Obesity
Population sequencing of two endocannabinoid metabolic genes identifies rare and common regulatory variants associated with extreme obesity and metabolite level.
Targeted re-sequencing of candidate genes in individuals at the extremes of a quantitative phenotype distribution is a method of choice to gain information on the contribution of rare variants to disease susceptibility. The endocannabinoid system mediates signaling in the brain and peripheral tissues involved in the regulation of energy balance, is highly active in obese patients, and thus a strong candidate pathway to examine for genetic association with BMI. We sequenced two intervals (totaling 188kb) encoding the endocannabinoid metabolic enzymes, FAAH and MGLL, in 147 normal controls (BMI=30 kg/m2) and 142 extremely obese cases (BMI=40 kg/m2). After applying quality filters, we called 1393 high quality single nucleotide variants, 55% of which are rare (MAF<0.01), and 143 indels. We used two different approaches, single marker tests and collapsed markers tests, to identify four intervals associated with BMI: the FAAH promoter, MGLL promoter, MGLL intron 2, and MGLL intron 3.  Two of these intervals are composed of rare (MAF<0.01) variants, one of rare and low frequencies (MAF˜0.03) variants and one of common (MAF˜0.15) variants. The majority of the associated variants are located in promoter sequences or in predicted transcriptional enhancers, suggesting a regulatory role.  Additionally, we demonstrate that the set of rare variants in the FAAH promoter associated with BMI are also associated with increased level of FAAH substrate anandamide, further implicating a functional role in obesity.  Our study, which is one of the first reports of a sequence based association study using next generation sequencing of candidate genes, provides insights into study design and analysis approaches and demonstrates the importance of examining regulatory elements rather than exclusively focusing on exon sequences.