Full-length isoform sequencing for resolving the molecular basis of Charcot-Marie-Tooth 2A.
Full-length isoform sequencing for resolving the molecular basis of Charcot-Marie-Tooth 2A.
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用于解析 Charcot-Marie-Tooth 2A 分子基础的全长异构体测序。
DOI：
10.1101/2023.02.07.526487
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发表时间：
2023
期刊：
影响因子：
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作者：
Stergachis,AndrewB;Blue,ElizabethE;Gillentine,MadelynA;Wang,Lee-Kai;Schwarze,Ulrike;Cortés,AdrianaSedeño;Ranchalis,Jane;Allworth,Aimee;Bland,AustinE;Chanprasert,Sirisak;Chen,Jingheng;Doherty,Daniel;Folta,AndrewB;Glass,Ian;
目的：对患者来源的样本进行转录测序已被证明可以提高疑似孟德尔病症病例的诊断率，但全长长读转录测序的额外好处在很大程度上尚未得到探索。方法：我们应用短读和全长转录本测序以及线粒体功能研究，对患者来源的成纤维细胞系进行研究，该细胞系来自以前缺乏分子诊断的神经病变患者。结果鉴定出一个内含子纯合子smfn2c。600-31T >g变体破坏了对内含子6剪接至关重要的分支点。用无义介导的mRNA衰变（NMD）抑制剂处理后，全长长读异构体互补DNA （cDNA）测序显示，该变异产生5种不同的剪接转录物改变。所有5个改变的剪接转录本都破坏了开放阅读框，并受到NMD的影响。此外，患者来源的成纤维细胞系显示异常脂滴形成，与MFN2功能障碍一致。尽管正确剪接的全长MFN2转录本仍然会产生，但这种分支点变异会导致MFN2水平不足和常染色体隐性的轴突型2A型夏科-玛丽-图斯病（CMT2A）。本病例强调了全长异构体测序在描述未确诊罕见疾病的分子机制方面的应用，并扩展了我们对CMT2A遗传基础的理解。
ObjectivesTranscript sequencing of patient-derived samples has been shown to improve the diagnostic yield for solving cases of suspected Mendelian conditions, yet the added benefit of full-length long-read transcript sequencing is largely unexplored.MethodsWe applied short-read and full-length transcript sequencing and mitochondrial functional studies to a patient-derived fibroblast cell line from an individual with neuropathy that previously lacked a molecular diagnosis.ResultsWe identified an intronic homozygousMFN2c.600-31T>G variant that disrupts the branch point critical for intron 6 splicing. Full-length long-read isoform complementary DNA (cDNA) sequencing after treatment with a nonsense-mediated mRNA decay (NMD) inhibitor revealed that this variant creates 5 distinct altered splicing transcripts. All 5 altered splicing transcripts have disrupted open reading frames and are subject to NMD. Furthermore, a patient-derived fibroblast line demonstrated abnormal lipid droplet formation, consistent with MFN2 dysfunction. Although correctly spliced full-lengthMFN2transcripts are still produced, this branch point variant results in deficient MFN2 levels and autosomal recessive Charcot-Marie-Tooth disease, axonal, type 2A (CMT2A).DiscussionThis case highlights the utility of full-length isoform sequencing for characterizing the molecular mechanism of undiagnosed rare diseases and expands our understanding of the genetic basis for CMT2A.