Harald Brumm

Lifestyle Intervention in Obese Children With Variations in the Melanocortin 4 Receptor Gene

Because information on weight changes after lifestyle intervention in children with mutations in the melanocortin 4 receptor (MC4R) gene is scarce, we compared weight changes after lifestyle intervention between children with and without MC4R variations. A group of 514 overweight children (aged 5–16 years), who presented to participate in a 1‐year lifestyle intervention based on exercise, behavior, and nutrition therapy were screened for MC4R mutations. For comparison, children with MC4R mutations leading to reduced receptor function (group A) were each of them randomly matched with five children of same age and gender without MC4R mutations (group B). Changes of weight status were analyzed as change of BMI standard deviation scores (BMI‐SDSs). Furthermore, 16 children (3.1%) harbored MC4R mutations leading to reduced receptor function, and 17 (3.3%) children carried variations not leading to reduced receptor function. Children with and without MC4R mutations reduced their overweight at the end of intervention to a similar degree (P = 0.318 between groups based on an intention‐to‐treat analysis). The maintenance of weight loss after intervention among children with MC4R mutations leading to reduced receptor function failed in contrast to children without such mutations (P < 0.001 adjusted for BMI‐SDS at baseline, age, and gender in an intention‐to‐treat analysis). In conclusion, children with MC4R mutations leading to reduced receptor function were able to lose weight in a lifestyle intervention but had much greater difficulties to maintain this weight loss supporting the impact of these mutations on weight status.

A heterozygous mutation in the third transmembrane domain causes a dominant-negative effect on signalling capability of the MC4R.

Background: Heterozygous MC4R mutation is the most frequent cause of monogenic obesity. For most MC4R mutations a gene dosage effect seems to be the underlying mechanism. However, a dominant negative effect of a heterozygous MC4R mutation was recently identified, pointing to an additional mechanism of MC4R inactivation. Methods: The complete loss-of-function mutation (Ser136Phe), identified in a cohort of obese Austrian patients, was characterized for cell surface expression, signal transduction and ligand binding properties. Co-transfection studies tested for a dominant negative effect. Dimerization was investigated by a sandwich ELISA and by fluorescence resonance energy transfer (FRET) approach. Potential intramolecular interactions of Ser136 were studied by homologous receptor modelling based on the crystal structure of the β2-adrenergic receptor. Results: The Ser136Phe mutation showed a dominant negative effect. The sandwich ELISA and FRET approach demonstrated dimerization of mutant and wild type receptor. Receptor modelling revealed an essential function of Ser136 at transmembrane helix 3 (TMH3) for establishing H-bonds between TMH2, TMH3, and TMH7. The mutation Ser136Phe most likely disrupts this network and leads to an incompetent helix-helix arrangement in the mutated receptor. Conclusion: Identification of dominant negative MC4R mutations is important to fully understand receptor function and to determine receptor regions that are involved in MC4R dimer activation.

Rescue of Melanocortin 4 Receptor (MC4R) Nonsense Mutations by Aminoglycoside-Mediated Read-Through

Aminoglycoside‐mediated read‐through of stop codons was recently demonstrated for a variety of diseases in vitro and in vivo. About 30 percent of human genetic diseases are the consequence of nonsense mutations. Nonsense mutations in obesity‐associated genes like the melanocortin 4 receptor (MC4R), expressed in the hypothalamus, show the impact of premature stop codons on energy homeostasis. Therefore, the MC4R could be a potential pharmaceutical target for obesity treatment and targeting MC4R stop mutations could serve as proof of principle for nonsense mutations in genes expressed in the brain. We investigated four naturally occurring nonsense mutations in the MC4R (W16X, Y35X, E61X, Q307X) located at different positions in the receptor for aminoglycoside‐mediated functional rescue in vitro. We determined localization and amount of full‐length protein before and after aminoglycoside treatment by fluorescence microscopy, cell surface and total enzyme linked immunosorbent assay (ELISA). Signal transduction properties were analyzed by cyclic adenosine monophosphate (cAMP) assays after transient transfection of MC4R wild type and mutant receptors into COS‐7 cells. Functional rescue of stop mutations in the MC4R is dependent on: (i) triplet sequence of the stop codon, (ii) surrounding sequence, (iii) location within the receptor, (iv) applied aminoglycoside and ligand. Functional rescue was possible for W16X, Y35X (N‐terminus), less successful for Q307X (C‐terminus) and barely feasible for E61X (first transmembrane domain). Restoration of full‐length proteins by PTC124 could not be confirmed. Future pharmaceutical applications must consider the potency of aminoglycosides to restore receptor function as well as the ability to pass the blood‐brain barrier.

Mutation screen and association studies in the Diacylglycerol O-acyltransferase homolog 2 gene ( DGAT2 ), a positional candidate gene for early onset obesity on chromosome 11q13

BackgroundDGAT2 is a promising candidate gene for obesity because of its function as a key enzyme in fat metabolism and because of its localization on chromosome 11q13, a linkage region for extreme early onset obesity detected in our sample.We performed a mutation screen in 93 extremely obese children and adolescents and 94 healthy underweight controls. Association studies were performed in samples of up to 361 extremely obese children and adolescents and 445 healthy underweight and normal weight controls. Additionally, we tested for linkage and performed family based association studies at four common variants in the 165 families of our initial genome scan.ResultsThe mutation screen revealed 15 DNA variants, four of which were coding non-synonymous exchanges: p.Val82Ala, p.Arg297Gln, p.Gly318Ser and p.Leu385Val. Ten variants were synonymous: c.-9447A > G, c.-584C > G, c.-140C > T, c.-30C > T, IVS2-3C > G, c.812A > G, c.920T > C, IVS7+23C > T, IVS7+73C > T and *22C > T. Additionally, the small biallelic trinucleotide repeat rs3841596 was identified. None of the case control and family based association studies showed an association of investigated variants or haplotypes in the genomic region of DGAT2.ConclusionIn conclusion, our results do not support the hypothesis of an important role of common genetic variation in DGAT2 for the development of obesity in our sample. Anyhow, if there is an influence of genetic variation in DGAT2 on body weight regulation, it might either be conferred by the less common variants (MAF < 0.1) or the detected, rare non-synonymous variants.

Increased constraints on MC4R during primate and human evolution

The melanocortin 4 receptor (MC4R) is routinely investigated for the role it plays in human obesity, as mutations in MC4R are the most common dominantly inherited form of the disease. As little is known about the evolutionary history of this locus, we investigated patterns of variation at MC4R in a worldwide sample of 1,015 humans from 51 populations, and in 8 central chimpanzees. There is a significant paucity of diversity at MC4R in humans, but not in chimpanzees. The spectrum of mutations in humans, combined with the overall low level of diversity, suggests that most (if not all) of the observed non-synonymous polymorphisms are likely to be transient deleterious mutations. The MC4R coding region was resequenced in 12 primate species and sequences from an additional 29 vertebrates were included in molecular evolutionary analyses. MC4R is highly conserved throughout vertebrate evolution, and has apparently been subject to high levels of continuous purifying selection that increased approximately threefold during primate evolution. Furthermore, the strong selection extends to codon usage bias, where most silent mutations are expected to be either quickly fixed or removed from the population, which may help explain the unusually low levels of silent polymorphisms in humans. Finally, there is a significant tendency for non-synonymous mutations that impact MC4R function to occur preferentially at sites that are identified by evolutionary analyses as being subject to very strong purifying selection. The information from this study should help inform future epidemiological investigations of MC4R.

A novel mutation Thr162Arg of the melanocortin 4 receptor gene in a Spanish children and adolescent population

Objective  The melanocortin 4 receptor gene (MC4R) is involved in body weight regulation. While many studies associated MC4R mutations with childhood obesity, information on MC4R mutations in Spanish children and adolescents is lacking. Our objective was to screen a population of children and adolescents from the north of Spain (Navarra) for MC4R mutations and to study the phenotypes of carriers and their families. In addition, functional assays were performed for a novel MC4R mutation.

Characterization of the melanocortin-4-receptor nonsense mutation W16X in vitro and in vivo

Several genetic diseases are triggered by nonsense mutations leading to the formation of truncated and defective proteins. Aminoglycosides have the capability to mediate a bypass of stop mutations during translation thus resulting in a rescue of protein expression. So far no attention has been directed to obesity-associated stop mutations as targets for nonsense suppression. Herein, we focus on the characterization of the melanocortin-4-receptor (MC4R) nonsense allele W16X identified in obese subjects. Cell culture assays revealed a loss-of-function of Mc4rX16 characterized by impaired surface expression and defect signaling. The aminoglycoside G-418 restored Mc4rX16 function in vitro demonstrating that Mc4rX16 is susceptible to nonsense suppression. For the evaluation of nonsense suppression in vivo, we generated a Mc4rX16 knock-in mouse line by gene targeting. Mc4rX16 knock-in mice developed hyperphagia, impaired glucose tolerance, severe obesity and an increased body length demonstrating that this new mouse model resembles typical characteristics of Mc4r deficiency. In a first therapeutic trial, the aminoglycosides gentamicin and amikacin induced no amelioration of obesity. Further experiments with Mc4rX16 knock-in mice will be instrumental to establish nonsense suppression for Mc4r as an obesity-associated target gene expressed in the central nervous system.

Do common variants separate between obese melanocortin-4 receptor gene mutation carriers and non-carriers? The impact of cryptic relatedness.

Background/Aims: Genome-wide association studies revealed associations of single nucleotide polymorphisms (SNPs) flanking MC4R with body mass index variability and obesity. We genotyped 28 SNPs, covering MC4R, and searched for haplotypes discriminating between obese mutation carriers and non-carriers. Methods: We analyzed all three-marker haplotype combinations of the 28 SNPs to discriminate between obese mutation carriers and non-carriers – overall and in functional categories for 25 different MC4R mutations: (a) ‘like wild type’, (b) ‘partial loss of function’, and (c) ‘complete loss of function’. We checked for the possible impact of ‘cryptic relatedness’ by sensitivity analyses including only 1 randomly selected patient per mutation. Results: Overall analyses revealed a haplotype of 3 SNPs downstream of the MC4R discriminating between obese mutation carriers and obese non-carriers. However, sensitivity analyses showed that the finding is most likely due to cryptic relatedness. Conclusion: Given a mutation prevalence of 1–5%, the sample size of 62 obese mutation carriers with overall 25 different MC4R mutations represents a unique feature of our study. Taking MC4R as an example, we demonstrate the impact of cryptic relatedness when trying to link non-coding SNPs to functionally relevant mutations. Hence, a thorough mutation screen can currently not be guided by SNP genotyping.