 del G; f) 1644 ins T; g) Deletion of exon 20.

As described above, the nucleotide positions described for the mutations are counted from the translation start position. The invention further provides a method of diagnosing autosomal dominant optic atrophy using a method according to the first or second aspect of the invention wherein binding of a mutant sequence is indicative of the disease.

Preferably the method is one wherein the sample is derived from a human foetus in utero .

In a preferred embodiment, the method according to the first or second aspect of the invention comprises the steps of providing a sample that has been obtained from subject; and providing a method for detecting the presence of a normal OPAl gene, a mutant OPAl gene or a mixture thereof in the sample.

According to a third aspect of the invention, there is provided a method of detecting an OPAl gene product in a sample obtained from a subject which method comprises contacting a sample with at least one antibody capable of binding to a peptide and determining whether binding occurs wherein the antibody is capable of binding to at least one of: a) a polypeptide which is encoded by the DNA sequence of Figure 2 (SEQ. ID.NO: 1) from nucleotide residue position 1 to position 2935, b) an OPAl polypeptide having the sequence according to Figure 3 (SEQ . ID.NO : 2) from amino acid residue position 1 to position 978, c) a polypeptide which is encoded by at least 15 sequential nucleotides of the sequence of a) , d) a polypeptide which comprises at least 5 amino acids and includes at least 5 sequential amino acids of the sequence of Figure 3 (SEQ. ID.NO: 2 ) from amino acid residue position 1 to position 978, e) an epitope encoded by at least 15 sequential nucleotides in the sequence of Figure 2 (SEQ. ID.NO: 1) from nucleotide residue position 1 to position 2935, and f) a polypeptide which is a mutant of a sequence according to a) , b) , c) , d) or e) .

In a fourth aspect of the invention, there is provided a method of detecting a mutant OPAl gene product in a sample obtained from a subject which method comprises contacting the sample with an antibody capable of binding to a peptide and determining whether binding occurs wherein the antibody is capable of binding to a mutant polypeptide (f) and the mutation in the polypeptide (f) is selected from the group consisting of those encoded by the following nucleic acid mutations: a) 869 G>A; b) 1016 del C; c) 1096 OT; d) Del 1296 CAT; e) 1354 del G; f) 1644 ins T; g) Deletion of exon 20.

As described above^ the nucleotide positions described for the mutations are counted from the translation start position.

The invention further provides a method of diagnosing autosomal dominant optic atrophy using the method according to the third or fourth aspect of the invention wherein binding of a mutant polypeptide is indicative of the disease. Preferably the sample is derived from a human foetus in utero .

In a preferred embodiment, the method according to the third or fourth aspect of the invention comprises the steps of providing a sample obtained from a subject; and providing a method for detecting, in the sample, the presence of a normal OPAl gene product, a mutant OPAl gene product or a mixture thereof .

The method according to the first, second, third or fourth aspect of the invention is carried out on a sample obtained from a subject. The sample may be of a body fluid, for example, whole blood, plasma or serum or may be saliva, urine, cerebrospinal fluid, joint fluid, sweat or tears. The sample may be derived from tissue, brought into an appropriate form, either solid or liquid, for the assay to be carried out. In methods in which oligonucleotides are used, the sample should contain nucleic acids. In methods in which antibodies are used, the sample should contain proteins.

The method according to the first or second aspect of the invention may comprise a hybridisation assay, preferably a hybridisation assay making use of a labelled nucleotide probe. In a suitable assay, a sample is contacted with an oligonucleotide probe and it is determined whether binding to the probe occurs. The assay can employ any suitable hybridisation method making use of a DNA having a sequence according to the invention. Such methods are well known and may include an amplification step, for example PCR (Polymerase Chain Reaction) , RT-PCR (Reverse Transcriptase PCR) , TMA (Transcription Mediated Amplification) or NASBA (Nucleic Acid Sequence-Based Amplification) . In the case where the method includes an amplification step, the oligonucleotide may a primer for the amplification.

The method according to the third or fourth aspect of the invention may comprise an immunological assay. Preferably, the method makes use of an antibody capable of binding to a normal OPAl polypeptide or an antibody capable of binding to a mutant OPAl polypeptide . Polyclonal antibodies may be used. The method may include at least one monoclonal antibody. Preferably the assay is a radioimmunoassay or an ELISA. Suitable assays are known in the art as described in, for example, Kemeny & Challacome (ELISA and other Solid Phase Immunoassays, Theoretical and Practical Aspects, John Wiley, 1988) and Tsu & Herzenberg ("Solid Phase Radioimmunoassay" in "Selected Methods in

Cellular Immunology", Mishell & Shiigi Eds., pages 373-397, Freeman, San Francisco, 1980) .

According to a fifth aspect of the invention, there is provided a kit for detecting the presence of an OPAl gene comprising an oligonucleotide according to the first or second aspect of the invention and other reagents required for carrying out a suitable assay. There is further provided a kit for detecting the presence of an OPAl gene product comprising an antibody according to the third or fourth aspect of the invention which is capable of binding to a gene product of an OPAl gene together with other reagents required for carrying out a suitable assay. Preferably a kit for the detection of an OPAl gene product comprises an antibody capable of binding to a gene product of an OPAl gene. The OPAl gene may be a normal OPAl gene or a mutant OPAl gene. According to a sixth aspect of the invention, there is provided an isolated, for example purified, OPAl gene comprising a DNA sequence encoding an amino acid sequence for a polypeptide, said polypeptide, if expressed in an altered, defective or non-functional form in cells of the human body, being associated with altered cell function which correlates with the genetic disease autosomal dominant optic atrophy. The OPAl gene may be a normal OPAl gene or a mutant OPAl gene. According to a seventh aspect of the invention, there is provided an isolated, for example purified, RNA molecule comprising an RNA sequence encoded by a DNA sequence according to the first or second aspect of the invention. According to an eighth aspect of the invention, there is provided an oligonucleotide comprising a DNA or RNA nucleotide having the sequence corresponding to at least one of: c) a DNA sequence which consists of at least 16 sequential nucleotides of the sequence of a) according to the first aspect of the invention, d) a DNA sequence which comprises at least 18 nucleotides and encodes a fragment of the amino acid sequence of Figure 3 (SEQ . ID.NO: 2) , e) a DNA sequence encoding an epitope encoded by at least 18 sequential nucleotides in the sequence of Figure 2 (SEQ. ID.NO :1) from nucleotide residue position 1 to 2935, f) a DNA sequence encoding a mutant of a sequence according to c) , d) or e) , and g) a DNA sequence complementary to a sequence according to c) , d) , e) or f) .

In a preferred embodiment, the oligonucleotide comprises a DNA or RNA nucleotide sequence corresponding to (i) a mutant DNA sequence (f) ; (ii) a DNA sequence complementary to a mutant DNA sequence (f) ; or (iii) an RNA sequence corresponding to a sequence according to (i) or (ii) and the mutation is selected from the group consisting of: a) 869 G>A; b) 1016 del C; c) 1096 OT; d) Del 1296 CAT; e) 1354 del G; f ) 1644 ins T; g) Deletion of exon 20.

The nucleotide positions described for the mutations are counted from the translation start position. The oligonucleotide according to the eighth aspect of the invention preferably comprises at least 18 nucleotides, more preferably it comprises at least 21 nucleotides.

The oligonucleotide according to the eighth aspect of the invention may be suitable for use as a probe or as a primer for a DNA amplification reaction, for example PCR.

The invention further provides a pair of primers for a DNA amplification reaction at least one of which has the sequence selected from: c) a DNA sequence which consists of sequential nucleotides of the sequence of a) according to the first aspect of the invention, d) a DNA sequence which comprises sequential nucleotides and encodes a fragment of the amino acid sequence of Figure 3 (SEQ. ID.NO: 2) , e) a DNA sequence encoding an epitope encoded by sequential nucleotides in the sequence of Figure 2

(SEQ. ID.NO: 1) from nucleotide residue position 1 to 2935, f) a DNA sequence encoding a mutant of a sequence according to c) , d) or e) , and g) a DNA sequence complementary to a sequence according to c) , d) , e) or f) .

In a preferred embodiment, the oligonucleotide comprises a DNA or RNA nucleotide sequence corresponding to

(i) a mutant DNA sequence (f) ; (ii) a DNA sequence complementary to a mutant DNA sequence

(f) ; or

(iii)an RNA sequence corresponding to a sequence according to

(i) or (ii) and the mutation is selected from the group consisting of: (a) 869 G>A;

(b) 1016 del C;

(c) 1096 OT;

(d) Del 1296 CAT; (e) 1354 del G;

(f) 1644 ins T;

(g) Deletion of exon 20.

The nucleotide positions described for the mutations are counted from the translation start position. The pair of primers may be suitable for any PCR reaction, including RT-PCR. Suitably the primers are at least 10 nucleotides long, preferably at least 12 nucleotides long, more preferably at least 13 nucleotides long, for example 14 nucleotides long. According to a ninth aspect of the invention there is provided a recombinant cloning or expression vector comprising a DNA molecule having a sequence as defined in the first, second or sixth aspect of the invention. Preferably said DNA molecule is operatively linked to an expression control sequence in the vector so that an OPAl polypeptide is expressed by the molecule, said expression control sequence being selected from sequences that control the expression of genes of prokaryotic cells, eukaryotic cells or viruses of prokaryotic or eukaryotic organsims, or combinations thereof. According to a tenth aspect of the invention, there is provided a host cell transformed with a vector according to the ninth aspect of the invention. The host cell may be a eukaryotic or a prokaryotic cell. Preferably the host cell is selected from the group consisting of bacterial cells, for example selected from strains of E. coli, Pseudomonas, Bacillus subtilis, Bacillus stearothermophilus or other bacilli; yeast or fungal cells; or plant or animal cells, for example insect, mouse, other animal or human cells. More preferably, the host cell is a human retinal cell. According to an eleventh aspect of the invention, there is provided a method of producing an OPAl polypeptide comprising the steps of: a) culturing a host cell transfected with a vector according to the ninth aspect of the invention in a medium and under conditions suitable for expression of the polypeptide and optionally b) isolating the expressed OPAl polypeptide.

According to a twelfth aspect of the invention there is provided an isolated, for example purified, normal or mutant OPAl polypeptide characterised by a molecular weight of about 112 kDa.

According to a thirteenth aspect of the invention, there is provided a normal or mutant OPAl polypeptide substantially free of other human proteins and encoded by a DNA sequence selected from: a) a DNA sequence which corresponds to the DNA sequence of Figure 2 (SEQ. ID.NO: 1) from nucleotide residue position 1 to position 2935, b) a DNA sequence encoding an OPAl polypeptide having the sequence according to Figure 3 (SEQ . ID.NO : 2) from amino acid residue position 1 to position 978, c) a DNA sequence which consists of at least 16 sequential nucleotides of the sequence of a) , d) a DNA sequence which comprises at least 18 nucleotides and encodes a fragment of the amino acid sequence of Figure 3 (SEQ . ID.NO : 2) , e) a DNA sequence encoding an epitope encoded by at least 18 sequential nucleotides in the sequence of Figure 2 (SEQ. ID.NO: 1) from nucleotide residue position 1 to 2935, and f) a DNA sequence encoding a mutant of a sequence according to a) , b) , c) , d) or e) .

Preferably a mutant DNA sequence (f) wherein the mutation is selected from the group consisting of: a) 869 G>A; b) 1016 del C; c) 1096 OT; d) Del 1296 CAT; e) 1354 del G; f) 1644 ins T; g) Deletion of exon 20.

The nucleotide positions described for the mutations are counted from the translation start position. (Position 1 of the coding sequence corresponds to position 56 on the sequence of Figure 2 (SEQ. ID.NO: 1) ) .

The invention further provides a normal or mutant OPAl polypeptide according to the twelfth or thirteenth aspect of the invention made by chemical synthesis or by in vi tro enzymic peptide synthesis. Fragments of said polypeptide may be prepared by such techniques .

The invention further provides a polypeptide fragment comprising a portion of the amino acid sequence of the thirteenth aspect of the invention. Preferably the fragment comprises 6 or more amino acids. More preferably the fragment comprises 10 or more amino acids. Still more preferably the fragment comprises 15 or more amino acids.

A polypeptide or polypeptide fragment according to the invention may be in the form of a fusion protein, that is to say, covalently linked via a peptide bond to another polypeptide sequence. For example a fusion protein may be formed with a polypeptide portion or a fragment of a polypeptide that facilitates isolation and optionally purification of the OPAl polypeptide or fragment thereof. According to a further aspect of the invention there is provided a polypeptide or polypeptide fragment according to the twelfth or thirteenth aspects of the invention for use as a medicament. According to a further aspect of the invention there is provided a polypeptide or polypeptide fragment according to the twelfth or thirteenth aspects of the invention for use in the treatment of a medical condition resulting from a defect in the OPAl gene.

According to a further aspect of the invention there is provided a polypeptide or polypeptide fragment according to the twelfth or thirteenth aspects of the invention for use in the treatment of autosomal dominant optic atrophy. According to a further aspect of the invention there is provided the use of a polypeptide or polypeptide fragment according to the twelfth or thirteenth aspects of the invention in the manufacture of a medicament for the treatment of a medical condition resulting from a a defect in the OPAl gene.

According to a further aspect of the invention there is provided the use of a polypeptide or polypeptide fragment according to the twelfth or thirteenth aspects of the invention in the manufacture of a medicament for the treatment of autosomal dominant optic atrophy.

According to a fourteenth aspect of the invention there is provided an antibody capable of binding to a polypeptide or polypeptide fragment according to the twelfth or thirteenth aspects of the invention. Preferably the antibody is a mouse or a human antibody. The antibody may be polyclonal or monoclonal . A non-human antibody may optionally be "humanised" and/or chimeric.

The invention further provides a kit for detecting the presence of an OPAl gene product comprising an antibody according to the thirteenth aspect of the invention. The

OPAl gene product may be a normal OPAl gene product or a mutant OPAl gene product .

According to a fifteenth aspect of the invention there is provided a DNA molecule comprising at least one of: a) a DNA sequence of Figure 2 (SEQ . ID.NO: 1) from nucleotide residue position 1 to 2935, b) a DNA sequence encoding an OPAl polypeptide having the sequence according to Figure 3 (SEQ. ID.NO: 2 ) from amino acid residue position 1 to position 978, c) a DNA sequence which consists of at least 16 sequential nucleotides of a) , d) a DNA sequence which comprises at least 18 nucleotides and encodes a fragment of the amino acid sequence of Figure 3 (SEQ . ID.NO: 2) , e) a DNA sequence encoding an epitope encoded by at least 18 sequential nucleotides in the sequence of Figure 2 (SEQ.ID.NO:l) from nucleotide residue position 1 to 2935, f) a DNA sequence encoding a mutant of a sequence according to a) , b) , c) , d) or e) , g) a DNA sequence complementary to a sequence according to a) , b) , c) , d) , e) or f) , or a vector according to the ninth aspect of the invention, for use in medicine. Preferably the DNA molecule is a mutant DNA sequence (f) selected from the group consisting of: a) 869 G>A; b) 1016 del C; c) 1096 OT; d) Del 1296 CAT; e) 1354 del G; f) 1644 ins T; g) Deletion of exon 20.

There is further provided a DNA molecule according to the fifteenth aspect of the invention for use in the treatment of a medical condition resulting from a defect in the OPAl gene. There is further provided a DNA molecule according to the fifteenth aspect of the invention for use in the treatment of autosomal dominant optic atrophy.

The invention further provides the use of a DNA molecule according to the fifteenth aspect of the invention in the manufacture of a medicament for the treatment of a medical condition resulting from a defect in the OPAl gene.

The invention further provides the use of a DNA molecule according to the fifteenth aspect of the invention in the manufacture of a medicament for the treatment of autosomal dominant optic atrophy.

According to a further aspect of the invention, there is provided a non-human animal comprising a heterologous cell system comprising a recombinant cloning vector according to the ninth aspect of the invention which induces autosomal dominant optic atrophy in the animal .

According to a further aspect of the invention, there is provided a transgenic mouse comprising a mutant OPAl gene and exhibiting autosomal dominant optic atrophy symptoms. According to a further aspect of the invention, there is provided a DNA molecule comprising an intronless DNA sequence selected from the group consisting of: a) a DNA sequence which corresponds to the DNA sequence of Figure 2 (SEQ . ID.NO: 1) from nucleotide residue position 1 to 2935 comprising one or more of the following mutations:

(i) 869 G>A

(ii) 1016 del C

(iii) 1096 OT

(iv) Del 1296 CAT (v) 1354 del G

(vi) 1644 ins T

(vii) Deletion of exon 20 b) a DNA sequence encoding a mutant OPAl polypeptide having the sequence according to Figure 3 (SEQ. ID.NO: 2) from amino acid residue position 1 to position 978 comprising one or more of the mutations defined in a) , c) a DNA sequence which consists of at least 16 sequential nucleotides of the sequence of a) or b) , d) a DNA sequence which comprises at least 16 nucleotides and encodes a fragment of the amino acid sequence coded for by the sequence of a) or b) including at least a part of the mutated portion, e) a DNA sequence encoding an epitope encoded by at least 18 sequential nucleotides in the sequence of a) or b) including at least a part of the mutated portion, and f) a DNA sequence complementary to a sequence according to a) , b) , c) , d) , or e) .

Preferably the DNA molecule is a cDNA molecule. According to a further aspect of the invention, there is provided a DNA molecule comprising an intronless DNA sequence selected from the group consisting of: a) a DNA sequence which corresponds to the DNA sequence of Figure 2 (SEQ . ID.NO: 1) from nucleotide residue position 1 to 2935, b) a DNA sequence encoding an OPAl polypeptide having the sequence according to Figure 3 (SEQ. ID.NO: 2) from amino acid residue position 1 to position 978, c) a DNA sequence which consists of at least 16 sequential nucleotides of the sequence of a) , d) a DNA sequence which comprises at least 18 nucleotides and encodes a fragment of the amino acid sequence of Figure 3 (SEQ. ID.NO: 2) , e) a DNA sequence encoding an epitope encoded by at least 18 sequential nucleotides in the sequence of Figure 2 (SEQ. ID.NO:l) from nucleotide residue position 1 to 2935, and f) a DNA sequence complementary to a sequence according to a) , b) , c) , d) or e) .

Preferably the DNA molecule is a cDNA molecule. According to a further aspect of the invention, there is provided a method for the diagnosis of autosomal dominant optic atrophy according to the first, second, third or fourth aspect of the invention wherein the presence of a mutation indicates the presence of, or propensity for, autosomal dominant optic atrophy.

According to a further aspect of the invention, there is provided a method of treating autosomal dominant optic atrophy comprising administering to a patient in need thereof a purified OPAl gene according to the sixth aspect of the invention, a purified RNA molecule according to the seventh aspect of the invention, a vector according to the ninth aspect of the invention, a host cell according the tenth aspect of the invention, or a polypeptide according to the twelfth or thirteenth aspect of the invention.

Brief Description of the Figures

Figure 1 shows a physical map of the OPAl interval and the genomic structure of the OPAl gene.

Figure 2 is the full length DNA sequence KIAA0567.

Figure 3 shows the amino acid sequence corresponding to the DNA sequence in Figure 2. Figure 4 shows Northern dot blot hybridisations showing the expression pattern of OPAl gene.

Figure 5 shows Northern blot hybridisations showing the expression pattern of OPAl gene.

Figure 6 shows electropherograms of various mutations of the OPAl gene.

Figures 7 and 8 show the cosegregation analysis of mutations within a pedigree.

Figure 9 shows the predicted mitochondrial import signal sequence of OPAl polypeptide. Figures 10 and 11 show a protein alignment depicting the similarity of human OPAl protein with dynamin-like proteins from other species and DYN2.

Figure 12 shows Table 2, giving Exon/Intron junctions of the human KIAA0567 gene. Intron sequences are shown by lower case letters and exon sequences by upper case letter . Approximate intron sizes were determined from PCR with exon specific primers.

Description

Isolation of the OPAl gene

The OPAl gene was identified using a positional cloning approach. A physical map of the OPAl interval and the genomic structure of OPAl are shown in Figure 1. The corresponding full length cDNA, KIAA0567, representing a gene of unknown function, had been isolated from a brain cDNA library (Nagase, T. et al . Prediction of the coding sequences of unidentified human genes. XII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res . 5 355-364 (1998)). That cDNA has been deposited in Genbank with the accession number AB011139. At the bottom of Figure 1 is an enlarged view of the genomic structure of OPAl. It consists of 28 coding exons between 54 and 319 bp in length, with the initiation and stop codon (ATG, TAA) present in exon 1 and exon 28, respectively. All exon/intron boundaries follow the GT/AG rule for consensus splice site sequences. Sizes of exons and introns are drawn to scale except for introns of unknown size, indicated by double slashes on the bar. The first in frame ATG codon is located in exon 1 leaving 56 bp of 5'UTR sequence. The 3'UTR is interrupted by at least one additional intron. The sequence

The nucleotide sequence of a cloned cDNA encoding OPAl polypeptide is shown in Figure 2 and SEQ. ID.NO: 1. Figure 3 and SEQ. ID.NO: 2 show the corresponding amino acid sequence. The translation start is at position 56 of the nucleotide sequence shown in Figure 2 (corresponding to amino acid position 19 in Figure 3) . The ATG codon starting at position 56 is indicated in bold in Figure 2 and the starting amino acid methionine is indicated in bold in Figure 3. Nucleotide and amino acid designations referred to herein (except in reference to the cDNA sequence in Figure 2 (SEQ . ID.NO: 1) or the amino acid sequence in Figure 3 (SEQ. ID.NO:2) ) commence at 1 at position 56 in the nucleotide sequence and at position 19 in the amino acid sequence respectively. The exon/intron boundaries are shown in Figure 12.

Sites of Expression

KIAA0567 has been found to be ubiquitously expressed albeit with varying abundancies (See Example 2 and Figures 4 and 5) . Strongest signals were present in the retina, followed by brain, heart, testis and skeletal muscle.

Nutations Patients from OPAl-defect linked families originating from Germany, the UK and Cuba were screened by direct sequencing of coding exons. Patients and families were recruited in different clinical centres. The diagnosis of ADOA was based on ophthalmological examination including visual acuity, visual field and colour testing, fundoscopy, electrophysiology and family history.

7 putative pathogenic mutations in index patients were identified including a non-conservative missense mutation (R290Q) , a stop codon mutation (R366X) , a 3bp in-frame deletion (I432del) , two 1 bp deletions (1016delC; 1354delG) , a deletion resulting in the complete loss of exon 20 and a lbp insertion (1644insT) . The mutations identified are shown in Table 1. Electropherogram sections illustrating selected mutations are shown in Figure 6. Analysis within the individual families revealed segregation of the mutations with the disease haplotype (see Figures 7 and 8) .

The preponderance of protein truncation mutations in defective OPAl genes (probably representing "null" alleles) suggests that the pathophysiological basis of ADOA may rely on the functional loss of one allele and may, thus result from haplo-insufficiency. Whilst not being bound by this theory, taking into account the high level of expression of OPAl gene in the retina, loss of one allele may decrease transcript level to a critical threshold in this tissue which may explain the restricted ocular phenotype. Although the number of families was small, no significant phenotypical differences between families with protein truncating mutations and those with missense mutations were noticed. Incomplete penetrance may occur in some families with ADOA and complicate genetic counselling. (Hoyt, C.S. Autosomal dominant optic atrophy. A spectrum of disability. Ophthalmology. 87,245-251 (1980), Votruba, M. et al . Clinical features in affected individuals from 21 pedigrees with dominant optic atrophy. Arch . Ophthalmol . 116,351-358 (1998) and Johnston, R.L., Seller, M.J., Behnam, J.T., Burdon, M.A. & Spalton, D.J. Dominant optic atrophy. Refining the clinical diagnostic criteria in light of genetic linkage studies. Ophthalmology. 106,123-128 (1999).) The cloning of OPAl genes, according to the present invention, allows molecular genetic diagnosis and identification of asymptomatic at risk family members. Furthermore, detailed psychophysical and electrophysiological investigation of those gene carriers may determine whether true incomplete penetrance or subtle ganglion cell dysfunction, indicating mild expression of the disease, applies.

Table 1 Mutations detected in the OPAl gene

Family Origin Exon/intron Nucleotide Predicted alteration* change

CI Cuba Exon 8 869G>A Arg290Gln Bl UK Exon 10 1016delC Frameshift, 19 novel aa then STOP

Gl Germany Exon 11 1096OT Arg366STOP B2 UK Exon 13 Dell296CAT Del432Ile G2 Germany Exon 14 1354delG Frameshift, 14 novel aa then STOP

G3 Germany Exon 17 1644insT Frameshift, 12 novel aa then STOP

B3 UK Exon 20 Deletion of Frameshift, entire exon 13 novel aa then STOP

*Nucleotide designation commencing 1 at position 56 (translation start of GenBank entry AB011139) .

OPAl polypeptide The polypeptide sequence is shown in Figure 3

(SEQ. ID.NO : 2) . Examination of the N-terminal leader sequence of the deduced protein revealed the typical features of a protein imported into the matrix space of mitochondria. This is based on 1.) an enrichment of basically charged amino acids and 2. ) the presence of the MPP/MIP cleavage consensus sequence RX(F/L/I)XX(G/S/T)XXXX. (Branda, S.et al . Yeast and human frataxin are processed to mature form in two sequential steps by the mitochondrial processing peptidase. J^". Biol . Chem. 274, 22763-22769 (1999).) Figure 9 shows the predicted mitochondrial import signal sequence of OPAl polypeptide. Three putative cleavage sites (residues 38-47, 80-89, 100- 109) matching the MPP/MIP consensus sequence are boxed.

OPAl polypeptide shows highest identity scores over the whole length to dynamin-related large GTPases from salmon, C.elegans, Drosophila and the RN protein. Figures 10 and 11 show the protein alignment depicting the similarity of human OPAl polypeptide with dynamin-like proteins from other species and DYN2. OPAl polypeptide shows highest homologies over its full length to salmon GTP-binding protein Mgl20 expressed in motor neurons of brain (75% over the entire protein) , rat RN protein (97% from residues 662-978) , a C.elegans dynamin-like GTP-binding protein (48% from residues 108-957) and Drosophila elanogaster CG8479 gene product (48% from residues 82-977) .

The C terminus of OPAl polypeptide differs from other dynamin family members in lacking a proline-rich region, a GED domain and a pleckstrin homology domain and may determine the specific functions of the protein. (Muhlberg, A.B., Warnock, D.E. & Schmid, S.L. Domain structure and intramolecular regulation ofdynamin GTPase. EMBO. 16,6676- 6683 (1997) and McNiven, M.A. , Cao, H. , Pitts, K.R. & Yoon, Y. The dynamin family of mechanoenzymes : pinching in new places. TIBS. 25, 115-120 (2000).) The GTPase domain, encompassing the core central region between amino acid residues 280-520, harbours the consensus tripartite GTP binding motif needed for phosphate binding (GXXXXGKS/T) , coordination of Mg²⁺ (DXXG) , nucleotide binding (T/NKXD) , and the dynamin sequence signature which are characteristically conserved in dynamin-related GTPases. Apart from Dynl, Dynll and Dynlll, only one dynamin-related large GTPase, Drpl has been identified in mammalian cells. This protein is located within the cytoplasm and controls mitochondrial distribution and vesicular transport. Studies in yeast have demonstrated that the dynamin-related large GTPases Dnml, MGMl and MSPl play an important role in the maintenance and inheritance of mitochondria. Of the yeast proteins, OPAl polypeptide shows greatest similarity at the primary level to MGMl (Figures 10 and 11) .

The pathophysiology and clinical symptoms observed in ADOA show remarkable overlap with those occurring in Leber's hereditary optic neuropathy. (Riordan-Eva, P. et al . The clinical features of Leber's hereditary optic neuropathy defined by the presence of a pathogenic mitochondrial mutation. Brain 118,319-337 (1995).) This disease is caused by mutations in mtDNA encoded genes for subunits of complex I of the respiratory chain. (Wallace, D.C. et al . Mitochondrial DNA mutations associated with Leber's hereditary optic neuropathy. Science 242, 1427-1430 (1988).) These mutations are believed to lead to insufficient energy supply in the highly energy-demanding neurons of the optic nerve (notably the papillomacular bundle) and to cause blindness by a catastrophic compromise of axonal transport in retinal ganglion cells. Furthermore, experimental work done in yeast indicates that impaired regulation of mitochondrial integrity can lead to loss of mitochondrial DNA and compromise respiratory competence. (Shepard, K.A. & Yaffe, M.P. The yeast dynamin-like protein, Mgmlp, functions on the mitochondrial outer membrane to mediate mitochondrial inheritance. J Cell . Biol . 144,711-720 (1999), Bleazard, W et al, The dynamin-related GTPase Dnml regulates mitochondrial fission in yeast. Nat . Cell . Biol . , 1, 298-304 (1999) and Pelloquin, L., Belenguer, P., Menon, Y. & Ducmmun, B, Identification of a fission yeast dynamin-related protein involved in mitochondrial DΝA maintenance, Biochem . Biophys . Res . Commun . 251, 720-726 (1998).) Without being bound by this theory, it is thus hypothesized that mutations in the OPAl gene affect mitochondrial integrity (perhaps exerting its function in miochondrial biogenesis or in stabilisation of mitochondrial membrane integrity) resulting in an impairment of energy supply. In the long term, this may affect normal metabolic processes in retinal ganglion cells and consequently their survival .

Methods of detection of an OPAl gene, an OPAl polypeptide or an OPAl gene product

The diagnosis method of the present invention, which is carried out on a sample isolated from a subject, can employ any suitable hybridisation method making use of a DNA having a sequence according to the invention. Such methods are well known and include PCR (Polymerase Chain Reaction) , RT-PCR (Reverse Transcriptase PCR) , TMA (Transcription Mediated Amplification) and NASBA (Nucleic Acid Sequence-Based

Amplification) . The diagnosis method of the invention can employ any suitable interaction method by using an antibody according to the invention. Such methods are well known and include ELISA (Enzyme-Linked ImmunoSorbent Assay) and methods related thereto.

Currently there exist no treatments for autosomal dominant optic atrophy. Accurate detection of the disease according to the present invention now enables a clinician to avoid the use of non-effective treatments. The discovery of the gene and gene product enables the new prophylactic and acute treatments as set out above.

Examples

Example 1: Isolation of the OPAl gene

The OPAl gene was identified using a positional cloning approach. A high density Phage Artificial Chromosome (PAC) contiguous map covering the entire OPAl gene candidate region of around 1 Mb (D343669 to D353562) was constructed. A physical map of the OPAl interval and the genomic structure of OPAl is shown in Figure 1. STSs and ESTs mapping within the refined linkage mapping interval D353669 and D353562 were used to construct the map. Vertical bars indicate the presence of STS and EST makers on the YAC and PAC clones . Only those PAC clones constituting the minimal tiling path are shown .

For the identification of candidate genes large scale sequence sampling on selected PACs representing the minimal tiling path for the OPAl interval was performed. The EST

SHGC37414 was localised to PAC H20545 (Figure 1) and found to be part of the Unigene cluster Hs . 147946 and the THC clusters 342414, 331187, and 379833. The corresponding full length cDNA, KIAA0567, representing a gene of unknown function, had been isolated from a brain cDNA library. (Nagase, T. et al . Prediction of the coding sequences of unidentified human genes. XII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res . 5 355-364 (1998)) The genomic structure of this corresponding gene was determined based on comparison of the cDNA with the genomic sequences obtained from the PAC sequencing, inter-exon PCRs, and vectorette-PCR using PAC DNA as template.

At the bottom of Figure 1 is an enlarged view of the genomic structure of OPAl covered by PAC clones J18270 and

H20545. It consists of 28 coding exons (spanning more than 40 kb of genomic sequence) each between 54 and 319 bp in length, with the initiation and stop codon (ATG, TAA) present in exon 1 and exon 28, respectively. All exon/intron boundaries follow the GT/AG rule for consensus splice site sequences. (Staden, R. , Beal, K.F. & Bonfield J .K. The Staden package, 1998. Methods Mol . Biol . 132, 115-130 (2000) and Breathnach, R. & Chambon, P. Organization and expression of eukaryotic split genes coding for proteins. Ann. .Rev. Biochem. 50,349- 383 (1981) .) Sizes of exons and introns are drawn to scale except for introns of unknown size, indicated by double slashes on the bar. The first in frame ATG codon is located in exon 1 leaving 56 bp of 5'UTR sequence. The 3'UTR is interrupted by at least one additional intron. The locations of the exon/intron boundaries are given in Figure 12.

PAC DNA was isolated using the alkaline lysis method, and the insert sizes determined by pulsed field gel electrophoretic separation of Nofcl digested PAC DΝA on a CHEF-DRIII system (Biorad) . For random subcloning, lOμg of PAC DΝA was sonicated for 3 x 20s with a Bandelin HD-70 sonicator, and the ends repaired by treatment with T4 DΝA polymerase and Klenow fragment in the presence of 200 μM dΝTPs . Fragments were size selected on agarose-gels prior to ligation with Smal linearized, dephosphorylated pUC19. The ligations were used for electro-transformation of E. coli DH10B and clones were selected on IPTG/X-Gal/ampicillin plates . Subclone DΝA was prepared from 1 ml cultures on a BioRobot 9600 (Quiagen) and sequenced with standard Ml3 forward/reverse primers using Big Dye Terminator chemistry (PE Biosystems) . Sequences were obtained on an ABI377 DΝA sequencer and the Staden Software Package (Staden, R. , Beal, K.F. & Bonfield J .K. The Staden package, 1998. Methods Mol . Biol . 132, 115-130 (2000) and Wallace, D.C. et al . Mitochondrial DΝA mutations associated with Leber's hereditary optic neuropathy. Science 242,1427-1430 (1988).) was employed for editing and assembling the raw data into sequence contigs . For database searches BLAST at ΝCBI and ΝIX application at the UK-HGMP, Hinxton were utilised. Exons and exon/intron boundaries were identified by analysis of sequences obtained from the original PAC sequence sampling with the KIAA0567 cD A as query. For exon sequences not covered by the sequence sampling approach inter-exon PCR was performed with primers designed from the cDΝA sequence applying the Expand Long Template PCR System (Boehringer Mannheim) . Amplification was performed with DNA of the PAC clones H20545 and J18270 as templates and products directly sequenced using the PCR primers. For the remaining exons vectorette libraries were established from PAC DNA digested with several blunt end and 5' overhang generating restriction enzymes. Nested PCRs were performed applying primers designed from the cDNA sequence and vectorette primers and the gel purified PCR products were sequenced.

Example 2 : Sites of Expression

Northern dot blot hybridisations showed that KIAA0567 is ubiquitously expressed albeit with varying abundancies (Fig 4) . A major transcript of -5.5 kb, corresponding roughly in size with the full-length cDNA, and minor species of ~4.5 and -4.0 kb were detected on Northern blots (Fig 5) . The highest transcript level was observed in retina, followed by brain, testis, heart and skeletal muscle. The high level of the transcript in the retina results from an increased abundance of the 5.5kb transcript.

In the Northern blots shown in Figure 5 , transcripts of ~5.5kb, ~4.5kb and ~4kb length are visible. Strongest signals were present in the retina, followed by brain, heart, testis and skeletal muscle. RT-PCR experiments on liver and kidney RNA using overlapping sets of primers covering the ORF ruled out the presence of alternatively spliced transcripts (data not shown) . Therefore, the shorter transcripts observed in the Northern blot may result from alternative polyadenylation sites . The Northern dot blot hybridisations were carried out with complete KIAA0567 cDNA against a commercial multiple tissue mRNA dot blot (ClonTech) . A Human Multiple Tissue mRNA Dot Blot (ClonTech #7775-1) and total RNA from human brain, heart, skeletal muscle, liver, testis and mammary gland were purchased from Clontech. In addition, total human retinal RNA was isolated from donor eyes using Trizol reagent (Gibco) . 6μg of total RNA each (adjusted by photometric measurement and a control gel) was separated on a 1% agarose 2.2M formaldehyde/MOPS gel and blotted onto a Hybond-N nylon membrane (Amersham) . The insert of the full-length KIAA0567 cDNA was labelled with -³²P-dCTP using the NEBlot Kit (New England Biolabs) and the probe was hybridised in ExpHyb solution (Clontech) for 15h at 65°C. Post-hybridisation washes were done twice in 1 x SSC, 0.15% SDS at 40°C and 0.1 x SSC, 0.15% SDS at 65°C. Finally, the blots were exposed against X-ray films for 3-24 hours at -80°C with intensifying screens. As seen in Figure 4, expression was found to be ubiquitous. The order and arrangement of samples (ranging from 110-75Ong mRNA/dot) is given below the blots. There were no signals in the control samples in the right hand column. The Northern dot blot hybridisations showed that KIAA0567 is ubiquitously expressed albeit with varying abundancies (Fig. 4) . For the Northern blots shown in Figure 5, the same amount of total RNA (6μg each) from the tissue of origin as noted above each respective lane was loaded on to a gel . After electrophoresis and transfer, the mRNA was hybridised with complete KIAA0567 cDNA. Autoradiographs of 2.5 hour and 6 hour exposure are shown on the left and the right of Figure 5.

Transcripts of ~5.5kb, -4.5kb and ~4kb length are visible. A major transcript of -5.5 kb, corresponding roughly in size with the full-length cDNA, and minor species of -4.5 and -4.0 kb were detected on Northern blots (Fig 5) .

Strongest signals were present in the retina, followed by brain, heart, testis and skeletal muscle. The high level of the transcript in the retina results from an increased abundance of the 5.5kb transcript. Example 3 : RT-PCR on liver and kidney RNA

One μg of human kidney and liver RNA was random primed and reverse-transcribed into single stranded cDNA with AMV Reverse Transcriptase according to the manufacturer's recommendations (RNA PCR Kit, Takara) and it was used for PCR amplification with overlapping primer pairs covering the complete coding sequence of OPAl gene. The PCR products were analysed on agarose gels and their identity was verified by DNA sequencing.

The RT-PCR experiments on liver and kidney RNA ruled out the presence of alternatively spliced transcripts (data not shown) . Therefore, the shorter transcripts observed in the Northern blot may result from alternative polyadenylation sites.

Example 4: Mutations

Patients from OPAl gene defect linked families originating from Germany the UK (Votruba, M. et al . Clinical features in affected individuals from 21 pedigrees with dominant optic atrophy. Arch . Ophthalmol . 116,351-358 (1998)) and Cuba (Lunkes et al . Refinement of the OPAl gene locus on chromosome 3q28-q29 to a region of 2-8 cM, in one Cuban pedigree with autosomal dominant optic atrophy Type Kjer. AM. J. Hum . Genet . 57,968-970 (1995)) were screened by direct sequencing of coding exons. Patients and families were recruited in different clinical centres. The diagnosis of ADOA was based on ophthalmological examination including visual acuity, visual field and colour testing, fundoscopy, electrophysiology and family history. (Votruba, M. et al . Clinical features in affected individuals from 21 pedigrees with dominant optic atrophy. Arch . Ophthalmol . 116,351-358 (1998).) Venous blood samples were taken after informed consent and extracted DNA according to standard procedures . Coding exons from patients' genomic DNA were amplified with primers located in flanking intron and UTR sequences. Standard 50μl PCRs were performed in 10 mM tris pH8.9, 50 mM KC1, 1.5-3 mM MgCl , 10 pmol of each primer and 200 μM each dNTP including 50-100 ng DNA and 1 U AmpliTaq Polymerase.

Cycling parameters were 4 min 94°C, 35 cycles of 30 s at 94°C, 30 s at 53°C and 30 s at 72°C and a final 7 min extension at 72°C. The PCR products were purified either by ultrafiltration (Centricon-100 cartridges, Amicon) or by Quiaquick columns (Quiagen) and the samples were sequenced using Big Dye Terminator chemistry. The sequences were edited and aligned using the Lasergene Software package (DNASTAR) .

Cosegregation analysis and screening of controls was done by either simple PCR amplification (deletion of exon 20 in pedigree B3), PCR/RFLP analysis (ntl096C>T/Arg366stop-loss of a Taql site in pedigree Gl; ntl354delG/frameshift - loss of a Tthllll site in pedigree G2) or PCR/SSCP analysis. For SSCP samples were separated on 10% non-denaturing polyacrylamide gels containing 10% glycerol with for 2Oh at 4°C and silver-staining was used for visualisation. 7 putative pathogenic mutations in index patients were identified including a non-conservative missense mutation (R290Q) , a stop codon mutation (R366X) , a 3bp in-frame deletion (I432del) , two 1 bp deletions (1016delC; 1354delG) , a deletion resulting in the complete loss of exon 20 and a lbp insertion (1644insT) (shown in Table 1) .

Figure 6 shows Electropherogram sections illustrating a missense mutation (top left) , a stop codon mutation (top right) , a 3bp deletion (bottom left) and a lbp insertion (bottom right) in selected families. An SNP was detected in intron 8 and is highlighted in the top left figure by * (8740T) . Analysis within the individual families revealed segregation of the mutations with the disease haplotype. Figure 7 shows segregation of the nt869G>A/Arg290Gln mutation in pedigree CI by SSCP analysis. The lane assignment (1-16) corresponds to samples of the respective individuals in the pedigree drawing. Figure 8 shows segregation of the ntl096C>T/Arg366stop mutation in pedigree Gl performed by RFLP analysis with Tagl . The mutation results in a loss of the restriction site on the mutated allele. The lane assignment was according to the pedigree drawing. Lanes 1 and 8 contain size standards. These sequence alterations were not present in at least 50 healthy subjects.

Example 5: OPAl polypeptide

The OPAl polypeptide sequence is shown in Figure 3 (SEQ. ID.NO: 2) . Examination of the N-terminal leader sequence of the deduced protein revealed the typical features of a protein imported into the matrix space of mitochondria. This is based on 1. an enrichment of basically charged amino acids and 2. the presence of the MPP/MIP cleavage consensus sequence RX(F/L/I) XX(G/S/T)XXXX. Figure 9 shows the predicted mitochondrial import signal sequence of OPAl polypeptide. The first 150 amino acids of the protein sequence are shown. Basic residues (R,K,H) are underlined. Acidic residues (D,E) are printed in bold. Three putative cleavage sites (residues 38-47, 80-89, 100-109) matching the MPP/MIP consensus sequence are boxed.

OPAl polypeptide shows highest identity scores over the whole length to dynamin-related large GTPases from salmon, (Kubakawa, K. , Miyashita, T. & Kubo, Y. Isolation of a cDNA for a novel 120-kDa GTP-binding protein expressed in motor neurons in the salmon brain. FEBS Letters . 431,231-235

(1998).) C.elegans, Drosophila and the RN protein. Figures 10 and 11 show the protein alignment depicting the similarity of human OPAl polypeptide with dynamin-like proteins from other species and DYN2. OPAl polypeptide shows highest homologies over its full length to salmon GTP-binding protein Mgl20 expressed in motor neurons of brain (Kubakawa, K. , Miyashita, T. & Kubo, Y. Isolation of a cDNA for a novel 120- kDa GTP-binding protein expressed in motor neurons in the salmon brain. FEBS Letters . 431,231-235 (1998)) (75% over the entire protein) , rat RN protein (97% from residues 662-978) , a C.elegans dynamin-like GTP-binding protein (48% from residues 108-957) and Drosophila melanogaster CG8479 gene product (48% from residues 82-977) . The dynamin-homologous region of OPAl polypeptide and its related proteins is shown which consists of amino acid residues 280-520 of OPAl polypeptide (exons 7-15). The three consensus GTP-binding motifs are indicated above the alignment in bold and the dynamin signature is overscored by * . Amino acid residues conserved only in putative orthologues of KIAA0567 are underlined in Figure 10, while those conserved also in characterised dynamin-like proteins are underlined in Figure 11, with those conserved across all members of the alignment shown in bold. Mutations altering one KIAA0567 amino acid residue found to segregate in ADOA pedigrees CI, Gl and B3 are indicated by an arrow.

The C terminus of OPAl polypeptide differs from other dynamin family members in lacking a proline-rich region, a GED domain and a pleckstrin homology domain and may determine the specific functions of the protein. (Muhlberg, A.B., Warnock, D.E. & Schmid, S.L. Domain structure and intramolecular regulation ofdynamin GTPase. EMBO. 16,6676- 6683 (1997) and McNiven, M.A. , Cao, H. , Pitts, K.R. & Yoon, Y. The dynamin family of mechanoenzymes : pinching in new places. TIBS. 25, 115-120 (2000).) The GTPase domain, encompassing the core central region between amino acid residues 280-520, harbours the consensus tripartite GTP binding motif needed for phosphate binding (GXXXXGKS/T) , coordination of Mg²⁺ (DXXG) , nucleotide binding (T/NKXD) , and the dynamin sequence signature which are characteristically conserved in dynamin-related GTPases. (Guan, K. , Farh, L., Marshall, T.K. & Deschenes, R.J. Normal mitochondrial structure and genome maintenance in yeast requires the dynamin-like product of the MGMl gene. Curr. Genet . 24, 141- 148 (1993) .) Apart from Dynl, Dynll and Dynlll, only one dynamin-related large GTPase, Drpl has been identified in mammalian cells. This protein is located within the cytoplasm and controls mitochondrial distribution and vesicular transport. (Kamimoto, T. et al . Dymple, a novel dynamin-like high molecular weight GTPase lacking a proline-rich carboxyl- ternlinal domain in mammalian cells. J Cell . Biol . Chem. 273,1044-1051 (1998) and Smirnova, E., Shurland, D.L., Ryazantsev, S.N. & van der Bliek, A. M. A human dynamin- related protein controls the distribution of mitochondria J^". Cell . Biol . 143,351-358 (1998).) Studies in yeast have demonstrated that the dynamin-related large GTPases Dnml, MGMl and MSPl play an important role in the maintenance and inheritance of mitochondria. Of the yeast proteins, OPAl polypeptide shows greatest similarity at the primary level to MGMl (Figures 10 and 11) .

Accession numbers

Genbank: KIAA0567 mRNA (AB011139); protein accession numbers: KIAA0567 protein (BAA25493), salmon GTP-binding protein (BAA32279), drosophila CG8479 gene product (AAF58275), C.elegans GTP-binding protein (CAA87771) , human dynamin 2 (NP_004936) , human dynamin-like protein Dymple isoform (AAC35283), yeast MGMl (P32266) , yeast MSPl (CAA69196) .

Claims

1. A method of detecting an OPAl gene in a sample obtained from a subject which method comprises contacting the sample with at least one oligonucleotide and determining whether binding occurs, wherein the at least one oligonucleotide comprises at least one of the following: a) the DNA sequence of Figure 2 (SEQ. ID.NO : 1) from nucleotide residue position 1 to position 2935, b) a DNA sequence encoding an OPAl polypeptide having the sequence according to Figure 3 (SEQ. ID.NO: 2) from amino acid residue position 1 to position 978, c) a DNA sequence which consists of at least 16 sequential nucleotides of the sequence of a) , d) a DNA sequence which comprises at least 18 nucleotides and encodes a fragment of the amino acid sequence of Figure 3 (SEQ . ID.NO: 2) , e) a DNA sequence encoding an epitope encoded by at least 18 sequential nucleotides in the sequence of Figure 2 (SEQ. ID.NO :1) from nucleotide residue position 1 to position 2935, f) a DNA sequence encoding a mutant of a sequence according to a) , b) , c) , d) or e) , g) a DNA sequence complementary to a sequence according to a) , b) , c) , d) , e) or f) , and h) an RNA sequence corresponding to a sequence according to a) , b) , c) , d) , e) , f) or g) .

2. A method as claimed in claim 1 wherein the oligonucleotide probe is

(i) a mutant DNA sequence (f) ;

(ii) a DNA sequence complementary to a mutant DNA sequence (f) ; or (iii) an RNA sequence corresponding to a sequence according to (i) or (ii)

and the mutation is selected from the group consisting of: (a) 869 OA;

(b) 1016 del C;

(c) 1096 OT;

(d) Del 1296 CAT;

(e) 1354 del G; (f) 1644 ins T;

(g) Deletion of exon 20;

3. A method of detecting a mutant OPAl gene in a sample obtained from a subject which method comprises contacting the sample with an oligonucleotide and determining whether binding occurs, wherein the at least one oligonucleotide comprises a mutant of at least one of the following: a) the DNA sequence which corresponds to the DNA sequence of Figure 2 (SEQ. ID.NO: 1) from nucleotide residue position 1 to position 2935, b) a DNA sequence encoding an OPAl polypeptide having the sequence according to Figure 3 (SEQ . ID.NO : 2 ) from amino acid residue position 1 to position 978, c) a DNA sequence which consists of at least 16 sequential nucleotides of the sequence of a) , d) a DNA sequence which comprises at least 18 nucleotides and encodes a fragment of the amino acid sequence of Figure 3 (SEQ . ID.NO : 2) , e) a DNA sequence encoding an epitope encoded by at least 18 sequential nucleotides in the sequence of Figure 2 (SEQ. ID.NO: 1) from nucleotide residue position 1 to position 2935, or a DNA sequence complementary to such a mutant DNA sequence or an RNA sequence corresponding to such a mutant DNA sequence or its complement.

4. A method as claimed in claim 3 wherein the mutation is selected from the group consisting of: a) 869 G>A; b) 1016 del C; c) 1096 OT; d) Del 1296 CAT; e) 1354 del G; f) 1644 ins T; g) Deletion of exon 20;

5. A method of diagnosing autosomal dominant optic atrophy using a method as claimed in any one of claims 1 to 4.

6. A method as claimed in any one of claims 1 to 5 wherein the sample is derived from a human foetus in utero .

7. A method as claimed in any one of claims 1 to 6 comprising the steps of: providing a sample that has been obtained from subject; and providing a method for detecting the presence of a normal OPAl gene, a mutant OPAl gene or a mixture thereof in the sample.

8. A method as claimed in any one of claims 1 to 7 comprising a hybridisation assay.

9. A method as claimed in claim 8 wherein the method makes use of a labelled nucleotide probe.

10. A method of detecting an OPAl gene product in a sample obtained from a subject which method comprises contacting a sample with at least one antibody capable of binding to a peptide and determining whether binding occurs, wherein the antibody is capable of binding to at least one of: a) a polypeptide which is encoded by the DNA sequence of Figure 2 (SEQ . ID.NO: 1) from nucleotide residue position 1 to position 2935, b) an OPAl polypeptide having the sequence according to Figure 3 (SEQ . ID.NO : 2 ) from amino acid residue position 1 to position 978, c) a polypeptide which is encoded by at least 15 sequential nucleotides of the sequence of a) , d) a polypeptide which comprises at least 5 amino acids and includes at least 5 sequential amino acids of the sequence of Figure 3 (SEQ. ID.NO: 2) from amino acid residue position 1 to position 978, e) an epitope encoded by at least 15 sequential nucleotides in the sequence of Figure 2 (SEQ. ID.NO: 1) from nucleotide residue position 1 to position 2935, and f) a polypeptide which is a mutant of a sequence according to a) , b) , c) , d) or e) .

11. A method as claimed in claim 10 wherein the mutant f) is selected from the group consisting of: a) 869 OA; b) 1016 del C; c) 1096 OT; d) Del 1296 CAT; e) 1354 del G; f) 1644 ins T; g) Deletion of exon 20;

12. A method of detecting a mutant OPAl gene product in a sample obtained from a subject which method comprises contacting the sample with an antibody capable of binding to a peptide and determining whether binding occurs wherein the antibody is capable of binding to a mutant polypeptide (f) and the mutation in the polypeptide (f) is selected from the group consisting of those encoded by the following nucleic acid mutations : a) 869 G>A; b) 1016 del C; c) 1096 OT; d) Del 1296 CAT; e) 1354 del G; f) 1644 ins T; g) Deletion of exon 20.

13. A method of diagnosing autosomal dominant optic atrophy using the method as claimed in any one of claims 10 to 12.

14. A method as claimed in any one of claims 10 to 13 wherein the sample is derived from a human foetus in utero .

15. A method as claimed in any one of claims 10 to 14 comprising the steps of providing a sample obtained from a subject; and providing a method for detecting, in the sample, the presence of a normal OPAl gene product, a mutant OPAl gene product or a mixture thereof ..

16. A method as claimed in any one of claims 10 to 15 wherein the assay comprises an immunological assay.

17. A method as claimed in any one of claims 10 to 16 wherein the assay makes use of an antibody capable of binding to a normal OPAl polypeptide .

18. A method as claimed in any one of claims 10 to 17 wherein the assay makes use of an antibody capable of binding to a mutant OPAl polypeptide.

19. A method as claimed in any one of claims 17 or 18 wherein the assay makes use of at least one monoclonal antibody.

20. A method as claimed in any one of claims 10 to 19 wherein the assay is a radioimmunoassay or an ELISA.

21. A kit for detecting the presence of an OPAl gene comprising an oligonucleotide as defined in any one of claims 1 to 4 and other reagents required for carrying out a suitable assay.

22. A kit for detecting the presence of an OPAl gene product comprising an antibody as defined in any one of claims 10 to 12 and other reagents required for carrying out a suitable assay.

23. A kit as claimed in claim 22 wherein the kit comprises an antibody capable of binding to a gene product of an OPAl gene.

24. A kit as claimed in any one of claims 21 to 23 wherein the OPAl gene is a normal OPAl gene.

25. A kit as claimed in any one of claims 21 to 23 wherein the OPAl gene is a mutant OPAl gene.

26. An isolated OPAl gene comprising a DNA sequence encoding an amino acid sequence for a polypeptide, said polypeptide, if expressed in an altered, defective or non-functional form in cells of the human body, being associated with altered cell function which correlates with the genetic disease autosomal dominant optic atrophy.

27. An isolated OPAl gene as claimed in claim 26 wherein the OPAl gene is a normal OPAl gene.

28. An isolated OPAl gene as claimed in claim 26 wherein the OPAl gene is a mutant OPAl gene.

29. An isolated RNA molecule comprising an RNA sequence corresponding to a DNA sequence as defined in any one of claims 1 to 4.

30. An oligonucleotide having the sequence corresponding to at least one of: c) a DNA sequence which consist of at least 16 sequential nucleotides of the sequence of a) according to the first aspect of the invention, d) a DNA sequence which comprises at least 18 nucleotides and encodes a fragment of the amino acid sequence of Figure 3 (SEQ. ID.NO: 2) , e) a DNA sequence encoding an epitope encoded by at least 18 sequential nucleotides in the sequence of Figure 2 (SEQ. ID.NO: 1) from nucleotide residue position 1 to 2935, f) a DNA sequence encoding a mutant of a sequence according to c) , d) or e) , and g) a DNA sequence complementary to a sequence according to c) , d) , e) or f) .

31. An oligonucleotide as claimed in claim 30 comprising (i) a mutant DNA sequence (f) ;

(ii) a DNA sequence complementary to a mutant DNA sequence (f) ; or (iii) an RNA sequence corresponding to a sequence according to (i) or (ii) whereby the mutation is selected from the group consisting of: (a) 869 G>A;

(b) 1016 del C;

(c) 1096 OT;

(d) Del 1296 CAT;

(e) 1354 del G; (f) 1644 ins T;

(g) Deletion of exon 20.

32. A recombinant cloning or expression vector comprising a DNA molecule having a DNA sequence as defined in any one of claims 1 to 4 or 26 to 28.

33. A recombinant vector as claimed in claim 32, wherein said DNA molecule is operatively linked to an expression control sequence in the vector so that an OPAl polypeptide is expressed by the molecule, said expression control sequence being selected from sequences that control the expression of genes of prokaryotic cells, eukaryotic cells or viruses of prokaryotic or eukaryotic organsims, or combinations thereof.

34. A host cell transformed with a vector as defined in claim 32 or 33.

35. A host cell as claimed in claim 34 selected from the group consisting of bacterial cells, for example selected from strains of E. coli, Pseudomonas, Bacillus subtilis,

Bacillus stearothermophilus or other bacilli; yeast or fungal cells; or plant or animal cells, for example insect, mouse, other animal or human cells.

36. A host cell as claimed in claim 35 wherein the cells are human retinal cells.

37. A method of producing an OPAl polypeptide comprising the steps of :

(a) culturing a host cell transfected with a vector as defined in claim 32 or 33 in a medium and under conditions suitable for expression of the polypeptide and optionally (b) isolating the expressed OPAl polypeptide.

38. An isolated normal or mutant OPAl polypeptide characterised by a molecular weight of about 112 kDa .

39. A normal or mutant OPAl polypeptide substantially free of other human proteins and encoded by a DNA sequence selected from: a) a DNA sequence which corresponds to the DNA sequence of Figure 2 (SEQ. ID.NO: 1) from nucleotide residue position 1 to position 2935, b) a DNA sequence encoding an OPAl polypeptide having the sequence according to Figure 3 (SEQ. ID.NO: 2) from amino acid residue position 1 to position 978, c) a DNA sequence which consists of at least 16 sequential nucleotides of the sequence of a) , d) a DNA sequence which comprises at least 18 nucleotides and encodes a fragment of the amino acid sequence of Figure 3 (SEQ. ID.NO: 2) , e) a DNA sequence encoding an epitope encoded by at least 18 sequential nucleotides in the sequence of Figure 2 (SEQ. ID.NO : 1) from nucleotide residue position 1 to 2935, and f) a DNA sequence encoding a mutant of a sequence according to a) , b) , c) , d) or e) .

40. An OPAl polypeptide as claimed in claim 39 wherein the DNA sequence is mutant DNA sequence (f) and the mutation is selected from the group consisting of:

(a) 869 G>A;

(b) 1016 del C;

(d) Del 1296 CAT;

(e) 1354 del G;

(f) 1644 ins T; (g) Deletion of exon 20.

41. An OPAl polypeptide as claimed in claim 39 or 40 made by chemical synthesis or by in vi tro enzymic peptide synthesis.

42. A polypeptide fragment comprising a portion of the amino acid sequence of claim 39 or 40.

43. A polypeptide fragment as claimed in claim 42 comprising 10 or more amino acids.

44. A polypeptide or polypeptide fragment as claimed in any one of claims 39 to 43 for use as a medicament.

45. A polypeptide or polypeptide fragment as claimed in any one of claims 39 to 44 for use in the treatment of a medical condition resulting from a defect in the OPAl gene.

46. A polypeptide or polypeptide fragment as claimed in any one of claims 39 to 44 for use in the treatment of autosomal dominant optic atrophy.

47. The use of a polypeptide or polypeptide fragment as claimed in any one of claims 39 to 44 in the manufacture of a medicament for the treatment of a medical condition resulting from a defect in the OPAl gene.

48. The use of a polypeptide or polypeptide fragment as claimed in any one of claims 39 to 44 in the manufacture of a medicament for the treatment of autosomal dominant optic atrophy.

49. An antibody capable of binding to a polypeptide or polypeptide fragment as claimed in any one of claims 39 to 44.

50. An antibody as claimed in claim 49 which is a mouse or a human antibody.

51. A kit for detecting the presence of an OPAl gene product comprising an antibody as claimed in claim 49 or 50.

52. A DNA molecule comprising a sequence as defined in claim 1 part a) , b) , c) , d) , e) , f) or g) , a DNA molecule comprising a sequence as defined in claim 2 or a vector as claimed in claim 32 or 33 for use in medicine.

53. A DNA molecule comprising a sequence as defined in claim 1 part a) , b) , c) , d) , e) , f) or g) , a DNA molecule comprising a sequence as defined in claim 2 or a vector as claimed in claim 32 or 33 for use in the treatment of a medical condition resulting from a defect in the OPAl gene.

54. A DNA molecule comprising a sequence as defined in claim 1 part a) , b) , c) , d) , e) , f) or g) , a DNA molecule comprising a sequence as defined in claim 2 or a vector as claimed in claim 32 or 33 for use in the treatment of autosomal dominant optic atrophy.

55. Use of a DNA molecule comprising a sequence as defined in claim 1 part a) , b) , c) , d) , e) , f) or g) , a DNA molecule comprising a sequence as defined in claim 2 or a vector as claimed in claim 32 or 33 in the manufacture of a medicament for the treatment of a medical condition resulting from a defect in the OPAl gene.

56. Use of a DNA molecule comprising a sequence as defined in claim 1 part a) , b) , c) , d) , e) , f) or g) , a DNA molecule comprising a sequence as defined in claim 2 or a vector as claimed in claim 32 or 33 in the manufacture of a medicament for the treatment of autosomal dominant optic atrophy.

57. A non-human animal comprising a heterologous cell system comprising a recombinant vector as claimed in claim 32 or 33 which induces autosomal dominant optic atrophy in the animal .

58. A transgenic mouse comprising a mutant OPAl gene and exhibiting autosomal dominant optic atrophy symptoms.

59. A DNA molecule comprising an intronless DNA sequence selected from the group consisting of : a) a DNA sequence which corresponds to the DNA sequence of Figure 2 (SEQ. ID.NO: 1) from nucleotide residue position 1 to 2935 comprising one or more of the following mutations:

(i) 869 G>A

(ii) 1016 del C

(iii) 1096 C>T (iv) Del 1296 CAT

(v) 1354 del G

(vi) 1644 ins T

(vii) Deletion of exon 20 b) a DNA sequence encoding a mutant OPAl polypeptide having the sequence according to Figure 3 (SEQ. ID.NO : 2) from amino acid residue position 1 to position 978 comprising one or more of the mutations defined in a) , c) a DNA sequence which consists of at least 16 sequential nucleotides of the sequence of a) or b) , d) a DNA sequence which comprises at least 16 nucleotides and encodes a fragment of the amino acid sequence coded for by the sequence of a) or b) including at least a part of the mutated portion, e) a DNA sequence encoding an epitope encoded by at least 18 sequential nucleotides in the sequence of a) or b) including at least a part of the mutated portion, and f) a DNA sequence complementary to a sequence according to a) , b) , c) , d) , or e) .

60. A DNA molecule as claimed in claim 59 wherein the DNA molecule is a cDNA molecule.

61. A DNA molecule comprising an intronless DNA sequence selected from the group consisting of: a) a DNA sequence which corresponds to the DNA sequence of Figure 2 (SEQ . ID.NO : 1) from nucleotide residue position 1 to 2935, b) a DNA sequence encoding an OPAl polypeptide having the sequence according to Figure 3 (SEQ. ID .NO : 2) from amino acid residue position 1 to position 978, c) a DNA sequence which consists of at least 16 sequential nucleotides of the sequence of a) , d) a DNA sequence which comprises at least 18 nucleotides and encodes a fragment of the amino acid sequence of Figure 3 (SEQ. ID.NO: 2) , e) a DNA sequence encoding an epitope encoded by at least 18 sequential nucleotides in the sequence of Figure 2 (SEQ. ID.NO:l) from nucleotide residue position 1 to 2935, and f) a DNA sequence complementary to a sequence according to a) , b) , c) , d) or e) .

62. A DNA molecule as claimed in claim 61 wherein the DNA molecule is a cDNA molecule.

63. A method for the diagnosis of autosomal dominant optic atrophy as claimed in claim 5 wherein the presence of a mutation indicates the presence of or propensity for autosomal dominant optic atrophy.

64. A method of screening for autosomal dominant optic atrophy using a method as claimed in any one of claims 1 to 4.

65. A cell line transformed with a vector as defined in claim 32 or 33.

66. A cell line comprising a gene as defined in any one of claims 26 to 28.

67. A cell line as claimed in claim 65 or 66 which is non- human .

68. A pair of primers for a DNA amplification reaction at least one of which has the sequence selected from: c) a DNA sequence which consists of sequential nucleotides of the sequence of a) according to the first aspect of the invention, d) a DNA sequence which comprises sequential nucleotides and encodes a fragment of the amino acid sequence of Figure 3

(SEQ. ID. NO: 2) , e) a DNA sequence encoding an epitope encoded by sequential nucleotides in the sequence of Figure 2

(SEQ. ID.NO : 1) from nucleotide residue position 1 to 2935, f) a DNA sequence encoding a mutant of a sequence according to c) , d) or e) , and g) a DNA sequence complementary to a sequence according to c) , d) , e) or f) .

69. The pair of primers as claimed in claim 68 wherein at least one of which has the sequence selected from the following: (i) a mutant DNA sequence (f) ;

(ii) a DNA sequence complementary to a mutant DNA sequence (f) ; or

(iii)an RNA sequence corresponding to a sequence according to

(i) or (ii) and the mutation is selected from the group consisting of:

(a) 869 G>A;

(b) 1016 del C;

(c) 1096 C>T;

(d) Del 1296 CAT; (e) 1354 del G;

(f) 1644 ins T;

(g) Deletion of exon 20.

70. The pair of primers as claimed in claim 68 or claim 69 which are each at least 10 nucleotides long.