Bryony A. Thompson

Correlation of tumour BRAF mutations and MLH1 methylation with germline mismatch repair (MMR) gene mutation status: a literature review assessing utility of tumour features for MMR variant classification

Colorectal cancer (CRC) that demonstrates microsatellite instability (MSI) is caused by either germline mismatch repair (MMR) gene mutations, or ‘sporadic’ somatic tumour MLH1 promoter methylation. MLH1 promoter methylation is reportedly correlated with tumour BRAF V600E mutation status. No systematic review has been undertaken to assess the value of BRAF V600E mutation and MLH1 promoter methylation tumour markers as negative predictors of germline MMR mutation status. A literature review of CRC cohorts tested for MMR mutations, and tumour BRAF V600E mutation and/or MLH1 promoter methylation was conducted using PubMed. Studies were assessed for tumour features, stratified by tumour MMR status based on immunohistochemistry or MSI where possible. Pooled frequencies and 95% CIs were calculated using a random effects model. BRAF V600E results for 4562 tumours from 35 studies, and MLH1 promoter methylation results for 2975 tumours from 43 studies, were assessed. In 550 MMR mutation carriers, the BRAF V600E mutation frequency was 1.40% (95% CI 0.06% to 3%). In MMR mutation-negative cases, the BRAF V600E mutation frequency was 5.00% (95% CI 4% to 7%) in 1623 microsatellite stable (MSS) cases and 63.50% (95% CI 47% to 79%) in 332 cases demonstrating MLH1 methylation or MLH1 expression loss. MLH1 promoter methylation of the ‘A region’ was reported more frequently than the ‘C region’ in MSS CRCs (17% vs 0.06%, p<0.0001) and in MLH1 mutation carriers (42% vs 6%, p<0.0001), but not in MMR mutation-negative MSI-H CRCs (40% vs 47%, p=0.12). Methylation of the ‘C region’ was a predictor of MMR mutation-negative status in MSI-H CRC cases (47% vs 6% in MLH1 mutation carriers, p<0.0001). This review demonstrates that tumour BRAF V600E mutation, and MLH1 promoter ‘C region’ methylation specifically, are strong predictors of negative MMR mutation status. It is important to incorporate these features in multifactorial models aimed at predicting MMR mutation status.

Tumor Mismatch Repair Immunohistochemistry and DNA MLH1 Methylation Testing of Patients With Endometrial Cancer Diagnosed at Age Younger Than 60 Years Optimizes Triage for Population-Level Germline Mismatch Repair Gene Mutation Testing

PURPOSE Clinicopathologic data from a population-based endometrial cancer cohort, unselected for age or family history, were analyzed to determine the optimal scheme for identification of patients with germline mismatch repair (MMR) gene mutations. PATIENTS AND METHODS Endometrial cancers from 702 patients recruited into the Australian National Endometrial Cancer Study (ANECS) were tested for MMR protein expression using immunohistochemistry (IHC) and for MLH1 gene promoter methylation in MLH1-deficient cases. MMR mutation testing was performed on germline DNA of patients with MMR-protein deficient tumors. Prediction of germline mutation status was compared for combinations of tumor characteristics, age at diagnosis, and various clinical criteria (Amsterdam, Bethesda, Society of Gynecologic Oncology, ANECS). RESULTS Tumor MMR-protein deficiency was detected in 170 (24%) of 702 cases. Germline testing of 158 MMR-deficient cases identified 22 truncating mutations (3% of all cases) and four unclassified variants. Tumor MLH1 methylation was detected in 99 (89%) of 111 cases demonstrating MLH1/PMS2 IHC loss; all were germline MLH1 mutation negative. A combination of MMR IHC plus MLH1 methylation testing in women younger than 60 years of age at diagnosis provided the highest positive predictive value for the identification of mutation carriers at 46% versus ≤ 41% for any other criteria considered. CONCLUSION Population-level identification of patients with MMR mutation-positive endometrial cancer is optimized by stepwise testing for tumor MMR IHC loss in patients younger than 60 years, tumor MLH1 methylation in individuals with MLH1 IHC loss, and germline mutations in patients exhibiting loss of MSH6, MSH2, or PMS2 or loss of MLH1/PMS2 with absence of MLH1 methylation.

The InSiGHT database: utilizing 100 years of insights into Lynch Syndrome

This article provides a historical overview of the online database (www.insight-group.org/mutations) maintained by the International Society for Gastrointestinal Hereditary Tumours. The focus is on the mismatch repair genes which are mutated in Lynch Syndrome. APC, MUTYH and other genes are also an important part of the database, but are not covered here. Over time, as the understanding of the genetics of Lynch Syndrome increased, databases were created to centralise and share the variants which were being detected in ever greater numbers. These databases were eventually merged into the InSiGHT database, a comprehensive repository of gene variant and disease phenotype information, serving as a starting point for important endeavours including variant interpretation, research, diagnostics and enhanced global collection. Pivotal to its success has been the collaborative spirit in which it has been developed, its association with the Human Variome Project, the appointment of a full time curator and its governance stemming from the well established organizational structure of InSiGHT.

A Multifactorial Likelihood Model for MMR Gene Variant Classification Incorporating Probabilities Based on Sequence Bioinformatics and Tumor Characteristics: A Report from the Colon Cancer Family Registry

Mismatch repair (MMR) gene sequence variants of uncertain clinical significance are often identified in suspected Lynch syndrome families, and this constitutes a challenge for both researchers and clinicians. Multifactorial likelihood model approaches provide a quantitative measure of MMR variant pathogenicity, but first require input of likelihood ratios (LRs) for different MMR variation‐associated characteristics from appropriate, well‐characterized reference datasets. Microsatellite instability (MSI) and somatic BRAF tumor data for unselected colorectal cancer probands of known pathogenic variant status were used to derive LRs for tumor characteristics using the Colon Cancer Family Registry (CFR) resource. These tumor LRs were combined with variant segregation within families, and estimates of prior probability of pathogenicity based on sequence conservation and position, to analyze 44 unclassified variants identified initially in Australasian Colon CFR families. In addition, in vitro splicing analyses were conducted on the subset of variants based on bioinformatic splicing predictions. The LR in favor of pathogenicity was estimated to be ∼12‐fold for a colorectal tumor with a BRAF mutation‐negative MSI‐H phenotype. For 31 of the 44 variants, the posterior probabilities of pathogenicity were such that altered clinical management would be indicated. Our findings provide a working multifactorial likelihood model for classification that carefully considers mode of ascertainment for gene testing.

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.

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.

Detection of Splicing Aberrations Caused by BRCA1 and BRCA2 Sequence Variants Encoding Missense Substitutions: Implications for Prediction of Pathogenicity

Missense substitutions in high‐risk cancer susceptibility genes create clinical uncertainty in the genetic counseling process. Multifactorial likelihood classification approaches and in vitro assays are useful for the classification of exonic sequence variants in BRCA1 and BRCA2, but these currently rely on the assumption that changes in protein function are the major biological mechanism of pathogenicity. This study investigates the potentially pathogenic role of aberrant splicing for exonic variants predicted to encode missense substitutions using patient‐derived RNA. No splicing aberrations were identified for BRCA1c.5054C>T and BRCA2c.7336A>G, c.8839G>A, and c.9154C>T. However, RT‐PCR analysis identified a major splicing aberration for BRCA1c.4868C>G(p.Ala1623Gly), a variant encoding a missense substitution considered likely to be neutral. Splicing aberrations were also observed for BRCA2c.7988A>T(p.Glu2663Val) and c.8168A>G(p.Asp2723Gly), but both variant and wildtype alleles were shown to be present in full‐length mRNA transcripts, suggesting that variant protein may be translated. BRCA2 protein function assays indicated that BRCA2p.Glu2663Val, p.Asp2723Gly and p.Arg3052Trp missense proteins have abrogated function consistent with pathogenicity. Multifactorial likelihood analysis provided evidence for pathogenicity for BRCA1 c.5054C>T(p.Thr1685Ile) and BRCA2c.7988A>T(p.Glu2663Val), c.8168A>G(p.Asp2723Gly) and c.9154C>T(p.Arg3052Trp), supporting experimentally derived evidence. These findings highlight the need for improved bioinformatic prediction of splicing aberrations and to refine multifactorial likelihood models used to assess clinical significance.

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.

Evaluation of CADD Scores in Curated Mismatch Repair Gene Variants Yields a Model for Clinical Validation and Prioritization.

Next‐generation sequencing in clinical diagnostics is providing valuable genomic variant data, which can be used to support healthcare decisions. In silico tools to predict pathogenicity are crucial to assess such variants and we have evaluated a new tool, Combined Annotation Dependent Depletion (CADD), and its classification of gene variants in Lynch syndrome by using a set of 2,210 DNA mismatch repair gene variants. These had already been classified by experts from InSiGHTs Variant Interpretation Committee. Overall, we found CADD scores do predict pathogenicity (Spearmans ρ = 0.595, P < 0.001). However, we discovered 31 major discrepancies between the InSiGHT classification and the CADD scores; these were explained in favor of the expert classification using population allele frequencies, cosegregation analyses, disease association studies, or a second‐tier test. Of 751 variants that could not be clinically classified by InSiGHT, CADD indicated that 47 variants were worth further study to confirm their putative pathogenicity. We demonstrate CADD is valuable in prioritizing variants in clinically relevant genes for further assessment by expert classification teams.

A review of mismatch repair gene transcripts: issues for interpretation of mRNA splicing assays.

Many mismatch repair (MMR) gene disease‐causing mutations identified in cancer patients result in aberrant messenger RNA (mRNA) splicing. However, mRNA assay interpretation can be complicated by the existence of naturally occurring alternative mRNA transcripts, most of which have not been formally described or fully characterized. Here, we provide a comprehensive catalogue of all MMR transcripts described to date, and a review of MMR nucleotide variants associated with an apparent upregulation of alternatively spliced transcripts. This work sets reference starting points for designing and interpreting MMR RNA analyses. Our database and literature searches retrieved 30 MLH1, 22 MSH2, 4 MSH6 and 9 PMS2 alternative transcripts, many predicted to introduce premature termination codons. Furthermore, we collected information on 66 MLH1, 24 MSH2 and 6 PMS2 nucleotide variants reported to be associated with altered expression of at least one of these alternative transcripts, and in many instances reported as splicing mutations. This review shows that there are many alternatively spliced MMR transcripts, which have potential to confound interpretation of splicing assays. These findings highlight the need to perform RNA analysis of patients systematically in parallel with control individuals, and call for the implementation of quantitative assessment of transcript levels for informed interpretation of mRNA assays.