Gender Differences in the Relationship of ENPP1/PC-1 Variants to Obesity in a Turkish Population

Abstract

The ectoenzyme ENPP1 (also termed membrane glycoprotein PC-1 or ENPP1/PC-1) is an inhibitor of insulin-induced activation of the insulin receptor. There is evidence from previous studies that coding variants of ENPP1/PC-1 (K121Q) are associated with type 2 diabetes (T2D) and obesity. Studies in the general Turkish population have demonstrated: unique plasma lipid characteristics; a high prevalence of cardiovascular risk factors; and an increased prevalence of obesity and T2D. We investigated, therefore, the association of ENPP1/PC-1 variants with obesity and T2D in Turkish individuals.

The TaqMan allelic discrimination assay was used for genotyping the relationship of ENPP1/PC-1 variants to obesity and T2D in a genetic association study of 1553 genotyped, randomly selected subjects from the Turkish Heart Study. The K121Q (rs1044498) variant, and other previously reported variants (rs997509, rs1799774, rs1044548, rs11964389, rs7754561) were analyzed.

In this cohort, the minor allele frequency of the K121Q variant was associated with obesity in male, but not female, subjects (male, OR 1.64, CI%95 1.004-2.698, p = 0.048; female, OR 1.003, CI%95 0.684-1.471, p=ns.). In addition, the previously reported ENPP1/PC-1 “risk haplotype” [Q (rs1044498), delT (rs1799774), and G (rs7754561) alleles] was found to be associated with obesity in male, but not female, subjects (p = 0.035). In contrast, there was no association of either the K121Q variant or the ENPP1/PC-1 haplotype with T2D.

We find evidence that variants of ENPP1/PC-1 are associated with obesity in the male Turkish population and thus these variants may contribute to the development of the obesity in these individuals.

INTRODUCTION

Several laboratories have observed that the ectoenzyme nucleotide pyrophosphate phosphodiesterase 1 (ENPP1/PC-1) is an inhibitor of the insulin receptor (IR), and is either overexpressed or overactive in muscle, adipose tissue, fibroblasts, and other tissues of insulin resistant individuals, both non-diabetic and diabetic[1,2]. In addition, there is evidence from previous studies that a coding variant of ENPP1/PC-1 (K121Q) is closely associated with insulin resistance, type 2 diabetes (T2D), obesity, and cardio- and nephrovascular diseases[3-6]. This variant is 2-3- fold more potent than the common ENPP1/PC-1 variant (K121K) in inhibiting the IR [7]. However, the relationship of this allele to T2D and obesity varies in different ethnic populations [6-9].

In the Turkish population, there is a high prevalence of cardiovascular risk factors including high triglyceride and low HDL cholesterol levels, and a high rate of cigarette smoking. Consequently, there is an increased rate of cardiovascular heart disease[10]. In Turkey, there is also an increasing prevalence of both obesity and T2D [11].

A total of seven potentially important ENPP1/PC-1 variants have been reported in the literature[12-14]. We selected 6 for genotyping purposes (rs997509, rs1044498, rs1799774, rs1044548, rs11964389, rs7754561). Since there is a strong linkage disequilibrium between rs1044548 and rs1044558 (r² = 1), only rs1044548 was analyzed. Therefore, we determined whether the K121Q variant and other variants were associated with either obesity or T2D in this population. We now report a significant association of ENPP1/PC-1 K121Q variant with obesity in male Turkish subjects.

METHODS

Study Subjects

DNA from a study population of 1553 Turkish adult volunteers from the Turkish Heart Study (10) was employed. Two different cohorts were analyzed. The first cohort consisted of 1386 subjects (101 had T2D) randomly obtained from the general Turkish population (Table 1). The second cohort consisted of 167 T2D patients from the same study population as a case series. T2D was defined either by fasting blood glucose levels >7 mmol/L, glucose levels >11.1 mmol/L during a 2-hour oral glucose test, or by a history of T2D and the use of antidiabetic medications [15] The Committee on Human Research of the University of California, San Francisco, approved the study protocols and they were in accordance with the Helsinki Declaration. All subjects gave informed consent.

TABLE 1.

Mean values and standard deviations of anthropometric and biochemical parameters in a random Turkish population

	Women	Men	p value
N	900	486
Age (years)	42.1 ± 12.7	41.7± 12.4	NS
BMI (kg/m2)	27.9 ± 5.1	27.6 ± 3.7	NS
Waist circumference (cm)	90.5 ± 12.4	98.9 ± 10.3	<0.01
Waist/hip ratio	0.83 ± 0.01	0.92 ± 0.01	<0.01
Blood pressure (mm Hg)
Systolic	130.7 ± 25.7	131.5 ± 17.9	NS
Diastolic	83.2 ± 13.8	84.6 ± 11.3	NS
Total cholesterol (mmol/l)	4.64 ± 1.07	4.63 ± 0.99	NS
HDL-C (mmol/l)	1.19 ± 0.31	0.99 ± 0.24	<0.01
LDL-C (mmol/l)	2.85 ± 0.9	2.86 ± 0.87	NS
Triglycerides (mmol/l)	1.28 ± 0.84	1.71 ± 1.33	<0.01
Glucose (mmol/l)	5.2 ± 1.82	5.39 ± 1.73	NS
Insulin (mIU/l)	11.4 ± 17.7	16.1 ± 23.7	<0.01

Genotyping of ENPP1/PC-1 variants

Genotyping was performed using the TaqMan allelic discrimination assay (Applied Biosystems, Inc., Foster City, CA). The TaqMan genotyping reaction was amplified on a GeneAmp PCR system 9700 (95° C for 10 min, 92° C for 15 sec, and 60° C for 1 min, for 40 cycles) and fluorescence was detected on an ABI PRISM 7700 sequence detector (Applied Biosystems, Inc.). To assess genotyping reproducibility, approximately 5% of the samples were regenotyped for all single nucleotide polymorphisms (SNPs); all repeated genotypes matched their initial designation. Haploview software was used to identify haplotypes (http://www.broad.mit.edu/mpg/haploview/index.php).

Statistical Analysis

Data were analyzed with SPSS (10.0 Chicago, IL, USA). Associations between genotypes, obesity and diabetes status were analyzed. Mean values of continuous traits were compared using the Student t-test according to genotype and haplotype groups; p < 0.05 (two-tailed) was considered significant. Fisher's exact test analysis was used to test differences for categorical variables: obese vs. lean (BMI <25 kg/m² vs. BMI ≥30 kg/m²); and T2D status (T2D vs. non T2D). All variants were in Hardy Weinberg equilibrium. We compared allele or haplotype frequencies after stratification by gender. Logistic regression analysis was performed to assess the effect of the Q121 allele on the obese (BMI ≥30 kg/m²) group versus the lean (BMI <25 kg/m²) group after adjusting for age and gender.

RESULTS

ENPP1/PC-1 K121Q allele frequency in obese subjects

In the random cohort of Turkish subjects, the minor allele frequencies (MAF) of the ENPP1/PC-1 K121Q variants are presented in supplementary Table 1. Only the K121Q variant gave significant results and results are discussed below. Samples from the diabetic case series were excluded in this analysis to keep the analyzed population random. The frequency of the 121Q variant was 13.6% in the lean population (BMI <25 kg/m²; n = 415), and was 14.7% in the obese population (BMI ≥30kg/m²; n = 489, p = 0.3). However, after gender stratification, the 121Q allele frequency was significantly higher in the obese male population than in the lean male population (17.3% vs. 11.5%, p = 0.034) (Table 2). In the male population, the MAF increased in a stepwise manner at increasing BMI cutoff values (Figure 1). In male subjects, mean BMI values were also significantly different in the 121KK and 121KQ vs. the 121QQ genotype groups (27.3 ± 3.7 kg/m² , 27.7±3.4 kg/m² vs. 29.9 ± 6.4 kg/m² p = 0.045). Employing a logistic regression analysis in male Q121 allele carriers, there was statistically significant association with obesity when comparing obese male subjects (BMI ≥30 kg/m²) to lean male subjects (BMI <25 kg/m²) (OR 1.646 [95% CI 1.004–2.698]; p = 0.048).

TABLE 2.

Relationship of obesity with K121Q minor allele frequency in Turkish population

	Total		Men		Woman
BMI	≥30 kg/m2	<25 kg/m2	≥30 kg/m2	<25 kg/m2	≥ 30kg/m2	<25 kg/m2
N	489	415	142	143	347	272
Genotype
K121K	317	317	99	112	260	205
%	(74.9)	(75.4)	(69.7)	(78.3)	(74.9)	(75.4)
K121Q	116	88	37	29	79	59
%	(23.7)	(21.2)	(26.1)	(20.3)	(22.8)	(21.7)
Q121Q	14	10	6	2	8	8
%	(2.9)	(2.4)	(4.2)	(1.4)	(2.3)	(2.3)
Minor allele frequency (%)	14.7	13.6	17.3a	11.5	13.7	13.8

*Comparison of allele frequencies between obese and lean Turks.

A significant difference was observed (Fisher's exact test, p = 0.034) in males.

FIG. 1. K121Q minor allele frequency for different BMI cut-off values in random Turkish male population.

Q121 allele frequency shows an increasing trend with BMI levels in the male Turkish population. BMI (kg/m²).

In contrast to male subjects, in female subjects, the K121Q MAF was 13.7% in obese individuals and it was 13.8% in lean individuals (p = ns). Mean BMI values were not significantly different in the 121KK and 121KQ vs. 121QQ genotype groups in female subjects (28.1 ± 5.3 kg/m² and 27.3±5.1 kg/m² vs. 28.4 ± 6.4 kg/m² p = ns). OR did not reach significance for the Q121 allele when obese female subjects were compared to lean female subjects [1.003(95% C.I. 0.684–1.471) p = ns] (Figure 2).

FIG. 2.

Age-adjusted logistic regression analysis showed Q121 allele has significant effect in obese Turkish male but not in female. Dotted line represents Turkish females and straight line represents males.

ENPP1/PC-1 haplotypes in obese subjects

Six variants were used to construct haplotype structure of ENPP1/PC-1 (Supplementary Table 2). Four haplotypes with a frequency >3% accounted for 83% of all haplotypes. Minor allele of three variants [rs1044548 and rs11964389 from Frittitta et. al.[13] and [rs997509 from Bochenski et. al.[12] were not represented in the common haplotypes; therefore, common haplotypes were defined by three variants (rs1044498, rs1799774 and rs7754561). These variants are the same ones reported by Meyre et. al.[14].

3 common marker haplotype, termed “risk haplotype”, including Q (rs1044498), delT (rs1799774), and G (rs7754561) alleles. This “risk haplotype” is associated with obesity, and T2D in French and Australian populations. We, therefore, examined this “risk haplotype” in our Turkish population. Our results indicated that the “risk haplotype” was more frequent in obese male subjects than in lean male subjects (14.2% vs. 8.7%, p = 0.035) (Table 3). However, there was no difference in this haplotype between obese and lean female subjects.

TABLE 3.

Haplotype distribution in 285 Turkish male samples

			Frequency	Case vs. Control	p value
Haplotype			%	%
K121	T	A	50.8	52.5 vs. 51.3	NS
K121	delT	G	17.2	15.2 vs. 17.3	NS
K121	T	G	15.7	13.8 vs. 18.5	NS
Q121	delT	G	11.8	14.2 vs. 8.7	0.035

The K121Q (rs1044498), IVS20delT-11 (rs1799774), and A–G 1044 (rs7754561) single nucleotide polymorphisms were used to determine haplotypes. 142 obese cases (BMI ≥30 kg/m²) and 143 lean control (BMI <25 kg/²) males were evaluated. Fisher's exact test analysis was performed to compare the haplotype frequency difference between the obese and lean groups.

ENPP1PC-1 121Q allele frequency and haplotype analysis in T2D subjects

In the random cohort of Turkish subjects the K121Q MAF was 11.7% in type 2 diabetic subjects (n = 101) and 14.7% in the non-diabetic population (n = 1285). This difference was not significant. Next, we analyzed both the random and the diabetic cohorts together. The Q allele frequency was 15.3% in type 2 diabetic subjects (n = 268) and 14.7 % in the non-diabetic subjects (n = 1285). In contrast to obesity, there was no significant association when data were analyzed separately by gender. In addition, there was no difference in the “risk haplotype” between T2D subjects and non-diabetic subjects even when analyzed by gender stratification.

ENPP1/PC-1 121Q allele frequency and risk haplotype analysis in subjects with elevated lipid parameters

There was no significant association among genotype groups of the K121Q variant with plasma lipid values including serum TG, HDL-C, and LDL-C when females and males were analyzed either separately or together. Further, there was no association between lipid parameters and the common ENPP1/PC-1 gene haplotypes mentioned above.

DISCUSSION

In the present study, we find that there is an association between the Q allele of K121Q variant of ENPP1/PC-1 and obesity in a random Turkish population. However, this association was only observed in male subjects. A similar association was observed with the ENPP1/PC-1 “risk haplotype” in males. In contrast, with both the K121Q variant and the “risk haplotype”, there was no association with T2D. Thus, in the male Turkish population, the presence of the K121Q variant is a risk factor for obesity.

The ENPP1/PC-1 Q121 allele has recently been reported to influence the risk of obesity[14,16,17]. However, conflicting data have been reported. Whereas some studies of individuals living in Europe have observed a significant association between the Q121 allele and higher BMI[14,16,18], just the opposite has been observed in the USA in both individuals of European descent[8,19] and in African descent[8]. In contrast, three recent studies reported no effect of several ENPP1/PC-1 gene variants, including K121Q variant, on body weight [6,20,21]. Discordant results in genotype-phenotype association studies are common in the evaluation of complex disorders[20]. The results of any study depend on the differences in the genetic and/or environmental background of the sampled population, as well as in the subject recruitment process; thus, discrepant findings among studies do not necessarily imply false results. The mechanism through which ENPP1/PC-1 might influence BMI has not yet been investigated. It is possible that carriers of the Q121 allele develop insulin resistance in hypothalamic neurons, where insulin has potent anorectic actions[22] and this resistance, in turn, increases the subject's appetite and body weight while impairing insulin inhibition of hepatic glucose production. Conversely, a reduced BMI in K carriers might be due to the effect of this allele on peripheral insulin resistance[23].

The previously defined “risk haplotype” for PC-1/ENPP1, including the Q121 allele and 2 non-coding variants (del T and A→G+1044TGA) showed an association with T2D and obesity. Since this haplotype has not been functionally tested, it is speculated that the Q121 allele plus the A→G+1044TGA on the 3UTR may have an additive affect on PC-1/ENPP1 gene expression such as increasing mRNA half-life. Thus PC-1/ENPP1 expression would be increased and the insulin signalling pathway further down regulated. In fact, studies of individuals with this haplotype have higher serum PC-1/ENPP1 levels than controls

A gender difference in association studies has been previously observed for PC-1/ENPP1. In a study of Han Chinese, a gender difference in the association between the Q allele and obesity has been observed [17]. In that study, in contrast to the present study, the Q allele frequency was higher in obese females. The reasons for this difference are unknown but most likely differences in the genetic background, and gender specific environmental factors, recruitment and evaluation of the study population such as lack of information about participants’ glycemic status, and inappropriate study randomization may have had an impact on the results.

Using a well-designed, randomized, study population and evaluating glycemic status of participants during the recruitment process minimizes false-positive associations. Therefore in the present study we studied a large number of random subjects and evaluated glycemic status to eliminate confounding factors. Gender differences in association studies are not uncommon in Turkish populations. For example, we previously reported one haplotype on the Apolipoprotein A5 gene that was associated with hypertriglyceridemia in Turkish males but not in females [24]. Most likely hormonal status plus environmental and nutritional factors caused this difference.

In summary, therefore, we find that the Q allele frequency of the K121Q variant of ENPP1/PC-1 is increased in obese male Turkish subjects. In view of the ability of ENPP1/PC-1 to inhibit insulin action, these data support a role for ENPP1/PC-1 in conditions of abnormal fat and carbohydrate metabolism. Further, this observed increase in male but not female subjects suggests that other genetic and/or environmental factors can modify the effects of ENPP1/PC-1.