SNPs In Linkage Disequilibrium Research Articles

Abstract 4607: Integration of multiethnic fine-mapping and genomic annotation to prioritize candidate functional SNPs at prostate cancer susceptibility regions

Abstract Background: Interpretation of biological mechanisms underlying genetic risk associations for prostate cancer is complicated by the relatively large number of risk variants (n = 100) and the thousands of surrogate SNPs in linkage disequilibrium (LD). Due to the varying LD patterns and allele frequencies observed across racial/ethnic groups, studies in multiple populations have been suggested to increase power for fine-mapping by reducing the number of surrogates that are correlated with the underlying functional allele. Methods: We examined 69 risk regions using genotyping and imputation-based fine-mapping in a large multiethnic sample comprised of 17,524 prostate cancer cases and 17,519 controls from populations of European (8,600 cases and 6,946 controls), African (5,327 cases and 5,136 controls), Asian (2,563 cases and 4,391 controls) and Latino (1,034 cases and 1,046 controls) ancestry. We then combined multiethnic fine-mapping results with detailed tissue-specific functional annotation (based upon epigenetic data and genome-encoded features) and expression quantitative trait loci (eQTL) analysis to localize and prioritize risk alleles for future laboratory testing. Results: Markers at 57 regions passed a region-specific significance threshold (p-value cutoff range: 5.6×10−3-3.9×10−4) and in 32 regions we identified markers that better capture the association signals in the multiethnic sample. Novel secondary signals (p<5.0×10−6) were also detected in two regions (rs13062436/3q21 and rs17181170/3p12). Among 668 variants in the 57 regions with p-values within one order of magnitude of the most-associated marker, 200 variants (30%) in 48 regions overlapped with epigenetic or other putative functional marks. In 12 of the 57 regions, cis-eQTLs were detected with nearby genes. For 12 of the 57 regions (21%), the most-associated marker represented the strongest candidate functional variant based on annotations; the number of regions increased to 20 (35%) and 27 (47%) when examining the 2 and 3 most significantly associated variants in each region, respectively. Conclusions: We have characterized 57 prostate cancer risk regions through statistical multiethnic fine-mapping, functional annotation and cis-eQTL analyses. The results have prioritized candidate variants for downstream functional evaluation to understand biological mechanisms at prostate cancer risk regions. Citation Format: Ying Han, Dennis J. Hazelett, Brian E. Henderson, David V. Conti, Gerhard A. Coetzee, Christopher A. Haiman, on behalf of the GAME-ON/ELLIPSE Consortium. Integration of multiethnic fine-mapping and genomic annotation to prioritize candidate functional SNPs at prostate cancer susceptibility regions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4607. doi:10.1158/1538-7445.AM2015-4607

Integration of multiethnic fine-mapping and genomic annotation to prioritize candidate functional SNPs at prostate cancer susceptibility regions

Interpretation of biological mechanisms underlying genetic risk associations for prostate cancer is complicated by the relatively large number of risk variants (n = 100) and the thousands of surrogate SNPs in linkage disequilibrium. Here, we combined three distinct approaches: multiethnic fine-mapping, putative functional annotation (based upon epigenetic data and genome-encoded features), and expression quantitative trait loci (eQTL) analyses, in an attempt to reduce this complexity. We examined 67 risk regions using genotyping and imputation-based fine-mapping in populations of European (cases/controls: 8600/6946), African (cases/controls: 5327/5136), Japanese (cases/controls: 2563/4391) and Latino (cases/controls: 1034/1046) ancestry. Markers at 55 regions passed a region-specific significance threshold (P-value cutoff range: 3.9 × 10(-4)-5.6 × 10(-3)) and in 30 regions we identified markers that were more significantly associated with risk than the previously reported variants in the multiethnic sample. Novel secondary signals (P < 5.0 × 10(-6)) were also detected in two regions (rs13062436/3q21 and rs17181170/3p12). Among 666 variants in the 55 regions with P-values within one order of magnitude of the most-associated marker, 193 variants (29%) in 48 regions overlapped with epigenetic or other putative functional marks. In 11 of the 55 regions, cis-eQTLs were detected with nearby genes. For 12 of the 55 regions (22%), the most significant region-specific, prostate-cancer associated variant represented the strongest candidate functional variant based on our annotations; the number of regions increased to 20 (36%) and 27 (49%) when examining the 2 and 3 most significantly associated variants in each region, respectively. These results have prioritized subsets of candidate variants for downstream functional evaluation.

Human aging in the post-GWAS era: further insights reveal potential regulatory variants.

Human aging involves a gradual decrease in cellular integrity that contributes to multiple complex disorders such as neurodegenerative disorders, cancer, diabetes, and cardiovascular diseases. Genome-wide association studies (GWAS) play a key role in discovering genetic variations that may contribute towards disease vulnerability. However, mostly disease-associated SNPs lie within non-coding part of the genome; majority of the variants are also present in linkage disequilibrium (LD) with the genome-wide significant SNPs (GWAS lead SNPs). Overall 600 SNPs were analyzed, out of which 291 returned RegulomeDB scores of 1-6. It was observed that just 4 out of those 291 SNPs show strong evidence of regulatory effects (RegulomeDB score <3), while none of them includes any GWAS lead SNP. Nevertheless, this study demonstrates that by combining ENCODE project data along with GWAS reported information will provide important insights on the impact of a genetic variant-moving from GWAS towards understanding disease pathways. It is noteworthy that both genome-wide significant SNPs as well as the SNPs in LD must be considered for future studies; this may prove to be crucial in deciphering the potential regulatory elements involved in complex disorders and aging in particular.

Validation of the 50k Illumina goat SNP chip in the South African Angora goat

Tools for the genomic evaluation of goats have generally lagged behind those for other species. However, the recent availability of the goat SNP50 consortium bead chip has marked a positive change for this small ruminant species. Polymorphic loci can differ greatly between breeds of the same species. Exclusion of fibre-producing breeds, such as the Angora goat, during the development of this genotyping array necessitates the validation of SNPs included on the chip to allow for genomic applications that would accelerate genetic progress in mohair yield and quality. Forty eight unrelated Angora goats, displaying phenotypic variation in two important price-determining traits, namely fibre diameter and fleece weight, were genotyped with the goat SNP50 consortium bead chip. Results revealed that 46 983 SNP (88.1%) of the 53 347 called SNPs were polymorphic (MAF&gt;0.05). After quality control, 3 960 SNP were filtered from further analysis for violating Hardy-Weinberg and call-rate parameter thresholds, leaving 43 759 (82%) of the 53 347 SNPs to be validated for downstream analysis. Observed and expected heterozygosity values of 0.365 and 0.370, respectively, were obtained for polymorphic SNPs. A total of 30 357 SNPs in linkage disequilibrium (LD) were removed to obtain a set of independent markers, resulting in a final SNP density of 1 SNP/ ~226 kb. Results indicate that the goat SNP50 bead chip was informative in the Angora goats that were studied, and should be useful in examining the underlying genetic variationKeywords: Call rate, Hardy-Weinberg equilibrium, linkage disequilibrium, minor allele frequency

Validation of the association of a functional microsatellite in macrophage migration factor with hip OA

Purpose: Microsatellites are not amenable for high-throughput genotyping and have been excluded from GWAS. However, several microsatellites have been found to affect expression of nearby genes and a couple of them, in the asporin and BMP5 genes, have been associated with susceptibility to OA in multiple studies. In a recent exploration of functional microsatellites we found association of the -794 CATT microsatellite in the MIF gene with hip OA in 1782 patients compared with 1878 healthy controls of European ancestry. Replication in other patient collections was hampered by the lack of a method to obtain the genotypes without actually performing the laboratory tests. Therefore, we aimed to develop an imputation methodology for this microsatellite using the genotypes of SNPs in linkage disequilibrium and to apply it for validation. Methods: In our previous study of the MIF microsatellite, we had included 1090 samples that were also in the arcOGEN GWAS. Genotypes of the 130 SNPs in the linkage disequilibrium region surrounding the microsatellite in these samples were used as source of haplotype information. Imputation was done with Impute2 with modifications allowing estimation of the probabilities for the number of copies of each microsatellite allele and their posterior combination. Performance of this procedure was evaluated in 10 replicates of training, with 90 % of the samples, and testing of accuracy in the remaining 10 % of the samples with known microsatellite genotypes. Once concordance of imputed genotypes with tested genotypes was established, the approach was applied for the imputation of the MIF microsatellite in 5667 population controls from Wellcome Trust Case-Control Consortium (WTCCC) and in 2466 hip OA cases from arcOGEN (all of them of European UK ancestry). In addition, we have validated the functional effect of the MIF microsatellite on the plasma levels of MIF in 361 healthy control samples by ELISA (R&D Systems) from subjects that were either homozygous for the 5 repeat or the 6 repeat alleles. MIF microsatellite allele frequencies were compared with POWERMARKER and differences in plasma levels of MIF were analyzed with ANOVA using Statistica 7.0 (StatSoft). Results: There was a microsatellite allele with frequency lower than 1% that was not included in the imputation. The genotypes of the other three alleles were imputed with sufficient accuracy (91.6 %) and call rate in the reference samples (98.8). However, other microsatellites showed that the procedure will require further refinements to attain this performance for microsatellites with more alleles. Application of this procedure to the WTCCC and arcOGEN samples produced genotypes for 99.0 % of the samples. Comparison of the allelic frequencies showed significant differences between hip OA and controls in women (Table 1) following the same pattern found in our previous study, with the five repeats allele less frequent in the patients than in the controls (OR = 0.88, [95% CI] 0.79 to 0.98, P = 0.018). However, no difference was appreciated in men or between control women and men as it was in our previous study. This contrast between studies could be attributed to the use of OA-free controls in the previous study and population controls here. Analysis of MIF in plasma of healthy controls showed higher levels in the homozygous for the 5 repeat allele (3.6 ng/mL) than for the 6 repeat allele (2.7 ng/mL; P = 0.00025) following the direction previously reported by other authors and without differences between women and men. Conclusions: A new method to impute genotypes of microsatellites with three alleles based on SNP genotypes has been developed. It has shown good call rate and accuracy. This procedure allowed us to validate the association of the MIF microsatellite with hip OA in a large number of cases and controls. Concordance with our previous study was obtained in women, but not in men. Higher MIF levels were found in the subjects with the hip OA protective allele. These results should contribute to define the role of MIF, an important cytokine, in the OA pathology.Tabled 1Comparison of the allele frequencies of the MIF microsatelliteControlsTHRWomenMenWomenMenAlleleCountFreqCountFreqCountFreqCountFreq5140225.4143925.367123.147424.36345362.5357462.7185363.7123763.5766912.168512.038613.323712.2Total5524569829101948 Open table in a new tab

Enrichment pathway analysis. The inflammatory genetic background in Bipolar Disorder

IntroductionThe pathophysiology of Bipolar Disorder (BD) is yet to be fully characterized. In the last years attention was focused on neurodevelopment or neurodegenerative events. In this context, hyper- and hypo- activation of inflammatory cascades may play a role in modulating the architecture and function of neuronal tissues. In the present paper we tested the enrichment of molecular pathways related to inflammatory cascades (IL-1, IL-2, IL-6, IL-8, TNF and INF) testing whether genes related to these systems hold more variations associated with the risk for BD than expected. Methods~7000 bipolar patients and controls with genome-wide data available from NIMH dataset were analyzed. SNPs were imputed, checked for quality control, pruned and tested for association (0.01<p). Fisher test was conducted to test the enrichment within the pathways and the association was permutated (105 times) to limit false positive findings. ResultsAs a result, IL-6, IL-8 and IFN related pathways held twice to thrice the number of expected variants associated with BD. These tests resisted the permutation analysis. LimitationsThe restricted number of inflammatory components included in the analysis and the lack of functional consequences for some of the SNPs analyzed may be biased; however, these choices helped the authors to lighten the statistical computational load for the analyses and at the same time included possibly hidden SNPs in linkage disequilibrium with the analyzed variations. ConclusionsWe bring evidence that the inflammatory cascades may be genetically varied in Bipolar patients. This genetic background may explain part of the pathophysiology of the disorder.

Validation of the 50k Illumina goat SNP chip in the South African Angora goat : short communication

Tools for the genomic evaluation of goats have generally lagged behind those for other species. However, the recent availability of the goat SNP50 consortium bead chip has marked a positive change for this small ruminant species. Polymorphic loci can differ greatly between breeds of the same species. Exclusion of fibre-producing breeds, such as the Angora goat, during the development of this genotyping array necessitates the validation of SNPs included on the chip to allow for genomic applications that would accelerate genetic progress in mohair yield and quality. Forty eight unrelated Angora goats, displaying phenotypic variation in two important price-determining traits, namely fibre diameter and fleece weight, were genotyped with the goat SNP50 consortium bead chip. Results revealed that 46 983 SNP (88.1%) of the 53 347 called SNPs were polymorphic (MAF>0.05). After quality control, 3 960 SNP were filtered from further analysis for violating Hardy-Weinberg and call-rate parameter thresholds, leaving 43 759 (82%) of the 53 347 SNPs to be validated for downstream analysis. Observed and expected heterozygosity values of 0.365 and 0.370, respectively, were obtained for polymorphic SNPs. A total of 30 357 SNPs in linkage disequilibrium (LD) were removed to obtain a set of independent markers, resulting in a final SNP density of 1 SNP/ ~226 kb. Results indicate that the goat SNP50 bead chip was informative in the Angora goats that were studied, and should be useful in examining the underlying genetic variation.

Characterization of population-based variation and putative functional elements for the multiple-cancer susceptibility loci at 5p15.33

Background: TERT encodes the telomerase reverse transcriptase, which is responsible for maintaining telomere ends by addition of (TTAGGG) n nucleotide repeats at the telomere. Recent genome-wide association studies have found common genetic variants at the TERT-CLPTM1L locus (5p15.33) associated with an increased risk of several cancers. Results: Data were acquired for 1627 variants in 1092 unrelated individuals from 14 populations within the 1000 Genomes Project. We assessed the population genetics of the 5p15.33 region, including recombination hotspots, diversity, heterozygosity, differentiation among populations, and potential functional impacts. There were significantly lower polymorphism rates, divergence, and heterozygosity for the coding variants, particularly for non-synonymous sites, compared with non-coding and silent changes. Many of the cancer-associated SNPs had differing genotype frequencies among ancestral groups and were associated with potential regulatory changes. Conclusions: Surrogate SNPs in linkage disequilibrium with the majority of cancer-associated SNPs were functional variants with a likely role in regulation of TERT and/or CLPTM1L. Our findings highlight several SNPs that future studies should prioritize for evaluation of functional consequences.

Characterization of population-based variation and putative functional elements for the multiple-cancer susceptibility loci at 5p15.33.

TERT encodes the telomerase reverse transcriptase, which is responsible for maintaining telomere ends by addition of (TTAGGG) n nucleotide repeats at the telomere. Recent genome-wide association studies have found common genetic variants at the TERT-CLPTM1L locus (5p15.33) associated with an increased risk of several cancers. Data were acquired for 1627 variants in 1092 unrelated individuals from 14 populations within the 1000 Genomes Project. We assessed the population genetics of the 5p15.33 region, including recombination hotspots, diversity, heterozygosity, differentiation among populations, and potential functional impacts. There were significantly lower polymorphism rates, divergence, and heterozygosity for the coding variants, particularly for non-synonymous sites, compared with non-coding and silent changes. Many of the cancer-associated SNPs had differing genotype frequencies among ancestral groups and were associated with potential regulatory changes. Surrogate SNPs in linkage disequilibrium with the majority of cancer-associated SNPs were functional variants with a likely role in regulation of TERT and/or CLPTM1L. Our findings highlight several SNPs that future studies should prioritize for evaluation of functional consequences.

Combinatorial effects of multiple enhancer variants in linkage disequilibrium dictate levels of gene expression to confer susceptibility to common traits

DNA variants (SNPs) that predispose to common traits often localize within noncoding regulatory elements such as enhancers. Moreover, loci identified by genome-wide association studies (GWAS) often contain multiple SNPs in linkage disequilibrium (LD), any of which may be causal. Thus, determining the effect of these multiple variant SNPs on target transcript levels has been a major challenge. Here, we provide evidence that for six common autoimmune disorders (rheumatoid arthritis, Crohn's disease, celiac disease, multiple sclerosis, lupus, and ulcerative colitis), the GWAS association arises from multiple polymorphisms in LD that map to clusters of enhancer elements active in the same cell type. This finding suggests a “multiple enhancer variant” hypothesis for common traits, where several variants in LD impact multiple enhancers and cooperatively affect gene expression. Using a novel method to delineate enhancer–gene interactions, we show that multiple enhancer variants within a given locus typically target the same gene. Using available data from HapMap and B lymphoblasts as a model system, we provide evidence at numerous loci that multiple enhancer variants cooperatively contribute to altered expression of their gene targets. The effects on target transcript levels tend to be modest and can be either gain- or loss-of-function. Additionally, the genes associated with multiple enhancer variants encode proteins that are often functionally related and enriched in common pathways. Overall, the multiple enhancer variant hypothesis offers a new paradigm by which noncoding variants can confer susceptibility to common traits.

THU0002 Comparison of Pathways Implicated in Anti-Citrillunated Peptide Antibody Positive and Negative Rheumatoid Arthritis Patients

BackgroundRheumatoid Arthritis (RA) is a chronic autoimmune disease characterised by inflammation and destruction of synovial joints affecting up to 1% of the adult population. GWAS have been successful in identifying...

Confirmation and fine‐mapping of clinical mastitis and somatic cell score QTL in Nordic Holstein cattle

A genome-wide association study of 2098 progeny-tested Nordic Holstein bulls genotyped for 36387 SNPs on 29 autosomes was conducted to confirm and fine-map quantitative trait loci (QTL) for mastitis traits identified earlier using linkage analysis with sparse microsatellite markers in the same population. We used linear mixed model analysis where a polygenic genetic effect was fitted as a random effect and single SNPs were successively included as fixed effects in the model. We detected 143 SNP-by-trait significant associations (P<0.0001) on 20 chromosomes affecting mastitis-related traits. Among them, 21 SNP-by-trait combinations exceeded the genome-wide significant threshold. For 12 chromosomes, both the present association study and the previous linkage study detected QTL, and of these, six were in the same chromosomal locations. Strong associations of SNPs with mastitis traits were observed on bovine autosomes 6, 13, 14 and 20. Possible candidate genes for these QTL were identified. Identification of SNPs in linkage disequilibrium with QTL will enable marker-based selection for mastitis resistance. The candidate genes identified should be further studied to detect candidate polymorphisms underlying these QTL.

Abstract TMP30: Genetic Variants on 9p21.3 are Associated with Brain Arteriovenous Malformations with Associated Arterial Aneurysms

Background: The 9p21.3 locus is associated with risk of coronary artery disease and intracranial and abdominal aortic aneurysms (rs10757278). We investigated whether previously reported 9p21.3 SNPs are associated with risk of brain arteriovenous malformations (BAVM), which often have accompanying flow-related arterial aneurysms. Methods: Genotypes for 6 SNPs, including rs10757278, were imputed using 1000 Genomes Phase 1 CEU data (R2&gt;0.9). We tested for association using logistic regression of imputed dosages adjusting for age, sex and top 3 components of ancestry in 338 BAVM cases vs. 504 controls. Of these, 205 cases had aneurysm data available for subgroup analysis: (a) 74 BAVM+(with) aneurysm cases vs. 504 controls, and (b) 131 BAVM-(without) aneurysm cases vs. 504 controls. Results: rs10757278-G was marginally associated with increased risk of BAVM (OR=1.23, 95% CI=0.99-1.53, P=0.064). Compared to controls, the association was stronger in BAVM+aneurysm (OR=1.52, 95% CI=1.03-2.22, P=0.032) than in BAVM-aneurysm cases (OR=0.98, 95% CI=0.72-1.34, P=0.91). Similar effects were observed for other SNPs in linkage disequilibrium (r2&gt;0.8) with rs10757278 (Table). Conclusion: Common 9p21.3 variants showed similar effect sizes as previously reported for aneurysmal disease. The SNP association with BAVM appears to be explained by known association with aneurysms, suggesting that BAVM associated aneurysms share similar vascular pathology mechanisms.

Effects of endothelial nitric oxide synthase tagSNPs haplotypes on nitrite levels in black subjects

Haplotypes formed by clinically relevant polymorphisms in the endothelial nitric oxide synthase (eNOS) gene have been associated with variations in endogenous nitric oxide (NO) formation in white and black subjects. We examined whether further genetic variation and haplotypes of the eNOS gene, represented by the rs3918188, rs743506 and rs7830 tagSNPs (polymorphisms that represent the information of neighboring SNPs in linkage disequilibrium) affect endogenous NO formation in 181 healthy black subjects. We measured whole blood nitrite concentration as a marker of endogenous NO formation for each subject. We found that the heterozygotes CA for the tagSNP rs7830 (P=0.0278, OR=0.4839, CI 95% 0.2567–0.9121), as well as the haplotype “C–G–A” (P=0.0068), were more common in subjects with low circulating whole blood nitrite concentrations compared with subjects with high circulating whole blood nitrite concentrations, although the genotype finding is not significant after correction for multiple comparisons. These findings suggest that these tagSNPs of the eNOS gene and haplotypes are associated with low levels of nitric oxide production in blacks, and could be used as a marker of cardiovascular risk.

Genome‐wide association study of N370S homozygous Gaucher disease reveals the candidacy of CLN8 gene as a genetic modifier contributing to extreme phenotypic variation

Mutations in GBA1 gene result in defective acid β-glucosidase and the complex phenotype of Gaucher disease (GD) related to the accumulation of glucosylceramide-laden macrophages. The phenotype is highly variable even among patients harboring identical GBA1 mutations. We hypothesize that modifier gene(s) underlie phenotypic diversity in GD and performed a GWAS study in Ashkenazi Jewish patients with type 1 GD (GD1), homozygous for N370S mutation. Patients were assigned to mild, moderate, or severe disease categories using composite disease severity scoring systems. Whole-genome genotyping for >500,000 SNPs was performed to search for association signals using OQLS algorithm in 139 eligible patients. Several SNPs in linkage disequilibrium within the CLN8 gene locus were associated with the GD1 severity: SNP rs11986414 was associated with GD1 severity at P value 1.26 × 10(-6) . Compared to mild disease, risk allele A at rs11986414 conferred an odds ratio of 3.72 for moderate/severe disease. Loss of function mutations in CLN8 causes neuronal ceroid-lipofuscinosis, but our results indicate that its increased expression may protect against severe GD1. In cultured skin fibroblasts, the relative expression of CLN8 was higher in mild GD compared to severely affected patients, in whom CLN8 risk alleles were overrepresented. In an in vitro cell model of GD, CLN8 expression was increased, which was further enhanced in the presence of bioactive substrate, glucosylsphingosine. Taken together, CLN8 is a candidate modifier gene for GD1 that may function as a protective sphingolipid sensor and/or in glycosphingolipid trafficking. Future studies should explore the role of CLN8 in pathophysiology of GD.

Lack of Association Between ABCC2 Gene Variants and Treatment Response in Epilepsy

The aim of this study was to replicate a previously reported association between drug resistance in epilepsy patients and the 24C>T variant of the ABCC2 gene that codes for the drug efflux transporter MRP2. We genotyped 381 Caucasian epileptic patients (337 drug resistant and 44 drug responsive) and 247 healthy controls for the ABCC2 gene -24C>T polymorphism (rs717620) and two other nearby SNPs in linkage disequilibrium (1249G>A and 3972C>T). Genotype, allele and three-SNP-haplotype frequencies were compared between groups. Patients were further stratified into four groups according to their degree of drug resistance (as measured by seizure frequency under medication) to perform regression analysis against genotypes and haplotpyes. We detected no significant differences in the distribution of any of the tested alleles, genotypes or haplotypes between the investigated groups. Neither was there an association between genotypes or haplotypes and degree of drug resistance. This study was adequately powered to detect genotype relative risks of above two. Although adequately powered to detect the previously reported effect size and although our definition of drug resistance, following the International League Against Epilepsy guidelines, was slightly stricter than in the original study, we failed to confirm an association between the 24C>T variant in the ABCC2 gene and drug resistance in epilepsy.

Atrial Fibrillation-Associated Locus 4Q25 Has the Cis-Regulatory Function toward the Neighboring Genes

Genome-wide association study (GWAS) of atrial fibrillation (AF) in Japanese population revealed that SNPs in 4q25 were strongly associated with AF. This region includes no protein-coding genes, but PITX2 exists in the neighborhood. Pitx2 is suggested for myocardial sleeve development in pulmonary vein and suppression of sinus node-like activities in left atrium, make us to assume that this gene-less regionmight include cis-regulatory elements toward neighboring genes such as PITX2. We tried to identify functional SNPs and to investigate pathogenesis of AF. We identified 155 variants including novel SNPs through re-sequencing the whole 4q25 region, selected 22 tag-SNPs, and analyzed association with AF. Among the 22 tag-SNPs, rs2200733 was the most strongly associated with AF. We regarded rs2200733 and other 22 SNPs in linkage disequilibrium as candidate functional SNPs. ChIPexperiments found that H3K4me1 and H3K27me3 bound to two specific SNPs, respectively. We performed reporter assay with reporter vectors, in which regions including each SNP were inserted with minimal promoter. We foundthesetwo regionselevated or suppressed transcriptional activities, respectively, and SNPs affected enhancement or suppression of transcriptional activities. These results demonstrate that cis-regulatory elements exist in 4q25 region and we assume that they control the expression of neighboring genes such as PITX2.

Genetic Determinants of Plasma Von Willebrand Factor Antigen Levels: A Target Gene SNP and Haplotype Analysis of the ARIC Cohort

AbstractAbstract 4310 Background and Objectives:Von Willebrand factor (VWF) is an essential component of hemostasis. It is known that multimer size and the amount of circulating VWF impact its hemostatic function. Genetic factors play a major role in regulating VWF synthesis and clearance and are reported to contribute to 66% of the variation in plasma VWF antigen level. We have analyzed 78 SNPs distributed throughout the VWF gene and haplotypes constructed from those SNPs for an association with VWF antigen level in 7,856 subjects of European descent in the Atherosclerosis Risk in Communities cohort. Methods and Results: Blood was drawn after an 8 hour fasting period and VWF antigen level was determined by a commercial ELISA kit. All subjects underwent analysis for 78 SNPs available from Affymetrix 6.0 chip in the region encompassing the VWF gene. We used the fastPHASE 1.2 program to resolve haplotypes from the unphased SNP genotype data. Using Haploview we determined SNPs in strong linkage disequilibrium (LD), their co-segregation rates and underlying haplotypes. Linear models were used to evaluate the association of actual or log VWF antigen levels with each SNP and haplotypes derived from those SNPs. Eighteen (16 are intronic) of the 78 SNPs were found to significantly associate with levels of VWF antigen (Table 1). All are clustered in a 50 kb region of the VWF gene in spite of the fact that the 78 SNPs studied are distributed throughout the VWF gene without apparent clusters. All 78 SNPs are co-segregated in 9 LD haplotype blocks, but a majority of positive SNPs (88.9%) are located in Block 5 and 6, which significantly correlate with VWF antigen level. The O blood type contributes to ∼ 15% of variation in VWF antigen levels. The association for SNPs and Haplotypes grows stronger after ABO effects are removed. Among non-genomic factors, age and BMI are the most significant factors and contribute to 4.8% and 1.4% of variation in VWF antigen level. Conclusions: We have found a strongly positive association between VWF level and SNPs and haplotypes from a strikingly concentrated region of the VWF gene. This region encodes the D2, D' and D3 domains of VWF, including the propeptide and multimerization and Factor VIII binding sites. The physiological significance of these clustered SNPs on VWF synthesis or clearance remains to be further investigated. Our data suggest that this region plays an important role in regulating VWF antigen level and support the idea that combinations of SNPs in LD can provide additive affects. Disclosures:No relevant conflicts of interest to declare.

Investigation of CLL-Susceptibility Loci with Monoclonal B-Cell Lymphocytosis (MBL) Risk and Confirmation of Recently Reported CLL-Susceptibility Loci

AbstractAbstract 2443 Background:There is strong and consistent evidence that a genetic component contributes to the etiology of chronic lymphocytic leukemia (CLL). A recent genome-wide association (GWA) study of CLL identified genetic variants located on chromosomes 2q13, 2q37.1, 6p25, 11q24, 15q23, and 19q13 that increased the risk of CLL within a European population. We replicated 5 of these 6 loci in an independent sample of CLL cases and controls from the United States. We now investigate whether these loci also influences MBL, a reported precursor condition of CLL. In addition, a follow-up analysis of the initial GWA study identified four more CLL-susceptibility loci on 2q37.3, 8q24.21, 15q21.3, and 16q24.1. Herein, we also evaluate the association of these four loci with risk of CLL. Methods:Peripheral blood samples were obtained from three ongoing studies: the Genetic Epidemiology CLL (GEC) Consortium, the Mayo Clinic non-Hodgkin lymphoma (NHL)/ CLL study, and the Mayo Clinic Biobank. We implemented rigorous genotyping quality-control measures, and successfully genotyped a total of 407 CLL patients, 965 controls, and 60 MBLs from these studies. Within each locus, the previously reported single nucleotide variants (SNPs) or variants in high linkage disequilibrium (LD) with the previously reported SNPs were evaluated with risk of MBL or CLL. Tests for association was done using the Cochran-Armitage trend test, and unconditional logistic regression was used to estimate odds ratios (ORs) and corresponding 95% confidence intervals (CIs) for CLL or MBL risk Results:In our evaluation of the six initially reported CLL-susceptibility loci (2q13, 2q37.1, 6p25, 11q24, 15q23, and 19q13) with MBL risk, we found three of the six had suggestive associations (p-value < 0.20) with ORs comparable to and in the same direction as those observed from CLL risk. Our strongest finding was with rs13397985 at locus 2q37.1 (OR= 1.56; 95% CI: 1.05, 2.31; p-trend = 0.041), followed by rs17483466 at locus 2q13 (OR= 1.49; 95% CI: 0.99, 2.24; p-trend = 0.074). As expected given our previously reported findings with CLL risk, the association between rs11083846 on chromosome 19q13 and MBL risk was not significant (p-trend = 0.70). Of the four recently reported CLL-susceptibility loci SNPs located on 2q37.3, 8q24.21, 15q21.3, and 16q24.1, we found all to be associated with CLL risk but one. Specifically, the strongest association was seen for locus 8q24.21 (best tagged SNP rs1021955; OR = 1.37; 95% CI: 1.10, 1.70; p-trend = 0.005), followed by locus 16q24.1 (best tagged SNP rs305065; OR= 0.77; 95% CI: 0.61, 0.97; p-trend = 0.024). However, we found no associations for locus 15q21.3 for the previously reported SNP nor for any SNPs in LD with the previously reported SNP. Conclusions:Our MBL results provide additional robust genetic evidence that MBL is a precursor to CLL and that it shares similar underlying genetic predisposition. Also our results confirm three of the four recently reported CLL-susceptibility loci and further support the role of a genetic basis in the etiology of CLL. More research is needed to elucidate the potential manner in which these genetic loci function in CLL or MBL. Disclosures:No relevant conflicts of interest to declare.

Association of SRD5A2 Variants and Serum Androstane-3α,17β-Diol Glucuronide Concentration in Chinese Elderly Men

Results of recent studies have demonstrated that genetic variants of the enzyme steroid 5α reductase type II (SRD5A2) are associated with serum concentrations of major androgen metabolites such as conjugates of androstane-3α,17β-diol-glucuronide (3α-diol-G). However, this association was not consistently found among different ethnic groups. Thus, we aimed to determine whether the association with SRD5A2 genetic variations exists in a cohort of healthy Chinese elderly men, by examining 2 metabolite conjugates: androstane-3α,l7β-diol-3-glucuronide (3α-diol-3G) and androstane-3α,17β-diol-17-glucuronide (3α-diol-17G). We used GC-MS and LC-MS to measure serum sex steroid concentrations, including testosterone and dihydrotestosterone, and 3α-diol-3G and 3α-diol-17G in 1182 Chinese elderly men age 65 and older. Genotyping of the 3 SRD5A2 tagSNPs [rs3731586, rs12470143, and rs523349 (V89L)] was performed by using melting-temperature-shift allele-specific PCR. The well-described SRD5A2 missense variant rs523349 (V89L) was modestly associated with the 3α-diol-17G concentration (P = 0.040). On the other hand, SNP rs12470143 was found to be significantly correlated with 3α-diol-3G concentration (P = 0.021). Results of haplotype analysis suggested that the presence of an A-C-G haplotype leads to an increased 3α-diol-3G concentration, a finding consistent with results of single SNP analysis. The genetic variation of SRD5A2 is associated with circulating 3α-diol-3G and 3α-diol-17G concentrations in Chinese elderly men. In addition, we showed that SRD5A2 haplotypic association, rather than a single SNP alone, might be a better predictor of the 3α-diol-G concentration. Thus, the effect of either the haplotype itself or of other ungenotyped SNPs in linkage disequilibrium with the haplotype is responsible for the interindividual variation of 3α-diol-G.