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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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