Defining the heterogeneity of unbalanced structural variation underlying breast cancer susceptibility by nanopore genome sequencing

Abstract

Germline structural variants (SVs) are challenging to resolve by conventional genetic testing assays. Long-read sequencing has improved the global characterization of SVs, but its sensitivity at cancer susceptibility loci has not been reported. Nanopore long-read genome sequencing was performed for nineteen individuals with pathogenic copy number alterations in BRCA1, BRCA2, CHEK2 and PALB2 identified by prior clinical testing. Fourteen variants, which spanned single exons to whole genes and included a tandem duplication, were accurately represented. Defining the precise breakpoints of SVs in BRCA1 and CHEK2 revealed unforeseen allelic heterogeneity and informed the mechanisms underlying the formation of recurrent deletions. Integrating read-based and statistical phasing further helped define extended haplotypes associated with founder alleles. Long-read sequencing is a sensitive method for characterizing private, recurrent and founder SVs underlying breast cancer susceptibility. Our findings demonstrate the potential for nanopore sequencing as a powerful genetic testing assay in the hereditary cancer setting.

Fig. 1. Recurrent deletions of BRCA1 exons 1–2 are mediated by sequence homology between BRCA1 intron 2 and adjacent loci.

Read alignments and contigs derived by local assembly of variant supporting reads at the BRCA1 locus. Telomeric breakpoints within intron 1 of the BRCA1 pseudogene were associated with deletions of 37,470 bp (P1) and 36,056 bp (P2), and breakpoints within NBR2 intron 1 were associated with a deletion of 6649 bp (P3). For clarity, mismatch bases and indels <50 bp are not shown.

Fig. 2. LRS reveals molecular heterogeneity of CHEK2 exons 9–10 deletions.

Read alignments and contigs derived by local assembly of variant supporting reads at the CHEK2 locus. Reads are colored in blue and pink by haplotype inferred from integrated read-based and reference-guided phasing. Reads that could not be assigned to a haplotype are colored in gray. Local haploid assemblies were generated from reads supporting each variant to refine the breakpoints of the CHEK2 del5395 European founder variant (P11) and the CHEK2 del6188 variant (P14) in representative carriers. SINEs short interspersed nuclear elements. For clarity, mismatch bases and indels <50 bp are not shown.