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.     

Common breast cancer-predisposition alleles are associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers

Germline mutations in BRCA1 and BRCA2 confer high risks of breast cancer. However, evidence suggests that these risks are modified by other genetic or environmental factors that cluster in families. A recent genome-wide association study has shown that common alleles at single nucleotide polymorphisms (SNPs) in FGFR2 (rs2981582), TNRC9 (rs3803662), and MAP3K1 (rs889312) are associated with increased breast cancer risks in the general population. To investigate whether these loci are also associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers, we genotyped these SNPs in a sample of 10,358 mutation carriers from 23 studies. The minor alleles of SNP rs2981582 and rs889312 were each associated with increased breast cancer risk in BRCA2 mutation carriers (per-allele hazard ratio [HR] = 1.32, 95% CI: 1.20-1.45, p(trend) = 1.7 x 10(-8) and HR = 1.12, 95% CI: 1.02-1.24, p(trend) = 0.02) but not in BRCA1 carriers. rs3803662 was associated with increased breast cancer risk in both BRCA1 and BRCA2 mutation carriers (per-allele HR = 1.13, 95% CI: 1.06-1.20, p(trend) = 5 x 10(-5) in BRCA1 and BRCA2 combined). These loci appear to interact multiplicatively on breast cancer risk in BRCA2 mutation carriers. The differences in the effects of the FGFR2 and MAP3K1 SNPs between BRCA1 and BRCA2 carriers point to differences in the biology of BRCA1 and BRCA2 breast cancer tumors and confirm the distinct nature of breast cancer in BRCA1 mutation carriers.     

Single nucleotide polymorphisms in clinical genetic testing: the characterization of the clinical significance of genetic variants and their application in clinical research for BRCA1

Clinical genetic testing is increasingly employed in the medical management of cancer patients. These tests support a variety of clinical decisions by providing results that indicate risk for future disease, confirmation of diagnoses, and more recently, therapeutic selection and prognosis. Most genetic variation detected during clinical testing involves single nucleotide polymorphisms (SNPs). Continued advances in the technologies of genetic analyses make these tests increasingly sensitive, cost-effective and timely, which contribute to their increased utilization. Conversely, it has proven difficult to characterize the clinical significance of genetic variants that do not obviously truncate the open reading frames of genes. These genetic variants of uncertain clinical significance diminish the value of genetic test results. This article highlights a variety of approaches that have emerged from research in diverse disciplines to solve the problem, including the application of information about common SNPs in multiple methods to better characterize clinically uncertain variants. Hereditary breast/ovarian cancer, and in particular BRCA1, provides a framework for this discussion. BRCA1 is particularly interesting in this respect since clinical genetic testing by direct DNA sequencing for over 50,000 patients in North America has revealed approximately 1500 genetic variants to date. This large data set combined with the clinical significance of BRCA1 have resulted in research groups selecting BRCA1 as a preferred gene to evaluate novel methods in this field. Finally, the lessons learned through work with BRCA1 are highly applicable to many other genes associated with cancer risk.     

The defining DNA methylation signature of Kabuki syndrome enables functional assessment of genetic variants of unknown clinical significance

Kabuki syndrome (KS) is caused by mutations in KMT2D, which is a histone methyltransferase involved in methylation of H3K4, a histone marker associated with DNA methylation. Analysis of >450,000 CpGs in 24 KS patients with pathogenic mutations in KMT2D and 216 controls, identified 24 genomic regions, along with 1,504 CpG sites with significant DNA methylation changes including a number of Hox genes and the MYO1F gene. Using the most differentiating and significant probes and regions we developed a “methylation variant pathogenicity (MVP) score,” which enables 100% sensitive and specific identification of individuals with KS, which was confirmed using multiple public and internal patient DNA methylation databases. We also demonstrated the ability of the MVP score to accurately reclassify variants of unknown significance in subjects with apparent clinical features of KS, enabling its potential use in molecular diagnostics. These findings provide novel insights into the molecular etiology of KS and illustrate that DNA methylation patterns can be interpreted as ‘epigenetic echoes’ in certain clinical disorders.     

Common genetic variants and modification of penetrance of BRCA2-associated breast cancer

The considerable uncertainty regarding cancer risks associated with inherited mutations of BRCA2 is due to unknown factors. To investigate whether common genetic variants modify penetrance for BRCA2 mutation carriers, we undertook a two-staged genome-wide association study in BRCA2 mutation carriers. In stage 1 using the Affymetrix 6.0 platform, 592,163 filtered SNPs genotyped were available on 899 young (<40 years) affected and 804 unaffected carriers of European ancestry. Associations were evaluated using a survival-based score test adjusted for familial correlations and stratified by country of the study and BRCA2*6174delT mutation status. The genomic inflation factor (λ) was 1.011. The stage 1 association analysis revealed multiple variants associated with breast cancer risk: 3 SNPs had p-values<10(-5) and 39 SNPs had p-values<10(-4). These variants included several previously associated with sporadic breast cancer risk and two novel loci on chromosome 20 (rs311499) and chromosome 10 (rs16917302). The chromosome 10 locus was in ZNF365, which contains another variant that has recently been associated with breast cancer in an independent study of unselected cases. In stage 2, the top 85 loci from stage 1 were genotyped in 1,264 cases and 1,222 controls. Hazard ratios (HR) and 95% confidence intervals (CI) for stage 1 and 2 were combined and estimated using a retrospective likelihood approach, stratified by country of residence and the most common mutation, BRCA2*6174delT. The combined per allele HR of the minor allele for the novel loci rs16917302 was 0.75 (95% CI 0.66-0.86, ) and for rs311499 was 0.72 (95% CI 0.61-0.85, ). FGFR2 rs2981575 had the strongest association with breast cancer risk (per allele HR = 1.28, 95% CI 1.18-1.39, ). These results indicate that SNPs that modify BRCA2 penetrance identified by an agnostic approach thus far are limited to variants that also modify risk of sporadic BRCA2 wild-type breast cancer.     

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.     

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.

     

Determining carrier probabilities for breast cancer-susceptibility genes BRCA1 and BRCA2.

Breast cancer-susceptibility genes BRCA1 and BRCA2 have recently been identified on the human genome. Women who carry a mutation of one of these genes have a greatly increased chance of developing breast and ovarian cancer, and they usually develop the disease at a much younger age, compared with normal individuals. Women can be tested to see whether they are carriers. A woman who undergoes genetic counseling before testing can be told the probabilities that she is a carrier, given her family history. In this paper we develop a model for evaluating the probabilities that a woman is a carrier of a mutation of BRCA1 and BRCA2, on the basis of her family history of breast and ovarian cancer in first- and second-degree relatives. Of special importance are the relationships of the family members with cancer, the ages at onset of the diseases, and the ages of family members who do not have the diseases. This information can be elicited during genetic counseling and prior to genetic testing. The carrier probabilities are obtained from Bayes's rule, by use of family history as the evidence and by use of the mutation prevalences as the prior distribution. In addressing an individual's carrier probabilities, we incorporate uncertainty about some of the key inputs of the model, such as the age-specific incidence of diseases and the overall prevalence of mutations. There is some evidence that other, undiscovered genes may be important in explaining familial breast cancer. Users of the current version of the model should be aware of this limitation. The methodology that we describe can be extended to more than two genes, should data become available about other genes.     

Characterization of common BRCA1 and BRCA2 variants

Many missense variants identified in BRCA1 and BRCA2, two genes responsible for the majority of hereditary breast and ovarian cancer, are of unclear clinical significance. Characterizing the significance of such variants is important for medical management of patients in whom they are identified. The aim of this study was to characterize eight of the most common reported missense mutations in BRCA1 and BRCA2 occurring in patients tested for hereditary risk of breast and ovarian cancers. The prevalence of each variant in a control population, co-segregation of the variant with cancer within families, location of the variant within the gene, the nature of the amino acid substitution and conservation of the wild-type amino acid among species were considered. In a control population, the BRCA1 variants M1652I, R1347G, and S1512I, were each observed at a frequency of 4.08%, 2.04%, and 2.04%, respectively, and the BRCA2 variants A2951T, V2728I, and D1420Y, were seen at 1.02%, 0.68%, and 0.34%, respectively. Although the BRCA2 variants T598A and R2034C were not seen in this group of controls, other clinical and published observations indicate that these variants are not deleterious. Based on epidemiological and biological criteria, we therefore conclude that the BRCA1 missense mutations R1347G, S1512I and M1652I, and the BRCA2 missense mutations T598A, D1420Y, R2034C, V2728I, and A2951T, are not deleterious mutations.     

Contribution of epigenetic alteration of BRCA1 and BRCA2 genes in breast carcinomas in Tunisian patients

Objective: The aim of this study was to evaluate the contribution of the BRCA1 and BRCA2 promoter methylation in the pathogenesis of sporadic breast cancer in Tunisian patients. Methods: Breast carcinoma tissues (n = 117) and available paired normal breast tissues (n = 65) from Tunisian women who had no family history were investigated for the methylation status of BRCA1 and BRCA2 promoters using methylation-specific PCR. Breast specimens from women without carcinoma (16 fibroadenomas and 5 mastopathies) were used as control. Results: Hypermethylation of BRCA1 and BRCA2 promoters was detected respectively in 60.7% and 69.2% of the carcinoma tissues, and in only 7.7% and 4.6% of the paired normal breast tissues. None of the fibroadenomas and mastopathies showed hypermethylation. Correlations were found between BRCA1 and BRCA2 hypermethylation and decrease in their mRNA expression (p = 0.02 and p = 0.009, respectively). Moreover, BRCA1 methylation correlates with patients age (p = 0.01) and triple negative (ER?, PR?, HER2?) tumors (p = 0.01). Patients with methylated BRCA1 and/or BRCA2 had a significant prolonged survivals compared to those with unmethylated tumors (p = 0.002). Conclusion: Our results suggest an important role of BRCA1 and BRCA2 promoter methylation in breast cancer development in the Tunisian population.     

BRCA1 and BRCA2 in hereditary breast cancer

The hereditary breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, have established roles in genome integrity maintenance and in the control of homologous recombination. Recent work has produced valuable insights into the mechanisms of action of the gene products. This review summarizes some of these advances, and attempts to place them in the context of known functions of the genes.     

Molecular characterization of germline mutations in the BRCA1 and BRCA2 genes from breast cancer families in Taiwan

A total of 18 families with multiple cases of breast cancer were identified from southern Taiwan, and 5 of these families were found to carry cancer-associated germline mutations in the BRCA1 and BRCA2 genes. One novel cryptic splicing mutation of the BRCA1 gene, found in two unrelated families, was shown to be a deletion of 10 bp near the branch site in intron 7. This mutation causes an insertion of 59 nucleotides derived from intron 7 and results in a frameshift, leading to premature translational termination of BRCA1 mRNA in exon 8. Deletions of 2670delC, 3073delT and 6696-7delTC in the BRCA2 gene were found in three other breast cancer families. All three deletions are predicted to generate frameshifts and to result in the premature termination of BRCA2 protein translation. Several genetic polymorphisms in both BRCA1 and BRCA2 genes were also detected in this investigation. Received: 28 September 1998 / Accepted: 20 November 1998     

Identification of sequence variants in genetic disease-causing genes using targeted next-generation sequencing

Background

Identification of gene variants plays an important role in research on and diagnosis of genetic diseases. A combination of enrichment of targeted genes and next-generation sequencing (targeted DNA-HiSeq) results in both high efficiency and low cost for targeted sequencing of genes of interest.

Methodology/Principal Findings

To identify mutations associated with genetic diseases, we designed an array-based gene chip to capture all of the exons of 193 genes involved in 103 genetic diseases. To evaluate this technology, we selected 7 samples from seven patients with six different genetic diseases resulting from six disease-causing genes and 100 samples from normal human adults as controls. The data obtained showed that on average, 99.14% of 3,382 exons with more than 30-fold coverage were successfully detected using Targeted DNA-HiSeq technology, and we found six known variants in four disease-causing genes and two novel mutations in two other disease-causing genes (the STS gene for XLI and the FBN1 gene for MFS) as well as one exon deletion mutation in the DMD gene. These results were confirmed in their entirety using either the Sanger sequencing method or real-time PCR.

Conclusions/Significance

Targeted DNA-HiSeq combines next-generation sequencing with the capture of sequences from a relevant subset of high-interest genes. This method was tested by capturing sequences from a DNA library through hybridization to oligonucleotide probes specific for genetic disorder-related genes and was found to show high selectivity, improve the detection of mutations, enabling the discovery of novel variants, and provide additional indel data. Thus, targeted DNA-HiSeq can be used to analyze the gene variant profiles of monogenic diseases with high sensitivity, fidelity, throughput and speed.     

The founder mutations in the BRCA1, BRCA2, and ATM genes in Moroccan Jewish women with breast cancer

To gain insight into the molecular mechanisms underlying the inherited predisposition to breast cancer in non-Ashkenazi Jews, we genotyped 54 Jewish Moroccan women with breast cancer, unselected for family history of cancer, for the predominant Jewish mutations in BRCA1, BRCA2, and ATM. One patient (2%) was found to have the 185de1AG BRCA1 mutation, none was a carrier of the 6174delT BRCA2 mutation, and 2/54 (4%) were heterozygous for the ATM mutation. These rates were not significantly different from the rates in the general non-Ashkenazi population. These preliminary data may indicate that the predominant Jewish mutations in BRCA1, BRCA2, and ATM genes contribute little, if any, to breast cancer predisposition and risk among Moroccan Jews.