Polymorphism of folate metabolism genes and thrombotic complications in patients with functionally single ventricle

Aim. To analyze the relationships between the carriage of polymorphic variants in the folate metabolism genes and the development of thrombotic complications in patients with single ventricle (SV) during surgical treatment.Material and Methods. A total of 102 children with SV were examined in the performed research. All patients underwent surgical hemodynamic correction of congenital heart disease (CHD). According to a retrospective chart review, thrombosis was diagnosed in 12.7 % of the examined patients with SV. The analysis of polymorphism in the MTR A2756G enzyme gene revealed significant differences between the groups of patients with a history of thrombosis and without it.Results. We found that the risk of developing thrombosis was associated with the carriage of homozygous genotype 2756AA of the MTR enzyme gene (OR = 11.21; 95% CI: 1.39–89.96; p = 0.023).

Polymorphism of methylenetetrahydrofolate reductase (MTHFR) folate metabolism gene and hyperhomocysteinemia in migraine

В обзоре обсуждается роль полиморфизма гена фолатного обмена метилентетрагидрофолат-редуктазы (MTHFR), ответственного за развитие гипергомоцистеинемии в патогенезе мигрени. Изложены общие данные о полиморфизме С677 гена фолатного цикла и метаболизме гомоцистеина. Представлен патогенетический механизм развития мигрени, связанный с провоспалительными, прокоагулянтными свойствами гомоцистеина, активацией процессов окислительного стресса, эндотелиальной дисфункцией и нейрогенным воспалением при повышении концентрации этой аминокислоты. Отражены перспективы и социальная значимость имплементации данных генетических исследований в клиническую практику, их роль в прогнозировании течения мигрени и оценке риска развития осложнений, а также коррекции фармакотерапевтических подходов. Методика. Для поиска данных в базах MEDLINE, SCOPUS и Web of Science использованы поисковые запросы: МТHFR, мигрень, патофизиология, гипергомоцистеинемия, таргетная терапия. The review discusses the role of polymorphism of the methylenetetrahydrofolate reductase (MTHFR) folate metabolism gene responsible for hyperhomocysteinemia in the pathogenesis of migraine. Data on the polymorphism of the folate cycle gene C677 and homocysteine metabolism are presented. The pathogenetic mechanism of migraine associated with proinflammatory, procoagulant properties of homocysteine and with the activation of oxidative stress, endothelial dysfunction, and neurogenic inflammation related with increased concentrations of homocysteine is described. Prospects and social significance of implementing data of genetic research into clinical practice are discussed. Included is the role of genetic research in predicting the course and complications of migraine, in assessment of risk for complications, and in pharmacotherapeutic approaches to migraine treatment. Methods. MEDLINE, SCOPUS and Web of Science databases were used to search for data: MTHFR, migraine, pathophysiology, hyperhomocysteinemia, targeted therapy

Precise Dose of Folic Acid Supplementation Is Essential for Embryonic Heart Development in Zebrafish

Folic acid, one of the 13 essential vitamins, plays an important role in cardiovascular development. Mutations in folic acid synthesis gene 5,10-methylenetetrahydrofolate reductase (MTHFR) is associated with the occurrence of congenital heart disease. However, the mechanisms underlying the regulation of cardiac development by mthfr gene are poorly understood. Here, we exposed zebrafish embryos to excessive folate or folate metabolism inhibitors. Moreover, we established a knock-out mutant of mthfr gene in zebrafish by using CRISPR/Cas9. The zebrafish embryos of insufficient or excessive folic acid and mthfr−/− mutant all gave rise to early pericardial edema and cardiac defect at 3 days post fertilization (dpf). Furthermore, the folic acid treated embryos showed abnormal movement at 5 dpf. The expression levels of cardiac marker genes hand2, gata4, and nppa changed in the abnormality of folate metabolism embryos and mthfr−/− mutant, and there is evidence that they are related to the change of methylation level caused by the change of folate metabolism. In conclusion, our study provides a novel model for the in-depth study of MTHFR gene and folate metabolism. Furthermore, our results reveal that folic acid has a dose-dependent effect on early cardiac development. Precise dosage of folic acid supplementation is crucial for the embryonic development of organisms.

Bacterial origin of thymidylate and folate metabolism in Asgard Archaea

Little is known about the evolution and biosynthetic function of DNA precursor and the folate metabolism in the Asgard group of archaea. As Asgard occupy a key position in the archaeal and eukaryotic phylogenetic trees, we have exploited very recently emerged genome and metagenome sequence information to investigate these central metabolic pathways. Our genome-wide analyses revealed that the recently cultured Asgard archaeon Candidatus Prometheoarchaeum syntrophicum strain MK-D1 (Psyn) contains a complete folate-dependent network for the biosynthesis of DNA/RNA precursors, amino acids and syntrophic amino acid utilization. Altogether our experimental and computational data suggest that phylogenetic incongruences of functional folate-dependent enzymes from Asgard archaea reflect their persistent horizontal transmission from various bacterial groups, which has rewired the key metabolic reactions in an important and recently identified archaeal phylogenetic group. We also experimentally validated the functionality of the lateral gene transfer of Psyn thymidylate synthase ThyX. This enzyme uses bacterial-like folates efficiently and is inhibited by mycobacterial ThyX inhibitors. Our data raise the possibility that the thymidylate metabolism, required for de novo DNA synthesis, originated in bacteria and has been independently transferred to archaea and eukaryotes. In conclusion, our study has revealed that recent prevalent lateral gene transfer has markedly shaped the evolution of Asgard archaea by allowing them to adapt to specific ecological niches.

Disruption of Folate Metabolism Causes Poor Alignment and Spacing of Mouse Conceptuses for Multiple Generations

Abnormal uptake or metabolism of folate increases risk of human pregnancy complications, though the mechanism is unclear. Here, we explore how defective folate metabolism influences early development by analysing mice with the hypomorphic Mtrrgt mutation. MTRR is necessary for methyl group utilisation from folate metabolism, and the Mtrrgt allele disrupts this process. We show that the spectrum of phenotypes previously observed in Mtrrgt/gt conceptuses at embryonic day (E) 10.5 is apparent from E8.5 including developmental delay, congenital malformations, and placental phenotypes. Notably, we report misalignment of some Mtrrgt conceptuses within their implantation sites from E6.5. The degree of misorientation occurs across a continuum, with the most severe form visible upon gross dissection. Additionally, some Mtrrgt/gt conceptuses display twinning. Therefore, we implicate folate metabolism in blastocyst orientation and spacing at implantation. Skewed growth likely influences embryo development since developmental delay and heart malformations (but not defects in neural tube closure or trophoblast differentiation) associate with severe misalignment of Mtrrgt/gt conceptuses. Typically, the uterus is thought to guide conceptus orientation. To investigate a uterine effect of the Mtrrgt allele, we manipulate the maternal Mtrr genotype. Misaligned conceptuses were observed in litters of Mtrr+/+, Mtrr+/gt, and Mtrrgt/gt mothers. While progesterone and/or BMP2 signalling might be disrupted, normal decidual morphology, patterning, and blood perfusion are evident at E6.5 regardless of conceptus orientation. These observations argue against a post-implantation uterine defect as a cause of conceptus misalignment. Since litters of Mtrr+/+ mothers display conceptus misalignment, a grandparental effect is explored. Multigenerational phenotype inheritance is characteristic of the Mtrrgt model, though the mechanism remains unclear. Genetic pedigree analysis reveals that severe conceptus skewing associates with the Mtrr genotype of either maternal grandparent. Moreover, the presence of conceptus skewing after embryo transfer into a control uterus indicates that misalignment is independent of the peri- and/or post-implantation uterus and instead is likely attributed to an embryonic mechanism that is epigenetically inherited. Overall, our data indicates that abnormal folate metabolism influences conceptus orientation over multiple generations with implications for subsequent development. This study casts light on the complex role of folate metabolism during development beyond a direct maternal effect.

SYNGAP1 and Methylenetetrahydrofolate in Cerebrospinal Fluid: Cognitive Development after Oral Folate (5-Methyltetrahydrofolate) Supplementation in a 5-Year-Old Girl

Synaptic Ras GTPase-activating protein 1 (SYNGAP1), also called Ras-GAP 1 or RASA5, is a cerebral protein with a role in brain synaptic function. Its expression affects the development, structure, function, and plasticity of neurons. Mutations in the gene cause a neurodevelopment disorder termed mental retardation-type 5, also called SYNGAP1 syndrome. This syndrome can cause many neurological symptoms including pharmaco-resistant epilepsy, intellectual disability, language delay, and autism spectrum disorder. The syndrome naturally evolves as epileptic encephalopathy with handicap and low intellectual level. A treatment to control epilepsy, limit any decrease in social capacities, and improve intellectual development is really a challenging goal for these patients. The etiologic investigation performed in a 5-year-old girl with early epileptic absence seizures (onset at 6 months) and psychomotor delay (language) revealed a low methylenetetrahydrofolate level in cerebrospinal fluid in a lumbar puncture, confirmed by a second one (35 nmol/L and 50 nmol/L vs. 60–100 nmol/L normal), associated with normal blood and erythrocyte folate levels. Hyperhomocysteinemia, de vivo disease, and other metabolic syndromes were excluded by metabolic analysis. No genetic disorders (like methylenetetrahydrofolate reductase and methenyltetrahydrofolate synthetase) with folate metabolism were found. The physical examination showed only a minor kinetic ataxia. An oral folate (5-methyltetrahydrofolate) supplementation was started with oral vitamin therapy. The child showed good progress in language with this new treatment; epilepsy was well balanced with only one antiepileptic drug. The SYNGAP1 mutation was identified in this patient's genetic analysis. Since the start of folate supplementation/vitamin therapy, the patient's neurologic development has improved. To our knowledge, no association between these two pathologies has been linked and no patient with this SYNGAP1 mutation has ever showed much intellectual progress. Low cerebral methylenetetrahydrofolate levels could be associated with SYNGAP1 mutations. One of the hypotheses is the link of folate metabolism with epigenetic changes including methylation process. One inborn metabolic activity in folate metabolism may be associated with SYNGAP1 disease with epigenetic repercussions. Further studies should assess the link of SYNGAP1 and methyltetrahydrofolate and the evolution of SYNGAP1 patients with oral folate supplementation or vitamin therapy.

MTHFD1 1958 G>A Genetic Polymorphism (rs2236225) Dichotomy in Schizophrenia: Lower Manifestation Risks, but More Severe Negative Symptoms

Despite a large amount of data on the association of folate metabolism disturbances with different aspects of schizophrenia, the role of the MTHFD1 1958 G>A polymorphism in this disorder is barely studied. The aim of this study was to assess the distribution of alleles and genotypes frequencies of MTHFD1 1958 G>A in patients with schizophrenia and healthy controls and to study the association of allele/genotype carriage of this SNP with biochemical markers of one-carbon metabolism and with the severity of schizophrenia symptoms. Methods: In 57 patients with schizophrenia and 37 healthy volunteers the carriage of alleles/genotypes of the MTHFD1 1958 G>A and biochemical markers of folate metabolism disturbances were evaluated. Clinical symptoms of schizophrenia and the severity of extrapyramidal side effects of therapy were assessed in patients. Results: an association of the wild GG genotype with schizophrenia was shown (GG versus AG / AA: χ2 = 7.31; p = 0.007). The serum folate level in carriers of the wild genotype GG is lower (in all participants p = 0.024, in patients p = 0.10), and the level of cobalamin in this subgroup is higher (in all participants p = 0.047, in patients p = 0.091) than in carriers of other genotypes. Patients carrying the G allele had less severe negative symptoms (p = 0.0041) and extrapyramidal side effects of antipsychotics (p = 0.054), than patients with AA genotype. The age of psychosis manifestation is the later, the more wild alleles G are present in the genotype (p = 0.00195).

Inhibition of Folate Metabolism Drives Autophagy-Dependent Differentiation and Reduces Survival of Therapy-Resistant Leukaemic Stem Cells

Metabolic rewiring is an important hallmark of cancer. The folate metabolism pathway, also known as one-carbon (1C) metabolism, allows for transfer of 1C units through folate intermediates for biosynthetic processes, including precursors for DNA synthesis. Recent studies have shown that enzymes involved in the mitochondrial arm of 1C metabolism are overexpressed in a subset of aggressive cancers and that their expression affects responses to anti-metabolite drug treatments. However, the role of 1C metabolism in therapy resistant leukemic stem cells (LSCs) is currently unknown. Therefore, we aimed to investigate the activity and the impact of genetic and pharmacological inhibition of folate enzymes in primitive chronic myeloid leukaemia (CML) cells. We initially performed transcriptomic analysis of CD34+38- cells, from individuals with chronic phase CML (E-MTAB-2581). This revealed a significant upregulation of folate metabolism genes in CML LSCs, including serine hydroxymethyltransferase (SHMT2; p≤0.05), a key mitochondrial enzyme. To assess the activity of 1C metabolism in primitive cells we performed gas chromatography-mass spectrometry-mediated secretomic analysis using patient-derived, c-KIT enriched CML cells, which revealed a significant increase in the exchange rate of formate (folate intermediate necessary for purine synthesis) in CML cells, when compared to the secretome of normal counterparts (p<0.05). This reinforced the idea that 1C metabolism may be a metabolic dependency in CML. Following CRISPR-Cas9-mediated SHMT2 knockout (KO) in CML cell line, we observed a significant decrease in growth rate, together with a decrease in glycolytic capacity and oxygen consumption rate (p<0.01), suggesting impairment in proliferation and central carbon metabolism. Further metabolic characterisation of CML SHMT2 KO cells using liquid chromatography-mass spectrometry demonstrated a significant increase in AICAR, a purine biosynthesis intermediate and an AMP activated kinase (AMPK) activator. This prompted us to investigate the effect of 1C metabolism inhibition on AMPK. We found that AMPK phosphorylation on the conserved Thr 172 (a site that is phosphorylated under energy stress) was increased in SHMT2 KO cells, with similar effect seen following pharmacological inhibition of both SHMT2 and its cytosolic counterpart SHMT1 using SHIN1,which also promoted AMPK-dependent phosphorylation of the autophagy-inducing kinase ULK1 and downstream ULK1 target ATG13. Moreover, analysis of mitochondrial fraction revealed accumulation of mitochondrial fission related protein DRP1 and the mitophagy receptor NIX on mitochondria, hinting towards cellular interplay between 1C metabolism and mitochondrial homeostasis. Phenotypically, both pharmacological and genetic inhibition of SHMT1/2 induced the expression of erythropoiesis markers CD71 and Glycophorin A, which was reversed following formate supplementation. CRISPR-Cas9-mediated double AMPKα1/α2 KO revealed that the increased expression of these erythropoiesis markers following SHMT1/2 inhibition was independent of AMPK activity. Conversely, while NIX KO had no effect, pharmacological inhibition of ULK1 kinase activity, or genetic inhibition of ULK1 and ATG7 (protein important for autophagosome formation), prevented increased expression of CD71/Glycophorin A following SHMT1/2 inhibition. We next investigated the effect of 1C metabolism inhibition on differentiation and survival of primary CML cells. Of clinical relevance, pharmacological inhibition of SHMT1/2 promoted erythroid maturation of CD34+ CML cells (measured by expression of CD71, CD44, CD36 and Glycophorin A) when challenged with erythropoietin, which sensitises primitive cells to erythroid lineage commitment. Lastly, pharmacological inhibition of 1C metabolism decreased the colony formation capacity of CD34+ CML by 50%, with minimum effect on normal CD34+ cells. Moreover, combination treatment of SHIN1 with imatinib, a frontline treatment for CML patients, further increased the sensitivity of primary CML cells to imatinib by 40%. Overall, our novel findings indicate that disruption of the folate metabolism pathway inhibits central carbon metabolism in CML cells, promotes autophagy dependent, but AMPK independent maturation phenotype and has detrimental effect on the survival of primitive CML cells. Disclosures No relevant conflicts of interest to declare.