247–252www.elsevier.com/locate/gene

Gene 392 (2007)

Polymorphism and chromosomal location of the MC4R(melanocortin-4 receptor) gene in the dog and red fox

Anna Skorczyk a, Monika Stachowiak a, Izabela Szczerbal a, Jolanta Klukowska-Roetzler b,Claude Schelling c, Gaudenz Dolf d, Marek Switonski a,⁎

a Department of Genetics and Animal Breeding, Agricultural University of Poznan, Polandb Department of Veterinary Clinical Studies, University of Berne, VETSUISSE–Faculty Berne, Switzerland

c Department of Animal Sciences, Swiss Federal Institute of Technology, Zurich and VETSUISSE–Faculty Zurich, Switzerlandd Institute of Genetics, University of Berne, VETSUISSE–Faculty Berne, Switzerland

Received 22 September 2006; received in revised form 20 December 2006; accepted 21 December 2006Available online 19 January 2007

Abstract

The melanocortin-4 receptor (MC4R) is expressed in the hypothalamus and regulates energy intake and body weight. In silico screening of thecanine chromosome 1 sequence and a comparison with the porcine MC4R sequence by BLAST were performed. The nucleotide sequence of thewhole coding region and 3′- and 5′-flanking regions of the dog (1214 bp) and red fox (1177 bp) MC4R gene was established and highconservation of the nucleotide sequences was revealed (99%). Five sets of PCR primers were designed and a search for polymorphism wasperformed by the SSCP technique in a group of 31 dogs representing nineteen breeds and 35 farm red foxes. Sequencing of DNA fragments,representing the identified SSCP patterns, revealed three single nucleotide polymorphisms (including a missense one) in dogs and four silent SNPsin red foxes. An average SNP frequency was approx. 1/400 bp in the dog and 1/300 bp in the red fox. We mapped the MC4R gene by FISH to thecanine chromosome 1 (CFA1q1.1) and to the red fox chromosome 5 (VVU5p1.2).© 2007 Elsevier B.V. All rights reserved.

Keywords: Dog; Red fox; MC4R; SNP; FISH

1. Introduction

Over the last several decades obesity has become a greatmedical problem. Searching for the genetic background ofhuman obesity is very important for its treatment and prevention.Phenotypic variability among dog breeds, including fat tissueamount, is a well known phenomenon and thus the dog can beconsidered as a model for human obesity. On the other hand, infur-bearing canids the accumulation of subcutaneous fat tissue isdesirable due to its influence on body weight, which is positivelycorrelated with the size of pelt (Gugolek et al., 2002).

Abbreviations: PCR, polymerase chain reaction; SNP, single nucleotidepolymorphism; SSCP, single strand conformation polymorphism; RFLP,restriction fragment length polymorphism.⁎ Corresponding author. Department of Genetics and Animal Breeding,

August Cieszkowski Agricultural University of Poznan, Wolynska 33, 60-637Poznan, Poland. Tel./fax: +48 61 8487246.

E-mail address: switonsk@jay.au.poznan.pl (M. Switonski).

0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.gene.2006.12.027

The melanocortin system plays an important role in energyhomeostasis and body-weight regulation (Benoit et al., 1998). Afamily of its receptors consists of five subtypes with differentpatterns of tissue expression (Gantz et al., 1993, 1994).Melanocortin-1 receptors (MC1R) are located exclusively inmelanocytes, MC2R in the adrenal cortex, MC5R in multipleexocrine tissues, while MC4R and MC3R are found extensivelyin the brain and MC3R is also expressed in the placenta and guttissues. The principal agonist to melanocortin receptors is the α-melanocyte-stimulating hormone (α-MSH), while the endoge-nous antagonist to these receptors is AgRP (agouti-relatedprotein).

The melanocortin-4 receptor (MC4R) is a G-protein coupledreceptor expressed in the brain, predominantly in the hypothal-amus. The adipocyte leptin hormone triggers melanocortin-4receptor response that modulates appetite and energy expendi-ture. Activation of the MC4R gene results in the inhibition offood intake and a targeted disruption of theMC4R gene in mice

248 A. Skorczyk et al. / Gene 392 (2007) 247–252

causes obesity associated with hyperphagia, hyperinsulinemiaand hyperglycinemia (Huszar et al., 1997).

The MC4R gene is encoded by a single exon and mutationsof this gene are the most common cause of hereditary humanobesity (Gantz et al., 1993). Over 50 mutations affecting theamino acid sequence of the human MC4R gene have beenreported to date (Perusse et al., 2005). Moreover, a significantassociation has been reported between MC4R genotypes andbackfat, growth rates and feed intake in a number of breeds andlines of the pig (Kim et al., 2000; Houston et al., 2004; Jokubkaet al., 2006; Stachowiak et al., 2006). The latest QTL analysesin this species have also supported the hypothesis of the MC4Rgene as a positional candidate gene for the fatness and meatnessQTL (Bruun et al., 2006). Moreover, it was shown that anadditive action of the MC4R and HMGA1 (high mobility groupAT-hook 1) genes may influence growth and lean meat mass(Kim et al., 2006).

Taking the above into account the MC4R gene can beconsidered as a candidate QTL for fat deposition traits also incanids. In this report we present comparative characteristics ofthe MC4R gene in two species of the family Canidae: the dog(Canis familiaris) and the red fox (Vulpes vulpes).

2. Materials and methods

2.1. Material

Altogether, 31 dogs representing nineteen different breeds(Afghan Hound, American Staffordshire Terrier, Beagle, Briard,Cane Corso, Central Asian Shepherd Dog, Cocker Spaniel,Dachshund, Doberman Pinscher, English Setter, GermanShepherd Dog, Great Dane, Greater Swiss Mountain Dog,Labrador Retriever, Miniature Pinscher, Miniature Poodle,Rottweiler, Scottish Terrier, Yorkshire Terrier) and 35 unrelated

Table 1Primer pairs used to screen MC4R gene for polymorphism

Primer pair Primer sequence (5′–3′) Size (bp) Location a

1 5′ GCA GGC CAG CTG GAT CCTCAG AA 3′

272 (−119)–153

5′ GAA CAC CTC CGG GGAGAC GAA GAG 3′

2 5′ ACG GCA ACG CCA CTGAGT CC 3′

457 71–527

5′ GCC GCC CAG ATG CAACTG AT 3′

3 5′ TAC GGATAC GGA CGCGCA GAG TTT 3′

297 327–623

5′ ATG AGG GCC AGC ATGGTG AAG AAC 3′

4 5′ GCA CGG TGT CAG GCATCT TGT TCA 3′

391 530–921

5′ TGG CTC CGG AGT GCATAA ATG AGA 3′

5 5′ CTT GTC CCC AGA ATCCAT ACT GTG 3′

288 809–(+97)

5′ ACA CTG AAG AAG CAGCTG TTG TCC 3′

a The numbers indicate initial position in the reference sequence (GenBankaccession no. DQ084210).

farm red foxes from a local fur animal farm were included in thisstudy. Genomic DNAwas extracted from peripheral blood withthe use of a Perfect gDNA Blood Mini Isolation Kit (A&ABiotechnology, Gdansk, Poland).

2.2. Primer design and PCR

In silico screening of the dog chromosome 1 sequence(GenBank accession no. NW876269) and a comparison withthe porcine MC4R sequence (GenBank accession no.AB021664) by BLAST (http://www.ncbi.nlm.gov/BLAST)were performed to search for the dog sequence (Lindblad-Tohet al., 2005). Five sets of PCR primers were designed to coverthe whole coding sequence and part of the 3′- and 5′-flankingregions. The sequences, locations of primer sets and the lengthof PCR products are shown in Table 1. Each PCR reactioncontained 100 ng of genomic DNA, 0.35 μM of each primer,0.1 mM dNTPs, 1× PCR buffer with 1.5 mM MgCl2 and 1 UTaq polymerase in a total volume of 20 μl. PCR was performedusing a Biometra thermocycler. The amplification conditionswere 5 min at 94 °C, 35 cycles of 30 s at 94 °C, 30 s at 63 °C(primer pair 1), 55 °C (primer pair 2), and 57 °C (primer pairs 3,4, 5) respectively, and 1 min at 72 °C, followed by a 5-min finalextension at 72 °C (Table 1).

2.3. SSCP

All the five amplified fragments (Table 1) of theMC4R genewere scanned for mutations using the SSCP approach. TheSSCP analysis was performed for 31 dogs and 35 farm redfoxes. The PCR products were denatured at 97 °C in a buffercontaining formamide. Electrophoresis was run on 9%polyacrylamide gels in 1× TBE buffer using an apparatus forvertical electrophoresis (C.B.S Scientific CO, Del Mar,California, USA, model DSG-250-02) at 10 °C and 13 °C at150 V for 19–21 h. Finally, silver staining was applied to detectelectrophoretic patterns.

2.4. DNA sequencing

The PCR products were isolated and purified from agarose gelwith the use of a Gene MATRIX Agarose-Out DNA PurificationKit (EURx, Gdansk, Poland) and then sequenced using each ofthe forward and reverse primers at the Institute of Biochemistryand Biophysics, Polish Academy of Sciences, Warsaw, Poland.The obtained sequences were verified using BLAST.

2.5. RFLP

To genotype all the studied animals (at three polymorphicsites) RFLP analyses were performed. Three endonucleaseswere used: TaiI, BtsI and SmaI. All restriction enzymedigestions were performed in 10 μl reaction mixture containing3 μl of the PCR products, 1× digestion buffer and 2.5 to 6 U ofthe respective endonuclease.

The amplified 391 bp fragments of the canine MC4R(genotyping for the G637T polymorphism) were digested

Fig. 1. RFLP (TaiI) genotyping of G637T SNP in canine MC4R gene. M —molecular marker (GeneRuler™ 100 bp DNA Ladder, Fermentas).

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overnight at 65 °C with 2.5 U of TaiI (Fermentas). Thefragments were separated on 1.5% agarose gel. Digestion withTaiI resulted in fragments: 282 bp and 109 bp for the G alleleand 391 bp for the T allele (Fig. 1).

To establish genotypes at the 5′-flanking (−62) site theamplified 272 bp fragment of the red fox MC4R was cut with4 U of SmaI (Promega, Madison, USA) at 25 °C, overnight. Thefragments separated on 2% agarose gel had the following sizes:214 bp and 58 bp (for the C allele), and 272 bp (G).

The amplified 457 bp fragment of the red fox MC4R wasdigested overnight at 37 °C with 4 U of BtsI (New EnglandBioLabs) to genotype at the polymorphic site T261A. Theexpected fragment sizes (separated on 2% agarose gel) were:367 bp and 90 bp for the A allele and 171 bp, 196 bp and 90 bp forthe T allele.

2.6. Chromosome preparations and fluorescence in situhybridization (FISH)

Chromosome preparations were obtained from short-termlymphocyte cultures and stained with the Q-banding techniqueprior to FISH. Chromosome nomenclature for the dog (Switonskiet al., 1996) and red fox (Makinen et al., 1985) was applied.

The canine genomic BAC library (Schelling et al., 2002) wasscreened for the MC4R gene. Canine primers (primer pair 2,

Fig. 2. SSCP scans for polymorphisms: (A) dog; 391 bp fragment; two SNPswere identified by further DNA sequencing: G637Tand T777C; genotypes: 1—GT/GT, 2 — TC/TC, 3 — GC/GT, 4 — GC/GC and 5 — GC/TC, (B) red fox;272 bp fragment; three SNPs by DNA sequencing were identified: A(−73)G, A(−65)G and C(−62)G; genotypes: 1 — AAC/GGG, 2 — AAC/AAC and 3 —GGG/GGG.

Table 1) for this gene were used. The clone SP069P09F9,containing theMC4R, was identified. The isolated DNA (500 ng)was labeled with biotin-16-dUTP by random priming (Prime-Itflour Fluorescence Labeling Kit, Promega). FISH was performedaccording to the standard protocol (Szczerbal et al., 2003). Thelabeled probes with an excess of salmon sperm DNA weredenatured for 7 min at 70 °C, preannealed for 15 min and appliedon denatured chromosome preparations. Hybridization wascarried out overnight at 37 °C. Standard protocols were followedfor posthybridizationwashing. Signal detection and amplificationwere done using avidin-FITC and anti-avidin. Staining wasperformed with propidium iodide. Slides were analyzed under afluorescence microscope (Nikon E 600 Eclipse) equipped with acooled digital camera, driven by computer-aided software Lucia.

3. Results

3.1. Sequencing and genomic organization of the MC4R gene

Nucleotide sequences of the whole coding region and a partof the 5′- and 3′-flanking regions of the dog (1214 bp) and redfox (1177 bp)MC4R gene were established (GenBank accessionnos. DQ084210 and DQ663625, respectively). The coding se-quence of both the dog and red fox genes consisted of 999 bp.In the dog the 5′- and the 3′-flanking sequences had 118 bp and97 bp, respectively. The length of these fragments in the red foxwas 81 bp and 97 bp, respectively. High conservation was foundfor the overall dog and red foxMC4R gene nucleotide sequencesand the predicted amino acid sequence (99%). Moreover, thepredicted amino acid sequence of the dog shares 95% identitywith the human and murine, and 96% identity with the porcineproteins.

3.2. Polymorphism analysis of canine MC4R gene

The SSCP technique revealed different electrophoreticpatterns in two canine fragments: 391 bp and 288 bp. Threesingle nucleotide polymorphisms (SNPs) were identified in thesequenced fragments. A missense substitution G637T (TaiI),changing valine to phenyloalanine at position 213 of thepredicted amino acid sequence, and a silent substitution T777C(BseSI) were found (Fig. 2A). Moreover, a C/G substitution

Table 2Frequencies of SNPs and haplotypes in the MC4R gene

Polymorphism Dog Red fox

SNP 637G (0.84) 637T (0.16) −73A (0.7) −73G (0.3)777T (0.26) 777C (0.74) −65A (0.7) −65G (0.3)+33C (0.26) +33G (0.74) −62C (0.7) −62G (0.3)– – 261T (0.64) 261A (0.36)

Haplotypes a GCG (0.48) AACT (0.63)TCG (0.16) GGGA (0.29)GTC (0.16) AACA (0.07)GTG (0.10) GGGT (0.01)GCC (0.10) –

a Haplotype at nucleotide positions: 637, 777, (+33) in the dog and (−73),(−65), (−62), 261 in the red fox.

Fig. 3. FISH mapping of BAC clone, harboring MC4R gene, on metaphases ofthe dog (A— Q-banding and ideogram of CFA1, B— FISH) and red fox (C—Q-banding and ideogram of VVU5, D — FISH).

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(BseSI) at nucleotide position +33 (downstream the terminationcodon) was revealed in the 3′-flanking region.

Allele frequencies for all the SNPs are listed in Table 2.Alleles 637G, 777C and (+33)G were significantly morefrequent (0.84, 0.74 and 0.74, respectively) than the oppositeones. The identified SNPs were also submitted to haplotypeanalysis (Table 2). The most frequent haplotype was GCG witha frequency of 0.47. The distribution of haplotypes wasanalyzed in groups of breeds classified based on commonancestry according to previous microsatellite markers study(Sutter and Ostrander, 2004). In hunting dogs (including alsoAmerican Staffordshire Terrier, English Setter, MiniaturePinscher, Miniature Poodle, Scottish Terrier and YorkshireTerrier) and Mastiff dogs (including also Cane corso) all fivehaplotypes were found. In herding dogs (including also Briardsand Central Asian Shepherd Dog) and in Asian/Ancient dogsonly some haplotypes were found: GCG, TCG and GCC, andGCG, respectively.

3.3. Polymorphism analysis of the red fox MC4R gene

Different SSCP electrophoretic patterns in two red foxfragments (272 bp and 456 bp) were observed and theirsequencing revealed four SNPs. Three of them occurred in the5′-flanking region: A(−73)G (BsaXI), A(−65)G, C(−62)G(SmaI) (upstream the start codon) (Fig. 2B). Alleles (−73)A,(−65)A and (−62)C occurred in this study with a similarfrequency — 0.7 (Table 2). As a result of SSCP and RFLP(SmaI) tests, as well as sequencing of two individualsrepresenting each SSCP pattern only two haplotypes wereidentified: AAC and GGG.

The fourth SNP was found in the 457 bp fragment of thecoding sequence. It was a silent T/A substitution at nucleotideposition 261. Genotypes at this polymorphic site were detectedby the RFLP test (BtsI). Allele 261T occurred with a higherfrequency (0.64).

A simultaneous analysis of all the SNPs showed the presenceof four haplotypes: AACA, AACT, GGGA and GGGT (Table2). The most frequent (0.63) haplotype was the AACT.

3.4. Mapping

For both species the used BAC probe hybridized clearly to asingle chromosome pair and hybridization signals werelocalized on Q-banded chromosomes. In the dog the MC4Rlocus was assigned to CFA1q1.1 (Fig. 3A and B) and in the redfox to VVU5p1.2 (Fig. 3C and D).

4. Discussion

In this study we characterized and mapped for the first timethe MC4R locus in two canids. The FISH localizations onCFA1q1.1 and VVU5p1.2 are in agreement with earlier dataobtained by the comparative chromosome painting for the dogand red fox genomes, since it was shown by Yang et al. (1999)that the pericentromeric region of the CFA1 and VVU5p isevolutionarily conserved. In the same study conservation of theHSA18q2 segment and the above mentioned dog and red foxchromosome fragments was revealed. The MC4R locus wasassigned to human HSA18q2.2 (Magenis et al., 1994;Sundaramurthy et al., 1998).

A comparative analysis of nucleotide sequences of theMC4R gene, including 5′- and 3′-flanking regions, revealedhigh identity of the MC4R amino acid sequence among thespecies compared (the human, mouse, pig, dog and red fox). Itis a strong indication of a high evolutionary conservation of thedog and red fox MC4R gene. On the other hand, the nucleotidesequence of the MC4R gene appeared to be quite polymorphicin both studied species, since SNP frequency was approx. 1/400 bp in the dog and 1/300 bp in the red fox. The current SNPmap of the dog contains more than 2.5 million distinct SNPswith an average density of SNPs 1/1000 bp (Lindblad-Toh et al.,2005). The SNP rate is 1/1.500 bp within breeds and 1/900 bpbetween breeds. The SNP rate between dog (boxer) and othercanids is approximately 1/500 bp. Analysis of the observedhaplotypes did not reveal any clear breed–haplotype relation-ships. It might be an effect of a small number of animalsrepresenting the groups.

It should be pointed out that in the human MC4R gene thenumber of the polymorphic sites was even higher than in thestudied two canids. In an extensive review on the humanMC4Rgene polymorphism altogether 51 SNPs and InDels weredescribed in a fragment of 2400 bp (Perusse et al., 2005). Thus,one can calculate that the frequency reached almost 1/50 bp.Higher SNP frequency in the human MC4R gene than in thedog and red fox may be caused by the fact, that in the studies onhuman MC4R gene many more patients were studied and amajority of them suffered from obesity. Studies on the porcine

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MC4R gene suggest that some species-specific differences interms of the SNP frequency within this gene may exist. Untilnow only two SNPs in the coding region have been reported inthe pig (Kim et al., 2000; Meidtner et al., 2006), although thisgene has been extensively studied as a promising candidateQTL for growth and fatness traits (Rothschild, 2004).

In our study we looked for polymorphism with the use of theSSCP technique which is considered as a useful tool forpreliminary DNA polymorphism studies. Sensitivity of thistechnique is negatively correlated with the length of the analyzedDNA fragment. Hayashi and Yandell (1993) reported that usingthe SSCP method, more than 80% mutations can be detected ifthe studied fragments are 300–350 bp. To enhance its sensitivityit is recommended to perform the gel separation in multipleconditions (Hestekin and Barron, 2006). Altogether, fivefragments (from 272 to 457 bp) were screened in two speciesapplying an optimized temperature (10 or 13 °C). Moreover, theSSCP technique was supplemented by DNA sequencing(altogether 112 fragments were sequenced) and always differentSSCP patterns resulted from DNA polymorphism. Thus, we canassume that this technique appeared to be efficient and reliable.Of course, it can not be excluded that also other SNPs, notdetected by SSCP, were present in the studied group of animals.

Only one SNP appeared to be a missense substitution in thedog (G637T). It caused a change of valine to phenyloalanine atposition 213 of the predicted amino acid sequence. This aminoacid substitution occurred in the fifth transmembrane domain ofthe predicted MC4R protein, in the region conserved betweenmelanocortins receptors (MC1R, MC2R, MC3R and MC4R). Itencompasses five amino acids— LYVHM (Gantz et al., 1993).The deletion of four nucleotides (211:del ‘CTCT’) in this regionof the human MC4R caused a complete loss of activity of theprotein (Yeo et al., 1998; Hinney et al., 2003; Ho andMacKenzie, 1999; Farooqi et al., 2003). It should be mentionedthat mutations in the region coding the fifth transmembranedomain of the human MC4R gene are not numerous – only twomissense substitutions — M200V (Branson et al., 2003) andF202L (Jacobson et al., 2002) – were described. Unfortunately,their effects were not studied. We assume that the valinephenyloalanine substitution identified in this study in the dogMC4R protein will probably be functionally neutral since bothamino acids have similar (hydrophobic) properties.

Our analysis on 35 farm red foxes suggests that three SNPsin the 5′-flanking region: A(−73)G (BsaXI), A(−65)G, C(−62)G (SmaI) of the red fox MC4R are inherited as two intragenehaplotypes: AAC and GGG. Interestingly, in silico studies withthe use of TESS software revealed that the substitution A(−73)G is located at potential consensus sequences for theglucocorticoide receptor element (GRE) and the progesteronereceptor element (PRE). The nucleotide sequence which canpotentially bind GR and PR is AGAACA. Such a sequence wasfound in the dog and red fox if adenine was present at position−73. Thus, the A(−73)G substitution in the red fox may have apotential effect on the regulation of the MC4R expression.Further analyses are needed to elucidate its functionality.

We sequenced and screened for polymorphisms only shortfragments of 5′-flanking (118 bp in the dog and 81 bp in the red

fox) and 3′-flanking nucleotide sequence (97 bp in both species)and we have found several polymorphic sites. The MC4Rpromoter was characterized only for the human gene (Lubrano-Berthelier et al., 2003). Based only on the comparison of thecanine 5′-flanking region with the human promoter we mayconclude that we have not received the sequence of the minimalpromoter. We found that the canine 5′-flanking fragment sharesonly 50% identity with the respective fragment of the humangene. In humans only few mutations in the predicted promoterand 5′-flanking region were described. Among them there weretwo deletions, −65delTG and −439delGC, and several sub-stitutions: G(−184)A, A(−178)C, A(−176)G and G(−360)T, C(−1042)T, C(−1005)T, C(−896)T and G(−719)A (Jacobson etal., 2002; Lubrano-Berthelier et al., 2003; Valli-Jaakola et al.,2004, 2006).

We also identified a single nucleotide polymorphism G(+33)C in the 3′UTR of the canine melanocortin receptor gene. Thereare not many polymorphisms reported in the human 3′-flankingregion. There have been described few substitutions: G(1101)A[G(+102)A] and T(1123)C [T(+124)C] (Jacobson et al., 2002),C(+60)T (Valli-Jaakola et al., 2004), A(1419)G [A(+420)G](Branson et al., 2003) and deletion of cytosine at the nucleotideposition 28 downstream of the stop codon, 3′UTR +28delC(Farooqi et al., 2000). The significance of this alterationsremains unclear.

The MC4R gene appeared to be quite polymorphic in bothstudied canids. Therefore, further studies of the polymorphismsin terms of their functionality may bring new information usefulin comparative analysis focused on the genetic background ofhuman obesity, as well as in fur-bearing animal breeding.

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