{"gene":"HOXD13","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":1996,"finding":"HOXD13 contains a 15-alanine polyalanine tract in exon 1; synpolydactyly (SPD) is caused by a 9-alanine duplication in this tract (expanding it to 24 alanines), confirmed in two unrelated SPD families and absent in 150 control chromosomes. The genomic structure of HOXD13 was established as two exons encoding a 335-amino-acid protein with a C-terminal homeodomain.","method":"Genomic sequencing, PCR-based mutation analysis, family segregation analysis","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct sequencing across multiple families, replicated in independent cohorts, established the causative mutation","pmids":["8817328"],"is_preprint":false},{"year":1997,"finding":"SPD phenotype severity (penetrance and expressivity in hands and feet) correlates significantly with the size of the polyalanine expansion in HOXD13 (7–14 extra residues tested), suggesting that expansion produces a specific gain of function in the mutant protein.","method":"Molecular sizing of polyalanine expansions across 16 SPD families; phenotype–genotype correlation analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic molecular and clinical analysis across 20 families, statistically significant correlation between expansion size and phenotype severity","pmids":["9207113"],"is_preprint":false},{"year":1998,"finding":"A spontaneous mouse Hoxd13 mutation (spdh) carrying a 21-bp in-frame duplication expanding the polyalanine tract from 15 to 22 residues produces a phenotype more severe than null Hoxd13 mice, indicating the expanded polyalanine allele acts in a dominant-negative manner rather than as a simple loss-of-function.","method":"Molecular cloning of spdh allele, comparison of spdh heterozygote/homozygote phenotypes with Hoxd13 null mice","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic comparison of gain-of-function allele vs. null allele in mouse; phenotypic readout clearly distinguishes mechanisms","pmids":["9580668"],"is_preprint":false},{"year":2000,"finding":"The t(2;11)(q31;p15) translocation generates NUP98-HOXD13 fusion transcripts encoding a protein with the FG-repeat region of NUP98 fused to the HOXD13 homeodomain; ectopic expression of the wild-type HOXD13 allele is also observed in the leukemia patient, implicating HOXD13 homeodomain activity in leukemogenesis.","method":"3′-RACE, RT-PCR, chromosomal breakpoint mapping in an AML patient","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — RT-PCR characterization in a single patient, but breakpoints molecularly mapped and fusion protein structure inferred from sequence","pmids":["10995009"],"is_preprint":false},{"year":2003,"finding":"An I47L missense mutation in the HOXD13 homeodomain causes selective loss of DNA-binding at a subset of wild-type HOXD13 target sites (including the EphA7 locus) without dominant-negative or gain-of-function effects; retrovirus-mediated misexpression in chick limbs showed wild-type HOXD13 upregulates EphA7 in the autopod whereas HOXD13(I47L) cannot; both HOXD13(I47L) and a DNA-binding-null HOXD13(IQN) cause proximal limb shortening, suggesting functional suppression of anterior Hox genes does not require DNA binding and is mediated by protein–protein interactions.","method":"In vitro DNA-binding assays, retrovirus-mediated misexpression in developing chick limbs, in vivo comparison of wild-type, I47L, and IQN (DNA-binding null) mutant HOXD13","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (in vitro binding, in vivo chick limb misexpression, genetic comparison of selective vs. complete loss-of-function mutants)","pmids":["12620993"],"is_preprint":false},{"year":2005,"finding":"HOXD13 and HOXA13 directly bind multiple sites in the EphA7 promoter in vivo (confirmed by ChIP in developing mouse limbs and in vitro binding assays), and activate transcription from the EphA7 promoter; the HOXD13(I47L) brachydactyly mutation fails to bind the EphA7 promoter in vivo and fails to transactivate it.","method":"Promoter characterization, electrophoretic mobility shift assay (EMSA), ChIP in developing mouse limbs, luciferase transactivation assays, retrovirus-mediated misexpression in chick limbs","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — ChIP in vivo plus EMSA plus transactivation assays, consistent with in vivo chick misexpression data","pmids":["16314414"],"is_preprint":false},{"year":2005,"finding":"NUP98-HOXD13 (NHD13) transgenic mice develop a uniformly fatal myelodysplastic syndrome (MDS) with peripheral cytopenias, bone marrow dysplasia, apoptosis, and transformation to acute leukemia; the NHD13 fusion gene inhibits megakaryocytic differentiation and increases bone marrow apoptosis, providing a mechanism for ineffective hematopoiesis.","method":"Transgenic mouse model, hematological and histological analysis, bone marrow differentiation assays","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — well-characterized transgenic mouse model with defined cellular phenotypes; replicated in multiple subsequent studies","pmids":["15755899"],"is_preprint":false},{"year":2007,"finding":"A Q317R missense mutation in the HOXD13 homeodomain (substituting the highly conserved glutamine critical for DNA-binding specificity) abolishes HOXD13-mediated transactivation of the human EPHA7 promoter in luciferase assays, causing syndactyly type V. A 21-bp deletion causing polyalanine contraction of seven residues leads to a different digital anomaly (brachydactyly-syndactyly), not synpolydactyly.","method":"Linkage analysis, sequencing, luciferase transactivation assays, molecular modeling","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional transactivation assay with mutant protein plus molecular modeling; single lab","pmids":["17236141"],"is_preprint":false},{"year":2007,"finding":"Retroviral insertional mutagenesis in NUP98-HOXD13 transgenic mice identifies collaborating genes including Meis1, Mn1, Gata2, Erg, and Epor, as well as the miR29a/miR29b1 locus, which are upregulated during leukemic transformation from MDS, defining the genetic landscape of NHD13-mediated leukemogenesis.","method":"Retroviral insertional mutagenesis screen in NHD13 transgenic mice, common insertion site mapping, expression analysis","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide insertional screen with expression validation; mechanistic insight into collaborating pathways, single lab","pmids":["17545593"],"is_preprint":false},{"year":2008,"finding":"HOXD13 directly regulates Fhl1 (skeletal muscle LIM protein 1) expression in developing rat embryo limbs: HOXD13 binds the Fhl1 promoter as shown by EMSA identifying a novel promoter element, luciferase transactivation assays, and ChIP of the endogenous Hoxd13–Fhl1 promoter complex from developing limbs.","method":"Immunofluorescence, luciferase assay, EMSA, chromatin immunoprecipitation (ChIP) in rat embryo limb tissue","journal":"Cytogenetic and genome research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus EMSA plus transactivation assay; single lab, single gene target","pmids":["18758158"],"is_preprint":false},{"year":2008,"finding":"Hoxd13 binds in vivo in developing limbs to the loci of Hand2, Meis1, Meis2, Sfrp1, Barx1, Fbn1, Dach1, Bmp2, Bmp4, and Emx2 (identified by ChIP-on-chip with 248 gene loci total); Hoxd13 misexpression in chick limbs alters expression of the majority of these genes, establishing them as direct transcriptional targets involved in limb AP/PD axis and skeletal patterning.","method":"ChIP-on-chip genome-wide binding analysis in developing mouse limbs; retrovirus-mediated Hoxd13 misexpression in chick limbs with expression validation","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — genome-wide ChIP-on-chip combined with in vivo misexpression validation for multiple target genes","pmids":["18407260"],"is_preprint":false},{"year":2008,"finding":"A G220V missense mutation outside the HOXD13 homeodomain (in the N-terminal transcription-regulating domain) significantly impairs HOXD13 DNA binding and transcriptional activation/repression through HOXD13-responsive elements, reduces protein stability within cells, and causes partial cytosolic accumulation of subtle aggregates; in vivo chick limb misexpression confirms impaired capacity to perturb proximal limb skeletal development and activate the Hand2 target gene. This represents a dominant loss-of-function revealing HOXD13 haploinsufficiency.","method":"Luciferase transactivation assays, EMSA, immunofluorescence of protein localization, retrovirus-mediated misexpression in chick limbs, protein stability assays","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (EMSA, transactivation, localization, in vivo misexpression) in a single rigorous study","pmids":["19060004"],"is_preprint":false},{"year":2008,"finding":"Mutant Hoxd13 (Spdh allele, polyalanine expansion) causes polydactyly in synpolydactyly by inducing ectopic interdigital chondrogenesis both directly and indirectly via reduction of retinoic acid synthesis: Raldh2 (rate-limiting enzyme for RA synthesis in the limb) is identified as a direct Hoxd13 transcriptional target, RA levels are decreased in Spdh/Spdh limbs, intrauterine RA treatment restores pentadactyly, and mutant Hoxd13 promotes chondrogenesis associated with increased Sox6/Sox9 expression. Transgenic and crossing experiments demonstrate the Spdh allele combines loss and gain of function.","method":"Transgenic mouse crossing experiments, intrauterine RA rescue, primary cell chondrogenesis assays, gene expression analysis, identification of Raldh2 as direct Hoxd13 target","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal approaches (genetic rescue, pharmacological rescue, direct target identification, primary cell assays) in a single study","pmids":["19075394"],"is_preprint":false},{"year":2009,"finding":"HOXD13 binds all characterized human replication origins in vivo, interacts with the CDC6 DNA replication loading factor, promotes pre-replication complex (pre-RC) protein assembly at origins, stimulates DNA synthesis in an in vivo replication assay, and accelerates DNA synthesis initiation when overexpressed. Geminin interacts with HOXD13 and blocks HOXD13-mediated pre-RC assembly and DNA replication induction.","method":"ChIP at replication origins, co-immunoprecipitation (CDC6 interaction, geminin interaction), in vivo replication assay, BrdU incorporation/flow cytometry for DNA synthesis timing","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (ChIP, Co-IP, functional replication assay, cell cycle analysis) establishing a novel replication-regulatory function","pmids":["19703996"],"is_preprint":false},{"year":2009,"finding":"NUP98-HOXD13 (NHD13) fusion gene expression impairs both B and T lymphoid differentiation: blocks pro-B to pre-B transition, causes a partial block at the DN2-to-DN3 thymocyte transition, and induces clonal expansion of thymocytes with incomplete TCRβ rearrangement (DJ but not VDJ). NHD13 thymi overexpress Hoxa cluster genes (Hoxa7, Hoxa9, Hoxa10).","method":"Flow cytometry of bone marrow and thymic compartments, degenerate RT-PCR for TCRβ rearrangement clonality, gene expression profiling in NHD13 transgenic mice","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — detailed immunophenotyping and molecular clonality analysis in transgenic mice; single lab","pmids":["19841179"],"is_preprint":false},{"year":2012,"finding":"A novel G11A mutation in HOXD13 (outside the homeodomain and polyalanine tract) affects the intracellular half-life of the protein; misexpression of HOXD13(G11A) in chick limbs phenocopies human SPD; in vitro studies demonstrate this mutation has a destabilizing effect on GLI3R, revealing that HOXD13 can influence limb patterning by modulating GLI3R stability.","method":"Identification of novel mutation, retrovirus-mediated chick limb misexpression, in vitro GLI3R stability assays","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro functional assay plus in vivo chick misexpression; single lab, two complementary methods","pmids":["22373878"],"is_preprint":false},{"year":2013,"finding":"HOXD13 regulates cell polarity in cartilage growth plate and perichondrium: spdh (Hoxd13 polyalanine expansion) metacarpal growth plates show defective chondrocyte polarization and failure of perichondrial cells to adopt flattened morphology. Wnt5a and Wnt5b are downregulated in spdh handplates; HOXD13 induces Wnt5a/5b expression in vitro; mislocalization of PCP components DVL2 and PRICKLE1 occurs in spdh metacarpals; WNT/β-CATENIN pathway is upregulated in the perichondral region. Non-cell autonomous rescue of cell polarity was achieved by providing HOXD13- or WNT5A-expressing cells to spdh limb explants.","method":"Mouse genetic analysis (spdh and Wnt5a knockout comparison), in vitro transactivation of Wnt5a/5b, immunofluorescence of PCP components, limb explant rescue experiments","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (genetic comparison, in vitro target activation, PCP protein localization, explant rescue) establishing HOXD13→WNT5A→PCP pathway","pmids":["24161848"],"is_preprint":false},{"year":2013,"finding":"Genome-wide ChIP-seq analysis of wild-type HOXD13 and a Q317K missense mutant in chicken mesenchymal stem cells reveals that the Q317K mutation shifts the genome-wide binding profile of HOXD13 toward a bicoid/PITX1 DNA-binding motif (due to substitution of the homeodomain glutamine conserved in most homeodomains). Gene expression analysis and in vivo overexpression studies confirm a partial conversion of HOXD13(Q317K) into a transcription factor with bicoid/PITX1 properties, which is not observed with the Q317R mutation associated with a milder phenotype.","method":"ChIP-seq in chicken mesenchymal stem cells, retroviral expression system, gene expression analysis, in vivo overexpression in chick","journal":"Genome research","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — genome-wide binding profiling combined with gene expression and in vivo functional validation; mechanistically rigorous","pmids":["23995701"],"is_preprint":false},{"year":2014,"finding":"Mutations p.R306Q and p.R306G in the HOXD13 homeodomain cause syndactyly type 1-c; luciferase assays demonstrated that both mutations impair the transcriptional activation ability of HOXD13, establishing a link between homeodomain mutations and specific syndactyly subtype.","method":"Linkage analysis, sequencing, luciferase transactivation assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — functional transactivation assay supports mechanistic claim; replicated across two families with two different mutations at same residue","pmids":["24789103"],"is_preprint":false},{"year":2015,"finding":"A homozygous missense mutation c.938C>G (p.T313R) in the HOXD13 DNA-binding domain prevents HOXD13 binding to DNA in vitro as demonstrated by EMSA; heterozygous carriers are unaffected (non-penetrant), while the homozygous state causes severe brachydactyly with metacarpal-to-carpal transformation, consistent with complete loss of functional HOXD13 protein.","method":"Whole exome sequencing, Sanger sequencing for cosegregation, electrophoretic mobility shift assay (EMSA) for DNA binding","journal":"American journal of medical genetics. Part A","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — EMSA directly demonstrates loss of DNA binding for the mutant protein; single lab, single patient","pmids":["26581570"],"is_preprint":false},{"year":2019,"finding":"NUP98-HOXD13 (NHD13) transgenic thymocytes exhibit self-renewal capacity (demonstrated by serial transplantation); NHD13-Tg thymocytes express a stem cell-like transcriptional programme including Lmo2 and its cofactor Lyl1; Lyl1 is essential for the stem cell-like gene expression programme and thymocyte self-renewal; loss of Lyl1 in NHD13-Tg mice accelerates T-ALL and eliminates AML transformation associated with loss of multipotent progenitors.","method":"Serial transplantation assays, transcriptome analysis, genetic cross of NHD13-Tg with Lyl1 knockout mice","journal":"Leukemia","confidence":"High","confidence_rationale":"Tier 2 / Strong — functional serial transplantation plus genetic epistasis (Lyl1 KO cross) plus transcriptome analysis establishing Lmo2/Lyl1 as effectors of NHD13-induced self-renewal","pmids":["30700838"],"is_preprint":false},{"year":2021,"finding":"HOXD13 suppresses prostate cancer metastasis by inhibiting SMAD1 transcription, thereby preventing BMP4-induced epithelial-mesenchymal transition (EMT); HOXD13 depletion increases bone metastasis in a mouse metastatic model.","method":"HOXD13 knockdown/knockout in prostate cancer cells, in vitro EMT assays, in vivo bone metastasis mouse model, gene expression analysis of SMAD1 transcription","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined in vitro and in vivo phenotypic readouts plus identification of SMAD1 as transcriptional target; single lab","pmids":["33521930"],"is_preprint":false},{"year":2007,"finding":"The intergenic region between Evx2 and Hoxd13 functions as a boundary element that restricts enhancer association to the Hoxd13 promoter in a spatially and temporally differential manner during development of limbs, genital bud, and brain; the boundary comprises at least two functional units: a constitutive boundary element blocking transcriptional regulatory influences, and a regulatory element controlling the constitutive boundary in time and space.","method":"ES cell-based genetic deletion and replacement experiments dissecting the Evx2-Hoxd13 intergenic region","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ES cell genetic analysis with functional readout of gene expression, single lab","pmids":["17245451"],"is_preprint":false},{"year":2009,"finding":"HOXD13 directly interacts with the Gli3 promoter in vivo in developing rat limb (confirmed by ChIP), and EMSA identifies a specific Hoxd13 binding site in the Gli3 promoter; low Hoxd13 expression correlates with increased Gli3 expression in an ICTEV (clubfoot) rat model, suggesting HOXD13 normally represses Gli3 transcription during limb development.","method":"Luciferase reporter assay, EMSA, ChIP from developing rat limb","journal":"BMC musculoskeletal disorders","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus EMSA plus luciferase assay; single lab, rat model","pmids":["19925654"],"is_preprint":false},{"year":2022,"finding":"In Ewing sarcoma, EWS::FLI1 creates a de novo GGAA microsatellite enhancer in the HOXD locus driving HOXD13 expression; HOXD13 binds at established EWS::FLI1 binding sites and activates EWS::FLI1-repressed genes (mesenchymal/migratory programme), directly antagonizing EWS::FLI1 transcriptional repression and defining a mesenchymal transcriptional continuum in tumor cells.","method":"CUT&RUN, RNA-seq, ChIP for EWS::FLI1 binding sites, CRISPR interference, single-cell transcriptomics, migration assays, flow cytometry","journal":"Clinical cancer research","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal genome-wide methods (CUT&RUN, RNA-seq, ChIP, scRNA-seq) plus functional assays establishing HOXD13 as a direct transcriptional antagonist of EWS::FLI1","pmids":["35653119"],"is_preprint":false}],"current_model":"HOXD13 is a homeodomain transcription factor that directly binds DNA at target gene promoters and replication origins, activates or represses transcription of downstream targets (including EphA7/EPHA7, Raldh2, Hand2, Meis1/2, Bmp2/4, Wnt5a, Fhl1, Gli3, SMAD1, and PTPRN2) to control distal limb patterning, chondrogenesis, and cell polarity via the WNT/PCP pathway; polyalanine expansion mutations act in a dominant-negative/gain-of-function manner to cause synpolydactyly by reducing retinoic acid synthesis and promoting ectopic interdigital chondrogenesis, while homeodomain missense mutations alter DNA-binding specificity or cause selective or complete loss of function leading to variable limb malformations; additionally, HOXD13 promotes pre-replication complex assembly at origins (inhibited by geminin), and NUP98-HOXD13 fusion proteins impair hematopoietic differentiation and drive MDS/leukemia partly through induction of Lmo2/Lyl1-dependent thymocyte self-renewal."},"narrative":{"mechanistic_narrative":"HOXD13 is a homeodomain transcription factor that directly binds DNA at target gene promoters to control distal limb (autopod) patterning, chondrogenesis, and cell polarity [PMID:16314414, PMID:18407260]. Through genome-wide and locus-specific binding it regulates a network of patterning and skeletal genes — activating EphA7/EPHA7 [PMID:12620993, PMID:16314414], Fhl1 [PMID:18758158], Hand2, Meis1/2, Bmp2/4 and other AP/PD axis loci [PMID:18407260], and the retinoic acid synthesis enzyme Raldh2 [PMID:19075394] — while repressing Gli3 [PMID:19925654] and, in cancer, SMAD1 [PMID:33521930]. HOXD13 also drives the WNT/PCP pathway by inducing Wnt5a/5b to establish chondrocyte and perichondrial polarization in the growth plate [PMID:24161848]. Its activity depends on a conserved homeodomain glutamine that dictates DNA-binding specificity: missense substitutions at this and other homeodomain residues abolish or re-target binding and transactivation, causing distinct syndactyly and brachydactyly phenotypes (I47L, Q317R/K, R306Q/G, T313R) [PMID:12620993, PMID:17236141, PMID:23995701, PMID:24789103, PMID:26581570]. The recurrent disease allele is an exon-1 polyalanine tract expansion that causes synpolydactyly; expansion size correlates with phenotypic severity, and the mutant allele acts in a combined dominant-negative/gain-of-function manner — more severe than the null — by reducing retinoic acid synthesis and promoting ectopic interdigital chondrogenesis [PMID:8817328, PMID:9207113, PMID:9580668, PMID:19075394]. Beyond development, HOXD13 binds replication origins, interacts with CDC6 to promote pre-replication complex assembly and accelerate DNA synthesis initiation, an activity blocked by geminin [PMID:19703996]. The t(2;11) NUP98-HOXD13 fusion impairs hematopoietic differentiation and drives myelodysplastic syndrome and leukemia, in part by conferring Lmo2/Lyl1-dependent thymocyte self-renewal [PMID:10995009, PMID:15755899, PMID:30700838].","teleology":[{"year":1996,"claim":"Establishing that a discrete polyalanine tract expansion in HOXD13 causes synpolydactyly defined the gene's first human disease link and its two-exon homeodomain structure.","evidence":"Genomic sequencing and family segregation analysis across two SPD families with control chromosomes","pmids":["8817328"],"confidence":"High","gaps":["Did not establish the molecular mechanism by which expansion perturbs protein function","No direct transcriptional readout"]},{"year":1997,"claim":"Correlating expansion size with phenotype severity argued the mutation is a specific gain of function rather than simple disruption.","evidence":"Molecular sizing of polyalanine expansions and genotype-phenotype correlation across 16 SPD families","pmids":["9207113"],"confidence":"High","gaps":["Correlative, not mechanistic","Did not distinguish gain-of-function from dominant-negative at the protein level"]},{"year":1998,"claim":"Comparing the spdh expansion allele to a null allele in mouse showed the expanded protein is more deleterious than absence, defining a dominant-negative mechanism.","evidence":"Cloning of the spdh allele and phenotypic comparison of heterozygote/homozygote vs Hoxd13 null mice","pmids":["9580668"],"confidence":"High","gaps":["Molecular target of the dominant-negative effect not identified","No biochemical demonstration of interference"]},{"year":2003,"claim":"An I47L homeodomain mutation that selectively loses binding at a subset of targets including EphA7 separated DNA-binding-dependent from interaction-dependent HOXD13 functions.","evidence":"In vitro DNA binding plus retroviral misexpression of WT, I47L, and DNA-binding-null IQN mutants in chick limbs","pmids":["12620993"],"confidence":"High","gaps":["Identity of protein partners mediating DNA-binding-independent suppression unknown","Direct EphA7 promoter occupancy not yet shown"]},{"year":2005,"claim":"Direct in vivo binding and transactivation of the EphA7 promoter, lost in the I47L mutant, confirmed EphA7 as a bona fide HOXD13 target underlying its limb phenotype.","evidence":"ChIP in developing mouse limbs, EMSA, and luciferase transactivation assays","pmids":["16314414"],"confidence":"High","gaps":["Cofactors at the EphA7 promoter not defined","Single target characterized"]},{"year":2000,"claim":"Discovery of the NUP98-HOXD13 fusion implicated HOXD13 homeodomain activity in leukemogenesis.","evidence":"3'-RACE, RT-PCR and breakpoint mapping in a single AML patient","pmids":["10995009"],"confidence":"Medium","gaps":["Single patient, no functional model","Causality not established at this stage"]},{"year":2005,"claim":"An NHD13 transgenic mouse demonstrated the fusion drives myelodysplastic syndrome with ineffective hematopoiesis and leukemic transformation, establishing causality.","evidence":"Transgenic mouse model with hematological/histological analysis and differentiation assays","pmids":["15755899"],"confidence":"High","gaps":["Downstream effectors of differentiation block not identified here","Mechanism of apoptosis induction unresolved"]},{"year":2007,"claim":"Insertional mutagenesis defined collaborating loci (Meis1, Mn1, Gata2, Erg, Epor, miR29) that cooperate with NHD13 during transformation.","evidence":"Retroviral insertional mutagenesis screen with common insertion site mapping in NHD13 mice","pmids":["17545593"],"confidence":"Medium","gaps":["Functional contribution of individual hits not validated","Single screen"]},{"year":2007,"claim":"A homeodomain Q317R mutation abolishing transactivation, and a polyalanine contraction allele, showed distinct HOXD13 lesions produce distinct digital phenotypes.","evidence":"Linkage, sequencing, luciferase transactivation assays and molecular modeling","pmids":["17236141"],"confidence":"Medium","gaps":["No in vivo developmental model for these alleles","Single lab"]},{"year":2008,"claim":"Genome-wide ChIP-on-chip identified a broad HOXD13 target network controlling limb AP/PD and skeletal patterning, moving beyond single-gene models.","evidence":"ChIP-on-chip in developing mouse limbs with chick misexpression validation","pmids":["18407260"],"confidence":"High","gaps":["Activation vs repression at individual loci not fully resolved","Cofactor requirements unknown"]},{"year":2008,"claim":"Direct regulation of Fhl1 added a muscle LIM protein to the HOXD13 target repertoire.","evidence":"EMSA, luciferase, and ChIP of endogenous Hoxd13-Fhl1 complex in rat limb","pmids":["18758158"],"confidence":"Medium","gaps":["Phenotypic consequence of Fhl1 regulation not tested","Single target, single lab"]},{"year":2008,"claim":"A G220V mutation outside the homeodomain that impairs binding, stability and localization revealed a dominant loss-of-function/haploinsufficiency mechanism distinct from the polyalanine alleles.","evidence":"EMSA, transactivation, localization, protein stability and chick misexpression assays","pmids":["19060004"],"confidence":"High","gaps":["Aggregation mechanism not detailed","Relationship to dominant-negative alleles not directly compared in vivo"]},{"year":2008,"claim":"Identifying Raldh2 as a direct target and rescuing synpolydactyly with retinoic acid pinned the polyalanine phenotype on reduced RA synthesis and ectopic interdigital chondrogenesis, demonstrating combined loss and gain of function.","evidence":"Mouse crossing, intrauterine RA rescue, primary chondrogenesis assays and direct target identification","pmids":["19075394"],"confidence":"High","gaps":["Mechanism of the gain-of-function chondrogenic activity not fully molecularly defined","Sox6/Sox9 induction pathway not mapped"]},{"year":2009,"claim":"Demonstrating HOXD13 binds replication origins, interacts with CDC6, and accelerates DNA synthesis revealed a transcription-independent role in pre-replication complex assembly antagonized by geminin.","evidence":"ChIP at origins, co-IP of CDC6 and geminin, in vivo replication assay and BrdU/flow cytometry","pmids":["19703996"],"confidence":"High","gaps":["Physiological context of origin licensing role unknown","Link to developmental function not established"]},{"year":2009,"claim":"Direct repression of Gli3 by HOXD13 connected the factor to hedgehog-pathway control during limb development.","evidence":"Luciferase, EMSA and ChIP in developing rat limb plus correlation in a clubfoot model","pmids":["19925654"],"confidence":"Medium","gaps":["Repression mechanism (cofactors) unknown","Correlative model data"]},{"year":2009,"claim":"NHD13 was shown to block both B- and T-lymphoid differentiation with Hoxa cluster overexpression, broadening the fusion's impact across lineages.","evidence":"Flow cytometry, TCRb clonality RT-PCR and expression profiling in NHD13 mice","pmids":["19841179"],"confidence":"Medium","gaps":["Causal driver among Hoxa genes not isolated","Single lab"]},{"year":2012,"claim":"A G11A allele that destabilizes both HOXD13 and GLI3R showed HOXD13 can influence patterning by modulating GLI3R stability.","evidence":"Novel mutation analysis, chick misexpression phenocopy, and in vitro GLI3R stability assays","pmids":["22373878"],"confidence":"Medium","gaps":["Direct physical mechanism of GLI3R destabilization unknown","Single lab"]},{"year":2013,"claim":"Linking HOXD13 to Wnt5a/5b induction and PCP component localization defined a HOXD13→WNT5A→PCP axis controlling chondrocyte and perichondrial polarity.","evidence":"spdh/Wnt5a genetic comparison, in vitro Wnt5a/5b activation, PCP immunofluorescence and explant rescue","pmids":["24161848"],"confidence":"High","gaps":["Direct vs indirect Wnt5a regulation not fully resolved","Mechanism of WNT/beta-catenin upregulation in perichondrium unclear"]},{"year":2013,"claim":"ChIP-seq of a Q317K mutant showed substitution of the conserved homeodomain glutamine re-targets HOXD13 toward a bicoid/PITX1 motif, demonstrating a neomorphic specificity switch distinct from milder Q317R loss-of-function.","evidence":"ChIP-seq in chicken mesenchymal stem cells with expression and in vivo overexpression validation","pmids":["23995701"],"confidence":"High","gaps":["Functional consequences of redirected binding on limb morphology not fully mapped","Cofactor contribution to motif preference unknown"]},{"year":2014,"claim":"R306Q/R306G homeodomain mutations impairing transactivation defined the molecular basis of syndactyly type 1-c.","evidence":"Linkage, sequencing and luciferase transactivation assays","pmids":["24789103"],"confidence":"Medium","gaps":["No in vivo model","Mechanism of partial vs complete loss not addressed"]},{"year":2015,"claim":"A homozygous T313R DNA-binding-null mutation, recessive in carriers, defined a complete loss-of-function allele causing severe brachydactyly with metacarpal-to-carpal transformation.","evidence":"Exome/Sanger sequencing with EMSA demonstrating loss of DNA binding","pmids":["26581570"],"confidence":"Medium","gaps":["Single family/patient","No in vivo functional confirmation"]},{"year":2019,"claim":"Demonstrating NHD13 confers thymocyte self-renewal via an Lmo2/Lyl1 stem-cell programme, with Lyl1 required for AML transformation, identified the effectors of fusion-driven leukemic stemness.","evidence":"Serial transplantation, transcriptome analysis and Lyl1 knockout genetic cross","pmids":["30700838"],"confidence":"High","gaps":["How NHD13 activates the Lmo2/Lyl1 programme mechanistically unresolved","Translation to human leukemia not established"]},{"year":2021,"claim":"Showing HOXD13 represses SMAD1 to block BMP4-induced EMT assigned it a metastasis-suppressor role in prostate cancer.","evidence":"Knockdown/knockout in prostate cancer cells, EMT assays and an in vivo bone metastasis model","pmids":["33521930"],"confidence":"Medium","gaps":["Direct SMAD1 promoter occupancy not shown","Single lab"]},{"year":2022,"claim":"Identifying HOXD13 as a direct transcriptional antagonist of EWS::FLI1 in Ewing sarcoma extended its role to oncogenic transcriptional networks.","evidence":"CUT&RUN, RNA-seq, ChIP, CRISPRi, single-cell transcriptomics and migration assays","pmids":["35653119"],"confidence":"High","gaps":["Therapeutic relevance not established","Mechanism of de-repression at shared sites not fully detailed"]},{"year":null,"claim":"The protein partners and cofactors that mediate HOXD13's DNA-binding-independent functions, the molecular basis of its neomorphic and dominant-negative disease alleles, and the physiological role of its replication-licensing activity remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No structural model of HOXD13-cofactor complexes","Physiological context of origin-binding/CDC6 interaction 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Part A","url":"https://pubmed.ncbi.nlm.nih.gov/26581570","citation_count":16,"is_preprint":false},{"pmid":"25829244","id":"PMC_25829244","title":"Decidual expression and localization of human surfactant protein SP-A and SP-D, and complement protein C1q.","date":"2015","source":"Molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/25829244","citation_count":16,"is_preprint":false},{"pmid":"33275228","id":"PMC_33275228","title":"GALNT10 promotes the proliferation and metastatic ability of gastric cancer and reduces 5-fluorouracil sensitivity by activating HOXD13.","date":"2020","source":"European review for medical and pharmacological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33275228","citation_count":15,"is_preprint":false},{"pmid":"20708683","id":"PMC_20708683","title":"Identification of genes downstream of the Shh signalling in the developing chick wing and syn-expressed with Hoxd13 using microarray and 3D computational analysis.","date":"2010","source":"Mechanisms of development","url":"https://pubmed.ncbi.nlm.nih.gov/20708683","citation_count":15,"is_preprint":false},{"pmid":"33721375","id":"PMC_33721375","title":"Soluble programmed cell death protein 1 (sPD-1) and the soluble programmed cell death ligands 1 and 2 (sPD-L1 and sPD-L2) in lymphoid malignancies.","date":"2021","source":"European journal of haematology","url":"https://pubmed.ncbi.nlm.nih.gov/33721375","citation_count":14,"is_preprint":false},{"pmid":"20974300","id":"PMC_20974300","title":"Polyalanine repeat expansion mutation of the HOXD13 gene in a Chinese family with unusual clinical manifestations of synpolydactyly.","date":"2010","source":"European journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/20974300","citation_count":14,"is_preprint":false},{"pmid":"33162825","id":"PMC_33162825","title":"A novel miR-7156-3p-HOXD13 axis modulates glioma progression by regulating tumor cell stemness.","date":"2020","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33162825","citation_count":13,"is_preprint":false},{"pmid":"22374128","id":"PMC_22374128","title":"A novel non-synonymous mutation in the homeodomain of HOXD13 causes synpolydactyly in a Chinese family.","date":"2012","source":"Clinica chimica acta; international journal of clinical chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22374128","citation_count":13,"is_preprint":false},{"pmid":"32355906","id":"PMC_32355906","title":"FaPAO5 regulates Spm/Spd levels as a signaling during strawberry fruit ripening.","date":"2020","source":"Plant direct","url":"https://pubmed.ncbi.nlm.nih.gov/32355906","citation_count":13,"is_preprint":false},{"pmid":"31171213","id":"PMC_31171213","title":"Quantification of surfactant protein D (SPD) in human serum by liquid chromatography-mass spectrometry (LC-MS).","date":"2019","source":"Talanta","url":"https://pubmed.ncbi.nlm.nih.gov/31171213","citation_count":13,"is_preprint":false},{"pmid":"22373878","id":"PMC_22373878","title":"An N-terminal G11A mutation in HOXD13 causes synpolydactyly and interferes with Gli3R function during limb pre-patterning.","date":"2012","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/22373878","citation_count":13,"is_preprint":false},{"pmid":"32652111","id":"PMC_32652111","title":"TLR2/6 signaling promotes the expansion of premalignant hematopoietic stem and progenitor cells in the NUP98-HOXD13 mouse model of MDS.","date":"2020","source":"Experimental hematology","url":"https://pubmed.ncbi.nlm.nih.gov/32652111","citation_count":13,"is_preprint":false},{"pmid":"22201901","id":"PMC_22201901","title":"SP-A and SP-D in host defense against fungal infections and allergies.","date":"2012","source":"Frontiers in bioscience (Elite edition)","url":"https://pubmed.ncbi.nlm.nih.gov/22201901","citation_count":12,"is_preprint":false},{"pmid":"30700838","id":"PMC_30700838","title":"The NUP98-HOXD13 fusion oncogene induces thymocyte self-renewal via Lmo2/Lyl1.","date":"2019","source":"Leukemia","url":"https://pubmed.ncbi.nlm.nih.gov/30700838","citation_count":12,"is_preprint":false},{"pmid":"32098834","id":"PMC_32098834","title":"SPD_1495 Contributes to Capsular Polysaccharide Synthesis and Virulence in Streptococcus pneumoniae.","date":"2020","source":"mSystems","url":"https://pubmed.ncbi.nlm.nih.gov/32098834","citation_count":12,"is_preprint":false},{"pmid":"28498426","id":"PMC_28498426","title":"Identification of a missense HOXD13 mutation in a Chinese family with syndactyly type I-c using exome sequencing.","date":"2017","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/28498426","citation_count":12,"is_preprint":false},{"pmid":"26421960","id":"PMC_26421960","title":"Expression of surfactant proteins SP-A and SP-D in murine decidua and immunomodulatory effects on decidual macrophages.","date":"2015","source":"Immunobiology","url":"https://pubmed.ncbi.nlm.nih.gov/26421960","citation_count":12,"is_preprint":false},{"pmid":"32325256","id":"PMC_32325256","title":"Functional characterization of the different oligomeric forms of human surfactant protein SP-D.","date":"2020","source":"Biochimica et biophysica acta. 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The genomic structure of HOXD13 was established as two exons encoding a 335-amino-acid protein with a C-terminal homeodomain.\",\n      \"method\": \"Genomic sequencing, PCR-based mutation analysis, family segregation analysis\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct sequencing across multiple families, replicated in independent cohorts, established the causative mutation\",\n      \"pmids\": [\"8817328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"SPD phenotype severity (penetrance and expressivity in hands and feet) correlates significantly with the size of the polyalanine expansion in HOXD13 (7–14 extra residues tested), suggesting that expansion produces a specific gain of function in the mutant protein.\",\n      \"method\": \"Molecular sizing of polyalanine expansions across 16 SPD families; phenotype–genotype correlation analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic molecular and clinical analysis across 20 families, statistically significant correlation between expansion size and phenotype severity\",\n      \"pmids\": [\"9207113\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"A spontaneous mouse Hoxd13 mutation (spdh) carrying a 21-bp in-frame duplication expanding the polyalanine tract from 15 to 22 residues produces a phenotype more severe than null Hoxd13 mice, indicating the expanded polyalanine allele acts in a dominant-negative manner rather than as a simple loss-of-function.\",\n      \"method\": \"Molecular cloning of spdh allele, comparison of spdh heterozygote/homozygote phenotypes with Hoxd13 null mice\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic comparison of gain-of-function allele vs. null allele in mouse; phenotypic readout clearly distinguishes mechanisms\",\n      \"pmids\": [\"9580668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The t(2;11)(q31;p15) translocation generates NUP98-HOXD13 fusion transcripts encoding a protein with the FG-repeat region of NUP98 fused to the HOXD13 homeodomain; ectopic expression of the wild-type HOXD13 allele is also observed in the leukemia patient, implicating HOXD13 homeodomain activity in leukemogenesis.\",\n      \"method\": \"3′-RACE, RT-PCR, chromosomal breakpoint mapping in an AML patient\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — RT-PCR characterization in a single patient, but breakpoints molecularly mapped and fusion protein structure inferred from sequence\",\n      \"pmids\": [\"10995009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"An I47L missense mutation in the HOXD13 homeodomain causes selective loss of DNA-binding at a subset of wild-type HOXD13 target sites (including the EphA7 locus) without dominant-negative or gain-of-function effects; retrovirus-mediated misexpression in chick limbs showed wild-type HOXD13 upregulates EphA7 in the autopod whereas HOXD13(I47L) cannot; both HOXD13(I47L) and a DNA-binding-null HOXD13(IQN) cause proximal limb shortening, suggesting functional suppression of anterior Hox genes does not require DNA binding and is mediated by protein–protein interactions.\",\n      \"method\": \"In vitro DNA-binding assays, retrovirus-mediated misexpression in developing chick limbs, in vivo comparison of wild-type, I47L, and IQN (DNA-binding null) mutant HOXD13\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (in vitro binding, in vivo chick limb misexpression, genetic comparison of selective vs. complete loss-of-function mutants)\",\n      \"pmids\": [\"12620993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"HOXD13 and HOXA13 directly bind multiple sites in the EphA7 promoter in vivo (confirmed by ChIP in developing mouse limbs and in vitro binding assays), and activate transcription from the EphA7 promoter; the HOXD13(I47L) brachydactyly mutation fails to bind the EphA7 promoter in vivo and fails to transactivate it.\",\n      \"method\": \"Promoter characterization, electrophoretic mobility shift assay (EMSA), ChIP in developing mouse limbs, luciferase transactivation assays, retrovirus-mediated misexpression in chick limbs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — ChIP in vivo plus EMSA plus transactivation assays, consistent with in vivo chick misexpression data\",\n      \"pmids\": [\"16314414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"NUP98-HOXD13 (NHD13) transgenic mice develop a uniformly fatal myelodysplastic syndrome (MDS) with peripheral cytopenias, bone marrow dysplasia, apoptosis, and transformation to acute leukemia; the NHD13 fusion gene inhibits megakaryocytic differentiation and increases bone marrow apoptosis, providing a mechanism for ineffective hematopoiesis.\",\n      \"method\": \"Transgenic mouse model, hematological and histological analysis, bone marrow differentiation assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — well-characterized transgenic mouse model with defined cellular phenotypes; replicated in multiple subsequent studies\",\n      \"pmids\": [\"15755899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"A Q317R missense mutation in the HOXD13 homeodomain (substituting the highly conserved glutamine critical for DNA-binding specificity) abolishes HOXD13-mediated transactivation of the human EPHA7 promoter in luciferase assays, causing syndactyly type V. A 21-bp deletion causing polyalanine contraction of seven residues leads to a different digital anomaly (brachydactyly-syndactyly), not synpolydactyly.\",\n      \"method\": \"Linkage analysis, sequencing, luciferase transactivation assays, molecular modeling\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional transactivation assay with mutant protein plus molecular modeling; single lab\",\n      \"pmids\": [\"17236141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Retroviral insertional mutagenesis in NUP98-HOXD13 transgenic mice identifies collaborating genes including Meis1, Mn1, Gata2, Erg, and Epor, as well as the miR29a/miR29b1 locus, which are upregulated during leukemic transformation from MDS, defining the genetic landscape of NHD13-mediated leukemogenesis.\",\n      \"method\": \"Retroviral insertional mutagenesis screen in NHD13 transgenic mice, common insertion site mapping, expression analysis\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide insertional screen with expression validation; mechanistic insight into collaborating pathways, single lab\",\n      \"pmids\": [\"17545593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"HOXD13 directly regulates Fhl1 (skeletal muscle LIM protein 1) expression in developing rat embryo limbs: HOXD13 binds the Fhl1 promoter as shown by EMSA identifying a novel promoter element, luciferase transactivation assays, and ChIP of the endogenous Hoxd13–Fhl1 promoter complex from developing limbs.\",\n      \"method\": \"Immunofluorescence, luciferase assay, EMSA, chromatin immunoprecipitation (ChIP) in rat embryo limb tissue\",\n      \"journal\": \"Cytogenetic and genome research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus EMSA plus transactivation assay; single lab, single gene target\",\n      \"pmids\": [\"18758158\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Hoxd13 binds in vivo in developing limbs to the loci of Hand2, Meis1, Meis2, Sfrp1, Barx1, Fbn1, Dach1, Bmp2, Bmp4, and Emx2 (identified by ChIP-on-chip with 248 gene loci total); Hoxd13 misexpression in chick limbs alters expression of the majority of these genes, establishing them as direct transcriptional targets involved in limb AP/PD axis and skeletal patterning.\",\n      \"method\": \"ChIP-on-chip genome-wide binding analysis in developing mouse limbs; retrovirus-mediated Hoxd13 misexpression in chick limbs with expression validation\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — genome-wide ChIP-on-chip combined with in vivo misexpression validation for multiple target genes\",\n      \"pmids\": [\"18407260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"A G220V missense mutation outside the HOXD13 homeodomain (in the N-terminal transcription-regulating domain) significantly impairs HOXD13 DNA binding and transcriptional activation/repression through HOXD13-responsive elements, reduces protein stability within cells, and causes partial cytosolic accumulation of subtle aggregates; in vivo chick limb misexpression confirms impaired capacity to perturb proximal limb skeletal development and activate the Hand2 target gene. This represents a dominant loss-of-function revealing HOXD13 haploinsufficiency.\",\n      \"method\": \"Luciferase transactivation assays, EMSA, immunofluorescence of protein localization, retrovirus-mediated misexpression in chick limbs, protein stability assays\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (EMSA, transactivation, localization, in vivo misexpression) in a single rigorous study\",\n      \"pmids\": [\"19060004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Mutant Hoxd13 (Spdh allele, polyalanine expansion) causes polydactyly in synpolydactyly by inducing ectopic interdigital chondrogenesis both directly and indirectly via reduction of retinoic acid synthesis: Raldh2 (rate-limiting enzyme for RA synthesis in the limb) is identified as a direct Hoxd13 transcriptional target, RA levels are decreased in Spdh/Spdh limbs, intrauterine RA treatment restores pentadactyly, and mutant Hoxd13 promotes chondrogenesis associated with increased Sox6/Sox9 expression. Transgenic and crossing experiments demonstrate the Spdh allele combines loss and gain of function.\",\n      \"method\": \"Transgenic mouse crossing experiments, intrauterine RA rescue, primary cell chondrogenesis assays, gene expression analysis, identification of Raldh2 as direct Hoxd13 target\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal approaches (genetic rescue, pharmacological rescue, direct target identification, primary cell assays) in a single study\",\n      \"pmids\": [\"19075394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"HOXD13 binds all characterized human replication origins in vivo, interacts with the CDC6 DNA replication loading factor, promotes pre-replication complex (pre-RC) protein assembly at origins, stimulates DNA synthesis in an in vivo replication assay, and accelerates DNA synthesis initiation when overexpressed. Geminin interacts with HOXD13 and blocks HOXD13-mediated pre-RC assembly and DNA replication induction.\",\n      \"method\": \"ChIP at replication origins, co-immunoprecipitation (CDC6 interaction, geminin interaction), in vivo replication assay, BrdU incorporation/flow cytometry for DNA synthesis timing\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (ChIP, Co-IP, functional replication assay, cell cycle analysis) establishing a novel replication-regulatory function\",\n      \"pmids\": [\"19703996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"NUP98-HOXD13 (NHD13) fusion gene expression impairs both B and T lymphoid differentiation: blocks pro-B to pre-B transition, causes a partial block at the DN2-to-DN3 thymocyte transition, and induces clonal expansion of thymocytes with incomplete TCRβ rearrangement (DJ but not VDJ). NHD13 thymi overexpress Hoxa cluster genes (Hoxa7, Hoxa9, Hoxa10).\",\n      \"method\": \"Flow cytometry of bone marrow and thymic compartments, degenerate RT-PCR for TCRβ rearrangement clonality, gene expression profiling in NHD13 transgenic mice\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — detailed immunophenotyping and molecular clonality analysis in transgenic mice; single lab\",\n      \"pmids\": [\"19841179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"A novel G11A mutation in HOXD13 (outside the homeodomain and polyalanine tract) affects the intracellular half-life of the protein; misexpression of HOXD13(G11A) in chick limbs phenocopies human SPD; in vitro studies demonstrate this mutation has a destabilizing effect on GLI3R, revealing that HOXD13 can influence limb patterning by modulating GLI3R stability.\",\n      \"method\": \"Identification of novel mutation, retrovirus-mediated chick limb misexpression, in vitro GLI3R stability assays\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro functional assay plus in vivo chick misexpression; single lab, two complementary methods\",\n      \"pmids\": [\"22373878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HOXD13 regulates cell polarity in cartilage growth plate and perichondrium: spdh (Hoxd13 polyalanine expansion) metacarpal growth plates show defective chondrocyte polarization and failure of perichondrial cells to adopt flattened morphology. Wnt5a and Wnt5b are downregulated in spdh handplates; HOXD13 induces Wnt5a/5b expression in vitro; mislocalization of PCP components DVL2 and PRICKLE1 occurs in spdh metacarpals; WNT/β-CATENIN pathway is upregulated in the perichondral region. Non-cell autonomous rescue of cell polarity was achieved by providing HOXD13- or WNT5A-expressing cells to spdh limb explants.\",\n      \"method\": \"Mouse genetic analysis (spdh and Wnt5a knockout comparison), in vitro transactivation of Wnt5a/5b, immunofluorescence of PCP components, limb explant rescue experiments\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (genetic comparison, in vitro target activation, PCP protein localization, explant rescue) establishing HOXD13→WNT5A→PCP pathway\",\n      \"pmids\": [\"24161848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Genome-wide ChIP-seq analysis of wild-type HOXD13 and a Q317K missense mutant in chicken mesenchymal stem cells reveals that the Q317K mutation shifts the genome-wide binding profile of HOXD13 toward a bicoid/PITX1 DNA-binding motif (due to substitution of the homeodomain glutamine conserved in most homeodomains). Gene expression analysis and in vivo overexpression studies confirm a partial conversion of HOXD13(Q317K) into a transcription factor with bicoid/PITX1 properties, which is not observed with the Q317R mutation associated with a milder phenotype.\",\n      \"method\": \"ChIP-seq in chicken mesenchymal stem cells, retroviral expression system, gene expression analysis, in vivo overexpression in chick\",\n      \"journal\": \"Genome research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — genome-wide binding profiling combined with gene expression and in vivo functional validation; mechanistically rigorous\",\n      \"pmids\": [\"23995701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Mutations p.R306Q and p.R306G in the HOXD13 homeodomain cause syndactyly type 1-c; luciferase assays demonstrated that both mutations impair the transcriptional activation ability of HOXD13, establishing a link between homeodomain mutations and specific syndactyly subtype.\",\n      \"method\": \"Linkage analysis, sequencing, luciferase transactivation assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — functional transactivation assay supports mechanistic claim; replicated across two families with two different mutations at same residue\",\n      \"pmids\": [\"24789103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A homozygous missense mutation c.938C>G (p.T313R) in the HOXD13 DNA-binding domain prevents HOXD13 binding to DNA in vitro as demonstrated by EMSA; heterozygous carriers are unaffected (non-penetrant), while the homozygous state causes severe brachydactyly with metacarpal-to-carpal transformation, consistent with complete loss of functional HOXD13 protein.\",\n      \"method\": \"Whole exome sequencing, Sanger sequencing for cosegregation, electrophoretic mobility shift assay (EMSA) for DNA binding\",\n      \"journal\": \"American journal of medical genetics. Part A\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — EMSA directly demonstrates loss of DNA binding for the mutant protein; single lab, single patient\",\n      \"pmids\": [\"26581570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NUP98-HOXD13 (NHD13) transgenic thymocytes exhibit self-renewal capacity (demonstrated by serial transplantation); NHD13-Tg thymocytes express a stem cell-like transcriptional programme including Lmo2 and its cofactor Lyl1; Lyl1 is essential for the stem cell-like gene expression programme and thymocyte self-renewal; loss of Lyl1 in NHD13-Tg mice accelerates T-ALL and eliminates AML transformation associated with loss of multipotent progenitors.\",\n      \"method\": \"Serial transplantation assays, transcriptome analysis, genetic cross of NHD13-Tg with Lyl1 knockout mice\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — functional serial transplantation plus genetic epistasis (Lyl1 KO cross) plus transcriptome analysis establishing Lmo2/Lyl1 as effectors of NHD13-induced self-renewal\",\n      \"pmids\": [\"30700838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HOXD13 suppresses prostate cancer metastasis by inhibiting SMAD1 transcription, thereby preventing BMP4-induced epithelial-mesenchymal transition (EMT); HOXD13 depletion increases bone metastasis in a mouse metastatic model.\",\n      \"method\": \"HOXD13 knockdown/knockout in prostate cancer cells, in vitro EMT assays, in vivo bone metastasis mouse model, gene expression analysis of SMAD1 transcription\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined in vitro and in vivo phenotypic readouts plus identification of SMAD1 as transcriptional target; single lab\",\n      \"pmids\": [\"33521930\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The intergenic region between Evx2 and Hoxd13 functions as a boundary element that restricts enhancer association to the Hoxd13 promoter in a spatially and temporally differential manner during development of limbs, genital bud, and brain; the boundary comprises at least two functional units: a constitutive boundary element blocking transcriptional regulatory influences, and a regulatory element controlling the constitutive boundary in time and space.\",\n      \"method\": \"ES cell-based genetic deletion and replacement experiments dissecting the Evx2-Hoxd13 intergenic region\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ES cell genetic analysis with functional readout of gene expression, single lab\",\n      \"pmids\": [\"17245451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"HOXD13 directly interacts with the Gli3 promoter in vivo in developing rat limb (confirmed by ChIP), and EMSA identifies a specific Hoxd13 binding site in the Gli3 promoter; low Hoxd13 expression correlates with increased Gli3 expression in an ICTEV (clubfoot) rat model, suggesting HOXD13 normally represses Gli3 transcription during limb development.\",\n      \"method\": \"Luciferase reporter assay, EMSA, ChIP from developing rat limb\",\n      \"journal\": \"BMC musculoskeletal disorders\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus EMSA plus luciferase assay; single lab, rat model\",\n      \"pmids\": [\"19925654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In Ewing sarcoma, EWS::FLI1 creates a de novo GGAA microsatellite enhancer in the HOXD locus driving HOXD13 expression; HOXD13 binds at established EWS::FLI1 binding sites and activates EWS::FLI1-repressed genes (mesenchymal/migratory programme), directly antagonizing EWS::FLI1 transcriptional repression and defining a mesenchymal transcriptional continuum in tumor cells.\",\n      \"method\": \"CUT&RUN, RNA-seq, ChIP for EWS::FLI1 binding sites, CRISPR interference, single-cell transcriptomics, migration assays, flow cytometry\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal genome-wide methods (CUT&RUN, RNA-seq, ChIP, scRNA-seq) plus functional assays establishing HOXD13 as a direct transcriptional antagonist of EWS::FLI1\",\n      \"pmids\": [\"35653119\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HOXD13 is a homeodomain transcription factor that directly binds DNA at target gene promoters and replication origins, activates or represses transcription of downstream targets (including EphA7/EPHA7, Raldh2, Hand2, Meis1/2, Bmp2/4, Wnt5a, Fhl1, Gli3, SMAD1, and PTPRN2) to control distal limb patterning, chondrogenesis, and cell polarity via the WNT/PCP pathway; polyalanine expansion mutations act in a dominant-negative/gain-of-function manner to cause synpolydactyly by reducing retinoic acid synthesis and promoting ectopic interdigital chondrogenesis, while homeodomain missense mutations alter DNA-binding specificity or cause selective or complete loss of function leading to variable limb malformations; additionally, HOXD13 promotes pre-replication complex assembly at origins (inhibited by geminin), and NUP98-HOXD13 fusion proteins impair hematopoietic differentiation and drive MDS/leukemia partly through induction of Lmo2/Lyl1-dependent thymocyte self-renewal.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"HOXD13 is a homeodomain transcription factor that directly binds DNA at target gene promoters to control distal limb (autopod) patterning, chondrogenesis, and cell polarity [#5, #10]. Through genome-wide and locus-specific binding it regulates a network of patterning and skeletal genes — activating EphA7/EPHA7 [#4, #5], Fhl1 [#9], Hand2, Meis1/2, Bmp2/4 and other AP/PD axis loci [#10], and the retinoic acid synthesis enzyme Raldh2 [#12] — while repressing Gli3 [#23] and, in cancer, SMAD1 [#21]. HOXD13 also drives the WNT/PCP pathway by inducing Wnt5a/5b to establish chondrocyte and perichondrial polarization in the growth plate [#16]. Its activity depends on a conserved homeodomain glutamine that dictates DNA-binding specificity: missense substitutions at this and other homeodomain residues abolish or re-target binding and transactivation, causing distinct syndactyly and brachydactyly phenotypes (I47L, Q317R/K, R306Q/G, T313R) [#4, #7, #17, #18, #19]. The recurrent disease allele is an exon-1 polyalanine tract expansion that causes synpolydactyly; expansion size correlates with phenotypic severity, and the mutant allele acts in a combined dominant-negative/gain-of-function manner — more severe than the null — by reducing retinoic acid synthesis and promoting ectopic interdigital chondrogenesis [#0, #1, #2, #12]. Beyond development, HOXD13 binds replication origins, interacts with CDC6 to promote pre-replication complex assembly and accelerate DNA synthesis initiation, an activity blocked by geminin [#13]. The t(2;11) NUP98-HOXD13 fusion impairs hematopoietic differentiation and drives myelodysplastic syndrome and leukemia, in part by conferring Lmo2/Lyl1-dependent thymocyte self-renewal [#3, #6, #20].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Establishing that a discrete polyalanine tract expansion in HOXD13 causes synpolydactyly defined the gene's first human disease link and its two-exon homeodomain structure.\",\n      \"evidence\": \"Genomic sequencing and family segregation analysis across two SPD families with control chromosomes\",\n      \"pmids\": [\"8817328\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish the molecular mechanism by which expansion perturbs protein function\", \"No direct transcriptional readout\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Correlating expansion size with phenotype severity argued the mutation is a specific gain of function rather than simple disruption.\",\n      \"evidence\": \"Molecular sizing of polyalanine expansions and genotype-phenotype correlation across 16 SPD families\",\n      \"pmids\": [\"9207113\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Correlative, not mechanistic\", \"Did not distinguish gain-of-function from dominant-negative at the protein level\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Comparing the spdh expansion allele to a null allele in mouse showed the expanded protein is more deleterious than absence, defining a dominant-negative mechanism.\",\n      \"evidence\": \"Cloning of the spdh allele and phenotypic comparison of heterozygote/homozygote vs Hoxd13 null mice\",\n      \"pmids\": [\"9580668\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular target of the dominant-negative effect not identified\", \"No biochemical demonstration of interference\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"An I47L homeodomain mutation that selectively loses binding at a subset of targets including EphA7 separated DNA-binding-dependent from interaction-dependent HOXD13 functions.\",\n      \"evidence\": \"In vitro DNA binding plus retroviral misexpression of WT, I47L, and DNA-binding-null IQN mutants in chick limbs\",\n      \"pmids\": [\"12620993\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of protein partners mediating DNA-binding-independent suppression unknown\", \"Direct EphA7 promoter occupancy not yet shown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Direct in vivo binding and transactivation of the EphA7 promoter, lost in the I47L mutant, confirmed EphA7 as a bona fide HOXD13 target underlying its limb phenotype.\",\n      \"evidence\": \"ChIP in developing mouse limbs, EMSA, and luciferase transactivation assays\",\n      \"pmids\": [\"16314414\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cofactors at the EphA7 promoter not defined\", \"Single target characterized\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Discovery of the NUP98-HOXD13 fusion implicated HOXD13 homeodomain activity in leukemogenesis.\",\n      \"evidence\": \"3'-RACE, RT-PCR and breakpoint mapping in a single AML patient\",\n      \"pmids\": [\"10995009\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single patient, no functional model\", \"Causality not established at this stage\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"An NHD13 transgenic mouse demonstrated the fusion drives myelodysplastic syndrome with ineffective hematopoiesis and leukemic transformation, establishing causality.\",\n      \"evidence\": \"Transgenic mouse model with hematological/histological analysis and differentiation assays\",\n      \"pmids\": [\"15755899\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream effectors of differentiation block not identified here\", \"Mechanism of apoptosis induction unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Insertional mutagenesis defined collaborating loci (Meis1, Mn1, Gata2, Erg, Epor, miR29) that cooperate with NHD13 during transformation.\",\n      \"evidence\": \"Retroviral insertional mutagenesis screen with common insertion site mapping in NHD13 mice\",\n      \"pmids\": [\"17545593\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional contribution of individual hits not validated\", \"Single screen\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"A homeodomain Q317R mutation abolishing transactivation, and a polyalanine contraction allele, showed distinct HOXD13 lesions produce distinct digital phenotypes.\",\n      \"evidence\": \"Linkage, sequencing, luciferase transactivation assays and molecular modeling\",\n      \"pmids\": [\"17236141\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo developmental model for these alleles\", \"Single lab\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Genome-wide ChIP-on-chip identified a broad HOXD13 target network controlling limb AP/PD and skeletal patterning, moving beyond single-gene models.\",\n      \"evidence\": \"ChIP-on-chip in developing mouse limbs with chick misexpression validation\",\n      \"pmids\": [\"18407260\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Activation vs repression at individual loci not fully resolved\", \"Cofactor requirements unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Direct regulation of Fhl1 added a muscle LIM protein to the HOXD13 target repertoire.\",\n      \"evidence\": \"EMSA, luciferase, and ChIP of endogenous Hoxd13-Fhl1 complex in rat limb\",\n      \"pmids\": [\"18758158\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phenotypic consequence of Fhl1 regulation not tested\", \"Single target, single lab\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"A G220V mutation outside the homeodomain that impairs binding, stability and localization revealed a dominant loss-of-function/haploinsufficiency mechanism distinct from the polyalanine alleles.\",\n      \"evidence\": \"EMSA, transactivation, localization, protein stability and chick misexpression assays\",\n      \"pmids\": [\"19060004\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Aggregation mechanism not detailed\", \"Relationship to dominant-negative alleles not directly compared in vivo\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identifying Raldh2 as a direct target and rescuing synpolydactyly with retinoic acid pinned the polyalanine phenotype on reduced RA synthesis and ectopic interdigital chondrogenesis, demonstrating combined loss and gain of function.\",\n      \"evidence\": \"Mouse crossing, intrauterine RA rescue, primary chondrogenesis assays and direct target identification\",\n      \"pmids\": [\"19075394\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of the gain-of-function chondrogenic activity not fully molecularly defined\", \"Sox6/Sox9 induction pathway not mapped\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrating HOXD13 binds replication origins, interacts with CDC6, and accelerates DNA synthesis revealed a transcription-independent role in pre-replication complex assembly antagonized by geminin.\",\n      \"evidence\": \"ChIP at origins, co-IP of CDC6 and geminin, in vivo replication assay and BrdU/flow cytometry\",\n      \"pmids\": [\"19703996\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological context of origin licensing role unknown\", \"Link to developmental function not established\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Direct repression of Gli3 by HOXD13 connected the factor to hedgehog-pathway control during limb development.\",\n      \"evidence\": \"Luciferase, EMSA and ChIP in developing rat limb plus correlation in a clubfoot model\",\n      \"pmids\": [\"19925654\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Repression mechanism (cofactors) unknown\", \"Correlative model data\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"NHD13 was shown to block both B- and T-lymphoid differentiation with Hoxa cluster overexpression, broadening the fusion's impact across lineages.\",\n      \"evidence\": \"Flow cytometry, TCRb clonality RT-PCR and expression profiling in NHD13 mice\",\n      \"pmids\": [\"19841179\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal driver among Hoxa genes not isolated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"A G11A allele that destabilizes both HOXD13 and GLI3R showed HOXD13 can influence patterning by modulating GLI3R stability.\",\n      \"evidence\": \"Novel mutation analysis, chick misexpression phenocopy, and in vitro GLI3R stability assays\",\n      \"pmids\": [\"22373878\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct physical mechanism of GLI3R destabilization unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Linking HOXD13 to Wnt5a/5b induction and PCP component localization defined a HOXD13→WNT5A→PCP axis controlling chondrocyte and perichondrial polarity.\",\n      \"evidence\": \"spdh/Wnt5a genetic comparison, in vitro Wnt5a/5b activation, PCP immunofluorescence and explant rescue\",\n      \"pmids\": [\"24161848\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect Wnt5a regulation not fully resolved\", \"Mechanism of WNT/beta-catenin upregulation in perichondrium unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"ChIP-seq of a Q317K mutant showed substitution of the conserved homeodomain glutamine re-targets HOXD13 toward a bicoid/PITX1 motif, demonstrating a neomorphic specificity switch distinct from milder Q317R loss-of-function.\",\n      \"evidence\": \"ChIP-seq in chicken mesenchymal stem cells with expression and in vivo overexpression validation\",\n      \"pmids\": [\"23995701\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequences of redirected binding on limb morphology not fully mapped\", \"Cofactor contribution to motif preference unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"R306Q/R306G homeodomain mutations impairing transactivation defined the molecular basis of syndactyly type 1-c.\",\n      \"evidence\": \"Linkage, sequencing and luciferase transactivation assays\",\n      \"pmids\": [\"24789103\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo model\", \"Mechanism of partial vs complete loss not addressed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"A homozygous T313R DNA-binding-null mutation, recessive in carriers, defined a complete loss-of-function allele causing severe brachydactyly with metacarpal-to-carpal transformation.\",\n      \"evidence\": \"Exome/Sanger sequencing with EMSA demonstrating loss of DNA binding\",\n      \"pmids\": [\"26581570\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single family/patient\", \"No in vivo functional confirmation\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrating NHD13 confers thymocyte self-renewal via an Lmo2/Lyl1 stem-cell programme, with Lyl1 required for AML transformation, identified the effectors of fusion-driven leukemic stemness.\",\n      \"evidence\": \"Serial transplantation, transcriptome analysis and Lyl1 knockout genetic cross\",\n      \"pmids\": [\"30700838\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How NHD13 activates the Lmo2/Lyl1 programme mechanistically unresolved\", \"Translation to human leukemia not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showing HOXD13 represses SMAD1 to block BMP4-induced EMT assigned it a metastasis-suppressor role in prostate cancer.\",\n      \"evidence\": \"Knockdown/knockout in prostate cancer cells, EMT assays and an in vivo bone metastasis model\",\n      \"pmids\": [\"33521930\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct SMAD1 promoter occupancy not shown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identifying HOXD13 as a direct transcriptional antagonist of EWS::FLI1 in Ewing sarcoma extended its role to oncogenic transcriptional networks.\",\n      \"evidence\": \"CUT&RUN, RNA-seq, ChIP, CRISPRi, single-cell transcriptomics and migration assays\",\n      \"pmids\": [\"35653119\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Therapeutic relevance not established\", \"Mechanism of de-repression at shared sites not fully detailed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The protein partners and cofactors that mediate HOXD13's DNA-binding-independent functions, the molecular basis of its neomorphic and dominant-negative disease alleles, and the physiological role of its replication-licensing activity remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural model of HOXD13-cofactor complexes\", \"Physiological context of origin-binding/CDC6 interaction unknown\", \"Direct molecular mechanism of polyalanine gain-of-function not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [4, 5, 10, 17, 21, 24]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [4, 5, 17, 19, 23]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [4, 10, 12, 16]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [5, 10, 17]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 6, 21, 24]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [16, 21]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CDC6\", \"GMNN\", \"HOXA13\", \"NUP98\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}