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

Evidence for three genetic loci involved in both anorexia nervosa risk and variation of body mass index

Palmer LJ ... GIANT collaborationThe maintenance of normal body weight is disrupted in patients with anorexia nervosa (AN) for prolonged periods of time. Prior to the onset of AN, premorbid body mass index (BMI) spans the entire range from underweight to obese. After recovery, patients have reduced rates of overweight and obesity. As such, loci involved in body weight regulation may also be relevant for AN and vice versa. Our primary analysis comprised a cross-trait analysis of the 1000 single-nucleotide polymorphisms (SNPs) with the lowest P-values in a genome-wide association meta-analysis (GWAMA) of AN (GCAN) for evidence of association in the largest published GWAMA for BMI (GIANT). Subsequently we performed sex-stratified analyses for these 1000 SNPs. Functional ex vivo studies on four genes ensued. Lastly, a look-up of GWAMA-derived BMI-related loci was performed in the AN GWAMA. We detected significant associations (P-values <5 × 10⁻⁵, Bonferroni-corrected P<0.05) for nine SNP alleles at three independent loci. Interestingly, all AN susceptibility alleles were consistently associated with increased BMI. None of the genes (chr. 10: CTBP2, chr. 19: CCNE1, chr. 2: CARF and NBEAL1; the latter is a region with high linkage disequilibrium) nearest to these SNPs has previously been associated with AN or obesity. Sex-stratified analyses revealed that the strongest BMI signal originated predominantly from females (chr. 10 rs1561589; Poverall: 2.47 × 10⁻⁰⁶/Pfemales: 3.45 × 10⁻⁰⁷/Pmales: 0.043). Functional ex vivo studies in mice revealed reduced hypothalamic expression of Ctbp2 and Nbeal1 after fasting. Hypothalamic expression of Ctbp2 was increased in diet-induced obese (DIO) mice as compared with age-matched lean controls. We observed no evidence for associations for the look-up of BMI-related loci in the AN GWAMA. A cross-trait analysis of AN and BMI loci revealed variants at three chromosomal loci with potential joint impact. The chromosome 10 locus is particularly promising given that the association with obesity was primarily driven by females. In addition, the detected altered hypothalamic expression patterns of Ctbp2 and Nbeal1 as a result of fasting and DIO implicate these genes in weight regulation.A Hinney ... GIANT ... et al

Non-synonymous, stop and frameshift variants¹ detected by NGS of the genes <i>FTO</i>, <i>TMEM18</i>, <i>SDCCAG8</i>, <i>TKNS</i>, <i>MSRA</i> and <i>TBC1D1</i> in 196 extremely obese children and adolescents and 176 lean adults.

<p>Non-synonymous, stop and frameshift variants<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147904#t002fn002" target="_blank">¹</a> detected by NGS of the genes <i>FTO</i>, <i>TMEM18</i>, <i>SDCCAG8</i>, <i>TKNS</i>, <i>MSRA</i> and <i>TBC1D1</i> in 196 extremely obese children and adolescents and 176 lean adults.</p

Phenotypes of heterozygous carriers of rare non-synonymous variants in the screening sample and extended <i>in silico</i> analyses for the respective variants.

<p>Phenotypes of heterozygous carriers of rare non-synonymous variants in the screening sample and extended <i>in silico</i> analyses for the respective variants.</p

Transmission disequilibrium test of the 22 variants detected by NGS in 355 German obesity trios.

<p>Transmission disequilibrium test of the 22 variants detected by NGS in 355 German obesity trios.</p

Flowchart of the experimental setup.

<p>Flowchart of the experimental setup.</p