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

Abstract

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.

Mutational spectrum of dystrophinopathies in Singapore: Insights for genetic diagnosis and precision therapy

“…Although the turnaround time for MLPA and Sanger sequencing is less than the commercial clinical panel gene testing, which ranges between 2‐6 weeks; a complete Sanger sequencing of DMD gene is tedious and requires around 94 PCR reactions followed by sequencing. Additionally, using a single NGS‐based platform to simultaneously detect copy number variations (CNVs) and small mutations involving single nucleotide variations (SNVs) reduces the actual cost of diagnosis compared to doing a MLPA followed by a Sanger sequencing approach (Alame et al, ; Schofield et al, ; Wei et al, ). The future use of whole genome sequencing methodology can also identify structural gene rearrangements in addition to CNVs and SNVs.…”

Evidence-Based Consensus and Systematic Review on Reducing the Time to Diagnosis of Duchenne Muscular Dystrophy

“…A + = 73%; A = 27% Strongly in favor Low 1C 3,11,12,35,43,[45][46][47][48][49][50][51][52][53][54] and expert opinion Statement 9: Delays in the initial clinical diagnosis/referral to a specialist, the sequential nature of the genetic testing process, and incomplete or nonexhaustive genetic testing should be addressed in order to prevent delays in reaching a complete genetic diagnosis for patients with DMD.…”

“…The DMD gene is one of the largest known human genes (2.2 Mb), containing 79 exons 34,104 with a relatively high mutation rate (~30% of cases are caused by a de novo mutation). 11,45,105 The approximate distribution of mutations in the DMD gene is as follows: deletion of 1 or more exons, 68%; duplication of 1 or more exons, 11%; small-scale mutations, 20% (small-scale deletions, 5%; small-scale insertions, 2%; splice-site, 3%; nonsense, 10%; missense, 0.4%); and deep intronic mutations, 0.3%. 35 This distribution is supported by a number of studies.…”

“…111 Deep intronic mutations often can be detected by next-generation sequencing if the full genomic sequence of the gene is available 37,112 ; however, it can be difficult to predict the impact of intronic mutations (or variations) on mRNA. If a muscle biopsy with dystrophin staining confirms dystrophinopathy, mRNA analysis should be performed to identify any impact on mRNA splicing 11,12,45,113 that escapes detection by both MLPA and DNA sequencing. RNA analysis is also crucial to determine the consequence of the mutation on the mRNA and can be considered for discordant phenotypes.…”

“…In the near future, newer assays such as next-generation sequencing (NGS) will be able to evaluate deletions, duplications, and point mutations in a single assay. 44,45 However, because routine NGS is not yet widely available, it was not included as part of this consensus statement.…”