{"gene":"MTHFR","run_date":"2026-06-10T02:59:51","timeline":{"discoveries":[{"year":1998,"finding":"The human MTHFR gene spans ~17 kb, contains 11 exons (102–432 bp each), with introns ranging from 250 bp to 4.2 kb. A 2.2 kb human cDNA was expressed and shown to produce a catalytically active enzyme of approximately 70 kDa. The mouse amino acid sequence is ~90% identical to human, with conserved exon sizes and intron boundary positions.","method":"Genomic clone isolation, cDNA expression in cell-free/cellular system, sequencing of human and mouse genomic clones","journal":"Mammalian genome","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct cDNA expression with catalytic activity confirmation, full gene structure determined, replicated in two species","pmids":["9680386"],"is_preprint":false},{"year":2001,"finding":"MTHFR catalyzes the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine, thereby directing folate species either toward DNA synthesis (thymidylate/purine) or toward homocysteine remethylation. The common C677T polymorphism (Ala222Val) reduces enzyme activity and thermostability, leading to altered intracellular folate distribution and elevated plasma homocysteine under low-folate conditions.","method":"Enzyme activity assays, thermolability studies, population metabolic studies correlating genotype with folate species and homocysteine levels","journal":"Trends in pharmacological sciences","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — enzymatic mechanism established by multiple labs; thermolability and activity reduction of C677T variant replicated across many independent studies","pmids":["11282420"],"is_preprint":false},{"year":2014,"finding":"The C677T polymorphism results in an amino acid change from alanine to valine at codon 222, which reduces enzyme activity and prevents optimal functioning at temperatures above 37°C (thermolability). Homozygous TT individuals have higher homocysteine levels than heterozygotes, who in turn have higher levels than wild-type CC individuals.","method":"Enzyme activity assays, genotyping, plasma homocysteine measurements across genotype groups","journal":"European journal of medical genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — biochemical phenotype (thermolability, reduced activity) replicated across many independent studies worldwide","pmids":["25449138"],"is_preprint":false},{"year":2014,"finding":"MTHFR is phosphorylated at threonine 34 (T34) in vivo, with phosphorylation peaking during mitosis. The CDK1/Cyclin B1 complex was identified as the kinase mediating this T34 phosphorylation. The MTHFR immunocomplex purified from mitotic cells exhibited lower enzymatic activity, indicating that mitotic phosphorylation by CDK1/Cyclin B1 inhibits MTHFR enzymatic activity. Furthermore, inhibition of MTHFR expression decreased H3K9me3 levels and increased transcription of centromeric heterochromatin markers, demonstrating a role for MTHFR in heterochromatin maintenance.","method":"Phospho-specific antibody generation, immunoprecipitation, cell-cycle synchronization, in vitro kinase assays, siRNA knockdown with H3K9me3 immunoblotting and RT-PCR of centromeric markers","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and phospho-specific antibody validation in single lab, multiple orthogonal methods (kinase assay, KD phenotype, chromatin marks)","pmids":["24769206"],"is_preprint":false},{"year":2021,"finding":"A comprehensive atlas of 98,336 variant functional-impact assays covering nearly all possible MTHFR amino acid substitutions in four folinate environments revealed complex environment- and genetic background-dependent variant effects. The atlas identified variants that enable escape from inhibition by S-adenosylmethionine (SAM), suggesting a regulatory role for SAM-mediated feedback inhibition of MTHFR. The atlas also suggests a role for a disordered loop in retaining the FAD cofactor at the active site. The p.Ala222Val (C677T) variant's functional impact depends on dietary folate levels, and atlas scores correlate with age of disease onset in severe MTHFR deficiency patients.","method":"Saturation genome editing / deep mutational scanning (98,336 variant functional assays), correlation of scores with clinical outcomes","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 1 / Strong — genome-scale saturation mutagenesis with functional readout in multiple environmental conditions, identifies active site features and regulatory mechanism","pmids":["34214447"],"is_preprint":false},{"year":2008,"finding":"Valproic acid (VPA) increases MTHFR promoter activity, mRNA expression (~2.5-fold), and protein levels (~3.7-fold) in HepG2 cells. Consistent with this upregulation, VPA-treated pregnant mice showed increased brain MTHFR enzyme activity and decreased plasma homocysteine. Mthfr(+/-) mice showed lower VPA teratogenicity than Mthfr(+/+) mice (similar resorption rates with/without VPA in heterozygotes vs. increased resorption in wild-type), indicating MTHFR deficiency modulates VPA-induced teratogenicity.","method":"MTHFR promoter-reporter assay, RT-PCR and Western blot in HepG2 cells, in vivo VPA injection in Mthfr(+/+) and Mthfr(+/-) pregnant mice, enzyme activity assay, plasma homocysteine measurement","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (promoter assay, mRNA, protein, in vivo enzymatic activity) in single lab","pmids":["18615588"],"is_preprint":false},{"year":2020,"finding":"In lymphoblastoid cell lines (LCLs) with low MTHFR activity (due to C677T/A1298C polymorphisms), supplementation with folic acid (FA) failed to increase intracellular 5-methyltetrahydrofolate (5-Me-THF) levels (no increase detected by LC-MS/MS), whereas direct supplementation with 5-Me-THF produced a 10-fold increase in intracellular 5-Me-THF. In normal MTHFR activity LCLs, FA supplementation produced a 2.5-fold increase in 5-Me-THF. Folate deprivation caused reversible S-phase cell cycle arrest.","method":"LC-MS/MS quantification of intracellular 5-Me-THF in genotyped lymphoblastoid cell lines, cell viability/proliferation assays under folate deprivation and supplementation conditions","journal":"Journal of clinical medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct metabolite measurement by LC-MS/MS in genotype-stratified cell lines, single lab with two orthogonal methods","pmids":["32887268"],"is_preprint":false},{"year":2022,"finding":"Heterozygous Mthfr-deficient (Mthfr+/-) mice show impaired endothelial function, reduced bleeding time, and enhanced wire-induced thrombus formation, associated with SIRT1 downregulation. Pharmacological activation of SIRT1 (by resveratrol or the synthetic activator ISIDE11) rescued endothelial vasorelaxation and reduced thrombus formation in Mthfr+/- mice without affecting homocysteine levels. Human MTHFR C677T carriers with normal homocysteine also showed endothelial dysfunction and enhanced platelet aggregation linked to SIRT1 downregulation, normalized by resveratrol.","method":"Flow Mediated Dilatation (FMD) in human subjects, light transmission aggregometry, wire-induced thrombosis model in Mthfr+/- mice, cell-based HTS screening for SIRT1 activators, SIRT1 inhibitor controls","journal":"Cellular and molecular life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic mouse model with functional vascular readouts, pharmacological rescue with mechanism (SIRT1 activity), single lab with multiple orthogonal assays","pmids":["35821533"],"is_preprint":false},{"year":2008,"finding":"Antisense-mediated inhibition of MTHFR in tumor-derived cell lines increased cytotoxicity in vitro and decreased tumor growth in vivo in xenograft models, demonstrating that MTHFR activity is required for tumor cell survival through its role in methionine biosynthesis. Tumor cells (unlike normal cells) are methionine-dependent and cannot efficiently utilize homocysteine as a methionine substitute.","method":"Antisense oligonucleotide inhibition of MTHFR in cell lines, in vitro cytotoxicity assays, in vivo tumor xenograft growth assays","journal":"Current pharmaceutical design","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined cellular phenotype (cytotoxicity, tumor growth) in vitro and in vivo, single lab","pmids":["18473861"],"is_preprint":false},{"year":2016,"finding":"MTHFR promoter hypermethylation was associated with lower MTHFR mRNA expression in placenta of preeclamptic women. The 3'-UTR rs1537514C>G polymorphism was associated with higher MTHFR mRNA expression and lower risk of preeclampsia; the rs1537514G allele correlated with higher expression in both PE and normotensive groups. No association was found between rs4846049C>A polymorphism and MTHFR expression.","method":"Methylation-specific PCR of MTHFR promoter in placental tissue, quantitative RT-PCR for MTHFR mRNA, PCR-RFLP genotyping of 3'-UTR SNPs","journal":"Journal of cellular biochemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single tissue type, correlative relationship between methylation and expression without functional validation of causality","pmids":["28722783"],"is_preprint":false},{"year":2015,"finding":"MTHFR gene promoter hypermethylation was detected at significantly higher frequency in placenta and peripheral venous blood of preeclamptic women compared to normotensive controls (placenta: 26.8% vs. 15.2%, p<0.05; peripheral blood: 22.8% vs. 12.1%, p<0.05), and was associated with elevated plasma homocysteine in preeclampsia.","method":"Methylation-specific PCR on placental and peripheral blood DNA, ELISA for plasma homocysteine","journal":"Genetics and molecular research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, MSP only, correlative without mechanistic proof of causal relationship","pmids":["26214484"],"is_preprint":false},{"year":2016,"finding":"MTHFR promoter methylation in spermatozoa of idiopathic infertile normozoospermic men was significantly higher (11%) than in fertile controls (4.3%), suggesting epigenetic silencing of MTHFR may contribute to idiopathic male infertility.","method":"Sodium bisulfite modification and pyrosequencing of MTHFR promoter in sperm DNA from infertile and fertile men","journal":"Andrologia","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, pyrosequencing only, correlative without functional consequence of silencing directly demonstrated","pmids":["27596009"],"is_preprint":false}],"current_model":"MTHFR is a key metabolic enzyme that irreversibly converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (catalytic mechanism confirmed by cDNA expression and in vitro activity assays), thereby channeling folate toward homocysteine remethylation to methionine and away from thymidylate/purine synthesis; its activity is feedback-inhibited by S-adenosylmethionine (SAM), requires FAD as a cofactor retained by a disordered active-site loop, and is regulated during mitosis by CDK1/Cyclin B1-mediated phosphorylation at T34 which reduces enzymatic activity; the common C677T (Ala222Val) polymorphism causes thermolability and reduced activity in a folate-dependent manner, and MTHFR additionally promotes centromeric heterochromatin maintenance (H3K9me3) and influences vascular function through a SIRT1-linked pathway independent of homocysteine."},"narrative":{"mechanistic_narrative":"MTHFR is the metabolic enzyme that irreversibly converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the folate co-substrate for homocysteine remethylation to methionine, thereby partitioning one-carbon units between DNA synthesis (thymidylate/purine) and methionine regeneration [PMID:11282420]. The catalytically active ~70 kDa enzyme is encoded by an 11-exon gene whose product was confirmed by cDNA expression [PMID:9680386]. Deep mutational scanning across folinate environments resolved its regulatory architecture, identifying variants that escape S-adenosylmethionine feedback inhibition and a disordered loop that retains the FAD cofactor at the active site [PMID:34214447]. Enzyme output is additionally controlled through the cell cycle: CDK1/Cyclin B1 phosphorylates MTHFR at Thr34, peaking in mitosis and lowering catalytic activity [PMID:24769206]. Beyond its canonical metabolic role, MTHFR supports centromeric heterochromatin maintenance, as its knockdown reduces H3K9me3 and de-represses centromeric transcription [PMID:24769206], and it influences vascular function through a SIRT1-linked pathway that operates independently of homocysteine levels [PMID:35821533]. The common C677T (Ala222Val) variant produces a thermolabile enzyme with reduced activity, elevating plasma homocysteine in a folate-dependent manner [PMID:11282420, PMID:25449138], and functional-impact scores correlate with age of onset in severe MTHFR deficiency [PMID:34214447].","teleology":[{"year":1998,"claim":"Establishing the gene structure and confirming that the cloned cDNA produces a catalytically active enzyme provided the molecular foundation for all subsequent mechanistic work.","evidence":"Genomic clone isolation and cDNA expression yielding an active ~70 kDa enzyme, with cross-species conservation between human and mouse","pmids":["9680386"],"confidence":"High","gaps":["No structural model of the active site at this stage","Regulatory mechanisms not addressed"]},{"year":2001,"claim":"Defining MTHFR as the committed, irreversible step directing folate toward homocysteine remethylation versus nucleotide synthesis placed it at a metabolic branch point and linked the C677T variant to elevated homocysteine.","evidence":"Enzyme activity and thermolability assays integrated with population metabolic studies correlating genotype, folate species, and homocysteine","pmids":["11282420"],"confidence":"High","gaps":["Structural basis of thermolability not resolved","Cofactor and feedback regulation not mechanistically defined"]},{"year":2008,"claim":"Loss-of-function studies tested whether MTHFR activity is required for cell survival, showing methionine-dependent tumor cells rely on it and that VPA can transcriptionally upregulate the enzyme.","evidence":"Antisense inhibition with cytotoxicity and xenograft assays (PMID 18473861); MTHFR promoter-reporter, mRNA/protein, and in vivo VPA studies in Mthfr+/- mice (PMID 18615588)","pmids":["18473861","18615588"],"confidence":"Medium","gaps":["Mechanism of VPA-driven promoter activation not mapped to specific elements","Generality of tumor methionine dependence beyond tested lines unestablished"]},{"year":2014,"claim":"Discovery of CDK1/Cyclin B1-mediated Thr34 phosphorylation and a heterochromatin phenotype revealed cell-cycle regulation of MTHFR activity and a moonlighting role beyond folate metabolism.","evidence":"Phospho-specific antibody, cell-cycle synchronization, in vitro kinase assays, and siRNA knockdown with H3K9me3 immunoblotting and centromeric RT-PCR","pmids":["24769206"],"confidence":"Medium","gaps":["Single-lab reciprocal Co-IP without independent confirmation","How reduced MTHFR activity mechanistically links to H3K9me3 maintenance is unresolved"]},{"year":2020,"claim":"Direct metabolite measurement in genotype-stratified cells clarified that low-activity MTHFR limits conversion of folic acid to 5-methyltetrahydrofolate, with implications for supplementation strategy.","evidence":"LC-MS/MS quantification of intracellular 5-Me-THF in genotyped lymphoblastoid lines under folic acid versus 5-Me-THF supplementation, with folate-deprivation cell-cycle assays","pmids":["32887268"],"confidence":"Medium","gaps":["Single cell-type system","In vivo relevance of supplementation findings not established"]},{"year":2021,"claim":"A genome-scale variant atlas resolved the enzyme's regulatory features—SAM feedback inhibition and FAD retention by a disordered loop—and made variant effects environment- and folate-dependent, linking functional scores to clinical onset.","evidence":"Saturation genome editing / deep mutational scanning of 98,336 variant assays across four folinate environments with correlation to disease onset","pmids":["34214447"],"confidence":"High","gaps":["Atomic-resolution structure of the disordered FAD-retaining loop not provided","Mechanism by which escape variants bypass SAM inhibition not structurally defined"]},{"year":2022,"claim":"A homocysteine-independent vascular axis was established, showing MTHFR deficiency causes endothelial dysfunction and thrombosis via SIRT1 downregulation that is pharmacologically reversible.","evidence":"Mthfr+/- mouse thrombosis and endothelial function models plus human C677T flow-mediated dilatation and aggregometry, with SIRT1 activator rescue and inhibitor controls","pmids":["35821533"],"confidence":"Medium","gaps":["Molecular link between MTHFR activity and SIRT1 expression not defined","Single-lab findings"]},{"year":null,"claim":"How epigenetic regulation of MTHFR (promoter hypermethylation in preeclampsia and sperm) causally affects expression and phenotype remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["Promoter methylation associations are correlative without functional proof of causality","No mechanistic link between methylation state and downstream pathology demonstrated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,1,4]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[1,4]}],"localization":[],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[1,6]}],"complexes":[],"partners":["CDK1","CCNB1","SIRT1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P42898","full_name":"Methylenetetrahydrofolate reductase (NADPH)","aliases":[],"length_aa":656,"mass_kda":74.6,"function":"Catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a cosubstrate for homocysteine remethylation to methionine (PubMed:29891918). Represents a key regulatory connection between the folate and methionine cycles (Probable)","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/P42898/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MTHFR","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MTHFR","total_profiled":1310},"omim":[{"mim_id":"614390","title":"PREGNANCY LOSS, RECURRENT, SUSCEPTIBILITY TO, 2; RPRGL2","url":"https://www.omim.org/entry/614390"},{"mim_id":"614389","title":"PREGNANCY LOSS, RECURRENT, SUSCEPTIBILITY TO, 1; RPRGL1","url":"https://www.omim.org/entry/614389"},{"mim_id":"613381","title":"CYSTATHIONINE BETA-SYNTHASE; CBS","url":"https://www.omim.org/entry/613381"},{"mim_id":"608516","title":"MAJOR DEPRESSIVE DISORDER; MDD","url":"https://www.omim.org/entry/608516"},{"mim_id":"608307","title":"CARBAMOYL PHOSPHATE SYNTHETASE I; CPS1","url":"https://www.omim.org/entry/608307"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cell Junctions","reliability":"Approved"},{"location":"Centrosome","reliability":"Additional"},{"location":"Basal body","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MTHFR"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P42898","domains":[{"cath_id":"3.20.20.220","chopping":"58-337","consensus_level":"high","plddt":92.7176,"start":58,"end":337},{"cath_id":"-","chopping":"364-645","consensus_level":"high","plddt":93.5576,"start":364,"end":645}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P42898","model_url":"https://alphafold.ebi.ac.uk/files/AF-P42898-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P42898-F1-predicted_aligned_error_v6.png","plddt_mean":88.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MTHFR","jax_strain_url":"https://www.jax.org/strain/search?query=MTHFR"},"sequence":{"accession":"P42898","fasta_url":"https://rest.uniprot.org/uniprotkb/P42898.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P42898/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P42898"}},"corpus_meta":[{"pmid":"11282420","id":"PMC_11282420","title":"Biological 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A 2.2 kb human cDNA was expressed and shown to produce a catalytically active enzyme of approximately 70 kDa. The mouse amino acid sequence is ~90% identical to human, with conserved exon sizes and intron boundary positions.\",\n      \"method\": \"Genomic clone isolation, cDNA expression in cell-free/cellular system, sequencing of human and mouse genomic clones\",\n      \"journal\": \"Mammalian genome\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct cDNA expression with catalytic activity confirmation, full gene structure determined, replicated in two species\",\n      \"pmids\": [\"9680386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"MTHFR catalyzes the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine, thereby directing folate species either toward DNA synthesis (thymidylate/purine) or toward homocysteine remethylation. The common C677T polymorphism (Ala222Val) reduces enzyme activity and thermostability, leading to altered intracellular folate distribution and elevated plasma homocysteine under low-folate conditions.\",\n      \"method\": \"Enzyme activity assays, thermolability studies, population metabolic studies correlating genotype with folate species and homocysteine levels\",\n      \"journal\": \"Trends in pharmacological sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — enzymatic mechanism established by multiple labs; thermolability and activity reduction of C677T variant replicated across many independent studies\",\n      \"pmids\": [\"11282420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The C677T polymorphism results in an amino acid change from alanine to valine at codon 222, which reduces enzyme activity and prevents optimal functioning at temperatures above 37°C (thermolability). Homozygous TT individuals have higher homocysteine levels than heterozygotes, who in turn have higher levels than wild-type CC individuals.\",\n      \"method\": \"Enzyme activity assays, genotyping, plasma homocysteine measurements across genotype groups\",\n      \"journal\": \"European journal of medical genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — biochemical phenotype (thermolability, reduced activity) replicated across many independent studies worldwide\",\n      \"pmids\": [\"25449138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MTHFR is phosphorylated at threonine 34 (T34) in vivo, with phosphorylation peaking during mitosis. The CDK1/Cyclin B1 complex was identified as the kinase mediating this T34 phosphorylation. The MTHFR immunocomplex purified from mitotic cells exhibited lower enzymatic activity, indicating that mitotic phosphorylation by CDK1/Cyclin B1 inhibits MTHFR enzymatic activity. Furthermore, inhibition of MTHFR expression decreased H3K9me3 levels and increased transcription of centromeric heterochromatin markers, demonstrating a role for MTHFR in heterochromatin maintenance.\",\n      \"method\": \"Phospho-specific antibody generation, immunoprecipitation, cell-cycle synchronization, in vitro kinase assays, siRNA knockdown with H3K9me3 immunoblotting and RT-PCR of centromeric markers\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and phospho-specific antibody validation in single lab, multiple orthogonal methods (kinase assay, KD phenotype, chromatin marks)\",\n      \"pmids\": [\"24769206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A comprehensive atlas of 98,336 variant functional-impact assays covering nearly all possible MTHFR amino acid substitutions in four folinate environments revealed complex environment- and genetic background-dependent variant effects. The atlas identified variants that enable escape from inhibition by S-adenosylmethionine (SAM), suggesting a regulatory role for SAM-mediated feedback inhibition of MTHFR. The atlas also suggests a role for a disordered loop in retaining the FAD cofactor at the active site. The p.Ala222Val (C677T) variant's functional impact depends on dietary folate levels, and atlas scores correlate with age of disease onset in severe MTHFR deficiency patients.\",\n      \"method\": \"Saturation genome editing / deep mutational scanning (98,336 variant functional assays), correlation of scores with clinical outcomes\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — genome-scale saturation mutagenesis with functional readout in multiple environmental conditions, identifies active site features and regulatory mechanism\",\n      \"pmids\": [\"34214447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Valproic acid (VPA) increases MTHFR promoter activity, mRNA expression (~2.5-fold), and protein levels (~3.7-fold) in HepG2 cells. Consistent with this upregulation, VPA-treated pregnant mice showed increased brain MTHFR enzyme activity and decreased plasma homocysteine. Mthfr(+/-) mice showed lower VPA teratogenicity than Mthfr(+/+) mice (similar resorption rates with/without VPA in heterozygotes vs. increased resorption in wild-type), indicating MTHFR deficiency modulates VPA-induced teratogenicity.\",\n      \"method\": \"MTHFR promoter-reporter assay, RT-PCR and Western blot in HepG2 cells, in vivo VPA injection in Mthfr(+/+) and Mthfr(+/-) pregnant mice, enzyme activity assay, plasma homocysteine measurement\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (promoter assay, mRNA, protein, in vivo enzymatic activity) in single lab\",\n      \"pmids\": [\"18615588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In lymphoblastoid cell lines (LCLs) with low MTHFR activity (due to C677T/A1298C polymorphisms), supplementation with folic acid (FA) failed to increase intracellular 5-methyltetrahydrofolate (5-Me-THF) levels (no increase detected by LC-MS/MS), whereas direct supplementation with 5-Me-THF produced a 10-fold increase in intracellular 5-Me-THF. In normal MTHFR activity LCLs, FA supplementation produced a 2.5-fold increase in 5-Me-THF. Folate deprivation caused reversible S-phase cell cycle arrest.\",\n      \"method\": \"LC-MS/MS quantification of intracellular 5-Me-THF in genotyped lymphoblastoid cell lines, cell viability/proliferation assays under folate deprivation and supplementation conditions\",\n      \"journal\": \"Journal of clinical medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct metabolite measurement by LC-MS/MS in genotype-stratified cell lines, single lab with two orthogonal methods\",\n      \"pmids\": [\"32887268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Heterozygous Mthfr-deficient (Mthfr+/-) mice show impaired endothelial function, reduced bleeding time, and enhanced wire-induced thrombus formation, associated with SIRT1 downregulation. Pharmacological activation of SIRT1 (by resveratrol or the synthetic activator ISIDE11) rescued endothelial vasorelaxation and reduced thrombus formation in Mthfr+/- mice without affecting homocysteine levels. Human MTHFR C677T carriers with normal homocysteine also showed endothelial dysfunction and enhanced platelet aggregation linked to SIRT1 downregulation, normalized by resveratrol.\",\n      \"method\": \"Flow Mediated Dilatation (FMD) in human subjects, light transmission aggregometry, wire-induced thrombosis model in Mthfr+/- mice, cell-based HTS screening for SIRT1 activators, SIRT1 inhibitor controls\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic mouse model with functional vascular readouts, pharmacological rescue with mechanism (SIRT1 activity), single lab with multiple orthogonal assays\",\n      \"pmids\": [\"35821533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Antisense-mediated inhibition of MTHFR in tumor-derived cell lines increased cytotoxicity in vitro and decreased tumor growth in vivo in xenograft models, demonstrating that MTHFR activity is required for tumor cell survival through its role in methionine biosynthesis. Tumor cells (unlike normal cells) are methionine-dependent and cannot efficiently utilize homocysteine as a methionine substitute.\",\n      \"method\": \"Antisense oligonucleotide inhibition of MTHFR in cell lines, in vitro cytotoxicity assays, in vivo tumor xenograft growth assays\",\n      \"journal\": \"Current pharmaceutical design\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined cellular phenotype (cytotoxicity, tumor growth) in vitro and in vivo, single lab\",\n      \"pmids\": [\"18473861\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MTHFR promoter hypermethylation was associated with lower MTHFR mRNA expression in placenta of preeclamptic women. The 3'-UTR rs1537514C>G polymorphism was associated with higher MTHFR mRNA expression and lower risk of preeclampsia; the rs1537514G allele correlated with higher expression in both PE and normotensive groups. No association was found between rs4846049C>A polymorphism and MTHFR expression.\",\n      \"method\": \"Methylation-specific PCR of MTHFR promoter in placental tissue, quantitative RT-PCR for MTHFR mRNA, PCR-RFLP genotyping of 3'-UTR SNPs\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single tissue type, correlative relationship between methylation and expression without functional validation of causality\",\n      \"pmids\": [\"28722783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"MTHFR gene promoter hypermethylation was detected at significantly higher frequency in placenta and peripheral venous blood of preeclamptic women compared to normotensive controls (placenta: 26.8% vs. 15.2%, p<0.05; peripheral blood: 22.8% vs. 12.1%, p<0.05), and was associated with elevated plasma homocysteine in preeclampsia.\",\n      \"method\": \"Methylation-specific PCR on placental and peripheral blood DNA, ELISA for plasma homocysteine\",\n      \"journal\": \"Genetics and molecular research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, MSP only, correlative without mechanistic proof of causal relationship\",\n      \"pmids\": [\"26214484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MTHFR promoter methylation in spermatozoa of idiopathic infertile normozoospermic men was significantly higher (11%) than in fertile controls (4.3%), suggesting epigenetic silencing of MTHFR may contribute to idiopathic male infertility.\",\n      \"method\": \"Sodium bisulfite modification and pyrosequencing of MTHFR promoter in sperm DNA from infertile and fertile men\",\n      \"journal\": \"Andrologia\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, pyrosequencing only, correlative without functional consequence of silencing directly demonstrated\",\n      \"pmids\": [\"27596009\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MTHFR is a key metabolic enzyme that irreversibly converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (catalytic mechanism confirmed by cDNA expression and in vitro activity assays), thereby channeling folate toward homocysteine remethylation to methionine and away from thymidylate/purine synthesis; its activity is feedback-inhibited by S-adenosylmethionine (SAM), requires FAD as a cofactor retained by a disordered active-site loop, and is regulated during mitosis by CDK1/Cyclin B1-mediated phosphorylation at T34 which reduces enzymatic activity; the common C677T (Ala222Val) polymorphism causes thermolability and reduced activity in a folate-dependent manner, and MTHFR additionally promotes centromeric heterochromatin maintenance (H3K9me3) and influences vascular function through a SIRT1-linked pathway independent of homocysteine.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MTHFR is the metabolic enzyme that irreversibly converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the folate co-substrate for homocysteine remethylation to methionine, thereby partitioning one-carbon units between DNA synthesis (thymidylate/purine) and methionine regeneration [#1]. The catalytically active ~70 kDa enzyme is encoded by an 11-exon gene whose product was confirmed by cDNA expression [#0]. Deep mutational scanning across folinate environments resolved its regulatory architecture, identifying variants that escape S-adenosylmethionine feedback inhibition and a disordered loop that retains the FAD cofactor at the active site [#4]. Enzyme output is additionally controlled through the cell cycle: CDK1/Cyclin B1 phosphorylates MTHFR at Thr34, peaking in mitosis and lowering catalytic activity [#3]. Beyond its canonical metabolic role, MTHFR supports centromeric heterochromatin maintenance, as its knockdown reduces H3K9me3 and de-represses centromeric transcription [#3], and it influences vascular function through a SIRT1-linked pathway that operates independently of homocysteine levels [#7]. The common C677T (Ala222Val) variant produces a thermolabile enzyme with reduced activity, elevating plasma homocysteine in a folate-dependent manner [#1, #2], and functional-impact scores correlate with age of onset in severe MTHFR deficiency [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing the gene structure and confirming that the cloned cDNA produces a catalytically active enzyme provided the molecular foundation for all subsequent mechanistic work.\",\n      \"evidence\": \"Genomic clone isolation and cDNA expression yielding an active ~70 kDa enzyme, with cross-species conservation between human and mouse\",\n      \"pmids\": [\"9680386\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structural model of the active site at this stage\",\n        \"Regulatory mechanisms not addressed\"\n      ]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Defining MTHFR as the committed, irreversible step directing folate toward homocysteine remethylation versus nucleotide synthesis placed it at a metabolic branch point and linked the C677T variant to elevated homocysteine.\",\n      \"evidence\": \"Enzyme activity and thermolability assays integrated with population metabolic studies correlating genotype, folate species, and homocysteine\",\n      \"pmids\": [\"11282420\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of thermolability not resolved\",\n        \"Cofactor and feedback regulation not mechanistically defined\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Loss-of-function studies tested whether MTHFR activity is required for cell survival, showing methionine-dependent tumor cells rely on it and that VPA can transcriptionally upregulate the enzyme.\",\n      \"evidence\": \"Antisense inhibition with cytotoxicity and xenograft assays (PMID 18473861); MTHFR promoter-reporter, mRNA/protein, and in vivo VPA studies in Mthfr+/- mice (PMID 18615588)\",\n      \"pmids\": [\"18473861\", \"18615588\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism of VPA-driven promoter activation not mapped to specific elements\",\n        \"Generality of tumor methionine dependence beyond tested lines unestablished\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovery of CDK1/Cyclin B1-mediated Thr34 phosphorylation and a heterochromatin phenotype revealed cell-cycle regulation of MTHFR activity and a moonlighting role beyond folate metabolism.\",\n      \"evidence\": \"Phospho-specific antibody, cell-cycle synchronization, in vitro kinase assays, and siRNA knockdown with H3K9me3 immunoblotting and centromeric RT-PCR\",\n      \"pmids\": [\"24769206\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab reciprocal Co-IP without independent confirmation\",\n        \"How reduced MTHFR activity mechanistically links to H3K9me3 maintenance is unresolved\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Direct metabolite measurement in genotype-stratified cells clarified that low-activity MTHFR limits conversion of folic acid to 5-methyltetrahydrofolate, with implications for supplementation strategy.\",\n      \"evidence\": \"LC-MS/MS quantification of intracellular 5-Me-THF in genotyped lymphoblastoid lines under folic acid versus 5-Me-THF supplementation, with folate-deprivation cell-cycle assays\",\n      \"pmids\": [\"32887268\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single cell-type system\",\n        \"In vivo relevance of supplementation findings not established\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A genome-scale variant atlas resolved the enzyme's regulatory features—SAM feedback inhibition and FAD retention by a disordered loop—and made variant effects environment- and folate-dependent, linking functional scores to clinical onset.\",\n      \"evidence\": \"Saturation genome editing / deep mutational scanning of 98,336 variant assays across four folinate environments with correlation to disease onset\",\n      \"pmids\": [\"34214447\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Atomic-resolution structure of the disordered FAD-retaining loop not provided\",\n        \"Mechanism by which escape variants bypass SAM inhibition not structurally defined\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"A homocysteine-independent vascular axis was established, showing MTHFR deficiency causes endothelial dysfunction and thrombosis via SIRT1 downregulation that is pharmacologically reversible.\",\n      \"evidence\": \"Mthfr+/- mouse thrombosis and endothelial function models plus human C677T flow-mediated dilatation and aggregometry, with SIRT1 activator rescue and inhibitor controls\",\n      \"pmids\": [\"35821533\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Molecular link between MTHFR activity and SIRT1 expression not defined\",\n        \"Single-lab findings\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How epigenetic regulation of MTHFR (promoter hypermethylation in preeclampsia and sperm) causally affects expression and phenotype remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Promoter methylation associations are correlative without functional proof of causality\",\n        \"No mechanistic link between methylation state and downstream pathology demonstrated\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [1, 4]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CDK1\", \"CCNB1\", \"SIRT1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}