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.
[1]Fudan Univ, Inst Reprod & Dev, State Key Lab Genet Engn, Obstet & Gynecol Hosp,Sch Life Sci, Shanghai 200011, Peoples R China.
[2]Fudan Univ, Key Lab Reprod Regulat NPFPC, Collaborat Innovat Ctr Genet & Dev, Shanghai 200032, Peoples R China.
[3]Shanghai Key Lab Female Reprod Endocrine Related, Shanghai 200011, Peoples R China.
[4]Peking Univ, Dept Pediat, Hosp 1, Beijing 100034, Peoples R China.
[5]Baylor Coll Med, Dept Mol & Human Genet, Houston, TX 77030 USA.
[6]China Med Univ, MOE Key Lab Med Cell Biol, Res Ctr Med Genom, Coll Basic Med Sci, Shenyang 110001, Peoples R China.
[7]Baylor Coll Med, Dept Pediat, Houston, TX 77030 USA.
[8]Texas Childrens Hosp, Houston, TX 77030 USA.
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