G.O.7: Multiple genetic variations in limb-girdle muscular dystrophies

Maria Flavia Savarese ,O De Concilio,Claudio Bruno ,Teresa Giugliano ,Arcomaria Garofalo ,Lucia Morandi ,Claudio Pisano ,Tiziana Mongini ,E Bertini ,Luisa Politano ,Gabriele Siciliano ,Annalaura Torella ,Francesca Del Vecchio Blanco ,Sabrina Sacconi ,Giuseppina Di Fruscio ,Adele D’amico ,Marina Mora ,Olimpia Musumeci ,Margherita Mutarelli ,Giorgio Tasca ,Filippo M Santorelli ,Giulio Piluso ,Elena Pegoraro ,Giacomo Pietro Comi ,Antonio Toscano ,Enzo Ricci ,Corrado Angelini ,Carlo Minetti ,Chiara Fiorillo ,Marina Fanin ,Vincenzo Nigro ,Vincenzo Nigro

doi:10.1016/j.nmd.2014.06.195

Abstract

Limb-girdle muscular dystrophies (LGMDs) are a highly heterogeneous group of muscle disorders affecting the pelvic and the shoulder girdle musculature. Until now, over thirty disease genes are known. Even if the age of onset and other clinical features could address the diagnosis towards the specific LGMD form, the phenotypic heterogeneity within each form hampers an easy and rapid molecular identification of causative mutations. We developed a unique and exhaustive platform, based on Next Generation Sequencing on Illumina HiSeq, to analyze 89 muscular disease genes at very high coverage. This regains 20–30% of missing sequences when compared with whole exome sequencing of the same DNA samples. Using this protocol, we have sequenced 312 patients with a LGMD phenotype. Most patients have been previously studied using a gene-by-gene approach without success. Thus, these cases are somewhat enriched for unknown and elusive mutations. Nevertheless, in about 20% of patients we found typical causative mutations in predicted genes, missed by Sanger sequencing or generally poorly studied. An additional 30% of patients carry other novel pathogenic variations. As easily predictable, the diagnostic rate sensibly increases in naïve samples not previously screened. Our extensive procedure allowed us to obtain a full comprehensive view of all sequence variants in these samples and to refine the genotype-phenotype correlation. More interestingly, we found in each patient at least five rare mutations (<0.01% in the general population); in about 35% of patients, we identified clearly damaging variants in addition to the causative ones. If we had detected these variants in the context of a single gene testing, we would have considered each of them as causative. In conclusion, our data suggest that "multiple troubles" may lead to complex phenotypes; in some patients, multiple defects in different genes could account for the variable expressivity and phenotypic divergence.

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