Classification of missense substitutions in the BRCA genes: a database dedicated to Ex-UVs

Unclassified sequence variants (UVs) arising from clinical mutation screening of cancer susceptibility genes present a frustrating issue to clinical genetics services and the patients that they serve. We created an open-access database holding missense substitutions from the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2. The main inclusion criterion is that each variant should have been assessed in a published work that used the Bayesian integrated evaluation of unclassified BRCA gene variants. Transfer of data on these substitutions from the original publications to our database afforded an opportunity to analyze the missense substitutions under a single model and to remove inconsistencies that arose during the evolution of the integrated evaluation over the last decade. This analysis also afforded the opportunity to reclassify these missense substitutions according to the recently published IARC 5-Class system. From an initial set of 248 missense substitutions, 31 were set aside due to nonnegligible probability to interfere with splicing. Of the remaining substitutions, 28 fell into one of the two pathogenic classes (IARC Class 4 or 5), 174 fell into one of the two nonpathogenic classes (IARC Class 1 or 2), and 15 remain in IARC Class 3, "Uncertain." The database is available at http://brca.iarc.fr/LOVD.     

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.     

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.     

A mutation analysis of the<Emphasis Type="Italic"> BRCA1</Emphasis> gene in 140 families from southeast France with a history of breast and/or ovarian cancer

A mutation analysis of the BRCA1 gene in 140 French families with a history of breast cancer or breast-ovarian cancer revealed several deleterious germline mutations, as well as rare sequence variants. The 19 genetics variants were of 15 different types, two of which had not been reported in the Breast cancer Information Core (BIC) database. Five distinct truncating mutations, leading to putative nonfunctional proteins, were identified out of 140 index cases (3.5%). One novel nonsense mutation, C4491T, was reported, whereas the four other BRCA1 deleterious mutations identified consisted of frequent frameshifts in the nucleotide sequence. One splice variant (331+3A>G) and thirteen missense variations leading to amino acid substitutions of unknown structural and functional importance were identified. Among these, two BRCA1 missense mutations, A120G and T243C could be considered as suspected deleterious. The first missense mutation modified the initiation codon (M1V) and the second (C39R) may have consequences on the structure and functioning of the BRCA1 protein by modifying cysteine ligands from the RING finger domain. As expected BRCA1 gene alteration, including missense mutations of unknown biological significance, were more frequent in families with a history of breast-ovarian-cancer (32%) than in breast-cancer-only families (12%).     

Genetic and bioinformatics analysis of four novel GCK missense variants detected in Caucasian families with GCK‐MODY phenotype

Heterozygous loss‐of‐function mutations in the glucokinase (GCK) gene cause maturity‐onset diabetes of the young (MODY) subtype GCK (GCK‐MODY/MODY2). GCK sequencing revealed 16 distinct mutations (13 missense, 1 nonsense, 1 splice site, and 1 frameshift‐deletion) co‐segregating with hyperglycaemia in 23 GCK‐MODY families. Four missense substitutions (c.718A>G/p.Asn240Asp, c.757G>T/p.Val253Phe, c.872A>C/p.Lys291Thr, and c.1151C>T/p.Ala384Val) were novel and a founder effect for the nonsense mutation (c.76C>T/p.Gln26*) was supposed. We tested whether an accurate bioinformatics approach could strengthen family‐genetic evidence for missense variant pathogenicity in routine diagnostics, where wet‐lab functional assays are generally unviable. In silico analyses of the novel missense variants, including orthologous sequence conservation, amino acid substitution (AAS)‐pathogenicity predictors, structural modeling and splicing predictors, suggested that the AASs and/or the underlying nucleotide changes are likely to be pathogenic. This study shows how a careful bioinformatics analysis could provide effective suggestions to help molecular‐genetic diagnosis in absence of wet‐lab validations.     

Variant effect on splicing regulatory elements,branchpoint usage,and pseudoexonization: Strategies to enhance bioinformatic prediction using hereditary cancer genes as exemplars

It is possible to estimate the prior probability of pathogenicity for germline disease gene variants based on bioinformatic prediction of variant effect/s. However, routinely used approaches have likely led to the underestimation and underreporting of variants located outside donor and acceptor splice site motifs that affect messenger RNA (mRNA) processing. This review presents information about hereditary cancer gene germline variants, outside native splice sites, with experimentally validated splicing effects. We list 95 exonic variants that impact splicing regulatory elements (SREs) in BRCA1, BRCA2, MLH1, MSH2, MSH6, and PMS2. We utilized a pre‐existing large‐scale BRCA1 functional data set to map functional SREs, and assess the relative performance of different tools to predict effects of 283 variants on such elements. We also describe rare examples of intronic variants that impact branchpoint (BP) sites and create pseudoexons. We discuss the challenges in predicting variant effect on BP site usage and pseudoexonization, and suggest strategies to improve the bioinformatic prioritization of such variants for experimental validation. Importantly, our review and analysis highlights the value of considering impact of variants outside donor and acceptor motifs on mRNA splicing and disease causation.     

Calibration of Multiple In Silico Tools for Predicting Pathogenicity of Mismatch Repair Gene Missense Substitutions

Classification of rare missense substitutions observed during genetic testing for patient management is a considerable problem in clinical genetics. The Bayesian integrated evaluation of unclassified variants is a solution originally developed for BRCA1/2. Here, we take a step toward an analogous system for the mismatch repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2) that confer colon cancer susceptibility in Lynch syndrome by calibrating in silico tools to estimate prior probabilities of pathogenicity for MMR gene missense substitutions. A qualitative five‐class classification system was developed and applied to 143 MMR missense variants. This identified 74 missense substitutions suitable for calibration. These substitutions were scored using six different in silico tools (Align‐Grantham Variation Grantham Deviation, multivariate analysis of protein polymorphisms [MAPP], MutPred, PolyPhen‐2.1, Sorting Intolerant From Tolerant, and Xvar), using curated MMR multiple sequence alignments where possible. The output from each tool was calibrated by regression against the classifications of the 74 missense substitutions; these calibrated outputs are interpretable as prior probabilities of pathogenicity. MAPP was the most accurate tool and MAPP + PolyPhen‐2.1 provided the best‐combined model (R² = 0.62 and area under receiver operating characteristic = 0.93). The MAPP + PolyPhen‐2.1 output is sufficiently predictive to feed as a continuous variable into the quantitative Bayesian integrated evaluation for clinical classification of MMR gene missense substitutions.     

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.     

Exceptions to the rule: Case studies in the prediction of pathogenicity for genetic variants in hereditary cancer genes

Based on current consensus guidelines and standard practice, many genetic variants detected in clinical testing are classified as disease causing based on their predicted impact on the normal expression or function of the gene in the absence of additional data. However, our laboratory has identified a subset of such variants in hereditary cancer genes for which compelling contradictory evidence emerged after the initial evaluation following the first observation of the variant. Three representative examples of variants in BRCA1, BRCA2 and MSH2 that are predicted to disrupt splicing, prematurely truncate the protein, or remove the start codon were evaluated for pathogenicity by analyzing clinical data with multiple classification algorithms. Available clinical data for all three variants contradicts the expected pathogenic classification. These variants illustrate potential pitfalls associated with standard approaches to variant classification as well as the challenges associated with monitoring data, updating classifications, and reporting potentially contradictory interpretations to the clinicians responsible for translating test outcomes to appropriate clinical action. It is important to address these challenges now as the model for clinical testing moves toward the use of large multi‐gene panels and whole exome/genome analysis, which will dramatically increase the number of genetic variants identified.     

Analysis of BRCA1 Variants in Double‐Strand Break Repair by Homologous Recombination and Single‐Strand Annealing

Missense substitutions of uncertain clinical significance in the BRCA1 gene are a vexing problem in genetic counseling for women who have a family history of breast cancer. In this study, we evaluated the functions of 29 missense substitutions of BRCA1 in two DNA repair pathways. Repair of double‐strand breaks by homology‐directed recombination (HDR) had been previously analyzed for 16 of these BRCA1 variants, and 13 more variants were analyzed in this study. All 29 variants were also analyzed for function in double‐strand break repair by the single‐strand annealing (SSA) pathway. We found that among the pathogenic mutations in BRCA1, all were defective for DNA repair by either pathway. The HDR assay was accurate because all pathogenic mutants were defective for HDR, and all nonpathogenic variants were fully functional for HDR. Repair by SSA accurately identified pathogenic mutants, but several nonpathogenic variants were scored as defective or partially defective. These results indicated that specific amino acid residues of the BRCA1 protein have different effects in the two related DNA repair pathways, and these results validate the HDR assay as highly correlative with BRCA1‐associated breast cancer.     

Functional Assays for Analysis of Variants of Uncertain Significance in BRCA2

Missense variants in the BRCA2 gene are routinely detected during clinical screening for pathogenic mutations in patients with a family history of breast and ovarian cancer. These subtle changes frequently remain of unknown clinical significance because of the lack of genetic information that may help establish a direct correlation with cancer predisposition. Therefore, alternative ways of predicting the pathogenicity of these variants are urgently needed. Since BRCA2 is a protein involved in important cellular mechanisms such as DNA repair, replication, and cell cycle control, functional assays have been developed that exploit these cellular activities to explore the impact of the variants on protein function. In this review, we summarize assays developed and currently utilized for studying missense variants in BRCA2. We specifically depict details of each assay, including variants of uncertain significance analyzed, and describe a validation set of (genetically) proven pathogenic and neutral missense variants to serve as a golden standard for the validation of each assay. Guidelines are proposed to enable implementation of laboratory‐based methods to assess the impact of the variant on cancer risk.     

Functional Analysis of BARD1 Missense Variants in Homology‐Directed Repair of DNA Double Strand Breaks

Genes associated with hereditary breast and ovarian cancer (HBOC) are often sequenced in search of mutations that are predictive of susceptibility to these cancer types, but the sequence results are frequently ambiguous because of the detection of missense substitutions for which the clinical impact is unknown. The BARD1 protein is the heterodimeric partner of BRCA1 and is included on clinical gene panels for testing for susceptibility to HBOC. Like BRCA1, it is required for homology‐directed DNA repair (HDR). We measured the HDR function of 29 BARD1 missense variants, 27 culled from clinical test results and two synthetic variants. Twenty‐three of the assayed variants were functional for HDR; of these, four are known neutral variants. Three variants showed intermediate function, and three others were defective in HDR. When mapped to BARD1 domains, residues crucial for HDR were located in the N‐ and C‐ termini of BARD1. In the BARD1 RING domain, critical residues mapped to the zinc‐coordinating amino acids and to the BRCA1‐BARD1 binding interface, highlighting the importance of interaction between BRCA1 and BARD1 for HDR activity. Based on these results, we propose that the HDR assay is a useful complement to genetic analyses to classify BARD1 variants of unknown clinical significance.     

Genetic analysis of BRCA1 ubiquitin ligase activity and its relationship to breast cancer susceptibility

The N-terminus of the Breast Cancer-1 predisposition protein (BRCA1) associates with the BRCA1-associated RING domain-1 protein (BARD1) to form a heterodimer, which exhibits ubiquitin ligase activity that is abrogated by known cancer-associated BRCA1 missense mutations. The majority of missense substitutions identified in patients with a personal or a family history of disease have not been followed in pedigrees, nor there is a functional understanding of their impact. We have examined, by extensive missense substitution, the interaction of BRCA1 with components that contribute to its ubiquitin ligase activity, BARD1 and the E2 ubiquitin-conjugating enzyme, UbcH5a. Selection from a randomly generated library of BRCA1 missense mutations for variants that inhibit the interaction with these components identified substitutions in residues found altered in patient DNA, indicating a correlation between loss of component-binding and propensity to disease development. We further show that the BRCA1:E2 interaction is sensitive to substitutions in all structural elements of the BRCA1 N-terminus, whereas the BARD1 interaction is sensitive to a subset of BRCA1 substitutions, which also inhibit E2-binding. Patient variants that inhibit the BRCA1:E2 interaction show loss of ubiquitin ligase activity and correlate with disease susceptibility and theoretical predictions of pathogenicity. These data link the loss of ubiquitin ligase activity, through loss of E2-binding, to the majority of non-polymorphic patient variants described within the N-terminus of BRCA1 and illustrate the likely significant role of BRCA1 ubiquitin ligase activity in tumour suppression.     

A quantitative model to predict pathogenicity of missense variants in the TP53 gene

Germline pathogenic variants in the TP53 gene cause Li‐Fraumeni syndrome, a condition that predisposes individuals to a wide range of cancer types. Identification of individuals carrying a TP53 pathogenic variant is linked to clinical management decisions, such as the avoidance of radiotherapy and use of high‐intensity screening programs. The aim of this study was to develop an evidence‐based quantitative model that integrates independent in silico data (Align‐GVGD and BayesDel) and somatic to germline ratio (SGR), to assign pathogenicity to every possible missense variant in the TP53 gene. To do this, a likelihood ratio for pathogenicity (LR) was derived from each component calibrated using reference sets of assumed pathogenic and benign missense variants. A posterior probability of pathogenicity was generated by combining LRs, and algorithm outputs were validated using different approaches. A total of 730 TP53 missense variants could be assigned to a clinically interpretable class. The outputs of the model correlated well with existing clinical information, functional data, and ClinVar classifications. In conclusion, these quantitative outputs provide the basis for individualized assessment of cancer risk useful for clinical interpretation. In addition, we propose the value of the novel SGR approach for use within the ACMG/AMP guidelines for variant classification.     

Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral

Background

Genetic testing for hereditary cancer syndromes contributes to the medical management of patients who may be at increased risk of one or more cancers. BRCA1 and BRCA2 testing for hereditary breast and ovarian cancer is one such widely used test. However, clinical testing methods with high sensitivity for deleterious mutations in these genes also detect many unclassified variants, primarily missense substitutions.

Methods

We developed an extension of the Grantham difference, called A‐GVGD, to score missense substitutions against the range of variation present at their position in a multiple sequence alignment. Combining two methods, co‐occurrence of unclassified variants with clearly deleterious mutations and A‐GVGD, we analysed most of the missense substitutions observed in BRCA1.

Results

A‐GVGD was able to resolve known neutral and deleterious missense substitutions into distinct sets. Additionally, eight previously unclassified BRCA1 missense substitutions observed in trans with one or more deleterious mutations, and within the cross‐species range of variation observed at their position in the protein, are now classified as neutral.

Discussion

The methods combined here can classify as neutral about 50% of missense substitutions that have been observed with two or more clearly deleterious mutations. Furthermore, odds ratios estimated for sets of substitutions grouped by A‐GVGD scores are consistent with the hypothesis that most unclassified substitutions that are within the cross‐species range of variation at their position in BRCA1 are also neutral. For most of these, clinical reclassification will require integrated application of other methods such as pooled family histories, segregation analysis, or validated functional assay.     

5′ splice site GC>GT and GT>GC variants differ markedly in terms of their functionality and pathogenicity

In the human genome, most 5′ splice sites (~99%) employ the canonical GT dinucleotide whereas a small minority (~1%) use the noncanonical GC dinucleotide. The functionality and pathogenicity of 5′ splice site GT>GC (+2T>C) variants have been extensively studied but we know very little about 5′ splice site GC>GT (+2C>T) variants. Herein, we have addressed this deficiency by performing a meta‐analysis of reported +2C>T “pathogenic” variants together with a functional analysis of engineered +2C>T substitutions using a cell culture‐based full‐length gene splicing assay. Our results establish proof of concept that +2C>T variants are qualitatively different from +2T>C variants in terms of their functionality and suggest that, in sharp contrast to +2T>C variants, most if not all +2C>T variants have no pathological relevance. Our findings have important implications for interpreting the clinical relevance of +2C>T variants and understanding the evolutionary switching between GT and GC 5′ splice sites in mammalian genomes.