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.

Evolutionary aspects in evaluating mutations in the melanocortin 4 receptor.

More than 70 missense mutations have been identified in the human melanocortin 4 receptor (MC4R), and many of them have been associated with obesity. In a number of cases, the causal link between mutations in MC4R and obesity is controversially discussed. Here, we mined evolution as an additional source of structural information that may help to evaluate the functional relevance of naturally occurring variations in MC4R. The sequence information of more than 60 MC4R orthologs enabled us to identify residues that are important for maintaining receptor function. More than 90% of all inactivating mutations found in obese patients were located at amino acid positions that are highly conserved during 450 million years of MC4R evolution in vertebrates. However, for a reasonable number of MC4R variants, we found no correlation between structural conservation of the mutated position and the reported functional consequence. By re-evaluating selected mutations in the MC4R, we demonstrate the usefulness of combining functional and evolutionary approaches.

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.

BACKGROUND: DGAT2 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. RESULTS: The 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. CONCLUSION: In 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.

Prevalence, spectrum, and functional characterization of melanocortin-4 receptor gene mutations in a representative population-based sample and obese adults from Germany.

CONTEXT: Autosomal dominant inheritance of mutations in the melanocortin-4 receptor gene (MC4R) is currently regarded as the most relevant genetic cause for extreme obesity and affects 2-4% of extremely obese individuals. OBJECTIVE: Our objective was to assess the relevance of MC4R mutations in a German population-based sample. DESIGN AND SETTING: We conducted a mutation screen of the MC4R gene by capillary electrophoresis-based single-strand conformation polymorphism analysis and denaturing HPLC. PARTICIPANTS: Subjects included 4068 individuals of a German population-based study group [Kooperative Gesundheitsforschung im Raum Augsburg, Survey 4 (KORA-S4); i.e. Cooperative Health Research in the Region of Augsburg] and 1003 German obese adults (body mass index >or= 30 kg/m(2)). MAIN OUTCOME MEASURES: Samples with aberrant capillary electrophoresis-based single-strand conformation polymorphism analysis/denaturing HPLC patterns were resequenced. Functional studies including agonistic receptor stimulation (Nle-D-Phe-alpha-, alpha-, and beta-MSH) and cell surface expression assays were performed. RESULTS: Sixteen (six novel) coding nonsynonymous mutations were detected in 27 heterozygous individuals of KORA-S4. Four of the mutation alleles led to impaired receptor function in vitro; however, none of these six heterozygous mutation carriers was obese (body mass index >or= 30 kg/m(2)). In the obese adults, six coding nonsynonymous and a nonsense mutation were detected in 13 individuals. Only the nonsense mutation allele entailed impaired receptor function. CONCLUSIONS: Our study depicts prevalence, spectrum, and functional characterization of MC4R mutations in the German population-based sample KORA-S4. In this epidemiological study group, individuals heterozygous for nonsynonymous MC4R mutation alleles entailing impaired function were not obese. Furthermore, nonsynonymous MC4R mutations causing impaired receptor function were rare in German obese adults (two in 1003 = 0.2%).

Congenital central hypothyroidism due to homozygous thyrotropin beta 313 Delta T mutation is caused by a Founder effect.

Neonatal TSH screening has been a major achievement for the early detection and treatment of primary congenital hypothyroidism. It nevertheless fails to reveal cases of central hypothyroidism caused by TSH levels in the low normal range. In the last 10 yr, homozygous mutations in the TSHbeta-subunit gene have been recognized as a cause of central hypothyroidism with isolated TSH deficiency. The most frequent TSHbeta mutation 313DeltaT (C105V) has been described in six apparently unrelated families. We investigated the frequency and possible monophyletic origin of the different TSHbeta 313DeltaT alleles of the three affected families. Haplotype analysis of five polymorphic single-nucleotide polymorphism loci in the TSHbeta region revealed the presence of seven different haplotypes in the general population. In all six parental lines, the mutation occurred on the same haplotype. Extending the haplotype by two flanking microsatellite markers led to a mutational age estimate of about 150 generations. In 500 unrelated individuals from the general population, we did not detect any TSHbeta 313DeltaT allele, suggesting a population heterozygote carrier frequency less than 1:170 with more than 95% probability. Accordingly, the disease risk in the general population because of homozygosity is low. Our data suggest a monophyletic origin of the TSHbeta 313DeltaT mutation from a common ancestor and no significant population prevalence. Therefore, identification and genetic counseling of heterozygous carriers in affected families seems to be more advisable than population-wide neonatal T(4) screening programs for an early detection of this rare condition.

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.

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.

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 novel melanocortin-4 receptor gene mutation in a female patient with severe childhood obesity.

This study targeted the identification of mutations of melanocortin-4 receptor gene (MC4R) in obese children. Fifty-one unrelated probands with early onset severe obesity (body mass index (BMI) > 99th percentile; 21 girls, mean age 10.6 +/- 3.6 years) were analyzed for nucleotide variations in the MC4R coding region, by the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method followed by direct DNA sequencing. MC4R variants were detected in three patients: the known I169S variant was found in heterozygote state in two patients and a novel heterozygous Y302F mutation was detected in one 12-year-old girl (BMI = 34 kg/m(2), BMI z-score 2.7) who has been overweight since the second year of life and suffered from hyperinsulinemia (at the age of 12: fasting insulin 45 mU/ml, after oral glucose load max. 300 mU/ml). The mutation also appears in the father, although both parents are obese (BMI father: 30.2 kg/m(2); mother: 31.9 kg/m(2)). This novel mutation is located in the functionally important NPXXY motif of the seventh transmembrane domain of the receptor. Functional characterization revealed reduction in cell surface expression and an alteration in signal transduction properties. These results add to the growing list of loss-of-function MC4R mutations in early onset obese patients and suggest an orexigenic effect of novel Y302F mutation.

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 beta2-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.