Proactive Variant Effect Mapping to Accelerate Genetic Diagnosis for Pediatric Cardiac Arrest

While genetic testing is becoming standard of care for patients with potentially inherited cardiovascular disease, the prevalence of uncertain results severely limits its utility. One promising approach is to generate variant effect maps that report the function of all possible variants in a gene prospectively. The proactive clinical application of these maps is nascent, and requires careful integration with current American College of Medical Genetics guidelines for variant interpretation. Here, we describe three pediatric cases of cardiac arrest or sudden cardiac death with variants of uncertain significance in calmodulin genes. We demonstrate the prospective clinical utility of a calmodulin variant effect map to inform variant interpretation, and therefore diagnosis and family care, in each case. This study was approved by the Stanford University and Vanderbilt University Medical Center IRBs. Consent was waived based on low risk of de-identified retrospective data collection per the IRB.

In-silico phenotype prediction by normal mode variant analysis in TUBB4A-related disease

TUBB4A-associated disorder is a rare condition affecting the central nervous system. It displays a wide phenotypic spectrum, ranging from isolated late-onset torsion dystonia to a severe early-onset disease with developmental delay, neurological deficits, and atrophy of the basal ganglia and cerebellum, therefore complicating variant interpretation and phenotype prediction in patients carrying TUBB4A variants. We applied entropy-based normal mode analysis (NMA) to investigate genotype–phenotype correlations in TUBB4A-releated disease and to develop an in-silico approach to assist in variant interpretation and phenotype prediction in this disorder. Variants included in our analysis were those reported prior to the conclusion of data collection for this study in October 2019. All TUBB4A pathogenic missense variants reported in ClinVar and Pubmed, for which associated clinical information was available, and all benign/likely benign TUBB4A missense variants reported in ClinVar, were included in the analysis. Pathogenic variants were divided into five phenotypic subgroups. In-silico point mutagenesis in the wild-type modeled protein structure was performed for each variant. Wild-type and mutated structures were analyzed by coarse-grained NMA to quantify protein stability as entropy difference value (ΔG) for each variant. Pairwise ΔG differences between all variant pairs in each structural cluster were calculated and clustered into dendrograms. Our search yielded 41 TUBB4A pathogenic variants in 126 patients, divided into 11 partially overlapping structural clusters across the TUBB4A protein. ΔG-based cluster analysis of the NMA results revealed a continuum of genotype–phenotype correlation across each structural cluster, as well as in transition areas of partially overlapping structural clusters. Benign/likely benign variants were integrated into the genotype–phenotype continuum as expected and were clearly separated from pathogenic variants. We conclude that our results support the incorporation of the NMA-based approach used in this study in the interpretation of variant pathogenicity and phenotype prediction in TUBB4A-related disease. Moreover, our results suggest that NMA may be of value in variant interpretation in additional monogenic conditions.

BRCA1 Norway: comparison of classification for BRCA1 germline variants detected in families with suspected hereditary breast and ovarian cancer between different laboratories

Pathogenic germline variants in Breast cancer susceptibility gene 1 (BRCA1) predispose carriers to hereditary breast and ovarian cancer (HBOC). Through genetic testing of patients with suspected HBOC an increasing number of novel BRCA1 variants are discovered. This creates a growing need to determine the clinical significance of these variants through correct classification (class 1–5) according to established guidelines. Here we present a joint collection of all BRCA1 variants of class 2–5 detected in the four diagnostic genetic laboratories in Norway. The overall objective of the study was to generate an overview of all BRCA1 variants in Norway and unveil potential discrepancies in variant interpretation between the hospitals, serving as a quality control at the national level. For a subset of variants, we also assessed the change in classification over a ten-year period with increasing information available. In total, 463 unique BRCA1 variants were detected. Of the 126 variants found in more than one hospital, 70% were interpreted identically, while 30% were not. The differences in interpretation were mainly by one class (class 2/3 or 4/5), except for one larger discrepancy (class 3/5) which could affect the clinical management of patients. After a series of digital meetings between the participating laboratories to disclose the cause of disagreement for all conflicting variants, the discrepancy rate was reduced to 10%. This illustrates that variant interpretation needs to be updated regularly, and that data sharing and improved national inter-laboratory collaboration greatly improves the variant classification and hence increases the accuracy of cancer risk assessment.

The Gene Curation Coalition: A global effort to harmonize gene-disease evidence resources

PURPOSE: Several groups and resources provide information that pertains to the validity of gene-disease relationships used in genomic medicine and research; however, universal standards and terminologies to define the evidence base for the role of a gene in disease, and a single harmonized resource were lacking. To tackle this issue, the Gene Curation Coalition (GenCC) was formed. METHODS: The GenCC drafted harmonized definitions for differing levels of gene-disease validity based on existing resources, and performed a modified Delphi survey with three rounds to narrow the list of terms. The GenCC also developed a unified database to display curated gene-disease validity assertions from its members. RESULTS: Based on 241 survey responses from the genetics community, a consensus term set was chosen for grading gene-disease validity and database submissions. As of December 2021, the database contains 15,241 gene-disease assertions on 4,569 unique genes from 12 submitters. When comparing submissions to the database from distinct sources, conflicts in assertions of gene-disease validity ranged from 5.3% to 13.4%. CONCLUSION: Terminology standardization, sharing of gene-disease validity classifications, and resolution of curation conflicts will facilitate collaborations across international curation efforts and in turn, improve consistency in genetic testing and variant interpretation.

A calibrated functional patch clamp assay to enhance clinical variant interpretation in KCNH2-related long QT syndrome

Purpose: Modern sequencing technologies have revolutionised our detection of gene variants. In most genes, including KCNH2, the majority of missense variants are currently classified as variants of uncertain significance (VUS). The aim of this study is to investigate the utility of an automated patch-clamp assay for aiding clinical variant classification in the KCNH2 gene. Methods: The assay was designed according to recommendations of the ClinGen sequence variant interpretation framework. Thirty-one control variants of known clinical significance (17 pathogenic/likely pathogenic, 14 benign/likely benign) were heterozygously expressed in Flp-In HEK293 cells. Variants were analysed for effects on current density and channel gating. A panel of 44 VUS was then assessed for reclassification. Results: All 17 pathogenic variant controls had reduced current density and 13/14 benign variant controls had normal current density, which enabled determination of normal and abnormal ranges for applying moderate or supporting evidence strength for variant classification. Inclusion of KCNH2 functional assay evidence enabled us to reclassify 6 out of 44 VUS as likely pathogenic. Conclusion: The high-throughput patch clamp assay can provide moderate strength evidence for clinical interpretation of clinical KCNH2 variants and demonstrates the value proposition for developing automated patch clamp assays for other ion channel genes.

Transcript-Specific Loss-of-Function Variants in VPS16 Are Enriched in Patients With Dystonia

Background and ObjectivesOur objective was to improve rare variant interpretation using statistical measures as well as publicly accessible annotation of expression levels and tissue specificity of different splice isoforms. We describe rare VPS16 variants observed in patients with dystonia and patients without dystonia, elaborate on our interpretation of VPS16 variants affecting different transcripts, and provide detailed clinical description of the movement disorder caused by VPS16 variants.MethodsIn-house exome and genome data sets (n = 11,539) were screened for rare heterozygous missense and putative loss-of-function (pLoF) variants in VPS16. Using pext (proportion expressed across transcripts) values from the Genome Aggregation Database (gnomAD), we differentiated variants affecting weakly and highly expressed exons/transcripts and applied statistical measures to systematically identify disease-associated genetic variation among patients with dystonia (n = 280).ResultsSix different heterozygous pLoFs in VPS16 transcripts were identified in 13 individuals. Three of these pLoFs occurred in 9 individuals with different phenotypes, and 3 pLoFs were identified in 4 unrelated individuals with early-onset dystonia. Although pLoFs were enriched in the dystonia cohort (n = 280; p = 2.04 × 10−4; 4/280 cases vs 9/11,259 controls; Fisher exact test), it was not exome-wide significant. According to the pext values in gnomAD, all 3 pLoFs observed in the patients with dystonia were located in the highly expressed canonical transcript ENST00000380445.3, whereas 2 of 3 pLoFs detected in 8 individuals without dystonia were located in the first exon of the noncanonical transcript ENST00000380443.3 that is weakly expressed across all tissues. Taking these biological implications into account, pLoFs involving the canonical transcript were exome-wide significantly enriched in patients with dystonia (p = 1.67 × 10−6; 4/280 cases vs 1/11,259 controls; Fisher exact test). All VPS16 patients showed mild progressive dystonia with writer's cramp as the presenting symptom between age 7 and 34 years (mean 20 years) that often progressed to generalized dystonia and was even accompanied by hyperkinetic movements and myoclonus in 1 patient.DiscussionOur data provide strong evidence for VPS16 pLoFs to be implicated in dystonia and knowledge on exon resolution expression levels as well as statistical measures proved to be useful for variant interpretation.

MitoVisualize: A resource for analysis of variants in human mitochondrial RNAs and DNA

We present MitoVisualize, a new tool for analysis of the human mitochondrial DNA (mtDNA). MitoVisualize enables visualization of: (1) the position and effect of variants in mitochondrial transfer RNA (tRNA) and ribosomal RNA (rRNA) secondary structures alongside curated variant annotations, (2) data across RNA structures, such as to show all positions with disease-associated variants or with post-transcriptional modifications, and (3) the position of a base, gene or region in the circular mtDNA map, such as to show the location of a large deletion. All visualizations can be easily downloaded as figures for reuse. MitoVisualize can be useful for anyone interested in exploring mtDNA variation, though is designed to facilitate mtDNA variant interpretation in particular. MitoVisualize can be accessed via https://www.mitovisualize.org/. The source code is available at https://github.com/leklab/mito_visualize/.

Genetic testing and diagnosis of inherited retinal diseases

Inherited retinal diseases (IRDs) are a diverse group of degenerative diseases of the retina that can lead to significant reduction in vision and blindness. Because of the considerable phenotypic overlap among IRDs, genetic testing is a critical step in obtaining a definitive diagnosis for affected individuals and enabling access to emerging gene therapy–based treatments and ongoing clinical studies. While advances in molecular diagnostic technologies have significantly improved the understanding of IRDs and identification of disease-causing variants, training in genetic diagnostics among ophthalmologists is limited. In this review, we will provide ophthalmologists with an overview of genetic testing for IRDs, including the types of available testing, variant interpretation, and genetic counseling. Additionally, we will discuss the clinical applications of genetic testing in the molecular diagnosis of IRDs through case studies.