Enhanced sensitivity of peripheral blood lymphocytes from women carrying a BRCA1 mutation towards the mutagenic effects of various cytostatics

We are studying the induction and repair of DNA damage in lymphocytes of women from families with familial breast cancer and mutations in the breast cancer susceptibility genes BRCA1 and BRCA2. Our previous results indicated a close relationship between the presence of a BRCA1 mutation and sensitivity for the induction of micronuclei by gamma irradiation and hydrogen peroxide (H2O2). To further characterize the mutagen sensitivity and to better understand the underlying mechanisms, we now tested the effect of various cytostatics on the micronucleus frequencies in lymphocytes of women with various BRCA1 mutations in comparison to controls. The results presented here indicate enhanced sensitivity towards bleomycin, cisplatin, cyclophosphamide and bischloroethylnitosurea (BCNU). However, mutagen sensitivity towards cisplatin and BCNU was not accompanied by enhanced induction of sister chromatid exchanges (SCE), suggesting that intrachromosomal recombination is not affected. In contrast to the various DNA-damaging agents, there was no clear difference in the response to vincristine and taxol. FISH analysis revealed that the two aneugens mainly induced centromere-positive micronuclei to a similar extent in lymphocytes with and without a BRCA1 mutation. We conclude that cells containing a heterozygous mutation in BRCA1 are more sensitive towards different kinds of DNA damage in accordance with the proposed central role of BRCA1 in maintaining genomic integrity. Although BRCA1 has been shown to interact with the mitotic spindle, spindle poisons do not cause enhanced induction of micronuclei. Since some of the DNA-damaging mutagens tested here are used as cytostatics in breast cancer chemotherapy, it might be that women with a BRCA1 mutation are at higher risk for the induction of mutations and secondary cancers by standard therapies.     

Mutagen sensitivity of human lymphoblastoid cells with a BRCA1 mutation in comparison to ataxia telangiectasia heterozygote cells

Our previous results indicated a close relationship between the presence of a BRCA1 mutation in lymphocytes and hypersensitivity for the induction of micronuclei by gamma irradiation and hydrogen peroxide (H(2)O(2)). Comparative investigations with the comet assay (single-cell gel electrophoresis) suggested a normal rate of damage removal and pointed to a disturbed fidelity of DNA repair as a direct or indirect consequence of a BRCA1 mutation. We now wanted to see whether similar results could be obtained with lymphoblastoid cell lines (LCLs) and whether such permanent cells are suitable as a model for the investigation of mechanisms involved in mutagen sensitivity. Our results show that LCLs with a BRCA1 mutation are also hypersensitive to the chromosome-damaging effects of gamma irradiation or H(2)O(2), as revealed by the micronucleus test. Interestingly, LCLs heterozygous for an ataxia telangiectasia (AT) mutation have similar characteristics as BRCA1 cells with respect to the induction and repair of DNA damage induced by either gamma irradiation or H(2)O(2). However, caffeine enhanced the induction of micronuclei by gamma irradiation only in normal and heterozygous AT cells but not in BRCA1 cells, thus indicating a difference in the pathways leading to mutagen sensitivity in cells with a BRCA1 or an AT mutation. Our results suggest that caffeine could be useful in discriminating AT heterozygotes from carriers of a BRCA1 mutation, as well as BRCA1 mutation carriers from normal individuals.     

Germline mutations in the BRCA1 gene predisposing to breast and ovarian cancers in Upper Silesia population

Germline mutations in the BRCA1 or BRCA2 genes predispose their carriers to breast or/and ovary cancers during their lifetime. The most frequent mutations: 5382insC, 185delAG, C61G and 4153delA in BRCA1, and 6174delT and 9631delC in BRCA2 were studied in a group of 148 probands admitted for genetic counseling, using allele-specific amplification (ASA) PCR test. Fifteen carriers of three different mutations: 5382insC, 185delAG and C61G in BRCA1 were found. Two families carried the 185delAG mutation and additional two C61G in BRCA1. Nobody carried the mutation 4153delA in BRCA1 nor 6174delT or 9631delC in BRCA2. Most of the carriers of a germline mutation were observed among the patients who developed bilateral breast cancer (17%). The lowest frequency of the germline mutations was found in the healthy persons who had two or more relatives affected with breast or ovarian cancer.     

BRCA2 gene mutations in Slovenian male breast cancer patients

Male breast cancer (MBC) is a rare disease, comprising less than 1% of breast cancer patients in Slovenia. Some inherited cases are due to the mutations of BRCA1 or BRCA2 genes. There is no information available about the frequency of BRCA gene mutations in Slovenian MBC population. The purpose of this study was to characterize BRCA germline mutations in Slovenian MBC patients. Forty-one patients who were diagnosed with breast cancer at the Institute of Oncology Ljubljana between 1970 and 2006 were proposed to take part in this study. Of them, 27 agreed to follow a genetic counseling session and 25 patients agreed to provide a blood sample for genetic testing. The BRCA1 and BRCA2 genes from the MBC patients were screened for four highly recurrent mutations in the Slovenian population. When an additional breast cancer case or an ovarian cancer was present in the family, a more extended analysis was performed. No BRCA1 mutations were found. A BRCA2 gene mutation was identified in four MBC patients. Three of them carried the Slovenian founder mutation IVS16-2A>G. All four mutations were confined to the patients with a family history of breast cancer. Among the MBC patients with a family history of breast cancer in the first- or second-degree relatives, the frequency of BRCA2 gene mutation was 50%. The median age of the patients with a BRCA2 gene mutation was 60 years, not significantly different from those without a mutation. The BRCA2 mutations were diagnosed in 16% of our MBC patients.     

Analysis of BRCA1/2 and CHEK2 mutations in ovarian cancer and primary multiple tumors involving the ovaries. Patients of Russian population using biochips

Ovarian cancer (OC) is one of the leading cause of cancer death in women. Inherited BRCA1 and BRCA2 mutations strikingly increase OC risk (with lifetime risk estimates ranging at 10-60%). Mutation 1100delC in CHEK2 gene was shown to be associated with breast cancer in women carrying this mutation. Knowledge of the nature and frequency of population-specific mutations in these genes is a critical step in the development of simple and inexpensive diagnostic approaches to DNA analysis. The frequencies of 185delAG, 300T>G, 4153delA, 4158A>G, 5382insC mutations in BRCA1 gene, 695insT and 6174delT mutations in BRCA2 gene and 1100delC mutation in CHEK2 gene were analyzed using biochips in Russian OC patients. We studied 68 women who received a diagnosis of epithelial OC and 19 women with primary multiple tumors involving the ovaries. The 185delAG, 300T>G, 4153delA and 5382insC in BRCA1 gene were identified. The most prevailing mutation was 5382insC in BRCA1 gene (87.5% of all BRCA1 mutations OC patients, 50.0% in patients with primary multiple tumors involving the ovaries). No mutations in BRCA2 and CHEK2 genes were detected.     

Genetic analyses of male breast cancer in Israel

Male breast cancer is a rare disorder, and little is known about the molecular mechanisms associated with the tumorigenic process. We genotyped 31 Jewish Israeli males with breast cancer for the predominant Jewish BRCA1 (185delAG, 5382InsC) and BRCA2 (6174delT) germline mutations: 11 individuals from high-risk families and 20 patients unselected for family history of cancer. Two patients of the high-risk group (18.2%) displayed germline mutations: one harbored the 185delAG BRCA1 mutation, and the other the 6174delT mutation in BRCA2. None of the unselected patients displayed any mutation. In 2 patients, complete mutation analysis of the BRCA2 gene did not reveal any disease-associated mutations. We conclude that the predominant Jewish germline mutations in BRCA1/BRCA2 contribute to male breast cancer in Israel, primarily in Ashkenazi individuals with a family history of cancer.     

Moderate frequency of BRCA1 and BRCA2 germ-line mutations in Scandinavian familial breast cancer.

Previous studies of high-risk breast cancer families have proposed that two major breast cancer-susceptibility genes, BRCA1 and BRCA2, may account for at least two-thirds of all hereditary breast cancer. We have screened index cases from 106 Scandinavian (mainly southern Swedish) breast cancer and breast-ovarian cancer families for germ-line mutations in all coding exons of the BRCA1 and BRCA2 genes, using the protein-truncation test, SSCP analysis, or direct sequencing. A total of 24 families exhibited 11 different BRCA1 mutations, whereas 11 different BRCA2 mutations were detected in 12 families, of which 3 contained cases of male breast cancer. One BRCA2 mutation, 4486delG, was found in two families of the present study and, in a separate study, also in breast tumors from three unrelated males with unknown family history, suggesting that at least one BRCA2 founder mutation exists in the Scandinavian population. We report 1 novel BRCA1 mutation, eight additional cases of 4 BRCA1 mutations described elsewhere, and 11 novel BRCA2 mutations (9 frameshift deletions and 2 nonsense mutations), of which all are predicted to cause premature truncation of the translated products. The relatively low frequency of BRCA1 and BRCA2 mutations in the present study could be explained by insufficient screening sensitivity to the location of mutations in uncharacterized regulatory regions, the analysis of phenocopies, or, most likely, within predisposed families, additional uncharacterized BRCA genes.     

<Emphasis Type="Italic">BRCA1</Emphasis> and <Emphasis Type="Italic">BRCA2</Emphasis> germline mutation analysis among Indian women from south India: identification of four novel mutations and high-frequency occurrence of 185delAG mutation

Mutations in the BRCA1 and BRCA2 genes profoundly increase the risk of developing breast and/or ovarian cancer among women. To explore the contribution of BRCA1 and BRCA2 mutations in the development of hereditary breast cancer among Indian women, we carried out mutation analysis of the BRCA1 and BRCA2 genes in 61 breast or ovarian cancer patients from south India with a positive family history of breast and/or ovarian cancer. Mutation analysis was carried out using conformation-sensitive gel electrophoresis (CSGE) followed by sequencing. Mutations were identified in 17 patients (28.0%); 15 (24.6%) had BRCA1 mutations and two (3.28%) had BRCA2 mutations. While no specific association between BRCA1 or BRCA2 mutations with cancer type was seen, mutations were more often seen in families with ovarian cancer. While 40% (4/10) and 30.8% (4/12) of families with ovarian or breast and ovarian cancer had mutations, only 23.1% (9/39) of families with breast cancer carried mutations in the BRCA1 and BRCA2 genes. In addition, while BRCA1 mutations were found in all age groups, BRCA2 mutations were found only in the age group of ≤40 years. Of the BRCA1 mutations, there were three novel mutations (295delCA; 4213T→A; 5267T→G) and three mutations that have been reported earlier. Interestingly, 185delAG, a BRCA1 mutation which occurs at a very high frequency in Ashkenazi Jews, was found at a frequency of 16.4% (10/61). There was one novel mutation (4866insT) and one reported mutation in BRCA2. Thus, our study emphasizes the importance of mutation screening in familial breast and/or ovarian cancers, and the potential implications of these findings in genetic counselling and preventive therapy.     

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.     

Prevalence of<Emphasis Type="Italic"> BRCA1</Emphasis> and<Emphasis Type="Italic"> BRCA2</Emphasis> mutations among clinic-based African American families with breast cancer

To define the prevalence and relative contributions of BRCA1 and BRCA2 mutations among African American families with breast cancer, we analyzed 28 DNA samples from patients identified through two oncology clinics. The entire coding regions of BRCA1 and BRCA2 were screened by protein truncation test, heteroduplex analysis, or single-stranded conformation polymorphism followed by DNA sequencing of variant bands. Deleterious protein-truncating BRCA1 and BRCA2 mutations were identified in five patients or 18% of the entire cohort. Only 8% (1 of 13) of women with a family history of breast cancer, but no ovarian cancer, had mutations. The mutation rates were higher for women from families with a history of breast cancer and at least one ovarian cancer (three of six, 50%). One woman with a family history of undocumented cancers was also found to carry a deleterious mutation in BRCA2. The spectrum of mutations was unique in that one novel BRCA1 mutation (1625del5) and three novel BRCA2 mutations (1536del4, 6696delTC, and 7795delCT) were identified. No recurrent mutations were identified in this cohort, although one BRCA2 (2816insA) mutation had been previously reported. In addition, two BRCA1 and four BRCA2 missense mutations of unknown significance were identified, one of which was novel. Taken together with our previous report on recurrent mutations seen in unrelated families, we conclude that African Americans have a unique mutation spectrum in BRCA1 and BRCA2 genes, but recurrent mutations are likely to be more widely dispersed and therefore not readily identifiable in this population.     

BRCA1 mutations in a population-based study of breast cancer in Stockholm County

The mutation frequency of BRCA1 and BRCA2 in women with breast cancer varies according to family history, age at diagnosis and ethnicity. The contribution of BRCA1 and BRCA2 mutations in breast cancer populations, unselected for age and family history, has been examined in several studies reporting mutation frequencies between 1% and 12% by screening methods, population sizes, and to what extent the gene/s were screened differed in the studies. We wanted to clarify the proportion of breast cancer attributable to mutations in BRCA1 in an unselected breast cancer population from the Stockholm region. All incident breast cancer patients treated surgically in a 19-month period were eligible for the study and 70% (489/696) participated. Exon 11 of BRCA1 was screened for mutations using the protein truncation test, and the mutation frequency was estimated from that. In previous studies on high-risk families from Stockholm, more than 70% of the mutations were detected in exon 11. Two mutations were found, both in patients with a family history or their own medical history of ovarian cancer, giving a mutation frequency in exon 11 of 0.4% and an estimated BRCA1 mutation frequency of <1%. Mutations in BRCA1 in unselected breast cancer cases in our region are rare and likely to be found only in high-risk families. Our BRCA1 prevalence is the lowest of all studies on unselected breast cancer patients, probably reflecting the comparatively low rates detected also in high-risk breast cancer families from the region.     

BRCA1 mutations and polymorphisms in a hospital-based consecutive series of breast cancer patients from Apulia, Italy

INTRODUCTION: Hereditary breast cancer has been partly attributed to germline mutations in the BRCA1 gene that are deleterious for BRCA1 protein activity. This paper analyzes the incidence and characteristics of detectable BRCA1 mutations and polymorphisms in a hospital-based consecutive series of breast cancer patients from southern Italy to investigate the incidence and the association of these molecular alterations with breast cancer biology and family history. METHODS: One hundred cases with familial characteristics were selected from a consecutive series of 511 patients with a first diagnosis of breast cancer. DNA from peripheral blood was screened for whole BRCA1 gene mutations utilizing dHPLC as a pre-screening analysis and automatic DNA sequencing for the identification of specific alterations. RESULTS: In the overall series of 511 patients, 100 had a family history of breast cancer and were investigated for BRCA1 mutations. Two types of BRCA1 mutations were identified, 5382insC in six cases and 4566delA in one case. The 5382insC mutation was present in two out of six cases with ovarian cancer while 4566delA in one case of male cancer. The most frequent missense polymorphisms were E1038G, P871L, K1183R in exon 11, S1613G, M1652I in exon 16 and D1778G in exon 22. Confirming what found in previous studies, patients in whom pathological BRCA1 mutations were detected had early-onset breast cancer (p=0.05), positive nodal status (p=0.05), lower ER (p=0.02) and PgR (p=0.01) content. Interestingly, the K1183R polymorphism and, less strongly, S1613G polymorphism were associated to mutational risk (K1183R: OR 0.1 p=0.03; S1613G: OR 2.7 p=0.08). CONCLUSION: Mutations in the BRCA1 gene are frequent also in our consecutive series of patients from southern Italy. An association between two detected single nucleotide polymorphisms (SNPs) and BRCA1 mutational risk was ascertained. Finally, we confirm the fact that peculiar clinical-pathological features seem to characterize patients with a family history of breast cancer and BRCA1 alterations.     

Mutation analysis of BRCA1 and BRCA2 in a male breast cancer population.

A population-based series of 54 male breast cancer cases from Southern California were analyzed for germ-line mutations in the inherited breast/ovarian cancer genes, BRCA1 and BRCA2. Nine (17%) of the patients had a family history of breast and/or ovarian cancer in at least one first-degree relative. A further seven (13%) of the patients reported breast/ovarian cancer in at least one second-degree relative and in no first-degree relatives. No germ-line BRCA1 mutations were found. Two male breast cancer patients (4% of the total) were found to carry novel truncating mutations in the BRCA2 gene. Only one of the two male breast cancer patients carrying a BRCA2 mutation had a family history of cancer, with one case of ovarian cancer in a first-degree relative. The remaining eight cases (89%) of male breast cancer with a family history of breast/ovarian cancer in first-degree relatives remain unaccounted for by mutations in either the BRCA1 gene or the BRCA2 gene.     

Full-exon pyrosequencing screening of BRCA germline mutations in Mexican women with inherited breast and ovarian cancer

Hereditary breast cancer comprises 10% of all breast cancers. The most prevalent genes causing this pathology are BRCA1 and BRCA2 (breast cancer early onset 1 and 2), which also predispose to other cancers. Despite the outstanding relevance of genetic screening of BRCA deleterious variants in patients with a history of familial cancer, this practice is not common in Latin American public institutions. In this work we assessed mutations in the entire exonic and splice-site regions of BRCA in 39 patients with breast and ovarian cancer and with familial history of breast cancer or with clinical features suggestive for BRCA mutations by massive parallel pyrosequencing. First we evaluated the method with controls and found 41-485 reads per sequence in BRCA pathogenic mutations. Negative controls did not show deleterious variants, confirming the suitability of the approach. In patients diagnosed with cancer we found 4 novel deleterious mutations (c.2805_2808delAGAT and c.3124_3133delAGCAATATTA in BRCA1; c.2639_2640delTG and c.5114_5117delTAAA in BRCA2). The prevalence of BRCA mutations in these patients was 10.2%. Moreover, we discovered 16 variants with unknown clinical significance (11 in exons and 5 in introns); 4 were predicted as possibly pathogenic by in silico analyses, and 3 have not been described previously. This study illustrates how massive pyrosequencing technology can be applied to screen for BRCA mutations in the whole exonic and splice regions in patients with suspected BRCA-related cancers. This is the first effort to analyse the mutational status of BRCA genes on a Mexican-mestizo population by means of pyrosequencing.     

Prevalence of the most frequent BRCA1 mutations in Polish population

The purpose of our study was to establish the frequency and distribution of the four most common BRCA1 mutations in Polish general population and in a series of breast cancer patients. Analysis of the population frequency of 5382insC (c.5266dupC), 300T >G (p.181T >G), 185delAG (c.68_69delAG) and 3819del5 (c.3700_3704del5) mutations of the BRCA1 gene were performed on a group of respectively 16,849, 13,462, 12,485 and 3923 anonymous samples collected at birth in seven Polish provinces. The patient group consisted of 1845 consecutive female breast cancer cases. The most frequent BRCA1 mutation in the general population was 5382insC found in 29 out of 16,849 samples (0.17%). 300T >G and 3819del5 mutations were found in respectively 11 of 13,462 (0.08%) and four of 3923 (0.1%) samples. The population prevalence for combined Polish founder 5382insC and 300T >G mutations was 0.25% (1/400). The frequencies of 5382insC and 300T >G carriers among consecutive breast cancer cases were, respectively, 1.9% (35/1845) and 1.2% (18/1486). Comparing these data with the population frequency, we calculated the relative risk of breast cancer for 5382insC mutation at OR = 17 and for 300T >G mutation at OR = 26. Our results, based on large population studies, show high frequencies of founder 5382insC and 300T >G BRCA1 mutations in Polish general population. Carriage of one of these mutations is connected with a very high relative risk of breast cancer.     

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.