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. Deleterious mutations in two tumor suppressor genes, BRCA1 and BRCA2, confer high risk for breast and/or ovarian cancer and are responsible for the majority of hereditary families. Recently, comprehensive clinical DNA sequencing revealed deleterious mutations in BRCA1 or BRCA2 in 17.2% of 10,000 patients. Women with a history of breast cancer or ovarian cancer had deleterious mutation rates of 20% and 34%, respectively. 1 The discovery of large genomic rearrangements within BRCA1 may partially explain the apparent discrepancy between expected mutation frequency and practical clinical sensitivity because existing clinical protocols using polymerase chain reaction (PCR)-based techniques designed to scan coding regions generally cannot detect these mutations. 2,3 The population distributions of rearrangement mutations mirror those of point mutations where some, such as the duplication of exon 13, are highly prevalent, whereas others appear restricted to certain ethnic groups or limited to single families. [3][4][5] Because both genes exhibit mutations distributed throughout their coding regions, clinical genetic tests have focused on mutation scanning encompassing their entire open reading frames. This approach has generated substantial data on the genetic variation within the coding regions of these genes (Breast Cancer Informatio...

Cross‐sectional analysis of germline BRCA1 and BRCA2 mutations in Japanese patients suspected to have hereditary breast/ovarian cancer

“…F, frameshift mutation; M, missense mutation; N, nonsense mutation; NR, not reported; P, genetic polymorphism; S, splice site mutation; Syn, synonymous mutation. 21, P = 0.01). All three mutations detected in Group I-1 were in BRCA2, of which two subjects showed the same mutational type, i.e.…”

“…This analysis also included the detection of the following five specific large genomic rearrangements of the BRCA1 gene (five-site rearrangement panel): 3.8-kb deletion of exon 13 and 510-bp deletion of exon 22 described in individuals of Dutch ancestry, (18) 6-kb duplication of exon 13 described in individuals of European (particularly British) ancestry, (19) 7.1-kb deletion of exons 8 and 9 described in individuals of European ancestry, (20) and 26-kb deletion of exons [14][15][16][17][18][19][20]. (21) Nucleotide positions of mutations were expressed according to GenBank entries U14680 and U43746. All variants were interpreted according to the following criteria.…”

Prevalence of five previously reported and recurrent BRCA1 genetic rearrangement mutations in 20,000 patients from hereditary breast/ovarian cancer families

“…Five rearrangement mutations were selected for study: a 6-kb duplication of exon 13 that was reported to be a recurrent founder mutation in breast cancer families of Celtic descent (Puget et al, 1999b; BRCA1 Exon 13 Duplication Screening Group, 2000); a 26-kb deletion of exons 14-20 identified in patients of Western European ancestry (Ward et al, 2005); a 510-bp deletion of exon 22 and a 3.4-kb deletion of exon 13 that represent founder mutations in Dutch patients (Petrij-Bosch et al, 1997) and may represent 23% of all BRCA1 mutations in that population (Hogervorst et al, 2003); and a 7.1-kb deletion of exons 8-9 that has been reported in two North American families of Western European descent (Rohlfs et al, 2000). PCR-based assays for detecting each specific mutation were developed (Fig.…”

“…Deletion of exons 14-20 is noteworthy because single-nucleotide polymorphism (SNP) haplotype analysis of clinical sequence results can sometimes indicate patients at greatly increased risk for this mutation, depending on the normal allele that is carried (Ward et al, 2005). Ten patients responded to letters informing them that they had the genotype that indicated this increased risk and offering them this newly available test.…”

Identification of rare complete BRCA1 gene deletion using a combination of SNP haplotype analysis, MLPA and array-CGH techniques

“…known SNPs (c.2082C > T, c.2311T > C, c.2612C > T, c.3113A > G, c.3548A > G in exon 11; c.4308T > C in exon 13; c.4837A > G in exon 16, IVS16 -68A > G in intron 16 and IVS18 + 65G > A in intron 18) may occur and were used to assign consensus and nonconsensus SNP haplotypes in the BRCA1 gene previously [8,10,11].…”