Jana Breitfeld

Genetics of adiponectin.

Anti-inflammatory, anti-atherogenic and anti-diabetic properties of adiponectin make this adipokine an attractive target in the metabolism research. Given its biological role, genetic variation in adiponectin affecting its function might consequently play a role in the pathophysiology of various metabolic disorders. In this light, genetic aspects of adiponectin including its gene structure, heritability of serum concentrations and the role of genetic variation have been addressed in multiple genetic studies. Here, we provide a brief summary of adiponectin genetics with focus on gene structure and genetic variation controlling circulating adiponectin levels. We summarize the main findings from genome-wide linkage and association studies that have revealed the major genetic determinants of serum adiponectin. Beside genetic variants in the adiponectin gene, several other genes/loci (ARL15, CDH13, KNG1, FER, ETV5) contributing to the variability in circulating adiponectin have been identified. The majority of these variants are significantly associated with metabolic phenotypes relevant to metabolic diseases (e.g. obesity or type 2 diabetes (T2D)). Considering the protective properties of adiponectin in diseases such as T2D, comprehensive analyses of genetic variants including rare as well as frequent polymorphisms might provide insights on the specific role of adiponectin in the pathophysiology of metabolic diseases.

Genetic and Evolutionary Analyses of the Human Bone Morphogenetic Protein Receptor 2 (BMPR2) in the Pathophysiology of Obesity

Objective Human bone morphogenetic protein receptor 2 (BMPR2) is essential for BMP signalling and may be involved in the regulation of adipogenesis. The BMPR2 locus has been suggested as target of recent selection in human populations. We hypothesized that BMPR2 might have a role in the pathophysiology of obesity. Research Design and Methods Evolutionary analyses (dN/dS, Fst, iHS) were conducted in vertebrates and human populations. BMPR2 mRNA expression was measured in 190 paired samples of visceral and subcutaneous adipose tissue. The gene was sequenced in 48 DNA samples. Nine representative single nucleotide polymorphisms (SNPs) were genotyped for subsequent association studies on quantitative traits related to obesity in 1830 German Caucasians. An independent cohort of 925 Sorbs was used for replication. Finally, relation of genotypes to mRNA in fat was examined. Results The evolutionary analyses indicated signatures of selection on the BMPR2 locus. BMPR2 mRNA expression was significantly increased both in visceral and subcutaneous adipose tissue of 37 overweight (BMI>25 and <30 kg/m2) and 80 obese (BMI>30 kg/m2) compared with 44 lean subjects (BMI<25 kg/m2) (P<0.001). In a case-control study including lean and obese subjects, two intronic SNPs (rs6717924, rs13426118) were associated with obesity (adjusted P<0.05). Combined analyses including the initial cohort and the Sorbs confirmed a consistent effect for rs6717924 (combined P = 0.01) on obesity. Moreover, rs6717924 was associated with higher BMPR2 mRNA expression in visceral adipose tissue. Conclusion Combined BMPR2 genotype-phenotype-mRNA expression data as well as evolutionary aspects suggest a role of BMPR2 in the pathophysiology of obesity.

Role of genetic variation in the human sodium–glucose cotransporter 2 gene (SGLT2) in glucose homeostasis

AIMS Mutations in the sodium-glucose cotransporter 2 (SGLT2), as well as treatment with SGLT2 inhibitors result in reduced fasting glucose levels, HbA(1c) and BMI. We therefore investigated the effects of common genetic variation in SGLT2 on human Type 2 diabetes and related traits. MATERIALS & METHODS Four HapMap tagging SNPs covering the common genetic variation in SGLT2 (r² > 0.8 and minor allele frequency > 0.01) were genotyped for subsequent association studies on BMI, Type 2 diabetes and related metabolic traits in 1013 Sorbs (Germany). An independent cohort from Berlin (n = 2042) was taken for replication. RESULTS The rs9934336 G-allele was nominally associated with increased 30-min plasma glucose, 120-min insulin concentrations and AUC120min(glucose) during oral glucose tolerance test in 907 nondiabetic Sorbs (p < 0.05). In the combined analysis including the Sorbs and the Berlin cohort, rs9934336 was nominally associated with 120-min insulin concentrations (adjusted p < 0.05) in nondiabetic subjects (n = 2590). CONCLUSION Our data suggest a role of SGLT2 genetic variation in the regulation of glucose homeostasis and promote pharmacogenomic studies to clarify the efficacy of antidiabetic treatment by SGLT2 inhibitors.

Role of genetic variation in the cannabinoid type 1 receptor gene (CNR1) in the pathophysiology of human obesity

AIMS The endocannabinoid system may contribute to the association of visceral fat accumulation with metabolic diseases. Here we investigated the effects of genetic variation in the cannabinoid type 1 receptor gene (CNR1) on its mRNA expression in adipose tissue from visceral and subcutaneous depots and on the development of obesity. MATERIALS & METHODS CNR1 was sequenced in 48 nonrelated German Caucasians to detect genetic variation. Five representative variants including HapMap tagging SNPs (rs12720071, rs806368, rs806370, rs1049353 and rs806369) were genotyped for subsequent association studies in two independent cohorts (total n = 2774) with detailed metabolic testing: subjects from the Leipzig Study (n = 1857) and a self-contained population of Sorbs from Germany (n = 917). RESULTS In a case-control study of lean (BMI <25 kg/m(2)) versus obese (BMI >30 kg/m(2)) subjects, rs806368 was found to be nominally associated with obesity in the Sorbian cohort (adjusted p < 0.05), but not in the Leipzig cohort. Also, several SNPs (rs806368, rs806370 and rs12720071) were nominally associated with serum leptin levels (p < 0.05 after adjusting for age, sex and BMI). However, none of these associations remained significant after accounting for multiple testing. Furthermore, none of the SNPs were related to CNR1 mRNA expression in visceral and subcutaneous fat. CONCLUSION The data suggest that common genetic variation in the CNR1 gene does not influence mRNA expression in adipose tissue nor does it play a significant role in the pathophysiology of obesity in German and Sorbian populations.

Eating Behaviour in the General Population: An Analysis of the Factor Structure of the German Version of the Three-Factor-Eating-Questionnaire (TFEQ) and Its Association with the Body Mass Index

The Three-Factor-Eating-Questionnaire (TFEQ) is an established instrument to assess eating behaviour. Analysis of the TFEQ-factor structure was based on selected, convenient and clinical samples so far. Aims of this study were (I) to analyse the factor structure of the German version of the TFEQ and (II)—based on the refined factor structure—to examine the association between eating behaviour and the body mass index (BMI) in a general population sample of 3,144 middle-aged and older participants (40–79 years) of the ongoing population based cohort study of the Leipzig Research Center for Civilization Diseases (LIFE Health Study). The factor structure was examined in a split-half analysis with both explorative and confirmatory factor analysis. Associations between TFEQ-scores and BMI values were tested with multiple regression analyses controlled for age, gender, and education. We found a three factor solution for the TFEQ with an ‘uncontrolled eating’, a ‘cognitive restraint’ and an ‘emotional eating’ domain including 29 of the original 51 TFEQ-items. Scores of the ‘uncontrolled eating domain’ showed the strongest correlation with BMI values (partial r = 0.26). Subjects with scores above the median in both ‘uncontrolled eating’ and ‘emotional eating’ showed the highest BMI values (mean = 29.41 kg/m²), subjects with scores below the median in all three domains showed the lowest BMI values (mean = 25.68 kg/m²; F = 72.074, p<0.001). Our findings suggest that the TFEQ is suitable to identify subjects with specific patterns of eating behaviour that are associated with higher BMI values. Such information may help health care professionals to develop and implement more tailored interventions for overweight and obese individuals.

Role of vaspin in human eating behaviour.

Objective The adipokine vaspin (visceral adipose tissue derived serine protease inhibitor, serpinA12) follows a meal-related diurnal variation in humans and intracerebroventricular vaspin administration leads to acutely reduced food intake in db/db mice. We therefore hypothesized that vaspin may play a role in human eating behaviour. Materials and Methods We measured serum vaspin concentrations in 548 subjects from a self-contained population of Sorbs (Germany) who underwent detailed metabolic testing including eating behaviour assessments using the three-factor eating questionnaire. In addition, genetic variation within vaspin was assessed by genotyping 28 single nucleotide polymorphisms (SNPs) in all study subjects. Results Serum vaspin concentrations correlated positively with restraint, disinhibition and hunger (all P<0.05), although the correlations did not withstand further adjustments for age, gender and BMI (all P>0.05). Independent of observed correlations, genetic variants in vaspin were associated with serum vaspin levels but showed no significant association with any of the eating behaviour phenotypes after accounting for multiple testing (P≥0.05 after adjusting for age, gender and BMI). Conclusion Our data suggest that serum vaspin concentrations might modulate human eating behaviour, which does not seem to be affected by common genetic variation in vaspin.

THOC5: a novel gene involved in HDL-cholesterol metabolism.

Although numerous genes are known to regulate serum lipid traits, identified variants explain only a small proportion of the expected heritability. We intended to identify further genetic variants associated with lipid phenotypes in a self-contained population of Sorbs in Germany. We performed a genome-wide association study (GWAS) on LDL-cholesterol, HDL-cholesterol (HDL-C), and triglyceride (TG) levels in 839 Sorbs. All single-nucleotide polymorphisms with a P value <0.01 were subjected to a meta-analysis, including an independent Swedish cohort (Diabetes Genetics Initiative; n = ∼3,100). Novel association signals with the strongest effects were subjected to replication studies in an additional German cohort (Berlin, n = 2,031). In the initial GWAS in the Sorbs, we identified 14 loci associated with lipid phenotypes reaching P values <10−5 and confirmed significant effects for 18 previously reported loci. The combined meta-analysis of the three study cohorts (n(HDL) = 6041; n(LDL) = 5,995; n(TG) = 6,087) revealed a novel association for a variant in THOC5 (rs8135828) with serum HDL-C levels (P = 1.78 × 10−7; Z-score = −5.221). Consistently, the variant was also associated with circulating APOA1 levels in Sorbs. The small interfering RNA-mediated mRNA silencing of THOC5 in HepG2 cells resulted in lower mRNA levels of APOA1, SCARB1, and ABCG8 (all P < 0.05). We propose THOC5 to be a novel gene involved in the regulation of serum HDL-C levels.

Genome Wide Meta-analysis Highlights the Role of Genetic Variation in RARRES2 in the Regulation of Circulating Serum Chemerin.

Chemerin is an adipokine proposed to link obesity and chronic inflammation of adipose tissue. Genetic factors determining chemerin release from adipose tissue are yet unknown. We conducted a meta-analysis of genome-wide association studies (GWAS) for serum chemerin in three independent cohorts from Europe: Sorbs and KORA from Germany and PPP-Botnia from Finland (total N = 2,791). In addition, we measured mRNA expression of genes within the associated loci in peripheral mononuclear cells by micro-arrays, and within adipose tissue by quantitative RT-PCR and performed mRNA expression quantitative trait and expression-chemerin association studies to functionally substantiate our loci. Heritability estimate of circulating chemerin levels was 16.2% in the Sorbs cohort. Thirty single nucleotide polymorphisms (SNPs) at chromosome 7 within the retinoic acid receptor responder 2 (RARRES2)/Leucine Rich Repeat Containing (LRRC61) locus reached genome-wide significance (p<5.0×10−8) in the meta-analysis (the strongest evidence for association at rs7806429 with p = 7.8×10−14, beta = −0.067, explained variance 2.0%). All other SNPs within the cluster were in linkage disequilibrium with rs7806429 (minimum r2 = 0.43 in the Sorbs cohort). The results of the subgroup analyses of males and females were consistent with the results found in the total cohort. No significant SNP-sex interaction was observed. rs7806429 was associated with mRNA expression of RARRES2 in visceral adipose tissue in women (p<0.05 after adjusting for age and body mass index). In conclusion, the present meta-GWAS combined with mRNA expression studies highlights the role of genetic variation in the RARRES2 locus in the regulation of circulating chemerin concentrations.

Signatures of Natural Selection at the FTO (Fat Mass and Obesity Associated) Locus in Human Populations

Background and aims Polymorphisms in the first intron of FTO have been robustly replicated for associations with obesity. In the Sorbs, a Slavic population resident in Germany, the strongest effect on body mass index (BMI) was found for a variant in the third intron of FTO (rs17818902). Since this may indicate population specific effects of FTO variants, we initiated studies testing FTO for signatures of selection in vertebrate species and human populations. Methods First, we analyzed the coding region of 35 vertebrate FTO orthologs with Phylogenetic Analysis by Maximum Likelihood (PAML, ω = dN/dS) to screen for signatures of selection among species. Second, we investigated human population (Europeans/CEU, Yoruba/YRI, Chinese/CHB, Japanese/JPT, Sorbs) SNP data for footprints of selection using DnaSP version 4.5 and the Haplotter/PhaseII. Finally, using ConSite we compared transcription factor (TF) binding sites at sequences harbouring FTO SNPs in intron three. Results PAML analyses revealed strong conservation in coding region of FTO (ω<1). Sliding-window results from population genetic analyses provided highly significant (p<0.001) signatures for balancing selection specifically in the third intron (e.g. Tajima’s D in Sorbs = 2.77). We observed several alterations in TF binding sites, e.g. TCF3 binding site introduced by the rs17818902 minor allele. Conclusion Population genetic analysis revealed signatures of balancing selection at the FTO locus with a prominent signal in intron three, a genomic region with strong association with BMI in the Sorbs. Our data support the hypothesis that genes associated with obesity may have been under evolutionary selective pressure.

Serum Adiponectin and Progranulin Levels are Associated with Gallstone Disease

BACKGROUND/AIM Aberrant adipokine serum concentrations are associated with a variety of obesity-related diseases. This study was designed to investigate the putative role of the adipokines adiponectin, chemerin, progranulin, vaspin, fibroblast growth factor 21 (FGF21) and adipocyte fatty acid binding protein (AFABP) in gallstone disease. METHODS Serum levels of adiponectin, chemerin, progranulin, vaspin, FGF21 and AFABP of 189 gallstone patients and 833 healthy controls were measured by enzyme-linked immunosorbent assays. RESULTS Increased adiponectin levels were nominally associated with lower gallstone risk in women (p=0.036, odds ratio (OR) 0.47, 95% confidence interval (CI) [0.23; 0.95]). Furthermore progranulin serum concentrations in men were significantly elevated in gallstone carriers in comparison to controls (p=0.012, OR 6.1, 95% CI [1.5; 24.9]). Serum levels of chemerin, vaspin, FGF21 and AFABP did not differ between controls and subjects with gallstones. CONCLUSION Our data further support a protective effect of adiponectin on gallstone risk and suggest a role of progranulin in the pathophysiology of cholelithiasis. Nevertheless, longitudinal data and functional analyses would be required to assess the pathogenetic link between gallstone formation and adipokine serum levels.