GFP-Fragment Reassembly Screens for the Functional Characterization of Variants of Uncertain Significance in Protein Interaction Domains of the BRCA1 and BRCA2 Genes.
ABSTRACT: Genetic testing for BRCA1 and BRCA2 genes has led to the identification of many unique variants of uncertain significance (VUS). Multifactorial likelihood models that predict the odds ratio for VUS in favor or against cancer causality, have been developed, but their use is conditioned by the amount of necessary data, which are difficult to obtain if a variant is rare. As an alternative, variants mapping to the coding regions can be examined using in vitro functional assays. BRCA1 and BRCA2 proteins promote genome protection by interacting with different proteins. In this study, we assessed the functional effect of two sets of variants in BRCA genes by exploiting the green fluorescent protein (GFP)-reassembly in vitro assay, which was set-up to test the BRCA1/BARD1, BRCA1/UbcH5a, and BRCA2/DSS1 interactions. Based on the findings observed for the validation panels of previously classified variants, BRCA1/UbcH5a and BRCA2/DSS1 binding assays showed 100% sensitivity and specificity in identifying pathogenic and non-pathogenic variants. While the actual efficiency of these assays in assessing the clinical significance of BRCA VUS has to be verified using larger validation panels, our results suggest that the GFP-reassembly assay is a robust method to identify variants affecting normal protein functioning and contributes to the classification of VUS.
Project description:Hereditary breast and ovarian cancers are mainly linked to variants in BRCA1/2 genes. Recently, data has shown that identification of BRCA variants has an immediate impact not only in cancer prevention but also in targeted therapeutic approaches. This prospective observational study characterized the overall germline BRCA variant and variant of uncertain significance (VUS) frequency and spectrum in individuals affected by breast (BC) or ovarian cancer (OC) and in healthy individuals at risk by sequencing the entire BRCA genes. Of the 363 probands analyzed, 50 (13.8%) were BRCA1/2 mutated, 28 (7.7%) at BRCA1 and 23 (6.3%) at BRCA2 gene. The variant c.5266dupC p.(Gln1756Profs) was the most frequent alteration, representing 21.4% of the BRCA1 variants and 12.0% of all variants identified. The variant c.6313delA p.(Ile2105Tyrfs) of BRCA2 was the most frequent alteration observed in 6 patients. Interestingly, two new variants were identified in BRCA2. In addition, 25 different VUS were identified; two were reported for the first time in BRCA1 and two in BRCA2. The number of triple-negative BCs was significantly higher in patients with the pathogenic BRCA1/2-variant (36.4%) than in BRCA1/2 VUS (16.0%) and BRCA1/2 wild-type patients (10.7%) (p < 0.001). Our study reveals that the overall frequency of BRCA germline variants in the selected high-risk Italian population is about 13.8%. We believe that our results could have significant implications for preventive strategies for unaffected BRCA-carriers and effective targeted treatments such as PARP inhibitors for patients with BC or OC.
Project description:In silico predictions of missense variants is an important consideration when interpreting variants of uncertain significance (VUS) in the BRCA1 and BRCA2 genes. We trained and evaluated hundreds of machine learning algorithms based on results from validated functional assays to better predict missense variants in these genes as damaging or neutral. This new optimal "BRCA-ML" model yielded a substantially more accurate method than current algorithms for interpreting the functional impact of variants in these genes, making BRCA-ML a valuable addition to data sources for VUS classification.
Project description:Objective:BRCA mutational status is important in the management of ovarian cancer, but there is a lack of evidence supporting genetic testing in Asian populations. This study was performed to investigate the prevalence and prognostic outcomes of BRCA1/2 mutation and variant of unknown significance (VUS) in Korean patients diagnosed with epithelial ovarian cancer (EOC). Methods:Among patients newly diagnosed with EOC between January 2007 and January 2017, those tested for germline BRCA1/2 mutation were studied, regardless of family history. Overall survival (OS) and progression-free survival (PFS) were compared between the patients with and without BRCA1/2 mutation and VUS. Results:A total of 313 patients underwent BRCA testing: 88 patients had a BRCA1/2 mutation and 48 patients had a BRCA1/2 VUS (28.1% and 15.3%, respectively). There were no significant associations between BRCA1/2 mutation, BRCA1/2 wild-type, or BRCA1/2 VUS with age at diagnosis, histologic distribution, or residual disease status after primary cytoreductive surgery. BRCA1 mutation, including BRCA1 VUS, showed no difference in PFS or OS compared to BRCA1 wild-type. In contrast, BRCA2 mutation showed longer PFS than that of BRCA2 wild-type (P=0.04) or BRCA2 VUS (P=0.02). BRCA2 mutation, including BRCA2 VUS, did not show any difference in OS compared to BRCA2 wild-type. Conclusion:BRCA mutation and BRCA VUS had similar clinical characteristics and survival outcomes, except that BRCA2 mutation showed better PFS. The results of this study will help to understand the prognostic significance of BRCA mutation and VUS in Korean patients.
Project description:Clinical genetic testing of BRCA1 and BRCA2 is commonly performed to identify specific individuals at risk for breast and ovarian cancers who may benefit from prophylactic therapeutic interventions. Unfortunately, it is evident that deleterious BRCA1 alleles demonstrate variable penetrance and that many BRCA1 variants of unknown significance (VUS) exist. In order to further refine hereditary risks that may be associated with specific BRCA1 alleles, we performed gene targeting to establish an isogenic panel of immortalized human breast epithelial cells harboring eight clinically relevant BRCA1 alleles. Interestingly, BRCA1 mutations and VUS had distinct, quantifiable phenotypes relative to isogenic parental BRCA1 wild type cells and controls. Heterozygous cells with known deleterious BRCA1 mutations (185delAG, C61G and R71G) demonstrated consistent phenotypes in radiation sensitivity and genomic instability assays, but showed variability in other assays. Heterozygous BRCA1 VUS cells also demonstrated assay variability, with some VUS demonstrating phenotypes more consistent with deleterious alleles. Taken together, our data suggest that BRCA1 deleterious mutations and VUS can differ in their range of tested phenotypes, suggesting they might impart varying degrees of risk. These results demonstrate that functional isogenic modeling of BRCA1 alleles could aid in classifying BRCA1 mutations and VUS, and determining BRCA allele cancer risk.
Project description:Background:The spectrum of BRCA mutations that predispose to development of breast/ovarian cancer in Indian population remains unexplored. We report incidence and various types of pathogenic, likely pathogenic and variants of unknown significance (VUS) mutations in BRCA1 and BRCA2 genes observed at a tertiary cancer center in North India. Materials and methods:A total of 206 unrelated breast and/or ovarian cancer patients, who met the National Comprehensive Cancer Network (NCCN) guidelines for genetic testing, were screened for germline BRCA1/BRCA 2 mutations on high-throughput sequencing platform; large genomic rearrangements were assessed by multiple ligation probe assay. Mutations were mined in mutational databases, PubMed, and discerned into classes. Furthermore, the clinicopathological correlation of BRCA mutation status with prognostic markers in breast cancer and tumor histology in ovarian cancer was performed. Results:In total, 45/206 and 17/206 cases showed positivity for BRCA1 and BRCA2 mutations, respectively, whereas 1/206 was positive for a mutation in both the genes. Altogether, 33 distinct BRCA1 mutations were observed, among which 27 were deleterious (12 frameshifts, 8 nonsense, 1 missense, 3 splice-site variants, 2 big deletions and 1 large duplication) and 6 were VUS. Five novel BRCA1 mutations (c.541G>T, c.1681delT, c.2295delG, c.4915C>T and exon 23 deletion) were identified. Seven mutations (c.2214_2215insT, c.2295delG, c.3607C>T,c.4158_4162delCTCTC, c.4571C>A, splicesite_3 (C>T) and exon 21-23 duplication) occurred more than once, whereas 16 distinct BRCA2 mutations were noted - 9 were lethal (6 frameshifts, 2 nonsense and 1 big deletion) and 7 VUS. One unique pathogenic BRCA2 mutation (c.932_933insT) was recognized. Two mutations (c.9976A>T and c.10089A>G) recurred twice. No significant difference in hormone receptor status was observed among BRCA1 carriers, BRCA2 carriers and noncarriers. Conclusion:We have documented various pathogenic and VUS mutations in BRCA1 and BRCA2 genes observed in the cohort. Six novel mutations were identified. The knowledge shared would assist genetic testing in enabling more focused site-specific screening for mutations in biological relatives.
Project description:Recent advances in DNA sequencing techniques have led to an increase in the identification of single nucleotide polymorphisms (SNPs) in BRCA1 and BRCA2 genes, but no further information regarding the deleterious probability of many of them is available (Variants of Unknown Significance/VUS). As a result, in the current study, different sequence- and structure-based computational tools including SIFT, PolyPhen2, PANTHER, SNPs&GO, FATHMM, SNAP, PhD-SNP, Align-GVGD, and I-Mutant were utilized for determining how resulted BRCA protein is affected by corresponding missense mutations. FoldX was used to estimate mutational effects on the structural stability of BRCA proteins. Variants were considered extremely deleterious only when all tools predicted them to be deleterious. A total of 10 VUSs in BRCA1 (Cys39Ser, Cys64Gly, Phe861Cys, Arg1699Pro, Trp1718Cys, Phe1761Ser, Gly1788Asp, Val1804Gly, Trp1837Gly, and Trp1837Cys) and 12 in BRCA2 (Leu2510Pro, Asp2611Gly, Tyr2660Asp, Leu2686Pro, Leu2688Pro, Tyr2726Cys, Leu2792Pro, Gly2812Glu, Gly2813Glu, Arg2842Cys, Asp3073Gly, and Gly3076Val) were considered as extremely deleterious. Results suggested that deleterious variants were mostly enriched in the N- and C-terminal domain of the BRCA1 and BRCA2 C-terminus. Utilizing evolutionary conservation analysis, we demonstrated that the majority of deleterious SNPs ensue in highly conserved regions of BRCA genes. Furthermore, utilizing FoldX, we demonstrated that alterations in the function of proteins are not always together with stability alterations.
Project description:Background:Pathogenic variants in BRCA1 and BRCA2 cause hereditary breast and ovarian cancer. Screening of these genes has become easily accessible in diagnostic laboratories. Sequencing and copy number analyses are used to detect pathogenic variants, but also lead to identification of variants of unknown clinical significance (VUS). If the effect of a VUS can be clarified, it has direct consequence for the clinical management of the patient and family members. A splicing assay is one of several tools that might help in the classification of VUS. We therefore established mRNA analyses for BRCA1 and BRCA2 in the diagnostic laboratory in 2015. We hereby report the results of mRNA analysis variants in BRCA1 and BRCA2 after three years. Methods:Variants predicted to alter splicing and variants within the canonical splice sites were selected for splicing analyses. Splicing assays were performed by reverse transcription-PCR of patient RNA. A biallalic expression analysis was carried out whenever possible. Results:Twenty-five variants in BRCA1 and BRCA2 were analyzed by splicing assays; nine showed altered transcripts and 16 showed normal splicing patterns. The two novel pathogenic variants in BRCA1 c.4484?+?3 A?>?C and c.5407-10G?>?A were characterized. Conclusions:We conclude that mRNA analyses are useful in characterization of variants that may affect splicing. The results can guide classification of variants from unknown clinical significance to pathogenic or benign in a diagnostic laboratory, and thus be of direct clinical importance.
Project description:Genetic testing allows for the identification of germline DNA variations, which are associated with a significant increase in the risk of developing breast cancer (BC) and ovarian cancer (OC). Detection of a BRCA1 or BRCA2 pathogenic variant triggers several clinical management actions, which may include increased surveillance and prophylactic surgery for healthy carriers or treatment with the PARP inhibitor therapy for carriers diagnosed with cancer. Thus, standardized validated criteria for the annotation of BRCA1 and BRCA2 variants according to their pathogenicity are necessary to support clinical decision-making and ensure improved outcomes. Upon detection, variants whose pathogenicity can be inferred by the genetic code are typically classified as pathogenic, likely pathogenic, likely benign, or benign. Variants whose impact on function cannot be directly inferred by the genetic code are labeled as variants of uncertain clinical significance (VUS) and are evaluated by multifactorial likelihood models that use personal and family history of cancer, segregation data, prediction tools, and co-occurrence with a pathogenic BRCA variant. Missense variants, coding alterations that replace a single amino acid residue with another, are a class of variants for which determination of clinical relevance is particularly challenging. Here, we discuss current issues in the missense variant classification by following a typical life cycle of a BRCA1 missense variant through detection, annotation and information dissemination. Advances in massively parallel sequencing have led to a substantial increase in VUS findings. Although the comprehensive assessment and classification of missense variants according to their pathogenicity remains the bottleneck, new developments in functional analysis, high throughput assays, data sharing, and statistical models are rapidly changing this scenario.
Project description:Germline mutations in BRCA1/2 genes are responsible for a large proportion of hereditary breast and/or ovarian cancers. Many highly penetrant predisposition alleles have been identified and include frameshift or nonsense mutations that lead to the translation of a truncated protein. Other alleles contain missense mutations, which result in amino acid substitution and intronic variants with splicing effect. The discovery of variants of uncertain/unclassified significance (VUS) is a result that can complicate rather than improve the risk assessment process. VUSs are mainly missense mutations, but also include a number of intronic variants and in-frame deletions and insertions. Over 2,000 unique BRCA1 and BRCA2 missense variants have been identified, located throughout the whole gene (Breast Cancer Information Core Database (BIC database)). Up to 10-20% of the BRCA tests report the identification of a variant of uncertain significance. There are many methods to discriminate deleterious/high-risk from neutral/low-risk unclassified variants (i.e., analysis of the cosegregation in families of the VUS, measure of the influence of the VUSs on the wild-type protein activity, comparison of sequence conservation across multiple species), but only an integrated analysis of these methods can contribute to a real interpretation of the functional and clinical role of the discussed variants. The aim of our manuscript is to review the studies on BRCA VUS in order to clarify their clinical relevance.
Project description:Background:It is often assumed any cancer in a germline BRCA1 or BRCA2 (collectively termed BRCA) mutation carrier was caused by that mutation. It is also often assumed the occurrence of breast or ovarian cancer in an individual with a variant of uncertain significance (VUS) suggests the VUS is pathogenic. These assumptions have profound management implications for cancer patients and healthy individuals. Methods:We compared the frequency of BRCA mutations, allele loss, and Signature 3 in 7632 individuals with 28 cancers and 1000 population controls. Because only increased frequency was the focus of the study, all statistical tests were one-sided. Results:Individuals with breast or ovarian cancer had increased germline BRCA pathogenic mutation frequencies compared to controls (P?=?1.0x10-10 and P?=?1.4x10-34, respectively). There was no increase in other cancer types. Wild-type allele loss and Signature 3 were statistically significantly higher in breast and ovarian cancers with BRCA mutations compared with other cancers with BRCA mutations (P?=?5.1x10-10 and P?=?3.7x10-9) and cancers without BRCA mutations (P?=?2.8x10-53 and P?=?1.0x10-134). There was no difference between non-breast and non-ovarian cancers with BRCA mutations and cancers without BRCA mutations. Allele loss and Signature 3 were statistically significantly higher in breast and ovarian cancers in individuals with BRCA pathogenic mutations compared to those with VUS (P?=?3.8x10-17 and P?=?1.6x10-8) or benign variants (P?=?1.2x10-28 and P?=?2.2x10-10). There was no difference between individuals with BRCA VUS and those with benign variants. Conclusions:These data show that non-breast and non-ovarian cancers in individuals with germline BRCA pathogenic mutations are often not causally related to the mutation and that BRCA VUS are highly unlikely to be pathogenic. These results should reduce inappropriate management of germline BRCA information.