MTHFR Protein Research Articles

Identification of common candidate genes and pathways for Spina Bifida and Wilm’s Tumor using an integrative bioinformatics analysis

Spina Bifida (SB) and Wilm’s Tumor (WT) are conditions, both associated with children. Several studies have shown that WT later develops in SB patients, which led us to elucidate common key genes and linked pathways of both conditions, aimed at their concurrent therapeutic management. For this, integrated bioinformatics analysis was employed. A comprehensive manual curation of genes identified 133 and 139 genes associated with SB and WT, respectively, which were used to construct a single protein-protein interaction (PPI) network. Topological parameters analysis of the network showed its scale-free and hierarchical nature. Centrality-based analysis of the network identified 116 hubs, of which, 6 were called the key genes attributed to being common between SB and WT besides being the hubs. Gene enrichment analysis of the 5 most essential modules, identified important biological processes and pathways possibly linking SB to WT. Additionally, miRNA-key gene-transcription factor (TF) regulatory network elucidated a few important miRNAs and TFs that regulate our key genes. In closing, we put forward TP53, DICER1, NCAM1, PAX3, PTCH1, MTHFR; hsa-mir-107, hsa-mir-137, hsa-mir-122, hsa-let-7d; and YY1, SOX4, MYC, STAT3; key genes, miRNAs and TFs, respectively, as the key regulators. Further, MD simulation studies of wild and Glu429Ala forms of MTHFR proteins showed that there is a slight change in MTHFR protein structure due to Glu429Ala polymorphism. We anticipate that the interplay of these three entities will be an interesting area of research to explore the regulatory mechanism of SB and WT and may serve as candidate target molecules to diagnose, monitor, and treat SB and WT, parallelly. Communicated by Ramaswamy H. Sarma

Modulation of blood pressure regulatory genes in the Agtrap-Plod1 locus associated with a deletion in Clcn6.

The AGTRAP‐PLOD1 locus is a conserved gene cluster containing several blood pressure regulatory genes, including CLCN6, MTHFR, NPPA, and NPPB. Previous work revealed that knockout of Clcn6 on the Dahl Salt‐Sensitive (SS) rat background (SS‐Clcn6) resulted in lower diastolic blood pressure compared to SS‐WT rats. Additionally, a recent study found sickle cell anemia patients with mutations in CLCN6 had improved survival and reduced stroke risk. We investigated whether loss of Clcn6 would delay the mortality of Dahl SS rats on an 8% NaCl (HS) diet. No significant difference in survival was found. The ability of Clcn6 to affect mRNA expression of nearby Mthfr, Nppa, and Nppb genes was also tested. On normal salt (0.4% NaCl, NS) diets, renal Mthfr mRNA and protein expression were significantly increased in the SS‐Clcn6 rats. MTHFR reduces homocysteine to methionine, but no differences in circulating homocysteine levels were detected. Nppa mRNA levels in cardiac tissue from SS‐Clcn6 rat in both normotensive and hypertensive conditions were significantly reduced compared to SS‐WT. Nppb mRNA expression in SS‐Clcn6 rats on a NS diet was also substantially decreased. Heightened Mthfr expression would be predicted to be protective; however, diminished Nppa and Nppb expression could be deleterious and by preventing or blunting vasodilation, natriuresis, and diuresis that ought to normally occur to offset blood pressure increases. The conserved nature of this genetic locus in humans and rats suggests more studies are warranted to understand how mutations in and around these genes may be influencing the expression of their neighbors.

5,10-Methylenetetrahydrofolate reductase becomes phosphorylated during meiotic maturation in mouse oocytes.

The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) links the folate cycle that produces one-carbon units with the methionine cycle that converts these into S-adenosylmethionine (SAM), the universal methyl donor for almost all methyltransferases. Previously, MTHFR has been shown to be regulated by phosphorylation, which suppresses its activity. SAM levels have been shown to increase substantially soon after initiation of meiotic maturation of the mouse germinal vesicle (GV) stage oocyte and then decrease back to their original low level in mature second meiotic metaphase (MII) eggs. As MTHFR controls the entry of one-carbon units into the methionine cycle, it is a candidate regulator of the SAM levels in oocytes and eggs. Mthfr transcripts are expressed in mouse oocytes and preimplantation embryos and MTHFR protein is present at each stage. In mature MII eggs, the apparent molecular weight of MTHFR was increased compared with GV oocytes, which we hypothesized was due to increased phosphorylation. The increase in apparent molecular weight was reversed by treatment with lambda protein phosphatase (LPP), indicating that MTHFR is phosphorylated in MII eggs. In contrast, LPP had no effect on MTHFR from GV oocytes, 2-cell embryos, or blastocysts. MTHFR was progressively phosphorylated after initiation of meiotic maturation, reaching maximal levels in MII eggs before decreasing again after egg activation. As phosphorylation suppresses MTHFR activity, it is predicted that MTHFR becomes inactive during meiotic maturation and is minimally active in MII eggs, which is consistent with the reported changes in SAM levels during mouse oocyte maturation.

Loss of Clcn6 Alters Expression of Nearby Regulatory Blood Pressure Genes but Does Not Affect High Salt Induced Mortality in Dahl Salt‐Sensitive Rats

Genome‐wide association studies in both humans and Dahl salt‐sensitive (SS) rats have identified the AGTRAP‐PLOD1 locus gene cluster as an area of interest that contains several genes with the ability to regulate blood pressure, including CLCN6, MTHFR, NPPA,and NPPB. We previously demonstrated that a functional knock‐out of Clcn6 on the Dahl SS rat background (SS‐Clcn6) had significantly reduced diastolic blood pressure (BP) compared to WT rats when challenged with a high 4% NaCl (HS) diet to induce hypertension. Increased stroke risk from high blood pressure is well established, and genetic variants, such as those in CLCN6 that reduce BP may also be expected to lower the risk of stroke within this population. Furthermore, a recent paper reported that loss of function mutations in CLCN6 were associated with long‐term survival in a small cohort of sickle cell anemia patients with lower stroke rates. We hypothesized that loss of Clcn6 would delay HS‐induced mortality of Dahl SS rats on a 8% NaCl diet by slowing or preventing lethal cerebrovascular events. However, we observed no significant differences in longevity or neuronal cell death between the SS‐Clcn6 knock‐out and SS‐WT groups. Further assessment led us to examine whether a mutation in Clcn6 could alter mRNA expression of nearby Mthfr, Nppa, and Nppb genes, thereby complicating the genetic phenotype of our knockout animal. We found that on normal salt (NS) diets, renal Mthfr mRNA and protein expression were significantly increased in the SS‐Clcn6 rats, while no differences were observed between WT and knockout rats on a HS diet. MTHFR encodes Methylene tetrahydrofolate reductase (MTHFR), which is involved in reducing homocysteine to methionine, and elevated levels of circulating homocysteine (tHcy) are associated with higher cardiovascular and thromboembolic risk. No significant differences in circulating tHCy between SS‐WT and SS‐Clcn6 (10.63 ± 0.47 vs 10.05 ± 1.12 nM/mL, respectively), were observed on a NS diet; however, there was a trend toward decreased tHcy in SS‐Clcn6 rats on a HS diet (11.85 ± 2.55 vs 6.90 ± 0.40 nM/mL, p=0.08). Genetic ablation of Clcn6 resulted in significant differences in NppamRNA levels in cardiac tissue from SS‐WT and SS‐Clcn6 rat in both normotensive (153.0 ± 23.3 vs 17.61 ±10.67 a.u., p<0.01) and hypertensive (614.9 ± 115.2 vs 153.0 ± 23.3 a.u., p<0.01) conditions. A substantial reduction in NppbmRNA expression in SS‐Clcn6 rats on a NS diet (66.04 ± 4.50 vs 24.01 ± 8.80 a.u., p<0.01) was also found. The observed genetc variances are controversial; while increased Mthfrexpression would be predicted to be protective, reduced Nppaand Nppbexpression could potentially be deleterious and prevent increased vasodilation, natriuresis, and diuresis that should normally occur to compensate for BP increases. Due to the similarity of this genetic locus in humans and rats, more studies are warranted to fully understand how genetic mutations in this cluster may be altering the expression of their neighbors as well as contributing to BP homeostasis.

Generation of a Human iPSC (ICSSUi002-A) with MTHFR SNP (rs1801133, TT) from Thoracic Aortic Dissection Patient

Thoracic aortic dissection is a devastating cardiovascular disease with an increasing annual incidence. The homozygous mutation in rs1801133 site has been accepted for decreased enzyme activity of mutant MTHFR protein, contributing to an accumulated homocysteine in blood. Recently, elevated homocysteine level is causally associated with an increased risk of cardiovascular disease. Conversely, the relationship between rs1801133 and thoracic aortic dissection is poorly understood. Here, the generated human induced pluripotent stem cell (iPSC) line provided a novel strategy for investigating the underlying mechanism of MTHFR mutation (rs1801133, TT) and its implication in the pathogenesis of thoracic aortic dissection.

MTHFR C677T Polymorphism and Serum Homocysteine Level as Risk Factors of Coronary Heart Disease in Patients with Androgenetic Alopecia: A Case Control Study

BackgroundAndrogenetic alopecia (AGA) is associated with a risk of coronary heart disease (CHD), although the causes underlying this association are not clear. Serum homocysteine (SH) is a known risk factor for CHD, and methylene tetrahydrofolate reductase enzyme (MTHFR) plays a crucial role in the remethylation of homocysteine to methionine. The polymorphism C677T that affects the catalytic domain of the MTHFR protein leads to a high levels of SH. Our hypothesis was that this polymorphism and SH level are risk factors for CHD in patients with AGA. Materials and methodsA total of 106 patients with AGA and 100 well-matched healthy controls were enrolled in the study. SH levels were estimated. DNA was extracted and polymerase chain reaction amplification, followed by restriction enzyme digestion for MTHFR (C677T) gene, was conducted. ResultsSH levels were significantly higher in the patient group and highest in those with the TT genotype. The mutant T allele was associated with hyperhomocysteinemia and an increased risk of CHD in patients with AGA. ConclusionsAGA is associated with a higher risk of developing CHD due to the associated higher level of SH that, in turn, depends on and is correlated with mutant MTHFR genotypes. Cardiac evaluation and follow-up of patients with AGA is recommended for early detection and treatment of CHD to avoid an overall detrimental course.

Testicular MTHFR deficiency may explain sperm DNA hypomethylation associated with high dose folic acid supplementation.

Supplementation with high doses of folic acid, an important mediator of one-carbon transfers for DNA methylation, is used clinically to improve sperm parameters in infertile men. We recently detected an unexpected loss of DNA methylation in the sperm of idiopathic infertile men after 6 months of daily supplementation with 5 mg folic acid (>10× the daily recommended intake-DRI), exacerbated in men homozygous for a common variant in the gene encoding an important enzyme in folate metabolism, methylenetetrahydrofolate reductase (MTHFR 677C>T). To investigate the epigenomic impact and mechanism underlying effects of folic acid on male germ cells, wild-type and heterozygote mice for a targeted inactivation of the Mthfr gene were fed high-dose folic acid (10× the DRI) or control diets (CDs) for 6 months. No changes were detected in general health, sperm counts or methylation of imprinted genes. Reduced representation bisulfite sequencing revealed sperm DNA hypomethylation in Mthfr+/- mice on the 10× diets. Wild-type mice demonstrated sperm hypomethylation only with a very high dose (20×) of folic acid for 12 months. Testicular MTHFR protein levels decreased significantly in wild-type mice on the 20× diet but not in those on the 10× diet, suggesting a possible role for MTHFR deficiency in sperm DNA hypomethylation. In-depth analysis of the folic acid-exposed sperm DNA methylome suggested mouse/human susceptibility of sequences with potential importance to germ cell and embryo development. Our data provide evidence for a similar cross-species response to high dose folic acid supplementation, of sperm DNA hypomethylation, and implicate MTHFR downregulation as a possible mechanism.

A family based triad study evaluating the role of MTHFR gene polymorphisms in spontaneous abortions

ObjectiveTo explore the combined association of MTHFR gene polymorphisms (C677T, A1298C) with spontaneous abortions in a triad group from Telangana State of South Indian population. MaterialsA total of 80 case families with spontaneous abortions and 100 control families with medically terminated pregnancies were considered. Fetal tissue with <20weeks of gestational age and peripheral blood from their respective parents were collected. MethodsDNA was isolated by phenol-chloroform method from both tissue and peripheral blood samples and then genotyped using Amplification Refractory Mutation System–Polymerase Chain Reaction followed by agarose gel electrophoresis. Genotype and allelic frequencies of MTHFR polymorphisms were determined by Odds Ratio with 95% Confidence Intervals and level of significance was considered at p value<0.05. The mRNA secondary structures were predicted and the transcription factor binding sites were determined. ResultsStatistically significant association was observed with 677C/T polymorphism in case fetal (p=0.006), maternal (p<0.001) and paternal (p=0.03) groups compared to their respective control groups. A significant difference in the genotypic distribution of 1298 A/C polymorphism was observed only in the maternal group (p=0.005) compared to controls. The combined frequencies of 677TT/1298AC genotypes revealed a significant association with an increased risk in all the case triad groups compared to their respective controls. The in silico analysis revealed the structural change and transcription factor binding site deviations in the variant allele compared to wild type allele in the mRNA regulation of MTHFR protein. ConclusionThe present study supports the major role of MTHFR gene polymorphisms in the etiology of spontaneous abortions.

Functional characterization of missense mutations in severe methylenetetrahydrofolate reductase deficiency using a human expression system.

5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADPH-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate using FAD as the cofactor. Severe MTHFR deficiency is the most common inborn error of folate metabolism, resulting in hyperhomocysteinemia and homocystinuria. Approximately 70 missense mutations have been described that cause severe MTHFR deficiency, however, in most cases their mechanism of dysfunction remains unclear. Few studies have investigated mutational specific defects; most of these assessing only activity levels from a handful of mutations using heterologous expression. Here, we report the in vitro expression of 22 severe MTHFR missense mutations and two known single nucleotide polymorphisms (p.Ala222Val, p.Thr653Met) in human fibroblasts. Significant reduction of MTHFR activity (<20% of wild-type) was observed for five mutant proteins that also had highly reduced protein levels on Western blot analysis. The remaining mutations produced a spectrum of enzyme activity levels ranging from 22-122% of wild-type, while the SNPs retained wild-type-like activity levels. We found increased thermolability for p.Ala222Val and seven disease-causing mutations all located in the catalytic domain, three of which also showed FAD responsiveness in vitro. By contrast, six regulatory domain mutations and two mutations clustering around the linker region showed increased thermostability compared to wild-type protein. Finally, we confirmed decreased affinity for NADPH in individual mutant enzymes, a result previously described in primary patient fibroblasts. Our expression study allows determination of significance of missense mutations in causing deleterious loss of MTHFR protein and activity, and is valuable in detection of aberrant kinetic parameters, but should not replace investigations in native material.

Identification of a functional SNP in the 3'-UTR of caprine MTHFR gene that is associated with milk protein levels.

Xinong Saanen (n=305) and Guanzhong (n=317) dairy goats were used to detect SNPs in the caprine MTHFR 3'-UTR by DNA sequencing. One novel SNP (c.*2494G>A) was identified in the said region. Individuals with the AA genotype had greater milk protein levels than did those with the GG genotype at the c.*2494G>A locus in both dairy goat breeds (P<0.05). Functional assays indicated that the MTHFR:c.2494G>A substitution could increase the binding activity of bta-miR-370 with the MTHFR 3'-UTR. In addition, we observed a significant increase in the MTHFR protein level of AA carriers relative to that of GG carriers. These altered levels of MTHFR protein may account for the association of the SNP with milk protein level.

A Functional Variant at the miR-214 Binding Site in the Methylenetetrahydrofolatereductase Gene Alters Susceptibility to Gastric Cancer in a Chinese Han Population

Background and Aims: Single nucleotide polymorphisms in miRNA binding sites, which are located in mRNA 3' untranslated regions (3'-UTRs), were recently found to influence microRNA-target interactions. Specifically, such polymorphisms can modulatebinding affinity or create or destroy miRNA-binding sites; such variants have also been found to be associated with cancer risk. In this study, we explored the effect of a functional variant at the miR-214 binding site in the methylenetetrahydrofolate reductase gene (rs114673809) on gastric cancer (GC) risk in a hospital-based case-control study in a Chinese Han population. Methods and Results: We genotyped the rs114673809 polymorphism in 345 gastric cancer patients and 376 cancer-free controls using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique. The functions of rs114673809 were investigated using a luciferase activity assay and validated by immunoblotting. We found that participants carrying the rs114673809 AA genotype or A allele had a significantly increased risk of gastric cancer (OR = 1.667, 95% CI = 1.044-2.660, P = 0.034; OR = 1.261, 95% CI = 1.017-1.563, P = 0.037, respectively) compared to those carrying the GG genotype and G allele. In addition, rs114673809 modified the binding of hsa-miR-214 to MTHFR as well as MTHFR protein levels in gastric cancer patients. Conclusion: Our data suggested that rs114673809, which is located at the miR-214 binding site in the 3'-UTR of MTHFR, may play an important role in the development of gastric cancer in a Chinese Han population.

PO-0406 The Contribution Of Prothrombotic Disorders To Perinatal Arterial Ischaemic Stroke (pais): A Study Of Case-control Parent-child Pairs

Background Prothrombotic risk factors (PRF) are suggested to be involved in the pathogenesis of PAIS. However, most of published studies are retrospective, they vary in the PRF tested and parents are not usually investigated. Objective To determine the impact of parental and infant thrombophilia on neonatal cases diagnosed of PAIS in a prospective case-control study. Methods Factor V (G1691A mutation), prothrombin G20210A variant, MTHFR C677T genotype, antithrombin, protein C, protein S, lipoprotein (a), homocystein (Hcy) and anticardiolipin antibodies were investigated in 45 infant-parent pairs with PAIS and in 85 controls. Blood samples were drawn within the first week of life. Results All thrombophilic factors investigated were similar or even less frequent among patients with PAIS and their parents compared to controls. The most frequent PFRs were Hcy > 11 and MTHFR homozygosity in cases (7.5% and 5.3%, respectively) and in controls (24.4% and 13.1%, respectively). Thirteen neonates diagnosed of PAIS (28.9%) had at least 1 PFR, compared to 39 subjects (44.3%) in the control group (OR/95% CI, 0.51/0.23 to 1.10) (p Conclusion Our data do not support that PFR play a major role in PAIS.

MTHFR gene mutation in an elderly patient that comes with portal vein thrombosis and ascites: a case report

Extrahepatic portal vein thrombosis is a common cause of presinusoidal portal hypertension and often the two entities connote each other. Portal vein thrombosis is reported in patients with underlying cirrhosis by 25%. Other than cirrhotic patients; malignancies, myeloproliferative disorders, collagen tissue disorders, pancreatitis, and thrombophilias can cause portal vein thrombosis. Conditions predisposing to thrombosis are mostly acquired. Hereditary disorders predisposing to thrombosis like FV Leiden mutation, prothrombin gene mutation, MTHFR gene mutation, protein C, S, and antithrombin 3 deficiency diseases are more rare. Sometimes with an accompanying factor provoking thrombosis, these abnormalities are usually under the age of 50 and present with venous thrombosis. Here, we present a case of homozygous MTHFR gene mutation that comes with portal vein thrombosis, liver failure and ascites.

Inherited Pro-Thrombotic Risk Factors in Children of South Moravia Region, Czech Republic

Abstract 5136 Introduction:Pro-thrombotic risk factors are associated with increased risk of thrombosis. Some of them are inherited, but clinical manifestation of thrombosis in childhood is rare. It is most likely to appear in the adulthood. When leading to thrombosis during childhood, risk factors must be either very strong (e. g. homozygous deficiency of natural coagulation inhibitors), or must act together with other, usually acquired factors. Aims:- To analyse data of children referred for thrombophilia screening to Thrombosis Haemostasis centre, Dpt. of Paediatric Haematology University Hospital Brno, Czech Republic in years 2005–09.- To specify the characteristics of that cohort and identify the reasons for referral.-To determine prevalence of respective risk factors (RF) in children within study cohort.-To analyse possible correlation between related RF findings (ProC Global and FV Leiden, Protein C, Protein S; homocystein and MTHFR C677T polymorphism). Methods:We have collected data of all consecutive children aged from 0 to 18 years referred to our out-patient clinic for assessment of inherited thrombophilia in years 2005–2009 for any reason. Data recorded for each patient were as follows: age, gender, reason for assessment, thrombotic event and its type (if applicable) and vascular anomalies. Laboratory examinations done: FV Leiden mutation, Prothrombin gene mutation G20210A, MTHFR C677T polymorphism, protein C, protein S and Antithrombin levels, coagulation activity of F VIII, ProC Global test, homocystein and lipoprotein (a) level. Data were statistically analyzed to reach the aims of the study. Results:Data of 849 Caucasian children aged 0–18 years were available for analysis, 475 girls (55. 9 %) and 374 boys (44. 1 %). Median age was 11 years at the time of referral, boys 10 years, girls 13 years. Reasons for thrombophilia screening were as follows: family history of thrombosis/thrombophilia (70. 6 %), vascular anomaly (10. 8 %), thrombotic event (8 %) and others (10. 6 %). Thrombophilia screening was in more than 70 % of children performed before 15 years of age. Thrombotic event has been recorded in 79 patients (9. 3 %), both arterial (37. 6 %) and venous (63. 3 %). Adolescent patients had significantly more thrombotic events than younger children. Most of them were venous thromboses. Ischemic strokes and other arterial events were relatively more frequent in younger children. Frequency of “positive” laboratory findings in the studied cohort were as follows: MTHFR 60. 3 %, positive ProC Global test findings 58. 1 %, FV Leiden (heterozygous)37 %, hyperlipoproteinemia (a) 28. 5 %, protein S deficiency 11. 3 %, high FVIII activity 9. 4 %, Prothrombin gene mutation (heterozygous) 7. 7 %, Antithrombin deficiency 2. 7 %, elevation of homocystein 1. 6 % and protein C deficiency 1 %. Findings have confirmed high sensitivity of ProC Global test to the pathology in protein C pathway, particularly to FV Leiden mutation (sensitivity 98. 4 %). There was no statistically significant relation between homocystein level and MTHFR polymorphism status. Prevalence of RF in study cohort was found to be similar to the literature data. Deficiencies of natural coagulation inhibitors (AT, PC and PS) were rare, pro-thrombotic mutations were more frequent. Data suggest, that role of inherited pro-thrombotic risk factors in the development of thrombotic event in children is not the major one. It is very likely, that more potent risk factors, often acquired ones, are often important for development of thrombotic event during childhood. Conclusions:In our centre, we assessed proximately 170 children referred for thrombophilia screening per year. The incidence is thus 8 children per 10000. During 2005–09the main reason for referral was positive family history, but children were often referred in younger age, than recommended. The evidence available to date suggests that it is neither necessary nor useful to examine asymptomatic children before they reach puberty. Finding of inherited thrombophilia, and thus possibility to use measures preventing eventual thrombotic event, are more important in adolescence and adulthood rather than in younger age. In children with thrombotic event, however, it is recommended to screen for inherited risks. Diagnosis and treatment of thrombotic event in children requires a specialised care and relevant expertise of the Paediatric Thrombosis Haemostasis centre. Disclosures:No relevant conflicts of interest to declare.

The human MTHFR rs4846049 polymorphism increases coronary heart disease risk through modifying miRNA binding

Background and aimsAbnormal functioning of 5,10-methylenetetrahydrofolate reductase (MTHFR) enhances the risk for coronary heart disease (CHD). Here, we tested whether a single-nucleotide polymorphism (SNP) located in the 3′ untranslated region (UTR) of MTHFR was associated with CHD susceptibility by affecting microRNAs binding. Methods and resultsWe first analyzed in silico the SNPs localized in the 3′ UTR of MTHFR for their ability to modify miRNA binding. We observed that rs4846049 (G > T) was a potential candidate SNP to modulate miRNAs:MTHFR mRNA complex, with the greatest changed binding free energy for has-miR-149. Based on luciferase analysis, hsa-miR-149 inhibited the activity of the reporter vector carrying -T allele, but not -G allele. We further conducted a case–control study (654 vs 455) in a Chinese Han population. rs4846049 was significantly associated with increased risk for CHD. In addition, the T allele was associated with decreased levels of HDL-cholesterol and apoA. Finally, we observed a reduced MTHFR protein level in peripheral blood mononuclear cells of CHD patients with TT carriers compared to GG carriers of rs4846049. ConclusionOur results suggest that rs4846049 (G > T) of MTHFR is associated with increased risk for CHD. We also identified a potentially pathogenetic mechanism of SNP-modified posttranscriptional gene regulation by miRNAs to MTHFR.

Homocysteine modulates the effect of simvastatin on expression of ApoA-I and NF- B/iNOS

Statins can ameliorate atherosclerosis by inhibition of cholesterol biosynthesis or by modulation of inflammation. In earlier work, we showed that homocysteine (Hcy) reduced synthesis of apolipoprotein A-I (ApoA-I). Our goal in this study was to determine whether Hcy could interfere with the ability of simvastatin to increase ApoA-I synthesis or to modify statin-dependent regulation of inflammatory factors. Human HepG2 hepatocarcinoma cells and murine RAW264.7 macrophages were treated with simvastatin, with and without Hcy, to examine the expression of ApoA-I and nuclear factor-kappaB (NF-kappaB) or the NF-kappaB target, inducible nitric-oxide synthase (iNOS), respectively. Mice with methylenetetrahydrofolate reductase (Mthfr) deficiency, an animal model of hyperhomocysteinemia, were administered simvastatin (in diets or by injection) for in vivo assessment of these interactions. In HepG2 cells, Hcy reduced the statin-dependent increases in ApoA-I protein, mRNA, and ApoA-I promoter activity. In RAW264.7 macrophages, simvastatin decreased, whereas Hcy increased, the expression of pro-inflammatory NF-kappaB protein; in the presence of both Hcy and simvastatin, the pro-inflammatory effect of Hcy prevailed. Hcy increased mRNA levels of iNOS in the macrophage line; the combination of Hcy and simvastatin resulted in a trend towards greater induction. In mouse studies, simvastatin decreased cholesterol levels, but levels of ApoA-I in Mthfr-deficient mice remained lower than those in Mthfr(+/+) mice. Simvastatin injection increased iNOS protein and mRNA levels in peripheral blood of hyperhomocysteinemic Mthfr-deficient mice, but not in Mthfr(+/+) mice. The drug also increased MTHFR protein in cells and mouse liver, an effect that was modified by Hcy. These findings provide a link between statins and folate-dependent Hcy metabolism, and suggest that Hcy interferes with some anti-atherogenic and anti-inflammatory properties of simvastatin. Our work may have clinical relevance for hyperhomocysteinemic individuals on statin therapy.

Endoplasmic Reticulum Stress Increases the Expression of Methylenetetrahydrofolate Reductase through the IRE1 Transducer

Methylenetetrahydrofolate reductase (MTHFR), an enzyme in folate and homocysteine metabolism, influences many cellular processes including methionine and nucleotide synthesis, methylation reactions, and maintenance of homocysteine at nontoxic levels. Mild deficiency of MTHFR is common in many populations and modifies risk for several complex traits including vascular disease, birth defects, and cancer. We recently demonstrated that MTHFR can be up-regulated by NF-kappaB, an important mediator of cell survival that is activated by endoplasmic reticulum (ER) stress. This observation, coupled with the reports that homocysteine can induce ER stress, prompted us to examine the possible regulation of MTHFR by ER stress. We found that several well characterized stress inducers (tunicamycin, thapsigargin, and A23187) as well as homocysteine could increase Mthfr mRNA and protein in Neuro-2a cells. The induction of MTHFR was also observed after overexpression of inositol-requiring enzyme-1 (IRE1) and was inhibited by a dominant-negative mutant of IRE1. Because IRE1 triggers c-Jun signaling, we examined the possible involvement of c-Jun in up-regulation of MTHFR. Transfection of c-Jun and two activators of c-Jun (LiCl and sodium valproate) increased MTHFR expression, whereas a reported inhibitor of c-Jun (SP600125) and a dominant-negative derivative of c-Jun N-terminal kinase-1 reduced MTHFR activation. We conclude that ER stress increases MTHFR expression and that IRE1 and c-Jun mediate this activation. These findings provide a novel mechanism by which the ER can regulate homeostasis and allude to an important role for MTHFR in cell survival.

Universal Thrombophilia Screening with Risk Stratification and Anticoagulation Management in Pediatric Renal Transplant Recipients.

Objective To identify renal transplant patients at a higher risk for allograft thrombosis and develop a risk stratification protocol for post-operative anticoagulation.Background According to the North American Pediatric Renal Transplant Cooperative Study data, vascular thrombosis is responsible for 12% of renal transplant failures. Congenital and acquired prothrombotic disorders have recently been found to play a major role in defining outcome of the renal transplants.Methods In 2007, we initiated universal screening for acquired and congenital thrombophilic risk factors for all renal transplant patients prior to transplant. They were assigned a risk category (Level I-IV) based on the presence of thrombophilic risk factors and their post-operative anticoagulation plan was pre-determined based on their risk level (Table). Low-dose unfractionated heparin drip was initiated a few hours after the transplant and titrated to achieve a 1.5 fold aPTT level. Anticoagulation was changed to low molecular weight heparin (LMWH) on day 2–4, when the risk of major bleed was over, and the serum creatinine had normalized.Result Nine patients have been screened and 8/9 were found to be at high risk for thrombosis (Level II or III). The prothrombotic risk factors identified were Prothrombin gene mutation (2), MTHFR (8), Lupus anticoagulant (2) and low protein S (2). Five patients have undergone renal transplant, and have been treated with LMWH post-operatively as per plan, with successful outcome. No patient has developed allograft thrombosis. 1/5 patients developed a peri-renal hematoma requiring evacuation, but completed prophylactic LMWH therapy.Conclusion Renal allograft thrombosis is preventable. Universal screening can stratify risk and allow an effective intervention to reduce that risk. Our Institution has developed a unique risk stratification protocol to identify patients at high risk for allograft thrombosis and prevent this complication by post-operative anticoagulation.Risk Categorization and Management PlanRisk LevelThrombophiliaManagementLevel IEvidence of Thrombophilia (other than MTHFR) and previous thrombosisHeparin postoperatively and then LMWH bid for 6 monthsLevel IIEvidence of Thrombophilia (other than MTHFR), No history of thrombosisHeparin postoperatively, LMWH bid for 3 months, then qd for 3 monthsLevel IIIMTHFR with normal homocysteineHeparin postoperatively, then LMWH qd for 3 monthsLevel IVNo evidence of thrombophiliaNo anticoagulation

FV Leiden mutation and risk of recurrent venous thromboembolism in Serbian population

The absolute rate of recurrence of venous thromboembolism (VTE) is approximately 5% per year. There is a lower rate of recurrence in provoked VTE, and higher in idiopathic one. So far, there is no consensus whether hereditary thrombophilia should be considered as a persistent risk factor, and whether it requires long-term anticoagulant therapy. The aim of our study was to estimate the risk of recurrent VTE in patients carrying FV Leiden mutation in Serbian population. In retrospective study (1994-2006), we have evaluated the risk of recurrent VTE in 56 patients who are carriers of FV Leiden mutation, in comparison to group consisting of 56 patients non-carriers of FV Leiden mutation. Patients with FII G20210A and MTHFR C677T mutations, antiphospholipid antibodies, antithrombin III, protein C or protein S deficiency, malignancies and diabetes were excluded from the study. Recurrent VTE occurred in 44.6% of the patients, carriers of the FV Leiden mutations, vs. 26.7% in non-carriers group (P<0.05). The incidence rate was 3.7 and 2.2% per year, respectively. The estimated relative risk of recurrence for FV Leiden carriers was 1.67 (95% CI 0.99-2.81, P=0.049). The 60% of patients with mutation and only 13% without mutation develop rethrombosis during first year after discontinuance of therapy (P<0.01). In our study patients with symptomatic VTE who are carriers of the FV Leiden gene mutations have a higher risk of recurrent VTE than non-carriers. Our data suggest the importance of the FV Leiden mutation detection and the estimation of the clinical condition for successful secondary prophylaxis of VTE.

Cloning and expression of 5, 10-Methylenetetrahydrofolate reductase (MTHFR) gene

Hyperhomocysteinemia is an independent risk factor of cardiovascular diseases and birth defects. One of the important factors causing hyperhomocysteinemia is decrease of 5,10-Methylenetetrahydrofolate reductase. Human and rat MTHFR cDNAs with RT-PCR were isolated, a prokaryodytic expression vector containing human MTHFR cDNA was constructed, and human MTHFR protein was expressed inE. coli. It was also found that the expression of rat MTHFR could be promoted by IL-1 and homocysteine.