LMNA Missense Mutation Causes Nonsense-Mediated mRNA Decay and Severe Dilated Cardiomyopathy
LMNA Missense Mutation Causes Nonsense-Mediated mRNA Decay and Severe Dilated Cardiomyopathy
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DOI：
10.1161/circgen.119.002853
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发表时间：
2020-10-01
影响因子：
7.4
通讯作者：
Horie, Minoru
中科院分区：
文献类型：
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作者：
Kato, Koichi;Ohno, Seiko;Horie, Minoru
背景：LMNA是扩张型心肌病和家族性传导障碍的致病基因。通常由无义突变引起的无义介导的mRNA衰变是保护细胞免受来自突变基因的不适当蛋白质的有害影响的保护过程。无义介导的mRNA衰变诱导的非终止密码子突变是罕见的。我们研究了在2个家族中发现的LMNA错义突变对心脏纤层蛋白病的影响。方法：从患者外周血淋巴细胞或心脏组织中提取基因组DNA和总RNA。通过直接测序筛选LMNA编码外显子。通过逆转录-聚合酶链反应从总RNA产生互补DNA。通过使用核纤层蛋白A和C的特异性引物进行定量聚合酶链反应以定量LMNA互补DNA的量。进行小基因剪接报告子实验以评估检测到的变体对RNA剪接的影响。结果：通过基因组DNA测序，在2个扩张型心肌病和心脏传导障碍家系中发现LMNA基因第5外显子末端存在c.936 G>C（p.Q312H）错义突变。这种变异先前在一个患有肌肉萎缩症和心脏传导障碍的法国家庭中报道。互补DNA测序表明突变等位基因不存在。通过定量聚合酶链反应测定，我们证实了90%的LMNA互补DNA减少。小基因剪接报告基因测定证明了变异体的剪接错误。结论：在2个家系中发现了LMNA错义突变，该突变破坏了正常的剪接位点，导致无义介导的mRNA降解，导致严重的心肌核纤层蛋白病。
Background:LMNA is a known causative gene of dilated cardiomyopathy and familial conduction disturbance. Nonsense-mediated mRNA decay, normally caused by nonsense mutations, is a safeguard process to protect cells from deleterious effects of inappropriate proteins from mutated genes. Nonsense-mediated mRNA decay induced by nonstop codon mutations is rare. We investigated the effect of an LMNA missense mutation identified in 2 families affected by cardiac laminopathy.Methods:Genomic DNA and total RNA were isolated from patients' peripheral blood lymphocytes or cardiac tissue. LMNA-coding exons were screened by direct sequencing. Complementary DNAs were generated by a reverse transcription-polymerase chain reaction from total RNA. Quantitative polymerase chain reaction was performed to quantify the LMNA complementary DNA amount by using specific primers for lamins A and C. A minigene splicing reporter experiment was performed to assess the effect of detected variants on RNA splicing. The protein expressions of both isoforms were analyzed by Western blotting.Results:We detected a missense variant c.936 G>C (p. Q312H) at the end of exon 5 of LMNA by genomic DNA sequencing in 2 unrelated families affected by dilated cardiomyopathy and cardiac conduction disturbance. This variant was previously reported in a French family suffering from muscular dystrophy and cardiac conduction disturbance. Sequencing of complementary DNA demonstrated that the mutated allele was absent. By quantitative polymerase chain reaction assay, we confirmed a 90% reduction in LMNA complementary DNA. The minigene splicing reporter assay demonstrated a splicing error by the variant. Western blot analysis revealed that lamin A and C expressions were reduced far >50%.Conclusions:We report an LMNA missense mutation found in 2 families, which disrupted a normal splicing site, led to nonsense-mediated mRNA decay, and resulted in severe cardiac laminopathy.