IFIH1 loss of function predisposes to inflammatory and SARS-CoV-2-related infectious diseases.

The IFIH1 gene, encoding melanoma differentiation-associated protein 5 (MDA5), is an indispensable innate immune regulator involved in the early detection of viral infections. Previous studies described MDA5 dysregulation in weakened immunological responses, and increased susceptibility to microbial infections and autoimmune disorders. Monoallelic gain-of-function of the IFIH1 gene has been associated with multisystem disorders, namely Aicardi-Goutieres and Singleton-Merten syndromes, while biallelic loss causes immunodeficiency. In this study, nine patients suffering from recurrent infections, inflammatory diseases, severe COVID-19 or multisystem inflammatory syndrome in children (MIS-C) were identified with putative loss-of-function IFIH1 variants by whole-exome sequencing. All patients revealed signs of lymphopaenia and an increase in inflammatory markers, including CRP, amyloid A, ferritin and IL-6. One patient with a pathogenic homozygous variant c.2807+1G>A was the most severe case showing immunodeficiency and glomerulonephritis. The c.1641+1G>C variant was identified in the heterozygous state in patients suffering from periodic fever, COVID-19 or MIS-C, while the c.2016delA variant was identified in two patients with inflammatory bowel disease or MIS-C. There was a significant association between IFIH1 monoallelic loss of function and susceptibility to infections in males. Expression analysis showed that PBMCs of one patient with a c.2016delA variant had a significant decrease in ISG15, IFNA and IFNG transcript levels, compared to normal PBMCs, upon stimulation with Poly(I:C), suggesting that MDA5 receptor truncation disrupts the immune response. Our findings accentuate the implication of rare monogenic IFIH1 loss-of-function variants in altering the immune response, and severely predisposing patients to inflammatory and infectious diseases, including SARS-CoV-2-related disorders.

Comprehensive interpretation of single-nucleotide substitutions in GJB2 reveals the genetic and phenotypic landscape of GJB2-related hearing loss.

Genetic variants in GJB2 are the most frequent cause of congenital and childhood hearing loss worldwide. The purpose of this study was to delineate the genetic and phenotypic landscape of GJB2 SNV variants. All possible single-nucleotide substitution variants of the coding region of GJB2 (N = 2043) were manually curated following the ACMG/AMP hearing loss guidelines. As a result, 60 (2.9%), 177 (8.7%), 1499 (73.4%), 301 (14.7%) and 6 (0.3%) of the variants were classified as pathogenic, likely pathogenic, variant of uncertain significance, likely benign, and benign, respectively. 53% (84/158) of the pathogenic/likely pathogenic missense variants were not present in ClinVar. The second transmembrane domain and the 310 helix were highly enriched for pathogenic missense variants, while the intracellular loops were tolerant to variation. The N-terminal tail and the extracellular loop showed high clustering of variants that are associated with syndromic or dominant non-syndromic hearing loss. In conclusion, our study interpreted all possible single-nucleotide substitution coding variants, characterized novel clinically significant variants in GJB2, and revealed significant genotype-phenotype correlations at this common hearing loss locus. Our work provides a prototype for other genes with similarly high genetic and phenotypic heterogeneity.

De Novo Variants Disturbing the Transactivation Capacity of POU3F3 Cause a Characteristic Neurodevelopmental Disorder.

POU3F3, also referred to as Brain-1, is a well-known transcription factor involved in the development of the central nervous system, but it has not previously been associated with a neurodevelopmental disorder. Here, we report the identification of 19 individuals with heterozygous POU3F3 disruptions, most of which are de novo variants. All individuals had developmental delays and/or intellectual disability and impairments in speech and language skills. Thirteen individuals had characteristic low-set, prominent, and/or cupped ears. Brain abnormalities were observed in seven of eleven MRI reports. POU3F3 is an intronless gene, insensitive to nonsense-mediated decay, and 13 individuals carried protein-truncating variants. All truncating variants that we tested in cellular models led to aberrant subcellular localization of the encoded protein. Luciferase assays demonstrated negative effects of these alleles on transcriptional activation of a reporter with a FOXP2-derived binding motif. In addition to the loss-of-function variants, five individuals had missense variants that clustered at specific positions within the functional domains, and one small in-frame deletion was identified. Two missense variants showed reduced transactivation capacity in our assays, whereas one variant displayed gain-of-function effects, suggesting a distinct pathophysiological mechanism. In bioluminescence resonance energy transfer (BRET) interaction assays, all the truncated POU3F3 versions that we tested had significantly impaired dimerization capacities, whereas all missense variants showed unaffected dimerization with wild-type POU3F3. Taken together, our identification and functional cell-based analyses of pathogenic variants in POU3F3, coupled with a clinical characterization, implicate disruptions of this gene in a characteristic neurodevelopmental disorder.

Genetic variant pathogenicity prediction trained using disease-specific clinical sequencing data sets.

Recent advances in DNA sequencing have expanded our understanding of the molecular basis of genetic disorders and increased the utilization of clinical genomic tests. Given the paucity of evidence to accurately classify each variant and the difficulty of experimentally evaluating its clinical significance, a large number of variants generated by clinical tests are reported as variants of unknown clinical significance. Population-scale variant databases can improve clinical interpretation. Specifically, pathogenicity prediction for novel missense variants can use features describing regional variant constraint. Constrained genomic regions are those that have an unusually low variant count in the general population. Computational methods have been introduced to capture these regions and incorporate them into pathogenicity classifiers, but these methods have yet to be compared on an independent clinical variant data set. Here, we introduce one variant data set derived from clinical sequencing panels and use it to compare the ability of different genomic constraint metrics to determine missense variant pathogenicity. This data set is compiled from 17,071 patients surveyed with clinical genomic sequencing for cardiomyopathy, epilepsy, or RASopathies. We further use this data set to demonstrate the necessity of disease-specific classifiers and to train PathoPredictor, a disease-specific ensemble classifier of pathogenicity based on regional constraint and variant-level features. PathoPredictor achieves an average precision >90% for variants from all 99 tested disease genes while approaching 100% accuracy for some genes. The accumulation of larger clinical variant training data sets can significantly enhance their performance in a disease- and gene-specific manner.

Evaluating the impact of in silico predictors on clinical variant classification.

PURPOSE: According to the American College of Medical Genetics and Genomics/Association of Medical Pathology (ACMG/AMP) guidelines, in silico evidence is applied at the supporting strength level for pathogenic (PP3) and benign (BP4) evidence. Although PP3 is commonly used, less is known about the effect of these criteria on variant classification outcomes. METHODS: A total of 727 missense variants curated by Clinical Genome Resource expert groups were analyzed to determine how often PP3 and BP4 were applied and their impact on variant classification. The ACMG/AMP categorical system of variant classification was compared with a quantitative point-based system. The pathogenicity likelihood ratios of REVEL, VEST, FATHMM, and MPC were calibrated using a gold standard set of 237 pathogenic and benign variants (classified independent of the PP3/BP4 criteria). RESULTS: The PP3 and BP4 criteria were applied by Variant Curation Expert Panels to 55% of missense variants. Application of those criteria changed the classification of 15% of missense variants for which either criterion was applied. The point-based system resolved borderline classifications. REVEL and VEST performed best at a strength level consistent with moderate evidence. CONCLUSION: We show that in silico criteria are commonly applied and often affect the final variant classifications. When appropriate thresholds for in silico predictors are established, our results show that PP3 and BP4 can be used at a moderate strength.

The genomic landscape of pediatric rheumatology disorders in the Middle East.

The landscape and clinical utility of comprehensive genomic investigations for a wide range of pediatric rheumatic disorders have not been fully characterized in the Middle East. Here, 71 pediatric patients, of diverse Arab origins, were clinically and genetically assessed for a spectrum of rheumatology-related diseases at the only dedicated tertiary children's hospital in the United Arab Emirates. Clinical genomic investigations included mainly (76%) next-generation sequencing-based gene panels and whole-exome sequencing, along with rapid sequencing in the intensive care unit and urgent setting. The overall positive yield was 46.5%, whereas dual diagnoses were made in two cases (3%). Although the majority (21/33, 64%) of positive findings involved the MEFV gene, the remaining (12/33, 36%) alterations were attributed to 11 other genes/loci. Copy number variants (CNVs) contributed substantially (5/33, 15.2%) to the overall diagnostic yield. Sequencing-based testing, specifically rapid sequencing, had a high positive rate and delivered timely results. Genetic findings guided clinical management plans and interventions in most cases (27/33, 81.8%). We highlight unique findings and provide additional evidence that heterozygous loss of function of the IFIH1 gene increases susceptibility to recurrent fevers. Our study provides new insights into the pathogenic variation landscape in pediatric rheumatic disorders.

VIP-HL: Semi-automated ACMG/AMP variant interpretation platform for genetic hearing loss.

The American College of Medical Genetics and Genomics, and the Association for Molecular Pathology (ACMG/AMP) have proposed a set of evidence-based guidelines to support sequence variant interpretation. The ClinGen hearing loss expert panel (HL-EP) introduced further specifications into the ACMG/AMP framework for genetic hearing loss. This study developed a tool named Variant Interpretation Platform for genetic Hearing Loss (VIP-HL), aiming to semi-automate the HL ACMG/AMP rules. VIP-HL aggregates information from external databases to automate 13 out of 24 ACMG/AMP rules specified by HL-EP, namely PVS1, PS1, PM1, PM2, PM4, PM5, PP3, BA1, BS1, BS2, BP3, BP4, and BP7. We benchmarked VIP-HL using 50 variants in which 82 rules were activated by the ClinGen HL-EP. VIP-HL concordantly activated 93% (76/82) rules, significantly higher than that of by InterVar (48%; 39/82). VIP-HL is an integrated online tool for reliable automated variant classification in hearing loss genes. It assists curators in variant interpretation and provides a platform for users to share classifications with each other. VIP-HL is available with a user-friendly web interface at http://hearing.genetics.bgi.com/.

Association of Dromedary Camels and Camel Ticks with Reassortant Crimean-Congo Hemorrhagic Fever Virus, United Arab Emirates.

We previously detected a potentially novel reassortant of Crimean-Congo hemorrhagic fever virus in camels at the largest livestock market in the United Arab Emirates. A broader survey of large mammals at the site indicated zoonotic transmission is associated with dromedaries and camel ticks. Seroprevalence in cattle, sheep, and goats is minimal.

Ca2+-Activated K+ Channels Reduce Network Excitability, Improving Adaptability and Energetics for Transmitting and Perceiving Sensory Information.

Ca2+-activated K+ channels (BK and SK) are ubiquitous in synaptic circuits, but their role in network adaptation and sensory perception remains largely unknown. Using electrophysiological and behavioral assays and biophysical modeling, we discover how visual information transfer in mutants lacking the BK channel (dSlo- ), SK channel (dSK- ), or both (dSK- ;; dSlo- ) is shaped in the female fruit fly (Drosophila melanogaster) R1-R6 photoreceptor-LMC circuits (R-LMC-R system) through synaptic feedforward-feedback interactions and reduced R1-R6 Shaker and Shab K+ conductances. This homeostatic compensation is specific for each mutant, leading to distinctive adaptive dynamics. We show how these dynamics inescapably increase the energy cost of information and promote the mutants' distorted motion perception, determining the true price and limits of chronic homeostatic compensation in an in vivo genetic animal model. These results reveal why Ca2+-activated K+ channels reduce network excitability (energetics), improving neural adaptability for transmitting and perceiving sensory information.SIGNIFICANCE STATEMENT In this study, we directly link in vivo and ex vivo experiments with detailed stochastically operating biophysical models to extract new mechanistic knowledge of how Drosophila photoreceptor-interneuron-photoreceptor (R-LMC-R) circuitry homeostatically retains its information sampling and transmission capacity against chronic perturbations in its ion-channel composition, and what is the cost of this compensation and its impact on optomotor behavior. We anticipate that this novel approach will provide a useful template to other model organisms and computational neuroscience, in general, in dissecting fundamental mechanisms of homeostatic compensation and deepening our understanding of how biological neural networks work.

Considerations for whole exome sequencing unique to prenatal care.

Whole exome sequencing (WES) is increasingly being used in the prenatal setting. The emerging data support the clinical utility of prenatal WES based on its diagnostic yield, which can be as high as 80% for certain ultrasound findings. However, detailed practice and laboratory guidelines, addressing the indications for prenatal WES and the surrounding technical, interpretation, ethical, and counseling issues, are still lacking. Herein, we review the literature and summarize the most recent findings and applications of prenatal WES. This review offers specialists and clinical genetic laboratorians a body of evidence and expert opinions that can serve as a resource to assist in their practice. Finally, we highlight the emerging technologies that promise a future of prenatal WES without the risks associated with invasive testing.

Using Machine Learning to Identify True Somatic Variants from Next-Generation Sequencing.

BACKGROUND: Molecular profiling has become essential for tumor risk stratification and treatment selection. However, cancer genome complexity and technical artifacts make identification of real variants a challenge. Currently, clinical laboratories rely on manual screening, which is costly, subjective, and not scalable. We present a machine learning-based method to distinguish artifacts from bona fide single-nucleotide variants (SNVs) detected by next-generation sequencing from nonformalin-fixed paraffin-embedded tumor specimens. METHODS: A cohort of 11278 SNVs identified through clinical sequencing of tumor specimens was collected and divided into training, validation, and test sets. Each SNV was manually inspected and labeled as either real or artifact as part of clinical laboratory workflow. A 3-class (real, artifact, and uncertain) model was developed on the training set, fine-tuned with the validation set, and then evaluated on the test set. Prediction intervals reflecting the certainty of the classifications were derived during the process to label "uncertain" variants. RESULTS: The optimized classifier demonstrated 100% specificity and 97% sensitivity over 5587 SNVs of the test set. Overall, 1252 of 1341 true-positive variants were identified as real, 4143 of 4246 false-positive calls were deemed artifacts, whereas only 192 (3.4%) SNVs were labeled as "uncertain," with zero misclassification between the true positives and artifacts in the test set. CONCLUSIONS: We presented a computational classifier to identify variant artifacts detected from tumor sequencing. Overall, 96.6% of the SNVs received definitive labels and thus were exempt from manual review. This framework could improve quality and efficiency of the variant review process in clinical laboratories.

SARS-CoV-2 Whole Genome Amplification and Sequencing for Effective Population-Based Surveillance and Control of Viral Transmission.

BACKGROUND: With the gradual reopening of economies and resumption of social life, robust surveillance mechanisms should be implemented to control the ongoing COVID-19 pandemic. Unlike RT-qPCR, SARS-CoV-2 whole genome sequencing (cWGS) has the added advantage of identifying cryptic origins of the virus, and the extent of community-based transmissions versus new viral introductions, which can in turn influence public health policy decisions. However, the practical and cost considerations of cWGS should be addressed before it is widely implemented. METHODS: We performed shotgun transcriptome sequencing using RNA extracted from nasopharyngeal swabs of patients with COVID-19, and compared it to targeted SARS-CoV-2 genome amplification and sequencing with respect to virus detection, scalability, and cost-effectiveness. To track virus origin, we used open-source multiple sequence alignment and phylogenetic tools to compare the assembled SARS-CoV-2 genomes to publicly available sequences. RESULTS: We found considerable improvement in whole genome sequencing data quality and viral detection using amplicon-based target enrichment of SARS-CoV-2. With enrichment, more than 99% of the sequencing reads mapped to the viral genome, compared to an average of 0.63% without enrichment. Consequently, an increase in genome coverage was obtained using substantially less sequencing data, enabling higher scalability and sizable cost reductions. We also demonstrated how SARS-CoV-2 genome sequences can be used to determine their possible origin through phylogenetic analysis including other viral strains. CONCLUSIONS: SARS-CoV-2 whole genome sequencing is a practical, cost-effective, and powerful approach for population-based surveillance and control of viral transmission in the next phase of the COVID-19 pandemic.

A mutation update for the PCDH19 gene causing early-onset epilepsy in females with an unusual expression pattern.

The PCDH19 gene consists of six exons encoding a 1,148 amino acid transmembrane protein, Protocadherin 19, which is involved in brain development. Heterozygous pathogenic variants in this gene are inherited in an unusual X-linked dominant pattern in which heterozygous females are affected, while hemizygous males are typically unaffected, although they pass on the pathogenic variant to each affected daughter. PCDH19-related disorder is known to cause early-onset epilepsy in females characterized by seizure clusters exacerbated by fever and in most cases, onset is within the first year of life. This condition was initially described in 1971 and in 2008 PCDH19 was identified as the underlying genetic etiology. This condition is the result of pathogenic loss-of-function variants that may be de novo or inherited from an affected mother or unaffected father and cellular interference has been hypothesized to be the culprit. Heterozygous females are symptomatic because of the presence of both wild-type and mutant cells that interfere with one another due to the production of different surface proteins, whereas nonmosaic hemizygous males produce a homogenous population of cells. Here, we review novel pathogenic variants in the PCDH19 gene since 2012 to date, and summarize any genotype-phenotype correlations.

Consensus interpretation of the p.Met34Thr and p.Val37Ile variants in GJB2 by the ClinGen Hearing Loss Expert Panel.

PURPOSE: Pathogenic variants in GJB2 are the most common cause of autosomal recessive sensorineural hearing loss. The classification of c.101T>C/p.Met34Thr and c.109G>A/p.Val37Ile in GJB2 are controversial. Therefore, an expert consensus is required for the interpretation of these two variants. METHODS: The ClinGen Hearing Loss Expert Panel collected published data and shared unpublished information from contributing laboratories and clinics regarding the two variants. Functional, computational, allelic, and segregation data were also obtained. Case-control statistical analyses were performed. RESULTS: The panel reviewed the synthesized information, and classified the p.Met34Thr and p.Val37Ile variants utilizing professional variant interpretation guidelines and professional judgment. We found that p.Met34Thr and p.Val37Ile are significantly overrepresented in hearing loss patients, compared with population controls. Individuals homozygous or compound heterozygous for p.Met34Thr or p.Val37Ile typically manifest mild to moderate hearing loss. Several other types of evidence also support pathogenic roles for these two variants. CONCLUSION: Resolving controversies in variant classification requires coordinated effort among a panel of international multi-institutional experts to share data, standardize classification guidelines, review evidence, and reach a consensus. We concluded that p.Met34Thr and p.Val37Ile variants in GJB2 are pathogenic for autosomal recessive nonsyndromic hearing loss with variable expressivity and incomplete penetrance.

Recommendations for interpreting the loss of function PVS1 ACMG/AMP variant criterion.

The 2015 ACMG/AMP sequence variant interpretation guideline provided a framework for classifying variants based on several benign and pathogenic evidence criteria, including a pathogenic criterion (PVS1) for predicted loss of function variants. However, the guideline did not elaborate on specific considerations for the different types of loss of function variants, nor did it provide decision-making pathways assimilating information about variant type, its location, or any additional evidence for the likelihood of a true null effect. Furthermore, this guideline did not take into account the relative strengths for each evidence type and the final outcome of their combinations with respect to PVS1 strength. Finally, criteria specifying the genes for which PVS1 can be applied are still missing. Here, as part of the ClinGen Sequence Variant Interpretation (SVI) Workgroup's goal of refining ACMG/AMP criteria, we provide recommendations for applying the PVS1 criterion using detailed guidance addressing the above-mentioned gaps. Evaluation of the refined criterion by seven disease-specific groups using heterogeneous types of loss of function variants (n = 56) showed 89% agreement with the new recommendation, while discrepancies in six variants (11%) were appropriately due to disease-specific refinements. Our recommendations will facilitate consistent and accurate interpretation of predicted loss of function variants.

Expert specification of the ACMG/AMP variant interpretation guidelines for genetic hearing loss.

Due to the high genetic heterogeneity of hearing loss (HL), current clinical testing includes sequencing large numbers of genes, which often yields a significant number of novel variants. Therefore, the standardization of variant interpretation is crucial to provide consistent and accurate diagnoses. The Hearing Loss Variant Curation Expert Panel was created within the Clinical Genome Resource to provide expert guidance for standardized genomic interpretation in the context of HL. As one of its major tasks, our Expert Panel has adapted the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for the interpretation of sequence variants in HL genes. Here, we provide a comprehensive illustration of the newly specified ACMG/AMP HL rules. Three rules remained unchanged, four rules were removed, and the remaining 21 rules were specified. These rules were further validated and refined using a pilot set of 51 variants assessed by curators and disease experts. Of the 51 variants evaluated in the pilot, 37% (19/51) changed category based upon application of the expert panel specified rules and/or aggregation of evidence across laboratories. These HL-specific ACMG/AMP rules will help standardize variant interpretation, ultimately leading to better care for individuals with HL.

AUDIOME: a tiered exome sequencing-based comprehensive gene panel for the diagnosis of heterogeneous nonsyndromic sensorineural hearing loss.

PURPOSE: Hereditary hearing loss is highly heterogeneous. To keep up with rapidly emerging disease-causing genes, we developed the AUDIOME test for nonsyndromic hearing loss (NSHL) using an exome sequencing (ES) platform and targeted analysis for the curated genes. METHODS: A tiered strategy was implemented for this test. Tier 1 includes combined Sanger and targeted deletion analyses of the two most common NSHL genes and two mitochondrial genes. Nondiagnostic tier 1 cases are subjected to ES and array followed by targeted analysis of the remaining AUDIOME genes. RESULTS: ES resulted in good coverage of the selected genes with 98.24% of targeted bases at >15 ×. A fill-in strategy was developed for the poorly covered regions, which generally fell within GC-rich or highly homologous regions. Prospective testing of 33 patients with NSHL revealed a diagnosis in 11 (33%) and a possible diagnosis in 8 cases (24.2%). Among those, 10 individuals had variants in tier 1 genes. The ES data in the remaining nondiagnostic cases are readily available for further analysis. CONCLUSION: The tiered and ES-based test provides an efficient and cost-effective diagnostic strategy for NSHL, with the potential to reflex to full exome to identify causal changes outside of the AUDIOME test.

Allele-Specific Droplet Digital PCR Combined with a Next-Generation Sequencing-Based Algorithm for Diagnostic Copy Number Analysis in Genes with High Homology: Proof of Concept Using Stereocilin.

BACKGROUND: Copy number variants (CNVs) can substantially contribute to the pathogenic variant spectrum in several disease genes. The detection of this type of variant is complicated in genes with high homology to other genomic sequences, yet such genomics regions are more likely to lead to CNVs, making it critical to address detection in these settings. METHODS: We developed a copy number analysis approach for high homology genes/regions that consisted of next-generation sequencing (NGS)-based dosage analysis accompanied by allele-specific droplet digital PCR (ddPCR) confirmatory testing. We applied this approach to copy number analysis in STRC, a gene with 98.9% homology to a nonfunctional pseudogene, pSTRC, and characterized its accuracy in detecting different copy number states by use of known samples. RESULTS: Using a cohort of 517 patients with hearing loss, we prospectively demonstrated the clinical utility of the approach, which contributed 30 of the 122 total positives (6%) to the diagnostic yield, increasing the overall yield from 17.6% to 23.6%. Positive STRC genotypes included homozygous (n = 15) or compound heterozygous (n = 8) deletions, or heterozygous deletions in trans with pathogenic sequence variants (n = 7). Finally, this approach limited ddPCR testing to cases with NGS copy number findings, thus markedly reducing the number of costly and laborious, albeit specific, ddPCR tests. CONCLUSIONS: NGS-based CNV detection followed by allele-specific ddPCR confirmatory testing is a reliable and affordable approach for copy number analysis in medically relevant genes with homology issues.

Prenatal DNA Sequencing: Clinical, Counseling, and Diagnostic Laboratory Considerations.

Clinical diagnostic laboratories are producing next-generation sequencing-based test results that are becoming increasingly incorporated into patient care. Whole genome and exome sequencing on fetal material derived from amniocytes, chorionic villi, or products of conception is starting to be offered clinically in specialized centers, but it has not yet become routine practice. The technical, interpretation, and ethical challenges are greatest in the area of prenatal medicine because the fetus has a limited health history, and the physical examination is only indirectly available via prenatal sonography. Here, we provide an overview of these challenges and highlight the clinical utility, reporting, and counseling issues associated with prenatal DNA sequencing. Future considerations are also discussed. © 2017 John Wiley & Sons, Ltd.