Variant Classification for Pompe disease; ACMG/AMP specifications from the ClinGen Lysosomal Diseases Variant Curation Expert Panel.

Accurate determination of the clinical significance of genetic variants is critical to the integration of genomics in medicine. To facilitate this process, the NIH-funded Clinical Genome Resource (ClinGen) has assembled Variant Curation Expert Panels (VCEPs), groups of experts and biocurators which provide gene- and disease- specifications to the American College of Medical Genetics & Genomics and Association for Molecular Pathology's (ACMG/AMP) variation classification guidelines. With the goal of classifying the clinical significance of GAA variants in Pompe disease (Glycogen storage disease, type II), the ClinGen Lysosomal Diseases (LD) VCEP has specified the ACMG/AMP criteria for GAA. Variant classification can play an important role in confirming the diagnosis of Pompe disease as well as in the identification of carriers. Furthermore, since the inclusion of Pompe disease on the Recommended Uniform Screening Panel (RUSP) for newborns in the USA in 2015, the addition of molecular genetic testing has become an important component in the interpretation of newborn screening results, particularly for asymptomatic individuals. To date, the LD VCEP has submitted classifications and supporting data on 243 GAA variants to public databases, specifically ClinVar and the ClinGen Evidence Repository. Here, we describe the ACMG/AMP criteria specification process for GAA, an update of the GAA-specific variant classification guidelines, and comparison of the ClinGen LD VCEP's GAA variant classifications with variant classifications submitted to ClinVar. The LD VCEP has added to the publicly available knowledge on the pathogenicity of variants in GAA by increasing the number of expert-curated GAA variants present in ClinVar, and aids in resolving conflicting classifications and variants of uncertain clinical significance.

Specifications of the ACMG/AMP guidelines for ACADVL variant interpretation.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is a relatively common inborn error of metabolism, but due to difficulty in accurately predicting affected status through newborn screening, molecular confirmation of the causative variants by sequencing of the ACADVL gene is necessary. Although the ACMG/AMP guidelines have helped standardize variant classification, ACADVL variant classification remains disparate due to a phenotype that can be nonspecific, the possibility of variants that produce late-onset disease, and relatively high carrier frequency, amongst other challenges. Therefore, an ACADVL-specific variant curation expert panel (VCEP) was created to facilitate the specification of the ACMG/AMP guidelines for VLCADD. We expect these guidelines to help streamline, increase concordance, and expedite the classification of ACADVL variants.

Evaluating the strength of evidence for genes implicated in peroxisomal disorders using the ClinGen clinical validity framework and providing updates to the peroxisomal disease nomenclature.

Peroxisomal disorders are heterogeneous in nature, with phenotypic overlap that is indistinguishable without molecular testing. Newborn screening and gene sequencing for a panel of genes implicated in peroxisomal diseases are critical tools for the early and accurate detection of these disorders. It is therefore essential to evaluate the clinical validity of the genes included in sequencing panels for peroxisomal disorders. The Peroxisomal Gene Curation Expert Panel (GCEP) assessed genes frequently included on clinical peroxisomal testing panels using the Clinical Genome Resource (ClinGen) gene-disease validity curation framework and classified gene-disease relationships as Definitive, Strong, Moderate, Limited, Disputed, Refuted, or No Known Disease Relationship. Subsequent to gene curation, the GCEP made recommendations to update the disease nomenclature and ontology in the Monarch Disease Ontology (Mondo) database. Thirty-six genes were assessed for the strength of evidence supporting their role in peroxisomal disease, leading to 36 gene-disease relationships, after two genes were removed for their lack of a role in peroxisomal disease and two genes were curated for two different disease entities each. Of these, 23 were classified as Definitive (64%), one as Strong (3%), eight as Moderate (23%), two as Limited (5%), and two as No known disease relationship (5%). No contradictory evidence was found to classify any relationships as Disputed or Refuted. The gene-disease relationship curations are publicly available on the ClinGen website (https://clinicalgenome.org/affiliation/40049/). The changes to peroxisomal disease nomenclature are displayed on the Mondo website (http://purl.obolibrary.org/obo/MONDO_0019053). The Peroxisomal GCEP-curated gene-disease relationships will inform clinical and laboratory diagnostics and enhance molecular testing and reporting. As new data will emerge, the gene-disease classifications asserted by the Peroxisomal GCEP will be re-evaluated periodically.

Assessing the strength of evidence for genes implicated in fatty acid oxidation disorders using the ClinGen clinical validity framework.

Newborn screening is an incredibly useful tool for the early identification of many metabolic disorders, including fatty acid oxidation (FAO) disorders. In many cases, molecular tests are necessary to reach a final diagnosis, highlighting the need for a thorough evaluation of genes implicated in FAO disorders. Using the ClinGen (Clinical Genome Resource) clinical validity framework, thirty genes were analyzed for the strength of evidence supporting their association with FAO disorders. Evidence was gathered from the literature by biocurators and presented to disease experts for review in order to assign a clinical validity classification of Definitive, Strong, Moderate, Limited, Disputed, Refuted, or No Reported Evidence. Of the gene-disease relationships evaluated, 22/30 were classified as Definitive, three as Moderate, one as Limited, three as No Reported Evidence and one as Disputed. Gene-disease relationships with a Limited, Disputed, and No Reported Evidence were found on two, six, and up to four panels out of 30 FAO disorder-specific panels, respectively, in the National Institute of Health Genetic Testing Registry, while over 70% of the genes on panels are definitively associated with an FAO disorder. These results highlight the need to systematically assess the clinical relevance of genes implicated in fatty acid oxidation disorders in order to improve the interpretation of genetic testing results and diagnosis of patients with these disorders.

Unique aspects of sequence variant interpretation for inborn errors of metabolism (IEM): The ClinGen IEM Working Group and the Phenylalanine Hydroxylase Gene.

The ClinGen Inborn Errors of Metabolism Working Group was tasked with creating a comprehensive, standardized knowledge base of genes and variants for metabolic diseases. Phenylalanine hydroxylase (PAH) deficiency was chosen to pilot development of the Working Group's standards and guidelines. A PAH variant curation expert panel (VCEP) was created to facilitate this process. Following ACMG-AMP variant interpretation guidelines, we present the development of these standards in the context of PAH variant curation and interpretation. Existing ACMG-AMP rules were adjusted based on disease (6) or strength (5) or both (2). Disease adjustments include allele frequency thresholds, functional assay thresholds, and phenotype-specific guidelines. Our validation of PAH-specific variant interpretation guidelines is presented using 85 variants. The PAH VCEP interpretations were concordant with existing interpretations in ClinVar for 69 variants (81%). Development of biocurator tools and standards are also described. Using the PAH-specific ACMG-AMP guidelines, 714 PAH variants have been curated and will be submitted to ClinVar. We also discuss strategies and challenges in applying ACMG-AMP guidelines to autosomal recessive metabolic disease, and the curation of variants in these genes.

Maternal oral intake mouse model for fetal alcohol spectrum disorders: ocular defects as a measure of effect.

BACKGROUND: This work was conducted in an effort to establish an oral intake model system in which the effects of ethanol insult that occur during early stages of embryogenesis can be easily examined and in which agents that may modulate ethanol's teratogenicity can be readily tested in vivo. The model system described utilizes the alcohol deprivation effect to obtain teratogenic levels of maternal ethanol intake on days 7 and 8 of pregnancy in C57Bl/6J mice. Ocular defects including microphthalmia and uveal coloboma, which have previously been shown to result from ethanol administered by gavage or via intraperitoneal injection on these days, served as the developmental end point for this study. The ocular defects are readily identifiable and their degree of severity is expected to correlate with concurrently developing defects of the central nervous system (CNS). METHODS: Female C57Bl/6J mice were maintained on an ethanol-containing (4.8% v/v) liquid diet for 14 days and then mated during a subsequent abstinence period. Mice were then reexposed to ethanol on days 7 and 8 of pregnancy only. Control as well as ethanol-exposed dams were killed on their 14th day of pregnancy. Fetuses were then weighed, measured for crown rump length, photographed, and analyzed for ocular abnormalities. Globe size, palpebral fissure length, and pupil size and shape were noted for both the right and left eyes of all fetuses and informative comparisons were made. RESULTS: This exposure paradigm resulted in peak maternal blood alcohol concentrations that ranged from 170 to 220 mg/dL on gestational day (GD) 8. Compared with the GD 14 fetuses from the normal control group, the pair-fed, acquisition controls, as well as the ethanol-exposed fetuses, were developmentally delayed and had reduced weights. Confirming previous studies, comparison of similarly staged control and treated GD 8 embryos illustrated reductions in the size of the forebrain in the latter. Subsequent ocular malformations were noted in 33% of the right eyes and 25% of the left eyes of the 103 GD 14 ethanol-exposed fetuses examined. This incidence of defects is twice that observed in the control groups. Additionally, it was found that the palpebral fissure length is directly correlated with globe size. CONCLUSIONS: The high incidence of readily identifiable ocular malformations produced by oral ethanol intake in this model and their relevance to human fetal alcohol spectrum disorders (FASD) makes this an excellent system for utilization in experiments involving factors administered to the embryo that might alter ethanol's teratogenic effects. Additionally, the fact that early ethanol insult yields ocular and forebrain abnormalities that are developmentally associated allows efficient specimen selection for subsequent detailed analyses of CNS effects in this in vivo mammalian FASD model.