An in‐frame pseudoexon activation caused by a novel deep‐intronic variant in the dysferlin gene

Figures

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  Figure 1. Muscle magnetic resonance imaging findings and pathologic features of the patient. (A and B) Axial muscle T1‐weighted images of the patient showing moderate fatty infiltration of the adductor longus, adductor magnus, and semimembranosus muscles in the right thigh, mild fatty infiltration of the adductor longus muscle in the left thigh, and no obvious fatty infiltration of the lower leg muscles. (C and D) Axial muscle short T1 inversion recovery images of the patient showing mild muscle edema of the right thigh muscles, moderate muscle edema of the left thigh muscles, and no obvious muscle edema of the lower leg muscles. (M) Hematoxylin–eosin staining showing a necrotizing myopathy pattern in the patient. Immunohistochemical staining showing positive expression of dystrophin‐N (N), dystrophin‐C (O), dystrophin‐R (P), α‐sarcoglycan (Q), β‐sarcoglycan (R), and γ‐sarcoglycan (S), and a partial reduction of dysferlin expression (T). (E–L) A normal control showing positive expression of dystrophin‐N, dystrophin‐C, dystrophin‐R, α‐sarcoglycan, β‐sarcoglycan, γ‐sarcoglycan, and dysferlin. 20× magnification.

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  Figure 2. Muscle‐derived mRNA studies and genomic DYSF sequencing of the patient. The entire DYSF mRNA (NM_003494.3) was divided into 12 overlapping cDNA fragments for RT‐PCR amplification. Gel electrophoresis analysis showed that the 12 overlapping cDNA fragments were successfully amplified in a normal control (A) and the patient (B). The upper band in F2 might be an alternative transcript of the DYSF gene. (C) Direct Sanger sequencing of the third cDNA fragment of the patient revealed aberrant DYSF transcripts, but could not recognize the overlapping sequences. (D) and (E) TA cloning of the aberrant DYSF transcripts revealed a 66‐bp insertion of intron 15 sequence between DYSF exons 15 and 16 (PE15 activation) and the normal splicing of DYSF exons 15 to 16. (F) Sanger sequencing of the genomic DYSF sequence around the PE15 detected a novel single‐nucleotide variant, the c.1397 + 649C > T variant. (G) The schematic of the in‐frame PE15 activation caused by the novel c.1397 + 649C > T variant in DYSF. RT‐PCR: reverse transcription‐polymerase chain reaction; F: fragment; bp: base pair; PE: pseudoexon.