Increased frequency of angiotensin-converting enzyme DD genotype in patients with type 2 diabetes in Taiwan
Ming-Chia Hsieh1,
Shiu-Ru Lin2,
Tusty-Jiuan Hsieh2,
Chin-Hsun Hsu2,
Hung-Chun Chen1,
Shyi-Jang Shin1 and
Juei-Hsiung Tsai1,
1 Departments of Internal Medicine and
2 Clinical Pathology, Kaohsiung Medical University, Kaohsiung, Taiwan
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Abstract
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Background. Diabetes is one of the major causes of end-stage renal failure in the Taiwanese population. Previous studies have shown that angiotensin-converting enzyme (ACE) inhibitor can improve glucose utilization and suppress hepatic glucose production and the reninangiotensin system may play an important role in the initiation and progression of diabetic nephropathy. Thus, ACE gene polymorphism may be associated with type 2 diabetes and diabetic nephropathy.
Methods. To investigate the distribution of ACE-I/D genotype in type 2 diabetes and diabetic nephropathy, we examined 336 patients with type 2 diabetes (157 without nephropathy and 179 with nephropathy) and 263 age-matched normal controls. The diagnosis of nephropathy was made when daily protein loss exceeded 500 mg. ACE gene polymorphism was analysed by use of polymerase chain reaction.
Results. Our study revealed that the frequency of the D allele of the ACE gene was 29.3% in normal controls. The frequency of ACE DD genotype was significantly higher in type 2 diabetics compared with normal controls (18.2 vs 9.1%, P<0.01). The frequency of ACE DD genotype in patients with diabetic nephropathy was significantly higher than in patients without nephropathy (22.3 vs 13.4%, P<0.05). To determine whether ACE gene polymorphism was associated with the severity of diabetic nephropathy, we divided patients with diabetic nephropathy into dialysis and non-dialysis groups. The frequency of ACE DD genotype in the dialysis group was significantly higher than in non-dialysis group (28.7 vs 15.3%, P<0.05).
Conclusion. Our results indicate that the frequency of ACE DD genotype is markedly higher in patients with type 2 diabetes, and the ACE DD genotype is significantly associated with diabetic nephropathy.
Keywords: ACE genotype; diabetic nephropathy; type 2 diabetes
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Introduction
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The prevalence of diabetes mellitus is 3.2% in mainland China [1] and 12.4% in Taiwan [2], and more than 90% of patients suffer from type 2 diabetes. The susceptibility to type 2 diabetes is strongly inherited, as evidenced by a high concordance in twins and the strong familial aggregation. A history of type 2 diabetes in a first-degree relative doubles an individual's risk of acquiring diabetes, and offspring of two diabetic parents have an 80% lifetime incidence of acquiring the disease [3,4]. Additional evidence for a genetic role is suggested by the wide variation in incidence of prevalence among different ethnic groups.
Diabetic nephropathy is an important cause of end-stage renal disease, accounting for 26.6% of patients on dialysis in Taiwan [5] and 4.7% in mainland China [6]. The pathogenesis of this complication is not clearly understood, but available data suggests that multiple factors such as haemodynamic alterations, metabolic abnormalities, various growth factors, and genetic factors contribute to this complication [7]. Genetic predisposition to diabetic nephropathy in patients with non-insulin-dependent diabetes mellitus (NIDDM) has been reported [8,9]. Prior studies have shown that angiotensin-converting enzyme (ACE) inhibitors can improve glucose utilization and suppress hepatic glucose production in patients with type 2 diabetes [10,11]. Also the reninangiotensin system may play a critical role in the initiation and progression of diabetic nephropathy [12]. ACE gene polymorphism is correlated to serum and tissue ACE activity [1317], thus, ACE polymorphism may be associated with type 2 diabetes and diabetic nephropathy.
The ACE gene consists of 26 exons and spans 21 kb on chromosome 17. The polymorphism consists of the presence (I allele) or absence (D allele) of a 287 bp Alu repeat sequence within intron 16, and the D allele is associated with higher serum ACE activity [1317]. According to previous studies, there are differences in the frequencies of I/D polymorphism in different ethnic groups [1823]: the frequency of the D allele is considerably lower in Asians than in Caucasians. In NIDDM patients there are conflicting results regarding the relationship between the ACE genotype and diabetic nephropathy [2429].
Although several studies on ACE gene polymorphism in type 2 diabetes have been performed in white and Japanese populations, the relationship between them has not been clarified. Moreover, no large-scale study of ACE gene polymorphism in type 2 diabetes had been performed in Taiwan. Therefore, our study was designed to determine whether ACE polymorphism is associated with type 2 diabetes and diabetic nephropathy in the Taiwanese population.
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Subjects and methods
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Patient population
The study included 263 age-matched normal controls and 336 patients with type 2 diabetes for more than 5 years. The normal controls were recruited from individuals who had a general health evaluation at the Kaohsiung Medical University Hospital. Diabetic patients were recruited from the diabetic clinic of the Metabolism Division of Kaohsiung Medical University Hospital and diabetic patients with dialysis-dependent diabetic nephropathy were recruited from the dialysis centre of Kaohsiung Medical University Hospital. All these patients received a baseline examination, blood biochemistry, urinalysis, renal sonography, chest X-ray, and retinal funduscopy. Diabetic nephropathy was defined as overt proteinuria (>500 mg/day) with or without elevated serum creatinine and hypertension. The patients with diabetic nephropathy were divided into dialysis and non-dialysis groups. Patients with renal disease other than diabetic nephropathy were excluded. All subjects were divided into four groups: group 1, normal controls; group 2, diabetes without nephropathy; group 3, diabetes with nephropathynon-dialysis group; and group 4, diabetes with nephropathydialysis group.
Determination of genotypes
In accordance with standard methodology, genomic DNA was extracted from peripheral blood with a blood DNA kit (Puregene Gentra System, Minneapolis, USA). Genomic DNA was suspended in 10 mmol/l TrisHCl, 1 mmol/l EDTA pH 8.0, and concentrations of DNA were measured by spectrophotometry.
To determine the ACE genotype of the patients, a genomic DNA fragment on intron 16 of the ACE gene was amplified by polymerase chain reaction (PCR) using a flanking primer pair and a primer pair that recognizes insertion-specific sequence. The sequence of flanking primer pair used was 5'-CTGGAGACCACTCCCATCCTTTCT-3' and 5'-GATGTGGCCATCACATTGGTCAGAT-3'. PCR amplification products were obtained using 50 µl reactions (1 pg genomic DNA, 500 pmol of primers, 0.5 mmol/l each of deoxy-ATP, GTP, CTP, TTP, and 3 mmol/l MgCl2; 1 U Taq DNA polymerase (Boehringer Mannhein); 50 mmol/l KCl; 0.001% gelatin; 10 mmol/l TrisHCl pH 8.3) with 4 min of denaturation at 94°C, followed by 35 cycles of 15 s at 94°C, 5 s at 67°C (annealing), and 30 s at 74°C (extension) in a thermal cycler (PTC-100, MJ Research, Watertown, MA, USA). Reaction was terminated at 72°C for 2 min. Fragments without insertion (D allele) and with insertion (I allele) of 199 and 479 bp were separately detected on a 3% agarose gel containing ethidium bromide. To increase the specificity of DD genotyping, PCR amplifications were performed with an insertion-specific primer pair (5'-TTTGAGACGGAGTCTCGCTC-3' and 5'-GATGTGGCCATCACATTGGTCAGAT-3') mixed in 25 µl reaction (0.5 µg genomic DNA, 500 pmol of primers; 0.5 mmol/l each of deoxy-ATP, GTP, CTP, TTP and 1.5 mmol/l MgCl2; 0.5 U Taq DNA polymerase (Boehringer Mannhein); 50 mmol/l KCl; 0.001% gelatin; and 10 mmol/l TrisHCl pH 8.3) with 4 min of denaturation at 94°C, followed by 35 cycles of 15 s at 94°C, 5 s at 62°C (annealing), and 30 s at 74°C (extension). Only the I allele produces a 335 bp amplicon. The 335 bp fragment was identified on a 3.0% agarose gel containing ethidium bromide. The reaction yields no product in the sample of DD genotype.
Statistical analysis
All values are reported as means±SEM. The levels of the variables between each group were compared by analysis of variance (ANOVA). Genotype distribution and allele frequencies between groups were analysed by a
2 test. A P value <0.05 was considered statistically significant.
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Results
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At baseline the four groups were similar with regard to sex, age, and body mass index (Table 1
). Diabetic patients had higher blood pressures, and diabetic patients with nephropathy had significantly higher systolic pressures than patients without nephropathy. The serum triglyceride level was higher in diabetic patients (groups 24) than in normal controls (P<0.001). The serum cholesterol concentration was higher in groups 2 and 3 than in group 1 (P<0.001). The HbA1c and duration of diabetes were not different between diabetic groups (groups 24).
Table 2
shows the ACE genotype distributions of normal controls and patients with type 2 diabetes. The frequency of the ACE D allele was 29.3% in normal controls, however, the ACE DD genotype was more frequent in diabetic patients (18.2 vs 9.1%, P<0.01). Table 3
shows that the ACE DD genotype (22.3 vs 13.4%, P<0.05) and D allele (38.8 vs 29.3%, P<0.05) were significantly more frequent in diabetic patients with nephropathy than those without nephropathy. The frequencies of DD genotype (28.7 vs 15.3%, P<0.05) and D allele (45.7 vs 34.7%) were also significantly higher in dialysis groups than in the non-dialysis group (Table 4
).
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Table 3. Distribution of ACE genotype and allele frequencies in patients with type 2 diabetes with or without nephropathy
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Table 4. Distribution of ACE genotype and allele frequencies in patients with type 2 diabetes with nephropathynon-dialysis and dialysis groups
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The characteristics of the patients with type 2 diabetes according to ACE genotype are shown in Table 5
. There were no significant differences with respect to age, body mass index, blood pressures, blood levels of cholesterol and triglyceride, HbA1c and duration of diabetes. The percentages of nephropathy (65.6 vs 48.2%, P<0.05) and dialysis (44.3 vs 21.1%, P<0.01) were higher in patients with ACE DD genotype than in patients with ACE II genotype.
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Discussion
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There are ethnic differences in the frequencies of ACE gene polymorphism. Our study reveals that the frequency of the D allele of the ACE gene was 29.3% in normal controls, which is considerably lower than the reported frequency of 55% in Caucasians [15,19,20].
Our data show that the frequency of the ACE DD genotype is significantly higher in Taiwanese patients with type 2 diabetes than in normal controls. Zingone et al. [30 found that the deletion polymorphism of the ACE gene is associated with elevating fasting blood glucose levels in a random sample of male subjects among the general population. Huang et al. [31] reported that NIDDM patients with the DD genotype have higher blood glucose levels and are more prone to glucose intolerance. Previous studies reported that the use of ACE inhibitors might improve glucose metabolism by increasing whole-body glucose elimination and glucose utilization rate in the peripheral tissues [10,11]. There has been no report investigating ACE DD genotype association with type 2 diabetes until now. Ethnicity might be a contributing factor. However, the reason why ACE gene polymorphism is related to Taiwanese patients with type 2 diabetes is unknown. Therefore, further studies are needed.
Our data also show an association between the ACE DD genotype and diabetic nephropathy in type 2 diabetic patients in Taiwan. In previous studies that included other ethnic groups, there have been conflicting data regarding the association between ACE gene polymorphism and diabetic nephropathy. Schmidt et al. [24] evaluated 247 IDDM and 455 NIDDM patients with a diabetic duration of greater than 10 years and revealed no association between the ACE genotype and diabetic nephropathy. The definition of diabetic nephropathy was urine albumin excretion >200 µg/min in their study. Ohno et al. [26] studied 132 unrelated Japanese NIDDM patients and showed that the D allele of the ACE gene was more frequent in micro- and macro-albuminuric groups than in normoalbuminuric group. Jeffers et al. [32] found an independent association between the DD genotype and development of overt albuminuria, but no significant association was obtained when performing the analysis using the definition of diabetic nephropathy as microalbuminuria. The inconsistency of the correlation between ACE gene polymorphism and diabetic nephropathy from several studies may be attributed to many factors. An important one is the definition of diabetic nephropathy. It has been noted that some diabetic patients have considerable glomerular lesions by morphology despite no albuminuria [33]. Moreover, particularly in individuals with type 2 diabetes, albuminuria may result from reasons other than diabetic nephropathy, such as congestive heart failure, urinary tract infection, or superimposed primary chronic glomerulonephritis [34,35]. After tracking patients with microalbuminuria for over 10 years, Mogensen [36] estimated the risk of clinical nephropathy was only 22% in NIDDM. In our study, we used the definition of diabetic nephropathy as overt proteinuria and tried to exclude renal disease other than diabetic nephropathy by detailed examination. This definition provides strong evidence to support the association between the ACE DD genotype and diabetic nephropathy in type 2 diabetes in Taiwan.
From two meta-analyses, Kunz et al. [37] revealed that the risk of nephropathy was increased in the presence of DD or ID genotype in Asian patients with type 2 diabetes but not in Caucasian patients. Also, Tarnow et al. [38] reported that ACE gene polymorphism contributed to the genetic susceptibility to diabetic nephropathy in Japanese type 2 diabetics but not in Caucasian diabetics. In our study, the ACE gene polymorphism was significantly associated with diabetic nephropathy in Taiwanese diabetics. The ethnic factor might contribute to some role between ACE gene polymorphism and diabetic nephropathy.
Yoshida et al. [39] studied 168 patients with NIDDM for over 10 years and has shown that the ACE DD genotype has a high prognostic value for progressive deterioration of renal function. Huang et al. [40] analysed 83 patients with NIDDM for 9 years and revealed that patients with ACE DD genotype tended to have a steeper decrease of glomerular filtration rate and increase of UAER. However, these changes were not statistically significant compared with ACE ID or II genotype. Because of the cross-sectional and case-control designs used in our study, we separated patients with diabetic nephropathy into dialysis and non-dialysis groups. The frequencies of ACE DD genotype and D allele were significantly higher in dialysis group than in non-dialysis group. ACE gene polymorphism may contribute to the progression of nephropathy in Taiwanese patients with type 2 diabetes; a large prospective study is necessary to clarify this.
The risk factors, including elevated blood pressure, an increased duration of diabetes, poor control of blood sugar, and hyperlipidaemia were associated with diabetic nephropathy. In the present study, the patients with diabetic nephropathy had higher systolic blood pressures and serum levels of cholesterol than patients without nephropathy. Stratification of patients according to ACE genotype was done and there was no significant difference in age, blood pressure, blood levels of cholesterol and triglyceride, HbA1c and duration of diabetes. The ACE DD genotype was independently related to diabetic nephropathy. Previous studies have revealed that the ACE DD genotype was associated with increased plasma and tissue ACE levels [1317]. Since ACE activity played an important role in the reninangiotensin and Kallikreinkinin systems in regulating renal haemodynamics, increased ACE activity associated with DD genotype in type 2 diabetes may contribute to increased intraglomerular pressure hyperfiltration [41,42]. These alterations might play an important role in the development and progression of diabetic nephropathy.
In conclusion, our study shows that the frequency of ACE DD genotype increases in Taiwanese patients with type 2 diabetes. This had not been reported previously. The relationship between the ACE gene polymorphism and type 2 diabetes might stem from the ethnic difference. We confirm that ACE DD genotype plays an important role in diabetic nephropathy of Taiwanese patients with type 2 diabetes.
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Notes
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Correspondence and offprint requests to: Dr Juei-Hsiung Tsai, Department of Internal Medicine Kaohsiung Medical University, No. 100 Shih-Chuan 1st Road, Kaohsiung, 80317, Taiwan. 
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Received for publication: 17. 7.99
Revision received 9. 2.00.