A multi-exonic BRCA1 deletion identified in multiple families through single nucleotide polymorphism haplotype pair analysis and gene amplification with widely dispersed primer sets

Abstract

The identification of intragenic rearrangements is important for a comprehensive understanding of mutations that occur in some clinically important genes. Single nucleotide polymorphism haplotypes obtained from clinical sequence data have been used to identify patients at high risk for rearrangement mutations. Application of this method identified a novel 26-kb deletion of BRCA1 exons 14 through 20 in patients from multiple families with hereditary breast and ovarian cancer. Clinical sequence data from 5911 anonymous patients were screened for genotypes that were inconsistent with known pairs of canonical haplotypes in BRCA1 that could be explained by hemizygous deletions involving exon 16. Long-range polymerase chain reaction demonstrated that two of six samples identified by this search contained a deletion in the expected region encompassing exons 14 through 20. The breakpoint was fully characterized by DNA sequencing and demonstrated that the deletion resulted from Alu-mediated recombination. This mutation was also identified twice in a set of 982 anonymous specimens that had negative clinical test results, but uninformative haplotypes. Three additional occurrences of this mutation were found by testing 10 other patients with the indicative genotype. An assay for this mutation was added to a comprehensive clinical breast/ovarian cancer test and eight more instances were found in 20,649 probands. This multiexon deletion has therefore been detected in 15 different North American families with hereditary breast/ovarian cancer. In conclusion, this primarily computational approach is highly effective and identifies specimens using existing data that are enriched for deletion mutations.

Figure 1

Detection of a large deletion mutation in patients by PCR. A fragment of ∼5 kb in length was produced in two (lanes 4 and 5) of six patients (lanes 1 through 6) with an unusual BRCA1 haplotype. PCR primers for these reactions annealed upstream from exon 13 (forward) and downstream from exon 21 (reverse). Lane 7 contains the PCR reaction from a control specimen with no family history of cancer. The lanes containing PCR product (lanes 1 to 7) are flanked on both sides by a HindIII digest of λ DNA as a size marker (left-hand column indicates marker fragment lengths in kb).

Figure 2

Schematic diagram of relevant regions of the BRCA1 locus. BRCA1 structure is depicted to scale as a horizontal black line (introns) with numbered gray boxes (exons 13 through 24). Distance is marked every 10 kb by vertical lines and the numbering conforms to GenBank accession no. L78833. Vertical arrows indicate the locations for 2 of the 14 polymorphisms used to define haplotypes that are assayed during clinical testing in exons 13 and 16 along with their base positions and the base change (consensus -> polymorphism). The dashed line depicts the region of BRCA1 encompassed by this deletion.

Figure 3

The sequence of the deletion breakpoint. Eighty bases centered about the breakpoint in the patient are flanked vertically by the wild-type sequences (numeric designations and sequence from GenBank accession no. L78833). Horizontal lines denote the region of identity (54 bases) between the sequences that have recombined.