Association of ACE and MTHFR genetic polymorphisms with type 2 diabetes mellitus: Susceptibility and complications

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, p<0.001), diabetic polyneuropathy (DPN) (OR=5.2, p<0.0001) and ischemic heart disease (IHD) (OR=2.9, p<0.05), while MTHFR 1298 C allele was a risk factor for DR (OR=4.2, p<0.001) and the ACE DD genotype was a risk factor for DPN (OR=3.1, p<0.001).

Conclusions:

The MTHFR 677 TT genotype was associated with T2DM susceptibility and complications (DR, DPN and IHD). The MTHFR 1298 CC, AC and ACE DD genotypes were associated with DR and DPN.

Keywords

type 2 diabetes diabetic neuropathy diabetic retinopathy

Introduction

Genetic polymorphisms related to the genes for angiotensin converting enzyme (ACE) and methylene-tetrahydrofolate reductase (MTHFR) were studied in terms of giving predisposition to the development of vascular complications of diabetes, nonetheless, with conflicting results.^1–5 It is known that ACE and angiotensinogen play an important role in blood pressure and blood volume homeostasis.⁶ Concentrations of plasma and tissue ACE are determined by the ACE gene which is located on chromosome 17q23. This gene manifests a 287-bp repeated Alu sequence insertion (I) or deletion (D) polymorphism in intron 16.⁷ The homozygous DD genotype, which is associated with a two- to threefold increase in levels of ACE, may cause a variety of adverse microvascular effects as DR, DPN and IHD that would modulate the course of T2DM.^5,8
–11

MTHFR is a key regulatory enzyme in folate and homocysteine metabolism. Deficiency of MTHFR may be associated with an increase in plasma homocysteine, which in turn is associated with an increased risk of vascular diseases among diabetic patients.^12
–14 The mutant polymorphisms include two variants; 677 C>T and 1298 A>C that were shown to affect the enzymatic activity of MTHFR.^15,16 The aim of this study was to check for the association of polymorphisms related to ACE I>D and MTHFR 677 C>T and 1298 A>C genes with the susceptibility to T2DM and its complications including DR, DPN and IHD among Egyptian cases.

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.

DNA extraction, amplification and digestion

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 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.

Statistical analysis

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 <0.05.

Results

Egyptian cases with T2DM showed a significantly higher frequency of the mutant homozygous MTHFR 677TT genotype compared to controls i.e. conforming to the recessive model (13.3% vs 6.4%, OR=2.2, p=0.01) with a significantly higher frequency of MTHFR 1298AA genotype (60.6% vs 45.5%, OR=1.84, p=0.001) and higher frequency of ACE I allele carriage (68.8% vs 52.5%, OR=2.0, p=0.0007) (see Table 1). This means that cases had a significantly lower frequency of the MTHFR 1298 C allele carriers including the AC+CC genotypes (39.4% vs 54.6%, OR=0.5, p=0.001) and also a lower frequency of the ACE DD homozygous variant (31.2% vs 47.5%, OR=0.5, p=0.0007). Testing for genetic equilibrium among controls showed that the distribution of frequencies of polymorphic variants of MTHFR 677 C>T and MTHFR 1298 A>C conformed with the Hardy-Weinberg Equilibrium, whereas those of the ACE I>D were not (p<0.05). On the other hand, cases showed non-conformity with the Hardy-Weinberg equilibrium for both the MTHFR 677 C>T and ACE I>D polymorphisms, probably due to the high frequency of the TT and DD homozygosity (p<0.05 and p<0.001, respectively).

Table 1.

Frequencies of genetic polymorphic variants of MTHFR 677 C>T, MTHFR 1298 A>C and ACE I>D in diabetic cases compared to controls.

	Diabetic cases	Controls
	n (%)	n (%)
MTHFR 677 C>T	n=203	n=311
CC	111 (54.7)	156 (50.2)
CT	65 (32.0)	135 (43.4)
TT	27 (13.3)	20 (6.4)
Genotypic model (CC vs CT vs TT) p=0.0043^a
Dominant model (CT+TT vs CC)	p=0.3, OR=0.8, 95% CI: 0.6–1.2
Recessive model (TT vs CT+CC)	p=0.01^a, OR=2.2, 95% CI: 1.2–4.1
HWE	p<0.05^a	p>0.05
MTHFR 1298 A>C	n=203	n=310
AA	123 (60.6)	141 (45.5)
AC	68 (33.5)	140 (45.2)
CC	12 (5.9)	29 (9.4)
Genotypic model (AA vs AC vs CC) p=0.0034^a
Dominant model (AC+CC vs AA)	p=0.001^a, OR=0.50, 95% CI: 0.4–0.8
(AA vs AC+CC)	p=0.001^a, OR=1.84, 95% CI: 1.3–2.6
Recessive model (CC vs AC+AA)	p=0.2, OR=0.6, 95% CI: 0.3–1.2
HWE	p>0.05	p>0.05
ACE I>D	n=202	n=238
II	9 (4.5)	12 (5.0)
ID	130 (64.3)	113 (47.5)
DD	63 (31.2)	113 (47.5)
Genotypic model (II vs ID vs DD) p=0.0015^a
Dominant model (ID+DD vs II)	p=0.9, OR=1.4, 95% CI: 0.5–2.8
Recessive model (DD vs ID+II)	p=0.0007^b, OR=0.5, 95% CI: 0.3–0.7
(ID+II vs DD)	p=0.0007^b, OR=2.0, 95% CI: 1.3–2.95
HWE	p<0.001^b	p<0.05^a

ACE: angiotensin converting enzyme; HWE: Hardy-Weinberg equilibrium; MTHFR: methylene-tetrahydrofolate reductase.

Significance level, p<0.05; ^bsignificance level, p<0.001.

Logistic regression multivariate analysis was done to assess the potential risk factors contributing to the development of diabetic complications, including the age of the patients, age of onset of diabetes, gender, family history, consanguinity, Hb A₁C level, total cholesterol, triglycerides, high density lipoprotein (HDL), low density lipoprotein (LDL), serum creatinine and hematologic parameters of hemoglobin (HB), white blood cells (WBCs) and platelets as well as genetic polymorphic variants of MTHFR 677 C>T, MTHFR 1298 A>C 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, p<0.001), DPN (OR=5.2, p<0.0001) and IHD (OR=2.9, p<0.05). On the other hand, MTHFR 1298C allele carriage (CC+AC) was shown to contribute to the risk of DR (OR=4.2, p<0.001), while the ACE DD genotype was shown to contribute to the risk of DPN (OR=3.1, p<0.001).

Table 2.

Multivariate logistic regression analysis of the potential risk factors contributing to retinopathy, neuropathy and ischemic heart disease in type 2 diabetes mellitus (T2DM) patients.

	Retinopathy	Neuropathy	Ischemic heart disease
	p, OR (95% CI)	p, OR (95% CI)	p, OR (95% CI)
Age	<0.001, 1.1 (1.04–1.2)^a	<0.0001, 1.2 (1.1–1.3)^a	<0.001, 1.1 (1.0–1.2)^a
Age of onset	<0.0001, 0.9 (0.8–0.95)^a	<0.0001, 0.9 (0.8–0.9)^a	<0.001, 0.9 (0.8–0.96)^a
Male sex	>0.05, 0.6 (0.3–1.4)	>0.05, 0.7 (0.3–1.6)	>0.05, 1.6 (0.7–4.0)
Positive family history	>0.05, 1.0 (0.4–2.4)	>0.05, 1.3 (0.6–3.2)	>0.05, 0.7 (0.3–1.8)
Positive consanguinity	>0.05, 1.8 (0.7–4.6)	>0.05, 1.7 (0.62–4.5)	>0.05, 0.96 (0.4–2.5)
MTHFR 677 C>T
TT vs CT+CC (recessive)	>0.05, 0.9 (0.3–2.7)	>0.05, 2.1 (0.6–7.6)	>0.05, 0.6 (0.2–2.1)
TT+CT vs CC (dominant)	<0.001, 3.5 (1.5–8.2)^a	<0.0001, 5.2 (2.1–12.99)^a	<0.05, 2.9 (1.3–7.0) ^b
MTHFR 1298 A>C
CC vs AC+AA (recessive)	>0.05, 0.9 (0.2–4.5)	>0.05, 3.2 (0.5–20.1)	>0.05, 2.6 (0.5–13.0)
CC+AC vs AA (dominant)	<0.001, 4.2 (1.8–9.4)^a	>0.05, 2.2 (0.96–5.3)	>0.05, 1.3 (0.6–3.1)
ACE I>D
DD vs ID+II (recessive)	>0.05, 2.1 (0.98–4.7)	<0.001, 3.1 (1.3–7.3)^a	>0.05, 1.6 (0.7–3.7)
DD+DI vs II (dominant)	>0.05, 0.7 (0.1–3.9)	>0.05, 3.5 (0.4–32.1)	>0.05, 2.5 (0.2–26.7)
HbA1c	>0.05, 1.0 (0.7–1.4)	>0.05, 1.2 (0.8–1.8)	>0.05, 0.9 (0.6–1.2)
Cholesterol	>0.05, 0.99 (0.9–1.1)	>0.05, 0.99 (0.94–1.1)	>0.05, 0.99 (0.9–1.1)
Triglyceride	>0.05, 0.99 (0.99–1.0)	>0.05, 1.0 (0.99–1.0)	>0.05, 1.0 (0.99–1.02)
HDL	>0.05, 1.03 (0.96–1.1)	>0.05, 1.0 (0.94–1.1)	>0.05, 1.0 (0.95–1.1)
LDL	>0.05, 1.02 (0.96–1.1)	>0.05, 1.0 (0.96–1.1)	>0.05, 1.0 (0.95–1.1)
Creatinine	>0.05, 0.99 (0.8–1.3)	>0.05, 0.9 (0.7–1.2)	>0.05, 1.1 (0.9–1.4)
HB	>0.05, 0.8 (0.6–1.1)	>0.05, 0.8 (0.6–1.1)	>0.05, 1.2 (0.9–1.5)
WBCs	>0.05, 1.0 (0.9–1.3)	>0.05, 1.0 (0.9–1.3)	>0.05, 1.1 (0.9–1.3)
Platelets	>0.05, 0.99 (0.99–1.0)	<0.001, 0.99 (0.98–0.99)^a	>0.05, 0.99 (0.99–1.0)

ACE: angiotensin converting enzyme; CI: confidence interval; OR: odds ratio; MTHFR: methylene-tetrahydrofolate reductase.

Significance level, p<0.001; ^bsignificance level, p<0.05.

Discussion

Various studies have been done concerning the association of genetic polymorphisms with diabetes and diabetic complications in various populations demonstrating controversial results. In a previous study, we have reported the association of MTHFR 677 C>T, 1298 A>C and ACE I>D polymorphisms with the development of diabetic nephropathy whether being presented with micro or macroalbuminuria.²⁰ On the other hand, this study investigated the association of these polymorphisms with the susceptibility and complications of T2DM mainly DR, DPN and IHD. The results of the present study have shown that susceptibility to T2DM in Egyptian cases was associated with the MTHFR 677 TT, MTHFR 1298 AA and ACE II and ID genotypes. Diabetic complications were associated with MTHFR 677 T allele carriage (DR, DPN and IHD), MTHFR 1298 C allele carriage (DR) and ACE DD genotype (DPN). To our knowledge, this study was the first to point to the association of the polymorphism MTHFR 1298 A>C with susceptibility to T2DM and its retinal complication. Similarly, MTHFR 766 T allele carriage was found to be associated with diabetes among Polish,²¹ Indian,²² Turkish,²³ and Chinese Han¹ populations. Also, the ACE I allele and II genotype were reported to be associated with T2DM among Russian cases.² Regarding diabetic complications, in accord with our results, others studies have reported that IHD was associated with the MTHFR 766 T allele among Polish,²¹ Czech²⁴ and Chinese^1,25 diabetics. DR was found to be associated with MTHFR 677 T mutations in a meta-analysis study²⁶ and in other studies among Chinese^27,28 and Japanese^29
–31 cases. Also in agreement with our results, DPN has been reported to be associated with the ACE I>D 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 Russians^2,33 and among Chinese^34,35 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

The diverse genetic association results shown in this study might point to the need for specific or ethnicity-based 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.

Footnotes

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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