Remethylation Pathway Of Homocysteine Research Articles

Methionine synthase deficiency: Variable clinical presentation and benefit of early diagnosis and treatment.

Methionine synthase deficiency (cblG complementation group) is a rare inborn error of metabolism affecting the homocysteine re-methylation pathway. It leads to a biochemical phenotype of hyperhomocysteinemia and hypomethioninemia. The clinical presentation of cblG is variable, ranging from seizures, encephalopathy, macrocytic anemia, hypotonia, and feeding difficulties in the neonatal period to onset of psychiatric symptoms or acute neurologic changes in adolescence or adulthood. Given the variable and nonspecific symptoms seen in cblG, the diagnosis of affected patients is often delayed. Medical management of cblG includes the use of hydroxocobalamin, betaine, folinic acid, and in some cases methionine supplementation. Treatment has been shown to lead to improvement in the biochemical profile of affected patients, with lowering of total homocysteine levels and increasing methionine levels. However, the published literature contains differing conclusions on whether treatment is effective in changing the natural history of the disease. Herein, we present five patients with cblG who have shown substantial clinical benefit from treatment with objective improvement in their neurologic outcomes. We demonstrate more favorable outcomes in our patients who were treated early in life, especially those who were treated before neurologic symptoms manifested. Given improved outcomes from treatment of presymptomatic patients, cblG warrants inclusion in newborn screening.

Clinical, Biochemical, Radiological, and Genetic Profile of Patients with Homocysteine Remethylation Pathway Defect and Spastic Paraplegia.

Objectives:The objective of this study is to describe the clinical, biochemical, radiological, and genetic profile of patients presenting with progressive spastic paraparesis due to homocysteine remethylation pathway defect.Methods:This was a retrospective study conducted by reviewing the medical records of patients with serum homocysteine levels >50 μmol/L between January 2015 and January 2019 at our hospital. We included patients presenting with progressive spastic paraparesis, having serum homocysteine >50 μmol/L with low or normal blood methionine suggesting disorders of homocysteine remethylation. Demographic details, clinical manifestations, biochemical abnormalities, neuroimaging findings, and genetic profile were analyzed.Results:A total of seven patients (M: F = 5:2) fulfilled the study eligibility criteria. The mean age at onset of the disease was 13.4 ± 2.4 years (range: 9–17 years). Spastic paraparesis was the presenting manifestation in 4/7 (57.1%) patients. Other manifestations included cognitive decline, poor scholastic performance, behavioral disturbances, seizures, and spastic bladder. Severe hyperhomocysteinemia (>100 μmol/L) was noted in 6/7 (85.7%) patients with median levels of serum homocysteine being 185.7 μmol/L (range: 85.78–338.5 μmol/L). Neuroimaging showed parieto-occipital predominant leukoencephalopathy in 5/7 (71.4%) and diffuse cerebral atrophy in 1/7 (14.2%). Genetic analysis in three patients revealed pathogenic missense variants c.459C >G (p.Ile153Met), c.973C >T (p.Arg325Cys), and c.1031G >T (p.Arg344Met) in MTHFR gene. All the patients received vitamin B12 (injection and oral), folic acid, and pyridoxine and two patients received betaine. At the last follow-up of a median duration of 12 months, there was a good clinical and biochemical response with reduction in the median value of serum homocysteine by 77.5 μmol/L.Conclusion:Evaluation of serum homocysteine and blood methionine in adolescents presenting with progressive spastic paraparesis gives clue to a treatable homocysteine remethylation disorders.

Association between Low Red Blood Cell 5-Methyltetrahydrofolate and Hyperhomocysteinaemia with Hypertension

Hyperhomocysteinaemia and other risk factors associated with blood pressure have been reported in large community-based studies in different populations. However, it is not fully established whether hypertension is associated with high plasma total homocysteine levels (tHcy) or components of the homocysteine re-methylation pathway including vitamin B(12), plasma 5-methyltetrahydrofolate (5-MTHF) or red blood cell (RBC) 5-MTHF. In this study we tested the hypothesis that RBC 5-MTHF could be a marker for long-term folate status in the blood and low RBC 5-MTHF may be associated with hypertension. This was a cross-sectional study in a community-based setting and 492 males and 431 females were investigated. Systolic and diastolic blood pressures were determined and fasting blood samples were taken for determination of plasma tHcy, creatinine, uric acid, lipids, plasma 5-MTHF, RBC 5-MTHF and vitamin B(12). In males, the risk of hypertension was significantly (95% CI 1.9, 8.7; p = 0.003) increased by 1.8-fold in the first quartile compared with the highest quartile of RBC 5-MTHF when adjusted for body mass index (BMI), age, dyslipidaemia, uric acid, creatinine, smoking, plasma tHcy and vitamin B(12). The risk of hypertension was also significantly increased (95% CI 1.1, 9.2; p = 0.03) by 1.1-fold in the highest quartile compared with the lowest quartile of plasma tHcy when adjusted for BMI, age, dyslipidaemia, uric acid, creatinine, smoking, plasma and RBC 5-MTHF and vitamin B(12). There were no associations of hypertension with plasma tHcy, and other components of the Hcy re-methylation pathway were observed in females. The association between hypertension and low RBC 5-MTHF was stronger than any other components of the homocysteine re-methylation pathway. Results from this study suggest that folate measurements in RBC seem to be the most reliable marker indicating 5-MTHF deficiency and disturbances in the Hcy re-methylation pathway in association with hypertension.

Oxidative stress and apoptosis in homocystinuria patients with genetic remethylation defects

Oxidative stress has been described as a putative disease mechanism in pathologies associated with an elevation of homocysteine. An increased reactive oxygen species (ROS) production and apoptosis rate have been associated with several disorders of cobalamin metabolism, particularly with methylmalonic aciduria (MMA) combined with homocystinuria cblC type. In this work, we have evaluated several parameters related to oxidative stress and apoptosis in fibroblasts from patients with homocystinuria due to defects in the MTR, MTRR, and MTHFR genes involved in the remethylation pathway of homocysteine. We have also evaluated these processes by knocking down the MTRR gene in cellular models, and complementation by transducing the wild-type gene in cblE mutant fibroblasts. All cell lines showed a significant increase in ROS content and in MnSOD expression level, and also a higher rate of apoptosis with similar levels to the ones in cblC fibroblasts. The amount of the active phosphorylated forms of p38 and JNK stress-kinases was also increased. ROS content and apoptosis rate increased in control fibroblasts and in a glioblastoma cell line by shRNA-mediated silencing of MTRR gene expression. In contrast, wild-type MTRR gene corrected mutant cell lines showed a decrease in ROS and apoptosis levels. To the best of our knowledge, this study provides the first evidence that an impaired remethylation capacity due to low MTRR and MTR activity might be partially responsible for stress response.

Spectrum of MMACHC mutations in Italian and Portuguese patients with combined methylmalonic aciduria and homocystinuria, cblC type

Methylmalonic aciduria (MMA) and homocystinuria, cblC type (MIM 277400) is the most frequent inborn error of vitamin B 12. The recent identification of the disease gene, MMACHC, has permitted preliminary genotype–phenotype correlations. We studied 24 Italian and 17 Portuguese patients with cblC defect to illustrate the spectrum of mutations in a southern European population and discuss the impact that mutation identification has on routine diagnostic procedures. Since the metabolic defect raises the serum levels of homocysteine, we also tested if variants in MTHFR—playing a key role in homocysteine remethylation pathway—could act as genetic modifier in cblC defect. We found that the c.271dupA (accounting for 55% of the MMACH alleles in our cohort) followed by c.394C > T (16%) and c.331C > T (9%) were the most frequent mutations. In our study we also identified a novel mutation (c.544T > C). On the other hand, the MTHFR genotype did not appear to influence age at onset, the clinical phenotype and outcome of patients with cblC defect. This study shows that mutation screening for the most common MMACH mutations occurring in early-onset forms (c.271dupA and c.331C > T) seems to have a high diagnostic yield in a southern European population with cblC defect. Although the identification of the gene defect per se does not predict completely time and severity of disease appearance, our data corroborate the importance of a molecular testing to offer accurate prenatal diagnosis to couples at high risk of having affected children.

A Ferritin-responsive Internal Ribosome Entry Site Regulates Folate Metabolism

Cytoplasmic serine hydroxymethyltransferase (cSHMT) enzyme levels are elevated by the expression of the heavy chain ferritin (H ferritin) cDNA in cultured cells without corresponding changes in mRNA levels, resulting in enhanced folate-dependent de novo thymidylate biosynthesis and impaired homocysteine remethylation. In this study, the mechanism whereby H ferritin regulates cSHMT expression was determined. cSHMT translation is shown to be regulated by an H ferritin-responsive internal ribosome entry site (IRES) located within the cSHMT mRNA 5'-untranslated region (5'-UTR). The cSHMT 5'-UTR exhibited IRES activity during in vitro translation of bicistronic mRNA templates, and in MCF-7 and HeLa cells transfected with bicistronic mRNAs. IRES activity was depressed in H ferritin-deficient mouse embryonic fibroblasts and elevated in cells expressing the H ferritin cDNA. H ferritin was shown to interact with the mRNA-binding protein CUGBP1, a protein known to interact with the alpha and beta subunits of eukaryotic initiation factor eIF2. Small interference RNA-mediated depletion of CUGBP1 decreased IRES activity from bicistronic templates that included the cSHMT 3'-UTR in the bicistronic construct. The identification of this H ferritin-responsive IRES represents a mechanism that accounts for previous observations that H ferritin regulates folate metabolism.

Association of red blood cell 5-methyltetrahydrfoate folate with bone mineral density in postmenopausal Iranian women

Recent studies have suggested that hyperhomocysteinemia and low plasma folate are associated with fracture and also bone mineral density (BMD) and that they may contribute to the pathogenicity of osteoporosis in postmenopausal women. However, as plasma total homocysteine (tHcy) and plasma folate can be regarded as short-term markers when compared to a long-term variable such as BMD, in this study we tested the hypothesis that low red blood cell 5-methyltetrahydrofolate (RBC 5-MTHFR) as a long-term marker of the folate status may be a better predictor of BMD than plasma 5-MTHF, and its deficiency may contribute to the pathogenicity of osteoporosis in postmenopausal Iranian women. The BMD at the femoral neck and lumbar spine (measured by dual-energy X-ray absorptiometry, DXA) together with anthropometric and biochemical components of the homocysteine re-methylation pathway including plasma tHcy, 5-MTHF and vitamin B12, RBC 5-MTHF and creatinine were determined in 366 postmenopausal women. RBC 5-MTHF was more highly correlated with BMD at the lumbar spine (r=0.21, P=0.001) and femoral neck (r=0.19, P=0.004) than was plasma 5-MTHF (lumbar spine; r=0.14, P=0.03 and femoral neck; r=0.17, P=0.006). Stepwise multiple linear regression analyses revealed that RBC 5-MTHF was one of the predictors of BMD explaining 4.3 and 4.0% variance of BMD at the lumbar spine and femoral neck, respectively, whereas plasma 5-MTHF was excluded in the model and not determined to be a predictor of BMD at both the lumbar spine and femoral neck when adjusted for age, BMI, years since menopause and RBC 5-MTHF. This study suggests that RBC 5-MTHF is a better predictor of BMD than plasma 5-MTHFR when compared to a long-term marker such as BMD, and its deficiency is associated with low BMD that may contribute to the pathogenicity of osteoporosis in postmenopausal women.

Association of Low Red Blood Cell Folate Concentrations with Coronary Artery Disease in Iranians: A Matched Case-Control Study

Background: It is not fully established whether the increasing risk of coronary artery disease (CAD) is associated with high plasma homocysteine levels or components of the homocysteine remethylation pathway, e.g. vitamin B₁₂ or 5-methyltetrahydrofolate (5-MTHF) in plasma and red blood cells (RBC). In this study, we tested the hypothesis that 5-MTHF in RBC, which represents the long-term folate status of individuals, may be a more reliable marker of homocysteine remethylation pathway disturbances, and its deficiency may be associated with CAD in Iranians. Methods: Plasma total homocysteine (tHcy), vitamin B₁₂, and plasma and RBC 5-MTHF were measured in 200 angiographically documented patients and 200 controls matched for sex and age. Results: In the plasma, tHcy levels were significantly higher in cases compared to controls (geometric mean 12.9 ± 6.5 vs. 10.6 ± 5.6 µmol/l, p = 0.04). However, RBC 5-MTHF (527.2 ± 185.9 vs. 461.3 ± 117.9 nmol/l, p = 0.007) and vitamin B₁₂ (254.2 ± 132.8 vs. 182.2 ± 110.4 pmol/l, p = 0.04) were significantly higher in controls than patients. RBC 5-MTHF was a strong and independent predictor of plasma tHcy (β = –0.01, p = 0.003, r² = 0.19). Subjects in the lowest quartile of red-cell 5-MTHF had a 2.5-fold increased prevalence of CAD compared to subjects in the highest quartile. The association of CAD in the first quartile with red-cell 5-MTHF remained significant when adjusted for plasma tHcy, vitamin B₁₂, hypertension and hypercholesterolemia (odds ratio, OR 2.3, confidence interval: 1.1–3.9, p = 0.01). However, the association between CAD in the highest quartile and plasma tHcy decreased and became insignificant when adjusted for red-cell 5-MTHF, vitamin B₁₂, hypertension and hypercholesterolemia (OR 1.27, confidence interval: 0.96–1.69, p = 0.11). Conclusion: In this study, the association between CAD and low RBC 5-MTHF was stronger than with plasma 5-MTHF and plasma tHcy levels, indicating that RBC 5-MTHF may be a more stable parameter to study disturbances in the homocysteine remethylation pathway in Iranians.

Red blood cell methylfolate and plasma homocysteine as risk factors for venous thromboembolism: a matched case-control study

Moderate hyperhomocysteinaemia is a risk factor for venous thromboembolism. We do not know whether this risk depends on homocysteine itself or on components of the homocysteine remethylation pathway, such as methylfolate. We did a case-control study to analyse the relation between the major components of the homocysteine remethylation pathway and risk of venous thromboembolism. We measured concentrations of homocysteine, methionine, and folate in plasma, total folate and methylfolate in red-blood cells, and 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T genotype and other known risk factors for venous thromboembolic disease in 243 patients with deep vein thrombosis or pulmonary embolism and controls matched for sex and age. Concentrations in plasma of homocysteine differed significantly between cases and controls. We noted a strong concentration-dependent association between concentrations of methylfolate in red-blood cells and risk of venous thromboembolism. The adjusted conditional odds ratio ranged from 1.0 for methylfolate 249 microg/L or greater to 7.1 (3.2-15.8) for methylfolate 141 microg/L or less. Methionine concentrations below the median were also independently associated with raised risk of venous thromboembolic disease, as were established risk factors such as high body-mass index, history of cancer, family history of thromboembolism, oral contraceptive use, and factor V Leiden mutation. Furthermore, the association between concentrations of methylfolate in red-blood cells and risk of thromboembolism varied according to MTHFR C677T genotype. Measurement of methylfolate concentrations in red-blood cells might help to identify people at risk of venous thromboembolism.

A Common Variant in Methionine Synthase Reductase Combined with Low Cobalamin (Vitamin B12) Increases Risk for Spina Bifida

Impairment of folate and cobalamin (vitamin B12) metabolism has been observed in families with neural tube defects (NTDs). Genetic variants of enzymes in the homocysteine remethylation pathway might act as predisposing factors contributing to NTD risk. The first polymorphism linked to increased NTD risk was the 677C→T mutation in methylenetetrahydrofolate reductase (MTHFR). We now report a polymorphism in methionine synthase reductase (MTRR), the enzyme that activates cobalamin-dependent methionine synthase. This polymorphorism, 66A→G (I22M), has an allele frequency of 0.51 and increases NTD risk when cobalamin status is low or when the MTHFR mutant genotype is present. Genotypes and cobalamin status were assessed in 56 patients with spina bifida, 58 mothers of patients, 97 control children, and 89 mothers of controls. Cases and case mothers were almost twice as likely to possess the homozygous mutant genotype when compared to controls, but this difference was not statistically significant. However, when combined with low levels of cobalamin, the risk for mothers increased nearly five times (odds ratio (OR) = 4.8, 95% CI 1.5–15.8); the OR for children with this combination was 2.5 (95% CI 0.63–9.7). In the presence of combined MTHFR and MTRR homozygous mutant genotypes, children and mothers had a fourfold and threefold increase in risk, respectively (OR = 4.1, 95% CI 1.0–16.4; and OR = 2.9, 95% CI 0.58–14.8). This study provides the first genetic link between vitamin B12 deficiency and NTDs and supports the multifactorial origins of these common birth defects. Investigation of this polymorphism in other disorders associated with altered homocysteine metabolism, such as vascular disease, is clearly warranted.

Is the Oral Methionine Loading Test Insensitive to the Remethylation Pathway of Homocysteine?

To the Editor: Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the remethylation pathway of homocysteine. The study by Girelli et al[1][1] showed that the C677T mutation of the gene encoding for MTHFR is common in Italy, is not associated with coronary atherosclerotic disease, and

Plasma homocysteine levels related to interactions between folate status and methylenetetrahydrofolate reductase: A study in 52 healthy subjects

Hyperhomocysteinemia, a risk factor for vascular disease, is related to vitamin B 12, vitamin B 6, and especially folate deficiency, or to genetic factors such as mutations in methylenetetrahydrofolate reductase (MTHFR), an enzyme involved in the remethylation pathway of homocysteine to methionine. Recently, a C677 → T mutation identified in the MTHFR gene was found to be frequently associated with decreased MTHFR activity and an elevated plasma homocysteine concentration. Since hyperhomocysteinemia seems to be determined by both genetic and environmental factors, we studied the interactions between MTHFR (phenotype and genotype) and folate status, including methyltetrahydrofolate (methylTHF), the product of MTHFR, on the homocysteine concentration in 52 healthy subjects, (28 women and 24 men; mean age, 32.7 years). MTHFR activity seems to be dependent on folate status, as shown by a lower activity in folate-deficient subjects and a return to normal values after supplementation with folic acid, and also by a decreased enzymatic activity on phytohemagglutinin (PHA)- stimulated lymphocytes grown in a folic acid-deficient medium. Conversely, the C677 → T mutation seems to influence folate metabolism. Subjects who were homozygous for this mutation (+/+) had significantly higher plasma homocysteine and lower plasma folate and total and methylfolate levels in red blood cells (RBCs) than heterozygous (+/−) and normal (−/−) subjects. The ratio of RBC methylfolate to RBC total folate was, respectively, 0.27 in +/+, 0.66 in +/−, and 0.71 in −/−. This mutation seems to have an impact on methylTHF generation. These data illustrate the interactions between nutritional and genetic factors.