A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase
Summary (1 min read)
A common mutation in methylenetetrahydrofolate reductase
- The three genotypes were all significantly different (P < .01) with respect to enzyme thermolability.
- Treatment of extracts at 46°C for five minutes revealed that the enzyme containing the substitution was significantly more thermolabile than the wild -type enzyme .
- This hypothesis is compatible with the well-documented influence of folate on homocysteine levels7,14 and with the reported correction of mild hyperhomocysteinaemia by folic acid in individuals with premature vascular disease14 and thermolabile MTHFR.15 Enzyme activity and plasma homocysteine were determined as previously reported.
- Each value represents mean ± standard error.
Methodology
- Primers were designed from the cDNA sequence to generate 250-300 bp fragments which overlapped 50-75 bp at each end.
- The individuals, all French Canadian, were not examined clinically or biochemically.
- Of the 13 cardiovascular patients, eight had cerebrovascular arteriosclerosis, and five had peripheral arteriosclerosis.
- The entire replacement fragment and the cloning sites were sequenced to verify that no additional changes were introduced by PCR.
- Western analysis was performed using the Amersham ECL kit with antiserum generated against purified porcine liver MTHFR.12 Enzymatic assays were performed by established procedures.
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Cites background from "A candidate genetic risk factor for..."
...mia under conditions of impaired folate status (29, 73, 74)....
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...In 1995, Frosst et al suggested that the C677T polymorphism in the MTHFR gene is a candidate risk factor for vascular disease (29)....
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...was later ascribed to a C677T mutation in MTHFR gene (29)....
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...The same year, the C677T mutation in the MTHFR gene was identified and proposed as a candidate risk factor for vascular disease (29)....
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...predictor of hyperhomocysteinemia in the general population (29, 73, 74), is...
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1,981 citations
1,953 citations
Cites background from "A candidate genetic risk factor for..."
...may well help to assess the nature of this association.(69,70) Individuals who have a...
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...gene that encodes the methylenetetrahydrofolate reductase enzyme have reduced enzyme activity and, as a consequence, have homocysteine levels that are about 25% higher than those with the CC genotype.(70,71) An accompanying article in THE JOURNAL (2002;288: 2023-2031) describes a meta-analysis of 40 studies of this genetic polymorphism in which individuals with the TT polymorphism have a 16% (95% CI, 5%28%) higher risk of IHD than those with the CC polymorphism....
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References
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Frequently Asked Questions (13)
Q2. What is the common inborn error of folate metabolism?
Severe MTHFR deficiency, the most common inborn error of folate metabolism, results in hyperhomocysteinaemia, homocystinuria, and hypomethioninaemia.
Q3. How many patients with MTHFR deficiency were first reported?
Individuals with 50% residual activity, due to a thermolabile form of the reductase, were first reported in approximately 17% of 212 North American patients with coronary artery disease.5
Q4. What is the significance of the mutant protein in E. coli?
It is possible that the mutant protein has increased stability in E. coli, or that inclusion bodies in their extracts contributed to differences in recovery of properly-assembled enzyme.
Q5. What is the risk of vascular complications?
Patients with severe MTHFR deficiency (0-20% residual activity in cultured fibroblasts) present in infancy or adolescence with developmental delay, motor and gait dysfunction, seizures, psychiatric disturbances, and other neurological abnormalities; they are also at risk for vascular complications.
Q6. What are the main characteristics of the MTHFR gene?
The presence of a thermolabile MTHFR is predictive of coronary artery stenosis, independent of other risk factors, such as age, smoking, hypercholesterolaemia, and hypertension.
Q7. What is the mean MTHFR activity for heterozygotes?
Heterozygotes had a mean MTHFR activity of 65% compared to (–/–) individuals, intermediate between values for (–/–) and (+/+) individuals.
Q8. What is the alanine residue in the DHFR gene?
The authors have also observed a region of homology in the human dihydrofolate reductase (DHFR) gene,11 although the alanine residue itself is not conserved; this region of amino acids 130-149 of DHFR contains Thr136, which has been implicated in folate binding of human DHFR.13
Q9. What is the cDNA used to isolate the MTHFR cDNA?
The authors have used the original MTHFR cDNA (1.3 kb) to isolate a 2.2 kb cDNA, which contained an additional 900 bp at the 3’ end; the latter contained a termination codon, 100 bp of 3’ UTR and a poly A tail (GenBank# UO9806).
Q10. What was the procedure used for the analysis of the cDNA from the porcine liver?
Western analysis was performed using the Amersham ECL kit with antiserum generated against purified porcine liver MTHFR.12 Enzymatic assays were performed by established procedures.
Q11. How did the authors identify the mutations in MTHFR?
Their recent isolation of a cDNA for human MTHFR10 has enabled us to identify nine mutations in this gene, in the severely-deficient group of patients, by SSCP analysis and direct sequencing of PCR fragments.
Q12. What is the phenotype of the porcine liver enzyme?
The purified porcine liver enzyme has been shown to have subunits of 77 kDa.12 Western analysis (Figure 2a) of several human tissues and of porcine liver reveals a polypeptide of 77 kDa, as well as an additional polypeptide of approximately 70 kDa in human fetal liver and in porcine liver, suggesting the presence of isozymes.
Q13. What is the corresponding hypothesis for the vascular disease?
This hypothesis is compatible with the well-documented influence of folate on homocysteine levels7,14 and with the reported correction of mild hyperhomocysteinaemia by folic acid in individuals with premature vascular disease14 and thermolabile MTHFR.15Enzyme activity and plasma homocysteine were determined as previously reported.