Variants Of Uncertain Significance In Genes Research Articles

The application value of whole exome sequencing technology in diagnosis of hereditary renal cysts

The data of 57 renal cyst patients who visited the First Affiliated Hospital of Zhengzhou University from January 2023 to March 2024 were retrospectively analyzed. The age of patients ranged from three months to 60 years old, with 31 males and 26 females. The whole exome sequencing (WES) detected pathogenic or suspected pathogenic (P/LP) variants in 48 renal cystic probands, with a detection rate of 84.2% (48/57), including PKD1, PKD2, PKHD1, LRP5, COL4A4 and ALG8 gene variants as well as copy number variations (CNV). In addition, four PKD1 gene variants of uncertain significance (VUS) were detected. In five WES negative families, one PKD1 nonsense variation was detected through long-range PCR (LR-PCR)+Oxford nanopore technologies, and one heterozygous deletion in exon 22 of PKD1 gene was detected through multiplex ligation-dependent probe amplification (MLPA). In summary, WES can detect multiple types of variations, which is helpful for early diagnosis and prognosis prediction of renal cyst patients. However, there is still a risk of failing to detect PKD1 gene by WES, therefore, healthcare practitioners should beware of the negative results of WES.

Two missense STK11 gene variations impaired LKB1/adenosine monophosphate-activated protein kinase signaling in Peutz-Jeghers syndrome.

Peutz-Jeghers syndrome (PJS) is a rare hereditary neoplastic disorder mainly associated with serine/threonine kinase 11 (STK11/LKB1) gene mutations. Preimplantation genetic testing can protect a patient's offspring from mutated genes; however, some variations in this gene have been interpreted as variants of uncertain significance (VUS), which complicate reproductive decision-making in genetic counseling. To identify the pathogenicity of two missense variants and provide clinical guidance. Whole exome gene sequencing and Sanger sequencing were performed on the peripheral blood of patients with PJS treated at the Reproductive and Genetic Hospital of Citic-Xiangya. Software was employed to predict the protein structure, conservation, and pathogenicity of the two missense variation sites in patients with PJS. Additionally, plasmids were constructed and transfected into HeLa cells to observe cell growth. The differences in signal pathway expression between the variant group and the wild-type group were compared using western blot and immunohistochemistry. Statistical analysis was performed using one-way analysis of variance. P < 0.05 was considered statistically significant. We identified two missense STK11 gene VUS [c.889A>G (p.Arg297Gly) and c.733C>T (p.Leu245Phe)] in 9 unrelated PJS families who were seeking reproductive assistance. The two missense VUS were located in the catalytic domain of serine/threonine kinase, which is a key structure of the liver kinase B1 (LKB1) protein. In vitro experiments showed that the phosphorylation levels of adenosine monophosphate-activated protein kinase (AMPK) at Thr172 and LKB1 at Ser428 were significantly higher in transfected variation-type cells than in wild-type cells. In addition, the two missense STK11 variants promoted the proliferation of HeLa cells. Subsequent immunohistochemical analysis showed that phosphorylated-AMPK (Thr172) expression was significantly lower in gastric, colonic, and uterine polyps from PJS patients with missense variations than in non-PJS patients. Our findings indicate that these two missense STK11 variants are likely pathogenic and inactivate the STK11 gene, causing it to lose its function of regulating downstream phosphorylated-AMPK (Thr172), which may lead to the development of PJS. The identification of the pathogenic mutations in these two clinically characterized PJS patients has been helpful in guiding them toward the most appropriate mode of pregnancy assistance. These two missense variants can be interpreted as likely pathogenic variants that mediated the onset of PJS in the two patients. These findings not only offer insights for clinical decision-making, but also serve as a foundation for further research and reanalysis of missense VUS in rare diseases.

The VUS Challenge in Cystic Kidney Disease: A Case-Based Review.

Genetic testing in nephrology is becoming increasingly important to diagnose patients and to provide appropriate care. This is especially true for autosomal dominant polycystic kidney disease (ADPKD) because this is a common cause of kidney failure and genetically complex. In addition to the major genes, PKD1 and PKD2 , there are at least six minor loci, and phenotypic, and in some cases, genetic overlap with other cystic disorders. Targeted next-generation sequencing, a low-cost, high-throughput technique, has made routine genetic testing viable in nephrology clinics. Appropriate pre- and post-testing genetic counseling is essential to the testing process. Carefully assessing variants is also critical, with the genetic report classifying variants in accordance with American College of Medical Genetics and Genomics guidelines. However, variant of uncertain significance (VUSs) may pose a significant challenge for the ordering clinician. In ADPKD, and particularly within PKD1 , there is high allelic heterogeneity; no single variant is present in more than 2% of families. The Mayo/Polycystic Kidney Disease Foundation variant database, a research tool, is the best current database of PKD1 and PKD2 variants containing over 2300 variants identified in individuals with polycystic kidney disease, but novel variants are often identified. In patients with a high pretest probability of ADPKD on the basis of clinical criteria, but no finding of a pathogenic (P) or likely pathogenic (LP) variant in a cystic kidney gene, additional evaluation of cystic gene VUS can be helpful. In this case-based review, we propose an algorithm for the assessment of such variants in a clinical setting and show how some can be reassigned to a diagnostic grouping. When assessing the relevance of a VUS, we consider both patient/family-specific and allele-related factors using population and variant databases and available prediction tools, as well as genetic expertise. This analysis plus further family studies can aid in making a genetic diagnosis.

Rifampin monotherapy for children with idiopathic infantile hypercalcemia

Idiopathic Infantile Hypercalcemia (IIH) is characterized by hypercalcemia and hypercalciuria owing to PTH-independent increases in circulating concentrations of 1,25(OH)2D. At least 3 forms of IHH can be distinguished genetically and mechanistically: infantile hypercalcemia-1 (Hypercalcemia, Infantile, 1; HCINF1) due to CYP24A1 mutations results in decreased inactivation of 1,25(OH)2D; HCINF2 due to SLC34A1 mutations results in excessive 1,25(OH)2D production; and HCINF3 in which a variety of gene variants of uncertain significance (VUS) have been identified and where the mechanism for increased 1,25 (OH)2D is unclear. Conventional management with dietary calcium and vitamin D restriction has only limited success. Induction of the P450 enzyme CYP3A4 by rifampin can provide an alternate pathway for inactivation of 1,25(OH)2D that is useful in HCINF1 and may be effective in other forms of IIH.We sought to assess the efficacy of rifampin to decrease levels of serum 1,25(OH)2D and calcium, and urinary calcium concentrations in subjects with HCINF3, and to compare the response to a control subject with HCINF1.Four subjects with HCINF3 and the control subject with HCINF1 completed the study using rifampin 5 mg/kg/day and 10 mg/kg/day each for 2 months separated by a 2-month washout period. Patients had age-appropriate intake of dietary calcium plus 200 IU vitamin D/day. Primary outcome was efficacy of rifampin to lower serum concentrations of 1,25(OH)2D. The secondary outcomes included the reduction of serum calcium, urinary calcium excretion (as random urine calcium: creatinine (ca:cr) ratio) and serum 1,25(OH)2D/PTH ratio.Rifampin was well tolerated and induced CYP3A4 at both doses in all subjects. The control subject with HCINF1 showed significant response to both rifampin doses with decreases in the serum concentration of 1,25(OH)2D and the 1,25(OH)2D/PTH ratio while the serum and urine ca:cr levels were unchanged. The four patients with HCINF3 showed reductions in 1,25(OH)2D and urinary ca:cr after 10 mg/kg/d, but hypercalcemia did not improve and there were variable responses in 1,25(OH)2D/PTH ratios. These results support further longer-term studies to clarify the usefulness of rifampin as a medical therapy for IIH.

Diagnosing Czech Patients with Inherited Platelet Disorders.

A single-center study was conducted on 120 patients with inherited disorders of primary hemostasis followed at our hematological center. These patients presented a variety of bleeding symptoms; however, they had no definitive diagnosis. Establishing a diagnosis has consequences for the investigation of probands in families and for treatment management; therefore, we aimed to improve the diagnosis rate in these patients by implementing advanced diagnostic methods. According to the accepted international guidelines at the time of study, we investigated platelet morphology, platelet function assay, light-transmission aggregometry, and flow cytometry. Using only these methods, we were unable to make a definitive diagnosis for most of our patients. However, next-generation sequencing (NGS), which was applied in 31 patients, allowed us to establish definitive diagnoses in six cases (variants in ANKRD26, ITGA2B, and F8) and helped us to identify suspected variants (NBEAL2, F2, BLOC1S6, AP3D1, GP1BB, ANO6, CD36, and ITGB3) and new suspected variants (GFI1B, FGA, GP1BA, and ITGA2B) in 11 patients. The role of NGS in patients with suspicious bleeding symptoms is growing and it changes the diagnostic algorithm. The greatest disadvantage of NGS, aside from the cost, is the occurrence of gene variants of uncertain significance.

A NOVEL PROTEIN KINASE, DNA-ACTIVATED, CATALYTIC SUBUNIT VARIANT IDENTIFIED IN T-B-NK+ SEVERE COMBINED IMMUNODEFICIENCY

<h3>Introduction</h3> Whole exome sequencing (WES) has led to advances in identifying genetic variants responsible for rare diseases. Here we report a novel protein kinase, DNA-activated, catalytic subunit (PRKDC) variant in a patient with T-B-NK+ severe combined immunodeficiency (SCID). <h3>Case Description</h3> A preterm male with intrauterine growth restriction (IUGR), Tetralogy of Fallot, horseshoe kidney, and numerous brain malformations presented with lymphopenia [absolute lymphocyte count (ALC) of 560 × 10³ / mcL]. Lymphocyte subpopulations demonstrated a T-B-NK+ phenotype. The infant died on day of life three from respiratory failure. Patient's NBS returned four days after he died and revealed absent T-cell receptor excision circles (TREC). The patient had a deceased older brother with similar congenital anomalies who also had absent TREC on NBS with a T-B-NK+ SCID phenotype in the setting of several variants of uncertain significance (VUS) on whole exome sequencing (WES), including a homozygosity for a variant in the PRKDC gene, p.Arg1155Gln which both parents were heterozygous for. Three months after patient's death, a post-mortem WES returned with homozygosity in the same PRKDC gene VUS identified in his older brother. <h3>Discussion</h3> The p.Arg1155Gin PRKDC gene variant found is a missense substitution. Multiple identified PRKDC variants lead to DNA repair defects which may be seen clinically as radiation-sensitive T-B-NK+ SCID. While the PRKDC gene variant in our case was identified as one of uncertain significance, the similarities between our patient and his brother strongly suggest that this is likely the pathogenic variant for this family. This PRKDC variant has yet to be reported in the literature.

Predictive functional assay-based classification of PMS2 variants in Lynch syndrome.

The large majority of germline alterations identified in the DNA mismatch repair (MMR) gene PMS2, a low‐penetrance gene for the cancer predisposition Lynch syndrome, represent variants of uncertain significance (VUS). The inability to classify most VUS interferes with personalized healthcare. The complete in vitro MMR activity (CIMRA) assay, that only requires sequence information on the VUS, provides a functional analysis‐based quantitative tool to improve the classification of VUS in MMR proteins. To derive a formula that translates CIMRA assay results into the odds of pathogenicity (OddsPath) for VUS in PMS2 we used a set of clinically classified PMS2 variants supplemented by inactivating variants that were generated by an in cellulo genetic screen, as proxies for cancer‐predisposing variants. Validation of this OddsPath revealed high predictive values for benign and predisposing PMS2 VUS. We conclude that the OddsPath provides an integral metric that, following the other, higher penetrance, MMR proteins MSH2, MSH6 and MLH1 can be incorporated as strong evidence type into the upcoming criteria for MMR gene VUS classification of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP).

P485Re-interpretation of variants of uncertain significance in inherited cardiovascular diseases-A pilot study

Abstract Background- Identification of variants of uncertain significance (VUSs) poses relevant challenges in counseling and managing patients. They have an unknown impact on health, making the genetic tests clinically irrelevant. Recent studies demonstrate that a routine reclassification analysis enables to reclassify from 20% to 80% of this type of variant, improving risk stratification. Purpose- We investigated whether, in the context of inherited cardiac conditions, a review of the updated literature, including new functional data, allele frequency (GnomAD) and segregation analysis may help in the variant reclassification. Methods- Retrospective review of all VUSs in genes associated with inherited cardiac conditions identified in our cardiogenetic clinic between 2016 and 2018. Results- Thirty-one VUSs, classified using ACMG guidelines, were identified in 26 cases with a confirmed or suspected diagnosis of inherited cardiovascular diseases, including Long QT syndrome, Brugada syndrome, Arrhythmogenic Cardiomyopathy and Hypertrophic Cardiomyopathy). Twentyfour variants were identified in well-defined causative genes (SCN5A, KCNQ1, KCNH2, KCNE1, DSP, DSG2, MYH7, TPM1, TNNI3, TNNT2, CACNA1C, MYL3) while the remaining variants were identified in minor genes with limited evidence to support their disease causation as, ANK2 and AKAP9 gene. Preliminary results of the reclassification analysis showed that two variants were downgraded to likely benign (LB) applying the BS1 criterion (allele frequency) and 4 variants were upgraded to likely pathogenic (LP) according to novel published data and family segregation studies. Moreover, further studies to assess cosegregation in other variants are still ongoing. Conclusion- Based on our experience, 25% of variants of uncertain significance in well-defined causative genes, identified in patients with a confirmed or suspected diagnosis of inherited cardiovascular disease, were reclassified. These findings suggest that re-evaluation of genetic test results should be performed routinely in all diagnostic labs, in order to improve risk stratification and identification of family members at high risk.

The use of fetal exome sequencing in prenatal diagnosis: a points to consider document of the American College of Medical Genetics and Genomics (ACMG)

The use of fetal exome sequencing in prenatal diagnosis: a points to consider document of the American College of Medical Genetics and Genomics (ACMG)

Detection of DNA mismatch repair deficient crypts in random colonoscopic biopsies identifies Lynch syndrome patients.

The hallmark of Lynch syndrome (LS)-associated neoplasia is DNA mismatch repair protein (MMR) deficiency. Recent studies have demonstrated that histologically normal colonic crypts in patients with LS can exhibit deficient MMR expression. The aim of this study was to determine the feasibility of detecting MMR deficient crypts in random colonoscopic biopsies of normal mucosa in patients with and without LS. Forty-nine patients, including 33 with LS, 12 without LS, and 4 with germline MMR gene variants of uncertain significance (VUS), were prospectively and blindly evaluated by immunohistochemistry for MMR deficient crypts within random normal-appearing mucosal biopsies obtained during surveillance colonoscopy. MMR deficient crypts were identified in 70% (23/33) of patients with LS and in no patients without LS (0/12) (p < 0.001). MMR deficient crypts were identified with nearly equal frequency in both LS patients with and without a cancer history and were associated with germline variants in all four MMR genes and EPCAM. MMR deficient crypts were also identified in LS patients with a history of non-colorectal cancer types, including patients with endometrial cancer, skin sebaceous neoplasms, and renal cancer. Two of the four patients with germline MMR gene VUS had MMR deficient crypts. In conclusion, MMR deficient crypts are a specific biomarker of LS and can be identified in random normal mucosal biopsies in LS patients. Evaluation for MMR deficient crypts in colonoscopic biopsies of normal mucosa can help identify LS patients.

Functional interrogation of Lynch syndrome‐associatedMSH2missense variants via CRISPR‐Cas9 gene editing in human embryonic stem cells

Lynch syndrome (LS) predisposes patients to cancer and is caused by germline mutations in the DNA mismatch repair (MMR) genes. Identifying the deleterious mutation, such as a frameshift or nonsense mutation, is important for confirming an LS diagnosis. However, discovery of a missense variant is often inconclusive. The effects of these variants of uncertain significance (VUS) on disease pathogenesis are unclear, though understanding their impact on protein function can help determine their significance. Laboratory functional studies performed to date have been limited by their artificial nature. We report here an in-cellulo functional assay in which we engineered site-specific MSH2 VUS using clustered regularly interspaced short palindromic repeats-Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild-type gene. We characterized the impact of the variants on cellular MMR functions including DNA damage response signaling and the repair of DNA microsatellites. We classified the MMR functional capability of eight of 10 VUS providing valuable information for determining their likelihood of being bona fide pathogenic LS variants. This human cell-based assay system for functional testing of MMR gene VUS will facilitate the identification of high-risk LS patients.

PO-206 Modelling T cell acute lymphoblastic leukaemia using CRISPR/Cas9 mediated genome editing in Xenopus tropicalis

IntroductionThe helicase BRIP1 is critically required for genomic maintenance, and BRIP1 helicase deficient germline variants are associated with Fanconi Anaemia (bi-allelic) and various cancers (mono-allelic), in particular ovarian and breast cancer. Cellular based assays and biochemical studies have elucidated the function of BRIP1 in replication stress responses, including interaction partners and enzymatic substrate specificity. However, functional characterisation of BRIP1 for these processes in vivo are largely unexplored, including the importance of BRIP1 as a tumour suppressor for breast cancer, and therapy responses. This project aims to elucidate the functional roles of BRIP1 as a tumour suppressor for breast cancer and in therapy responses.Material and methodsWe have developed breast tissue-specific knockout mice for BRIP1, and will monitor the development of breast tumours. In addition, we have developed mice deficient for BRIP1 helicase activity (K52R) to further investigate the functional role of BRIP1 as a tumour suppressor. In addition, we will use the mouse models for intervention studies, testing known and novel therapies for BRIP1 deficient breast cancer. Moreover, we will investigate BRIP1 germline variants identified in high-risk breast cancer families by large scale sequencing efforts. This comprehensive list of patient derived mutations will be introduced endogenously in a primary epithelial cell line using a CRISPR-CAS9 approach. To monitor the effect on BRIP1 activity, these cell lines will be tested in cancer relevant functional assays for which BRIP1 deficient cells are known to have a phenotype.Results and discussionsThis project is at an early stage, and we will present our preliminary data from our BRIP1 mouse models, and from our in vitro CRISPR assay for testing BRIP1 gene variants of uncertain significance (VUS).ConclusionThis project is expected to yield novel insight into the regulation of critical genomic stability pathways and provide important knowledge for developing improved germ line variant risk prediction and treatment opportunities for patients.

Structural Biology Helps Interpret Variants of Uncertain Significance in Genes Causing Endocrine and Metabolic Disorders.

ContextVariants of uncertain significance (VUSs) lack sufficient evidence, in terms of statistical power or experimental studies, to allow unequivocal determination of their damaging effect. VUSs are a major burden in performing genetic analysis. Although in silico prediction tools are widely used, their specificity is low, thus urgently calling for methods for prioritizing and characterizing variants.ObjectiveTo assess the frequency of VUSs in genes causing endocrine and metabolic disorders, the concordance rate of predictions from different in silico methods, and the added value of three-dimensional protein structure analysis in discerning and prioritizing damaging variants.ResultsA total of 12,266 missense variants reported in 641 genes causing endocrine and metabolic disorders were analyzed. Among these, 4123 (33.7%) were VUSs, of which 2010 (48.8%) were predicted to be damaging and 1452 (35.2%) were predicted to be tolerated according to in silico tools. A total of 5383 (87.7%) of 6133 disease-causing variants and 823 (55.8%) of 1474 benign variants were correctly predicted. In silico predictions were noninformative in 5.7%, 14.4%, and 16% of damaging, benign, and VUSs, respectively. A damaging effect on 3D protein structure was present in 240 (30.9%) of predicted damaging and 40 (9.7%) of predicted tolerated VUSs (P < 0.001). An in-depth analysis of nine VUSs occurring in TSHR, LDLR, CASR, and APOE showed that they greatly affect protein stability and are therefore strong candidates for disease.ConclusionsIn our dataset, we confirmed the high sensitivity but low specificity of in silico predictions tools. 3D protein structural analysis is a compelling tool for characterizing and prioritizing VUSs and should be a part of genetic variant analysis.

PO-205 Functional characterisation of BRIP1/FANCJ in tumour suppression and therapy response

Introduction The helicase BRIP1 is critically required for genomic maintenance, and BRIP1 helicase deficient germline variants are associated with Fanconi Anaemia (bi-allelic) and various cancers (mono-allelic), in particular ovarian and breast cancer. Cellular based assays and biochemical studies have elucidated the function of BRIP1 in replication stress responses, including interaction partners and enzymatic substrate specificity. However, functional characterisation of BRIP1 for these processes in vivo are largely unexplored, including the importance of BRIP1 as a tumour suppressor for breast cancer, and therapy responses. This project aims to elucidate the functional roles of BRIP1 as a tumour suppressor for breast cancer and in therapy responses. Material and methods We have developed breast tissue-specific knockout mice for BRIP1, and will monitor the development of breast tumours. In addition, we have developed mice deficient for BRIP1 helicase activity (K52R) to further investigate the functional role of BRIP1 as a tumour suppressor. In addition, we will use the mouse models for intervention studies, testing known and novel therapies for BRIP1 deficient breast cancer. Moreover, we will investigate BRIP1 germline variants identified in high-risk breast cancer families by large scale sequencing efforts. This comprehensive list of patient derived mutations will be introduced endogenously in a primary epithelial cell line using a CRISPR-CAS9 approach. To monitor the effect on BRIP1 activity, these cell lines will be tested in cancer relevant functional assays for which BRIP1 deficient cells are known to have a phenotype. Results and discussions This project is at an early stage, and we will present our preliminary data from our BRIP1 mouse models, and from our in vitro CRISPR assay for testing BRIP1 gene variants of uncertain significance (VUS). Conclusion This project is expected to yield novel insight into the regulation of critical genomic stability pathways and provide important knowledge for developing improved germ line variant risk prediction and treatment opportunities for patients.

Invasive breast carcinomas with ATM gene variants of uncertain significance share distinct histopathologic features.

The increasing availability of next-generation sequencing for clinical research dramatically improved our understanding of breast cancer genetics and resulted in detection of new mutation variants. Cancer risk data relating to some of these variants are insufficient, prompting the designation of variants of uncertain significance (VUS). The histopathologic characteristics of these variants have not been previously described. We propose to depict these characteristics and determine if invasive carcinomas with similar VUS genes share similar histomorphologic features. In total, 28 invasive breast cancers with VUS were retrospectively identified. Tumor sections were reviewed and a predefined set of histopathologic characteristics were documented and compared. Nine of the 28 cases were variants in the ATM gene and were found to share similar histologic characteristics; all had tumor cells with low nuclear grade, absent tumor infiltrating lymphocytes, as well as a marked desmoplastic response. A subset of the above findings were identified in variants of other genes but none had all findings collectively. Furthermore, variants of ATM gene had smaller tumor size, lower pathologic T stage at presentation, and more favorable surrogate molecular subtype compared to variants of other genes. These findings could potentially be used to reclassify VUS and predict which patients may harbor ATM mutations, and hence could have implications in triaging toward ATM variant identification for potential future targeted therapy.

Germline Analysis from Tumor-Germline Sequencing Dyads to Identify Clinically Actionable Secondary Findings.

To evaluate germline variants in hereditary cancer susceptibility genes among unselected cancer patients undergoing tumor-germline sequencing. Germline sequence data from 439 individuals undergoing tumor-germline dyad sequencing through the LCCC1108/UNCseq™ (NCT01457196) study were analyzed for genetic variants in 36 hereditary cancer susceptibility genes. These variants were analyzed as an exploratory research study to determine whether pathogenic variants exist within the germline of patients undergoing tumor-germline sequencing. Patients were unselected with respect to indicators of hereditary cancer predisposition. Variants indicative of hereditary cancer predisposition were identified in 19 (4.3%) patients. For about half (10/19), these findings represent new diagnostic information with potentially important implications for the patient and their family. The others were previously identified through clinical genetic evaluation secondary to suspicion of a hereditary cancer predisposition. Genes with pathogenic variants included ATM, BRCA1, BRCA2, CDKN2A, and CHEK2 In contrast, a substantial proportion of patients (178, 40.5%) had Variants of Uncertain Significance (VUS), 24 of which had VUS in genes pertinent to the presenting cancer. Another 143 had VUS in other hereditary cancer genes, and 11 had VUS in both pertinent and nonpertinent genes. Germline analysis in tumor-germline sequencing dyads will occasionally reveal significant germline findings that were clinically occult, which could be beneficial for patients and their families. However, given the low yield for unexpected germline variation and the large proportion of patients with VUS results, analysis and return of germline results should adhere to guidelines for secondary findings rather than diagnostic hereditary cancer testing. Clin Cancer Res; 22(16); 4087-94. ©2016 AACRSee related commentary by Mandelker, p. 3987.

Classification of genetic variants in genes associated with Lynch syndrome using a clinical history weighting algorithm.

BackgroundLynch syndrome is a hereditary cancer syndrome associated with high risks of colorectal and endometrial cancer that is caused by pathogenic variants in the mismatch repair genes (MLH1, MSH2, MSH6, PMS2, EPCAM). Accurate classification of variants identified in these genes as pathogenic or benign enables informed medical management decisions. Previously, we developed a clinical History Weighting Algorithm (HWA) for the classification of variants of uncertain significance (VUSs) in BRCA1 and BRCA2. The BRCA1/2 HWA is based on the premise that pathogenic variants in these genes will be identified more often in individuals with strong personal and/or family histories of breast and/or ovarian cancer, while the identification of benign variants should be independent of cancer history. Here we report the development of a similar HWA to allow for classification of VUSs in genes associated with Lynch syndrome using data collected through both syndrome-specific and pan-cancer panel testing.MethodsUpon completion of algorithm development, the HWA was tested using simulated variants constructed from 79,214 probands, as well as 379 true variants. Positive (PPV) and negative predictive values (NPV) were calculated on a per gene basis.Results25,500 pathogenic and 50,500 benign simulated variants were analyzed using the HWA and the PPVs and NPVs for each gene were greater than 0.997 and 0.999, respectively. The HWA was also evaluated using 100 trials for each of the 379 true variants. PPVs of >0.998 and NPVs of >0.999 were obtained for all genes.ConclusionsWe have developed and implemented a HWA to aid in the classification of VUSs in genes associated with Lynch syndrome. The work presented here demonstrates that this HWA is able to classify MLH1, MSH2, and MSH6 VUSs as either benign or pathogenic with high accuracy.

Mismatch repair analysis of inherited MSH2 and/or MSH6 variation pairs found in cancer patients

Mismatch repair (MMR) malfunction causes the accumulation of mismatches in the genome leading to genomic instability and cancer. The inactivation of an MMR gene (MSH2, MSH6, MLH1, or PMS2) with an inherited mutation causes Lynch syndrome (LS), a dominant susceptibility to cancer. MMR gene variants of uncertain significance (VUS) may be pathogenic mutations, which cause LS, may result in moderately increased cancer risks, or may be harmless polymorphisms. Our study suggests that an inherited MMR VUS individually assessed as proficient may, however, in a pair with another MMR VUS found in the same colorectal cancer (CRC) patient have a concomitant contribution to the MMR deficiency. Here, eight pairs of MMR gene variants found in cancer patients were functionally analyzed in an in vitro MMR assay. Although the other pairs do not suggest a compound deficiency, the MSH2 VUS pair c.380A>G/c.982G>C (p.Asn127Ser/p.Ala328Pro), which nearly halves the repair capability of the wild-type MSH2 protein, is presumed to increase the cancer risk considerably. Moreover, two MSH6 variants, c.1304T>C (p.Leu435Pro) and c.1754T>C (p.Leu585Pro), were shown to be MMR deficient. The role of one of the most frequently reported MMR gene VUS, MSH2 c.380A>G (p.Asn127Ser), is especially interesting because its concomitant defect with another variant could finally explain its recurrent occurrence in CRC patients.