Prevalence of angiotensin converting enzyme (ACE) gene insertion/deletion polymorphism in South Indian population with hypertension and chronic kidney disease.

CONTEXT: Chronic Kidney Disease (CKD) is associated with a high risk of developing further severe complications such as, cardiovascular disease and eventually End Stage Renal Disease (ESRD) leading to death. Hypertension plays a key role in the progression of renal failure and is also a chief risk factor for the occurrence of End Stage Renal Disease (ESRD). AIM: This study investigates the possible association of insertion (I) and deletion (D) polymorphism of ACE gene in patients of Chronic Kidney Disease (CKD) with and without hypertension (HT). SETTINGS AND
DESIGN: Total 120 participants with 30 members in each group (Control, HT, CKD and CKD-HT) were chosen followed by informed consent.
MATERIALS AND METHODS: Blood samples were collected and subjected to biochemical analyses and nested PCR amplification was performed to genotype the DNA, for ACE I/D using specific primers. STATISTICAL ANALYSIS: Statistical analyses were performed using SPSS version 13. Allele and genotypic frequency was calculated by direct gene counting method. Comparison of the different genotypes was done by using Chi square test. Odd's ratios were calculated with a 95% confidence interval limit.
RESULTS: The ACE genotype were distributed as II, 27 (90%); DD, 2 (6.67%) and ID, 1 (3.33%) in control, II, 1 (3.33%); DD, 5 (16.67%) and ID, 24 (80%) in HT, II, 4 (13.33%); DD, 24 (80%) and ID, 2 (6.67%) in CKD and II, 0 (0%); DD, 2 (6.67%) and ID, 28 (93.33%) in CKD-HT group.
CONCLUSIONS: D allele of ACE gene confers a greater role in genetic variations underlying CKD and hypertension. This result suggest that CKD patients should be offered analysis for defects in ACE I/D polymorphisms, especially if they are hypertensive.

Introduction

Currently, the incidence of Chronic Kidney Disease (CKD) is around 10% worldwide.[123] CKD patients own a high risk of developing further severe complications such as, cardiovascular disease and eventually End Stage Renal Disease (ESRD) leading to death.[4] It has already been well documented that genetic factors, including ethnicity[5] and family history of disease[67] govern the pathogenesis of CKD. Hence, profiling the influence of genetic variations on the development of renal complications has garnered great attention in recent years.[8]

Hypertension plays a key role in the progression of renal failure and is also a chief risk factor for the occurrence of End Stage Renal Disease (ESRD).[9] Several epidemiological studies have put forth that the genetic aspects account for just about 30% of the discrepancy in blood pressure in diverse study groups.[10] Renin Angiotensin System (RAS) is the chief regulator of both blood pressure and functions of the kidney and their interface.[1112] Obviously, it is enviable to discover the candidate genes of RAS and assess their effects. Among those, the angiotensin converting enzyme (ACE) gene is apparent to be predominantly biologically and clinically pertinent to CKD.

ACE gene is located on chromosome 17q23 and contains 26 exons and 25 introns.[13] The insertion/deletion (I/D) polymorphism of ACE gene determines the plasma and tissue ACE levels.[8] One of the most significant polymorphisms of ACE gene is a 287-bp insertion/deletion in intron 16 (ACE I/D), and an earlier study has unravelled the impact of this polymorphism on ACE gene expression.[14] In this context, exploration of polymorphism in the ACE gene will bestow an extensive awareness about the progression of renal diseases and will pave way for enhanced treatment options.[9] Hence, this work aimed to study the distribution of the I/D polymorphism of ACE in Indian patients with CKD or hypertension and with both.

Materials and Methods

Ethics

Study protocol was approved by the Independent Ethics Committee and written, informed consent was taken from all participants.

Study subjects

A total of 120 prospective participants (convenience sampling) were recruited from a private nephrology OPD clinic, Tiruchirappalli, Tamilnadu, India. The subjects were divided into four groups: Healthy individuals without any renal complications or hypertension (group 1, control, n = 30), patients with hypertension without chronic kidney disease (group 2, n = 30), chronic kidney disease without hypertension (group 3, n = 30), both chronic kidney disease and hypertension (group 4, n = 30).

CKD is defined as kidney damage for three or more months, as defined by structural or functional abnormalities of the kidney, with or without decreased GFR, manifested by pathologic abnormalities or markers of kidney damage, including abnormalities in the composition of the blood or urine or abnormalities in imaging tests either kidney damage or GFR <60 ml/min/1.73 m2 for >3 months[15] and it was primary and of stages III-V (<15-59 ml/min/1.73 m2) in the present study. Informed consent was obtained from all the participants. The protocol for this project was approved by the ethics committee and in this study, hypertension was defined as >140 mmHg systolic blood pressure and >90 mmHg diastolic blood pressure or the use of antihypertensive therapy.[16] Blood pressure was measured on the right arm with an automated blood pressure monitor while the subject was seated and allowed resting for at least 10 minutes.

DNA extraction

Blood samples (5.0 ml) were drawn from the peripheral vein of all study population into ethylene diamine tetra acetic acid (EDTA) coated tubes. Genomic DNA extraction from the samples was performed by the standard salting out method.[17] DNA was then checked for quality and stored at 4°C for further studies.

Determination of ACE genotypes

Polymerase chain reaction (PCR) was used to detect I/D polymorphism of the ACE gene using thermo cycler (Eppendorf Master Cycler, USA) with a nested polymerase chain reaction (PCR) protocol. In this method, the polymorphism status was first assessed, and then to increase accuracy, another reaction was performed. PCR amplification of deletions (D) and insertions (I) of ACE1 were evaluated in a 20 μl reaction mixture containing 200 ng of the template DNA, 7.5 pmol/l of each primer, 0.2 mM of each dNTP, 1.5 mM MgCl2, 2.5 μL10X buffer and 1 U Taq polymerase. The cycling conditions for PCR were initial denaturation at 94°C for 1 minute 1 cycle, followed by 35 cycles of 94°C for 1 minute (melting), 58°C for 30 seconds (annealing), 72°C for 1 minute (extension) and final extension at 72°C for 8 minutes. The amplified PCR products were analyzed on 2% agarose gel (w/v) containing ethidium bromide. Agarose gel was visualized using gel documentation system (BioRad, USA). The primers used were as follows:

Forward Primer 1 - 5’-CTGGAGACCACCCATCCTTTCT-3’ (GC: 54.6, Tm: 56.7)

Reverse Primer 1 - 5’-GATGTGGCCATCACATTCGTCAGAT-3’ (GC: 48, Tm: 57.7)

Forward Primer 2 - 5’-TCGGACCACAGCGCCCGCCACTAC-3’ (GC: 70.8, Tm: 65.9)

Reverse Primer 2 - 5’-CGCCAGCCCTCCCATGCCCATAA-3’ (GC: 65.2, Tm: 62.4)

Statistical analyses

Statistical analyses were performed using SPSS version 13. Data were summarized as mean ± SD, range or percentage. Allele and genotypic frequency was calculated by direct gene counting method. Comparison of the different genotypes was done by using Chi square test. Odd's ratios were calculated with a 95% confidence interval limit. Clinical characteristics of CKD patients with different ACE genotypes were compared using independent t test. P-value <0.05 was considered significant.

Results

As indicated in Table 1, the study groups of patients were well matched for parameters under consideration. CKD and CKD-HT groups showed significantly higher (P < 0.05) levels of urea and creatinine values as compared to control and HT groups [Table 1]. The genotypic and allelic frequencies of the ACE gene I/D polymorphism in groups of patients and controls are given in Table 2. The ACE genotype were distributed as II, 27 (90%); DD, 2 (6.67%) and ID, 1 (3.33%) in control group. The HT patients represented II, 1 (3.33%); DD, 5 (16.67%) and ID, 24 (80%). The CKD group has shown II, 4 (13.33%); DD, 24 (80%) and ID, 2 (6.67%). In CKD-HT group, there were II, 0 (0%); DD, 2 (6.67%) and ID, 28 (93.33%). It identifies that the D allele distribution is significantly higher in patients of HT, CKD and CKD-HT compared to control group [Table 2 and Figure 1].

Table 1

Characteristics of study groups

Parameter	Control	HT	CKD	CKD-HT
Age (years)	42.14±12.81	48.77±12.14	50.61±15.83	55.90±17.26
Sex (M/F)	17/13	20/10	18/12	22/8
Urea (mg/dl)	27.83±6.10	38.19±3.70	110.73±30.80	97.18±35.47
Creatinine (mg/dl)	1.1±0.97	1.3±0.65	6.75±2.32	7±3.81
HB (gm%)	14.71±1.83	13.10±1.03	9.53±2.19	9.21±2.34
Sugar (mg/dl)	130.98±12.31	159.05±20.67	241.81±39.42	237.20±42.53
Sodium (meq/dl)	140.30±2.54	142.13±1.97	147.61±2.83	146.85±2.68
Potassium (meq/dl)	4±0.56	4.6±.97	4.9±0.90	4.5±1.02
Systolic blood pressure (mmHg)	122±8	141±9	146±8	145±5
Diastolic blood pressure (mmHg)	78±4	95±2	90±5	93±4
Total cholesterol (mg/dl)	178.50±23.14	203.71±40.54	191.23±32.59	213.84±50.96
Triglycerides (mg/dl)	63.35±16.17	146.23±21.30	125.17±20.83	160.03±41.73

Values are mean ± SD

Table 2

Distribution of ACE genotypes and allele frequencies in study groups

Study group	II (%)	DD (%)	ID (%)	I	D
Control	27 (90)	2 (6.67)	1 (3.33)	0.917	0.083
HT	1 (3.33)	5 (16.67)	24 (80)	0.433	0.567
CKD	4 (13.33)	24 (80)	2 (6.67)	0.167	0.833
CKD+HT	0 (0)	2 (6.67)	28 (93.33)	0.467	0.533

Figure 1

Lane 1 indicates 100bp DNA ladder; Lane 2, 3 (Control), 335 bp: Homozygous II; Lanes 5-7 (HT), 335 bp, 190 bp: Heterozygous ID; Lanes 8-10 (CKD), 190 bp: Homozygous DD; Lanes 11-13 (HT-CKD), 335 bp, 190 bp: Heterozygous ID

The genotypic (II and DD) and allelic (I and D) frequency distributions in the study groups were also compared and presented in Table 3. Significantly different II and DD genotypes and I and D allele distributions were observed in cases as compared to controls [Table 3].

Table 3

Comparison of genotype and allele frequencies in study groups

Study groups	Yate’s Chi-square value	Degree of freedom	P-Value	Odds ratio	95% confidence interval
Control Vs HT	13.887	1	<0.05 (0.0002)	14.467	3.278-63.835
Control Vs CKD	31.036	1	<0.05 (0.001)	55.109	10.979-276.442
Control Vs CKD-HT	12.218	1	<0.05 (0.00047331)	12.610	2.866-55.479

Discussion

Hypertension is a polygenic disorder involving the genetic predisposition of the genes associated with various components of RAS. Variations in the ACE gene of the RAS and the resultant impact on complications like hypertension, cardiovascular diseases, and nephropathy have already been reported.[18] Allelic differences in ACE gene determine the rates of progression of hypertension and interrelated diseases in particular, chronic kidney diseases.[19] The data obtained from the current study manifested the association between the ACE gene polymorphisms and hypertension and CKD. These results were in accordance with those of the previous studies, which have linked the I/D polymorphism of the ACE gene to the incidence and progression of chronic renal diseases of diverse etiologies.[12202122]

Our study has demonstrated the remarkable differences in the prevalence of ID genotype and D allele of ACE gene in hypertensive patients than in controls. Moreover, a high prevalence of the ACE DD genotype and D allele in CKD group and ACE ID genotype and D allele in CKD with hypertension group was observed.[23] D allele of ACE gene might bestow a potential risk of suffering from renal diseases.

The DD genotype had commonly been revealed to have enhanced the serum ACE levels and activity whereas II and ID genotypes were associated with low and intermediate levels of ACE respectively.[19] The lower ACE activity in the II genotypes is because the I allele has a sequence similar to a silencer sequence.[24] Previous studies have documented the role of the DD genotype at cellular level ending up in hypertension and renal diseases.[19252627] Moreover, a higher level of systolic pressure was observed in ACE-DD genotype cases while compared to II and ID genotypes among 105 CKD patients.[9] However, the present study exhibited the higher prevalence of ID genotype in cases with hypertension and in those having hypertension with CKD. The direct correlation between some gene polymorphisms and the diseases remains a controversy among various human ancestries reported. Some previous studies approved the importance of human genetic variation in complex disease which can cause alleles to occur at a greater frequency in people from specific geographic regions.[2829]

The limitations of the current study include limited sample size, absence of a replication cohort, non-estimation of serum ACE levels and probable less impact of the investigated polymorphism while compared to other environmental and genetic factors, in causing renal diseases. The limited sample size might have led to low statistical power and trivial difference in between study groups with respect to ACE I/D polymorphism. Moreover, the study has involved a single centre. Hence, a detailed multicentre study on the population from various renal centres has to be performed for better elucidation of the role of ACE gene polymorphism on the progression of renal failure. Also the present study has not looked at ACE levels in study groups and the control group that adds another dimension which needs to be understood in the context of the results and their interpretation. It has been shown through several elegant experiments that even though the plasma levels of ACE are remarkably stable within an individual there are marked differences between individuals and at variance with sodium balance. Hence population studies linking ACE gene to hypertension, CAD and CKD need to study ACE gene aberrations in the context of ethnicity, age, gender, environmental and geographic factors, and importantly, to the duration of hypertension.

Conclusion

In conclusion, the present study proposes that D allele of ACE gene confers a greater role in genetic variations underlying CKD and hypertension. It is also revealed that DD genotype seems to be of prognostic importance in CKD patients whereas ID genotype is apparent as a prognostic marker for CKD patients with hypertension. This phenomenon might have been the major factor behind the association of ACE genotypes with CKD, hypertension and duo. However, the results have to be further validated in a large population.

Financial support and sponsorship

Nil.

Conflict of interest

There are no conflicts of interest.