Introduction
We have established different adult (KORA surveys, diabetes family study) and children cohorts with a wealth on information concerning obesity and related phenotypes. Our contribution to the Network is fourfold: A) The major aim of this subproject is to confirm candidate genes for obesity (leanness). B) Furthermore, the results of our studies will show the epidemiological relevance of the tested DNA variants both for adults and children, thus allowing determination of their frequencies as well as assessment of the quantitative effects of the respective genotypes on BMI at these two developmental stages. C) Additionally, the relevance of confirmed obesity genes will be tested for related phenotypes such as hypertension, waist hip ratio, or type 2 diabetes. D) Finally, we will analyse genotypes confirmed to be relevant for obesity in independently ascertained type 2 diabetes families.
Description of the available populations
Population based KORA surveys, adult population: The GSF has established the population-based KORA platform (Cooperative health research in the Region of Augsburg). KORA has evolved from the WHO MONICA study (Monitoring of Trends and Determinants of Cardiovascular Disease). In total, four population based health surveys have been conducted during 1984/85-1999/2000 with 4.000-5.000 participants each in the age of 25 to 74 years. For this project two of these studies KORA S3 and KORA S4, where follow up information (F3, F4) and samples with excellent DNA quality are available (Table 1), are to be utilised. In KORA S3/S4 several phenotypical, medical, laboratory and interview data are available (Table 2), which are relevant for obesity and related phenotypes. All participants underwent an extensive standardized medical examination including collection of a non fasting blood sample. Among other parameters blood glucose, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides and HbA1c analyses were determined. Anthropometric measurements were performed to calculate body mass index. Systolic and diastolic blood pressures were measured under standardized conditions. For further and follow up examinations the GSF maintains the KORA Study Center located in the centre of Augsburg. A biological specimen repository including, a laboratory for DNA isolation, a semi automatic system to fill serum and plasma into synthetic straws, a big cell culture lab for the establishment of EBV immortalized cell lines as well as nitrogen tanks and -80°C freezers for storage of the materials has been established. Serum/plasma and genomic DNA were processed from blood samples of the 18 000 participants from KORA, as well as from the other big studies. EBV immortalized cell lines are available from 1 600 subjects. From KORA S3 and S4 more than 9 000 DNA samples are available.
Augsburg diabetes family study (ADFS)
Between 2001 and 2002 we collected 600 nuclear families with at least one proband suffering from type 2 diabetes. The phenotype was confirmed by the treating physician. For all family members who self-reported to have no diabetes, the phenotype was confirmed by an oral glucose tolerance test (OGTT) performed in the Augsburg KORA examination center. For all non-diabetic family members, who were unable to come to the examination center, the phenotype was confirmed by the treating physician. All participants underwent an extensive standardized medical examination including collection of a non-fasting blood sample. Among other things blood glucose, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides and HbA1c analyses were carried out. Anthropometric measurements were performed to calculate body mass index. Systolic and diastolic blood pressure was measured under standardized
conditions. Each study participant signed the consent form. Genomic DNA was isolated from fresh blood using the Puregene DNA Isolation kit (Gentra).
Children cohorts: A population-based cohort of 5.500 school children aged 6 to 12 years has been established by the GSF Research Center in Eastern Germany (Sachsen-Anhalt). The children have been phenotyped for body weight, respiratory endpoints and allergies. A further cohort of 5.000 newborns from Munich, Leipzig and Wesel has been recruited, together with partners. These children have now been re-examined at the age of 6 years for general health outcomes, especially obesity, respiratory and allergic symptoms, also including questions pertaining to the family medical history. Biological materials for both studies are available in a frozen state. Isolation of genomic DNA is being performed at the moment.
Candidate gene analysis
Candidate genes will be identified in various subprojects, by associated partners and by external groups. Candidate genes or candidate DNA variants showing positive association findings in extreme groups have been and will be evaluated for their relevance in our above described large representative adult as well as children cohorts. High-throughput genotyping will be performed in our genotyping center located at the GSF. Results are statistically analysed at the GEM Munich.
Results/Project Status
The melanocortin-4-receptor gene (MC4R) is part of the melanocortinergic pathway that controls energy homeostasis. In a recent meta-analysis, the MC4R V103I (rs2229616) polymorphism was shown to be associated with body weight regulation. Although no functional differences between the isoleucine comprising receptor and the wild type receptor have been detected as yet, this meta-analysis of 14 case–control studies reported a mild negative association with obesity (odds ratio (OR) 0.69, p = 0.03) (Geller at al., 2004). However, evidence in a large population based study in a homogeneous population and a significant estimate of the change in quantitative measures of obesity was lacking. Therefore we analysed the data of two surveys of the KORA population with the same high quality study protocol, giving a total of 7937 participants. By linear regression, we found a significant decrease of 0.52 body mass index (BMI) units (95% confidence interval (CI) -0.02 to -1.03, p = 0.043) for carriers of the heterozygote rs2229616 G/A genotype, which was observed in 3.7% of the participants. Logistic regression yielded a significantly negative association of the MC4R variant with ‘‘above average weight’’ (BMI > median BMI) yielding an OR of 0.75 (95% CI 0.59 to 0.95, p = 0.017). We obtained similar results comparing obese (BMI >30 kg/m2, World Health Organization results for 1997) with non-obese (BMI <30 kg/m2) participants. The results were found for both sexes and each survey separately, and did not depend on the modelling of age, sex, or survey effects (Heid/Vollmert et al., 2005).
A second gene being an interesting candidate for obesity (leanness) is the adiponectin gene (APM1). Reports on DNA variants of APM1 showed numerous, but inconsistent associations with parameters of the metabolic syndrome, which is a cluster of risk factors related to cardiovascular disease and type 2 diabetes. We performed a systematic investigation based on fine-mapped SNPs highlighting the genetic architecture of APM1 and its role in modulating adiponectin plasma concentrations in a particularly healthy population of 1727 Caucasians avoiding secondary effects from disease processes.
Genotyping 53 SNPs (average spacing of 0.7 kb) in the APM1 gene region in 81 Caucasians revealed a two-block LD structure and enabled comprehensive tagSNP selection. We found particularly strong association with circulating adiponectin for 11 out of the 18 tagSNPs genotyped subsequently in the 1727 well-phenotyped subjects up to a difference of 3 µg/ml adiponectin concentration (increased levels: SNP 14811, p<0.000001; SNP-11388 p<0.000001; SNP 45, p=0.0006; SNP 276, p=0.00007; SNP 712, p<0.000001; Y111H, p=0.04; SNP 3639, p=0.001; SNP 4545, p=0.01; decreased levels: SNP 10066, p<0.000001; SNP 8564, p=0.00002; and SNP -450, p=0.0003). Haplotype analysis provided a thorough differentiation of adiponectin levels with 9 out of 17 showing significant association (5 with p-values < 0.0001). A minimal spanning net of haplotypes showed branches with “good haplotypes” and “bad haplotypes”. The variants in the APM1 explained 8% of the adiponectin variance. No significant association was found for any of the parameters of the metabolic syndrome. We thus present the first study based on fine-mapped APM1 SNPs in combination with a haplotype tagging SNP approach showing strong associations of APM1 gene variants with adiponectin plasma concentrations in healthy subjects. The revealed two-block structure may point towards two causal regions, one including the promoter SNPs and Exon 1 and a second the rest of the exons. Our data on a large number of healthy subjects further suggests that the modulation of adiponectin concentrations by variants in the APM1 gene is not merely a concomitant effect in the course of disease but most apparent already in a healthy status (Heid/Wagner et al., submitted).
Outlook
After successful analysis of functional candidate genes the next goal of the subproject is the identification up to now unknown obesity/ leaness related genes or chromosomal regions. Therefore in October 2005 a genome wide association study using 500 k chips from Affymetrix will be started. More than 1000 persons from the population based KORA surveys will be genotyped. This huge approach is financed by several institutions and partners. Firstly, the GSF has brought up a major investment to make this study possible. The rest of the resources was added by Affymetrix and partners from different networks of the NGFN.
Lit.: 1.Geller F et al. Melanocortin-4 receptor gene variant I103 is negatively associated with obesity. 2004, Am J Hum Gen, 74:572–581. 2. Heid IM/Vollmert C et al. Association of the 103I MC4R Allele with Decreased Body Mass in 7937 Participants of Two Population-Based Surveys, 2005, J Med Genet, 42:e21, 1-6. 3. Heid IM/Wagner SA et al. Genetic architecture of the APM1 gene and its influence on adiponectin plasma levels and parameters of the metabolic syndrome in 1727 healthy Caucasians, Diabetes, submitted.
