Screening for RAD51 and BRCA2 BRC repeat mutations in breast and ovarian cancer families

Together, germline mutations in the two major susceptibility genes BRCA1 and BRCA2 account for approximately 20-30% and 70-80% of the familial breast and ovarian cancer cases, respectively. This indicates involvement of additional susceptibility genes, perhaps in combination with a polygenic effect. However, it is also possible that part of the mutations disrupting BRCA1 and BRCA2 function still remains to be discovered. In response to double-strand DNA damage the co-operation between RAD51 and BRCA2 is of great importance, and the conserved BRC repeat motifs in BRCA2 are crucial for this interaction. In the current study, patients belonging to 126 breast and/or ovarian cancer families were screened for RAD51 and BRCA2 BRC repeat mutations in order to uncover aberrations that may contribute to hereditary cancer susceptibility. The performed study revealed several novel alterations, however, none of them appeared to be disease-related. Thus, it seems likely that germline mutations in the highly conserved RAD51 gene are extremely rare and generally poorly tolerated.     

Early detection of breast and ovarian cancer in families with BRCA mutations

Women at risk of breast and ovarian cancer due to a genetic predisposition may opt for preventive surgery or surveillance. The aim of this study was to determine the effectiveness of surveillance in families with a BRCA mutation. Sixty-eight BRCA-families underwent surveillance using annual mammography, transvaginal ultrasound, and estimation of CA125. Two hundred and two women had at least one breast examination, and 138 at least one examination of the ovaries. After a mean follow-up of 33 months, breast cancer was detected in 21 women, four with lymph node metastases. After a mean follow-up of 37 months, six advanced ovarian cancers were detected. The percentage of metastatic breast cancers in the current study appeared to be acceptable. However, because these women have a high-risk of developing breast cancer, they still have a substantial risk of developing metastatic disease under surveillance. Surveillance for ovarian cancer was not effective.     

Rad52 gene mutations in breast/ovarian cancer families and sporadic ovarian carcinoma patients

Rad52 encodes a protein which is required for recombinational repair of double-strand breaks. It is also associated with breast cancer predisposition genes BRCA1 and BRCA2. Mutations in the genes Rad51 or Rad52 result in severe defects in genetic recombination and the repair of double-strand DNA breaks. In order to examine if Rad52 mutations might be involved in sporadic ovarian cancer, we analyzed two stop mutations (Ser346ter and Tyr415ter) in 142 Austrian ovarian carcinoma patients and 128 healthy volunteers. In addition, we analyzed these two mutations in 105 breast/ovarian cancer families (160 members) to examine if the mutations in Rad52 are associated with the occurrence of cancer and with mutations in the BRCA1 and BRCA2 genes. Our results show that these two mutations are rare in all three groups examined. There are no statistically significant differences in the frequencies of the Rad52 mutations between the control group and sporadic ovarian cancer patients and between the control groups and familial breast/ovarian cancer patients, indicating that these two mutations of the Rad52 do not play a major role in the initiation of sporadic ovarian carcinoma and familial breast/ovarian cancer.     

BRCA1 mutation analysis in 83 Spanish breast and breast/ovarian cancer families.

A group of 83 Spanish BC/OC families were analysed for BRCA1 germ-line mutations. Analysis of the entire coding sequence was carried out by SSCP and PTT. We identified 5 frameshift mutations: 185delAG (2 times), 189insTGTC, 1241delAC, and 5537delA and 3 missense mutations in BRCA1: 330A > G, 1240C > T, and 5263G > A. The 185delAG mutation was identified in 2 families. One of them was the only Gypsy family participating in our study. All carriers shared the known founder haplotype, although they did not have any Jewish ancestry. Three frameshift mutations were found among the 14 families with 3 or more BC cases and 1 or more OC, which results in a higher percentage (21.4%) of BRCA1 mutations in this group of high risk families. Two missense mutations were found in families with 2 or 3 BC and no OC, indicating a low proportion (4.8%) of mutations in these families. There was no association between bilateral BC and a mutation carrier status. The frequency of BRCA1 involvement is lower than that of any other country hitherto reviewed. Our results highlight the influence of geographical and ethnic origin of the population studied.     

Risk of ovarian cancer in BRCA1 and BRCA2 mutation-negative hereditary breast cancer families

Women from site-specific hereditary breast cancer families who carry a BRCA1 or BRCA2 mutation are at increased risk for ovarian cancer. It is less clear, however, whether individuals from hereditary breast cancer families who do not carry such a mutation are also at increased ovarian cancer risk. To determine whether women from BRCA mutation-negative hereditary breast cancer families are at increased risk for ovarian cancer, 199 probands from BRCA mutation-negative, site-specific breast cancer kindreds who consented to prospective follow-up at the time of genetic testing were identified. The incidence of new breast and ovarian cancers in probands and their families since receipt of their genetic test results was determined by questionnaire. The expected number of cancers and standardized incidence ratios (SIRs) were determined from age-specific cancer incidence rates from the Surveillance, Epidemiology, and End Results (SEER) program by using the method of Byar. All statistical tests were two-sided. During 2534 women-years of follow-up in 165 kindreds, 19 new cases of breast cancer were diagnosed, whereas only 6.07 were expected (SIR = 3.13, 95% confidence interval [CI] = 1.88 to 4.89; P < .001), and one case of ovarian cancer was diagnosed, whereas only 0.66 was expected (SIR = 1.52, 95% CI = 0.02 to 8.46; P = .48). These results suggest that women from BRCA mutation-negative, site-specific breast cancer families are not at increased risk for ovarian cancer.     

Screening of 1331 Danish breast and/or ovarian cancer families identified 40 novel BRCA1 and BRCA2 mutations

Germ-line mutations in the tumour suppressor genes BRCA1 and BRCA2 predispose to breast and ovarian cancer. Since 1999 we have performed mutational screening of breast and/or ovarian cancer patients in East Denmark. During this period we have identified 40 novel sequence variations in BRCA1 and BRCA2 in high risk breast and/or ovarian cancer families. The mutations were detected via pre-screening using dHPLC or high-resolution melting and direct sequencing. We identified 16 variants in BRCA1, including 9 deleterious frame-shift mutations, 2 intronic variants, 4 missense mutations, and 1 synonymous variant. The remaining 24 variants were identified in BRCA2, including 10 deleterious mutants (6 frame-shift and 4 nonsense), 2 intronic variants, 10 missense mutations and 2 synonymous variants. The frequency of the variants of unknown significance was examined in control individuals. Moreover, the presumed significance of the missense mutations was predicted in silico using the align GVGD algorithm. In conclusion, the mutation screening identified 40 novel variants in the BRCA1 and BRCA2 genes and thereby extends the knowledge of the BRCA1/BRCA2 mutation spectrum. Nineteen of the mutations were interpreted as pathogenic, 3 missense mutations were suggested to be pathogenic based on in silico analysis, 6 mutations were suggested to be benign since they were identified in patients together with a well-known disease-causing BRCA1/BRCA2 mutation, while 12 were variants of unknown significance.     

Mutation screening of RAD51C in high-risk breast and ovarian cancer families

Deleterious mutations in the RAD51C gene, which encodes a DNA double-strand break repair protein, have been reported to confer high-penetrance susceptibility to both breast and ovarian cancer. To confirm this we conducted a mutation screen of the RAD51C gene in 192 probands from high-risk breast and/or ovarian cancer families that do not carry BRCA1 or BRCA2 mutations. The nine exons of the RAD51C gene containing protein coding sequence were screened for mutations in genomic DNA from family probands by high-resolution melting analysis and direct DNA sequencing. Four missense variants, p.Ser364Gly, p.Ala126Thr, p.Val169Ala, and p.Thr287Ala were detected in six patients. The p.Ser364Gly variant is a novel variant predicted to have little influence on RAD51C activity. The p.Ala126Thr and p.Val169Ala variants have been reported to have no association with risk of breast cancer in a case–control study. However, p.Thr287Ala disrupts the DNA repair activity of RAD51C, suggesting some influence on risk. Consistent with published results from similar follow-up studies, we suggest that RAD51C mutations are rare events among high-risk breast cancer and breast/ovarian cancer families. Large population-based studies will be needed to reliably assess the prevalence and penetrance of inactivating mutations in the RAD51C susceptibility gene.     

Germline BRCA1 mutation analysis in Indian breast/ovarian cancer families

Most of the predisposition to hereditary breast and ovarian cancer has been attributed to inherited defects in two tumor suppressor genes BRCA1 and BRCA2. To explore the contribution of BRCA1 mutations to hereditary breast cancer among Indian women, we examined the coding sequence of the BRCA1 gene in 14 breast cancer patients 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. Three mutations (21%) in the BRCA1 gene were identified. Two of them are novel mutations of which one is a missense mutation in exon 7 near the RING finger domain, while the other is a one base pair deletion in exon 11 which results in protein truncation. The third mutation, 185 delAG, has been previously described in Ashkenazi Jewish families. To our knowledge this is the first report of a study of germline BRCA1 mutation analysis in familial breast cancer in India. Our data from 14 different families suggests a lower prevalence but definite involvement of germline mutations in the BRCA1 gene among Indian women with breast cancer and a family history of breast cancer.     

Risk perception and cancer worries in families at increased risk of familial breast/ovarian cancer

While families at increased risk for familial breast/ovarian cancer continue to overestimate their cancer risk with increased cancer worries about the future, few studies have examined factors that affect inherited cancer risk perception and cancer worries in both survivors and unaffected female relatives. The purpose of this study was to examine variables that may affect cancer worries and risk perceptions from a family-based perspective in a racially diverse, community-based, random sample of 146 dyads consisting of adult female breast and/or ovarian cancer survivors and their unaffected female relatives (N=292). Results indicated that coping style, self-efficacy, partner's income, family role relationship, and cancer risk perception were significant contributors to the survivors' and their unaffected relatives' cancer worries. Significant variables for perception of cancer risk for both survivors and relatives included income, race, family history of cancer, and cancer worries. Relatives had a higher perception of cancer risk, whereas survivors had more cancer worries. Additionally, the level of cancer worries reported by one member of the dyad was related to the amount of worries reported by the other. The results from this study underscore the importance of clinicians addressing concerns of both affected and unaffected members of families at increased risk of cancer to assist them in managing cancer worries and having realistic risk appraisals to make informed decisions about their own and their family's health surveillance options.     

Germline alterations in the 53BP1 gene in breast and ovarian cancer families

The conserved TP53-binding protein 1 (53BP1) is a central mediator of the DNA damage checkpoint and appears to be one of the sensors of DNA double-strand breaks (DSBs). Improper processing of DSBs can result in loss or rearrangement of genetic information, leading to cell death or tumorigenesis. 53BP1 interacts with both TP53 and ATM, key proteins involved in the monitoring of genomic integrity and regulation of apoptosis. 53BP1 is also required for the formation of BRCA1 foci and the C-terminal part of these two proteins display significant homology. Based on its biological function, the 53BP1 gene is a good candidate for being involved in cancer susceptibility. Consequently, in the current study patients belonging to 126 breast and/or ovarian cancer families were screened for germline mutations in the entire coding region of the 53BP1 gene. A number of sequence variants were found, but none of them appeared to associate with cancer predisposition. To our knowledge this is the first comprehensive screening of 53BP1 mutations in familial breast and ovarian cancer cases.     

BRCA1 and BRCA2 mutations in Turkish breast/ovarian families and young breast cancer patients

To date, BRCA1 and BRCA2 mutations in breast and/or ovarian patients have not been characterized in the Turkish population. We investigated the presence of BRCA mutations in 53 individuals with a personal and family history of breast and/or ovarian cancer, and 52 individuals with a personal history of breast cancer diagnosed below age 50 without additional family history. We have identified 11 mutations (nine BRCA1 and two BRCA2) using combined techniques involving protein truncation test, direct sequencing and heteroduplex analysis. We found eight out of 53 patients (15.1%) with a family history to carry BRCA gene mutations (seven BRCA1 and one BRCA2). Of these, four were found in 43 families presenting only breast cancer histories, and four were found in families presenting ovarian cancer with or without breast cancer. We also demonstrated two BRCA1 and one BRCA2 mutations in three out of 52 (5.8%) early-onset breast cancer cases without additional family history. Three of nine BRCA1 and both BRCA2 mutations detected in this study were not reported previously. These mutations may be specific to the Turkish population. The BRCA1 5382insC mutation, specific to Ashkenazi and Russian populations, was found twice in our study group, representing a possible founder mutation in the Turkish population.     

BRCA1-related breast cancer in Austrian breast and ovarian cancer families: Specific BRCA1 mutations and pathological characteristics

We identified 17 BRCA1mutations in 86 Austrian breast and ovarian cancer families (20%) that were screened for mutations by denaturing high-performance liquid chromatography (DHPLC) and the protein<0B> <0R>truncation test (PTT). Eleven distinct mutations were detected, 4 of them (962del4, 2795del4, 3135del4 and L3376stop) not previously reported in families of non-Austrian origin. In addition, 6 rare missense mutations (allele frequency < 1%) with unknown biological effects were identified. Four mutations occurred more than once in the Austrian population: 2795del4 (3 times), Cys61Gly (3 times) 5382insC (2 times) and Q1806stop (2 times). Haplotype analysis of the 4 recurrent mutations suggested a common ancestor for each of these. Thirty-four breast cancer cases from 17 families with BRCA1 mutations were further analyzed. We observed a low median age of onset (39.5 years). Sixty-eight percent of all BRCA1 breast cancer cases had negative axillary lymph nodes. This group showed a significant prevalence of a negative estrogen and progesterone receptor status and stage I tumors compared with an age-related, node-negative control group. The prevalence of grade III tumors was marginally significant . Survival analysis either with a control group matched for age (within 5 years), grade, histologic subtype and estrogen receptor status, or with an age-related, node-negative comparison group, showed no statistical difference. Int. J. Cancer 77:354–360, 1998. © 1998 Wiley-Liss, Inc.     

Comprehensive genetic characterization of hereditary breast/ovarian cancer families from Slovakia

Germline mutations in the BRCA1/2 genes account for the majority of hereditary breast ovarian cancer (HBOC). Identification of causal mutations may have significant impact on clinical management of such families. Despite high mutation detection rate, many HBOC cases remain without identified cause. These cases warrant use of several analysis methods, such as those for large genomic rearrangements and DNA copy number changes, or analysis other genes, shown to be associated with increased HBOC risk. We assessed 585 Slovak HBOC for the presence of mutations in BRCA genes. Sequencing revealed mutations in 100 families, representing 17.1% (88 and 12% of mutations were located in BRCA1 and BRCA2, respectively). Four of the mutations, c.80+4del4, c.1938_1947del10 and c.1166delG in BRCA1 and c.6589delA in BRCA2 gene have been described only in Slovak population. Using MLPA analysis, we detected two large genomic rearrangements in three families, a deletion of exons 21 and 22, and a rare deletion of a whole BRCA1 gene. Twenty-seven different variants of uncertain clinical effect (four novel) and 14 distinct SNP BRCA1 haplotypes were detected. Their potential effect was considered using the prediction software packages Align-GVGD, Pmut and Polyphen. We observed that the best clinical criterion for the initiation of BRCA1 analysis is the presence of breast cancer at 40 years of age in the association with the presence of ovarian cancer diagnosed around the age of 50. Conversely, the best clinical criterion for starting with BRCA2 analysis is the presence of breast cancer diagnosed in older age (above 50), or the presence of breast cancer in conjunction with carcinomas at different sites e.g., prostate, colorectum, ovary and uterus. Finally we have seen that the analyses of other HBOC risk gene TP53 and specific mutation in CHEK2*c.1100delC in Slovak HBOC families were not efficient since no mutations were found in these genes.     

Ovarian and breast cancer risks to women in families with two or more cases of ovarian cancer

There are few published estimates of the risk of developing breast or ovarian cancer in women with a strong family history of ovarian cancer. As these women commonly present to family cancer clinics, accurate cancer risk estimates are needed. We have estimated these risks in women from families with 2 or more confirmed ovarian cancers in first-degree relatives using data from the UKCCCR Familial Ovarian Cancer Register. The number of cancers observed in more than 10,000 person years of follow-up was compared with the number expected based on national-, age-, sex- and period-specific incidence rates. The relative risk of ovarian cancer was found to be 7.18 (95% CI 3.82-12.3), declining from 16.0 (6.40-32.9) in women under 50 to 4.38 (1.60-9.52) in women 50 years of age and older. For breast cancer, the relative risk for women under 50 was 3.74 (2.04-6. 28) and 1.79 (1.02-2.90) for women 50 years of age and older (average RR 2.36, 1.59-3.37). These correspond to absolute risks by age 70 of 11% for ovarian cancer and 15% for breast cancer. When the analyses were restricted to families that had been negative for mutations in the breast/ovarian cancer susceptibility genes, BRCA1 and BRCA2, the ovarian cancer risk was 11.59 (3.12-29.7) and that of breast cancer 3.32 (1.52-6.31). As well as having clinical relevance, our finding may suggest that other breast/ovarian cancer genes are segregating in these families, though the possibility of undetected BRCA1/2 mutations must also be considered.     

Low incidence of BRCA1 mutations among Italian families with breast and ovarian cancer

Most familial breast or ovarian cancers are thought to be due to highly penetrant mutations in the predisposing genes BRCA1 and BRCA2. The cloning of these genes has opened a new era for the genetic counseling of women with a family history of breast or ovarian cancer. To estimate the incidence of detectable BRCA1 mutations and to define the eligibility criteria for genetic testing in the Italian population, a total of 53 patients belonging to 46 families clustering multiple cases of breast and/or ovarian cancer were investigated. Seven families presented with ovarian cancer only, 16 had both ovarian and breast cancers, and 23 were characterized by breast cancer only. Using a combination of protein truncation test (PTT) and single strand conformational polymorphism (SSCP) analysis followed, when necessary, by direct sequencing, we found 8 distinct mutations, 2 of these not reported before. Five frameshift and 2 nonsense mutations led to a truncated protein. One mutation was a missense substitution involving a cysteine in the zinc finger domain. One variant creating an ETS binding site in intron 1 was found but its role was not defined. The percentage of families carrying mutations was 17%. Among the families characterized by ovarian cancer only and by breast and ovarian cancer, the percentage of BRCA1 mutations was 57% and 12.5%, respectively. In contrast, the percentage of altered BRCA1 in families with only breast cancers was 9%. In the 46 Italian families studied, BRCA1 mutations were detected in fewer kindreds than those previously hypothesized based on linkage analysis, especially when these were characterized by breast cancers only. Our results indicate that families with a low number of cancer patients should be referred for BRCA1 genetic testing mainly when ovarian cancer is present. Int. J. Cancer 78:581–586, 1998. © 1998 Wiley-Liss, Inc.     

Screening for ovarian cancer

Ovarian cancer frequently presents late, when chances for long-term survival are poor. The increased survival advantage for patients diagnosed with early-stage ovarian cancer suggests that screening to detect early-stage disease might have an impact on disease mortality. Attempts are being made to develop effective screening procedures for early ovarian cancer in symptom-free women, using a variety of serum tumor markers, proteomic patterns, and ovarian morphological and vascular features. Two distinct screening strategies have emerged, one utilizing transvaginal scanning as the primary test, and the other involving measurement of the serum tumor marker CA125 as the primary test with transvaginal ultrasonography as the secondary test (multimodal screening). Large randomized trials are now underway to provide definitive data on the impact of screening on mortality and address morbidity, health economics and psychosocial issues.     

Screening for ovarian cancer

Ovarian cancer frequently presents late, when chances for long-term survival are poor. The increased survival advantage for patients diagnosed with early-stage ovarian cancer suggests that screening to detect early-stage disease might have an impact on disease mortality. Attempts are being made to develop effective screening procedures for early ovarian cancer in symptom-free women, using a variety of serum tumor markers, proteomic patterns, and ovarian morphological and vascular features. Two distinct screening strategies have emerged, one utilizing transvaginal scanning as the primary test, and the other involving measurement of the serum tumor marker CA125 as the primary test with transvaginal ultrasonography as the secondary test (multimodal screening). Large randomized trials are now underway to provide definitive data on the impact of screening on mortality and address morbidity, health economics and psychosocial issues.