Figure 1. Big_mac mice exhibited increased body weights. Scaled weight data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

The Big_mac phenotype was identified among G3 mice of the pedigree R6807, some of which showed increased body weights compared to wild-type littermates (Figure 1).

Whole exome HiSeq sequencing of the G1 grandsire identified 72 mutations. The increased body weight phenotype was linked to a mutation in Mc4r: an A to T transversion at base pair 66,859,856 (v38) on chromosome 18, or base pair 632 in the GenBank genomic region NC_000084 for the Mc4r gene. Linkage was found with an additive model of inheritance (P = 1.234 x 10^-7), wherein two variant homozygotes and 18 heterozygous mice departed phenotypically from eight homozygous reference mice (Figure 2).

The mutation corresponds to residue 632 in the mRNA sequence NM_016977 within exon 1 of 1 total exons. 

The mutated nucleotide is indicated in red. The mutation results in an asparagine to isoleucine substitution at position 62 (N62I) in the MC4R protein, and is strongly predicted by Polyphen-2 to cause loss of function (score = 1.000).

MC4R belongs to the family of melanocortin receptors, which are seven transmembrane (TM) spanning G-protein coupled receptors (GPCRs) (Figure 3 & 4). MC4R activates the heterotrimeric G-protein G_s, which stimulates adenylyl cyclase production of cAMP from ATP (1). GPCRs have seven transmembrane helices connected by loops, and ligand binding occurs at extracellular loops facilitated by specific transmembrane helices. Based on a pure modeling approach modeled upon the crystal structure of bovine rhodopsin, another GPCR, residues in transmembrane domain (TM)3, TM4, TM5 and TM6 were predicted to flank the ligand binding site. Extracellular loops 2 and 3 also participate in docking of ligand (2). Interestingly, TM1 and TM7 were not predicted to contribute to ligand binding, although F284 was found at the edge of the ligand-binding pocket (2). The third intracellular loop of MC4R is predicted to form an α-helical segment, and play an important role in coupling the receptor to G_s.

The Big_mac mutation results in an asparagine to isoleucine substitution at position 62 (N62I); Asn62 is within the first transmembrane domain.

Please see the record for Southbeach for more information about Mc4r.

A main mechanism of energy balance regulation involves the control of signaling by the central melanocortin receptors (MCRs) MC3R and MC4R within a defined hypothalamic neural network. Two sets of neurons in the arcuate nucleus (a region surrounding the third ventricle in the most ventral portion of the hypothalamus) act as sensors of whole-body energy status and initiate signals to maintain energy stores at a constant level. The Agrp/Npy neurons (producing Agrp and neuropeptide Y) are inhibited by the leptin peptide (see the record for Potbelly) by signaling through the leptin receptor (see the record for Business_class), while Pomc/Cart neurons (producing Pomc; its proteolytic products and cocaine- and amphetamine-regulated transcript) are stimulated by leptin (3-5). Both Agrp/Npy and Pomc/Cart neurons synapse onto MC4R-expressing neurons (3;6). Thus, when leptin levels are low, Agrp/Npy neurons are activated and Pomc/Cart neurons are inhibited, producing Agrp but not Pomc, and resulting in inhibition of MC4R and increased food intake.

In humans, mutations in MC4R are associated with obesity (OMIM #601665). Human patients with MC4R mutations exhibit increased body mass index, increased appetite, increased height, increased lean mass, increased bone mineral density and hyperinsulinemia (7). With the exception of increased bone mineral density, these phenotypes are recapitulated in Mc4r null mice (8).

The obesity phenotype observed in the Big_mac mice mirrored that of other ENU-induced mutations attributed to Mc4r, including Southbeach and Fatboy (MGI:2671841) (9). The localization, expression, and function of the MC4R^Big_mac protein have not been determined; however, the obesity phenotype of the Big_mac mice indicates that the mutation results in loss of MC4R function.

PCR program

1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40x
6) 72°C 10:00
7) 4°C hold

The following sequence of 400 nucleotides is amplified (chromosome 18, - strand):

1   tcagttcaag gaggactcaa atccagctgc tgcaggaaga tgaactccac ccaccaccat
61  ggcatgtata cttccctcca cctctggaac cgcagcagct acgggctgca cggcaatgcc
121 agcgagtcgc tggggaaggg ccacccggac ggaggatgct atgagcaact ttttgtttcc
181 cccgaggtgt ttgtgactct gggtgtcata agcctgttgg agaacattct agtgatcgtg
241 gcgatagcca agaacaagaa cctgcactca cccatgtact ttttcatctg tagcctggct
301 gtggcagata tgctggtgag cgtttcgaat gggtcggaaa ccatcgtcat taccctgtta
361 aacagtacgg atacggatgc ccagagcttc accgtgaaca 

Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.