In the present study, we attempted to replicate the important recent findings of Boutin et al. [18
], which implicated three SNPs in GAD2
(the −243 A>G allele and a haplotype of the +61450 C>A and +83897 T>A SNPs) in the predisposition to class III human obesity. To replicate their findings, we first performed family-based tests of association for all three SNPs in 693 nuclear families segregating severe obesity (2,359 participants, nearly four times as many participants as in the original report). This group of individuals included 89 families found to have linkage of severe obesity to Chromosome 10p12 [16
]. No evidence for excess transmission of any GAD2
alleles or haplotypes from parents to affected offspring was obtained. Next, we conducted an adequately powered case-control study to test the association between class III obesity and the GAD2
−243 A>G variant in Caucasians. Consistent with the family-based association results, we did not observe any association between the −243 G variant and class III obesity in 680 cases and 1,186 lean controls. These findings were also obtained in a meta-analysis for the association between the −243 A>G SNP and class III obesity. Lastly, we obtained results from the reporter gene and DNA binding experiments for the −243 A>G variant that were inconsistent with the original report. Overall, we found that (i) a haplotype consisting of the WT alleles at SNPs +61450 C>A and +83897 T>A does not appear to protect against severe, early-onset obesity, (ii) the −243 A>G SNP is not associated with class III obesity in adults, (iii) other haplotypes in the region of GAD2
are not associated with severe obesity, and (iv) the −243 A>G SNP does not elicit detectable effects on transcription of a luciferase reporter gene in βTC3 murine insulinoma cells.
Irreproducibility of positive findings has been a common criticism leveled at association studies investigating the common genetic basis of complex diseases [19
]. The reasons cited are numerous, and include a lack of statistical power to detect small to moderate effects, lack of control over the Type I error rate, overinterpretation of marginal data, population stratification, and poor biological plausibility [27
]. Regarding the conflicting results obtained by Boutin et al. [18
] and the current study, it is likely that the lack of replication could be ascribed to any of these causes, which are discussed below. The inconsistencies between association studies may also reflect the complex interactions between multiple population-specific genetic and environmental factors.
The lack of statistical power to detect alleles of minor effect is likely to have contributed to the differences between the study by Boutin et al. [18
] and the current investigation. Based on the findings of the initial report, we conducted an adequately powered, ethnically matched, case-control study. Although our results overlapped with the size of the initial effect, they did not show a significant association between the −243 G allele and class III obesity (). We estimate that we had 60% power to detect a significant difference (α of 0.05) in allele frequency between our pooled groups of cases and controls, assuming that the −243 G allele (frequency of 0.18) was the disease allele, a genotype relative risk of 1.25, and a prevalence of class III obesity in the general population of 5% [29
]. The family-based association tests had a similar amount of power (~60%), given the same assumptions. Under these conditions, the original study [18
] may have been underpowered. Moreover, it must be pointed out that the marginally significant association (p
= 0.04) they observed between the −243 G allele and class III obesity was observed in only one of their two groups of participants, and did not reach nominal significance in their family-based analysis (p
= 0.06). Although the lack of statistical significance does not exclude the possibility of an association (as we cannot rule out smaller effects), the data do not support a relationship between this SNP and class III obesity.
The interpretation of results from genetic association studies is frequently complicated by other statistical issues, such as a failure to control for multiple hypothesis testing, overinterpretation of marginal data as positive trends, and the well-documented tendency for initial positive findings to overestimate the strength of the association [21
]. This “jackpot” phenomenon [24
] can be readily observed in our meta-analysis ().
Population stratification may also account for some of the inconsistencies observed between association studies, though its importance may have been overestimated [19
]. Population stratification is usually controlled for by careful matching of cases and controls by ethnicity, using family-based tests of association (such as the TDT) or studying multiple case-control populations [30
]. Considering the marked differences in allele frequency that we observed between ethnic groups for the GAD2
SNPs (the −243 A>G and +61450 C>A SNPs in particular), as well as the known differences in the prevalence of class III obesity between Caucasian Americans and African Americans [31
], it is plausible that a small difference in ancestry between cases and controls could lead to spurious claims of association. Naturally, future studies of the GAD2
gene should carefully take this into consideration.
There is no obvious explanation for the differences in results obtained for the EMSA and reporter gene assays. Regarding the EMSA, a major problem with these experiments is that most random DNA sequences will be bound by a nuclear extract from any cell line (Figures S4
and Figure 4 in [18
]). It is likely that the introduction of single base-pair differences into this DNA sequence will interfere with the binding pattern observed. Moreover, while an allele-specific difference in the binding of βTC3 cell nuclear extract definitely occurs for the −243 A>G polymorphism, this observation is of limited physiological significance, because: (i) it appears to be restricted to this cell type (and there is no apparent difference in allele-specific binding for nuclear extract derived from a neuronal cell line); and (ii) the binding of this nuclear protein does not appear to affect transcription of a luciferase reporter gene in βTC3 cells. Finally, even if the −243 A>G SNP did affect transcription of the reporter gene in this context, there is no prior biological evidence to suggest that perturbation of GAD2
expression in β cells could exert detectable effects on long-term energy homeostasis.
This latest point raises the issue of biological plausibility. GAD2
encodes the 65-kDa isoform of the enzyme glutamate decarboxylase, which catalyzes the production of γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter, from glutamic acid. The biological evidence implicating GAD2
as a candidate gene (and by extension, hypothalamic GABA levels as causative) in severe obesity is as follows: GAD2
mRNA is co-expressed with neuropeptide Y in neurons of the hypothalamic arcuate nucleus that act in the nearby paraventricular nucleus and other hypothalamic areas to stimulate food intake [32
]. Concomitantly, these arcuate neuropeptide Y neurons inhibit the parallel and opposing effects of neighboring pro-opiomelanocortin/cocaine- and amphetamine-regulated transcript neurons via GABA-ergic collateral inputs [33
]. In rats, administration of muscimol, a GABAA
receptor agonist, into either the third ventricle or the hypothalamic paraventricular nucleus stimulates feeding in a dose-dependent manner [34
]. Similarly, inhibition of GABA synthesis in the ventromedial hypothalamus, by injection of antisense GAD-65 and GAD-67 oligonucleotides, has been shown to suppress food intake [35
However, enthusiasm for GAD2
as a candidate gene for severe obesity is dampened somewhat by the observation that GAD2
-deficient mice appear normal with respect to behavior, locomotion, reproduction, and glucose homeostasis, but suffer from epileptic seizures [36
]. Also, levels of GAD2
mRNA in the arcuate nucleus of the rat do not change in response to 48 hr of food deprivation, as do levels of prepro–neuropeptide Y mRNA [37
]. Furthermore, the notion that hypothalamic GABA levels are proportional to food intake may be an oversimplification; although microinjection of GABA into the paraventricular nucleus and ventromedial hypothalamus stimulates feeding [38
], injection of GABA, muscimol [39
], or an adenovirus expressing GAD2
], into the lateral hypothalamus of rats has been observed to have the opposite effect.
While these experimental results do not exclude GAD2
as a candidate gene for human obesity, it remains possible that the linkage signal could be due to variation in a neighboring gene. Certainly GAD2
is the leading candidate in this region, due to some of the biological evidence presented above and the location of D10S197 within one of its introns. However, in light of the large number of genes involved in energy homeostasis (recently reviewed in [41
] and [42
]), the multiple tissue-specific roles of each gene, and the readily available information regarding the homology and expression pattern of uncharacterized genes, it now seems possible to make a tenuous case for almost any single gene in the regulation of body weight. For example, a preliminary glance at the Chromosome 10p12 region yielded several interesting genes: TPRT
(trans-prenyltransferase), the enzyme that elongates the prenyl side-chain of coenzyme Q, one of the key elements of the respiratory chain within mitochondria; GPR158
, which encodes a metabotropic glutamate, GABAB
–like G-protein-coupled receptor; and PTF1A,
which encodes pancreas-specific transcription factor 1a. Although only a little is known about each of these genes, it is possible to speculate on the potential role of each in obesity. GAD2
is no exception. At present, however, there is insufficient genetic or biological evidence to implicate genetic variation in GAD2
in the predisposition to severe obesity in humans.