Supplementary Material for: Clinical and Functional Relevance of Melanocortin-4 Receptor Variants in Obese German Children

<b><i>Background:</i></b> Variants in the melanocortin-4 receptor <i>(MC4R)</i> gene are the most frequent cause of monogenic obesity. The relevance of <i>MC4R</i> variations with respect to clinical phenotype and biochemical function remains controversial. <b><i>Methods:</i></b> We sequenced the <i>MC4R</i> gene in 510 overweight/obese children. The clinical phenotype was assessed in a case-control setting matched for age, gender, puberty and body mass index. Identified <i>MC4R</i> variants were functionally characterized in vitro. <b><i>Results:</i></b> The frequency of <i>MC4R</i> variants was 5.3%, with functionally relevant mutations (D<sup>90</sup>N, V<sup>103</sup>I/S<sup>127</sup>L, R<sup>165</sup>W, G<sup>181</sup>D) occurring in 1.2% (confidence interval 0.26–2.15) of our sample. 4.1% were carriers of variants (Y<sup>35</sup>Y, V<sup>103</sup>I, T<sup>112</sup>M, M<sup>200</sup>V, I<sup>251</sup>L) with preserved receptor function in vitro. We did not detect large heterozygous deletions by multiple-ligand probe amplification assay. There were no differences in anthropometric or metabolic parameters between children with loss-of-function mutations and noncarriers. Carriers of the V<sup>103</sup>I or I<sup>251</sup>L variant had higher high-density lipoprotein cholesterol and HbA1c levels than matched noncarriers of <i>MC4R</i> variants. <b><i>Conclusions:</i></b> In our data set of childhood obesity in central Germany, we identified functionally relevant mutations in the <i>MC4R</i> gene in only 1.2% of the children. There were no major significant phenotypic differences between obese children with and without <i>MC4R</i> mutations. Hence, the diagnosis of genetically caused obesity due to <i>MC4R</i> mutation should be made with caution.