{"gene":"HSD3B2","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":1999,"finding":"Functional characterization of 25 HSD3B2 missense mutations by transient expression in 293 cells demonstrated that severe salt-wasting forms result from complete loss of enzyme activity, while non-salt-losing forms retain residual enzymatic activity. Additionally, some mutations cause disease through protein instability rather than direct catalytic impairment.","method":"Site-directed mutagenesis, transient expression in 293 cells, [14C]-DHEA substrate assay, Northern/Western blot, in vitro transcription/translation","journal":"The Journal of clinical endocrinology and metabolism","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic assay with mutagenesis across 25 mutant enzymes, multiple orthogonal methods (activity assay, Northern/Western, reticulocyte translation), single comprehensive study","pmids":["10599696"],"is_preprint":false},{"year":2000,"finding":"The A10E mutation in the NAD-binding domain of HSD3B2 abolishes all detectable enzymatic activity when expressed in transfected Ad293 cells, establishing that Ala10 is critical for catalytic function.","method":"Direct sequencing, transient expression in Ad293 cells, enzymatic activity assay","journal":"The Journal of clinical endocrinology and metabolism","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro activity assay with mutagenesis, single lab, single mutation characterized","pmids":["10843183"],"is_preprint":false},{"year":2012,"finding":"Metformin inhibits HSD3B2 enzymatic activity and decreases HSD3B2 expression in NCI-H295R steroidogenic cells. This inhibition is dependent on organic cation transporters for metformin uptake and is mediated through inhibition of mitochondrial complex I (not AMPK, ERK1/2, or atypical PKC signaling). Direct inhibition of complex I by rotenone also inhibits HSD3B2 activity, establishing the mechanistic link.","method":"Enzymatic activity assay, mRNA/protein expression analysis, pharmacological inhibition (rotenone), AMPK/ERK signaling analysis, organic cation transporter dependency assay in NCI-H295R cells","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (activity assay, expression, pharmacological epistasis), single lab","pmids":["22778212"],"is_preprint":false},{"year":2004,"finding":"The transcription factor YY1 binds to two sites within HSD3B2 intron 1 and is required for maximal basal promoter activity. YY1 binding to the second intron 1 site (35 bp downstream of the 3beta1-A element) strongly activates HSD3B2 basal promoter, as abrogating this binding causes a ~50% decrease in transcription. Complete loss of YY1 binding within intron 1 reduces promoter activity to the same level as constructs lacking the entire intron 1.","method":"Reporter gene assay, gel shift assay, anti-YY1 antibody supershift, competition analysis, mutational analysis, transient transfection","journal":"Journal of molecular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal gel shift with antibody and mutagenesis plus functional reporter assays, single lab, multiple orthogonal methods","pmids":["15291746"],"is_preprint":false},{"year":2010,"finding":"HSD3B2 and cytochrome b5 (CYB5A) are co-expressed in hybrid cortical cells at the zona fasciculata/zona reticularis border in human adrenal glands and together contribute to androstenedione production; inhibition of HSD3B2 with trilostane or siRNA knockdown of CYB5A both significantly reduced androstenedione production in H295R cells.","method":"Immunohistochemistry/immunolocalization, siRNA knockdown, pharmacological inhibition with trilostane, steroid hormone measurement in H295R cells","journal":"The Journal of steroid biochemistry and molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization combined with functional siRNA and pharmacological inhibition experiments, single lab, two orthogonal approaches","pmids":["21185375"],"is_preprint":false},{"year":2015,"finding":"Decanoic acid (DA) inhibits HSD3B2 expression and androgen biosynthesis in NCI-H295R cells through a cAMP-dependent mechanism: both DA and metformin reduce cAMP-stimulated recruitment of the orphan nuclear receptor Nur77 to the HSD3B2 promoter, decreasing HSD3B2 transcription and protein expression.","method":"Reporter gene assay (dual luciferase), chromatin immunoprecipitation/promoter recruitment assay, RT-PCR, Western blot, testosterone ELISA in NCI-H295R cells and rat PCOS model","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter recruitment assay combined with expression analysis and in vivo model, single lab, multiple orthogonal methods","pmids":["26465200"],"is_preprint":false},{"year":2019,"finding":"GATA1 is an activating transcription factor that binds the HSD3B2 promoter to drive its expression and androgen biosynthesis. Baicalin inhibits androgen production by reducing GATA1 recruitment to the HSD3B2 promoter, identified via gene expression profiling, dual luciferase assay, RNA interference, and genetic mutations in NCI-H295R cells.","method":"Gene expression profiling, dual luciferase assay, RNA interference (siRNA), site-directed mutagenesis of promoter, ChIP (promoter recruitment), Western blot, ELISA","journal":"The Journal of endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (luciferase, RNAi, mutagenesis, promoter recruitment), single lab","pmids":["30650063"],"is_preprint":false},{"year":2014,"finding":"Two novel HSD3B2 missense mutations (Y190C and S218P), located adjacent to the predicted substrate-binding pocket in a structural model, severely impair enzymatic activity but with substrate-specific residual activity: both mutants retain higher residual activity toward 17-OH pregnenolone than toward other Δ5-steroids, suggesting these residues influence substrate-binding pocket conformation differentially for different substrates.","method":"In vitro enzymatic activity assay in transfected cells, 3D structural homology modeling, site-directed mutagenesis","journal":"Clinical endocrinology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro enzymatic assay with mutagenesis plus structural modeling, single lab, two mutations","pmids":["24372086"],"is_preprint":false},{"year":2015,"finding":"The G250V mutation in HSD3B2 reduces Vmax for progesterone synthesis without affecting Km for pregnenolone, and does not alter protein expression or intracellular localization. Molecular modeling predicts that G250V disrupts an L239-Q251 loop adjacent to a β-sheet in the NAD+-binding domain, establishing this loop as important for catalytic activity.","method":"In vitro enzymatic activity assay in COS-7 cells, Michaelis-Menten kinetics (Km/Vmax), Western blot, immunofluorescence, molecular homology modeling","journal":"The Journal of clinical endocrinology and metabolism","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro kinetic characterization with mutagenesis, localization imaging, and structural modeling, single lab","pmids":["25322271"],"is_preprint":false},{"year":2011,"finding":"Sunitinib inhibits HSD3B2 by downregulating its mRNA and protein expression in adrenocortical carcinoma cells (NCI-H295 and SW13), but does NOT directly inhibit HSD3B2 enzyme activity as tested in yeast microsomes expressing HSD3B2.","method":"RT-PCR, Western blot, gas chromatography-mass spectrometry steroid measurement, yeast microsome enzymatic activity assay","journal":"Frontiers in endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct enzymatic assay in yeast microsomes (negative for direct inhibition) plus expression analysis, single lab, multiple orthogonal methods","pmids":["22654799"],"is_preprint":false},{"year":2016,"finding":"DNA methylation is not involved in the zona reticularis-specific downregulation of HSD3B2 in the human adrenal cortex, as HSD3B2 lacks CpG islands and no zone-specific methylation differences were detected.","method":"RT-qPCR, methylation analysis, microdissected adrenal zone samples","journal":"Molecular and cellular endocrinology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — expression and methylation analysis only, single lab, negative finding regarding mechanism","pmids":["27670690"],"is_preprint":false},{"year":1994,"finding":"A homozygous frameshift mutation (273 delta AA, deletion of two adenosines at codon 273) in HSD3B2 leads to a premature stop codon at position 279, establishing loss-of-function as the molecular basis of severe salt-wasting 3β-HSD deficiency in affected patients.","method":"Direct sequencing of PCR products, haplotype analysis","journal":"Human molecular genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — sequencing only, no functional expression assay performed, single lab","pmids":["8004103"],"is_preprint":false}],"current_model":"HSD3B2 encodes a bifunctional NAD+-dependent microsomal enzyme (3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase type 2) that converts Δ5-steroids to Δ4-steroids in adrenal glands and gonads; its catalytic activity depends critically on the NAD+-binding domain (including residues such as Ala10 and the L239-Q251 loop) and a substrate-binding pocket (near residues Y190 and S218), loss-of-function mutations cause complete or partial enzymatic activity loss correlating with disease severity, its transcription is activated by YY1 binding within intron 1 and by GATA1 at the promoter, repressed by Nur77 pathway modulation, and its activity can be suppressed indirectly through mitochondrial complex I inhibition (e.g., by metformin or rotenone), while co-expression with CYB5A in hybrid adrenocortical cells enables androstenedione production."},"narrative":{"mechanistic_narrative":"HSD3B2 encodes a NAD+-dependent 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase that catalyzes the conversion of Δ5-steroids to Δ4-steroids in adrenal and gonadal steroidogenesis, and loss-of-function mutations cause 3β-HSD deficiency with a genotype–phenotype correlation in which severe salt-wasting forms reflect complete loss of catalytic activity while non-salt-losing forms retain residual activity [PMID:10599696]. Catalytic competence depends on an intact NAD+-binding domain: the A10E substitution at Ala10 abolishes activity [PMID:10843183], and the G250V substitution disrupts an L239-Q251 loop adjacent to a β-sheet in this domain, lowering Vmax for progesterone synthesis without altering substrate affinity, protein level, or localization [PMID:25322271]. A separate substrate-binding pocket governs substrate handling, as the Y190C and S218P mutations adjacent to this pocket impair activity in a substrate-specific manner, retaining greater residual activity toward 17-OH pregnenolone than other Δ5-steroids [PMID:24372086]. Some disease alleles act through protein instability rather than direct catalytic impairment [PMID:10599696]. In human adrenal cortex, HSD3B2 is co-expressed with cytochrome b5 (CYB5A) in hybrid cells at the zona fasciculata/zona reticularis border, and together they drive androstenedione production [PMID:21185375]. HSD3B2 transcription is governed by multiple factors: YY1 binding at two sites within intron 1 is required for maximal basal promoter activity [PMID:15291746], GATA1 binds the promoter to activate expression and androgen biosynthesis [PMID:30650063], and cAMP-stimulated recruitment of the orphan nuclear receptor Nur77 to the promoter is suppressed by decanoic acid and metformin [PMID:26465200]. Enzyme output is additionally modulated indirectly through mitochondrial complex I, since metformin and rotenone inhibit HSD3B2 activity via complex I inhibition rather than AMPK or ERK signaling [PMID:22778212].","teleology":[{"year":1994,"claim":"Established that loss of HSD3B2 protein underlies severe salt-wasting 3β-HSD deficiency by identifying a frameshift truncating allele in affected patients.","evidence":"Direct sequencing and haplotype analysis of a homozygous 273-delta-AA frameshift producing a premature stop at codon 279","pmids":["8004103"],"confidence":"Low","gaps":["No functional expression assay was performed to confirm the predicted null","Does not address milder allelic forms or residual-activity mutations"]},{"year":1999,"claim":"Resolved the genotype–phenotype basis of 3β-HSD deficiency by showing that disease severity tracks with quantitative residual enzyme activity, and that some mutations act through protein instability.","evidence":"Site-directed mutagenesis of 25 missense mutants, transient expression in 293 cells, [14C]-DHEA substrate assay, plus Northern/Western and in vitro translation","pmids":["10599696"],"confidence":"High","gaps":["Activity measured in cell expression systems, not purified reconstituted enzyme","Does not map which structural domains each mutation perturbs"]},{"year":2000,"claim":"Pinpointed a single NAD+-binding-domain residue (Ala10) as essential for catalysis by showing A10E abolishes all detectable activity.","evidence":"Direct sequencing and transient expression with enzymatic activity assay in Ad293 cells","pmids":["10843183"],"confidence":"Medium","gaps":["Single mutation in one lab","No structural model linking Ala10 to cofactor binding in this study"]},{"year":2004,"claim":"Identified the first transcriptional driver of HSD3B2 by mapping YY1 binding within intron 1 as required for maximal basal promoter activity.","evidence":"Reporter assays, gel shift with anti-YY1 supershift, competition and mutational analysis in transient transfection","pmids":["15291746"],"confidence":"Medium","gaps":["Basal promoter context only; hormonal regulation not addressed","In vivo occupancy in adrenal tissue not demonstrated"]},{"year":2010,"claim":"Defined a functional partnership in which HSD3B2 co-expressed with CYB5A in zona fasciculata/reticularis hybrid cells produces androstenedione.","evidence":"Immunolocalization in human adrenal plus trilostane inhibition and CYB5A siRNA knockdown with steroid measurement in H295R cells","pmids":["21185375"],"confidence":"Medium","gaps":["Does not establish direct physical interaction between HSD3B2 and CYB5A","Functional contribution quantified only in a cell line"]},{"year":2012,"claim":"Revealed an indirect metabolic route of enzyme regulation by linking metformin-mediated HSD3B2 inhibition to mitochondrial complex I rather than AMPK/ERK/PKC signaling.","evidence":"Activity and expression assays, rotenone pharmacological epistasis, and organic cation transporter dependency in NCI-H295R cells","pmids":["22778212"],"confidence":"Medium","gaps":["Molecular link between complex I status and HSD3B2 catalysis/expression unresolved","Single cell-line model"]},{"year":2014,"claim":"Distinguished substrate-binding from cofactor-binding determinants by showing Y190C and S218P mutations near the substrate pocket impair activity in a substrate-selective manner.","evidence":"In vitro enzymatic assays in transfected cells, site-directed mutagenesis, and 3D homology modeling","pmids":["24372086"],"confidence":"Medium","gaps":["Pocket conformation inferred from homology model, no experimental structure","Only two mutations characterized"]},{"year":2015,"claim":"Localized a catalytically critical loop in the NAD+-binding domain by showing G250V lowers Vmax for progesterone without changing Km, expression, or localization.","evidence":"Michaelis-Menten kinetics in COS-7 cells, Western blot, immunofluorescence, and homology modeling of the L239-Q251 loop","pmids":["25322271"],"confidence":"Medium","gaps":["Loop importance inferred from modeling, not crystal structure","Single mutation"]},{"year":2015,"claim":"Connected cAMP signaling to HSD3B2 transcription by showing decanoic acid and metformin reduce cAMP-stimulated Nur77 recruitment to the promoter.","evidence":"Dual luciferase reporter, ChIP promoter recruitment, RT-PCR, Western blot, and testosterone ELISA in NCI-H295R cells and a rat PCOS model","pmids":["26465200"],"confidence":"Medium","gaps":["Whether Nur77 acts directly or via cofactors at the promoter not resolved","Single lab"]},{"year":2016,"claim":"Excluded DNA methylation as the mechanism of zona reticularis-specific HSD3B2 downregulation, narrowing the search for the responsible regulator.","evidence":"RT-qPCR and methylation analysis on microdissected adrenal zones showing absence of CpG islands and zone-specific methylation","pmids":["27670690"],"confidence":"Low","gaps":["Negative finding; the actual zone-specific repressor remains unidentified","Limited to methylation as a candidate mechanism"]},{"year":2019,"claim":"Identified GATA1 as a promoter-binding activator of HSD3B2 driving androgen biosynthesis, targetable pharmacologically.","evidence":"Expression profiling, dual luciferase, RNAi, promoter mutagenesis, ChIP recruitment, Western blot and ELISA in NCI-H295R cells","pmids":["30650063"],"confidence":"Medium","gaps":["Interplay between GATA1, YY1, and Nur77 at the locus not integrated","Single cell-line system"]},{"year":null,"claim":"An experimentally determined structure of HSD3B2 and the identity of the zone-specific repressor controlling its adrenal expression pattern remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["Catalytic-domain assignments rest on homology models, not solved structures","The factor responsible for zona reticularis-specific downregulation is unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,7,8]},{"term_id":"GO:0016853","term_label":"isomerase activity","supporting_discovery_ids":[0]}],"localization":[],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,4]}],"complexes":[],"partners":["CYB5A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P26439","full_name":"3 beta-hydroxysteroid dehydrogenase/Delta 5-->4-isomerase type 2","aliases":["3 beta-hydroxysteroid dehydrogenase/Delta 5-->4-isomerase type II","3-beta-HSD II","3-beta-HSD adrenal and gonadal type"],"length_aa":372,"mass_kda":42.1,"function":"3-beta-HSD is a bifunctional enzyme, that catalyzes the oxidative conversion of Delta(5)-ene-3-beta-hydroxy steroid, and the oxidative conversion of ketosteroids. The 3-beta-HSD enzymatic system plays a crucial role in the biosynthesis of all classes of hormonal steroids","subcellular_location":"Endoplasmic reticulum membrane; Mitochondrion membrane","url":"https://www.uniprot.org/uniprotkb/P26439/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HSD3B2","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/HSD3B2","total_profiled":1310},"omim":[{"mim_id":"613890","title":"3-@BETA-HYDROXYSTEROID DEHYDROGENASE 2; HSD3B2","url":"https://www.omim.org/entry/613890"},{"mim_id":"604453","title":"NUCLEAR RECEPTOR SUBFAMILY 5, GROUP A, MEMBER 2; NR5A2","url":"https://www.omim.org/entry/604453"},{"mim_id":"201810","title":"ADRENAL HYPERPLASIA, CONGENITAL, DUE TO 3-BETA-HYDROXYSTEROID DEHYDROGENASE 2 DEFICIENCY","url":"https://www.omim.org/entry/201810"},{"mim_id":"139139","title":"NUCLEAR RECEPTOR SUBFAMILY 4, GROUP A, MEMBER 1; NR4A1","url":"https://www.omim.org/entry/139139"},{"mim_id":"109715","title":"3-@BETA-HYDROXYSTEROID DEHYDROGENASE 1; HSD3B1","url":"https://www.omim.org/entry/109715"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Endoplasmic reticulum","reliability":"Approved"},{"location":"Primary cilium","reliability":"Approved"},{"location":"Nucleoli","reliability":"Additional"},{"location":"Microtubules","reliability":"Additional"},{"location":"Cytokinetic bridge","reliability":"Additional"},{"location":"Primary cilium transition zone","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"adrenal gland","ntpm":2103.4}],"url":"https://www.proteinatlas.org/search/HSD3B2"},"hgnc":{"alias_symbol":["SDR11E2"],"prev_symbol":[]},"alphafold":{"accession":"P26439","domains":[{"cath_id":"3.40.50.720","chopping":"2-191_223-258","consensus_level":"high","plddt":96.2851,"start":2,"end":258}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P26439","model_url":"https://alphafold.ebi.ac.uk/files/AF-P26439-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P26439-F1-predicted_aligned_error_v6.png","plddt_mean":94.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=HSD3B2","jax_strain_url":"https://www.jax.org/strain/search?query=HSD3B2"},"sequence":{"accession":"P26439","fasta_url":"https://rest.uniprot.org/uniprotkb/P26439.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P26439/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P26439"}},"corpus_meta":[{"pmid":"22778212","id":"PMC_22778212","title":"Metformin inhibits human 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factor.","date":"2004","source":"Journal of molecular endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/15291746","citation_count":6,"is_preprint":false},{"pmid":"33180036","id":"PMC_33180036","title":"Late diagnosis of 3β-Hydroxysteroid dehydrogenase deficiency: the pivotal role of gas chromatography-mass spectrometry urinary steroid metabolome analysis and a novel homozygous nonsense mutation in the HSD3B2 gene.","date":"2020","source":"Journal of pediatric endocrinology & metabolism : JPEM","url":"https://pubmed.ncbi.nlm.nih.gov/33180036","citation_count":6,"is_preprint":false},{"pmid":"25619355","id":"PMC_25619355","title":"[A novel homozygous mutation p.E25X in the HSD3B2 gene causing salt wasting 3β-hydroxysteroid dehydrogenases deficiency in a Chinese pubertal girl: a delayed diagnosis until recurrent ovary cysts].","date":"2014","source":"Zhonghua er ke za zhi = Chinese journal of pediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/25619355","citation_count":6,"is_preprint":false},{"pmid":"29803408","id":"PMC_29803408","title":"Up regulation of the steroid hormone synthesis regulator HSD3B2 is linked to early PSA recurrence in prostate cancer.","date":"2018","source":"Experimental and molecular pathology","url":"https://pubmed.ncbi.nlm.nih.gov/29803408","citation_count":5,"is_preprint":false},{"pmid":"27626911","id":"PMC_27626911","title":"Non-Virilizing Congenital Adrenal Hyperplasia in a Female Patient with a Novel HSD3B2 Mutation.","date":"2016","source":"Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/27626911","citation_count":5,"is_preprint":false},{"pmid":"39387578","id":"PMC_39387578","title":"Immunohistochemical expression of CYP11A1, CYP11B, CYP17, and HSD3B2 in functional and nonfunctional canine adrenocortical tumors.","date":"2024","source":"Journal of veterinary internal medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39387578","citation_count":4,"is_preprint":false},{"pmid":"32506065","id":"PMC_32506065","title":"Co-Existence of Congenital Adrenal Hyperplasia and Bartter Syndrome due to Maternal Uniparental Isodisomy of HSD3B2 and CLCNKB Mutations.","date":"2020","source":"Hormone research in paediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/32506065","citation_count":4,"is_preprint":false},{"pmid":"37384334","id":"PMC_37384334","title":"Identification of a novel candidate HSD3B2 gene variant for familial hypospadias by whole-exome sequencing.","date":"2023","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/37384334","citation_count":3,"is_preprint":false},{"pmid":"27670690","id":"PMC_27670690","title":"DNA methylation is not involved in specific down-regulation of HSD3B2, NR4A1 and RARB genes in androgen-secreting cells of human adrenal cortex.","date":"2016","source":"Molecular and cellular endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/27670690","citation_count":2,"is_preprint":false},{"pmid":"37274334","id":"PMC_37274334","title":"High carrier frequency of a nonsense p.Trp230* variant in HSD3B2 gene in Ossetians.","date":"2023","source":"Frontiers in endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/37274334","citation_count":2,"is_preprint":false},{"pmid":"36553457","id":"PMC_36553457","title":"Co-Occurrence of a Pathogenic HSD3B2 Variant and a Duplication on 10q22.3-q23.2 Detected in Newborn Twins with Salt-Wasting Congenital Adrenal Hyperplasia.","date":"2022","source":"Genes","url":"https://pubmed.ncbi.nlm.nih.gov/36553457","citation_count":2,"is_preprint":false},{"pmid":"39839754","id":"PMC_39839754","title":"Ambiguous Genitalia Due to 3β-Hydroxysteroid Dehydrogenase Type 2 Deficiency: Clinical, Genetic, and Functional Characterization of Two Novel HSD3B2 Variants.","date":"2025","source":"JCEM case reports","url":"https://pubmed.ncbi.nlm.nih.gov/39839754","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":26137,"output_tokens":3075,"usd":0.062268,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10527,"output_tokens":3427,"usd":0.069155,"stage2_stop_reason":"end_turn"},"total_usd":0.131423,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"Functional characterization of 25 HSD3B2 missense mutations by transient expression in 293 cells demonstrated that severe salt-wasting forms result from complete loss of enzyme activity, while non-salt-losing forms retain residual enzymatic activity. Additionally, some mutations cause disease through protein instability rather than direct catalytic impairment.\",\n      \"method\": \"Site-directed mutagenesis, transient expression in 293 cells, [14C]-DHEA substrate assay, Northern/Western blot, in vitro transcription/translation\",\n      \"journal\": \"The Journal of clinical endocrinology and metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic assay with mutagenesis across 25 mutant enzymes, multiple orthogonal methods (activity assay, Northern/Western, reticulocyte translation), single comprehensive study\",\n      \"pmids\": [\"10599696\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The A10E mutation in the NAD-binding domain of HSD3B2 abolishes all detectable enzymatic activity when expressed in transfected Ad293 cells, establishing that Ala10 is critical for catalytic function.\",\n      \"method\": \"Direct sequencing, transient expression in Ad293 cells, enzymatic activity assay\",\n      \"journal\": \"The Journal of clinical endocrinology and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro activity assay with mutagenesis, single lab, single mutation characterized\",\n      \"pmids\": [\"10843183\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Metformin inhibits HSD3B2 enzymatic activity and decreases HSD3B2 expression in NCI-H295R steroidogenic cells. This inhibition is dependent on organic cation transporters for metformin uptake and is mediated through inhibition of mitochondrial complex I (not AMPK, ERK1/2, or atypical PKC signaling). Direct inhibition of complex I by rotenone also inhibits HSD3B2 activity, establishing the mechanistic link.\",\n      \"method\": \"Enzymatic activity assay, mRNA/protein expression analysis, pharmacological inhibition (rotenone), AMPK/ERK signaling analysis, organic cation transporter dependency assay in NCI-H295R cells\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (activity assay, expression, pharmacological epistasis), single lab\",\n      \"pmids\": [\"22778212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The transcription factor YY1 binds to two sites within HSD3B2 intron 1 and is required for maximal basal promoter activity. YY1 binding to the second intron 1 site (35 bp downstream of the 3beta1-A element) strongly activates HSD3B2 basal promoter, as abrogating this binding causes a ~50% decrease in transcription. Complete loss of YY1 binding within intron 1 reduces promoter activity to the same level as constructs lacking the entire intron 1.\",\n      \"method\": \"Reporter gene assay, gel shift assay, anti-YY1 antibody supershift, competition analysis, mutational analysis, transient transfection\",\n      \"journal\": \"Journal of molecular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal gel shift with antibody and mutagenesis plus functional reporter assays, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"15291746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"HSD3B2 and cytochrome b5 (CYB5A) are co-expressed in hybrid cortical cells at the zona fasciculata/zona reticularis border in human adrenal glands and together contribute to androstenedione production; inhibition of HSD3B2 with trilostane or siRNA knockdown of CYB5A both significantly reduced androstenedione production in H295R cells.\",\n      \"method\": \"Immunohistochemistry/immunolocalization, siRNA knockdown, pharmacological inhibition with trilostane, steroid hormone measurement in H295R cells\",\n      \"journal\": \"The Journal of steroid biochemistry and molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization combined with functional siRNA and pharmacological inhibition experiments, single lab, two orthogonal approaches\",\n      \"pmids\": [\"21185375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Decanoic acid (DA) inhibits HSD3B2 expression and androgen biosynthesis in NCI-H295R cells through a cAMP-dependent mechanism: both DA and metformin reduce cAMP-stimulated recruitment of the orphan nuclear receptor Nur77 to the HSD3B2 promoter, decreasing HSD3B2 transcription and protein expression.\",\n      \"method\": \"Reporter gene assay (dual luciferase), chromatin immunoprecipitation/promoter recruitment assay, RT-PCR, Western blot, testosterone ELISA in NCI-H295R cells and rat PCOS model\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter recruitment assay combined with expression analysis and in vivo model, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"26465200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GATA1 is an activating transcription factor that binds the HSD3B2 promoter to drive its expression and androgen biosynthesis. Baicalin inhibits androgen production by reducing GATA1 recruitment to the HSD3B2 promoter, identified via gene expression profiling, dual luciferase assay, RNA interference, and genetic mutations in NCI-H295R cells.\",\n      \"method\": \"Gene expression profiling, dual luciferase assay, RNA interference (siRNA), site-directed mutagenesis of promoter, ChIP (promoter recruitment), Western blot, ELISA\",\n      \"journal\": \"The Journal of endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (luciferase, RNAi, mutagenesis, promoter recruitment), single lab\",\n      \"pmids\": [\"30650063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Two novel HSD3B2 missense mutations (Y190C and S218P), located adjacent to the predicted substrate-binding pocket in a structural model, severely impair enzymatic activity but with substrate-specific residual activity: both mutants retain higher residual activity toward 17-OH pregnenolone than toward other Δ5-steroids, suggesting these residues influence substrate-binding pocket conformation differentially for different substrates.\",\n      \"method\": \"In vitro enzymatic activity assay in transfected cells, 3D structural homology modeling, site-directed mutagenesis\",\n      \"journal\": \"Clinical endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro enzymatic assay with mutagenesis plus structural modeling, single lab, two mutations\",\n      \"pmids\": [\"24372086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The G250V mutation in HSD3B2 reduces Vmax for progesterone synthesis without affecting Km for pregnenolone, and does not alter protein expression or intracellular localization. Molecular modeling predicts that G250V disrupts an L239-Q251 loop adjacent to a β-sheet in the NAD+-binding domain, establishing this loop as important for catalytic activity.\",\n      \"method\": \"In vitro enzymatic activity assay in COS-7 cells, Michaelis-Menten kinetics (Km/Vmax), Western blot, immunofluorescence, molecular homology modeling\",\n      \"journal\": \"The Journal of clinical endocrinology and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro kinetic characterization with mutagenesis, localization imaging, and structural modeling, single lab\",\n      \"pmids\": [\"25322271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Sunitinib inhibits HSD3B2 by downregulating its mRNA and protein expression in adrenocortical carcinoma cells (NCI-H295 and SW13), but does NOT directly inhibit HSD3B2 enzyme activity as tested in yeast microsomes expressing HSD3B2.\",\n      \"method\": \"RT-PCR, Western blot, gas chromatography-mass spectrometry steroid measurement, yeast microsome enzymatic activity assay\",\n      \"journal\": \"Frontiers in endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct enzymatic assay in yeast microsomes (negative for direct inhibition) plus expression analysis, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"22654799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"DNA methylation is not involved in the zona reticularis-specific downregulation of HSD3B2 in the human adrenal cortex, as HSD3B2 lacks CpG islands and no zone-specific methylation differences were detected.\",\n      \"method\": \"RT-qPCR, methylation analysis, microdissected adrenal zone samples\",\n      \"journal\": \"Molecular and cellular endocrinology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — expression and methylation analysis only, single lab, negative finding regarding mechanism\",\n      \"pmids\": [\"27670690\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"A homozygous frameshift mutation (273 delta AA, deletion of two adenosines at codon 273) in HSD3B2 leads to a premature stop codon at position 279, establishing loss-of-function as the molecular basis of severe salt-wasting 3β-HSD deficiency in affected patients.\",\n      \"method\": \"Direct sequencing of PCR products, haplotype analysis\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — sequencing only, no functional expression assay performed, single lab\",\n      \"pmids\": [\"8004103\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HSD3B2 encodes a bifunctional NAD+-dependent microsomal enzyme (3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase type 2) that converts Δ5-steroids to Δ4-steroids in adrenal glands and gonads; its catalytic activity depends critically on the NAD+-binding domain (including residues such as Ala10 and the L239-Q251 loop) and a substrate-binding pocket (near residues Y190 and S218), loss-of-function mutations cause complete or partial enzymatic activity loss correlating with disease severity, its transcription is activated by YY1 binding within intron 1 and by GATA1 at the promoter, repressed by Nur77 pathway modulation, and its activity can be suppressed indirectly through mitochondrial complex I inhibition (e.g., by metformin or rotenone), while co-expression with CYB5A in hybrid adrenocortical cells enables androstenedione production.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"HSD3B2 encodes a NAD+-dependent 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase that catalyzes the conversion of Δ5-steroids to Δ4-steroids in adrenal and gonadal steroidogenesis, and loss-of-function mutations cause 3β-HSD deficiency with a genotype–phenotype correlation in which severe salt-wasting forms reflect complete loss of catalytic activity while non-salt-losing forms retain residual activity [#0]. Catalytic competence depends on an intact NAD+-binding domain: the A10E substitution at Ala10 abolishes activity [#1], and the G250V substitution disrupts an L239-Q251 loop adjacent to a β-sheet in this domain, lowering Vmax for progesterone synthesis without altering substrate affinity, protein level, or localization [#8]. A separate substrate-binding pocket governs substrate handling, as the Y190C and S218P mutations adjacent to this pocket impair activity in a substrate-specific manner, retaining greater residual activity toward 17-OH pregnenolone than other Δ5-steroids [#7]. Some disease alleles act through protein instability rather than direct catalytic impairment [#0]. In human adrenal cortex, HSD3B2 is co-expressed with cytochrome b5 (CYB5A) in hybrid cells at the zona fasciculata/zona reticularis border, and together they drive androstenedione production [#4]. HSD3B2 transcription is governed by multiple factors: YY1 binding at two sites within intron 1 is required for maximal basal promoter activity [#3], GATA1 binds the promoter to activate expression and androgen biosynthesis [#6], and cAMP-stimulated recruitment of the orphan nuclear receptor Nur77 to the promoter is suppressed by decanoic acid and metformin [#5]. Enzyme output is additionally modulated indirectly through mitochondrial complex I, since metformin and rotenone inhibit HSD3B2 activity via complex I inhibition rather than AMPK or ERK signaling [#2].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Established that loss of HSD3B2 protein underlies severe salt-wasting 3β-HSD deficiency by identifying a frameshift truncating allele in affected patients.\",\n      \"evidence\": \"Direct sequencing and haplotype analysis of a homozygous 273-delta-AA frameshift producing a premature stop at codon 279\",\n      \"pmids\": [\"8004103\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No functional expression assay was performed to confirm the predicted null\", \"Does not address milder allelic forms or residual-activity mutations\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Resolved the genotype–phenotype basis of 3β-HSD deficiency by showing that disease severity tracks with quantitative residual enzyme activity, and that some mutations act through protein instability.\",\n      \"evidence\": \"Site-directed mutagenesis of 25 missense mutants, transient expression in 293 cells, [14C]-DHEA substrate assay, plus Northern/Western and in vitro translation\",\n      \"pmids\": [\"10599696\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Activity measured in cell expression systems, not purified reconstituted enzyme\", \"Does not map which structural domains each mutation perturbs\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Pinpointed a single NAD+-binding-domain residue (Ala10) as essential for catalysis by showing A10E abolishes all detectable activity.\",\n      \"evidence\": \"Direct sequencing and transient expression with enzymatic activity assay in Ad293 cells\",\n      \"pmids\": [\"10843183\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single mutation in one lab\", \"No structural model linking Ala10 to cofactor binding in this study\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified the first transcriptional driver of HSD3B2 by mapping YY1 binding within intron 1 as required for maximal basal promoter activity.\",\n      \"evidence\": \"Reporter assays, gel shift with anti-YY1 supershift, competition and mutational analysis in transient transfection\",\n      \"pmids\": [\"15291746\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Basal promoter context only; hormonal regulation not addressed\", \"In vivo occupancy in adrenal tissue not demonstrated\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined a functional partnership in which HSD3B2 co-expressed with CYB5A in zona fasciculata/reticularis hybrid cells produces androstenedione.\",\n      \"evidence\": \"Immunolocalization in human adrenal plus trilostane inhibition and CYB5A siRNA knockdown with steroid measurement in H295R cells\",\n      \"pmids\": [\"21185375\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not establish direct physical interaction between HSD3B2 and CYB5A\", \"Functional contribution quantified only in a cell line\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Revealed an indirect metabolic route of enzyme regulation by linking metformin-mediated HSD3B2 inhibition to mitochondrial complex I rather than AMPK/ERK/PKC signaling.\",\n      \"evidence\": \"Activity and expression assays, rotenone pharmacological epistasis, and organic cation transporter dependency in NCI-H295R cells\",\n      \"pmids\": [\"22778212\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between complex I status and HSD3B2 catalysis/expression unresolved\", \"Single cell-line model\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Distinguished substrate-binding from cofactor-binding determinants by showing Y190C and S218P mutations near the substrate pocket impair activity in a substrate-selective manner.\",\n      \"evidence\": \"In vitro enzymatic assays in transfected cells, site-directed mutagenesis, and 3D homology modeling\",\n      \"pmids\": [\"24372086\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Pocket conformation inferred from homology model, no experimental structure\", \"Only two mutations characterized\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Localized a catalytically critical loop in the NAD+-binding domain by showing G250V lowers Vmax for progesterone without changing Km, expression, or localization.\",\n      \"evidence\": \"Michaelis-Menten kinetics in COS-7 cells, Western blot, immunofluorescence, and homology modeling of the L239-Q251 loop\",\n      \"pmids\": [\"25322271\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Loop importance inferred from modeling, not crystal structure\", \"Single mutation\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected cAMP signaling to HSD3B2 transcription by showing decanoic acid and metformin reduce cAMP-stimulated Nur77 recruitment to the promoter.\",\n      \"evidence\": \"Dual luciferase reporter, ChIP promoter recruitment, RT-PCR, Western blot, and testosterone ELISA in NCI-H295R cells and a rat PCOS model\",\n      \"pmids\": [\"26465200\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Nur77 acts directly or via cofactors at the promoter not resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Excluded DNA methylation as the mechanism of zona reticularis-specific HSD3B2 downregulation, narrowing the search for the responsible regulator.\",\n      \"evidence\": \"RT-qPCR and methylation analysis on microdissected adrenal zones showing absence of CpG islands and zone-specific methylation\",\n      \"pmids\": [\"27670690\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Negative finding; the actual zone-specific repressor remains unidentified\", \"Limited to methylation as a candidate mechanism\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified GATA1 as a promoter-binding activator of HSD3B2 driving androgen biosynthesis, targetable pharmacologically.\",\n      \"evidence\": \"Expression profiling, dual luciferase, RNAi, promoter mutagenesis, ChIP recruitment, Western blot and ELISA in NCI-H295R cells\",\n      \"pmids\": [\"30650063\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interplay between GATA1, YY1, and Nur77 at the locus not integrated\", \"Single cell-line system\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"An experimentally determined structure of HSD3B2 and the identity of the zone-specific repressor controlling its adrenal expression pattern remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Catalytic-domain assignments rest on homology models, not solved structures\", \"The factor responsible for zona reticularis-specific downregulation is unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 7, 8]},\n      {\"term_id\": \"GO:0016853\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CYB5A\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}