Population sequencing of two endocannabinoid metabolic genes identifies rare and common regulatory variants associated with extreme obesity and metabolite level

We sequenced two intervals (covering 188 kb) encoding the endocannabinoid metabolic enzymes fatty-acid amide hydrolase (FAAH) and monoglyceride lipase (MGLL) in 147 normal controls and 142 extremely obese cases. After applying quality filters, we called 1,393 high quality single nucleotide variants, 55% of which are rare, and 143 indels. Using single marker tests and collapsed marker tests, we identified four intervals associated with BMI: the FAAH promoter, the MGLL promoter, MGLL intron 2, and MGLL intron 3. Two of these intervals are composed of rare variants and the majority of the associated variants are located in promoter sequences or in predicted transcriptional enhancers, suggesting a regulatory role. The set of rare variants in the FAAH promoter associated with BMI is 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.During the past decade, the search for the underlying genetic basis of complex traits and diseases in humans has been focused on common DNA variants with a minor allele frequency (MAF) > 0.05. This approach is based on the common variant common disease hypothesis [1], our increased knowledge of common variants [2], and improved genotyping methods [3]. The effort of the human genetics community has led, through genome-wide association studies (GWASs), to the identification of over 400 genetic loci associated with complex traits. However, GWASs have uncovered only a small fraction of the estimated heritability underlying complex phenotypes. The missing heritability is potentially accounted for by rare variants or variants in epistasis, both of which are difficult to identify