Zhang Ling, Wang Jingmin, Zhang Cheng, Li Dongxiao, Carvalho Claudia M B, Ji Haoran, Xiao Jianqiu, Wu Ye, Zhou Weichen, Wang Hongyan, Jin Li, Luo Yang, Wu Xiru, Lupski James R, Zhang Feng, Jiang Yuwu
Obstetrics and Gynecology Hospital, State Key Laboratory of Genetic Engineering at School of Life Sciences, Institute of Reproduction and Development, Fudan University, Shanghai 200011, China.
Key Laboratory of Reproduction Regulation of NPFPC, Collaborative Innovation Center of Genetics and Development, Fudan University, Shanghai 200032, China.
Hum Mol Genet. 2017 May 15;26(10):1927-1941. doi: 10.1093/hmg/ddx102.
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.
基因组疾病是由亚微观基因组重排（包括拷贝数变异，CNV）所表现出的临床病症。CNV可通过基于微阵列的比较基因组杂交（aCGH）来识别，这是用于基因组疾病分子诊断的最常用技术。然而，临床aCGH仅能告知探针 interrogated区域中的CNV。既不提供方向信息，也不提供由此产生的基因组重排结构，而这是揭示基因组疾病潜在突变和致病机制的关键。长距离聚合酶链反应（PCR）是获取CNV断点连接的传统方法，但当面对诸如与佩利措伊斯 - 默茨巴赫病（PMD）相关的PLP1位点常见的结构复杂性时，该方法效率低下。在此，我们引入了“捕获和单分子实时测序”（cap-SMRT-seq）以及新开发的“不对称接头介导的巢式PCR步移”（ALN-walking），用于对49例与PMD相关的CNV患者进行CNV断点测序。值得注意的是，31个常规长距离PCR无法获得的CNV断点连接中有29个（94%）通过cap-SMRT-seq和ALN-walking得以解析。值得注意的是，高效的断点测序揭示了意想不到的CNV复杂性，包括aCGH无法解析的染色体间重排。这些基于序列的与PMD相关的CNV结构进一步支持了DNA复制机制在CNV诱变中的作用，并促进了基因型 - 表型相关性研究。有趣的是，CNV获得片段的长度与PMD的临床严重程度密切相关，这可能反映了除PLP1之外其他剂量敏感基因的功能贡献。我们的研究为CNV断点测序提供了新的高效实验方法（尤其是ALN-walking），并强调了它们在揭示基因组疾病中CNV诱变和发病机制方面的重要性。
