Identification of fifteen novel germline variants in the BRCA1 3'UTR reveals a variant in a breast cancer case that introduces a functional miR-103 target site.

Mutations in the BRCA1 gene confer a substantial increase in breast cancer risk, yet routine clinical genetic screening is limited to the coding regions and intron-exon boundaries, precluding the identification of mutations in noncoding and untranslated regions (UTR). As 3'UTR mutations can influence cancer susceptibility by altering protein and microRNA (miRNA) binding regions, we screened the BRCA1 3'UTR for mutations in a large series of BRCA-mutation negative, population and clinic-based breast cancer cases, and controls. Fifteen novel BRCA1 3'UTR variants were identified, the majority of which were unique to either cases or controls. Using luciferase reporter assays, three variants found in cases, c.* 528G>C, c.* 718A>G, and c.* 1271T>C and four found in controls, c.* 309T>C, c.* 379G>A, c.* 823C>T, and c.* 264C>T, reduced 3'UTR activity (P < 0.02), whereas two variants found in cases, c.* 291C>T and c.* 1139G>T, increased 3'UTR activity (P < 0.01). Three case variants, c.* 718A>G, c.* 800T>C, and c.* 1340_1342delTGT, were predicted to create new miRNA binding sites and c.* 1340_1342delTGT caused a reduction (25%, P = 0.0007) in 3'UTR reporter activity when coexpressed with the predicted targeting miRNA, miR-103. This is the most comprehensive identification and analysis of BRCA1 3'UTR variants published to date.

Allelic imbalance in BRCA1 and BRCA2 gene expression is associated with an increased breast cancer risk.

The contribution of BRCA1 and BRCA2 to familial and non-familial forms of breast cancer has been difficult to accurately estimate because of the myriad of potential genetic and epigenetic mechanisms that can ultimately influence their expression and involvement in cellular activities. As one of these potential mechanisms, we investigated whether allelic imbalance (AI) of BRCA1 or BRCA2 expression was associated with an increased risk of developing breast cancer. By developing a quantitative approach utilizing allele-specific real-time PCR, we first evaluated AI caused by nonsense-mediated mRNA decay in patients with frameshift mutations in BRCA1 and BRCA2. We next measured AI for BRCA1 and BRCA2 in lymphocytes from three groups: familial breast cancer patients, non-familial breast cancer patients and age-matched cancer-free females. The AI ratios of BRCA1, but not BRCA2, in the lymphocytes from familial breast cancer patients were found to be significantly increased as compared to cancer-free women (BRCA1: 0.424 versus 0.211, P = 0.00001; BRCA2: 0.206 versus 0.172, P = 0.38). Similarly, the AI ratios were greater for BRCA1 and BRCA2 in the lymphocytes of non-familial breast cancer cases versus controls (BRCA1: 0.353, P = 0.002; BRCA2: 0.267, P = 0.03). Furthermore, the distribution of under-expressed alleles between cancer-free controls and familial cases was significantly different for both BRCA1 and BRCA2 gene expression (P < 0.02 and P < 0.02, respectively). In conclusion, we have found that AI affecting BRCA1 and to a lesser extent BRCA2 may contribute to both familial and non-familial forms of breast cancer.

Intronic alterations in BRCA1 and BRCA2: effect on mRNA splicing fidelity and expression.

Germline mutations in the human breast cancer susceptibility genes BRCA1 and BRCA2 account for the majority of hereditary breast and ovarian cancer. In spite of the large number of sequence variants identified in BRCA1 and BRCA2 mutation analyses, many of these genetic alterations are still classified as variants of unknown significance (VUS). In this study, we evaluated 12 BRCA1/2 intronic variants in order to differentiate their pathogenic or polymorphic effects on the mRNA splicing process. We detected the existence of aberrant splicing in three BRCA1 variants (c.301-2delA/IVS6-2delA, c.441+1G>A/IVS7+1G>A, and c.4986+6T>G/IVS16+6T>G) and two BRCA2 variants (c.8487+1G>A/IVS19+1G>A and c.8632-2A>G/IVS20-2A>G). All but one of the aberrant transcripts arise from mutations affecting the conserved splice acceptor or donor sequences and all would be predicted to result in expression of truncated BRCA1 or BRCA2 proteins. However, we demonstrated that four of these splice-site mutations (i.e., c.301-2delA, c.441+1G>A, c.4986+6T>G, and c.8632-2A>G) with premature termination codons were highly unstable and were unlikely to encode for abundant expression of a mutant protein. Three variants of BRCA1 (c.212+3A>G/IVS5+3A>G, c.593+8A>G/IVS9+8A>G, and c.4986-20A>G/IVS16-20A>G) and four variants of BRCA2 (c.516-19C>T/IVS6-19C>T, c.7976-4_7976_3delTT/IVS17-4delTT, c.8487+19A>G/IVS19+19A>G, and c.9256- 18C>A/IVS24- 18C>A) in our studies show no effects on the normal splicing process, and they are considered to be benign polymorphic alterations. Our studies help to clarify the aberrant splicing in BRCA1 and BRCA2 as well as provide information that can be used clinically to help counsel breast/ovarian cancer prone families.

Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations.

A number of methods are used for mutational analysis of BRCA1, a large multi-exon gene. A comparison was made of five methods to detect mutations generating premature stop codons that are predicted to result in synthesis of a truncated protein in BRCA1. These included four DNA-based methods: two-dimensional gene scanning (TDGS), denaturing high performance liquid chromatography (DHPLC), enzymatic mutation detection (EMD), and single strand conformation polymorphism analysis (SSCP) and an RNA/DNA-based protein truncation test (PTT) with and without complementary 5' sequencing. DNA and RNA samples isolated from 21 coded lymphoblastoid cell line samples were tested. These specimens had previously been analyzed by direct automated DNA sequencing, considered to be the optimum method for mutation detection. The set of 21 cell lines included 14 samples with 13 unique frameshift or nonsense mutations, three samples with two unique splice site mutations, and four samples without deleterious mutations. The present study focused on the detection of protein-truncating mutations, those that have been reported most often to be disease-causing alterations that segregate with cancer in families. PTT with complementary 5' sequencing correctly identified all 15 deleterious mutations. Not surprisingly, the DNA-based techniques did not detect a deletion of exon 22. EMD and DHPLC identified all of the mutations with the exception of the exon 22 deletion. Two mutations were initially missed by TDGS, but could be detected after slight changes in the test design, and five truncating mutations were missed by SSCP. It will continue to be important to use complementary methods for mutational analysis.

Altered gene expression in morphologically normal epithelial cells from heterozygous carriers of BRCA1 or BRCA2 mutations.

We hypothesized that cells bearing a single inherited "hit" in a tumor suppressor gene express an altered mRNA repertoire that may identify targets for measures that could delay or even prevent progression to carcinoma. We report here on the transcriptomes of primary breast and ovarian epithelial cells cultured from BRCA1 and BRCA2 mutation carriers and controls. Our comparison analyses identified multiple changes in gene expression, in both tissues for both mutations, which were validated independently by real-time reverse transcription-PCR analysis. Several of the differentially expressed genes had been previously proposed as cancer markers, including mammaglobin in breast cancer and serum amyloid in ovarian cancer. These findings show that heterozygosity for a mutant tumor suppressor gene can alter the expression profiles of phenotypically normal epithelial cells in a gene-specific manner; these detectable effects of "one hit" represent early molecular changes in tumorigenesis that may serve as novel biomarkers of cancer risk and as targets for chemoprevention.

Perceived stress is associated with impaired T-cell response to HPV16 in women with cervical dysplasia.

BACKGROUND: Infection with high-risk subtypes of human papillomavirus (HPV) is a central factor in the development of cervical neoplasia. Cell-mediated immunity against HPV16 plays an important role in the resolution of HPV infection and in controlling cervical disease progression. Research suggests that stress is associated with cervical disease progression, but few studies have examined the biological mechanisms that may be driving this association. PURPOSE: This study examines whether stress is associated with immune response to HPV16 among women with cervical dysplasia. METHODS: Seventy-four women presenting for colposcopy completed measures of health behaviors, stressful life events and perceived stress. A blood sample was obtained to evaluate proliferative T-cell response to HPV16, and a cervical sample was obtained during gynecologic exam for HPV-typing. RESULTS: More than 55% tested positive for one or more HPV subtypes. Women who did not show proliferative responses to HPV (i.e. non-responders) were more likely to be HPV(+) compared to women who had a response (i.e. responders). Consistent with study hypotheses, logistic regression revealed that higher levels of perceived stress were associated with a non-response to HPV16, controlling for relevant covariates. Stressful life events were not associated with T-cell response to HPV. CONCLUSIONS: Higher levels of perceived stress are associated with impaired HPV-specific immune response in women with cervical dysplasia, suggesting a potential mechanism by which stress may influence cervical disease progression.

DNA array-based method for detection of large rearrangements in the BRCA1 gene.

In most families with multiple cases of breast and ovarian cancer, the cancer appears to be associated with germline alterations in BRCA1 or BRCA2. However, somatic mutations in BRCA1 and BRCA2 in sporadic breast and ovarian tumors are rare, even though loss of heterozygosity in BRCA1 and BRCA2 loci in these tumors appears frequently. This may be attributed to mutation detection assays that detect alterations in the coding regions and splice site junctions, but that miss large gene rearrangements. To look specifically for mutations such as large gene rearrangements that span several kilobases (kb) of genomic DNA, we have developed a fluorescence DNA microarray assay. This assay rapidly and simultaneously screens for such rearrangements along the entire gene. In our screen of 15 malignant ovarian tumors, we found one sample with a novel 3-kb deletion encompassing exon 17 of BRCA1 that leads to a frameshift mutation. This deletion was not detected in the corresponding constitutive DNA. Our results indicate that, whereas somatic mutations in BRCA1 appear to be rare in ovarian cancers, the search for large gene rearrangements should be included in any BRCA1 mutational analysis. Furthermore, the method described in this report has the potential to screen clinical tumor samples for genomic rearrangements simultaneously in a large number of cancer-associated genes.

Communication of BRCA1 and BRCA2 results to at-risk relatives: a cancer risk assessment program's experience.

We describe results from a survey designed to assess patterns of communication within families shortly after an individual receives results of BRCA1 and BRCA2 mutation carrier status. Shortly after disclosure of BRCA1 and BRCA2 genetic test results, the proband was contacted by phone to administer the post disclosure survey. Questions asked included whether they had shared their results with their siblings or adult children, if there were difficulties in communicating the test results, and if there was any distress associated with the sharing of results. A total of 162 women who have received results from BRCA1 and BRCA2 genetic testing participated in the survey. The probands shared their results more often with their female than their male relatives (P < 0.001). Probands who had tested positive for a mutation in the BRCA1 or BRCA2 gene shared their results more often with their relatives than did probands who were not carriers (P = 0.002). Probands reported more often that their siblings rather than their adult children had difficulties understanding the results (P = 0.001). The probands who were carriers more often reported having difficulties explaining their results to their relatives (P < 0.001) and their relatives were upset on hearing the result more often than were the relatives of probands who were not carriers (P < 0.001). The probands who were carriers reported more often that they were upset explaining their results to their relatives than did the probands who were not carriers (P < 0.001). Individuals are disclosing their test results to their relatives. Probands who are BRCA1- or BRCA2-positive are more likely to experience difficulty and distress with the communication of their test results to family members.