Biochemical and structural characterization of two variants of uncertain significance in the PMS2 gene

Lynch syndrome (LS) is an autosomal dominant inherited disorder that is associated with an increased predisposition to certain cancers caused by loss‐of‐function mutations in one of four DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6, or PMS2). The diagnosis of LS is often challenged by the identification of missense mutations where the functional effects are not known. These are termed variants of uncertain significance (VUSs) and account for 20%–30% of noncoding and missense mutations. VUSs cause ambiguity during clinical diagnosis and hinder implementation of appropriate medical management. In the current study, we focus on the functional and biological consequences of two nonsynonymous VUSs in PMS2. These variants, c.620G>A and c.123_131delGTTAGTAGA, result in the alteration of glycine 207 to glutamate (p.Gly207Glu) and the deletion of amino acid residues 42–44 (p.Leu42_Glu44del), respectively. While the PMS2 p.Gly207Glu variant retains in vitro MMR and ATPase activities, PMS2 p.Leu42_Glu44del appears to lack such capabilities. Structural and biophysical characterization using circular dichroism, small‐angle X‐ray scattering, and X‐ray crystallography of the N‐terminal domain of the PMS2 variants indicate that the p.Gly207Glu variant is properly folded similar to the wild‐type enzyme, whereas p.Leu42_Glu44del is disordered and prone to aggregation.     

Phenotypic characterization of Familial Mediterranean Fever patients harboring variants of uncertain significance

Background/aimFamilial Mediterranean Fever (FMF) is the prototype of hereditary autoinflammatory disorders and caused by mutations on the MEFV gene located on the short arm of chromosome 16. Although some MEFV variants are clearly associated with disease phenotype, there are numerous variants with unknown clinical association which are termed as variants of uncertain significance (VUS). Here, we present clinical correlations of VUS in a large cohort of adult FMF patients from three tertiary centers located in Central Anatolia.Materials and methodsAll patients were recruited from FMF in Central Anatolia (FiCA) cohort. Demographic (sex, age at disease onset) and clinical features (disease characteristics, attack frequency, mean colchicine dose, colchicine nonresponsiveness, amyloidosis, and persistent inflammation) of patients with VUS were compared with those harboring pathogenic variants. Disease severity and damage were also evaluated using international severity score for FMF (ISSF) and autoinflammatory disease damage index (ADDI), respectively.ResultsAmong 971 participants included, MEFV gene analysis results were available for 814 patients. Twenty-six (3.2%) patients had single heterozygous VUS and 54 (6.6%) had pathogenic/VUS complex heterozygous variants. Patients with single heterozygous VUS had similar demographic/clinical features, ISSF and ADDI scores compared to those with single heterozygous pathogenic variant (p > 0.05 for all). No difference was observed in the demographic and clinical features of patients with single heterozygous pathogenic mutation and pathogenic/VUS complex heterozygous variant (p > 0.05 for all). ISSF and ADDI scores were lower in pathogenic/VUS complex heterozygous patients than those harboring single pathogenic mutation (p = 0.006 and 0.004, respectively).ConclusionOur findings suggest that patients with single heterozygous VUS has mild FMF phenotype similar to those with single pathogenic mutation. Pathogenic/VUS complex heterozygosity does not lead to a more severe clinical phenotype than having a single pathogenic variant.     

The functional impact of variants of uncertain significance in BRCA2

PurposeGenetic testing has uncovered large numbers of variants in the BRCA2 gene for which the clinical significance is unclear. Cancer risk prediction of these variants of uncertain significance (VUS) can be improved by reliable assessment of the extent of impairment of the tumor suppressor function(s) of BRCA2.MethodsHere, we evaluated the performance of the mouse embryonic stem cell (mESC)-based functional assay on an extensive set of BRCA2 missense variants.ResultsWhereas all 20 nonpathogenic (class 1/2) variants were able to complement the cell lethal phenotype induced by loss of endogenous mouse Brca2, only 1 out of 15 pathogenic (class 4/5) variants (p.Gly2609Asp) was able to do so. However, in this variant the major tumor suppressive activity of BRCA2, i.e., homology directed repair (HDR), was severely abrogated. Among 43 evaluated VUS (class 3), 7 were unable to complement the lethal phenotype of mouse Brca2 loss while 7 other variants displayed a more severe reduction of HDR activity than observed for class 1/ 2 variants.ConclusionThe mESC-based BRCA2 functional assay can reliably determine the functional impact of VUS, distinguish between pathogenic and nonpathogenic variants, and may contribute to improved cancer risk estimation for BRCA2 VUS carriers.     

PALB2 variants in hereditary and unselected Finnish Prostate cancer cases

Background  

PALB2 1592delT mutation is associated with increased breast cancer and suggestive prostate cancer (PRCA) risk in Finland. In this study we wanted to assess if any other PALB2 variants associate to increased PRCA risk and clinically describe patients with formerly found PALB2 1592delT mutation.     

Functional characterization of CHEK2 variants in a Saccharomyces cerevisiae system

Genetic testing for cancer predisposition leads to the identification of a number of variants with uncertain significance. To some extent, variants of BRCA1/2 have been classified, in contrast to variants of other genes. CHEK2 is a typical example, in which a large number of variants of unknown clinical significance were identified and still remained unclassified. Herein, the CHEK2 variant assessment was performed through an in vivo, yeast‐based, functional assay. In total, 120 germline CHEK2 missense variants, distributed along the protein sequence, and two large in‐frame deletions were tested, originating from genetic test results in breast cancer families, or selected from the ClinVar database. Of these, 32 missense and two in‐frame deletions behaved as non‐functional, 73 as functional, and 15 as semi‐functional, after comparing growth rates of each strain with positive and negative controls. The majority of non‐functional variants were localized in the CHK2 kinase and forkhead‐associated domains. In vivo results from the non‐functional variants were in agreement with in silico predictions, and, where available, with strong breast cancer family history, to a great extent. The results of the largest, to date, yeast‐based assay, evaluating CHEK2 variants, can complement and assist in the classification of rare CHEK2 variants with unclear clinical significance.     

Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay

Single-nucleotide substitutions and small in-frame insertions or deletions identified in human breast cancer susceptibility genes BRCA1 and BRCA2 are frequently classified as variants of unknown clinical significance (VUS) due to the availability of very limited information about their functional consequences. Such variants can most reliably be classified as pathogenic or non-pathogenic based on the data of their co-segregation with breast cancer in affected families and/or their co-occurrence with a pathogenic mutation. Biological assays that examine the effect of variants on protein function can provide important information that can be used in conjunction with available familial data to determine the pathogenicity of VUS. In this report, we have used a previously described mouse embryonic stem (mES) cell-based functional assay to characterize eight BRCA2 VUS that affect highly conserved amino acid residues and map to the N-terminal PALB2-binding or the C-terminal DNA-binding domains. For several of these variants, very limited co-segregation information is available, making it difficult to determine their pathogenicity. Based on their ability to rescue the lethality of Brca2-deficient mES cells and their effect on sensitivity to DNA-damaging agents, homologous recombination and genomic integrity, we have classified these variants as pathogenic or non-pathogenic. In addition, we have used homology-based modeling as a predictive tool to assess the effect of some of these variants on the structural integrity of the C-terminal DNA-binding domain and also generated a knock-in mouse model to analyze the physiological significance of a residue reported to be essential for the interaction of BRCA2 with meiosis-specific recombinase, DMC1.     

A guide for functional analysis of BRCA1 variants of uncertain significance

Germline mutations in the tumor suppressor gene BRCA1 confer an estimated lifetime risk of 56–80% for breast cancer and 15–60% for ovarian cancer. Since the mid 1990s when BRCA1 was identified, genetic testing has revealed over 1,500 unique germline variants. However, for a significant number of these variants, the effect on protein function is unknown making it difficult to infer the consequences on risks of breast and ovarian cancers. Thus, many individuals undergoing genetic testing for BRCA1 mutations receive test results reporting a variant of uncertain clinical significance (VUS), leading to issues in risk assessment, counseling, and preventive care. Here, we describe functional assays for BRCA1 to directly or indirectly assess the impact of a variant on protein conformation or function and how these results can be used to complement genetic data to classify a VUS as to its clinical significance. Importantly, these methods may provide a framework for genome‐wide pathogenicity assignment. Hum Mutat 33:1526–1537, 2012. © 2012 Wiley Periodicals, Inc.     

Disclosure of cardiac variants of uncertain significance results in an exome cohort

This study examined the impact of disclosing subclassifications of genetic variants of uncertain significance (VUS) on behavioral intentions. We studied return of VUS results to 79 individuals with a cardiomyopathy‐associated VUS, subclassified into VUS‐high or VUS‐low. Primary outcomes were perceived risk (absolute and comparative), perceived severity, perceived value of information, self‐efficacy, decision regret, and behavioral intentions to share results and change behaviors. There was no significant difference between the 2 subclasses in overall behavioral intentions (t = 0.023, P = .982) and each of the individual items on the behavioral intentions scale; absolute (t = ?1.138, P = .259) or comparative (t = ?0.463, P = .645) risk perceptions; perceived value of information (t = 0.582, P = .563) and self‐efficacy (t = ?0.733, P = .466). Decision regret was significantly different (t = 2.148, P = .035), with VUS‐low (mean = 17.24, SD = 16.08) reporting greater regret. Combining the subclasses, perceived value of information was the strongest predictor of behavioral intentions (β = 0.524, P < .001). Participants generally understood the meaning of a genetic VUS result classification and reported satisfaction with result disclosure. No differences in behavioral intentions were found, but differences in decision regret suggest participants distinguish subclasses of VUS results. The perceived value of VUS may motivate recipients to pursue health‐related behaviors.     

Reporting practices for variants of uncertain significance from next generation sequencing technologies

The nature of next generation sequencing technologies (NGS) results in the generation of large amounts of data and the identification of numerous variants, for some of which the clinical significance may be difficult to ascertain based on our current knowledge. These Variants of Uncertain Significance (VUS) may be identified in genes in which the function is known or unknown and which may or may not be related to the original rationale for sequencing the patient. Little is known about whether laboratories report VUS to clinicians and current guidelines issued by some of the most notable professional bodies do not provide specific recommendations on this point. To address this, 26 interviews were conducted with 27 laboratory personnel, representing 24 laboratories in Europe (12), Canada (5) and Australasia (7) in order to explore their reporting practices. Participants highlighted that the classification of variants is a real challenge despite the presence of classification guidelines. We identified variation in the reporting practices of VUS across the laboratories within the study. While some laboratories limit their reporting to variants that are pathogenic and thought to be causative of the phenotype, more commonly laboratories report VUS when they are identified in genes related to the clinical question. Some laboratories will also report VUS in candidate genes. VUS that are secondary findings are generally not reported. While it is unclear whether uniformity in reporting is desirable, exploring the perspectives of laboratory personnel undertaking these analyses are critical to ensure the feasibility of any future reporting recommendations.     

Outcomes of 92 patient-driven family studies for reclassification of variants of uncertain significance

PurposeFamily studies are an important but underreported source of information for reclassification of variants of uncertain significance (VUS). We evaluated outcomes of a patient-driven framework that offered familial VUS reclassification analysis to any adult with any clinically ascertained VUS from any laboratory in the United States.MethodsWith guidance from FindMyVariant.org, participants recruited their own relatives for study participation. We genotyped relatives, calculated quantitative cosegregation likelihood ratios, and evaluated variant classifications using Tavtigian’s unified framework for Bayesian analysis with American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria. We report participation and VUS reclassification rates from the 50 families enrolled for at least one year and reclassification results for 112 variants from the larger 92-family cohort.ResultsFor the 50-family cohort, 6.7 relatives per family were invited to participate and 67% of relatives returned samples for genotyping. Sixty-one percent of VUS were reclassified, 84% of which were classified as benign or likely benign. Genotyping relatives identified a de novo variant, phase variants, and relatives with phenotypes highly specific for or incompatible with specific classifications.ConclusionsMotivated families can contribute to successful VUS reclassification at substantially higher rates than those previously published. Clinical laboratories could consider offering family studies to all patients with VUS.     

Impact of variants of uncertain significance of LDL receptor on phenotypes of familial hypercholesterolemia

BackgroundData on the effect of variants of uncertain significance (VUS) of LDL receptor (LDLR) on familial hypercholesterolemia (FH) phenotype is limited.ObjectiveTo investigate the associations between genotypes and phenotypes, including low-density lipoprotein (LDL) cholesterol level and occurrence of major adverse cardiac events (MACEs), in FH patients (N = 1050, male/female = 490/560).MethodsWe retrospectively assessed the data of patients with FH admitted at Kanazawa University Hospital between 1990 and 2020. Based on genotype, the patients were divided into patients without variants, with VUS of LDLR, and with pathogenic variants. Cox proportional hazard model was used to identify the factors associated with MACEs.ResultsThe median follow-up duration was 12.6 years (interquartile range: 9.5–17.9 years). Altogether, 777 patients had FH mutation and 273 had pathogenic mutation, with 92 having VUS. Over the follow-up duration, 175 MACEs were observed. LDL cholesterol level was found to be significantly higher in patients with pathogenic variants (251 mg/dL) than in patients with VUS (225 mg/dL) and without variants (203 mg/dL). Pathogenic variants and VUS are significantly associated with MACEs (hazard ratio [HR] = 1.52, 95% confidence interval [CI] = 1.02–2.02, P = 0.033 and HR = 3.18, 95% CI = 2.00–4.36, P = 1.9 × 10^?5, relative to patients without any variants, respectively), independent of classical risk factors.ConclusionVUS of LDLR was significantly associated with poor outcomes in FH patients. Genetic testing is useful for the diagnosis and risk stratification of FH patients.     

Functional characterization of the splicing variants of human norepinephrine transporter

Human norepinephrine transporter (NET) displays three splicing variants having different carboxy terminals, hNET, hNET C-t var1 and hNET C-t var2. Functional characterization of these isoforms was performed with transient expression system in COS-7 cells. Cells transfected with hNET C-t var2, but not hNET C-t var1, revealed a significant increase in [(3)H]norepinephrine (NE) uptake and [(3)H]nisoxetine binding as well as hNET, in association with their different cellular localization indicated by immunostaining using NET-specific antisera. Kinetic and pharmacological analyses of [(3)H]NE uptake revealed different characteristics between hNET and hNET C-t var2. These results suggest that hNET C-t var2 may participate in NE transport in a manner different from hNET at noradrenergic synapses or in other tissues including placenta where NET variants were found to exist.     

Tumor characteristics as an analytic tool for classifying genetic variants of uncertain clinical significance

It is important to identify a germline mutation in a patient with an inherited cancer syndrome to allow mutation carriers to be included in cancer surveillance programs, which have been proven to save lives. Many of the mutations identified result in premature termination of translation, and thus in loss-of-function of the encoded mutated protein. However, the significance of a large proportion of the sequence changes reported is unknown. Some of these variants will be associated with a high risk of cancer and have direct clinical consequence. Many criteria can be used to classify variants with unknown significance; most criteria are based on the characteristics of the amino acid change, on segregation data and appearance of the variant, on the presence of the variant in controls, or on functional assays. In inherited cancers, tumor characteristics can also be used to classify variants. It is worthwhile to examine the clinical, morphological and molecular features of a patient, and his or her family, when assessing whether the role of a variant is likely to be neutral or pathogenic. Here we describe the advantages and disadvantages of using the tumor characteristics of patients carrying germline variants of uncertain significance (VUS) in BRCA1, BRCA2, or in one of the mismatch repair (MMR) genes, MLH1, MSH2, or MSH6, to infer pathogenicity.     

Health behaviors among unaffected participants following receipt of variants of uncertain significance in cardiomyopathy-associated genes

PurposeStudies on returning variants of uncertain significance (VUS) results have predominantly included patients with a personal or family history of cancer and cancer-associated gene VUS. This study examined health behaviors among participants with cardiomyopathy-associated gene VUS, but without a personal history of cardiomyopathy.MethodsSixty-eight eligible participants without apparent cardiomyopathy but with VUS in cardiomyopathy-associated genes completed a survey of health behaviors, disclosure, distress, uncertainty, positive experiences, decisional conflict, and perceived value. The medical records of participants who reported cardiac testing because of their VUS were reviewed for testing indication(s).ResultsTwo participants had cardiac testing due to their VUS alone. Four had cardiac testing because of their VUS and other clinical indications. Twelve changed health behaviors, including one participant who was subsequently diagnosed with cardiomyopathy. Distress, uncertainty, and decisional conflict were low (means = 1.2, 4.2, and 24.5 (scale ranges = 0–30, 0–45, and 15–75), respectively), and positive experiences and perceived value were moderate (means = 12.4 and 14.4 (scale ranges = 0–20 and 4–20), respectively). Greater perceived value was associated with greater likelihood to engage in health behaviors (P = 0.04).ConclusionPositive VUS results can be returned to apparently unaffected individuals with modest use of healthcare resources, minimal behavioral changes, and favorable psychological reactions.     

Functional characterization of BRCA1 gene variants by mini-gene splicing assay

Mutational screening of the breast cancer susceptibility gene BRCA1 leads to the identification of numerous pathogenic variants such as frameshift and nonsense variants, as well as large genomic rearrangements. The screening moreover identifies a large number of variants, for example, missense, silent, and intron variants, which are classified as variants of unknown clinical significance owing to the lack of causal evidence. Variants of unknown clinical significance can potentially have an impact on splicing and therefore functional examinations are warranted to classify whether these variants are pathogenic or benign. Here we validate a mini-gene splicing assay by comparing the results of 24 variants with previously published data from RT-PCR analysis on RNA from blood samples/lymphoblastoid cell lines. The analysis showed an overall concordance of 100%. In addition, we investigated 13 BRCA1 variants of unknown clinical significance or putative variants affecting splicing by in silico analysis and mini-gene splicing assay. Both the in silico analysis and mini-gene splicing assay classified six BRCA1 variants as pathogenic (c.80+1G>A, c.132C>T (p.=), c.213−1G>A, c.670+1delG, c.4185+1G>A, and c.5075−1G>C), whereas six BRCA1 variants were classified as neutral (c.-19-22_-19-21dupAT, c.302−15C>G, c.547+14delG, c.4676−20A>G, c.4987−21G>T, and c.5278−14C>G) and one BRCA1 variant remained unclassified (c.670+16G>A). In conclusion, our study emphasizes that in silico analysis and mini-gene splicing assays are important for the classification of variants, especially if no RNA is available from the patient. This knowledge is crucial for proper genetic counseling of patients and their family members.     

Incorporating truncating variants in PALB2, CHEK2, and ATM into the BOADICEA breast cancer risk model

PurposeThe proliferation of gene panel testing precipitates the need for a breast cancer (BC) risk model that incorporates the effects of mutations in several genes and family history (FH). We extended the BOADICEA model to incorporate the effects of truncating variants in PALB2, CHEK2, and ATM.MethodsThe BC incidence was modeled via the explicit effects of truncating variants in BRCA1/2, PALB2, CHEK2, and ATM and other unobserved genetic effects using segregation analysis methods.ResultsThe predicted average BC risk by age 80 for an ATM mutation carrier is 28%, 30% for CHEK2, 50% for PALB2, and 74% for BRCA1 and BRCA2. However, the BC risks are predicted to increase with FH burden. In families with mutations, predicted risks for mutation-negative members depend on both FH and the specific mutation. The reduction in BC risk after negative predictive testing is greatest when a BRCA1 mutation is identified in the family, but for women whose relatives carry a CHEK2 or ATM mutation, the risks decrease slightly.ConclusionsThe model may be a valuable tool for counseling women who have undergone gene panel testing for providing consistent risks and harmonizing their clinical management. A Web application can be used to obtain BC risks in clinical practice (http://ccge.medschl.cam.ac.uk/boadicea/).Genet Med 18 12, 1190–1198.     

Familial wild-type gastrointestinal stromal tumour in association with germline truncating variants in both SDHA and PALB2

Gastrointestinal stromal tumour (GIST) is a mesenchymal neoplasm arising in the gastrointestinal tract. A rare subset of GISTs are classified as wild-type GIST (wtGIST) and these are frequently associated with germline variants that affect the function of cancer predisposition genes such as the succinate dehydrogenase subunit genes (SDHA, SDHB, SDHC, SDHD) or NF1. However, despite this high heritability, familial clustering of wtGIST is extremely rare. Here, we report a mother–son diad who developed wtGIST at age 66 and 34 years, respectively. Comprehensive genetic testing revealed germline truncating variants in both SDHA (c.1534C>T (p.Arg512*)) and PALB2 (c.3113G>A (p.Trp1038*)) in both affected individuals. The mother also developed breast ductal carcinoma in-situ at age 70 years. Immunohistochemistry and molecular analysis of the wtGISTs revealed loss of SDHB expression and loss of the wild-type SDHA allele in tumour material. No allele loss was detected at PALB2 suggesting that wtGIST tumourigenesis was principally driven by succinate dehydrogenase deficiency. However, we speculate that the presence of multilocus inherited neoplasia alleles syndrome (MINAS) in this family might have contributed to the highly unusual occurrence of familial wtGIST. Systematic reporting of tumour risks and phenotypes in individuals with MINAS will facilitate the clinical interpretation of the significance of this diagnosis, which is becoming more frequent as strategies for genetic testing for hereditary cancer becomes more comprehensive.Subject terms: Cancer genetics, Genetic testing