Efficient CNV breakpoint analysis reveals unexpected structural complexity and correlation of dosage-sensitive genes with clinical severity in genomic disorders
Efficient CNV breakpoint analysis reveals unexpected structural complexity and correlation of dosage-sensitive genes with clinical severity in genomic disorders
复制标题
高效的 CNV 断点分析揭示了意想不到的结构复杂性以及剂量敏感基因与基因组疾病临床严重程度的相关性
DOI：
10.1093/hmg/ddx102
复制
发表时间：
2017-05-15
影响因子：
3.5
通讯作者：
Jiang, Yuwu
中科院分区：
文献类型：
--
作者：
Zhang, Ling;Wang, Jingmin;Jiang, Yuwu
基因组疾病是由包括拷贝数变异（CNV）在内的亚显微基因组重排表现的临床病症。CNV可以通过基于阵列的比较基因组杂交（aCGH）来鉴定，aCGH是基因组病症的分子诊断中最常用的技术。然而，临床aCGH仅告知探针询问区域中的CNV。既不提供方向信息，也不提供所得到的基因组重排结构，这是揭示基因组疾病的突变和致病机制的关键。长距离聚合酶链反应（PCR）是获得CNV断点连接的传统方法，但是当受到结构复杂性的挑战时，例如经常在与Pelizaeus-Merzbacher病（PMD）相关的PLP 1位点发现时，该方法效率低下。在这里，我们介绍了“捕获和单分子实时测序”（cap-SMRT-seq）和新开发的“不对称接头介导的巢式PCR步行”（ALN-walking），用于49例PMD相关CNV受试者的CNV断点测序。值得注意的是，31个CNV断点连接中的29个（94%）通过常规长距离PCR无法获得，通过cap-SMRT-seq和ALN-walking解析。值得注意的是，通过有效的断点测序揭示了意想不到的CNV复杂性，包括不能通过aCGH解决的染色体间重排。PMD相关CNV的这些基于序列的结构进一步支持DNA复制机制在CNV诱变中的作用，并促进基因型-表型相关性研究。有趣的是，CNV获得的片段长度与PMD的临床严重程度密切相关，可能反映了除PLP 1外的其他剂量敏感基因的功能贡献。我们的研究为CNV断裂点测序提供了新的有效的实验方法（特别是ALN步行），并强调了它们在揭示基因组疾病中CNV突变和发病机制方面的重要性。
Genomic disorders are the clinical conditions manifested by submicroscopic genomic rearrangements including copy number variants (CNVs). The CNVs can be identified by array-based comparative genomic hybridization (aCGH), the most commonly used technology for molecular diagnostics of genomic disorders. However, clinical aCGH only informs CNVs in the probe-interrogated regions. Neither orientational information nor the resulting genomic rearrangement structure is provided, which is a key to uncovering mutational and pathogenic mechanisms underlying genomic disorders. Long-range polymerase chain reaction (PCR) is a traditional approach to obtain CNV breakpoint junction, but this method is inefficient when challenged by structural complexity such as often found at the PLP1 locus in association with Pelizaeus-Merzbacher disease (PMD). Here we introduced 'capture and single-molecule real-time sequencing' (cap-SMRT-seq) and newly developed 'asymmetry linker-mediated nested PCR walking' (ALN-walking) for CNV breakpoint sequencing in 49 subjects with PMD-associated CNVs. Remarkably, 29 (94%) of the 31 CNV breakpoint junctions unobtainable by conventional long-range PCR were resolved by cap-SMRT-seq and ALN-walking. Notably, unexpected CNV complexities, including inter-chromosomal rearrangements that cannot be resolved by aCGH, were revealed by efficient breakpoint sequencing. These sequence-based structures of PMD-associated CNVs further support the role of DNA replicative mechanisms in CNV mutagenesis, and facilitate genotype-phenotype correlation studies. Intriguingly, the lengths of gained segments by CNVs are strongly correlated with clinical severity in PMD, potentially reflecting the functional contribution of other dosage-sensitive genes besides PLP1. Our study provides new efficient experimental approaches (especially ALN-walking) for CNV breakpoint sequencing and highlights their importance in uncovering CNV mutagenesis and pathogenesis in genomic disorders.