Résumé :
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Nemaline myopathy (NM) (OMIM 161800, 256030 and 605355) is a rare, heterogeneous muscle disorder defined on the basis of ultrastructural abnormalities, i.e. nemaline bodies in the muscle fibres. Typical features of NM are muscle weakness and hypotonia. Inheritance in the majority of cases is autosomal recessive, although some families have been described with an autosomal dominant mode of inheritance. Prior to this study, mutations in four different genes encoding thin filament proteins have been shown to cause NM. Mutations in the nebulin (NEB) and the skeletal muscle actin (ACTA1) genes are common causes of NM, whereas mutations in troponin T1 (TNNT1) and γ tropomyosin (TPM3) genes are rare. One of the main objectives of this study was to screen a novel candidate gene, TPM2, for mutations causing NM. Mutations in TPM3 had been shown to cause NM, and γ tropomyosin encoded by this gene was known to function in the thin filaments of the muscle sarcomere in close proximity to β-tropomyosin. Therefore, we speculated that mutations in the β tropomyosin gene, TPM2, could be causing NM. Mutation analysis of samples from 66 unrelated patients, using single strand conformation polymorphism analysis and sequencing, revealed four polymorphisms and two heterozygous missense mutations in two different families. The regions in which the TPM2 mutations were identified are homologous between different tropomyosin isoforms expressed in skeletal muscle, and affect amino acids that are conserved between species. Another main objective of this study was to clarify the molecular basis of nebulin as a cause of NM. A prerequisite for this was to elucidate the structure of NEB, which encodes a giant structural protein of the skeletal muscle sarcomere. One nebulin molecule spans the length of the thin filament. It has been speculated that during myofibrillogenesis nebulin might determine the length of the thin filaments. A search through GenBank revealed two BAC clones homologous to NEB. Altogether 183 exons were identified in the genomic sequence. The translation initiation codon is in exon 3, the stop codon and the 3'UTR in exon 183. The total length of the genomic sequence spanned by NEB is 249 kb. There are four regions with alternatively spliced exons, i.e. exons 63-66, 82-105, 143-144 and 166-177, giving rise to a number of different transcripts. These transcripts were studied with the aid of RT PCR, cloning, and sequencing. To study the expression of mouse exons 127 and 128, corresponding to human exons 143 and 144, we used quantitative real-time PCR. Exons 143 144 in humans and 127-128 in mice give rise to two different transcripts varying between muscle types and between muscles at different developmental stages. The lengths of the transcripts encoded by exons 63-66 differ between fetal and adult human muscles. Exons 166 177 express at least twenty different transcripts in adult human tibialis anterior muscle alone. Preliminary results indicate that alternative splicing of exons 82-105 also give rise to a number of different transcripts. Extensive alternative splicing of NEB may explain why NM patients with homozygous truncating mutations, contrary to expectations, show expression of the carboxy terminus of the nebulin protein. When this study was initiated mutations in NEB had already been identified as a common cause of NM. Screening for new mutations in NEB continued and new disease-causing mutations were found almost monthly. Single strand conformation polymorphism analysis and sequencing were used for mutation screening of the last 42 exons of the gene in 77 NM patients. Twelve novel recessive mutations were identified in 13 families. Affected individuals were homozygous for the mutations in five families and compound heterozygous in two, while in the remaining cases only one heterozygous mutation was identified. Most of the mutations are predicted to result in truncated or internally deleted proteins. Mutations in the differentially expressed exons are expected to reduce the nebulin isoform diversity necessary for normal muscle development.
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