Thermolabile Variant Research Articles

Mutations C677T and A1298C of the 5,10-methylenetetrahydrofolate reductase gene and fasting plasma homocysteine levels are not associated with the increased risk of venous thromboembolic disease.

Mild hyperhomocysteinemia is associated with homozygosity for the thermolabile variant of 5,10-methylenetetrahydrofolate reductase (MTHFR) and could increase the risk of venous thromboembolic disease (VTD). Recently, the second A1298C mutation of the MTHFR gene was described. The present study aimed to analyze both mutations of the MTHFR gene and plasma homocysteine levels in subjects with VTD. The study groups comprised 146 patients with VTD and 100 healthy subjects. There were no statistical differences in carrier frequency and allelic frequency for both A1298C and C677T mutations, nor were there any differences encountered between subjects with VTD and controls in either plasma homocysteine levels or according to C677T or A1298C genotypes of MTHFR. In our VTD patients and controls, neither MTHFR 677CT/1298CC nor MTHFR 677TT/1298CC combined genotypes were observed; double heterozygotes (A1298C/C677T) were represented only in 11% of VTD patients, and in 15% of the controls. In conclusion, the polymorphisms C677T and A1298C of MTHFR and fasting plasma homocysteine levels do not seem to be significant risk factors for venous thromboembolic disease.

A1298C methylenetetrahydrofolate reductase mutation and coronary artery disease: relationships with C677T polymorphism and homocysteine/folate metabolism.

5, 10-Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme in homocysteine/methionine metabolism. The most-studied C677T polymorphism in the MTHFR gene results in a thermolabile variant with reduced activity, and is associated with increased levels of total plasma homocysteine, a risk factor for coronary artery disease. A new mutation in the MTHFR gene (A1298C) has also been reported to lower enzyme activity. Whether A1298C is a risk factor for coronary artery disease, separately or in combination with C677T, and/or relative to total plasma homocysteine and folate status, is unclear to date. We evaluated this hypothesis in 470 angiographically characterized subjects, 302 with coronary artery disease, and 168 with normal coronary arteries. The frequency of the 1298C allele was 0.33 and that of combined heterozygosity 0.315. No difference was found in the frequency of the genotypes or when analyzed for combined heterozygosity between patients with coronary artery disease and normals. Independent of folate status, the 1298C allele was not associated with increased total plasma homocysteine. No additional effect of A1298C on total plasma homocysteine was observed in 148 combined heterozygotes compared with 98 heterozygotes for the C677T alone. These findings do not support a major role for the A1298C mutation in homocysteine metabolism and emphasize the hypothesis that MTHFR genotypes may interfere with coronary artery disease risk only when an unbalanced nutritional status leads to raised total plasma homocysteine levels.

5-methyltetrahydrofolate restores endothelial function in uraemic patients on convective haemodialysis.

Hyperhomocysteinaemia is an independent risk factor for the development of atherosclerosis. In patients with chronic renal failure, the administration of folic acid or its metabolites reduces but does not normalize plasma homocysteine concentrations. Furthermore, homocysteine induces endothelial dysfunction by an increased inactivation of nitric oxide. We examined the effect of the active metabolite of folic acid, 5-methyltetrahydrofolate (5-MTHF), 45 mg/week i.v. for 10 weeks, combined during the last 2 weeks with vitamin B12, 500 microg s.c. twice weekly, on homocysteinaemia and endothelial function in 15 patients undergoing convective haemodialysis. Endothelial function was evaluated by B-mode ultrasonography on the brachial artery. Flow-mediated dilation (FMD) was recorded during reactive hyperaemia produced by inflation of a pneumatic tourniquet. Nitroglycerine-mediated dilation (NMD) was recorded after administration of isosorbide dinitrate. Finally, the presence of the thermolabile variant of methyltetrahydrofolate reductase (t-MTHFR) was assessed by genotype analysis. Plasma homocysteine concentrations fell by 47% after treatment with 5-MTHF alone and by a further 13.6% after the addition of vitamin B12. The reduction was more marked in homo- and heterozygous patients than in normal genotypes for t-MTHFR. Flow-mediated endothelial vasodilation, measured by ultrasonography of the brachial artery, improved after administration of 5-MTHF (12.52+/- 2.47% vs. 7.03+/-1.65%; P<0.05), but there were no further changes following the addition of vitamin B12. Our study demonstrated that 5-MTHF administration not only reduced plasma homocysteine but also improved endothelial function in uraemic patients undergoing convective haemodialysis.

HLA antigenes in schizophrenia: relation to eye movement disturbances

Background and aims: Homozygosity for the thermolabile variant of 5,10-methylenetetrahydrofolate reductase (C677T) that causes hyperhomocysteinemia has been reported in 5–15% of general populations. This mutation has also been suggested to be positively associated with the risk of vascular disease and neural tube defects. It has also been suggested that present dietary reference values may need to be altered for people heterozygote or homozygote for this mutation as tissue folate status has been reported to be compromised by these genetic variants. The aims of this study were to investigate the distribution of 5,10-methylenetetrahydrofolate reductase (C677T) polymorphism in a population of Shiraz, south west of Iran and to test the hypothesis that folate status is compromised by this mutation in our population.Methods: In this study age, body mass index, plasma and red blood cell 5-methytetrahydrofolate, plasma total homocysteine and vitamin B12 of 391 healthy Iranians (198 men and 193 women) together with methylenetetrahydrofolate reductase C667T genotypes were determined. The correlates of methylenetetrahydrofolate reductase polymorphism were determined using univariate and multivariate statistical analysis.Results: The frequencies of CC, CT and TT genotypes were 56.2%, 38.7% and 5.1%, respectively. The C and T allele frequencies were determined to be 0.76 and 0.24, respectively and this polymorphism was compatible with Hardy-Weinberg equilibrium (X2=1.54, df=2, P=0.46). Among all the variables examined, red blood cell 5-methyltetrahydrofolate (P=0.007, ANOVA) and plasma 5-methyltetrahydrofolate (P=0.012, ANOVA) were significantly lower in individuals with TT genotype than those with either CC or CT genotype. Plasma total homocysteine was significantly higher in individuals with TT than those with either CC or CT genotype at below the median levels of red blood cell 5-methylterahydrofolate (P=0.03, ANOVA) and plasma 5-methylterahydrofolate (P=0.04, ANOVA). Univariate (r=-0.16, P=0.002) and multivariate analysis (β=-0.0005, P=0.003) showed that red blood cell 5-methylterahydrofolate was the strongest correlates of methylenetetrahydrofolate reductase genotypes.Conclusions: Results from this study suggest that methyltetrahydrofoate reductase C667T genotypes are strongly and independently associated with low red blood cell 5-methylterahydrofolate that has been reported to be a more reliable and long-term marker for body's folate status among Iranians. These results may suggest that substantial minority of people in general populations may have increased folate needs and this may place doubts on the validity of assuming “normality” for nutrient requirements in any general population.

Hyperhomocysteinemia and venous thromboembolism: a risk factor more prevalent in the elderly and in idiopathic cases

Fasting plasma homocysteine level and the related clinical findings were analysed in 240 consecutive patients with venous thromboembolism. Hyperhomocysteinemia, defined as a plasma level above 20 μmol/l (corresponding to the percentile 95th in the controls), was present in 11.2% of the patients. Plasma homocysteine level was similar in patients presenting with either deep venous thrombosis, pulmonary embolism or both conditions. It was significantly higher in patients with primary (unprovoked) VTE than in patients with secondary disease (associated with at least one risk factor): 12.3 vs. 9.55 μmol/l ( p<0.005). Mean homocysteine was higher in male than in female patients (14.51 vs. 12.9 μmol/l, p<0.05) and increased significantly with age. Hyperhomocysteinemia was more frequent in patients with relapsing disease (14 of 76, 18.4%) than in those presenting with a single episode (13 of 164, 7.9%) ( p=0.034). Furthermore, hyperhomocysteinemia was correlated with reduced protein C level ( p=0.013). In a multivariate analysis, two factors were significantly associated with hyperhomocysteinemia: older age ( p<0.0001) and idiopathic occurrence ( p<0.02). Since the frequency of homozygous MTHFR thermolabile variant was rather similar in patients and controls, testing for C677T mutation was not helpful in screening VTE patients. However, the homozygous mutation was significantly more prevalent among hyperhomocysteinemia patients, confirming its role in the genesis of hyperhomocysteinemia. According to its prevalence, to the putative role in venous and arterial disease and the availability of an effective and low-cost corrective therapy, hyperhomocysteinemia deserves interest, especially in the elderly and in the patients with idiopathic VTE disease.

Inherited thrombophilias in pregnant patients: detection and treatment paradigm1,2

Inherited thrombophilias are the leading cause of maternal thromboembolism and are associated with an increased risk of certain adverse pregnancy outcomes including second- and third-trimester fetal loss, abruptions, severe intrauterine growth restriction, and early-onset, severe preeclampsia. Current information suggests that all patients with a history of prior venous thrombotic events and those with these characteristic adverse pregnancy events should be evaluated for thrombophilias. The most common, clinically significant, inherited thrombophilias are heterozygosity for the factor V Leiden and prothrombin G20210A mutations. The autosomal-dominant deficiencies of protein C and protein S are of comparable thrombogenic potential but are far less common. Homozygosity for the 4G/4G mutation in the type-1 plasminogen activator inhibitor gene and the thermolabile variant of the methylenetetrahydrofolate reductase gene, the leading cause of hyperhomocysteinemia, although relatively common, confer a low risk of thrombosis. In contrast, autosomal-dominant antithrombin deficiency and homozygosity or compound heterozygosity (ie, carriers of one copy of each mutant allele) for the factor V and prothrombin mutations are very rare but highly thrombogenic states. Regardless of their antecedent histories, pregnant patients with these highly thrombogenic conditions are at very high risk for both thromboembolism and characteristic adverse pregnancy outcomes, require full therapeutic heparin therapy throughout pregnancy, and need at least 6 weeks of postpartum oral anticoagulation. There is also compelling evidence that patients with the less thrombogenic thrombophilias and a history of venous thrombotic events or characteristic adverse pregnancy outcomes require prophylactic anticoagulant therapy during pregnancy and, in the case of prior thromboembolism, during the puerperium. Antepartum anticoagulation does not appear warranted among patients with less thrombogenic thrombophilias who are without a history of venous thromboembolism, characteristic adverse pregnancy outcomes, or other high risk factors for venous thrombosis.

Hyperhomocysteinaemia, Coagulation Pathway Activation and Thrombophilia in Patients with Inflammatory Bowel Disease

Background : The 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C → T polymorphism encoding the thermolabile variant is, when present as homozygote type (TT variant), a known genetic cause of mild hyperhomocysteinaemia (HHCY). This polymorphism has been observed in increased numbers in patients with inflammatory bowel disease (IBD). Coagulation and fibrinolysis are activated in patients with active IBD, but it is not known whether raised plasma homocysteine (HCY) found in patients with IBD significantly contributes to this activation. The aim of this study was to investigate if HHCY or presence of the TT variant significantly induces a hypercoagulable state in IBD patients receiving anti-inflammatory therapy during active disease, and to study if genetic determinants for thromboembolic disease are more frequent in these patients. Methods : The study was designed as a cross-sectional study in an outpatient clinic comprising 106 IBD patients receiving anti-inflammatory therapy. Markers of coagulation were measured in order to elucidate whether patients with HHCY or the MTHFR TT variant were hypercoagulant compared with patients with no impairment of HCY metabolism. In addition, markers of inflammation and acute-phase reactants were measured in order to compare activity during active disease and during remission. Genetic determinants of thromboembolic disease in patients with IBD and in relevant controls were investigated in the expectation of a more frequent occurrence of these markers of thrombophilia if hypercoagulability could be a primary or contributory factor in IBD. Results : No significant difference could be found in coagulation activity, acute-phase reactants or inflammatory markers in IBD patients with the TT variant of the 677C → T polymorphism or high (>15 μmol/L) plasma HCY levels, compared with IBD patients with no impairment of HCY metabolism. In patients with IBD, the coagulation activity was significantly increased during active disease compared with a state of remission. As expected, a significant difference regarding interleukin 6, C-reactive protein and erythrocyte sedimentation rate was present in IBD, comparing active disease with a state of remission. No significant complement activation was present in either of the groups or during active disease. Neither of the allele frequencies of genetic determinants for thrombophilia (coagulation factor V 1691G → A (factor V Leiden) and factor II 20210G → A polymorphisms) in the background population differed significantly from that in IBD patients. Conclusions : This study found no correlation between the MTHFR TT variant or HHCY and a hypercoagulable state in IBD patients receiving anti-inflammatory treatment. This coagulation activity is high during exacerbations of disease, but a considerable reduction is seen in patients on anti-inflammatory therapy compared with non-treated patients. Coagulation activation in IBD is probably a consequence of the inflammatory nature of the disease. That thrombophilia could be a contributory or primary factor in the development of IBD is not supported by the present study, as the frequencies for the genetic determinants for thrombophilia are similar in IBD patients and controls.

New Enzymatic Colorimetric Assay for Total Homocysteine

In 1969, McCully (1) observed that increased plasma homocysteine (Hcy) was linked with vascular disease. Subsequent studies demonstrated that even mild hyperhomocysteinemia is an independent risk factor for cardiovascular, cerebrovascular, and peripheral vascular disease (2)(3)(4)(5)(6)(7)(8). Increased plasma Hcy is associated with a thermolabile variant of 5,10-methylenetetrahydrofolate reductase, smoking, lack of exercise, and excessive use of alcohol and coffee. Plasma or serum total Hcy (tHcy) concentrations are most commonly measured by HPLC (9), which is time- consuming and expensive, and by immunochemical (10)(11)(12) or enzymatic (13) methods, which may not be applicable to all colorimetric-based clinical chemistry analyzers. The thiol group of Hcy allows it to form a disulfide bond with other thiol-containing molecules, such as Hcy itself, cysteine, and the cysteine residue of plasma proteins. Biologic fluids may often contain both reduced and oxidized species of Hcy, and the sum of all the forms of Hcy is usually called total Hcy (tHcy) (14). Most clinical studies concerning Hcy have relied on the measurement of tHcy. An initial chemical reduction step of the sample is inevitable in the tHcy assay. Because reducing agents can interfere with the oxidation of redox indicators, such as Trinder’s reagents and derivatives of methylene blue generally used in diagnostic reagents, methods for tHcy that use these indicators have not been developed. The present method is a new enzymatic colorimetric assay for tHcy in biologic samples. The principle is as follows. In the first step, samples are reduced by dithiothreitol …

Homocysteine levels in pregnant women who smoke cigarettes

Homocysteine is a sulfur-containing amino acid produced when methionine is demethylated (Figure 1, Pathway 1). The majority of homocysteine undergoes transsulfuration to cysteine by cystathionine -synthase (CBS), of which vitamin B6 (pyridoxine) is an essential cofactor (Figure 1, Pathway 2). The remainder of homocysteine is remethylated by methionine synthase, of which vitamin B12 (cobalamin) is an essential cofactor along with methylenetetrahydrofolate (MTHF) (Figure 1, Pathway 3). MTHF is generated by the enzyme MTHF-reductase (MTHFR) (Figure 1, pathway 4) (1). High levels of homocysteine, or hyperhomocysteinemia, can result from a variety of genetic and environmental factors. Enzyme defects in either of the above pathways can cause hyperhomocysteinemia. One particular mutation, the thermolabile variant of MTHFR, is found in 5±15% of the Canadian population in a homozygous state (2). More than 33 mutations of the CBS gene have been identified. Heterozygous cystathionine synthase (CBS) mutations occur in 0.5±1.5% of the general population (3). Hyperhomocysteinemia may also be secondary to deficiencies in vitamin B12, B6, and folate (4,5).

Major and Potential Prothrombotic Genotypes in a Cohort of Patients With Venous Thromboembolism

Factor V Leiden (FVL) and the prothrombin 20210A (PT-20210A) variant are well-known risk factors for venous thromboembolism (VT). The thermolabile variant (TT) of the methylenetetrahydrofolate reductase (MTHFR) gene, and homozygosity for the 4G allele of the promoter region of the plasminogen activator inhibitor-1 (PAI-1) are potential genetic polymorphisms that have not been consistently associated with increased risk of VT. A case-control study was performed on 192 consecutive unrelated patients referred for evaluation of thrombophilia because of VT and 200 healthy controls. FVL was found in 10.4% of patients compared to 3.0% of controls, while 6.3% of patients were carriers of the PT-20210A allele compared to 2.0% of controls. The adjusted odds ratios (OR) were 5.92 and 4.03 for FVL ( P=.001) and the PT-20210A ( P=.033), respectively. The prevalence of homozygotes for MTHFR (TT) and PAI-1 (4G/4G) among patients and controls were 13.7% versus 13.0% and 21.6% versus 23.5%, respectively ( P=ns). A total of 121 patients underwent a complete screening for FVL, the PT-20210A, protein C (PC), protein S (PS), antithrombin III (ATIII), levels of factor VIII, and antiphospholipid antibodies (aPL). In 59 patients (48.8%) at least one defect was found, being a single defect in 55 and combined defects in 4 patients. Plasma levels of homocysteine (Hcy) were measured in 138 patients and 144 controls. Subjects from both groups carrying the MTHFR-TT variant had higher Hcy levels than those with the normal genotype. Hyperhomocysteinemia (HHcy) by itself is a risk factor for VT (OR 4.92, P<.0001). We conclude that FVL and the PT-20210A are risk factors for VT as well as Hcy levels, but the MTHFR and PAI-1 polymorphisms do not appear to be associated with VT in our country.

Relevance of folate metabolism in the pathogenesis of colorectal cancer

The purpose of this review is to outline the principal mechanisms involved in folate metabolism and how they may relate to the pathogenesis of colorectal cancer (CRC). In recent years, mild folate depletion (low normal level) has been associated with an increased risk of developing certain cancers, in particular colorectal neoplasia. The epidemiologic and mechanistic evidence linking folate deficiency with carcinogenesis is reviewed, with a particular emphasis on colorectal neoplasia. Methylenetetrahydrofolate reductase (MTHFR) is a critical folate metabolizing enzyme, and a functional polymorphic variant of this enzyme, the so-called thermolabile variant, caused by a C677T transition in the MTHFR gene, is common in the general population. This review critically examines the evidence that suggests that carriers of this C677T variant may be at increased risk of developing colorectal neoplasia. Although folate depletion may predispose to the initiation of the neoplastic process, folate supplementation, on the other hand, might potentiate the progression of an already established early neoplastic clone (eg, a colorectal adenoma). This could have potential public health implications, given an increasingly widespread policy of folate supplementation of food staples. (J Lab Clin Med 2001;138:164-76)

Serum C-Reactive Protein in Canadian Inuit and Its Association with Genetic Variation on Chromosome 1q21

Mortality from cardiovascular disease among Inuit living in the far north of Canada is ∼40% lower than in the rest of the country (1). This might be attributable to the protection resulting from environmental factors such as dietary ω-3 fatty acids in Arctic fish (1). Genetic factors may also be important. For example, the thermolabile variant of methylenetetrahydrofolate reductase is much less prevalent among Inuit than among subjects of European origin (2). However, there are also paradoxical genetic findings in these people. For example, some common genetic variants associated with a higher risk of cardiovascular disease, such as the APOE E4 and AGT T235 alleles, are more prevalent among Inuit than among subjects of European origin (3). These apparent inconsistencies may be related to the fact that there are numerous determinants of susceptibility to cardiovascular disease and that these determinants may differ among ethnic groups (4). As newer determinants of cardiovascular disease risk are identified, these can be evaluated in the Inuit. C-Reactive protein (CRP), an acute-phase reactant originally detected through its interaction with pneumococcal C polysaccharide (5), has been proposed to be a risk factor for cardiovascular disease (6). When detected with a high-sensitivity assay (6), increased serum CRP could be related to increased vascular disease risk either directly through its association with inflammation (7) or indirectly through its association with obesity and insulin resistance (8). The serum CRP concentration in the Inuit has not been reported. Furthermore, the role of possible genetic …

Reduction of the Homocysteine Plasma Concentration by Intravenously Administered Folinic Acid and Vitamin B12 in Uraemic Patients on Maintenance Haemodialysis

Background: Hyperhomocysteinaemia is an independent cardiovascular risk factor which can induce vascular lesions, thus contributing to the early development of atherosclerosis. Low-dose folic acid supplementation reduces the pretreatment homocysteine plasma levels by 25–35%. Recent studies report that higher intravenous or oral administration of the active form of folic acid reduces the homocysteine plasma concentration by nearly 70%. The reduction could also be influenced by the thermolabile variant of methylenetetrahydrofolate reductase (tMTHFR) and by the dialysis modality. Methods: A cross-sectional clinical study was performed to evaluate the effect of a drug containing folinic acid and vitamin B₁₂ on the plasma homocysteine concentration and whether this variable could also be influenced by the presence of a genetic variant of the methionine pathway and the use of different dialysis modalities. The plasma homocysteine concentration was measured in 55 patients undergoing haemodialysis, 27 of whom have been treated intravenously for megaloblastic anaemia using a drug containing low concentrations of folinic acid and vitamin B₁₂ at the end of each dialysis session for 6 months. The presence of tMTHFR was sought by molecular analysis, and the role of the dialysis modality was also investigated. Results: The patients given the folic acid treatment had lower homocysteine plasma levels than those not so treated. The plasma homocysteine concentration was significantly higher in the tMRHFR homozygotes than in the patients with a normal genotype, significantly lower in the treated than in the untreated homozygotes, and significantly higher in the untreated homozygotes than in the untreated subgroup with a normal genotype. The homocysteine level was also significantly lower in the patients who underwent convective haemodialysis than in those who received standard bicarbonate dialysis. Conclusions: A drug containing low concentrations of folinic acid combined with vitamin B₁₂ using an intermittent intravenous regimen is effective in reducing the homocysteine plasma concentration in uraemic patients. The homocysteine levels seem also to depend on genotype and dialysis modality.

Homozygous thermolabile variant of the methylenetetrahy-drofolate reductase gene: a potential risk factor for hyperhomo-cysteinaemia, CVD, and stroke in childhood

In this study of 118 children (median age 5.1 years; range 6 months to 17 years) with ischaemic stroke or transient ischaemic attack (TIA), 22 children (19%) were homozygous for the thermolabile variant of the methylenetetrahydrofolate reductase allele (t-MTHFR), compared with nine of 78 (12%) of a reference population (p=0.18, OR 1.76, 95% CI 0.76 to 4.04). Of those with cerebrovascular disease (CVD), 17 of 84 were homozygous for the t-MTHFR allele (p=0.13 compared with the reference population (OR 1.95, 95% CI 0.81 to 4.65). There was a significant (p<0.025) increment of plasma total homocysteine concentration in homozygotes for the t-MTHFR allele compared with heterozygotes, negatives for the t-MTHFR allele, and control children with no history of stroke. In four of 12 homozygotes for the t-MTHFR allele, plasma homocysteine levels were raised, compared with three of 38 of those who were negative or heterozygous (p=0.047; OR 5.8, 95% CI 1.1 to 31.2). Homozygotes for the t-MTHFR allele were significantly more likely to have a recurrent event than those who were negative or heterozygous (Cox regression p=0.031, hazard ratio 2.18, 95% CI 1.08 to 4.42). These data suggest that homozygosity for the t-MTHFR allele is associated with raised homocysteine levels in children and is a risk factor for primary and secondary stroke and TIA.

Homozygous thermolabile variant of the methylenetetrahy‐drofolate reductase gene: a potential risk factor for hyperhomo‐cysteinaemia, CVD, and stroke in childhood

In this study of 118 children (median age 5.1 years; range 6 months to 17 years) with ischaemic stroke or transient ischaemic attack (TIA), 22 children (19%) were homozygous for the thermolabile variant of the methylenetetrahydrofolate reductase allele (t‐MTHFR), compared with nine of 78 (12%) of a reference population (p=0.18, OR 1.76, 95% CI 0.76 to 4.04). Of those with cerebrovascular disease (CVD), 17 of 84 were homozygous for the t‐MTHFR allele (p=0.13 compared with the reference population (OR 1.95, 95% CI 0.81 to 4.65). There was a significant (p&lt;0.025) increment of plasma total homocysteine concentration in homozygotes for the t‐MTHFR allele compared with heterozygotes, negatives for the t‐MTHFR allele, and control children with no history of stroke. In four of 12 homozygotes for the t‐MTHFR allele, plasma homocysteine levels were raised, compared with three of 38 of those who were negative or heterozygous (p=0.047; OR 5.8, 95% CI 1.1 to 31.2). Homozygotes for the t‐MTHFR allele were significantly more likely to have a recurrent event than those who were negative or heterozygous (Cox regression p=0.031, hazard ratio 2.18, 95% CI 1.08 to 4.42). These data suggest that homozygosity for the t‐MTHFR allele is associated with raised homocysteine levels in children and is a risk factor for primary and secondary stroke and TIA.

5,10-methylenetetrahydrofolate reductase polymorphism and early organ damage in primary hypertension

Hyperhomocyst(e)inemia is a known risk factor for the development of atherosclerotic vascular damage. Plasma homocyst(e)ine levels are influenced by nutritional and hereditary factors. A point mutation (cytosine to thymidine substitution; C677T) in the gene encoding 5,10-methylenetetrahydrofolate reductase (MTHFR) makes the enzyme thermolabile and has been associated with elevated homocyst(e)ine levels in homozygous carriers (TT genotypes). We evaluated the relationship between the T allele encoding for the thermolabile variant of MTHFR and several biochemical risk factors and early signs of hypertensive and atherosclerotic organ damage in 206 untreated patients with primary hypertension. The MTHFR genotype was evaluated by polymerase chain reaction. Albuminuria was measured as albumin-to-creatinine ratio in three nonconsecutive first morning urine samples (negative urine culture). Persistent Mi (Alb+) was defined as an average albumin-to-creatinine ratio between 2.38 and 19 (men) and 2.96 and 20 (women). Left ventricular (LV) mass index (LVMI) was assessed by M-B mode echocardiography (LV hypertrophy, LVH = LVMI ≥125g/m 2), carotid geometry by high-resolution ultrasound scan, and retinal vascular changes by direct ophthalmoscopy (Keith-Wagener classification). The prevalence of Mi, LVH, and retinopathy was 14%, 45%, and 42%, respectively. The prevalence of carotid plaque was 25%. Allele frequencies for C (wild-type allele) and T allele (mutant allele) were 56% and 44%, respectively. Genotype frequencies were CC 29%, CT 54%, TT 17% according to Hardy Weinberg equilibrium. There were no differences as for age, sex, body mass index, blood pressure levels, lipid profile, smoking habits, and alcohol intake, and LVMI and urinary albumin excretion on the basis of MTHFR genotype. Patients with TT polymorphism showed a higher prevalence of retinal vascular changes (TT, 61% v CT + CC, 38%; P < .02) and carotid plaque (TT, 42% v CT + CC, 21%; P < .05) compared to patients with CC and CT polymorphism. Moreover, patients with T allele showed increased carotid artery size as demonstrated by intima plus media thickness (TT, 0.79 ± 0.05 mm v CT + CC, 0.67 ± 0.02 mm; P < .02), relative wall thickness (TT, 0.23 ± 0.01 mm v CT + CC, 0.20 ± 0.005 mm; P < .02), and surface area (TT, 19 ± 1.9 mm 2 v CT + CC, 15 ± 0.55 mm 2; P < .05). Multiple linear regression analysis demonstrated that MTHFR genotype and systolic blood pressure independently influence intima-media thickness and together account for about 11% of its variations (r 2 = 0.11, F = 9.7, dF = 1–205, P < .0001). Homozygosity for the T allele of the MTHFR gene is an independent risk factor for the development of early atherosclerotic organ damage in hypertensive patients.

Frequency of the 677 C-->T mutation of the methylenetetrahydrofolate reductase gene among Kuwaiti sickle cell disease patients.

Sickle cell disease (SCD) is relatively mild among Kuwaiti Arabs. However, an atypical subset of patients exists with frequent, severe vaso-occlusive crisis and osteonecrosis. The thermolabile variant of MTHFR, resulting from a C-->T mutation at nucleotide 677, has been shown to be associated with hyperhomocysteinemia, which is an important risk factor for premature vascular disease. We have screened an unselected group of 41 Kuwaiti SCD patients (33 SS and 8 Sbeta(0)-thal) attending the Hematology Clinic of Kuwait University Teaching Hospital for the MTHFR mutation, using a PCR-RFLP method. The patients were aged 2-41 years (mean of 12.8 +/- 8.6). One (2.4%) individual was homozygous for the mutation while 15 (36.6%) were heterozygous, giving an allele frequency of 20.7%. Twenty-one patients (14 SS and 7 Sbeta(0)-thal) were screened for osteonecrosis using MRI of the hip (spin-echo T1- and T2-weighted images). Seven (33.3%) had varying degrees of osteonecrosis, among whom the frequency of the 677 C-->T allele was 21.4%. The frequency was identical among those without osteonecrosis. Although the allele frequency is higher among our patients compared to American SS patients, our results do not suggest an association with osteonecrosis.

THE METHYLENE TETRAHYDROFOLATE REDUCTASE (C677T) MUTATION AS A POTENTIAL RISK FACTOR FOR AVASCULAR NECROSIS IN SICKLE CELL DISEASE

Avascular necrosis (AVN) of the humeral and femoral heads is a frequent and debilitating complication of sickle cell disease. Some of the risk factors for AVN are a-thalassemia and age. Recently, newly discovered thrombophilia mutations have been associated with AVN in patients without sickle cell disease. We studied the frequency of the thermolabile methylene tetrahydrofolate reductase (MTHFR) variant (C677T) in adult sickle cell patients with and without AVN. The frequency of the MTHFR mutation was 35.6% in patients with AVN and 12.9% in those without AVN (p=0.006). These data suggest that the thermolabile MTHFR variant may be a contributing risk factor for AVN in some populations with sickle cell disease.

Incidence of thrombophilia in patients with Gaucher disease.

An inherited risk for thrombosis, including mutant thermolabile variant of methylenetetrahydrofolate reductase (MTHFR), factor V Leiden, or prothrombin may be the co-factor(s) for avascular necrosis (AVN) in patients with sickle cell disease. Similarly, heterozygosity for factor V Leiden is sufficient to explain the increased blood viscosity observed in children with Legg-Calve-Perthes disease who develop AVN. Because there are no laboratory tests or clinical markers that are helpful in predicting which patients with Gaucher disease may develop AVN, the current study was undertaken to ascertain if there exists an inherited predilection to hypercoagulability in patients with Gaucher disease and AVN. Analysis was performed on genomic DNA extracted from 56 adult patients with type I Gaucher disease. In this cohort of Ashkenazi Jewish patients, the frequency of mutations in the MTHFR, prothrombin, and factor V Leiden genes was found to be low, as was the presence of anticardiolipin antibodies; and none was correlated with increased incidence of AVN. Splenectomy, that may be a predisposing factor to AVN in patients with Gaucher disease, was factored out. Hence the presence of any of the above thrombophilic factors, and which by extension may be risk factors for AVN in other diseases, are not more common in patients with Gaucher disease who develop AVN. Studies in larger cohorts and possibly inclusion of additional factors may be needed to ascertain whether a correlation exists.

The number of dichorionic twin pregnancies is reduced by the common MTHFR 677C→T mutation

In multiple pregnancies, demands for folic acid are considerably increased. The most common inborn error of folate metabolism is mild methylenetetrahydrofolate reductase (MTHFR) deficiency due to the synthesis of a thermolabile variant of the enzyme with impaired catalytic activity which leads to reduced 5-methyltetrahydrofolate (5-methyl-THF) and mildly elevated homocysteine plasma concentrations when folate status is inadequate. To investigate whether the number of offspring is influenced by this mutation, we determined the frequency of the 677C-->T substitution in 156 singleton and 40 twin mothers with dichorionic placentation. The T allele frequency in singleton (0.30) and twin mothers (0.16) was significantly different (P = 0.011). Mothers with the 677C-->T mutation had a 2.28 times lower risk of having a twin pregnancy than those without (95% confidence interval = 1.18-4.66; P = 0.008). Our observation would explain, at least in part, the hereditary trait of multiple gestations and is in agreement with the ethnic distribution pattern of the T allele which has been found to be inversely correlated with the incidence of dichorionic twins. Our findings suggest that the MTHFR 677C-->T mutation interferes with human brood size, probably by influencing the proliferation of rapidly dividing embryonic and maternal cells.