Abstract
Purpose: Exon deletions make up to 80% of mutations in the DMD gene, which cause Duchenne and Becker muscular dystrophy. Exon 45-55 regions were reported as deletion hotspots and intron 44 harbored more than 25% of deletion start points. We aimed to investigate the fine structures of breakpoints in intron 44 to find potential mechanisms of large deletions in intron 44.Methods: Twenty-two dystrophinopathy patients whose deletion started in intron 44 were sequenced using long-read sequencing of a DMD gene capture panel. Sequence homology, palindromic sequences, and polypyrimidine sequences were searched at the breakpoint junctions. RepeatMasker was used to analyze repetitive elements and Mfold was applied to predict secondary DNA structure.Results: With a designed DMD capture panel, 22 samples achieved 2.25 gigabases and 1.28 million reads on average. Average depth was 308× and 99.98% bases were covered at least 1×. The deletion breakpoints in intron 44 were scattered and no breakpoints clustered in any region less than 500 bp. A total of 72.7% of breakpoints located in distal 100 kb of intron 44 and more repetitive elements were found in this region. Microhomologies of 0–1 bp were found in 36.4% (8/22) of patients, which corresponded with non-homologous end-joining. Microhomologies of 2–20 bp were found in 59.1% (13/22) of patients, which corresponded with microhomology-mediated end-joining. Moreover, a 7 bp insertion was found in one patient, which might be evidence of aberrant replication origin firing. Palindromic sequences, polypyrimidine sequences, and small hairpin loops were found near several breakpoint junctions. No evidence of large hairpin loop formation in deletion root sequences was observed.Conclusion: This study was the first to explore possible mechanisms underlying exon deletions starting from intron 44 of the DMD gene based on long-read sequencing. Diverse mechanisms might be associated with deletions in the DMD gene.
Highlights
The Duchenne muscular dystrophy (DMD) gene spans 2.3 Mb in chromosome X and is composed of 79 exons and lengthy introns (2.1 Mb) (Ahn and Kunkel, 1993)
Mutations on DMD can be divided into structural variations (SV) which include copy number variations (CNVs) and other complex rearrangements, and single nucleotide variations (SNVs)
Clinical data of the involved patients were extracted from the National Rare Diseases Registry System of China program (NRDRSDMD/Becker muscular dystrophy (BMD) database), which was described in detail before (Tong et al, 2020)
Summary
The DMD gene spans 2.3 Mb in chromosome X and is composed of 79 exons and lengthy introns (2.1 Mb) (Ahn and Kunkel, 1993). Among the DMD mutation spectrum, deletions of one or more exons account for the majority, ranging from 43% to 80% in previous literature (Flanigan et al, 2009; Takeshima et al, 2010; Yang J. et al, 2013; Bladen et al, 2015; Tong et al, 2020). Intron 44 spans the largest length (12%) of all introns in the DMD gene, while harboring more than 25% of the deletion start points (Tong et al, 2020). The percentage is far more out of proportion to its length, which may suggest that intron 44 may harbor specific sequence and structural features which are predisposed to large deletion
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