Ma Hou-Shi, Gong Xiu-Li, Li Wen-Xiu, Cai Qin, Chen Yan-Wen, Guo Xin-Bing, Ren Zhao-Rui, Zeng Fanyi, Yan Jing-Bin
Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, School of medicine, Shanghai Jiao Tong University, Shanghai, 200040, China.
NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai, 200040, China.
Clin Genet. 2023 Jun;103(6):663-671. doi: 10.1111/cge.14330. Epub 2023 Mar 31.
Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a specific LGMD caused by a gene mutation encoding the calcium-dependent neutral cysteine protease calpain-3 (CAPN3). In our study, the compound heterozygosity with two missense variants c.635 T > C (p.Leu212Pro) and c.2120A > G (p.Asp707Gly) was identified in patients with LGMDR1. However, the pathogenicity of c.635 T > C has not been investigated. To evaluate the effects of this novel likely pathogenic variant to the motor system, the mouse model with c.635 T > C variant was prepared by CRISPR/Cas9 gene editing technique. The pathological results revealed that a limited number of inflammatory cells infiltrated the endomyocytes of certain c.635 T > C homozygous mice at 10 months of age. Compared with wild-type mice, motor function was not significantly impaired in Capn3 c. 635 T > C homozygous mice. Western blot and immunofluorescence assays further indicated that the expression levels of the Capn3 protein in muscle tissues of homozygous mice were similar to those of wild-type mice. However, the arrangement and ultrastructural alterations of the mitochondria in the muscular tissues of homozygous mice were confirmed by electron microscopy. Subsequently, muscle regeneration of LGMDR1 was simulated using cardiotoxin (CTX) to induce muscle necrosis and regeneration to trigger the injury modification process. The repair of the homozygous mice was significantly worse than that of the control mice at day 15 and day 21 following treatment, the c.635 T > C variant of Capn3 exhibited a significant effect on muscle regeneration of homozygous mice and induced mitochondrial damage. RNA-sequencing results demonstrated that the expression levels of the mitochondrial-related functional genes were significantly downregulated in the mutant mice. Taken together, the results of the present study strongly suggested that the LGMDR1 mouse model with a novel c.635 T > C variant in the Capn3 gene was significantly dysfunctional in muscle injury repair via impairment of the mitochondrial function.
肢带型肌营养不良隐性1型（LGMDR1），以前称为LGMD2A，是一种由编码钙依赖性中性半胱氨酸蛋白酶钙蛋白酶-3（CAPN3）的基因突变引起的特定肢带型肌营养不良。在我们的研究中，在LGMDR1患者中鉴定出具有两个错义变体c.635T>C（p.Leu212Pro）和c.2120A>G（p.Asp707Gly）的复合杂合性。然而，c.635T>C的致病性尚未得到研究。为了评估这种新的可能致病变体对运动系统的影响，通过CRISPR/Cas9基因编辑技术制备了具有c.635T>C变体的小鼠模型。病理结果显示，在10月龄时，有限数量的炎性细胞浸润了某些c.635T>C纯合小鼠的肌内膜细胞。与野生型小鼠相比，Capn3 c.635T>C纯合小鼠的运动功能没有显著受损。蛋白质免疫印迹和免疫荧光分析进一步表明，纯合小鼠肌肉组织中Capn3蛋白的表达水平与野生型小鼠相似。然而，通过电子显微镜证实了纯合小鼠肌肉组织中线粒体的排列和超微结构改变。随后，使用心脏毒素（CTX）模拟LGMDR1的肌肉再生，以诱导肌肉坏死和再生，从而触发损伤修复过程。在治疗后的第15天和第21天，纯合小鼠的修复明显比对照小鼠差，Capn3的c.635T>C变体对纯合小鼠的肌肉再生有显著影响，并诱导线粒体损伤。RNA测序结果表明，突变小鼠中线粒体相关功能基因的表达水平显著下调。综上所述，本研究结果强烈表明，Capn3基因中具有新的c.635T>C变体的LGMDR1小鼠模型通过线粒体功能受损在肌肉损伤修复中存在显著功能障碍。
