Association of MTHFR C677T and A1298C gene polymorphisms with hypertension.

OBJECTIVES
To check for the association of genetic polymorphisms related to the methylenetetrahydrofolate reductase (MTHFR) gene namely C677T and A1298C with hypertension in Saudi affected subjects from Qassim region.


SUBJECTS AND METHODS
Participants included 123 Saudi hypertensive cases (83 males and 40 females) in addition to 250 (142 males and 108 females) unrelated healthy controls from the same locality. Their age mean ±SD was 50.93 ± 15.43 years. For all subjects, DNA was extracted followed by real-time PCR amplifications for characterization of genotypes and alleles related to MTHFR C677T and A1298C gene polymorphisms


RESULTS
Total cases showed significantly higher carriage rate for the mutant allele 677T compared to controls (40.7% vs. 26%, OR=1.9, 95% CI= 1.2-3.1) with a lower frequency of the wild type 677CC genotype (59.3% vs. 74%, p=0.004). The same was observed among cases-subgroups of hypertension associated with obesity with a notably higher odds ratio (OR=2.6, 95% CI=1.3-5.01, p=0.004). Total cases showed also significantly higher frequency of mutant 1298 C allele carriage rate compared to controls (59.3% vs. 42.4%, OR=1.98, 95% CI= 1.3-3.1) with a lower frequency of the normal AA genotype (40.7% vs. 57.6%, p=0.003). The same was observed among cases-subgroups of hypertension associated with both diabetes and obesity and among cases of hypertension with obesity, also with higher odds ratio (OR=2.6 and 2.2 respectively).


CONCLUSION
This work showed that genetic polymorphisms related to the MTHFR gene are associated with the risk of hypertension particularly when accompanied with obesity and diabetes among Saudi subjects.


Introduction
The development of hypertension is believed to originate from an interaction of lifestyle exposures, such as high dietary sodium, excess weight, and alcohol consumption, with multiple genetic factors. (1,2) In addition, in many hypertensive patients, there is strong inherited evidence derived from twin, adoption and family studies indicating that about one third to one-half of the interindividual variation of blood pressure levels is heritable. (1,3) Despite considerable efforts to study the molecular genetics of hypertension, the inherently complex nature is somewhat an obstacle in elucidating the involved genes. (4) The case-control association study of candidate genes remain an efficient approach for detecting the "modest-effect" genetic variants underlying disease susceptibility provided the study is adequately powered. (5) Many case-control studies have addressed in particular the putative role of C to T transition at nucleotide 677 (C677T) and A to C transition at nucleotide 1298 (A1298C) in the methylenetetrahydrofolate reductase (MTHFR) gene in the development of hypertension. MTHFR is partly controlling the folate metabolism through catalyzing the reduction of 5, 10-methylenetetrahydrofolate to 5methyltetrahydrofolate; (6) a co substrate for the conversion of homocysteine to methionine; (5) thus resulting into a state of hyperhomocysteinemia; (3,7) particularly in subjects with low plasma folate levels. (8) Both homozygosity for MTHFR C677T (TT) and A1298C (CC), and compound heterozygosity for C677T and A1298C (677CT/1298AC) genotypes were found to be associated with a reduced MTHFR enzyme activity. (3,7,9) Homozygous MTHFR TT genotype has also been found to be associated with an increased risk of hypertension, (8) which may due to in increased homocysteine levels that were also found in patients with complicated type I and II diabetes mellitus. (10,11) Hypertension is a well known prevalent disorder among Saudi population especially in Qassim Region which is a tribal region characteristically having high frequency of consanguinity plus a high aggregation rate of familial diseases as hypertension, obesity, and diabetes. (12)(13)(14) In spite of this fact, little data were -so far-published concerning the genetic background of Saudi subjects in terms of their susceptibility to hypertension.
The objective of this study was to test for the association of the MTHFR (C667T and A1298C) gene polymorphisms with hypertension, obesity and diabetes among Saudi subjects from Qassim region.

Subjects and Methods
Participants included 123 cases (83 males and 40 females) with an age mean ±SD of 50.93 ± 15.43 years. They were known to have longstanding essential hypertension i.e. with systolic blood pressure (SBP) between 140 and 200 mmHg, or diastolic blood pressure (DBP) between 90 and 120 mmHg. Their minimum duration of hypertension is one year that was confirmed during at least two visits at the outpatient clinic of Buraidah Central Hospital, Qassim region, Saudi Arabia. Of these cases, 24.1% had a positive family history of hypertension, 32.4% had positive parental consanguinity, 12.8 % were smokers, 84.6% were obese and 46.2% had diabetes. For comparison, 250 normal healthy unrelated subjects (142 males and 108 females) with an age mean and SD of 47.65 ± 11.15 were taken from the same locality as controls.
Obesity was diagnosed on the basis of the most commonly used definitions, established by the World Health Organization (WHO) in 1997 and published in 2000, that defines the body mass index (BMI) of obesity as being 30 or greater. (15) On the other hand, diabetestype 2 was diagnosed on the basis of blood sugar during fasting time (12-14hr), being higher than 126 mg/dL at least for two times; or with symptoms of hyperglycemia and a casual (random) plasma glucose is higher than 200 mg/dl (11.1 mmol/l). (16) An informed consent was obtained from all participants as well as an ethical approval from the local Ethics and Scientific Committees.

DNA extraction and amplification
The isolation of genomic DNA was done on a MagNA Pure LC instrument (Roche Molecular Biochemicals, Mannheim, Germany), using the manufacturer's standard protocol. Oligonucleotide primers and fluorescence-labeled hybridization probes (Table 1.) were designed for amplification and sequence-specific detection of corresponding polymorphism (TIB MolBiol; Berlin, Germany) as described before. (17,18)  The master mixture contained 2 μl of a 10 X mixture of Light Cycler Fast-Start DNA master hybridization probes (Roche Diagnostics), 5 mM MgCl2 , a 1 μM MTHFR primers including 0.075 μM of specific primers, and a 0.2 μM of hybridization probes. Fluorescence curves were analyzed with the LightCycler software (version 3.5.3). Automated calculation of crossing points was done by the secondderivative maximum method. The fluorescence of each capillary was measured at wavelengths of 640 and 705 nm (dual-color option). Each run contained positive control, wild type (WT), mutant type (MT), heterozygous type (HT) and one negative control (blank reagent and water). Each result was confirmed by the specific peak in the corresponding melting curve.

Statistical analysis
Allelic and genotypic frequencies were compared and statistically analyzed using the Fisher's exact test and Odds ratio (OR) with 95% confidence intervals (95% CI). Conformity of genotype distributions with Hardy-Weinberg (HW) equilibrium was evaluated by chi-square analysis. For all tests, a p-value < 0.05 was considered to be statistically significant.

Results
Compared to controls, total hypertensive cases showed significantly higher frequency of mutant allele 677T carriage (TT + CT) (40.7% vs. 26%, OR=1.9, 95% CI= 1.2-3.1) with a lower frequency of the normal 677CC genotype (59.3% vs. 74%, p=0.004). The same was observed among cases-subgroup of hypertension associated with obesity notably with higher odds ratio (OR=2.6, 95% CI=1.3-5.01, p=0.004). Although cases-subgroup of hypertension with obesity and diabetes and cases-subgroup with hypertension alone showed the same phenomena of higher frequency of mutant T allele carriage and lower normal CC genotype than controls, yet it did not reach a statistical significance (p>0.05) ( Table 2).
Compared to controls, total hypertensive cases showed significantly higher frequency of mutant 1298 C allele carriage (CC + CA) (59.3% vs. 42.4%, OR=1.98, 95% CI= 1.3-3.1) with a lower frequency of the normal AA genotype (40.7% vs. 57.6%, p=0.003). The same was observed among cases-subgroup of hypertension associated with diabetes and obesity and cases-subgroup of hypertension and obesity, with higher odds ratio (OR=2.6 and 2.2 respectively). Although cases of hypertension alone showed the same phenomena of higher frequency of mutant C allele carriage and lower normal AA genotype than controls, yet it did not reach a statistical significance (p=1) ( Table 3). Comparing case subgroups in terms of gender, age, smoking, consanguinity and family history showed non-significant difference related to all studied genotypes and alleles (Table 3).

Discussion
The current results showed that Saudi hypertensive cases had significantly higher frequency of mutant alleles; MTHFR 677T particularly in obese cases; and MTHFR 1298C particularly among obese and diabetic cases. An interesting previous finding was that reported by Settin et al. showing no statistical significance of the association of MTHFR alleles with obesity among healthy Saudi controls. (19) This might give a more confirmation to our finding here of an association of these alleles with hypertension particularly if accompanied by obesity and diabetes among affected Saudi subjects compared to healthy controls.
These results are consistent with those of Markan et al., who stated that MTHFR 677T and 1298C alleles and the co-occurrence of MTHFR 677 CT/MTHFR 1298 CC genotypes were associated with increased risk of hypertension among Indian patients. (20) In another study, Koupepidou et al., have proposed that MTHFR 677TT and 677CT/1298AC genotypes may be predisposing the hypertensive patients to hypertensive nephrosclerosis and chronic renal failure. (21) It was also reported that MTHFR 677C/T polymorphism might be significant risk factors for hypertension in Taiwanese population.
(22) A recent large scale study conducted by Inamoto et al. reported that the TT genotype of the MTHFR gene was associated with an increased risk of hypertension among Japanese subjects conferring a small, but significant risk in Caucasians. (23) However, a modest significant association was reported between MTHFR C677T mutation and essential hypertension. (8) Regarding association with diabetes, a previous study conducted on Czech subjects concluded that the C allele of the 677C/T MTHFR polymorphism is associated with type II diabetes mellitus in women. (24) Previous researches could also elicit positive association of obesity and premature coronary artery disease in hypertensive adolescents with MTHFR C677T mutations. (25,26) Conversely, no significant association was reported between C677T variant of MTHFR polymorphism and metabolic syndrome, hypertension, and diabetes in a study among Iranians.
(27) Similarly Russo et al. found no association between MTHFR C677T polymorphism and metabolic syndrome in a group of type II diabetic subjects with mild hyperhomocysteinemia. (28) Also, Yamada et al., observed no association between MTHFR C677T polymorphism and metabolic syndrome. (29) This discrepancy may be postulated to be due to the effect of epigenetic mechanisms controlling gene expression influenced by environmental conditions as life style and diet. In this respect, it is hypothesized that folic acid fortification could overcome metabolic block caused by thermolabile MTHFR mutation, and consequently affects DNA methylation and gene expression. (27,30) Interestingly, comparing case subgroups in terms of gender, age, smoking, consanguinity, and positive family history of hypertension; showed non-significant difference related to the frequency of all studied genotypes and alleles. In fact consanguinity is known to increases the incidence of autosomal and multifactorial disorders. On our series of cases, mutant 677 TT and 1298 CC genotypes were present in only 2.9% and 14.3% of positive consanguineous cases respectively while it was still present in 1.3% and 5.1% of negative consanguineous ones with a final statistically nonsignificant association with the susceptibility for hypertension (p>0.05). This implies that these polymorphisms predispose to hypertension regardless of the effect of age, gender or family status. These results were also reported previously by other researchers. (31)(32)(33)(34) However, we recommend another wide scale study including the detailed data of the amount of exposure to smoking or other pollutants and also, the detailed data of diet and exercise to get a big view about the gene environmental interactive factors into the causation of hypertension, diabetes and obesity.
In this respect, researchers recommended programs of screening for serum homocystein; serum and RBC folate, vitamin B6, B12 and folate supplementations to subjects with risk genotypes and/or other environmental risk factors. (35)(36)(37) In conclusion; this study demonstrated the association of MTHFR gene polymorphisms with hypertension, obesity and diabetes in Saudi subjects from Qassim Region. These polymorphisms can be used as markers for the susceptibility to hypertension among affected families who can be counseled for potential prevention and management.