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

Abstract

Background: 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 represents a strong candidate pathway to examine for genetic association with body mass index (BMI).

Results: 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.

Conclusions: 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.

Figure 1

BMI distribution in the CRESCENDO cohort (grey) and in the selected controls (147 samples with BMI ≤ 30 kg/m², blue) and cases (142 samples with BMI ≥40 kg/m², red).

Figure 2

Sequence coverage distribution. (a,b) Genome Browser tracks showing locations of the 40 LR-PCR amplicons (black rectangles), the number of samples with coverage below 20× (blue histogram, 100-bp windows) and GC percent (red histogram, 10-bp windows) along the FAAH (a) and MGLL (b) re-sequenced intervals. The ends of the intervals have lower coverage due to the fact they were amplified by a single amplicon. The 5' end of the FAAH gene was successfully amplified but coverage is low due to difficulty sequencing high GC content regions. The high GC content at the 5' end of the MGLL gene resulted in an inability to successfully design PCR primer pairs despite several attempts. (c) Distribution of the fraction of bases (y-axis) sequenced at increasing usable coverage (x-axis) for sequence-based association studies. Usable coverage is defined at each base as the minimum coverage reached by 90% or more of the samples.

Figure 3

Quality control of SNV identification. Distribution of the matching status of 1,697 genotypes obtained from the 9 replicated samples.

Figure 4

Association with BMI. (a) Significance of the association with BMI identified by single marker tests (-log10(chi-square P-value)) for all SNVs located in the MGLL interval (x-axis, NCBI36 coordinates). SNPs with a P-value < 0.01 are highlighted in red. The recombination rate [56] in the HapMap CEU population for this region is indicated by a blue line and measured on the right axis. (b,c) Significance of the association with BMI for all locus-variants identified by RareCover (see Materials and methods) in the MGLL (a) and FAAH (b) sequenced intervals. For both genes, locus-variants with a P-value < 0.01 are highlighted in red. The MGLL and FAAH gene structures are aligned based on their genomic positions.

Figure 5

Functional annotation of the associated variants in the MGLL interval. Track A: variants identified by the single marker tests (blue bars) or the merge of the locus-variants identified by the collapsed marker test RareCover (red boxes) are indicated. Track B: predicted intervals for promoters (green) or transcriptional enhancers (orange) in HeLa cells [45]. Tracks C and D: the distribution of chromatin binding proteins from the ENCODE data obtained from the UCSC genome browser (15 November 2009). Track C: Broad/MGH ENCODE group chromatin signatures corresponding to enhancers (H3K4me1, H3K27ac) and promoters (H3K4me1+3, Pol2) in various cell types (GM12878, HUVEC, K562, Keratinocytes) [46]. Track D: Yale/UCD/Harvard ENCODE group identified AP2α and γ, c-Myc, Max, c-Fos and Pol2 binding sites in HeLa cells, and STAT1 binding sites in HeLa treated with IFNγ [46].