{"gene":"FECH","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2001,"finding":"An intronic SNP (IVS3-48T/C) in FECH modulates use of a constitutive aberrant acceptor splice site; the aberrantly spliced mRNA is degraded by nonsense-mediated decay (NMD), producing decreased steady-state FECH mRNA and additional enzyme deficiency required for EPP phenotypic expression.","method":"Haplotype segregation analysis combined with molecular characterization of aberrant splicing and NMD mechanism","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct molecular mechanism established (aberrant splice site + NMD), replicated across multiple families, multiple orthogonal methods (haplotype analysis, mRNA quantification, NMD characterization)","pmids":["11753383"],"is_preprint":false},{"year":2022,"finding":"The flavonoid DMC directly inhibits FECH enzymatic activity (confirmed by thermal proteome profiling and enzymatic assay); FECH inhibition combined with HMOX1 upregulation causes iron overload and triggers ferroptosis in cancer cells.","method":"Thermal proteome profiling (TPP) to identify FECH as DMC target, enzymatic activity assay, knockdown/overexpression in cancer cells","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — TPP target identification plus enzymatic activity confirmation plus cellular phenotype, single lab","pmids":["35697292"],"is_preprint":false},{"year":2020,"finding":"HIF-1α acts downstream of MEK signaling to regulate FECH activity; HIF-1α inhibition decreases FECH activity and increases PpIX accumulation, placing FECH in the HIF-1α-FECH axis downstream of Ras/MEK.","method":"MEK inhibitor treatment, HIF-1α inhibition, RasV12-transformed cell lines and transgenic mice, PpIX accumulation assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via inhibitor/knockdown in multiple cell models including in vivo transgenic mice, single lab","pmids":["33335181"],"is_preprint":false},{"year":2011,"finding":"5-aza-2'-deoxycytidine promotes nuclear translocation of c-Myc and its binding to Max, enabling c-Myc to bind E-boxes in the Fech promoter (outside CpG islands), thereby increasing Fech transcription and heme biosynthesis in erythroid cells.","method":"Promoter E-box identification, methylation analysis, ChIP/binding assays for c-Myc and Max, nuclear translocation imaging in murine erythroid leukemia cells and erythroblasts","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct promoter binding and methylation analysis with c-Myc nuclear translocation confirmed, single lab with two cell model systems","pmids":["21903580"],"is_preprint":false},{"year":2018,"finding":"The FECH c.315-48T>C (IVS3-48T/C) variant increases the proportion of aberrantly spliced FECH mRNA in a dose-dependent manner; homozygosity for this variant doubles the percentage of aberrant splice insertions and is sufficient to reduce FECH expression to pathological levels, establishing it as pathogenic on its own.","method":"Digital PCR (dPCR) absolute quantification of aberrantly spliced and total FECH mRNA molecules in a cohort stratified by genotype","journal":"Molecular genetics and metabolism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct quantitative measurement of aberrant splicing by dPCR across a comprehensive cohort, single lab","pmids":["29941360"],"is_preprint":false},{"year":2007,"finding":"A 10,376 bp deletion encompassing the FECH promoter, exon 1, and part of intron 1 abolishes expression of the mutated allele entirely (no transcript produced), reducing total FECH gene expression by half, as confirmed by RNA analysis.","method":"Long-PCR to define deletion breakpoints, RNA analysis to demonstrate absent expression from deleted allele","journal":"Blood cells, molecules & diseases","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct RNA analysis demonstrating absent transcript from deleted allele, single lab","pmids":["17888693"],"is_preprint":false},{"year":2019,"finding":"A novel deep intronic FECH variant abolishes an exonic splicing silencer site and creates a new methylated CpG dinucleotide, causing pseudo-exon insertion with a stop codon into the mature FECH transcript via a methylation-dependent splicing mechanism.","method":"High-throughput resequencing, qualitative RNA analysis, quantitative DNA methylation examination","journal":"Genetics in medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct RNA and methylation analysis demonstrating mechanism, single lab with cohort validation","pmids":["31273344"],"is_preprint":false},{"year":2024,"finding":"CRISPR/Cas9-mediated fech knockout in zebrafish causes protoporphyrin IX accumulation, apoptosis (elevated bax/bcl2 ratio), and increased macrophage/neutrophil production, establishing fech loss-of-function phenotype in vivo; UDCA treatment reduced PPIX fluorescence and suppressed the intrinsic apoptosis pathway in fech−/− larvae.","method":"CRISPR/Cas9 knockout zebrafish, acridine orange staining, qRT-PCR bax/bcl2 ratio, neutral red and Sudan black staining, PPIX fluorescence quantification","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct genetic knockout with multiple orthogonal cellular readouts in zebrafish model, single lab","pmids":["39409147"],"is_preprint":false},{"year":2021,"finding":"Knockdown of IGHG1 in colorectal cancer cells reduced phosphorylated ERK and FECH expression, resulting in decreased hemin biosynthesis and increased PpIX accumulation, placing FECH downstream of an MEK-ERK axis regulated by IGHG1.","method":"shRNA-mediated knockdown of IGHG1, western blot for pERK and FECH, PpIX and hemin quantification","journal":"Open life sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, indirect pathway placement via knockdown of upstream regulator, no direct FECH mechanistic assay","pmids":["34553073"],"is_preprint":false}],"current_model":"FECH (ferrochelatase) catalyzes the terminal step of heme biosynthesis (insertion of iron into protoporphyrin IX); its expression is transcriptionally regulated by c-Myc binding to promoter E-boxes in a methylation-dependent manner, and post-transcriptionally regulated by an intronic IVS3-48T/C SNP that modulates aberrant splicing followed by NMD of the mis-spliced transcript; enzymatically, FECH activity is controlled downstream of HIF-1α within the Ras/MEK pathway, and FECH inhibition (genetic or pharmacological) blocks PpIX-to-heme conversion leading to PpIX accumulation and ferroptosis."},"narrative":{"mechanistic_narrative":"FECH (ferrochelatase) catalyzes the terminal step of heme biosynthesis, the insertion of iron into protoporphyrin IX (PpIX), and its loss or inhibition causes PpIX accumulation with downstream cell-death consequences [PMID:35697292, PMID:39409147]. Expression of FECH is controlled at multiple levels: transcriptionally, c-Myc translocates to the nucleus, binds Max, and occupies E-boxes in the FECH promoter (outside CpG islands) to drive transcription in erythroid cells, a process modulated by promoter methylation [PMID:21903580]; post-transcriptionally, the intronic IVS3-48T/C (c.315-48T>C) variant increases use of a constitutive aberrant acceptor splice site, and the mis-spliced transcript is degraded by nonsense-mediated decay, dose-dependently lowering steady-state FECH and producing the enzyme deficiency required for erythropoietic protoporphyria (EPP) [PMID:11753383, PMID:29941360]. Additional pathogenic alleles act through allele-specific transcript loss (a promoter/exon-1 deletion) or a deep-intronic methylation-dependent pseudo-exon insertion, converging on the same outcome of reduced functional FECH [PMID:17888693, PMID:31273344]. Enzymatically, FECH activity is positioned downstream of Ras/MEK signaling via HIF-1α, such that pathway inhibition lowers FECH activity and raises PpIX [PMID:33335181]. Pharmacological FECH inhibition blocks PpIX-to-heme conversion and, when combined with HMOX1 upregulation, drives iron overload and ferroptosis in cancer cells [PMID:35697292].","teleology":[{"year":2001,"claim":"Established why a low-penetrance intronic polymorphism, rather than a coding mutation, is required for EPP phenotype expression by defining a post-transcriptional mechanism that lowers FECH dosage.","evidence":"Haplotype segregation analysis with molecular characterization of aberrant splicing and NMD in EPP families","pmids":["11753383"],"confidence":"High","gaps":["Does not quantify the absolute fraction of mis-spliced transcript needed to cross the pathological threshold","Mechanism of how the SNP biases splice-site choice not resolved at the spliceosome level"]},{"year":2007,"claim":"Showed that large structural deletions removing the promoter and exon 1 abolish transcription from the affected allele, defining an allele-loss route to FECH haploinsufficiency.","evidence":"Long-PCR breakpoint mapping plus RNA analysis demonstrating absent transcript from the deleted allele","pmids":["17888693"],"confidence":"Medium","gaps":["Single family/lab observation","Does not address whether residual allele expression is itself modulated by trans factors"]},{"year":2011,"claim":"Identified the transcriptional driver of FECH in erythroid cells, linking promoter methylation status to c-Myc/Max E-box occupancy and heme output.","evidence":"Promoter E-box mapping, methylation analysis, c-Myc/Max ChIP and nuclear translocation imaging in murine erythroid leukemia cells and erythroblasts","pmids":["21903580"],"confidence":"Medium","gaps":["Single lab","Direct demonstration in human erythroid cells not shown","Quantitative contribution of c-Myc relative to other promoter factors unclear"]},{"year":2018,"claim":"Resolved whether the IVS3-48T/C variant is merely a modifier or independently pathogenic by quantifying aberrant splicing across genotypes.","evidence":"Digital PCR absolute quantification of aberrant and total FECH mRNA in a genotype-stratified cohort","pmids":["29941360"],"confidence":"Medium","gaps":["Single lab","Does not link transcript-level changes to enzyme activity measurements in the same individuals"]},{"year":2019,"claim":"Extended the mutational spectrum by showing a deep-intronic variant can act through a methylation-dependent pseudo-exon insertion, coupling DNA methylation to aberrant splicing.","evidence":"High-throughput resequencing with qualitative RNA analysis and quantitative DNA methylation assays","pmids":["31273344"],"confidence":"Medium","gaps":["Single lab","Causal contribution of the new CpG methylation versus the silencer loss not fully separated"]},{"year":2020,"claim":"Placed FECH enzymatic activity within oncogenic signaling by showing HIF-1α operates downstream of MEK to control FECH and PpIX levels.","evidence":"MEK and HIF-1α inhibition in RasV12-transformed cells and transgenic mice with PpIX accumulation assays","pmids":["33335181"],"confidence":"Medium","gaps":["Single lab","Whether HIF-1α regulates FECH transcription, translation, or activity directly not distinguished"]},{"year":2022,"claim":"Demonstrated FECH as a druggable enzymatic target whose inhibition, combined with HMOX1 upregulation, drives ferroptotic cell death in cancer.","evidence":"Thermal proteome profiling to identify FECH as the DMC target, enzymatic activity assay, and knockdown/overexpression in cancer cells","pmids":["35697292"],"confidence":"Medium","gaps":["Single lab","DMC selectivity for FECH versus other heme-pathway enzymes not exhaustively excluded"]},{"year":2024,"claim":"Defined the in vivo loss-of-function phenotype of FECH, linking PpIX accumulation to intrinsic apoptosis and altered myeloid output, and showed pharmacological rescue.","evidence":"CRISPR/Cas9 fech knockout zebrafish with apoptosis, PpIX fluorescence, and immune-cell readouts plus UDCA treatment","pmids":["39409147"],"confidence":"Medium","gaps":["Single lab","Mechanism by which UDCA reduces PpIX and apoptosis not defined","Relationship between apoptosis here and ferroptosis reported in cancer models unresolved"]},{"year":2021,"claim":"Implicated an upstream MEK-ERK regulator (IGHG1) in controlling FECH expression in colorectal cancer.","evidence":"shRNA knockdown of IGHG1 with western blot for pERK and FECH and PpIX/hemin quantification","pmids":["34553073"],"confidence":"Low","gaps":["Indirect pathway placement via upstream knockdown with no direct FECH mechanistic assay","Single lab","Does not establish whether IGHG1 acts transcriptionally or post-transcriptionally on FECH"]},{"year":null,"claim":"How the multiple regulatory inputs (c-Myc transcription, splicing/NMD dosage control, HIF-1α/MEK signaling) are integrated to set FECH activity in a given cell type, and the structural basis of iron insertion, remain unresolved in this corpus.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural/catalytic mechanism of iron insertion characterized in the timeline","Cross-talk between transcriptional and splicing regulation not integrated","Whether ferroptosis versus apoptosis dominates upon FECH loss is context-dependent and unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[1,2,7]}],"localization":[],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[1,2,7]}],"complexes":[],"partners":[],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P22830","full_name":"Ferrochelatase, mitochondrial","aliases":["Heme synthase","Protoheme ferro-lyase"],"length_aa":423,"mass_kda":47.9,"function":"Catalyzes the ferrous insertion into protoporphyrin IX and participates in the terminal step in the heme biosynthetic pathway","subcellular_location":"Mitochondrion inner membrane","url":"https://www.uniprot.org/uniprotkb/P22830/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/FECH","classification":"Common Essential","n_dependent_lines":520,"n_total_lines":1208,"dependency_fraction":0.4304635761589404},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CALM1","stoichiometry":0.2},{"gene":"CALM2","stoichiometry":0.2},{"gene":"CALM3","stoichiometry":0.2},{"gene":"DNAJB6","stoichiometry":0.2},{"gene":"MINK1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/FECH","total_profiled":1310},"omim":[{"mim_id":"616860","title":"ANEMIA, SIDEROBLASTIC, 3, PYRIDOXINE-REFRACTORY; SIDBA3","url":"https://www.omim.org/entry/616860"},{"mim_id":"615317","title":"IRON-SULFUR CLUSTER ASSEMBLY 2; ISCA2","url":"https://www.omim.org/entry/615317"},{"mim_id":"615316","title":"IRON-SULFUR CLUSTER ASSEMBLY FACTOR IBA57; IBA57","url":"https://www.omim.org/entry/615316"},{"mim_id":"614981","title":"ATPase INHIBITORY FACTOR 1; ATPIF1","url":"https://www.omim.org/entry/614981"},{"mim_id":"612386","title":"FERROCHELATASE; FECH","url":"https://www.omim.org/entry/612386"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":36.8}],"url":"https://www.proteinatlas.org/search/FECH"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P22830","domains":[{"cath_id":"3.40.50.1400","chopping":"70-229_375-421","consensus_level":"medium","plddt":96.4769,"start":70,"end":421},{"cath_id":"3.40.50.1400","chopping":"232-368","consensus_level":"medium","plddt":97.8447,"start":232,"end":368}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P22830","model_url":"https://alphafold.ebi.ac.uk/files/AF-P22830-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P22830-F1-predicted_aligned_error_v6.png","plddt_mean":86.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FECH","jax_strain_url":"https://www.jax.org/strain/search?query=FECH"},"sequence":{"accession":"P22830","fasta_url":"https://rest.uniprot.org/uniprotkb/P22830.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P22830/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P22830"}},"corpus_meta":[{"pmid":"11753383","id":"PMC_11753383","title":"The penetrance of dominant erythropoietic protoporphyria is modulated by expression of wildtype FECH.","date":"2001","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/11753383","citation_count":197,"is_preprint":false},{"pmid":"35697292","id":"PMC_35697292","title":"Flavonoid 4,4'-dimethoxychalcone induced ferroptosis in cancer cells by synergistically activating Keap1/Nrf2/HMOX1 pathway and inhibiting FECH.","date":"2022","source":"Free radical biology & medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35697292","citation_count":71,"is_preprint":false},{"pmid":"1783383","id":"PMC_1783383","title":"Assignment of the human ferrochelatase gene (FECH) and a locus for protoporphyria to chromosome 18q22.","date":"1991","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/1783383","citation_count":62,"is_preprint":false},{"pmid":"17597821","id":"PMC_17597821","title":"Gene dosage analysis identifies large deletions of the FECH gene in 10% of families with erythropoietic protoporphyria.","date":"2007","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/17597821","citation_count":30,"is_preprint":false},{"pmid":"33335181","id":"PMC_33335181","title":"MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes.","date":"2020","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/33335181","citation_count":21,"is_preprint":false},{"pmid":"21903580","id":"PMC_21903580","title":"5-aza-2'-deoxycytidine activates iron uptake and heme biosynthesis by increasing c-Myc nuclear localization and binding to the E-boxes of transferrin receptor 1 (TfR1) and ferrochelatase (Fech) genes.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21903580","citation_count":21,"is_preprint":false},{"pmid":"8034322","id":"PMC_8034322","title":"Molecular analysis of functional and nonfunctional genes for human ferrochelatase: isolation and characterization of a FECH pseudogene and its sublocalization on chromosome 3.","date":"1994","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/8034322","citation_count":13,"is_preprint":false},{"pmid":"31273344","id":"PMC_31273344","title":"Targeted resequencing of FECH locus reveals that a novel deep intronic pathogenic variant and eQTLs may cause erythropoietic protoporphyria (EPP) through a methylation-dependent mechanism.","date":"2019","source":"Genetics in medicine : official journal of the American College of Medical Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/31273344","citation_count":12,"is_preprint":false},{"pmid":"34686726","id":"PMC_34686726","title":"Nkx3-1 and Fech genes might be switch genes involved in pituitary non-functioning adenoma invasiveness.","date":"2021","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/34686726","citation_count":7,"is_preprint":false},{"pmid":"20337824","id":"PMC_20337824","title":"A homoallelic FECH mutation in a patient with both erythropoietic protoporphyria and palmar keratoderma.","date":"2010","source":"Journal of the European Academy of Dermatology and Venereology : JEADV","url":"https://pubmed.ncbi.nlm.nih.gov/20337824","citation_count":7,"is_preprint":false},{"pmid":"35470464","id":"PMC_35470464","title":"The role of the genetic variant FECH rs11660001 in the occurrence of anti-tuberculosis drug-induced liver injury.","date":"2022","source":"Journal of clinical pharmacy and therapeutics","url":"https://pubmed.ncbi.nlm.nih.gov/35470464","citation_count":6,"is_preprint":false},{"pmid":"17888693","id":"PMC_17888693","title":"A 10376 bp deletion of FECH gene responsible for erythropoietic protoporphyria.","date":"2007","source":"Blood cells, molecules & diseases","url":"https://pubmed.ncbi.nlm.nih.gov/17888693","citation_count":6,"is_preprint":false},{"pmid":"19888946","id":"PMC_19888946","title":"A novel splicing mutation and haplotype analysis of the FECH gene in a Chinese family with erythropoietic protoporphyria.","date":"2009","source":"Journal of the European Academy of Dermatology and Venereology : JEADV","url":"https://pubmed.ncbi.nlm.nih.gov/19888946","citation_count":5,"is_preprint":false},{"pmid":"24751349","id":"PMC_24751349","title":"Comparative uptake of ¹⁸F-FEN-DPAZn2, ¹⁸F-FECH, ¹⁸F-fluoride, and ¹⁸F-FDG in fibrosarcoma and aseptic inflammation.","date":"2014","source":"Applied radiation and isotopes : including data, instrumentation and methods for use in agriculture, industry and medicine","url":"https://pubmed.ncbi.nlm.nih.gov/24751349","citation_count":5,"is_preprint":false},{"pmid":"29941360","id":"PMC_29941360","title":"Digital PCR (dPCR) analysis reveals that the homozygous c.315-48T>C variant in the FECH gene might cause erythropoietic protoporphyria (EPP).","date":"2018","source":"Molecular genetics and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/29941360","citation_count":5,"is_preprint":false},{"pmid":"34553073","id":"PMC_34553073","title":"Down-regulation of IGHG1 enhances Protoporphyrin IX accumulation and inhibits hemin biosynthesis in colorectal cancer by suppressing the MEK-FECH axis.","date":"2021","source":"Open life sciences","url":"https://pubmed.ncbi.nlm.nih.gov/34553073","citation_count":5,"is_preprint":false},{"pmid":"19055472","id":"PMC_19055472","title":"A novel large deletion and three polymorphisms in the FECH gene associated with erythropoietic protoporphyria.","date":"2009","source":"Clinical chemistry and laboratory medicine","url":"https://pubmed.ncbi.nlm.nih.gov/19055472","citation_count":5,"is_preprint":false},{"pmid":"39409147","id":"PMC_39409147","title":"CRISPR/Cas9-Mediated fech Knockout Zebrafish: Unraveling the Pathogenesis of Erythropoietic Protoporphyria and Facilitating Drug Screening.","date":"2024","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/39409147","citation_count":4,"is_preprint":false},{"pmid":"27704751","id":"PMC_27704751","title":"Identification of FECH gene multiple variations in two Chinese patients with erythropoietic protoporphyria and a review.","date":"2016","source":"Journal of Zhejiang University. Science. B","url":"https://pubmed.ncbi.nlm.nih.gov/27704751","citation_count":3,"is_preprint":false},{"pmid":"26789144","id":"PMC_26789144","title":"A Novel Mutation in the FECH Gene in a Czech Family with Erythropoietic Protoporphyria and a Population Study of IVS3-48C Variant Contributing to the Disease.","date":"2015","source":"Folia biologica","url":"https://pubmed.ncbi.nlm.nih.gov/26789144","citation_count":3,"is_preprint":false},{"pmid":"21659066","id":"PMC_21659066","title":"Erythropoietic protoporphyria: a family study and report of a novel mutation in the FECH gene.","date":"2011","source":"European journal of dermatology : EJD","url":"https://pubmed.ncbi.nlm.nih.gov/21659066","citation_count":2,"is_preprint":false},{"pmid":"31304091","id":"PMC_31304091","title":"Characterization of a novel pathogenic variant in the FECH gene associated with erythropoietic protoporphyria.","date":"2019","source":"Molecular genetics and metabolism reports","url":"https://pubmed.ncbi.nlm.nih.gov/31304091","citation_count":2,"is_preprint":false},{"pmid":"40582371","id":"PMC_40582371","title":"Effects of FECH Gene Polymorphisms and Serum Ferrochelatase Levels on Antituberculosis Drug-Induced Liver Injury in China.","date":"2025","source":"Fundamental & clinical pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40582371","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13194,"output_tokens":2115,"usd":0.035653,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9250,"output_tokens":2964,"usd":0.060175,"stage2_stop_reason":"end_turn"},"total_usd":0.095828,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"An intronic SNP (IVS3-48T/C) in FECH modulates use of a constitutive aberrant acceptor splice site; the aberrantly spliced mRNA is degraded by nonsense-mediated decay (NMD), producing decreased steady-state FECH mRNA and additional enzyme deficiency required for EPP phenotypic expression.\",\n      \"method\": \"Haplotype segregation analysis combined with molecular characterization of aberrant splicing and NMD mechanism\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct molecular mechanism established (aberrant splice site + NMD), replicated across multiple families, multiple orthogonal methods (haplotype analysis, mRNA quantification, NMD characterization)\",\n      \"pmids\": [\"11753383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The flavonoid DMC directly inhibits FECH enzymatic activity (confirmed by thermal proteome profiling and enzymatic assay); FECH inhibition combined with HMOX1 upregulation causes iron overload and triggers ferroptosis in cancer cells.\",\n      \"method\": \"Thermal proteome profiling (TPP) to identify FECH as DMC target, enzymatic activity assay, knockdown/overexpression in cancer cells\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — TPP target identification plus enzymatic activity confirmation plus cellular phenotype, single lab\",\n      \"pmids\": [\"35697292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HIF-1α acts downstream of MEK signaling to regulate FECH activity; HIF-1α inhibition decreases FECH activity and increases PpIX accumulation, placing FECH in the HIF-1α-FECH axis downstream of Ras/MEK.\",\n      \"method\": \"MEK inhibitor treatment, HIF-1α inhibition, RasV12-transformed cell lines and transgenic mice, PpIX accumulation assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via inhibitor/knockdown in multiple cell models including in vivo transgenic mice, single lab\",\n      \"pmids\": [\"33335181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"5-aza-2'-deoxycytidine promotes nuclear translocation of c-Myc and its binding to Max, enabling c-Myc to bind E-boxes in the Fech promoter (outside CpG islands), thereby increasing Fech transcription and heme biosynthesis in erythroid cells.\",\n      \"method\": \"Promoter E-box identification, methylation analysis, ChIP/binding assays for c-Myc and Max, nuclear translocation imaging in murine erythroid leukemia cells and erythroblasts\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct promoter binding and methylation analysis with c-Myc nuclear translocation confirmed, single lab with two cell model systems\",\n      \"pmids\": [\"21903580\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The FECH c.315-48T>C (IVS3-48T/C) variant increases the proportion of aberrantly spliced FECH mRNA in a dose-dependent manner; homozygosity for this variant doubles the percentage of aberrant splice insertions and is sufficient to reduce FECH expression to pathological levels, establishing it as pathogenic on its own.\",\n      \"method\": \"Digital PCR (dPCR) absolute quantification of aberrantly spliced and total FECH mRNA molecules in a cohort stratified by genotype\",\n      \"journal\": \"Molecular genetics and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct quantitative measurement of aberrant splicing by dPCR across a comprehensive cohort, single lab\",\n      \"pmids\": [\"29941360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"A 10,376 bp deletion encompassing the FECH promoter, exon 1, and part of intron 1 abolishes expression of the mutated allele entirely (no transcript produced), reducing total FECH gene expression by half, as confirmed by RNA analysis.\",\n      \"method\": \"Long-PCR to define deletion breakpoints, RNA analysis to demonstrate absent expression from deleted allele\",\n      \"journal\": \"Blood cells, molecules & diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct RNA analysis demonstrating absent transcript from deleted allele, single lab\",\n      \"pmids\": [\"17888693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"A novel deep intronic FECH variant abolishes an exonic splicing silencer site and creates a new methylated CpG dinucleotide, causing pseudo-exon insertion with a stop codon into the mature FECH transcript via a methylation-dependent splicing mechanism.\",\n      \"method\": \"High-throughput resequencing, qualitative RNA analysis, quantitative DNA methylation examination\",\n      \"journal\": \"Genetics in medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct RNA and methylation analysis demonstrating mechanism, single lab with cohort validation\",\n      \"pmids\": [\"31273344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CRISPR/Cas9-mediated fech knockout in zebrafish causes protoporphyrin IX accumulation, apoptosis (elevated bax/bcl2 ratio), and increased macrophage/neutrophil production, establishing fech loss-of-function phenotype in vivo; UDCA treatment reduced PPIX fluorescence and suppressed the intrinsic apoptosis pathway in fech−/− larvae.\",\n      \"method\": \"CRISPR/Cas9 knockout zebrafish, acridine orange staining, qRT-PCR bax/bcl2 ratio, neutral red and Sudan black staining, PPIX fluorescence quantification\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct genetic knockout with multiple orthogonal cellular readouts in zebrafish model, single lab\",\n      \"pmids\": [\"39409147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Knockdown of IGHG1 in colorectal cancer cells reduced phosphorylated ERK and FECH expression, resulting in decreased hemin biosynthesis and increased PpIX accumulation, placing FECH downstream of an MEK-ERK axis regulated by IGHG1.\",\n      \"method\": \"shRNA-mediated knockdown of IGHG1, western blot for pERK and FECH, PpIX and hemin quantification\",\n      \"journal\": \"Open life sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, indirect pathway placement via knockdown of upstream regulator, no direct FECH mechanistic assay\",\n      \"pmids\": [\"34553073\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FECH (ferrochelatase) catalyzes the terminal step of heme biosynthesis (insertion of iron into protoporphyrin IX); its expression is transcriptionally regulated by c-Myc binding to promoter E-boxes in a methylation-dependent manner, and post-transcriptionally regulated by an intronic IVS3-48T/C SNP that modulates aberrant splicing followed by NMD of the mis-spliced transcript; enzymatically, FECH activity is controlled downstream of HIF-1α within the Ras/MEK pathway, and FECH inhibition (genetic or pharmacological) blocks PpIX-to-heme conversion leading to PpIX accumulation and ferroptosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"FECH (ferrochelatase) catalyzes the terminal step of heme biosynthesis, the insertion of iron into protoporphyrin IX (PpIX), and its loss or inhibition causes PpIX accumulation with downstream cell-death consequences [#1, #7]. Expression of FECH is controlled at multiple levels: transcriptionally, c-Myc translocates to the nucleus, binds Max, and occupies E-boxes in the FECH promoter (outside CpG islands) to drive transcription in erythroid cells, a process modulated by promoter methylation [#3]; post-transcriptionally, the intronic IVS3-48T/C (c.315-48T>C) variant increases use of a constitutive aberrant acceptor splice site, and the mis-spliced transcript is degraded by nonsense-mediated decay, dose-dependently lowering steady-state FECH and producing the enzyme deficiency required for erythropoietic protoporphyria (EPP) [#0, #4]. Additional pathogenic alleles act through allele-specific transcript loss (a promoter/exon-1 deletion) or a deep-intronic methylation-dependent pseudo-exon insertion, converging on the same outcome of reduced functional FECH [#5, #6]. Enzymatically, FECH activity is positioned downstream of Ras/MEK signaling via HIF-1\\u03b1, such that pathway inhibition lowers FECH activity and raises PpIX [#2]. Pharmacological FECH inhibition blocks PpIX-to-heme conversion and, when combined with HMOX1 upregulation, drives iron overload and ferroptosis in cancer cells [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established why a low-penetrance intronic polymorphism, rather than a coding mutation, is required for EPP phenotype expression by defining a post-transcriptional mechanism that lowers FECH dosage.\",\n      \"evidence\": \"Haplotype segregation analysis with molecular characterization of aberrant splicing and NMD in EPP families\",\n      \"pmids\": [\"11753383\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not quantify the absolute fraction of mis-spliced transcript needed to cross the pathological threshold\", \"Mechanism of how the SNP biases splice-site choice not resolved at the spliceosome level\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Showed that large structural deletions removing the promoter and exon 1 abolish transcription from the affected allele, defining an allele-loss route to FECH haploinsufficiency.\",\n      \"evidence\": \"Long-PCR breakpoint mapping plus RNA analysis demonstrating absent transcript from the deleted allele\",\n      \"pmids\": [\"17888693\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single family/lab observation\", \"Does not address whether residual allele expression is itself modulated by trans factors\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified the transcriptional driver of FECH in erythroid cells, linking promoter methylation status to c-Myc/Max E-box occupancy and heme output.\",\n      \"evidence\": \"Promoter E-box mapping, methylation analysis, c-Myc/Max ChIP and nuclear translocation imaging in murine erythroid leukemia cells and erythroblasts\",\n      \"pmids\": [\"21903580\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct demonstration in human erythroid cells not shown\", \"Quantitative contribution of c-Myc relative to other promoter factors unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Resolved whether the IVS3-48T/C variant is merely a modifier or independently pathogenic by quantifying aberrant splicing across genotypes.\",\n      \"evidence\": \"Digital PCR absolute quantification of aberrant and total FECH mRNA in a genotype-stratified cohort\",\n      \"pmids\": [\"29941360\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Does not link transcript-level changes to enzyme activity measurements in the same individuals\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended the mutational spectrum by showing a deep-intronic variant can act through a methylation-dependent pseudo-exon insertion, coupling DNA methylation to aberrant splicing.\",\n      \"evidence\": \"High-throughput resequencing with qualitative RNA analysis and quantitative DNA methylation assays\",\n      \"pmids\": [\"31273344\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Causal contribution of the new CpG methylation versus the silencer loss not fully separated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed FECH enzymatic activity within oncogenic signaling by showing HIF-1\\u03b1 operates downstream of MEK to control FECH and PpIX levels.\",\n      \"evidence\": \"MEK and HIF-1\\u03b1 inhibition in RasV12-transformed cells and transgenic mice with PpIX accumulation assays\",\n      \"pmids\": [\"33335181\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Whether HIF-1\\u03b1 regulates FECH transcription, translation, or activity directly not distinguished\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated FECH as a druggable enzymatic target whose inhibition, combined with HMOX1 upregulation, drives ferroptotic cell death in cancer.\",\n      \"evidence\": \"Thermal proteome profiling to identify FECH as the DMC target, enzymatic activity assay, and knockdown/overexpression in cancer cells\",\n      \"pmids\": [\"35697292\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"DMC selectivity for FECH versus other heme-pathway enzymes not exhaustively excluded\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the in vivo loss-of-function phenotype of FECH, linking PpIX accumulation to intrinsic apoptosis and altered myeloid output, and showed pharmacological rescue.\",\n      \"evidence\": \"CRISPR/Cas9 fech knockout zebrafish with apoptosis, PpIX fluorescence, and immune-cell readouts plus UDCA treatment\",\n      \"pmids\": [\"39409147\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Mechanism by which UDCA reduces PpIX and apoptosis not defined\", \"Relationship between apoptosis here and ferroptosis reported in cancer models unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Implicated an upstream MEK-ERK regulator (IGHG1) in controlling FECH expression in colorectal cancer.\",\n      \"evidence\": \"shRNA knockdown of IGHG1 with western blot for pERK and FECH and PpIX/hemin quantification\",\n      \"pmids\": [\"34553073\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Indirect pathway placement via upstream knockdown with no direct FECH mechanistic assay\", \"Single lab\", \"Does not establish whether IGHG1 acts transcriptionally or post-transcriptionally on FECH\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple regulatory inputs (c-Myc transcription, splicing/NMD dosage control, HIF-1\\u03b1/MEK signaling) are integrated to set FECH activity in a given cell type, and the structural basis of iron insertion, remain unresolved in this corpus.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural/catalytic mechanism of iron insertion characterized in the timeline\", \"Cross-talk between transcriptional and splicing regulation not integrated\", \"Whether ferroptosis versus apoptosis dominates upon FECH loss is context-dependent and unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [1, 2, 7]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [1, 2, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":5,"faith_total":5,"faith_pct":100.0}}