SNPs In High Linkage Disequilibrium Research Articles

The 9p21.3 coronary artery disease risk locus drives vascular smooth muscle cells to an osteochondrogenic state.

Genome-wide association studies have identified common genetic variants at ~400 human genomic loci linked to coronary artery disease (CAD) susceptibility. Among these genomic regions, the most impactful is the 9p21.3 CAD risk locus, which spans a 60 kb gene desert and encompasses ~80 SNPs in high linkage disequilibrium. Despite nearly two decades since its discovery, the functional mechanism of this genomic region remains incompletely resolved. To investigate the transcriptional gene programs mediated by 9p21.3 risk locus, we applied a model of induced pluripotent stem cells (iPSCs) from risk and non-risk donors at 9p21.3, as well as isogenic lines with a full haplotype deletion. Upon differentiation to vascular smooth muscle cells (VSMC), single-cell transcriptomic profiling demonstrated iPSC-VSMC phenotypes resembling those from native human coronary arteries, confirming the robustness of this model. Remarkably, our analyses revealed that VSMCs harboring the 9p21.3 risk haplotype preferentially adopt an osteochondrogenic state. Importantly, we identified LIMCH1 and CRABP1 as signature genes critical for defining this transcriptional program. Our study provides new insights into the mechanism at the 9p21.3 risk locus and defines its role in driving a disease-prone transcriptional state in VSMCs.

Abstract 2072: The 9p21.3 Coronary Artery Disease Risk Locus Induces Cell State Transitions In Vascular Smooth Muscle Cells

Background: Genome-wide association studies have identified common genetic variants (Single Nucleotide Polymorphisms - SNPs) at ~300 human genomic loci linked to coronary artery disease (CAD) susceptibility. Among these genomic regions, the most impactful is the 9p21.3 CAD risk locus, which spans a 60 kb gene desert and encompasses ~80 SNPs in high linkage disequilibrium. We have previously generated isogenic induced Pluripotent Stem Cells (iPSCs) lines from risk and non-risk donors at 9p21.3 and performed a complete deletion of the locus. By using iPSC-derived vascular smooth muscle cells (iPSC-VSMCs) we demonstrated that the risk haplotype at 9p21.3 causes altered expression of numerous genes essential for muscle function, including contraction and adhesion. Notably, deletion of the risk haplotype restores the non-risk phenotype, suggesting a gain of function effect. Hypothesis: The 9p21.3 risk haplotype alters VSMCs plasticity and induces dedifferentiation and alternative lineage acquisition. Aims: Identify transcriptomic signatures and trajectories of VSMCs induced by the 9p21.3 risk haplotype Methods: We have used iPSCs from individuals carrying the risk and non-risk haplotype at 9p21.3 and isogenic knockout lines. After VSMCs differentiation induction, mature VSMCs were used for single cell RNA sequencing using 10X Chromium platform. Results: Our analysis revealed that the 9p21.3 risk haplotype prompts VSMCs to acquire a novel cellular state showing reduced plasticity and divergent from other VSMC states previously linked to CAD. We found markers of alternative lineages, including osteogenic and neuronal, coupled with altered expression of genes within other CAD loci. We identified a set of new molecular markers crucial to define risk-associated VSMCs and uncovered their upstream regulators. Conclusions: Our study provides insights into CAD pathogenesis driven by the 9p21.3 risk locus and identified new gene regulatory networks essential for maintaining the normal functionality of the muscle layer of the arteries. Leveraging the power of iPSCs we present novel concepts about the biology of VSMCs and shed light on the impact of CAD genetic risk factors in preserving a healthy cellular state within the vasculature.

Abstract 5217: Genome-wide interactions of processed and red meat intake on colorectal cancer risk

Abstract Introduction: Colorectal cancer (CRC) is the third most common cancer, and second cause of cancer death worldwide. High red meat and/or processed meat consumption are established risk factors for CRC. Genome-wide association studies have identified over 200 genetic variants that explain ~20% of the variability in CRC risk. However, few large studies have evaluated genome-wide gene by environment (GxE) interactions with meat in CRC. Using the largest CRC pooled dataset available to date we conducted a genome-wide GxE analysis to identify possible interactions between common variants and red meat and/or processed meat intake and CRC risk. Methods: A pooled sample of 29,912 CRC cases and 39,704 unaffected controls of European (EUR) ancestry from 27 studies was analyzed. Sex- and study-specific quartiles for red meat and processed meat intake were constructed from harmonized questionnaire data on meat consumption. Genotyping arrays were imputated to the Haplotype Reference Consortium. To identify novel CRC interaction loci we used the two-step EDGE method, standard GxE analyses and a 1-3 degrees of freedom (DF) test. We removed previously known GWAS hits and SNPs in high linkage disequilibrium with them. Results: Our Meta-analyses confirmed a positive association between quartile consumption increase of red meat and processed meat with CRC risk (Red meat OR = 1.30; 95%CI = 1.21-1.41; Processed meat OR = 1.40; 95% CI = 1.20 -1.63). Greater magnitude in point estimates were observed for case-control studies compared to cohort studies, for processed meat consumption among men compared to women, and for red meat consumption and distal colon localization compared to proximal colon or rectum. Two DF GxE tests of red meat consumption revealed two SNPs in chromosome (Chr) 8 (rs4871179 & rs75212442) and a SNP in Chr10 (rs117674361) that map downstream HAS2, COLEC10 and downstream CCSER2. In addition, the two-step EDGE GxE method identified the rs35352860 SNP that maps to the SMAD7 gene in Chr18. Genotype-stratified analyses showed that the red meat and CRC risk association was restricted to homozygous carriers of major alleles for rs4871179, rs75212442 & rs117674361. Moreover, we observed dose-response increase in the red meat and CRC risk association with additional copies of the T allele of the SNP in Chr18, with homozygous carriers of the T allele having 46% higher chance of developing CRC associated with high red meat intake; this in contrast to homozygous carriers of the reference C allele, that had an 18% higher risk of developing CRC. No statistically significant GXE interactions with processed meat intake were found. Conclusion: In this large-scale genome-wide GxE analysis we identified interactions between 4 SNPs and red meat consumption affecting CRC risk. The interaction with the SMAD7 rs35352860 SNP provides supportive evidence for a role of heme iron in the carcinogenic pathway of red meat intake in CRC development. Citation Format: Joel Sanchez Mendez, Mariana C. Stern, Andre Kim, John Morrison, Juan P. Lewinger, Li Hsu, Eric Kawaguchi, Ulrike Peters, William J. Gauderman, on behalf of GECCO, CCFR and CORECT. Genome-wide interactions of processed and red meat intake on colorectal cancer risk. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5217.

Integrated glycomics and genetics analyses reveal a potential role for N-glycosylation of plasma proteins and IgGs, as well as the complement system, in the development of type 1 diabetes

Aims/hypothesisWe previously demonstrated that N-glycosylation of plasma proteins and IgGs is different in children with recent-onset type 1 diabetes compared with their healthy siblings. To search for genetic variants contributing to these changes, we undertook a genetic association study of the plasma protein and IgG N-glycome in type 1 diabetes.MethodsA total of 1105 recent-onset type 1 diabetes patients from the Danish Registry of Childhood and Adolescent Diabetes were genotyped at 183,546 genetic markers, testing these for genetic association with variable levels of 24 IgG and 39 plasma protein N-glycan traits. In the follow-up study, significant associations were validated in 455 samples.ResultsThis study confirmed previously known plasma protein and/or IgG N-glycosylation loci (candidate genes MGAT3, MGAT5 and ST6GAL1, encoding beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase, alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase and ST6 beta-galactoside alpha-2,6-sialyltransferase 1 gene, respectively) and identified novel associations that were not previously reported for the general European population. First, novel genetic associations of IgG-bound glycans were found with SNPs on chromosome 22 residing in two genomic intervals close to candidate gene MGAT3; these include core fucosylated digalactosylated disialylated IgG N-glycan with bisecting N-acetylglucosamine (GlcNAc) (pdiscovery=7.65 × 10−12, preplication=8.33 × 10−6 for the top associated SNP rs5757680) and core fucosylated digalactosylated glycan with bisecting GlcNAc (pdiscovery=2.88 × 10−10, preplication=3.03 × 10−3 for the top associated SNP rs137702). The most significant genetic associations of IgG-bound glycans were those with MGAT3. Second, two SNPs in high linkage disequilibrium (missense rs1047286 and synonymous rs2230203) located on chromosome 19 within the protein coding region of the complement C3 gene (C3) showed association with the oligomannose plasma protein N-glycan (pdiscovery=2.43 × 10−11, preplication=8.66 × 10−4 for the top associated SNP rs1047286).Conclusions/interpretationThis study identified novel genetic associations driving the distinct N-glycosylation of plasma proteins and IgGs identified previously at type 1 diabetes onset. Our results highlight the importance of further exploring the potential role of N-glycosylation and its influence on complement activation and type 1 diabetes susceptibility.Graphical abstract

Brain MRI Phenotypes Associated with Polymorphisms at or near the MAPT (Microtubule Associated Protein Tau) and COASY (Coenzyme A Synthase) Genes

Abstract Objectives Impaired metal homeostasis in the brain has been reported in neurodegenerative diseases such as Alzheimer's Disease and Parkinson's Disease and can be detected using magnetic resonance imaging (MRI). Many factors, including genetics, affect brain metal accumulation and the risk of neurodegeneration or premature brain aging. Our objective is to uncover independent association signals at chromosome 17 associated with susceptibility-weighted MRI (swMRI) intensities in the caudate and putamen. Methods Conditional and joint association analysis by GCTA-COJO was performed on the summary statistics dataset from a genome-wide association study (GWAS) on brain swMRI phenotypes from 7778 healthy adults of European descent aged 40–69 years from the UK BioBank. Independent association signals (single nucleotide polymorphisms or SNPs) found by GCTA-COJO were grouped further by SNPclip to identify additional candidate SNPs in high linkage disequilibrium (LD) (R2 > 0.8, P < 5.0 × 10–8) with the independent lead/index SNP at each signal. Correlations were performed to show the similarity of impacts (beta) of SNPs significantly associated with MRI differences in both caudate and putamen at one locus. Results There are 2 independent and distinct loci at chromosome 17 associated with MRI differences in the putamen, whereas there is only 1 locus for the caudate. One locus for the putamen is associated with the MAPT H2 haplotype, which has been linked to different neurological diseases. Another locus for both putamen and caudate is located near the genes COASY, NAGLU, and MLX, which are all plausible candidate genes for brain metal accumulation. For one locus identified by GCTA-COJO, there are 19 SNPs in LD (R2 > 0.8) and overlapping in both the caudate and the putamen. The effects (beta) of the SNPs in the putamen and caudate are highly correlated across both brain regions (R2 = 0.907, P-value = 0.000921). Conclusions There is likely 1 signal from the COASY/NAGLU locus on chromosome 17 affecting both brain regions. The signal from the MAPT locus only affects the MRI pattern seen in the putamen. These results connect genetics with brain MRI patterns and an individual's risk for neurodegenerative diseases. Funding Sources This work was funded by grants from the Oklahoma Center for the Advancement of Science & Technology and the Oklahoma Agricultural Experiment Station.

Variations and expression features of CYP2D6 contribute to schizophrenia risk

Genome-wide association studies (GWAS) have successfully identified 145 loci implicated in schizophrenia (SCZ). However, the underlying mechanisms remain largely unknown. Here, we analyze 1497 RNA-seq data in combination with their genotype data and identify SNPs that are associated with expression throughout the genome by dissecting expression features to genes (eGene) and exon–exon junctions (eJunction). Then, we colocalize eGene and eJunction with SCZ GWAS using SMR and fine mapping. Multiple ChIP-seq data and DNA methylation data generated from brain were used for identifying the causal variants. Finally, we used a hypothesis-free (no SCZ risk loci considered) enrichment analysis to determine implicated pathways. We identified 171 genes and eight splicing junctions located within four genes (SNX19, ARL6IP4, APOPT1, and CYP2D6) that potentially contribute to SCZ susceptibility. Among the genes, CYP2D6 is significantly associated with SCZ SNPs in eGene and eJunction. In-depth examination of the CYP2D6 region revealed that a nonsynonymous single nucleotide variant rs16947 is strongly associated with a higher abundance of CYP2D6 exon 3 skipping junctions. While we found rs133377 and other functional SNPs in high linkage disequilibrium with rs16947 (r2 = 0.9539), histone acetylation analysis showed they are located within active transcription start sites. Furthermore, our data-driven enrichment analysis showed that CYP2D6 is significantly involved in drug metabolism of codeine, tamoxifen, and citalopram. Our study facilitates an understanding of the genetic architecture of SCZ and provides new drug targets.

Stratified analyses of genome wide association study data reveal haplotypes for a candidate gene on chromosome 2 (KIAA1211L) is associated with opioid use in patients of Arabian descent

BackgroundGenome Wide Association Studies (GWAS) have been conducted to identify genes and pathways involved in development of opioid use disorder. This study extends the first GWAS of substance use disorder (SUD) patients from the United Arab Emirates (UAE) by stratifying the study group based on opioid use, which is the most common substance of use in this cohort.MethodsThe GWAS cohort consisted of 512 (262 case, 250 controls) male participants from the UAE. The samples were genotyped using the Illumina Omni5 Exome system. Data was stratified according to opioid use using PLINK. Haplotype analysis was conducted using Haploview 4.2.ResultsTwo main associations were identified in this study. Firstly, two SNPs on chromosome 7 were associated with opioid use disorder, rs118129027 (p-value = 1.23 × 10 − 8) and rs74477937 (p-value = 1.48 × 10 − 8). This has been reported in Alblooshi et al. (Am J Med Genet B Neuropsychiatr Genet 180(1):68–79, 2019). Secondly, haplotypes on chromosome 2 which mapped to the KIAA1211L locus were identified in association with opioid use. Five SNPs in high linkage disequilibrium (LD) (rs2280142, rs6542837, rs12712037, rs10175560, rs11900524) were arranged into haplotypes. Two haplotypes GAGCG and AGTTA were associated with opioid use disorders (p-value 3.26 × 10− 8 and 7.16 × 10− 7, respectively).ConclusionThis is the first GWAS to identify candidate genes associated with opioid use disorder in participants from the UAE. The lack of other genetic data of Arabian descent opioid use patients has hindered replication of the findings. Nevertheless, the outcomes implicate new pathways in opioid use disorder that requires further research to assess the role of the identified genes in the development of opioid use disorder.

Single Nucleotide Polymorphisms in MIR143 Contribute to Protection Against Non-Hodgkin Lymphoma (NHL) in Caucasian Populations.

Recent studies show an association of microRNA (miRNA) polymorphisms (miRSNPs) in different cancer types, including non-Hodgkin lymphoma (NHL). The identification of miRSNPs that are associated with NHL susceptibility may provide biomarkers for early diagnosis and prognosis for patients who may not respond well to current treatment options, including the immunochemotherapy drug combination that includes rituximab, cyclophosphamide, doxorubicin, vincristine and prednisome (R-CHOP). We developed a panel of miRSNPs for genotyping while using multiplex PCR and chip-based mass spectrometry analysis in an Australian NHL case-control population (300 cases, 140 controls). Statistical association with NHL susceptibility was performed while using Chi-square (χ2) and logistic regression analysis. We identified three SNPs in MIR143 that are to be significantly associated with reduced risk of NHL: rs3733846 (odds ratio (OR) [95% confidence interval (CI)] = 0.54 [0.33–0.86], p = 0.010), rs41291957 (OR [95% CI] = 0.61 [0.39–0.94], p = 0.024), and rs17723799 (OR [95% CI] = 0.43 [0.26–0.71], p = 0.0009). One SNP, rs17723799, remained significant after correction for multiple testing (p = 0.015). Subsequently, we investigated an association between the rs17723799 genotype and phenotype by measuring target gene Hexokinase 2 (HKII) expression in cancer cell lines and controls. Our study is the first to report a correlation between miRSNPs in MIR143 and a reduced risk of NHL in Caucasians, and it is supported by significant SNPs in high linkage disequilibrium (LD) in a large European NHL genome wide association study (GWAS) meta-analysis.

Genetic Determinants of Glycated Hemoglobin in Type 1 Diabetes.

Glycated hemoglobin (HbA1c) is an important measure of glycemia in diabetes. HbA1c is influenced by environmental and genetic factors both in people with and in people without diabetes. We performed a genome-wide association study (GWAS) for HbA1c in a Finnish type 1 diabetes (T1D) cohort, FinnDiane. Top results were examined for replication in T1D cohorts DCCT/EDIC, WESDR, CACTI, EDC, and RASS, and a meta-analysis was performed. Three SNPs in high linkage disequilibrium on chromosome 13 near relaxin family peptide receptor 2 (RXFP2) were associated with HbA1c in FinnDiane at genome-wide significance (P < 5 × 10−8). The minor alleles of rs2085277 and rs1360072 were associated with higher HbA1c also in the meta-analysis with RASS (P < 5 × 10−8), where these variants had minor allele frequencies ≥1%. Furthermore, these SNPs were associated with HbA1c in an East Asian population without diabetes (P ≤ 0.013). A weighted genetic risk score created from 55 HbA1c-associated variants from the literature was associated with HbA1c in FinnDiane but explained only a small amount of variation. Understanding the genetic basis of glycemic control and HbA1c may lead to better prevention of diabetes complications.

SU1 - EXPLORING GENETIC VARIATION THAT INFLUENCES BRAIN METHYLATION IN ATTENTION-DEFICIT/HYPERACTIVITY DISORDER (ADHD)

Background Allele-Specific Methylation (ASM) is a common epigenetic mechanism that involves SNPs correlating with differential levels of methylation at CpG sites. Epigenetic changes play a crucial role in driving lasting changes in gene expression in several tissues, including the brain. As alteration of DNA methylation levels has recently been linked to ADHD symptoms, our aim is to explore the contribution of ASM to ADHD through a case-control association study using different GWAS datasets. Methods We performed a primary SNP selection based on two previous studies that identified ASM variants in different brain regions of post-mortem human samples. Subsequently, from those variants we selected a total of 3,896 SNPs following these criteria: associations in cis between the SNP and methylation at a CpG site, correlation of methylation levels with gene expression (R2) ≥ 0.2, tagSNPs for each CpG site (LD; r2≥0.85). These candidates were inspected in summary statistics data from the first GWAS meta-analysis of adult and childhood ADHD, performed by iPSYCH and the Psychiatric Genomics Consortium (PGC) in 20,183 cases and 35,191 controls. We applied False Discovery Rate (FDR) correction for multiple testing (5% FDR, corrected P≤ 6.78e-05) to determine the top hits. These signals were followed-up to retrieve (i) SNPs located within the same CpG site and thereafter (ii) SNPs in high linkage disequilibrium with the associated SNPs (using the same FDR threshold). Impact of the variants on gene expression levels were further explored in the GTEx database. Results The case-control association study identified eight tagSNPs surviving FDR, which correlate with differential methylation of six CpG sites. Top hits were followed-up to retrieve other SNPs that correlate with methylation of the same CpG sites, making a total of 39 candidate SNPs. The six CpG sites are located in the promoter regions of five genes, not previously related to ADHD. Notably, the risk SNPs associated with three of the genes are eQTLs for those genes in various brain tissues, including the cerebellar hemisphere and the cerebellum. Discussion We have directed existing knowledge of ASM to evaluate its possible contribution to ADHD both at genetic and epigenetic levels. We followed a systematic approach by employing cis-acting ASM variants to pinpoint candidate genes for ADHD. Interestingly, several of the identified variants are eQTLs for genes that are expressed in brain. Our aim is now to further investigate the role of these genes in the development of the ADHD phenotype.

Natural variation of ZmHKT1 affects root morphology in maize at the seedling stage.

Eight variants in ZmHKT1 promoter were significantly associated with root diameter, four haplotypes based on these significant variants were found, and Hap2 has the largest root diameter. Roots play an important role in uptake of water, nutrients and plant anchorage. Identification of gene and corresponding SNPs associated with root traits would enable develop maize lines with better root traits that might help to improve capacity for absorbing nutrients and water acquisition. The genomic sequences of a salt tolerance gene ZmHKT1 was resequenced in 349 maize inbred lines, and the association between nucleotide polymorphisms and seedling root traits was detected. A total of 269 variants in ZmHKT1 were identified, including 226 single nucleotide polymorphisms and 43 insertions and deletions. The gene displayed high level of nucleotide diversity, especially in non-genic regions. A total of 19 variations in untranslated region of ZmHKT1 were found to be associated with six seedling traits. Eight variants in promoter region were significantly associated with average root diameter (ARD), four haplotypes were found based on these significant variants, and Hap2 has the largest ARD. Two SNPs in high-linkage disequilibrium (SNP-415 and SNP 2169) with pleiotropic effects were significantly associated with plant height, root surface area, root volume, and shoot dry weight. This result revealed that ZmHKT1 was an important contributor to the phenotypic variations of seedling root traits in maize, these significant variants could use to develop functional markers to improve root traits.

Positional integration of lung adenocarcinoma susceptibility loci with primary human alveolar epithelial cell epigenomes.

To identify functional lung adenocarcinoma (LUAD) risk SNPs. Eighteen validated LUAD risk SNPs (p≤5×10-8) and 930 SNPs in high linkage disequilibrium (r2>0.5) were integrated with epigenomic information from primary human alveolar epithelial cells. Enhancer-associated SNPs likely affecting transcription factor-binding sites were predicted. Three SNPs were functionally investigated using luciferase assays, expression quantitative trait loci and cancer-specific expression. Forty-seven SNPs mapped to putative enhancers; 11 located to open chromatin. Of these, seven altered predicted transcription factor-binding motifs. Rs6942067 showed allele-specific luciferase expression and expression quantitative trait loci analysis indicates that it influences expression of DCBLD1, a gene that encodes an unknown membrane protein and is overexpressed in LUAD. Integration of candidate LUAD risk SNPS with epigenomic marks from normal alveolar epithelium identified numerous candidate functional LUAD risk SNPs including rs6942067, which appears to affect DCBLD1 expression. Data deposition: Data are provided in GEO record GSE84273.

Genome-wide association study reveals a QTL and strong candidate genes for umbilical hernia in pigs on SSC14

BackgroundUmbilical hernia is one of the most prevalent congenital defect in pigs, causing economic losses and substantial animal welfare problems. Identification and implementation of genomic regions controlling umbilical hernia in breeding is of great interest to reduce incidences of hernia in commercial pig production. The aim of this study was to identify such regions and possibly identify causative variation affecting umbilical hernia in pigs. A case/control material consisting of 739 Norwegian Landrace pigs was collected and applied in a GWAS study with a genome-wide distributed panel of 60 K SNPs. Additionally candidate genes were sequenced to detect additional polymorphisms that were used for single SNP and haplotype association analyses in 453 of the pigs.ResultsThe GWAS in this report detected a highly significant region affecting umbilical hernia around 50 Mb on SSC14 (P < 0.0001) explaining up to 8.6% of the phenotypic variance of the trait. The region is rather broad and includes 62 significant SNPs in high linkage disequilibrium with each other. Targeted sequencing of candidate genes within the region revealed polymorphisms within the Leukemia inhibitory factor (LIF) and Oncostatin M (OSM) that were significantly associated with umbilical hernia (P < 0.001).ConclusionsA highly significant QTL for umbilical hernia in Norwegian Landrace pigs was detected around 50 Mb on SSC14. Resequencing of candidate genes within the region revealed SNPs within LIF and OSM highly associated with the trait. However, because of extended LD within the region, studies in other populations and functional studies are needed to determine whether these variants are causal or not. Still without this knowledge, SNPs within the region can be used as genetic markers to reduce incidences of umbilical hernia in Norwegian Landrace pigs.

Genome-wide association study of subcortical brain volume in PTSD cases and trauma-exposed controls

Depending on the traumatic event, a significant fraction of trauma survivors subsequently develop PTSD. The additional variability in PTSD risk is expected to arise from genetic susceptibility. Unfortunately, several genome-wide association studies (GWAS) have failed to identify a consistent genetic marker for PTSD. The heritability of intermediate phenotypes such as regional brain volumes is often 80% or higher. We conducted a GWAS of subcortical brain volumes in a sample of recent military veteran trauma survivors (n = 157), grouped into PTSD (n = 66) and non-PTSD controls (n = 91). Covariates included PTSD diagnosis, sex, intracranial volume, ancestry, childhood trauma, SNP×PTSD diagnosis, and SNP×childhood trauma. We identified several genetic markers in high linkage disequilibrium (LD) with rs9373240 (p = 2.0 × 10−7, FDR q = 0.0375) that were associated with caudate volume. We also observed a significant interaction between rs9373240 and childhood trauma (p-values = 0.0007–0.002), whereby increased trauma exposure produced a stronger association between SNPs and increased caudate volume. We identified several SNPs in high LD with rs34043524, which is downstream of the TRAM1L1 gene that were associated with right lateral ventricular volume (p = 1.73 × 10−7; FDR q = 0.032) and were also associated with lifetime alcohol abuse or dependence (p = 2.49 × 10−7; FDR q = 0.0375). Finally, we identified several SNPs in high LD with rs13140180 (p = 2.58 × 10−7; FDR q = .0016), an intergenic region on chromosome 4, and several SNPs in the TMPRSS15 associated with right nucleus accumbens volume (p = 2.58 × 10−7; FDR q = 0.017). Both TRAM1L1 and TMPRSS15 have been previously implicated in neuronal function. Key results survived genome-wide multiple-testing correction in our sample. Leveraging neuroimaging phenotypes may offer a shortcut, relative to clinical phenotypes, in mapping the genetic architecture and neurobiological pathways of PTSD.

Gene-environment Interaction in Pediatric Eosinophilic Esophagitis

Introduction: The rapid increase in the incidence of eosinophilic esophagitis (EoE) may be due to interactions between a genetic predisposition and changing environmental factors. While EoE has been associated with several early life factors, gene-environment interactions have not been explored. Here, we test for interactions between SNPs or SNPs in high linkage disequilibrium with SNPs previously associated with EoE (TSLP, LOC283710/ KLF13, CAPN14, and CCL26) and early life factors (antibiotic use in infancy, cesarean delivery, breastfeeding, NICU admission, and absence of pets in the home). Methods: We conducted a case-control study using hospital-based cases identified from a registry of EoE patients (n=127) and population-based controls representative of the hospital catchment area (n=121). Early life environmental factors were assessed through a self-administered questionnaire provided to mothers. Cases and controls had been previously genotyped. We computed case-only interaction tests and where there was evidence of possible interaction, secondary analyses were used to describe the combined and independent effects of genotype and environmental factor on risk of EoE. Models were adjusted for maternal education and population stratification. Results: The case-only analyses identified interactions between CAPN14 and breastfeeding (p=0.02), TSLP and NICU admission (p=0.06), TSLP and not having a furry pet in infancy (p=0.05), and LOC283710/ KLF13 and NICU admission (p=0.02), but not with CCL26 and any of the early life factors examined. Case-control analyses of these associations showed the interactive effects for all associations were antagonistic; specifically the risk of having EoE for those with both the susceptibility genotype and early life factor was attenuated as compared to the risk of disease among those with only the genotype or early life factor (Figure). Examination of the effect of early life factors, among those with the CAPN14 susceptibility SNP, identified a strong protective association for breastfeeding (OR: 0.09, 95% CI: 0.01-0.63). Conclusion: In this first study of gene-environment interactions in EoE, there is preliminary evidence that certain early life factors may interact with susceptibility genotypes to modify risk of disease. In particular, interactions between CAPN14 and breastfeeding, LOC283710/ KLF13 and NICU admission, TSLP and NICU admission, and TSLP and pets merit further examination.Figure 1

A Candidate Trans-Acting Modulator of Fetal Hemoglobin Gene Expression in the Arab-Indian Haplotype of Sickle Cell Anemia

In the Arab-Indian (AI) β-globin gene (HBB) haplotype of sickle cell anemia, fetal hemoglobin (HbF) levels are higher and the disease phenotype milder than in African HBB haplotypes. To study the genetic basis of HbF regulation in the AI haplotype, whole genome sequencing (WGS) was done in 14 unrelated Saudi AI haplotype HbS homozygotes; 7 selected for low HbF (7.9±1.9%) and 7 for high HbF (18.5±3.2%). WGS was also done in 3 Indians with the AI haplotype (HbF 26.0±4.5%), 3 African Americans with the Benin haplotype selected because of the uncommonly high HbF for this haplotype (19.8±0.4%) and 1 African American homozygote with the Senegal haplotype (HbF 16.0%). Association of SNPs with HbF was tested using simple linear regression with an additive genetic model, and the Sequence Kernel Association Test using sliding windows of 5 SNPs. We found 465 SNPs that were significantly associated with HbF in a single SNP analysis (p≤10-5). Following annotation using SNPper and ENCODE RegulomeDB, PLINK was employed to remove all SNPs in high linkage disequilibrium (r2>0.8) ending with 128 SNPs for follow-up analysis. These prioritized variants were further investigated using functional evaluation tools such as SCAN, Hembase, ErythronDB and the results of WGS were followed by genome-wide SNP analysis, imputation of genotypes to 1000genomes, and targeted direct genotyping in the following cohorts with sickle cell anemia: 110 AI haplotype cases (HbF 18.0±7.0%); 71 Saudi Benin haplotype cases (HbF 11.4±4.1%); 44 Indian AI haplotype homozygotes (HbF 23.0±4.8%); 894 African Americans with diverse HBB haplotypes (HbF 5.2±5.6%). All cases were examined when the HbF levels had stabilized. None of the patients were using hydroxyurea when HbF was measured. Based on WGS, 2 SNPs, rs4527238 and rs35685045 (D'=1) in intron 9 of ANTXR1 (anthrax toxin receptor 1, 2p13.1) remained associated with HbF after correction for multiple comparisons. This result was replicated in the cohort of 110 additional AI haplotype Saudi patients but not in cohorts with other HBB haplotypes. The alternative alleles of these 2 SNPs had nearly equal frequencies in Saudis with the Saudi AI haplotype and lower frequencies in Indians, Saudi Benin haplotype and African Americans with sickle cell anemia. The T allele of rs4527238 and the C allele of rs35685045 are associated with high HbF. ANTXR1 SNPs explained 10% of the HbF variability compared with 8% for BCL11A; these genes had independent, additive effects on HbF and explained about 15% of HbF variability. RNA sequencing and gene expression microarray analysis at 3 time points during the erythroid differentiation of CD34+ and iPSCs isolated from the same subjects showed that only the long splice variant of ANTXR1 was expressed. Expression increased over time in CD34+ erythroid cells and decreased over time in iPSC-derived erythroid progenitors in a pattern similar to BCL11A expression, with increasing expression as CD34+ cells matured and HbF expression fell and declining expression as iPSCs differentiated and HbF expression increased. Immunofluorescence co-staining for ANTXR1 and HbF during the erythroid differentiation of sickle iPSCs showed that reduced expression of ANTXR1 was paralleled by increased expression of HbF. By GTEx analysis, eQTLs at rs4527238 and rs35685045 showed a trend for increased expression according to genotype with rs4527238 T/T having the lowest level of expression and T/C and C/C genotypes, higher expression levels (p= 0.08) and for rs35685045, C/C the lowest expression levels and C/T and T/T higher expression levels (p= 0.07); rs4527238 and rs35685045 had no effect on the expression of BCL11A, a repressor of HbF expression located at 2p16 (p=0.8). STRING 9.1 predicted protein interactions of ANTXR1 directly with LRP6, HDAC2, BCLX and BRAC1 with the highest level of confidence (0.95) with LRP6. The HbF phenotype in AI haplotype sickle cell anemia is unique and ANTXR1 is the first trans-acting element associated with HbF whose effects appear to be limited to the Saudi AI haplotype. ANTXR1 might have dual effects on HbF, indirectly affecting hematopoiesis via interactions with LRP6 and Wnt signaling, and directly modulating HBG expression through its effects on HDAC2. A dual effect on HbF expression has been suggested for MYB. Whether targeting ANTXR1 is a therapeutic option will require further studies. DisclosuresNo relevant conflicts of interest to declare.

Genome wide association study identifies a novel putative mammographic density locus at 1q12-q21.

Mammographic density (MD) is an intermediate phenotype for breast cancer. Previous studies have identified genetic variants associated with MD; however, much of the genetic contribution to MD is unexplained. We conducted a two-stage genome-wide association analysis among the participants in the "Determinants of Density in Mammographies in Spain" study, together with a replication analysis in women from the Australian MD Twins and Sisters Study. Our discovery set covered a total of 3,351 Caucasian women aged 45 to 68 years, recruited from Spanish breast cancer screening centres. MD was blindly assessed by a single reader using Boyd's scale. A two-stage approach was employed, including a feature selection phase exploring 575,374 SNPs in 239 pairs of women with extreme phenotypes and a verification stage for the 183 selected SNPs in the remaining sample (2,873 women). Replication was conducted in 1,786 women aged 40 to 70 years old recruited via the Australian Twin Registry, where MD were measured using Cumulus-3.0, assessing 14 SNPs with a p value <0.10 in stage 2. Finally, two genetic variants in high linkage disequilibrium with our best hit were studied using the whole Spanish sample. Evidence of association with MD was found for variant rs11205277 (OR = 0.74; 95% CI = 0.67-0.81; p = 1.33 × 10(-10) ). In replication analysis, only a marginal association between this SNP and absolute dense area was found. There were also evidence of association between MD and SNPs in high linkage disequilibrium with rs11205277, rs11205303 in gene MTMR11 (OR = 0.73; 95% CI = 0.66-0.80; p = 2.64 × 10(-11) ) and rs67807996 in gene OTUD7B (OR = 0.72; 95% CI = 0.66-0.80; p = 2.03 × 10(-11)). Our findings provide additional evidence on common genetic variations that may contribute to MD.

CD33: increased inclusion of exon 2 implicates the Ig V-set domain in Alzheimer's disease susceptibility

We previously demonstrated that the Alzheimer's disease (AD) associated risk allele, rs3865444(C), results in a higher surface density of CD33 on monocytes. Here, we find alternative splicing of exon 2 to be the primary mechanism of the genetically driven differential expression of CD33 protein. We report that the risk allele, rs3865444(C), is associated with greater cell surface expression of CD33 in both subjects of European and African-American ancestry and that there is a single haplotype influencing CD33 surface expression. A meta-analysis of the two populations narrowed the number of significant SNPs in high linkage disequilibrium (LD) (r(2) > 0.8) with rs3865444 to just five putative causal variants associated with increased protein expression. Using gene expression data from flow-sorted CD14(+)CD16(-) monocytes from 398 healthy subjects of three populations, we show that the rs3865444(C) risk allele is strongly associated with greater expression of CD33 exon 2 (pMETA = 2.36 × 10(-60)). Western blotting confirms increased protein expression of the full-length CD33 isoform containing exon 2 relative to the rs3865444(C) allele (P < 0.0001). Of the variants in strong LD with rs3865444, rs12459419, which is located in a putative SRSF2 splice site of exon 2, is the most likely candidate to mediate the altered alternative splicing of CD33's Immunoglobulin V-set domain 2 and ultimately influence AD susceptibility.

Gene-based multiple regression association testing for combined examination of common and low frequency variants in quantitative trait analysis

Multi-marker methods for genetic association analysis can be performed for common and low frequency SNPs to improve power. Regression models are an intuitive way to formulate multi-marker tests. In previous studies we evaluated regression-based multi-marker tests for common SNPs, and through identification of bins consisting of correlated SNPs, developed a multi-bin linear combination (MLC) test that is a compromise between a 1 df linear combination test and a multi-df global test. Bins of SNPs in high linkage disequilibrium (LD) are identified, and a linear combination of individual SNP statistics is constructed within each bin. Then association with the phenotype is represented by an overall statistic with df as many or few as the number of bins. In this report we evaluate multi-marker tests for SNPs that occur at low frequencies. There are many linear and quadratic multi-marker tests that are suitable for common or low frequency variant analysis. We compared the performance of the MLC tests with various linear and quadratic statistics in joint or marginal regressions. For these comparisons, we performed a simulation study of genotypes and quantitative traits for 85 genes with many low frequency SNPs based on HapMap Phase III. We compared the tests using (1) set of all SNPs in a gene, (2) set of common SNPs in a gene (MAF ≥ 5%), (3) set of low frequency SNPs (1% ≤ MAF < 5%). For different trait models based on low frequency causal SNPs, we found that combined analysis using all SNPs including common and low frequency SNPs is a good and robust choice whereas using common SNPs alone or low frequency SNP alone can lose power. MLC tests performed well in combined analysis except where two low frequency causal SNPs with opposing effects are positively correlated. Overall, across different sets of analysis, the joint regression Wald test showed consistently good performance whereas other statistics including the ones based on marginal regression had lower power for some situations.

Genetic Variation in the SLC19A1 gene and Methotrexate Toxicity in Rheumatoid Arthritis Patients

We investigated the clinical relevance of SLC19A1 genetic variability for methotrexate (MTX) toxicity in rheumatoid arthritis patients using a haplotype-based approach. Two hundred and twelve unrelated rheumatoid arthritis patients and 89 lymphoblastoid cell lines were used to investigate the effect of SLC19A1 SNPs and haplotypes on MTX adverse events and treatment discontinuation. Two putatively functional SNPs in high linkage disequilibrium, rs1051266 and rs1131596, were associated with protection (hazard ratio: 0.33; 95% CI: 0.16-0.69; adjusted p = 0.021 and hazard ratio: 0.38; 95% CI: 0.17-0.27; adjusted p = 0.021, respectively) of discontinuation of MTX treatment owing to toxicity. These SNPs were also associated with protection from infections. SLC19A1 haplotype analysis found significant associations with MTX discontinuation owing to toxicity (p = 0.025). Quantification of SLC19A1 mRNA in cell lines suggested that rs1131596 was not a major causal variant. Individual SNP and haplotype analyses suggest that rs1051266 could be a functional variant altering MTX toxicity; however, validation in independent studies is needed.