CAPN3基因非经典剪接位点突变致肢带型肌营养不良2A型患者3例的临床、肌肉病理和遗传学特点分析

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

目的探讨CAPN3基因非经典剪接位点突变致肢带型肌营养不良2A(LGMD2A)患者的临床、肌肉病理和遗传学特点。方法选取2016年7月至2018年7月于山东大学齐鲁医院就诊的3例LGMD2A患者为研究对象。收集其家系的临床资料,对先证者进行全外显子组测序,利用Sanger测序对候选变异进行家系验证。从先证者肌肉组织中提取RNA,利用逆转录聚合酶链反应验证剪接突变对先证者CAPN3基因前体mRNA剪接的影响。从先证者肌肉组织中提取蛋白,用蛋白质印迹法检测calpain 3蛋白的含量。结果3例先证者均存在四肢肌无力症状,近端重于远端。肌肉活组织检查结果均提示肌源性损害。基因检测表明先证者1携带CAPN3基因c.2185-14T>G和c.2305C>T(p.R769W)复合杂合突变,先证者2携带CAPN3基因c.1193+30G>A和c.20692070delAC(p.H690Rfs*9)复合杂合突变,先证者3携带CAPN3基因c.1194-9A>G纯合突变。剪接实验表明c.2185-14T>G突变位于内含子20,导致内含子20整体保留,c.1193+30G>A位于内含子9,导致内含子9前31个碱基保留,c.1194-9A>G突变位于内含子9,导致内含子9最后8个碱基保留。蛋白质印迹法结果表明先证者1和先证者2存在calpain 3蛋白缺失。结论LGMD2A患者以四肢近端肌力下降为主要临床症状,肌肉病理多表现为肌源性损害,非经典剪切位点突变致前体mRNA剪接异常在该病中发挥了致病作用。

Objective To 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.Methods For 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.Results All 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.Conclusions The 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.