Long-Term Weight-Loss in Gastric Bypass Patients Carrying Melanocortin 4 Receptor Variants Bryn S. Moore 1 , Uyenlinh L. Mirshahi 1 , Evan A. Yost 1 , Ann N. Stepanchick 1 , Michael D. Bedrin 1 , Amanda M. Styer 1 , Kathryn K. Jackson 1 , Christopher D. Still 2 , Gerda E. Breitwieser 1 , Glenn S. Gerhard 1 , David J. Carey 1 , Tooraj Mirshahi 1,2 * 1 Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania, United States of America, 2 Geisinger Obesity Institute, Geisinger Clinic, Danville, Pennsylvania, United States of America Abstract Background: The melanocortin 4 receptor (MC4R) critically regulates feeding and satiety. Rare variants in MC4R are predominantly found in obese individuals. Though some rare variants in MC4R discovered in patients have defects in localization, ligand binding and signaling to cAMP, many have no recognized defects. Subjects/Methods: In our cohort of 1433 obese subjects that underwent Roux-en-Y Gastric Bypass (RYGB) surgery, we found fifteen variants of MC4R. We matched rare variant carriers to patients with the MC4R reference alleles for gender, age, starting BMI and T2D to determine the variant effect on weight-loss post-RYGB. In vitro, we determined expression of mutant receptors by ELISA and western blot, and cAMP production by microscopy. Results: While carrying a rare MC4R allele is associated with obesity, carriers of rare variants exhibited comparable weight- loss after RYGB to non-carriers. However, subjects carrying three of these variants, V95I, I137T or L250Q, lost less weight after surgery. In vitro, the R305Q mutation caused a defect in cell surface expression while only the I137T and C326R mutations showed impaired cAMP signaling. Despite these apparent differences, there was no correlation between in vitro signaling and pre- or post-surgery clinical phenotype. Conclusions: These data suggest that subtle differences in receptor signaling conferred by rare MC4R variants combined with additional factors predispose carriers to obesity. In the absence of complete MC4R deficiency, these differences can be overcome by the powerful weight-reducing effects of bariatric surgery. In a complex disorder such as obesity, genetic variants that cause subtle defects that have cumulative effects can be overcome after appropriate clinical intervention. Citation: Moore BS, Mirshahi UL, Yost EA, Stepanchick AN, Bedrin MD, et al. (2014) Long-Term Weight-Loss in Gastric Bypass Patients Carrying Melanocortin 4 Receptor Variants. PLoS ONE 9(4): e93629. doi:10.1371/journal.pone.0093629 Editor: Miguel Lo ´ pez, University of Santiago de Compostela School of Medicine - CIMUS, Spain Received December 18, 2013; Accepted February 6, 2014; Published April 4, 2014 Copyright: ß 2014 Moore 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. Funding: This work was supported by the Geisinger Clinic and National Institutes of Health DK092775 to TM, and DK072488 to GSG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: tmirshahi@geisinger.edu Introduction Obesity is a worldwide epidemic that contributes to comorbid- ities such as diabetes and heart disease [1]. Severe obesity unresponsive to medication and dieting can be effectively treated with bariatric surgery. Roux-en Y gastric bypass (RYGB), vertical sleeve gastrectomy and gastric banding are the most common bariatric surgeries [2]. Importantly, type 2 diabetes (T2D) often remits following RYGB, before significant weight-loss occurs [3,4]. RYGB improves blood glucose levels more rapidly and completely than caloric restricted weight-loss or other common bariatric procedures [2,5]. Regulation of feeding and satiety, essential for maintaining healthy weight, occurs in the hypothalamus. In the fed state, insulin and leptin stimulate neurons expressing proopiomelano- cortin (POMC) to release a-melanocyte stimulating hormone (a- MSH) and b-MSH [6]. a-MSH binds and activates melanocortin 4 receptor (MC4R), resulting in an increase in cAMP, inducing the sensation of satiety. Insulin and leptin also inhibit neurons expressing neuropeptide Y (NPY) and agouti-related protein (AgRP). AgRP is a biased agonist of MC4R that stimulates appetite [7]. The balance between the signals from POMC and AgRP neurons critically regulates feeding behavior and energy homeostasis. The development of obesity, as well as the degree of weight-loss following RYGB surgery can be greatly impacted by genetic variants [8,9]. For example, extreme obesity can be due to mutations in genes such as MC4R [9–12]. Common missense variants in MC4R, occurring in both lean and obese people at equal frequencies, have also been described [8,12–14]. Recently, we reported that having the common MC4R variant I251L leads to better weight-loss and weight maintenance following RYGB [8]. Patients with an I251L allele also resolved their T2D more quickly than patients with two copies of the MC4R reference allele [15]. Numerous rare MC4R variants have also been reported, primarily identified in cohorts of obese individuals [16–18]. While, some of PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e93629