Risks of breast and ovarian cancer for women harboring pathogenic missense variants in BRCA1 and BRCA2 compared with those harboring protein truncating variants

PurposeGermline genetic testing for BRCA1 and BRCA2 variants has been a part of clinical practice for >2 decades. However, no studies have compared the cancer risks associated with missense pathogenic variants (PVs) with those associated with protein truncating (PTC) variants.MethodsWe collected 582 informative pedigrees segregating 1 of 28 missense PVs in BRCA1 and 153 pedigrees segregating 1 of 12 missense PVs in BRCA2. We analyzed 324 pedigrees with PTC variants in BRCA1 and 214 pedigrees with PTC variants in BRCA2. Cancer risks were estimated using modified segregation analysis.ResultsEstimated breast cancer risks were markedly lower for women aged >50 years carrying BRCA1 missense PVs than for the women carrying BRCA1 PTC variants (hazard ratio [HR] = 3.9 [2.4-6.2] for PVs vs 12.8 [5.7-28.7] for PTC variants; P = .01), particularly for missense PVs in the BRCA1 C-terminal domain (HR = 2.8 [1.4-5.6]; P = .005). In case of BRCA2, for women aged >50 years, the HR was 3.9 (2.0-7.2) for those heterozygous for missense PVs compared with 7.0 (3.3-14.7) for those harboring PTC variants. BRCA1 p.[Cys64Arg] and BRCA2 p.[Trp2626Cys] were associated with particularly low risks of breast cancer compared with other PVs.ConclusionThese results have important implications for the counseling of at-risk women who harbor missense PVs in the BRCA1/2 genes.     

BRCA1 and BRCA2 pathogenic sequence variants in women of African origin or ancestry

BRCA1 and BRCA2 (BRCA1/2) pathogenic sequence variants (PSVs) confer elevated risks of multiple cancers. However, most BRCA1/2 PSVs reports focus on European ancestry individuals. Knowledge of the PSV distribution in African descent individuals is poorly understood. We undertook a systematic review of the published literature and publicly available databases reporting BRCA1/2 PSVs also accessed the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) database to identify African or African descent individuals. Using these data, we inferred which of the BRCA PSVs were likely to be of African continental origin. Of the 43,817 BRCA1/2 PSV carriers in the CIMBA database, 469 (1%) were of African descent. Additional African descent individuals were identified in public databases (n = 291) and the literature (n = 601). We identified 164 unique BRCA1 and 173 unique BRCA2 PSVs in individuals of African ancestry. Of these, 83 BRCA1 and 91 BRCA2 PSVs are of likely or possible African origin. We observed numerous differences in the distribution of PSV type and function in African origin versus non‐African origin PSVs. Research in populations of African ancestry with BRCA1/2 PSVs is needed to provide the information needed for clinical management and decision‐making in African descent individuals worldwide.     

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.     

Comprehensive profiling of BRCA1 and BRCA2 variants in breast and ovarian cancer in Chinese patients

The identification and interpretation of germline BRCA1/2 variants become increasingly important in breast and ovarian cancer (OC) treatment. However, there is no comprehensive analysis of the germline BRCA1/2 variants in a Chinese population. Here we performed a systematic review and meta‐analysis on such variants from 94 publications. A total of 2,128 BRCA1/2 variant records were extracted, including 601 from BRCA1 and 632 from BRCA2. In addition, 414, 734, 449, and 307 variants were also recorded in the BIC, ClinVar, ENIGMA, and UMD databases, respectively, and 579 variants were newly reported. Subsequent analysis showed that the overall germline BRCA1/2 pathogenic variant frequency was 5.7% and 21.8% in Chinese breast and OC, respectively. Populations with high‐risk factors exhibited a higher pathogenic variant percentage. Furthermore, the variant profile in Chinese is distinct from that in other ethnic groups with no distinct founder pathogenic variants. We also tested our in‐house American College of Medical Genetics‐guided pathogenicity interpretation procedure for Chinese BRCA1/2 variants. Our results achieved a consistency of 91.2–97.6% (5‐grade classification) or 98.4–100% (2‐grade classification) with public databases. In conclusion, this study represents the first comprehensive meta‐analysis of Chinese BRCA1/2 variants and validates our in‐house pathogenicity interpretation procedure, thereby providing guidance for further PARP inhibitor development and companion diagnostics in the Chinese population.     

Prevalence,spectrum, and founder effect of BRCA1 and BRCA2 mutations in epithelial ovarian cancer from the Middle East

Germline mutations in breast cancer susceptibility gene 1 and 2 have previously been estimated to contribute to 13–18% of all epithelial ovarian cancer (EOC). To characterize the prevalence and effect of BRCA1 and BRCA2 mutations in Middle Eastern EOC patients, BRCA mutation screening was performed in 407 unselected ovarian cancer patients using targeted capture and/or Sanger sequencing. A total of 19 different pathogenic variants (PVs) were identified in 50 (12.3%) women. Nine PVs were recurrent accounting for 80% of cases with PVs (40/50) in the entire cohort. Founder mutation analysis revealed only two mutations (c.4136_4137delCT and c.1140dupG) sharing the same haplotypes thus representing founder mutations in the Middle Eastern population. Identification of the mutation spectrum, prevalence, and founder effect in Middle Eastern population facilitates genetic counseling, risk assessment, and development of a cost‐effective screening strategy.     

Comprehensive prediction of mRNA splicing effects of BRCA1 and BRCA2 variants

Variants of uncertain significance (VUS) in the BRCA1 and BRCA2 genes potentially affecting coding sequence as well as normal splicing activity have confounded predisposition testing in breast cancer. Here, we apply information theory to analyze BRCA1/2 mRNA splicing mutations categorized as VUS. The method was validated for 31 of 36 mutations known to cause missplicing in BRCA1/2 and all 26 that do not alter splicing. All single-nucleotide variants in the Breast Cancer Information Resource (BIC; Breast Cancer Information Core Database; http://research.nhgri.nih.gov/bic; last access June 1, 2010) were then analyzed. Information analysis is similar in sensitivity to other predictive methods; however, the thermodynamic basis of the theory also enables splice-site affinity to be determined accurately, which is important for assessing mutations that render natural splice sites partially functional and competition between cryptic and natural splice sites. We report 299 of 2,071 single-nucleotide BIC mutations that are predicted to significantly weaken natural sites and/or strengthen cryptic splice sites, 171 of which are not designated as splicing mutations in the database. Splicing alterations are predicted for 68 of 690 BRCA1 and 60 of 958 BRCA2 mutations designated as VUS. These analyses should be useful in prioritizing suspected mutations for downstream expression studies and for predicting aberrantly spliced isoforms generated by these mutations.     

A yeast recombination assay to characterize human BRCA1 missense variants of unknown pathological significance

The BRCA1 tumor suppressor gene is found mutated in familial breast cancer. Although many of the mutations are clearly pathological because they give rise to truncated proteins, several missense variants of uncertain pathological consequences have been identified. A novel functional assay to screen for BRCA1 missense variants in a simple genetic system could be very useful for the identification of potentially deleterious mutations. By using two prediction computer programs, Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen), seven nonsynonymous missense BRCA1 variants likely disrupting the gene function were selected as potentially deleterious. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) was used to test these cancer-related missense mutations for their ability to affect cell growth and homologous recombination (HR) at the HIS3 and ADE2 loci. The variants localized in the BRCA1 C-Terminus (BRCT) domain did not show any growth inhibition when overexpressed in agreement with previous results. Overexpression of either wild-type BRCA1 or two neutral missense variants did not increase yeast HR but when cancer-related variants were overexpressed a significant increase in recombination was observed. Results clearly showed that this genetic system can be useful to discriminate between neutral and deleterious BRCA1 missense variants.     

Estimating survival rates after ovarian cancer among women tested for BRCA1 and BRCA2 mutations

Several studies have reported that women with ovarian cancer and a BRCA1 or BRCA2 mutations have better survival than women with ovarian cancer and no mutation. Potential reasons for this include possible differences in histologic subtype, stage, grade and response to chemotherapy, but some of the difference in survival may be due to systematic bias, i.e. a difference in survival rates for women who do and who do not undergo genetic testing. We estimated the survival rate in 1423 ovarian cancer patients from Ontario who had genetic testing and compared this with the survival rate for all 3367 ovarian cancer patients from the province from whom the tested sample was derived. Tested women had a 10‐year survival of 54.5%, compared to 35.8% for all patients in the province. We evaluated the extent to which three different methods of adjustment eliminated the observed difference. The adjusted rates for the tested cohort were closer to the provincial average, but each adjustment method resulted in a modest over‐estimate of 10‐year survival, ranging from 6.1% to 10.0%. The mortality advantage for tested women was due, in part, to a lower than expected mortality rate of tested women in the period following genetic testing.     

Founder BRCA1 and BRCA2 mutations in French Canadian ovarian cancer cases unselected for family history

The breast cancer susceptibility genes, BRCA1 and BRCA2, differ in their contribution to ovarian cancer. Recently, founder mutations in each of these genes were identified in Canadian breast cancer and breast ovarian cancer families of French ancestry. We have examined the prevalence of the founder mutations in a series of 113 French Canadian women with ovarian cancer unselected for family history. Germline mutations were found in eight of 99 invasive carcinomas and in none of the 14 tumors of borderline malignancy. Five cases carried the BRCA1 C4446T mutation and two cases carried the BRCA2 8765delAG mutation which are the most common mutations that have been described in French Canadian breast cancer and breast ovarian cancer families. All of these cases reported a family history of at least one first-degree relative with breast cancer, diagnosed below age 60 years, or with ovarian cancer. The identification of founder BRCA1 and BRCA2 mutations in ovarian cancer cases unselected for family history can facilitate carrier detection when the expected yield of a comprehensive screen may be low.     

Contribution of BRCA1 and BRCA2 mutations to inherited ovarian cancer

A total of 283 epithelial ovarian cancer families from the United Kingdom (UK) and the United States (US) were screened for coding sequence changes and large genomic alterations (rearrangements and deletions) in the BRCA1 and BRCA2 genes. Deleterious BRCA1 mutations were identified in 104 families (37%) and BRCA2 mutations in 25 families (9%). Of the 104 BRCA1 mutations, 12 were large genomic alterations; thus this type of change represented 12% of all BRCA1 mutations. Six families carried a previously described exon 13 duplication, known to be a UK founder mutation. The remaining six BRCA1 genomic alterations were previously unreported and comprised five deletions and an amplification of exon 15. One of the 25 BRCA2 mutations identified was a large genomic deletion of exons 19-20. The prevalence of BRCA1/2 mutations correlated with the extent of ovarian and breast cancer in families. Of 37 families containing more than two ovarian cancer cases and at least one breast cancer case with diagnosis at less than 60 years of age, 30 (81%) had a BRCA1/2 mutation. The mutation prevalence was appreciably less in families without breast cancer; mutations were found in only 38 out of 141 families (27%) containing two ovarian cancer cases only, and in 37 out of 59 families (63%) containing three or more ovarian cancer cases. These data indicate that BRCA1 and BRCA2 are the major susceptibility genes for ovarian cancer but that other susceptibility genes may exist. Finally, it is likely that these data will be of clinical importance for individuals in families with a history of epithelial ovarian cancer, in providing accurate estimates of their disease risks.     

BRCA1 5272-1G>A and BRCA2 5374delTATG are founder mutations of high relevance for genetic counselling in breast/ovarian cancer families of Spanish origin

Infante M, Durán M, Acedo A, Pérez-Cabornero L, Sanz DJ, García-González M, Beristain E, Esteban-Cardeñosa E, de la Hoya M, Teulé A, Vega A, Tejada M-I, Lastra E, Miner C, Velasco EA. BRCA1 5272-1G>A and BRCA2 5374delTATG are founder mutations of high relevance for genetic counselling in breast/ovarian cancer families of Spanish origin.
The distribution of BRCA1 and BRCA2 germ line mutations in breast/ovarian cancer families varies among different populations, which typically present a wide spectrum of unique mutations. Splicing mutation 5272-1G>A of BRCA1 and frameshift mutation 5374delTATG of BRCA2 are highly prevalent mutations in Castilla-León (Spain), accounting for 18.4% and 13.6% of BRCA1 and BRCA2 positive families, respectively. To test the presence of founder effects, 9 Spanish 5272-1G>A and 13 5374delTATG families were genotyped with polymorphic markers linked to BRCA1 or BRCA2 . All the 5272-1G>A families shared a common haplotype in eight markers (1.1 Mb region) and the mutation age was estimated in 15 generations (∼380 years). A conserved haplotype associated to 5374delTATG was observed in four markers (0.82 Mb). The mutation occurred approximately 48 generations ago (∼1200 years). Each mutation likely arose from a common ancestor that could be traced to a small area of Castilla-León and expanded to other Spanish regions. They can have a significant impact on the clinical management of asymptomatic carriers as well as on the genetic screening strategy to be followed in populations with Spanish ancestries.     

Recurrent BRCA1 and BRCA2 germline mutations in ovarian cancer: a founder mutation of BRCA1 identified in the Chinese population

Previous mutational analysis for BRCA gene mutations in sporadic ovarian cancer occurring in Chinese patients in Hong Kong identified six germline BRCA1 mutations and one germline BRCA2 mutation, six of which were novel (Khoo et al., 2000). Knowledge of BRCA gene mutations in the Chinese population is relatively scant. In this study, we focussed on whether any of these mutations could be recurrent in our Chinese population, making use of archival paraffin embedded tissue. A consecutive series of 214 ovarian cancer cases, half of Southern Chinese origin from Hong Kong whilst the other half of Northern Chinese origin from Beijing were used for the study. We identified one further novel mutation, 1081delG, in BRCA1. This was found to occur in two unrelated individuals with shared haplotype as revealed by allelotype analysis, thus demonstrating founder effect. Two other recurrent mutations were also identified, the 2371-2372delTG mutation in BRCA1 and the 3337C>T mutation in BRCA2 recurring in two and three unrelated individuals respectively, giving an overall prevalence 4.7% of recurrent BRCA mutations in ovarian cancer in the Southern Chinese population. Most importantly, all our recurrent mutation carriers were identified from Southern Chinese patients from Hong Kong whilst such mutations were absent in samples from the Northern Chinese. Our findings indicate possible heterogeneity in the BRCA genotype between Northern and Southern Chinese. The identification of a founder mutation and two recurrent mutations moreover, has important implications towards screening strategies for breast and ovarian cancer among Chinese of southern ancestral origin who are now dispersed throughout the world.     

BRCA1 and BRCA2 mutations in breast/ovarian cancer patients from central Italy

We report on the screening of the entire BRCA1/BRCA2 coding sequence by SSCP, PTT, and direct sequencing in 68 Italian families with recurrent breast or ovarian cancer. For each investigated proband, the probability of being carrier of a BRCA1/BRCA2 mutation was evaluated using the BRCAPRO software. We detected BRCA1/BRCA2 mutations in 8 patients (11.7%). However, if considering only patients with a carrier probability >10%, the detection rate was 36.8%, confirming the usefulness of the BRCAPRO software. One change (BRCA1 4172insT) was a novel mutation not reported in BIC database.     

Splicing and multifactorial analysis of intronic BRCA1 and BRCA2 sequence variants identifies clinically significant splicing aberrations up to 12 nucleotides from the intron/exon boundary

Clinical management of breast cancer families is complicated by identification of BRCA1 and BRCA2 sequence alterations of unknown significance. Molecular assays evaluating the effect of intronic variants on native splicing can help determine their clinical relevance. Twenty-six intronic BRCA1/2 variants ranging from the consensus dinucleotides in the splice acceptor or donor to 53 nucleotides into the intron were identified in multiple-case families. The effect of the variants on splicing was assessed using HSF matrices, MaxEntScan and NNsplice, followed by analysis of mRNA from lymphoblastoid cell lines. A total of 12 variants were associated with splicing aberrations predicted to result in production of truncated proteins, including a variant located 12 nucleotides into the intron. The posterior probability of pathogenicity was estimated using a multifactorial likelihood approach, and provided a pathogenic or likely pathogenic classification for seven of the 12 spliceogenic variants. The apparent disparity between experimental evidence and the multifactorial predictions is likely due to several factors, including a paucity of likelihood information and a nonspecific prior probability applied for intronic variants outside the consensus dinucleotides. Development of prior probabilities of pathogenicity incorporating bioinformatic prediction of splicing aberrations should improve identification of functionally relevant variants and enhance multifactorial likelihood analysis of intronic variants.     

Intronic variants in BRCA1 and BRCA2 that affect RNA splicing can be reliably selected by splice-site prediction programs

A large number of sequence variants identified in BRCA1 and BRCA2 cannot be distinguished as either disease-causing mutations or neutral variants. These so-called unclassified variants (UVs) include variants that are located in the intronic sequences of BRCA1 and BRCA2. The purpose of this study was to assess the use of splice-site prediction programs (SSPPs) to select intronic variants in BRCA1 and BRCA2 that are likely to affect RNA splicing. We performed in vitro molecular characterization of RNA of six intronic variants in BRCA1 and BRCA2. In four cases (BRCA1, c.81-6T>A and c.4986+5G>T; BRCA2, c.7617+2T>G and c.8754+5G>A) a deleterious effect on RNA splicing was seen, whereas the c.135-15_-12del variant in BRCA1 showed no effect on RNA splicing. In the case of the BRCA2 c.68-7T>A variant, RNA analysis was not sufficient to establish the clinical significance. Six SSPPs were used to predict whether an effect on RNA splicing was expected for these six variants as well as for 23 intronic variants in BRCA1 for which the effect on RNA splicing has been published. Out of a total of 174 predictions, 161 (93%) were informative (i.e., the wild-type splice-site was recognized). No false-negative predictions were observed; an effect on RNA splicing was always predicted by these programs. In four cases (2.5%) a false-positive prediction was observed. For DNA diagnostic laboratories, these programs are therefore very useful to select intronic variants that are likely to affect RNA splicing for further analysis.     

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%.     

Probability of BRCA1/2 mutation varies with ovarian histology: results from screening 442 ovarian cancer families

While there are many reports in the literature of mutation testing of BRCA1 and BRCA2 in breast/ovarian cancer families, the question of which type of ovarian cancers are relevant still pertains. We have undertaken whole gene screening including multiple ligation-dependent probe amplification in an affected individual within 442 unrelated non-Jewish families containing at least one reported ovarian cancer diagnosed less than 50 years or at any age with family history of breast or ovarian cancer for mutations in BRCA1 and BRCA2. A total of 166 mutations were identified 110 (25%) in BRCA1 and 56 (13%) in BRCA2 . In families without confirmation of ovarian diagnosis, the detection rate drops significantly. In families fulfilling Breast Cancer Linkage Consortium (BCLC) criteria with confirmed ovarian cancer cases, the mutation detection frequency was 80%. If only BCLC families with unconfirmed ovarian cancers were included, the detection rate dropped to 36% when a relevant ovarian cancer diagnosis was not confirmed. In BCLC families containing only one ovarian cancer, BRCA2 accounted for 45% of identified mutations. No mutations were identified in affected individuals with borderline or mucinous tumours. Detection rates dropped below the 10/20% international thresholds in a number of families with unconfirmed ovarian cancers. Borderline/mucinous pathology substantially reduces the likelihood of identifying a BRCA1/2 mutation. Strenuous efforts should be made to confirm ovarian pathology if the lack of confirmation or refuting the diagnosis would decrease a family's likelihood of mutation detection below screening thresholds. In the UK, a higher proportion of families harbour BRCA2 pathogenic mutations than predicted from previous studies.     

Contribution of bioinformatics predictions and functional splicing assays to the interpretation of unclassified variants of the BRCA genes

A large fraction of sequence variants of unknown significance (VUS) of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 may induce splicing defects. We analyzed 53 VUSs of BRCA1 or BRCA2, detected in consecutive molecular screenings, by using five splicing prediction programs, and we classified them into two groups according to the strength of the predictions. In parallel, we tested them by using functional splicing assays. A total of 10 VUSs were predicted by two or more programs to induce a significant reduction of splice site strength or activation of cryptic splice sites or generation of new splice sites. Minigene-based splicing assays confirmed four of these predictions. Five additional VUSs, all at internal exon positions, were not predicted to induce alterations of splice sites, but revealed variable levels of exon skipping, most likely induced by the modification of exonic splicing regulatory elements. We provide new data in favor of the pathogenic nature of the variants BRCA1 c.212+3A>G and BRCA1 c.5194−12G>A, which induced aberrant out-of-frame mRNA forms. Moreover, the novel variant BRCA2 c.7977−7C>G induced in frame inclusion of 6 nt from the 3′ end of intron 17. The novel variants BRCA2 c.520C>T and BRCA2 c.7992T>A induced incomplete skipping of exons 7 and 18, respectively. This work highlights the contribution of splicing minigene assays to the assessment of pathogenicity, not only when patient RNA is not available, but also as a tool to improve the accuracy of bioinformatics predictions.