O.17 Mutation spectrum of limb-girdle muscular dystrophies by New Generation Sequencing approaches

The traditional gene-to-gene approach is too expensive and time-consuming for the molecular diagnosis of genetically heterogeneous disorders. We developed a cost-effective protocol for the DNA testing of many genes in large groups of limb-girdle muscular dystrophy (LGMD) patients based on next generation technologies. We defined this process in a flow-chart, composed of four steps: (a) to identify qualitative DNA sequence variations by targeted NGS; (b) to identify copy number variations (such as deletions or duplications  >100 bp); (c) to sequence the mRNA and study splicing in diseased muscles; (d) to perform “whole exome sequencing”. For the first step we collected  >400 DNA samples from unsolved LGMD patients from a number of Italian reference clinical centres. DNA samples were pooled and enriched for 493,598 bp, covering the exons of 93 genes (Motor Haloplex 3.0). In comparison with the previous design, new genes were added and others removed, considering the ease of differential diagnosis. We obtained  >0.1 billion reads/sample and identified a large number of mutations. We preferred an asymmetrical pooling of 5 and 16 samples, in which the 5-plex pools are used to identify the variations, while the 16-plex to assign them to the single DNA samples. Samples negative for causative mutations were also studied by the Agilent MotorChip CGH array version 3.0 to identify deletions and duplications at the single exon resolution. Third, in selected cases, we performed targeted RNA-Seq starting from a muscle biopsy sample. The targeting probes had a 4× coverage with a total target size of 1.41 Mbp of sequences/sample, with a coverage of at 100×. Fourth, we performed whole exome sequencing using the novel Haloplex enrichment. On average, three rare and potentially damaging variations were detected per each patient. Our results confirm that there is a very high genetic heterogeneity, non-penetrance, and complexity in LGMDs.