DNA methylation profiling to assess pathogenicity of BRCA1 unclassified variants in breast cancer

Germline pathogenic mutations in BRCA1 increase risk of developing breast cancer. Screening for mutations in BRCA1 frequently identifies sequence variants of unknown pathogenicity and recent work has aimed to develop methods for determining pathogenicity. We previously observed that tumor DNA methylation can differentiate BRCA1-mutated from BRCA1-wild type tumors. We hypothesized that we could predict pathogenicity of variants based on DNA methylation profiles of tumors that had arisen in carriers of unclassified variants. We selected 150 FFPE breast tumor DNA samples [47 BRCA1 pathogenic mutation carriers, 65 BRCAx (BRCA1-wild type), 38 BRCA1 test variants] and analyzed a subset (n=54) using the Illumina 450K methylation platform, using the remaining samples for bisulphite pyrosequencing validation. Three validated markers (BACH2, C8orf31, and LOC654342) were combined with sequence bioinformatics in a model to predict pathogenicity of 27 variants (independent test set).  Predictions were compared with standard multifactorial likelihood analysis. Prediction was consistent for c.5194-12G>A (IVS 19-12 G>A) (P>0.99); 13 variants were considered not pathogenic or likely not pathogenic using both approaches. We conclude that tumor DNA methylation data alone has potential to be used in prediction of BRCA1 variant pathogenicity but is not independent of estrogen receptor status and grade, which are used in current multifactorial models to predict pathogenicity.     

BRCA1 and BRCA2 missense variants of high and low clinical significance influence lymphoblastoid cell line post-irradiation gene expression

The functional consequences of missense variants in disease genes are difficult to predict. We assessed if gene expression profiles could distinguish between BRCA1 or BRCA2 pathogenic truncating and missense mutation carriers and familial breast cancer cases whose disease was not attributable to BRCA1 or BRCA2 mutations (BRCAX cases). 72 cell lines from affected women in high-risk breast ovarian families were assayed after exposure to ionising irradiation, including 23 BRCA1 carriers, 22 BRCA2 carriers, and 27 BRCAX individuals. A subset of 10 BRCAX individuals carried rare BRCA1/2 sequence variants considered to be of low clinical significance (LCS). BRCA1 and BRCA2 mutation carriers had similar expression profiles, with some subclustering of missense mutation carriers. The majority of BRCAX individuals formed a distinct cluster, but BRCAX individuals with LCS variants had expression profiles similar to BRCA1/2 mutation carriers. Gaussian Process Classifier predicted BRCA1, BRCA2 and BRCAX status, with a maximum of 62% accuracy, and prediction accuracy decreased with inclusion of BRCAX samples carrying an LCS variant, and inclusion of pathogenic missense carriers. Similarly, prediction of mutation status with gene lists derived using Support Vector Machines was good for BRCAX samples without an LCS variant (82–94%), poor for BRCAX with an LCS (40–50%), and improved for pathogenic BRCA1/2 mutation carriers when the gene list used for prediction was appropriate to mutation effect being tested (71–100%). This study indicates that mutation effect, and presence of rare variants possibly associated with a low risk of cancer, must be considered in the development of array-based assays of variant pathogenicity.     

The BRCA1 Variant p.Ser36Tyr Abrogates BRCA1 Protein Function and Potentially Confers a Moderate Risk of Breast Cancer

The identification of variants of unknown clinical significance (VUS) in the BRCA1 gene complicates genetic counselling and causes additional anxiety to carriers. In silico approaches currently used for VUS pathogenicity assessment are predictive and often produce conflicting data. Furthermore, functional assays are either domain or function specific, thus they do not examine the entire spectrum of BRCA1 functions and interpretation of individual assay results can be misleading. PolyPhen algorithm predicted that the BRCA1 p.Ser36Tyr VUS identified in the Cypriot population was damaging, whereas Align-GVGD predicted that it was possibly of no significance. In addition the BRCA1 p.Ser36Tyr variant was found to be associated with increased risk (OR = 3.47, 95% CI 1.13-10.67, P = 0.02) in a single case-control series of 1174 cases and 1109 controls. We describe a cellular system for examining the function of exogenous full-length BRCA1 and for classifying VUS. We achieved strong protein expression of full-length BRCA1 in transiently transfected HEK293T cells. The p.Ser36Tyr VUS exhibited low protein expression similar to the known pathogenic variant p.Cys61Gly. Co-precipitation analysis further demonstrated that it has a reduced ability to interact with BARD1. Further, co-precipitation analysis of nuclear and cytosolic extracts as well as immunofluorescence studies showed that a high proportion of the p.Ser36Tyr variant is withheld in the cytoplasm contrary to wild type protein. In addition the ability of p.Ser36Tyr to co-localize with conjugated ubiquitin foci in the nuclei of S-phase synchronized cells following genotoxic stress with hydroxyurea is impaired at more pronounced levels than that of the p.Cys61Gly pathogenic variant. The p.Ser36Tyr variant demonstrates abrogated function, and based on epidemiological, genetic, and clinical data we conclude that the p.Ser36Tyr variant is probably associated with a moderate breast cancer risk.     

Trans-activation-based risk assessment of <Emphasis Type="Italic">BRCA1</Emphasis> BRCT variants with unknown clinical significance

Background

Deleterious variants in the tumour suppressor BRCA1 are known to cause hereditary breast and ovarian cancer syndrome (HBOC). Missense variants in BRCA1 pose a challenge in clinical care, as their effect on protein functionality often remains unknown. Many of the pathogenic missense variants found in BRCA1 are located in the BRCA1 C-terminal (BRCT) domains, domains that are known to be vital for key functions such as homologous recombination repair, protein-protein interactions and trans-activation (TA). We investigated the TA activity of 12 BRCA1 variants of unknown clinical significance (VUSs) located in the BRCT domains to aid in the classification of these variants.

Results

Twelve BRCA1 VUSs were investigated using a modified version of the dual luciferase TA activity assay (TA assay) that yielded increased sensitivity and sample throughput. Variants were classified according to American College of Medical Genetics and Genomics (ACMG) criteria using TA assay results and available data. In combining our TA-assay results and available data, in accordance with the ACMG guidelines for variant classification, we proposed the following variant classifications: c.5100A>G, c.5326C>T, c.5348T>C and c.5477A>T as likely benign (class 2) variants. c.5075A>C, c.5116G>A and c.5513T>G were likely pathogenic (class 4), whereas c.5096G>A likely represents a likely pathogenic variant with moderate penetrance. Variants c.5123C>T, c.5125G>A, c.5131A>C and c.5504G>A remained classified as VUSs (class 3).

Conclusions

The modified TA assay provides efficient risk assessment of rare missense variants found in the BRCA1 BRCT-domains. We also report that increased post-transfection incubation time yielded a significant increase in TA assay sensitivity.

     

Development and Validation of a Next-Generation Sequencing Assay for BRCA1 and BRCA2 Variants for the Clinical Laboratory

The objective of this study was to design and validate a next-generation sequencing assay (NGS) to detect BRCA1 and BRCA2 mutations. We developed an assay using random shearing of genomic DNA followed by RNA bait tile hybridization and NGS sequencing on both the Illumina MiSeq and Ion Personal Gene Machine (PGM). We determined that the MiSeq Reporter software supplied with the instrument could not detect deletions greater than 9 base pairs. Therefore, we developed an alternative alignment and variant calling software, Quest Sequencing Analysis Pipeline (QSAP), that was capable of detecting large deletions and insertions. In validation studies, we used DNA from 27 stem cell lines, all with known deleterious BRCA1 or BRCA2 mutations, and DNA from 67 consented control individuals who had a total of 352 benign variants. Both the MiSeq/QSAP combination and PGM/Torrent Suite combination had 100% sensitivity for the 379 known variants in the validation series. However, the PGM/Torrent Suite combination had a lower intra- and inter-assay precision of 96.2% and 96.7%, respectively when compared to the MiSeq/QSAP combination of 100% and 99.4%, respectively. All PGM/Torrent Suite inconsistencies were false-positive variant assignments. We began commercial testing using both platforms and in the first 521 clinical samples MiSeq/QSAP had 100% sensitivity for BRCA1/2 variants, including a 64-bp deletion and a 10-bp insertion not identified by PGM/Torrent Suite, which also suffered from a high false-positive rate. Neither the MiSeq nor PGM platform with their supplied alignment and variant calling software are appropriate for a clinical laboratory BRCA sequencing test. We have developed an NGS BRCA1/2 sequencing assay, MiSeq/QSAP, with 100% analytic sensitivity and specificity in the validation set consisting of 379 variants. The MiSeq/QSAP combination has sufficient performance for use in a clinical laboratory.     

A new bioinformatics tool to help assess the significance of <Emphasis Type="Italic">BRCA1</Emphasis> variants

Background

Germline pathogenic variants in the breast cancer type 1 susceptibility gene BRCA1 are associated with a 60% lifetime risk for breast and ovarian cancer. This overall risk estimate is for all BRCA1 variants; obviously, not all variants confer the same risk of developing a disease. In cancer patients, loss of BRCA1 function in tumor tissue has been associated with an increased sensitivity to platinum agents and to poly-(ADP-ribose) polymerase (PARP) inhibitors. For clinical management of both at-risk individuals and cancer patients, it would be important that each identified genetic variant be associated with clinical significance. Unfortunately for the vast majority of variants, the clinical impact is unknown. The availability of results from studies assessing the impact of variants on protein function may provide insight of crucial importance.

Results and conclusion

We have collected, curated, and structured the molecular and cellular phenotypic impact of 3654 distinct BRCA1 variants. The data was modeled in triple format, using the variant as a subject, the studied function as the object, and a predicate describing the relation between the two. Each annotation is supported by a fully traceable evidence. The data was captured using standard ontologies to ensure consistency, and enhance searchability and interoperability. We have assessed the extent to which functional defects at the molecular and cellular levels correlate with the clinical interpretation of variants by ClinVar submitters. Approximately 30% of the ClinVar BRCA1 missense variants have some molecular or cellular assay available in the literature. Pathogenic variants (as assigned by ClinVar) have at least some significant functional defect in 94% of testable cases. For benign variants, 77% of ClinVar benign variants, for which neXtProt Cancer variant portal has data, shows either no or mild experimental functional defects. While this does not provide evidence for clinical interpretation of variants, it may provide some guidance for variants of unknown significance, in the absence of more reliable data.The neXtProt Cancer variant portal (https://www.nextprot.org/portals/breast-cancer) contains over 6300 observations at the molecular and/or cellular level for BRCA1 variants.

     

MAT2A Mutations Predispose Individuals to Thoracic Aortic Aneurysms

Up to 20% of individuals who have thoracic aortic aneurysms or acute aortic dissections but who do not have syndromic features have a family history of thoracic aortic disease. Significant genetic heterogeneity is established for this familial condition. Whole-genome linkage analysis and exome sequencing of distant relatives from a large family with autosomal-dominant inheritance of thoracic aortic aneurysms variably associated with the bicuspid aortic valve was used for identification of additional genes predisposing individuals to this condition. A rare variant, c.1031A>C (p.Glu344Ala), was identified in MAT2A, which encodes methionine adenosyltransferase II alpha (MAT IIα). This variant segregated with disease in the family, and Sanger sequencing of DNA from affected probands from unrelated families with thoracic aortic disease identified another MAT2A rare variant, c.1067G>A (p.Arg356His). Evidence that these variants predispose individuals to thoracic aortic aneurysms and dissections includes the following: there is a paucity of rare variants in MAT2A in the population; amino acids Glu344 and Arg356 are conserved from humans to zebrafish; and substitutions of these amino acids in MAT Iα are found in individuals with hypermethioninemia. Structural analysis suggested that p.Glu344Ala and p.Arg356His disrupt MAT IIα enzyme function. Knockdown of mat2aa in zebrafish via morpholino oligomers disrupted cardiovascular development. Co-transfected wild-type human MAT2A mRNA rescued defects of zebrafish cardiovascular development at significantly higher levels than mRNA edited to express either the Glu344 or Arg356 mutants, providing further evidence that the p.Glu344Ala and p.Arg356His substitutions impair MAT IIα function. The data presented here support the conclusion that rare genetic variants in MAT2A predispose individuals to thoracic aortic disease.     

Three novel BRCA1/BRCA2 mutations in breast/ovarian cancer families in Croatia

BRCA1 and BRCA2 genes from 167 candidates (145 families) were scanned for mutations. We identified 14 pathogenic point mutations in 17 candidates, 9 in BRCA1 and 5 in BRCA2. Of those, 11 have been previously described and 3 were novel (c.5335C>T in BRCA1 and c.4139_4140dupTT and c.8175G>A in BRCA2). No large deletions or duplications involving BRCA1 and BRCA2 genes were identified. No founder mutations were detected for the Croatian population. Croatia shares most of the mutations with neighboring Slovenia and also with Germany, Austria and Poland. Two common sequence variants in BRCA1, c.2077G>A and c.4956G>A, were found more frequently in mutation carriers compared to healthy controls. No difference in BRCA2 variants was detected between the groups. Haplotype inference showed no difference in haplotype distributions between deleterious mutation carriers and non-carriers in neither BRCA1 nor BRCA2. In silico analyses identified one BRCA1 sequence variant (c.4039A>G) and two BRCA2 variants (c.5986G>A and c.6884G>C) as harmful with high probability, and inconclusive results were obtained for our novel BRCA2 variant c.3864_3866delTAA. Combination of QMPSF and HRMA methods provides high detection rate and complete coverage of BRCA1/2 genes. Benefit of BRCA1/2 mutation testing is clear, since we detected mutations in young unaffected women, who will be closely monitored for breast and ovarian cancer.     

Integrated evaluation of DNA sequence variants of unknown clinical significance: application to BRCA1 and BRCA2

Many sequence variants in predisposition genes are of uncertain clinical significance, and classification of these variants into high- or low-risk categories is an important problem in clinical genetics. Classification of such variants can be performed by direct epidemiological observations, including cosegregation with disease in families and degree of family history of the disease, or by indirect measures, including amino acid conservation, severity of amino acid change, and evidence from functional assays. In this study, we have developed an approach to the synthesis of such evidence in a multifactorial likelihood-ratio model. We applied this model to the analysis of three unclassified variants in BRCA1 and three in BRCA2. The evidence strongly suggests that two variants (C1787S in BRCA1 and D2723H in BRCA2) are deleterious, three (R841W in BRCA1 and Y42C and P655R in BRCA2) are neutral, and one (R1699Q in BRCA1) remains of uncertain significance. These results provide a demonstration of the utility of the model.     

Progress of a Comprehensive Familial Cancer Genetic Counseling Program in the Era of BRCA1 and BRCA2

BRCA1 and BRCA2 mutation carriers have an increased risk of developing breast and/or ovarian cancer. Technical advances in genetic testing have increased the need for genetic counseling services; therefore, we have developed a counseling program for these individuals. The purpose of this study is to characterize this population, assess level of interest in genetic testing, and evaluate our program over a 5-year period. Our Familial Cancer Genetic Counseling Program was established in November, 1994. Information was collected prospectively, with comprehensive evaluation including complete pedigree, risk assessment, and counseling by a genetic counselor, geneticist, and oncologist. Data were collected on risk level, and subsequent recommendations for screening and/or genetic testing. There were 824 contacts recorded from November, 1994, through August, 1999. To date, 162 families have undergone comprehensive genetic evaluation and counseling. 90 (56%) were seen for a concerning family history and 72 (44%) were seen due to a personal history of malignancy. The majority of families had a significant level of risk with 126 (78%) families having two and 70 (43%) families having three affected first-degree relatives. Of the 162 families who received full counseling, 125 (77%) met criteria to recommend BRCA1/BRCA2 genetic testing. At this time, 30 of the 162 (18%) have had genetic testing. A brief phone contact or clinic visit is useful to screen individuals so that counseling can be directed toward truly high-risk families. In our program, the majority of families counseled were eligible for BRCA1/BRCA2 testing, but only 18% have elected to proceed at this time.     

BRCA1 mutations in African Americans

The breast cancer predisposing gene, BRCA1, was analyzed for germline mutations in 45 African American families at high-risk for hereditary breast cancer. Patients were considered high-risk if they had a family history of the disease, early onset breast cancer, bilateral breast cancer, or breast and ovarian cancer. The entire BRCA1 coding and flanking intron regions have been examined by single stranded conformation polymorphism analysis followed by sequencing of variant bands. Eleven different BRCA1 germline mutations/variations were identified in 7 patients from the 45 high-risk families. Two pathogenic, protein-truncating mutations were detected in exon 11. A ten base pair tandem duplication, 943ins10, was present in a woman with breast and ovarian cancer whose first-degree relatives had prostate cancer. A four base pair deletion, 3450del4, was detected in a breast cancer patient with five cases of breast cancer in the family; two of the proband's sisters with breast cancer also carried the same mutation. Four amino acid substitutions (Lys1183Arg, Leu1564Pro, Gln1785His, and Glu1794Asp) and four nucleotide substitutions in intron 22 (IVS22+78 C/A, IVS22+67 T/C, IVS22+8 T/A and IVS22+7 T/C) were observed in patients and not in control subjects. One early onset breast cancer patient carried five distinct BRCA1 variations, two amino acid substitutions and three substitutions in intron 22. An amino acid substitution in exon 11, Ser1140Gly, was identified in 3 different unrelated patients and in 6 of 92 control samples. The latter probably represents a benign polymorphism. Electronic Publication     

Evidence for a pathogenic role of BRCA1 L1705P and W1837X germ-line mutations

BRCA1 L1705P (c.5114T>C) has been classified in the NCBI SNP database as the variant with uncertain significance and is absent in major BRCA1 databases. BRCA1 W1837X (c.5511G>A) results in a loss of only last 27 residues of BRCA1 protein, thus its pathogenic role still requires a confirmation. This report describes two breast cancer (BC) patients carrying BRCA1 L1705P and W1837X germ-line mutations, respectively. Significant evidence for BC-predisposing impact of the mentioned mutations have been obtained: (1) both index cases presented with the triple-negative receptor status of BC disease; (2) complete segregation with BRCA1-related cancers was observed in the families of these patients; (3) somatic loss of the remaining (wild-type) BRCA1 allele was detected in tumor tissues of the affected women. The results of this study have to be taken into account while providing genetic counseling to cancer patients and while considering the use of BRCA1-specific therapeutic compounds for BC treatment.     

Detection of New Point Mutations of BRCA1 and BRCA2 in Breast Cancer Patients

This study included 20 selected female patients with breast cancer, 30 of their female relatives (sisters and daughters), and 10 healthy females as a control group. Genomic DNA was extracted from peripheral blood lymphocytes of all the subjects, and the polymerase chain reaction was carried out using specific primers for BRCA1 (exons 2 and 8) and BRCA2 (exons 9, 11, and 21). The mutations were detected using a single-strand conformation polymorphism assay and heteroduplex analysis. Finally, the sample variants and their controls were sequenced. Mutations were detected in 44% of the study population, with 18% found in the BRCA1 gene and 26% attributed to BRCA2. Five sequence variants were identified, including two frameshift mutations, one nonsense mutation, and two missense mutations. Therefore, we conclude that germline mutations in two major genes, BRCA1 and BRCA2, may have an important influence on the predisposition and development of familial breast cancer.     

Analysis of PALB2 Gene in BRCA1/BRCA2 Negative Spanish Hereditary Breast/Ovarian Cancer Families with Pancreatic Cancer Cases

Background

The PALB2 gene, also known as FANCN, forms a bond and co-localizes with BRCA2 in DNA repair. Germline mutations in PALB2 have been identified in approximately 1% of familial breast cancer and 3–4% of familial pancreatic cancer. The goal of this study was to determine the prevalence of PALB2 mutations in a population of BRCA1/BRCA2 negative breast cancer patients selected from either a personal or family history of pancreatic cancer.

Methods

132 non-BRCA1/BRCA2 breast/ovarian cancer families with at least one pancreatic cancer case were included in the study. PALB2 mutational analysis was performed by direct sequencing of all coding exons and intron/exon boundaries, as well as multiplex ligation-dependent probe amplification.

Results

Two PALB2 truncating mutations, the c.1653T>A (p.Tyr551Stop) previously reported, and c.3362del (p.Gly1121ValfsX3) which is a novel frameshift mutation, were identified. Moreover, several PALB2 variants were detected; some of them were predicted as pathological by bioinformatic analysis. Considering truncating mutations, the prevalence rate of our population of BRCA1/2-negative breast cancer patients with pancreatic cancer is 1.5%.

Conclusions

The prevalence rate of PALB2 mutations in non-BRCA1/BRCA2 breast/ovarian cancer families, selected from either a personal or family pancreatic cancer history, is similar to that previously described for unselected breast/ovarian cancer families. Future research directed towards identifying other gene(s) involved in the development of breast/pancreatic cancer families is required.     

Targeted Sequencing of the Mitochondrial Genome of Women at High Risk of Breast Cancer without Detectable Mutations in BRCA1/2

Breast Cancer is a complex multifactorial disease for which high-penetrance mutations have been identified. Approaches used to date have identified genomic features explaining about 50% of breast cancer heritability. A number of low- to medium penetrance alleles (per-allele odds ratio < 1.5 and 4.0, respectively) have been identified, suggesting that the remaining heritability is likely to be explained by the cumulative effect of such alleles and/or by rare high-penetrance alleles. Relatively few studies have specifically explored the mitochondrial genome for variants potentially implicated in breast cancer risk. For these reasons, we propose an exploration of the variability of the mitochondrial genome in individuals diagnosed with breast cancer, having a positive breast cancer family history but testing negative for BRCA1/2 pathogenic mutations. We sequenced the mitochondrial genome of 436 index breast cancer cases from the GENESIS study. As expected, no pathogenic genomic pattern common to the 436 women included in our study was observed. The mitochondrial genes MT-ATP6 and MT-CYB were observed to carry the highest number of variants in the study. The proteins encoded by these genes are involved in the structure of the mitochondrial respiration chain, and variants in these genes may impact reactive oxygen species production contributing to carcinogenesis. More functional and epidemiological studies are needed to further investigate to what extent variants identified may influence familial breast cancer risk.