Nadja Knoll

Chipping away the 'missing heritability': GIANT steps forward in the molecular elucidation of obesity - but still lots to go.

Although heritability of human body weight is assumed to be high, only a small fraction of the variance can as yet be attributed to molecular genetic factors. Single monogenic forms of obesity have been identified. Functionally relevant coding mutations in the melanocortin-4 receptor gene occur in 1–6% of extremely obese children and adolescents and thus represent the most common major gene effect. Genome-wide association studies (GWAS) had previously identified 14 obesity loci with genome-wide significant (p < 5 x 10–8) associations. Many of the respective genes are expressed in the central nervous system. The GIANT (Genetic Investigation of ANtropometric Traits) Consortium has now performed a meta-analysis of GWAS data based on 123,865 individuals of European ancestry followed by confirmatory analyses for the 42 best independent loci in up to 125,931 independent individuals (Speliotes et al: Association analyses of 249,796 individuals reveal eighteen new loci associated with body mass index. Nature Genetics; epub October 2010 [1]). Apart from confirming the 14 known loci, 18 novel BMI-associated loci (p < 5 x 10–8) were identified. Several of the new loci point to genes involved in key hypothalamic pathways of energy balance. The identified variants mostly have small to very small effect sizes; only 1–2% of the BMI variance is explained. Currently, a consensus explanation for this ‘missing heritability’ in complex diseases has not yet emerged.

Genome-wide association study in German patients with attention deficit/hyperactivity disorder

The heritability of attention deficit hyperactivity disorder (ADHD) is approximately 0.8. Despite several larger scale attempts, genome‐wide association studies (GWAS) have not led to the identification of significant results. We performed a GWAS based on 495 German young patients with ADHD (according to DSM‐IV criteria; Human660W‐Quadv1; Illumina, San Diego, CA) and on 1,300 population‐based adult controls (HumanHap550v3; Illumina). Some genes neighboring the single nucleotide polymorphisms (SNPs) with the lowest P‐values (best P‐value: 8.38 × 10−7) have potential relevance for ADHD (e.g., glutamate receptor, metabotropic 5 gene, GRM5). After quality control, the 30 independent SNPs with the lowest P‐values (P‐values ≤ 7.57 × 10−5) were chosen for confirmation. Genotyping of these SNPs in up to 320 independent German families comprising at least one child with ADHD revealed directionally consistent effect‐size point estimates for 19 (10 not consistent) of the SNPs. In silico analyses of the 30 SNPs in the largest meta‐analysis so far (2,064 trios, 896 cases, and 2,455 controls) revealed directionally consistent effect‐size point estimates for 16 SNPs (11 not consistent). None of the combined analyses revealed a genome‐wide significant result. SNPs in previously described autosomal candidate genes did not show significantly lower P‐values compared to SNPs within random sets of genes of the same size. We did not find genome‐wide significant results in a GWAS of German children with ADHD compared to controls. The second best SNP is located in an intron of GRM5, a gene located within a recently described region with an infrequent copy number variation in patients with ADHD.

Molecular Genetic Aspects of Weight Regulation

BACKGROUND Family and twin studies have empirically revealed a 40% to 70% heritability of body-mass index, yet only a few hereditary factors have been identified to date that increase the risk of being overweight. METHODS We present the current state of molecular genetic research on obesity with a selective review of the literature. RESULTS A number of monogenic recessive mutations causing obesity have been identified, but these are rare. Various dominant mutations of the melanocortin-4 receptor gene are found in about 1% to 4% of all markedly obese persons. Current molecular genetic research focuses on the identification of common DNA variants affecting body weight; the genetic material of hundreds of thousands of people from around the world has now been investigated in genome-wide association studies. More than 30 variants conferring an increased risk have been identified, most of which are single nucleotide polymorphisms (SNPs) of no immediately clear functional significance. On average, these variants raise body weight by 500 g (range, 180 to 1400 g). Aside from SNPs, variations in the number of copies of specific DNA sequences have also been linked to obesity, as well as to subnormal weight. All the hereditary factors that have been identified to date account for about 5% of the variability of BMI. Extrapolation yields figures ranging from 10% to 15%. CONCLUSIONS The amount of genetic variability seen to date at the DNA level accounts only for a small fraction of the inter-individual variability of BMI. Obesity is thought to be a largely hereditary condition; the fact that its genetic basis has not yet been demonstrated may be due to various genetic or experimental factors.

Mutation screen in the GWAS derived obesity gene SH2B1 including functional analyses of detected variants

BackgroundThe SH2B1 gene (Src-homology 2B adaptor protein 1 gene) is a solid candidate gene for obesity. Large scale GWAS studies depicted markers in the vicinity of the gene; animal models suggest a potential relevance for human body weight regulation.MethodsWe performed a mutation screen for variants in the SH2B1 coding sequence in 95 extremely obese children and adolescents. Detected variants were genotyped in independent childhood and adult study groups (up to 11,406 obese or overweight individuals and 4,568 controls). Functional implications on STAT3 mediated leptin signalling of the detected variants were analyzed in vitro.ResultsWe identified two new rare mutations and five known SNPs (rs147094247, rs7498665, rs60604881, rs62037368 and rs62037369) in SH2B1. Mutation g.9483C/T leads to a non-synonymous, non-conservative exchange in the beta (βThr656Ile) and gamma (γPro674Ser) splice variants of SH2B1. It was additionally detected in two of 11,206 (extremely) obese or overweight children, adolescents and adults, but not in 4,506 population-based normal-weight or lean controls. The non-coding mutation g.10182C/A at the 3’ end of SH2B1 was only detected in three obese individuals. For the non-synonymous SNP rs7498665 (Thr484Ala) we observed nominal over-transmission of the previously described risk allele in 705 obesity trios (nominal p = 0.009, OR = 1.23) and an increased frequency of the same allele in 359 cases compared to 429 controls (nominal p = 0.042, OR = 1.23). The obesity risk-alleles at Thr484Ala and βThr656Ile/γPro674Ser had no effect on STAT3 mediated leptin receptor signalling in splice variants β and γ.ConclusionThe rare coding mutation βThr656Ile/γPro674Ser (g.9483C/T) in SH2B1 was exclusively detected in overweight or obese individuals. Functional analyzes did not reveal impairments in leptin signalling for the mutated SH2B1.

Mitochondrial DNA Variants in Obesity

Heritability estimates for body mass index (BMI) variation are high. For mothers and their offspring higher BMI correlations have been described than for fathers. Variation(s) in the exclusively maternally inherited mitochondrial DNA (mtDNA) might contribute to this parental effect. Thirty-two to 40 mtDNA single nucleotide polymorphisms (SNPs) were available from genome-wide association study SNP arrays (Affymetrix 6.0). For discovery, we analyzed association in a case-control (CC) sample of 1,158 extremely obese children and adolescents and 435 lean adult controls. For independent confirmation, 7,014 population-based adults were analyzed as CC sample of n = 1,697 obese cases (BMI≥30 kg/m2) and n = 2,373 normal weight and lean controls (BMI<25 kg/m2). SNPs were analyzed as single SNPs and haplogroups determined by HaploGrep. Fishers two-sided exact test was used for association testing. Moreover, the D-loop was re-sequenced (Sanger) in 192 extremely obese children and adolescents and 192 lean adult controls. Association testing of detected variants was performed using Fishers two-sided exact test. For discovery, nominal association with obesity was found for the frequent allele G of m.8994G/A (rs28358887, p = 0.002) located in ATP6. Haplogroup W was nominally overrepresented in the controls (p = 0.039). These findings could not be confirmed independently. For two of the 252 identified D-loop variants nominal association was detected (m.16292C/T, p = 0.007, m.16189T/C, p = 0.048). Only eight controls carried the m.16292T allele, five of whom belonged to haplogroup W that was initially enriched among these controls. m.16189T/C might create an uninterrupted poly-C tract located near a regulatory element involved in replication of mtDNA. Though follow-up of some D-loop variants still is conceivable, our hypothesis of a contribution of variation in the exclusively maternally inherited mtDNA to the observed larger correlations for BMI between mothers and their offspring could not be substantiated by the findings of the present study.

Gene Set of Nuclear-Encoded Mitochondrial Regulators Is Enriched for Common Inherited Variation in Obesity

There are hints of an altered mitochondrial function in obesity. Nuclear-encoded genes are relevant for mitochondrial function (3 gene sets of known relevant pathways: (1) 16 nuclear regulators of mitochondrial genes, (2) 91 genes for oxidative phosphorylation and (3) 966 nuclear-encoded mitochondrial genes). Gene set enrichment analysis (GSEA) showed no association with type 2 diabetes mellitus in these gene sets. Here we performed a GSEA for the same gene sets for obesity. Genome wide association study (GWAS) data from a case-control approach on 453 extremely obese children and adolescents and 435 lean adult controls were used for GSEA. For independent confirmation, we analyzed 705 obesity GWAS trios (extremely obese child and both biological parents) and a population-based GWAS sample (KORA F4, n = 1,743). A meta-analysis was performed on all three samples. In each sample, the distribution of significance levels between the respective gene set and those of all genes was compared using the leading-edge-fraction-comparison test (cut-offs between the 50th and 95th percentile of the set of all gene-wise corrected p-values) as implemented in the MAGENTA software. In the case-control sample, significant enrichment of associations with obesity was observed above the 50th percentile for the set of the 16 nuclear regulators of mitochondrial genes (pGSEA,50 = 0.0103). This finding was not confirmed in the trios (pGSEA,50 = 0.5991), but in KORA (pGSEA,50 = 0.0398). The meta-analysis again indicated a trend for enrichment (pMAGENTA,50 = 0.1052, pMAGENTA,75 = 0.0251). The GSEA revealed that weak association signals for obesity might be enriched in the gene set of 16 nuclear regulators of mitochondrial genes.

Comparison of metabolic profiles of acutely ill and short-term weight recovered patients with anorexia nervosa reveals alterations of 33 out of 163 metabolites

Starvation represents an extreme physiological state and entails numerous endocrine and metabolic adaptations. The large-scale application of metabolomics to patients with acute anorexia nervosa (AN) should lead to the identification of state markers characteristic of starvation in general and of the starvation specifically associated with this eating disorder. Novel metabolomics technology has not yet been applied to this disorder. Using a targeted metabolomics approach, we analysed 163 metabolite concentrations in 29 patients with AN in the acute stage of starvation (T0) and after short-term weight recovery (T1). Of the 163 metabolites of the respective kit, 112 metabolites were quantified within restrictive quality control limits. We hypothesized that concentrations are different in patients in the acute stage of starvation (T0) and after weight gain (T1). Furthermore, we compared all 112 metabolite concentrations of patients at the two time points (T0, T1) with those of 16 age and gender matched healthy controls. Thirty-three of the metabolite serum levels were found significantly different between T0 and T1. At the acute stage of starvation (T0) serum concentrations of 90 metabolites differed significantly from those of healthy controls. Concentrations of controls mostly differed even more strongly from those of AN patients after short-term weight recovery than at the acute stage of starvation. We conclude that AN entails profound and longer lasting alterations of a large number of serum metabolites. Further studies are warranted to distinguish between state and trait related alterations and to establish diagnostic sensitivity and specificity of the thus altered metabolites.

The fatty acid amide hydrolase (FAAH) gene variant rs324420 AA/AC is not associated with weight loss in a 1-year lifestyle intervention for obese children and adolescents.

Adult obese carriers of the A allele of SNP rs324420 in the fatty acid amide hydrolase (FAAH) gene lose more weight and improve associated phenotypes better than non-carriers during an intervention. We aimed to replicate this finding in obese children and adolescents undergoing a one year lifestyle intervention (Obeldicks program). A total of 453 overweight and obese children and adolescents (10.8±2.6 years, BMI-SDS 2.4±0.5; 55% girls) were genotyped for rs324420 (C/A) by restriction fragment length polymorphism (RFLP) analysis. Participants were prescribed a balanced diet, containing 55 En% carbohydrates, 30 En% fat, and 15 En% proteins. Moreover, they took part in an exercise therapy once a week. Blood was taken at baseline and after 1 year of intervention. Anthropometric (height, weight, BMI, and BMI-SDS) and plasma parameters (total cholesterol, LDL-cholesterol, HDL-cholesterol, triacylglycerides, glucose, insulin, and HOMA) as well as blood pressure were measured. Both mean BMI and BMI-SDS improved significantly. The mean systolic blood pressure was also lowered and concentrations of HDL-cholesterol increased significantly. However, none of the measured changes were associated with FAAH rs324420 AA/AC genotype. We did not detect evidence for an association of FAAH genotypes with weight reduction in overweight and obese children and adolescents. Hence, the previous finding in adults could not be confirmed. As the length (1 year as compared to 3 months) and mode of treatment (hypocaloric diet in adults vs. physical activity plus balanced meals) of the interventions varied, these parameters might have influenced the inconsistent results.

Analysis of Genes Involved in Body Weight Regulation by Targeted Re-Sequencing

Introduction Genes involved in body weight regulation that were previously investigated in genome-wide association studies (GWAS) and in animal models were target-enriched followed by massive parallel next generation sequencing. Methods We enriched and re-sequenced continuous genomic regions comprising FTO, MC4R, TMEM18, SDCCAG8, TKNS, MSRA and TBC1D1 in a screening sample of 196 extremely obese children and adolescents with age and sex specific body mass index (BMI) ≥ 99th percentile and 176 lean adults (BMI ≤ 15th percentile). 22 variants were confirmed by Sanger sequencing. Genotyping was performed in up to 705 independent obesity trios (extremely obese child and both parents), 243 extremely obese cases and 261 lean adults. Results and Conclusion We detected 20 different non-synonymous variants, one frame shift and one nonsense mutation in the 7 continuous genomic regions in study groups of different weight extremes. For SNP Arg695Cys (rs58983546) in TBC1D1 we detected nominal association with obesity (pTDT = 0.03 in 705 trios). Eleven of the variants were rare, thus were only detected heterozygously in up to ten individual(s) of the complete screening sample of 372 individuals. Two of them (in FTO and MSRA) were found in lean individuals, nine in extremely obese. In silico analyses of the 11 variants did not reveal functional implications for the mutations. Concordant with our hypothesis we detected a rare variant that potentially leads to loss of FTO function in a lean individual. For TBC1D1, in contrary to our hypothesis, the loss of function variant (Arg443Stop) was found in an obese individual. Functional in vitro studies are warranted.

Fine Mapping of a GWAS-Derived Obesity Candidate Region on Chromosome 16p11.2

Introduction Large-scale genome-wide association studies (GWASs) have identified 97 chromosomal loci associated with increased body mass index in population-based studies on adults. One of these SNPs, rs7359397, tags a large region (approx. 1MB) with high linkage disequilibrium (r²>0.7), which comprises five genes (SH2B1, APOBR, sulfotransferases: SULT1A1 and SULT1A2, TUFM). We had previously described a rare mutation in SH2B1 solely identified in extremely obese individuals but not in lean controls. Methods The coding regions of the genes APOBR, SULT1A1, SULT1A2, and TUFM were screened for mutations (dHPLC, SSCP, Sanger re-sequencing) in 95 extremely obese children and adolescents. Detected non-synonymous variants were genotyped (TaqMan SNP Genotyping, MALDI TOF, PCR-RFLP) in independent large study groups (up to 3,210 extremely obese/overweight cases, 485 lean controls and 615 obesity trios). In silico tools were used for the prediction of potential functional effects of detected variants. Results Except for TUFM we detected non-synonymous variants in all screened genes. Two polymorphisms rs180743 (APOBR p.Pro428Ala) and rs3833080 (APOBR p.Gly369_Asp370del9) showed nominal association to (extreme) obesity (uncorrected p = 0.003 and p = 0.002, respectively). In silico analyses predicted a functional implication for rs180743 (APOBR p.Pro428Ala). Both APOBR variants are located in the repetitive region with unknown function. Conclusion Variants in APOBR contributed as strongly as variants in SH2B1 to the association with extreme obesity in the chromosomal region chr16p11.2. In silico analyses implied no functional effect of several of the detected variants. Further in vitro or in vivo analyses on the functional implications of the obesity associated variants are warranted.