Methylenetetrahydrofolate Reductase and Angiotensin-Converting Enzyme Gene Polymorphisms Among Saudi Population from Qassim Region

Abstract

Background: Hypertension and cardiac diseases are multifactorial disorders with genetic background determined by multiple gene polymorphisms. Methods: This work included 273 healthy unrelated subjects ethnically belonging to the Qassim region. Their age ranged from 18 to 60 years, with a median age of 20 years. They included 152 (55.7%) men and 121 (44.3%) women. Their DNAs were analyzed for genetic polymorphisms of the methylenetetrahydrofolate reductase (MTHFR; 677C/T and 1298 A/C) as well as angiotensin-converting enzyme genes (ACE; insertion/deletion [I/D]) using real-time polymerase chain reaction. Results: Carriers of the mutant MTHFR 677 T allele (CT + TT) and that of the 1298 C allele (CC + AC) constituted 33.7% and 48.9% of studied subjects, respectively, whereas carriers of ACE gene mutant D allele (DD + ID) represented 93.3% of subjects. The allele frequencies of MTHFR 677T, 1298C, and ACE D alleles were 18.7%, 29.45%, and 72.5%, respectively. Haplotype analysis of characterized chromosomes revealed that 2.5% were likely to carry the three mutant alleles together, 30.91% were likely to carry two of the three mutant alleles, and 51.92% were likely to carry one mutant allele. Conclusion: The Saudi population from the Qassim region is a carrier of a relatively large number of genetic alleles predisposing them to hypertension and cardiac diseases. This gives a warning to local health authorities for adoption of competent programs for prevention as well as early diagnosis and management.

Introduction

Methylenetetrahydrofolate reductase (MTHFR) enzyme plays a central role in folate metabolism by irreversibly catalyzing the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the primary circulating form of folate and a cosubstrate for homocysteine (Hcy) methylation to methionine. Its involvement in the regulation of Hcy concentration causes it to be a risk factor for cardiovascular disorders (CVDs) (Wald et al., 2002). Additionally, folates provide methyl groups for de novo deoxynucleotide synthesis and for intracellular methylation reactions (Rozen, 1997). Thus, a high dietary folate intake reduces risk of carcinogenesis by limiting aberrant DNA methylation especially in situations of impaired folate status. Lastly, folic acid seems to have a preventive effect against neural tube defects and other congenital anomalies in pregnant women (Charles et al., 2005).

In the MTHFR gene located in chromosome 1, a common single nucleotide polymorphism is associated with reduced enzyme activity: C → T in exon 4 at nucleotide 677, leading to Ala222Val, with the T allele frequency being around 44% in Caucasians (Botto and Yang, 2000); and A → C in exon 7 at nucleotide 1298, leading to Glu429Ala, with the C allele frequency around 20% in Caucasians (van der Put et al., 1998). Individuals who are homozygous for the MTHFR 677 less frequent variant (TT) have 30% of the expected enzyme activity in vitro, compared with those who are homozygous for the common variant (CC), with a reduced folate status and higher serum Hcy levels, whereas heterozygous carriers have 65% activity (Rozen, 1997). The TT genotype of the 677C/T is therefore likely to be associated with essential hypertension (EH) and coronary artery disease (CAD). On the other hand, individuals carrying the 677CC together with the 1298CC genotypes have 60% activity compared with subjects carrying the 1298AA variant (van der Put et al., 1998).

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) is a chloride- and zinc-dependent dipeptidyl carboxypeptidase ubiquitously existing in mammalian species. As a bioactive component of the renin-angiotensin system and the kallikrein-kinin system, ACE plays a significant role in blood pressure regulation, fluid and electrolyte balancing, cardiovascular system development, and vascular remodeling by hydrolyzing angiotensin I into a potent vasopressor peptide angiotensin II and deactivating the vasodepressor peptide bradykinin (Zhao and Xu, 2008).

ACE gene has been mapped on chromosome 17. The insertion/deletion (I/D) polymorphism of the ACE gene was characterized by the presence or absence of a 278-bp Alu repetitive sequence in intron 16. This polymorphism accounts for a large proportion of serum ACE activity variability with the DD genotype having the highest and the II genotype having the lowest ACE activity (Rigat et al., 1990). ACE polymorphism is reported to be associated with hypertension, CVDs, left ventricle hypertrophy, myocardial infarction, and diabetes (Morshed et al., 2002).

Hypertension is a well-known prevalent disorder among the Saudi population especially in the Qassim region (Soyannwo et al., 1997, 1998). This is a tribal region characteristically having high consanguinity and high aggregation rates for familial diseases. Therefore, this work has been planned to assess the frequency of MTHFR and ACE gene polymorphisms as potential genetic risk factors for hypertension and CVDs among this population.

Subjects and Methods

This work included 273 healthy unrelated subjects ethnically belonging to the Qassim region. They were selected from those visiting the Qassim University-affiliated clinics for routine checkups. Their age ranged from 18 to 60 years, with a mean age of 25.3 years and a median age of 20 years. They included 152 (55.7%) men and 121 (44.3%) women. Informed consents were obtained from all subjects in addition to an authorized approval from the scientific and ethical committees of the Qassim University. For all subjects, genomic DNA was extracted from 1 mL peripheral blood using the MagNA Pure LV blood reagent set (Roche Diagnostics, Riyadh, Saudi Arabia), according to the manufacturer's instruction. Real-time polymerase chain reaction amplifications for C677T, A1298C gene polymorphisms of MTHFR and I/D polymorphism of ACE genes were done using Light Cycler apparatus (Roche Diagnostics). An amplification mix including specific primers and probes (Light Mix, TIB MOLBIOL, and Light Cycler Fast Start DNA Master hybridization probes) was used in an optimized polymerase chain reaction under conditions recommended by the manufacturer (Roche Diagnostics).

Results

Genotype and allele frequencies of the studied gene polymorphisms related to median age, sex, and median blood pressure tested by Hardy-Weinberg law of genetic equilibrium are shown in Table 1. The mutant MTHFR 677T, 1298C and ACE D allele frequencies were 18.7%, 29.45%, and 72.5%, respectively, of examined chromosomes. MTHFR 677 T allele carriers represented 33.7% of examined subjects (3.7% being TT homozygotes and 30% being CT heterozygotes), whereas the 1298C allele carriers were 48.9% of them (10% being CC homozygotes and 38.9% being AC heterozygotes). On the other hand, ACE D allele carriers represented 93.3% of examined subjects (51.7% being DD homozygotes and 41.6% being ID heterozygotes).

Table 1.

Genotype and Allele Frequencies of the Studied Gene Polymorphisms Related to Median Age, Sex, and Median Blood Pressure Tested by Hardy-Weinberg Law of Genetic Equilibrium

Genotypes	n (%)	Median age (years)	Sex (M/F)	Median blood pressure	HWE χ² (p)
MTHFR 677C/T (n = 270)^a
CC	179 (66.3)	26	101/78	113/73	0.0016 (p > 0.05)
CT	81 (30)	25	42/39	115/72	0.0148 (p > 0.05)
TT	10 (3.7)	29.5	6/4	118/76	0.0311 (p > 0.05)
C allele	439/540 (81.3)
T allele	101/540 (18.7)
MTHFR 1298A/C (n = 270)^a
AA	138 (51.1)	25	82/56	116/75	0.122 (p > 0.05)
AC	105 (38.9)	28	49/56	115/73	0.438 (p > 0.05)
CC	27 (10)	29.5	18/9	118/73	0.499 (p > 0.05)
A allele	381/540 (70.56)
C allele	159/540 (29.44)
ACE D/I (n = 269)^a
DD	139 (51.7)	24	66/73	115/72	0.041 (p > 0.05)
ID	112 (41.6)	28	69/43	115/75	0.200 (p > 0.05)
II	18 (6.7)	25.5	13/5	114/76	0.305 (p > 0.05)
D allele	390/538 (72.5)
I allele	148/538 (27.5)

Some of the cases were omitted as being not accurately genotyped.

HWE χ² (p), Hardy-Weinberg test; MTHFR, methylenetetrahydrofolate reductase; ACE, angiotensin-converting enzyme.

Analysis of chromosomal haplotypes using the maximum likelihood method (Table 2) revealed that 2.53% of the characterized chromosomes were likely to carry the three mutant alleles together. On the other hand, two mutant alleles were carried by 30.91% of chromosomes and only one mutant allele was carried by 51.92%, whereas the rest (14.69%) had only normal alleles.

Table 2.

Haplotype Frequencies of the Studied Gene Polymorphisms Among Saudi Subjects

Subject haplotypes	Number (%)
Total	524^a
Haplotypes with three mutant alleles (n = 13, 2.48%)
MTHFR_677T/1298C/ACE_D	13 (2.48%)
Haplotypes with two mutant alleles (n = 162, 30.91%)
MTHFR_677C/1298C/ACE_D	96 (18.32%)
MTHFR_677T/1298C/ACE_I	5 (0.95%)
MTHFR_677T/1298A/ACE_D	61 (11.64%)
Haplotypes with one mutant allele (n = 272, 51.92%)
MTHFR_677T/1298A/ACE_I	20 (3.82%)
MTHFR_677C/1298C/ACE_I	42 (8.02%)
MTHFR_677C/1298A/ACE_D	210 (40.08%)
Haplotypes with normal alleles (n = 77, 14.69%)
MTHFR_677C/1298A/ACE_I	77 (14.69%)

Total number corresponds to the number of characterized chromosomes.

Discussion

Polymorphisms in genes involved in the Hcy pathway (MTHFR) and in ACE are not only associated with EH and CAD but also can modify the efficacy of therapy in reducing the risk of cardiovascular events (Ilhan et al., 2008; Maitland-van der Zee et al., 2008; Ramachandran et al., 2008).

The Saudi population particularly in the Qassim region has been reported to be affected by hypertension and CADs (Soyannwo et al., 1997, 1998; Al-Sheikh et al., 2007). So far, little data were published concerning the genetic background of Saudi subjects in terms of their susceptibility to hypertension and CADs. To our knowledge, this is the first report of analysis of three interactive gene polymorphisms that have a probable role in the susceptibility for EH and CAD among the Saudi population.

This study showed that the mutant allele MTHFR 677T frequency (18.7%) in the studied subjects from the Qassim region was higher than that reported previously in the Riadh region (14.8%) (Mammo et al., 2007), Bahrain (12.6%) (Ameen et al., 2005), and Jordan (16%) (Eid and Rihani, 2004). However, it is much lower than that reported in other Middle Eastern and Mediterranean countries such as Iran (32.5%) (Rahimi et al., 2008), Turkey (27.8%) (Sazci et al., 2005), Lebanon (30.4%) (Ameen et al., 2005), Tunisia (29.2%) (Ameen et al., 2005), Morocco (28.9%) (Guéant et al., 2007), Greece (35%), and Italy (45%) (Antoniadi et al., 1999, Margaglione et al., 1999, Angelopoulou et al., 2000; Pallaud et al., 2001).

This study showed also that the mutant allele MTHFR 1298C frequency in the studied subjects from the Qassim region (29.45%) was lower than that reported in Bahrain (34%) (Almawi et al., 2004), Lebanon (49%) (Shaw et al., 1999), Turkey (33.1%) (Sazci et al., 2005), and among Jews (34%) (Friedman et al., 1999). However, it was higher than was reported in other Mediterranean countries such as Italy (25%) (De Marco et al., 2002; Chiusolo et al., 2004), and Asian countries such as Japan (16%) and India (10%) (Markan et al., 2007).

Regarding the ACE gene polymorphism, this study showed that the ACE DD genotype frequency in the Qassim region (51.7%) was higher than that reported in other Mediterranean and Asian countries such as Egypt (48.9%) (Ulu et al., 2006), Turkey (27.5%) (Eroglu et al., 2008), France (30.6%) (Marre et al., 1994), Japan (29.03%) (Mizuiri et al., 1995), and Korea (25%) (Oh et al., 1996).

Analysis of chromosomal haplotypes revealed that the three studied mutant alleles were likely to be carried by 2.5% of the characterized chromosomes, whereas two mutant alleles were carried by 30.91%. This actually represents a substantial risk for the development of vascular disorders among Saudi subjects, particularly when integrated with other risk factors such as smoking, hyperlipedimia, and diabetes.

From these results, we can conclude that the Saudi population from the Qassim region is a carrier of a relatively high amount of genetic alleles predisposing them to hypertension and cardiac diseases. We recommend adoption of competent programs for prevention and/or early detection of CVDs. In this respect we suggest screening for serum Hcy levels. We also recommend raising awareness for folate supplementation either from dietary sources or from medications especially to women during pregnancy. In addition, these polymorphisms can be used as markers for the susceptibility to CVDs among affected families which should be counseled to avoid other interactive environmental risk factors.

Footnotes

Disclosure Statement

No competing financial interests exist.

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