516172 research-article2014
JRA0010.1177/1470320313516172Journal of the Renin-Angiotensin-Aldosterone System 0(0)Settin et al.
Original Article
Association of ACE and MTHFR genetic polymorphisms with type 2 diabetes mellitus: Susceptibility and complications
Journal of the Renin-AngiotensinAldosterone System 1–6 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1470320313516172 jra.sagepub.com
Ahmad Settin1, Rizk El-Baz1, Azza Ismaeel2, Wafaa Tolba2 and Wafaa A Allah2
Abstract Hypothesis/introduction: Polymorphisms of angiotensin converting enzyme (ACE) and methylene-tetrahydrofolate reductase (MTHFR) genes have been proposed to be associated with type 2 diabetes mellitus (T2DM) with conflicting results. This work was planned in order to check for the association of these polymorphisms with the susceptibility for and complications of T2DM among Egyptian cases. Materials and methods: This is a case controlled study involving 203 patients with T2DM and 311 healthy controls. Polymorphic variants of ACE I>D and MTHFR (677 C>T and 1298 A>C) were determined using the polymerase chain reaction (PCR) restriction analysis technique. Results: The susceptibility to T2DM was higher among subjects having the MTHFR 677TT (odds ratio (OR)=2.2, p=0.01), MTHFR 1298 AA (OR=1.84, p=0.001) and ACE (ID+II) (OR=2.0, p=0.0007) genotypes. Logistic regression analysis showed that MTHFR 677T allele was a risk factor for diabetic retinopathy (DR) (OR=3.47, pC genes with the susceptibility to T2DM and its complications including DR, DPN and IHD among Egyptian cases.
1Genetics 2Zoology
Unit, Mansoura University, Egypt Department, Mansoura University, Egypt
Corresponding author: Ahmad Settin, Qassim University, BO 6655 Buraydah 51452, Saudi Arabia. Email: [email protected]
Downloaded from jra.sagepub.com by guest on November 14, 2015
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Journal of the Renin-Angiotensin-Aldosterone System
Subjects and method This study was a case-controlled study involving 202 cases with T2DM in addition to 311 healthy controls. Cases were recruited from the Diabetes and Endocrinology Departments, Internal Medicine Specialized Hospital, Mansoura University, Egypt. The T2DM cases included 84 (41.6%) males and 118 (58.4%) females with age mean±standard deviation (SD) of 55.48±8.09 years. Diabetes diagnosis was based upon World health Organization (WHO) criteria with a fasting plasma glucose ≥7.0 mmol/l.17 Inclusion criteria were cases with Egyptian origin, age at onset of diabetes >35 years, diabetes duration of ≥10 years, C-peptide ≥0.3 nmol/l and glutamic acid decarboxylase (GAD) antibody negativity. Of these cases, 67 (33.2%) were complicated with DR, 93 (46%) with DPN and 47 (23.3%) with IHD. These cases were compared to 311 healthy non-diabetic unrelated controls from the same locality. They were 129 males and 182 females with age mean±SD of 51.17±1.07 years. Before the start of the study, ethical approval was obtained from the scientific and ethical committees of the local health authorities in addition to informed consent that was obtained from all the participants.
a preliminary denaturation at 95ºC for 3 min, 5 cycles: denaturation at 94ºC for 1 min, annealing at 64ºC for 1 min, synthesis at 72ºC for 30 s, next 30 cycles were run as: denaturation at 94ºC for 45 s, annealing at 62ºC for 45 s, synthesis at 72ºC for 25 s and final synthesis at 72ºC for 7 min. Digestion was performed by the enzyme HinfI (Fermentas, UK) at 37ºC for four hours. For evaluation of the other MTHFR 1298 A>C polymorphism, the following primers were used: Forward: (5`-CTT TGG GGA GGT GAA GGA CTA CTA C-3`) and Reverse: (5`-CAC TTT GTG AGC ATT CCG GTT TG-3`) (Gibeo BRL). The PCR procedure was composed of preliminary denaturation at 95ºC for 2 min, 5 cycles: denaturation at 95ºC for 1 min, annealing at 55ºC for 2 min, synthesis at 72ºC for 2 min, next 32 cycles were run: denaturation at 95ºC for 75 s, annealing at 55ºC for 75 s, synthesis at 72ºC for 90 s and final synthesis at 72ºC for 6 min. Restrictase MboII (Fermentas, UK) was used for digestion at 37ºC for seven hours. Each final reaction volume plus 5 µl of bromophenol blue track dye were loaded into 2% agarose gel (Boehringer Mannheim, Germany) containing ethidium bromide. Gels were electrophoresced for 30 min at 100 V, photographed under UV light (320 nm) and then scored for the presence or absence of an allele specific bands.
DNA extraction, amplification and digestion
Statistical analysis
DNA was extracted and purified using a the kit of Gentra Systems, USA according to the manufacturer’s instructions. Characterization of the ACE I/D polymorphism was done using PCR amplification of the respective fragments from intron 16 of the ACE gene according to the method described previously.18 An optimized primer pair was used to amplify the D and I alleles (5’-GCC CTG CAG GTG TCT GCA GCA TGT-3’ and 5’-GGA TGG CTC TCC CCG CCT TGT CTC-3’) resulting in 319-bp and 597-bp amplicons respectively. The cycling profile consisted of denaturation at 94ºC for 30 s, annealing at 56ºC for 45 s, and synthesis at 72ºC for 2 min, repeated for 35 cycles, followed by a final synthesis at 72ºC for 7 min. Because the D allele in heterozygous samples is preferentially amplified, each sample found to have the DD genotype was subjected to a second, independent PCR amplification with a primer pair that recognizes an insertion-specific sequence (5’-TGG GAC CAC AGC GCC CGC CAC TAC-3’ and 5’-TCG CCA GCC CTC CCA TGC CCA TAA-3’), with identical PCR conditions except for an annealing temperature of 67ºC. The reaction yields a 335 bp amplicon only in the presence of an I allele, and no product in samples was homozygous for DD. MTHFR 677 C>T and 1298 A>C genotypes were characterized using the PCR-RFLP technique described previously.19 The primer sequences for 677 C>T genotypes were: Forward: (5`-TGA AGG AGA AGG TGT CTG CGG GA-3`) and Reverse: (5`-AGG ACG GTG CGG TGA GAG TG-3`) (Gibeo BRL). PCR protocol was in the form of
Data were processed and analyzed using the Statistical Package of Social Science (SPSS, version 17.0). Frequencies of studied genotypic and allelic polymorphisms among cases were compared to those of controls and tested for positive association using chi-square (χ2) test or Fisher exact tests and odds ratio (OR) with a 95% confidence interval (CI). The distribution of alleles and genotypes in the studied groups was tested for fitting to the Hardy-Weinberg equilibrium through comparing the observed and expected frequencies of genetic variants using the χ2 test. Binary logistic regression analysis was done to assess the genetic and demographic risk factors contributing to each type of diabetic complication. A minimum level of significance was considered if p was T, MTHFR 1298 A>C and ACE I>D in diabetic cases compared to controls. Diabetic cases
Controls
n (%)
n (%)
MTHFR 677 C>T CC CT TT Genotypic model (CC vs CT vs TT) p=0.0043a Dominant model (CT+TT vs CC) Recessive model (TT vs CT+CC) HWE MTHFR 1298 A>C AA AC CC Genotypic model (AA vs AC vs CC) p=0.0034a Dominant model (AC+CC vs AA) (AA vs AC+CC) Recessive model (CC vs AC+AA) HWE ACE I>D II ID DD Genotypic model (II vs ID vs DD) p=0.0015a Dominant model (ID+DD vs II) Recessive model (DD vs ID+II) (ID+II vs DD) HWE
n=203 111 (54.7) 65 (32.0) 27 (13.3)
n=311 156 (50.2) 135 (43.4) 20 (6.4)
p=0.3, OR=0.8, 95% CI: 0.6–1.2 p=0.01a, OR=2.2, 95% CI: 1.2–4.1 p0.05 n=310 141 (45.5) 140 (45.2) 29 (9.4)
p=0.001a, OR=0.50, 95% CI: 0.4–0.8 p=0.001a, OR=1.84, 95% CI: 1.3–2.6 p=0.2, OR=0.6, 95% CI: 0.3–1.2 p>0.05 n=202 9 (4.5) 130 (64.3) 63 (31.2)
p>0.05 n=238 12 (5.0) 113 (47.5) 113 (47.5)
p=0.9, OR=1.4, 95% CI: 0.5–2.8 p=0.0007b, OR=0.5, 95% CI: 0.3–0.7 p=0.0007b, OR=2.0, 95% CI: 1.3–2.95 pD polymorphisms, probably due to the high frequency of the TT and DD homozygosity (pC and ACE I>D (see Table 2). An older age of the patient and a
younger starting age of diabetes were statistically significant risk factors for all types of diabetes complications. Polymorphic MTHFR 677T allele carriage (TT+CT) contributed significantly to the risk of DR (OR=3.47, pC CC vs AC+AA (recessive) CC+AC vs AA (dominant) ACE I>D DD vs ID+II (recessive) DD+DI vs II (dominant) HbA1c Cholesterol Triglyceride HDL LDL Creatinine HB WBCs Platelets
Neuropathy
p, OR (95% CI) (1.04–1.2)a
Ischemic heart disease
p, OR (95% CI)
p, OR (95% CI)
0.05, 1.0 (0.4–2.4) >0.05, 1.8 (0.7–4.6)
0.05, 1.3 (0.6–3.2) >0.05, 1.7 (0.62–4.5)
0.05, 0.7 (0.3–1.8) >0.05, 0.96 (0.4–2.5)
>0.05, 0.9 (0.3–2.7) 0.05, 2.1 (0.6–7.6) 0.05, 0.6 (0.2–2.1) 0.05, 0.9 (0.2–4.5) 0.05, 3.2 (0.5–20.1) >0.05, 2.2 (0.96–5.3)
>0.05, 2.6 (0.5–13.0) >0.05, 1.3 (0.6–3.1)
>0.05, 2.1 (0.98–4.7) >0.05, 0.7 (0.1–3.9) >0.05, 1.0 (0.7–1.4) >0.05, 0.99 (0.9–1.1) >0.05, 0.99 (0.99–1.0) >0.05, 1.03 (0.96–1.1) >0.05, 1.02 (0.96–1.1) >0.05, 0.99 (0.8–1.3) >0.05, 0.8 (0.6–1.1) >0.05, 1.0 (0.9–1.3) >0.05, 0.99 (0.99–1.0)
0.05, 3.5 (0.4–32.1) >0.05, 1.2 (0.8–1.8) >0.05, 0.99 (0.94–1.1) >0.05, 1.0 (0.99–1.0) >0.05, 1.0 (0.94–1.1) >0.05, 1.0 (0.96–1.1) >0.05, 0.9 (0.7–1.2) >0.05, 0.8 (0.6–1.1) >0.05, 1.0 (0.9–1.3) 0.05, 1.6 (0.7–3.7) >0.05, 2.5 (0.2–26.7) >0.05, 0.9 (0.6–1.2) >0.05, 0.99 (0.9–1.1) >0.05, 1.0 (0.99–1.02) >0.05, 1.0 (0.95–1.1) >0.05, 1.0 (0.95–1.1) >0.05, 1.1 (0.9–1.4) >0.05, 1.2 (0.9–1.5) >0.05, 1.1 (0.9–1.3) >0.05, 0.99 (0.99–1.0)
(1.1–1.3)a
ACE: angiotensin converting enzyme; CI: confidence interval; OR: odds ratio; MTHFR: methylene-tetrahydrofolate reductase. aSignificance level, pD polymorphism among
Japanese diabetic patients32 while, the II genotype of the ACE gene has been found to have a protective effect against the development of DPN in Pakistani9 and Spanish11 T2DM patients. Also, ACE I>D was reported to be not associated with DR among Russians2,33 and among Chinese34,35 diabetic patients. In contrast with our results, MTHFR and ACE polymorphisms were not found to be associated with the pathogenesis or complications of diabetes among Taiwanese3 and Polish36 patients. Also in another study, the MTHFR 677 C (rather than the T) allele was associated with diabetes among Czech women.24 Other researchers have reported a non-association between the MTHFR 677 TT genotype with IHD in Caucasian37, Finnish38 and French39 diabetic patients. Others reported a non-association of the MTHFR 677 TT genotype with DR in Euro-Brazilian,40 Brazilian,41 Turkish4 and Japanese42 diabetic patients. Also the, MTHFR 677 T allele was reported to be not associated with DPN among Turkish diabetics.4 On the other hand, the DD variant of the ACE gene polymorphism was reported to be associated with increased IHD in Chinese,10,34,43 Russian33 and Slovak44 patients with T2DM. The ACE DD variant was also reported to be associated with DR in the Chinese population.5
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Settin et al. The diverse genetic association results shown in this study might point to the need for specific or ethnicitybased genomic markers and also a wider-scale genomic microarray analysis for possible gene-gene interactions. Other minor factors contributing to the apparently controversial results include the potential lack of precision due to the relatively small sample size, poorly defined inclusion and exclusion criteria, and environmental interactions. Although this work might also suffer from some of these limitations, it has clearly confirmed the association of MTHFR 677 C>T and 1298 A>C and ACE I>D polymorphisms with susceptibility to T2DM and its micro-vascular complications among Egyptian patients. Acknowledgements The authors are very grateful to the team of the Endocrinology and Diabetes Department, Internal Medicine Specialized Hospital, Mansoura University, Egypt for their great help and support throughout this study.
Conflicts of interest The authors declare that this work was absolutely free from all issues related to conflicts of interest.
Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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is frequent in type 2 diabetic patients with normal fasting homocysteine levels. J Intern Med 2005; 257: 446–453. 24. Benes P, Kanková K, Muzík J, et al. Methylenetetrahydrofolate reductase polymorphism, type II diabetes mellitus, coronary artery disease, and essential hypertension in the Czech population. Mol Genet Metab 2001; 73: 188–195. 25. Sun J, Xu Y, Xue J, et al. Methylenetetrahydrofolate reductase polymorphism associated with susceptibility to coronary heart disease in Chinese type 2 diabetic patients. Mol Cell Endocrinol 2005; 229: 95–101. 26. Niu W and Qi Y. An updated meta-analysis of methylenetetrahydrofolate reductase gene 677C/T polymorphism with diabetic nephropathy and diabetic retinopathy. Diabetes Res Clin Pract 2012; 95: 110–118. 27. Hu S, Gan P, Li J, et al. [The relationship between the mutation of methylenetetrahydrofolate reductase gene 677C–>T and the diabetic microangiopathy]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2001;18: 118–121. 28. Sun J, Xu Y, Zhu Y, et al. The relationship between MTHFR gene polymorphisms, plasma homocysteine levels and diabetic retinopathy in type 2 diabetes mellitus. Chin Med J (Engl) 2003; 116: 145–147. 29. Maeda M, Yamamoto I, Fukuda M, et al. MTHFR gene polymorphism as a risk factor for diabetic retinopathy in type 2 diabetic patients without serum creatinine elevation. Diabetes Care 2003; 26: 547–548. 30. Maeda M, Fujio Y and Azuma J. MTHFR gene polymorphism and diabetic retinopathy. Curr Diabetes Rev 2006; 2: 467–476. 31. Maeda M, Yamamoto I, Fukuda M, et al. MTHFR gene polymorphism is susceptible to diabetic retinopathy but not to diabetic nephropathy in Japanese type 2 diabetic patients. J Diabetes Complications 2008; 22: 119–125. 32. Ito H, Tsukui S, Kanda T, et al. Angiotensin-converting enzyme insertion/deletion polymorphism and polyneuropathy in type 2 diabetes without macroalbuminuria. J Int Med Res 2002; 30: 476–482. 33. Chistiakov DA, Demurov LM, Kondrat’ev IaIu, et al. [Polymorphism of the angiotensin I-converting enzyme gene in diabetic retinopathy patients and type 2 diabetes mellitus patients with myocardial infarction]. Genetika. 1998; 34(12): 1699–703. 34. Wu SS, Guo QM, Liu GL, et al. [The relationship of angiotensin I-converting enzyme gene polymorphism with
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diabetic retinopathy and diabetes myocardial infarction]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2004; 21: 283–285. Liao L, Lei MX, Chen HL, et al. [Angiotensin converting enzyme gene polymorphism and type 2 diabetic retinopathy]. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2004; 29: 410–413. Moleda P, Majkowska L, Safranow K, et al. [Relationship between I/D polymorphism of angiotensin I converting enzyme gene and microvascular complications in type 2 diabetic patients]. Przegl Lek 2007; 64: 134–139. Böger CA, Stubanus M, Haak T, et al. Effect of MTHFR 677 C>T genotype on survival in type 2 diabetes patients with end-stage diabetic nephropathy. Nephrol Dial Transplant 2007; 22: 154–162. Wirta V, Huang XH, Wirta O, et al. Mutation 677 C>T of methylenetetrahydrofolate reductase gene is not associated with coronary artery disease, but possibly with albuminuria, in type 2 diabetic patients. Clin Chem Lab Med 1998; 36: 625–628. Brulhart MC, Dussoix P, Ruiz J, et al. The (Ala-Val) mutation of methylenetetrahydrofolate reductase as a genetic risk factor for vascular disease in non-insulin-dependent diabetic patients. Am J Hum Genet 1997; 60: 228–229. Santos KG, Tschiedel B, Schneider J, et al. Diabetic retinopathy in Euro-Brazilian type 2 diabetic patients: Relationship with polymorphisms in the aldose reductase, the plasminogen activator inhibitor-1 and the methylenetetrahydrofolate reductase genes. Diabetes Res Clin Pract 2003; 61: 133–136. Errera FI, Silva ME, Yeh E, et al. Effect of polymorphisms of the MTHFR and APOE genes on susceptibility to diabetes and severity of diabetic retinopathy in Brazilian patients. Braz J Med Biol Res 2006; 39: 883–888. Yoshioka K, Yoshida T, Takakura Y, et al. No association between the MTHFR gene polymorphism and diabetic retinopathy in type 2 diabetic patients without overt nephropathy. Diabetes Care 2003; 26: 1947–1948; author reply 1948. Xiang K, Zheng T, Sun D, et al. [The relationship between angiotensin II type 1 receptor gene and coronary heart disease, hypertension and diabetes mellitus in Chinese]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 1998; 15: 9–12. Mosorjáková D, Kyslanová L, Kozárová M, et al. [Relation of polymorphism of genes controlling endothelial function and blood pressure and the occurrence of vascular complications in type 2 diabetes]. Vnitr Lek 2002; 48: 749–754.
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JRA0010.1177/1470320313516172Journal of the Renin-Angiotensin-Aldosterone System 0(0)Settin et al.
Original Article
Association of ACE and MTHFR genetic polymorphisms with type 2 diabetes mellitus: Susceptibility and complications
Journal of the Renin-AngiotensinAldosterone System 1–6 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1470320313516172 jra.sagepub.com
Ahmad Settin1, Rizk El-Baz1, Azza Ismaeel2, Wafaa Tolba2 and Wafaa A Allah2
Abstract Hypothesis/introduction: Polymorphisms of angiotensin converting enzyme (ACE) and methylene-tetrahydrofolate reductase (MTHFR) genes have been proposed to be associated with type 2 diabetes mellitus (T2DM) with conflicting results. This work was planned in order to check for the association of these polymorphisms with the susceptibility for and complications of T2DM among Egyptian cases. Materials and methods: This is a case controlled study involving 203 patients with T2DM and 311 healthy controls. Polymorphic variants of ACE I>D and MTHFR (677 C>T and 1298 A>C) were determined using the polymerase chain reaction (PCR) restriction analysis technique. Results: The susceptibility to T2DM was higher among subjects having the MTHFR 677TT (odds ratio (OR)=2.2, p=0.01), MTHFR 1298 AA (OR=1.84, p=0.001) and ACE (ID+II) (OR=2.0, p=0.0007) genotypes. Logistic regression analysis showed that MTHFR 677T allele was a risk factor for diabetic retinopathy (DR) (OR=3.47, pC genes with the susceptibility to T2DM and its complications including DR, DPN and IHD among Egyptian cases.
1Genetics 2Zoology
Unit, Mansoura University, Egypt Department, Mansoura University, Egypt
Corresponding author: Ahmad Settin, Qassim University, BO 6655 Buraydah 51452, Saudi Arabia. Email: [email protected]
Downloaded from jra.sagepub.com by guest on November 14, 2015
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Journal of the Renin-Angiotensin-Aldosterone System
Subjects and method This study was a case-controlled study involving 202 cases with T2DM in addition to 311 healthy controls. Cases were recruited from the Diabetes and Endocrinology Departments, Internal Medicine Specialized Hospital, Mansoura University, Egypt. The T2DM cases included 84 (41.6%) males and 118 (58.4%) females with age mean±standard deviation (SD) of 55.48±8.09 years. Diabetes diagnosis was based upon World health Organization (WHO) criteria with a fasting plasma glucose ≥7.0 mmol/l.17 Inclusion criteria were cases with Egyptian origin, age at onset of diabetes >35 years, diabetes duration of ≥10 years, C-peptide ≥0.3 nmol/l and glutamic acid decarboxylase (GAD) antibody negativity. Of these cases, 67 (33.2%) were complicated with DR, 93 (46%) with DPN and 47 (23.3%) with IHD. These cases were compared to 311 healthy non-diabetic unrelated controls from the same locality. They were 129 males and 182 females with age mean±SD of 51.17±1.07 years. Before the start of the study, ethical approval was obtained from the scientific and ethical committees of the local health authorities in addition to informed consent that was obtained from all the participants.
a preliminary denaturation at 95ºC for 3 min, 5 cycles: denaturation at 94ºC for 1 min, annealing at 64ºC for 1 min, synthesis at 72ºC for 30 s, next 30 cycles were run as: denaturation at 94ºC for 45 s, annealing at 62ºC for 45 s, synthesis at 72ºC for 25 s and final synthesis at 72ºC for 7 min. Digestion was performed by the enzyme HinfI (Fermentas, UK) at 37ºC for four hours. For evaluation of the other MTHFR 1298 A>C polymorphism, the following primers were used: Forward: (5`-CTT TGG GGA GGT GAA GGA CTA CTA C-3`) and Reverse: (5`-CAC TTT GTG AGC ATT CCG GTT TG-3`) (Gibeo BRL). The PCR procedure was composed of preliminary denaturation at 95ºC for 2 min, 5 cycles: denaturation at 95ºC for 1 min, annealing at 55ºC for 2 min, synthesis at 72ºC for 2 min, next 32 cycles were run: denaturation at 95ºC for 75 s, annealing at 55ºC for 75 s, synthesis at 72ºC for 90 s and final synthesis at 72ºC for 6 min. Restrictase MboII (Fermentas, UK) was used for digestion at 37ºC for seven hours. Each final reaction volume plus 5 µl of bromophenol blue track dye were loaded into 2% agarose gel (Boehringer Mannheim, Germany) containing ethidium bromide. Gels were electrophoresced for 30 min at 100 V, photographed under UV light (320 nm) and then scored for the presence or absence of an allele specific bands.
DNA extraction, amplification and digestion
Statistical analysis
DNA was extracted and purified using a the kit of Gentra Systems, USA according to the manufacturer’s instructions. Characterization of the ACE I/D polymorphism was done using PCR amplification of the respective fragments from intron 16 of the ACE gene according to the method described previously.18 An optimized primer pair was used to amplify the D and I alleles (5’-GCC CTG CAG GTG TCT GCA GCA TGT-3’ and 5’-GGA TGG CTC TCC CCG CCT TGT CTC-3’) resulting in 319-bp and 597-bp amplicons respectively. The cycling profile consisted of denaturation at 94ºC for 30 s, annealing at 56ºC for 45 s, and synthesis at 72ºC for 2 min, repeated for 35 cycles, followed by a final synthesis at 72ºC for 7 min. Because the D allele in heterozygous samples is preferentially amplified, each sample found to have the DD genotype was subjected to a second, independent PCR amplification with a primer pair that recognizes an insertion-specific sequence (5’-TGG GAC CAC AGC GCC CGC CAC TAC-3’ and 5’-TCG CCA GCC CTC CCA TGC CCA TAA-3’), with identical PCR conditions except for an annealing temperature of 67ºC. The reaction yields a 335 bp amplicon only in the presence of an I allele, and no product in samples was homozygous for DD. MTHFR 677 C>T and 1298 A>C genotypes were characterized using the PCR-RFLP technique described previously.19 The primer sequences for 677 C>T genotypes were: Forward: (5`-TGA AGG AGA AGG TGT CTG CGG GA-3`) and Reverse: (5`-AGG ACG GTG CGG TGA GAG TG-3`) (Gibeo BRL). PCR protocol was in the form of
Data were processed and analyzed using the Statistical Package of Social Science (SPSS, version 17.0). Frequencies of studied genotypic and allelic polymorphisms among cases were compared to those of controls and tested for positive association using chi-square (χ2) test or Fisher exact tests and odds ratio (OR) with a 95% confidence interval (CI). The distribution of alleles and genotypes in the studied groups was tested for fitting to the Hardy-Weinberg equilibrium through comparing the observed and expected frequencies of genetic variants using the χ2 test. Binary logistic regression analysis was done to assess the genetic and demographic risk factors contributing to each type of diabetic complication. A minimum level of significance was considered if p was T, MTHFR 1298 A>C and ACE I>D in diabetic cases compared to controls. Diabetic cases
Controls
n (%)
n (%)
MTHFR 677 C>T CC CT TT Genotypic model (CC vs CT vs TT) p=0.0043a Dominant model (CT+TT vs CC) Recessive model (TT vs CT+CC) HWE MTHFR 1298 A>C AA AC CC Genotypic model (AA vs AC vs CC) p=0.0034a Dominant model (AC+CC vs AA) (AA vs AC+CC) Recessive model (CC vs AC+AA) HWE ACE I>D II ID DD Genotypic model (II vs ID vs DD) p=0.0015a Dominant model (ID+DD vs II) Recessive model (DD vs ID+II) (ID+II vs DD) HWE
n=203 111 (54.7) 65 (32.0) 27 (13.3)
n=311 156 (50.2) 135 (43.4) 20 (6.4)
p=0.3, OR=0.8, 95% CI: 0.6–1.2 p=0.01a, OR=2.2, 95% CI: 1.2–4.1 p0.05 n=310 141 (45.5) 140 (45.2) 29 (9.4)
p=0.001a, OR=0.50, 95% CI: 0.4–0.8 p=0.001a, OR=1.84, 95% CI: 1.3–2.6 p=0.2, OR=0.6, 95% CI: 0.3–1.2 p>0.05 n=202 9 (4.5) 130 (64.3) 63 (31.2)
p>0.05 n=238 12 (5.0) 113 (47.5) 113 (47.5)
p=0.9, OR=1.4, 95% CI: 0.5–2.8 p=0.0007b, OR=0.5, 95% CI: 0.3–0.7 p=0.0007b, OR=2.0, 95% CI: 1.3–2.95 pD polymorphisms, probably due to the high frequency of the TT and DD homozygosity (pC and ACE I>D (see Table 2). An older age of the patient and a
younger starting age of diabetes were statistically significant risk factors for all types of diabetes complications. Polymorphic MTHFR 677T allele carriage (TT+CT) contributed significantly to the risk of DR (OR=3.47, pC CC vs AC+AA (recessive) CC+AC vs AA (dominant) ACE I>D DD vs ID+II (recessive) DD+DI vs II (dominant) HbA1c Cholesterol Triglyceride HDL LDL Creatinine HB WBCs Platelets
Neuropathy
p, OR (95% CI) (1.04–1.2)a
Ischemic heart disease
p, OR (95% CI)
p, OR (95% CI)
0.05, 1.0 (0.4–2.4) >0.05, 1.8 (0.7–4.6)
0.05, 1.3 (0.6–3.2) >0.05, 1.7 (0.62–4.5)
0.05, 0.7 (0.3–1.8) >0.05, 0.96 (0.4–2.5)
>0.05, 0.9 (0.3–2.7) 0.05, 2.1 (0.6–7.6) 0.05, 0.6 (0.2–2.1) 0.05, 0.9 (0.2–4.5) 0.05, 3.2 (0.5–20.1) >0.05, 2.2 (0.96–5.3)
>0.05, 2.6 (0.5–13.0) >0.05, 1.3 (0.6–3.1)
>0.05, 2.1 (0.98–4.7) >0.05, 0.7 (0.1–3.9) >0.05, 1.0 (0.7–1.4) >0.05, 0.99 (0.9–1.1) >0.05, 0.99 (0.99–1.0) >0.05, 1.03 (0.96–1.1) >0.05, 1.02 (0.96–1.1) >0.05, 0.99 (0.8–1.3) >0.05, 0.8 (0.6–1.1) >0.05, 1.0 (0.9–1.3) >0.05, 0.99 (0.99–1.0)
0.05, 3.5 (0.4–32.1) >0.05, 1.2 (0.8–1.8) >0.05, 0.99 (0.94–1.1) >0.05, 1.0 (0.99–1.0) >0.05, 1.0 (0.94–1.1) >0.05, 1.0 (0.96–1.1) >0.05, 0.9 (0.7–1.2) >0.05, 0.8 (0.6–1.1) >0.05, 1.0 (0.9–1.3) 0.05, 1.6 (0.7–3.7) >0.05, 2.5 (0.2–26.7) >0.05, 0.9 (0.6–1.2) >0.05, 0.99 (0.9–1.1) >0.05, 1.0 (0.99–1.02) >0.05, 1.0 (0.95–1.1) >0.05, 1.0 (0.95–1.1) >0.05, 1.1 (0.9–1.4) >0.05, 1.2 (0.9–1.5) >0.05, 1.1 (0.9–1.3) >0.05, 0.99 (0.99–1.0)
(1.1–1.3)a
ACE: angiotensin converting enzyme; CI: confidence interval; OR: odds ratio; MTHFR: methylene-tetrahydrofolate reductase. aSignificance level, pD polymorphism among
Japanese diabetic patients32 while, the II genotype of the ACE gene has been found to have a protective effect against the development of DPN in Pakistani9 and Spanish11 T2DM patients. Also, ACE I>D was reported to be not associated with DR among Russians2,33 and among Chinese34,35 diabetic patients. In contrast with our results, MTHFR and ACE polymorphisms were not found to be associated with the pathogenesis or complications of diabetes among Taiwanese3 and Polish36 patients. Also in another study, the MTHFR 677 C (rather than the T) allele was associated with diabetes among Czech women.24 Other researchers have reported a non-association between the MTHFR 677 TT genotype with IHD in Caucasian37, Finnish38 and French39 diabetic patients. Others reported a non-association of the MTHFR 677 TT genotype with DR in Euro-Brazilian,40 Brazilian,41 Turkish4 and Japanese42 diabetic patients. Also the, MTHFR 677 T allele was reported to be not associated with DPN among Turkish diabetics.4 On the other hand, the DD variant of the ACE gene polymorphism was reported to be associated with increased IHD in Chinese,10,34,43 Russian33 and Slovak44 patients with T2DM. The ACE DD variant was also reported to be associated with DR in the Chinese population.5
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Settin et al. The diverse genetic association results shown in this study might point to the need for specific or ethnicitybased genomic markers and also a wider-scale genomic microarray analysis for possible gene-gene interactions. Other minor factors contributing to the apparently controversial results include the potential lack of precision due to the relatively small sample size, poorly defined inclusion and exclusion criteria, and environmental interactions. Although this work might also suffer from some of these limitations, it has clearly confirmed the association of MTHFR 677 C>T and 1298 A>C and ACE I>D polymorphisms with susceptibility to T2DM and its micro-vascular complications among Egyptian patients. Acknowledgements The authors are very grateful to the team of the Endocrinology and Diabetes Department, Internal Medicine Specialized Hospital, Mansoura University, Egypt for their great help and support throughout this study.
Conflicts of interest The authors declare that this work was absolutely free from all issues related to conflicts of interest.
Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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