Bariatric surgery in a patient with complete MC4R deficiency - Nature

International Journal of Obesity (2011) 35, 457–461 & 2011 Macmillan Publishers Limited All rights reserved 0307-0565/11 www.nature.com/ijo

SHORT COMMUNICATION Bariatric surgery in a patient with complete MC4R deficiency IR Aslan1, SA Ranadive2, BA Ersoy3, SJ Rogers4, RH Lustig1 and C Vaisse3 1 Division of Endocrinology, Department of Pediatrics, University of California San Francisco (UCSF), San Francisco, CA, USA; 2Department of Endocrinology, Children’s Hospital and Research Center Oakland, Oakland, CA, USA; 3Department of Medicine and Diabetes Center, University of California San Francisco, San Francisco, CA, USA and 4Department of Surgery, University of California San Francisco, San Francisco, CA, USA

Bariatric surgery is often successful for treatment of severe obesity. The mechanisms of weight loss after bariatric surgery and the role of central energy homeostatic pathways in this weight loss process are not well understood. The study of individuals with complete loss of function of genes important in the leptin–melanocortin system may help establish the significance of these pathways for weight loss after bariatric surgery. We describe the outcome of bariatric surgery in an adolescent with compound heterozygosity and complete functional loss of both alleles of the melanocortin 4 receptor (MC4R). The patient underwent 8 laparoscopic adjustable gastric banding and truncal vagotomy at 1812 years of age, which resulted in initial, but not long-term weight loss. Our experience with this patient suggests that complete MC4R deficiency impairs response to gastric banding and results in poor weight loss after this surgery. International Journal of Obesity (2011) 35, 457–461; doi:10.1038/ijo.2010.168; published online 24 August 2010 Keywords: melanocortin-4 receptor; adolescent; gastric banding; vagotomy; bariatric surgery

Introduction Bariatric surgery, laparoscopic gastric bypass or gastric banding, is the most effective therapy for severe obesity in adults. Lifestyle modification and behavioral therapy remain the preferable first options for adolescents. Gastric banding, however, should be considered for severely obese adolescents in whom nonsurgical approaches have failed.1 How bariatric surgery alters the compensatory response to weight loss is not well understood. Specifically, it is unknown whether the integrity of central neuroendocrine systems regulating energy homeostasis is required for the success of bariatric surgery. The major neuroendocrine pathway involved in long-term energy homeostasis is the leptin–melanocortin system. This system integrates information about peripheral energy stores, relayed primarily by the adipocyte-secreted hormone leptin, leading to adaptive changes in food intake and energy

Correspondence: Dr C Vaisse, Department of Medicine and Diabetes Center, University of California San Francisco, 513 Parnassus Avenue, Room HSW1113, San Francisco, CA 94143-0573, USA. E-mail: [email protected] Received 21 March 2010; revised 9 July 2010; accepted 18 July 2010; published online 24 August 2010

expenditure.2 Bariatric surgery lowers both adiposity and leptin levels, inferring improved leptin sensitivity postoperatively. A major mediator of the central effect of leptin is the melanocortin 4 receptor (MC4R). MC4R is a G-protein-coupled receptor expressed in neurons of the paraventricular nucleus of the hypothalamus,3 which regulates food intake and maintains long-term energy homeostasis by integrating signals provided by its agonist, a-melanocyte-stimulating hormone, and antagonist, Agouti-related peptide, from leptin-sensitive neurons within the arcuate nucleus.4 Heterozygous mutations in MC4R are the most common monogenic form of obesity, found in 2.5% of severely obese children and adults.5,6 These patients present with very early-onset severe obesity and hyperphagia without other physical, hormonal or developmental consequences.5 In contrast, patients with homozygous or compound heterozygous MC4R mutations are rare with fewer than 10 patients described.7–10 The study of MC4R null mutants has highlighted the role of the melanocortin system in energy homeostasis. The clinical outcome of such patients after bariatric surgery may provide information on the role of the leptin–melanocortin pathway in weight loss after bariatric surgery. Here, we describe the first patient with complete MC4R deficiency who underwent bariatric surgery.

Bariatric surgery in MC4R deficiency IR Aslan et al

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Subject and methods Case report A 17-year-old Caucasian male with severe obesity was referred for hyperphagia and abnormal weight gain since infancy. At presentation, his weight was 103.4 kg (Z-score of 2.2), height was 175 cm (Z-score of –0.04) and body mass 8 years of age, index was 33.8 kg m–2 (Z-score of 2.2). At 1812 his weight was 166.2 kg and body mass index was 54.2 kg m–2 (Figure 1). The patient’s parents are not consanguineous. His birth weight was 3.25 kg. His parents report he had insatiable hunger since 2 months of age, gaining 11 pounds between 3 and 5 months of age. He progressed through puberty normally, but had a history of depression, gastroesophageal reflux and obstructive sleep apnea requiring tonsillectomy and adenoidectomy. The patient was not taking any medications. At 18 years of age, his serum leptin was 37.9 ng ml–1 (appropriate for his body fat), ghrelin was 452 pg ml–1 (appropriate for his level of obesity), fasting low-density lipoprotein 141 mg per 100 ml, total cholesterol 210 mg day–1, triglycerides 160 mg per 100 ml, insulin 50.3 U l–1, glucose 85 mg per 100 ml, aspartate aminotransferase 29 U l–1 and alanine transaminase 58 U l–1. His daily caloric intake was approximately 2500 kcal.

MC4R functional studies HEK293 cells expressing the luciferase reporter under the control of a cyclic adenosine monophosphate-responsive promoter11,12 were maintained in a-minimum essential medium supplemented with 10% calf serum (Invitrogen, San Diego, CA, USA), L-glutamine, non-essential amino acids and penicillin/streptomycin. Transfection and dose response assays were performed using a-melanocyte-stimulating hormone (Sigma, St Louis, MO, USA) added to the medium at the desired concentrations.11,12 Luciferase activity, representing cAMP production through MC4R activation, was assessed using the Steady-Glo Luciferase Assay System (Promega, Madison, WI, USA) and a microplate luminescence counter (Packard Instrument, Downers Grove, IL, USA). Luciferase activity on MC4R activation was normalized for transfection efficiency by co-transfection with Renilla luciferase, and the results were expressed as percentage of maximum stimulation of the wild-type receptor.

Genetic studies Genomic DNA was extracted from white blood cells using standard methods. Forward (50 -ATCAATTCAGGGGGACA CTG-30 ) and reverse (50 -TGCATGTTCCTATATTGCGTG-30 ) primers were used to amplify the coding region of MC4R as

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International Journal of Obesity


459 previously described.13 PCR products were sequenced using the above and two internal primers: 50 -TGTAGCTCCTT GCTTGCATC-30 and 50 -GGCCATCAGGAACATGTGGA-30 . Sequencing was performed on an ABI PRISM 3700 automated DNA sequencer using a BigDye terminator kit according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA). Sequencing of the MC4R gene from the patient and his parents showed that the patient was compound heterozygous for 750–751delGA (Ile251fsX34) inherited from his mother, and 873–875delCAT (Ile291del) inherited from his father (Figure 2a). Wild-type and mutant alleles of MC4R were amplified and cloned into the vector pcDNA 3.1 (Invitrogen). All expression vectors were sequenced to establish the presence of the mutation and absence of any induced mutations. The maternal 750–751delGA frameshift mutation, which results in a receptor that is truncated at the sixth transmembrane domain, has been previously described.8 The paternal 873–875delCAT mutation, which results in an in-frame deletion of isoleucine 291, is novel. To assess the function of this novel mutant, we assayed its activation by a-melanocytestimulating hormone in transfected cells; both mutations encoded proteins devoid of activity (Figures 2b and c).

age. The LTV was performed as part of a research protocol approved by the Committee on Human Research at UCSF. The posterior vagal trunk was identified on the posterior aspect of the esophagus. The anterior vagal trunk and accessory fibers were identified on the anterior surface of the esophagus, gastroesophageal junction and proximal stomach. A 2 cm portion of the posterior vagal nerve trunk, and a 1 cm portion of the anterior vagal nerve trunk were resected and sent for pathologic confirmation. The gastric band was placed using a pars flacida technique and secured in place with a standard fundoplication around the band using permanent seromuscular sutures, leaving an approximately 30 ml reservoir gastric pouch proximal to the band. The patient was instructed with the standard restrictive dietary protocol including ingestion of liquids only for 1 week following surgery, followed by a mechanical soft diet for 1 week, and then a calorie-restricted diet of o1000 kcal per day. He was instructed to eat food slowly and chew thoroughly to prevent dyspepsia or dysphagia. The patient was to avoid high energy density foods and liquids, and record daily calorie count. To improve caloric restriction, his gastric band was adjusted twice. These adjustments occurred at 3 weeks ( þ 1.5 ml) and at 10 months ( þ 2.0 ml) after surgery (Figure 1). Total weight loss (kg) and percentage of excess weight lost (EWL) after surgery were calculated using Centers for Disease Control and Prevention growth charts and defined excess weight as the weight above the 85% percentile of body mass index for age and sex.14 The patient initially lost 11.6 kg over 4 months following surgery (Figure 1), for an EWL of 12%. However, by 12 months postoperatively he had regained his

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Surgery and outcome Before surgery, the patient gained 65 kg, from 100.5 to 166.2 kg, because of excessive eating in an unrestricted college environment. The patient underwent laparoscopic truncal vagotomy 8 years of (LTV) and adjustable gastric banding (LAGB) at 1812

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Figure 2 (a) Patient’s pedigree showing direct MC4R sequencing from parents and patient. The mutant genes were cloned into pcDNA 3.1: clone 1, MC4R 750–751delGA; clone 2, MC4R 873–875delCAT. (b, c) Functional analysis of mutant MC4Rs. Dose-response to a-MSH in mutant and wild-type receptor. Data points represent means±s.e.m. of at least three experiments performed in triplicate. The data are expressed as a percentage of the maximal wild-type activity.



460 preoperative weight and gained an additional 6.5 kg (EWL 7%) and his percentage of excess body mass index lost was 7.5%.15 One could argue that our patient’s weight stabilized after bariatric surgery and despite the fact that he did not lose weight, the surgery might have helped him to maintain his weight and to prevent even greater weight gain. One year after surgery, his fasting low-density lipoprotein was 151 mg per 100 ml, total cholesterol 214 mg day–1, triglycerides 157 mg per 100 ml, insulin 35.5 U l–1, glucose 78 mg per 100 ml, aspartate aminotransferase 25 U l–1 and alanine transaminase 47 U l–1. The patient still describes insatiable hunger. He has not followed a postoperative caloric restriction program and has missed several followup appointments.

This report provides insight into the underlying physiology of the therapeutic effects of LAGB. Larger studies will be required to draw clear conclusions about the role of the leptin–melanocortin system in the response to bariatric surgery, optimal time for surgical intervention and whether routine screening for MC4R mutations would be cost effective on these patients. As the homozygous MC4R null patients are rare, a case–control study is not feasible. Animal studies using homozygous MC4R knockout mice may help us understand if MC4R has a role in resetting the metabolic state after bariatric surgery.

Conflict of interest The authors declare no conflict of interest.

Discussion Our patient exhibits compound heterozygosity for two null MC4R mutations, which likely compromise his satiety response significantly and affect his response to bariatric surgery. However, other factors such as non-compliance with follow-up appointments and poor adherence to diet and physical activity modifications may also have contributed to failure of weight loss after surgery. Before surgery, our patient had an abnormally excessive weight gain when his environment was not restricted, re-enforcing the importance of lifestyle modifications in patients with severe obesity. Reinehr et al.16 showed that MC4R carriers can reduce their body weight to a similar degree at the end of 1 year of intense lifestyle intervention, but have difficulty maintaining weight loss. Our patient underwent LTV combined with LAGB. LAGB is a gastric restrictive procedure that lacks a neurohormonal mechanism to alter satiation.17 LAGB in obese adolescent males average 59% EWL and 40% EWL 2 and 3 years postoperatively.18,19 LTV for obesity is currently performed as a research protocol. The vagus nerve is downstream of the MC4R, mediates hunger and is crucial for energy storage.20 The leptin–melanocortin system is influenced by signals from the GI tract.21 Appetite and gut hormone changes are reported within days following Roux-en-Y gastric bypass (RYGB),22 with decreased appetite. Our patient might have had a better response to Roux-en-Y gastric bypass, given that the weight loss after LAGB is dependent on mechanical restriction without effects on neurohormonal appetite regulation (although the LTV was used to alter satiety). Currently, data on bariatric surgery outcome in patients with genetic obesity are limited. Bariatric surgery has also been attempted in patients with hypothalamic obesity after craniopharyngioma.23–25 Inge et al.24 described a 14-yearold male with hypothalamic obesity who experienced significant weight loss, correction of hyperinsulinemia, reduced ghrelin, improved satiety and amelioration of co-morbidities after Roux-en-Y gastric bypass. International Journal of Obesity

Acknowledgements This publication was supported by NIH/NCRR UCSF-CTSI UL1 RR024131. IA is supported by ADA mentor-based postdoctoral fellowship award, American Heart Association post-doctoral fellowship and Genentech clinical fellowship. SAR was supported by Pediatric Endocrinology Training Grant T32-DK07161. CV is supported by NIH RO1 DK DK60540 and DK068152 as well as an established investigator award from the American Heart Association: AHA#0740041N.

References 1 O’Brien PE, Sawyer SM, Laurie C, Brown WA, Skinner S, Veit F et al. Laparoscopic adjustable gastric banding in severely obese adolescents: a randomized trial. JAMA 2010; 303: 519–526. 2 Ranadive SA, Vaisse C. Lessons from extreme human obesity: monogenic disorders. Endocrinol Metab Clin North Am 2008; 37: 733–751, x. 3 Mountjoy KG, Mortrud MT, Low MJ, Simerly RB, Cone RD. Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain. Mol Endocrinol 1994; 8: 1298–1308. 4 Schwartz MW, Woods SC, Porte Jr D, Seeley RJ, Baskin DG. Central nervous system control of food intake. Nature 2000; 404: 661–671. 5 Lubrano-Berthelier C, Dubern B, Lacorte JM, Picard F, Shapiro A, Zhang S et al. Melanocortin 4 receptor mutations in a large cohort of severely obese adults: prevalence, functional classification, genotype-phenotype relationship, and lack of association with binge eating. J Clin Endocrinol Metab 2006; 91: 1811–1818. 6 Calton MA, Ersoy BA, Zhang S, Kane JP, Malloy MJ, Pullinger CR et al. Association of functionally significant melanocortin-4 but not melanocortin-3 receptor mutations with severe adult obesity in a large North-American case control study. Hum Mol Genet 2009; 18: 1140–1147. 7 Farooqi IS, Keogh JM, Yeo GS, Lank EJ, Cheetham T, O’Rahilly S. Clinical spectrum of obesity and mutations in the melanocortin 4 receptor gene. N Engl J Med 2003; 348: 1085–1095. 8 Lubrano-Berthelier C, Le Stunff C, Bougneres P, Vaisse C. A homozygous null mutation delineates the role of the


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10

11

12

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melanocortin-4 receptor in humans. J Clin Endocrinol Metab 2004; 89: 2028–2032. Dubern B, Bisbis S, Talbaoui H, Le Beyec J, Tounian P, Lacorte JM et al. Homozygous null mutation of the melanocortin-4 receptor and severe early-onset obesity. J Pediatr 2007; 150: 613–617, 7 e1. Kobayashi H, Ogawa Y, Shintani M, Ebihara K, Shimodahira M, Iwakura T et al. A Novel homozygous missense mutation of melanocortin-4 receptor (MC4R) in a Japanese woman with severe obesity. Diabetes 2002; 51: 243–246. Lubrano-Berthelier C, Durand E, Dubern B, Shapiro A, Dazin P, Weill J et al. Intracellular retention is a common characteristic of childhood obesity-associated MC4R mutations. Hum Mol Genet 2003; 12: 145–153. Lubrano-Berthelier C, Cavazos M, Le Stunff C, Haas K, Shapiro A, Zhang S et al. The human MC4R promoter: characterization and role in obesity. Diabetes 2003; 52: 2996–3000. Vaisse C, Clement K, Durand E, Hercberg S, Guy-Grand B, Froguel P. Melanocortin-4 receptor mutations are a frequent and heterogeneous cause of morbid obesity. J Clin Invest 2000; 106: 253–262. Kuczmarski RJ, Ogden CL, Grummer-Strawn LM, Flegal KM, Guo SS, Wei R et al. CDC growth charts: United States. Adv Data 2000; 8: 1–27. Deitel M, Gawdat K, Melissas J. Reporting weight loss 2007. Obes Surg 2007; 17: 565–568. Reinehr T, Hebebrand J, Friedel S, Toschke AM, Brumm H, Biebermann H et al. Lifestyle intervention in obese children with variations in the melanocortin 4 receptor gene. Obesity (Silver Spring) 2009; 17: 382–389.

17 Maggard MA, Shugarman LR, Suttorp M, Maglione M, Sugerman HJ, Livingston EH et al. Meta-analysis: surgical treatment of obesity. Ann Intern Med 2005; 142: 547–559. 18 Dolan K, Fielding G. A comparison of laparoscopic adjustable gastric banding in adolescents and adults. Surg Endosc 2004; 18: 45–47. 19 Angrisani L, Favretti F, Furbetta F, Paganelli M, Basso N, Doldi SB et al. Obese teenagers treated by Lap-Band system: the Italian experience. Surgery 2005; 138: 877–881. 20 Hellstrom PM, Geliebter A, Naslund E, Schmidt PT, Yahav EK, Hashim SA et al. Peripheral and central signals in the control of eating in normal, obese and binge-eating human subjects. Br J Nutr 2004; 92 (Suppl 1): S47–S57. 21 Ellacott KL, Halatchev IG, Cone RD. Interactions between gut peptides and the central melanocortin system in the regulation of energy homeostasis. Peptides 2006; 27: 340–349. 22 Pournaras DJ, le Roux CW. Obesity, gut hormones, and bariatric surgery. World J Surg 2009; 33: 1983–1988. 23 Rottembourg D, O’Gorman CS, Urbach S, Garneau PY, Langer JC, Van Vliet G et al. Outcome after bariatric surgery in two adolescents with hypothalamic obesity following treatment of craniopharyngioma. J Pediatr Endocrinol Metab 2009; 22: 867–872. 24 Inge TH, Pfluger P, Zeller M, Rose SR, Burget L, Sundararajan S et al. Gastric bypass surgery for treatment of hypothalamic obesity after craniopharyngioma therapy. Nat Clin Pract Endocrinol Metab 2007; 3: 606–609. 25 Muller HL, Gebhardt U, Wessel V, Schroder S, Kolb R, Sorensen N et al. First experiences with laparoscopic adjustable gastric banding (LAGB) in the treatment of patients with childhood craniopharyngioma and morbid obesity. Klin Padiatr 2007; 219: 323–325.
