Multitrait genetic association analysis identifies 50 new risk loci for gastro-oesophageal reflux, seven new loci for Barrett's oesophagus and provides insights into clinical heterogeneity in reflux diagnosis.

OBJECTIVE: Gastro-oesophageal reflux disease (GERD) has heterogeneous aetiology primarily attributable to its symptom-based definitions. GERD genome-wide association studies (GWASs) have shown strong genetic overlaps with established risk factors such as obesity and depression. We hypothesised that the shared genetic architecture between GERD and these risk factors can be leveraged to (1) identify new GERD and Barrett's oesophagus (BE) risk loci and (2) explore potentially heterogeneous pathways leading to GERD and oesophageal complications. DESIGN: We applied multitrait GWAS models combining GERD (78 707 cases; 288 734 controls) and genetically correlated traits including education attainment, depression and body mass index. We also used multitrait analysis to identify BE risk loci. Top hits were replicated in 23andMe (462 753 GERD cases, 24 099 BE cases, 1 484 025 controls). We additionally dissected the GERD loci into obesity-driven and depression-driven subgroups. These subgroups were investigated to determine how they relate to tissue-specific gene expression and to risk of serious oesophageal disease (BE and/or oesophageal adenocarcinoma, EA). RESULTS: We identified 88 loci associated with GERD, with 59 replicating in 23andMe after multiple testing corrections. Our BE analysis identified seven novel loci. Additionally we showed that only the obesity-driven GERD loci (but not the depression-driven loci) were associated with genes enriched in oesophageal tissues and successfully predicted BE/EA. CONCLUSION: Our multitrait model identified many novel risk loci for GERD and BE. We present strong evidence for a genetic underpinning of disease heterogeneity in GERD and show that GERD loci associated with depressive symptoms are not strong predictors of BE/EA relative to obesity-driven GERD loci.

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.

Genome-wide association study of Hirschsprung disease detects a novel low-frequency variant at the RET locus.

Hirschsprung disease (HSCR) is a congenital disorder with a population incidence of ~1/5000 live births, defined by an absence of enteric ganglia along variable lengths of the colon. HSCR genome-wide association studies (GWAS) have found common associated variants at RET, SEMA3, and NRG1, but they still fail to explain all of its heritability. To enhance gene discovery, we performed a GWAS of 170 cases identified from the Danish nationwide pathology registry with 4717 controls, based on 6.2 million variants imputed from the haplotype reference consortium panel. We found a novel low-frequency variant (rs144432435), which, when conditioning on the lead RET single-nucleotide polymorphism (SNP), was of genome-wide significance in the discovery analysis. This conditional association signal was replicated in a Swedish HSCR cohort with discovery plus replication meta-analysis conditional odds ratio of 6.6 (P = 7.7 × 10-10; 322 cases and 4893 controls). The conditional signal was, however, not replicated in two HSCR cohorts from USA and Finland, leading to the hypothesis that rs144432435 tags a rare haplotype present in Denmark and Sweden. Using the genome-wide complex trait analysis method, we estimated the SNP heritability of HSCR to be 88%, close to estimates based on classical family studies. Moreover, by using Lasso (least absolute shrinkage and selection operator) regression we were able to construct a genetic HSCR predictor with a area under the receiver operator characteristics curve of 76% in an independent validation set. In conclusion, we combined the largest collection of sporadic Hirschsprung cases to date (586 cases) to further elucidate HSCR's genetic architecture.

Trans-ethnic meta-analysis of genome-wide association studies for Hirschsprung disease.

Hirschsprung disease (HSCR) is the most common cause of neonatal intestinal obstruction. It is characterized by the absence of ganglia in the nerve plexuses of the lower gastrointestinal tract. So far, three common disease-susceptibility variants at the RET, SEMA3 and NRG1 loci have been detected through genome-wide association studies (GWAS) in Europeans and Asians to understand its genetic etiologies. Here we present a trans-ethnic meta-analysis of 507 HSCR cases and 1191 controls, combining all published GWAS results on HSCR to fine-map these loci and narrow down the putatively causal variants to 99% credible sets. We also demonstrate that the effects of RET and NRG1 are universal across European and Asian ancestries. In contrast, we detected a European-specific association of a low-frequency variant, rs80227144, in SEMA3 [odds ratio (OR) = 5.2, P = 4.7 × 10-10]. Conditional analyses on the lead SNPs revealed a secondary association signal, corresponding to an Asian-specific, low-frequency missense variant encoding RET p.Asp489Asn (rs9282834, conditional OR = 20.3, conditional P = 4.1 × 10-14). When in trans with the RET intron 1 enhancer risk allele, rs9282834 increases the risk of HSCR from 1.1 to 26.7. Overall, our study provides further insights into the genetic architecture of HSCR and has profound implications for future study designs.