GWAS Candidate Research Articles

Nicotine Motivated Behavior in C. elegans.

To maximize the advantages offered by Caenorhabditis elegans as a high-throughput (HTP) model for nicotine dependence studies, utilizing its well-defined neuroconnectome as a robust platform, and to unravel the genetic basis of nicotine-motivated behaviors, we established the nicotine conditioned cue preference (CCP) paradigm. Nicotine CCP enables the assessment of nicotine preference and seeking, revealing a parallel to fundamental aspects of nicotine-dependent behaviors observed in mammals. We demonstrated that nicotine-elicited cue preference in worms is mediated by nicotinic acetylcholine receptors and requires dopamine for CCP development. Subsequently, we pinpointed nAChR subunits associated with nicotine preference and validated human GWAS candidates linked to nicotine dependence involved in nAChRs. Functional validation involves assessing the loss-of-function strain of the CACNA2D3 ortholog and the knock-out (KO) strain of the CACNA2D2 ortholog, closely related to CACNA2D3 and sharing human smoking phenotypes. Our orthogonal approach substantiates the functional conservation of the α2δ subunit of the calcium channel in nicotine-motivated behavior. Nicotine CCP in C. elegans serves as a potent affirmation of the cross-species functional relevance of GWAS candidate genes involved in nicotine seeking associated with tobacco abuse, providing a streamlined yet comprehensive system for investigating intricate behavioral paradigms within a simplified and reliable framework.

GWAS and comparative genomics reveal candidate antibiotic resistance genes in the avian pathogen Gallibacterium anatis for six widespread antibiotics

Gallibacterium anatis is a Gram-negative bacterium found in the respiratory and genital tracts of various animals, primarily poultry. Its association with septicemia and high mortality in poultry, along with the rise in multidrug-resistant strains, has amplified concerns. Recent research uncovered significant variability in antibiotic resistance profiles among G. anatis isolates from different Austrian flocks, and even between different organs within the same bird. In response, in the present study 60 of these isolates were sequenced and a combination of comparative genomics and genome-wide association study (GWAS) analysis was applied to understand the genetic variability of G. anatis across flocks and organs and to identify genes related to antibiotic resistance. The results showed that each flock harbored one or two strains of G. anatis with only a few strains shared between flocks, demonstrating a great variability among flocks. We identified genes associated with resistance to nalidixic acid, trimethoprim, cefoxitin, tetracycline, ampicillin and sulfamethoxazole. Our findings revealed that G. anatis may develop antibiotic resistance through two mechanisms: single-nucleotide mutations and the presence of specific genes that confer resistance. Unexpectedly, some tetracycline-resistant isolates lacked all known tetracycline-associated genes, suggesting the involvement of novel mechanisms of tetracycline resistance that require additional exploration. Furthermore, our functional annotation of resistance genes highlighted the citric acid cycle pathway as a potential key modulator of antibiotic resistance in G. anatis. In summary, this study describes the first application of GWAS analysis to G. anatis and provides new insights into the acquisition of multidrug resistance in this important avian pathogen.

LocusDivergence analysis of GWAS candidates identified for root:shoot ratio changes under salt stress in Arabidopsis

LocusDivergence analysis of GWAS candidates identified for root:shoot ratio changes under salt stress in Arabidopsis

Abstract 10199: Establishing Human Ipsc-Derived Vascular Organoids as Angiogenesis Model

Recent large-scale GWA meta-analyses identified more than 240 risk loci associated with coronary artery disease (CAD) on a genome-wide significance level. Pathway analyses confirmed established pathomechanisms contributing to the progression of CAD like lipid metabolism, inflammation, extracellular matrix remodelling, or neoangiogenesis. Human induced pluripotent stem cells (iPSCs) offer a great source to derive 3D models, so-called organoids, allowing a more physiological environment to be analyzed in vitro . We aim to establish vascular organoids from iPSCs as 3D model of angiogenesis to investigate the role of CAD- and angiogenesis-associated risk genes like ADAMTS7 , ARNTL , and TBX20 .Four iPSC lines (UCSD18i-3-6, female, healthy; WC028i-5807-6, female, healthy; UCSD167i-99-1, female, healthy; UCSD179i-27-1, male, healthy; all from skin fibroblasts) will be differentiated into vascular organoids following the protocol published by Wimmer et al (2019). The first iPSC lines were differentiated to estimate the optimal seeding density for each cell line, and organoids were characterized. During embedding of aggregates for vascular sprouting organoids will be used for angiogenesis assay, and sprout length and number will be analysed. Different inhibitors and stimulators of angiogenesis can easily be applied proving that vascular organoids can be used as angiogenesis model. Using siRNA we will then knock-down (KD) above-mentioned risk genes, assessing their effect on angiogenesis. Additionally, cells will be edited using CRISPR/Cas to knockout the same risk genes.The first organoids showed expression of pericyte (PC) and endothelial cell (EC) markers on RNA level, while smooth muscle cell markers were not expressed. These results were confirmed in immunofluorescence stainings in whole organoids. Additionally, we performed cryosections which we also stained with immunofluorescence, showing an even distribution of cells. Staining of the cryosections further confirmed expression of PC- and EC markers. In conclusion, we successfully established iPSC-derived vascular organoids from iPSCs. We have now a 3D cell model in our hand to study GWAS candidate genes and their role in angiogenesis in the context of CAD.

Comprehensive Genetic Analysis of 128 Candidate Genes in a Cohort With Idiopathic, Severe, or Familial Osteoporosis.

ContextThe genetic bases of osteoporosis (OP), a disorder with high heritability, are poorly understood at an individual level. Cases of idiopathic or familial OP have long puzzled clinicians as to whether an actionable genetic cause could be identified.ObjectiveWe performed a genetic analysis of 28 cases of idiopathic, severe, or familial osteoporosis using targeted massively parallel sequencing.DesignTargeted sequencing of 128 candidate genes was performed using Illumina NextSeq. Variants of interest were confirmed by Sanger sequencing or SNP array.Patients and SettingThirty-seven patients in an academic tertiary hospital participated (54% male; median age, 44 years; 86% with fractures), corresponding to 28 sporadic or familial cases.Main Outcome MeasureThe identification of rare stop-gain, indel, splice site, copy-number, or nonsynonymous variants altering protein function.ResultsAltogether, we identified 28 variants of interest, but only 3 were classified as pathogenic or likely pathogenic variants: COL1A2 p.(Arg708Gln), WNT1 p.(Gly169Asp), and IDUA p.(His82Gln). An association of variants in different genes was found in 21% of cases, including a young woman with severe OP bearing WNT1, PLS3, and NOTCH2 variants. Among genes of uncertain significance analyzed, a potential additional line of evidence has arisen for GWAS candidates GPR68 and NBR1, warranting further studies.ConclusionsWhile we hope that continuing efforts to identify genetic predisposition to OP will lead to improved and personalized care in the future, the likelihood of identifying actionable pathogenic variants in intriguing cases of idiopathic or familial osteoporosis is seemingly low.

Functional validation of the osteoporosis GWAS candidate FUBP3 in knockout mice

Functional validation of the osteoporosis GWAS candidate FUBP3 in knockout mice

Integrating GWAS and Transcriptomics to Identify the Molecular Underpinnings of Thermal Stress Responses in Drosophila melanogaster.

Thermal tolerance of an organism depends on both the ability to dynamically adjust to a thermal stress and preparatory developmental processes that enhance thermal resistance. However, the extent to which standing genetic variation in thermal tolerance alleles influence dynamic stress responses vs. preparatory processes is unknown. Here, using the model species Drosophila melanogaster, we used a combination of Genome Wide Association mapping (GWAS) and transcriptomic profiling to characterize whether genes associated with thermal tolerance are primarily involved in dynamic stress responses or preparatory processes that influence physiological condition at the time of thermal stress. To test our hypotheses, we measured the critical thermal minimum (CTmin) and critical thermal maximum (CTmax) of 100 lines of the Drosophila Genetic Reference Panel (DGRP) and used GWAS to identify loci that explain variation in thermal limits. We observed greater variation in lower thermal limits, with CTmin ranging from 1.81 to 8.60°C, while CTmax ranged from 38.74 to 40.64°C. We identified 151 and 99 distinct genes associated with CTmin and CTmax, respectively, and there was strong support that these genes are involved in both dynamic responses to thermal stress and preparatory processes that increase thermal resistance. Many of the genes identified by GWAS were involved in the direct transcriptional response to thermal stress (72/151 for cold; 59/99 for heat), and overall GWAS candidates were more likely to be differentially expressed than other genes. Further, several GWAS candidates were regulatory genes that may participate in the regulation of stress responses, and gene ontologies related to development and morphogenesis were enriched, suggesting many of these genes influence thermal tolerance through effects on development and physiological status. Overall, our results suggest that thermal tolerance alleles can influence both dynamic plastic responses to thermal stress and preparatory processes that improve thermal resistance. These results also have utility for directly comparing GWAS and transcriptomic approaches for identifying candidate genes associated with thermal tolerance.

Evaluation of Gene-Based Family-Based Methods to Detect Novel Genes Associated With Familial Late Onset Alzheimer Disease.

Gene-based tests to study the combined effect of rare variants on a particular phenotype have been widely developed for case-control studies, but their evolution and adaptation for family-based studies, especially studies of complex incomplete families, has been slower. In this study, we have performed a practical examination of all the latest gene-based methods available for family-based study designs using both simulated and real datasets. We examined the performance of several collapsing, variance-component, and transmission disequilibrium tests across eight different software packages and 22 models utilizing a cohort of 285 families (N = 1,235) with late-onset Alzheimer disease (LOAD). After a thorough examination of each of these tests, we propose a methodological approach to identify, with high confidence, genes associated with the tested phenotype and we provide recommendations to select the best software and model for family-based gene-based analyses. Additionally, in our dataset, we identified PTK2B, a GWAS candidate gene for sporadic AD, along with six novel genes (CHRD, CLCN2, HDLBP, CPAMD8, NLRP9, and MAS1L) as candidate genes for familial LOAD.

Attempts to replicate genetic associations with schizophrenia in a cohort from north India

Schizophrenia is a chronic, severe, heritable disorder. Genome-wide association studies, conducted predominantly among Caucasians, have indicated > 100 risk alleles, with most significant SNPs on chromosome 6. There is growing interest as to whether these risk alleles are relevant in other ethnic groups as well. Neither an Indian genome-wide association studies nor a systematic replication of GWAS findings from other populations are reported. Thus, we analyzed 32 SNPs, including those associated in the Caucasian ancestry GWAS and other candidate gene studies, in a north Indian schizophrenia cohort (n = 1009 patients; n = 1029 controls) using a Sequenom mass array. Cognitive functioning was also assessed using the Hindi version of the Penn Computerized Neuropsychological Battery in a subset of the sample. MICB (rs6916394) a previously noted Caucasian candidate, was associated with schizophrenia at the p = 0.02 level. One SNP, rs2064430, AHI1 (6q23.3, SZ Gene database SNP) was associated at the p = 0.04 level. Other candidates had even less significance with rs6932590, intergenic (p = 0.07); rs3130615, MICB (p = 0.08); rs6916921, NFKBIL1 (p = 0.08) and rs9273012, HLA-DQA1 (p = 0.06) and haplotypic associations (p = 0.01–0.05) of 6p SNPs were detected. Of note, nominally significant associations with cognitive variables were identified, after covarying for age and diagnostic status. SNPs with p < 0.01 were: rs3130375, with working memory (p = 0.007); rs377763, with sensorimotor (p = 0.004); rs6916921, NFKBIL1 with emotion (p = 0.01). This relative lack of significant positive associations is likely influenced by the sample size and/or differences in the genetic architecture of schizophrenia across populations, encouraging population specific studies to identify shared and unique genetic risk factors for schizophrenia.

Systems genetics of intravenous cocaine self-administration in the BXD recombinant inbred mouse panel.

Cocaine addiction is a major public health problem with a substantial genetic basis for which the biological mechanisms remain largely unknown. Systems genetics is a powerful method for discovering novel mechanisms underlying complex traits, and intravenous drug self-administration (IVSA) is the gold standard for assessing volitional drug use in preclinical studies. We have integrated these approaches to identify novel genes and networks underlying cocaine use in mice. Mice from 39 BXD strains acquired cocaine IVSA (0.56mg/kg/infusion). Mice from 29 BXD strains completed a full dose-response curve (0.032-1.8mg/kg/infusion). We identified independent genetic correlations between cocaine IVSA and measures of environmental exploration and cocaine sensitization. We identified genome-wide significant quantitative trait loci (QTL) on chromosomes 7 and 11 associated with shifts in the dose-response curve and on chromosome 16 associated with sessions to acquire cocaine IVSA. Using publicly available gene expression data from the nucleus accumbens, midbrain, and prefrontal cortex of drug-naïve mice, we identified Aplp1 and Cyfip2 as positional candidates underlying the behavioral QTL on chromosomes 7 and 11, respectively. A genome-wide significant trans-eQTL linking Fam53b (a GWAS candidate for human cocaine dependence) on chromosome 7 to the cocaine IVSA behavioral QTL on chromosome 11 was identified in the midbrain; Fam53b and Cyfip2 were co-expressed genome-wide significantly in the midbrain. This finding indicates that cocaine IVSA studies using mice can identify genes involved in human cocaine use. These data provide novel candidate genes underlying cocaine IVSA in mice and suggest mechanisms driving human cocaine use.

EHFPI: a database and analysis resource of essential host factors for pathogenic infection.

High-throughput screening and computational technology has greatly changed the face of microbiology in better understanding pathogen–host interactions. Genome-wide RNA interference (RNAi) screens have given rise to a new class of host genes designated as Essential Host Factors (EHFs), whose knockdown effects significantly influence pathogenic infections. Therefore, we present the first release of a manually-curated bioinformatics database and analysis resource EHFPI (Essential Host Factors for Pathogenic Infection, http://biotech.bmi.ac.cn/ehfpi). EHFPI captures detailed article, screen, pathogen and phenotype annotation information for a total of 4634 EHF genes of 25 clinically important pathogenic species. Notably, EHFPI also provides six powerful and data-integrative analysis tools, i.e. EHF Overlap Analysis, EHF-pathogen Network Analysis, Gene Enrichment Analysis, Pathogen Interacting Proteins (PIPs) Analysis, Drug Target Analysis and GWAS Candidate Gene Analysis, which advance the comprehensive understanding of the biological roles of EHF genes, as in diverse perspectives of protein–protein interaction network, drug targets and diseases/traits. The EHFPI web interface provides appropriate tools that allow efficient query of EHF data and visualization of custom-made analysis results. EHFPI data and tools shall keep available without charge and serve the microbiology, biomedicine and pharmaceutics research communities, to finally facilitate the development of diagnostics, prophylactics and therapeutics for human pathogens.

Cellular dissection of psoriasis for transcriptome analyses and the post-GWAS era.

BackgroundGenome-scale studies of psoriasis have been used to identify genes of potential relevance to disease mechanisms. For many identified genes, however, the cell type mediating disease activity is uncertain, which has limited our ability to design gene functional studies based on genomic findings.MethodsWe identified differentially expressed genes (DEGs) with altered expression in psoriasis lesions (n = 216 patients), as well as candidate genes near susceptibility loci from psoriasis GWAS studies. These gene sets were characterized based upon their expression across 10 cell types present in psoriasis lesions. Susceptibility-associated variation at intergenic (non-coding) loci was evaluated to identify sites of allele-specific transcription factor binding.ResultsHalf of DEGs showed highest expression in skin cells, although the dominant cell type differed between psoriasis-increased DEGs (keratinocytes, 35%) and psoriasis-decreased DEGs (fibroblasts, 33%). In contrast, psoriasis GWAS candidates tended to have highest expression in immune cells (71%), with a significant fraction showing maximal expression in neutrophils (24%, P < 0.001). By identifying candidate cell types for genes near susceptibility loci, we could identify and prioritize SNPs at which susceptibility variants are predicted to influence transcription factor binding. This led to the identification of potentially causal (non-coding) SNPs for which susceptibility variants influence binding of AP-1, NF-κB, IRF1, STAT3 and STAT4.ConclusionsThese findings underscore the role of innate immunity in psoriasis and highlight neutrophils as a cell type linked with pathogenetic mechanisms. Assignment of candidate cell types to genes emerging from GWAS studies provides a first step towards functional analysis, and we have proposed an approach for generating hypotheses to explain GWAS hits at intergenic loci.

Functional validation of GWAS gene candidates for abnormal liver function during zebrafish liver development

SUMMARYGenome-wide association studies (GWAS) have revealed numerous associations between many phenotypes and gene candidates. Frequently, however, further elucidation of gene function has not been achieved. A recent GWAS identified 69 candidate genes associated with elevated liver enzyme concentrations, which are clinical markers of liver disease. To investigate the role of these genes in liver homeostasis, we narrowed down this list to 12 genes based on zebrafish orthology, zebrafish liver expression and disease correlation. To assess the function of gene candidates during liver development, we assayed hepatic progenitors at 48 hours post fertilization (hpf) and hepatocytes at 72 hpf using in situ hybridization following morpholino knockdown in zebrafish embryos. Knockdown of three genes (pnpla3, pklr and mapk10) decreased expression of hepatic progenitor cells, whereas knockdown of eight genes (pnpla3, cpn1, trib1, fads2, slc2a2, pklr, mapk10 and samm50) decreased cell-specific hepatocyte expression. We then induced liver injury in zebrafish embryos using acetaminophen exposure and observed changes in liver toxicity incidence in morphants. Prioritization of GWAS candidates and morpholino knockdown expedites the study of newly identified genes impacting liver development and represents a feasible method for initial assessment of candidate genes to instruct further mechanistic analyses. Our analysis can be extended to GWAS for additional disease-associated phenotypes.

Abstract 154: GWAS Candidate Gene Knockout in Rats Reveals a Role for Erk/mapk Signaling in Hypertension

The data revealed by GWAS studies provide a wealth of candidate human disease genes now requiring functional validation in animal models. In two years, we disrupted a large set of GWAS candidate genes for human hypertension and chronic kidney disease by Zinc Finger Nuclease (ZFN)-mediated gene targeting in the SS (Dahl salt-sensitive) strain background. Phenotyping male rats from gene-disrupted strains revealed five genes ( Sh2b3 , Rasgrp3 , Gpr73 , Ulk3 , and Wdr72 ) significantly altering the salt-induced hypertension in and renal damage phenotypes in this model strain of human hypertension compared to age matched wild type controls. Ingenuity Pathway Analysis revealed a putative connection for four of these genes ( Sh2b3 , Rasgrp3 , Gpr73 , and Ulk3 ) to the ERK/MAPK signaling pathway and suggested that decreased ERK signaling would exacerbate disease in the SS model. To test this hypothesis, we measured the response of SS rats to a 4% salt diet with daily IP injections of the MEK inhibitor PD98059 or vehicle control. Wild type SS animals treated with PD98059 show a significant increase in mean arterial pressure compared to vehicle-injected controls (n=10, 142±7 vs. 126±6 mmHg respectively, P&lt;0.05) suggesting that ERK signaling plays an important role in the pathogenesis of this model. In conclusion, we have used gene knockout in a hypertensive rat model to identify genes and associated pathway mechanisms of BP regulation. The gene disruption phenotype and participation of multiple genes in the ERK/MAPK signaling pathway indicates that regulation of this pathway plays a major role in determining blood pressure.