|Year : 2017 | Volume
| Issue : 2 | Page : 261-267
|Associations between clinical characteristics and angiotensin-converting enzyme gene insertion/deletion polymorphism in Moroccan population with Type-2 diabetic nephropathy
Maria Mansouri1, Amal Zniber2, Lamia Boualla1, Ghizlane El Badaoui2, Mariam Benkacem3, Kaoutar Rifai3, Abdelmjid Chraibi3, Loubna Benamar2, Abdelaziz Sefiani1, Rabia Bayahia2
1 Human Genome Center, Faculty of Medicine and Pharmacy, University Mohammed V; Department of Medical Genetics, National Institute of Health, Rabat, Morocco
2 Department of Nephrology, Dialysis and Kidney Transplantation, Faculty of Medicine and Pharmacy, Ibn Sina Hospital, University Mohammed V, Rabat, Morocco
3 Department of Endocrinology and Metabolic Diseases, Faculty of Medicine and Pharmacy, Ibn Sina Hospital, University Mohammed V, Rabat, Morocco
Click here for correspondence address and email
|Date of Web Publication||23-Mar-2017|
| Abstract|| |
Diabetic nephropathy (DN) is one of the severe complications of Type-2 diabetes mellitus (T2DM) and a major cause of end-stage renal disease in these patients. Results from published studies on the relationship between angiotensin-converting enzyme (ACE) insertion/ deletion (I/D) gene polymorphism and patients with DN are still conflicting. We compared the clinical characteristics and the genotype frequencies of ACE polymorphism in 130 T2DM Moroccan patients with DN and 85 T2DM Moroccan patients without DN (controls) using specific primers in a polymerase chain reaction. The degenerative complications of diabetes were significantly higher in the group with nephropathy. The distribution of the I/D genotypes was in Hardy–Weinberg equilibrium. The D allele was the most frequent allele in the Moroccan population in both groups studied (P = 0.68), however, there was no significant difference between the genotypes in T2DM patients with or without DN (P = 0.78). The ACE gene I/D polymorphism was not associated with an increased risk of DN in the Moroccan population.
|How to cite this article:|
Mansouri M, Zniber A, Boualla L, El Badaoui G, Benkacem M, Rifai K, Chraibi A, Benamar L, Sefiani A, Bayahia R. Associations between clinical characteristics and angiotensin-converting enzyme gene insertion/deletion polymorphism in Moroccan population with Type-2 diabetic nephropathy. Saudi J Kidney Dis Transpl 2017;28:261-7
|How to cite this URL:|
Mansouri M, Zniber A, Boualla L, El Badaoui G, Benkacem M, Rifai K, Chraibi A, Benamar L, Sefiani A, Bayahia R. Associations between clinical characteristics and angiotensin-converting enzyme gene insertion/deletion polymorphism in Moroccan population with Type-2 diabetic nephropathy. Saudi J Kidney Dis Transpl [serial online] 2017 [cited 2017 Oct 18];28:261-7. Available from: http://www.sjkdt.org/text.asp?2017/28/2/261/202792
| Introduction|| |
The incidence of diabetes mellitus has increased over the last several decades, resulting in an increase in different diabetic complications, principally kidney complications. Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes [type-1 and type-2 diabetes mellitus (T2DM)] and is the leading cause of end-stage renal disease.,,
Among the possible etiological factors associated with the development of DN in diabetic patients, some genetic factors should be noted; especially the genetic polymorphism in angiotensin-converting enzyme (ACE) gene. The presence of ACE insertion/deletion (I/D) polymorphism affects the plasma levels of ACE.
ACE DD genotype is associated with the highest systemic and renal ACE levels, compared with the lowest ACE activity in carriers of II genotype. Most studies have confirmed that ACE I/D polymorphism is involved in the susceptibility of having nephropathy with protective role of ACE II genotype against the disease in both Type-1 and T2DM.,,,,
The potential role of this polymorphism in influencing the risk of developing DN has not been studied yet in the Moroccan population. Therefore, studying the functional allelic differences in the ACE I/D polymorphism may be a determining factor in the onset and pathogenesis of DN in diabetic patients.
The aim of the present study was to investigate the distribution of ACE I/D gene polymorphism among T2DM Moroccan population and to evaluate the association between the ACE I/D polymorphism and DN.
| Patients and Methods|| |
We conducted a prospective study, including Moroccan patients with T2DM recruited from diabetes and nephrology outpatient clinics at the Ibn Sina Hospital. The duration of the study was from January 2012 to December 2013. T2DM was diagnosed according to the American Diabetes Association criteria: glycosylated hemoglobin (HbA1C) ≥6.5% or fasting plasma glucose ≥7.0 mmol/L or 2-h plasma glucose ≥11.1 mmol/L. The subjects were divided into two groups:
The study group
A total of 130 patients with DN defined by persistent albuminuria (> 30 mg/g creatinine in urine) in two of three consecutive measurements of sterile urine samples, with or without renal failure, defined as estimated glomerular filtration rate <60 mL/mn/1.73 m2 according to the modification of diet in renal disease (MDRD) formula.
The control group
Eighty-five patients without DN, but with T2DM for at least 15 years without proteinuria or renal insufficiency, constituted the control group.
Demographic and clinical characteristics
Anthropometric data such as age, sex, ethnicity, smoking, consanguinity and family history of diabetes, DN or blood pressure were obtained from each patient. Clinical and metabolic parameters, including duration of diabetes, systolic (SBP) and diastolic blood pressure (DBP), body mass index (BMI), urine albumin excretion (UAE), serum creatinine, fasting blood sugar, HbA1C, total cholesterol, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, and triglyceride measurements were recorded. Degenerative complications related to diabetes, such as retinopathy, neuropathy, hypertension, cardiovascular disease, stroke, and diabetic foot were also recorded.
Informed consent was obtained from the patients before implementation of the genetic studies reported here. Peripheral blood was collected in ethylenediaminetetraacetic acid L300 anticoagulated tubes. Genomic DNA was extracted from blood by salting out method.
ACE I/D polymorphism was performed by polymerase chain reaction (PCR). Primers that were used in the assay were forward primer (5’-CTGGAGACCACTCCCATCCTTTCT-3’) and reverse primer (5’-GATGTGGCCATCACATTCGTCAGAT-3’). In 50 PCR volumes, the following components were added with the final concentration of MgCl2 1.5 mmol/L, KCl 50 mmol/L, ×1 PCR Buffer (Bioline Reagents, London, UK), 1 mMol/L each of deoxynucleotide triphosphate, 126 pmol each primer, 1 U of Taq polymerase, and 100 ng of DNA. DNA was denatured at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 56.5°C for 45 s, and extension at 72° for 45 s, with a final extension at 72° for 10 min. PCR products were separated on 2% agarose gel containing ethidium bromide.
The ACE, I/D polymorphism, detected by PCR was evident as a 490-bp product in the presence of the insertion (I allele) and as a 190-bp fragment in the absence of the insertion (D allele). Thus, for each DNA sample presented, one of three possible patterns were available after electrophoresis: 190-bp band (genotype DD), both a 190- and a 490-bp band (genotype ID) or a 490-bpband (genotype II).
Genotype distribution and Hardy–Weinberg equilibrium
Genotype frequencies in our two groups were calculated in accordance with the Hardy–Weinberg equilibrium. Hardy–Weinberg equilibrium states that both allele and genotype frequencies in a population remain constant, that is, they are in equilibrium from generation to generation unless specific disturbing influences are introduced.
Hardy–Weinberg formula was calculated as follows: expected II = p2n; expected ID = 2pqn; expected DD = q2n; where, p = 2 (II) + (ID)/2 (II + ID +DD) and q = 1-p.n. = Number of samples.
Statistical analysis was performed using the Statistical Package for the Social Science (SPSS) software version 19.0 (SPSS Inc.,Chicago, IL, USA). The data are presented as mean ± standard deviation. Nonparametric measures were used to calculate Chi-square values. P <0.05 was considered statistically significant. Odds ratios with 95% confidence intervals were calculated.
| Results|| |
A total of 215 patients with T2DM were recruited (85 patients without DN and 130 patients with DN) during the study.
Characteristics of the patient sample and clinical findings
DN was in the stage of microalbuminuria, macroalbuminuria and renal insufficiency in 18.5%, 4.6% and 76.9% of cases, respectively. A family history of diabetes and DN was more frequent in the DN group [Table 1].
|Table 1: Characteristics of Type-2 diabetic patients with and without nephropathy.|
Click here to view
The patients with DN were older (P = 0.005) and had higher systolic (SBP) (P < 0.001) and diastolic blood pressure (DBP) (P = 0.017) and more dyslipidemia than the control group (P <0.05); there was a male predominance in the case group (P = 0.001). The control group had longer duration of diabetes compared with patients with DN and the degenerative complications of diabetes, including retinopathy, neuropathy, stroke and diabetic foot, were significantly higher in the group with nephropathy [Table 2].
|Table 2: Degenerative complications of diabetes in patients with and without nephropathy.|
Click here to view
Genotype distribution and Hardy–Weinberg equilibrium
Genotype frequencies in both groups were in accordance with the Hardy–Weinberg equilibrium, showing that the study sample excluded selection bias for the genotypes. No significant differences were observed between the observed and expected values relative to genotype distribution (χ2 = 1.98; P =0.159).
Allele frequencies and genotypes
The allele and genotype distributions of the ACE I/D polymorphism in T2DM individuals with DN and those without DN are shown in [Table 3]. The genotype DD is the most frequent genotype in the Moroccan population; however, there were no differences in the distribution of genotypes of ACE I/D polymorphism between the groups studied, with a 58.5% of DD genotype frequency in the DN group and 55.3% in control group.
|Table 3: Genotype frequencies of angiotensin-converting enzyme insertion/deletion polymorphism in T2DM patients with and without nephropathy.|
Click here to view
The odds ratio (95% CI) for the allele II was 1.29 with 95% CI (0.38–4.38) for T2DM patients with DN compared with T2DM patients without DN, and it was 1.13 with 95% CI (0.59–2.16) for the allele DD in the T2DM patients with DN compared with controls.
| Discussion|| |
DN is a complex pathophysiological process which accounts for reduced life expectancy in different populations; it involves the contribution of several etiologies, both environmental as well as genetic. The ACE gene is one of the most studied genes in the pathogenesis of DN. The ACE gene is located on the long arm of chromosome 17 (17q23) and has 21 kilo bases long containing 26 exons encoding the ACE, which is a member of the renin-angiotensin-aldosterone system and plays a role in the regulation of renal and systemic vascular pressure through the formation of angiotensin II and kinin metabolism., More than 160 ACE gene polymorphisms are known, principally the single nucleotide polymorphisms. The ACE I/D polymorphism was reported the first time by Rigat et al to be involved in the development of DN. It is a 287 bp in intron 16 of the ACE gene which has been found to affect plasma ACE activity; the subjects with the D allele have higher levels of ACE and thus, will produce more angiotensin II which will lead consequently to more tissue and joint destruction unlike the subject with the I allele which has a protective effect. Accumulated evidence highlights the role of genetic factors in the etiology of DN.
Ethnicity is also one of the most important factors in favor of this genetic predisposition. The frequency of ACE I/D varies in different ethnic groups, relating this polymorphism to DN., This polymorphism has been studied in the Caucasian population (French, Danes, and Finns), and the Asian and Tunisian population, objectifying the strong liaison between the D allele of ACE and the DN.,,,,,,
The protective role of genotype II against DN was also investigated and confirmed mainly in the Asian population (Chinese, Japanese, and Koreans) than in the Caucasian population.,, In our study, we present the distribution of ACE genotypes as well as their allele frequencies among the Moroccan population and also its association with DN. The study population as a whole showed a high frequency of the DD genotype (56.8%) than the ID (34.95%) and II (8.15%) genotypes. These findings indicate that the DD genotype is the most common genotype in Moroccan patients. Our results were comparable to that reported by all studies of the ACE I/D polymorphism. More importantly, this study has found that the I/D polymorphism in the ACE gene was not a major risk factor for the onset and progression of DN among the Moroccan population. In particular, the DD genotype, which has previously been implicated in DN in Type-I and Type-II diabetes, is not associated with an increased risk of developing DN. Despite the fact that the ACE gene is one of the most studied genes in the pathogenesis of DN, the results are inconsistent. We studied the role of ACE gene, especially the I/D polymorphism, in the Moroccan population, to evaluate the phenotypes, to prove that there is no association between the ACE I/D polymorphism and DN in our population and to try and understand some of the pathogenic mechanisms of diabetic renal diseases.
| Conclusion|| |
In this study, we compared the clinical data of diabetic patients with and without DN; we evaluate the frequency of alleles of ACE polymorphism and we found that there was no association between the I/D polymorphism of ACE and a greater occurrence of DN in the Moroccan individuals with diabetes. To the best our knowledge, this is the first study that evaluates and compares the ACE I/D polymorphism in diabetic patients with DN in our population.
Conflict of interest: None declared.
| References|| |
Hoffmann F, Haastert B, Koch M, Giani G, Glaeske G, Icks A. The effect of diabetes on incidence and mortality in end-stage renal disease in Germany. Nephrol Dial Transplant 2011;26:1634-40.
Fassett RG, Robertson IK, Mace R, Youl L, Challenor S, Bull R. Palliative care in end-stage kidney disease. Nephrology (Carlton) 2011;16:4-12.
Grace BS, Clayton P, McDonald SP. Increases in renal replacement therapy in Australia and New Zealand: Understanding trends in diabetic nephropathy. Nephrology (Carlton) 2012;17:76-84.
McDonough CW, Palmer ND, Hicks PJ, et al. A genome-wide association study for diabetic nephropathy genes in African Americans. Kidney Int 2011;79:563-72.
Alkhalaf A, Bakker SJ, Bilo HJ, et al. A polymorphism in the gene encoding carnosinase (CNDP1) as a predictor of mortality and progression from nephropathy to end-stage renal disease in type 1 diabetes mellitus. Diabetologia 2010;53:2562-8.
El-Din Bessa SS, Hamdy SM. Impact of nitric oxide synthase Glu298Asp polymorphism on the development of end-stage renal disease in type 2 diabetic Egyptian patients. Ren Fail 2011;33:878-84.
Hubacek JA, Viklicky O, Dlouha D, et al. The FTO gene polymorphism is associated with end-stage renal disease: Two large independent case-control studies in a general population. Nephrol Dial Transplant 2012;27: 1030-5.
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.
American Diabetes Association. Standards of Medical Care in Diabetes-2016. Diabetes Care 2016;39 Suppl 1:S1-106.
Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215.
Uddin M, Azam M, Chowdhury N, Akhteruzzaman S. Angiotensin I-converting enzyme gene polymorphism in type 2 diabetic patients with nephropathy. J Med Sci 2007; 7:682-5.
Giacchetti G, Sechi LA, Rilli S, Carey RM. The renin-angiotensin-aldosterone system, glucose metabolism and diabetes. Trends Endocrinol Metab 2005;16:120-6.
Ferrario CM, Strawn WB. Role of the renin-angiotensin-aldosterone system and proinflammatory mediators in cardiovascular disease. Am J Cardiol 2006;98:121-8.
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.
Gürkan A, Emingil G, Saygan BH, et al. Angiotensin-converting enzyme (ACE), angiotensinogen (AGT), and angiotensin II type 1 receptor (AT1R) gene polymorphisms in generalized aggressive periodontitis. Arch Oral Biol 2009;54:337-44.
Felehgari V, Rahimi Z, Mozafari H, Vaisi-Raygani A. ACE gene polymorphism and serum ACE activity in Iranians type II diabetic patients with macroalbuminuria. Mol Cell Biochem 2011;346:23-30.
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.
Rahimi Z, Felehgari V, Rahimi M, et al. The frequency of factor V leiden mutation, ACE gene polymorphism, serum ACE activity and response to ACE inhibitor and angiotensin II receptor antagonist drugs in Iranians type II diabetic patients with microalbuminuria. Mol Biol Rep 2011;38:2117-23.
Nikzamir A, Nakhjavani M, Esteghamati A, Rashidi A. Correlates of ACE activity in macroalbuminuric type 2 diabetic patients treated with chronic ACE inhibition. Nephrol Dial Transplant 2008;23:1274-7.
Nikzamir A, Esteghamati A, Feghhi M, Nakhjavani M, Rashidi A, Reza JZ. The insertion/deletion polymorphism of the angiotensin-converting enzyme gene is associated with progression, but not development, of albuminuria in Iranian patients with type 2 diabetes. J Renin Angiotensin Aldosterone Syst 2009;10:109-14.
Hadjadj S, Tarnow L, Forsblom C, et al. Association between angiotensin-converting enzyme gene polymorphisms and diabetic nephropathy: Case-control, haplotype, and family-based study in three European populations. J Am Soc Nephrol 2007;18:1284-91.
Ahluwalia TS, Ahuja M, Rai TS, et al. ACE variants interact with the RAS pathway to confer risk and protection against type 2 diabetic nephropathy. DNA Cell Biol 2009; 28:141-50.
Ezzidi I, Mtiraoui N, Kacem M, Chaieb M, Mahjoub T, Almawi WY. Identification of specific angiotensin-converting enzyme variants and haplotypes that confer risk and protection against type 2 diabetic nephropathy. Diabetes Metab Res Rev 2009;25:717-24.
Staessen JA, Wang JG, Ginocchio G, et al. The deletion/insertion polymorphism of the angiotensin converting enzyme gene and cardiovascular-renal risk. J Hypertens 1997;15 (12 Pt 2):1579-92.
Ng D, Ramli SN, Chia KS, Koh D, Tai BC. Genetic varation at the angiotensin-I converting enzyme locus and the risk for diabetic nephropathy: An update based on 53 studies and 17 791 subjects. Buenos Aires: Sociedad Iberoamericana de Información Científica (SIIC); 2007.
Ng DP, Tai BC, Koh D, Tan KW, Chia KS. Angiotensin-I converting enzyme insertion/ deletion polymorphism and its association with diabetic nephropathy: A meta-analysis of studies reported between 1994 and 2004 and comprising 14,727 subjects. Diabetologia 2005;48:1008-16.
Movva S, Alluri RV, Komandur S, et al. Relationship of angiotensin-converting enzyme gene polymorphism with nephropathy associated with type 2 diabetes mellitus in Asian Indians. J Diabetes Complications 2007;21: 237-41.
Lee YJ, Tsai JC. ACE gene insertion/deletion polymorphism associated with 1998 World Health Organization definition of metabolic syndrome in Chinese type 2 diabetic patients. Diabetes Care 2002;25:1002-8.
Human Genome Center, Faculty of Medicine and Pharmacy, University Mohammed V, Rabat
[Table 1], [Table 2], [Table 3]
| Article Access Statistics|
| Viewed||755 |
| Printed||10 |
| Emailed||0 |
| PDF Downloaded||163 |
| Comments ||[Add] |