Long-Term Weight-Loss in Gastric Bypass Patients Carrying Melanocortin 4 Receptor Variants

<div><p>Background</p><p>The melanocortin 4 receptor (MC4R) critically regulates feeding and satiety. Rare variants in <i>MC4R</i> are predominantly found in obese individuals. Though some rare variants in <i>MC4R</i> discovered in patients have defects in localization, ligand binding and signaling to cAMP, many have no recognized defects.</p><p>Subjects/Methods</p><p>In our cohort of 1433 obese subjects that underwent Roux-en-Y Gastric Bypass (RYGB) surgery, we found fifteen variants of <i>MC4R</i>. We matched rare variant carriers to patients with the <i>MC4R</i> reference alleles for gender, age, starting BMI and T2D to determine the variant effect on weight-loss post-RYGB. <i>In vitro</i>, we determined expression of mutant receptors by ELISA and western blot, and cAMP production by microscopy.</p><p>Results</p><p>While carrying a rare <i>MC4R</i> 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, <i>V95I</i>, <i>I137T</i> or <i>L250Q</i>, lost less weight after surgery. <i>In vitro</i>, 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 <i>in vitro</i> signaling and pre- or post-surgery clinical phenotype.</p><p>Conclusions</p><p>These data suggest that subtle differences in receptor signaling conferred by rare <i>MC4R</i> variants combined with additional factors predispose carriers to obesity. In the absence of complete <i>MC4R</i> 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.</p></div

cAMP Assay of MC4R mutants.

<p>cAMP production of MC4R mutants after stimulation with 10 nmol/L melanotan II (MTII) and then 100 µmol/L forskolin to activate maximum receptor-independent cAMP response. MTII stimulation was normalized to baseline cAMP production and plotted as a percentage of forskolin in the same cell. Mutants similar to wild-type (Black ○) are designated with the symbol (Gray ○). MC4R novel mutant G34A (Red ○) and those that have different statistically altered cAMP signaling (p<0.01 compared to wild-type by a one way ANOVA with Dunnet's post hoc test) (I137T (Green ○), D90N (Blue ○) and C326R (Purple ○)) are highlighted with different colored symbols. The D90N variant was not found in this cohort, but included as a control.</p

BMIs of patients with rare variants only found in obese populations.

<p>The pre-surgery BMIs of patients with rare variants were matched with non-carrier patients of the same gender, T2D status, insulin medication status and similar age (within 5 years) (black symbols). The variant carrier's age, sex and T2D status is also listed (○). *The starting BMI range for the matched patient was extended to ±2.</p

Expression of MC4R mutants.

<p>A) ELISA of HEK293 cells expressing wild-type MC4R or mutations. The cell surface expression was normalized to total expression for each mutant and then to wild-type receptor for that batch (* denotes p<0.05 compared to wild-type by a one way ANOVA with Dunnet's post-hoc test). □ MC4R mutant D90N was not found in our cohort. B) Cell surface localization of HEK-293 cells expressing mutant or wild-type (BBS-MC4R) constructs labeled with Bungarotoxin-Texas Red. C) Normalized GFP loading reveals no differences in HA-MC4R mutant lifespan. HA-MC4R expression was normalized to GFP expression and plotted as a percentage of wild-type for each blot (n≥3).</p

Clinical variables for Roux-en Y gastric bypass patients with rare <i>MC4R</i> variants.

<p>Non-carrier patients are separated into non-diabetics, type 2 diabetics (T2D) and T2Ds taking insulin. Pre-surgery BMIs and blood pressure are reported as three month averages. No data were collected after surgery for the S4F (denoted ¶).</p><p>*The total number of patients differ in the six month remission data because those patients that did not have enough follow-up data collected were excluded from analysis (T2D-25 patients, T2D insulin dependent-9 patients).</p

BMIs of patients with rare variants reported in both obese and lean populations.

<p>The pre-surgery BMIs of patients with rare variants were matched with non-carrier patients of the same gender, T2D status, insulin medication status and similar age (within 5 years) (black symbols). The variant carrier's age, sex and T2D status is also listed (○). *The starting BMI range for the matches was extended to ±2. **The starting BMI range for the matches was extended to ±3 or the age difference extended to ±8 years.</p