Biology subjects

Chung, W. K.

Publications and source records attributed to Chung, W. K..

At least 19 recordsLinked to original sources

bioRxiv2026

Pathogenic MAPK8IP3 variants drive distinct motor and behavioral phenotypes in humans and mice

Pathogenic variants in MAPK8IP3 (JIP3) cause Neurodevelopmental Disorder with or without variable Brain Abnormalities (NEDBA), characterized by cognitive impairment, global developmental delay, motor dysfunction, abnormal muscle tone, behavioral dysregulation including autism and attention deficit hyperactivity disorder (ADHD), seizures, and structural brain abnormalities. While more than 30 largely de novo MAPK8IP3 variants have been reported, the functional impact of variants across JIP3 protein structural domains is poorly defined. To address this knowledge gap, we compare clinical features of individuals with a truncating (p.E27X) or one of two missense (p.R578C and p.R1146C) variants from distinct JIP3 functional protein domains to corresponding knock-in mouse models. Our findings showed that all individuals, regardless of variant type, exhibited delays in language and gross motor function, but individual variants were associated with distinct motor, cognitive, and psychiatric symptoms. Corresponding homozygous p.E27X and p.R1147C variant mice resulted in embryonic lethality, consistent with essential roles for JIP3 in early neurodevelopment. Behavioral characterization of viable heterozygous mice revealed variant-specific locomotor, motor coordination, and hindlimb clasping defects that closely recapitulate clinical observations. Heterozygous p.R579C mice exhibited reduced locomotion, impaired motor performance, and hypertonia-like clasping, mirroring human motor deficits. In contrast, p.R1147C mice displayed hyperactivity, hindlimb clasping, and decreased brain weight, paralleling human clinical features. Together, our findings demonstrate that while distinct MAPK8IP3 variants lead to some shared phenotypes, they are also associated with distinct phenotypes that could reveal domain-specific aspects of JIP3 function. This work establishes the first domain-resolved in vivo rodent models of NEDBA and provides a validated translational platform for mechanistic investigation and preclinical therapeutic testing.

neuroscience↗

bioRxiv2026

Fetal MRI reveals altered prenatal cortical surface area in fetuses later diagnosed with autism spectrum disorder

Autism spectrum disorder (ASD) is increasingly conceptualized as a condition rooted in altered prenatal neurodevelopment, yet in vivo evidence from fetal brain imaging remains limited. Using retrospective fetal MRI and surface-based morphometry, we investigated cortical development in 15 fetuses later diagnosed with ASD (77%; mean gestational age [GA] = 26.7 weeks) without major structural brain abnormalities and compared them with 60 typically developing controls (57% male; mean GA = 28.4 weeks). Fetuses later diagnosed with ASD showed significantly reduced whole-brain inner cortical plate surface area compared with controls ({beta} = -0.08 {+/-} 0.02 SE, p = 0.002, partial {superscript 2} = 0.13), corresponding to an estimated [~]7.7% reduction (predicted at GA = 28.1 weeks). Lobar mixed-effects analyses demonstrated broadly distributed reductions across all cortical lobes (FDR-corrected p = <0.001-0.024; Cohens d = -0.06 to -0.10), with modest regional heterogeneity indicating relatively greater frontal and insular involvement (groupxlobe: F = 19.31, p = 0.002, {superscript 2} = 0.08). Surface area findings remained directionally stable across sensitivity analyses, including restriction to neurodevelopmentally confirmed controls and models accounting for image quality variability, although effect sizes were attenuated after quality adjustment. Normative modeling further demonstrated subtle negative deviations from typical prenatal cortical surface area trajectories in ASD (mean Z = -0.27, p = 0.018). These findings suggest that aspects of cortical morphogenesis may diverge prenatally in individuals later diagnosed with ASD and suggest the feasibility of fetal MRI-based surface morphometry for studying early neurodevelopmental variation associated with ASD risk.

neuroscience↗

bioRxiv2026

Modeling patient variants of Cnot1 and Cdc42bpb results in distinct forms of congenital diaphragmatic hernia in mice

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly characterized by impairment of both diaphragm and lung development in utero. CDH presents as a spectrum of forms and severities, with diaphragm defects arising in the dorsal/posterior region typically correlating with more severe pulmonary disease and higher risk of mortality than those appearing in ventral/anterior regions. The genetic etiology underlying CDH is complex, with many genes implicated showing variable expressivity and incomplete penetrance in both human patients and mouse models. Here we present in vivo validation of two genes previously unassociated with CDH: the CDC42-interacting kinase CDC42BPB; and CNOT1, a scaffolding protein of the CCR4-NOT protein complex, critical for mRNA regulation through modifications such as deadenylation. Each gene was found to have a damaging, de novo missense variant in a recent large-scale CDH patient sequencing screen. Loss of Cdc42bpb leads to ventral diaphragmatic hernias, heart septal defects and minor lung epithelial differentiation defects in mouse embryos. Installation of the orthologous patient-specific missense variant through CRISPR/Cas9 editing leads to less severe ventral diaphragm defects. Mouse embryos with either one or two copies of the orthologous Cnot1 variant, c.1867C>T (p.R623W), develop dorsal diaphragmatic hernias with low (<50%) penetrance, and mutants showed alterations in mRNA isoform expression consistent with the molecular role of Cnot1 in RNA splicing. These results underscore the power of in vivo functional modeling to validate genes and patient-specific variants uncovered by patient sequencing, reveal two previously unrecognized genetic causes of CDH, and highlight the heterogeneity of different patient anatomic presentations.

genetics↗

bioRxiv2026

Dysregulated TGFb-ERK Signaling Drives Aberrant Extracellular Matrix Production in Noonan Syndrome-Associated Pulmonary Valve Stenosis

Pulmonary valve stenosis (PVS) is the most common congenital heart defect in Noonan syndrome (NS) and related RASopathies, yet the molecular mechanisms linking pathogenic variants to the valve pathology remain poorly defined. Here, we utilized a human iPSC-based valve differentiation platform to generate the cardiac valve cell lineages--including fibrosa and spongiosa valve interstitial cell (VIC) subtypes. CRISPR-edited iPSCs harboring NS gain-of-function RAS/MAPK and Noonan syndrome with multiple lentigines (NSML) dominant-negative RAS/MAPK variants exhibited early defects in mesodermal and endocardial specification in all genotypes. Additionally, NS-iPSC endocardial cells exhibited defects in endothelial-to-mesenchymal transition (EndMT) specifically towards fibrosa VICs, which was most pronounced in PTPN11N308D (N308D) cells. Single-cell transcriptomics revealed widespread dysregulation of extracellular matrix (ECM) programs in N308D fibrosa VICs, including increased expression of collagens and proteoglycans, as well as dysregulation of multiple genes involved in ECM remodeling. We also detected activation of RAS-MAPK, TGF{beta}, and fibrosis-associated pathways in our transcriptional dataset. Mass spectrometry-based phosphoproteomics confirmed coordinated increases in ERK, PKC, and stress-related kinases, as well as enhanced activity of the TGF{beta} receptor. Functionally, N308D fibrosa VICs exhibited exaggerated upregulation of ECM genes in the presence of TGF{beta}2 ligand, suggesting that these cells are hypersensitive to TGF{beta} stimulation. Furthermore, we demonstrated that this pathological ECM-program occurs independently of BAMBI, a negative regulator of TGF{beta} signaling that was found to be decreased in N308D fibrosa VICs. Lastly, we performed histopathological analyses of stenotic pulmonary valves from two NS infants, which demonstrated marked overproduction and disorganization of ECM, mirroring the findings from our iPSC-based disease model. Together, our data reveal a central mechanism where NS-associated alleles sensitize fibrosa VICs to TGF{beta}, which leads to aberrant downstream signaling and drives the pathological ECM program in NS-associated PVS.

cell biology↗

bioRxiv2025

Recessive but damaging alleles of muscle-specific ribosomal protein gene RPL3L drive neonatal dilated cardiomyopathy

The heart employs a specialized ribosome in its muscle cells to translate genetic information into proteins, a fundamental adaptation with an elusive physiological role1-3. Its significance is underscored by the discovery of neonatal patients suffering from often fatal heart failure caused by severe dilated cardiomyopathy when both copies of the gene RPL3L are mutated4-9. RPL3L is a muscle-specific paralog1-3 of the ubiquitous ribosomal protein L3 (RPL3), which makes the closest contact of any protein to the ribosomes RNA-based catalytic center10. RPL3L-linked heart failure represents the only known human disease associated with tissue-specific ribosomes, yet the underlying pathogenetic mechanisms remain poorly understood. Intriguingly, disease is linked to a large number of mostly missense variants in RPL3L, and RPL3L-knockout resulted in no severe heart defect in either human or mice3, 11-13, challenging the prevailing view that autosomal recessive diseases are caused by loss-of-function mutations. Here, we report three new cases of RPL3L-linked severe neonatal heart failure and present a unifying pathogenetic mechanism by which a large number of variants in the muscle-specific ribosome led to disease. Specifically, affected families often carry one of two recurrent toxic gain-of-function variants alongside a family-specific putative loss-of-function variant. While the non-recurrent variants often trigger partial compensation of RPL3 similar to Rpl3l-knockout mice, both recurrent variants exhibit increased affinity for the RPL3/RPL3L chaperone GRWD114-16 and 60S biogenesis factors, sequester 28S rRNA in the nucleus, disrupt ribosome biogenesis, and trigger severe cellular toxicity that extends beyond the loss of ribosomes. These findings provide critical insights for genetic screening and therapeutic development of neonatal heart failure. Our results suggest that gain-of-toxicity mechanisms may be more prevalent in autosomal recessive diseases, and a combination of gain-of-toxicity and loss-of-function mechanisms could underlie many diseases involving genes with paralogs.

molecular biology↗

bioRxiv2024

Role of Forkhead box F1 in the Pathobiology of Pulmonary Arterial Hypertension

RationaleApproximately 80% of patients with non-familial pulmonary arterial hypertension (PAH) lack identifiable pathogenic genetic variants. While most genetic studies of PAH have focused on predicted loss-of-function variants, recent approaches have identified ultra-rare missense variants associated with the disease. FOXF1 encodes a highly conserved transcription factor, essential for angiogenesis and vasculogenesis in human and mouse lungs. ObjectivesWe identified a rare FOXF1 missense coding variant in two unrelated probands with PAH. FOXF1 is an evolutionarily conserved transcription factor required for lung vascular development and vascular integrity. Our aims were to determine the frequency of FOXF1 variants in larger PAH cohorts compared to the general population, study FOXF1 expression in explanted lung tissue from PAH patients versus control (failed-donor) lungs, and define potential downstream targets linked to PAH development. MethodsThree independent, international, multicenter cohorts were analyzed to evaluate the frequency of FOXF1 rare variants. Various composite prediction models assessed the deleteriousness of individual variants. Bulk RNA sequencing datasets from human explanted lung tissues were compared to failed-donor controls to determine FOXF1 expression. Bioinformatic tools identified putative FOXF1 binding targets, which were orthogonally validated using mouse ChIP-seq datasets. Measurements and Main ResultsSeven novel or ultra-rare missense coding variants were identified across three patient cohorts in different regions of the FOXF1 gene, including the DNA binding domain. FOXF1 expression was dysregulated in PAH lungs, correlating with disease severity. Histological analysis showed heterogeneous FOXF1 expression, with the lowest levels in phenotypically abnormal endothelial cells within complex vascular lesions in PAH samples. A hybrid bioinformatic approach identified FOXF1 downstream targets potentially involved in PAH pathogenesis, including BMPR2. ConclusionsLarge genomic and transcriptomic datasets suggest that decreased FOXF1 expression or predicted dysfunction is associated with PAH.

genomics↗

bioRxiv2024

GATA6 regulates WNT and BMP programs to pattern precardiac mesoderm during the earliest stages of human cardiogenesis

Haploinsufficiency for GATA6 is associated with congenital heart disease (CHD) with variable comorbidity of pancreatic or diaphragm defects, although the etiology of disease is not well understood. Here, we used cardiac directed differentiation from human embryonic stem cells (hESCs) as a platform to study GATA6 function during early cardiogenesis. GATA6 loss-of-function hESCs had a profound impairment in cardiac progenitor cell (CPC) specification and cardiomyocyte (CM) generation due to early defects during the mesendoderm and lateral mesoderm patterning stages. Profiling by RNA-seq and CUT&RUN identified genes of the WNT and BMP programs regulated by GATA6 during early mesoderm patterning. Furthermore, interactome analysis detected GATA6 binding with developmental transcription factors and chromatin remodelers suggesting cooperative regulation of cardiac lineage gene accessibility. We show that modulating WNT and BMP inputs during the first 48 hours of cardiac differentiation is sufficient to partially rescue CPC and CM defects in GATA6 heterozygous and homozygous mutant hESCs. This study provides evidence of the regulatory functions for GATA6 directing human precardiac mesoderm patterning during the earliest stages of cardiogenesis to further our understanding of haploinsufficiency causing CHD and the co-occurrence of cardiac and other organ defects caused by human GATA6 mutations.

developmental biology↗

bioRxiv2024

Automated Identification of Germline de novo Mutations in Family Trios: A Consensus-Based Informatic Approach

Accurate identification of germline de novo variants (DNVs) remains a challenging problem despite rapid advances in sequencing technologies as well as methods for the analysis of the data they generate, with putative solutions often involving ad hoc filters and visual inspection of identified variants. Here, we present a purely informatic method for the identification of DNVs by analyzing short-read genome sequencing data from proband-parent trios. Our method evaluates variant calls generated by three genome sequence analysis pipelines utilizing different algorithms--GATK HaplotypeCaller, DeepTrio and Velsera GRAF--exploring the assumption that a requirement of consensus can serve as an effective filter for high- quality DNVs. We assessed the efficacy of our method by testing DNVs identified using a previously established, highly accurate classification procedure that partially relied on manual inspection and used Sanger sequencing to validate a DNV subset comprising less confident calls. The results show that our method is highly precise and that applying a force-calling procedure to putative variants further removes false-positive calls, increasing precision of the workflow to 99.6%. Our method also identified novel DNVs, 87% of which were validated, indicating it offers a higher recall rate without compromising accuracy. We have implemented this method as an automated bioinformatics workflow suitable for large- scale analyses without need for manual intervention.

bioinformatics↗

bioRxiv2024

PreMode predicts mode of action of missense variants by deep graph representation learning of protein sequence and structural context

Accurate prediction of the functional impact of missense variants is important for disease gene discovery, clinical genetic diagnostics, therapeutic strategies, and protein engineering. Previous efforts have focused on predicting a binary pathogenicity classification, but the functional impact of missense variants is multi-dimensional. Pathogenic missense variants in the same gene may act through different modes of action (i.e., gain/loss-of-function) by affecting different aspects of protein function. They may result in distinct clinical conditions that require different treatments. We developed a new method, PreMode, to perform gene-specific mode-of-action predictions. PreMode models effects of coding sequence variants using SE(3)-equivariant graph neural networks on protein sequences and structures. Using the largest-to-date set of missense variants with known modes of action, we showed that PreMode reached state-of-the-art performance in multiple types of mode-of-action predictions by efficient transfer-learning. Additionally, PreModes prediction of G/LoF variants in a kinase is consistent with inactive-active conformation transition energy changes. Finally, we show that PreMode enables efficient study design of deep mutational scans and optimization in protein engineering.

bioinformatics↗

bioRxiv2023

Disrupted endosomal trafficking of the Vangl-Celsr polarity complex causes congenital anomalies in trachea-esophageal separation

Disruptions in foregut morphogenesis can result in life-threatening conditions where the trachea and esophagus fail to separate properly, such as esophageal atresia (EA) and tracheoesophageal fistulas (TEF). The developmental basis of these congenital anomalies is poorly understood, but recent genome sequencing reveals that de novo variants in intracellular trafficking genes are enriched in EA/TEF patients. Here, we confirm that mutation of orthologous genes in Xenopus disrupts trachea-esophageal separation similar to EA/TEF patients. We show that the Rab11a recycling endosome pathway is required to localize Vangl-Celsr polarity complexes at the luminal cell surface where opposite sides of the foregut tube fuse. Partial loss of endosome trafficking or Vangl-Celsr complexes disrupts epithelial polarity and mutant cells accumulate at the fusion point, fail to downregulate Cadherin, and do not separate into distinct trachea and esophagus. These data provide insights into the mechanisms of congenital anomalies and general paradigms of tissue fusion during organogenesis.

developmental biology↗

bioRxiv2022

Context-dependent functions of mitochondria protein quality control in lung

Aside from its role as the universal energy source of the cell, mitochondria also control many aspects of cell behavior. In an intact tissue, whether all cells require mitochondria function to the same extent, and how mitochondria insufficiency impacts cell behavior are poorly understood. Here we show that in the mouse lung epithelium, inactivation of LONP1, an energy ATP-dependent protease that functions in the mitochondria to degrade unfolded and misfolded proteins, led to mitochondria deficiency. In the naive epithelium of the developing lung, loss of Lonp1 obliterated cell proliferation and differentiation. In the adult airway epithelium during homeostasis, loss of Lonp1 led to selective death of terminally differentiated multiciliated cells, leading to a cascade of progenitor activation to replace lost cells. In the adult airway epithelium following influenza infection, loss of Lonp1 led to failure of airway progenitor migration into the damaged alveolar region. Bulk and single cell transcriptomic analysis revealed that one branch of the ER stress pathways, namely integrated stress response (ISR), is ectopically upregulated in mutants under all three conditions. Inactivation of core ISR transcription factor ATF4 in the Lonp1 mutant airway reversed abovementioned phenotypes. Taken together, our findings demonstrate that depending on a cellular context, intact mitochondria function is required in either progenitor or progeny cells, and is essential for cell proliferation, survival or migration in the mammalian lung.

cell biology↗

bioRxiv2022

Combination of an inject-and-transfer system for serial femtosecond crystallography

Serial femtosecond crystallography (SFX) enables the determination of the room-temperature crystal structure of macromolecules without causing radiation damage and provides time-resolved molecular dynamics by pump-probe experiments. In the SFX experiment, the injector-based sample delivery method continuously provides fresh crystals to X-rays, and the fixed-target scanning method can be programmed to move the crystals to the desired location. This study introduces a combination of the inject-and-transfer system (BITS) method for sample delivery for SFX experiments, a hybrid injection, and a fixed-target scanning method. BITS allows solution samples to be reliably deposited on an ultraviolet ozone (UVO)-treaed polyimide films at flow rates as low as 1 nl/min. In application of BITS in SFX experiment, the lysozyme crystal samples were embedded in a viscous lard medium and injected at a 50-100 nl/min flow rate through a syringe needle onto an UVO-treated polyimide film mounted on a fixed-target scan stage. The deposited crystal sample on film were raster scanned to XFEL by motion stage in the horizontal and vertical directions. Using this method, we successfully determined the room-temperature structure of lysozyme at 2.1 [A] resolution. This method can be applied to the SFX experiments.

biophysics↗

bioRxiv2021

Environmental carcinogens disproportionally mutate genes implicated in neurodevelopmental disorders

De novo mutations contribute to a large proportion of sporadic psychiatric and developmental disorders, yet the potential role of environmental carcinogens as drivers of causal de novo mutations in neurodevelopmental disorders is poorly studied. We demonstrate that several mutagens, including polycyclic aromatic hydrocarbons (PAHs), disproportionately mutate genes related to neurodevelopmental disorders including autism spectrum disorders (ASD), schizophrenia, and attention deficit hyperactivity disorder (ADHD). Other disease genes including amyotrophic lateral sclerosis (ALS), Alzheimers disease, congenital heart disease, orofacial clefts, and coronary artery disease were generally not mutated more than expected. Our findings support a new paradigm of neurodevelopmental disease etiology driven by a contribution of environmentally induced rather than random mutations.

bioinformatics↗

bioRxiv2021

Predicting functional effect of missense variants using graph attention neural networks

Accurate prediction of damaging missense variants is critically important for interpreting genome sequence. While many methods have been developed, their performance has been limited. Recent progress in machine learning and availability of large-scale population genomic sequencing data provide new opportunities to significantly improve computational predictions. Here we describe gMVP, a new method based on graph attention neural networks. Its main component is a graph with nodes capturing predictive features of amino acids and edges weighted by coevolution strength, which enables effective pooling of information from local protein context and functionally correlated distal positions. Evaluated by deep mutational scan data, gMVP outperforms published methods in identifying damaging variants in TP53, PTEN, BRCA1, and MSH2. Additionally, it achieves the best separation of de novo missense variants in neurodevelopmental disorder cases from the ones in controls. Finally, the model supports transfer learning to optimize gain- and loss-of-function predictions in sodium and calcium channels. In summary, we demonstrate that gMVP can improve interpretation of missense variants in clinical testing and genetic studies.

genomics↗

bioRxiv2021

Human stem cell model of HNF1A deficiency shows uncoupled insulin to C-peptide secretion with accumulation of abnormal insulin granules

Mutations in HNF1A cause Maturity Onset Diabetes of the Young type 3 (MODY3), the most prevalent form of monogenic diabetes. We generated stem cell-derived pancreatic endocrine cells from human embryonic stem cells (hESCs) with induced hypomorphic mutations in HNF1A. Using these cells, we show that HNF1A orchestrates a transcriptional program required for distinct aspects of {beta}-cell fate and function. During islet cell differentiation, HNF1A deficiency biases islet endocrine cells towards an -cell fate associated with PAX4 down-regulation. HNF1A- deficient {beta}-cells display impaired basal and glucose stimulated-insulin secretion in association with a reduction in CACNA1A and intracellular calcium levels, and impaired insulin granule exocytosis in association with SYT13 down-regulation. Knockout of PAX4, CACNA1A and SYT13 reproduce the relevant phenotypes. Reduction of insulin secretion is associated with accumulation of enlarged secretory granules, and altered stoichiometry of secreted insulin to C-peptide. In HNF1A deficient {beta}-cells, glibenclamide, a sulfonylurea drug used in the treatment of MODY3 patients, increases intracellular calcium to levels beyond those achieved by glucose, and restores C-peptide and insulin secretion to a normal stoichiometric ratio. To study HNF1A deficiency in the context of a human disease model, we also generated stem cell-derived pancreatic endocrine cells from two MODY3 patients induced pluripotent stem cells (iPSCs). While insulin secretion defects are constitutive in cells with complete HNF1A loss of function, {beta}-cells heterozygous for hypomorphic HNF1A mutations are initially normal, but lose the ability to secrete insulin and acquire abnormal stoichiometric secretion ratios. Importantly, the defects observed in these stem cell models are also seen in circulating proportions of insulin:C-peptide in nine MODY3 patients. One sentence of summaryDeficiency of the transcription factor HNF1A biases islet endocrine cell fate towards -cells, impairs intracellular calcium homeostasis and insulin exocytosis, alters the stoichiometry of insulin to C-peptide release, and leads to an accumulation of abnormal insulin secretory granules in {beta}-cells.

cell biology↗

bioRxiv2020

Rare variant analysis of 4,241 pulmonary arterial hypertension cases from an international consortium implicate FBLN2, PDGFD and rare de novo variants in PAH

BackgroundGroup 1 pulmonary arterial hypertension (PAH) is a lethal vasculopathy characterized by pathogenic remodeling of pulmonary arterioles leading to increased pulmonary pressures, right ventricular hypertrophy and heart failure. Recent high-throughput sequencing studies have identified additional PAH risk genes and suggested differences in genetic causes by age of onset. However, known risk genes explain only 15-20% of non-familial idiopathic PAH cases. MethodsTo identify new risk genes, we utilized an international consortium of 4,241 PAH cases with 4,175 sequenced exomes (n=2,572 National Biological Sample and Data Repository for PAH; n=469 Columbia University Irving Medical Center, enriched for pediatric trios) and 1,134 sequenced genomes (UK NIHR Bioresource - Rare Diseases Study). Most of the cases were adult-onset disease (93%), and 55% idiopathic (IPAH) and 35% associated with other diseases (APAH). We identified protein-coding variants and performed rare variant association analyses in unrelated participants of European ancestry, including 2,789 cases and 18,819 controls (11,101 unaffected parents from the Simons Powering Autism Research for Knowledge study and 7,718 gnomAD individuals). We analyzed de novo variants in 124 pediatric trios. ResultsSeven genes with rare deleterious variants were significantly associated (false discovery rate <0.1) with IPAH, including three known genes (BMPR2, GDF2, and TBX4), two recently identified candidate genes (SOX17, KDR), and two new candidate genes (FBLN2, fibulin 2; PDGFD, platelet-derived growth factor D). The candidate genes exhibit expression patterns in lung and heart similar to that of known PAH risk genes, and most of the variants occur in conserved protein domains. Variants in known PAH gene, ACVRL1, showed association with APAH. Predicted deleterious de novo variants in pediatric cases exhibited a significant burden compared to the background mutation rate (2.5x, p=7.0E-6). At least eight novel candidate genes carrying de novo variants have plausible roles in lung/heart development. ConclusionsRare variant analysis of a large international consortium identifies two new candidate genes - FBLN2 and PDGFD. The new genes have known functions in vasculogenesis and remodeling but have not been previously implicated in PAH. Trio analysis predicts that ~15% of pediatric IPAH may be explained by de novo variants.

genetics↗

bioRxiv2020

Deep whole genome sequencing of multiple proband tissues and parental blood reveals the complex genetic etiology of congenital diaphragmatic hernias

The diaphragm is a mammalian muscle critical for respiration and separation of the thoracic and abdominal cavities. Defects in the development of the diaphragm are the cause of congenital diaphragmatic hernia (CDH), a common birth defect. In CDH, weaknesses in the developing diaphragm allow abdominal contents to herniate into the thoracic cavity and impair lung development, leading to a high neonatal mortality. The genetic etiology of CDH is complex. Single nucleotide variants (SNVs), insertion/deletions (indels), and structural/copy number variants in more than 150 genes have been associated with CDH, although few genes are recurrently mutated in multiple patients and recurrently mutated genes can be incompletely penetrant. This suggests that multiple genetic variants in combination, other not yet investigated classes of variants, and/or nongenetic factors contribute to CDH susceptibility. However, to date no studies have comprehensively investigated the contribution of all possible classes of variants throughout the genome to the etiology of CDH. In our study, we used a unique cohort of four patients with isolated CDH with samples from blood, skin, and diaphragm connective tissue and parental blood samples and deep whole genome sequencing to assess germline and somatic de novo and inherited variants of various sizes (SNVs, indels, and structural variants) in exons, introns, UTRs, and intergenic regions. In each patient we found a different mutational landscape that included germline de novo, and inherited SNVs and indels in multiple genes. We also found in two patients an inherited 343 bp deletion interrupting an annotated enhancer of the CDH associated gene, GATA4, and we hypothesize that this common deletion (found in 1-2% of the population) acts as a sensitizing allele for CDH. Overall, our comprehensive reconstruction of the genetic architecture of four CDH individuals demonstrates that the etiology of CDH is heterogeneous and multifactorial. AUTHOR SUMMARYDeep whole genome sequencing of family trios shows that etiology of congenital diaphragmatic hernias is heterogeneous and multifactorial.

genetics↗

bioRxiv2020

Functional modeling of NMIHBA-causing PRUNE1 variants reveals a requirement for its exopolyphosphatase activity.

Neurodevelopmental disorder with microcephaly, hypotonia, and variable brain anomalies (NMIHBA) is an autosomal recessive neurodevelopmental and neurodegenerative disorder characterized by global developmental delay and severe intellectual disability. Microcephaly, progressive cortical atrophy, cerebellar hypoplasia and delayed myelination are neurological hallmarks in affected individuals. NMIHBA is caused by biallelic variants in PRUNE1 encoding prune exopolyphosphatase 1. We provide in-depth clinical description of two affected siblings harboring compound heterozygous variant alleles, c.383G>A (p.Arg128Gln), c.520G>T (p.Gly174*) in PRUNE1. To gain insights into disease biology, we biochemically characterized missense variants within the conserved N-terminal aspartic acid-histidine-histidine (DHH) motif and provide evidence that they result in the destabilization of protein structure and/or loss of exopolyphosphatase activity. Genetic ablation of Prune1 results in midgestational lethality in mice, associated with perturbations to embryonic growth and vascular development. Our findings suggest that NMIHBA results from hypomorphic variant alleles in humans and underscore the potential key role of PRUNE1 exopolyphoshatase activity in neurodevelopment.

genetics↗