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The epilepsies affect around 65 million people worldwide and have a substantial missing heritability component. We report a genome-wide mega-analysis involving 15,212 individuals with epilepsy and 29,677 controls, which reveals 16 genome-wide significant loci, of which 11 are novel. Using various prioritization criteria, we pinpoint the 21 most likely epilepsy genes at these loci, with the majority in genetic generalized epilepsies. These genes have diverse biological functions, including coding for ion-channel subunits, transcription factors and a vitamin-B6 metabolism enzyme. Converging evidence shows that the common variants associated with epilepsy play a role in epigenetic regulation of gene expression in the brain. The results show an enrichment for monogenic epilepsy genes as well as known targets of antiepileptic drugs. Using SNP-based heritability analyses we disentangle both the unique and overlapping genetic basis to seven different epilepsy subtypes. Together, these findings provide leads for epilepsy therapies based on underlying pathophysiology., SCOPUS: ar.j, info:eu-repo/semantics/published

Background Heart failure (HF) with mid-range ejection fraction (HFmrEF, EF 40-49%) accounts for ∼15% of HF patients and these are a heterogeneous group with a phenotypic character that reflects aspects of both preserved and reduced EF HF. There are no proven treatments for this group but identifying subsets of patients that will respond to conventional therapies such as beta blockers (BB) would be desirable. We hypothesized that HFmrEF patients with elevated Nt-proBNP or ST2 would have more favorable response to BB therapy. Methods A prospective registry was conducted that enrolled 1760 HF patients who met Framingham HF criterion and who received care through our health system and insurance product. Participants donated blood for biomarker measurements at enrollment and their medication exposure was quantified over time using pharmacy claims data. The current study utilized all participants who had EF 40-49% and baseline biomarker measurement (n=338). The primary endpoint was all-cause mortality. Biomarker categories were dichotomized at Nt-BNP >1000ng/L and ST2 ≥35ng/mL. We quantified the HR for BB exposure overall and divided by biomarker level using Cox models adjusted for MAGGIC score and BB propensity score. Results Vital status and Nt-BNP measurements were available for all 338 patients, while 279 had ST2 measured, and 201 had both markers (Table 1). For Nt-BNP, 200 (58%) had elevated levels while 143 (42%) had low values. BB use in patients with elevated Nt-BNP levels had a hazard ratio (HR) of 0.43 (p=0.1) compared to HR 0.97 (p=0.97) in the low Nt-BNP group. For ST2, 195 patients (70%) had levels 0.99) while in patients with either ST2 or Nt-BNP elevated the BB HR was 0.06 (p=0.03). Conclusions In this observational study of HFmrEF patients, those with low ST2 and Nt-BNP have low risk of death and little benefit of BB therapy whereas among the approximately two-thirds of patients with either marker elevated a substantial BB benefit association was seen

Heart failure (HF) is a morbid condition associated with impaired exercise capacity. Exercise training is an effective strategy to improve functional capacity and quality of life. However, response to exercise training is highly variable and whether there are genetic factors that may impact response

Biomarkers are critical for modern heart failure (HF) care. There are several established prognostic markers such as N-terminal pro-b-type natriuretic peptide (NTproBNP) and soluble suppressor of tumorgenicity 2 (ST2). Recent advances in multiplexing/multi-marker platforms offer faster and broader

Background: Heart failure with reduced ejection fraction (HFrEF) continues to be a global health burden. We recently reported a strong association of plasma metabolite profile (thirteen different metabolites) with survival among patients with HFrEF but the mechanism of this association is unclear. We studied the genetic associations of plasma metabolite to try to illuminate the disease mechanisms at work. Methods: A prospective genetic registry including 1032 HFrEF with genome-wide genotyping and imputation of single nucleotide polymorphisms (SNP; ~7 millions) and targeted metabolomics profiling. Genome-wide (GW) association analyses were performed to investigate the genetic impact of each SNP on the 13 metabolites of interest. Analyses were performed using linear models stratified by race (European Americans [N=516] and African Americans [N=516] and including the top five principal components (PC) for each population to avoid spurious associations due to population stratification. A fixed-effect meta-analysis was then used to combine results from the two populations. Threshold for GW significance was p Results: We found at least one SNP reaching GW significance for 6 metabolites metabolites (a-KG, C5.Isovaleryl, Fumarate, Leucine, Succinate and X3.HBA). The most significant SNP (rs77836784, an intronic variant within PACRG) was associated with X3.HBA in African American HF patients (Beta estimate=22.82, P-value=7.14E-14). A cross-checking of SNP associations on both metabolites and HF survival identified 13 independent SNPs that may partially explain the contribution of the metabolites on HF mortality (Table 1) and seem to have biologic plausibility including for example a G-protein coupled receptor (GPR68) and protein kinase C (PRKC1). Conclusion: Genetic variants are associated with predictive plasma metabolites and survival. This may be useful to understand the underlying pathways connecting metabolites to HF progression. A complete investigation of genomic and metabolomics determinants including causal mediation and pathway analyses are on-going.

BACKGROUND: Although inhaled corticosteroid (ICS) medication is considered the cornerstone treatment for patients with persistent asthma, few ICS pharmacogenomic studies have involved non-white populations. OBJECTIVE: To identify genetic predictors of ICS response in multiple population groups with asthma. METHODS: The discovery group comprised African American participants from the Study of Asthma Phenotypes and Pharmacogenomic Interactions by Race-ethnicity (SAPPHIRE) who underwent 6 weeks of monitored ICS therapy (n=244). A genome-wide scan was performed to identify single nucleotide polymorphism (SNP) variants jointly associated (i.e., the combined effect of the SNP and SNP x ICS treatment interaction) with changes in asthma control. Top associations were validated by assessing the joint association with asthma exacerbations in three additional groups – African Americans (n=803 and n=563) and Latinos (n=1,461). RNA-seq data from 408 asthma cases and 405 controls were used to examine whether genotype was associated with gene expression. RESULTS: One variant, rs3827907, was significantly associated with ICS-mediated changes in asthma control in the discovery set (P=7.79×10(−8)) and was jointly associated with asthma exacerbations in three validation cohorts (P=0.023, P=0.029, and P=0.041). RNA-seq analysis found the rs3827907 C-allele to be associated with lower RNASE2 expression (P=6.10×10(−4)). RNASE2 encodes eosinophil-derived neurotoxin (EDN), and the rs3827907 C-allele appeared to particularly influence ICS treatment response in the presence of eosinophilic inflammation (i.e., high pre-treatment EDN levels or blood eosinophil counts). CONCLUSION: We identified a variant, rs3827907, which appears to influence response to ICS treatment in multiple population groups, and likely mediates its effect through eosinophils. CLINICAL IMPLICATIONS: African Americans and Latinos are disproportionately affected by asthma and its complications. Here we identify a pharmacogenomic variant that may assist in identifying individuals from these groups who will respond to ICS treatment. CAPSULE SUMMARY: This is the first study to use a non-white study population for the discovery of pharmacogenomic predictors of asthma controller response. Variant rs3827907 genotype appeared to influenced ICS response especially in the setting of eosinophilic inflammation.

In the version of this article initially published, in Fig. 3, the y-axis numbering did not match the log scale indicated in the axis label. The error has been corrected in the HTML and PDF version of the article.

Allergic rhinitis is the most common clinical presentation of allergy, affecting 400 million people worldwide, with increasing incidence in westernized countries1,2. To elucidate the genetic architecture and understand the underlying disease mechanisms, we carried out a meta-analysis of allergic rhinitis in 59,762 cases and 152,358 controls of European ancestry and identified a total of 41 risk loci for allergic rhinitis, including 20 loci not previously associated with allergic rhinitis, which were confirmed in a replication phase of 60,720 cases and 618,527 controls. Functional annotation implicated genes involved in various immune pathways, and fine mapping of the HLA region suggested amino acid variants important for antigen binding. We further performed genome-wide association study (GWAS) analyses of allergic sensitization against inhalant allergens and nonallergic rhinitis, which suggested shared genetic mechanisms across rhinitis-related traits. Future studies of the identified loci and genes might identify novel targets for treatment and prevention of allergic rhinitis. An Author Correction to this article was published on 16 August 2018. https://www.nature.com/articles/s41588-018-0197-6

Nearly 100 loci have been identified for pulmonary function, almost exclusively in studies of European ancestry populations. We extend previous research by meta-analyzing genome-wide association studies of 1000 Genomes imputed variants in relation to pulmonary function in a multiethnic population of 90,715 individuals of European (N = 60,552), African (N = 8429), Asian (N = 9959), and Hispanic/Latino (N = 11,775) ethnicities. We identify over 50 additional loci at genome-wide significance in ancestry-specific or multiethnic meta-analyses. Using recent fine-mapping methods incorporating functional annotation, gene expression, and differences in linkage disequilibrium between ethnicities, we further shed light on potential causal variants and genes at known and newly identified loci. Several of the novel genes encode proteins with predicted or established drug targets, including KCNK2 and CDK12. Our study highlights the utility of multiethnic and integrative genomics approaches to extend existing knowledge of the genetics of lung function and clinical relevance of implicated loci., Supported in part by the Intramural Research Program of the National Institutes of Health, NIEHS. Infrastructure for the CHARGE Consortium is supported in part by the National Heart, Lung, and Blood Institute Grant R01HL105756

An unstable epigenome is implicated in the pathophysiology of neurodevelopmental disorders such as schizophrenia and autism. This is important because the epigenome is potentially modifiable. We have previously reported that adult offspring exposed to maternal immune activation (MIA) prenatally have significant global DNA hypomethylation in the hypothalamus. However, what genes had altered methylation state, their functional effects on gene expression and whether these changes can be moderated, have not been addressed. In this study, we used next-generation sequencing (NGS) for methylome profiling in a MIA rodent model of neurodevelopmental disorders. We assessed whether differentially methylated regions (DMRs) affected the chromatin state by mapping known DNase I hypersensitivity sites (DHSs), and selected overlapping genes to confirm a functional effect of MIA on gene expression using qPCR. Finally, we tested whether methylation differences elicited by MIA could be limited by post-natal dietary (omega) n-3 polyunsaturated fatty acid (PUFA) supplementation. These experiments were conducted using hypothalamic brain tissue from 12-week-old offspring of mice injected with viral analogue PolyI:C on gestation day 9 of pregnancy or saline on gestation day 9. Half of the animals from each group were fed a diet enriched with n-3 PUFA from weaning (MIA group, n = 12 units, n = 39 mice; Control group, n = 12 units, n = 38 mice). The results confirmed our previous finding that adult offspring exposed to MIA prenatally had significant global DNA hypomethylation. Furthermore, genes linked to synaptic plasticity were over-represented among differentially methylated genes following MIA. More than 80% of MIA-induced hypomethylated sites, including those affecting chromatin state and MECP2 binding, were stabilised by the n-3 PUFA intervention. MIA resulted in increased expression of two of the ‘top five’ genes identified from an integrated analysis of DMRs, DHSs and MECP2 binding sites, namely Abat (t = 2.46, p Gnas9 (t = 2.96, p

Background It remains unclear whether beta‐blockade is similarly effective in black patients with heart failure and reduced ejection fraction as in white patients, but self‐reported race is a complex social construct with both biological and environmental components. The objective of this study was to compare the reduction in mortality associated with beta‐blocker exposure in heart failure and reduced ejection fraction patients by both self‐reported race and by proportion African genetic ancestry. Methods and Results Insured patients with heart failure and reduced ejection fraction (n=1122) were included in a prospective registry at Henry Ford Health System. This included 575 self‐reported blacks (129 deaths, 22%) and 547 self‐reported whites (126 deaths, 23%) followed for a median 3.0 years. Beta‐blocker exposure ( BB exp) was calculated from pharmacy claims, and the proportion of African genetic ancestry was determined from genome‐wide array data. Time‐dependent Cox proportional hazards regression was used to separately test the association of BB exp with all‐cause mortality by self‐reported race or by proportion of African genetic ancestry. Both sets of models were evaluated unadjusted and then adjusted for baseline risk factors and beta‐blocker propensity score. BB exp effect estimates were protective and of similar magnitude both by self‐reported race and by African genetic ancestry (adjusted hazard ratio=0.56 in blacks and adjusted hazard ratio=0.48 in whites). The tests for interactions with BB exp for both self‐reported race and for African genetic ancestry were not statistically significant in any model ( P >0.1 for all). Conclusions Among black and white patients with heart failure and reduced ejection fraction, reduction in all‐cause mortality associated with BB exp was similar, regardless of self‐reported race or proportion African genetic ancestry.

© The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved. AIM: To investigate the role of IKBKAP (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein) in the development of enteric nervous system (ENS) and Hirschsprung disease (HSCR). METHODS: In this study, we injected a morpholino that blocked the translation of ikbkap protein to 1-cell stage zebrafish embryos. The phenotype in the ENS was analysed by antibody staining of the pan-neuronal marker HuC/D followed by enteric neuron counting. The mean numbers of enteric neurons were compared between the morphant and the control. We also studied the expressions of ret and phox2bb, which are involved in ENS development, in the ikbkap morpholino injected embryos by quantitative reverse transcriptase polymerase chain reaction and compared them with the control. RESULTS: We observed aganglionosis (χ2, P < 0.01) and a reduced number of enteric neurons (38.8 ± 9.9 vs 50.2 ± 17.3, P < 0.05) in the zebrafish embryos injected with ikbkap translation-blocking morpholino (morphant) when compared with the control embryos. Specificity of the morpholino was confirmed by similar results obtained using a second non-overlapping morpholino that blocked the translation of ikbkap. We further studied the morphant by analysing the expression levels of genes involved in ENS development such as ret, phox2bb and sox10, and found that phox2bb, the ortholog of human PHOX2B, was significantly down-regulated (0.51 ± 0.15 vs 1.00 ± 0, P < 0.05). Although we also observed a reduction in the expression of ret, the difference was not significant. CONCLUSION: Loss of IKBKAP contributed to HSCR as demonstrated by functional analysis in zebrafish embryos., Link_to_subscribed_fulltext

Evidence from genome-wide association studies (GWAS) suggest that pleiotropic effects on human complex phenotypes are very common. Recently, an atlas of genetic correlations among complex phenotypes has broadened our understanding of human diseases and traits. Here, we examine genetic overlap, from a gene-centric perspective, among the same 24 phenotypes previously investigated for genetic correlations. After adopting the multilevel pipeline (freely available at http://grass.cgs.hku.hk/limx/kgg/), which includes intragenic single nucleotide polymorphisms (SNPs), genes, and gene-sets, to estimate genetic similarities across phenotypes, a large amount of sharing of several biologically related phenotypes was confirmed. In addition, significant genetic overlaps were also found among phenotype pairs that were previously unidentified by SNP-level approaches. All these pairs with new genetic links are supported by earlier epidemiological evidence, although only a few of them have pleiotropic genes in the GWAS Catalog. Hence, our gene and gene-set analyses are able to provide new insights into cross-phenotype connections. The investigation on genetic sharing at three different levels presents a complementary picture of how common DNA sequence variations contribute to disease comorbidities and trait manifestations.

Background Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human. Results We performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS. Conclusions Our data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1174-6) contains supplementary material, which is available to authorized users.

Summary To adopt an efficient approach of identifying rare variants possibly related to Hirschsprung disease (HSCR), a pilot study was set up to evaluate the performance of a newly designed protocol for next generation targeted resquencing. In total, 20 Chinese HSCR patients and 20 Chinese sex-matched individuals with no HSCR were included, for which coding sequences (CDS) of 62 genes known to be in signaling pathways relevant to enteric nervous system development were selected for capture and sequencing. Blood DNAs from eight pools of five cases or controls were enriched by PCR-based RainDance technology (RDT) and then sequenced on a 454 FLX platform. As technical validation, five patients from case Pool-3 were also independently enriched by RDT, indexed with barcode and sequenced with sufficient coverage. Assessment for CDS single nucleotide variants showed DNA pooling performed well (specificity/sensitivity at 98.4%/83.7%) at the common variant level; but relatively worse (specificity/sensitivity at 65.5%/61.3%) at the rare variant level. Further Sanger sequencing only validated five out of 12 rare damaging variants likely involved in HSCR. Hence more improvement at variant detection and sequencing technology is needed to realize the potential of DNA pooling for large-scale resequencing projects.

Epilepsy and schizophrenia are common and typical neurological or mental illness respectively, and sometimes they comorbid in the same patients, however the underlying genetic relationship between the two brain diseases is still not fully understood. To investigate the possible genetic contribution to their comorbidity, we performed polygenic risk score (PRS) analyses and genetic correlation estimation so as to identify the overall genetic overlap between the two diseases. The global schizophrenia PRS is strongly associated with schizophrenia phenotype in Hong Kong population (odds ratio = 1.7, p = 2.26E-16), and focal epilepsy PRS is moderately associated with epilepsy phenotype in Hong Kong population (odds ratio = 1.14, p = 0.013). However the disease-specific PRS can only predict its own well-matched phenotype but not the other ones (p > 0.05). This pattern is further supported by non-significant pairwise genetic correlation and insufficient statistical power for PRS association from the cross-phenotype analyses. Our study reveals there's limited shared genetic aetiology between schizophrenia and epilepsy, and thus supports a model of shared environmental factors to explain the comorbidity between the two phenotypes.

Background We recently reported a strong association of plasma metabolite profile with survival in patients with heart failure and reduced ejection fraction (HFrEF). Mitochondrial function is perturbed in HFrEF, but whether plasma metabolite levels are linked to mitochondrial dysfunction is unknown. Elamipretide, a 4 amino acid peptide, normalizes mitochondrial energy production and thus provides a tool to test the association of mitochondrial function with the plasma metabolome in HFrEF. Methods Dogs (n=14) underwent intracoronary micro-embolization to induce HFrEF (pre-treatment EF 35%) and then were randomized 1:1 to elamipretide or placebo for 3 months. Targeted plasma metabolomic profiling was performed to quantitate 8 organic acids (OA), 23 amino acids (AA), and up to 59 acylcarnitines (AC) at pre-treatment and end of study. The association of changes in metabolite levels with treatment were tested using the Wilcoxon rank sum test on the principal components (PC) of each of the three metabolite classes. We also tested the correlation of metabolite levels with mitochondrial maximal rate of ATP synthesis in HF dog myocardium. We then attempted to confirm associations of interest in a human HFrEF cohort (n=1060) using bivariate Cox models. Result There was no association of plasma AA or AC changes with elamipretide treatment. However, there was a near-significant association of the first PC of OA changes (p=0.073), with treatment leading to lower plasma levels of OA compared to placebo (Figure). The OA also trended towards inverse relationship to maximal rate of ATP synthesis in dogs (correlation coef -0.09 to -0.67, mean -0.29), pyruvate having the strongest association (Coef =-0.67, p=0.012). In humans with HFrEF, directionally congruent and statistically significant associations with mortality risk were seen for 6 of these 8 OA (Table). Conclusion In dogs with HFrEF improving mitochondrial function using elamipretide resulted in favorable changes in plasma metabolites, specifically OA, which were risk-associated in human HFrEF. Our data suggest that mitochondrial dysfunction is the mechanism linking these plasma metabolomic abnormalities with worse survival in HFrEF.

Beta-blockers (BB) reduce the risk of mortality in most patients with heart failure with reduced ejection fraction (HFrEF), but not all patients benefit. Genome-wide association studies (GWAS) discovered variants influencing BB benefit in patients with hypertension, but a GWAS evaluating BB benefit

Hirschsprung disease (HSCR, aganglionic megacolon) is a complex genetic disorder of the enteric nervous system (ENS) characterized by the absence of enteric neurons along a variable length of the intestine. While rare variants (RVs) in the coding sequence (CDS) of several genes involved in ENS development lead to disease, the association of common variants (CVs) with HSCR has only been reported for RET (the major HSCR gene) and NRG1. Importantly, RVs in the CDS of these two genes are also associated with the disorder. To assess independent and joint effects between the different types of RET and NRG1 variants identified in HSCR patients, we used 254 Chinese sporadic HSCR patients and 143 ethnically matched controls for whom the RET and/or NRG1 variants genotypes (rare and common) were available. Four genetic risk factors were defined and interaction effects were modeled using conditional logistic regression analyses and pair-wise Kendall correlations. Our analysis revealed a joint effect of RET CVs with RET RVs, NRG1 CVs or NRG1 RVs. To assess whether the genetic interaction translated into functional interaction, mouse neural crest cells (NCCs; enteric neuron precursors) isolated from embryonic guts were treated with NRG1 (ErbB2 ligand) or/and GDNF (Ret ligand) and monitored during the subsequent neural differentiation process. Nrg1 inhibited the Gdnf-induced neuronal differentiation and Gdnf negatively regulated Nrg1-signaling by down-regulating the expression of its receptor, ErbB2. This preliminary data suggest that the balance neurogenesis/gliogenesis is critical for ENS development.

Background/Purpose Hirschsprung disease (HSCR; megacolon, congenital aganglionosis) is a congenital disorder characterized by the absence of ganglion cells along variable segments of the gut. Both rare (RV) and common variants of the RET gene are associated with HSCR. The aim of this study is to assess, for the first time, the variation in the RET gene of Vietnamese HSCR patients. Methods We used Sanger sequencing to screen the coding sequence of the RET gene of 97 Vietnamese HSCR patients of Southern Chinese ancestry. The healthy population consisted of 250 Southern Chinese individuals with no diagnosis of HSCR. Results We detected 8 heterozygous RVs distributed among 13 patients (13.40%) and that were not present in healthy individuals. Among those variants, there were 2 novel and deleterious (R133C [c.397 C>T]; R144C [c.430 C>T]) missense amino acid substitutions, 2 novel silent variants (P667P [c.2001 A>T]; Y809Y [c.2427 C>T]), and 4 previously described missense substitutions (R114H [c.341 G>A]; V292M [c.874 G>A]; G533S [c.1597 G>A]; R982C [c.2944 C>T]). As expected, the common RET coding sequence variants rs1800858 (A45A [c.135 G>A]) and rs1800861 (L769L [c.2307 T>G]) were highly associated with the disease. Conclusions The identification of novel deleterious variants together with the fact RET RVs are virtually unique to HSCR patients indicates that the RET gene is a target for mutations among Hirschsprung patients of any population.

Introduction Protein kinase C α (PRKCA) is a fundamental regulator of beta-adrenergic signaling and cardiac contractility. Previous studies suggested that increased cardiac expression of PRKCA is associated with adverse cardiac outcomes. Cardiac expression of PRKCA is partly genetically regulated, with the recently identified rs9909004 (T→C) as the most likely regulatory variant in failing human heart tissue, but the clinical impact of rs9909004 genotype remains unknown. Therefore the objectives of this study were to determine whether PRKCA rs9909004 genotype is associated with mortality in patients with heart failure and reduced ejection fraction (HFrEF), and if so, whether the genotype effects are modified by beta-blocker (BB) exposure. Hypothesis The T allele of rs9909004, associated with increased cardiac expression of PRKCA, will be associated with increased risk for all-cause mortality in HFrEF patients, and its effect will be less pronounced in HFrEF patients treated with higher BB exposure. Methods Insured HFrEF patients (n = 1,122) were enrolled from a single health care system into a prospective registry beginning in 2007, and they were genotyped with the Affymetrix Axiom® biobank array. BB exposure was quantified using pharmacy claims. Time-updating Cox models were used to test the independent association of rs9909004 genotype (additive genetic model for T allele) with all-cause mortality with adjustment for the following covariates: Meta-Analysis Global Group in Chronic Heart Failure (MAGGIC) risk score; BB exposure; N-terminal pro B-type natriuretic peptide (NT-proBNP) level; and the patients’ self-reported race. The interaction of rs9909004 genotype with BB exposure was tested in similarly adjusted models stratified by BB status (yes/no for any exposure) and then in the total cohort using a multiplicative interaction term. Results Patients were mean age 68 ± 12 years with left ventricular ejection fraction 35% ± 11; 51% were African-Americans, and 35% were women. Follow-up was median 3.0 years with 255 deaths (23%). The T allele frequency was 59% in African-Americans and 53% in whites. The T allele significantly associated with increased risk for all-cause mortality with an adjusted hazard ratio (HR) = 1.34 (95% CI = 1.06-1.71, p = 0.016). Interestingly, the association of this genotype with all-cause mortality was not modified BB exposure. The adjusted HRs were similar regardless of BB status (no BB, HR = 1.46, p = 0.146; yes BB, HR = 1.33, p = 0.041), and the formal test of interaction with BB was also not significant (p = 0.306, β=0.35). Conclusions The T allele of rs9909004 in PRKCA is a significant risk factor for mortality in patients with HFrEF, independent of other risk predictors, and its effect was not modified by BB exposure.

ObjectiveWe investigated the role of rare genetic variants and of de novo variants in the pathogenesis of mesial temporal lobe epilepsy related to hippocampal sclerosis (MTLE-HS).MethodsWhole-exome sequencing (WES) was performed in patients with MTLE-HS and their unaffected parents (trios). Genes or gene sets that were enriched with predicted damaging rare variants in the patients as compared to population controls were identified. Patients and their parents were compared to identify whether the variants were de novo or inherited.ResultsAfter quality control, WES data from 47 patients (26 female), including 23 complete trios, were available for analysis. Compared with population controls, significant enrichment of rare variants was observed in SEC24B. Integration of gene set data describing neuronal functions and psychiatric disorders showed enrichment signal on fragile X mental retardation protein (FMRP) targets. Twenty-one de novo variants were identified, with many known to cause neuropsychiatric disorders. The FMRP-targeted genes also carried more de novo variants. Inherited compound heterozygous and homozygous variants were identified.ConclusionsThe genetic architecture underlying MTHE-HS is complex. Multiple genes carrying de novo variants and rare variants among FMRP targets were identified, suggesting a pathogenic role. MTLE-HS and other neuropsychiatric disorders may have shared biology.

Heart failure (HF) is a global health burden despite effective therapies, and patients hospitalized for HF are particularly at risk. Identifying genetic associates of death or rehospitalization may identify new targets for therapy or especially high risk patient subgroups. The Acute Study of

The drivers of symptom burden in heart failure (HF) are poorly understood. Small studies have suggested racial disparities, but data are sparse, and the extent to which these disparities may have genetic underpinnings is unclear. A prospective genetic registry enrolled 1765 patients with HF. All

Background & Aims Hirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis. Methods We performed deep-target sequencing of DNA from 20 patients with Hirschsprung disease (16 men, 4 women), and 20 individuals without (controls), and searched for mutation(s) in GLI1, GLI2, GLI3 , SUFU , and SOX10 . Biological effects of GLI mutations were tested in luciferase reporter assays using HeLa or neuroblastoma cell lines. Development of the enteric nervous system was studied in Sufu f/f , Gli3 Δ699 , Wnt1-Cre , and Sox10 NGFP mice using immunohistochemical and whole-mount staining procedures to quantify enteric neurons and glia and analyze axon fasciculation, respectively. NCC migration was studied using time-lapse imaging. Results We identified 3 mutations in GLI in 5 patients with Hirschsprung disease but no controls; all lead to increased transcription of SOX10 in cell lines. SUFU, GLI, and SOX10 form a regulatory loop that controls the neuronal vs glial lineages and migration of NCCs. Sufu mutants mice had high Gli activity, due to loss of Sufu, disrupting the regulatory loop and migration of enteric NCCs, leading to defective axonal fasciculation, delayed gut colonization, or intestinal hypoganglionosis. The ratio of enteric neurons to glia correlated inversely with Gli activity. Conclusions We identified mutations that increase GLI activity in patients with Hirschsprung disease. Disruption of the SUFU−GLI−SOX10 regulatory loop disrupts migration of NCCs and development of the enteric nervous system in mice.

Epilepsy is a common neurological disease characterized by recurrent unprovoked seizures. Evidence suggested that abnormal activity of brain-derived neurotrophic factor (BDNF) contributes to the pathogenesis of epilepsy. Some previous studies identified association between genetic variants of BDNF and risk of epilepsy. In this study, this association has been examined in the Hong Kong and Malaysian epilepsy cohorts. Genomic DNA of 6047 subjects (1640 patients with epilepsy and 4407 healthy individuals) was genotyped for rs6265, rs11030104, rs7103411, and rs7127507 polymorphisms by using Sequenom MassArray and Illumina HumanHap 610-Quad or 550-Duo BeadChip arrays techniques. Results showed significant association between rs6265 T, rs7103411 C, and rs7127507 T and cryptgenic epilepsy risk (p = 0.00003, p = 0.0002, and p = 0.002, respectively) or between rs6265 and rs7103411 and symptomatic epilepsy risk in Malaysian Indians (TT vs. CC, p = 0.004 and T vs. C, p = 0.0002, respectively) as well as between rs6265 T and risk of cryptogenic epilepsy in Malaysian Chinese (p = 0.005). The Trs6265-Crs7103411-Trs7127507 was significantly associated with cryptogenic epilepsy in Malaysian Indians (p = 0.00005). In conclusion, our results suggest that BDNF polymorphisms might contribute to the risk of epilepsy in Malaysian Indians and Chinese.

Hirschsprung disease (HSCR; OMIM 142623) is a developmental disorder characterized by aganglionosis along variable lengths of the distal gastrointestinal tract, which results in intestinal obstruction. Interactions among known HSCR genes and/or unknown disease susceptibility loci lead to variable severity of phenotype. Neither linkage nor genome-wide association studies have efficiently contributed to completely dissect the genetic pathways underlying this complex genetic disorder. We have performed whole exome sequencing of 16 HSCR patients from 8 unrelated families with SOLID platform. Variants shared by affected relatives were validated by Sanger sequencing. We searched for genes recurrently mutated across families. Only variations in the FAT3 gene were significantly enriched in five families. Within-family analysis identified compound heterozygotes for AHNAK and several genes (N = 23) with heterozygous variants that co-segregated with the phenotype. Network and pathway analyses facilitated the discovery of polygenic inheritance involving FAT3, HSCR known genes and their gene partners. Altogether, our approach has facilitated the detection of more than one damaging variant in biologically plausible genes that could jointly contribute to the phenotype. Our data may contribute to the understanding of the complex interactions that occur during enteric nervous system development and the etiopathology of familial HSCR.

Background & Aims Hirschsprung disease is caused by failure of enteric neural crest cells (ENCCs) to fully colonize the bowel, leading to bowel obstruction and megacolon. Heterozygous mutations in the coding region of the RET gene cause a severe form of Hirschsprung disease (total colonic aganglionosis). However, 80% of HSCR patients have short-segment Hirschsprung disease (S-HSCR), which has not been associated with genetic factors. We sought to identify mutations associated with S-HSCR, and used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system to determine how mutations affect ENCC function. Methods We created induced pluripotent stem cell (iPSC) lines from 1 patient with total colonic aganglionosis (with the G731del mutation in RET ) and from 2 patients with S-HSCR (without a RET mutation), as well as RET +/− and RET −/− iPSCs. IMR90-iPSC cells were used as the control cell line. Migration and differentiation capacities of iPSC-derived ENCCs were analyzed in differentiation and migration assays. We searched for mutation(s) associated with S-HSCR by combining genetic and transcriptome data from patient blood- and iPSC-derived ENCCs, respectively. Mutations in the iPSCs were corrected using the CRISPR/Cas9 system. Results ENCCs derived from all iPSC lines, but not control iPSCs, had defects in migration and neuronal lineage differentiation. RET mutations were associated with differentiation and migration defects of ENCCs in vitro. Genetic and transcriptome analyses associated a mutation in the vinculin gene ( VCL M209L) with S-HSCR. CRISPR/Cas9 correction of the RET G731del and VCL M209L mutations in iPSCs restored the differentiation and migration capacities of ENCCs. Conclusions We identified mutations in VCL associated with S-HSCR. Correction of this mutation in iPSC using CRISPR/Cas9 editing, as well as the RET G731del mutation that causes Hirschsprung disease with total colonic aganglionosis, restored ENCC function. Our study demonstrates how human iPSCs can be used to identify disease-associated mutations and determine how they affect cell functions and contribute to pathogenesis.

To adopt an efficient approach of identifying rare variants possibly related to Hirschsprung disease (HSCR), a pilot study was set up to evaluate the performance of a newly designed protocol for next generation targeted resquencing. In total, 20 Chinese HSCR patients and 20 Chinese sex-matched individuals with no HSCR were included, for which coding sequences (CDS) of 62 genes known to be in signaling pathways relevant to enteric nervous system development were selected for capture and sequencing. Blood DNAs from eight pools of five cases or controls were enriched by PCR-based RainDance technology (RDT) and then sequenced on a 454 FLX platform. As technical validation, five patients from case Pool-3 were also independently enriched by RDT, indexed with barcode and sequenced with sufficient coverage. Assessment for CDS single nucleotide variants showed DNA pooling performed well (specificity/sensitivity at 98.4%/83.7%) at the common variant level; but relatively worse (specificity/sensitivity at 65.5%/61.3%) at the rare variant level. Further Sanger sequencing only validated five out of 12 rare damaging variants likely involved in HSCR. Hence more improvement at variant detection and sequencing technology is needed to realize the potential of DNA pooling for large-scale resequencing projects.

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.

Exome sequencing strategy is promising for finding novel mutations of human monogenic disorders. However, pinpointing the casual mutation in a small number of samples is still a big challenge. Here, we propose a three-level filtration and prioritization framework to identify the casual mutation(s) in exome sequencing studies. This efficient and comprehensive framework successfully narrowed down whole exome variants to very small numbers of candidate variants in the proof-of-concept examples. The proposed framework, implemented in a user-friendly software package, named KGGSeq (http://statgenpro.psychiatry.hku. hk/kggseq), will play a very useful role in exome sequencing-based discovery of human Mendelian disease genes. © 2011 The Author(s)., published_or_final_version

Hypertension is caused by the interaction of environmental and genetic factors. The condition which is very common, with about 18% of the adult Hong Kong Chinese population and over 50% of older individuals affected, is responsible for considerable morbidity and mortality. To identify genes influencing hypertension and blood pressure, we conducted a combined linkage and association study using over 500,000 single nucleotide polymorphisms (SNPs) genotyped in 328 individuals comprising 111 hypertensive probands and their siblings. Using a family-based association test, we found an association with SNPs on chromosome 5q31.1 (rs6596140; P, published_or_final_version

Cognitive decline is a reduction in cognitive ability usually associated with aging, and those with more extreme cognitive decline either have or are at risk of progressing to mild cognitive impairment and dementia including Alzheimer's disease (AD). We hypothesized that genetic variants predisposing to AD should be predictive of cognitive decline in elderly individuals. We selected 1325 subjects with extreme cognitive decline and 1083 well-matched control subjects from the Guangzhou Biobank Cohort Study in which more than 30,000 southern Chinese older people have been recruited and followed up. Thirty single-nucleotide polymorphisms in 29 AD-associated genes were genotyped. No statistically significant allelic associations with cognitive decline were found by individual variant analysis. At the level of genotypic association, we confirmed that the APOE ε4 homozygote significantly accelerated cognitive decline and found that carriers of the ACE rs1800764_C allele were more likely to show cognitive decline than noncarriers, particularly in those without college education. However, these effects do not survive after multiple testing corrections, and together they only explain 1.7% of the phenotypic variance in cognitive score change. This study suggests that AD risk variants and/or genes are not powerful predictors of cognitive decline in our Chinese sample.

The gene has been proposed as an attractive unit of analysis for association studies, but a simple yet valid, powerful, and sufficiently fast method of evaluating the statistical significance of all genes in large, genome-wide datasets has been lacking. Here we propose the use of an extended Simes test that integrates functional information and association evidence to combine the p values of the single nucleotide polymorphisms within a gene to obtain an overall p value for the association of the entire gene. Our computer simulations demonstrate that this test is more powerful than the SNP-based test, offers effective control of the type 1 error rate regardless of gene size and linkage-disequilibrium pattern among markers, and does not need permutation or simulation to evaluate empirical significance. Its statistical power in simulated data is at least comparable, and often superior, to that of several alternative gene-based tests. When applied to real genome-wide association study (GWAS) datasets on Crohn disease, the test detected more significant genes than SNP-based tests and alternative gene-based tests. The proposed test, implemented in an open-source package, has the potential to identify additional novel disease-susceptibility genes for complex diseases from large GWAS datasets. © 2011 The American Society of Human Genetics., link_to_OA_fulltext

Table S1. Loci from of other GWAS studies tested in HFrEF patients. Table S2. Additional GWAS results of potential interest (P

Table S1. Loci from of other GWAS studies tested in HFrEF patients. Table S2. Additional GWAS results of potential interest (P