Analysis of insertion/deletion polymorphisms of the angiotensin converting enzyme gene in Malaysian end-stage renal disease patients

Abstract

Introduction:

Insertion/deletion (I/D) polymorphisms found in the angiotensin converting enzyme (ACE) gene have been associated with hypertension, diabetes and renal disease. The present study sought to determine the association of I/D polymorphisms of the ACE gene with end-stage renal disease (ESRD) patients in Malaysia.

Materials and methods:

A total of 380 subjects were recruited to determine the genotypes of I/D polymorphisms of the ACE gene. Genotyping was performed using a PCR method. Statistical analyses were carried out using statistical software, and a level of p < 0.05 was considered statistically significant.

Results:

The frequencies for II, ID and DD genotypes of the ACE gene were 24.7%, 65.80% and 9.47%, respectively, in ESRD patients, and in control subjects were 45.26%, 47.37% and 7.37% respectively. The frequency for the D allele was found to be higher (42.40%) in ESRD patients compared to control subjects (31.05%). The genotypic and allelic frequencies of I/D polymorphisms of the ACE gene differed significantly (p < 0.05) between ESRD patients and control subjects in the Malaysian population.

Conclusion:

The findings of this study indicate that I/D polymorphisms of the ACE gene are a useful marker and are likely to play a major role in determining genetic susceptibility to ESRD in the Malaysian population.

Keywords

Angiotensin converting enzyme end-stage renal disease insertion/deletion PCR polymorphism Malaysia

Introduction

End-stage renal disease (ESRD) is a multifactorial disease and an advanced form of chronic renal failure. ESRD is a complex phenotypic structure of renal diseases affected by different etiologies.¹ Although ethnic, social and environ-mental factors play a part in development of the disease, to a large extent the cause of the disease is determined by genetic factors.² The genes responsible for the development and rate of progression to ESRD have not been identified. However, several studies have shown that renin–angi-otensin–aldosterone system (RAAS) gene polymorphisms are highly associated with renal complications.³ The RAAS plays an important role in regulating blood pressure, electro-lyte balance and renal haemodynamics.⁴ RAAS gene poly-morphisms have been extensively studied to determine the genetic susceptibility to essential hypertension,⁵ type 2 dia-betes mellitus,⁶ cardiovascular disease⁷ and renal disease.³ Among the RAAS genes, angiotensin-converting enzyme (ACE) is said to be a major component and it has been extensively studied as a candidate gene for various disor-ders. ACE polymorphisms appear to have significant impact on the progression of ESRD.⁸ ACE plays an important role in modulating the vascular tone, catalysing generation of angiotensin II, a potent vasoconstrictor.⁹ A 287-bp Alu inser-tion/deletion (I/D) polymorphism at intron 16 of ACE was found to be susceptible to essential hypertension,¹⁰ type 2 diabetes mellitus,¹¹ diabetic complications¹² and ESRD¹³ in various populations.

Several studies have reported that the DD homozygote of I/D polymorphism of the ACE gene is associated with an increased risk of developing ESRD,¹³ diabetic nephropa-thy¹⁴ and polycystic kidney disease.¹⁵ However, in contrast to those studies, some reports have shown a negative asso-ciation with the D allele in ESRD¹ and hypertension¹⁶ in various populations.¹⁷ The conflicting results for I/D poly-morphisms of the ACE gene might be due to consideration of different ethnic groups.¹⁸ Taking this into account, we sought to determine the association of I/D polymorphisms of the ACE gene in the Malaysian ESRD population.

Materials and methodology

Subjects

Following ethical approval from the Faculty of Medicine and Health Science, Universiti Putra Malaysia (UPM) and given permission from National Kidney Foundation of Malaysia, 190 ESRD patients and 190 control subjects were recruited. All the patients were undergoing dialysis treat-ment following diagnosis of stage 5 chronic kidney disease by nephrologists. Decisive factors such as age, gender, race, body mass index (BMI), systolic blood pressure (SBP) and diastolic blood pressure (DBP), urea level, lipid profiles and serum electrolytes were identified from the patients’ medi-cal records. Using a cytology brush, buccal cells were col-lected from the ESRD patients. For the control subjects, SBP and DBP were measured using a sphygmomanometer. All the control subjects were recruited based on the absence of any history of kidney disease and the presence of normal serum creatinine level. To determine the serum creatinine and blood glucose levels, and the lipid profiles, 4–5 ml of blood were collected from the subjects by a qualified phle-botomist. All biochemical analyses were carried out using the Hitachi-912 Autoanalyser (Hitachi, Germany) using kits supplied by Roche Diagnostics (Mannheim, Germany). Written consent was obtained from all the subjects who par-ticipated in this study.

Genotyping

For the patients, genomic DNA was extracted from buccal cells using a Puregene Buccal cell core kit (Qiagen, Germany), whereas for the control subjects, genomic DNA was extracted from peripheral blood using an Accuprep Genomic DNA Extraction kit (Bioneer, Korea). The extracted DNA from both sources was quantified and quali-fied. PCR was carried out to determine the genotypes of ACE I/D using specific flanking primers, which has been described elsewhere.¹⁰ The amplified PCR fragments show three different genotypes on gel electrophoresis; however, DD genotypes were confirmed using an insertion-specific primer pair to identify the I allele of the ACE gene. All the amplified products were separated using 2% agarose gel electrophoresis and the gel was stained using Gel Red (Biotium, USA). The stained gel was visualised under UV light using an Alpha Imager (Alpha Innotech, USA).

Statistical analysis

Statistical analysis for continuous data variables and other analyses, such as t-test, ANOVA were carried out using SPSS (SPSS, USA) software version 14.0 for Microsoft Windows. Allelic frequencies were calculated using the gene counting method, and the genotype distribution with Hardy–Weinberg expectations by a chi-square test. Odds ratios (OR) with 95% confidence intervals (CI) were esti-mated for the effects of high-risk alleles. A level of p < 0.05 was considered statistically significant.

Results

A total of 400 subjects were approached to participate in this study; 20 of the subjects were excluded due to incon-sistent results and extreme values, leaving a final study group of 380 subjects. Within the study group, 190 ESRD patients were recruited from the various National Kidney Foundation Dialysis centres in Malaysia, whereas 190 control subjects were collected randomly by conducting a health screening programme at various places in and around UPM.

The clinical parameters of the ESRD patients and control subjects are shown in table 1. The majority of the total subjects were female; however, there were more male ESRD patients (54.7%) than female (45.3%). The age of the ESRD patients ranged from 31 to 75 years old, with a mean age of 54.68 ± 12.20 years, whereas the age of the control subjects ranged from 25 to 74 years old, with a mean age of 45.82 ± 13.39 years. There were significant differences observed between the ESRD patients and con-trol subjects in age, SBP, BMI, creatinine levels, triglycer-ide and total cholesterol levels (p < 0.05), but not in DBP, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels.

Table 1.

Clinical characteristics of ESRD patients and control subjects

Parameter	ESRD patients (190)	Control subjects (190)	p-value
Gender, M/F	104/86	75/115	-
H/D/ H+D	77/30/83	-	-
Age (years)	54.68 ± 12.20	45.60 ± 13.62	0.001*
SBP (mm Hg)	149.14 ± 27.95	132.16 ± 19.1	0.001*
DBP (mm Hg)	77.59 ± 16.64	78.08 ± 10.81	0.737
BMI (kg/m²)	24.71 ± 3.59	27.91 ± 4.01	0.001*
Creatinine (mg/dL)	13.26 ± 5.09	0.62 ± 0.23	0.001*
LDL (mmol/L)	2.99 ± 1.12	3.10 ± 1.08	0.323
HDL (mmol/L)	1.09 ± 0.33	1.16 ± 0.45	0.138
Triglycerides (mmol/L)	2.34 ± 1.21	1.16 ± 0.81	0.001*
T. Cholesterol (mmol/L)	5.08 ± 1.28	4.72 ± 1.31	0.007*

ESRD, end-stage renal disease; H, hypertension; D, type 2 diabetes mellitus. *Significant p < 0.05. Values shown are mean ± SD.

Genotypes of I/D polymorphism of the ACE gene were determined using a conventional PCR method, and calculated based on the pattern of bands on gel electrophoresis (figure 1). The absence/presence of a 287-bp Alu repeated sequence I/D polymorphism represents the DD genotype (190 bp), whereas II indicates the homozygous (490 bp) and ID heterozygote genotypes (490 and 190 bp). Figure 2 shows the mistyping of ID heterozygotes of I/D polymor-phism of the ACE gene. Random blind samples were chosen and genotyped twice under similar conditions, and the results were identical to the previous results.

Figure 1.

Detection of I/D polymorphism of the ACE gene in 2% agarose gel electrophoresis. Lanes 3, 7, 8 and 9 show homozygous II genotypes; lanes 1, 2, 4, 6 and 10 show heterozygous ID genotypes; lane 5 shows homozygous DD genotypes of I/D polymorphism. M represents a 100-bp DNA ladder plus (Bioline) and N represents a negative control.

Figure 2.

Detection of mistyping of ID heterozygotes in 2% agarose gel electrophoresis. Lanes 1, 2, 4 and 5 show mistyping of ID heterozygotes as D homozygotes, which show the presence of the I allele (335 bp), whereas lanes 3, 6 and 7 show no products, which means there are confirmed homozygous DD genotypes of I/D polymorphism. M represents a 100-bp DNA ladder plus (Bioline) and N represents a negative control.

The findings of this study showed that the D allele is highly associated with Malaysian ESRD patients as com-pared to control subjects (p < 0.05). Table 2 shows the dis-tribution of I/D polymorphic genotypes of the ACE gene between the ESRD patients and control subjects. The geno-typic frequencies of I/D polymorphism of the ACE gene found in ESRD patients were 24.7%, 65.80% and 9.47%, and in the control subjects 45.26%, 47.37% and 7.37% for the II, ID and DD genotypes respectively. The D allele was found to be higher (42.40%) in the ESRD patients as com-pared to the control subjects (31.05%), and statistically sig-nificant (p < 0.05)

Table 2.

Distribution of genotypes and allele frequencies between ESRD patients and control subjects

	ESRD patients (n = 190) n (%)	Control subjects (n = 190) n (%)
Genotypes
II	47 (24.70%)	86 (45.26%)
ID	125 (65.80%)	90 (47.37%)
DD	18 (9.50%)	14 (7.37%)
p-value	0.000*
Alleles
I	219 (57.60%)	262 (68.95%)
D	161 (42.40%)	118 (31.05%)
p-value	0.001*
Odds ratio (95% confidence interval)	0.613 (0.455–0.825)

Significant value (p < 0.05) obtained using chi-square test as compared with controls. Data are reported as number of subjects with per cent in parentheses. ESRD, end-stage renal disease.

Discussion

According to the 16th Report of the Malaysian Dialysis and Transplant Registry 2008, the intake of new dialysis patients increased rapidly from 1559 in 1999 to 3874 in 2007.¹⁹ The prevalence of dialysis patients increased from 244 per million population in 1999 to more than 626 per million at year end 2007. However, in the last 2 years the transplant numbers and rates have decreased. This drastic change is predominantly due to the unhealthy lifestyle of Malaysians, with increased prevalence of obesity, hyper-tension and diabetes.^20,21 This is reflected in the present study as the control subjects are in the obesity category as compared to the ESRD patients who are in the overweight category (Clinical Practice Guidelines on Management of Obesity 2004).²² Moreover, significant differences were observed in triglycerides and total cholesterol between the groups, showing that an elevation in triglycerides and a reduction in the HDL-C increased chances of development of early occurrence of the disease.²³ Hence, there is a need to know the etiological factors in relation to renal disease among the Malaysian population.

Genetic variations can be used as a marker to identify the individuals at risk. Many association studies have been carried out to identify genetic risk factors that predispose to various diseases. The RAAS regulates blood pressure, elec-trolyte and fluid homeostasis.²⁴ Genetic variations found in RAAS genes influence the activity of the system and may predispose to development of hypertension and renal fail-ure. The role of genetic polymorphisms has been inten-sively studied in development or progression of renal diseases, with conflicting results in various population.^25

–29 Among the RAAS genes, the ACE gene is involved in the conversion of angiotensin I to angiotensin II and the degra-dation of bradykinin.¹⁰ The ACE gene is located on chro-mosome 17q23 and consists of 26 exons in a 21-kb section. The presence or absence of a 287-bp repeat sequence at intron 16 has been used as a common marker in susceptibil-ity to various disorders.²⁹

Various studies have been carried out to determine the susceptibility gene that predisposes to ESRD and other dis-eases.³⁰ To determine the candidate gene, association stud-ies have been carried out in various populations with contradictory findings.^31
–33 The present association study was undertaken to determine the association of I/D poly-morphisms of the ACE gene in Malaysian ESRD patients as compared with control subjects. To the best of the authors’ knowledge, there are no previous reports based on I/D polymorphisms in Malaysian ESRD subjects; this is the first comprehensive report.

According to the third National Health Morbidity Survey 2004 in Malaysia, among adults the prevalence of hyper-tension was found to be 42.5% and the prevalence of type 2 diabetes mellitus 11.6%.¹⁹ In this study, the prevalence of hypertension with diabetes was higher (43.70%) than of hypertension (40.50%) and type 2 diabetes mellitus (15.7%) alone. This indicates that cholesterol and other environ-mental factors influence the development of ESRD.¹²

As all the patients having dialysis were not advised to give blood samples, buccal cells were collected and the genomic DNA extracted, whereas peripheral blood was col-lected from the healthy individuals to measure the biochem-ical parameters as well as for downstream applications. Moreover, the yields of genomic DNA extracted from both sources were similar, as measured using a biophotometer. Increased plasma and serum ACE levels were genetically determined by a 287-bp fragment of I/D polymorphism in the 16th intron of the ACE gene at chromosome 17.¹⁰ The I/D polymorphism of the ACE gene was amplified and ana-lysed using PCR and electrophoresis. However, there is a chance of mistyping of ID heterozygotes as D homozygotes may occur. To increase the specificity of DD genotyping, PCR amplifications were also performed with an insertion-specific primer to identify the presence of the I allele (335 bp), whereas there were no products in samples that were homozygous for the DD genotype.

The ACE gene plays an important role both biologically and clinically in development of ESRD.¹ Several studies have shown the association of the D allele with an increased risk of essential hypertension,⁸ diabetes and its complica-tions;³¹ however, DD genotypes failed to show an associa-tion with essential hypertension³² and type 2 diabetes³³ in various populations. These controversial findings com-pelled us to determine I/D polymorphisms of the ACE gene in Malaysian ESRD patients.

In this study, the allelic frequency of the D allele of I/D polymorphisms of the ACE gene was found to be higher in ESRD patients (42.40%) than in control subjects (31.05%), and was similar to the frequency reported in North Indian¹³ and Switzerland³⁴ populations. To support our findings, we searched databases to find details of the genotypic and allelic frequencies among Asian populations in reaction with I/D polymorphisms of the ACE gene with ESRD and its complications. We compiled the search results in table 3. From the table, it can be clearly seen that I/D polymor-phisms of the ACE gene were not consistently associated with ESRD and its complications among and within the populations. From the findings of Ramachandran et al.,¹⁰ I/D polymorphism of the ACE gene is a risk factor for hypertension and type 2 diabetes mellitus in Malaysian subjects. Our study showed a significant relationship for the D allele of I/D polymorphism of the ACE gene in ESRD patients and supports the previous findings of Ramachandran et al.¹⁰ However, our results are contradictory to some reports.^35-37 This discrepancy might be due to genetic heterogeneity, environmental background or dif-ferent study designs.

Table 3.

Genotypes and allele frequency distribution of insertion/deletion polymorphism of the ACE gene in Asian populations

Population	Disease	No.	Genotypes n (%)				Allele n (%)
			II	ID	DD	P	I	D	p	Study
Malaysia	ESRD	190	47	125	18	***	219	161	***	Present study
			(24.7)	(65.8)	(9.5)		(57.6)	(42.4)
Malaysia	HPT	65	24	34	7	*	82	48	**	Ramachandran et al.¹⁰
			(36.9)	(52.3)	(10.8)		(63.1)	(36.9)
Malaysia	T2DM	60	24	25	11	**	73	47	**	Ramachandran et al.¹⁰
			(40.0)	(41.7)	(18.3)		(60.8)	(39.2)
North Indian	ESRD	184	56	74	54	***	182	186	***	Gaurav et al.¹³
			(30.4)	(40.2)	(29.4)		(49.5)	(50.5)
Chinese	ESRD+Cardiovascular	153	28	44	81	*	100	206	**	Feng et al.³⁸
			(18.3)	(28.8)	(52.9)		(32.7)	(67.3)
Chinese	ESRD+T2DM	129	56	60	13	NS	172	86	NS	Hung et al.³⁵
			(43.4)	(46.5)	(10.1)		(66.7)	(33.3)
South Indian	CKD	118	27	58	33	NS	112	124	NS	Kolandaswamy et al.³⁶
			(23.0)	(49.0)	(28.0)		(47.5)	(52.5)
South Indian	DN	86	45	24	17	*	114	58	*	Vijay et al.³⁷
			(52.3)	(27.9)	(19.8)		(66.3)	(33.7)

ESRD, end-stage renal disease; CKD, chronic kidney disease; DN, diabetic nephropathy; HPT, hypertension; T2DM, type 2 diabetes mellitus.

*: p < 0.05, **: p < 0.01, ***: p < 0.001, at 5% level of significance, NS: Not significant (p > 0.05).

Limitations

The present study has provided only a genetic association for I/D polymorphism of the ACE gene among Malaysian ESRD patients as compared to control subjects. However, we failed to analyse the renal ACE mRNA levels in both ESRD patients and control subjects. The control subjects were not age- and sex-matched with the ESRD patients. Moreover, our population is not homogenous as compared to the populations studied by Feng et al.³⁸ and Monika et al.¹ Other than I/D polymorphisms of ACE, we are analysing other polymorphisms such as rs4331, rs4334 and rs4341 found in the ACE gene. Also, we are studying other RAAS gene polymorphisms, such as M235T and T174M polymor-phisms of the angiotensinogen gene, A1166C polymorphism of the angiotensin II type 1 receptor gene, T344C polymor-phism of the aldosterone synthase gene and Gly460Trp polymorphism of the adducin gene to investigate the role of genetic polymorphisms involved in development of ESRD among Malaysian subjects. However, replication studies with larger number of samples on a homogenous study popu-lation are strongly recommended to confirm the association and to avoid population stratification.

Conclusions

I/D polymorphisms of the ACE gene are strongly associated with ESRD in Malaysian patients, and these can be used as a genetic marker for susceptibility to ESRD in this population.

Footnotes

Acknowledgements

The authors would like to extend their gratitude to National Kidney Foundation Dialysis centres and all the volunteers involved in this study. We also appreciate the assistance of Miss Mimi Soraya Binti Mansor and Miss Rusni Mohd Jas in collecting the samples.

Funding

This study was supported by the RUGS project number 91104.

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