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

Area of Science:

• Genetics
• Molecular Biology
• Biochemistry

Background:

• Biallelic pathogenic variants in the CAPN3 gene are the cause of limb-girdle muscular dystrophy type R1/2A (LGMDR1/2A).
• Investigating noncanonical intronic variants is crucial for understanding disease mechanisms.

Purpose of the Study:

• To investigate the RNA mis-splicing effects of five noncanonical intronic variants in the CAPN3 gene.
• To determine the impact of these variants on calpain 3 protein levels.

Main Methods:

• RNA extraction from skeletal muscle samples.
• Reverse-transcription PCR, DNA electrophoresis, and sequencing.
• Western blotting to assess protein levels.

Main Results:

• Three variants induced CAPN3 pre-mRNA mis-splicing by activating cryptic splice sites.
• One variant caused pseudoexonization of intron 20, and another led to intron 6 retention.
• All splicing variants introduced premature termination codons, and protein analysis showed reduced calpain 3 levels.

Conclusions:

• Aberrant mRNA splicing caused by intronic variants in CAPN3 contributes to LGMDR1/2A.
• This study expands the known spectrum of splicing defects in calpainopathy.