Brian E. Ward

Sequence analysis of BRCA1 and BRCA2: correlation of mutations with family history and ovarian cancer risk.

PURPOSE Previous studies of mutations in BRCA1 or BRCA2 have used detection methods that may underestimate the actual frequency of mutations and have analyzed women using heterogeneous criteria for risk of hereditary cancer. PATIENTS AND METHODS A total of 238 women with breast cancer before age 50 or ovarian cancer at any age and at least one first- or second-degree relative with either diagnosis underwent sequence analysis of BRCA1 followed by analysis of BRCA2 (except for 27 women who declined analysis of BRCA2 after a deleterious mutation was discovered in BRCA1). Results were correlated with personal and family history of malignancy. RESULTS Deleterious mutations were identified in 94 (39%) women, including 59 of 117 (50%) from families with ovarian cancer and 35 of 121 (29%) from families without ovarian cancer. Mutations were identified in 14 of 70 (20%) women with just one other relative who developed breast cancer before age 50. In women with breast cancer, mutations in BRCA1 and BRCA2 were associated with a 10-fold increased risk of subsequent ovarian carcinoma (P = .005). CONCLUSION Because mutations in BRCA1 and BRCA2 in women with breast cancer are associated with an increased risk of ovarian cancer, analysis of these genes should be considered for women diagnosed with breast cancer who have a high probability of carrying a mutation according to the statistical model developed with these data.

BRCA1 and BRCA2 mutations in women of different ethnicities undergoing testing for hereditary breast-ovarian cancer

In women at increased risk for breast and ovarian cancer, the identification of a mutation in breast cancer gene 1 (BRCA1) and BRCA2 has important implications for screening and prevention counseling. Uncertainty regarding the role of BRCA1 and BRCA2 testing in high‐risk women from diverse ancestral backgrounds exists because of variability in prevalence estimates of deleterious (disease‐associated) mutations in non‐white populations. In this study, the authors examined the prevalence of BRCA1 and BRCA2 mutations in an ethnically diverse group of women who were referred for genetic testing.

BRCA1/BRCA2 Germline Mutations in Locally Recurrent Breast Cancer Patients After Lumpectomy and Radiation Therapy: Implications for Breast-Conserving Management in Patients With BRCA1/BRCA2 Mutations

PURPOSE Breast cancer patients treated conservatively with lumpectomy and radiation therapy (LRT) have an estimated lifetime risk of local relapse (ipsilateral breast tumor recurrence [IBTR]) of 10% to 15%. For breast cancer patients carrying BRCA1 or BRCA2 (BRCA1/2) mutations, the outcome of treatment with LRT with respect to IBTR has not been determined. In this study, we estimate the frequency of BRCA1/2 mutations in a study of breast cancer patients with IBTR treated with LRT. PATIENTS AND METHODS Between 1973 and 1994, there were 52 breast cancer patients treated with LRT who developed an IBTR within the prior irradiated breast and who were willing to participate in the current study. From our database, we also identified 52 control breast cancer patients treated with LRT without IBTR. The control patients were individually matched to the index cases with respect to multiple clinical and pathologic parameters. Lymphocyte DNA specimens from all 52 locally recurrent patients and 15 of the matched control patients under age 40 were used as templates for polymerase chain reaction amplification and dye-primer sequencing of exons 2 to 24 of BRCA1, exons 2 to 27 of BRCA2, and flanking intron sequences. RESULTS After LRT, eight (15%) of 52 breast cancer patients had IBTR with deleterious BRCA1/2 mutations. By age, there were six (40%) of 15 patients with IBTR under age 40 with BRCA1/2 mutations, one (9.0%) of 11 between ages 40 and 49, and one (3.8%) of 26 older than age 49. In comparison to the six (40%) of 15 of patients under age 40 with IBTR found to have BRCA1/2 mutations, only one (6.6%) of 15 matched control patients without IBTR and had a BRCA1/2 mutation (P =.03). The median time to IBTR for patients with BRCA1/2 mutations was 7.8 years compared with 4.7 years for patients without BRCA1/2 mutations (P =.03). By clinical and histologic criteria, these relapses represented second primary tumors developing in the conservatively treated breast. All patients with BRCA1/2 mutations and IBTR underwent successful surgical salvage mastectomy at the time of IBTR and remain alive without evidence of local or systemic progression of disease. CONCLUSION In this study, we found an elevated frequency of deleterious BRCA1/2 mutations in breast cancer patients treated with LRT who developed late IBTR. The relatively long time to IBTR, as well as the histologic and clinical criteria, suggests that these recurrent cancers actually represent new primary breast cancers. Early onset breast cancer patients experiencing IBTR have a disproportionately high frequency of deleterious BRCA1/2 mutations. This information may be helpful in guiding management in BRCA1 or BRCA2 patients considering breast-conserving therapy.

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

Many rearrangement mutations in the BRCA1 gene have been identified. It is becoming clear that some of these mutations are prevalent, and therefore their detection is necessary in order for clinical genetic tests to have high sensitivity. Published information on particular rearrangements is frequently limited to a single patient, small groups of patients, or patients of a particular ethnicity. The objectives of this work included characterizing the prevalence of five specific rearrangement mutations in a large North American patient population. A mutation‐specific multiplex PCR assay was used for determining the prevalence of five BRCA1 rearrangement mutations that previously had been reported to occur in unrelated patients. The mutation status of these rearrangements, which came from 20,712 patients at high risk for hereditary breast and/or ovarian cancers who had submitted specimens for clinical genetic testing, is presented. The results, obtained from 2,634 mutation carriers, showed a 6‐kb duplication of exon 13, identified in 53 patients (2.01%); a 26‐kb deletion encompassing exons 14–20, detected in seven patients (0.27%); a 510‐bp deletion of exon 22, detected in 5 patients (0.19%); and a 3.4‐kb deletion of exon 13, detected in one patient (0.04%). A previously reported 7.1‐kb deletion of exons 8–9 was not found. The high frequency of the exon 13 duplication makes it the fourth most prevalent mutation in these patients. These results provide an accurate picture of the prevalence of these mutations in hereditary breast/ovarian cancer patients undergoing genetic testing in North America.

Application of Embryonic Lethal or Other Obvious Phenotypes to Characterize the Clinical Significance of Genetic Variants Found in Trans with Known Deleterious Mutations

This work describes an approach to characterize the clinical significance of genetic variants detected during the genetic testing of BRCA1 in patients from hereditary breast/ovarian cancer families. Results from transgenic mice and extensive clinical testing support the hypothesis that biallelic BRCA1 mutations result in embryonic lethality. Therefore, it is reasonable to conclude that variants of uncertain clinical significance found to reside in trans with known deleterious mutations impart reduced risk for cancer. This approach was applied to a large data set of 55,630 patients who underwent clinical BRCA1 screening by whole gene direct DNA sequencing. Fourteen common single nucleotide polymorphisms (SNPs) were used to assign 10 previously defined common, recurrent, or canonical haplotypes in 99% of these cases. From a total of 1,477 genetic variants detected in these patients, excluding haplotype-tagging SNPs, 877 (59%) could be unambiguously assigned to one or more haplotypes. In 41 instances, variants previously classified as being of uncertain clinical significance, mostly missense variants, were excluded as fully penetrant mutations due to their coincidence in trans with known deleterious mutations. From a total of 1,150 patients that harbored these 41 variants, 956 carried one as the sole variant of uncertain clinical significance reported. This approach could have widespread application to other disease genes where compound heterozygous mutations are incompatible with life or result in obvious phenotypes. This largely computational technique is advantageous because it relies upon existing clinical data and is likely to prove informative for prevalent genetic variants in large data sets.

The BRCA2 genetic variant IVS7 + 2T → G is a mutation

AbstractBiochemical and genetic characterizations that support the conclusion that the variant BRCA2 IVS7 + 2T → G represents a deleterious mutation are presented. RNA analysis from a breast cancer patient with BRCA2 IVS7 + 2T → G showed that the productive message was produced from only one chromosome. A haplotype analysis confirmed that the intronic variant resides on the chromosome that does not produce the normal mRNA. Additionally, an RNA splicing product that deletes exon 7 was produced by the chromosome that carries BRCA2 IVS7 + 2T → G. The deletion of exon 7 from the RNA alters the open reading frame by removing residues 249–287 and incorporating 18 abnormal amino acids before terminating with an opal stop codon. The experimental approach presented produces strong evidence of the presence of a deleterious mutation, because the contribution by both chromosomes to each RNA species analyzed was tracked using a coding region polymorphism as a marker. Furthermore, a single nucleotide polymorphism (SNP) haplotype analysis that confirms the location of the intronic variant and an associated family history that shows a high incidence of cancer supported these biochemical data.

A Biochemical analysis demonstrates that the BRCA1 intronic variant IVS10-2A→C is a mutation

AbstractSequence analysis of cDNA from an asymptomatic patient belonging to a high-risk breast cancer family carrying the genetic variant BRCA1 IVS10-2A→C revealed that functional BRCA1 mRNA was derived from only one of the patients chromosomes. The other chromosome produced an aberrant RNA splicing transcript that deleted exon 11. Analysis of the patients genomic DNA demonstrated that the chromosome producing the non-functional mRNA carried the genotype BRCA1 IVS10-2A→C. This transversion disrupts a highly conserved base in the consensus splice acceptor motif. These results support the conclusion that BRCA1 IVS10-2A→C is a mutation that confers predisposition to breast and ovarian cancer.

A characterization of genetic variants in BRCA1 intron 8 identifies a mutation and a polymorphism

The biochemical and genetic characterizations of two variants that occur in BRCA1 intron 8 are presented. The variant IVS8+2T-->C induces an aberrant transcript that deletes exon 8. This exon-skipping deletion disrupts the open reading frame by juxtaposing exon 7 and exon 9 in the aberrant splice product. Theoretically, 50 abnormal residues from reading frame 2 are translated following exon 7 before a stop codon is encountered. The chromosomal contribution to the relevant RNA species was tracked using a silent polymorphism at codon 694 (serine AGC or AGT). Nucleotide sequencing of this polymorphic codon demonstrated that the aberrant transcript was derived solely from the chromosome encoding AGT. The normally spliced productive transcript also displayed loss of heterozygosity and was derived solely from the chromosome encoding AGC at codon 694. Also, a haplotype analysis using a breast cancer patient database showed that the chromosome bearing serine 694-AGT carried IVS8+2T-->C. A second more common variant, IVS8-58delT, was characterized as a polymorphism. Analysis of RNA from patient samples used the same silent polymorphism at codon 694 and showed that the normal message was derived from both chromosomes.

Biochemical and genetic characterisation shows that the BRCA1 IVS20 insertion is a polymorphism

Editor—Two breast cancer susceptibility genes, BRCA1 and BRCA2, have been identified.1-3 Combined, these large and complex genes have over 800 reported genetic variants. More than 50 variants occurring within the introns of these genes are known. The clinical significance of these intronic variants, which could potentially impact RNA splicing, is largely undetermined.4-7 A variant within intron 20 of BRCA1 occurs through the duplication of 12 base pairs (bp) (GTATTCCACTCC) 48 bp from the donor junction. This variant has been observed in patients from cancer families.8-10 Furthermore, biochemical analysis showed the loss of contribution by one chromosome to the normal RNA in a breast cancer patient with IVS20ins12.11 This was shown by sequencing cDNA which showed the loss of a heterozygous base at the silent polymorphism at codon 1436 (serine TCT or TCC). These authors concluded that IVS20ins12 could represent a clinically significant regulatory mutation, although this variant was also present in a control specimen. More recently, IVS20ins12 was reported in four controls (with varying family histories of cancer, but otherwise healthy) and one early onset breast/ovarian cancer patient in a Polish BRCA1 study.12 No aberrant splicing products were detected in these samples, but mRNA abundance and possible loss of transcript have not been assayed. These authors concluded that the issue merits a more extensive study. Here, a patient diagnosed with breast cancer at the age of 40 and with …

Recurrent intragenic rearrangement mutations in the tumor suppressor gene BRCA1: Prevalence results from 12,272 patients at high risk for breast and/or ovarian cancers and methods of biochemical analysis

9533 Background: Rearrangement mutations may represent 5-10% of clinically significant variants in the BRCA1 tumor suppressor gene. Several of these mutations occur recurrently in unrelated families or in specific ethic groups. Previously, we described a multiplexed PCR based method that accurately and efficiently detects several of these recurrent mutations (Nielsen et al; Proc ASCO 2002). Inclusion of this assay as part of a comprehensive genetic analysis for hereditary breast cancer in 12,272 patients revealed the prevalence of five rearrangement mutations in a predominantly North American sample set. METHODS Genomic DNA, isolated from whole blood, was inoculated into a multiplexed PCR assay that detects the previously characterized deletions of BRCA1 exons 8-9, 13, 14-20, 22 and a duplication of exon 13. RESULTS Clinical testing identified 1,646 patients (13.4%) with clinically significant mutations, 43 (2.6%) being rearrangements detected by the assay. The duplication of exon 13 was identified in 36 patients (2.2% of mutation carriers), making this the most prevalent non-Ashkenazi BRCA1 mutation in our test population. The deletions of exon 22 and exons 14-20 were found in four and three patients, respectively. No occurrences of the deletions of exons 8-9 or exon 13 were identified in this group. CONCLUSIONS Mutation-specific, as well as more generalized methods to detect large rearrangement mutations improve the sensitivity of clinical tests to determine breast/ovarian cancer risk. [Table: see text].