Claudia Lamina

Meta-Analysis of 28,141 Individuals Identifies Common Variants within Five New Loci That Influence Uric Acid Concentrations

Elevated serum uric acid levels cause gout and are a risk factor for cardiovascular disease and diabetes. To investigate the polygenetic basis of serum uric acid levels, we conducted a meta-analysis of genome-wide association scans from 14 studies totalling 28,141 participants of European descent, resulting in identification of 954 SNPs distributed across nine loci that exceeded the threshold of genome-wide significance, five of which are novel. Overall, the common variants associated with serum uric acid levels fall in the following nine regions: SLC2A9 (p = 5.2×10−201), ABCG2 (p = 3.1×10−26), SLC17A1 (p = 3.0×10−14), SLC22A11 (p = 6.7×10−14), SLC22A12 (p = 2.0×10−9), SLC16A9 (p = 1.1×10−8), GCKR (p = 1.4×10−9), LRRC16A (p = 8.5×10−9), and near PDZK1 (p = 2.7×10−9). Identified variants were analyzed for gender differences. We found that the minor allele for rs734553 in SLC2A9 has greater influence in lowering uric acid levels in women and the minor allele of rs2231142 in ABCG2 elevates uric acid levels more strongly in men compared to women. To further characterize the identified variants, we analyzed their association with a panel of metabolites. rs12356193 within SLC16A9 was associated with DL-carnitine (p = 4.0×10−26) and propionyl-L-carnitine (p = 5.0×10−8) concentrations, which in turn were associated with serum UA levels (p = 1.4×10−57 and p = 8.1×10−54, respectively), forming a triangle between SNP, metabolites, and UA levels. Taken together, these associations highlight additional pathways that are important in the regulation of serum uric acid levels and point toward novel potential targets for pharmacological intervention to prevent or treat hyperuricemia. In addition, these findings strongly support the hypothesis that transport proteins are key in regulating serum uric acid levels.

SNP-Based Analysis of Genetic Substructure in the German Population

Objective: To evaluate the relevance and necessity to account for the effects of population substructure on association studies under a case-control design in central Europe, we analysed three samples drawn from different geographic areas of Germany. Two of the three samples, POPGEN (n = 720) and SHIP (n = 709), are from north and north-east Germany, respectively, and one sample, KORA (n = 730), is from southern Germany. Methods: Population genetic differentiation was measured by classical F-statistics for different marker sets, either consisting of genome-wide selected coding SNPs located in functional genes, or consisting of selectively neutral SNPs from ‘genomic deserts’. Quantitative estimates of the degree of stratification were performed comparing the genomic control approach [Devlin B, Roeder K: Biometrics 1999;55:997–1004], structured association [Pritchard JK, Stephens M, Donnelly P: Genetics 2000;155:945–959] and sophisticated methods like random forests [Breiman L: Machine Learning 2001;45:5–32]. Results: F-statistics showed that there exists a low genetic differentiation between the samples along a north-south gradient within Germany (FST(KORA/POPGEN): 1.7 · 10–4; FST(KORA/SHIP): 5.4 · 10–4; FST(POPGEN/SHIP): –1.3 · 10–5). Conclusion: Although the FST -values are very small, indicating a minor degree of population structure, and are too low to be detectable from methods without using prior information of subpopulation membership, such as STRUCTURE [Pritchard JK, Stephens M, Donnelly P: Genetics 2000;155:945–959], they may be a possible source for confounding due to population stratification.

Lifelong reduction of LDL-cholesterol related to a common variant in the LDL-receptor gene decreases the risk of coronary artery disease--a Mendelian Randomisation study.

Background Rare mutations of the low-density lipoprotein receptor gene (LDLR) cause familial hypercholesterolemia, which increases the risk for coronary artery disease (CAD). Less is known about the implications of common genetic variation in the LDLR gene regarding the variability of cholesterol levels and risk of CAD. Methods Imputed genotype data at the LDLR locus on 1 644 individuals of a population-based sample were explored for association with LDL-C level. Replication of association with LDL-C level was sought for the most significant single nucleotide polymorphism (SNP) within the LDLR gene in three European samples comprising 6 642 adults and 533 children. Association of this SNP with CAD was examined in six case-control studies involving more than 15 000 individuals. Findings Each copy of the minor T allele of SNP rs2228671 within LDLR (frequency 11%) was related to a decrease of LDL-C levels by 0.19 mmol/L (95% confidence interval (CI) [0.13–0.24] mmol/L, p = 1.5×10−10). This association with LDL-C was uniformly found in children, men, and women of all samples studied. In parallel, the T allele of rs2228671 was associated with a significantly lower risk of CAD (Odds Ratio per copy of the T allele: 0.82, 95% CI [0.76–0.89], p = 2.1×10−7). Adjustment for LDL-C levels by logistic regression or Mendelian Randomisation models abolished the significant association between rs2228671 with CAD completely, indicating a functional link between the genetic variant at the LDLR gene locus, change in LDL-C and risk of CAD. Conclusion A common variant at the LDLR gene locus affects LDL-C levels and, thereby, the risk for CAD.

Genome-Wide Association Analysis of High-Density Lipoprotein Cholesterol in the Population-Based KORA Study Sheds New Light on Intergenic Regions

Background—High-density lipoprotein cholesterol (HDLC) is a strong risk factor for atherosclerosis and is assumed to be under considerable genetic control. We aimed to identify gene regions that influence HDLC levels by a genome-wide association analysis in the population-based KORA (Cooperative Health Research in the Region of Augsburg) study. Methods and Results—In KORA S3/F3 (n=1643), we analyzed 377 865 quality-checked single-nucleotide polymorphisms (SNPs; 500K, Affymetrix, Santa Clara, Calif), complemented by the publicly available genome-wide association results from the Diabetes Genetics Initiative (n=2631) and by replication data from KORA S4 (n=4037) and the Copenhagen City Heart Study (n=9205). Among the 13 SNPs selected from the KORA S3/F3 500K probability value list, 3 showed consistent associations in subsequent replications: 1 SNP 10 kb upstream of CETP (pooled probability value=8.5×10−27), 1 SNP approximately 40 kb downstream of LIPG (probability value=4.67×10−10), both independent of previously reported SNPs, and 1 from an already reported region of LPL (probability value=2.82×10−11). Bioinformatical analyses indicate a potential functional relevance of the respective SNPs. Conclusions—The present genome-wide association study identified 2 interesting HDLC-relevant regions upstream of CETP and downstream of LIPG. This draws attention to the importance of long-range effects of intergenic regions, which have been underestimated so far, and may impact future candidate-gene–association studies toward extending the region analyzed. Furthermore, the present study reinforced CETP and LPL as HDLC genes and thereby underscores the power of this type of genome-wide association approach to pinpoint associations of common polymorphisms with effects explaining as little as 0.5% of the HDLC variance in the general population.

Mendelian Randomization Studies Do Not Support a Causal Role for Reduced Circulating Adiponectin Levels in Insulin Resistance and Type 2 Diabetes

Adiponectin is strongly inversely associated with insulin resistance and type 2 diabetes, but its causal role remains controversial. We used a Mendelian randomization approach to test the hypothesis that adiponectin causally influences insulin resistance and type 2 diabetes. We used genetic variants at the ADIPOQ gene as instruments to calculate a regression slope between adiponectin levels and metabolic traits (up to 31,000 individuals) and a combination of instrumental variables and summary statistics–based genetic risk scores to test the associations with gold-standard measures of insulin sensitivity (2,969 individuals) and type 2 diabetes (15,960 case subjects and 64,731 control subjects). In conventional regression analyses, a 1-SD decrease in adiponectin levels was correlated with a 0.31-SD (95% CI 0.26–0.35) increase in fasting insulin, a 0.34-SD (0.30–0.38) decrease in insulin sensitivity, and a type 2 diabetes odds ratio (OR) of 1.75 (1.47–2.13). The instrumental variable analysis revealed no evidence of a causal association between genetically lower circulating adiponectin and higher fasting insulin (0.02 SD; 95% CI −0.07 to 0.11; N = 29,771), nominal evidence of a causal relationship with lower insulin sensitivity (−0.20 SD; 95% CI −0.38 to −0.02; N = 1,860), and no evidence of a relationship with type 2 diabetes (OR 0.94; 95% CI 0.75–1.19; N = 2,777 case subjects and 13,011 control subjects). Using the ADIPOQ summary statistics genetic risk scores, we found no evidence of an association between adiponectin-lowering alleles and insulin sensitivity (effect per weighted adiponectin-lowering allele: −0.03 SD; 95% CI −0.07 to 0.01; N = 2,969) or type 2 diabetes (OR per weighted adiponectin-lowering allele: 0.99; 95% CI 0.95–1.04; 15,960 case subjects vs. 64,731 control subjects). These results do not provide any consistent evidence that interventions aimed at increasing adiponectin levels will improve insulin sensitivity or risk of type 2 diabetes.

Functional Characterization of Promoter Variants of the Adiponectin Gene Complemented by Epidemiological Data

OBJECTIVE Adiponectin (APM1, ACDC) is an adipocyte-derived protein with downregulated expression in obesity and insulin-resistant states. Several potentially regulatory single nucleotide polymorphisms (SNPs) within the APM1 gene promoter region have been associated with circulating adiponectin levels. None of them have been functionally characterized in adiponectin-expressing cells. Hence, we investigated three SNPs (rs16861194, rs17300539, and rs266729) for their influence on adiponectin promoter activity and their association with circulating adiponectin levels. RESEARCH DESIGN AND METHODS Basal and rosiglitazone-induced promoter activity of different SNP combinations (haplotypes) was analyzed in 3T3-L1 adipocytes using luciferase reporter gene assays and DNA binding studies comparing all possible APM1 haplotypes. This functional approach was complemented with analysis of epidemiological population-based data of 1,692 participants of the MONICA/KORA S123 cohort and 696 participants from the KORA S4 cohort for SNP and haplotype association with circulating adiponectin levels. RESULTS Major to minor allele replacements of the three SNPs revealed significant effects on promoter activity in luciferase assays. Particularly, a minor variant in rs16861194 resulted in reduced basal and rosiglitazone-induced promoter activity and hypoadiponectinemia in the epidemiological datasets. The haplotype with the minor allele in all three SNPs showed a complete loss of promoter activity, and no subject carried this haplotype in either of the epidemiological samples (combined P value for statistically significant difference from a random sample was 0.006). CONCLUSIONS Our results clearly demonstrate that promoter variants associated with hypoadiponectinemia in humans substantially affect adiponectin promoter activity in adipocytes. Our combination of functional experiments with epidemiological data overcomes the drawback of each approach alone.

A common variant in the adiponutrin gene influences liver enzyme values

Background Two recent genome-wide association studies identified the liver expressed transmembrane protein adiponutrin to be associated with liver related phenotypes such as non-alcoholic fatty liver disease and liver function enzymes. These associations were not uniformly reported for various ethnicities. The aim of this study was to investigate a common non-synonymous variant within adiponutrin (rs738409, exon 3) with parameters of liver function in three independent West Eurasian study populations including a total of 4290 participants. Methods The study was performed in (1) the population based Bruneck Study (n=783), (2) the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk Study from Austria based on a healthy working population (n=1705), and the Utah Obesity Case–Control Study including a group of 1019 severely obese individuals (average body mass index 46.0 kg/m2) and 783 controls from the same geographical region of Utah. Liver enzymes measured were alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl transferase (GGT). Results A strong recessive association of this polymorphism was found with age and gender adjusted ALT and AST concentrations: being homozygous for the minor allele resulted in a highly significant increase of ALT concentration of 3.53 U/l (p=1.86×10−9) and of AST concentration of 2.07 U/l (p=9.58×10−6), respectively. The associations were consistently found in all three study populations. Conclusion The highly significant associations of this transversion polymorphism within the adiponutrin gene with increased ALT and AST concentrations support a role for adiponutrin as a susceptibility gene for hepatic dysfunction.

Risk gene variants for nicotine dependence in the CHRNA5–CHRNA3–CHRNB4 cluster are associated with cognitive performance†‡§¶

Recent studies strongly support an association of the nicotinic acetylcholine receptor gene cluster CHRNA5–CHRNA3–CHRNB4 with nicotine dependence (ND). However, the precise genotype–phenotype relationship is still unknown. Clinical and epidemiological data on smoking behavior raise the possibility that the relevant gene variants may indirectly contribute to the development of ND by affecting cognitive performance in some smokers who consume nicotine for reasons of “cognition enhancement.” Here, we tested seven single nucleotide polymorphisms (SNPs) rs684513, rs637137, rs16969968, rs578776, rs1051730, rs3743078, rs3813567 from the CHRNA5–CHRNA3–CHRNB4 gene cluster for association with ND, measures of cognitive performance and gene expression. As expected, we found all SNPs being associated with ND in three independent cohorts (KORA, NCOOP, ESTHER) comprising 5,561 individuals. In an overlapping sample of 2,186 subjects we found three SNPs (rs16969968, rs1051730, rs3743078) being associated with cognitive domains from the Wechsler‐Adult‐Intelligence Scale (WAIS‐R)—most notably in the performance subtest “object assembly” and the verbal subtest “similarities.” In a refined analysis of a subsample of 485 subjects, two of these three SNPs (rs16969968, rs1051730) were associated with n‐back task performance/Continuous Performance Test. Furthermore, two CHRNA5 risk alleles (rs684513, rs637137) were associated with CHRNA5 mRNA expression levels in whole blood in a subgroup of 190 subjects. We here report for the first time an association of CHRNA5–CHRNA3–CHRNB4 gene variants with cognition possibly mediating in part risk for developing ND. The observed phenotype–genotype associations may depend on altered levels of gene expression.

Association of nicotinic acetylcholine receptor subunit α4 polymorphisms with nicotine dependence in 5500 Germans

Polymorphisms in the CHRNA4 gene coding the nicotinic acetylcholine receptor subunit α4 have recently been suggested to play a role in the determination of smoking-related phenotypes. To examine this hypothesis, we conducted a genetic association study in three large samples from the German general population (N1=1412; N2=1855; N3=2294). Five single-nucleotide polymorphisms in CHRNA4 were genotyped in 5561 participants, including 2707 heavily smoking cases (regularly smoking at least 20 cigarettes per day) and 2399 never-smoking controls (⩽100 cigarettes over lifetime). We examined associations of the polymorphisms with smoking case–control status and with the extent of nicotine dependence as measured by the Fagerstrom test of nicotine dependence (FTND) score (N=1030). The most significant association was observed between rs2236196 and FTND (P=0.0023), whereas the closely linked rs1044396 had most statistical support in the case–control models (P=0.0080). The consistent effect estimates across three independent cohorts elaborate on recently published functional studies of rs2236196 from the CHRNA4 3′-untranslated region and seem to converge with accumulating evidence to firmly implicate the variant G allele of this polymorphism in the intensification of nicotine dependence.

Genetic evidence for a role of adiponutrin in the metabolism of apolipoprotein B-containing lipoproteins

Adiponutrin (PNPLA3) is a predominantly liver-expressed transmembrane protein with phospholipase activity that is regulated by fasting and feeding. Recent genome-wide association studies identified PNPLA3 to be associated with hepatic fat content and liver function, thus pointing to a possible involvement in the hepatic lipoprotein metabolism. The aim of this study was to examine the association between two common variants in the adiponutrin gene and parameters of lipoprotein metabolism in 23,274 participants from eight independent West-Eurasian study populations including six population-based studies [Bruneck (n = 800), KORA S3/F3 (n = 1644), KORA S4/F4 (n = 1814), CoLaus (n = 5435), SHIP (n = 4012), Rotterdam (n = 5967)], the SAPHIR Study as a healthy working population (n = 1738) and the Utah Obesity Case-Control Study including a group of 1037 severely obese individuals (average BMI 46 kg/m2) and 827 controls from the same geographical region of Utah. We observed a strong additive association of a common non-synonymous variant within adiponutrin (rs738409) with age-, gender-, and alanine-aminotransferase-adjusted lipoprotein concentrations: each copy of the minor allele decreased levels of total cholesterol on average by 2.43 mg/dl (P = 8.87 x 10(-7)), non-HDL cholesterol levels by 2.35 mg/dl (P = 2.27 x 10(-6)) and LDL cholesterol levels by 1.48 mg/dl (P = 7.99 x 10(-4)). These associations remained significant after correction for multiple testing. We did not observe clear evidence for associations with HDL cholesterol or triglyceride concentrations. In conclusion, our study suggests that adiponutrin is involved in the metabolism of apoB-containing lipoproteins.