Variants Of Unknown Significance In Genes Research Articles

Sequence variants of unknown significance in small fiber neuropathy : Characterization of a heterogeneous patient population

In almost half of patients suffering from small fiber neuropathies (SFN), the etiology remains elusive. For these patients with "idiopathic SFN", symptomatic analgesic therapy is the only option. Reports on apotential genetic background of neuropathic pain syndromes are increasing and particularly in SFN patients, several genetic variants were found mainly located in genes encoding voltage-gated sodium channels. Although up to 30% of SFN patients show genetic alterations, most of these remain of "unknown pathogenic significance" and little is known about "genetic SFN". The study aimed to determine clinical characteristics of SFN patients carrying arare genetic variant of unknown significance in pain-associated genes. From 2015 to 2020, 66patients with primarily idiopathic SFN were examined and rare gene variants of unknown significance detected in 13/66 (20%) of these. Adetailed medical history with focus on pain was recorded and patients filled in standardized questionnaires to assess physical and emotional burden due to pain. The authors found 13/66 (20%) patients with rare variants of unknown significance located in pain-associated genes who reported pain refractory to analgesic treatment, ahigher number of external factors influencing clinical symptoms, and ahigher level of physical impairment and emotional stress due to pain compared with patients without such genetic variants. Early genetic assessment is recommended to optimize the management of patients with potentially hereditary SFN. Early access to rehabilitation and mental support as well as aconsequent elimination of external triggering factors should be granted.

Abstract 18478: Association of Family History and Cardiomyopathy-Associated Gene Variants of Unknown Significance in Ethnic Minority Patients With Early Onset Atrial Fibrillation

Introduction: Underrepresentation of ethnic minorities in genetic studies can complicate interpretation of variants of unknown significance (VUS) in these groups. Likely pathogenic or pathogenic (LP/P) cardiomyopathy (CM) gene variants are associated with early onset-atrial fibrillation (EOAF) in a cohort of predominantly European ancestry, but prevalence in minorities, and the role of VUS, is unclear. Three-generation family history (FH) may provide insight into the potential pathogenicity of VUS in ethnic minorities. Hypothesis: In patients with EOAF and low comorbidity burden carrying CM VUS, does positive FH suggest variant pathogenicity? Aims: To determine whether CM gene VUS in ethnic minority probands with EOAF are associated with a FH of arrhythmia, stroke, or heart failure. Methods: Of 301 individuals from the University of Illinois Health Multi-Ethnic AF Registry who underwent whole exome sequencing, we identified 89 with either LP/P variant, positive FH from medical records, or AF diagnosis age ≤55 years and ≤2 comorbidities. We examined 22 genes strongly associated with CM and graded pathogenicity according to American College of Medical Genetics criteria. A three-generation FH was obtained by contacting probands or family members. Results: A FH was obtained in 36 patients (Figure 1a; median age 52 years, 56% female, 83% African-American, 17% Hispanic/Latinx). LP/P variants were present in 3 (8%) probands, VUS in 10 (28%), and likely benign/benign (LB/B) or no variant in 23 (64%). A FH of arrhythmia, heart failure, or stroke was present in 27 subjects (75%) with a FH of heart failure more likely in VUS carriers compared to those with LB/B or no variant (70% vs 26%, p=0.02), with no difference in FH of arrhythmia or stroke. Conclusion: Probands with EOAF who carry CM VUS have greater rates of FH of heart failure than those without disease-associated variants. This suggests pathogenicity of CM VUS in ethnic minorities with EOAF.

Genetic testing outcome disparities in minority racial and ethnic sub-populations within a hereditary breast and ovarian cancer program serving a diverse, urban-based population (191)

Genetic testing outcome disparities in minority racial and ethnic sub-populations within a hereditary breast and ovarian cancer program serving a diverse, urban-based population (191)

Feasibility of Follow-Up Studies and Reclassification in Spinocerebellar Ataxia Gene Variants of Unknown Significance.

Spinocerebellar ataxia (SCA) is a heterogeneous group of neurodegenerative disorders with autosomal dominant inheritance. Genetic testing for SCA leads to diagnosis, prognosis and risk assessment for patients and their family members. While advances in sequencing and computing technologies have provided researchers with a rapid expansion in the genetic test content that can be used to unravel the genetic causes that underlie diseases, the large number of variants with unknown significance (VUSes) detected represent challenges. To minimize the proportion of VUSes, follow-up studies are needed to aid in their reclassification as either (likely) pathogenic or (likely) benign variants. In this study, we addressed the challenge of prioritizing VUSes for follow-up using (a combination of) variant segregation studies, 3D protein modeling, in vitro splicing assays and functional assays. Of the 39 VUSes prioritized for further analysis, 13 were eligible for follow up. We were able to reclassify 4 of these VUSes to LP, increasing the molecular diagnostic yield by 1.1%. Reclassification of VUSes remains difficult due to limited possibilities for performing variant segregation studies in the classification process and the limited availability of routine functional tests.

Gene variants of unknown significance in Fabry disease: Clinical characteristics of c.376A>G (p.Ser126Gly).

BackgroundAnderson–Fabry disease (FD) is an X‐linked lysosomal storage disorder with varying organ involvement and symptoms, depending on the underlying mutation in the alpha‐galactosidase A gene (HGNC: GLA). With genetic testing becoming more readily available, it is crucial to precisely evaluate pathogenicity of each genetic variant, in order to determine whether there is or might be not a need for FD‐specific therapy in affected patients and relatives at the time point of presentation or in the future.MethodsThis case series investigates the clinical impact of the specific GLA gene variant c.376A>G (p.Ser126Gly) in five (one heterozygous and one homozygous female, three males) individuals from different families, who visited our center between 2009 and 2021. Comprehensive neurological, nephrological and cardiac examinations were performed in all cases. One patient received a follow‐up examination after 12 years.ResultsIndex events leading to suspicion of FD were mainly unspecific neurological symptoms. However, FD‐specific biomarkers, imaging examinations (i.e., brain MRI, heart MRI), and tissue‐specific diagnostics, including kidney and skin biopsies, did not reveal evidence for FD‐specific symptoms or organ involvement but showed normal results in all cases. This includes findings from 12‐year follow‐up in one patient with renal biopsy.ConclusionThese findings suggest that p.Ser126Gly represents a benign GLA gene variant which per se does not cause FD. Precise clinical evaluation in individuals diagnosed with genetic variations of unknown significance should be performed to distinguish common symptoms broadly prevalent in the general population from those secondary to FD.

25. Large scale analysis in Von Hippel-Lindau disease

Introduction: Von Hippel-Lindau (VHL) disease is a rare hereditary cancer syndrome where individuals are at risk of developing tumors in many organs, including the brain, adrenal gland, pancreas and kidneys. It is caused by deleterious mutations in the VHL tumor suppressor gene, although gene variants of unknown significance are often seen in clinical practice. The diversity of VHL patients has made it difficult to collect standardized disease information.

Identification of ZBTB26 as a Novel Risk Factor for Congenital Hypothyroidism.

Congenital primary hypothyroidism (CH; OMIM 218700) is characterized by an impaired thyroid development, or dyshormonogenesis, and can lead to intellectual disability and growth retardation if untreated. Most of the children with congenital hypothyroidism present thyroid dysgenesis, a developmental anomaly of the thyroid. Various genes have been associated with thyroid dysgenesis, but all known genes together can only explain a small number of cases. To identify novel genetic causes for congenital hypothyroidism, we performed trio whole-exome sequencing in an affected newborn and his unaffected parents. A predicted damaging de novo missense mutation was identified in the ZBTB26 gene (Zinc Finger A and BTB Domain containing 26). An additional cohort screening of 156 individuals with congenital thyroid dysgenesis identified two additional ZBTB26 gene variants of unknown significance. To study the underlying disease mechanism, morpholino knock-down of zbtb26 in Xenopus laevis was carried out, which demonstrated significantly smaller thyroid anlagen in knock-down animals at tadpole stage. Marker genes expressed in thyroid tissue precursors also indicated a specific reduction in the Xenopus ortholog of human Paired-Box-Protein PAX8, a transcription factor required for thyroid development, which could be rescued by adding zbtb26. Pathway and network analysis indicated network links of ZBTB26 to PAX8 and other genes involved in thyroid genesis and function. GWAS associations of ZBTB26 were found with height. Together, our study added a novel genetic risk factor to the list of genes underlying congenital primary hypothyroidism and provides additional support that de novo mutations, together with inherited variants, might contribute to the genetic susceptibility to CH.

Predicting Ovarian/Breast Cancer Pathogenic Risks of Human BRCA1 Gene Variants of Unknown Significance.

High-throughput sequencing is gaining popularity in clinical diagnoses, but more and more novel gene variants with unknown clinical significance are being found, giving difficulties to interpretations of people's genetic data, precise disease diagnoses, and the making of therapeutic strategies and decisions. In order to solve these issues, it is of critical importance to figure out ways to analyze and interpret such variants. In this work, BRCA1 gene variants with unknown clinical significance were identified from clinical sequencing data, and then, we developed machine learning models so as to predict the pathogenicity for variants with unknown clinical significance. Through performance benchmarking, we found that the optimized random forest model scored 0.85 in area under receiver operating characteristic curve, which outperformed other models. Finally, we applied the best random forest model to predict the pathogenicity of 6321 BRCA1 variants from both sequencing data and ClinVar database. As a result, we obtained the predictive pathogenic risks of BRCA1 variants of unknown significance.

The Postmortem Interpretation of Cardiac Genetic Variants of Unknown Significance in Sudden Death in the Young: A Case Report and Review of the Literature.

Sudden cardiac death (SCD) in adolescents and young adults is a major traumatic event for families and communities. In these cases, it is not uncommon to have a negative autopsy with structurally and histologically normal heart. Such SCD cases are generally attributed to channelopathies, which include long QT syndrome, short QT syndrome, Brugada syndrome, and catecholaminergic polymorphic ventricular tachycardia. Our understanding of the causes for SCDs has changed significantly with the advancements in molecular and genetic studies, where many mutations are now known to be associated with certain channelopathies. Postmortem analysis provides great value in informing decision-making with regard to screening tests and prophylactic measures that should be taken to prevent sudden death in first degree relatives of the decedent. As this is a rapidly advancing field, our ability to identify genetic mutations has surpassed our ability to interpret them. This led to a unique challenge in genetic testing called variants of unknown significance (VUS). VUSs present a diagnostic dilemma and uncertainty for clinicians and patients with regard to next steps. Caution should be exercised when interpreting VUSs since misinterpretation can result in mismanagement of patients and their families. A case of a young adult man with drowning as his proximate cause of death is presented in circumstances where cardiac genetic testing was indicated and undertaken. Eight VUSs in genes implicated in inheritable cardiac dysfunction were identified and the interpretation of VUSs in this scenario is discussed.

Sorting Rare ALS Genetic Variants by Targeted Re-Sequencing Panel in Italian Patients: OPTN, VCP, and SQSTM1 Variants Account for 3% of Rare Genetic Forms.

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disease due to motor neuron loss variably associated with frontotemporal dementia (FTD). Next generation sequencing technology revealed an increasing number of rare and novel genetic variants and interpretation of their pathogenicity represents a major challange in the diagnosis of ALS. We selected 213 consecutive patients with sporadic or familial (16%) ALS, tested negative for SOD1, FUS, TARDBP, and C9orf72 mutations. To reveal rare forms of genetic ALS, we performed a comprehensive multi-gene panel screening including 46 genes associated with ALS, hereditary motor neuronopathies, spastic paraplegia, and FTD. Our study allowed the identification of pathogenic or likely pathogenic variants in 4.2% of patients. The genes with the highest percentage of pathogenic variants were OPTN (1%), VCP (1%) SQSTM1(1%), SETX (0.4%), FIG4 (0.4%), and GARS1 (0.4%) genes. We also found 49 novel or rare gene variants of unknown significance in 30 patients (14%), 44 unlikely pathogenic variants (39%), and 48 variants in ALS susceptibility genes. The results of our study suggest the screening of OPTN, VCP, and SQSTM1 genes in routine diagnostic investigations for both sporadic and familial cases, and confirm the importance of diagnosis and couselling for patients and their relative family members.

Prevalence of Inherited Cancer Predisposition Mutations in a Cohort of Older AML Patients Enrolled on the Beat AML Master Trial

Prevalence of Inherited Cancer Predisposition Mutations in a Cohort of Older AML Patients Enrolled on the Beat AML Master Trial

Disease Modeling and Phenotype Rescue Using Inducible Pluripotent Stem Cells from Patients with Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a congenital ribosomopathy and bone marrow failure syndrome manifesting typically in infancy with erythroid hypoplasia. Approximately half of affected individuals also have developmental anomalies. Over time, additional cytopenias can develop, including reduced hematopoietic stem and progenitor cells (HSPC). Heterozygous loss-of-function mutations in over 20 ribosomal protein (RP) genes cause approximately 70% of DBA cases, although only 7 genes (RPS19, RPL5, RPS26, RPL11, RPL35a, RPS24 and RPS7) account for over 90% of patients with a known DBA genotype. Medical therapies including steroids, chronic transfusions are partially effective but have considerable side effects. Hematopoietic stem cell transplantation (HSCT) from matched related or unrelated donors is curative with recently reported good outcomes, although many patients lack a suitable donor and/or have serious treatment-related comorbidities that increase HSCT-related toxicities. Case reports of spontaneous genetic reversion in DBA suggest that RP gene-corrected HSPC have competitive advantage over RP-deficient cells, thus providing the rationale for gene therapy as a feasible therapeutic approach. Induced pluripotent stem cell (iPSC) technology provides a robust model of human disease and can recapitulate hematopoietic defects encountered in bone marrow failure syndromes. The goals of this study was to establish a culture system from patient-derived iPSCs that can recapitulate key aspects of DBA pathophysiology and provide a preclinical model for gene manipulation to correct the abnormal phenotype (Figure 1). We developed iPSCs from individuals with DBA who were enrolled on INSIGHT (NCT02720679), an IRB-approved, prospective study that includes biobanking of peripheral blood mononuclear cells (PBMNC) from patients with bone marrow failure syndromes. We first reprogrammed these DBA PBMNC into iPSCs using non-integrating Sendai virus to establish lines with pathogenic mutations in RPS19 (c.191>T, p.Leu64Pro), RPS19 (c.184C>T, p.Arg62Trp), RPL11 (c.61dupT, p.Cys21Leufs*13), and a variant of uncertain significance (VUS) in RPS7 (c.277_279delGTC, pVal93del). Undifferentiated iPSC lines exhibited abnormal ribosomal biogenesis revealed by polysome profiling and pre-rRNA analysis. Upon in vitrodifferentiation to hematopoietic lineages, the mutant iPSCs recapitulated DBA phenotypes with reduced CD34+ HSPCs, near absence of erythroid colonies (BFU-E and CFU-E) colonies and failure to produce erythroid cells in liquid culture. We used two methods to correct single nucleotide RP mutations in DBA iPSCs (Figure 1): i) CRISPR/Cas9-mediated homology-directed repair, and ii) base-editing, which utilizes catalytically inactive Cas9 fused to a deaminase that interconverts nucleotides directly in the absence of double-stranded DNA breaks. Corrected "isogenic" lines showed phenotype similar to wild type controls, with restored erythroid differentiation, and normal polysome maturation and pre-rRNA ratios. Because some patients carry large intragenic or whole RP gene deletions that are not amenable to gene correction, we also explored the feasibility of gene rescue by inserting a wild type copy of the defective gene (Figure 1). Using zinc-finger nuclease (ZFN), we inserted wild type RP cDNA constructs into the "safe harbor" AAVS1 locus on chromosome 19, thereby rescuing abnormal phenotypes of patient-derived iPSC lines with RPS19(p.Arg62Trp) and RPL11(p.Cys21Leufs*34) mutations. Additionally, we explored lentiviral gene delivery as an alternative method for RP gene replacement. We compared different promoters including MND, PGK and EF1a and found that the latter was most effective at rescuing RP gene expression in iPSC cells. Transduction of lentiviral vectors with wild type RPS19or RPL11fused to the EF1a promoter into three iPSC lines with RPS19or RPL11mutations resulted in stable transgene expression of RPS19or RPL11genes and phenotypic rescue. This study supports the feasibility of establishing iPSCs from DBA subjects with different genotypes. These iPSC lines provide a useful resource for numerous studies of DBA including preclinical approaches to gene therapy, evaluating the pathogenicity of RP gene variants of unknown significance and examining the pathophysiology of RP haploinsufficiency. Disclosures Estepp: Esperion: Consultancy; Forma Therapeutics: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Pfizer: Research Funding; Eli Lilly and Co: Research Funding; Daiichi Sankyo: Consultancy. Weiss:GlaxoSmithKline: Consultancy; Rubius Inc.: Consultancy; Cellarity Inc.: Consultancy; Beam Therapeutics: Consultancy; Esperion: Consultancy.

A method of high-throughput functional evaluation of EGFR gene variants of unknown significance in cancer.

Numerous variants of unknown significance (VUS) have been identified through large-scale cancer genome projects, although their functional relevance remains uninvestigated. We developed a mixed-all-nominated-mutants-in-one (MANO) method to evaluate the transforming potential and drug sensitivity of oncogene VUS in a high-throughput manner and applied this method to 101 nonsynonymous epidermal growth factor receptor (EGFR) mutants. We discovered a number of mutations conferring resistance to EGFR tyrosine kinase inhibitors (TKIs), including gefitinib- and erlotinib-insensitive missense mutations within exon 19 and other gefitinib-resistant mutations, such as L833V, A839T, V851I, A871T, and G873E. L858R-positive tumors (12.8%) harbored compound mutations primarily in the cis allele, which decreased the gefitinib sensitivity of these tumors. The MANO method further revealed that some EGFR mutants that are highly resistant to all types of TKIs are sensitive to cetuximab. Thus, these data support the importance of examining the clinical relevance of uncommon mutations within EGFR and of evaluating the functions of such mutations in combination. This method may become a foundation for the in vitro and in vivo assessment of variants of cancer-related genes and help customize cancer therapy for individual patients.

Management of Gene Variants of Unknown Significance: Analysis Method and Risk Assessment of the VHL Mutation p.P81S (c.241C>T).

Background: Evaluation of the pathogenicity of a gene variant of unknown significance (VUS) is crucial for molecular diagnosis and genetic counseling, but can be challenging. This is especially so in phenotypically variable diseases, such as von Hippel-Lindau disease (vHL). vHL is caused by germline mutations in the VHL gene, which predispose to the development of multiple tumors such as central nervous system hemangioblastomas and renal cell carcinoma (RCC).Objective: We propose a method for the evaluation of VUS pathogenicity through our experience with the VHL missense mutation c.241C>T (p.P81S).Method: 1) Clinical evaluation of known variant carriers: We evaluated a family of five VHL p.P81S carriers, as well as the clinical characteristics of all the p.P81S carriers reported in the literature; 2) Evaluation of tumor tissue via genetic analysis, histology, and immunohistochemistry (IHC); 3) Assessment of the variant’s impact on protein structure and function, using multiple databases, in silico algorithms, and reports of functional studies.Results: Only one family member had clinical signs of vHL with early-onset RCC. IHC analysis showed no VHL protein expressed in the tumor, consistent with biallelic VHL inactivation. The majority of in silico algorithms reported p.P81S as possibly pathogenic in relation to vHL or RCC, but there were discrepancies. Functional studies suggest that p.P81S impairs the VHL protein’s function.Conclusion: The VHL p.P81S mutation is most likely a low-penetrant pathogenic variant predisposing to RCC development. We suggest the above-mentioned method for VUS evaluation with use of different methods, especially a variety of in silico methods and tumor tissue analysis.

The role of testing for BRCA1 and BRCA2 mutations in cancer prevention.

The role of testing for BRCA1 and BRCA2 mutations in cancer prevention.

End‐Recovery QTc: A Useful Metric for Assessing Genetic Variants of Unknown Significance in Long‐QT syndrome

Genetic variants represent benign single-nucleotide polymorphisms, disease causing mutations or variants of unknown significance (VUS). Resting, exercise, and recovery QTc intervals have been utilized to detect long-QT syndrome (LQTS) mutations. We sought to provide clinical data that may assist in classifying the presented VUS as disease causing/benign and to determine whether resting and/or end-recovery QT parameters can evaluate the significance of VUS. Twenty-six patients with a VUS in genes associated with LQTS (15 females, age 38 ± 16 years) and 26 age and gender matched controls (age 37 ± 20 years) were included. There were 10 VUS (5 KCNQ1, 4 KCNH2, 1 KCNE1) in 12 families. All but 1 VUS was associated with sudden cardiac death (SCD), aborted SCD or Torsade de pointes. A Schwartz score of ≥3.5 was observed in at least 1 family member with each VUS. Resting QTc was marginally longer in VUS patients compared with controls (458 ± 48 vs 437 ± 25, P = 0.052). A prolonged resting QTc (>470 ms males, >480 ms females) identified 6 VUS carriers and 1 control. VUS carriers had a substantially longer end-recovery QTc (502 ± 68 vs 427 ± 17, P < 0.01) with an end-recovery QTc > 445 ms in 20/26 VUS patients compared to 2/26 controls (P < 0.01). The area under the receiver operating characteristic curve for resting QTc was 0.68 (95% CI, 0.53-0.83, P = 0.03) compared to the end-recovery QTc of 0.88 (95% CI, 0.76-0.99, P < 0.0001). Variants in the current study appear to be disease causing. The end-recovery QTc is a useful metric when interpreting LQT VUS.

Interobserver variability in the evaluation of mismatch repair protein immunostaining

Immunohistochemical staining for mismatch repair proteins has during recent years been established as a routine analysis in many pathology laboratories with the aim to identify tumors linked to the hereditary nonpolyposis colorectal cancer syndrome. Despite widespread application, data on reliability are lacking. We therefore evaluated interobserver variability among 6 pathologists, 3 experienced gastrointestinal pathologists and 3 residents. In total, 225 immunohistochemically stained colorectal cancers were evaluated as having normal, weak, loss of, or nonevaluable mismatch repair protein staining. Full consensus was achieved in 51% of the stainings for MLH1, 61% for PMS2, 83% for MSH2, and 45% for MSH6. Weak stainings were the main cause of reduced consensus, whereas contradictory evaluations with normal as well as loss of staining were reported in 2% to 6% of the tumors. Interobserver variability was considerable, though experienced pathologists and residents reached the same level of consensus. Because results from immunohistochemical mismatch repair protein stainings are used for decisions on mutation analysis and as an aid in the interpretation of gene variants of unknown significance in hereditary nonpolyposis colorectal cancer, the interobserver variability identified highlights the need for quality assessment programs, including guidelines for classification of different expression patterns.