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

RESEARCH ARTICLE Analysis of Genes Involved in Body Weight Regulation by Targeted Re-Sequencing Anna-Lena Volckmar1☯, Chung Ting Han2,3☯, Carolin Pütter4, Stefan Haas2, Carla I. G. Vogel5, Nadja Knoll1, Christoph Struve1, Maria Göbel1, Katharina Haas1, Nikolas Herrfurth1, Ivonne Jarick6, Harald Grallert7, Annette Schürmann8,9, Hadi AlHasani9,10, Johannes Hebebrand1, Sascha Sauer2,11☯, Anke Hinney1☯* 1 Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, University Hospital Essen, Essen, Germany, 2 Nutrigenomics and Gene Regulation, Max-Planck-Institute for Molecular Genetics, Berlin, Germany, 3 Genomics, Core Facilities and Technology, Institute of Molecular Biology (IMB), Mainz, Germany, 4 Institute for Medical Informatics, Biometry and Epidemiology (IMIBE), University Hospital Essen, Essen, Germany, 5 Department of Animal and Food Production, Santa Catarina State University, Lages, Brazil, 6 Institute of Medical Biometry and Epidemiology, Philipps-University of Marburg, Marburg, Germany, 7 Institute of Epidemiology, Helmholtz-Zentrum Munich, Munich, Germany, 8 Institute of Experimental Diabetology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany, 9 German Center for Diabetes Research (DZD), München-Neuherberg, Germany, 10 Institute of Pathobiochemistry, German Diabetes Center and German Center for Diabetes Research, Düsseldorf, Germany, 11 CU Systems Medicine, University of Wuerzburg, Wuerzburg, Germany OPEN ACCESS Citation: Volckmar A-L, Han CT, Pütter C, Haas S, Vogel CIG, Knoll N, et al. (2016) Analysis of Genes Involved in Body Weight Regulation by Targeted ReSequencing. PLoS ONE 11(2): e0147904. doi:10.1371/journal.pone.0147904 ☯ These authors contributed equally to this work. * Abstract Editor: Juergen Eckel, GDC, GERMANY Received: September 2, 2015 Accepted: January 11, 2016 Published: February 1, 2016 Copyright: © 2016 Volckmar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The ascertainment of study groups belonging to different weight extremes was supported by the Deutsche Forschungsgemeinschaft (DFG). All other analyses were supported by the Deutsche Forschungsgesellschaft (HI865/2-1, AH) and the Bundesministerium für Bildung und Forschung (01KU0903, AH; 0315082/01EA1303, SS; NGFNplus: 01GS0820, JH) and the IFORES program of the University of Duisburg-Essen (AH, JH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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 PLOS ONE | DOI:10.1371/journal.pone.0147904 February 1, 2016 1 / 16 Next Generation Sequencing in Genes for Weight Regulation Competing Interests: The authors have declared that no competing interests exist. 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. Introduction Obesity is one of the major health problems, which is associated with increased mortality and morbidity [1]. To date more than 100 body mass index (BMI) associated loci have been published from GWAS [2, 3, 4]. On the other hand, murine models have shown other relevant genes for weight regulation which were not detected by GWAS (e.g. Tbc1d1 [5]). The aim of this study is to identify functionally relevant mutations in genes involved in body weight regulation derived from either GWAS or murine models. The rationale for the chosen genes is briefly delineated in the following: TBC1D1 In SJL mice a specific mutation in the Tbc1d1 gene (fsAla4047 4119) results in a truncated protein lacking the TBC Rab-GTPase-activating protein domain. The mutation led to resistance to diet-induced obesity [5] and its causality for the phenotype was confirmed in Tbc1d1 knockout mice [6, 7]. In mouse skeletal muscle cells, knockdown of Tbc1d1 increased fatty acid uptake and oxidation whereas overexpression of Tbc1d1 had the opposite effect [5]. Mutations in the human TBC1D1 gene are associated with increased risk for familial obesity [8, 9]. Polygenic effects on BMI and waist circumference in humans were also described for some TBC1D1 SNP alleles [10]. FTO The fat mass and obesity associated gene (FTO) harbors GWAS derived polygenic variants with the largest effect size on BMI (FTO; [3, 11]). The body weight of carriers of one risk allele is increased by approximately 1.5 kgs. The effect of the risk alleles in intron 1 of FTO on body weight had been replicated in most analyzed study groups, either across the whole life span [3, 11–14] or in all analyzed ethnic groups [11, 15, 16]. FTO is highly expressed in the hypothalamus which is the key region for control of food intake [17]. FTO belongs to a gene family that is involved in post-translational modifications, DNA repair and fatty acid metabolism [18–20]. Fto deficient mice are lean as a consequence of increased energy expenditure [21] and show an improvement in metabolic syndrome in comparison to leptin-deficient mice, wild type or heterozygotes for Fto [22]. Mice over expressing Fto showed a dose-dependent and diet-independent increase in body weight and fat mass [23]. This is consistent with the finding tha (...truncated)