LGMD GENE CODING FOR A CALCIUM DEPENDENT PROTEASE

Abstract: A nucleic acid sequence comprising: 1) the sequence represented in figure 8; or 2) the sequence represented in figure 2; or 3) a part of the sequence of figure 2 with the proviso that it is able to code for a protein having a calcium dependant protease activity involved in a LGMD2; or 4) a sequence derived from a sequence defined in 1), 2) or 3) by substitution, deletion or addition of one or more nucleotides with the proviso that said sequences still codes for said protease.

Description: LGMD gene coding for a calcium dependent protease The invention relates to the isolated gene coding for a calcium dependent protease belonging to the Calpaϊn family which, when it is mutated, is a cause of a disease called Limb-Girdle Muscular Dystrophy (LGMD). The term limb-girdle muscular dystrophy (LGMD) was first proposed by Walton and Nattrass (1954) as part of a classification of muscular dystrophies. LGMD is characterised by progressive symmetrical atrophy and weakness of the proximal limb muscles and by elevated serum creatine kinase. Muscle biopsies demonstrate dystrophic lesions and electromyograms show myopathic features. The symptoms usually begin during the first two decades of life and the disease gradually worsens, often resulting in loss of walking ability 10 or 20 years after onset (Bushby, 1994). Yet, the precise nosological definition of LGMD still remains unclear. Consequently, various neuromuscular diseases such as facioscapulohumeral, Becker muscular dystrophies and especially spinal muscular atrophies have been occasionally classified under this diagnosis. For example, a recent study (Arikawa et al., 1991) reported that 17% (out of 41) of LGMD patients showed a dystrophinopathy. These issues highlight the difficulty in undertaking an analysis of the molecular and genetic defect(s) involved in this pathology. Attempts to identify the genetic basis of this disease go back over 35 years. Morton and Chung (1959) estimated that "the frequency of heterozygous carrier ... is 16 per thousand persons". The same authors also stated that "the segregation analysis gives no evidence on whether these genes in different families are allelic or at different loci". Both autosomal dominant and recessive transmission have been reported, the latter being more common with an estimated prevalence of 10 -5 (Emery, 1991). The localisation of a gene for a recessive form on chromosome 15 (LGMD2A, MIM 253600; Beckmann et al., 1991) provided the definitive proof that LGMD is a specific genetic entity. Subsequent genetic analyses confirmed this chromosome 15 localisation (Young et al., 1992; Passos-Bueno et al., 1993), the latter group demonstrating genetic heterogeneity of this disease. Although a recent study localised a second mutant 1 2 gene to chromosome 2 (LGMD2B, MIM 253601 ; Bashir et al.. 1994). there is evidence that at least one other locus can be involved. Genetic analyses of the LGMD2 kindreds revealed unexpected findings First genetic heterogeneity was demonstrated in the highly inbred Indiana Amish community. Second although the Isle of la Reunion families were thought to represent a genetic isolate, at least 6 different disease haplotypes were observed , providing evidence against the hypothesis of a single founder effect (Beckmann et al., 1991 ) in this inbred population. The nonspecific nosological definition, the relatively low prevalence and genetic heterogeneity of this disorder limit the number of families which can be used to restrict the genetic boundaries of the LGMD2A interval. Cytogenetic abnormalities, which could have helped to focus on a particular region, have not been reported. Immunogenetic studies of dystrophin-associated proteins (Matsumura et al., 1993) and cytoskeletal or extracellular matrix proteins such as a merosin (Tome et al., 1994) failed to demonstrate any deficiency. In addition, there is no known specific physiological feature or animal model that could help to identify a candidate gene. Thus, there is no alternative to a positional cloning strategy. It is establisheα that the LGMD2 chromosomal region is localized on chromosome 15 as 15q15.1 - 15q21.1 region ( Fougerousse et al., 1994). Construction and analysis of a 10-12 Mb YAC contig (Fougerousse et al., 1994) permitted the mapping of 33 polymorphic markers within this interval and to further narrow the LGMD2A region to between D15S514 and D15S222. Furthermore, extensive analysis of linkage disequilibrium suggested a likely position for the gene in the proximal part of the contig. The invention results from the construction of a partial cosmid map and the screening by cDNA selection (Lovett et al., 1991 ; Tagle et al., 1993) for muscle- expressed sequences encoded by this interval led to the identification of a number of potential candidate genes. One of these, previously cloned by Sorimachi et al. (1989). encodes a muscle specific protein, nCL1 (novel Calpain Large subunit 1 ), which belongs to the calpain family (CANP, calcium-activated neutral protease; EC 3.4.22.17), and appeared to be a functional candidate gene for this disease. 96/16175 3 Calpains are non-lysosomal intracellular cysteine proteases which require calcium for their catalytic activities (for a review see Croall D E et al, 1991 ) The mammalian calpains include two ubiquitous proteins CANP1 and CANP2 as well as tissue-specific proteins In addition to the muscle specific nCLl , stomach specific nCL2 and nCL2' proteins have also been described these are derived from the same gene by alternative splicing The ubiquitous enzymes consist of heterodimers with distinct large subunits associated with an common small subunit , the association of tissue-specific large subunits with a small subunit has not yet been demonstrated The large subunits of calpains can be subdivided into 4 protein domains Domains I and III, whose functions remain unknown, show no homology with known proteins Domain I, however, seems important for the regulation of the proteolytic activity Domain II shows similarity with other cysteine proteases, sharing histidme, cysteine and asparagme residues at its active sites Domain IV comprises four EF-hand structures which are potential calcium binding sites In addition, three unique regions with no known homology are present in the muscle-specific nCL1 protein, namely NS, IS1 and IS2, the latter containing a nuclear translocation signal These regions may be important for the muscle specific function of nCL1 It is usually accepted that muscular dystrophies are associated with excess or deregulated calpains, and all the known approaches for curing these diseases are the use of antagonists of these proteases , examples are disclosed in EP 359309 or EP 525420 The invention results from the finding that, on the opposite to all these hypothesis, the LGMD2 disease is strongly correlated to the defect of a calpain which is expressed in healthy people The invention relates to the nucleic acid sequence such as represented in Figure 2 coding for a Ca ** dependent protease, or calpain, which is involved in LGMD2 disease, and more precisely LGMD2A It also relates to a part of this sequence provided it is able to code for a protein having a calcium-dependent protease activity involved in LGMD2, or a sequence derived from one of the above sequences by substitution, deletion or addition of one or more nucleotides provided that said sequence is still coding for said protein, all the nucleic acids yielding a sequence complementary to a sequence as defined above 4 The genomic organisation of the human nCL1 gene has been determined by the inventors, and consists of 24 exons and extends over 40 kb as represented in Figure 8, and is also a part of the invention About 35 kb of this gene have been sequenced A systematic screening of this gene in LGMD2A families led to the identification of 14 different mutations, establishing that a number of independent mutational events in nCL1 are responsible for LGMD2A Furthermore, this is the first demonstration of a muscular dystrophy resulting from an enzymatic rather than a structural defect In the present specification, CANP3 means the protein which is a Ca dependent protease, or calpain, and coded by the nCL1 gene on chromosome 15 The invention relates also to a protein, called CANP3, consisting in the ammo acid sequence such as represented in figure 2 and which is involved, when mutated, in the LGMD2 disease The cDNA of the gene coding for CANP3, which is coding for the protein, is also represented in Figure 2, and is a part of the invention The protein coded by this DNA is CANP3, a calcium-dependent protease belonging to the Calpain family Are also included in the present invention the nucleic acid sequences derived from the cDNA of Figure 2 by one or more substitutions, deletions, insertions, or by mutations in 5' or 3' non coding regions or in splice sites, provided that the translated protein has the protease, calcium-dependent activity, and when mutated, induce LGMD2 disease The nucleic acid sequence encoding the protein might be DNA or RNA and be complementary to the nucleic and sequence represented in Figure 2 The invention also relates to a recombinant vector including a DNA sequence of the invention, under the control of a promoter allowing the expression of the calpain in an appropriate host cell A procaryotic or eucaryotic host cell transformed by or transfected with a DNA sequence comprising all or part of the sequence of Figure 2 is a part of the invention Such a host cell might be either 6/16175 5 - a cell which is able to secrete the protein and this recombinant protein might be used as a drug to treat the LGMD2, or - a packaging cell line transfected by a viral or retroviral vector the cell lines bearing recombinant vector might be used as a drug for gene therapy of LGMD2 All the systems used today for gene therapy including adenoviruses and retroviruses and others described for example in « I'ADN medicament », (John Libbey, Eurotext, 1993), and bearing one of the DNA sequence of the invention are included herein by reference The examples hereunder and attached figures indicate how the structure of the gene was established, and how relationship between the gene and the LGMD was established Legend of the figures Figure 1 A) Genomic organisation of the nCL1 gene The gene covers a 40 kb region of which 35 were sequenced (Accession number pending) Introns and exons are drawn to scale, the latter being indicated by numbered vertical bars The first intron is the largest one and remains to be fully sequenced Position of intragenic microsatellites are indicated by asterisks Arrows indicate the orientation of Alu (closed) and of Mer2 (greyed) repeat sequences B) EcoR\ restriction map An EcoR\ (E) restriction map of this region was established with the help of cosmids from this region The location of nCL1 gene is indicateα as a black bar The size of the corresponding fragments are indicated and are underlined when determined by sequence analysis C) Cosmid map of the nCL1 gene region Cosmids were from a cosmid library constructed by subcloning YAC 774G4 (Richard in preparation) and are presented as lines Dots on lines indicate positive STSs (indicated in boxed rectangles) A minimum of three cosmids cover the entire gene T3.T7 6/16175 6 Figure 2: Sequence of the human nCL1 cDNA (B) , and the flanking 5 ' (A) and 3 ' (C) genomic regions A) and C) The polyadenylation signal and putative CAAT, TATAA sites are boxed. Putative Sp1 (position -477 to -472), MEF2 binding sites (-364 to -343) and CArG box (-685 to -672) are in bold. The Alu sequence present in the 5' region is underlined B) The corresponding amino acids are shown below the sequence. The coding sequence between the ATG initiation codon and the TGA stop codon is 2466 bp, encoding for a 821 amino acid protein. The adenine in the first methionine codon has been assigned position 1. Locations of introns within the nCL1 gene are indicated by arrowheads. Nucleotides which differ from the previously published ones are indicated by asterisks. Figure.3. Alignments of ammo acid sequences of the muscle-specific calpains. The human nCL1 protein is shown on the first line. The 3 muscle-specific sequences (NS, IS1 and IS2) are underlined. The second line corresponds to the rat sequence (Accession no P). The third and fourth lines show the deduced ammo acid sequences encoded by pig and bovine Expressed Sequences Tagged (GenBank accession no U05678 and no U07858, respectively) The ammo acids residues which are conserved among all known members of the calpains are in reverse letters A period indicates that the same ammo acid is present in the sequence. Letters refer to the variant amino acid found in the homologous sequence. Position of missense mutations are given as numbers above the mutated ammo acid. Figure 4: Distribution of the mutations along nCL1 protein structure. A) Positions of the 23 introns are indicated by vertical bars in relation to the corresponding ammo acid coordinates. B) The nCL1 protein is depicted showing the four domains (I, II, III, IV) and the muscle specific sequences (NS, IS1 and IS2). The position of missense mutations within nCL1 domain are indicated by black dots. The effect of nonsense and frameshift mutations are illustrated as truncated lines, representing the extent of protein synthesised. Name of the corresponding families are indicated on the left of the line. The out of frame ORF is given by hatched lines Figure 5 Northern blot hybridisation of a nCL1 clone A mRNA blot (Clontech) containing 2 μg of poly(A)+ RNA from each of eight human tissues was hybridised with a nCL1 genomic clone spanning exons 20 and 21 The latter detects a 3.6 kb mRNA present only in a line corresponding to the skeletal muscle mRNA Figure 6 Representative mutations identified by heteroduplex analysis Examples of mutation screening by heteroduplex analysis Pedigree B505 shows the segregation of two different mutations in exon 22 Figure 7 Homozygous mutations in the nCL1 gene Detection by sequencing of mutations in exons 2 (a), 8 (b), 13 (c) and 22 (d) Sequences from a healthy control are shown above each mutant sequence Asterisks indicate the position of the mutated nucleotides The consequences on codon and am o acid residues are indicated on the left of the figure together with the name of the family Figure 8 Structure of nCL1 gene Figure 8A represents the 5' part of the gene with exon 1 Figure 8B represents the part of the gene including exons 2 to 8, Figure 8C represents the part of the gene including exon 9, Figure 8D represents the part of the gene including exons 10 to 24 including the 3' non transcribed region EXAMPLES EXAMPLE 1 Localisation of the nCL1 within the LGMD2A interval Detailed genetic and physical maps of the LGMD2A region were constructed (Fougerousse et al , 1994), following the primary linkage assignment to 15q (Beckmann et al , 1991 ) The disease locus was bracketed between the D15S129 and D15S143 markers, defining the cytogenetic boundaries of the LGMD2A region as 15q15 1-15q21 1 (Fougerousse et al , 1994) Construction and analysis of a 10-12 Mb YAC contig (Fougerousse et al., 1994) permitted us to map 33 polymorphic markers within this interval and to further narrow the LGMD2A region to between D15S514 and D15S222 The nCL1 gene had been localised to chromosome 15 by hybridisation with sorted chromosomes and by Southern hybridisation to DNA from human-mouse cell hybrids (Ohno et al., 1989).cDNA capture using YACs from the LGMD2A interval allowed the identification of thirteen positional candidate genes. nCL1 was one of the two transcripts identified that showed muscle-specific expression as evidenced by northen blot analysis.The localisation was further confirmed by STS (for Sequence Tagged Site) assays. Primers used for the localisation of the nCL1 gene are P94in2, P94in13 and pcr6a3, as shown in Figure 1 and their characteristics being defined in Table 1. Table 1 : PCR rimer for l ion f h n L1 ene. These primers are designed from different parts of the published human cDNA sequence (Sorimachi et al., 1989), and were used for an STS content screening on DNA from three chromosome 15 somatic cell hybrids and YACs from the LGMD2A contig The results positioned the gene in a region previously defined as 15q15 1-q21 1 and on 3 YACs (774G4, 926G10, 923G7) localised in this region The relative positions of STSs along the LGMD2A contig allowed to localise the gene between D15S512 and D15S488, in a candidate region suggested by linkage disequilibrium studies. The same primers as above were used to screen a cosmid library from YAC 774G4 A group of 5 cosmids was identified (Fig 1 ). Experiments with another nCL1 primer pair (P94exlter, Table 1 ) established that these cosmids cover all nCL1 exons except number 1 , and that a second group of 4 cosmids contain this 6/16175 9 exon (Fig 1 ) A minimal set of three overlapping cosmids (2G8-2B11-1F11 ) covers the entire gene (Figure 1 ) DNA from these cosmids was used to construct an EcoR\ restriction map of this region (Figure 1B) EXAMPLE 2 Determination of the nCL1 gene sequence Most of the sequences were obtained through shotgun sequencing of partial digests of cosmid 1F11 subcloned in M13 and bluescπpt vectors, and by walking with internal primers The sequence assembly was made using the XBAP software of the Staden package (Staden) and was in agreement with the restriction map of the cosmids Sequences of exon 1 and adjacent regions were obtained by sequencing cosmid DNA or PCR products from human genomic DNA The first intron is still not fully sequenced, but there is evidence that it may be between 10 to 16 kb in length (based on hybridisation of restriction fragments, data not shown) The entire gene, including its 5" and 3' regions, is more than 40 kb long, and shown in Figure 8 a) the cDNA seguence The used technology allows the implementation of the published human cDNA sequence of nCL1 (Sorimachi 1989) It contains the missing 129 bases corresponding to the N-termmal 43 am o acids (Figure 2) It also differs from it at 12 positions Three of which occur at third base positions of codons and preserve the encoded ammo acid sequence The other 9 differences lead to changes in ammo-acid composition (Figure 2) As these different exons were sequenced repeatedly on at least 10 distinct genomes, we are confident that the sequence of Fig 2 represents an authentic sequence and does not contain minor polymorphic variants Furthermore, these modifications increase the local similarity with the rat nCL1 ammo acid sequence (Sorimachi), although the overall similarity is still 94 % The ATG numbered 1 in Figure 2 is the translation initiation site based on homology with the rat nCL1 , and is within a sequence with only 5 nucleotides out of 8 in common with the Kosak consensus sequence (Kosak M, 1984) Putative CCAAT and TATA boxes were observed 590, 324, (CCAAT) and 544 or 33 bp (TATA) upstream of the initiating ATG codon, respectively (Bucher, 1990) A GC- box binding the Sp1 protein (Dynan et al , 1983) was identified at position -477 6/16175 10 Consensus sequences corresponding to potential muscle-specific regulatory elements were identified (Fig 2) These include a myocyte-specific enhancer- binding factor 2 (MEF2) binding site (Cserjesi P 1991 ), a CArG box (Minty A 1986) and 6 E-boxes (binding sites for basic Helix-Loop-Helix proteins frequently found in members of MyoD family, Blackwell et Weintraub, 1990) The functional significance of these putative transcription factor binding sites in the regulation of nCL1 gene expression remains to be established. Two potential AAUAAA polyadenylation signals, were identified 520 and 777 bp downstream of the TGA stop codon The sequencing of a partial nCL1 cDNA containing a polyA tail, demonstrated that the first AAUAAA is the polyadenylation signal The latter is embedded in a region well conserved with the rat nCL1 sequence and is followed after 4 bp by a G/T cluster, present in most genes 3' of the polyadenylation site (Birnstiel et al , 1985) The 3'- untraπslated region of the nCL1 mRNA is 565 bp long The predicted length of the cDNA should therefore be approximately 3550 or 3000 bp b) Comparison with calpain The sequence of the human nCL1 gene was compared to those of other calpains thereof (Figure 3) The most telling comparisons are with the homologous rat (Accession no J05121 ), bovine (Accession no U07858) and porcine (Accession no U05678) sequences The accession numbers refers to those or international genebanks, such as GeneBank (N I.H ) or EMBL Database (EMBL, Heidelberg) High local similarities between the human and rat DNA sequences are even observed in the 5' (75%) or in different parts of the 3' untranslated regions (over 60%) (data not shown) The high extent of sequence homology manifested by the human and rat nCL1 gene in their untranslated regions is suggestive of evolutionary pressures on common putative regulatory sequences c) Genomic organisation of the nCL1 gene A comparison of the published nCL1 human cDNA (Sorimachi et al , 1989) with the corresponding genomic sequence led to the identification of 24 exons ranging in length from 12 bp (exon 13) to 309 bp (exon 1 ), with a mean size of 100 bp (Figure 1 ) The size of introns ranges from 86 bp to about 10-16 kb for mtroπ 1 The i ntron-exon boundaries as shown -m Table 2 exhibit close adherence to 5' and 3 ' spl i ce site consensus sequences (Shapiro and Senapathy , 1987) Table 2: Sequences at the intron-exon junctions. A score expressing adherence to the consensus was calculated for each site according to Shapiro and Senapathy (1987). Sequences of exons and introns are in upper and lower cases, respectively. Size of exons are given in parenthesis. splice donor sue score Intron score splice acceptor site Exon Exon 1 (309 bp) -> ...ttmgmcacagGAAAT.. Exon 2 (70 bp ) -> ...gtgtctgcctgcagGGGAC... Exon 3 (119 bp) -> ...acgcttctgigcagTTCTG... Exon 4 (134 bp) -> ...atcctctctctaagGCTCC... Exon 5 (169 bp) -> ...ccatcgggcctcagGATGG.. Exon 6 (144 bp) -> ...ttacigctctacagACAAT. Exon 7 (84 bp ) -> ...tctgigtgcttaagGTCCC Exon 8 (86 bp ) -> ...cattttcccaccagATGGA Exon 9 (78 bp ) -> ...ttccaacctctcagGATGT... Exon 10 (161 bp) -> ...UctgggggigcagATACT.. Exon 11 (170 bp ) -> ...tgtttcttctcaagGTTCC. Exon 12 (12 bp) -> ...tccccatαctcagATGCA . Exon 13 (209 bp) -> ...tgtaucctcacagGGAAG. Exon 14 (37 bp) -> ...ctmcttatgcag AA AAA.. Exon 15 ( 18 bp) -> ...cctcctαctccagCCCAT.. Exon 16 (114 bp ) -> ..ugigcctccacagCCACA... Exon 17 (78 bp ) -> ..cccnc ccicagGACAT... Exon 18 (58 bp ) -> ..ctccatccccccagACAAC. Exon 19 (65 bp ) -> ..cctccctcctccagACAGA... Exon 20 (69 bp ) -> .mtctattgccagAAATA.. Exon 21 (79 bp) -> .gg t cccctccacagGATTC. Exon 22 (117 bp ) -> ...GTTCAgtaagt.. 79 <-lmron 22-> 93.5 ...gcattctttcacagGAGCT.. Exon 23 (5 bp) - ■ TGGAGgtaaag.. 81 <-lmron 23-> 79 ...ggeacttctucagTGGCT Exon 24 (27 bp) - When the genomic sequence was submitted to GRAIL analysis (Uberbacher et al., 1991 ), 11 exons were correctly recognised, 4 were not identified, 6 were inadequately defined and 2 were too small to be recognised (data not shown). As already noted, the nCL1 gene has three unique sequence blocks, NS (amino acid residues 1 to 61 ), IS1 (residues 267 to 329) and IS2 (residues 578 to 653). It is interesting to note that each of these sequences, as well as the nuclear translocation signal inside IS2, are essentially flanked by introns (Fig. 4). The exon-intron organisation of the human nCL1 is similar to that reported for the chicken CANP (the only other large subunit calpain gene whose genomic structure is known; (Emori et al., 1986). Four microsatellite sequences were identified. Two of them are in the distal part of the first intron: an (AT)1 and an previously identified mixed-pattern microsatellite, S774G4B8. which was demonstrated to be non polymorphic (Fougerousse et al., 1994). A (TA)7(CA)4(GA)13 was identified in the second intron and genotyping of 64 CEPH unrelated individuals revealed two alleles (with frequencies of 0.10 and 0.90). The fourth microsatellite is a mixed (CA)n(TA)m repeat present in the 9th intron. The latter and the (AT)14 repeat have not been investigated for polymorphism. Fourteen repetitive sequences of the Alu family and one Mer2 repeat were identified in the nCL1 gene (Fig. 1C), which has, thus, on the average one Alu element per 2.5 kb. Southern blot experiments (Ohno et al., 1989) and STS screening (data not shown) suggest that there is but one copy per genome of this member of the calpain family. 5 EXAMPLE 3 Expression of the nCL1 gene The pattern of tissue-specificity was investigated by northern blot hybridisation with a genomic subclone probe from cosmid 1 F11 spanning exons 20 and 21 There is no evidence for the existence of an alternatively spliced form o of nCL , although this cannot be excluded. A transcript of about 3.4-3.6 kb was 6/16175 detected in skeletal muscle mRNA (Figure 5). This size therefore favours that the position -544 is the functional TATA box. Transcription studies suggested that it is an active gene rather than a pseudogene and its muscle-specific pattern of expression is consistent with the phenotype of this disorder (Sorimachi et al., 1989 and Figure 5). EXAMPLE 4 Mutation screening nCL1 fulfils both positional and functional criteria to be a candidate gene for LGMD2A. To evaluate its role in the etiology of this disorder, nCL1 was systematically screened in 38 LGMD2 families for the presence of nucleotide changes using a combination of heteroduplex (Keen et al., 1991) and direct sequence analyses. PCR primers were designed to specifically amplify the exons and splice junctions and also the regions containing the putative CAT, TATA boxes and the polyadenylation signal of the gene as shown in Table 3. Table 3: PCR primers used for the analysis of the nCL1 gene in LGMD patients. amplified region Primer sequences (5'-3 * ) Size (bp) Annealing lemp (°C) promotor TTCAGTACCTCCCGTTCACC GATGCTTGAGCCAGGAAAAC exon 1 CTTTCCTTGAAGGTAGCTGTAT GAGGTGCTGAGTGAGAGGAC exon2 ACTCCGTCTCAAAAAAATACCT ATTGTCCCTTTACCTCCTGG exon 3 TGGAAGTAGGAGAGTGGGCA GGGTAGATGGGTGGGAAGTT exon4 GAGGAATGTGGAGGAAGGAC TTCCTGTGAGTGAGGTCTCG exon 5 GGAACTCTGTGACCCCAAAT TCCTCAAACAAAACATTCGC exon6 GTTCCCTACATTCTCCATCG GTTATTTCAACCCAGACCCTT exon 7 AATGGGTTCTCTGGTTACTGC AGCACGAAAAGCAAAGATAAA exon 8 GTAAGAGATTTGCCCCCCAG TCTGCGGATCATTGGTTTTG exon 9 CCTTCCCTTCTTCCTGCTTC CTCTCTTCCCCACCCTTACC exon 10 CCTCCTCACCTGCTCCCATA TTTTTCGGCTTAGACCCTCC exon 11 TGTGGGGAATAGAAATAAATGG CCAGGAGCTCTGTGGGTCA exon 12 GGCTCCTCATCCTCATTCACA GTGGAGGAGGGTGAGTGTGC exon 13 TGTGGCAGGACAGGACGTTC 1 TTCAACCTCTGGAGTGGGCC exon 14 CACCAGAGCAAACCGTCCAC ACAGCCCAGACTCCCATTCC exon 15 TTCTCTTCTCCCTTCACCCT ACACACTTCATGCTCTCTACCC exon 16 CCGCCTATTCCTTTCCTCTT GACAAACTCCTGGGAAGCCT c\on 17 ACCTCTGACCCCTGTGAACC TGTGGATTTGTGTGCTACGC exon 18 CATAAATAGCACCGACAGGGA GGGATGGAGAAGAGTGAGGA exon 19 TCCTCACTCTTCTCCATCCC ACCCTGTATGTTGCCTTGG exons 20-21 GGGGATTTTGCTGTGTGCTG ATTCCTGCTCCCACCGTCTC exon 22 CACAGAGTGTCCGAGAGGCA GGAGATTATCAGGTGAGATGCC 22-23 CAGAGTGTCCGAGAGGCAGGG CGTTGACCCCTCCACCTTGA exon 24 GGGAAAACATGCACCTTCTT TAGGGGGTAAAATGGAGGAG polyadenylation signal ACTAACTCAGTGGAATAGGG GGAGCTAGGATAGCTCAAT PCR products made on DNA from blood of specific LGMD2A patients were then subjected either to heteroduplex analysis or to direct sequencing, depending on whether the mutation, based on haplotype analysis, was expected to be homozygous or heterozygous, respectively. It was occasionally necessary to clone the PCR products to precisely identify the mutations (i.e., for microdeletions or insertions and for some heterozygotes). Disease-associated mutations are summarised in Table 4 hereunder and their position along the protein is shown in Fig 4 Table 4 nCL1 mutations in LGMD2A families. Codons and am o acid positions are numbered on the basis of the cDNA sequence starting from ATG The first letter of the family code refers to the origin of the population B= Brazil, M= metropolitan France, R = Isle of La Rέunion, A= Amish. Each mutation was confirmed by heteroduplex analysis, by sequencing of both strands in several members of the family or by enzymatic digestion when the mutation resulted in the modification of a restriction site. Segregation analyses of the mutations, performed on DNAs from all available members of the families, confirmed that these sequence variations are on the parental chromosome carrying the LGMD2A mutation. To exclude the possibility that the missense substitutions might be polymoφhisms, their presence was systematically tested in a control population: none of these mutations was seen among 120 control chromosomes from the CEPH reference families. EXAMPLE 5 : Analysis of families genes, chromosome-15 ascertained families The initial screening for causative mutations was performed on families, each containing a LGMD gene located on chromosome 15. These included families from the Island of La Reunion (Beckmann et al., 1991 ), from the Old Order Amish from northern Indiana (Young et al., 1992.) and 2 Brazilian families (Passos Bueno et al., 1993). a) Reunion Island families Genealogical studies and geographic isolation of the families from the Isle of La Reunion were suggestive of a single founder effect. Genetic analyses are, however, inconsistent with this hypothesis as the families present haplotype heterogeneity. At least, six different carrier chromosomes are encountered, (with affected individuals in several families being compound heterozygotes) Distinct mutations corresponding to four of these six haplotypes have been identified thus far In family R14, exons 13, 21 and 22 showed evidence for sequence variation upon heteroduplex analysis (Fig. 6) Sequencing of the associated PCR products revealed (i) a polymorphism in exon 13, (ii) a missense mutation (A->G) in exon 21 transforming the Ser 744 residue to a glycine in the loop of the second EF- hand in domain IV of the protein (Figure 4), and (iii) a frameshift mutation in exon 22 The exon 21 mutation and the polymorphism in exon 13 form an haplotype which is also encountered in family R17. Subcloning of the PCR products was necessary to identify the exon 22 mutation. Sequencing of several clones revealed a replacement of AG by TCATCT (data not shown) This frameshift mutation causes premature termination at nucleotide 2400 where an in frame stop codon occurs (Figure 4). The affected individuals in family R12 are homozygous for all markers of the LGMD2A interval (Allamand, submitted) Sequencing of the PCR products of exon 13 revealed a G to A transition at base 1715 of the cDNA resulting in a substitution of glutamine for Arg572 (Figure 7) within domain III, a residue which is highly conserved throughout all known calpains This mutation, detectable by loss of Mspl restriction site, is present only in this family and in no other examined LGMD2A families or unrelated controls In family R27, heteroduplex analysis followed by sequencing of the PCR products of an affected child revealed a two base pair deletion in exon 19 (Figure. 6 and table 4). One AC out of three is missing at this position of the sequence, producing a stop codon at position 2069 of the cDNA sequence (Figure 4) b) Amish families As expected, due to multiple consanguineous links, the examined LGMD2A Northern Indiana Amish patients were homozygous for the haplotype on the chromosome bearing the mutant allele (Allamand, submitted). A (G->A) missense mutation was identified at nucleotide 2306 within exon 22 (Fig. 7) The 96/16175 17 resulting codon change is CGG to CAG, transforming Arg 769 to glutamine This residue, which is conserved throughout all members of the calpain family in all species, is located in domain IV of the protein within the 3rd EF-hand at the helix-loop junction (ref). This mutation was encountered in a homozygous state in all patients from 12 chromosome 15-linked Amish families, in agreement with the haplotype analysis. We also screened six Southern Indiana Amish LGMD families, for which the chromosome 15 locus was excluded by linkage analyses (Allamand ESHG, submitted, ASHG 94). As expected, this nucleotide change was not present in any of the patients from these families, thus confirming the genetic heterogeneity of this disease in this genetically related isolate c) Brazilian families As a result of consanguineous marriages, two Brazilian families (B501 , B519) are homozygous for extended LGMD2A carrier haplotypes (data not shown) Sequencing PCR products from affected individuals of these families demonstrated that family B501 has the same exon 22 mutation found in northern Indiana Amish patients (Figure 7), but embedded in a completely different haplotype In family B519, the patients carry a C to T transition in exon 2, replacing Arg328 , h a TGA stop codon (Figure 7), thus leading, presumably, to a very truncated protein (Figure 4) d) Analysis of other LGMD families Having validated the role of the candidate gene in the chromosome 15 ascertained families, we next examined by heteroduplex analysis LGMD families for which linkage data were not informative These included one Brazilian (B505) and 13 metropolitan French pedigrees Heteroduplex bands were revealed for exons 1 , 3, 4, 5, 6, 8, 11 , 22 of one or more patients (Figure 6) Of all sequence variants, 10 were identified as possible pathogenic mutations (5 missense, 1 nonsense and 4 frameshift mutations) and 3 as polymorphisms with no change of am o acid of the protein All causative mutations identified are listed in Table 4 here-above Identical mutations were uncovered in apparently unrelated families The mutations shared by families M35 and M37, and M2888 and M1394, respectively, are likely to be the consequence of independent events since they are embedded in different marker haplotypes In contrast, it is likely that the point mutation in exon 96/16175 18 22 of the Amish and in the M32 kindreds corresponds to the same mutational event as both chromosomes share a common four marker haplotype (774G4A1 - 774G4A10-774G454D-774G4A2) around nCL1 (data not shown) possibly reflecting a common ancestor The same holds true for the AG to TCATCT substitution mutation encountered in exon 22 in families B505 and R14 The exon 8 (T->G) transversion is present in the two carrier chromosomes of M2407, the only metropolitan family homozygous by haplotype, possibly reflecting an undocumented consanguinity For some families, no disease-causing mutation has been detected thus far (M40 for example). In addition to the polymorphism present in exon 13 in families R14 and R17 (position 668) and in the intragenic microsatellites, four additional neutral variations were detected a (T->C) transition at position 96, abolishing a Ddel restriction site in exon 1 in M31 , a (C->T) transition in exon 3 (position 495) in M40 and in M37 forming a haplotype with the exon 5 mutation (in the former family, this polymorphism does not cosegregate with the disease), a (T->C) transition in the paternally derived promotor in M42 at position -428, which was also evidenced in healthy controls, and a variable poly(G) in intron 22 close to the splice site in families R20, R11 , R19, M35 and M37 The latter is also present in the members of the CEPH families, but is not useful as a genetic marker as the visualisation and interpretation of mononucleotide repeat alleles is difficult In total, sixteen independent mutational events representing fourteen different mutations were identified All mutations cosegregate with the disease in LGMD2A families The characterised morbid calpain alleles contain nucleotide changes which were not found in alleles from normal individual The discovery of two nonsense and five frameshift mutations in nCL1 supports the hypothesis that a deficiency of this product causes LGMD2A All seven mutations result in a premature in-frame stop codon leading to the production of truncated and presumably inactive proteins (Figure 4) Evidences for the morbidity of the missense mutations come from (1 ) the relative high incidence of such mutations among LGMD2A patients , although it is difficult in the absence of functional assays to differentiate between a polymorphism and a morbid mutation, the occurrence of different "missense" mutations in this gene cannot all be 6/16175 19 accounted for as rare private polymorphisms: (2) the failure to observe these mutations in control chromosomes, and (3) the occurrence of mutations in evolutionarily conserved residues and/or in regions of documented functional importance. Four of seven missense mutations change an am o acid which is conserved in all known members of the calpain family in all species (Figure 3) Two of the remaining mutations affect less conserved am o acid residues, but are located in important functional domains. The substitution V354G in exon 8 is 4 residues before the asparagine at the active site and S744G in exon 21 is within the loop of the second EF-hand and may impair the calcium-dependent regulation of calpain activity or the interaction with a small subunit (Figure 4) Several missense mutations change a hydrophobic residue to a polar one, or vice versa (Table 4) possibly disrupting higher order structures. METHODS Description of the patients The LGMD2A families analysed were from 4 different geographic origins. They included 3 Brazilian families, 13 interrelated nuclear families from the Isle of la Reunion, 10 French metropolitan families and 12 US Amish families The majority of these families were previously ascertained to belong to the chromosome 15 group by linkage analysis (Beckmann, 1991 , Young, Passos- Bueno et al., 1993) However, some families from metropolitan France as well as one Brazilian family, B505, had non significant lodscores for chromosome 15 Genomic DNA was obtained from peripheral blood lymphocytes Seguencmg of cosmid c774G4-1F11 and EcoRI restriction map of cosmids Cosmid 1F11 (Figure 1C) was subcloned following DNA preparation through Qiagen procedure (Qiagen Inc., USA) and partial digestion with either Sau3A, Rsa\ or Alu\. Size-selected restriction fragments were recovered fom low-melting agarose and eventually ligated with M13 or Bluescript (Stratagene, USA) vectors. After electroporation in E.coli, recombinant colonies were picked in 100 μl of LB/ampicillin media PCR reactions were performed on 1 μl of the culture in 10 mM Tris-HCI, pH 9.0, 50 mM KCI, 1.5 mM MgCI2, 0.1% Triton X-100, 0.01 gelatine, 200μM of each dNTP, 1 U of Taq Polymerase (Amersham) with 100 ng of each vectors primers. Amplification was initiated by 5 mm denaturation at 95°C, followed by 30 cycles of 40 sec denaturation at 92 β C and 30 sec annealing 20 at 50°C. PCR products were purified through Microcon devices (Amicon, USA) and sequenced using the dideoxy chain termination method on an ABI sequencer (Applied Biosystems, Foster City, USA). The sequences were analysed and alignments performed using the XBAP software of the Staden package, version 93.9 (Staden, 1982). Gaps between sequence contigs were filled by walking with internal primers. EcoR\ restriction map of cosmids was performed essentially as described in Sambrook et al. (1989). Northern Blot analysis The probes were labelled by random priming with dCTP-(a32p). Hybridisation was performed to human multiple tissue northern blots as recommended by the manufacturer (Clontech, USA). Analysis of PCR products from LGMD2A families One hundred ng of human DNA were used per PCR under the buffer and cycle conditions described in Fougerousse (1994) (annealing temperature shown in Table 3). Heteroduplex analysis (Keene et al., 1991 ) was performed by electrophoresis of ten μl of PCR products on a 1.5 mm-thick Hydrolink MDE gels (Bioprobe) at 500-600 volt for 12-15 h depending of the fragment length Migration profile was visualised under UV after ethidium bromide staining. For sequence analysis, the PCR products were subjected to dye-dideoxy sequencing, after purification through microcon devices (Amicon, USA). When necessary, depending on the nature of the mutations (e.g., frameshift mutation or for some heterozygotes), the PCR products were cloned using the TA cloning kit from Invitrogeπ (UK) One μl of product was ligated to 25 ng of vector at 12°C overnight. After electroporation into XL1-blue bacteria, several independent clones were analysed by PCR and sequenced as described above The invention results from the finding that the nCL1 gene when it is mutated is involved in the etiology of LGMD2A. It is exactly the contrary to what is stated in the litterature, e.g that the disease is accompanied by the presence of a deregulated calpain Identification of nCL1 as the defective gene in LGMD2A represents the first example of muscular dystrophy caused by mutation affecting a gene which is not a structural component of muscle tissue, in contrast with previously identified muscular dystrophies such as Duchenne and Becker (Bonilla et al., 1988), severe childhood autosomal recessive (Matsumara et al., 1992), Fukuyama (Matsumara et al , 1993) and merosin-deficieπt congenital muscular dystrophies (Tome et al , 1994) The understanding of the LGMD2A phenotype needs to take into account the fact that there is no active nCL1 protein in several patients, a loss compatible with the recessive manifestation of this disease. Simple models in which this protease would be involved in the degradation or destabilisation of structural components of the cytoskeleton, extracellular matrix or dystrophin complex can therefore be ruled out Furthermore, there are no signs of such alterations by immunocytogenetic studies on LGMD2 muscle biopsies (Matsumara et al., 1993, Tome et al., 1994). Likewise, since LGMD2A myofibers are apparently not different from other dystrophic ones, it seems unlikely that this calpain plays a role in myoblast fusion, as proposed for ubiquitous calpains (Wang et al., 1989) All the data disclosed in these examples confirm that the nCL1 gene is a major gene involved in the disease when mutated The fact that morbidity results from the loss of an enzymatic activity raises hopes for novel pharmaco-therapeutic prospects The availability of transgenic models will be an invaluable tool for these investigations The invention is also relative to the use of a nucleic acid or a sequence of nucleic acid of the indention, or to the use of a protein coded by the nucleic acid for the manufacturing of a drug in the prevention or treatment of LGMD2 The finding that a defective calpain underlies the pathogenesis of LGMD2A may prove useful for the identification of the other loci involved in the LGMDs Other forms of LGMD may indeed be caused by mutations in genes whose products are the CANP substrates or in genes involved in the regulation of nCL1 expression Techniques such as the two-hybrid selection system (Fields et al , 1989) could lend themselves to the isolation of the natural protein substrate(s) of this calpain, and thus potentially help to identify other LGMD loci The invention also relates to the use of all or a part of the peptidic sequence of the enzyme, or of the enzyme, product of nCL1 gene, for the screening of the ligands of this enzyme, which might be also involved in the etiology and the morbidity of LGMD2 The ligands which might be involved are for example substrate(s), activators or inhibitors of the enzyme The nucleic acids of the invention might also be used in a screening method for the determination of the components which may act on the regulation of the gene expression. A process of screening using either the enzyme or a host recombinant cell, containing the nCL1 gene and expressing the enzyme, is also a part of the invention. The pharmacological methods, and the use of nucleic acid and peptidic sequences of the invention are very potent applications. The methods used for such screenings of ligands or regulatory elements are those described for example for the screening of ligands using cloned receptors. The identification of mutations in the nCL1 gene provides the means for direct prenatal or presymptomatic diagnosis and carrier detection in families in which both mutations have been identified. Gene-based accurate classification of LGMD2A families should prove useful for the differential diagnosis of this disorder. The invention relates to a method of detection of a predisposition to LGMD2 in a family or a human being, such method comprising the steps of : - selecting one or more exons or flanking sequences which are sensitive in said family, - selecting the primers specific for the or these exons or their flanking sequences, a specific example being the PCR primers of Table 3, or an hybrid thereof, - amplifying the nucleic acid sequence, the substrate for this amplification being the DNA of the human being to be checked for the predisposition, and - comparing the amplified sequence to the corresponding sequence derived from Figure 2 or Figure 8. Table 2 indicates the sequences of the introns-exons junctions, and primers comprising in their structure these junctions are also included in the invention. All other primers suitable for such RNA or DNA amplification may be used in the method of the invention. In the same way, any suitable amplification method : PCR (for Polymerase Chain Reaction ®) NASBA ® (for Nucleic acid Sequence Based Amplification), or others might be used The methods usually used in the detection of one site mutations, like ASO (Allele specific PCR), LCR, or ARMS (Amplification Refactory Mutation System) may be implemented with the specific primers of the invention The primers, such as described in Tables 1 and 3, or including junctions of Table 2, or more generally including the flanking sequences of one of the 24 exons are also a part of the invention The kit for the detection of a predisposition to LGMD2 by nucleic acid amplification is also in the scope of the invention, such a kit comprises a least PCR primers selected from the group of : a) in those described in table 1 b) in those described in table 3 c) those including the introns-exons junctions of Table 2. d) derived from primers defined in a),b) or c). The nucleic acid sequence of claim 1 to 3 might be inserted in a viral or a retroviral vector, said vector being able to transfect a packaging cell line The packaging transfected cell line, might be used as a drug for gene therapy of LGMD2 The treatment of LGMD2 disease by gene therapy is implemented by a pharmaceutical composition containing a component selected from the group of a) a nucleic acid sequence according to claims 1 to 4, b) a cell line according to claim 24, c) an aminoacid sequence according to claims 5 to 9 96/16175 24 REFERENCES Aπkawa, E , Hoffman, E P Kaido, M , Nonaka, I , Sugita H and Arahata K (1991 ) The frequency of patients with dystrophin abnormalities in a limb-girdle patient population Neurology 41, 1491-1496 Bashir, R , Strachan, T , Keers, S , Stephenson, A , Mahjneh, I , Marconi, G , Nashef, L and Bushby, K M D (1994) A gene for autosomal recessive hmb- girdle muscular dystrophy maps to chromosome 2p Hum Mol Genet 3, 455- 457 Beckmann, J S , Richard, I , Hillaire, D , Broux, 0 , Antignac, C , Bois, E , Cann, H , Cottmgham, R W , Jr , Fe gold, N , Feingold, J , Kalil, J , Lathrop, G M , Marcadet, A , Masset, M , Mignard, C , Passos-Bueno, M R , Pelleram N , Zatz, M , Dausset, J , Fardeau, M and Cohen, D (1991 ) A gene for limb-girdle muscular dystrophy maps to chromosome 15 by linkage C R Acad Sci Paris III 372, 141-148 Bimstiel, M L , Busslmger, M and Sturb, K (1985) Transcription termination and 3' processing The end is in site' Cell 41, 349-359 Blackwell, T K and Wemtraub, H (1990) Differences and similarities in DNA- bindmg preferences of MyoD and E2A protein complexes revealed by binding site selection Science 250, 1104-1110 Bonilla, E , Samitt, C E , Miranda, A F , Hays, A P , Salviati, G , DiMauro, S , Kunkel, L M , Hoffman, E P and Rowland L P (1988) Duchenne muscular dystrophy deficiency of dystrophin at the muscle cell surface Cell 54, 447-452 Bucher, P (1990) Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences J Mol Biol 212, 563-578 96/16175 25 Bushby, K. 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Sambrook, J., Fritsh, E. F. and Maniatis, T. (1989). Molecular cloning a laboratory manual. Cold spring Harbor Laboratory Press, Cold spring Harbor, USA. Shapiro, M. and Senapathy, P. (1987). RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res. 15, 7155-7174. Sorimachi, H., Imajoh-Ohmi, S., Emori, Y., Kawasaki, H., Ohno, S., Minami, Y. and Suzuki K. (1989). Molecular cloning of a novel mammalian calcium- dependant protease distinct from both m- and mu- type. Specific expression of the mRNA in skeletal muscle. J. Biol Chem. 264, 20106-20111 Sorimachi, H., Ishiura, S. and Suzuki, K. (1993a). A novel tissue-specific calpain species expressed predominantly in the stomach comprises two alternative splicing products with and without Ca2 + -bιndιng domain. J. Biol Chem 268, 19476-19482 Sorimachi, H., Toyama-Soπmachi, N., Saido, T. 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Claims: CLAIMS 1. A nucleic acid sequence comprising : 1 ) the sequence represented in Figure 8; or 2) the sequence represented in Figure 2; or 5 3) a part of the sequence of Figure 2 with the proviso that it is able to code for a protein having a calcium dependant protease activity involved in a LGMD2 disease ; or 4) a sequence derived from a sequence defined in 1), 2) or 3) by substitution, deletion or addition of one or more nucleotides with the proviso that 0 said sequence still codes for said protease. 2. A nucleic acid sequence that is complementary to a nucleic acid sequence according to claim 1. 3. A nucleic acid sequence comprising in its structure a nucleotidic sequence according to claim 1 or 2, under the control of regulatory elements, 5 and involved in the expression of calpain activity in a LGMD2 disease. 4. A nucleic acid sequence encoding the aminoacid sequence represented in Figure 2. 5. An amino acid sequence which is coded by a nucleic acid sequence according to claims 1 to 4, characterized in that it is a calcium dependent 0 protease enzyme belonging to the calpain family, involved in the etiology of LGMD2. 6. An aminoacid sequence according to claim 5 or 6, characterized in that either it contains the sequence such as represented in Figure 2, or the amino acid sequence of Figure 2 modified by deletion, insertion and/or replacement of 5 one or more amino acids with the proviso that such aminoacid sequence has the calpain activity involved in LGMD2 disease. 7. An amino acid sequence according to claim 5 or 6, characterized in that LGMD2 is LGMD2A. 8. A host cell unable to express a calpain enzyme activity, characterized in o that it is transformed or transfected with a nucleic acid sequence comprising all or part of the nucleic acid sequence according to any one of claims 1 to 4. 9. Use of a nucleic acid according to one of claims 1 to 4 or a host cell according to claim 8 in the manufacturing of a drug for the prevention or the treatment of an LGMD2 disease. 10. Use of an amino acid sequence according to claims 5 to 6 in the manufacturing of a drug for the prevention or the treatment of an LGMD2 disease. 11. Use according to claims 10 or 11, characterized in that LGMD2 is LGMD2A. 12. Use of an amino acid sequence according to claims 5 to 7 for the screening of the ligands of said amino acid sequence, said ligand being selected in a group consisting of substrate(s), co-factors or regulatory components. 13. Use of a nucleic acid sequence according to one of claims 1 to 4 in a screening method for the determination of the components which may act on the regulation of gene expression of calpain. 14. Use of an host cell according to claim 8 in a screening method for the determination of components active on the expression of the calpain. 15. A method for detecting of a predisposition to a LGMD2 disease in a family or a human being, such method comprising the steps of : - selecting one or more exons or their flanking sequences of the gene, - selecting primers specific for these exons, or their flanking sequences, or an hybrid thereof, - amplifying the nucleic acid sequences with these primers, the substrate for this amplification being the DNA of a human being; and - comparing the amplified sequence to the corresponding sequence derived from Figure 2 or Figure 8. 16. The method according to claim 15, characterized in that the primers are those selected from the group of : a) those described in Table 1 ; b) those described in Table 3; and c) those including the introns-exons junctions of Table 2; d) those derived from the primers in a), b), or c). 17. The method according to claim 15 or 16, characterized in that LGMD2 is LGMD2A. 18. A kit for the detection of a predisposition to LGMD2 by nucleic and amplification characterized in that it comprises primers selected from the group of : a) those described in Table 1 ; b) those described in Table 3; and c) those including the introns-exons junctions of Table 2; d) those derived from the primers in a), b) or c). 19. Use of a host cell according to claim 8 in a manufacturing of a drug for gene therapy of an LGMD2 disease. 20. Pharmaceutical composition for the treatment of an LGMD2 disease characterized in that in contains a component selected from the group of : a) a nucleic acid sequence according to claims 1 to 4, b) a host cell according to claim 8, c) an aminoacid sequence according to claims 5 to 7.

Inventors: BECKMANN JACQUES, RICHARD ISABELLE, BECKMANN, JACQUES, RICHARD, ISABELLE
Applicants: ASS FR CONTRE LES MYOPATHIES, BECKMANN JACQUES, RICHARD ISABELLE, ASSOCIATION FRANCAISE CONTRE LES MYOPATHIES, BECKMANN, JACQUES, RICHARD, ISABELLE
Application year: 1995
Main classification: C12N15
IPC codes: A61K38, A61K48, A61P43, C07K14, C12N5, C12N9, C12N15, C12Q1, G01N33