Long-read whole-genome sequencing for the genetic diagnosis of dystrophinopathies

Abstract

The precise genetic diagnosis of dystrophinopathies can be challenging, largely due to rare deep intronic variants and more complex structural variants (SVs). We report on the genetic characterization of a dystrophinopathy patient. He remained without a genetic diagnosis after routine genetic testing, dystrophin protein and mRNA analysis, and short- and long-read whole DMD gene sequencing. We finally identified a novel complex SV in DMD via long-read whole-genome sequencing. The variant consists of a large-scale (~1Mb) inversion/deletion-insertion rearrangement mediated by LINE-1s. Our study shows that long-read whole-genome sequencing can serve as a clinical diagnostic tool for genetically unsolved dystrophinopathies.

Figure 1

Graphic representation of the aberrant splicing of DMD caused by the complex structural variant. The complex structural variant, 982,323bp inversion flanked by 3,719bp deletion‐insertion, caused the skipping of exons 8–51 from the mature mRNA. The aberrant transcript was predicted to create a frameshift and premature termination codon, which was consistent with the absent expression of dystrophin observed on immunostaining. (A) reference genome; (B) patient genome (NC_000023.10); (C) dystrophin pre‐mRNA; (D) dystrophin mRNA (NM_004006.2). (E) and (I) hematoxylin and eosin staining (×20); (F) and (J) immunohistochemical staining for dystrophin‐N (×20); (G) and (K) dystrophin‐C (×20); (H) and (L) dystrophin‐R (×20). (E)–(H), a healthy control; (I)–(L), the patient. L1, LINE‐1.

Figure 2

Identification and validation of the complex structural variant in DMD. (A) An integrative genomics viewer screenshot of long‐read whole‐genome sequencing showing that 13 reads indicating an inversion and a deletion event around the 5′ breakpoint region (indicated in reads with black frame). (B) Gel electrophoresis analysis of the PCR products of the genomic breakpoint regions using primers designed for the complex structural variant in the patient (positive) and healthy control (negative). Successful Sanger validation of the 3′ (C) and 5′ (D) breakpoint regions. Ctr1, a healthy control; Ctr2, a reagent control (blank).