GAD2 on chromosome 10p12 is a candidate gene for human obesity

The gene GAD2 encoding the glutamic acid decarboxylase enzyme (GAD65) is a positional candidate gene for obesity on Chromosome 10p11&ndash;12, a susceptibility locus for morbid obesity in four independent ethnic populations. GAD65 catalyzes the formation of &gamma;-aminobutyric acid (GABA), which interacts with neuropeptide Y in the paraventricular nucleus to contribute to stimulate food intake. A case-control study (575 morbidly obese and 646 control subjects) analyzing GAD2 variants identified both a protective haplotype, including the most frequent alleles of single nucleotide polymorphisms (SNPs) +61450 C&gt;A and +83897 T&gt;A (OR = 0.81, 95% CI [0.681&ndash;0.972], p = 0.0049) and an at-risk SNP (&minus;243 A&gt;G) for morbid obesity (OR = 1.3, 95% CI [1.053&ndash;1.585], p = 0.014). Furthermore, familial-based analyses confirmed the association with the obesity of SNP +61450 C&gt;A and +83897 T&gt;A haplotype (&chi;2 = 7.637, p = 0.02). In the murine insulinoma cell line &beta;TC3, the G at-risk allele of SNP &minus;243 A&gt;G increased six times GAD2 promoter activity (p &lt; 0.0001) and induced a 6-fold higher affinity for nuclear extracts. The &minus;243 A&gt;G SNP was associated with higher hunger scores (p = 0.007) and disinhibition scores (p = 0.028), as assessed by the Stunkard Three-Factor Eating Questionnaire. As GAD2 is highly expressed in pancreatic &beta; cells, we analyzed GAD65 antibody level as a marker of &beta;-cell activity and of insulin secretion. In the control group, &minus;243 A&gt;G, +61450 C&gt;A, and +83897 T&gt;A SNPs were associated with lower GAD65 autoantibody levels (p values of 0.003, 0.047, and 0.006, respectively). SNP +83897 T&gt;A was associated with lower fasting insulin and insulin secretion, as assessed by the HOMA-B% homeostasis model of &beta;-cell function (p = 0.009 and 0.01, respectively). These data support the hypothesis of the orexigenic effect of GABA in humans and of a contribution of genes involved in GABA metabolism in the modulation of food intake and in the development of morbid obesity.<br /

Ln GAD65Ab Levels Adjusted for Age and BMI According to the Genotypes at SNPs −243 A>G, +61450 C>A, and +83897 T>A

<p>Mean level values ± SD are displayed for each genotype. Wild-type are AA, CC, TT; heterozygous are AG, CA, TA; and homozygous are GG, AA, AA, respectively, for each SNP. (A) Nonobese normoglycemic subjects. (B) Morbidly obese patients.</p

EMSA of the the −243 A>G Polymorphism with βTC3 Nuclear Proteins

<p>Specific complex formation is indicated by an arrow. Lanes (−) are radiolabeled probes without nuclear extract. βTC3 nuclear proteins have an higher affinity for the G allele variant than for the A allele at SNP −243 A>G.</p

Mapping of Chromosome 10p and the <i>GAD2</i> Gene

<div><p>(A) Fine mapping of the Chromosome 10p locus between markers D10S548 and D10S220 in 188 nuclear families (620 individuals). Multipoint analysis for obesity phenotype.</p> <p>(B) SNP map of the <i>GAD2</i> gene. Positions were assigned according the location to the A of the ATG. The 15 SNPs selected for association studies are indicated in red. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000068#pbio.0000068-t001" target="_blank">Table 1</a>.</p></div

Pairwise LD between SNPs of <i>GAD2</i> and <i>MYO3</i> Genes in the French Obese Population

<p>Pairwise LD between SNPs is measured by triangles (color scale). Regions of high and low LD are represented by red and blue shading, respectively. The graph is not to scale; indeed, the SNPs are equidistant to highlight the detailed pattern of LD.</p

Effect of the −243 A>G , −1600 G>A, and −2004 A>T SNPs on Transcriptional Activity

<p>G, A, and T alleles at −243 A>G, −1600 G>A, and −2004 A>T SNPs were generated into the wild-type construct (wt). βTC3 cells were transfected with equivalent amounts of pGL3, wild-type, Gad-2004T, Gad-1600A, and Gad-243G constructs. pRLTK was cotransfected with each construct. Luciferase and <i>Renilla</i> activities were assayed and normalized. Each experiment was performed in duplicate and replicated five times. A 6-fold increase of transcriptional activity was observed for the Gad-243 as compared to the wild-type construct.</p

Fasting Insulin and HOMA

<p>Fasting insulin (A) and assessment of HOMA-B% (B) in 376 nonobese normoglycemic subjects according to the genotypes at SNPs −243 A>G, +61450 C>A, and +83897 T>A. Mean level values ± SD are displayed for each genotype. Wild-type are AA, CC, TT; heterozygous are AG, CA, TA; and homozygous are GG, AA, AA, respectively, for each SNP.</p

Association Studies of the −243 A>G Variant with Food Intake Behavior Parameters in Morbidly Obese Patients

<p>The three stable factors—cognitive restraint of eating, disinibition, and hunger—were assessed in 464 morbidly obese patients to fill in the TFEQ established by <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000068#pbio.0000068-Stunkard1" target="_blank">Stunkard and Messick (1985</a>). Mean score values ± SD for each genotype are represented.</p