Correction of pseudoexon splicing caused by a novel intronic dysferlin mutation

Abstract

Objective Dysferlin is a large transmembrane protein that functions in critical processes of membrane repair and vesicle fusion. Dysferlin‐deficiency due to mutations in the dysferlin gene leads to muscular dystrophy (Miyoshi myopathy ( MM ), limb girdle muscular dystrophy type 2B ( LGMD 2B), distal myopathy with anterior tibial onset ( DMAT )), typically with early adult onset. At least 416 pathogenic dysferlin mutations are known, but for approximately 17% of patients, one or both of their pathogenic variants remain undefined following standard exon sequencing methods that interrogate exons and nearby flanking intronic regions but not the majority of intronic regions. Methods We sequenced RNA from myogenic cells to identify a novel dysferlin pathogenic variant in two affected siblings that previously had only one disease‐causing variant identified. We designed antisense oligonucleotides ( AON s) to bypass the effects of this mutation on RNA splicing. Results We identified a new pathogenic point mutation deep within dysferlin intron 50i. This intronic variant causes aberrant mRNA splicing and inclusion of an additional pseudoexon ( PE , we term PE 50.1) within the mature dysferlin mRNA . PE 50.1 inclusion alters the protein sequence, causing premature translation termination. We identified this mutation in 23 dysferlinopathy patients (seventeen families), revealing it to be one of the more prevalent dysferlin mutations. We used AON ‐mediated exon skipping to correct the aberrant PE 50.1 splicing events in vitro, which increased normal mRNA production and significantly restored dysferlin protein expression. Interpretation Deep intronic mutations can be a common underlying cause of dysferlinopathy, and importantly, could be treatable with AON ‐based exon‐skipping strategies.

Sequential targeted exome sequencing of 1001 patients affected by unexplained limb-girdle weakness

“…In addition, we identified an intronic deletion (c.585–31_585–24delTCTGCTGA) in one of the SGCA carriers, which results in partial expression of the α-sarcoglycan protein. 21 Conversely, none of the reported pathogenic intronic variants in DYSF 13 were found in the DYSF carrier.…”

“…In addition, for the DYSF carriers we Sanger sequenced two previously reported pathogenic intronic variants. 13 Finally, one patient was also subjected to genome sequence (GS) and RNA sequencing (RNA-Seq), as described elsewhere. 14 …”

The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements

“…Splicing mutations for many disorders have been tackled successfully, but with mostly subtherapeutic efficiencies, with antisense oligonucleotide approaches, for instance, for Leber congenital amaurosis [50,51,52], Stargardt disease [54], choroideremia [54,65], Miyoshi myopathy [55], and erythropoietic protoporphyria [57]. Similar to what has been achieved for HBB IVSI-110(G>A) thalassemia by moving from the antisense oligonucleotide application [66] to DARE [16], those same mutations and many more may instead be addressed by potentially highly efficient, disruption-based curative therapies.…”

The genetic profile of dysferlinopathy in a cohort of 209 cases: Genotype–phenotype relationship and a hotspot on the inner DysF domain

“…Korea (Shin et al, 2015), China (Jin et al, 2016;Xi et al, 2014), Iran (Fatehi et al, 2015), France (Krahn et al, 2009), as well as from an international multicenter study (Harris et al, 2016), demonstrating the genetic character in each population including the existence of have been reported to be located deep in intronic regions (Dominov et al, 2019). Hence, RNA analysis combined with genomic DNA will be needed to characterize undiagnosed cases.…”