|Year : 2015 | Volume
| Issue : 2 | Page : 43-50
Prognostic significance of ACE and PAI-1 genes polymorphisms with diabetic retinopathy and diabetic non-retinopathy in Type 2 diabetes
Sami Ullah Khan1, Abdul Qayyum2, Syed Sajjad Hussain3
1 PhD grant Holder, Department of Medical Microbiology, Kazan Federal University, Russian Federation, Pakistan
2 Associate Professor, Department of Languages, Government Degree College, Pakistan
3 Syed Sajjad Hussain, Australian Concept Infertility Medical Centre, Pakistan
|Date of Web Publication||5-Jul-2017|
Sami Ullah Khan
PhD Grant Holder, Department of Medical Microbiology, Kazan Federal University, Russian Federation
Source of Support: None, Conflict of Interest: None
Background: ACE a renin-angiotensin system that regulates blood pressure, balance of fluids and salts in body and PAI-1 is a serine protease inhibitor, which inhibits tissue plasminogen activator andurokinase.They are thought to play an important role in pathophysiology of kidney disease in diabetes. Aim: In our present study, we studied the association of altered ACE-gene and PAI-1 gene with diabetic retinopathy (DR) and NDR in 592 samples consisted of (cohort I; 196 DR patients, cohort II; 200 diabetic non-retinopathy (DNR) and cohort III, 196 respective controls. Methods: For genotyping of ACE-gene and PAI-1 gene, genomic DNA was isolated and purified which was then amplified by PCR, and thePCR products analyzedwere by Agarose gel electrophoresis. Results: In first part, the ACE genotype and allele frequency distribution was studied. For ACE gene polymorphism, the genotype and allele frequency distribution were analyzed in DR subjects and respective controls. The results indicated that there is no statistically significant difference between DR males and females compared to respective controls. The results were significantly high between genotype frequencies of DR and DNR in males. The recessive model was found to be significantly associated with the DR male subjects (OR=0.45 [95% CI=0.20-0.99], p<0.05), whereas in females these are non-significant as compared to respective controls individuals. In second part of study, the disease status analysis of ACE gene on basis of DR stages (NPDR and PDR) was observed. The χ2 analysis indicated that results are significantly different between NPDR and respective controls (χ2=8.75, p=0.01) .And in third part of present study, disease status analysis for PAI-1 gene on the basis of DR stages (NPDR and PDR) was studied, which indicated statistically non-significance. The χ2 analysis values for DNR and NPDR and for DNR and PDR was (χ2=0.48, p>0.05)(χ2 =2.00, p>0.05) respectively, Conclusion: Our present study suggests that changes in genetic polymorphisms of ACE-gene and PAI-1 gene in DR, DNR and T2D Patients are risk factors, which may serve as useful prognostic markers.
Keywords: Genetic polymorphisms, ACE-gene, DNR, T2D, Prognostic markers
|How to cite this article:|
Khan SU, Qayyum A, Hussain SS. Prognostic significance of ACE and PAI-1 genes polymorphisms with diabetic retinopathy and diabetic non-retinopathy in Type 2 diabetes. Acta Med Int 2015;2:43-50
| Introduction|| |
Type 2 diabetes (T2D) is a metabolic disease associated with serious micro- and macro-vascular complications, including diabetic retinopathy (DR), a major cause of blindness among diabetic adults that are aggravated by poor glycaemic control, hypertension and longer disease duration. DR is associated with a strong genetic predisposition, highlighted by familial clustering of DR, and the association of several gene polymorphisms with DR.,,,,,,, These include aldose reductase,, advanced glycation end-products receptor,, adhesion molecules, and coagulation and fibrinolytic system gene polymorphisms,including plasminogen activator system (PAS), and angiotensin converting enzyme.,, PAS includes distinct serine proteases and their inhibitors plasminogen activator inhibitor that control plasminogen activation., High PAI-1 activity is associated with atherosclerosis and thromboembolism, and several polymorphisms in PAI-1 gene stimulus PAI- 1 levels. Higher levels of the PAI-1 are detected in serum of diabetic individuals and experimental animals, and are implicated in retinal micro-vascular occlusion.,, Insofar as 4G/4G genotype is associated with increased PAI-1 gene transcription and elevated PAI-1 serum levels, it may be that 4G/5G polymorphism is allied to DR pathogenesis. Previous studies of association between 4G allele and DR have yielded conflicting results, ranging from strong links, to no association,, while others suggest that high PAI-1 gene activity, independent of 4G/4G genotype, may beimplicated in DR pathogenesis.
ACE is a zinc metallo-peptidase usuallydispersed on surface of endothelial and epithelial cells. This enzyme havedistinct role in regulation of blood pressure and electrolyte balance thus it is an important regulator of blood homeostasis and is also involved in deactivating a potent vasodilator, bradykinin. Studies have shown that increased intraocular and serum ACE, prorenin angiotensin II correlated with severity and progression of DR.,,
ACE gene has 25 coding exons and 25 introns. Already reported literatures suggest a strong genetic association of 287 bp Alu-repeat polymorphism (rs4646994) in intron 16 of the ACE gene with significant raise in protein level in cell as well as in plasma circulation. The present study was aimed to determine genetic association of ACE-gene (rs4646994) and PAI-1 gene (rs1799768) polymorphisms with DR andDNR in T2D patients. The incidence of disease is among the most prevalent micro-vascular complication of diabetes and seriously affects quality of life in individuals, especially in working age group, as it can lead to irreversible loss of vision and thus impairment in normal daily routines of patient.
| Materials and Methods|| |
Diabetic Retinopathy patients were consulted and diagnosed at Australian Concept Infertility Medical Centre, Pakistan. All patients were tested for presence of T2D followed byscreening for diabetic retinopathy. The present study was approved by the Ethics Committee and Departmental Review Board of Australian Concept Infertility Medical Centre, Pakistan. All participants were informed about purpose of study. In addition their complete demographic data and clinical history were also obtained. In addition, blood samples of healthy individuals, free from any disease were also collected.
Genomic DNA Extraction
ForDNA extraction, 6ml of blood was collected in 8.5ml vacutainer™ tubes (BD, Franklin Lakes, NJ) containing EDTA. A standard organic method (Phenol/Chloroform) was used for DNA extraction to obtain high yield genomic DNA (Sambrook and Russell, 2001) . Anucleated red blood cells (RBCs) were lysisedand cellular debris was removed by using RBC lysis buffer solution.Similarly, white blood cells were lysisedandcellular debris was removed by using nuclear lysis buffer solution. These were washed by Phenol/ Chloroform and removed cellular debris. The DNA was triggered by isopropanol in presence of sodium acetate, followed by washing of genomic DNA with 70% ice-cold ethanol.
For RBC and nuclear lysis3ml of blood was taken and to this 12ml of RBC lysis buffer (pH 7.4) was added. The tubes were then incubated for 5 min and during incubation they were vortexed several times in order to lyse all the RBCs completely. The tubes were then centrifuged for 3 min at 4100rpm, and supernatant was descanted from the pallet. The cell pellet was then washed by re-suspending it in 4ml RBC Lysis buffer solution followed by centrifugation at 4100rpm for 3 min. The supernatant was descanted and cell pellet was re-suspended in 400 μl nuclear lysis buffer solution (pH 8.2). 30 μl SDS (20%) and 10 μl Proteinase K were added to this mixture and incubated at 55°C in a water bath with constant shaking for overnight.To overnight inculated mixture 3 ml of phenol was added, followed by addition of an equal volume of TE buffer solution and incubated at room temperature for 5 min with vortexed several times. It was then centrifuged for 10 min at 4500 rpm resulting in separation of two phases; lower organic phase, containing digested proteins and other cellular debris and the upper aqueous phase; containing dissolved DNA. The aqueous phase was transferred to a new 15ml falcon tube and to this an equal volume of chloroform/isoamyl alcohol was added. The tubes were then mixed by inversion and centrifuged for 10 min at 4500 rpm.The upper aqueous phase was then transferred to a new 15ml falcon tube.
To aqueous phase obtained 1/10th volume 3M sodium acetate and 2 volumes of ice-cold isopropanol were added. The tubes were vortexedmany times to precipitate DNA, and followed by centrifugation at 4500 rpm (-2°C) for 10 min.
The DNA pellet formed was washed with ice cold 70% ethanol and then centrifugedat 4500 rpm for 10 min. The supernatant was descended and pellet was air dried or dried in an incubator at 37°C. After completely dryness, the pellet was re-suspended in 400μl TE buffer solution and the DNA was re-suspended by incubating them at 37°C for 30 min. The samples were labeled and stored at -20°C.
ACE and PAI-1 polymorphism
The patients and respective controls were genotyped for ACE Insertion/Deletion (I/D) polymorphism and PAI-14/5G polymorphism using sets of primers [Table 1]. The genotypes were calculated to determine association of ACE (I/D) and PAI-1 (4G/5G) polymorphisms with DR. All PCR reactions were performed with 100ng genomic DNA in a final volume of 25 μl PAI-1 4G/5G amplification mixture contained 2.5 μl 10X Taq Buffer solution (750mM Tris-HCl (pH 8.8), 0.2mM of each dNTPs (Fermentas®Opelstrasse, Germany), 0.1% Tween 20 and 200mM (NH4) SO4. The reaction mixture also contained 1U of Taq DNA Polymerase, 1.5mM MgCl2, 0.12 μM internal control primer and 0.24 μM of each forward (4G or 5G) and reverse primer. For genotyping of ACE-gene polymorphism 0.2 μM of each forward and reverse primer were used and concentrations of rest of the reagents were same as for PAI-1 gene genotyping.
|Table 1: The primer sequences of ACE gene (rs4646994) and PAI-1 gene (rs1799768) and their product size|
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Polymerase Chain Reaction (PCR)
PCR for both ACE-gene and PAI-1 gene polymorphism were performed in Thermo Electron Corporation System (PXE 0.2 Thermal Cycler) .
Agarose gel electrophoresis
PCR amplified products were electrophoretically separated for 30 min at 120 V on either a 2% or 3% agarose gel for the ACE or PAI-1 gene polymorphism respectively. To stain and visualize DNA upon UV transillumination in gel 5-10 μl of Ethidium Bromide was also added. ACE (I/D) amplification gave a band size of 312 bp (Deletion) and 599 bp (Insertion) . PAI-1 (4G/5G) polymorphism gave a band of 139 bp and 140 bp for the 4G and 5G polymorphisms respectively, along with an internal control band of 256 bp. The PCR amplified products were then loaded into wells of agarose gel. Electrophoresis was carried out in 1X TBE Buffer and the gel was visualized by UV transilluminator and image was capturedwith help of a digital camera and analyzed by software BioCap MW (ver 11.01) from VilberLourmat (Marne-La-Vallee Cedex1, France) .
The means and percentages of genotypes and allele frequencies in DR cases DNR cases and respective controls were statistically analyzed by chi square (χ2) test and p-values were calculated by Epi Info Version 6.0 stats calculator. Z-test was performed using SPSSvol 13.0. The dominant and recessive models for risk allele were calculated by Java Stat 2-way contingency table analysis (http://statpages.org/ctab2x2.html) .
| Results|| |
In present study, 592 samples were studied consists of (cohort I; 196 DR patients,cohort II; 200 diabetic non-retinopathy (DNR) and cohort III, 196 respective controls. In case of ACE-gene polymorphism band size of 599 bp indicates insertion polymorphism (I), whilethe 312 bp band designates deletion polymorphism of rs4646994 [Figure 1]. In case of the “4G” allele of PAI-1 gene, a 139 bp band was obtained along with an internal control band of 256 bp [Figure 2] .
|Figure 1: Gel picture of 2% agarose gel showing the ACE gene (rs4646994) I/D polymorphism with upper band size of 599bp in case of Insertion (I) and lower band size of 312bp in case of Deletion (D)|
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|Figure 2: Gel picture of 3% agarose gel showing the allele pattern obtained with 4G allele of PAI-1 gene with upper internal control (IC) band of 256bp and lower allele specific (4G) band of 139bp|
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The statistical χ2test was performed for comparison of genotype frequency differences between patients and respective controls. The rs4646994 genotype frequencies are figured in [Table 2].
|Table 2: The genotype and allele frequency of the ACE gene I/D polymorphism in the DR cases, DNR cases and unaffected controls|
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In first part, the ACE genotype and allele frequency distribution was studied. For ACE gene polymorphism, the genotype and allele frequency distribution were analyzedin DR subjects and respective controls rs4646994. The results indicate that there is no statistically significant difference between DR males and females compared to respective controls. The results were significantly high between genotype frequencies of DR and DNR in males. The recessive model was found to be significantly associated with the DR male subjects (OR=0.45 [95% CI=0.20-0.99], p<0.05), whereas in females these are nonsignificant as compared to respective controls individuals. Genetic analysis for PAI-1 gene polymorphism indicates that there is no significance in PAI-1 gene rs1799768 genotype frequencies in DR and DNR as compared to respective control shown in [Table 3].There is no significance difference in PAI-1 genotypes between patients and respective control individuals. The genotype frequencies of DR and DNR inmale patients are non-significant (χ2=0.54, p>0.05). PAI-1 genotype allele frequency distribution in DR females was 43 (44.3%) heterozygous 4G/5G, 29 (29.9%) homozygous 5G/5G, and 25 (25.8%) homozygous 4G/4G. While, in respective control females, the frequencies are 43 (43.8%) heterozygous 4G/5G, 30 (30.6%) homozygous 5G/5G, and 25 (25.5%) homozygous 4G/4G, The frequencies observed are non-significantbetween two groups (χ2=0.01, p>0.05) . There is no difference among frequencies of 4G/4G genotype (z=0.04, p>0.05), 5G/5G genotype (z=0.11, p>0.05), and 4G/5G genotype (z=0.06, p>0.05). These results also indicates that the PAI-1 genotype allele frequency distribution in DNR patientsare 44 (43%) heterozygous 4G/5G, 33 (32.5%) homozygous 5G/5G, and 25 (24.5%) homozygous 4G/4G, which are statistically non-significant when compared to respective controls (χ2=0.07, p>0.05) . Logistic regression analysis also showed statistically non-significant genotype association with disease (DM: OR=0.92 [95% CI=0.49-1.75], p>0.05; RM: OR=0.95 [95% CI=0.48-1.89], p>0.05, respectively).
|Table 3: The genotype and allele frequency of the PAI-1 gene 4G/5G polymorphism in the DR cases, DNR cases and unaffected controls|
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In second part of the study the Disease status analysis of ACE gene on the basis of DR stages (NPDR and PDR) was observed. The genotype frequencies for ACE gene polymorphism in NPDR and respective controls were observed as 58 (58.6%) heterozygous I/D, 24 (24.2%) homozygous I/I, and 17 (17.2%) homozygous D/D. The χ2 analysis indicated that the results are significantly different between NPDR and respective controls (χ2=8.75, p=0.01) . The homozygous I/I genotype frequency was significantly lower as compared to respective control individuals (z=2.43, p<0.05) . In contrast the heterozygous I/D genotype frequency was significantly higher compared to respective controls (z=2.87, p<0.01) . Homozygous D/D genotype frequency distribution was non-significant when to control individuals (z=0.72, p>0.05) . Under dominant model the odds ratio indicated statistically significant genotype association to NPDR patients (OR=1.91 [95% CI=1.10-3.47], p<0.05) and in recessive model the odds ratio was statistically non-significant related to disease (OR=0.80 [95% CI=0.41-1.55], p>0.05). In PDR cases compare torespective controls, the observations are 37 (47.4%) heterozygous I/D, 24 (30.8%) homozygous I/I, and 17 (21.8%) homozygous D/D. The χ2 analysis indicates statistically non-significant PDR cases compared to controls (χ2=1.48, p>0.05) . The odds ratio under dominant model (OR=1.40 [95% CI=0.77-2.55], p>0.05) and recessive model (OR=1.07 [95% CI=0.54-2.11], p>0.05) was statisticallynon-insignificantly associated with disease. Statistical analysis among DNR, NPDR and PDR were shown in [Table 4]. And in third part of the present study,disease status analysis for PAI-1 gene on the basis of DR stages (NPDR and PDR) was studied, which are statistically non-significant. The χ2 analysis values for DNR and NPDR and for DNR and PDR was (χ2=0.48, p>0.05) (χ2 =2.00, p>0.05) respectively.
|Table 4: The genotype and allele frequency of the ACE gene I/D polymorphism in DNR cases, NPDR cases and PDR cases|
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| Discussion|| |
Diabetic Retinopathy (DR) is most detrimental micro-vascular impediments of diabetes mellitus and remains animportant cause of visual morbidity among developed and under developing countries worldwide. It is a progressive disease that severely damages cellular and structural composition of retinal vasculature resulting in severe vision impairment problems often leading to blindness. DR is a complex disorder involving different biochemical pathways in pathogenesis of disease, probably due to altered glucose metabolism and genetic factors associated with those pathways. Clinically DR manifests in two forms; Non Proliferative Diabetic Retinopathy (NPDR) and Proliferative Diabetic Retinopathy (PDR) .
Polymorphisms and mutation in various genes and chromosomal regions could be a factor in development of DR. Linkage analysis has indicated a linkage between chromosome 3 and 9,which is associatedonset and progression of retinopathy in Pima Indians with T2D. Various studies on polymorphisms in several genes have also been carried out with respect to micro-vascular complications of diabetes but very few associations have been reported with retinopathy.
ACE is a key regulator of the RAS, which accelerateproduction of angiotensin II from angiotensin I. Several components of RAS such as ACE, pro-renin, renin, angiotensinogen, angiotensin I and angiotensin II have been shown to express in human eye, but underlying biochemical mechanismsin RAS with respect to DR are not known yet. Since angiotensin II is a strong vasoconstrictor with angiogenic properties, it is assumed that local RAS may be involved in pathogenesis of proliferative retinopathy and promotes neovascularization in patients withdiabetes.,,, This assumption is further strengthened by fact that levels of pro-renin are raised in ocular vitreous fluid and plasma of individuals with PDR. Therefore, it might be possible that activated RAS is an important factor involved in development of blood vessels in retina causing vascular injury leading to loss of vision. Patients suffering from heart and kidney disorders as well as T2D showed presence of insertion allele or absence of deletion allele within intron 16 of ACE gene indicating that ACE plasma levels to be under regulation of genetic factors.,, Polymorphism (rs4646994) was responsible for about 50% of inter-individual variability in the plasma ACE levels and individuals with D/D genotype had significantly higher levels as compared to those with I/I or I/D genotypes. The reported risk allele is suggested to be “D” of this polymorphism as evident from numerous studies and has been suggested to be involved in retinal complications ofdiabetes.
The trend of increased levels of ACE within plasma is not related to diabetes but also to other severe micro-vascular complications of diabetes particularly with retinopathy leading to retinal vascular damage in patients suffering from proliferative retinopathy.,, The data available regarding association of I/D polymorphism depict diverse results, however, this ACE gene polymorphism is strong indicator for DR.
In our present study, thegenotype and allele frequency distribution was found to be insignificantly associated with severity and progression of DR. Results in our population for rs4646994 polymorphism was observed to be similar with findings in other populations that also reported association of ACE gene with development and progression of retinopathy in subjects with T1D or T2D., It has beenshowedinsignificant association of the rs4646994 polymorphismin Caucasian population, suffering from any retinopathy (NPDR or PDR) among 186 T1D and 363 T2D cases when compared with 98 healthy non-diabetic controls.
However, in our study a significant distribution of genotype frequency was observed due to higher frequency of heterozygous I/D genotype in DR cases as compared to DNR. But this was not found to be associated with DR susceptibility after logistic regression analysis under recessive, dominant or co-dominant models. The current findings are in opposition to work reported by in Iranian population comprising of 178 DR and 206 DNR cases, those authors have shown that frequency of homozygous genotype for risk “D” allele was statistically higheramong DR subjects as compared to DNR subjects.
In current study, gender-wise analysis gave a significant association of rs4646994 polymorphism of ACE gene in male DR cases when compared to DNR cases, where D/D genotype frequency was found to be higher in DNR males under recessive model for “D” allele as compared to DR males. These findings point to a possible role of “D” allele in this polymorphism to be linked with incidence of DNR but not with development and progression of DR. However, the heterozygous genotype I/D frequency was observed to be greater among DR males when compared with DNR males and controls, indicating that individuals with intermediate plasma ACE levels are at a higher risk of developing DR. Although a number of studies have shown higher plasma ACE activity in individuals with “D” allele but exact mechanism through which “D” allele is involved in pathogenesis of DR needs to be determined.
In our study, we found ACE geners 4646994 polymorphism was statistically more related with subjects with NPDR as compared to those with PDR. Hence, our results are contrary to other studies where they found significant association of I/D polymorphism with PDR subjects i.e. Chinese and Iranian populations suffering from T2D.,
Results obtained in our population suggest a significant function of this polymorphism in development of early stage of retinopathy (NPDR), which is in contrast to other studies, where significant association was found with advance stage of retinopathy i.e. PDR. Apart from ethnic variations between Pakistani and other populations worldwide, there might be some other pathophysiological mechanisms that are also implicated in association of RAS with advance stages of DR.
The present study showed no significant association of the PAI-1 gene rs1799768 with development and progression of DR in T2D subjects. Inpresent study the gender-wise analysis pointed towards the fact that there may be no role of rs1799768 with onset and advancing of DR. Similarly, subgroup analysis based on staging of retinopathy, showed no significant association of rs1799768 in PAI-1 gene with incidence and progression of DR. Our results are in accordance with Tunisian T2D patients and Caucasians with T1D and Caucasians with T1D and T2D, which also showed no significant association between PAI-1 gene polymorphism in subjects with NPDR or PDR. Similarly, in another study conducted by, it was shown that 4G/5G polymorphism did not predispose genetic susceptibility to DR, DNR, NPDR or PDR cases in Caucasian Slovenian patients with T2D.
The prominence of current study is to determine genetic association of ACE (rs4646994) and PAI-1 genes (rs1799768) polymorphisms respectively with DR in T2D. In conclusion, we found astatistically significant role of “D” allele (ACE gene) as risk for diabetic male population having no retinopathy. However, polymorphism in PAI-1 gene is statistically not associated with diabetes and its complications in any of groups under present study.Our study suggests that ACE (rs4646994) and PAI-1 genes (rs1799768) polymorphisms may play role in development and progression of DR and DNR, which may serve as useful prognostic markers.
| Acknowledgements|| |
All authors wish to acknowledge the patients and control individuals who contributed to this research work. We are also thankful to hospital staff (Australian Concept Infertility Medical Centre Pakistan) for their help and cooperation. This study was supported by Australian Concept Infertility Medical Centre Pakistan. The authors declare that they have no conflict of interests.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]