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

<p>Abstract</p> <p>Background</p> <p><it>DGAT2 </it>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.</p> <p>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.</p> <p>Results</p> <p>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 <it>DGAT2</it>.</p> <p>Conclusion</p> <p>In conclusion, our results do not support the hypothesis of an important role of common genetic variation in <it>DGAT2 </it>for the development of obesity in our sample. Anyhow, if there is an influence of genetic variation in <it>DGAT2 </it>on body weight regulation, it might either be conferred by the less common variants (MAF < 0.1) or the detected, rare non-synonymous variants.</p

Aminoglycoside induced rescue of signal transduction properties of melanocortin-4 receptors with a premature terminating codon

GesamtdissertationDie stetige Zunahme der Anzahl übergewichtiger und adipöser Personen stellt bedingt durch assoziierte Erkrankungen wie Typ-2-Diabetes und koronare Herz¬defi¬zienz weltweit eines der größten Gesundheitsprobleme dar. Zurzeit stellen heterozygote inaktivierende Mutationen im MC4R die häufigste molekulare Ursache der Adipositas. Die häufigste europäische MC4R Mutation ist eine Stopp-Mutation im N-Terminus des Rezeptors, Y35X, welche zur Translation eines vorzeitig terminierten Proteins und somit zu einem kompletten Funktionsverlust des Rezeptors führt. Seit einigen Jahren ist bekannt, daß Aminoglykoside zu einem Überlesen von mutationsbedingten Stopp-Codons führen können. In der hier vorliegenden Arbeit wird erstmalig ein Überlesen von Stopp-Mutationen des MC4R (W16X, Y35X, E61X, W258X) durch Aminoglykoside in vitro gezeigt. Es wurden die Signaltransduktionseigenschaften, die Oberflächenexpression und die Gesamt¬expression der Rezeptorvarianten von den angegebenen Mutationen im Vergleich zum Wildtyp¬rezeptor sowohl mit als auch ohne Aminoglykosid-Behandlung untersucht. Durch Untersuchung der Gesamtexpression wurde gezeigt, daß in Abhängigkeit von der Konzentration der eingesetzten Aminoglykoside ein Überlesen aller Stopp-Mutationen möglich ist. Die Effizienz des Überlesens hängt von der Art der Stopp-Mutation und der Nukleotide um die Stopp-Mutation herum ab, wobei sich TGA Mutationen (W16X, W258X) besser überlesen lassen als die Varianten TAG (E61X) und TAA (Y35X). Außerdem spielt die Art des ver¬wendeten Aminoglykosids eine erhebliche Rolle, wobei sich Geneticin (G418) als effizienter gegenüber Gentamycin, Puromycin, Hygromycin und Streptomycin erwies. Dieses wurde zusätzlich durch Untersuchung der ligandeninduzierten Signal¬trans¬duktions¬eigenschaften der Rezeptorvarianten durch Stimulation mit den endogenen Liganden alpha-MSH und beta-MSH sowie dem hochpotenten Agonisten NDP-alpha-MSH bestätigt. Hier zeigte sich ein weiterer wichtiger Aspekt des aminoglykosid¬vermittelten Überlesens in Form der Lage des Stoppcodons innerhalb der MC4R-Sequenz. Stoppvarianten im N-Terminus des Rezeptors (W16X und Y35X), welcher keine entscheidende Rolle für die Funktion des Rezeptors spielt, ließen sich in höherem Maße funktionell wiederherstellen als Mutationen im Rezeptorbereich der Transmembrandomänen (E61X, W258X).The melanocortin 4 receptor (MC4R) plays a crucial role in hypothalamic weight regulation as a key component of the leptin-melanocortin signalling pathway. This makes the MC4R an attractive target of pharmceutical intervention to treat obesity. In 1985 it was shown for the first time that aminoglycosides are able to read through stop codons in mammalian cells. Approximately 5 to 10 percent of all genetic diseases are caused by nonsense mutations. Aminoglycoside-mediated read through was recently demonstrated for a variety of diseases including cystic fibrosis and muscular dystrophy Duchenne in vitro and in vivo. This study investigated four nonsense mutations of the MC4R gene two located in the N-terminus of the receptor (W16X and Y35X), one mutation in the first transmembrane domain (E61X) and one in the sixth transmembrane membrane domain for aminoglycoside-mediated functional (re-induction of signal transduction properties) rescue. This work shows that in dependence of the location of the stop mutation in the receptor and the kind of the stop mutation functional rescue is possible for stop mutations in the N-terminus but not or only very slightly for the stop mutation in the first transmembrane domain which seems to be functionally more important for signal transduction properties. These data clearly demonstrate that after aminoglycoside treatment the determination of full-length protein together with functional studies will help to calculate which kind of stop mutation in the MC4R gene is suitable for an aminoglycoside-mediated rescue approach