CONGENITAL MYOPATHIES 1 – NEMALINE: P.26 A new intronic mutation in nebulin myopathy

Abstract

The aim of this study was to demonstrate functional and clinical presentation of a new homozygous intronic mutation in nebulin. Nebulin encodes for a giant protein component of the cytoskeletal matrix that coexists with the thick and thin filaments within the sarcomeres of skeletal muscle. Of the 183 exons in the nebulin gene, at least 43 are alternatively spliced, although exons 143 and 144 are not found in the same transcript. We report the case of a 6 yo boy, born from consanguineous parents with typical phenotypic presentation for nemaline myopathy, proven on muscle biopsy. Muscle biopsy showed a significant amount of nemaline bodies. Muscle MRI was strongly suggestive of nebulin related myopathy but gene panel for congenital myopathies was negative. Exome sequencing was performed and did not reveal specific findings. Approach with SNP to show loss of heterozygosity demonstrates that nebulin gene was of interest. Deep sequencing of nebulin revealed an intronic homozygous splicing mutation between exon 144 and exon 145. Cloning of the biopsy PCR fragment revealed that a large proportion of the isoform with exon 144 contained an 117 additional base pairs probably due to the creation of a new splicing site as predicted by in silico analysis. Exon 143 and 144 cannot coexist in the same isoform but in our patient functional studies demonstrated an altered exon 144/143 isoform ratio. Nebulin protein sequence encoded by exon 143 is highly conserved across species, suggesting that it may have a special function, such as species interaction with another protein. For example, the binding site for KLHL40 is within S21, but its precise location is not known. Therefore this intronic mutation alters nebulin protein by direct functional alteration of the transcript and by destabilizing other transcripts, particularly exon 143, whose interaction in KLHL40 binding site could explain the high amount of nemaline inclusion bodies in the muscle biopsy as seen in KLHL40 related nemaline myopathy. Some unresolved cases of nemaline myopathy could be explained by intronic mutations in nebulin. New diagnostic approaches could help to explain these cases.