Implementation of a Reliable Next-Generation Sequencing Strategy for Molecular Diagnosis of Dystrophinopathies

Diagnosis of dystrophinopathies needs to combine several techniques for detecting copy number variations (CNVs; two-thirds of mutations) and single nucleotide variations (SNVs). We participated in the design of an amplicon-based PCR kit (Multiplicom) for sequencing with a GS-Junior instrument (Roche) and later with a MiSeq instrument (Illumina). We compared two different software programs, MiSeq Reporter (Illumina) and SeqNext (JSI Medical Systems) for data analyses. Testing of six patient DNA samples carrying 72 SNVs in the DMD gene showed an experimental sensitivity of 91.7% with MiSeq Reporter, 98.6% with SeqNext, and >99.9% with both, demonstrating the need to use two different software programs. Analytical specificity was >98%. Fifty-eight additional patient DNAs were analyzed, and 25 deleterious mutations were identified, without false-negative results. We also tested the possibility for our protocol to identify CNVs. We performed additional next-generation sequencing experiments on 50 DNAs and identified 28 CNVs, all confirmed by multiple ligation probe amplification. Statistical analyses on amplicons without CNV (n = 3797), amplicons with heterozygous deletions (n = 51) or duplications (n = 191), and with hemizygous duplications (n = 63) showed a sensitivity and specificity of >99.9%. We implemented a strategy to simultaneously detect SNVs and CNVs in the DMD gene with one comprehensive technique, allowing considerable reduction of time and cost burden for diagnosis of dystrophinopathies.