Mutation screen and association studies for the fatty acid amide hydrolase (FAAH) gene and early onset and adult obesity.

BACKGROUND: The orexigenic effects of cannabinoids are limited by activation of the endocannabinoid degrading enzyme fatty acid amide hydrolase (FAAH). The aim of this study was to analyse whether FAAH alleles are associated with early and late onset obesity. METHODS: We initially assessed association of five single nucleotide polymorphisms (SNPs) in FAAH with early onset extreme obesity in up to 521 German obese children and both parents. SNPs with nominal p-values 0.05). CONCLUSIONS: As we observed some evidence for an association of the FAAH variants rs2295632 rs324420 with early onset but not adult obesity, we conclude that the FAAH variants analyzed here at least do not seem to play a major role in the etiology of obesity within our samples.

The association of a SNP upstream of INSIG2 with body mass index is reproduced in several but not all cohorts.

A SNP upstream of the INSIG2 gene, rs7566605, was recently found to be associated with obesity as measured by body mass index (BMI) by Herbert and colleagues. The association between increased BMI and homozygosity for the minor allele was first observed in data from a genome-wide association scan of 86,604 SNPs in 923 related individuals from the Framingham Heart Study offspring cohort. The association was reproduced in four additional cohorts, but was not seen in a fifth cohort. To further assess the general reproducibility of this association, we genotyped rs7566605 in nine large cohorts from eight populations across multiple ethnicities (total n = 16,969). We tested this variant for association with BMI in each sample under a recessive model using family-based, population-based, and case-control designs. We observed a significant (p < 0.05) association in five cohorts but saw no association in three other cohorts. There was variability in the strength of association evidence across examination cycles in longitudinal data from unrelated individuals in the Framingham Heart Study Offspring cohort. A combined analysis revealed significant independent validation of this association in both unrelated (p = 0.046) and family-based (p = 0.004) samples. The estimated risk conferred by this allele is small, and could easily be masked by small sample size, population stratification, or other confounders. These validation studies suggest that the original association is less likely to be spurious, but the failure to observe an association in every data set suggests that the effect of SNP rs7566605 on BMI may be heterogeneous across population samples.

Gastric inhibitory polypeptide receptor: association analyses for obesity of several polymorphisms in large study groups.

BACKGROUND: Gastric inhibitory polypeptide (GIP) is postulated to be involved in type 2 diabetes mellitus and obesity. It exerts its function through its receptor, GIPR. We genotyped three GIPR SNPs (rs8111428, rs2302382 and rs1800437) in German families with at least one obese index patient, two case-control studies and two cross-sectional population-based studies. METHODS: Genotyping was performed by MALDI-TOF, ARMS-PCR and RFLP. The family-study: 761 German families with at least one extremely obese child or adolescent (n = 1,041) and both parents (n = 1,522). Case-control study: (a) German obese children (n = 333) and (b) obese adults (n = 987) in comparison to 588 adult lean controls. The two cross-sectional population-based studies: KORA (n = 8,269) and SHIP (n = 4,310). RESULTS: We detected over-transmission of the A-allele of rs2302382 in the German families (pTDT-Test = 0.0089). In the combined case-control sample, we estimated an odd ratio of 1.54 (95%CI 1.09;2.19, pCA-Test = 0.014) for homozygotes of the rs2302382 A-allele compared to individuals with no A-allele. A similar trend was found in KORA where the rs2302382 A-allele led to an increase of 0.12 BMI units (p = 0.136). In SHIP, however, the A-allele of rs2302382 was estimated to contribute an average decrease of 0.27 BMI units (p-value = 0.031). CONCLUSION: Our data suggest a potential relevance of GIPR variants for obesity. However, additional studies are warranted in light of the conflicting results obtained in one of the two population-based studies.

Preferential reciprocal transfer of paternal/maternal DLK1 alleles to obese children: first evidence of polar overdominance in humans.

DLK1 is part of the Notch signalling pathway that controls various developmental processes. A functional role for DLK1 in adipogenesis is suggested by several animal models. Interestingly, the DLK1 gene is imprinted in eutherian mammals. To study whether variations in DLK1 affect body weight in humans, we analysed 32 polymorphisms in a 109 kb genomic region encompassing DLK1 on human chromosome 14. In a study sample of 1025 French and German trio families comprised of both parents and extremely obese offspring we found a single nucleotide polymorphism (rs1802710) associated with child and adolescent obesity. Analysis of the allelic transmission pattern indicated the existence of polar overdominance, an unusual mode of non-Mendelian inheritance in humans previously known from the callipyge mutation in sheep.

Gain-of-function screen for genes that affect Drosophila muscle pattern formation.

This article reports the production of an EP-element insertion library with more than 3,700 unique target sites within the Drosophila melanogaster genome and its use to systematically identify genes that affect embryonic muscle pattern formation. We designed a UAS/GAL4 system to drive GAL4-responsive expression of the EP-targeted genes in developing apodeme cells to which migrating myotubes finally attach and in an intrasegmental pattern of cells that serve myotubes as a migration substrate on their way towards the apodemes. The results suggest that misexpression of more than 1.5% of the Drosophila genes can interfere with proper myotube guidance and/or muscle attachment. In addition to factors already known to participate in these processes, we identified a number of enzymes that participate in the synthesis or modification of protein carbohydrate side chains and in Ubiquitin modifications and/or the Ubiquitin-dependent degradation of proteins, suggesting that these processes are relevant for muscle pattern formation.

The 103I variant of the melanocortin 4 receptor is associated with low serum triglyceride levels.

CONTEXT: The melanocortin 4 receptor (MC4R) is an essential regulator of energy intake and body weight. Recently, the V103I polymorphism of MC4R has been shown to be negatively associated with body mass index. This suggests that serum lipids and blood pressure in individuals carrying the 103I allele might be influenced as well. OBJECTIVE: The objective of this study was to determine whether the most common polymorphism of the MC4R, V103I, affects serum lipid levels and/or blood pressure. DESIGN, SETTING, AND PARTICIPANTS: The study participants were 1173 consecutive patients undergoing cardiac catheterization; they were genotyped for the rs2229616 G-->A substitution at codon 103 (V103I polymorphism) of the MC4R gene. Patients had strictly fasted for at least 12 h before blood samples were drawn. The average age of the patients was 60.9 yr; 72% were males. MAIN OUTCOME MEASURES: The main outcome measures were body mass index, serum lipids, aortic and systolic blood pressure, and MC4R polymorphism V103I. RESULTS: Heterozygous carriers of the 103I allele had significantly lower triglyceride levels than individuals homozygous for the wild-type allele (127 vs. 168 mg/dl mean total triglyceride; P = 0.001 or 0.009 after Bonferroni adjustment for seven tests). No homozygous carriers of the 103I allele were present in the study population. CONCLUSIONS: Our study suggests an influence of MC4R activity on triglyceride levels in cardiovascular patients.

Association of the MC4R V103I polymorphism with the metabolic syndrome: the KORA Study.

OBJECTIVE: Epidemiological studies showing an association between the melanocortin-4-receptor (MC4R) 103I variant (rs2229616) and decreased BMI are complemented by functional studies; these suggest a mechanism for appetite regulation and a linkage signal for physical activity and dietary intake for the region encompassing the MC4R. This study aims to provide epidemiological evidence for showing the association of this polymorphism with features of the metabolic syndrome and with parameters related to energy expenditure and dietary habits as potential mediators. METHODS AND PROCEDURES: We analyzed this polymorphism in 7,888 adults of a population-based cross-sectional study applying regression-based statistical models. RESULTS: Carriers of the MC4R 103I (3.7%) exhibited a significantly decreased waist circumference (-1.46 cm, P = 0.020), decreased glycosylated hemoglobin (HbA(1c)) (-0.09%, P = 0.040), and increased HDL-cholesterol (HDL-C) (+1.76 mg/dl, P = 0.056), but no change in blood pressure. The odds of having three or more components of the metabolic syndrome were substantially reduced among carriers of MC4R 103I (odds ratio (OR) = 0.46, P = 0.003). Controlling for BMI reduced the HbA(1c) and HDL-C association. Mediator analyses revealed a borderline association of MC4R 103I with carbohydrate intake (OR = 1.26, P = 0.059) possibly mediating association with leanness. DISCUSSION: Our representative study of well-phenotyped Europeans is the first to describe the association of the MC4R V103I with the metabolic syndrome and with a nutrient-related phenotype. Our data support the idea that this polymorphism plays a role in appetite regulation that not only affects BMI, but also other features of the metabolic syndrome. It further establishes that the association of the MC4R V103I with obesity and related phenotypes is genuine.

Lack of association of genetic variants in genes of the endocannabinoid system with anorexia nervosa.

BACKGROUND: Several lines of evidence indicate that the central cannabinoid receptor 1 (CNR1) as well as the major endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH), N-acylethanolamine-hydrolyzing acid amidase (NAAA) and monoglyceride lipase (MGLL) are implicated in mediating the orexigenic effects of cannabinoids. The aim of this study was to analyse whether nucleotide sequence variations in the CNR1, FAAH, NAAA and MGLL genes are associated with anorexia nervosa (AN). METHODS: We analysed the association of a previously described (AAT)n repeat in the 3' flanking region of CNR1 as well as a total of 15 single nucleotide polymorphisms (SNPs) representative of regions with restricted haplotype diversity in CNR1, FAAH, NAAA or MGLL in up to 91 German AN trios (patient with AN and both biological parents) using the transmission-disequilibrium-test (TDT). One SNP was additionally analysed in an independent case-control study comprising 113 patients with AN and 178 normal weight controls. Genotyping was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, ARMS-PCR or using 3730xl capillary sequencers. RESULTS: The TDT revealed no evidence for association for any of the SNPs or the (AAT)n repeat with AN (all two-sided uncorrected p-values > 0.05). The lowest p-value of 0.11 was detected for the A-allele of the CNR1 SNP rs1049353 for which the transmission rate was 59% (95% confidence interval 47%...70%). Further genotyping of rs1049353 in 113 additional independent patients with AN and 178 normal weight controls could not substantiate the initial trend for association (p = 1.00). CONCLUSION: As we found no evidence for an association of genetic variation in CNR1, FAAH, NAAA and MGLL with AN, we conclude that genetic variations in these genes do not play a major role in the etiology of AN in our study groups.

INS VNTR is not associated with childhood obesity in 1,023 families: a family-based study.

Previous studies have described genetic associations of the insulin gene variable number tandem repeat (INS VNTR) variant with childhood obesity and associated phenotypes. We aimed to assess the contribution of INS VNTR genotypes to childhood obesity and variance of insulin resistance, insulin secretion, and birth weight using family-based design. Participants were either French or German whites. We used transmission disequilibrium tests (TDTs) for assessing binary traits and quantitative pedigree disequilibrium tests for assessing continuous traits. In contrast to previous findings, we did not observe any familial association with childhood obesity (T = 50%, P = 0.77) in the 1,023 families tested. In French obese children, INS VNTR did not associate with fasting insulin levels (P = 0.23) and class I allele showed only borderline association with increased insulin secretion index at 30 min (P = 0.03). INS VNTR did not associate with birth weight in obese children (P = 0.98) and TDT analyses in 350 French families with history of low birth weight (LBW) showed no association with this condition (P = 0.92). In summary, our study, the largest performed so far, does not support the previously reported associations between INS VNTR and childhood obesity, insulin resistance, or birth weight, and does not suggest any major role for this variant in modulating these traits.

No evidence for an involvement of variants in the cannabinoid receptor gene (CNR1) in obesity in German children and adolescents.

Studies in rodent models demonstrated that the central cannabinoid receptor (Cnr1) mediates the orexigenic effects of cannabinoids. To analyze whether genetic variation in the cannabinoid receptor gene (CNR1) is implicated in human obesity, we initially genotyped 8 single nucleotide polymorphisms (SNPs) located in the 5' region (rs9353527, rs754387, rs6454676), intron 2 (rs806379, rs1535255), exon 3 (rs2023239), intron 3 (rs806370) and the coding region (rs1049353) in up to 364 German obesity trios (extremely obese child or adolescent and both parents). The transmission disequilibrium test (TDT) was negative for these SNPs (p>0.05). However, there was a slight trend towards preferential transmission of the A-allele of rs1049353 (p=0.12). We therefore genotyped this SNP in 235 independent German obesity families (at least two obese sibs and both parents) and in parallel screened the CNR1 coding region for sequence variations in 120 German extremely obese children and adolescents who mainly contributed to the initial trend observed for rs1049353. The trend for preferential transmission of the A-allele could not be substantiated (pedigree disequilibrium test, PDT p=0.15; A-allele less frequently transmitted). In the mutation screen we detected two rare variations, one novel non-conservative mutation (c.1256C>A; A419E) and the known variant 1419+1G>C. In addition, we confirmed the presence of rs1049353. As these variants could not explain the initial TDT, we conclude that there is no evidence for an association of CNR1 alleles with obesity in our study groups.

Genome wide association (GWA) study for early onset extreme obesity supports the role of fat mass and obesity associated gene (FTO) variants.

BACKGROUND: Obesity is a major health problem. Although heritability is substantial, genetic mechanisms predisposing to obesity are not very well understood. We have performed a genome wide association study (GWA) for early onset (extreme) obesity. METHODOLOGY/PRINCIPAL FINDINGS: a) GWA (Genome-Wide Human SNP Array 5.0 comprising 440,794 single nucleotide polymorphisms) for early onset extreme obesity based on 487 extremely obese young German individuals and 442 healthy lean German controls; b) confirmatory analyses on 644 independent families with at least one obese offspring and both parents. We aimed to identify and subsequently confirm the 15 SNPs (minor allele frequency > or =10%) with the lowest p-values of the GWA by four genetic models: additive, recessive, dominant and allelic. Six single nucleotide polymorphisms (SNPs) in FTO (fat mass and obesity associated gene) within one linkage disequilibrium (LD) block including the GWA SNP rendering the lowest p-value (rs1121980; log-additive model: nominal p = 1.13 x 10(-7), corrected p = 0.0494; odds ratio (OR)(CT) 1.67, 95% confidence interval (CI) 1.22-2.27; OR(TT) 2.76, 95% CI 1.88-4.03) belonged to the 15 SNPs showing the strongest evidence for association with obesity. For confirmation we genotyped 11 of these in the 644 independent families (of the six FTO SNPs we chose only two representing the LD bock). For both FTO SNPs the initial association was confirmed (both Bonferroni corrected p<0.01). However, none of the nine non-FTO SNPs revealed significant transmission disequilibrium. CONCLUSIONS/SIGNIFICANCE: Our GWA for extreme early onset obesity substantiates that variation in FTO strongly contributes to early onset obesity. This is a further proof of concept for GWA to detect genes relevant for highly complex phenotypes. We concurrently show that nine additional SNPs with initially low p-values in the GWA were not confirmed in our family study, thus suggesting that of the best 15 SNPs in the GWA only the FTO SNPs represent true positive findings.

Genetic factors for overweight and CAD.

Obesity is a growing clinical problem reaching epidemic proportions in developed and developing countries. It is associated with several comorbidities including cardiovascular diseases. There is strong evidence for the association of excess weight and established metabolic risk factors for coronary artery disease (CAD) such as hyperlipidemia, high blood pressure and type 2 diabetes. In addition, obesity may promote cardiovascular disease independent of these factors, possibly via hormones secreted by adipocytes, promotion of proinflammatory processes or other as yet unrecognized mechanisms. In this article the authors will outline the heredity of body weight and CAD/myocardial infarction and describe genetic factors involved in the etiology of these diseases. The methods commonly applied for the detection of such factors are described (e. g., animal models, linkage studies, association studies, etc.). These methods are discussed either in the paragraphs on obesity or on CAD; the described principles apply to both phenotypes.

Control of triglyceride storage by a WD40/TPR-domain protein.

Obesity is a metabolic disorder related to improper control of energy uptake and expenditure, which results in excessive accumulation of body fat. Initial insights into the genetic pathways that regulate energy metabolism have been provided by a discrete number of obesity-related genes that have been identified in mammals. Here, we report the identification of the adipose (adp) gene, the mutation of which causes obesity in Drosophila. Loss of adp activity promotes increased fat storage, which extends the lifespan of mutant flies under starvation conditions. By contrast, adp gain-of-function causes a specific reduction of the fat body in Drosophila. adp encodes an evolutionarily conserved WD40/tetratricopeptide-repeat-domain protein that is likely to represent an intermediate in a novel signalling pathway.