RNA Mis-Splicing Effects of Noncanonical Splicing Variants in Limb-Girdle Muscular Dystrophy Type R1/2A

Biallelic pathogenic variants in the CAPN3 gene cause limb-girdle muscular dystrophy type R1/2A (LGMDR1/2A). Our study investigated RNA mis-splicing effects of 5 noncanonical intronic variants in patients with LGMDR1/2A.

Total RNA was obtained from the skeletal muscle samples of patients with LGMDR1/2A. Reverse-transcription PCR, DNA electrophoresis, agarose gel extraction, pMD18-T vector cloning, and sequencing were conducted.

Transcriptional analysis revealed that three of these 5 variants (c.1193 + 30G > A, c.1194-9A > G, and c.1354 + 5G > A) induced CAPN3 pre-mRNA mis-splicing through recognition of cryptic donor or acceptor splice sites. In addition, the c.2185-14T > G variant in the polypyrimidine tract of intron 20 caused the pseudoexonization of the entire intron 20 while the c.946-29T > C variant in the branch point sequence (BPS) of intron 6 led to the retention of the last 390 bp of intron 6 through disruption of original BPS and recognition of cryptic BPS and acceptor splice site. All of these noncanonical splicing variants triggering pre-mRNA mis-splicing were predicted to introduce premature termination codons. Western blotting showed deficiency of full-length (94-kDa) and 60-kDa autolytic fragments of the calpain 3 protein in skeletal muscle samples from 4 probands.

Our study broadens the spectrum of aberrant mRNA splicing caused by intronic variants in calpainopathy.