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Saudi Journal of Kidney Diseases and Transplantation
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Year : 2017  |  Volume : 28  |  Issue : 2  |  Page : 325-329
Association between angiotensin-converting enzyme insertion/deletion gene polymorphism and end-stage renal disease in lebanese patients with diabetic nephropathy


1 Department of Biological and Environmental Sciences, Faculty of Science, Beirut Arab University, Beirut, Lebanon
2 Department of Biochemistry, Faculty of Science, Alexandria University, Alexandria, Egypt
3 Department of Biochemistry and Molecular Medicine, College of Medicine, AlFaisal University, Riyadh, Saudi Arabia
4 Faculty of Health Sciences, Beirut Arab University, Beirut, Lebanon

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Date of Web Publication23-Mar-2017
 

   Abstract 

Diabetic nephropathy (DN) is one of the leading causes of end-stage renal disease (ESRD). The development and progression of nephropathy is strongly determined by genetic factors, and few genes have been shown to contribute to DN. An insertion/deletion (I/D) polymorphism of the gene encoding angiotensin-converting enzyme (ACE) was reported as a candidate gene predisposing to DN and ESRD. Accordingly, we investigated the frequency of ACE I/D polymorphism in 50 patients with DN, of whom 33 had ESRD and compared them with 64 patients with type 2 diabetes mellitus (T2DM) but with normal renal function. Polymerase chain reaction amplification, using specific primers, was performed to genotype ACE I/D. Chi-square test was used to assess the differences between the groups. The frequencies of the ACE genotypes were as follows: 48% D/D, 40% I/D, and 12% I/I in patients with DN in contrast to 32.8% D/D, 45.3% I/D, and 21.9% I/I in T2DM. The distribution of the D/D, D/I, and I/I genotypes did not significantly differ between T2DM and DN. However, having the D allele carried a risk for the development of DN [odds ratio (OD), 1.71, P = 0.054]. On the other hand, the distribution of the D/D, D/I, and I/I genotypes was significantly different between T2DM and ESRD patients, χ2 = 7.23, P = 0.027. This was reflected by the D allele which carried a risk for the development of ESRD (OR, 2.51, P = 0.0057). These findings suggest that the D allele may be considered as a risk factor for both the development of DN and the progression of DN to ESRD in Lebanese population with T2DM.

How to cite this article:
Fawwaz S, Balbaa M, Fakhoury H, Borjac J, Fakhoury R. Association between angiotensin-converting enzyme insertion/deletion gene polymorphism and end-stage renal disease in lebanese patients with diabetic nephropathy. Saudi J Kidney Dis Transpl 2017;28:325-9

How to cite this URL:
Fawwaz S, Balbaa M, Fakhoury H, Borjac J, Fakhoury R. Association between angiotensin-converting enzyme insertion/deletion gene polymorphism and end-stage renal disease in lebanese patients with diabetic nephropathy. Saudi J Kidney Dis Transpl [serial online] 2017 [cited 2019 Apr 25];28:325-9. Available from: http://www.sjkdt.org/text.asp?2017/28/2/325/202789

   Introduction Top


Angiotensin-converting enzyme (ACE) is one of the major key enzymes in the reninangio-tensin system (RAS), which plays a crucial role in the development of high blood pressure.[1] The human ACE gene is located on chromosome 17 (17q23) and comprises 26 exons and 25 introns.[2] The most studied variant of the ACE gene is characterized by an insertion or deletion (I/D) of a 287bp fragment of an Alu sequence in intron 16 that appears to affect the level of serum ACE activity. Rigat et al have shown that the deletion genotype resulted in higher plasma ACE levels while the insertion genotype is associated with lower ACE levels.[3]

Diabetic nephropathy (DN) is one of the leading causes of end-stage renal disease (ESRD),[4] the pathogenesis of which is not clearly understood.[5] Administration of ACE inhibitors and angiotensin-II receptor blockers has been shown to have beneficial effects in retarding the development and progression of DN.[6] Studies have suggested that genetic predisposition plays a role in the development of DN as only a portion of patients with diabetes will have renal disease.[7] Indeed, ACE I/D polymorphism is the first and the most extensively studied candidate gene of RAS for predisposition of DN.[8],[9],[10],[11]

The aim of this study was to assess the association between ACE I/D polymorphism in DN and ESRD, in comparison with type-2 diabetes mellitus (T2DM) patients without DN.


   Materials and Methods Top


Subjects

Patients with T2DM were recruited from Sibleen Government Hospital and Al-Zahraa University Hospital. The study included 114 Lebanese individuals. There were 64 patients with T2DM (history of more than 10 years of diabetes with normal renal function) and 50 patients with DN, of whom 33 patients had ESRD receiving dialysis three times a week. Consent forms were signed by the study participants or their guardians.

DNA Extraction Whole blood was collected in EDTA tubes, and genomic DNA was extracted using GenElute Blood Genomic DNA kit as described by the manufacturer Sigma-Aldrich (St. Louis, Missouri, USA).

Genotyping

ACE I/D polymorphism was examined using polymerase chain reaction (PCR). The following designed primers were purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and were used for the conventional determination of the genotypes:

Forward:

5CTGGAGACCACTCCCATCCTTTCT-3’.

Reverse:

5GATGTGGCCATCACATTCGTCAGAT-3’.

Reaction was performed in a total volume of 25 μL containing 12.5 μL Taq Polymerase ReadyMix (Sigma-Aldrich), 16 pmoles of forward and reverse primer, and 10 ng extracted DNA. DNA was initially denatured at 94°C for 3 min followed by 40 cycles consisting of 45 s of denaturation at 94°C, 45 s of annealing at 60°C, and 45 s of extension at 72°C and a final extension at 72°C for 9 min.

Due to mistyping of around 4–5% I/D genotype as D/D genotype, an additional confirmatory PCR using a new sense insertion-specific primer was introduced. Therefore, all the D/D genotypes were subjected to an additional PCR using the insertion-specific primers as follows:

Forward: 5’ TGG GAC CAC AGC GCC CGC CAC TAC 3’.

Reverse: 5’ TCG CCA GCC CTC CCA TGC CCA TAA 3’.

The PCR cycle was slightly modified, and annealing was performed at 67°C. The resulting PCR products were separated on a 2% agarose gel and visualized using ultraviolet transilluminator.


   Statistical Analysis Top


Data were analyzed using the Statistical Package for the Social Science (SPSS) software version 19.0 (SPSS Inc., Chicago, Illinois, USA). Mean, standard deviation, and frequency were calculated using one-way analysis of variance. ACE genotypes were computed and tested for Hardy–Weinberg equilibrium. Differences in allele frequencies were calculated by odds ratios (OR). The statistical difference in genotype distribution was assessed by Pearson’s Chi-square test. P <0.05 was considered significant.


   Results Top


Demographic and clinical data of the patients included in this study are shown in [Table 1]. Patients were aged between 53 and 75 years. The number of female patients in the T2DM group was three times higher than male patients. However, there were no significant differences in age, smoking habits, fasting blood sugar, triglycerides, or high-density lipoprotein levels between T2DM and DN groups (P >0.05). As expected, urinary albumin excretion, serum creatinine, and blood urea nitrogen levels were significantly higher in patients with DN compared with patients with T2DM. On the other hand, total cholesterol and LDL levels were significantly higher in patients with T2DM compared to patients with DN (P<0.05).
Table 1: Demographic and clinical data of patients with type-2 diabetes mellitus and diabetic nephropathy.

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The ACE genotype frequencies were as follows: 48% D/D, 40% I/D, and 12% I/I in DN group compared to 32.8% D/D, 45.3% I/D, and 21.9% I/I in T2DM [Table 2]. The distribution of the D/D, D/I, and I/I genotypes did not significantly differ between T2DM and DN. However, having the D allele carried a risk for the development of DN (OR, 1.71, P = 0.054). On the other hand, the distribution of the D/D, D/I, and I/I genotypes was significantly different between T2DM group and the group with ESRD, χ2 = 7.23, P = 0.027 [Table 3]. This was reflected by the D allele which carried a risk for the development of ESRD (OR, 2.51, P = 0.0057).
Table 2: Distribution of angiotensin-converting enzyme genotypes and alleles in type-2 diabetes mellitus and diabetic nephropathy patients.

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Table 3: Angiotensin-converting enzyme genotype distribution between type-2 diabetes mellitus and endstage renal disease patients.

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   Discussion Top


In this study, we investigated the frequency of ACE I/D polymorphism in Lebanese patients with T2DM with and without DN. In the literature, the genetic association between ACE I/D polymorphism and DN, with or without ESRD, has been extensively studied in different populations around the world.[12] However, few studies have been conducted in the Arab countries. One of the very first association studies in Arab countries was performed on Tunisian patients with DN, which found no evidence that ACE gene polymorphism might contribute to DN.[13] This was also the case in a Bahraini population.[14] The results were different in Egypt, where the D/D genotype was more prevalent in DN.[15] When analyzing the link between ACE gene polymorphism and the progression of nephropathy to ESRD, the ACE I/D polymorphism was strongly associated with susceptibility to ESRD as defined in a recent meta-analysis.[10] A Japanese study compared the ACE I/D polymorphism in different stages of DN including ESRD.[16] They concluded that the D/D genotype is associated with progression of renal disease in Japanese patients with DN. The Japanese and other studies showed that the D/D genotype of patients with diabetes has a high prognostic value for the progressive worsening of renal function.[17] However, Wong et al[18] and Schmidt et al[19] reported that the ACE gene polymorphism did not influence either the development or the progression of renal failure in patients with diabetes. This discrepancy in association studies may be attributed to ethnic differences.[11]

It is noteworthy that the small sample size was a major limitation in our study. Further large-scale studies are required to confirm the suggested associations in the current study.

Conflict of interest:

None declared.

 
   References Top

1.
Davis GK, Roberts DH. Molecular genetics of the renin-angiotensin system: Implications for angiotensin II receptor blockade. Pharmacol Ther 1997;75:43-50.  Back to cited text no. 1
    
2.
Rigat B, Hubert C, Corvol P, Soubrier F. PCR detection of the insertion/deletion polymorphism of the human angiotensin converting enzyme gene (DCP1) (dipeptidyl carboxypeptidase 1). Nucleic Acids Res 1992;20:1433.  Back to cited text no. 2
    
3.
Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 1990;86: 1343-6.  Back to cited text no. 3
    
4.
de Boer IH, Rue TC, Hall YN, Heagerty PJ, Weiss NS, Himmelfarb J. Temporal trends in the prevalence of diabetic kidney disease in the United States. JAMA 2011;305:2532-9.  Back to cited text no. 4
    
5.
Tarnow L. Genetic pattern in diabetic nephropathy. Nephrol Dial Transplant 1996;11:410-2.  Back to cited text no. 5
    
6.
Lewis EJ, Hunsicker LG, Clarke WR, et al. Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes. N Engl J Med 2001;345:851-60.  Back to cited text no. 6
    
7.
Freedman BI, Tuttle AB, Spray BJ. Familial predisposition to nephropathy in African-Americans with non-insulin-dependent diabetes mellitus. Am J Kidney Dis 1995;25:710-3.  Back to cited text no. 7
    
8.
Marre M, Bernadet P, Gallois Y, et al. Relationships between angiotensin I converting enzyme gene polymorphism, plasma levels, and diabetic retinal and renal complications. Diabetes 1994; 43:384-8.  Back to cited text no. 8
    
9.
Wang F, Fang Q, Yu N, et al. Association between genetic polymorphism of the angiotensin-converting enzyme and diabetic nephropathy: A meta-analysis comprising 26,580 subjects. J Renin Angiotensin Aldosterone Syst 2012;13:161-74.  Back to cited text no. 9
    
10.
Yu ZY, Chen LS, Zhang LC, Zhou TB. Meta-analysis of the relationship between ACE I/D gene polymorphism and end-stage renal disease in patients with diabetic nephropathy. Nephrology (Carlton) 2012;17:480-7.  Back to cited text no. 10
    
11.
Grimm R, Rettig R. Association studies between the angiotensin-converting enzyme insertion/deletion polymorphism and hypertension: Still interesting? J Hypertens 2002;20: 1049-51.  Back to cited text no. 11
    
12.
Krolewski AS, Ng DP, Canani LH, Warram JH. Genetics of diabetic nephropathy: How far are we from finding susceptibility genes? Adv Nephrol Necker Hosp 2001;31:295-315.  Back to cited text no. 12
    
13.
Arfa I, Abid A, Nouira S, et al. Lack of association between the angiotensin-converting enzyme gene (I/D) polymorphism and diabetic nephropathy in Tunisian type 2 diabetic patients. J Renin Angiotensin Aldosterone Syst 2008;9: 32-6.  Back to cited text no. 13
    
14.
Al-Harbi EM, Farid EM, Gumaa KA, Singh J. Genotypes and allele frequencies of angiotensin-converting enzyme (ACE) insertion/deletion polymorphism among Bahraini population with type 2 diabetes mellitus and related diseases. Mol Cell Biochem 2012;362:219-23.  Back to cited text no. 14
    
15.
El-Baz R, Settin A, Ismaeel A, et al. MTHFR C677T, A1298C and ACE I/D polymorphisms as risk factors for diabetic nephropathy among type 2 diabetic patients. J Renin Angiotensin Aldosterone Syst 2012;13:472-7.  Back to cited text no. 15
    
16.
Gohda T, Makita Y, Shike T, et al. Association of the DD genotype and development of Japanese type 2 diabetic nephropathy. Clin Nephrol 2001;56:475-80.  Back to cited text no. 16
    
17.
Yoshida H, Kuriyama S, Atsumi Y, et al. Angiotensin I converting enzyme gene polymorphism in non-insulin dependent diabetes mellitus. Kidney Int 1996;50:657-64.  Back to cited text no. 17
    
18.
Wong TY, Chan JC, Poon E, Li PK. Lack of association of angiotensin-converting enzyme (DD/II) and angiotensinogen M235T gene polymorphism with renal function among Chinese patients with type II diabetes. Am J Kidney Dis 1999;33:1064-70.  Back to cited text no. 18
    
19.
Schmidt S, Strojek K, Grzeszczak W, Bergis K, Ritz E. Excess of DD homozygotes in haemo-dialysed patients with type II diabetes. The Diabetic Nephropathy Study Group. Nephrol Dial Transplant 1997;12:427-9.  Back to cited text no. 19
    

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Correspondence Address:
Hana Fakhoury
Department of Biochemistry and Molecular Medicine, College of Medicine, AlFaisal University, P. O. Box 50927, Riyadh 11533
Saudi Arabia
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DOI: 10.4103/1319-2442.202789

PMID: 28352015

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  [Table 1], [Table 2], [Table 3]



 

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