A novel non-synonymous mutation in the melanocortin-4 receptor gene (MC4R) in a 2-year-old Austrian girl with extreme obesity

Background: Functionally relevant mutations in the melanocortin-4 receptor gene (MC4R) currently display the most common major gene/allele effect on extreme obesity. Objective: Mutation screen of the MC4R in consecutively ascertained Austrian children and adolescents with severe obesity, to analyse the phenotype of mutation carriers and to functionally characterise novel mutations. Subjects and Methods: 102 unrelated extremely obese children and adolescents (mean BMI 33.5 +/- 7.1 kg/m(2), > 97th centile; mean age 13.8 +/- 4.1yr) and 109 parents (79 mothers/30 fathers) of 88 of these patients were studied. The MC4P coding region was screened using denaturing high-performance liquid chromatography (dHPLC) PCR products of aberrant dHPLC pattern were re-sequenced. Signal transduction properties of mutant MC4R was investigated by challenge with the highly potent agonist NDP-alpha-MSH. Cell surface expression was determined by ELISA. Magnetic resonance imaging (MRI) of the central nervous system (CNS) was applied to a 2.3 year old index patient. Body fat and bone mineral content were assessed in three of the five mutation carriers by dual energy x-ray absorptiometry (DEXA). Oral glucose tolerance test (OGTT) was applied to some mutation carriers. Results: Heterozygous carriers of two non-synonymous mutations, two polymorphisms and a silent variation were identified within the study group. (1) A novel MC4P non-synonymous mutation (S136F) was detected in a 2.3 year old girl with extreme obesity (BMI 33.2 kg/m(2), > 99th centile); (2) a previously described non-synonymous mutation (V253I) was identified in an obese mother (BMI 28.1 kg/m(2)) who did not transmit this mutation to her extremely obese son; (3) two known polymorphisms (V103I and I251L) were also identified; and (4) one obese mother was carrier of a silent variation (c.594C > T; 1198). Co-segregation of S136F with the obesity phenotype was shown for three generations. In vitro functional studies revealed a complete loss of signal transduction activity of the mutant receptor while cell surface expression was only slightly reduced compared to the wild-type receptor. Conclusions: We detected a novel non-synonymous mutation (S136F) that leads to a complete loss of MC4R function in vitro.