Analysis of variation in the melanocortin-4 receptor gene (mc4r) in golden retriever dogs

Abstract

Source/description: The melanocortin-4 receptor plays a central role in the regulation of energy balance. Activation of the receptor leads to decreased energy intake and increased energy expenditure.1 Genetic variation at the melanocortin-4 receptor locus (mc4r) is associated with body measures in several species.2–4 The purebred dog population is a collection of genetic isolates with highly diverging morphology.5 Dog breeds also appear to differ in their tendency to gain weight. It would be interesting to study the role of mc4r variation in this phenotypic diversity. The objective of this study was to identify polymorphisms in mc4r in the Golden Retriever breed for future genetic association studies. Samples: We selected the 23 least related dogs (mean genetic relationship 0.0035) from a birth cohort of Golden Retrievers with known genealogy. Genomic DNA was isolated from blood using standard methodology. Polymorphism detection: A 1101-bp region encompassing the mc4r coding sequence was amplified in four overlapping polymerase chain reactions and analysed using an ABI3130xl Genetic Analyzer. Experimental procedures are described in Appendix S1; primer sequences are listed in Table S1. Functional effects of the detected variants were predicted using a bioinformatic approach (see Appendix S2). Detected polymorphisms: We detected four single nucleotide polymorphisms in the canine mc4r gene: c.637G>T (minor allele frequency, MAF = 0.33), c.777T>C (MAF = 0.15), c.868C>T (MAF = 0.33), and c.*33C>G (in the 3′-untranslated region, MAF = 0.15). c.868C>T was not reported previously; the other three were reported in other dog breeds.6 The variant c.637G>T is expected to affect receptor function because (i) it is a missense variant (p.V213F); (ii) the involved amino acid residue is highly conserved; (iii) M3 class of this residue was 0, i.e. it is under strong purifying selection; and (iv) the polymorphism was predicted to be possibly damaging by PolyPhen. Additional studies, including in vitro assays, are required to confirm the impact of this polymorphism on receptor function. The other variants are not expected to affect receptor function (Table S2). Comments: Our work provides a starting point for studies of the association between mc4r variation and morphological measures in dogs. In a preliminary analysis of 187 Golden Retrievers, we found no association between the detected mc4r polymorphisms and morphological measures (Appendix S3 and Table S3).