Association of Angiotensin Converting Enzyme Gene Insertion/Deletion Polymorphism with Type 2 Diabetic Nephropathy in Gaza Strip-Palestine

Abstract

Introduction: The insertion/deletion (I/D) polymorphism of 287 bp Alu repeat sequence in intron 16 of Angiotensin Converting Enzyme (ACE) gene resulting in three genotypes I/D, D/D and I/I. ACE gene expression is associated with ACE levels in cells and in the plasma. It indicated that the polymorphism may modulate the expression of the ACE gene. The D/D genotype is believed to confer deleterious effect to many pathogenesis, also, it might be a cause-effect for type 2 diabetic nephropathy (T2DN). In this study, we evaluated the frequency of the different genotypes of ACE gene and investigated if there is an association between ACE gene polymorphism and T2DN by comparing the genotypes results of T2DN patients to healthy control, Type 2 diabetes mellitus (T2DM) and nephropathy patients in Gaza Strip.

Methodology: The study included 170 subjects, consisting of 43 T2DM patients undergoing dialysis "T2DN" compared to 41 T2DM patients who were not undergoing dialysis, 43 patients undergoing dialysis without T2DM and 43 healthy individuals. Blood samples were collected in EDTA tubes for DNA extraction. Polymerase Chain Reaction (PCR) was used to detect ACE gene polymorphism. All subjects were asked to fill the questionnaire interview.

Results and Conclusion: The initial results showed that there is no statistically significant association between ACE genotypes and T2DN, also between the T2DM and nephropathy when compared to the healthy control (p> 0.05). The (D/D) genotype was the most frequent in all study groups. Moreover, no association was observed between ACE genotypes and gender, diabetic nephropathy, nephropathy, diabetes, hypertension, obesity and various other diabetes complications.

 

INTRODUCTION

Type 2 diabetes mellitus (T2DM) is metabolic disease which can arise from different causes and characterized by insulin resistance in peripheral tissues beside to impaired secretion of insulin from pancreatic β -cells 1 . The estimated global burden of T2DM is 438 million by 2030 which was 285 million people in 2010, this indicates that an increase of 65% will happen 2 . T2DM complexity can be related to many factors which include the heterogeneity, interactions between genes and environment role 3 . T2DM, hypertension (HTN) and obesity along with dyslipidemia is termed metabolic syndrome, which is prevalent in the populations of modernized nations 4 . The genetic susceptibility of T2DM has been found to be modulated by obesity status. Insulin resistance-related genetic variants were only associated with T2DM in the obese population, while insulin secretory variants conferred a greater T2DM risk only in non-obese individuals, suggesting a potential interaction of these variants with obesity status in T2DM incidence 5 .

Type 2 Diabetic Nephropathy (T2DN) is a progressive kidney disease caused by angiopathy of capillaries in the kidney glomeruli. Microangiopathy is the earliest detectable change in the course of T2DN. The development of T2DN is characterized by glomerular hyperfiltration and an increased albumin excretion rate 6 . The amount of albumin lost in the urine has important clinical connotations, where excretion of amounts in excess of 300 mg in 24h is termed macro-albuminuria and excretion of lesser amounts of albumin, between 30 and 300 mg in 24 h, is termed microalbuminuria 7 . T2DN affects approximately one-third of diabetic patients, and it is the leading cause of end-stage renal disease (ESRD) which require dialysis or transplantation in developed countries 8 , as well as in rapidly developing countries in Asia 9 . One-third of T2DM patients with excellent blood glucose control can develop to T2DN, while in most patients, even with anti-hypertensive therapy and suboptimal blood glucose control, T2DN is not noted. Therefore, genetic susceptibility to diabetic nephropathy has been proposed 10 11 .

ACE gene is mapped on chromosome 17q23 and consists of 26 exons and 25 introns 12 . Insertion/deletion of Alu repeat sequence in introns 16 of the ACE gene leading to three genotypes " I/I, I/D and D/D genotypes" 13 . These genotypes showed different plasma expression, whereas the D/D genotype showed the highest plasma expression and I/I genotype showed the lowest plasma expression. Also, its believed that these genotypes can contribute to deleterious or protective effects. D/D genotype could contributes to deleterious effects on various disease path-genetic mechanisms. On the other hand, I/I genotype is thought to has protective benefits 14 . Therefore, ACE I/D gene polymorphism has been suggested to be used to make a decision about treatment regimens in antihypertensive patients 15 .

Many researchers tried to explain the association of ACE I/D polymorphism with T2DM risk and its related renal and cardiovascular complications in different ethnic groups, but with highly inconsistent findings. D allele was found to be more common in T2DM and related complications in the Tunisian 16 , Indian 17 and Iranian populations 18 , while others studied demonstrated no association of either allele with T2DM or related cardiovascular disease (CVD) and renal disease in Malaysia and Indonesia 19 . These differences are mostly due to multifactorial or polygenic disorders, as evidenced by the varying disease outcomes modulated by the gene-gene or gene-environment interactions 20 . Other researchers examined the response of drugs in T2DM patients and compared it through ACE different genotypes, D/D genotype patients showed less response of drugs than patients have I/I allele of the ACE gene. It seems that diabetes-associated kidney disease risk is magnified by inheriting risk alleles at several susceptibility loci 21 .

METHODOLOGY

Sampling

EDTA whole blood samples were collected from 170 subjects, where 43 T2DN patients were the case group that compared to three control groups. The control groups were 43 nephropathy " undergoing dialysis" patients, 41 T2DM patients were been collected from health care centers and hospitals in the Gaza Strip and 43 healthy individuals. Self-administered questionnaires were completed by the participants. DNA extraction was performed using a Promega kit and GeneAll Kit for human DNA isolation.

PCR amplification of ACE gene

The sequence of the Primers for ACE gene used in this study was as reported 13 . PCR reaction was used to amplify the genomic DNA fragments on the intron 16 of ACE gene. About 150ng of extracted DNA (3 μL) was added to 7 μL master mix and 0.5 μL of each primer where all were placed in a 0.2 ml thin-walled micro-centrifuge PCR tube. The tubes were centrifuged and placed in a thermal cycler. PCR thermal cycles were performed as follow: 1): denaturation for 1 minute at 95 Cᵒ. 2): 36 cycles of melting for 15 seconds at 95 Cᵒ, Annealing for 15 seconds at 59 Cᵒ, extension for 10 seconds at 72 Cᵒ. 3): final elongation for 10 min at 72°C. PCR product was analyzed on 2% ethidium bromide-stained agarose gel electrophoresis to determine the polymorphism of the gene 22 .

The amplicon generated from ACE gene should yield a 490 bp with I/I genotype and 190 bp with D/D genotype. Nuclease-free water (instead of the DNA template) was used as a negative control. Amplicon (PCR product) size was estimated by comparing it with DNA marker (DNA ladder 50 bp) run on the same gel.

Data Analysis

Statistical Package for Social Sciences (SPSS) program, version (20.0), was used to analyze data. Independent Samples T-test and Chi-square test were used. P -values < 0.05 were considered statistically significant.

RESULTS

General characteristics of study population

The study was conducted by using 170 individuals, where 43 T2DN patients (mean age 60.60±8.25) were the case group that compared to three control groups. The control groups were 43 nephropathy "undergoing dialysis" patients (mean age 58.73±11.54), 41 T2DM patients (mean age 58.73±11.54) were been collected from health care centers and hospitals in the Gaza Strip and 43 healthy individuals (mean age 49.44±7.33). In total, 80.0% of the study population were non-smokers, while 20.0% were smokers. Moreover, 48.8% of the participants were males, while about 51.2% were females. 44.1% of subjects were non-hypertensive, while 55.9% of them were hypertensive. 50.6% of the participants were non-diabetic, while 49.4% were diabetic. All of the healthy population were non-hypertensive and non-diabetic.

Table 1 Demographic characteristics of T2DN, T2DM, Nephropathy patients and healthy subjects in the studied population

	T2DN		Healthy		Nephropathy		T2DM
	N0	%	N0		N0	%	N0	%
No. of participant	43	100	43	100	43	100	41	100
Male	24	55.8	22	51.2	13	30.2	24	58.5
Female	19	44.2	21	48.8	30	69.8	17	41.5
Age (mean)	60.60±8.25		49.44±7.33		58.73±11.54		58.73±11.54

PCR Results

PCR amplification products

Once the amplicons were obtained, they were introduced to 2% agarose gel electrophoresis with ethidium bromide and the bands were visualized under UV light. With the help of a DNA ladder, D allele and I allele were identified at 190 and 490 bp fragments respectively.

Figure 1 . PCR amplification products ; M 1 490 bp: and M 2: 190 bp; DD indicates to the deletion deletion polymorphism, ID indicates to the insertion deletion polymorphism and II indicates to the insertion insertion polymorphism.

ACE Genotypes

ACE genotypes distribution in all study population

ACE gene D/D genotype was the most frequent genotype (shown in Figure 2 ) .

Figure 2 . ACE genotypes distribution in all study population.

Distribution of ACE genotypes in study subjects

ACE genotypes distribution in all subjects was as shown in Table 2 . No statistical significant difference was found in ACE genotypes among study subjects ( p > 0.05).

Table 2 Distribution of ACE genotype in subjects

		T2DN		Healthy		Nephropathy		T2DM		Total
		N0		N0		N0	%	N0	%	N0	%
ACE Genotype	I/D	14	32.5	17	39.5	15	34.9	13	31.7	59	34.7
	D/D	28	65.1	26	60.5	25	58.1	28	68.3	107	62.9
	I/I	1	2.3	0	0.0	3	7.0	0	0.0	4	2.4
Total		43		43		43		41		170
p-value		0.344

ACE gene allele frequency in all study subjects

Our study showed that the D allele was the most frequent among the studied groups as shown in Table 3 .

Table 3 ACE gene allele frequency in all study subjects

		T2DN	Healthy	Nephropathy	T2DM	Total (%)
Allele	D	7081.4%	6980.2%	4571.4%	6984.1%	80.3
	I	1618.6%	1719.8%	1828.6%	1315.9%	19.7
Total		86	86	63	82	170

The relationship between ACE genotype and gender in all study subjects

The initial results showed that there was no statistically significant relationship between ACE genotypes and gender ( p > 0.05), as shown in Table 4 .

Table 4 Relationship between ACE genotype and gender in all study subjects

		MaleNo. (%)	FemaleNo. (%)	Total	p-value
ACE Genotype		30 (36.1%)	29 (33.3%)	59	0.14
		53 (63.9%)	54 (62.1%)	107
		0	4 (4.6%)	4
		83	87	170

The relationship between ACE genotypes with diabetes duration in the diabetic population

The initial data in Table 5 showed that there is no statistically significant association between the distribution of ACE genotypes and diabetes duration, where ( p >0.05).

Table 5 Relationship between ACE genotypes with diabetes duration in diabetic population compared to the non-diabetic population

		Non-Diabetic		Diabetic (years)
				5-9		10-15		>15
		n	%	n	%	n	%	n	%
ACE Genotype	I/D	15	35.7	8	19.0	6	14.3	13	31.0
	D/D	25	30.9	18	22.2	15	18.5	23	28.4
	I/I	3	75.0	0	0.0	0	0.0	1	25.0
p-value		0.635
Allele	D	65	75.6%	44	84.6%	36	85.7%	59	79.7%
	I	21	24.4%	8	15.4%	6	14.3%	15	20.3%
Total		86		52		42		74

The relationship between ACE genotypes with HTN and obesity state

The initial results showed that there was no statistically significant association between ACE genotypes with HTN and obesity state ( p > 0.05). The D/D genotype was the most frequent among the hypertensive and obese population (65.3% and 55.0%, respectively), as shown in Table 6 .

Table 6 Relationship between ACE genotypes with HTN and obesity state in case groups

		HTN					Obesity
		Yes		No			Yes			No
			%			%		%		%
ACE Genotype	I/D	29	30.5	13		40.6	15	37.5	27	31.0
	D/D	62	65.3	19		59.4	22	55.0	59	67.8
	I/I	4	4.2	0		0.0	3	7.5	1	1.1
p-value		0.330					0.102
Allele	D	15380.5%			5179.7%		5973.7%		14583.3%
	I	3719.5%			1320.3%		2126.3%		2916.7%
	Total	190			64		80		174

The relationship between ACE genotypes with nephropathy, kidney diseases, neuropathy and retinopathy in case groups subjects

Our results found that there is no statistically significant association between ACE genotypes with nephropathy and kidney disease, retinopathy and neuropathy ( p > 0.05). The D/D genotype was the most frequent among the nephropathy and kidney diseases, retinopathy and neuropathy patients (61.4%, 58.9%, 80.6%, respectively), as shown in Table 7 .

Table 7 Relationship between ACE genotypes in case group subjects with nephropathy and kidney diseases, retinopathy and neuropathy

		Nephropathyand kidney problems				Retinopathy				Neuropathy
		Yes		No		Yes		No		Yes		No
		N0	%	N0	%	N0	%	N0	%	N0	%	N0	%
ACE Genotype	I/D	30	34.1	12	30.8	27	37.0	15	27.8	6	19.4	36	37.5
	D/D	54	61.4	27	69.2	43	58.9	38	70.4	25	80.6	56	58.3
	I/I	4	4.5	0	0.0	3	4.1	1	1.9	0	0.0	4	4.2
p-value		0.346			0.379				0.065
Allele	D	13878.4%	66 84.6%		113 77.4%		91 84.3%		56 90.3%		148 77.1%
	I	3821.6%	12 15.4%		33 22.6%		17 15.7%		6 9.7%		44 22.9%
	Total	176	78		146		108		62		192

The relationship between ACE genotypes with CVD and Heart attack in case group subjects

The initial results showed that there is no statistically significant association between ACE genotypes with CVD and heart attack ( p > 0.05), as shown in Table 8 .

Table 8 Relationship between ACE genotypes with CVD and heart attack in case group subjects

		CVD					Heart attack
		Yes		No			Yes		No
			%	N0		%	N0	%	N0	%
ACE Genotype	I/D	13	26.5	29		37.2	10	38.5	32	31.7
	D/D	35	71.4	46		59.0	16	61.5	65	64.4
	I/I	1	2.0	3		3.8	0	0.0	4	4.0
p-value		0.354					0.511
Allele	D	8384.7%			12177.6%		4280.8%		16280.2%
	I	1515.3%			3522.4%		1019.2%		4019.8%
Total		98			156		52		202

The relationship between ACE genotypes and medical family history

The results showed that there is no statistically significant relationship between ACE genotypes and medical family history in all subjects ( p > 0.05), as shown in Table 9 .

Table 9 Relationship between ACE genotypes and family history to diabetes and HTN in all subjects

		Family history
		Diabetes				HTN
		Yes		No		Yes		No
		N0	%	N0	%	N0	%	N0	%
ACE Genotype	I/D	28	47.5	31	52.5	23	39.0	36	61.0
	D/D	48	44.9	59	55.1	53	49.5	54	50.5
	I/I	1	25.0	3	75.0	1	25.0	3	75.0
Total		77		93		77		93
p -value		0.676				0.303

Odds Ratio (OR) and 95% Confidence Interval (CI) of ACE genotypes between the studied groups

Table 10 OR and 95% CI in the genotype of the ACE gene I/D polymorphism in each medical condition

Group	ACE genotype	Cases	Control group	p - value	OR	95% CI
T2DN	D/D	28	26	0.655	1.221	0.508-2.930
	I/I+I/D	15	17
T2DM	D/D	28	26	0.454	1.408	0.574-3.457
	I/I+I/D	13	17
Nephropathy	D/D	25	26	0.826	0.908	0.384-2.148
	I/I+I/D	18	17
HTN	D/D	62	45	0.480	1.253	0.670-2.342
	I/I+I/D	33	30
Obesity	D/D	28	79	0.319	0.709	0.36-01.397
	I/I+I/D	21	42

Table 11 OR and 95% CI in the genotype of the ACE gene I/D polymorphism between T2DM and T2DN subjects

Group	ACE genotype	No.	p-value	OR	95% CI
T2DM	D/D	28	0.758	1.154	0.465-2.863
	I/I+I/D	13
T2DN	D/D	28
	I/I+I/D	15

Table 12 OR and 95% CI in the genotype of the ACE gene I/D polymorphism between T2DN and nephropathy subjects

Group	ACE genotype	No.	p -value	OR	95% CI
Nephropathy	D/D	25	0.506	0.744	0.311-1.780
	I/I+I/D	18
T2DN	D/D	28
	I/I+I/D	15

Table 13 OR and 95% CI in the genotype of the ACE gene I/D polymorphism between T2DN and healthy subjects

Group	ACE genotype	No.	p-value	OR	95% CI
Healthy	D/D	26	0.655	1.221	0.508-2.930
	I/I+I/D	17
T2DN	D/D	28
	I/I+I/D	15

DISCUSSION

Genotype Frequencies of the ACE gene in the Gaza Strip

ACE Genotype frequencies were 34.7% for I/D , 62.9% for D/D and 2.4% for I/I genotype in this study. In terms of the frequency of I/D polymorphism of the ACE gene in the current study compared to different ethnic groups in various studies, D/D genotype has the highest frequency in the Gaza Strip, Lebanon and Iraq, while the I/D genotype has the highest frequency in Egypt, the UAE, China, and Pakistan, as shown in Table 13 .

Table 14 Distribution of ACE gene in different human population

Egypt	39.2	31.6	29.2	23
Lebanon	34.8	57.5	7.7	24
Emiratis	52.6	41.1	6.3	19
Iraq	31	50	19	25
China	45	13.3	41.7	26
Pakistan	47.9	22.3	29.8	27
Palestine (Gaza strip)	38.2	57.5	4.7	22
Palestine (Gaza strip)	38.85	53.85	7.3	28
Palestine (Gaza strip)	34.7	62.9	2.4	The Present Study

The relationship between ACE genotype with diabetic nephropathy

According to our initial results, there was no statistically significant association between ACE genotypes and T2DN, and this is similar to the data from other studies conducted in China 26 , Iran 9 , Tunisia 29 , Turkey 30 , South India 31 and others who have all failed to confirm the association of the ACE gene I/D polymorphism with diabetic nephropathy. On the other hand, a strong association between the ACE D/D genotype and/or the D allele and the risk for nephropathy in T2DM was found in Egypt 23 , Iraq 25 , Pakistan 27 , South Indian 32 , Bahrain 33 and Japan 34 .

The relationship between ACE genotype and diabetes

The current study did not find a statistically significant association between ACE genotypes and T2DM in the Gaza Strip, although Palestinian diabetic patients with ACE D/D genotype have a higher prevalence of diabetes. To our knowledge, this is the first report showing the association of ACE gene polymorphism with diabetes and these results are in agreement with different studies. Such as a study in the UAE 19 and with a meta-analysis study in China 26 . On the other hand, the association of D/D genotype with increased T2DM risk was reported in Tunisia 15 , Iran 17 and others.

The relationship between ACE genotype and Nephropathy

As we showed in our previous study 22 , according to the initial results of the study no association between ACE genotype and nephropathy in the Gaza Strip can be approved, and this result again obtained with a different population while in a meta-analysis study by Schena et al . (2001), who found that the D/D genotype could be associated with the development of nephropathy (1.96 of risk) 35 .

The relationship between ACE genotype and family history to diabetes and HTN

Our study showed that there is no statistically significant association between ACE genotypes and either Diabetes or HTN family history, but this does not conform with a study in Iraq 26 as well as a study on British Caucasians 36 , which showed a significant association between genotype and a family history of diabetes ( p = 0.03) The OR for a family history of diabetes in D/D compared to I/I subjects was 1.52 [0.89–2.60], and this disagreement may be due to the size of the sample.

CONCLUSION

The present study focused on the association of ACE gene ( I/D ) polymorphism in the Gaza Strip and the relationship between those genotypes and T2DN in a comparison to healthy, T2DM and nephropathy subjects. D/D genotype was the most common in all groups. Finally, our results did not reveal any statistical association between T2DN, T2DM and nephropathy with ACE I /D polymorphism.

LIMITATIONS OF THE STUDY

The original PCR method 13 has been reported to sometimes miss extension of I allele, particularly in heterozygotes. Improved methods, which include a second, nested extension with allele-specific primer, have been designed for this reason. In a future study, the genotypes should be checked with such a method.

RECOMMENDATIONS

To the best of our knowledge, this is the first study to investigate the relationship between ACE I/D polymorphism with Diabetic Nephropathy in the Gaza Strip. Hence, additional studies considering gene-gene and gene-environment interactions should be conducted to estimate the overall risk of the ACE gene in the pathogenesis of diabetic nephropathy by a using larger sample size. The steady increase in the number of T2DM patients will impose a significant burden on healthcare systems, so the early identification and treatment of diabetic complications are one of the primary goals in the efforts to solve this problem. Also, we recommend that the ACE levels in plasma should be measured for all subjects and compared with the genotypes, which will help in the selection of treatment and dosage, particularly for HTN patients.

Competing Interests

The authors declare no conflict of interest in this study.

Authors' Contributions

All authors participated in drafting the article and revising it critically for important intellectual content, also they all gave a final approval of the version to be submitted.

Dedication

The authors dedicate this work to the soul of the Palestinian Martyr " Nabil Shahwan" who kept supporting science and scientists all over his life.