Methylenetetrahydrofolate reductase gene polymorphisms in patients with cerebral hemorrhage

Methylenetetrahydrofolate reductase gene polymorphisms in patients with cerebral hemorrhage

Fang, Xin


Cerebral vascular disease (stroke) is the third leading cause of death and the most common basis for long-time disability. Because no efficient therapies for neurological deficits after stroke are expected, preventive medicine aiming at reducing known risk factors is more important. Hyperhomocysteinemia has been regarded as an independent risk factor not only for the development of coronary heart disease1 and myocardial infarction2,3, but also for the development of ischemie stroke besides many other traditional factors such as hypertension, hyperlipidemia, diabetes mellitus and smoking. Moderate increases in plasma HCY concentrations are common in adults, especially elderly adults, with cerebrovascular diseases4,5. The 5,10-methylenetetrahydrofolate reductase (MTHFR) catalyses the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominant circulating form of folate and carbon donor for the remethylation of HCY to methionine. Functional abnormalities of MTHFR are regarded as one of the factors attributed to moderately raised plasma HCY levels6,7.

In 1994, the human cDNA of 2.2 kb in length for the MTHER was isolated and assigned to chromosome 1p36.3(8). Then a common mutation that decreased thermotolerance of the MTHFR enzyme was detected9. The genetic change underlying thermolabile MTHFR is a C to T base transition at the nucleotide 667 (C667T ) in the MTHFR cDNA, which results in the substitution of valine for alanine6. The C667T mutation resulted in decreased activity of the MTHFR enzyme and then elevated plasma HCY level6. Jacques and colleagues, however, found that plasma HCY levels in subjects with homozygous mutant genotype was increased only at lower plasma folate concentrations and suggested that folate supplementation may be beneficial to prevent fasting hyperhomocysteinemia10.

In this study, we analysed MTHFR gene polymorphisms by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method in the patients with cerebral ischemia (Cl) and cerebral hemorrhage (CH), as well as in control subjects. Plasma levels of HCY and folate were also measured to investigate the relationship between their plasma levels and MTHFR gene polymorphism.



We studied 86 subjects of Mongolian races, including 42 Cl cases (66.5 ± 0.1 years, 24 males and 18 females), 22 CH cases (64.1±3.1 years, 14 males and eight females), and 24 non-stroke controls (64.5 ± 8.3 years, 13 males and 11 females) from the same age group and geographical area. None of the control subjects had history of stroke, which was also confirmed by computed tomography brain scan or magnetic resonance imaging. The cases with mixed type stroke and lacunar infarction, and those with abnormal renal and hepatic functions were excluded from the study. The patients who had received thrombolysis therapy were also excluded. Written informed consent was obtained from each subject.

Genetic analysis

Polymorphisms were analysed by the PCR-RFLP. Total DNA was extracted by the method described previously11. All the DNA samples were preserved at -40°C. Primers for the MTHFR gene were designed according to the literature6 as follows: P1: 5′-TGAAGGAGAAGGTGTCTGCGGCA-3′ and P2: 5′-AGGACGGTGCGGTGAGAGTG-3′, which corresponded to nucleotide sequence of human cDNA positions 667. PCR was performed with primers for 30 cycles with cycle times of 30 seconds at 94°C, 45 seconds at 62°C, 50 seconds at 72°C and, after the 30 cycles, extended by 10 minutes at 70°C, which yielded a PCR product of 198 bp. The PCR products were detected by electrophoresis with 2% sepharose with 0.5 µg ml^sup -1^ EB at 110 V for 30 minutes.

The amplified fragments were digested with Hinfl (Takara Shuzo Co. Ltd., Osaka, Japan), which can recognize the C-T substitution of the fragment. After reacting at 37°C for 32 hours, the terminal products were resolved by electrophoresis on 3% agarose prestained with ethidium bromide at 11OV for 2 hours. The results were visualized by ultraviolet trans-illumination and photographed using Pharmacia Biotech gelatum photograph system.

Measurement of the plasma levels of HCY and folate

Non-fasting venous blood samples were collected. The plasma total HCY levels were detected using highperformance liquid chromatography (HP1100 Hewlett Packard USA) at fluorescence at excitation wavelength of 390 nm and an echo wavelength of 470 nm^sup 12^. The reaction mixture contained 1 µl boric buffer, 2 µl patients plasma, 1 µl mercaptoethanol, 2 µl 1 × 10^sup -4^ mmol 1^sup -1^ ophthalaldehyde (OPA, Sigma USA). Standard HCY reagent (1 ×; 10^sup -4^ mmol l^sup -1^, Sigma USA) was used for quantifying plasma HCY.

The plasma folate levels were measured by radioimmunoassay. All the exponents were reserved at -20°C. We added 2 µl plasma to the folate reagent box (Sigma USA), then measured the folate levels using β-auto counter (WLA Finland).

Data analysis

Results are expressed as the mean± SD. The u tests were used for comparison of measurement data. Differences between groups were examined with the use of the χ^sup 2^ test or the unpaired Student’s t-test. The Hardy-We in berg genetic equilibrium test was performed to the control group in order to clarify the representatives of the MTHFR allele.


Analysis of the PCR product

The PCR products yielded 198 bp band by electrophoresis. After digestion with Hinfl, three different genotypes were observed; homozygous mutation ( + /+) with only 175 bp band, heterozygous mutation ( + /-) with both 198 and 175 bp bands, and wild-type (-/-) with only 198 bp band. Genotype distribution of the 24 controls was not significantly different from the Hardy-Weinberg equilibrium.

Distribution of MTHFR genotypes in the CH, Cl and control group

The prevalence of C677T mutation in the MTHFR gene ( + /+ and +/-) was examine in patients with Cl and CH, as well as in control subjects (Table 1). The incidences of both homozygous (26%) and heterozygous (43%) mutations in the Cl group were significantly higher than those in the controls (8 and 25%; χ^sup 2^ = 8.24, p0.05).

The C677T allele frequencies of the Cl, CH, and control groups were 0.48, 0.55 and 0.21, respectively (Table 7). This difference was significant between the Cl group and control (χ^sup 2^=4.65, p0.05).

MTHFR genotype and plasma HCY and folate levels

The plasma levels of HCY and folate were listed in Table 2. No significant differences in the HCY concentration were observed among +/ + , +/- and -/ – in CH, Cl, and control groups.

The plasma folate concentrations tend to decrease as C677T alleles increase (+/+


Distribution of MTHFR genotypes in the CH, Cl and control group

In the present study, we observed that genotype frequencies in the Cl group were 26% for homozygous mutation (+/ + ), 43% for heterozygous mutation (+/-) and 31% for wild-type (-/-), and those in the controls were 8, 25 and 67%, respectively (Table 7). The frequencies of +/+ and +/- in the Cl group were significantly higher than those in the controls. The C677T allele frequency in the Cl group was 0.48 and significant higher than the controls. Though the numbers of subjects were small, those results coincided well with the data observed in Japanese population13. Two European studies on white subjects showed that the C677T allele frequency was 0.3-0.4 in both stroke and control groups, and there was no significant difference between the groups7,14. Since there were marked ethnic variations in common vascular candidate gene polymorphisms15, coincided data of the present study on Mongolian subjects with those in Japanese population would be meaningful.

We also found, in the present study, that MTHFR gene frequencies in CH group of +/ + , + /-, -/- were 25, 60 and 15%, respectively. This result coincided with the previous observation in 35 Japanese CH patients (34, 52 and 14%), which was the sole report studying MTHFR gene polymorphisms in CH patients16. The C677T allele frequency of 55% in CH group is significantly higher than that in the control group, but not different from Cl group. It is concluded that MTHFR gene mutation at the 667 base is closely correlated with onset of stroke, both Cl and CH. The fact that there is no difference in the mutation distribution between Cl and CH group suggests that there is concordance of the mechanism of the gene mutation effect on the onset of Cl and CH.

MTHFR genotype and plasma HCY and folate levels

In the present study, the plasma HCY and folate levels were also measured (Table 2). We found that plasma folate levels in the homozygous MTHFR gene mutation were lower than those in wild-type in stroke patients (especially patients with CH), while plasma HCY levels were not different among any genotypes and any of the study groups. A majority of the previous reports on homozygous MTHFR mutation have suggested an inverse relationship between plasma levels of HCY and folate3,10,13,14,16. Since there exists other metabolic pathways of HCY and HCY metabolism is also influenced by many factors besides MTHFR gene mutations, we could not observe a clear relationship between plasma HCY levels and MTHFR genotypes in this study.

In the population of homozygous mutation, plasma folate levels in the CH group was significantly lower than those in the Cl and control groups, while such a tendency was not observed in the other genotypes (Figure 1). Although the number of subjects is too small to draw a firm conclusion, it could be possible that +/ + genotype subjects with low plasma folate levels have a predisposition to intracerebral bleeding. A recent study by Bezzano and colleagues showed an inverse relationship between dietary intake of folate and subsequent risk of stroke in 9764 US population17. On the other hand, a more recent randomized controlled trial showed that moderate reduction of total HCY by administration of folate, pyridoxine and cobalamine in 3680 adults with non-disabling cerebral infarction had no effect on vascular outcomes during the 2 years of follow-up18. Though there is still controversy in folate supplementation and stroke prevention, the results of the present study suggest the possibility of folate supplementation in preventing CH. Further studies in larger populations are needed to clarify the possible mechanism of the effect of MTHFR gene mutation on plasma HCY and folate levels and development of stroke.

Copyright Maney Publishing Jan 2005

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