Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 K173Q Polymorphism Is Associated with Diabetic Nephropathy in the Taiwanese Population

Abstract

Diabetic nephropathy is the leading cause of end-stage renal disease in the world. The cause of diabetic nephropathy seems to be multifactorial, and about one-third of patients with diabetes eventually develop this complication. The gene encoding ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is a candidate susceptibility gene for obesity and type 2 diabetes. We assessed rs1044498 (K173Q) located in the ENPP1 gene for association with diabetes nephropathy among 201 diabetic subjects without nephropathy and 215 diabetic subjects with nephropathy in the Taiwanese population. The single-nucleotide polymorphism (SNP) rs1044S498 in ENPP1 was associated with diabetes nephropathy in our study subjects. The AC+CC genotype of the rs1044498 SNP was a risk factor for the development of nephropathy in diabetic patients. Further, the AC+CC genotype of rs1044498 was a genetic risk factor in obese (defined by waist circumference) diabetic patients, but not in nonobese diabetic patients. We confirmed the association between the rs1044498 SNP in ENPP1 and diabetic nephropathy, especially among obese diabetic patients, in the Taiwanese population.

Introduction

Diabetic nephropathy is the leading cause of end-stage renal disease in the United States (Excerpts from the United States Renal Data System, 2001), western Europe (Rychlik et al., 1998), and Japan (Patient Registration Committee, 2002). Although the precise mechanisms of pathogenesis are unknown, the cause of diabetic nephropathy seems to be multifactorial. Not all diabetes patients develop nephropathy, but about one-third of patients with diabetes eventually develop this complication (Dronavalli et al., 2003). There is evidence for a genetic susceptibility to diabetic nephropathy, but it has been proven difficult to identify the causative genes (Conway and Maxwell, 2009).

Obesity is one of the main factors affecting the prevalence as well as the development and severity of type 2 diabetes (Dedoussis et al., 2007). Recent studies (Vasseur et al., 2005; Bochenski et al., 2006; Cauchi et al., 2008) indicated that obesity may play a key role in modulating genetic predispositions to type 2 diabetes by investigating candidate genes, including ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). It has been reported that single-nucleotide polymorphisms (SNPs) of ENPP1 such as rs1044498 (K173Q, also known as K121Q) and rs997509 are associated with greater type 2 diabetes susceptibility in obese populations (Vasseur et al., 2005; Bochenski et al., 2006; Cauchi et al., 2008).

ENPP1 K173Q polymorphism was also reported to associate with insulin resistance (Pizzuti et al., 1999; Frittitta et al., 2001; Abate et al., 2003; Hamaguchi et al., 2004; Meyre et al., 2005), type 2 diabetes (Hamaguchi et al., 2004; Kubaszek et al., 2004), and obesity (Meyre et al., 2005; Bottcher et al., 2006) in many populations. Some studies indicated that ENPP1 K173Q polymorphism was associated with diabetes nephropathy in Taiwanese (in type 2 diabetes) (Wu et al., 2009) and Caucasian (in type 1 diabetes) (Canani et al., 2002). However, the role of obesity is unclear from the studies on diabetes nephropathy. How the Q173 allele increases the risk of type 2 diabetes nephropathy directly or through its effect on obesity is unknown.

In this study, we selected the rs1044498 SNP of ENPP1, which is located in the exonic region and found with a higher minor allele frequency in the Asian population, and investigated its association with diabetic nephropathy.

Patients and Methods

Clinical sample collection

Diabetic patients were recruited from the Chang Gung Memorial Hospital at Keelung, Taiwan. The patients were divided into two groups: (a) 215 type 2 diabetic patients with overt diabetic nephropathies and (b) 201 type 2 diabetes patients without nephropathies. The Institutional Review Board of Chang Gung Memorial Hospital approved the study protocol and informed, written consent was obtained from each subject before conducting the study.

Clinical evaluation

Patients diagnosed with type 2 diabetes by doctors received a series of clinical examinations including blood pressure, blood biochemistry (fasting plasma glucose, glycosylated hemoglobin [HbA1c], serum triglyceride, serum total cholesterol, low-density lipoprotein, high-density lipoprotein, and C-peptide), oral glucose tolerance testing, routine urinalysis (dipstick test), urine albumin, and retinopathy status to ascertain the status of disease. Diabetic nephropathy was diagnosed when the following criteria were met: (a) age 30-70 years, (b) classic symptoms of diabetes, (c) fasting plasma glucose >126 mg/dL, (d) diabetes diagnosis more than 5 years previously, (e) positive dipstick test for protein, (f ) macroalbuminuria (two tests of spot urinary albumin >300 μg/mg of creatinine), (g) HbA1c >6%, and (h) retinopathy. On the other hand, diabetes without nephropathy was defined when the following criteria were met: (a) age 30-70 years, (b) classic symptoms of diabetes, (c) fasting plasma glucose >126 mg/dL, (d) diabetes diagnosis more than 5 years ago, (e) negative dipstick test for protein, (f ) negative dipstick test for microalbuminuria, (g) normoalbuminuria (two spot urinary albumin tests <30 μg/mg of creatinine), (h) blood urea nitrogen <20 mg/dL, and (i) HbA1c >6%. The demographic and clinical information of study subjects is shown in Table 1.

Table 1.

Demographic and Clinical Information of the Subjects

	DM	DN	p-Value
Mean age (SD), years	56.49 (9.477)	58.2 (9.965)	0.110
Gender, M/F	88/113	118/97	0.025
Body mass index	24.82 (3.870)	25.71 (3.778)	0.019
Hemoglobin Alc (%)	8.52 (1.471)	8.87 (1.967)	0.079
Fasting plasma glucose (mg/dL)	165.64 (46.905)	177.03 (66.835)	0.044
Waist circumference^a	Normal: 94	Normal: 88	0.197
	Obese: 103	Obese: 127

Waist circumference, normal: in men <90 cm and in women <80 cm; obese: in men >90 cm and in women >80 cm.

DM, diabetes only; DN, diabetes nephropathy.

DNA preparation

Genomic DNA was extracted from each blood sample of the 185 diabetic Taiwanese subjects using the QIAamp DNA Blood kit (QIAGEN), according to the manufacturer's instructions. The extracted genomic DNA was checked by agarose gel electrophoresis, quantified by spectrophotometry, and stored at −80°C until use.

SNP genotyping

The rs1044498 (ENPP1 K173Q) SNP genotyping was performed using Taqman^® SNP genotyping assay (ABI, Applied Biosystems). The primers and probes for rs1044498 were included in the ABI Assay on Demand Kit (ABI). Reactions were performed according to the manufacturer's protocol. The fluorescent probe was detected using the ABI Prism 7900 real-time polymerase chain reaction system.

Statistical analysis

The quality of the genotype data was evaluated using Hardy-Weinberg equilibrium proportion tests and single-point association analyses using the χ² test. Further evaluation used logistic regressions adjusted with other factors in odds ratio analysis. The logistic regression and odds ratio analysis were performed by SPSS version 17 (SPSS, Inc.).

Results

Demographics of study subjects

Unrelated cases and controls had similar distributions of age, but significant differences in gender and duration of diabetes were apparent (Table 1). Fasting plasma glucose and HbA1c were also similar in both groups. The mean of body mass index (BMI) in diabetic patients with nephropathy was larger than in diabetic patients without nephropathy. Urinary albumin and the blood urea nitrogen differed significantly between the two groups, as expected (data not shown).

Association of the SNP rs1044498 in ENPP1 with diabetes nephropathy

Genotype distributions of the rs1044498 SNP did not deviate from the Hardy-Weinberg equilibrium in the study subjects (data not shown). Strengths of associations and genotype frequencies of the rs1944498 SNP with diabetes nephropathy are summarized in Table 2. The odds ratio analysis showed that the risk of diabetic nephropathy was associated with the rs1044498 SNP genotypes AC+CC. The strength of the associations and odds ratios were slightly diluted by statistically adjusting for BMI, gender, and duration of diabetes.

Table 2.

Genotype Distribution of ENPP1 rs1044498 in Diabetes with and Without Nephropathy Groups

Genotype	DM	DN	p-Value	p-Value (adj.)^a
AA	140	116	0.002	0.008
AC	50	79
CC	11	20
AA (ref.)^b	140	116	OR (95% CI)	OR (95% CI)
AC+CC	61	99	1.96 (1.31, 2.93)	1.81 (1.19, 2.76)
			p = 0.001	p (adj.)^a = 0.005

Adjusted by gender, body mass index (BMI), and duration of diabetes.

Ref., reference group.

Association of the rs1044498 SNP in ENPP1 with diabetes nephropathy in obese diabetic patients

The interaction of waist circumference (1 defined as >90 cm in men and >80 cm in women; 0 defined as ≤90 cm in men and ≤80 cm in women) and the rs1044498 SNP genotypes (AA vs. AC+CC) was tested by logistical regression. After adjusting for BMI, gender, and duration of diabetes, the genotype, waist circumference, and interaction did not reach statistical significance; p-values were 0.278, 0.584, and 0.218, respectively. The result should indicate the part of the association determined by waist circumference and rs1044498 SNP genotype interaction.

The study subjects were further divided into obese and nonobese groups. The obese group was defined by a waist circumference of >90 cm in men and >80 cm in women. The odds ratio analysis showed that the risk of diabetic nephropathy was associated with the AC+CC genotype of rs1044498 in obese diabetic patients, but not in nonobese diabetic patients (Table 3). After adjusting for gender, BMI, and duration of diabetes, the statistical results remained significant.

Table 3.

Odds Ratio Analysis of ENPP1 rs1044498 in Obese and Nonobese Diabetes Patients

	DM	DN	OR (95% CI)	OR (95% CI)
Obese^a
AA (ref.)	76	31	2.41 (1.40, 4.16)	2.23 (1.27, 3.90)
AC+CC	64	63	p = 0.001	p = 0.005
Nonobese^a
AA (ref.)	64	52	1.48 (0.81, 2.71)	1.35 (0.71, 2.58)
AC+CC	30	36	p = 0.208	p = 0.258

Obese is defined by waist circumference of >90 cm in men and >80 cm in women.

Discussion

In this study, we investigated the association between obesity/diabetes susceptibility gene ENPP1 K173Q polymorphism (rs1044498) and diabetes nephropathy. We found that association between diabetes nephropathy and ENPP1 K173Q was significant before and after adjusting by gender, duration of diabetes, and BMI. The association between diabetes nephropathy and ENPP1 K173Q was further analyzed using obese and nonobese subjects, which showed significant association in obese diabetes patients. The results of this study indicated a possibility that the polymorphism K173Q of ENPP1 gene may confer risk for diabetes nephropathy in the Taiwanese population, especially in obese (defined by waist circumference) diabetic patients.

Polymorphisms of ENPP1, excluding K173Q, were reported to associate with diabetes nephropathy (both type 1 and type 2 diabetes) in African Americans (Keene et al., 2008). ENPP1 K173Q polymorphism was associated with diabetes nephropathy in Taiwanese type 2 diabetes patients (Wu et al., 2009) and Caucasian type 1 diabetes patients (Canani et al., 2002). The same polymorphism was reported to associate with glomerular filtration rate in both type I (De Cosmo et al., 2000; Canani et al., 2002) and type 2 (De Cosmo et al., 2009) diabetes. Glomerular filtration rate is an important measurement for renal function, as it is reduced in several kidney diseases and by nephrotoxic drugs. ENPP1 K173Q should associate with renal function and involve mechanisms related to development of kidney disease. Although the association results of ENPP1 K173Q polymorphism and diabetes nephropathy were inconsistent in different study populations (Keshavarz et al., 2006), the genetic polymorphism(s) in ENPP1 gene apparently act as risk factors in the development of diabetes nephropathy.

Obesity is remarkably heterogeneous as some obese patients are insulin sensitive, whereas others are insulin resistant (Abbasi et al., 2002; Reaven et al., 2004; Reaven 2006). Even some massively obese patients show a normal plasma lipoprotein-lipid profile despite their very significant excess of body fat (Drapeau et al., 2006; Lemieux et al., 2006). It is with this heterogeneity in mind that measuring an index of abdominal adiposity such as the waist circumference is clinically relevant, as it allows the identification of subgroups of abdominally obese patients who are more likely to be insulin resistant. Our preliminary result may indicate that diabetes patients with ENPP1 Q173 allele under the abdominal adiposity and/or insulin-resistant status should be considered as having high risk to develop diabetes nephropathy. On the other hand, the diabetes patients with ENPP1 Q173 allele should maintain normal waist circumference to prevent or delay the process of diabetes nephropathy. Although a significant association was detected in this study, the statistical power of this study was not adequate because of small sample size. Independent replication with larger sample size is needed to confirm the results.

The results of this study may suggest that ENPP1 is a candidate gene and K173Q polymorphism confers the genetic susceptibility for diabetes nephropathy in the Taiwanese population. Further, the results show a genetic basis that the ENPP1 K131Q is involved in diabetes nephropathy pathological mechanism, especially in obese diabetes patients. To further understand the interaction of ENPP1 K131Q function and characters of metabolic syndrome (for example, waist circumference) in the development of diabetes nephropathy, future investigations are needed.

Footnotes

Disclosure Statement

No competing financial interests exist.

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