Faculty Bibliography

Despite extensive genetic heterogeneity, 72% of pathogenic alleles for Hirschsprung disease (HSCR) arise from coding and regulatory variants in genes of the RET and EDNRB gene regulatory network (GRN) in the enteric nervous system (ENS). To elucidate the mechanisms leading to enteric neuronal loss from these genetic defects, we generated four strains of mice carrying reduced function alleles at Ret or Ednrb or both, along with their wild-type alleles. ENS tissue- and single-cell gene expression profiling of the developing and postnatal gastrointestinal tract in these five mouse models revealed three major insights: i) Ret and Ednrb deficiency, rather than complete loss, is sufficient to induce HSCR, ii) Ret and Ednrb demonstrate strong trans interactions, and iii) disruption of this interaction leads to cellular fate changes to compensate for neuronal loss. Critically, we show the combined reduction of signaling of these two receptors below a threshold in enteric neural crest-derived cells (ENCDCs) leads to a molecular tipping point at which otherwise lesser cellular defects result in aganglionosis. This study of targeted mouse models of a multifactorial disorder reveals how increasing dosage of genetic defects within a GRN leads to quantifiably increasing dysregulation from genotype to gene expression to cellular identity to function. Importantly, our studies establish that aganglionosis results only with severely reduced gene expression at both receptor genes and their consequent disruption of normal and compensatory cell fate trajectories.

Autism is highly heritable and diagnosed more frequently in males than females. To identify neurodevelopmental processes that might present sex-biased vulnerability, we generated transcriptomic and epigenomic profiles of cell types present in the prenatally developing human cerebral cortex of 27 males and 21 females. By intersecting sex-biased molecular signatures and genes with de novo mutations in male and female autistic probands, we reveal two points of vulnerability contributing to the sex-biased penetrance in neurodevelopmental disorders (NDDs). First, we show that NDD risk genes are biased towards higher expression in females, identifying the NDD gene MEF2C as a critical transcription factor for female-biased expression. Second, we identify a significant contribution of X chromosome genes to NDD pathobiology. We construct a gene regulatory map of X-linked risk genes to enable functional studies of genetic variants that likely disrupt gene expression in the developing brains of autistic males. Together, these results point towards an outsized contribution of the X-chromosome to both the origin of sex differences in the developing human cortex and NDD vulnerability. We propose a model where female-biased vulnerability is driven by coding variation within genes while male-biased vulnerability is driven by noncoding variation in regulatory elements that affect gene expression.

Coding and enhancer variants of the RET receptor tyrosine kinase gene contribute to ~50% of Hirschsprung disease (HSCR) risk, a congenital disorder of disrupted enteric nervous system (ENS) development. The greatest contribution of this risk is from a common variant (rs2435357) in an ENS-active, SOX10-bound RET enhancer (MCS+9.7) that reduces RET gene expression in vivo and triggers expression changes in other ENS genes in the human fetal gut. To uncover the cellular basis of RET-mediated aganglionosis, we used CRISPR/Cas9 to delete (Δ) the homologous mouse enhancer (mcs+9.7). We used single cell RNA sequencing and high-resolution immunofluorescence to demonstrate four significant features of the developing E14.5 gut of Δmcs+9.7/Δmcs+9.7 embryos: (1) a small (5%) yet significant reduction in Ret gene expression in only two major cell types - early differentiating neurons and fate-restricted inhibitory motor neurons; (2) no significant cellular loss in the ENS; and, (3) loss of expression of 19 cell cycle regulator genes suggesting a proliferative defect. To identify the Ret functional threshold for normal ENS development, we also generated, in combination with the Ret CFP null allele, (4) Δmcs+9.7/CFP double heterozygote mice which reduced Ret gene expression in the ENS to 42% with severe loss of inhibitory motor neurons, an effect restricted to the hindgut and driven by proliferative loss. Thus, Ret gene expression drives proliferation of ENS progenitor cells and hindgut-specific inhibitory motor neuron development, and that HSCR aganglionosis arises from a cascade of cellular defects triggered by >50% loss of Ret function.

Hirschsprung disease (HSCR) exhibits extensive genetic heterogeneity, with 72% of cases involving pathogenic variants in 10 genes forming a gene regulatory network (GRN) essential for enteric nervous system (ENS) development. The receptor tyrosine kinase gene RET is the most significant contributor, implicated in 12%-50% of individuals depending on the phenotype. RET plays a critical role in ENS precursor proliferation and migration, and defects in these processes lead to HSCR. However, the functional impact of RET pathogenic variants and their mechanisms of disease remain poorly understood. To address this, we investigated proliferative and migratory phenotypes in a RET-dependent neural crest-derived cell line harboring one of five missense (c.166C>A [p.Leu56Met]; c.532G>C [p.Glu178Gln]; c.2372A>T [p.Tyr791Phe]; c.2765C>A [p.Ser922Tyr]; or c.2994T>A [p.Phe998Leu]) or three nonsense (c.612C>A, c.2308C>T, or c.2943C>G) heterozygous pathogenic RET variants. Using cDNA- and CRISPR-based prime reverse insertion mechanism engineering (PRIME) editing coupled with quantitative proliferation and migration assays, we observed significant losses in proliferation and migration in three missense (c.612C>A [p.Tyr204^∗]; c.2308C>T [p.Arg770^∗]; and c.2943C>G [p.Tyr981^∗]) and all nonsense variants. Notably, the c.2372A>T (p.Tyr791Phe) missense variant, whose pathogenicity has been debated, appears benign. Importantly, the severity of migration loss did not consistently correlate with proliferation defects, and the phenotypic severity of nonsense variants was independent of their position within the RET protein. This study highlights the necessity of targeted functional assays to accurately assess the pathogenicity of HSCR-associated variants rather than relying solely on bioinformatics predictions, which could be refined by incorporating functional data.

Despite the extensive genetic heterogeneity of Hirschsprung disease (HSCR; congenital colonic aganglionosis) 72% of patients harbor pathogenic variants in 10 genes that form a gene regulatory network (GRN) controlling the development of the enteric nervous system (ENS). Among these genes, the receptor tyrosine kinase gene RET is the most significant contributor, accounting for pathogenic variants in 12%-50% of patients depending on phenotype. RET plays a critical role in the proliferation and migration of ENS precursors, and defects in these processes lead to HSCR. However, despite the gene's importance in HSCR, the functional consequences of RET pathogenic variants and their mechanism of disease remain poorly understood. To address this, we investigated the proliferative and migratory phenotypes in a RET-dependent neural crest-derived cell line harboring one of five missense (L56M, E178Q, Y791F, S922Y, F998L) or three nonsense (Y204X, R770X, Y981X) pathogenic heterozygous variants. Using a combination of cDNA-based and CRISPR-based PRIME editing coupled with quantitative proliferation and migration assays, we detected significant losses in cell proliferation and migration in three missense (E178Q, S922Y, F998L) and all nonsense variants. Our data suggests that the Y791F variant, whose pathogenicity has been debated, is likely not pathogenic. Importantly, the severity of migration loss did not consistently correlate with proliferation defects, and the phenotypic severity of nonsense variants was independent of their position within the RET protein. This study highlights the necessity and feasibility of targeted functional assays to accurately assess the pathogenicity of HSCR-associated variants, rather than relying solely on machine learning predictions, which could themselves be refined by incorporating such functional data.

BACKGROUND/UNASSIGNED:Inter-individual variation in blood pressure (BP) arises in part from sequence variants within enhancers modulating the expression of causal genes. We propose that these genes, active in tissues relevant to BP physiology, can be identified from tissue-level epigenomic data and genotypes of BP-phenotyped individuals. METHODS/UNASSIGNED:We used chromatin accessibility data from the heart, adrenal, kidney, and artery to identify cis-regulatory elements (CREs) in these tissues and estimate the impact of common human single-nucleotide variants within these CREs on gene expression, using machine learning methods. To identify causal genes, we performed a gene-wise association test. We conducted analyses in 2 separate large-scale cohorts: 77 822 individuals from the Genetic Epidemiology Research on Adult Health and Aging and 315 270 individuals from the UK Biobank. RESULTS/UNASSIGNED:<0.0001). These results enabled tissue expression prediction of these 988 to 2875 putative BP genes in individuals of both cohorts to construct an expression polygenic score. This score explained ≈27% of the reported single-nucleotide variant heritability, substantially higher than expected from prior studies. CONCLUSIONS/UNASSIGNED:Our work demonstrates the power of tissue-restricted comprehensive CRE analysis, followed by CRE-based expression prediction, for understanding BP regulation in relevant tissues and provides dual-modality supporting evidence, CRE and expression, for the causality genes.

BACKGROUND:We examined effects of single-nucleotide variants (SNVs) of IL1RN, the gene encoding the anti-inflammatory interleukin 1 receptor antagonist (IL-1Ra), on the cytokine release syndrome (CRS) and mortality in patients with acute severe respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS:IL1RN CTA haplotypes formed from 3 SNVs (rs419598, rs315952, rs9005) and the individual SNVs were assessed for association with laboratory markers of inflammation and mortality. We studied 2589 patients hospitalized with SARS-CoV-2 between March 2020 and March 2021. RESULTS:Mortality was 15.3% and lower in women than men (13.1% vs 17.3%, P = .0003). Carriers of the CTA-1/2 IL1RN haplotypes exhibited decreased inflammatory markers and increased plasma IL-1Ra. Evaluation of the individual SNVs of the IL1RN, carriers of the rs419598 C/C SNV exhibited significantly reduced inflammatory biomarker levels and numerically lower mortality compared to the C/T-T/T genotype (10.0% vs 17.8%, P = .052) in men, with the most pronounced association observed in male patients ≤74 years old, whose mortality was reduced by 80% (3.1% vs 14.0%, P = .030). CONCLUSIONS:The IL1RN haplotype CTA and C/C variant of rs419598 are associated with attenuation of the CRS and decreased mortality in men with acute SARS-CoV-2 infection. The data suggest that the IL1RN pathway modulates the severity of coronavirus disease 2019 (COVID-19) via endogenous anti-inflammatory mechanisms.

Genome-wide association studies (GWASs) have identified numerous variants associated with polygenic traits and diseases. However, with few exceptions, a mechanistic understanding of which variants affect which genes in which tissues to modulate trait variation is lacking. Here, we present genomic analyses to explain trait heritability of blood pressure (BP) through the genetics of transcriptional regulation using GWASs, multiomics data from different tissues, and machine learning approaches. Approximately 500,000 predicted regulatory variants across four tissues explain 33.4% of variant heritability: 2.5%, 5.3%, 7.7%, and 11.8% for kidney-, adrenal-, heart-, and artery-specific variants, respectively. Variation in the enhancers involved shows greater tissue specificity than in the genes they regulate, suggesting that gene regulatory networks perturbed by enhancer variants in a tissue relevant to a phenotype are the major source of interindividual variation in BP. Thus, our study provides an approach to scan human tissue and cell types for their physiological contribution to any trait.

Genome-wide association studies have identified sequence polymorphisms in a functional enhancer of the NOS1AP gene as the most common genetic regulator of QT interval and human cardiac NOS1AP gene expression in the general population. Functional studies based on in vitro overexpression in murine cardiomyocytes and ex vivo knockdown in zebrafish embryonic hearts, by us and others, have also demonstrated that NOS1AP expression levels can alter cellular electrophysiology. Here, to explore the role of NOS1AP in cardiac electrophysiology at an organismal level, we generated and characterized constitutive and heart muscle-restricted Nos1ap knockout mice to assess whether NOS1AP disruption alters the QT interval in vivo. Constitutive loss of Nos1ap led to genetic background-dependent variable lethality at or right before birth. Heart muscle-restricted Nos1ap knockout, generated using cardiac-specific alpha-myosin heavy chain promoter-driven tamoxifen-inducible Cre, resulted in tissue-level Nos1ap expression reduced by half. This partial loss of expression had no detectable effect on the QT interval or other electrocardiographic and echocardiographic parameters, except for a small but significant reduction in the QRS interval. Given that challenges associated with defining the end of the T wave on murine electrocardiogram can limit identification of subtle effects on the QT interval and that common noncoding NOS1AP variants are also associated with the QRS interval, our findings support the role of NOS1AP in regulation of the cardiac electrical cycle.

Hirschsprung disease (HSCR) is associated with deficiency of the receptor tyrosine kinase RET, resulting in loss of cells of the enteric nervous system (ENS) during fetal gut development. The major contribution to HSCR risk is from common sequence variants in RET enhancers with additional risk from rare coding variants in many genes. Here, we demonstrate that these RET enhancer variants specifically alter the human fetal gut development program through significant decreases in gene expression of RET, members of the RET-EDNRB gene regulatory network (GRN), other HSCR genes, with an altered transcriptome of 2,382 differentially expressed genes across diverse neuronal and mesenchymal functions. A parsimonious hypothesis for these results is that beyond RET's direct effect on its GRN, it also has a major role in enteric neural crest-derived cell (ENCDC) precursor proliferation, its deficiency reducing ENCDCs with relative expansion of non-ENCDC cells. Thus, genes reducing RET proliferative activity can potentially cause HSCR. One such class is the 23 RET-dependent transcription factors enriched in early gut development. We show that their knockdown in human neuroblastoma SK-N-SH cells reduces RET and/or EDNRB gene expression, expanding the RET-EDNRB GRN. The human embryos we studied had major remodeling of the gut transcriptome but were unlikely to have had HSCR: thus, genetic or epigenetic changes in addition to those in RET are required for aganglionosis.