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ENPP1 Variants and Haplotypes Predispose to Early Onset Obesity and Impaired Glucose and Insulin Metabolism in German Obese Children

Medical Department III (Y.B., B.E., M.S., P.K.), University Hospital for Children and Adolescents (A.K., W.K.), University of Leipzig, D-04103 Leipzig, Germany; and Vestische Hospital for Children and Adolescents Datteln (T.R.), University of Witten/Herdecke, D- 45711 Datteln, Germany

Address all correspondence and requests for reprints to: Peter Kovacs, Ph.D., Medical Department III, University of Leipzig, Philipp-Rosenthal-Strasse 27, D-04103 Leipzig, Germany. E-mail: peter.kovacs{at}medizin.uni-leipzig.de.

	Abstract

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Context: ENPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) encodes a membrane-bound glycoprotein that inhibits the insulin-receptor tyrosine kinase activity, resulting in reduced insulin sensitivity. Hence, variants in this gene may be related to obesity and insulin resistance.

Objective: Therefore, in this study, we aimed to explore the role of ENPP1 genetic variants in obesity and related traits in a representative population of Caucasian children and in cohorts of obese children with detailed metabolic characteristics including oral glucose tolerance test.

Design: We genotyped the K121Q, IVS20delT-11, and A/G+1044TGA ENPP1 genetic variants for association analyses in 712 schoolchildren (346 boys and 366 girls; mean age 12 ± 3 yr; mean body mass index-SD score 0.09 ± 0.04) and in independent cohorts of 205 obese children from Leipzig and 195 obese children from Datteln, Germany.

Results: We identified a significantly increased risk of obesity in Leipzig children carrying the 121Q variant (adjusted odds ratio, 1.82; 95% confidence interval, 1.30–2.56; P = 0.0005) or the [Q-delT-G] haplotype [1.75 (1.17–2.62), P = 0.006] as compared with a lean control group. This was replicated in another independent obesity/overweight cohort from Leipzig as well as obese children from Datteln. In addition, obese children from Leipzig with the [Q-delT-G] haplotype were characterized by impaired glucose metabolism, whereas the [K-delT-G] and [K-insT-A] haplotypes were significantly associated with improved insulin sensitivity and glucose metabolism (all P < 0.05 after adjusting for age, gender, and body mass index).

Conclusions: In conclusion, our study suggests a potential role of the K121Q polymorphism or derived ENPP1 haplotypes in increased susceptibility to obesity and early impairment of glucose and insulin metabolism in children.

	Introduction

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THE ENPP1 (NUCLEOTIDE pyrophosphatase/phosphodiesterase-1) gene encodes a membrane-bound glycoprotein that inhibits the insulin-receptor tyrosine kinase activity, resulting in reduced insulin sensitivity (1). Given the role of the protein in insulin signaling, the ENPP1 gene has been proposed as a susceptibility gene for insulin resistance and type 2 diabetes mellitus (T2DM). Pizzuti et al. (2) identified a K121Q polymorphism in exon 4 of the ENPP1 that was strongly associated with insulin resistance in healthy nonobese, nondiabetic Caucasians in Sicily. Subsequently, effects of the K121Q on insulin resistance and T2DM have been demonstrated in a number of association studies in adults (3, 4, 5, 6, 7). Further supporting evidence comes from positional cloning studies, where the chromosomal location of ENPP1 on 6q16.3-q24.2 was linked to childhood obesity in French families, and an association between a three-allele ENPP1 haplotype and childhood obesity was found (8, 9). The risk-haplotype includes the K121Q, IVS20delT-11, and A/G+1044TGA genetic variants (8) and associates with a higher risk of glucose intolerance and T2DM to obese children as well as increased serum levels of soluble ENPP1 protein in children (8).

Hence, ENPP1 is a promising candidate gene for early onset obesity and related sequelae. Children represent a particularly interesting study population for identifying primary genetic determinants involved in susceptibility to complex polygenic diseases such as obesity and related traits, because unlike in adults, phenotypes are less influenced by comorbidities and prolonged exposure to environmental factors.

Therefore, the aim of the present study was to explore the association of the three ENPP1 genetic variants (K121Q, IVS20delT-11, and A/G+1044TGA) and their haplotypes with obesity, insulin resistance, and hypertension, as components of the metabolic syndrome, in schoolchildren and in independent cohorts of obese children from Germany.

	Subjects and Methods

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Leipzig schoolchildren cohort

This cohort is part of the Leipzig Schoolchildren Project that investigated anthropometric and clinical parameters in 2500 children aged 6–17 yr from 1999–2001 and is representative of a normal population of Caucasian children. DNA was available in 712 children [346 boys and 366 girls; mean age 12 ± 3 yr; mean body mass index (BMI)-SD score (SDS) 0.09 ± 0.04]. The BMI was standardized referring to national reference data (10). Consensus guidelines of the German Working Group for Pediatric Obesity and representative reference ranges of Germany (10) were used to classify criteria for normal weight and obesity in children. Children with a BMI 1.88 or greater SDS (97th percentile) were considered obese.

Healthy lean children

A total of 492 children and adolescents (237 boys and 255 girls; mean age 12.0 ± 0.12 yr; mean BMI-SDS, –0.05 ± 0.02) were selected from the 712 schoolchildren to serve as healthy normal weight control group. The selection criterion was a BMI between –1.0 SDS and 1.0 SDS.

Obesity cohorts

Leipzig obesity cohort. A total of 205 Caucasian children and adolescents (97 boys and 108 girls; 11.6 ± 0.24 yr; BMI-SDS 2.70 ± 0.04) were consecutively recruited from the obesity clinic at the University Hospital for Children and Adolescents (Leipzig, Germany). All obese children had a detailed metabolic work-up including an oral glucose tolerance test and blood pressure measurements as described in detail elsewhere (11). The study was approved by the ethical committee of the University of Leipzig.

Datteln replication obesity cohort. A total of 195 Caucasian children and adolescents (89 boys and 106 girls; 11.2 ± 0.18 yr; BMI-SDS 2.41 ± 0.03) with detailed metabolic testing including fasting plasma insulin and blood glucose, who were recruited from the Vestische Hospital for Children and Adolescents Datteln, University of Witten/Herdecke, Germany, served as a replication obesity cohort.

Written informed consent had been obtained from all children’s guardians.

Genotyping of ENPP1 variants

Genotyping of all three variants was done 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, 95 C for 15 sec, and 62 C for 1 min, for 38 cycles) and fluorescence was detected on an ABI PRISM 7700 sequence detector (Applied Biosystems, Inc.). To assess genotyping reproducibility, a random approximately 10% selection of the sample was regenotyped in all single nucleotide polymorphisms (SNPs); all genotypes matched initial designated genotypes. Measures of linkage disequilibrium (D' and r²) were calculated by the EMLD program (https://epi.mdanderson.org/qhuang/Software/pub.htm), and the PHASE version 2.1 software was used to identify haplotypes (12, 13).

Statistical analyses

Before statistical analysis, nonnormally distributed parameters were transformed logarithmically to approximate a normal distribution. Three studies were performed: 1) association study of genotypes/haplotypes and anthropometric measures in German schoolchildren; 2) case-control study comparing genotype/haplotype frequencies between obese and lean children; 3) association of genotypes/haplotypes and obesity-related traits in a group of obese children. Differences in genotype frequencies between the obese and lean healthy controls were compared using logistic regression. The effects of genetic variants and their haplotypes on anthropometric characteristics in schoolchildren as well as obesity-related phenotypes in obese children were evaluated using generalized linear regression models. Statistical analyses were performed using the SPSS software package (version 11.5) (SPSS, Inc., Chicago, IL) and the statistical analysis system of the SAS Institute (Cary, NC). P < 0.05 was considered to be of statistical significance.

	Results

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ENPP1 variants and haplotypes

The genotypic distribution of all three ENPP1 variants (K121Q, IVS20delT-11, and A/G+1044TGA) in 712 schoolchildren and obese children cohorts was in Hardy-Weinberg equilibrium (all P > 0.01). Measures of linkage disequilibrium between variants were as follows: D' = 0.44 and r² = 0.12 between K121Q and IVSdelT-11; D' = 0.46 and r² = 0.10 between K121Q and A/G+1044TGA; D' = 0.70 and r² = 0.38 between IVSdelT-11 and A/G+1044TGA. We identified six haplotypes among the three different variants that accounted for more than 95% of the observed haplotypes: haplotype [K-InsT-A] (67.3%), [K-insT-G] (9.3%), [K-delT-A] (4.7%), [K-delT-G] (7.6%), [Q-insT-A] (4.8%), and [Q-delT-G] (6.3%), where haplotypes are defined by the composition of alleles at each variant in the order: [K121Q]-[IVS20delT-11]-[A/G+1044TGA].

Association with childhood obesity

We first evaluated the association between the ENPP1 variants and BMI in a representative population of 712 schoolchildren. Children with the 121K allele (KK homozygotes + KQ heterozygotes) had a lower mean BMI-SDS (KK = 0.07 ± 0.04 and KQ = 0.10 ± 0.07) than 121Q homozygotes (0.66 ± 0.55), but this did not reach statistical significance (P = 0.14 in a dominant mode of inheritance after adjusting for age and sex). We further performed a comparative analysis between Leipzig obese children (n = 205) and a selected group of lean children (n = 492). Under logistic regression analysis, we identified a significantly higher 121Q allele frequency in obese children compared with lean controls (17.6 vs. 11.2%) conferring a higher risk of obesity in both additive [adjusted odds ratio (OR) = 1.82, 95% confidence interval (95%CI) 1.30–2.56, P = 0.0005] and recessive mode of inheritance (adjusted OR = 1.85, 95%CI 1.28–2.63, P = 0.0009).

To confirm this finding, an independent sample of overweight/obese cases (defined as BMI 1.28 SDS corresponding to the 90th percentile) was selected from the cohort of 712 schoolchildren. Again, we identified a significantly higher 121Q allele frequency in overweight/obese children (n = 96) compared with lean controls (16.1 vs. 11.2%) conferring a higher risk of obesity in additive mode of inheritance (adjusted OR = 1.59, 95%CI 1.02–2.49, P = 0.04). The 121Q allele frequency was also significantly higher in a third independent cohort of obese children from Datteln (14.9 vs. 11.2%; adjusted OR = 1.49, 95%CI 1.03–2.14, P = 0.03 in additive mode of inheritance), hence confirming the association of this SNP with early onset obesity.

In addition, the [Q-delT-G] haplotype frequency was significantly higher in Leipzig obese children vs. controls (10.5 vs. 6.3%; OR = 1.75, 95%CI 1.17–2.62, P = 0.006) and showed a similar trend in overweight/obese group vs. controls (9.9 vs. 6.3%; P = 0.066, OR = 1.66, 95%CI 0.97–2.86) as well as in obese children from Datteln (8.7 vs. 6.3%; P = 0.09, OR = 1.47, 95%CI 0.94–2.30). No association of childhood obesity with IVS20delT-11 or A/G+1044TGA was found (data not shown). There was also no association of the haplotypes with anthropometric parameters in 712 schoolchildren (data not shown).

Association with extended phenotypes in German obese children

In 205 obese children from Leipzig, in whom detailed metabolic testing including oral glucose tolerance test had been performed, the IVS20delT-11 variant was associated with insulin resistance [homeostasis model assessment of insulin resistance (HOMA-IR)] (P < 0.05; Table 1). No relationship between the SNPs and remaining metabolic characteristics (fasting and 2-h blood glucose, fasting plasma insulin), anthropometric parameters [waist-to-hip ratio (WHR), BMI] or blood pressure (systolic and diastolic) was found (Table 1). In haplotype analyses, three of six identified haplotypes showed significant associations with parameters of glucose and insulin metabolism in obese children from Leipzig. Children with the [Q-delT-G] obesity risk haplotype had significantly increased 2-h plasma glucose concentrations as compared with noncarriers (P < 0.05; Table 2). The [K-insT-A] carriers had significantly lower plasma insulin and increased insulin sensitivity as compared with noncarriers, and the [K-delT-G] haplotype was associated with a significant decrease in fasting and 2-h plasma glucose (all P < 0.05; Table 2). No significant association of the metabolic parameters with either ENPP1 variants or their haplotypes was observed in obese children from Datteln (data not shown).

	Discussion

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Noteworthy, the [K-insT-A] haplotype was associated with fasting plasma insulin and insulin resistance in obese children from Leipzig. The frequency of this haplotype in obese (62.7%) and lean German children (67.3%) was similar to that observed in French children (60.3% in obese vs. 64.0% in lean; P = 0.003) (8), suggesting its protective effect against obesity. However, most likely due to a smaller sample size, this did not reach statistical significance in our study (P = 0.16 in the Leipzig cohort and P = 0.11 in the Datteln cohort). Nevertheless, in addition to the French data, we identified a significant decrease in fasting plasma insulin and HOMA-IR in obese children from Leipzig carrying the [K-insT-A] haplotype, which is consistent with a higher frequency of this haplotype in our lean control children.

In conclusion, our study suggests a potential role of the K121Q polymorphism and/or ENPP1 haplotypes in susceptibility to obesity in German children. ENPP1 haplotypes also may have a significant influence on the variation in parameters of glucose and insulin metabolism in obese children, potentially conferring increased risk of T2DM or other comorbidities related to insulin resistance. These findings are compatible with impaired insulin signaling proposed for the 121Q allele variant (6), but does not preclude the possibility of modified gene expression through noncoding variants (8).

	Acknowledgments

 
We thank all children who participated in the studies. We appreciate the help of the nurses and physicians who performed the clinical examinations and data collection.

	Footnotes

 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (KFO-152 to A.K., W.K., and M.S.), from the Interdisciplinary Center for Clinical Research at the University of Leipzig (Z14 to M.S.; B21 to A.K.), from the German Diabetes Association (to A.K. and P.K.), and from the European Community "PIONEER" (to W.K.). The Leipzig schoolchildren project was supported by unrestricted grants from Pfizer Pharma GmbH and Novo Nordisk GmbH (to W.K.). Part of the study was supported by a research grant from the German Hypertension Association and Tanita Community Health Trust (to A.K.).

Disclosure statement: The authors have nothing to disclose.

First Published Online September 12, 2006

¹ Y.B. and A.K. contributed equally to this work.

Abbreviations: BMI, Body mass index; 95%CI, 95% confidence interval; HOMA-IR, homeostasis model assessment of insulin resistance; OR, odds ratio; SDS, SD score; SNP, single nucleotide polymorphism; T2DM, type 2 diabetes mellitus; WHR, waist-to-hip ratio.

Received March 9, 2006.

Accepted September 5, 2006.

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