Faculty Bibliography
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Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2019:116(52):26697-708.DOI: 10.1073/pnas.1908756116
Gene- and tissue-level interactions in normal gastrointestinal development and Hirschsprung disease
The development of the gut from endodermal tissue to an organ with multiple distinct structures and functions occurs over a prolonged time during embryonic days E10.5-E14.5 in the mouse. During this process, one major event is innervation of the gut by enteric neural crest cells (ENCCs) to establish the enteric nervous system (ENS). To understand the molecular processes underpinning gut and ENS development, we generated RNA-sequencing profiles from wild-type mouse guts at E10.5, E12.5, and E14.5 from both sexes. We also generated these profiles from homozygous Ret null embryos, a model for Hirschsprung disease (HSCR), in which the ENS is absent. These data reveal 4 major features: 1) between E10.5 and E14.5 the developmental genetic programs change from expression of major transcription factors and its modifiers to genes controlling tissue (epithelium, muscle, endothelium) specialization; 2) the major effect of Ret is not only on ENCC differentiation to enteric neurons but also on the enteric mesenchyme and epithelium; 3) a muscle genetic program exerts significant effects on ENS development; and 4) sex differences in gut development profiles are minor. The genetic programs identified, and their changes across development, suggest that both cell autonomous and nonautonomous factors, and interactions between the different developing gut tissues, are important for normal ENS development and its disorders.
PMID: 31818953
ISSN: 1091-6490
CID: 4238762
Sequence characterization of RET in 117 Chinese Hirschsprung disease families identifies a large burden of de novo and parental mosaic mutations
BACKGROUND:Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by the absence of enteric ganglia in the distal part of the gut. RET is the major causative gene and contains > 80% of all known disease-causing mutations. RESULTS:To determine the incidence of RET pathogenic variants, be they Mendelian inherited, mosaic in parents or true de novo variants (DNVs) in 117 Chinese families, we used high-coverage NGS and droplet digital polymerase chain reaction (ddPCR) to identify 15 (12.8%) unique RET coding variants (7 are novel); one was inherited from a heterozygous unaffected mother, 11 were DNVs (73.3%), and 3 full heterozygotes were inherited from parental mosaicism (2 paternal, 1 maternal): two clinically unaffected parents were identified by NGS and confirmed by ddPCR, with mutant allele frequency (13-27%) that was the highest in hair, lowest in urine and similar in blood and saliva. An extremely low-level paternal mosaicism (0.03%) was detected by ddPCR in blood. Six positive-controls were examined to compare the mosaicism detection limit and sensitivity of NGS, amplicon-based deep sequencing and ddPCR. CONCLUSION/CONCLUSIONS:Our findings expand the clinical and molecular spectrum of RET variants in HSCR and reveal a high frequency of RET DNVs in the Chinese population.
PMID: 31666091
ISSN: 1750-1172
CID: 4163352
A gene regulatory network explains RET-EDNRB epistasis in Hirschsprung disease
Disruptions in gene regulatory networks (GRNs), driven by multiple deleterious variants, potentially underlie complex traits and diseases. Hirschsprung disease (HSCR), a multifactorial disorder of enteric nervous system (ENS) development, is associated with at least 24 genes and 7 chromosomal loci, with RET and EDNRB as its major genes. We previously demonstrated that RET transcription in the ENS is controlled by an extensive GRN involving the transcription factors (TF) RARB, GATA2 and SOX10 and other HSCR genes. We now demonstrate, using human and mouse cellular and animal models, that EDNRB is transcriptionally regulated in the ENS by GATA2, SOX10 and NKX2.5 TFs. Significantly, RET and EDNRB expression is regulated by their shared use of GATA2 and SOX10 and, in turn, these TFs are controlled by EDNRB and RET in a dose-dependent manner. This study expands the ENS development GRN to include both RET and EDNRB, uncovers the mechanistic basis for RET-EDNRB epistasis and emphasizes how functionally different genes associated with a complex disorder can be united through a common GRN.
PMID: 31313802
ISSN: 1460-2083
CID: 3977902
Journal of pediatric gastroenterology & nutrition. JPGN. 2019:69(3):299-305.DOI: 10.1097/MPG.0000000000002392
High Levels of Interest in Reproductive Genetic Information in Parents of Children and Adults with Hirschsprung Disease
OBJECTIVES/OBJECTIVE:Families affected by Hirschsprung disease (HSCR) have opportunities to learn recurrence risks to their children from statistical genetic and empiric studies and, in some cases, prenatal genetic testing or preimplantation genetic diagnosis (PGD). This study aimed to assess interest in reproductive genetic information for HSCR and factors that predict this interest in two groups with elevated risk of having a child with HSCR. METHODS:Adult HSCR patients and parents of children with HSCR were surveyed about their interest in learning reproductive genetic information regarding HSCR through genetic counseling, prenatal testing, and PGD. Covariates assessed in this cross-sectional study included quality of life, illness perceptions, depressive symptoms, and adaptation to the condition. Bivariate analyses assessed differences between affected adults and parents. Logistic modeling was used to identify predictors of interest in reproductive genetic information. RESULTS:Study participants (n = 368) reported high interest in reproductive genetic information through genetic counseling (yes = 60%/unsure = 16%), prenatal testing (yes = 59%/unsure = 16%), and PGD (yes = 43%/unsure = 18%). Illness perceptions differed between affected adults and parents, but perceived severity of HSCR was high among all participants (μ = 3.42, SD = 0.67, 4 point scale). Interest in reproductive information was associated with being an affected adult, not having a family history of HSCR, negative emotional representations, and adaptation to the condition. CONCLUSIONS:Findings from this study support a desire among the surveyed HSCR patient groups to attend genetic counseling and be offered testing when available. Exploration of perceptions and experiences with the condition should be incorporated into the counseling to insure informed preference-based decision making.
PMID: 31107799
ISSN: 1536-4801
CID: 3978432
The gastrointestinal development ‘parts list’: transcript profiling of embryonic gut development in wildtype and Ret-deficient mice [PrePrint]
ORIGINAL:0014922
ISSN: 2692-8205
CID: 4772442
A multi-ancestry genome-wide study incorporating gene-smoking interactions identifies multiple new loci for pulse pressure and mean arterial pressure
Elevated blood pressure (BP), a leading cause of global morbidity and mortality, is influenced by both genetic and lifestyle factors. Cigarette smoking is one such lifestyle factor. Across five ancestries, we performed a genome-wide gene-smoking interaction study of mean arterial pressure (MAP) and pulse pressure (PP) in 129 913 individuals in stage 1 and follow-up analysis in 480 178 additional individuals in stage 2. We report here 136 loci significantly associated with MAP and/or PP. Of these, 61 were previously published through main-effect analysis of BP traits, 37 were recently reported by us for systolic BP and/or diastolic BP through gene-smoking interaction analysis and 38 were newly identified (P < 5 × 10-8, false discovery rate < 0.05). We also identified nine new signals near known loci. Of the 136 loci, 8 showed significant interaction with smoking status. They include CSMD1 previously reported for insulin resistance and BP in the spontaneously hypertensive rats. Many of the 38 new loci show biologic plausibility for a role in BP regulation. SLC26A7 encodes a chloride/bicarbonate exchanger expressed in the renal outer medullary collecting duct. AVPR1A is widely expressed, including in vascular smooth muscle cells, kidney, myocardium and brain. FHAD1 is a long non-coding RNA overexpressed in heart failure. TMEM51 was associated with contractile function in cardiomyocytes. CASP9 plays a central role in cardiomyocyte apoptosis. Identified only in African ancestry were 30 novel loci. Our findings highlight the value of multi-ancestry investigations, particularly in studies of interaction with lifestyle factors, where genomic and lifestyle differences may contribute to novel findings.
PMID: 31127295
ISSN: 1460-2083
CID: 3967142
Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2019:116(22):10636-10645.DOI: 10.1073/pnas.1808734116
Multiple SCN5A variant enhancers modulate its cardiac gene expression and the QT interval
The rationale for genome-wide association study (GWAS) results is sequence variation in cis-regulatory elements (CREs) modulating a target gene's expression as the major cause of trait variation. To understand the complete molecular landscape of one of these GWAS loci, we performed in vitro reporter screens in cardiomyocyte cell lines for CREs overlapping nearly all common variants associated with any of five independent QT interval (QTi)-associated GWAS hits at the SCN5A-SCN10A locus. We identified 13 causal CRE variants using allelic reporter activity, cardiomyocyte nuclear extract-based binding assays, overlap with human cardiac tissue DNaseI hypersensitive regions, and predicted impact of sequence variants on DNaseI sensitivity. Our analyses identified at least one high-confidence causal CRE variant for each of the five sentinel hits that could collectively predict SCN5A cardiac gene expression and QTi association. Although all 13 variants could explain SCN5A gene expression, the highest statistical significance was obtained with seven variants (inclusive of the five above). Thus, multiple, causal, mutually associated CRE variants can underlie GWAS signals.
PMID: 31068470
ISSN: 1091-6490
CID: 3909312
Molecular Genetic Anatomy and Risk Profile of Hirschsprung's Disease
BACKGROUND:Hirschsprung's disease, or congenital aganglionosis, is a developmental disorder of the enteric nervous system and is the most common cause of intestinal obstruction in neonates and infants. The disease has more than 80% heritability, including significant associations with rare and common sequence variants in genes related to the enteric nervous system, as well as with monogenic and chromosomal syndromes. METHODS:We genotyped and exome-sequenced samples from 190 patients with Hirschsprung's disease to quantify the genetic burden in patients with this condition. DNA sequence variants, large copy-number variants, and karyotype variants in probands were considered to be pathogenic when they were significantly associated with Hirschsprung's disease or another neurodevelopmental disorder. Novel genes were confirmed by functional studies in the mouse and human embryonic gut and in zebrafish embryos. RESULTS:). For individual patients, the estimated risk of Hirschsprung's disease ranged from 5.33 cases per 100,000 live births (approximately 1 per 18,800) to 8.38 per 1000 live births (approximately 1 per 120). CONCLUSIONS:. For individual patients, the genotype-specific odds ratios varied by a factor of approximately 67, which provides a basis for risk stratification and genetic counseling. (Funded by the National Institutes of Health.).
PMID: 30970187
ISSN: 1533-4406
CID: 3826322
Leveraging linkage evidence to identify low-frequency and rare variants on 16p13 associated with blood pressure using TOPMed whole genome sequencing data
In this study, we investigated low-frequency and rare variants associated with blood pressure (BP) by focusing on a linkage region on chromosome 16p13. We used whole genome sequencing (WGS) data obtained through the NHLBI Trans-Omics for Precision Medicine (TOPMed) program on 395 Cleveland Family Study (CFS) European Americans (CFS-EA). By analyzing functional coding variants and non-coding rare variants with CADD score > 10 residing within the chromosomal region in families with linkage evidence, we observed 25 genes with nominal statistical evidence (burden or SKAT p < 0.05). One of the genes is RBFOX1, an evolutionarily conserved RNA-binding protein that regulates tissue-specific alternative splicing that we previously reported to be associated with BP using exome array data in CFS. After follow-up analysis of the 25 genes in ten independent TOPMed studies with individuals of European, African, and East Asian ancestry, and Hispanics (N = 29,988), we identified variants in SLX4 (p = 2.19 × 10-4) to be significantly associated with BP traits when accounting for multiple testing. We also replicated the associations previously reported for RBFOX1 (p = 0.007). Follow-up analysis with GTEx eQTL data shows SLX4 variants are associated with gene expression in coronary artery, multiple brain tissues, and right atrial appendage of the heart. Our study demonstrates that linkage analysis of family data can provide an efficient approach for detecting rare variants associated with complex traits in WGS data.
PMID: 30671673
ISSN: 1432-1203
CID: 3610552
Combined linkage and association analysis identifies rare and low frequency variants for blood pressure at 1q31
High blood pressure (BP) is a major risk factor for cardiovascular disease (CVD) and is more prevalent in African Americans as compared to other US groups. Although large, population-based genome-wide association studies (GWAS) have identified over 300 common polymorphisms modulating inter-individual BP variation, largely in European ancestry subjects, most of them do not localize to regions previously identified through family-based linkage studies. This discrepancy has remained unexplained despite the statistical power differences between current GWAS and prior linkage studies. To address this issue, we performed genome-wide linkage analysis of BP traits in African-American families from the Family Blood Pressure Program (FBPP) and genotyped on the Illumina Human Exome BeadChip v1.1. We identified a genomic region on chromosome 1q31 with LOD score 3.8 for pulse pressure (PP), a region we previously implicated in DBP studies of European ancestry families. Although no reported GWAS variants map to this region, combined linkage and association analysis of PP identified 81 rare and low frequency exonic variants accounting for the linkage evidence. Replication analysis in eight independent African ancestry cohorts (N = 16,968) supports this specific association with PP (P = 0.0509). Additional association and network analyses identified multiple potential candidate genes in this region expressed in multiple tissues and with a strong biological support for a role in BP. In conclusion, multiple genes and rare variants on 1q31 contribute to PP variation. Beyond producing new insights into PP, we demonstrate how family-based linkage and association studies can implicate specific rare and low frequency variants for complex traits.
PMCID:6336803
PMID: 30262922
ISSN: 1476-5438
CID: 3975192
