Twenty-three novel BRCA1 and BRCA2 sequence variations identified in a cohort of Swiss breast and ovarian cancer families

BRCA1 and BRCA2 are the major genes predisposing to breast-ovarian cancer (i.e., breast or ovarian cancer or both). Since 1994, hundreds of distinct germline alterations have been reported in these two genes. Besides pathogenic mutations resulting in loss of function of the protein, an increased number of variants of unknown clinical significance have been described. In a cohort of 350 Swiss breast-ovarian cancer families, the systematic search for BRCA1/BRCA2 germline mutations was carried out using denaturating high-performance liquid chromatography as the first screening procedure. The screening strategy resulted in the identification of 23 alterations not previously reported: 9 in BRCA1 and 14 in BRCA2. By using the available tools to assign a functional role to newly identified sequence variations, 5 (22%) of these were classified as new disease-causing mutations, 5 (22%) were classified as benign polymorphisms, and the remaining 13 (56%) alterations were considered as unclassified variants. These data illustrate the major challenge for clinical oncologists currently facing the interpretation of alterations identified in BRCA1 or BRCA2. The key points are to classify these genetic variations as pathogenic mutations, benign polymorphisms, or variants of unknown clinical significance and to adequately use this information for the management of high-risk individuals and their families.     

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.     

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.     

Founder BRCA1 mutations and two novel germline BRCA2 mutations in breast and/or ovarian cancer families from North-Eastern Poland

Germline mutations in the BRCA1 and BRCA2 genes account for the majority of high-risk breast/ovarian cancer families, depending on the population studied. Previously, BRCA1 mutations were described in women from Western Poland. To further characterize the spectrum of BRCA1 mutations and the impact of BRCA2 mutations in Poland, we have analyzed 25 high-risk breast and/or ovarian cancer families from North-Eastern Poland for mutations in all coding exons of the BRCA1 and BRCA2 genes, using combined heteroduplex analysis/SSCP followed by direct DNA sequence analysis. Out of 25 probands a total of five (20%) carried three recurrent BRCA1 mutations (300T>G, 3819del5, 5382insC). The 300T>G mutation accounted for 60% (3/5) of BRCA1 mutations and allelotyping suggested a common founder of this mutation. No unique mutations were found. In addition, we identified three BRCA2 (12%) mutations, one recurrent 4075delGT, and two novel frameshift mutations, 7327ins/dupl19 and 9068delA. We conclude that 30% of high-risk families from North-Eastern Poland may be due to recurrent BRCA1 and unique BRCA2 mutations. Intriguingly, the BRCA1 mutation spectrum seems to be different within subregions of Poland.     

BRCA1 and BRCA2 sequence variants in Chinese breast cancer families

A series of 45 high-risk breast cancer patients, consisting of 25 affected individuals from 16 families in China with at least two cases of breast cancer and 20 cases of breast cancer diagnosed under age 35 without reported family history, were studied for germline mutations of the BRCA1 and BRCA2 genes. Thirteen of the 16 families contained at least one case diagnosed under age 50. Three distinct protein truncating sequence variants, likely to be disease-associated, were identified: two novel mutations in BRCA1 (1584G>T and 5028delC), and a previously reported mutation in BRCA2 (7883delTTAA). Additional sequence variants identified included common polymorphisms, and several variants of unknown clinical significance, including a novel BRCA1 alteration. Based on models for predictive testing using allele frequencies and risks estimated in Western populations, our results suggest that BRCA1/2 mutations account for a somewhat smaller fraction of breast cancer cases in Tianjin than in the Caucasian populations studied. This difference could be the result of a lower penetrance of BRCA1/2 mutations due to the surrounding environmental and hormonal milieu, or a lower frequency of mutations in this population. Larger, more detailed, studies will be necessary to determine which factors underlie this difference.     

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.

     

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%.     

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.     

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.     

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.     

Application of BRCA1 and BRCA2 mutation carrier prediction models in breast and/or ovarian cancer families of French Canadian descent

The BRCAPRO, Couch, Myriad I and II, Ontario Family History Assessment Tool (FHAT), and Manchester models have been used to predict BRCA1 or BRCA2 mutation carrier status of women at high risk for developing the heritable form of breast and ovarian cancers. We have evaluated these models for their accuracy in classifying 224 French Canadian families with at least three cases of breast cancer (diagnosed before the age of 65 years), ovarian cancer, or male breast cancer where mutation status was known for an index affected case used to assess the model. This series includes 44 BRCA1 and 52 BRCA2 mutation-positive families. Using receiver operator characteristics analyses, the C-statistics were found to be 0.81, 0.80, 0.79, and 0.74 for the BRCAPRO, FHAT, Manchester, and Myriad II models, respectively, when incorporating both BRCA1 and BRCA2 mutation carrier predictions. For the BRCAPRO model, 75% scored greater than a 0.43 probability in the mutation-positive group and 75% scored less than 0.50 in the mutation-negative group. Only 38 of 128 (30%) mutation-negative group had a probability greater than 0.43 with the BRCAPRO model. While all models were highly predictive of carrier status, the BRCAPRO model was the most accurate where a cut-off of 10% would have eliminated 60 of 128 (47%) mutation-negative families for genetic testing and only miss 10 of 96 (10%) mutation-positive families. A review of the cancer phenotypes with high BRCAPRO probabilities showed that significantly more metachronous bilateral breast cancer cases occurred in BRCA1/2 mutation carrier families in comparison to mutation-negative families, a feature which is not discriminated in the BRCAPRO model.     

Contribution of germline BRCA1 and BRCA2 sequence alterations to breast cancer in Northern India

Background

A large number of distinct mutations in the BRCA1 and BRCA2 genes have been reported worldwide, but little is known regarding the role of these inherited susceptibility genes in breast cancer risk among Indian women. We investigated the distribution and the nature of BRCA1 and BRCA2 germline mutations and polymorphisms in a cohort of 204 Indian breast cancer patients and 140 age-matched controls.

Method

Cases were selected with regard to early onset disease (≤40 years) and family history of breast and ovarian cancer. Two hundred four breast cancer cases along with 140 age-matched controls were analyzed for mutations. All coding regions and exon-intron boundaries of the BRCA1 and BRCA2 genes were screened by heteroduplex analysis followed by direct sequencing of detected variants.

Results

In total, 18 genetic alterations were identified. Three deleterious frame-shift mutations (185delAG in exon 2; 4184del4 and 3596del4 in exon 11) were identified in BRCA1, along with one missense mutation (K1667R), one 5'UTR alteration (22C>G), three intronic variants (IVS10-12delG, IVS13+2T>C, IVS7+38T>C) and one silent substitution (5154C>T). Similarly three pathogenic protein-truncating mutations (6376insAA in exon 11, 8576insC in exon19, and 9999delA in exon 27) along with one missense mutation (A2951T), four intronic alterations (IVS2+90T>A, IVS7+75A>T, IVS8+56C>T, IVS25+58insG) and one silent substitution (1593A>G) were identified in BRCA2. Four previously reported polymorphisms (K1183R, S1613G, and M1652I in BRCA1, and 7470A>G in BRCA2) were detected in both controls and breast cancer patients. Rare BRCA1/2 sequence alterations were observed in 15 out of 105 (14.2%) early-onset cases without family history and 11.7% (4/34) breast cancer cases with family history. Of these, six were pathogenic protein truncating mutations. In addition, several variants of uncertain clinical significance were identified. Among these are two missense variants, one alteration of a consensus splice donor sequence, and a variant that potentially disrupts translational initiation.

Conclusion

BRCA1 and BRCA2 mutations appear to account for a lower proportion of breast cancer patients at increased risk of harboring such mutations in Northern India (6/204, 2.9%) than has been reported in other populations. However, given the limited extent of reported family history among these patients, the observed mutation frequency is not dissimilar from that reported in other cohorts of early onset breast cancer patients. Several of the identified mutations are unique and novel to Indian patients.     

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.     

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.     

早发性乳腺癌/卵巢癌BRCA1基因突变分析

目的探讨中国北方地区汉族人群早发性乳腺癌和卵巢癌与BRCA1基因的关系.方法应用聚合酶链反应-单链构象多态性分析和DNA直接测序法检测BRCA1基因外显子2和外显子11部分序列.结果对12例早发性乳腺癌/卵巢癌患者PCR-SSCP的检测中,在外显子2发现1例突变,经DNA测序证实突变为第166碱基插入碱基"C".结论 BRCA1基因突变可能与北方地区汉族人群早发性乳腺癌有关.     

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.