BRCA1 mutation analysis in breast/ovarian cancer families from Greece

Germline mutations in BRCA1 gene account for varying proportions of breast/ovarian cancer families, and demonstrate considerable variation in mutational spectra coincident with ethnic and geographical diversity. We have screened for mutations the entire coding sequence of BRCA1 in 30 breast/ovarian cancer women with family history of two or more cases of breast cancer under age 50 and/or ovarian cancer at any age. Genomic DNA from patient was initially analyzed for truncating mutations in exon 11 with PTT followed by DNA sequencing. In the cases where no frameshift mutation was observed in exon 11, all other exons were screened with direct sequencing. Two novel (3099delT, 3277insG) and three already described (3741insA, 1623del5-TTAAA, 5382insC-twice) truncating mutations were identified. In addition, 6 point mutations (L771L, P871L, E1038G, K1183R, S1436S, S1613G) which are already classified as polymorphisms were identified. Three unclassified intronic variants (IVS16-68 G>A, IVS16-92 G>A, IVS18+65G>A) were also detected. These results show that BRCA1 deleterious mutations are present in a fraction (20%) of Greek breast/ovarian cancer families similar to other European countries. Mutations were detected in high- (>/=3 members) as well as in moderate-risk (2 members) families. This is the first report of BRCA1 mutation analysis in Greece.     

The role of targeted BRCA1/BRCA2 mutation analysis in hereditary breast/ovarian cancer families of Portuguese ancestry

We report the analysis of altogether 1050 suspected hereditary breast/ovarian cancer (HBOC) families, 524 fully screened for BRCA1/BRCA2 mutations and 526 tested only for the most common mutations. Of the 119 families with pathogenic mutations, 40 (33.6%) had the BRCA2 c.156_157insAlu rearrangement and 15 (12.6%) the BRCA1 c.3331_3334del mutation, the former being specific of Portuguese ancestry and the latter showing a founder effect in Portugal. Interestingly, the two most common mutations were found in a significant proportion of the HBOC families with an a priori BRCAPRO mutation probability <10%. We recommend that all suspected HBOC families from Portugal or with Portuguese ancestry, even those fulfilling moderately stringent clinical‐criteria for genetic testing, should be specifically analyzed for the two most common BRCA1/BRCA2 founder mutations, and we here present a simple method for this first tier test. Screening of the entire coding regions of BRCA1 and BRCA2 should subsequently be offered to those families with a mutation probability ≥10% if none of those founder mutations are found.     

Screening for BRCA1 and BRCA2 mutations in Eastern Finnish breast/ovarian cancer families

Familial aggregation is thought to account for 5-10% of all breast cancer cases, and high penetrance breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 explain < or =20% of these. Hundreds of mutations among breast/ovarian cancer families have been found in these two genes. The mutation spectrum and prevalence, however, varies widely among populations. Thirty-six breast/ovarian cancer families were identified from a population sample of breast and ovarian cancer cases among a relatively isolated population in Eastern Finland, and the frequency of BRCA1/BRCA2 germline mutations were screened using heteroduplex analysis, protein truncation test and sequencing. Five different mutations were detected in seven families (19.4%). Two mutations were found in BRCA1 and three in BRCA2. One of the mutations (BRCA2 4088insA) has not been detected elsewhere in Finland while the other four, 4216-2nt A-->G and 5370 C-->T in BRCA1 and 999del5 and 6503delTT in BRCA2, are recurrent Finnish founder mutations. These results add to the evidence of the geographical differences in distribution of Finnish BRCA1/BRCA2 mutations. This screen also provides further evidence for the presumption that the majority of Finnish BRCA1/BRCA2 founder mutations have been found and that the proportion of BRCA1/BRCA2 mutations in Finnish breast/ovarian cancer families is around 20%.     

Haplotype analysis of the BRCA2 9254delATCAT recurrent mutation in breast/ovarian cancer families from Spain

A frame-shift 9254del5 mutation was independently identified in 12 families, eleven of them with Spanish ancestors, in a BRCA2 screening performed in 841 breast and/or ovarian cancer families and in 339 women with breast cancer diagnosed before the age of 40 at different centers in France and Spain. We sought to analyze in detail the haplotype and founder effects of the 9254del5 and to estimate the time of origin of the mutation. Eight polymorphic microsatellite markers and two BRCA2 polymorphisms were used for the haplotype analyses. The markers were located flanking the BRCA2 gene spanning a region of 6.1 cM. Our results suggest that these families shared a common ancestry with BRCA2 9254del5, which is a founder mutation originating in the Northeast Spanish, with an estimated age of 92 (95% CI 56-141) generations.     

Novel germline BRCA1 and BRCA2 mutations in breast and breast/ovarian cancer families from the Czech Republic

Germline mutations in breast cancer susceptibility genes, BRCA1 and BRCA2, are responsible for a substantial proportion of high‐risk breast and breast/ovarian cancer families. To characterize the spectrum of BRCA1 and BRCA2 mutations, we screened Czech families with breast/ovarian cancer using the non‐radioactive protein truncation test, heteroduplex analysis and direct sequencing. In a group of 100 high‐risk breast and breast/ovarian cancer families, four novel frame shift mutations were identified in BRCA1 and BRCA2 genes. In BRCA1, two novel frame shift mutations were identified as 3761‐3762delGA and 2616‐2617ins10; in BRCA2, two novel frame shift mutations were identified as 5073‐5074delCT and 6866delC. Furthermore, a novel missense substitution M18K in BRCA1 gene in a breast/ovarian cancer family was identified which lies adjacent just upstream of the most highly conserved C3HC4 RING zinc finger motif. To examine the tertiary structure of the RING zinc finger domain and possible effects of M18K substitution on its stability, we used threading techniques according to the crystal structure of RAG1 dimerization domain of the DNA‐binding protein. © 2000 Wiley‐Liss, Inc.     

Germline mutations of BRCA1 and BRCA2 in Korean breast and/or ovarian cancer families

Germline mutations in the BRCA1 and BRCA2 genes are responsible for the predisposition and development of familial breast and/or ovarian cancer. Most mutations of BRCA1 and BRCA2 associated with breast and/or ovarian cancer result in truncated proteins. To investigate the presence of BRCA1 and BRCA2 germline mutations in Korean breast and/or ovarian cancer families, we screened a total of 27 cases from 21 families including two or more affected first- or second-degree relatives with breast and/or ovarian cancer. PTT, PCR-SSCP, and DHPLC analysis, followed by sequencing were used in the screening process. In nine families, we found BRCA1 and BRCA2 germline mutations that comprised four frameshift mutations and five nonsense mutations. All nine mutations led to premature termination producing shortened proteins. Among the nine mutations, three novel BRCA1 mutations (E1114X, Q1299X, 4159delGA) and two novel BRCA2 mutations (K467X, 8945delAA) were identified in this work.     

BRCA1 and BRCA2 unclassified variants and missense polymorphisms in Algerian breast/ovarian cancer families

Background: BRCA1 and BRCA2 germline mutations predispose heterozygous carriers to hereditary breast/ovarian cancer. However, unclassified variants (UVs) (variants with unknown clinical significance) and missense polymorphisms in BRCA1 and BRCA2 genes pose a problem in genetic counseling, as their impact on risk of breast and ovarian cancer is still unclear. The objective of our study was to identify UVs and missense polymorphisms in Algerian breast/ovarian cancer patients and relatives tested previously for BRCA1 and BRCA2 genes germline mutations analysis. Methods: We analyzed 101 DNA samples from 79 breast/ovarian cancer families. The approach used is based on BRCA1 and BRCA2 sequence variants screening by SSCP or High-Resolution Melting (HRM) curve analysis followed by direct sequencing. In silico analyses have been performed using different bioinformatics programs to individualize genetics variations that can disrupt the BRCA1 and BRCA2 genes function. Results: Among 80 UVs and polymorphisms detected in BRCA1/2 genes (33 BRCA2 and 47 BRCA2), 31 were new UVs (10 BRCA2 and 21 BRCA2), 7 were rare UVs (4 BRCA2 and 3 BRCA2) and 42 were polymorphic variants (19 BRCA2 and 23 BRCA2). Moreover, 8 new missense UVs identified in this study: two BRCA1 (c.4066C>A/p.Gln1356Lys, c.4901G>T/p.Arg1634Met) located respectively in exons 11 and 16, and six BRCA2 (c.1099G>A/p.Asp367Asn, c.2636C>A/p.Ser879Tyr, c.3868T>A/p.Cys1290Ser, c.5428G>T/p.Val1810Phe, c.6346C>G/p.His2116Asp and c.9256G>A/p.Gly3086Arg) located respectively in exons 10, 11 and 24, show a damaging PSIC score yielded by PolyPhen2 program and could be pathogenic. In addition, 5 new BRCA2 missense UVs out of six that were found to be damaging by PolyPhen2 program, also were deleterious according to SIFT program. The rare BRCA2 UV c.5332G>A/p.Asp1778Asn was found here for the first time in co-occurrence in trans with the deleterious BRCA1 mutation c.798_799delTT/p.Ser267LysfsX19 in young breast cancer patient. Moreover, 10 new identified intronic variants with unknown clinical significance (3 BRCA1 and 7 BRCA2) in the present study, could be considered as benign, because GeneSplicer, SpliceSiteFinder and MaxEntScan prediction programs show no splice site alteration for these variants. Several missense polymorphisms of BRCA1 c.2612C>T/p.Pro871Leu, c.3548A>G/p.Lys1183Arg, c.4837A>G/p.Ser1613Gly and BRCA2 c.865A>C/p.Asn289His, c.1114A>C/p.Asn372His, c.2971A>G/p.Asn991Asp, c.7150C>A/p.Gly2384Lys have been identified with high frequency in patients who were tested negative for BRCA1 and BRCA2 mutations. These missense polymorphisms could have a role as susceptibility breast cancer markers in Algerian breast/ovarian cancer families where pathological BRCA1 and BRCA2 mutations were not present. Conclusions: For the first time, UVs and missense polymorphisms in BRCA1 and BRCA2 genes have been identified in Algerian breast/ovarian cancer families. Evaluation of breast/ovarian cancer risk induced by the eight new missense UVs and common polymorphisms detected in our present work is on going in a larger study.     

Analysis of BRCA1 and BRCA2 in breast and breast/ovarian cancer families shows population substructure in the Iberian peninsula

An estimated 5–10% of all breast and ovarian cancers are due to an inherited predisposition, representing a rather large number of patients. In Spain 1/13–1/14 women will be diagnosed with breast cancer during their lifetime. Two major breast cancer genes, BRCA1 and BRCA2 , have been identified. To date, several hundred pathogenic mutations in these two genes have been published or reported to the Breast Cancer Information Core, BIC database ( http://www.nhgri.nih.gov/Intramural_research_Labtransfer/Bic/index.html ). In the present study, 30 Spanish breast and breast/ovarian cancer families (29 from Galicia, NW Spain, and 1 from Catalonia, NE Spain) were screened for mutations in the BRCA1 and BRCA2 genes. The analysis of these genes was carried out by SSCP for shorter exons and direct sequencing in the case of longer ones. Mutations were found in 8 of the 30 families studied (26.66%). It is important to note that all mutations were detected within the BRCA1 gene: 330 A>G, 910_913delGTTC, 2121 C>T, 3958_3962delCTCAGinsAGGC, and 5530 T>A. The BRCA1 330 A>G mutation was found in four unrelated families and accounted for 50% of all identified mutations.     

Pre-test prediction models of BRCA1 or BRCA2 mutation in breast/ovarian families attending familial cancer clinics

Objective: To test whether statistical models developed to calculate pre-test probability of being a BRCA1/2 carrier can differentiate better between the breast/ovarian families to be referred to the DNA test laboratory.

Study design: A retrospective analysis was performed in 109 Spanish breast/ovarian families previously screened for germline mutations in both the BRCA1 and BRCA2 genes. Four easy to use logistic regression models originally developed in Spanish (HCSC model), Dutch (LUMC model), Finnish (HUCH model), and North American (U Penn model) families and one model based on empirical data of Frank 2002 were tested. A risk counsellor was asked to assign a subjective pre-test probability for each family. Sensitivity, specificity, negative and positive predictive values, and areas under receiver operator characteristics (ROC) curves were calculated in each case. Correlation between predicted probability and mutation prevalence was tested. All statistical tests were two sided.

Results: Overall, the models performed well, improving the performances of a genetic counsellor. The median ROC curve area was 0.80 (range 0.77-0.82). At 100% sensitivity, the median specificity was 30% (range 25-33%). At 92% sensitivity, the median specificity was 42% (range 33.3-54.2%) and the median negative predictive value was 93% (range 89.7-98%). BRCA1 families tended to score higher risk than BRCA2 families in all models tested.

Conclusions: All models increased the discrimination power of an experienced risk counsellor, suggesting that their use is valuable in the context of clinical counselling and genetic testing to optimise selection of patients for screening and allowing for more focused management. Models developed in different ethnic populations performed similarly well in a Spanish series of families, suggesting that models targeted to specific populations may not be necessary in all cases. Carrier probability as predicted by the models is consistent with actual prevalence, although in general models tend to underestimate it. Our study suggests that these models may perform differently in populations with a high prevalence of BRCA2 mutations.

     

Psychosocial factors predicting BRCA1/BRCA2 testing decisions in members of hereditary breast and ovarian cancer families

Although BRCA1/2 testing has increasingly entered clinical practice, much is to be learned about the most effective ways to provide counseling to persons potentially interested in receiving test results. The purpose of this study was to identify factors affecting genetic testing decisions in a cohort of hereditary breast and ovarian cancer (HBOC) families presented with the choice to undergo testing. Relatives in these families are known to carry BRCA1 or BRCA2 mutations. Sociodemographics, personality traits, and family functioning were self-assessed using validated psychometric instruments at baseline. Among 172 individuals who participated in pretest education and counseling, 135 (78%) chose to undergo genetic testing and 37 (22%) chose not to be tested. Individuals who chose to undergo genetic testing were more likely to be older (> or =40 years), to have lower levels of optimism, and to report higher levels of cohesiveness in their families. A better understanding of factors that influence interest in predictive testing may help to inform the counseling that occurs prior to genetic testing.     

Ten novel BRCA1 and BRCA2 mutations in breast and/or ovarian cancer families from northern Germany

Germline mutations in the BRCA1 and BRCA2 gene account for the majority of high-risk breast/ovarian cancer families. We have screened such families from Northern Germany by using DHPLC analysis and subsequent direct sequencing techniques. In ten families we identified six novel BRCA1 and 4 novel BRCA2 mutations comprising four frame shift mutations, one nonsense and one splice site mutation in the BRCA1 gene as well as three frameshift mutations and one nonsense mutation in the BRCA2 gene. Our analysis contributes to the further characterisation of the mutational spectrum of BRCA1 and BRCA2.     

Twenty-three novel BRCA1 and BRCA2 sequence alterations in breast and/or ovarian cancer families in Southern Germany

BRCA1 and BRCA2 germline mutations cause a substantially increased life time risk of both breast and ovarian cancer. Mutational screening of these genes by means of Denaturing High Performance Liquid Chromatography (DHPLC) in breast and/or ovarian cancer-prone families from Southern Germany revealed 15 novel BRCA1 and 8 novel BRCA2 sequence variants. Predictions on the BRCA1/BRCA2 protein functions lead to the identification of 11 novel deleterious cancer predisposing mutations. Mutation types and their functional relevances are discussed. Our data contribute to phenotype-genotype correlation studies and to the characterisation of the mutation spectrum of BRCA1/BRCA2.     

BRCA1 and BRCA2 mutation analysis in 86 early onset breast/ovarian cancer patients.

Eighty-six women fulfilling specific selection criteria were studied for germline mutations in two breast cancer susceptibility genes, BRCA1 and BRCA2, using the protein truncation test (PTT). Nine germline mutations were identified, six in BRCA1 and three in BRCA2. Of the six BRCA1 mutations, three have previously been described and three are new, and for BRCA2, one is a new mutation and the other two appear to occur at a site that has been described several times. Four kindreds were breast cancer families, one a breast/ovarian cancer family, and the sixth an ovarian cancer family. The three kindreds with BRCA2 mutations were classified as one breast/ovarian cancer family, one breast cancer family, and one family which harboured one early onset breast cancer patient and two melanoma patients. The mutations in BRCA1 were either insertions, deletions, or transitions which all resulted in a premature stop codon. Mutations in BRCA2 were all frameshift mutations as a result of either 2 or 4 bp deletions. Two BRCA2 mutations were identical, suggesting a Swiss founder effect which was confirmed by haplotype sharing. The 10% mutation detection rate is compatible with the relaxed criteria used for patient selection. Considering the relative ease with which coding sequences can be screened by PTT, this assay is useful as a first screen for BRCA1 and BRCA2 mutations.     

Frequency of BRCA1 and BRCA2 mutations in a clinic-based series of breast and ovarian cancer families

Mutations in BRCA1 and BRCA2 account for a significant proportion of hereditary breast and ovarian cancer cases. In this study, we sought to determine the frequency of BRCA1- and BRCA2-mutation carrier families in a hospital-based cancer family registry. The frequency of families with germline truncating mutations in BRCA1 and BRCA2 was 17.3% (18/104) and 1.9% (2/104), respectively. Two novel truncating mutations, BRCA1 1848delGA and BRCA2 5694insT, were identified. We also sought to determine the carrier frequency of other affected family members for which the mutation lineage could be established within these families. Not including the probands, 72% (18/25) of the affected family members within the BRCA1 mutation-associated families were carriers, and all four affected members of the BRCA2 families were carriers. These data imply that risk evaluation based on cancer family history alone may result in inaccurate estimates, and where possible, mutation testing should be considered in other affected family members to verify carrier status.     

BRCA1/2 testing in hereditary breast and ovarian cancer families II: impact on relationships

Members of hereditary breast and ovarian cancer (HBOC) families often express concern during genetic counseling about the impact of BRCA1/2 testing on close relatives. Yet whether there are likely to be adverse effects of either the decision to undergo genetic testing or the results of testing on family relationships is unknown. One purpose of this study was to assess the impact on close family relationships. Within a randomized trial of breast cancer genetic counseling methods, members of 13 HBOC families were offered BRCA1/2 testing for a known family mutation. The Family Relationship Index (FRI) of the Family Environment Scale (FES) was used to measure perceived family cohesion, conflict, and expressiveness at baseline and again 6-9 months following the receipt of test results, or at the equivalent time for those who declined testing. Participants (n = 212) completed baseline and follow-up questionnaires. Comparisons were made between testers and non-testers as well as between those who tested positive and negative for the family mutation. One hundred eighty-one participants elected to undergo genetic testing (85%) and 47 (26%) were identified to have a mutation. After adjusting for baseline family relationship scores, counseling intervention, gender and marital status, non-testers reported a greater increase in expressiveness (P = 0.006) and cohesion (P = 0.04) than testers. Individuals who tested positive reported a decrease in expressiveness (P = 0.07), although as a trend. Regardless of test decision or test result, those who were randomized to a client-centered counseling intervention reported a decrease in conflict (P = 0.006). Overall, study results suggest that undergoing genetic testing and learning ones BRCA1/2 status may affect family relationships. Those individuals who declined testing reported feeling closer to family members and more encouraged to express emotions to other family members demonstrating potential benefit from the offer of testing. Since those who tested positive reported feeling less encouraged to express their emotions within the family, we recommend helping clients to identify others with whom they feel comfortable sharing their thoughts and feelings about their positive gene status and increased cancer risk.