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Effect of the rs997509 Polymorphism on the Association between Ectonucleotide Pyrophosphatase Phosphodiesterase 1 and Metabolic Syndrome and Impaired Glucose Tolerance in Childhood Obesity

Department of Pediatrics "F. Fede" Seconda Università degli Studi di Napoli, 80138, Napoli, Italy

Address all correspondence and requests for reprints to: Dr. Emanuele Miraglia del Giudice, Dipartimento di Pediatria, Seconda Università di Napoli, Via Luigi De Crecchio No. 2, 80138 Napoli, Italy. E-mail: emanuele.miraglia{at}unina2.it.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Variants on the nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP-1) gene have been associated with obesity and insulin resistance. Because insulin resistance is a pivotal factor in the development of metabolic syndrome (MS) and impaired glucose tolerance (IGT), we aimed to test the association between the K121Q and rs997509 ENPP-1 variants with obesity, MS and IGT in obese children and adolescents.

Methods: We screened 809 children, 409 obese and 400 lean controls. Obese subjects underwent a standard oral glucose tolerance test, whole body insulin sensitivity index (WBISI) and homeostasis model assessment (HOMA) were calculated.

Results: No difference in prevalence for K121Q and rs997509 polymorphisms between obese and controls (P > 0.05) were observed. Obese children carrying the rs997509 rare allele showed higher insulin (P = 0.001), HOMA (P < .001) and lower WBISI values (P = 0.04) compared with common allele homozygous. A similar observation was done for K121Q variant, with 121Q allele carriers showing higher insulin (P = 0.03) and HOMA (P = 0.04) values than 121K homozygotes. Moreover, subjects carrying the rs997509 rare allele had higher risk of MS (odds ratio 2.4, 95% confidence interval: 1.3–4.3) and IGT (odds ratio 4.7, 95% confidence interval: 1.9–11.4) than common allele homozygotes. Evaluating combined effects of both polymorphisms, which are in strong linkage disequilibrium, we showed that the effect on insulin sensitivity was due to the rs997509 T variant.

Conclusion: We conclude that the ENPP1 rs997509T allele can predispose obese children to MS and IGT and that this variant might drive the association between the ENPP1 121Q allele and insulin resistance.

	Introduction

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Children are becoming increasingly vulnerable to overweight and obesity around the world. At least 155 million school-age children worldwide are overweight or obese, according to the latest estimates from the International Obesity Task Force (1). One of the most frequent complications of obesity is the early occurrence of metabolic syndrome, defined as the cooccurrence of dyslipidaemia, hypertension and impaired glucose homeostasis (2). Insulin resistance represents the link between obesity and these metabolic alterations (3). A crucial step in the pathogenesis of insulin resistance is represented by the free fatty acids (FFA) accumulation in the liver, fat cells and, particularly, skeletal muscle of obese patients, that, interfering with the normal insulin signaling cascade, appears as the primary determinant of insulin resistance (4). As consequence of insulin resistance, pancreas needs to increase its insulin production to maintain normal values of glycaemia, but the progressive fat accumulation in pancreatic β cells leads to β cells failure causing a defective insulin secretion, inadequate to maintain glycaemia in the normal range. This condition appears as clinically evident when impaired glucose tolerance (IGT) or type 2 diabetes (T2D) occurs. In fact, along with metabolic syndrome, IGT and T2D are nowadays much more common that in the past years among obese children and adolescents, with a range of incidence between 0.5% and 4% for T2D and 5% and 25% for IGT (5, 6). The plasma membrane enzyme termed ENPP1 (ectonucleotide pyrophosphatase phosphodiesterase 1), has been shown to inhibit insulin receptor function by affecting its tyrosine kinase activity in peripheral tissues, including liver, muscle and fat (7). It is a class II transmembrane glycoprotein located both on plasma membrane and in the endoplasmatic reticulum (8). Although its physiological function is not completely understood, there is evidence that ENPP1 plays a major role in insulin resistance development (9). Several studies have reported that the locus where ENPP1 gene maps (6q22-q23) is linked to both insulin resistance and T2D, highlighting the potential role of this gene in modulating susceptibility to these metabolic issues (9). The most widely investigated ENPP1 polymorphism is the substitution of a lysine with a glutamine at codon 121 (K121Q), with the 121Q variant associated with type 2 diabetes (9). This amino acid change is located in the second somatomedin-B-like domain of ENPP1 and may interfere with protein-protein interactions (10). Human studies demonstrated that obese adults carrying the Q variant show lower insulin sensitivity than noncarriers (11). Moreover, association studies have shown that ENPP1 121Q variant may influence the susceptibility to develop obesity and IGT during childhood (12, 13). Although the majority of the studies dealing with the ENPP1 gene have focused their attention on the K121Q variant obtaining discordant results (9), a recent report by Bochenski et al. examining the associations of tagging single nucleotide polymorphisms (SNPs) and haplotypes of the linkage disequilibrium block containing K121Q with T2D, showed that another variant, the rs997509, lying in the intron 1 and consisting in a substitution of a cytosine (C) with a timine (T), had a strong correlation with T2D (14). The authors suggested that the T allele of the new identified SNP, is sufficed to distinguish the 121Q carrying haplotype that was significantly more associated with T2D.

Given these evidences we decided to screen the K121Q and rs997509 in a population of obese children and adolescents to try to replicate previous results and to verify which relationship intercourse between the two polymorphisms and the predisposition to obesity and insulin resistance in our population.

	Subjects and Methods

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Cohort description and clinical evaluation

Four hundred and nine Caucasian obese children and adolescents, referred to our ward (childhood obesity service) since the 1999 have been enrolled. Subjects between 2 and 16 yr of age and with a body mass index (BMI) exceeding the 95^th percentile for their age and sex were eligible. Subjects using medications that alter blood pressure, glucose or lipid metabolism were excluded. Moreover, all subjects missing the complete data set needed for the analysis were excluded. The ethical committee of the Second University of Study of Naples approved the study. Informed consent was obtained by parents and, where appropriate, by children.

Of the 409 subjects enrolled, 208 were girls. This sample was representative of the 2482 children referred to our ward from 1999 to 2005; in fact, no differences in mean age, sex distribution and pubertal stage were observed between the study sample and the sample of excluded subjects. Weight and height were measured and BMI was calculated. SDs scores for BMI were calculated by using the LMS method (15). The population mean age was 11.5 ± 3.0 yr; the mean z-score BMI was 3.6 ± 1.0. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured three times while the subjects were seated and the two last measurements were averaged for the analysis (16). Pubertal stage was assessed using Tanner criteria (17). Thirty one percent of children included in the study were pubertal (71 girls). All measurements were taken twice by the same operator.

To test the ENPP1 allelic distribution a group of controls, composed by 400 (200 girls) lean age and sex matched children (27% pubertal, 63 girls), was recruited as previously described (18). Briefly, unrelated lean children who were age and sex matched and belonged to the same geographic area were recruited as controls (mean age, 10.5 ± 2.3 yr; mean BMI z-score, 0.4 ± 0.4). They consulted the Department of Pediatrics of the Second University of Naples for presumed diseases and were found to be normal. Informed consent from parents and assent from children were obtained before participation in the study. The dosage of fasting insulin, serum glucose, plasma lipids and blood pressure measurement has been performed also in 200 controls.

Metabolic evaluation

After informed consent, a blood sample was drawn from each patient after an overnight fast. The serum was frozen at –20C until analyzed. Triglycerides and total cholesterol levels were determined by an enzymatic colorimetric test with lipid clearing factor and high density lipoprotein (HDL)-cholesterol was measured by precipitation. To underwent an oral glucose tolerance test (OGTT) by assuming 1.75 g of glucose per kilogram of body weight, subjects were evaluated at 8 a.m. after an overnight fast; they consumed a diet containing at least 250 g of carbohydrates per day for three days before the study and refrained from vigorous physical activity; insulin and glucose levels were measured during the OGTT at baseline and later every 30 min for 120 min.

Given the changes occurring in body composition during growth, to diagnose the metabolic syndrome the criteria of the National Cholesterol Education Program’s Adult Treatment Panel (19) and the World Health Organization (20) were modified according to Weiss et al. (2). Consequently, children and adolescents in our study were classified as having the metabolic syndrome if they met three of the following criteria: BMI exceeding the 95^th percentile, systolic and/or diastolic blood pressure exceeding the 95^th percentile, triglycerides levels higher than 110 mg/dl, HDL cholesterol lower than 40 mg/dl for males and 50 mg/dl for females, impaired glucose tolerance (glucose level greater than 140 mg/dl, but less than 200 mg/dl after two h from the beginning of the OGTT).

Insulin resistance was assessed using the homeostasis model assessment (HOMA) as follows: fasting insulin (pmol/liter) x fasting glucose (mmol/liter)/135. The degree of insulin sensitivity was assessed by using the whole body insulin sensitivity index (WBISI). The composite WBISI is based on values of insulin and glucose obtained from the OGTT and the corresponding fasting values, as originally described (21). It represents good estimates for clamp-derived insulin sensitivity and it has been demonstrated to be correlated with intramyocellular lipid content (22). It has been obtained according to the following formula: 10.000/square root of [(fasting insulin x fasting glycaemia) x (mean insulin concentration during OGTT) x (mean glycaemia during OGTT)] (22).

Genotyping

All the patients and controls were genotyped for the K121Q and rs997509 variants. For the rs997509 C/T substitution the following primers were used, F: 5'-ATTTTTTCCTTCAGTGTATA-3' and R: 5'-ACACCCTACAACCCCTAAGA-3'. The AciI restriction enzyme was used to identify the variant, because the T allele eliminates an AciI restriction site. To detect the K121Q variant the couple of primers used were: F: 5'-GCAATTCTGTGTTCACTTTGG-3' and R: 5'-GAGCACCTGTTGACACA-3'. AvaII restriction enzyme was used to detect the variant.

Statistical analysis

Power calculations were performed using the genetic power calculator. http://pngu.mgh.harvard.edu/purcell/gpc/. Because previous studies showed an association between childhood obesity and K121Q variant (12, 13), before analyze the association of Q variant with obesity we calculated that the statistical power in the proband – control data set was 98% at P = 0.05 to detect an association using the control frequencies and Odds Ratios (OR) shown in the German population (13) (allele frequency 0.11. OR=1.82 for T allele). To reach a statistical power of 98% at P = 0.05 we screened 400 age and sex matched lean controls.

Chi Square test was used to verify whether the genotypes were in Hardy-Weinberg equilibrium and to compare allele frequencies between obese and nonobese subjects and metabolic syndrome and IGT prevalence between the different genotypes. Linkage disequilibrium between markers was assessed as described (23, 24). A general linear model (GLM) was used to evaluate the differences between groups of genotype and the effect of genotypes interaction on phenotype. Because of the low prevalence of rare allele homozygotes for the K121Q and rs997509 variants, to test genotype/phenotype associations rare allele homozygotes and heterozygotes were merged. When necessary the variables were adjusted for age, sex, BMI and pubertal stage. A logistic regression was generated to calculate the odds of developing the metabolic syndrome or IGT for a given genotype. Although raw values are shown, nonnormally distributed variables were log-transformed before performing the analysis. Data are expressed as means and SDs. P values <0.05 were considered statistically significant. The SAS Statistical Software Package version 8.2 (SAS institute, Clary, NC) was used to analyze data.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
One hundred and forty three (35%) patients (66 girls) showed the criteria to make diagnosis of metabolic syndrome, three patients (0.7%) showed impaired fasting glucose (IFG) and 23 subjects (5.6%) had impaired glucose tolerance (IGT); all IFG patients had also IGT. None showed type 2 diabetes. Subjects with metabolic syndrome had higher insulin levels, HOMA, triglycerides, z-score systolic blood pressure (SBP), z-score diastolic blood pressure (DBP) and lower WBISI and HDL levels than subjects who did not meet the criteria for the diagnosis of metabolic syndrome (Table 1). Subjects with IGT showed higher serum insulin levels (40.6 ± 32 vs. 27.4 ± 18; P < 0.001), higher HOMA (9.40 ± 6.2 vs. 5.6 ± 3.8; P < 0.001) and lower WBISI (1.4 ± 0.7 vs. 2.5 ± 2.0; P = 0.007) than those with a normal glucose tolerance. None of the lean controls showed dyslipidemia, hypertension or IFG and the serum insulin levels (8.8 ± 3.3) and the HOMA values (0.9 ± 0.6) were significantly different from those observed in obese (both P < 0.001).

When compared for the clinical features, subjects carrying the 121Q allele showed significantly higher serum insulin levels and HOMA values than subjects homozygotes for the common allele and subjects carrying the rs997509T allele showed higher serum insulin levels, higher HOMA and lower WBISI than children homozygotes for the common allele (Table 2).

View larger version (13K): [in this window] [in a new window]   FIG. 1. Serum insulin levels (A) and serum glucose levels (B) during OGTT according to the rs997509 SNP genotype. The squares represent the rare allele carriers; the circle represent the common allele homozygotes. Data are shown as means and deviation scores.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Serum insulin levels (A) and serum glucose levels (B) during OGTT according to the K121Q genotype. The squares represent the rare allele carriers; the circle represent the common allele homozygotes. Data are shown as means and deviation scores.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Conflicting results exist concerning the role played in humans by ENPP1 variants and particularly the K121Q polymorphism in predisposing to the quantitative traits related to insulin resistance: obesity and type 2 diabetes (9). We did not show any association between obesity and K121Q. This observation is in contrast with several studies ruled out in Europe and in USA (12, 13, 28, 29), but it is consistent with three recent large multicentric studies in European and African-American individuals (30, 31, 32). Although it is difficult to establish the reasons of these different results, it has been suggested that differences in the genetic and/or environmental background and in recruitment procedures may have played a role (9).

Another important clinical outcome we examined was the occurrence of insulin resistance, IGT and MS according to K121Q and rs997509, the latter being a polymorphism which has been suggested to drive the association between K121Q and diabetes. Particularly, Bochenski et al. by studying five ENPP1 gene variants identified a risk haplotype for developing type 2 diabetes. The haplotype contained both the rs997509 T allele and the 121Q allele. The authors observed that when the carriers of the T allele of rs997509 were excluded from the analysis, the frequency of the remaining 121Q carriers was not different in the type 2 diabetic cases and controls (14).

We report that rs997509 variant has an effect on glucose metabolism and MS independently of the presence of 121Q and that the highest degree of insulin resistance in our population was apparent only when the rs997509 T allele was present (Table 3). Our results suggest that rs997509 T allele is strongly associated with insulin resistance and related abnormalities in obese children and adolescents and that the T allele drives also the 121Q allele association with insulin resistance. In fact, the association observed between 121Q and insulin resistance disappeared when we compared only subjects not carrying the rs997509 T allele. This observation can be ascribed to the strong linkage disequilibrium existing between the two variants and can explain why contrasting results concerning the association between K121Q and type 2 diabetes have been reported. Moreover, the observation that this association was not present in lean subjects means that obesity is essential for revealing the phenotype, with variant’s effect being noticed only when, as usually happens in obese children, an increasing insulin production in response to fat accumulation is required.

Remarkably, in obese subjects, the higher degree of insulin resistance seemed to influence also serum glucose levels and consequently insulin secretion during OGTT. Subjects carrying the rs997509 T allele, in fact, showed during the curve constantly higher glucose levels and consequently higher insulin secretion than C homozygotes. The progressive inadequateness of insulin response to hyperglicemia of subjects carrying rs997509 T allele is documented also by the higher prevalence of IGT in this group of patients respect to obese subjects homozygotes for the C allele. Children with the T allele, in fact, showed, not only a higher prevalence of IGT, but also of metabolic syndrome, that is consequent to insulin resistance (3). Likely, in subjects carrying the rs997509 T allele, hyperinsulinemia causes not only the worsening of the metabolic status, but also a progressive impairment of β cell function, via fat deposition in the pancreas.

Unfortunately, functional studies exploring the effect of the rs997509 variant on ENPP1 synthesis and function are not available. The rs997509 is located in the 3' end of intron 1 in a region containing a regulatory element, as reported previously (14) and suggested by the Five Regulatory track of the University of California Santa Cruz genome browser (14). Moreover, another polymorphism, rs9493114, which is in complete linkage disequilibrium with the rs997509, has been described in intron 8 and may be the functional responsible of this association, although other in vivo and in vitro reports on this variant, at our knowledge, do not exist (14).

In conclusion, consistently with previous reports this study supports a genetic link between ENPP1 gene variants and metabolic abnormalities in obese children. Moreover, we suggest that the ENPP1 rs997509 variant is strongly associated with insulin resistance, metabolic syndrome and IGT in obese children and adolescents and that it drives the known association between the 121Q variant and insulin resistance.

	Footnotes

 
Disclosure Statement: The authors have nothing to disclose.

First Published Online October 21, 2008

Abbreviations: BMI, Body mass index; C, cytosine; DBP, diastolic blood pressure; ENPP1, ectonucelotide pyrophosphate phosphodiesterase 1; ENPP-1, pyrophosphtase/phosphodiesterase-1; FFA, free fatty acids; GLM, general linear model; HDL, high density lipoprotein; HOMA, homeostasis model assessment; IFG, impaired glucose tolerance; IGT, impaired glucose tolerance; K121Q, codon 121; MS, metabolic syndrome; OGTT, oral glucose tolerance test; OR, odds ratio; SBP, systolic blood pressure; SNP, single nucleotide polymorphisms; T, timine; T2D, type 2 diabetes; WBISI, whole body insulin sensitivity index.

Received July 30, 2008.

Accepted October 15, 2008.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints, Permissions and Rights Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Santoro, N. Articles by del Giudice, E. M. Search for Related Content PubMed PubMed Citation Articles by Santoro, N. Articles by del Giudice, E. M. Related Collections Pediatric Endocrinology Diabetes and Insulin Metabolism Obesity