This work has identified a common mutation in MTHFR which alters a highly-conserved amino acid; the substitution occurs at a frequency of approximately 38% of unselected chromosomes and may represent an important genetic risk factor in vascular disease.
Abstract:
Hyperhomocysteinaemia has been identified as a risk factor for cerebrovascular, peripheral vascular and coronary heart disease. Elevated levels of plasma homocysteine can result from genetic or nutrient-related disturbances in the trans-sulphuration or re-methylation pathways for homocysteine metabolism. 5, 10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominant circulatory form of folate and carbon donor for the re-methylation of homocysteine to methionine. Reduced MTHFR activity with a thermolabile enzyme has been reported in patients with coronary and peripheral artery disease. We have identified a common mutation in MTHFR which alters a highly-conserved amino acid; the substitution occurs at a frequency of approximately 38% of unselected chromosomes. The mutation in the heterozygous or homozygous state correlates with reduced enzyme activity and increased thermolability in lymphocyte extracts; in vitro expression of a mutagenized cDNA containing the mutation confirms its effect on thermolability of MTHFR. Finally, individuals homozygous for the mutation have significantly elevated plasma homocysteine levels. This mutation in MTHFR may represent an important genetic risk factor in vascular disease.
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Q1. What have the authors contributed in "A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase" ?
8 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. A recent study of the Netherlands population identified the thermolabile variant in different forms of premature vascular disease,6 and estimated its incidence to be 7 % of vascular patients. The frequency of the three genotypes were as follows: –/–, 37 % ; +/–, 51 % ; and +/+, 12 % ( + indicates the presence of the HinfI site and a valine residue ). The authors next performed genotypic analysis and measured enzyme activity and thermolability in a total of 40 lymphocyte pellets from patients with premature vascular disease and controls ( Table I ). The authors selected 13 vascular patients from their previous study, among whom five were considered to have thermolabile MTHFR. From a large reference group of 89 controls, the authors studied all seven individuals who had thermolabile MTHFR, and selected at random an additional 20 controls with normal MTHFR from the same reference group. The Hague, The Netherlands Corresponding author — R. Rozen 111 Figure 1. The differences among genotypes for plasma homocysteine were maintained when homocysteine was measured following six hours of methionine loading. 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. 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.
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.