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

IF 3.7 3区医学 Q2 CLINICAL NEUROLOGY Neurology-Genetics Pub Date : 2025-04-01 eCollection Date: 2025-06-01 DOI:10.1212/NXG.0000000000200259

Guangyu Wang, Haoyang Liu, Guiguan Yang, Shen Gu, Chuanzhu Yan, Pengfei Lin

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Abstract

Background and objectives: 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.

Methods: 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.

Results: 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.

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

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肢腰肌营养不良症 R1/2A 型非规范剪接变异的 RNA 错剪接效应

背景与目的：CAPN3基因双等位致病变异可导致R1/2A型肢体带状肌营养不良症（LGMDR1/2A）。我们的研究调查了LGMDR1/2A患者中5种非规范内含子变异的RNA错剪接效应。方法：从LGMDR1/2A患者的骨骼肌样本中提取总RNA。逆转录PCR、DNA电泳、琼脂糖凝胶提取、pMD18-T载体克隆及测序。结果：转录分析显示，这5个变异体中的3个（c.1193 + 30G > A、c.1194-9A > G和c.1354 + 5G > A）通过识别隐秘的供体或受体剪接位点诱导CAPN3前mrna错误剪接。此外，内含子20多嘧啶区C .2185- 14t > G变异导致整个内含子20假外显化，而内含子6分支点序列（BPS）中的C .946- 29t > C变异通过破坏原始BPS和识别隐BPS和受体剪接位点导致内含子6最后390 bp的保留。据预测，所有这些触发前mrna错误剪接的非规范剪接变体都会引入过早终止密码子。Western blotting结果显示，4个先证者骨骼肌样品中缺乏全长（94-kDa）和60-kDa的钙蛋白酶3蛋白自溶片段。讨论：我们的研究拓宽了钙痛症中由内含子变异引起的异常mRNA剪接的范围。

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来源期刊

Neurology-Genetics Medicine-Neurology (clinical)

CiteScore

6.30

自引率

3.20%

发文量

107

审稿时长

15 weeks

期刊介绍： Neurology: Genetics is an online open access journal publishing peer-reviewed reports in the field of neurogenetics. Original articles in all areas of neurogenetics will be published including rare and common genetic variation, genotype-phenotype correlations, outlier phenotypes as a result of mutations in known disease-genes, and genetic variations with a putative link to diseases. This will include studies reporting on genetic disease risk and pharmacogenomics. In addition, Neurology: Genetics will publish results of gene-based clinical trials (viral, ASO, etc.). Genetically engineered model systems are not a primary focus of Neurology: Genetics, but studies using model systems for treatment trials are welcome, including well-powered studies reporting negative results.