Identification of the Molecular Components of Enhancer-Mediated Gene Expression Variation in Multiple Tissues Regulating Blood Pressure.

BACKGROUND: 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: 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: We identified 309, 259, 331, and 367 genes (false discovery rate <0.05) for diastolic BP and 191, 184, 204, and 204 genes for systolic BP in the artery, kidney, heart, and adrenal, respectively, in Genetic Epidemiology Research on Adult Health and Aging; 50% to 70% of these genes were replicated in the UK Biobank, significantly higher than the 12% to 15% expected by chance (P<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: 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.

RET enhancer haplotype-dependent remodeling of the human fetal gut development program.

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.

Tissue-specific and tissue-agnostic effects of genome sequence variation modulating blood pressure.

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.

Cardiac muscle-restricted partial loss of Nos1ap expression has limited but significant impact on electrocardiographic features.

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.

Ret deficiency decreases neural crest progenitor proliferation and restricts fate potential during enteric nervous system development.

The receptor tyrosine kinase RET plays a critical role in the fate specification of enteric neural crest-derived cells (ENCDCs) during enteric nervous system (ENS) development. RET loss of function (LoF) is associated with Hirschsprung disease (HSCR), which is marked by aganglionosis of the gastrointestinal (GI) tract. Although the major phenotypic consequences and the underlying transcriptional changes from Ret LoF in the developing ENS have been described, cell type- and state-specific effects are unknown. We performed single-cell RNA sequencing on an enriched population of ENCDCs from the developing GI tract of Ret null heterozygous and homozygous mice at embryonic day (E)12.5 and E14.5. We demonstrate four significant findings: 1) Ret-expressing ENCDCs are a heterogeneous population comprising ENS progenitors as well as glial- and neuronal-committed cells; 2) neurons committed to a predominantly inhibitory motor neuron developmental trajectory are not produced under Ret LoF, leaving behind a mostly excitatory motor neuron developmental program; 3) expression patterns of HSCR-associated and Ret gene regulatory network genes are impacted by Ret LoF; and 4) Ret deficiency leads to precocious differentiation and reduction in the number of proliferating ENS precursors. Our results support a model in which Ret contributes to multiple distinct cellular phenotypes during development of the ENS, including the specification of inhibitory neuron subtypes, cell cycle dynamics of ENS progenitors, and the developmental timing of neuronal and glial commitment.

Interleukin-1 receptor antagonist gene ( IL1RN ) variants modulate the cytokine release syndrome and mortality of SARS-CoV-2.

Objective: To explore the regulation of the inflammatory response in acute SARS-CoV-2 infection, we examined effects of single nucleotide variants (SNVs) of IL1RN , the gene encoding the anti-inflammatory IL-1 receptor antagonist (IL-1Ra), on the cytokine release syndrome and mortality. Methods: We studied 2589 patients hospitalized with SARS-CoV-2 between March 2020 and March 2021 at NYU Langone's Tisch Hospital. CTA and TTG haplotypes formed from three SNVs (rs419598, rs315952, rs9005) and the individual SNVs of the IL1RN gene were assessed for association with laboratory markers of the cytokine release syndrome (CRS) and mortality. Results: Mortality in the population was 15.3%, and was lower in women than men (13.1% vs.17.3%, p<0.0003). Carriers of the CTA-1/2 IL1RN haplotypes exhibited decreased inflammatory markers and increased plasma IL-1Ra relative to TTG carriers. Decreased mortality among CTA-1/2 carriers was observed in male patients between the ages of 55-74 [9.2% vs. 17.9%, p=0.001]. Evaluation of individual SNVs of the IL1RN gene (rs419598, rs315952, rs9005) indicated that carriers of the IL1RN rs419598 CC SNV exhibited lower inflammatory biomarker levels, and was associated with reduced mortality compared to the CT/TT genotype in men (OR 0.49 (0.23 - 1.00); 0.052), with the most pronounced effect observed between the ages of 55-74 [5.5% vs. 18.4%, p<0.001]. Conclusion: The IL1RN haplotype CTA, and sequence variant of rs419598 are associated with attenuation of the cytokine release syndrome and decreased mortality in males with acute SARS-CoV2 infection. The data suggest that IL1RN modulates the COVID-19 cytokine release syndrome via endogenous " anti-inflammatory" mechanisms. Significance statement: We provide evidence that variants of IL1RN modulate the severity of SARS-CoV-2 infection. The IL1RN CTA haplotype and rs419598 CC single nucleotide variant are associated with decreased plasma levels of inflammatory markers, interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), interleukin-2 (IL-2), C-reactive protein (CRP), D-dimer, ferritin, and procalcitonin, in association with higher levels of IL-1Ra and IL-10, anti-inflammatory proteins. Both haplotype CTA and rs419598 CC genotype are associated with a significant reduction in the mortality of men. These data provide genetic evidence that inflammasome activation and the IL-1 pathway plays an important role in the mortality and morbidity associated with severe SARS-CoV-2 infection, and that genetic regulation of inflammatory pathways by variants of IL1RN merits further evaluation in severe SARS-CoV-2 infection.

Quality assessment and refinement of chromatin accessibility data using a sequence-based predictive model.

Chromatin accessibility assays are central to the genome-wide identification of gene regulatory elements associated with transcriptional regulation. However, the data have highly variable quality arising from several biological and technical factors. To surmount this problem, we developed a sequence-based machine learning method to evaluate and refine chromatin accessibility data. Our framework, gapped k-mer SVM quality check (gkmQC), provides the quality metrics for a sample based on the prediction accuracy of the trained models. We tested 886 DNase-seq samples from the ENCODE/Roadmap projects to demonstrate that gkmQC can effectively identify "high-quality" (HQ) samples with low conventional quality scores owing to marginal read depths. Peaks identified in HQ samples are more accurately aligned at functional regulatory elements, show greater enrichment of regulatory elements harboring functional variants, and explain greater heritability of phenotypes from their relevant tissues. Moreover, gkmQC can optimize the peak-calling threshold to identify additional peaks, especially for rare cell types in single-cell chromatin accessibility data.

Interferon pathway lupus risk alleles modulate risk of death from acute COVID-19.

Type I interferon (IFN) is critical in our defense against viral infections. Increased type I IFN pathway activation is a genetic risk factor for systemic lupus erythematosus (SLE), and a number of common risk alleles contribute to the high IFN trait. We hypothesized that these common gain-of-function IFN pathway alleles may be associated with protection from mortality in acute COVID-19. We studied patients admitted with acute COVID-19 (756 European-American and 398 African-American ancestry). Ancestral backgrounds were analyzed separately, and mortality after acute COVID-19 was the primary outcome. In European-American ancestry, we found that a haplotype of interferon regulatory factor 5 (IRF5) and alleles of protein kinase cGMP-dependent 1 (PRKG1) were associated with mortality from COVID-19. Interestingly, these were much stronger risk factors in younger patients (OR = 29.2 for PRKG1 in ages 45-54). Variants in the IRF7 and IRF8 genes were associated with mortality from COVID-19 in African-American subjects, and these genetic effects were more pronounced in older subjects. Combining genetic information with blood biomarker data such as C-reactive protein, troponin, and D-dimer resulted in significantly improved predictive capacity, and in both ancestral backgrounds the risk genotypes were most relevant in those with positive biomarkers (OR for death between 14 and 111 in high risk genetic/biomarker groups). This study confirms the critical role of the IFN pathway in defense against COVID-19 and viral infections, and supports the idea that some common SLE risk alleles exert protective effects in antiviral immunity.

Identifying Needs, Challenges, and Benefits Among Adults and Parents of Children With Hirschsprung Disease.

ABSTRACT: Adults with Hirschsprung disease (HD), unaffected parents of children with HD, and affected adults with an affected child completed a cross-sectional survey with open-ended questions about greatest needs at diagnosis and at current time, greatest challenges encountered, and any benefits of having HD or having a child with HD. In the 297 respondents, information and good medical care were common needs at diagnosis and at the time of survey, but the information needed evolved with time. Managing ongoing symptoms was a frequently cited need and challenge, along with managing medical care and the social and emotional impact of HD. Perceived benefits included empathy for others and new perspectives on life. The needs and challenges identified in this study can guide healthcare providers in discussions with families. Provision of information, recommendations, and referrals based on each individual family's needs can support families with HD throughout the lifecycle and facilitate adaptation.

Genome-wide pleiotropy analysis identifies novel blood pressure variants and improves its polygenic risk scores.

Systolic and diastolic blood pressure (S/DBP) are highly correlated modifiable risk factors for cardiovascular disease (CVD). We report here a bidirectional Mendelian Randomization (MR) and horizontal pleiotropy analysis of S/DBP summary statistics from the UK Biobank (UKB)-International Consortium for Blood Pressure (ICBP) (UKB-ICBP) BP genome-wide association study and construct a composite genetic risk score (GRS) by including pleiotropic variants. The composite GRS captures greater (1.11-3.26 fold) heritability for BP traits and increases (1.09- and 2.01-fold) Nagelkerke's R2 for hypertension and CVD. We replicated 118 novel BP horizontal pleiotropic variants including 18 novel BP loci using summary statistics from the Million Veteran Program (MVP) study. An additional 219 novel BP signals and 40 novel loci were identified after a meta-analysis of the UKB-ICBP and MVP summary statistics but without further independent replication. Our study provides further insight into BP regulation and provides a novel way to construct a GRS by including pleiotropic variants for other complex diseases.

Multi-Ancestry Genome-wide Association Study Accounting for Gene-Psychosocial Factor Interactions Identifies Novel Loci for Blood Pressure Traits.

Psychological and social factors are known to influence blood pressure (BP) and risk of hypertension and associated cardiovascular diseases. To identify novel BP loci, we carried out genome-wide association meta-analyses of systolic, diastolic, pulse, and mean arterial BP taking into account the interaction effects of genetic variants with three psychosocial factors: depressive symptoms, anxiety symptoms, and social support. Analyses were performed using a two-stage design in a sample of up to 128,894 adults from 5 ancestry groups. In the combined meta-analyses of Stages 1 and 2, we identified 59 loci (p value <5e-8), including nine novel BP loci. The novel associations were observed mostly with pulse pressure, with fewer observed with mean arterial pressure. Five novel loci were identified in African ancestry, and all but one showed patterns of interaction with at least one psychosocial factor. Functional annotation of the novel loci supports a major role for genes implicated in the immune response (PLCL2), synaptic function and neurotransmission (LIN7A, PFIA2), as well as genes previously implicated in neuropsychiatric or stress-related disorders (FSTL5, CHODL). These findings underscore the importance of considering psychological and social factors in gene discovery for BP, especially in non-European populations.

A multi-enhancer RET regulatory code is disrupted in Hirschsprung disease.

The major genetic risk factors for Hirschsprung disease (HSCR) are three common polymorphisms within cis-regulatory elements (CREs) of the receptor tyrosine kinase gene RET, which reduce its expression during enteric nervous system (ENS) development. These risk variants attenuate binding of the transcription factors RARB, GATA2, and SOX10 to their cognate CREs, reduce RET gene expression, and dysregulate other ENS and HSCR genes in the RET-EDNRB gene regulatory network (GRN). Here, we use siRNA, ChIP, and CRISPR-Cas9 deletion analyses in the SK-N-SH cell line to ask how many additional HSCR-associated risk variants reside in RET CREs that affect its gene expression. We identify 22 HSCR-associated variants in candidate RET CREs, of which seven have differential allele-specific in vitro enhancer activity, and four of these seven affect RET gene expression; of these, two enhancers are bound by the transcription factor PAX3. We also show that deleting multiple variant-containing enhancers leads to synergistic effects on RET gene expression. These, coupled with our prior results, show that common sequence variants in at least 10 RET enhancers affect HSCR risk, seven with experimental evidence of affecting RET gene expression, extending the known RET-EDNRB GRN to reveal an extensive regulatory code modulating disease risk at a single gene.

Interferon pathway lupus risk alleles modulate risk of death from acute COVID-19.

Type I interferon (IFN) is critical in our defense against viral infections. Increased type I IFN pathway activation is a genetic risk factor for systemic lupus erythematosus (SLE), and a number of common risk alleles contribute to the high IFN trait. We hypothesized that these common gain-of-function IFN pathway alleles may be associated with protection from mortality in acute COVID-19. We studied patients admitted with acute COVID-19 (756 European-American and 398 African-American ancestry). Ancestral backgrounds were analyzed separately, and mortality after acute COVID-19 was the primary outcome. In European-American ancestry, we found that a haplotype of interferon regulatory factor 5 (IRF5) and alleles of protein kinase cGMP-dependent 1 (PRKG1) were associated with mortality from COVID-19. Interestingly, these were much stronger risk factors in younger patients (OR=29.2 for PRKG1 in ages 45-54). Variants in the IRF7 and IRF8 genes were associated with mortality from COVID-19 in African-American subjects, and these genetic effects were more pronounced in older subjects. Combining genetic information with blood biomarker data such as C-reactive protein, troponin, and D-dimer resulted in significantly improved predictive capacity, and in both ancestral backgrounds the risk genotypes were most relevant in those with positive biomarkers (OR for death between 14 and 111 in high risk genetic/biomarker groups). This study confirms the critical role of the IFN pathway in defense against COVID-19 and viral infections, and supports the idea that some common SLE risk alleles exert protective effects in anti-viral immunity. BACKGROUND: We find that a number of IFN pathway lupus risk alleles significantly impact mortality following COVID-19 infection. These data support the idea that type I IFN pathway risk alleles for autoimmune disease may persist in high frequency in modern human populations due to a benefit in our defense against viral infections. TRANSLATIONAL SIGNIFICANCE: We develop multivariate prediction models which combine genetics and known biomarkers of severity to result in greatly improved prediction of mortality in acute COVID-19. The specific associated alleles provide some clues about key points in our defense against COVID-19.

Magnitude of Mendelian versus complex inheritance of rare disorders.

In medical genetics, the vast majority of patients with a currently known genetic basis harbor a rare deleterious allele explaining its Mendelian inheritance. Increasingly, for these phenotypes, we recognize exceptions to Mendelian expectations from non-penetrance of clinical disease to significant inter-individual variation in clinical manifestations, likely reflecting the actions of additional modifier genes. Despite recent progress, we still remain ignorant about the molecular basis for many rare disorders presumed to be Mendelian. The molecular evidence increasingly suggests a role for multiple genes in some of these cases, but for how many? In this article, I discuss why equating a phenotype as Mendelian or complex may be short-sighted or even erroneous. As we learn more about the functions of the human genome with its genes in networks, we should view the phenotype of an individual patient as arising from his or her total genomic deleterious burden in a set of functionally inter-related genes affecting that phenotype. This can sometimes arise from deleterious allele(s) at a single gene (Mendelian inheritance) creating a specific biochemical deficiency (or excess) but could just as frequently arise from the cumulative effects of multiple disease alleles (complex inheritance) leading to the same biochemical deficiency (or excess).

Sequence-based correction of barcode bias in massively parallel reporter assays.

Massively parallel reporter assays (MPRAs) are a high-throughput method for evaluating in vitro activities of thousands of candidate cis-regulatory elements (CREs). In these assays, candidate sequences are cloned upstream or downstream from a reporter gene tagged by unique DNA sequences. However, tag sequences may themselves affect reporter gene expression and lead to major potential biases in the measured cis-regulatory activity. Here, we present a sequence-based method for correcting tag-sequence-specific effects and show that our method can significantly reduce this source of variation and improve the identification of functional regulatory variants by MPRAs. We also show that our model captures sequence features associated with post-transcriptional regulation of mRNA. Thus, this new method helps not only to improve detection of regulatory signals in MPRA experiments but also to design better MPRA protocols.

Multiple, independent, common variants at RET, SEMA3 and NRG1 gut enhancers specify Hirschsprung disease risk in European ancestry subjects.

PURPOSE: Hirschsprung disease (HSCR) is a developmental disorder of the enteric nervous system (ENS) characterized by congenital aganglionosis arising from coding variants in ENS genes causing partial or total loss-of-function. Low-penetrance, common, noncoding variants at RET, SEMA3 and NRG1 loci are also associated with HSCR, with small-to-moderate loss of gene expression mediated through sequence variants in cis-regulatory elements (CRE) as another causal mechanism. Since these latter variants are common, many individuals carry multiple risk variants. However, the extent and combinatorial effects of all putative CRE variants within and across these loci on HSCR is unknown. METHODS: Using 583 HSCR subjects, one of the largest samples of European ancestry studied, and genotyping 56 tag variants, we evaluated association of all common variants overlapping putative gut CREs and fine-mapped causal variants at RET, SEMA3 and NRG1. RESULTS: We demonstrate that 28 and 8 tag variants, several of which are genetically independent, overlap putative-enhancers at the RET and SEMA3 loci, respectively, as well as two fine-mapped tag variants at the NRG1 locus, are significantly associated with HSCR. Importantly, disease risk increases with increasing numbers of risk alleles from multiple variants within and across these loci, varying >25-fold across individuals. CONCLUSION: This increasing allele number-dependent risk, we hypothesize, arises from HSCR-relevant ENS cells sensing the reduced gene expression at multiple ENS genes since their developmental effects are integrated through gene regulatory networks.

Pathogenic alleles in microtubule, secretory granule and extracellular matrix-related genes in familial keratoconus.

Keratoconus is a common corneal defect with a complex genetic basis. By whole exome sequencing of affected members from 11 multiplex families of European ancestry, we identified 23 rare, heterozygous, potentially pathogenic variants in 8 genes. These include nonsynonymous single amino acid substitutions in HSPG2, EML6 and CENPF in two families each, and in NBEAL2, LRP1B, PIK3CG and MRGPRD in three families each; ITGAX had nonsynonymous single amino acid substitutions in two families and an indel with a base substitution producing a nonsense allele in the third family. Only HSPG2, EML6 and CENPF have been associated with ocular phenotypes previously. With the exception of MRGPRD and ITGAX, we detected the transcript and encoded protein of the remaining genes in the cornea and corneal cell cultures. Cultured stromal cells showed cytoplasmic punctate staining of NBEAL2, staining of the fibrillar cytoskeletal network by EML6, while CENPF localized to the basal body of primary cilia. We inhibited the expression of HSPG2, EML6, NBEAL2 and CENPF in stromal cell cultures and assayed for the expression of COL1A1 as a readout of corneal matrix production. An upregulation in COL1A1 after siRNA inhibition indicated their functional link to stromal cell biology. For ITGAX, encoding a leukocyte integrin, we assayed its level in the sera of 3 affected families compared with 10 unrelated controls to detect an increase in all affecteds. Our study identified genes that regulate the cytoskeleton, protein trafficking and secretion, barrier tissue function and response to injury and inflammation, as being relevant to keratoconus.

The road ahead in genetics and genomics.

In celebration of the 20th anniversary of Nature Reviews Genetics, we asked 12 leading researchers to reflect on the key challenges and opportunities faced by the field of genetics and genomics. Keeping their particular research area in mind, they take stock of the current state of play and emphasize the work that remains to be done over the next few years so that, ultimately, the benefits of genetic and genomic research can be felt by everyone.

Analysis of putative cis-regulatory elements regulating blood pressure variation.

Hundreds of loci have been associated with blood pressure (BP) traits from many genome-wide association studies. We identified an enrichment of these loci in aorta and tibial artery expression quantitative trait loci in our previous work in ~100 000 Genetic Epidemiology Research on Aging study participants. In the present study, we sought to fine-map known loci and identify novel genes by determining putative regulatory regions for these and other tissues relevant to BP. We constructed maps of putative cis-regulatory elements (CREs) using publicly available open chromatin data for the heart, aorta and tibial arteries, and multiple kidney cell types. Variants within these regions may be evaluated quantitatively for their tissue- or cell-type-specific regulatory impact using deltaSVM functional scores, as described in our previous work. We aggregate variants within these putative CREs within 50 Kb of the start or end of 'expressed' genes in these tissues or cell types using public expression data and use deltaSVM scores as weights in the group-wise sequence kernel association test to identify candidates. We test for association with both BP traits and expression within these tissues or cell types of interest and identify the candidates MTHFR, C10orf32, CSK, NOV, ULK4, SDCCAG8, SCAMP5, RPP25, HDGFRP3, VPS37B and PPCDC. Additionally, we examined two known QT interval genes, SCN5A and NOS1AP, in the Atherosclerosis Risk in Communities Study, as a positive control, and observed the expected heart-specific effect. Thus, our method identifies variants and genes for further functional testing using tissue- or cell-type-specific putative regulatory information.

MicroRNA-4516-mediated regulation of MAPK10 relies on 3' UTR cis-acting variants and contributes to the altered risk of Hirschsprung disease.

BACKGROUND: Hirschsprung disease (HSCR) is a life-threatening congenital disorder in which the enteric nervous system is completely missing from the distal gut. Recent studies have shown that miR-4516 markedly inhibits cell migration, and as one of its potential targets, MAPK10 functions as a modifier for developing HSCR. We thus aimed to evaluate the role of miR-4516 and MAPK10 in HSCR and how they contribute to the pathogenesis of HSCR. METHODS: We examined 13 genetic variants using the MassArray system in a case-control study (n=1015). We further investigated miR-4516-mediated regulation of MAPK10 in HSCR cases and human neural cells, the effects of cis-acting elements in MAPK10 on miR-4516-mediated modulation and cell migration process. RESULTS: Three positive 3' UTR variants in MAPK10 were associated with altered HSCR susceptibility. We also showed that miR-4516 directly regulates MAPK10 expression, and this regulatory mechanism is significantly affected by the 3' UTR cis-acting elements of MAPK10. In addition, knock-down of MAPK10 rescued the effect of miR-4516 on the migration of human neural cells. CONCLUSION: Our findings indicate a key role of miR-4516 and its direct target MAPK10 in HSCR risk, and highlight the general importance of cis- and posttranscriptional modulation for HSCR pathogenesis.