Anke Toenjes

A Genome-Wide Screen for Interactions Reveals a New Locus on 4p15 Modifying the Effect of Waist-to-Hip Ratio on Total Cholesterol

Recent genome-wide association (GWA) studies described 95 loci controlling serum lipid levels. These common variants explain ∼25% of the heritability of the phenotypes. To date, no unbiased screen for gene–environment interactions for circulating lipids has been reported. We screened for variants that modify the relationship between known epidemiological risk factors and circulating lipid levels in a meta-analysis of genome-wide association (GWA) data from 18 population-based cohorts with European ancestry (maximum N = 32,225). We collected 8 further cohorts (N = 17,102) for replication, and rs6448771 on 4p15 demonstrated genome-wide significant interaction with waist-to-hip-ratio (WHR) on total cholesterol (TC) with a combined P-value of 4.79×10−9. There were two potential candidate genes in the region, PCDH7 and CCKAR, with differential expression levels for rs6448771 genotypes in adipose tissue. The effect of WHR on TC was strongest for individuals carrying two copies of G allele, for whom a one standard deviation (sd) difference in WHR corresponds to 0.19 sd difference in TC concentration, while for A allele homozygous the difference was 0.12 sd. Our findings may open up possibilities for targeted intervention strategies for people characterized by specific genomic profiles. However, more refined measures of both body-fat distribution and metabolic measures are needed to understand how their joint dynamics are modified by the newly found locus.

Widely used commercial ELISA for human Zonulin reacts with Complement C3 rather than pre-Haptoglobin 2

BACKGROUND There is increasing evidence for the role of impaired intestinal permeability in obesity and associated metabolic diseases. Zonulin is an established serum marker for intestinal permeability and supposedly identical to pre-haptoglobin2. Here, we aimed to investigate the relationship between intestinal permeability represented by circulating zonulin and metabolic traits related to obesity. METHODS Serum zonulin was measured by using a widely used commercially ELISA kit in 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In addition, haptoglobin genotype was determined in DNA samples from all study subjects. RESULTS The genotype frequencies for haptoglobin genotypes HP1/1, HP1/2 and HP2/2 were 15.8%, 47.6% and 36.6% respectively. Since zonulin concentrations did not correspond to the haptoglobin genotypes we investigated the specificity of the zonulin ELISA assay using immunoprecipitation experiments, mass spectrometry and Western blot analysis. Thus we demonstrated by mass spectrometry that the kit is mainly capturing complement factor C3 and derived fragments. Circulating complement factor C3 was significantly increased in patients with diabetes and obesity and correlated strongly with markers of the lipid and glucose metabolism. CONCLUSIONS Our study supports the role of the complement C3 in the pathophysiology of obesity and necessitates critical revision of published data on zonulin as a marker for altered intestinal permeability from studies that have used the ELISA kit investigated here.Background: There is increasing evidence for the role of impaired intestinal permeability in obesity and associated metabolic diseases. Zonulin is an established serum marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to investigate the relationship between circulating zonulin and metabolic traits related to obesity. Methods: Serum zonulin was measured by using a widely used commercial ELISA kit in 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In addition, haptoglobin genotype was determined in DNA samples from all study subjects. Results: As zonulin concentrations did not correlate to the haptoglobin genotypes, we investigated the specificity of the zonulin ELISA assay using antibody capture experiments, mass spectrometry, and Western blot analysis. Using serum samples that gave the highest or lowest ELISA signals, we detected several proteins that are likely to be captured by the antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. We used increasing concentrations of recombinant pre-haptoglobin2 and complement C3 as one of the representative captured proteins and the ELISA kit did not detect either. Western blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies rose against zonulin showed a similar protein recognition pattern but with different intensity of detection. The protein(s) measured using the ELISA kit was (were) significantly increased in patients with diabetes and obesity and correlated strongly with markers of the lipid and glucose metabolism. Combining mass spectrometry and Western blot analysis using the polyclonal antibodies used in the ELISA kit, we identified properdin as another member of the zonulin family. Conclusion: Our study suggests that the zonulin ELISA does not recognize pre-haptoglobin2, rather structural (and possibly functional) analog proteins belonging to the mannose-associated serine protease family, with properdin being the most likely possible candidate.

Sequence variants at CHRNB3-CHRNA6 and CYP2A6 affect smoking behavior

Smoking is a common risk factor for many diseases. We conducted genome-wide association meta-analyses for the number of cigarettes smoked per day (CPD) in smokers (n = 31,266) and smoking initiation (n = 46,481) using samples from the ENGAGE Consortium. In a second stage, we tested selected SNPs with in silico replication in the Tobacco and Genetics (TAG) and Glaxo Smith Kline (Ox-GSK) consortia cohorts (n = 45,691 smokers) and assessed some of those in a third sample of European ancestry (n = 9,040). Variants in three genomic regions associated with CPD (P < 5 × 10−8), including previously identified SNPs at 15q25 represented by rs1051730[A] (effect size = 0.80 CPD, P = 2.4 × 10−69), and SNPs at 19q13 and 8p11, represented by rs4105144[C] (effect size = 0.39 CPD, P = 2.2 × 10−12) and rs6474412-T (effect size = 0.29 CPD, P = 1.4 × 10−8), respectively. Among the genes at the two newly associated loci are genes encoding nicotine-metabolizing enzymes (CYP2A6 and CYP2B6) and nicotinic acetylcholine receptor subunits (CHRNB3 and CHRNA6), all of which have been highlighted in previous studies of smoking and nicotine dependence. Nominal associations with lung cancer were observed at both 8p11 (rs6474412[T], odds ratio (OR) = 1.09, P = 0.04) and 19q13 (rs4105144[C], OR = 1.12, P = 0.0006).