Obesity has become a major health problem in modern societies, with a prevalence of up to 25% in western societies and an increasing incidence in children (1). Obesity present in adolescence has been shown to be associated with increased overall mortality in adults (2).
There is strong evidence for a genetic component to the risk of obesity, including prevalence differences between racial groups (3,4). Both the familial occurrence of obesity and higher concordance for fat mass among monozygotic twins has been long noted (5,6).
The MC4R gene, encoding the melanocortin 4 receptor, was the first locus at which mutations were associated with dominantly inherited morbid human obesity and was the commonest genetic cause of human obesity described before the era of genome-wide association (GWA) studies.
A common genetic variant located upstream of insulin-induced gene 2 (INSIG2) was described in 2006 to be associated with both adult and childhood obesity from the first GWA study published for this phenotype (7); however, this has proven controversial, with three subsequent technical reports refuting the observation (8,9,10). The publication of a second obesity gene, FTO (11), almost a year later has been more robust, with independent studies coming out around the same time drawing similar conclusions (12,13,14). Indeed, we have reported replication to the FTO gene in our pediatric obesity cohort, together with a successful refinement of the signal in African Americans (AAs) (15).
To identify additional common variants influencing BMI, Loos et al. (16) analyzed GWA data from ∼17,000 individuals of European descent, derived from multiple efforts. After the FTO gene, the strongest association signal (rs17782313) mapped 188 kb downstream of the MC4R gene. They then went on to confirm the BMI association in ∼60,000 adults and ∼6,000 children, with the latter showing higher odds ratios and higher levels of significance of association with variants 3′ to MC4R in comparison to the other analyses.
We elected to analyze this signal in the context of our ongoing GWA study of childhood obesity. The previously published single-nucleotide polymorphism (SNP), rs17782313, was not included on the Illumina BeadChip that are used in this study, but three other SNPs present were perfect surrogates for this SNP i.e., r2 = 1, in the CEU HapMap sample, namely rs571312, rs10871777, and rs476828. Using the allelic χ2 association test, we observed significant or borderline association between these SNPs and risk for childhood obesity in our current European-American (EA) cohort, consisting of 728 obese children (BMI ≥95th percentile) and 3,960 controls (BMI <95th percentile). The minor allele frequencies of rs571312, rs10871777, and rs476828 in the cases were 0.249, 0.249, and 0.257, respectively, while they were 0.225, 0.226, and 0.232 in controls, respectively, yielding odds ratios of 1.142 (95% confidence interval 1.003–1.301; P = 0.045), 1.137 (95% confidence interval 0.998–1.294; P = 0.054), and 1.145 (95% confidence interval 1.005–1.305; P = 0.042) (Table 1).
As such, from this interim analysis of our ongoing GWA study, we could observe replication in the childhood form of the disorder in EA. The three surrogate SNPs conferred risk for the disorder with a comparable magnitude to that previously observed in this ethnicity. However, it should be noted that only rs10871777 gave a P value of 0.054 as a consequence of a lower genotyping yield than the other two SNPs, rs571312 and rs476828, both of which were statistically significant.
We went on to analyze 27 additional SNPs on the BeadChip in the region of linkage disequilibrium (LD) harboring the association signal. Table 1 shows that three other SNPs (rs633265, rs2051311, and rs1350341) that are in strong, but not perfect, LD with rs17782313 are also nominally associated with childhood obesity in EA.
Variants found in populations of both African and European ancestry may represent more universally important genes to the disorder. A cohort of African ancestry can also potentially aid in refining associations made with the GWA approach due to differing LD in this ethnicity, as was the case in our study of the FTO gene and its role in childhood obesity (15) (see Supplementary Figure S1 online for a direct comparison of LD patterns (r2) at this locus, 3′ to MC4R, between the CEU and YRI HapMap sample sets). As such, we also analyzed rs571312, rs10871777, and rs476828 in our AA cohort, consisting of 1,008 obese children (BMI ≥95th percentile) and 2,715 controls. Of these three SNPs, which are in complete LD with rs17782313 in CEU HapMap sample, only rs10871777 is in strong LD with this marker in the YRI HapMap sample (r2 = 0.927) while rs571312 and rs476828 are in weak to moderate LD (r2 = 0.149 and 0.526, respectively). The resulting genomic inflation factor was only 1.05; however, there was no significant association observed with these SNPs in this cohort (Table 2). With respect to all 30 SNPs present on the BeadChip in this region, although rs9966951 and rs12457166 yielded nominally significant association (and also rs1942880 when reanalyzing this data adjusting for admixture), there was no significant association with any of these SNPs in this ethnicity when correcting for the number of tests employed (significance threshold P = 0.0017) (Table 2).
Therefore, we failed to show evidence of association in the AA cohort, despite the fact that the AA case cohort was larger than the EA set. However, we may have missed a bona fide association at this locus in AA due to the fact that the SNPs assayed, using the BeadChip employed in this study, were not selected for optimal haplotype tagging for the YRI HapMap sample; as such, additional SNP genotyping would be required for a more comprehensive appraisal of this locus in this ethnicity. As we did not observe association at this locus in AA, we were unable to refine this signal working with this ethnicity. It should, however, be noted that rs10871777, which was the only SNP in strong LD with rs17782313 in both ethnicities, did yield the same direction of effect in the AA cohort, albeit nonsignificantly, with a very modest odds ratio.
In conclusion, we have demonstrated that SNPs 3′ to the MC4R locus confer a similar magnitude of risk for obesity in our pediatric white cohort as previously reported in both adults and children with the same phenotype. This observation further supports the notion that this pathway is causally linked to the disorder in children, over and above the previously described role of this gene in the rarer syndromic form of obesity, suggesting that interventions at this pathway level may be of value in patients who suffer from the more general form of the disease. The variants that we observe association to may directly dictate expression levels or some other regulatory mechanism, but are more likely to be in LD with the causative variant(s). However, unlike with the FTO gene (15), we were unable to observe association at this locus in AAs with the genotyping platform we employed.