Pathogenicity Of Variants Research Articles

Abstract 4136581: Shared Genetic Burden of Ischemic and Non-Ischemic Dilated Cardiomyopathy

Introduction: Ischemic dilated cardiomyopathy (IDCM) is defined as DCM caused by severe stenosis of one or more coronary arteries. Patients with non-ischemic dilated cardiomyopathy (NIDCM) have normal coronary arteries and may possess rare disease-causing variants in cardiac proteins such as TTN . However, IDCM and NIDCM share echocardiographic and pathologic features, possibly suggesting a shared etiology. Hypothesis: There is a similar prevalence of pathogenic (P)/likely pathogenic (LP) variants in patients with IDCM compared to patients with NIDCM. Aims: We compared the prevalence of P/LP genetic variants in DCM-associated genes in a cohort of patients with NIDCM or IDCM who received a heart transplant. Methods: We conducted a retrospective cohort study of patients who received a heart transplant between May 2017 and October 2023 at Cedars Sinai Medical Center in Los Angeles, CA. All subjects had LVEF ≤ 50% and LVEDD ≥ 95 th percentile for sex and height. Whole exome sequencing was performed on explanted heart tissue. We assessed for rare variants (minor allele frequency <0.1%) in genes with a definite, strong, or moderate rating in the ClinGen DCM Gene Curation Expert Panel. Variant pathogenicity was interpreted using the American College of Genetics and Genomics criteria for DCM. Ischemic cardiomyopathy was defined by the Felker criteria (Felker et al., JACC 2002). Results: We included 60 subjects: 16 with IDCM and 44 with NIDCM. There was no difference in LVEF or LVEDD between groups (TABLE). 2/16 (12.5%) patients with IDCM carried a P/LP variant, both of which were in TTN . 11/44 (25.0%) patients with NIDCM carried a P/LP variant: 6 in TTN and 1 each in FLNC, ILK, LMNA, MYH7 , and RBM20 . The prevalence of TTN variants in each group was similar (12.5% in IDCM vs. 13.6% in NIDCM) and higher than the expected frequency in the general population (0.2%). There was no significant difference in the prevalence of P/LP variants between the groups (chi-squared p-value 0.493, Bayes factor 0.45). Conclusion: Patients with IDCM exhibited a similar prevalence of P/LP variants to NIDCM patients, which suggests a shared genetic burden. If further studies confirm our findings, genetic testing for individuals with IDCM may be warranted.

Abstract 4127919: Disease-associated variants in DSP -encoded desmoplakin are common, yet low penetrant alleles associated with development of myocarditis and cardiomyopathy at a population level

Intro: DSP- encoded desmoplakin is implicated in a distinct form of arrhythmic cardiomyopathy (ACM), often involving the LV and characterized by myocardial inflammation. Conventional ACM diagnostic criteria has poor sensitivity for DSP -associated ACM, thus highlighting the need to identify the full spectrum of phenotypic risk associated with disease-associated DSP variants. Objective: To analyze the genotypic and phenotypic spectrum of DSP -mediated disease at a large population level. Methods: UK Biobank participants with exome sequencing (ES) were included. Variants were filtered by gene evidence category, yielding 3 groups: predicted deleterious (pDel); subset with predicted loss of function (LOF); and ultra-filtered subset by ClinVar 2* pathogenic/likely pathogenic (P/LP). Phenotypic penetrance was analyzed with DSP -neg individuals as control. Variant location analysis assessed LOFs by susceptibility to nonsense mediated decay region and missenses by amino acid sequence to identify mutational hotspots where P/LPs localize. Results: Out of 200,580 with ES, 1407 DSP carriers had pDel, 168 with LOF, and 44 with ClinVar 2* P/LP. DSP carriers had higher burden of myocarditis, cardiomyopathy (CM), and heart failure. A progressive enrichment in myocarditis and CM was observed by more stringent variant filtering in DSP carriers compared to control. A higher proportion of DSP carriers had myocarditis – 0.28% (4) of the pDels, 1.8% (3) of LOFs, and 4.5% (2) compared to control 0.07% (p<0.05). A parallel trend noted the proportion of DSP carriers with CM growing from 1.4% (19) to 5.4% (9) and 6.8% (3) in the pDel, LOF, and ClinVar 2* P/LPs respectively compared to 0.6% in the control group (p<0.01). Location analysis of missense variants revealed that P/LPs localized in higher proportions (74%) to key domains compared to non-P/LPs (50%; p<0.001), highlighting prognostic value of predicting pathogenicity of missense DSP variants by mutation hotspots. Conclusions: While DSP variants are relatively common in the population, variant presence is associated with low penetrance. Enhancing clinical penetrance with increasingly stringent filtering of DSP variants emphasizes the importance of variant evaluation in clinical risk assessment. This first-in-kind population study of DSP provides insights into novel risk prediction factors, allowing us to predict pathogenicity by type and location of mutation and using variant filtering strategy to better assess phenotypic penetrance.

Abstract 4120763: Predicting Disease Risk Among Variants in Cardiomyopathy-Associated Genes Across Diverse Genetic Ancestries

Background: Many cases of cardiomyopathy (CM) are genetically inherited, and the expansion of broad genomic sequencing has opened the door for predicting those at risk for developing disease. However, genetic tools that help determine variant pathogenicity employ genetic data from predominantly European genetic ancestries. Understanding the full diversity of genetic variants in cardiomyopathy genes is central to identifying which variants will cause disease. Hypothesis: Participants of non-European genetic ancestry will have a higher burden of cardiomyopathy gene variants with "VUS" designation in ClinVar but will have equivalent/less penetrance of clinical cardiomyopathy compared to a European ancestry group. Methods: Participants in the AllofUs database with exome sequencing (ES) were queried and a cohort of 230,013 participants were retained after sample quality control filters. Participant genetic ancestry was based on the designated ancestry group determined via AllofUs as previously described (1). Participants were queried for variants in genes with definitive or moderate evidence of hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic cardiomyopathy (ACM) in ClinGen. Variants were filtered by ClinVar status (1* and above P/LP, 1* and above B/LB, and VUS). Participant CM disease status was determined via ICD-9/10 code. Results: For each ancestry group, prevalence of cardiomyopathy ranged from 0.34% in the Latino/admixed American ancestry group to 1.09% in the African group, with an average prevalence of 0.91% across all groups. The European group had a statistically significant lower percentage of ClinVar VUS variants at 26%, while all other non-European ancestry groups had a combined VUS prevalence rate of 35% (****p<0.0001). The penetrance of CM in participants with CM VUS variants was lower in combined non-European ancestry groups (0.84%) than European ancestry (1.02%) (*p=0.0156). Conclusion: Prevalence of ClinVar VUS variants varies across different genetic ancestry groups, with European ancestry having a lower burden of VUS variants compared to non-European groups. However, non-European ancestry groups with increased burden of VUS variants have lower disease penetrance. Citations: (1) The All of Us Research Program Genomics Investigators. Genomic data in the All of Us Research Program. Nature 627 , 340–346 (2024). https://doi.org/10.1038/s41586-023-06957-x

Assessing the Utility of ColabFold and AlphaMissense in Determining Missense Variant Pathogenicity for Congenital Myasthenic Syndromes

Background/Objectives: Congenital myasthenic syndromes (CMSs) are caused by variants in >30 genes with increasing numbers of variants of unknown significance (VUS) discovered by next-generation sequencing. Establishing VUS pathogenicity requires in vitro studies that slow diagnosis and treatment initiation. The recently developed protein structure prediction software AlphaFold2/ColabFold has revolutionized structural biology; such predictions have also been leveraged in AlphaMissense, which predicts ClinVar variant pathogenicity with 90% accuracy. Few reports, however, have tested these tools on rigorously characterized clinical data. We therefore assessed ColabFold and AlphaMissense as diagnostic aids for CMSs, using variants of the CHRN genes that encode the nicotinic acetylcholine receptor (nAChR). Methods: Utilizing a dataset of 61 clinically validated CHRN variants, (1) we evaluated the possibility of a ColabFold metric (either predicted structural disruption, prediction confidence, or prediction quality) that distinguishes variant pathogenicity; (2) we assessed AlphaMissense’s ability to differentiate variant pathogenicity; and (3) we compared AlphaMissense to the existing pathogenicity prediction programs AlamutVP and EVE. Results: Analyzing the variant effects on ColabFold CHRN structure prediction, prediction confidence, and prediction quality did not yield any reliable pathogenicity indicative metric. However, AlphaMissense predicted variant pathogenicity with 63.93% accuracy in our dataset—a much greater proportion than AlamutVP (27.87%) and EVE (28.33%). Conclusions: Emerging in silico tools can revolutionize genetic disease diagnosis—however, improvement, refinement, and clinical validation are imperative prior to practical acquisition.

Investigating the role of non-synonymous variant D67N of ADGRE2 in chronic myeloid leukemia

BackgroundChronic myeloid leukaemia (CML) is a type of blood cancer that begins in the hematopoietic stem cells. It is primarily characterized by a specific chromosomal aberration, the Philadelphia chromosome. While the fusion gene is a major contributor to CML, several other genes including ADGRE2, that are reported as highly expressed in hematopoietic stem cells and could be utilized as a therapeutic marker in leukemic patients are implicated in the disease’s progression. Until recently, little research had been conducted to identify single nucleotide polymorphisms (SNPs) associated with CML. Therefore, this study aims to investigate the influence of non-synonymous variants on the structure and function of the gene encoding adhesion G protein-coupled receptor E2, ADGRE2, and to evaluate their association with CML and its clinical and pathological characteristics.MethodsNon-synonymous SNPs of ADGRE2 were retrieved from the ENSEMBL, COSMIC, and gnomAD genome browsers, and the pathogenicity of deleterious variants was assessed using several established computational tools, including SIFT, CADD, REVEL, PolyPhen, and MetaLR.ResultsVarious in silico analyses explored the impact of damaging SNP on the function, stability, and structure of EGF-like modules containing mucin-like hormone receptor-like2 (EMR2) protein encoded by the ADGRE2 gene. Genotype analysis was performed on collected blood samples, revealing that altered genotype TT of variant rs765071211 (C/T) was associated significantly with CML patients compared to the control. Further in vitro and in vivo analyses suggest that this SNP holds potential for clinical translation.

Molecular and Functional Assessment of TSC1 and TSC2 in Individuals with Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is an autosomal dominant neurodevelopmental disorder and multisystem disease caused by pathogenic DNA alterations in the TSC1 and TSC2 tumor suppressor genes. A molecular genetic diagnosis of TSC confirms the clinical diagnosis, facilitating the implementation of appropriate care and surveillance. TSC1 and TSC2 encode the core components of the TSC, a negative regulator of the mechanistic target of rapamycin (MTOR) complex 1 (TORC1). Functional analysis of the effects of TSC1 and TSC2 variants on TORC1 activity can help establish variant pathogenicity. We searched for pathogenic alterations to TSC1 and TSC2 in DNA isolated from 116 individuals with a definite clinical diagnosis of TSC. Missense variants and in-frame deletions were functionally assessed. Pathogenic DNA alterations were identified in 106 cases (91%); 18 (17%) in TSC1 and 88 (83%) in TSC2. Of these, 35 were novel. Disruption of TSC activity was demonstrated for seven TSC2 variants. Molecular diagnostics confirms the clinical diagnosis of TSC in a large proportion of cases. Functional assessment can help establish variant pathogenicity and is a useful adjunct to DNA analysis.

The cumulative effect of compound heterozygous variants in TRPV3 caused Olmsted syndrome

BackgroundOlmsted syndrome (OS) is a rare genodermatosis predominantly inherited in an autosomal dominant manner, typically arising from gain-of-function (GOF) variants in the transient receptor potential channel vanilloid 3 (TRPV3). ObjectiveThis study aims to investigate potential mechanisms underlying OS in two cases presenting with an autosomal recessive inheritance pattern. MethodsNext-generation sequencing panel was employed to identify TRPV3 variants. TRPV3 plasmids carrying specific point variations were generated and transiently transfected into HEK293T cells. Electrophysiological patch-clamp techniques were utilized to record voltage-activated and ligand-activated currents. Celltiter-Glo luminescent assay was employed to analyze the cell viabilities. ResultsCompound heterozygous variants, c.1563G>C (p.W521C) and c.1376C>T (p.S459L), as well as c.1773G>C (p.L591F) and c.2186G>A (p.R729Q), were identified in the two OS patients respectively. Electrophysiological analysis of ligand-induced activation of TRPV3 variants demonstrated the closest correlation with clinical manifestations. All four variants displayed GOF channel activity characterized by increased sensitivity. Notably, W521C and L591F exhibited both heightened sensitivity and lower EC50 values for the TRPV3 agonist. Co-transfection with wild-type TRPV3 plasmids significantly rescued these effects. Cells co-transfected with the corresponding compound heterozygous variants exhibited intermediate electrophysiological characteristics. ConclusionsIn this study, we present two cases of OS by autosomal-recessive inheritance of TPRV3 variants. This study presents a notable observation of compound heterozygous GOF variants in TRPV3, highlighting their cumulative impact on clinical manifestations. Additionally, we advocate for the use of ligand-dependent ion channel activity assays to assess the pathogenicity of TRPV3 variants in OS.

Updating probability of pathogenicity for RYR1 and CACNA1S exon variants in individuals without malignant hyperthermia after exposure to triggering anesthetics

Objectives We aimed to classify genetic variants in RYR1 and CACNA1S associated with malignant hyperthermia using biobank genotyping data in patients exposed to triggering anesthetics without malignant hyperthermia phenotype. Methods We identified individuals who underwent surgery and were exposed to triggering anesthetics without malignant hyperthermia phenotype and who had RYR1 or CACNA1S genotyping data available in our biobank. We classified all variants in the cohort using a Bayesian framework of the American College of Medical Genetics and Genomics and the Association of Molecular Pathologists guidelines for variant classification and updated the posterior probabilities from this model with the new information from our biobank cohort. Results We identified 253 patients with 95 RYR1 variants and 12 CACNA1S variants. After applying a Bayesian framework, we classified 17 variants as benign (B), 31 as likely benign (LB), 57 as uncertain (VUS), and 2 as likely pathogenic (LP). When we incorporated evidence about unique exposures to malignant hyperthermia triggering anesthetic agents, 48 of 107 (45%) variants were downgraded (9 to B, 37 to LB, and 2 to VUS). Notably, 41 (72%) of 57 VUSs were downgraded to B or LB. When repeat anesthetics in the same individual were counted as one exposure, 42 of 107 (39%) of variants were downgraded (5 to B, 35 to LB, and 2 to VUS). Specifically, 37 (65%) of 57 VUSs were downgraded to LB. Conclusion Deidentified biorepositories linked with anesthetic data offer a new method of integrating clinical evidence into the assessment of variant probability of pathogenicity.

Cardiovascular Disease Pathogenicity Predictor (CVD-PP): A Tissue-Specific In Silico Tool for Discriminating Pathogenicity of Variants of Unknown Significance in Cardiovascular Disease Genes.

Interpretation of variants of uncertain significance (VUSs) remains a challenge in the care of patients with inherited cardiovascular diseases (CVDs); 56% of variants within CVD risk genes are VUS, and machine learning algorithms trained upon large data resources can stratify VUS into higher versus lower probability of contributing to a CVD phenotype. We used ClinVar pathogenic/likely pathogenic and benign/likely benign variants from 47 CVD genes to build a predictive model of variant pathogenicity utilizing measures of evolutionary constraint, deleteriousness, splicogenicity, local pathogenicity, cardiac-specific expression, and population allele frequency. Performance was validated using variants for which the ClinVar pathogenicity assignment changed. Functional validation was assessed using prior studies in >900 identified VUS. The model utility was demonstrated using the Catheterization Genetics cohort. We identified a top-ranked model that accurately prioritized variants for which ClinVar clinical significance had changed (n=663; precision-recall area under the curve, 0.97) and performed well compared with conventional in silico methods. This model (CVD pathogenicity predictor) also had high accuracy in prioritizing VUS with functional effects in vivo (precision-recall area under the curve, 0.58). In Catheterization Genetics, there was a greater burden of higher CVD pathogenicity predictor scored VUS in individuals with dilated cardiomyopathy compared with controls (P=8.2×10-15). Of individuals in Catheterization Genetics who harbored highly ranked CVD pathogenicity predictor VUS meeting clinical pathogenicity criteria, 27.6% had clinical evidence of disease. Variant prioritization using this model increased genetic diagnosis in Catheterization Genetics participants with a known clinical diagnosis of hypertrophic cardiomyopathy (7.8%-27.2%). We present a cardiac-specific model for prioritizing variants underlying CVD syndromes with high performance in discriminating the pathogenicity of VUS in CVD genes. Variant review and phenotyping of individuals carrying VUS of pathogenic interest support the clinical utility of this model. This model could also have utility in filtering variants as part of large-scale genomic sequencing studies.

Prognostic role of gene variants in sudden cardiac death risk associated to paediatric hypertrophic cardiomyopathy

Abstract Introduction Genetic testing plays an important role in diagnosis of hypertrophic cardiomyopathy (HCM), being sarcomeric genes commonly involved, particularly MYBPC3. However, arrhythmic risk related to genotype remains unclear in adults and almost unexplored in children. Hence, current sudden cardiac death (SCD) risk calculators do not include genetic information. Purpose To analyse risk of arrhythmic events associated with causal variants in MYBPC3 respecting to other genes in a paediatric cohort with non-syndromic HCM from a cardiomyopathy referral centre. Methods This retrospective study included patients with non-syndromic HCM 0-18 years old between 2009-2022. Demographic, clinical, and genetic data were collected. Genetic variants pathogenicity followed American College of Medical Genetics criteria. A positive genetic test was considered if a likely pathogenic or pathogenic variant in HCM related genes was found. Considered outcomes were SCD or a major arrhythmic cardiac event (MACE) within 5 years follow-up: recovered SCD, appropriate ICD therapy, or sustained ventricular tachycardia. Some SCD-risk estimation models (in 5 years) as HCM Risk-SCD model for patients >=15 y.o and/or 55 kg, ESC Guidelines paediatric algorithm, and HCM Risk-Kids model were applied. Results 77 patients were diagnosed with non-syndromic HCM. A causal variant was found in 45/73 (62%). No significant differences in cardiac phenotype were found related to genes. 10/77 patients (13%) presented a MACE in 5 years follow-up. A positive genetic study was significantly associated with having an event (p=0.011). Most patients with a positive genetic test harboured MYBPC3 variants (21/45, 47%), however only 2 patients who suffered a MACE had a causal variant in MYBPC3. Those with causal variants in genes other than MYBPC3 presented more and earlier events (p=0.022). Risk of having a MACE during follow-up was higher when presenting causal variants in genes other than MYBPC3: Hazard Ratio 18.5 (95% CI 2.31, 148), p=0.006. Applying HCM Risk-SCD calculator, model had better predictive discrimination when variable "causal variant in gene other than MYBPC3" was added in multivariate analysis, with high-risk category >=6% (C-statistic =0.931 vs 0.844) and intermediate-risk category 4-6% (C-statistic =0.845 vs 0.514). Same occurred with ESC Guidelines algorithm, adding that variable to 2 risk factors category (C-statistic =0.881 vs 0.742) or 1 risk factor (C-statistic =0.844 vs 0.635). HCM Risk-Kids also improved its predictive discrimination adding the genetic value to high-risk category >=6% (C-statistic =0.896 vs 0.796) and intermediate-risk (C-statistic =0.821 vs 0.506). Conclusions Genotype may have prognostic value in SCD risk in HCM in children. Our results suggests that causal genetic variants in genes other than MYBPC3 can lead to higher risk of SCD. Genetic information might be considered for future models of arrhythmic risk in children with HCM.

Phenotype in Individuals with Heterozygous Rare Variants in LIPC Encoding Hepatic Lipase.

Hepatic lipase deficiency is a rare genetic condition caused by biallelic loss-of-function variants in the LIPC gene encoding hepatic lipase. These variants reduce or abolish the protein's lipolytic activity, resulting in elevated plasma lipids. The condition is classified as autosomal recessive, since dyslipidemia is inconsistently observed in heterozygous patients with only one LIPC variant. However, this has been concluded from historical studies encompassing a few families and having very small sample sizes. Here, we conduct a retrospective chart review of 46 heterozygous subjects, each harboring one rare pathogenic LIPC variant. We compare plasma levels of total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides, and apolipoprotein B to those of matched controls without LIPC variants. Variant pathogenicity is classified according to the guidelines of the American College of Medical Genetics and Genomics. We observe that levels of total cholesterol, LDL-C, and triglycerides are significantly elevated in the LIPC variant heterozygotes, but HDL-C and apolipoprotein B are not. When filtering solely with respect to pathogenic or likely pathogenic variants, all lipid variables emerge as significantly elevated compared to controls. Thus, hepatic lipase deficiency may not necessarily be a recessive condition, but perhaps semi-dominant since individuals with one variant are phenotypically distinct from the controls. These hypothesis-generating findings regarding LIPC genetic variations observed in a clinical cohort should be evaluated in larger populations and databases.

Evaluating novel in silico tools for accurate pathogenicity classification in epilepsy-associated genetic missense variants.

Determining the pathogenicity of missense variants in clinical genetic tests for individuals with epilepsy is crucial for guiding personalized treatment. However, achieving a definitive pathogenic classification remains challenging, with most missense variants still classified as variants of uncertain significance (VUS) and with the availability of many computational tools which may provide conflicting predictions. Here, we aim to evaluate the performance of state-of-the-art computational tools in pathogenicity prediction of missense variants in epilepsy-associated genes. This will assist in selecting the most appropriate tool and critically assess their use in clinical setting. We assessed the performance of nine in silico pathogenicity prediction tools for missense variants in epilepsy-associated genes on three carefully curated data sets. The first two data sets comprise missense variants in epilepsy associated genes that have been uploaded to ClinVar in the last year and were, therefore, not part of the training set of any of the nine considered tools. These two data sets are based on two different lists of epilepsy-associated genes and comprise ~700 and ~ 250 missense variants, respectively. The third data set includes ~400 missense variants within epilepsy-associated genes for which the functional effects have been determined experimentally and are therefore used here to infer pathogenicity. These three data sets represent the best available approximation to blind and independent test sets. Among the nine assessed tools, AlphaMissense (area under the curve [AUC]: .93, .88, and .95) and REVEL (AUC: .93, .88, and .93) showed the best classification performance, also outperforming other tools in the number of classified variants. We show which recently developed prediction tools achieve higher performance in epilepsy-associated genes and should be integrated, therefore, into the American College of Medical Genetics and Genomics/Association of Molecular Pathology (AGMC/AMP) variant classification process. Periodic reevaluation of genetic test results with newly developed or updated tools should be incorporated into standard clinical practice to improve diagnostic yield and better inform precision medicine.

Phenotypic evaluation of deep learning models for classifying germline variant pathogenicity.

Deep learning models for predicting variant pathogenicity have not been thoroughly evaluated on real-world clinical phenotypes. Here, we apply state-of-the-art pathogenicity prediction models to hereditary breast cancer gene variants in UK Biobank participants. Model predictions for missense variants in BRCA1, BRCA2 and PALB2, but not ATM and CHEK2, were associated with breast cancer risk. However, deep learning models had limited clinical utility when specifically applied to variants of uncertain significance.

Slowly progressive autosomal dominant Alport Syndrome due to COL4A3 splicing variant.

Alport syndrome is a rare genetic kidney disease caused by variants in the COL4A3/A4/A5 genes. It's characterised by progressive kidney failure, though therapies targeting Renin-Angiotensin System can delay its progression. Additionally, extrarenal manifestations may sometimes coexist. Recent advances in genetic analysis and the necessity to better clarify genotype-phenotype correlations in affected patients raises the importance of detecting even cryptic splicing variants, lying in both canonical and non-canonical splice sites variants such as last exonic nucleotide variants. These variants, often, do not cause an amino acid change but alter the snRNP proteins binding. We studied a big Italian family with Alport syndrome showing a clear dominant pattern of transmission with younger family members having only haematuria and older individuals presenting with End-Stage Kidney Failure (ESKF). Kidney biopsy showed the typical disease hallmarks. We deeply mined the data for SNV and CNV through exome sequencing on DNA from both peripheral blood samples and patients' podocytes-lineage cells. We identified an already reported synonymous variant, c.765G>A (p.(Thr255Thr)), in the last exonic nucleotide of exon 13 of the COL4A3 gene. Employing the patient's podocytes we demonstrated that this variant results in exon skipping leading to an in-frame deletion of 28 amino acids without leaky effect. According to the pattern of transmission, to the kidney biopsy and to the exome data analysis we provided further evidence that autosomal dominant Alport syndrome is a well-defined clinical entity. We also confirmed the pathogenicity of the synonymous COL4A3 variant for the first time demonstrating its role in a dominant pattern of transmission.

Clinical and genetic landscape of optic atrophy in 826 families: insights from 50 nuclear genes.

Hereditary optic neuropathies (HON) are a group of diseases due to genetic defects either in mitochondria or in nuclear genomes. The increasing availability of genetic testing has expanded a broader genetic and phenotypic spectrum of HON than previously recognized. To provide systematic insight into the genetic and phenotypic landscape of HON attributed to 50 nuclear genes, we conducted genetic analysis on part of 4776 index patients with clinical diagnosis of HON following our previous study on 1516 probands with Leber hereditary optic neuropathy (LHON) and mitochondrial DNA variants. Exome sequencing was performed in 473 patients diagnosed with nuclear gene-related HON (nHON) and 353 cases with unsolved LHON. Sequencing and variant interpretation in 50 causative nuclear genes indicated that the diagnostic yield of exome sequencing for nHON was 31.50% (149/473), while it was markedly lower at 1.42% (5/353) for LHON patients without primary mtDNA mutations. The top five implicated genes causing nHON in our in-house cohort, OPA1, WFS1, FDXR, ACO2, and AFG3L2, account for 82.46% of mutations. Although OPA1 was the most prevalent causative gene of nHON in both our cohort (53.25%) and literature review (37.09%), the prevalence of OPA1, WFS1, and FDXR differed significantly between our in-house cohort and the literature review (P-adjusted<0.001). Fundus changes in nHON could be stratified into three categories, the most common is optic atrophy at the examination (78.79%), the rarest is LHON-like optic atrophy (3.64%), and the intermediate is optic atrophy with concurrent retinal degeneration (17.57%), which was an independent risk factor for visual prognosis in nHON. A systematic genotype-phenotype analysis highlighted different genetic contributions for ocular, extraocular neurological, and extraocular non-neurological phenotypes. In addition, systemic variant analysis at the individual gene level suggested a revised interpretation of the pathogenicity of a WFS1 heterozygous truncation variant. This study provides a panoramic summary of both the genetic and phenotypic profiles of HON in real-world studies and literature. The category for nHON fundus phenotypes is built for future studies on molecular mechanisms underlying HON and targeted therapies. In addition to routine ophthalmic examinations, careful examination of the extraocular symptoms and meaningful genetic counseling are warranted for patients with nHON.

Promise and Peril of a Genotype-First Approach to Mendelian Cardiovascular Disease.

Precision medicine, which among other aspects includes an individual's genomic data in diagnosis and management, has become the standard-of-care for Mendelian cardiovascular disease (CVD). However, early identification and management of asymptomatic patients with potentially lethal and manageable Mendelian CVD through screening, which is the promise of precision health, remains an unsolved challenge. The reduced costs of genomic sequencing have enabled the creation of biobanks containing in-depth genetic and health information, which have facilitated the understanding of genetic variation, penetrance, and expressivity, moving us closer to the genotype-first screening of asymptomatic individuals for Mendelian CVD. This approach could transform health care by diagnostic refinement and facilitating prevention or therapeutic interventions. Yet, potential benefits must be weighed against the potential risks, which include evolving variant pathogenicity assertion or identification of variants with low disease penetrance; costly, stressful, and inappropriate diagnostic evaluations; negative psychological impact; disqualification for employment or of competitive sports; and denial of insurance. Furthermore, the natural history of Mendelian CVD is often unpredictable, making identification of those who will benefit from preventive measures a priority. Currently, there is insufficient evidence that population-based genetic screening for Mendelian CVD can reduce adverse outcomes at a reasonable cost to an extent that outweighs the harms of true-positive and false-positive results. Besides technical, clinical, and financial burdens, ethical and legal aspects pose unprecedented challenges. This review highlights key developments in the field of genotype-first approaches to Mendelian CVD and summarizes challenges with potential solutions that can pave the way for implementing this approach for clinical care.

A new-disease-causing dominant-negative variant in CARD11 gene in a Chinese case with recurrent fever

Immunodeficiency 11B with atopic dermatitis (IMD11B, OMIM:617638) is rare primary immunodeficiency disease caused by germline dominant negative (DN) mutations in the CARD11 gene. Affected patients present with immune dysfunction, recurrent infections and atopic dermatitis. In this study, we sought to identify and characterize the genetic variant in one patient with periodic fever, recurrent infections, and eczema. Trio whole-exome sequencing (WES) was employed in this patient and her parents, and Sanger sequencing validated the potential pathogenic variant. In vitro functional study was performed to evaluate the pathogenicity of genetic variant identified. A very rare missense mutation (c.2324C > T, p.S775L) in CARD11 gene (NM_032415) was identified by WES in the patient but not her parents. Luciferase reporter assays and co-immunoprecipitation demonstrated mutation exerts a dominant-interfering effect on wild-type CARD11, inhibiting the activity of NF-κB. RNA sequencing analysis also confirmed that mutant CARD11 inhibited down-stream transcriptional activity of NF-κB. A review of literature doesn’t found significant genotype–phenotype correlation. We identified a vary rare DN CARD11 mutation, expanding the genetic and phenotypic spectrum of CARD11.

Monitoring Calcium-Sensing Receptor (CaSR)-Induced Intracellular Calcium Flux Using an Indo-1 Flow Cytometry Assay.

The calcium-sensing receptor (CaSR) has a critical role in maintaining serum calcium concentrations within the normal physiological range, and mutations in the receptor, or components of its signaling and trafficking pathway, cause disorders of calcium homeostasis. Inactivating mutations cause neonatal severe hyperparathyroidism or familial hypocalciuric hypercalcemia (FHH), while gain-of-function mutations cause autosomal dominant hypocalcemia (ADH). Characterizing the functional impact of mutations of the CaSR, and components of the CaSR-signaling pathway, is clinically important to enable correct diagnoses of FHH and ADH, optimize management, and prevent inappropriate parathyroidectomy or vitamin D supplementation. CaSR signals predominantly by activating the G-alpha subunit-11 to mobilize calcium release from intracellular stores. Thus, measurement of CaSR-induced intracellular calcium (Ca2+i) signaling is the gold standard method to investigate the pathogenicity of CaSR genetic variants. This protocol describes a method to assess CaSR-induced Ca2+I signaling using the Indo-1 calcium indicator dye and flow cytometry. This method has been used to assess multiple genetic variants in CaSR and components of its signaling and trafficking pathway in HEK293 cells.

Anakinra-Dependent Recurrent Pericarditis: The Role of the R202Q Variant of the MEFV Gene.

Background: the role of the R202Q (c.605G>A, p.Arg202Gln) missense variant of the MEFV gene has been debated as either a benign polymorphism or a potentially pathogenic mutation. We report and discuss here the case of a young female with corticosteroid-dependent recurrent pericarditis carrying the homozygous R202Q variant, exhibiting distinctive clinical features possibly influenced by this genetic variant. Methods: a 30-year-old woman with a previous diagnosis of cancer and recent respiratory infection presented with severe pleuritic chest pain, hypotension, tachycardia, and fever. Initial diagnostic evaluation indicated cardiac tamponade, and emergent pericardiocentesis was performed. Despite initial treatment with NSAIDs, colchicine, and corticosteroids, the patient experienced multiple recurrences. Genetic testing identified homozygous R202Q variant in the MEFV gene. Given the corticosteroid dependency and recurrent nature of her condition, IL-1 inhibitor anakinra was introduced, leading to significant improvement, although tapering below 150 mg per week failed to prevent recurrences. Results: the introduction of anakinra resulted in rapid symptom relief and resolution of pericardial effusion. However, attempts to taper or discontinue anakinra led to pericarditis recurrences. Ultimately, a maintenance dose of 50 mg every three days was established, which maintained remission for 18 months without recurrence. Despite multiple tapering attempts, further reduction in anakinra dosage was unsuccessful without triggering relapses. Conclusions: the R202Q variant, although typically considered benign, may contribute to an autoinflammatory phenotype resembling familial Mediterranean fever. This case underscores the potential pathogenicity of the homozygous R202Q variant in recurrent pericarditis and its responsiveness to IL-1 inhibition. In patients with corticosteroid-dependent recurrent pericarditis, genetic testing for the R202Q variant should be considered when anti-IL-1 drugs cannot be withdrawn. Further studies are warranted to elucidate the variant's role in pericardial inflammation and guide personalized treatment strategies.

Performance of a Protein Language Model for Variant Annotation in Cardiac Disease.

Genetic testing is a cornerstone in the assessment of many cardiac diseases. However, variants are frequently classified as variants of unknown significance, limiting the utility of testing. Recently, the DeepMind group (Google) developed AlphaMissense, a unique artificial intelligence-based model, based on language model principles, for the prediction of missense variant pathogenicity. We aimed to report on the performance of AlphaMissense, accessed by VarCardio, an open web-based variant annotation engine, in a real-world cardiovascular genetics center. All genetic variants from an inherited arrhythmia program were examined using AlphaMissense via VarCard.io and compared with the ClinVar variant classification system, as well as another variant classification platform (Franklin by Genoox). The mutation reclassification rate and genotype-phenotype concordance were examined for all variants in the study. We included 266 patients with heritable cardiac diseases, harboring 339 missense variants. Of those, 230 (67.8%) were classified by ClinVar as either variants of unknown significance or nonclassified. Using VarCard.io, 198 variants of unknown significance (86.1%, 95% CI, 80.9-90.3) were reclassified to either likely pathogenic or likely benign. The reclassification rate was significantly higher for VarCard.io than for Franklin (86.1% versus 34.8%, P<0.001). Genotype-phenotype concordance was highly aligned using VarCard.io predictions, at 95.9% (95% CI, 92.8-97.9) concordance rate. For 109 variants classified as pathogenic, likely pathogenic, benign, or likely benign by ClinVar, concordance with VarCard.io was high (90.5%). AlphaMissense, accessed via VarCard.io, may be a highly efficient tool for cardiac genetic variant interpretation. The engine's notable performance in assessing variants that are classified as variants of unknown significance in ClinVar demonstrates its potential to enhance cardiac genetic testing.