Members of the pancreatic polypeptide family and their receptors have been implicated in the control of food intake in rodents and humans. To investigate whether nucleotide changes in these candidate genes result in abnormal weight in humans, we sequenced the coding exons and splice sites of seven family members (NPY, PYY, PPY, NPY1R, NPY2R, NPY4R, and NPY5R) in a large cohort of extremely obese (n=379) and lean (n=378) individuals. In total we found eleven rare non-synonymous variants, four of which exhibited familial segregation, NPY1R L53P and PPY P63L with leanness and NPY2R D42G and PYY Q62P with obesity. Functional analysis of the obese variants revealed NPY2R D42G to have reduced cell surface expression, while previous cell culture based studies indicated variant PYY Q62P to have altered receptor binding selectivity and we show that it fails to reduce food intake through mouse peptide injection experiments. These results support that rare non-synonymous variants within these genes can alter susceptibility to human body mass index extremes.

Background

Methods and findings

Conclusions

Body weight is a quantitative trait with significant heritability in humans. To identify potential genetic contributors to this phenotype, we resequenced the coding exons and splice junctions of 58 genes in 379 obese and 378 lean individuals. Our 96Mb survey included 21 genes associated with monogenic forms of obesity in humans or mice, as well as 37 genes that function in body weight-related pathways. We found that the monogenic obesity-associated gene group was enriched for rare nonsynonymous variants unique to the obese (n=46) versus lean (n=26) populations. Computational analysis further predicted a significantly greater fraction of deleterious variants within the obese cohort. Consistent with the complex inheritance of body weight, we did not observe obvious familial segregation in the majority of the 28 available kindreds. Taken together, these data suggest that multiple rare alleles with variable penetrance contribute to obesity in the population and provide a deep medical sequencing based approach to detect them.

Demonstration of association between common genetic variants and chronic human diseases such as obesity could have profound implications for the prediction, prevention and treatment of these conditions. Unequivocal proof of such an association, however, requires adherence to established methodological guidelines, which include independent replication of initial positive findings. Recently, single nucleotide polymorphisms (SNPs) within GAD2 were found to be associated with class III obesity (BMI > 40 kg/m2) in 188 families (612 individuals) segregating the condition and a case-control study of 575 cases and 646 lean controls. Functional data supporting a pathophysiological role for one of the SNPs (-243A>G) were also presented. In the present study, we attempted to replicate this association in larger groups of subjects, and to extend the functional studies of the -243A>G SNP. In 2,327 subjects comprising 692 German nuclear families with severe, early-onset obesity, we found no evidence for a relationship between the three GAD2 SNPs and obesity, whether SNPs were studied individually or as haplotypes. In two independent case-control studies (a total of 680 class III obesity cases and 1,186 lean controls), there was no significant relationship between the -243A>G SNP and obesity (odds ratio (OR) = 0.99, 95 percent CI 0.83 - 1.18, in the pooled sample). These negative findings were reinforced by a meta-analysis for the association between the 243G allele and class III obesity, which yielded an OR of 1.11 (95 percent CI 0.90 - 1.36) in a total sample of 1,252 class III obese cases and 1,800 lean controls. Finally, we were unable to confirm or extend the functional data pertaining to th e -243A>G variant. Potential confounding variables in association studies invo lving common variants and complex diseases (low power to detect modest genetic effects, over-interpretation of marginal data, population stratification and biological plausibility) are also discussed in the context of GAD2 and severe obesity.