Neuromuscular Diseases
Analysis of clinical, pathological and genetic features of 3 patients with limb girdle muscular dystrophy type 2A caused by non-canonical splice site mutations in the CAPN3 gene
Wang Guangyu, Liu Haoyang, Lyu Xiaoqing, Yan Chuanzhu, Lin Pengfei
Published 2023-12-08
Cite as Chin J Neurol, 2023, 56(12): 1341-1348. DOI: 10.3760/cma.j.cn113694-20230912-00154
Abstract
ObjectiveTo investigate the clinical, pathological and genetic features of 3 cases of limb-girdle muscular dystrophy 2A (LGMD2A) caused by non-canonical splice site mutations in the CAPN3 gene.
MethodsFor the 3 LGMD2A patients admitted to Qilu Hospital of Shandong University from July 2016 to July 2018 were selected as the subjects. Clinical data were collected, whole exome sequencing was conducted, and the candidate variants were verified by Sanger sequencing. Total RNA was extracted from the skeletal muscle tissue of 3 probands and effects of splicing mutations on pre-mRNA splicing in the CAPN3 gene were verified by reverse-transcription polymerase chain reaction. Total protein was extracted from the muscle tissue of the probands and expression level of calpain 3 protein was detected by Western blotting.
ResultsAll the 3 probands presented muscle weakness in upper and lower limbs, and muscle weakness in proximal limbs was more severe. Muscle biopsies all indicated myogenic impairment. Genetic sequencing showed proband 1 carried compound heterozygous c.2185-14T>G and c.2305C>T (p.R769W) mutations in the CAPN3 gene, proband 2 carried compound heterozygous c.1193+30G>A and c.2069_2070delAC (p.H690Rfs*9) mutations, and proband 3 carried homozygous c.1194-9A>G mutations in the CAPN3 gene. Splicing assay showed the c.2185-14T>G mutation located in intron 20 induced retention of the entire intron 20, the c.1193+30G>A mutation in intron 9 induced retention of the first 31 nucleotides of intron 9, and the c.1194-9A>G mutation in intron 9 induced retention of the last eight nucleotides of intron 9. Western blotting revealed deficiency of calpain 3 protein in skeletal muscle of proband 1 and proband 2.
ConclusionsThe clinical manifestation of LGMD2A is muscle weakness predominantly in proximal limbs, and the muscle pathology is mostly characterized by myogenic impairment.Moreover, aberrant splicing of pre-mRNA caused by non-canonical splice site mutations plays a pathogenic role in this disease.
Key words:
Muscular dystrophies, limb-girdle; Biopsy; Mutation; CAPN3 gene
Contributor Information
Wang Guangyu
Department of Neurology, Qilu Hospital of Shandong University, Jinan 250012, China
Liu Haoyang
Department of Neurology, Qilu Hospital of Shandong University, Jinan 250012, China
Lyu Xiaoqing
Department of Neurology, Qilu Hospital of Shandong University, Jinan 250012, China
Yan Chuanzhu
Department of Neurology, Qilu Hospital of Shandong University, Jinan 250012, China
Lin Pengfei
Department of Neurology, Qilu Hospital of Shandong University, Jinan 250012, China
