BRCA1 and BRCA2 germline mutations in Malaysian women with early-onset breast cancer without a family history

Background

In Asia, breast cancer is characterised by an early age of onset: In Malaysia, approximately 50% of cases occur in women under the age of 50 years. A proportion of these cases may be attributable, at least in part, to genetic components, but to date, the contribution of genetic components to breast cancer in many of Malaysia''s ethnic groups has not been well-characterised.

Methodology

Given that hereditary breast carcinoma is primarily due to germline mutations in one of two breast cancer susceptibility genes, BRCA1 and BRCA2, we have characterised the spectrum of BRCA mutations in a cohort of 37 individuals with early-onset disease (≤40 years) and no reported family history. Mutational analysis of BRCA1 and BRCA2 was conducted by full sequencing of all exons and intron-exon junctions.

Conclusions

Here, we report a total of 14 BRCA1 and 17 BRCA2 sequence alterations, of which eight are novel (3 BRCA1 and 5 BRCA2). One deleterious BRCA1 mutation and 2 deleterious BRCA2 mutations, all of which are novel mutations, were identified in 3 of 37 individuals. This represents a prevalence of 2.7% and 5.4% respectively, which is consistent with other studies in other Asian ethnic groups (4–9%).     

Prevalence and penetrance of germline BRCA1 and BRCA2 mutations in a population series of 649 women with ovarian cancer

A population-based series of 649 unselected incident cases of ovarian cancer diagnosed in Ontario, Canada, during 1995-96 was screened for germline mutations in BRCA1 and BRCA2. We specifically tested for 11 of the most commonly reported mutations in the two genes. Then, cases were assessed with the protein-truncation test (PTT) for exon 11 of BRCA1, with denaturing gradient gel electrophoresis for the remainder of BRCA1, and with PTT for exons 10 and 11 of BRCA2. No mutations were found in all 134 women with tumors of borderline histology. Among the 515 women with invasive cancers, we identified 60 mutations, 39 in BRCA1 and 21 in BRCA2. The total mutation frequency among women with invasive cancers, 11.7% (95% confidence interval [95%CI] 9.2%-14.8%), is higher than previous estimates. Hereditary ovarian cancers diagnosed at age <50 years were mostly (83%) due to BRCA1, whereas the majority (60%) of those diagnosed at age >60 years were due to BRCA2. Mutations were found in 19% of women reporting first-degree relatives with breast or ovarian cancer and in 6.5% of women with no affected first-degree relatives. Risks of ovarian, breast, and stomach cancers and leukemias/lymphomas were increased nine-, five-, six- and threefold, respectively, among first-degree relatives of cases carrying BRCA1 mutations, compared with relatives of noncarriers, and risk of colorectal cancer was increased threefold for relatives of cases carrying BRCA2 mutations. For carriers of BRCA1 mutations, the estimated penetrance by age 80 years was 36% for ovarian cancer and 68% for breast cancer. In breast-cancer risk for first-degree relatives, there was a strong trend according to mutation location along the coding sequence of BRCA1, with little evidence of increased risk for mutations in the 5' fifth, but 8.8-fold increased risk for mutations in the 3' fifth (95%CI 3.6-22.0), corresponding to a carrier penetrance of essentially 100%. Ovarian, colorectal, stomach, pancreatic, and prostate cancer occurred among first-degree relatives of carriers of BRCA2 mutations only when mutations were in the ovarian cancer-cluster region (OCCR) of exon 11, whereas an excess of breast cancer was seen when mutations were outside the OCCR. For cancers of all sites combined, the estimated penetrance of BRCA2 mutations was greater for males than for females, 53% versus 38%. Past studies may have underestimated the contribution of BRCA2 to ovarian cancer, because mutations in this gene cause predominantly late-onset cancer, and previous work has focused more on early-onset disease. If confirmed in future studies, the trend in breast-cancer penetrance, according to mutation location along the BRCA1 coding sequence, may have significant impact on treatment decisions for carriers of BRCA1-mutations. As well, BRCA2 mutations may prove to be a greater cause of cancer in male carriers than previously has been thought.     

Contribution of BRCA1 and BRCA2 mutations to breast and ovarian cancer in Pakistan

The population of Pakistan has been reported to have the highest rate of breast cancer of any Asian population (excluding Jews in Israel) and one of the highest rates of ovarian cancer worldwide. To explore the contribution that genetic factors make to these high rates, we have conducted a case-control study of 341 case subjects with breast cancer, 120 case subjects with ovarian cancer, and 200 female control subjects from two major cities of Pakistan (Karachi and Lahore). The prevalence of BRCA1 or BRCA2 mutations among case subjects with breast cancer was 6.7% (95% confidence interval [CI] 4.1%-9.4%), and that among case subjects with ovarian cancer was 15.8% (95% CI 9.2%-22.4%). Mutations of the BRCA1 gene accounted for 84% of the mutations among case subjects with ovarian cancer and 65% of mutations among case subjects with breast cancer. The majority of detected mutations are unique to Pakistan. Five BRCA1 mutations (2080insA, 3889delAG, 4184del4, 4284delAG, and IVS14-1A-->G) and one BRCA2 mutation (3337C-->T) were found in multiple case subjects and represent candidate founder mutations. The penetrance of deleterious mutations in BRCA1 and BRCA2 is comparable to that of Western populations. The cumulative risk of cancer to age 85 years in female first-degree relatives of BRCA1-mutation-positive case subjects was 48% and was 37% for first-degree relatives of the BRCA2-mutation-positive case subjects. A higher proportion of case subjects with breast cancer than of control subjects were the progeny of first-cousin marriages (odds ratio [OR] 2.1; 95% CI 1.4-3.3; P=.001). The effects of consanguinity were significant for case subjects with early-onset breast cancer (age <40 years) (OR=2.7; 95% CI 1.5-4.9; P=.0008) and case subjects with ovarian cancer (OR=2.4; 95% CI 1.4-4.2; P=.002). These results suggest that recessively inherited genes may contribute to breast and ovarian cancer risk in Pakistan.     

Molecular characterization of germline mutations in the BRCA1 and BRCA2 genes from breast cancer families in Taiwan

A total of 18 families with multiple cases of breast cancer were identified from southern Taiwan, and 5 of these families were found to carry cancer-associated germline mutations in the BRCA1 and BRCA2 genes. One novel cryptic splicing mutation of the BRCA1 gene, found in two unrelated families, was shown to be a deletion of 10 bp near the branch site in intron 7. This mutation causes an insertion of 59 nucleotides derived from intron 7 and results in a frameshift, leading to premature translational termination of BRCA1 mRNA in exon 8. Deletions of 2670delC, 3073delT and 6696-7delTC in the BRCA2 gene were found in three other breast cancer families. All three deletions are predicted to generate frameshifts and to result in the premature termination of BRCA2 protein translation. Several genetic polymorphisms in both BRCA1 and BRCA2 genes were also detected in this investigation. Received: 28 September 1998 / Accepted: 20 November 1998     

Founder BRCA1 and BRCA2 mutations in French Canadian breast and ovarian cancer families.

We have identified four mutations in each of the breast cancer-susceptibility genes, BRCA1 and BRCA2, in French Canadian breast cancer and breast/ovarian cancer families from Quebec. To identify founder effects, we examined independently ascertained French Canadian cancer families for the distribution of these eight mutations. Mutations were found in 41 of 97 families. Six of eight mutations were observed at least twice. The BRCA1 C4446T mutation was the most common mutation found, followed by the BRCA2 8765delAG mutation. Together, these mutations were found in 28 of 41 families identified to have a mutation. The odds of detection of any of the four BRCA1 mutations was 18.7x greater if one or more cases of ovarian cancer were also present in the family. The odds of detection of any of the four BRCA2 mutations was 5.3x greater if there were at least five cases of breast cancer in the family. Interestingly, the presence of a breast cancer case <36 years of age was strongly predictive of the presence of any of the eight mutations screened. Carriers of the same mutation, from different families, shared similar haplotypes, indicating that the mutant alleles were likely to be identical by descent for a mutation in the founder population. The identification of common BRCA1 and BRCA2 mutations will facilitate carrier detection in French Canadian breast cancer and breast/ovarian cancer families.     

Three novel BRCA1/BRCA2 mutations in breast/ovarian cancer families in Croatia

BRCA1 and BRCA2 genes from 167 candidates (145 families) were scanned for mutations. We identified 14 pathogenic point mutations in 17 candidates, 9 in BRCA1 and 5 in BRCA2. Of those, 11 have been previously described and 3 were novel (c.5335C>T in BRCA1 and c.4139_4140dupTT and c.8175G>A in BRCA2). No large deletions or duplications involving BRCA1 and BRCA2 genes were identified. No founder mutations were detected for the Croatian population. Croatia shares most of the mutations with neighboring Slovenia and also with Germany, Austria and Poland. Two common sequence variants in BRCA1, c.2077G>A and c.4956G>A, were found more frequently in mutation carriers compared to healthy controls. No difference in BRCA2 variants was detected between the groups. Haplotype inference showed no difference in haplotype distributions between deleterious mutation carriers and non-carriers in neither BRCA1 nor BRCA2. In silico analyses identified one BRCA1 sequence variant (c.4039A>G) and two BRCA2 variants (c.5986G>A and c.6884G>C) as harmful with high probability, and inconclusive results were obtained for our novel BRCA2 variant c.3864_3866delTAA. Combination of QMPSF and HRMA methods provides high detection rate and complete coverage of BRCA1/2 genes. Benefit of BRCA1/2 mutation testing is clear, since we detected mutations in young unaffected women, who will be closely monitored for breast and ovarian cancer.     

RAD51C germline mutations in breast and ovarian cancer cases from high-risk families

BRCA1 and BRCA2 are the most well-known breast cancer susceptibility genes. Additional genes involved in DNA repair have been identified as predisposing to breast cancer. One such gene, RAD51C, is essential for homologous recombination repair. Several likely pathogenic RAD51C mutations have been identified in BRCA1- and BRCA2-negative breast and ovarian cancer families. We performed complete sequencing of RAD51C in germline DNA of 286 female breast and/or ovarian cancer cases with a family history of breast and ovarian cancers, who had previously tested negative for mutations in BRCA1 and BRCA2. We screened 133 breast cancer cases, 119 ovarian cancer cases, and 34 with both breast and ovarian cancers. Fifteen DNA sequence variants were identified; including four intronic, one 5' UTR, one promoter, three synonymous, and six non-synonymous variants. None were truncating. The in-silico SIFT and Polyphen programs were used to predict possible pathogenicity of the six non-synonomous variants based on sequence conservation. G153D and T287A were predicted to be likely pathogenic. Two additional variants, A126T and R214C alter amino acids in important domains of the protein such that they could be pathogenic. Two-hybrid screening and immunoblot analyses were performed to assess the functionality of these four non-synonomous variants in yeast. The RAD51C-G153D protein displayed no detectable interaction with either XRCC3 or RAD51B, and RAD51C-R214C displayed significantly decreased interaction with both XRCC3 and RAD51B (p<0.001). Immunoblots of RAD51C-Gal4 activation domain fusion peptides showed protein levels of RAD51C-G153D and RAD51C-R214C that were 50% and 60% of the wild-type, respectively. Based on these data, the RAD51C-G153D variant is likely to be pathogenic, while the RAD51C- R214C variant is hypomorphic of uncertain pathogenicity. These results provide further support that RAD51C is a rare breast and ovarian cancer susceptibility gene.     

Founding BRCA1 mutations in hereditary breast and ovarian cancer in southern Sweden.

Nine different germ-line mutations in the BRCA1 breast and ovarian cancer susceptibility gene were identified in 15 of 47 kindreds from southern Sweden, by use of SSCP and heteroduplex analysis of all exons and flanking intron region and by a protein-truncation test for exon 11, followed by direct sequencing. All but one of the mutations are predicted to give rise to premature translation termination and include seven frameshift insertions or deletions, a nonsense mutation, and a splice acceptor site mutation. The remaining mutation is a missense mutation (Cys61Gly) in the zinc-binding motif. Four novel Swedish founding mutations were identified: the nucleotide 2595 deletion A was found in five families, the C 1806 T nonsense mutation in three families, the 3166 insertion TGAGA in three families, and the nucleotide 1201 deletion 11 in two families. Analysis of the intragenic polymorphism D17S855 supports common origins of the mutations. Eleven of the 15 kindreds manifesting BRCA1 mutations were breast-ovarian cancer families, several of them with a predominant ovarian cancer phenotype. The set of 32 families in which no BRCA1 alterations were detected included 1 breast-ovarian cancer kindred manifesting clear linkage to the BRCA1 region and loss of the wild-type chromosome in associated tumors. Other tumor types found in BRCA1 mutation/haplotype carriers included prostatic, pancreas, skin, and lung cancer, a malignant melanoma, an oligodendroglioma, and a carcinosarcoma. In all, 12 of 16 kindreds manifesting BRCA1 mutation or linkage contained ovarian cancer, as compared with only 6 of the remaining 31 families (P<.001). The present study confirms the involvement of BRCA1 in disease predisposition for a subset of hereditary breast cancer families often characterized by ovarian cancers.     

New germline mutations in non-BRCA genes among breast cancer women of Mongoloid origin

Molecular Biology Reports - In accordance with the Asian BRCA Consortium data, there is a significant difference in incidence rate of breast cancer depending on age, as well as spectrum and...     

Genotyping of BRCA1, BRCA2, and CHEK2 germline mutations in Russian breast cancer patients using diagnostic biochips

The identification of BRCA1/2 and CHEK2 germline mutations is central to the molecular diagnostics of susceptibility to breast or/and ovarian cancer. A microarray-based rapid genotyping technique has been developed for identifying BRCA1 (185delAG, 300T>G, 4153delA, 5382insC, and 4158 A>G, 5382insC), BRCA2 (695insT and 6174delT), and CHEK2 (1100delC) mutations. It was applied for 412 randomly collected breast-cancer specimens from central Russia. In 25 (6.0%) patients, breast cancer was associated with other tumors of, e.g., ovarian, cervical, or colorectal cancer. BRCA1/2 and CHEK2 mutations were detected in 33 breast-cancer patients (8.0%). The most frequent mutations were BRCA1 5382insC, which was found in 16 patients (3.9%), and CHEK2 1100delC, which was detected in seven patients (1.7%). The suggested diagnostic microarray proved to be an efficient means of identifying BRCA1/2 and CHEK2 founder mutations most frequent in central Russia and can be proposed as a high-throughput diagnostic tool for clinical genetic testing.     

BRCA2 mutations and triple-negative breast cancer

Recently, BRCA1 germline mutations were found in a high proportion (14-34%) of patients with triple-negative breast cancer (TNBC). BRCA2 was either not analyzed or showed much lower mutation frequencies. Therefore, we screened a group of TNBC patients (n = 30) of white European descent for mutations in BRCA2 as well as in BRCA1. Cases were unselected for age of disease-onset (median age at breast cancer diagnosis was 58 years, ranging from 37 to 74 years), family history of cancer and BRCA1 and BRCA2 mutation status. Half of the patients (15/30) showed a family history of breast and/or ovarian cancer. A high frequency of deleterious germline mutations was observed in BRCA2 (5/30; 16.7%), and only one case showed a BRCA1 mutation (3.3%). Although the study group was small, these results point to BRCA2 mutations being important in TNBC.     

Contribution of BRCA1 germline mutation in patients with sporadic breast cancer

Hereditary artifacts in BRCA1 gene have a significant contributory role in familial cases of breast cancer. However, its germline mutational penetrance in sporadic breast cancer cases with respect to Pakistani population has not yet been very well defined. This study was designed to assess the contributory role of germline mutations of this gene in sporadic cases of breast cancer. 150 cases of unilateral breast cancer patients, with no prior family history of breast cancer and no other disorders or diseases in general with age range 35–75 yrs, were included in this study.Mutational analysis for hot spots on Exon 2, 3 and 13 of BRCA1 was done by using Single Strand Conformational Polymorphism (SSCP). Sequence analysis revealed five variants (missense) and one novel splice site mutation at exon 13. No germline mutation was observed on the remaining exons with respect sporadic breast cancer cases in Pakistani population. A vast majority of breast cancer cases are sporadic; the present study may be helpful for designing a better genetic screening tool for germline BRCA mutations in sporadic breast cancer patients of Pakistani population. Further studies involving a screening of entire coding region of BRCA1 is required to explore the merits of genetic diagnosis and counseling in breast cancer patients.     

The founder mutations in the BRCA1, BRCA2, and ATM genes in Moroccan Jewish women with breast cancer

To gain insight into the molecular mechanisms underlying the inherited predisposition to breast cancer in non-Ashkenazi Jews, we genotyped 54 Jewish Moroccan women with breast cancer, unselected for family history of cancer, for the predominant Jewish mutations in BRCA1, BRCA2, and ATM. One patient (2%) was found to have the 185de1AG BRCA1 mutation, none was a carrier of the 6174delT BRCA2 mutation, and 2/54 (4%) were heterozygous for the ATM mutation. These rates were not significantly different from the rates in the general non-Ashkenazi population. These preliminary data may indicate that the predominant Jewish mutations in BRCA1, BRCA2, and ATM genes contribute little, if any, to breast cancer predisposition and risk among Moroccan Jews.     

Proteome changes in ovarian epithelial cells derived from women with BRCA1 mutations and family histories of cancer

Malignant transformation of the ovarian surface epithelium (OSE) accounts for most ovarian carcinoma. Detection of preneoplastic changes in the OSE leading to overt malignancy is important in prevention and management of ovarian cancer. We identified OSE proteins with altered expression derived from women with a family history (FH) of ovarian and/or breast cancer and mutations in the BRCA1 tumor suppressor gene. Proteins from SV-40-transformed FH-OSE cell lines and control OSE lines derived from women without such histories (non-family history) were separated by two-dimensional PAGE. Gels were analyzed, a protein data base was created, and proteins were characterized according to their molecular weight, isoelectric point, and relative abundance. Mass spectrometry was performed on tryptic protein digests, and data bases were searched for known proteins with the same theoretical tryptic peptide masses. Several proteins showed altered expression in the FH-OSE cells. Beta-tubulin and to a lesser extent ubiquitin carboxyl-terminal hydrolase and glyoxalase 1 appeared to be up-regulated. In contrast, proteins suppressed in FH lines include the 27-kDa heat shock protein, translationally controlled tumor protein, and several proteins associated with actin modification such as actin prepeptide, F-actin capping protein alpha subunit, and cofilin. Sequencing of several cofilin gel spots revealed phosphorylation of serine 3, a post-translational modification associated with decreased actin binding and cytoskeletal reorganization. Two-dimensional Western blots probed with cofilin antibody showed multiple protein spots with isoelectric points of 6-9 pH units. Blots of one-dimensional gels showed a significant reduction in cofilin expression in three FH lines when compared with three non-family history lines (p < or = 0.05). Identification of these and other OSE proteins may be useful in detecting changes suggestive of increased risk of developing preneoplastic disease and defining the possible role(s) of the BRCA1 gene in regulation of OSE cell function.     

Detection of eight BRCA1 mutations in 10 breast/ovarian cancer families, including 1 family with male breast cancer.

Genetic epidemiological evidence suggests that mutations in BRCA1 may be responsible for approximately one half of early onset familial breast cancer and the majority of familial breast/ovarian cancer. The recent cloning of BRCA1 allows for the direct detection of mutations, but the feasibility of presymptomatic screening for cancer susceptibility is unknown. We analyzed genomic DNA from one affected individual from each of 24 families with at least three cases of ovarian or breast cancer, using SSCP assays. Variant SSCP bands were subcloned and sequenced. Allele-specific oligonucleotide hybridization was used to verify sequence changes and to screen DNA from control individuals. Six frameshift and two missense mutations were detected in 10 different families. A frameshift mutation was detected in a male proband affected with both breast and prostate cancer. A 40-bp deletion was detected in a patient who developed intra-abdominal carcinomatosis 1 year after prophylactic oophorectomy. Mutations were detected throughout the gene, and only one was detected in more than a single family. These results provide further evidence that inherited breast and ovarian cancer can occur as a consequence of a wide array of BRCA1 mutations. These results suggests that development of a screening test for BRCA1 mutations will be technically challenging. The finding of a mutation in a family with male breast cancer, not previously thought to be related to BRCA1, also illustrates the potential difficulties of genetic counseling for individuals known to carry mutations.     

Average age-specific cumulative risk of breast cancer according to type and site of germline mutations in BRCA1 and BRCA2 estimated from multiple-case breast cancer families attending Australian family cancer clinics

If the risk of disease is not the same for all germline mutations in a given gene, or if there are other familial modifiers of risk in carriers, then family-history-based estimates of average risk for detected mutations in that gene will depend on how carriers are sampled. Risk may also depend on the site or type of mutation. We studied 51 families with strong histories of breast cancer who attended Australian family cancer clinics and in which a germline mutation in BRCA1 or BRCA2 had been identified (28 and 23 families, respectively). Breast cancer risk in carriers was estimated under maximum likelihood theory, using information from all family members including those not tested, with adjustment for ascertainment by conditioning on genotype of the proband and family phenotype. The average cumulative risk of breast cancer for mutations in either BRCA1 or BRCA2 was 27% (95% confidence interval 16-43%) to age 50 and 64% (44-83%) to age 70. When grouped, the incidence in carriers was on average 17 (10-30) times that in non-carriers, independent of gene or mutation type (hazard ratios: 11 (4-29) for BRCA1, 23 (12-43) for BRCA2 (P for difference = 0.23); 13 (6-29) for protein-truncating mutations, 30 (9-104) for missense mutations and 30 (10-90) for splice-site mutations). For missense mutations, this was equivalent to a cumulative risk to age 70 of 83% (40-100%) and was due in part, but not totally, to the missense mutations 300 T>G in BRCA1 and 4486 G>T in BRCA2, which were individually found to be associated with high risk (P<0.001). Mutations in the central region of BRCA1 may be associated with a lower risk. The issue of the pathogenicity of specific variants may be addressed analytically providing there are one or more suitably informative families with that mutation.