|Year : 2017 | Volume
| 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
Sarah Fawwaz1, Mahmoud Balbaa2, Hana Fakhoury3, Jamila Borjac1, Rajaa Fakhoury4
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 Publication||23-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|| |
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. The human ACE gene is located on chromosome 17 (17q23) and comprises 26 exons and 25 introns. 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.
Diabetic nephropathy (DN) is one of the leading causes of end-stage renal disease (ESRD), the pathogenesis of which is not clearly understood. Administration of ACE inhibitors and angiotensin-II receptor blockers has been shown to have beneficial effects in retarding the development and progression of DN. 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. Indeed, ACE I/D polymorphism is the first and the most extensively studied candidate gene of RAS for predisposition of DN.,,,
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|| |
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).
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:
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|| |
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|| |
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|| |
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. 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. This was also the case in a Bahraini population. The results were different in Egypt, where the D/D genotype was more prevalent in DN. 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. A Japanese study compared the ACE I/D polymorphism in different stages of DN including ESRD. 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. However, Wong et al and Schmidt et al 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.
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:
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Department of Biochemistry and Molecular Medicine, College of Medicine, AlFaisal University, P. O. Box 50927, Riyadh 11533
[Table 1], [Table 2], [Table 3]
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