Lucia Guidugli

Inherited Mutations in 17 Breast Cancer Susceptibility Genes Among a Large Triple-Negative Breast Cancer Cohort Unselected for Family History of Breast Cancer

PURPOSE Recent advances in DNA sequencing have led to the development of breast cancer susceptibility gene panels for germline genetic testing of patients. We assessed the frequency of mutations in 17 predisposition genes, including BRCA1 and BRCA2, in a large cohort of patients with triple-negative breast cancer (TNBC) unselected for family history of breast or ovarian cancer to determine the utility of germline genetic testing for those with TNBC. PATIENTS AND METHODS Patients with TNBC (N = 1,824) unselected for family history of breast or ovarian cancer were recruited through 12 studies, and germline DNA was sequenced to identify mutations. RESULTS Deleterious mutations were identified in 14.6% of all patients. Of these, 11.2% had mutations in the BRCA1 (8.5%) and BRCA2 (2.7%) genes. Deleterious mutations in 15 other predisposition genes were detected in 3.7% of patients, with the majority observed in genes involved in homologous recombination, including PALB2 (1.2%) and BARD1, RAD51D, RAD51C, and BRIP1 (0.3% to 0.5%). Patients with TNBC with mutations were diagnosed at an earlier age (P < .001) and had higher-grade tumors (P = .01) than those without mutations. CONCLUSION Deleterious mutations in predisposition genes are present at high frequency in patients with TNBC unselected for family history of cancer. Mutation prevalence estimates suggest that patients with TNBC, regardless of age at diagnosis or family history of cancer, should be considered for germline genetic testing of BRCA1 and BRCA2. Although mutations in other predisposition genes are observed among patients with TNBC, better cancer risk estimates are needed before these mutations are used for clinical risk assessment in relatives.

A review of a multifactorial probability-based model for classification of BRCA1 and BRCA2 variants of uncertain significance (VUS).

Clinical mutation screening of the BRCA1 and BRCA2 genes for the presence of germline inactivating mutations is used to identify individuals at elevated risk of breast and ovarian cancer. Variants identified during screening are usually classified as pathogenic (increased risk of cancer) or not pathogenic (no increased risk of cancer). However, a significant proportion of genetic tests yields variants of uncertain significance (VUS) that have undefined risk of cancer. Individuals carrying these VUS cannot benefit from individualized cancer risk assessment. Recently, a quantitative “posterior probability model” for assessing the clinical relevance of VUS in BRCA1 or BRCA2, which integrates multiple forms of genetic evidence has been developed. Here, we provide a detailed review of this model. We describe the components of the model and explain how these can be combined to calculate a posterior probability of pathogenicity for each VUS. We explain how the model can be applied to public data and provide tables that list the VUS that have been classified as not pathogenic or pathogenic using this method. While we use BRCA1 and BRCA2 VUS as examples, the method can be used as a framework for classification of the pathogenicity of VUS in other cancer genes. Hum Mutat 33:8–21, 2012.

BRCA1 R1699Q variant displaying ambiguous functional abrogation confers intermediate breast and ovarian cancer risk

Background Clinical classification of rare sequence changes identified in the breast cancer susceptibility genes BRCA1 and BRCA2 is essential for appropriate genetic counselling of individuals carrying these variants. We previously showed that variant BRCA1 c.5096G>A p.Arg1699Gln in the BRCA1 transcriptional transactivation domain demonstrated equivocal results from a series of functional assays, and proposed that this variant may confer low to moderate risk of cancer. Methods Measures of genetic risk (report of family history, segregation) were assessed for 68 BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) families recruited through family cancer clinics, comparing results with 34 families carrying the previously classified pathogenic BRCA1 c.5095C>T p.Arg1699Trp (R1699W) mutation at the same residue, and to 243 breast cancer families with no BRCA1 pathogenic mutation (BRCA-X). Results Comparison of BRCA1 carrier prediction scores of probands using the BOADICEA risk prediction tool revealed that BRCA1 c.5096G>A p.Arg1699Gln variant carriers had family histories that were less ‘BRCA1-like’ than BRCA1 c.5095C>T p.Arg1699Trp mutation carriers (p<0.00001), but more ‘BRCA1-like’ than BRCA-X families (p=0.0004). Further, modified segregation analysis of the subset of 30 families with additional genotyping showed that BRCA1 c.5096G >A p.Arg1699Gln had reduced penetrance compared with the average truncating BRCA1 mutation penetrance (p=0.0002), with estimated cumulative risks to age 70 of breast or ovarian cancer of 24%. Conclusions Our results provide substantial evidence that the BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) variant, demonstrating ambiguous functional deficiency across multiple assays, is associated with intermediate risk of breast and ovarian cancer, highlighting challenges for risk modelling and clinical management of patients of this and other potential moderate-risk variants.

A Classification Model for BRCA2 DNA Binding Domain Missense Variants Based on Homology-Directed Repair Activity

The relevance of many BRCA2 variants of uncertain significance (VUS) to breast cancer has not been determined due to limited genetic information from families carrying these alterations. Here, we classified six new variants as pathogenic or nonpathogenic by analysis of genetic information from families carrying 64 individual BRCA2 DNA binding domain (DBD) missense mutations using a multifactorial likelihood model of cancer causality. Next, we evaluated the use of a homology-directed DNA break repair (HDR) functional assay as a method for inferring the clinical relevance of VUS in the DBD of BRCA2 using 18 established nonpathogenic missense variants and all 13 established pathogenic missense mutations from the BRCA2 DBD. Compared with the known status of these variants based on the multifactorial likelihood model, the sensitivity of the HDR assay for pathogenic mutations was estimated at 100% [95% confidence interval (CI): 75.3%-100%] and specificity was estimated at 100% (95% CI: 81.5%-100%). A statistical classifier for predicting the probability of pathogenicity of BRCA2 DBD variants was developed using these functional results. When applied to 33 additional VUS, the classifier identified eight with 99% or more probability of nonpathogenicity and 18 with 99% or more probability of pathogenicity. Thus, in the absence of genetic evidence, a cell-based HDR assay can provide a probability of pathogenicity for all VUS in the BRCA2 DBD, suggesting that the assay can be used in combination with other information to determine the cancer relevance of BRCA2 VUS.

BRCA2 Localization to the Midbody by Filamin A Regulates CEP55 Signaling and Completion of Cytokinesis

Disruption of the BRCA2 tumor suppressor is associated with structural and numerical chromosomal defects. The numerical abnormalities in BRCA2-deficient cells may partly result from aberrations in cell division caused by disruption of BRCA2 during cytokinesis. Here we show that BRCA2 is a component of the midbody that is recruited through an interaction with Filamin A actin-binding protein. At the midbody, BRCA2 influences the recruitment of endosomal sorting complex required for transport (ESCRT)-associated proteins, Alix and Tsg101, and formation of CEP55-Alix and CEP55-Tsg101 complexes during abscission. Disruption of these BRCA2 interactions by cancer-associated mutations results in increased cytokinetic defects but has no effect on BRCA2-dependent homologous recombination repair of DNA damage. These findings identify a specific role for BRCA2 in the regulation of midbody structure and function, separate from DNA damage repair, that may explain in part the whole-chromosomal instability in BRCA2-deficient tumors.

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.

Evaluation of ACMG-Guideline-Based Variant Classification of Cancer Susceptibility and Non-Cancer-Associated Genes in Families Affected by Breast Cancer.

Sequencing tests assaying panels of genes or whole exomes are widely available for cancer risk evaluation. However, methods for classification of variants resulting from this testing are not well studied. We evaluated the ability of a variant-classification methodology based on American College of Medical Genetics and Genomics (ACMG) guidelines to define the rate of mutations and variants of uncertain significance (VUS) in 180 medically relevant genes, including all ACMG-designated reportable cancer and non-cancer-associated genes, in individuals who met guidelines for hereditary cancer risk evaluation. We performed whole-exome sequencing in 404 individuals in 253 families and classified 1,640 variants. Potentially clinically actionable (likely pathogenic [LP] or pathogenic [P]) versus nonactionable (VUS, likely benign, or benign) calls were 95% concordant with locus-specific databases and Clinvar. LP or P mutations were identified in 12 of 25 breast cancer susceptibility genes in 26 families without identified BRCA1/2 mutations (11%). Evaluation of 84 additional genes associated with autosomal-dominant cancer susceptibility identified LP or P mutations in only two additional families (0.8%). However, individuals from 10 of 253 families (3.9%) had incidental LP or P mutations in 32 non-cancer-associated genes, and 9% of individuals were monoallelic carriers of a rare LP or P mutation in 39 genes associated with autosomal-recessive cancer susceptibility. Furthermore, 95% of individuals had at least one VUS. In summary, these data support the clinical utility of ACMG variant-classification guidelines. Additionally, evaluation of extended panels of cancer-associated genes in breast/ovarian cancer families leads to only an incremental clinical benefit but substantially increases the complexity of the results.

Rare variants in XRCC2 as breast cancer susceptibility alleles

Background Recently, rare germline variants in XRCC2 were detected in non-BRCA1/2 familial breast cancer cases, and a significant association with breast cancer was reported. However, the breast cancer risk associated with these variants needs further evaluation. Methods The coding regions and exon–intron boundaries of XRCC2 were scanned for mutations in an international cohort of 3548 non-BRCA1/2 familial breast cancer cases and 1435 healthy controls using various mutation scanning methods. Predictions on functional relevance of detected missense variants were obtained from three different prediction algorithms. Results The only protein-truncating variant detected was found in a control. Rare non-protein-truncating variants were detected in 20 familial cases (0.6%) and nine healthy controls (0.6%). Although the number of variants predicted to be damaging or neutral differed between prediction algorithms, in all instances these categories were evenly represented among cases and controls. Conclusions Our data do not confirm an association between XRCC2 variants and breast cancer risk, although a relative risk smaller than two could not be excluded. Variants in XRCC2 are unlikely to explain a substantial proportion of familial breast cancer.

Comparison of mRNA Splicing Assay Protocols across Multiple Laboratories: Recommendations for Best Practice in Standardized Clinical Testing

BACKGROUND Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting. METHODS We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design. RESULTS PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1G>T Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp). CONCLUSIONS We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.

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. Hum Mutat 32:1–10, 2011.