Genetic Variation in the Human Winged Helix/Forkhead Transcription Factor Gene FOXC2 in Pima Indians

The forkhead box C2 (FOXC2; also known as homologue-like 14 or mesenchyme forkhead 1) is a winged helix transcription factor whose clinical relevance was first recognized in the context of hereditary lymphedema. Several loss-of-function mutations were initially identified in families with lymphedema distichiasis (1), a condition characterized not only by lymphedema of the limbs but also by double rows of eyelashes (distichiasis), cardiac defects, cleft palate, extradural cysts, and photophobia, illustrating the developmental pleiotropy of this transcription factor.

The role of FOXC2 as a key regulator of adipocyte metabolism has recently been described (2). In mice overexpressing FOXC2 in adipocytes, the intra-abdominal white adipose tissue depot was reduced and had acquired a histology similar to brown adipose tissue, whereas interscapular brown adipose tissue was hypertrophic. Increased adipocyte FOXC2 expression had a pleiotropic effect on the expression of genes influencing cellular differentiation and metabolism, insulin action, β-adrenergic sensitivity, and intracellular signaling. The net effect of these FOXC2-related alterations was consistent with protection against obesity. Furthermore, increased FOXC2 expression induced by a high-fat diet seemed to counteract most of the symptoms associated with obesity, including hypertriglyceridemia and diet-induced insulin resistance, suggesting a protective effect also against type 2 diabetes. These findings in mice suggest that FOXC2 is a plausible candidate gene for obesity, insulin resistance, and type 2 diabetes in humans.

The prevalence of obesity and type 2 diabetes in the Pima Indian population is among the highest in the world. Both of these diseases have a substantial genetic component (3). To determine whether variation in the FOXC2 contributes to the genetic basis of either type 2 diabetes or obesity in humans, we screened the gene for prevalent and functionally relevant variants and genotyped two informative single nucleotide polymorphisms (SNPs) in a large Pima cohort for association analyses.

The single exon of the FOXC2 gene, as well as an additional 638 bp of the flanking 5′ region and 300 bp of the 3′ region, was sequenced in 24 DNA samples from full-blooded Pima Indians. Four SNPs were identified (Fig. 1). Two SNPs were found in the putative promoter region, a C-512T transition (allelic frequency T = 23%) and a G-350T (allelic frequency T = 28%). In addition, two SNPs were found in the 3′ region, a C1548T (allelic frequency T = 4.5%) and a C1702T (allelic frequency T = 28%). The G-350T and the C1702T variants were in complete linkage disequilibrium, and the C1548T variant was rare; therefore, only the C-512T and the G-350T variants were additionally genotyped in 937 Pima Indians. The genotype distributions for all SNPs were consistent with Hardy-Weinberg equilibrium (P > 0.13 vs. expected frequency, χ² test).

There was no association of either the C-512T or the G-350T with type 2 diabetes in 937 full-blooded Pima Indians (611 diabetic and 326 nondiabetic subjects), which is consistent with a recent Japanese study (4). However,the C-512T SNP was associated with BMI in 644 Pimas with normal glucose tolerance who were at least 18 years of age. Individuals homozygous for the −512T allele (T/T) had a lower BMI than the homozygous C/C and heterozygous C/T individuals (35.4 ± 0.4 kg/m² in C/C, 35.9 ± 0.5 kg/m² in CT, and 32.4 ± 0.9 kg/m² in TT, P = 0.03 or P = 0.009 under a recessive model, adjusted for age, sex, date of birth, and family membership). The association with BMI was substantiated by an association with percent body fat (P = 0.02, adjusted for age, sex, and family membership) in 215 Pima Indians with normal glucose tolerance who had undergone further metabolic testing to exam body composition (Table 1). However, the C-512T variant was not associated with an insulin secretory defect or with insulin resistance—metabolic characteristics previously determined to be risk factors for type 2 diabetes in this population. To estimate the likelihood of erroneously assuming no difference, a power calculation was performed for insulin action (glucose disposal for low-dose insulin clamp). Based on the measured variance of this parameter, the power to detect a 20 or 15% difference between two genotype groups was 0.83 or 0.61, respectively.

In a study from a Swedish cohort, the T allele at position −512 was associated with enhanced insulin sensitivity (based on homeostasis model analysis) in 82 female sibpairs discordant for the variant (5). The same researchers reported that muscle FOXC2 mRNA levels increased in response to insulin in subjects carrying the protective T allele, while no significant change was seen in carriers of the C allele during a hyperinsulinemic-euglycemic clamp. Therefore, in contrast to our results in Pima Indians, the Swedish data clearly suggest an involvement of FOXC2 in insulin resistance, as well as suggest a potential mechanism of differential expression of FOXC2. Since the Swedish study provided evidence for female-specific effects of the −512 polymorphism, we analyzed men and women separately (Table 2). Basal glucose turnover, which in the steady state is equal to glucose disposal, was significantly greater in women with the −512T allele as compared with women with the −512C allele. This difference was not observed in men. In the presence of similar fasting insulin, a difference in basal glucose turnover may suggest greater insulin sensitivity only detectable at low insulin levels, in agreement with the Swedish report, but not during experimental hyperinsulinemia. Also consistent with the Swedish study, we found a sex-specific association of fasting plasma triglycerides with the C-512T variant, where women carrying the −512T allele had lower plasma triglycerides. No difference in triglycerdies was seen in Pima Indian men (Table 2). The G-350T variant also showed a sex-specific effect in that there was a significant association with plasma free fatty acids in men only (P = 0.04) (Table 2). Although both our study and the Swedish study identified sex-specific effects, the physiologic basis of these sex differences remains unknown. It is also noteworthy that these sex differences were not observed when we performed association analyses in men and women together, and simply adjusted for sex.

In Pima Indians with normal glucose tolerance, the G-350T variant was associated with fasting plasma glucose concentrations (P = 0.006) (Table 1). However, we view this association with caution, since none of the traits implicated in the regulation of fasting glucose, such as insulin action, insulin secretion, glucose production, or 2-h plasma glucose, significantly differed between the genotypic groups (Table 1). Moreover, the association with fasting plasma glucose concentrations was only due to the difference in the mean from a small group of T/T homozygotes (n = 23). Therefore, we cannot exclude the possibility that the association of G-350T with fasting plasma glucose concentrations is due to a statistical type 1 error.

In conclusion, variation in the FOXC2 does not appear to have an important role in the etiology of type 2 diabetes in Pima Indians. However, the C-512T variant is associated with obesity and percent body fat in Pima Indians and seems to be involved in the regulation of basal glucose turnover and plasma triglyceride levels in women.