Null association between ACE gene I/D polymorphism and diabetic nephropathy among multiethnic Malaysian subjects

BACKGROUND: Wide inter-ethnic allelic variations of the Angiotensin Converting Enzyme (ACE) i nsertion-deletion (I/D) gene polymorphism were thought to be responsible for the conflicting gene–diabetic nephropathy disease association worldwide. We have investigated the genetic susceptibility of the ACE gene to diabetic nephropathy in the multiethnic Malaysian population. MATERIALS AND METHODS: A total of 137 healthy (control) and 256 diabetic subjects were recruited. The diabetic subjects were further subdivided according to their nephropathy status based on urinary albumin-creatinine ratio (ACR) and glomerular filtration rate (GFR). Triple primer polymerase chain reaction (PCR) was used for ACE I/D genotyping. Subsequently, populationwide genetic analysis and gene-disease association studies were performed. RESULTS: The genotype frequencies in all subgroups were in Hardy-Weinberg equilibrium. Similar allelic and genotypic frequency of ACE I/D gene polymorphism was observed between healthy controls versus pooled type 2 diabetes mellitus (T2DM) subjects, and normoalbuminuria versus microalbuminuria, macroalbuminuria and End Stage Renal Failure (ESRF) (P > 0.05). Neither ethnicity nor gender exerted any influence on the ACE I/D gene polymorphism (P > 0.05), with the exception of the Chinese ethnic group which exhibited a higher frequency of ID genotype (P = 0.042). A multinomial logistic regression model showed that predictive factors including age, systolic blood pressure (SBP), high density lipoprotein (HDL) and glycosylated hemoglobin (HbA1C) were independently associated with diabetic nephropathy, in that order. CONCLUSION: The I/D polymorphism of the ACE gene is not significantly associated with both T2DM and/or diabetic nephropathy in this Malaysian population regardless of ethnicity and gender.


Introduction
Diabetic nephropathy is a syndrome characterized by albuminuria in which the urinary albumin excretion rate rises progressively over time leading to declining glomerular filtration rate (GFR) and renal fibrosis with loss of renal function, coupled with arterial hypertension, increased cardiovascular disease risk and eventually end stage renal disease (ESRD). [1,2] This disease is marked by the initial appearance of microalbuminuria. [3] Annual ESRD incidence worldwide has been reported to escalate at an alarming rate, in particular, due to diabetic kidney disease. [4,5] In addition to other recognized promoters of diabetic nephropathy including chronic hyperglycemia, [6] systemic and intra-renal hypertension, [7] dyslipidemia, [8] smoking, [9] obesity, aging, high degree of insulin resistance, male sex, race, possibly high dietary protein intake [1] and familial clustering of diabetic nephropathy, [10] the effects of genetic polymorphisms cannot be overlooked. Polymorphisms in genes encoding PPAR-γ, [11] eNOS, [12] GLUT-1, [13] aldose reductase, [14] MTHFR, [15] apo-E [16] and components of the renin-angiotensin system (RAS) including angiotensinogen, Ang-II receptor type 1, [17] and particularly, the ACE gene, [18] have been implicated in the pathogenesis of diabetic nephropathy. genotypes II, ID and DD. [19] Polymorphism was thought to affect ACE-mediated physiological functions [20] and to cause differences in plasma ACE expression levels. [21] Conflicting findings in different populations on the relationship between ACE I/D gene polymorphism and diabetic nephropathy led us to investigate ACE I/D gene polymorphism as a risk factor for the development of diabetic nephropathy in the multiethnic Malaysian population.

Subject selection and recruitment
Informed consent was obtained from all subjects with approval granted by the Medical Ethics Committee, prior to sample collection.
Non-diabetic, non-nephropathic, normotensive healthy unrelated control subjects were randomly selected and recruited from local community centers (n = 137). Patients enrolled in the study consisted of type 2 diabetes mellitus (T2DM) patients with various stages of nephropathy on follow-up at the outpatient diabetes clinic (n = 196) and those undergoing dialysis treatment (n = 60). The subjects were initially grouped into four categories based on the urinary albumin-creatinine ratio (ACR): normoalbuminuria (<29 μg/mg creatinine), microalbuminuria (30-299 μg/mg creatinine), proteinuria (>300 μg/mg creatinine) while ESRF subjects were on dialysis. [3] These subjects were further re-grouped into two subgroups using urinary ACR as the cut-off point: Group 1: T2DM without significant nephropathy (ACR ≤ 300 μg/mg creatinine); Group 2: T2DM with significant nephropathy (ACR ≥ 300 μg/mg creatinine), to facilitate statistical analysis according to ethnicity and gender.

Demographic and clinical characteristics
Anthropometric data such as age and sex were obtained for each subject and relevant clinical parameters including duration (dx) of diabetes, ACR, serum creatinine, GFR, oral glucose tolerance test (OGTT), glycosylated hemoglobin (HbA1C), systolic (SBP) and diastolic blood pressure (DBP), triglyceride (TG) and high density lipoprotein-C (HDL-C) measurement were recorded.
ACR was calculated based on the microalbumin level in the urine sample and serum creatinine level measured by immunoturbidimetric assay [22,23] and modified Jaffe's kinetic reaction, [24] respectively. The Modified Diet in Renal Disease (MDRD) equation was used to estimate the GFR. [25] OGTT was performed on control subjects following the guidelines described. [26] The HbA 1C was measured as the ratio of total Hb to HbA 1C determined by the cyanidefree colorimetric method and immunoturbidimetric assay, [27,28] respectively. At least two readings of SBP and DBP were measured from the subjects who had rested adequately before measurement, using a digital electronic blood pressure (Omron Model M5, Omron Healthcare, Singapore). The measurement of serum TG was based on the kinetic bichromatic enzymatic technique [29] whilst the accelerator selective detergent methodology (Siemens Healthcare Diagnostics, Camberley, UK) was used to measure the HDL-C concentration.

ACE I/D genotyping
Genomic DNA was isolated using a Wizard ® Genomic DNA purification kit (Promega, Madison, WI, USA). Triple primer polymerase chain reaction (PCR) and subsequent agarose gel electrophoresis were essentially employed as described. [30] Distinct banding patterns enabled the genotypic determination of the ACE I/D allele as II, ID and DD genotypes. Genotypes were validated by single pass DNA sequencing (Research Biolabs, Singapore) on randomly selected samples. All samples were analyzed in duplicate and were stored at −20°C.

Genepop DOS version 3.3 Population Genetics
Software Package was used to test genetic equilibrium of the ACE gene. [31] Statistical Package for Social Sciences (SPSS) version 14.0 (Chicago, IL, US) was used to perform the statistical analysis. Parametric one-way analysis of variance (ANOVA) test was used to compare means of a quantitative variable between two or more groups when equal variances were assumed, while the nonparametric Kruskal-Wallis test was used when the assumption for equal variances was not met. Levene's test of homogeneity was used to verify the assumption.
Subsequently, Tukey's HSD and Dunnett's T3 post hoc test were used, respectively, to ensure that the overall probability of declaring any significant differences between all possible pairs of groups is maintained at a significant level of 0.05. Chi-square test was used for association studies between the ACE I/D gene polymorphism and disease etiology, ethnicity and gender.
The stepwise multinomial logistic regression procedure was used to assess and identify the significant predictors of diabetic nephropathy in our population. P values less than 0.05 were considered significant.

Demographic and clinical characteristics
Demographic and clinical characteristics of the subjects are shown in Table 1. All subjects included were characterized according to the nephropathy status.
ACR ranges observed between the subgroups were in agreement with the ranges outlined. [3] Similarly, there was no overlap between the ACR categories, thus providing a clear demarcation of subgroups. Significant mean age and HDL level differences between the groups were observed. The healthy subjects were younger and had higher levels of HDL-C compared to the type 2 diabetic nephropathy subgroups (P < 0.05).
We have also observed significant mean rank differences in serum creatinine, GFR, HbA 1C , SBP, DBP and TG between the groups (P < 0.05). The lowest mean serum creatinine concentration was exhibited by 1.01 ± 0.41 n = number of subjects; M = Malay; C = Chinese; I = Indian; NA = not applicable; All were expressed in mean ± SD except ACR in median (10 th -90 th percentile); *One-way ANOVA and Tukey post hoc test; † Kruskal-Wallis and Dunnett's T3 post hoc test; a P < 0.05 compared with groups N and E; b P < 0.05 compared with groups N and E; c P < 0.05 compared with groups N and E; d P < 0.05 compared with groups N and E; e P < 0.05 compared with group N and E; f P < 0.05 compared with groups N and E; g P < 0.05 compared with groups P and E; h P < 0.05 compared with groups N and E; i P < 0.05 compared with groups M and E; j P < 0.05 compared with group E; k P < 0.05 compared with groups N and E the healthy controls, followed by the patient subgroups in an ascending manner, according to the severity of nephropathy. On the other hand, the healthy controls were observed to have the highest mean GFR, followed by normoalbuminuric, microalbuminuric, proteinuric and the ESRF subjects. Also, mean HbA 1C , SBP, DBP and TG measured among the healthy controls were significantly lower compared to patient subgroups (P < 0.05).

Influence of ethnicity and gender on diabetic nephropathy
Regrouping of the patient subjects using ACR at a cut-off point at 300 μg/mg creatinine resulted in Group1 (n = 129) and Group2 (n = 127). Table 3  the groups, demonstrating a null association between ACE genotype and diabetic nephropathy. The Chinese ethnic group however, showed a significant difference in the ACE genotypic distribution between groups. A greater incidence of ID genotype (66.7%) among the Group 2 subjects was observed. All ethnic groups demonstrated comparable ACE allelic distribution, thus demonstrating a null association between ACE allele and diabetic nephropathy. Table 4 shows the influence of gender on ACE genotypic and allelic distribution in healthy, Group 1 and Group 2 subjects. The distribution of ACE genotypes and alleles was not different between the groups for both male and female categories.

Potential predictors of diabetic nephropathy
All potential risk factors for diabetic nephropathy including age, ethnicity, blood pressure, HDL, triglyceride, gender, ACE genotype and HbA 1C were assessed by stepwise logistic regression procedure. T2DM patients with proteinuria and ESRF were evaluated with reference to healthy controls by this model. The results of fitting the stepwise logistic regression model for diabetic nephropathy are presented in Table 5. Factors that are independently associated with diabetic nephropathy are age, SBP, HDL, and HbA 1C , in that order.

Discussion
The true genetic susceptibility of ACE I/D gene polymorphism on disease pathogenesis remains unelucidated due to contrasting outcomes worldwide. Table 6 shows the various findings linking the influence of ACE I/D gene polymorphism to diabetic nephropathy. Publication bias, differences in study design, patient selection, case and control definition, effect of disease progression or regression, [32] mistyping of genotypes, small sample size and particularly, ethnic differences were thought to be the causes of these outcomes. [33] A number of meta-analyses have been conducted    [34] failed to demonstrate a conclusive correlation in his meta-analysis due to methodological limitations, while Tarnow et al., [35] supported the association of ACE I/D gene polymorphism with diabetic nephropathy only among Northeast Asians. In contrast, Fujisawa et al., [36] concluded that ACE I/D gene polymorphism was a universal risk factor for diabetic nephropathy.
However, Ng et al., [37] presented a comprehensive report  Table 3]. In contrast, Ramachandran et al., [49] reported a positive association between ACE I/D gene polymorphism with diabetes and hypertension among Malaysian subjects.

Demarcation of subjects according to ethnicity
shows that the Malay, Chinese and Indian ethnic groups with both T2DM and diabetic nephropathy were not independently associated with ACE I/D gene polymorphism, disputing the finding of Ng et al. [37] To our knowledge, there have not been any previous studies in the Malay ethnic group on the association between the ACE gene and diabetic nephropathy. Sasongko et al., [50] conducted a study on the Javanese-Indonesians demonstrating a negative association between ACE gene polymorphism, however, with idiopathic nephrotic syndrome. Therefore, our current study provides a novel finding demonstrating a null association between ACE I/D gene polymorphism and diabetic nephropathy, with regard to the Malay ethnic group.
Our findings among the Indian ethnic group were similar to that of a previous study by Kumar et al., [46] but were not in agreement with that of Viswanathan et al., [45] even though both the latter studies were conducted on  [38] 509 DN Healthy control NA WN Positive Caucasian-Polish [39] 108 DN Healthy control NA WN Negative Taiwanese [40] 599 T2DM Healthy control Positive DN WN Positive Japanese [41] 467 T2DM Healthy control Negative DN WN Positive Korean [42] 191 T2DM Healthy control NA DN WN Positive Chinese [43] 168 DN Healthy control NA WN Negative Chinese [44] 1281 DN Healthy control NA WN Positive South Indian [18] 460 DN Healthy control Negative WN Positive South Indian [45] 109 DN Healthy control NA WN Positive North Indian [46] 117 DN Healthy control Negative WN NA Turkish [47] 377 DN Healthy control NA WN Negative Turkish [48] 112 T2DM Healthy control Positive DN Negative *Case and control definition for diabetic nephropathy (DN) and T2DM without nephropathy (WN), respectively, may be different between studies. WN = T2DM subjects without nephropathy; NA = not applicable Asian Indians. Later, however, Movvaa et al., [18] resolved the dispute between the latter two studies using a larger sample size and appropriate case-control definition, hence ascertaining the D alleles as a risk factor for renal complication in T2DM subjects among Asian Indians.
Similarly, the discrepancies found among the Indian ethnic group in this study could most likely be due to errors in the technical/methodological aspects rather than real genetic differences. [51] Our observation with regard to the Chinese ethnic group is in contrast with the findings on Southern Chinese and other Far East Chinese populations, which demonstrated strong evidence of the deleterious effect of the D allele in the etiology of T2DM [40,52,53] and diabetic nephropathy. [40][41][42]44] Nevertheless, our findings are in agreement with those of Wong et al. [43] who Interestingly, we also found an independent association between ID genotype and diabetic nephropathy among the Chinese when patients were further subgrouped according to the severity of nephropathy. Although the ID genotype is generally the most frequent type of ACE genotype, [54] the plausibility of ID genotype as a potential predictor for diabetic nephropathy is highly unlikely.
The D allele was thought to behave as a recessive trait, requiring the presence of two alleles to contribute to the progression of ESRF. [42] In addition, the II, ID and DD genotypes were previously demonstrated to display lowest, intermediate and highest levels of serum ACE levels, respectively. [19] In line with this, the DD genotype has always been held accountable for exerting its deleterious effects on various disease pathogenesis including diabetic nephropathy.
We did not observe any gender-dependent association between the ACE I/D gene polymorphism and T2DM or diabetic nephropathy. Although an increase in D allele frequencies was observed among the females, it was not significant enough to alter the genotypic presentation that was thought to be a predisposing factor for the progression of renal disease. The influence of gender on the deterioration of renal function remains controversial.
Many have suggested that decline in renal function of patients with chronic renal disease is more rapid in men than in women. [55] The receptor-mediated effects of sex hormone estrogen was thought to exert potent antioxidant actions contributing to renoprotective effects in females. In contrast, other reports point to females being predisposed to diabetic renal disease. [56] We conclude that in this study the role of ACE I/D gene polymorphism susceptibility to both T2DM and/or diabetic nephropathy was not demonstrated. Factors including ethnicity and gender did not alter the null influences of ACE I/D gene polymorphism on disease patterns in our population. Although statistically sufficient sample sizes were used in the present study, it would be beneficial to extend the sample size to confirm and validate our present findings. In addition, profiling of polymorphic genes encoding for components of the RAS might confer relevant insight into genetic predisposition to diabetic nephropathy.