Efficient CNV breakpoint analysis reveals unexpected structural complexity and correlation of dosage-sensitive genes with clinical severity in genomic disorders

• Published in: Human molecular genetics Vol. 26; no. 10; pp. 1927 - 1941
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 15.05.2017
• Subjects: Base Sequence; Comparative Genomic Hybridization - methods; DNA Copy Number Variations - genetics; DNA Copy Number Variations - physiology; DNA Replication; Female; Gene Dosage - genetics; Gene Duplication - genetics; Gene Rearrangement - genetics; Genetic Association Studies - methods; Genome, Human; Genomics - methods; Humans; Male; Mutation; Pelizaeus-Merzbacher Disease - blood; Pelizaeus-Merzbacher Disease - genetics; Sequence Analysis, DNA - methods
• ISSN: 0964-6906; 1460-2083; 1460-2083
• 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.