{"gene":"KHDC3L","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":2011,"finding":"Biallelic loss-of-function mutations in C6orf221 (KHDC3L) cause familial biparental hydatidiform mole (FBHM), implicating KHDC3L as a regulator of genomic imprinting in the human oocyte; the gene family's biological properties suggest KHDC3L and NLRP7 may interact as components of an oocyte complex required for determination of epigenetic status on the oocyte genome.","method":"Sanger sequencing of affected families; identification of biallelic mutations segregating with FBHM phenotype (genome-wide failure to specify/maintain maternal epigenotype at imprinted loci)","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via biallelic mutations with defined phenotype, but molecular interaction with NLRP7 proposed rather than directly demonstrated","pmids":["21885028"],"is_preprint":false},{"year":2007,"finding":"ECAT1 (KHDC3L) belongs to a eutherian-specific gene family characterized by an atypical KH RNA-binding domain; the protein is specifically expressed in oocytes and/or embryonic stem cells, absent in fish, bird, and marsupial genomes, and encoded in a syntenic genomic region that has undergone rapid evolution in eutherians.","method":"Comparative genomics, phylogenetic analysis, domain structure analysis, RT-PCR expression profiling across species","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — multiple orthogonal approaches (comparative genomics, domain analysis, expression) but no direct functional assay of the KH domain","pmids":["17913455"],"is_preprint":false},{"year":2012,"finding":"KHDC3L protein displays a juxta-perinuclear signal and co-localizes with NLRP7 in lymphoblastoid cell lines from normal subjects; protein-truncating mutations in KHDC3L do not alter this subcellular localization in hematopoietic cells.","method":"Immunofluorescence microscopy in lymphoblastoid cell lines from normal subjects and KHDC3L-mutant patients","journal":"European journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct immunofluorescence localization with functional context (mutant vs. wild-type), single lab, single method","pmids":["23232697"],"is_preprint":false},{"year":2014,"finding":"NLRP7 and KHDC3L co-localize to the oocyte cytoskeleton and cortical region in growing human oocytes; after first cell division, both proteins become asymmetrically confined to the outer cortical region and excluded from the cell-to-cell contact region until the blastocyst stage, at which point KHDC3L redistributes to the nucleus.","method":"High-resolution confocal immunofluorescence and electron microscopy on 164 human oocytes and preimplantation embryos","journal":"Human reproduction (Oxford, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization with functional context, high-resolution confocal + electron microscopy, large sample size (164 oocytes/embryos), single lab with multiple orthogonal imaging methods","pmids":["25358348"],"is_preprint":false},{"year":2019,"finding":"KHDC3L is a component of the subcortical maternal complex (SCMC) whose integrity is essential for de novo DNA methylation genome-wide in the female germline; oocytes from a patient homozygous for an inactivating KHDC3L mutation show genome-wide deficit of DNA methylation including at germline differentially methylated regions (gDMRs) of imprinted genes, with no selectivity toward imprinted genes. Post-fertilization, methylation defects at imprinted genes persist while most non-imprinted regions recover near-normal methylation.","method":"Single-cell bisulphite sequencing (scBS-seq) of oocytes from a KHDC3L c.1A>G homozygous patient; genome-wide methylation analysis of preimplantation embryo and molar tissue from the same patient","journal":"Genome medicine","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-coverage genome-wide single-cell methylation sequencing in patient-derived oocytes with matched embryo and molar tissue; multiple genomic features assessed; inactivating mutation with clear functional readout","pmids":["31847873"],"is_preprint":false},{"year":2019,"finding":"KHDC3L is expressed in human epiblast cells and ensures genome stability and viability; KHDC3L binds to PARP1 and stimulates its enzymatic activity. In response to DNA damage, KHDC3L localizes to DNA damage sites and facilitates homologous recombination (HR)-mediated DNA repair. ATM kinase phosphorylates KHDC3L at Thr145 and Thr156, which is essential for KHDC3L's functions. RPL-associated deletions p.E150_V160del and p.E150_V172del both lack Thr156 and are impaired in PARP1 activation and HR repair.","method":"Co-immunoprecipitation (KHDC3L–PARP1 binding), in vitro PARP1 activity assays, DNA damage localization by immunofluorescence, HR repair assays, site-directed mutagenesis of Thr145/Thr156, analysis of patient-derived deletion mutants","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, enzymatic assay, mutagenesis, DNA repair assay, patient mutation analysis), direct mechanistic readouts; single lab but comprehensive experimental coverage","pmids":["31609975"],"is_preprint":false},{"year":2016,"finding":"ECAT1 (KHDC3L) is a subunit of the subcortical maternal complex required for spindle assembly in human oocytes; RNAi-mediated knockdown of ECAT1 impairs spindle assembly, reduces oocyte maturation and fertilization rates, and decreases cleavage rate of resulting zygotes. ECAT1 is localized to the cytoplasm with enrichment in the cortical region of oocytes and preimplantation embryos.","method":"RNA interference (RNAi) in human oocytes; immunofluorescence for spindle assembly and protein localization; RT-PCR and quantitative expression analysis across developmental stages","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function with direct cellular phenotype (spindle assembly defect, maturation rate, fertilization rate, cleavage rate) combined with localization in human oocytes; multiple functional readouts in the relevant cell type","pmids":["27917907"],"is_preprint":false},{"year":2019,"finding":"KHDC3L is essential for integrity of the subcortical maternal complex (SCMC) in human oocytes; loss of KHDC3L function causes a genome-wide failure of de novo DNA methylation in oocytes, establishing SCMC integrity as a prerequisite for normal oocyte epigenome establishment.","method":"Single-cell bisulphite sequencing of patient oocytes; genome-wide methylation profiling","journal":"Genome medicine","confidence":"High","confidence_rationale":"Tier 1 / Strong — genome-wide single-cell functional assay in patient-derived material with inactivating mutation; mechanistic conclusion directly supported by data","pmids":["31847873"],"is_preprint":false},{"year":2014,"finding":"Bovine Ecat1 (ortholog of KHDC3L) protein localizes to the cytoplasm of oocytes and its transcript peaks at the 8- to 16-cell embryo stage, consistent with a maternal-to-embryonic transition role.","method":"Immunofluorescence, RT-PCR, quantitative real-time PCR across bovine oocyte and embryo developmental stages; 3' RACE-PCR","journal":"Reproduction in domestic animals","confidence":"Low","confidence_rationale":"Tier 3 / Weak — expression and localization data in a non-human model (bovine), single lab, no functional perturbation","pmids":["25366560"],"is_preprint":false}],"current_model":"KHDC3L (also known as ECAT1/C6orf221) is a eutherian oocyte-specific protein with an atypical KH RNA-binding domain that functions as a component of the subcortical maternal complex (SCMC): it co-localizes with NLRP7 at the oocyte cytoskeleton and cortical region, is essential for de novo genome-wide DNA methylation in the oocyte (required for genomic imprinting), supports spindle assembly and meiotic progression, and in epiblast cells binds and activates PARP1 while facilitating ATM-dependent (Thr145/Thr156 phosphorylation) homologous recombination repair of DNA damage; loss-of-function mutations cause familial biparental hydatidiform mole and recurrent pregnancy loss."},"narrative":{"mechanistic_narrative":"KHDC3L (ECAT1/C6orf221) is an oocyte- and embryo-expressed protein that functions as a subunit of the subcortical maternal complex (SCMC), where it governs establishment of the maternal epigenome and meiotic competence [PMID:31847873, PMID:27917907]. In growing human oocytes it co-localizes with NLRP7 at the cytoskeleton and cortical region, becomes asymmetrically confined to the outer cortex after the first cell division, and redistributes to the nucleus by the blastocyst stage [PMID:25358348]. SCMC integrity dependent on KHDC3L is a prerequisite for genome-wide de novo DNA methylation in the female germline: oocytes from a patient with an inactivating KHDC3L mutation show a non-selective, genome-wide methylation deficit, and after fertilization the defect persists specifically at imprinted germline differentially methylated regions while most non-imprinted regions recover [PMID:31847873]. KHDC3L is also required for spindle assembly, oocyte maturation, fertilization, and early cleavage, consistent with a cytoplasmic cortical role in meiotic progression [PMID:27917907]. Beyond the oocyte, KHDC3L is expressed in epiblast cells where it binds and stimulates PARP1, localizes to sites of DNA damage, and facilitates homologous-recombination repair; ATM-dependent phosphorylation at Thr145 and Thr156 is essential for these activities, and recurrent-pregnancy-loss deletions that remove Thr156 abolish PARP1 activation and HR repair [PMID:31609975]. Biallelic loss-of-function mutations in KHDC3L cause familial biparental hydatidiform mole [PMID:21885028].","teleology":[{"year":2007,"claim":"Established KHDC3L (ECAT1) as a member of a eutherian-specific gene family with an atypical KH RNA-binding domain restricted in expression to oocytes and embryonic stem cells, framing it as a candidate germline-specific regulator.","evidence":"Comparative genomics, phylogenetic and domain analysis, and cross-species RT-PCR expression profiling","pmids":["17913455"],"confidence":"Medium","gaps":["No functional assay of the KH domain or demonstration of RNA binding","Molecular role in oocytes not addressed"]},{"year":2011,"claim":"Identified KHDC3L as a disease gene by showing biallelic loss-of-function mutations cause familial biparental hydatidiform mole, linking it to maternal imprinting establishment and predicting a functional partnership with NLRP7.","evidence":"Sanger sequencing of affected families with mutation segregation against the FBHM imprinting phenotype","pmids":["21885028"],"confidence":"Medium","gaps":["NLRP7 interaction proposed but not directly demonstrated","Molecular mechanism connecting mutation to imprinting failure unknown"]},{"year":2012,"claim":"Provided first direct evidence that KHDC3L co-localizes with NLRP7 (juxta-perinuclear signal) and showed truncating mutations do not relocalize the protein in hematopoietic cells.","evidence":"Immunofluorescence in patient and control lymphoblastoid cell lines","pmids":["23232697"],"confidence":"Medium","gaps":["Co-localization in a non-germline surrogate cell type","No co-immunoprecipitation confirming physical interaction"]},{"year":2014,"claim":"Resolved the dynamic localization of KHDC3L in the relevant cell type, showing cortical/cytoskeletal co-localization with NLRP7 in oocytes, asymmetric cortical confinement after first division, and nuclear redistribution at blastocyst stage.","evidence":"High-resolution confocal and electron microscopy on 164 human oocytes and preimplantation embryos","pmids":["25358348"],"confidence":"High","gaps":["Functional consequence of stage-specific redistribution not tested","Mechanism driving nuclear relocalization unknown"]},{"year":2016,"claim":"Demonstrated a causal cellular function: KHDC3L as an SCMC subunit is required for spindle assembly, oocyte maturation, fertilization, and early cleavage.","evidence":"RNAi knockdown in human oocytes with immunofluorescence spindle/localization readouts and developmental rate analysis","pmids":["27917907"],"confidence":"High","gaps":["Molecular link between KHDC3L and spindle machinery not defined","How cortical SCMC controls a spindle process is unresolved"]},{"year":2019,"claim":"Established SCMC integrity dependent on KHDC3L as a prerequisite for genome-wide de novo oocyte DNA methylation, showing a non-selective methylation deficit that resolves to an imprinted-locus-specific defect after fertilization.","evidence":"Single-cell bisulphite sequencing of patient oocytes with matched embryo and molar tissue methylation profiling","pmids":["31847873"],"confidence":"High","gaps":["Mechanism by which a cortical complex controls nuclear DNA methylation machinery is unknown","Why imprinted loci selectively fail to recover post-fertilization unresolved"]},{"year":2019,"claim":"Defined a distinct DNA-repair function in epiblast cells: KHDC3L binds and stimulates PARP1, localizes to damage sites, and promotes homologous recombination, with ATM phosphorylation at Thr145/Thr156 essential and RPL deletions removing Thr156 abolishing these activities.","evidence":"Co-IP, in vitro PARP1 activity assays, DNA-damage immunofluorescence, HR repair assays, site-directed mutagenesis, and patient deletion-mutant analysis","pmids":["31609975"],"confidence":"High","gaps":["Whether this DNA-repair role operates in oocytes or only epiblast unclear","Relationship between SCMC function and PARP1/HR activity not integrated"]},{"year":null,"claim":"How a cortically localized SCMC subunit mechanistically directs nuclear processes — de novo DNA methylation and HR repair — and whether its KH domain binds RNA in vivo remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No demonstrated RNA target despite the predicted KH domain","No structural model of KHDC3L within the SCMC","Mechanistic connection between cortical SCMC and nuclear methylation/repair machinery undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,6]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[3]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3,5]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[5]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[4,7]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[6]}],"complexes":["subcortical maternal complex (SCMC)"],"partners":["NLRP7","PARP1","ATM"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q587J8","full_name":"KH domain-containing protein 3","aliases":["ES cell-associated transcript 1 protein","KHDC3-like protein"],"length_aa":217,"mass_kda":24.3,"function":"Component of the subcortical maternal complex (SCMC), a multiprotein complex that plays a key role in early embryonic development (By similarity). The SCMC complex is a structural constituent of cytoplasmic lattices, which consist in fibrous structures found in the cytoplasm of oocytes and preimplantation embryos (By similarity). They are required to store maternal proteins critical for embryonic development, such as proteins that control epigenetic reprogramming of the preimplantation embryo, and prevent their degradation or activation (By similarity). KHDC3 ensures proper spindle assembly by regulating the localization of AURKA via RHOA signaling and of PLK1 via a RHOA-independent process (By similarity). Required for the localization of MAD2L1 to kinetochores to enable spindle assembly checkpoint function (By similarity). As part of the OOEP-KHDC3 scaffold, recruits BLM and TRIM25 to DNA replication forks, thereby promoting the ubiquitination of BLM by TRIM25, enhancing BLM retainment at replication forks and therefore promoting stalled replication fork restart (By similarity). Regulates homologous recombination-mediated DNA repair via recruitment of RAD51 to sites of DNA double-strand breaks, and sustainment of PARP1 activity, which in turn modulates downstream ATM or ATR activation (PubMed:31609975). Activation of ATM or ATR in response to DNA double-strand breaks may be cell-type specific (By similarity). Its role in DNA double-strand break repair is independent of its role in restarting stalled replication forks (By similarity). Promotes neural stem cell neurogenesis and neuronal differentiation in the hippocampus (By similarity). May regulate normal development of learning, memory and anxiety (By similarity). Capable of binding RNA (By similarity)","subcellular_location":"Cytoplasm; Cytoplasm, cell cortex; Nucleus; Mitochondrion; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Chromosome","url":"https://www.uniprot.org/uniprotkb/Q587J8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/KHDC3L","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/KHDC3L","total_profiled":1310},"omim":[{"mim_id":"618432","title":"HYDATIDIFORM MOLE, RECURRENT, 4; HYDM4","url":"https://www.omim.org/entry/618432"},{"mim_id":"618431","title":"HYDATIDIFORM MOLE, RECURRENT, 3; HYDM3","url":"https://www.omim.org/entry/618431"},{"mim_id":"618421","title":"REC114 MEIOTIC RECOMBINATION PROTEIN; REC114","url":"https://www.omim.org/entry/618421"},{"mim_id":"616109","title":"TOP6B-LIKE INITIATOR OF MEIOTIC DOUBLE STRAND BREAKS; TOP6BL","url":"https://www.omim.org/entry/616109"},{"mim_id":"614293","title":"HYDATIDIFORM MOLE, RECURRENT, 2; HYDM2","url":"https://www.omim.org/entry/614293"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"brain","ntpm":1.3},{"tissue":"testis","ntpm":1.0}],"url":"https://www.proteinatlas.org/search/KHDC3L"},"hgnc":{"alias_symbol":["ECAT1"],"prev_symbol":["C6orf221"]},"alphafold":{"accession":"Q587J8","domains":[{"cath_id":"3.30.1370.10","chopping":"14-117","consensus_level":"high","plddt":87.5088,"start":14,"end":117}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q587J8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q587J8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q587J8-F1-predicted_aligned_error_v6.png","plddt_mean":66.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=KHDC3L","jax_strain_url":"https://www.jax.org/strain/search?query=KHDC3L"},"sequence":{"accession":"Q587J8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q587J8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q587J8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q587J8"}},"corpus_meta":[{"pmid":"21885028","id":"PMC_21885028","title":"Mutations causing familial biparental hydatidiform mole implicate c6orf221 as a possible regulator of genomic imprinting in the human oocyte.","date":"2011","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/21885028","citation_count":185,"is_preprint":false},{"pmid":"17913455","id":"PMC_17913455","title":"Atypical structure and phylogenomic evolution of the new eutherian oocyte- and embryo-expressed KHDC1/DPPA5/ECAT1/OOEP gene family.","date":"2007","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/17913455","citation_count":66,"is_preprint":false},{"pmid":"23232697","id":"PMC_23232697","title":"Report of four new patients with protein-truncating mutations in C6orf221/KHDC3L and colocalization with NLRP7.","date":"2012","source":"European journal of human genetics : EJHG","url":"https://pubmed.ncbi.nlm.nih.gov/23232697","citation_count":64,"is_preprint":false},{"pmid":"31847873","id":"PMC_31847873","title":"A KHDC3L mutation resulting in recurrent hydatidiform mole causes genome-wide DNA methylation loss in oocytes and persistent imprinting defects post-fertilisation.","date":"2019","source":"Genome medicine","url":"https://pubmed.ncbi.nlm.nih.gov/31847873","citation_count":64,"is_preprint":false},{"pmid":"25358348","id":"PMC_25358348","title":"NLRP7 and KHDC3L, the two maternal-effect proteins responsible for recurrent hydatidiform moles, co-localize to the oocyte cytoskeleton.","date":"2014","source":"Human reproduction (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/25358348","citation_count":55,"is_preprint":false},{"pmid":"31609975","id":"PMC_31609975","title":"KHDC3L mutation causes recurrent pregnancy loss by inducing genomic instability of human early embryonic cells.","date":"2019","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/31609975","citation_count":49,"is_preprint":false},{"pmid":"23125094","id":"PMC_23125094","title":"Mutations in NLRP7 and KHDC3L confer a complete hydatidiform mole phenotype on digynic triploid conceptions.","date":"2012","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/23125094","citation_count":46,"is_preprint":false},{"pmid":"27621838","id":"PMC_27621838","title":"Two novel mutations in the KHDC3L gene in Asian patients with recurrent hydatidiform mole.","date":"2016","source":"Human genome variation","url":"https://pubmed.ncbi.nlm.nih.gov/27621838","citation_count":28,"is_preprint":false},{"pmid":"23963444","id":"PMC_23963444","title":"NLRP7 or KHDC3L genes and the etiology of molar pregnancies and recurrent miscarriage.","date":"2013","source":"Molecular human reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/23963444","citation_count":21,"is_preprint":false},{"pmid":"35643636","id":"PMC_35643636","title":"Novel genetic variants of KHDC3L and other members of the subcortical maternal complex associated with Beckwith-Wiedemann syndrome or Pseudohypoparathyroidism 1B and multi-locus imprinting disturbances.","date":"2022","source":"Clinical epigenetics","url":"https://pubmed.ncbi.nlm.nih.gov/35643636","citation_count":15,"is_preprint":false},{"pmid":"32210488","id":"PMC_32210488","title":"Founder Effect of KHDC3L, p.M1V Mutation, on Iranian Patients with Recurrent Hydatidiform Moles.","date":"2020","source":"Iranian journal of medical sciences","url":"https://pubmed.ncbi.nlm.nih.gov/32210488","citation_count":12,"is_preprint":false},{"pmid":"25376457","id":"PMC_25376457","title":"No evidence for mutations in NLRP7, NLRP2 or KHDC3L in women with unexplained recurrent pregnancy loss or infertility.","date":"2014","source":"Human reproduction (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/25376457","citation_count":12,"is_preprint":false},{"pmid":"22909446","id":"PMC_22909446","title":"Mosaic moles and non-familial biparental moles are not caused by mutations in NLRP7, NLRP2 or C6orf221.","date":"2012","source":"Molecular human reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/22909446","citation_count":12,"is_preprint":false},{"pmid":"24105752","id":"PMC_24105752","title":"No evidence for mutations in NLRP7 and KHDC3L in women with androgenetic hydatidiform moles.","date":"2013","source":"Prenatal diagnosis","url":"https://pubmed.ncbi.nlm.nih.gov/24105752","citation_count":12,"is_preprint":false},{"pmid":"27917907","id":"PMC_27917907","title":"ECAT1 is essential for human oocyte maturation and pre-implantation development of the resulting embryos.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27917907","citation_count":11,"is_preprint":false},{"pmid":"33639414","id":"PMC_33639414","title":"KH domain containing 3 like (KHDC3L) frame-shift mutation causes both recurrent pregnancy loss and hydatidiform mole.","date":"2021","source":"European journal of obstetrics, gynecology, and reproductive biology","url":"https://pubmed.ncbi.nlm.nih.gov/33639414","citation_count":7,"is_preprint":false},{"pmid":"31220306","id":"PMC_31220306","title":"NLRP7 and KHDC3L variants in Chinese patients with recurrent hydatidiform moles.","date":"2019","source":"Japanese journal of clinical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/31220306","citation_count":6,"is_preprint":false},{"pmid":"25366560","id":"PMC_25366560","title":"Expression of bovine Ecat1 gene in immature and in vitro matured oocytes as well as during early embryonic development.","date":"2014","source":"Reproduction in domestic animals = Zuchthygiene","url":"https://pubmed.ncbi.nlm.nih.gov/25366560","citation_count":5,"is_preprint":false},{"pmid":"24215781","id":"PMC_24215781","title":"Absence of KHDC3L mutations in Chinese patients with recurrent and sporadic hydatidiform moles.","date":"2013","source":"Cancer genetics","url":"https://pubmed.ncbi.nlm.nih.gov/24215781","citation_count":5,"is_preprint":false},{"pmid":"39763182","id":"PMC_39763182","title":"A Maternal Loss-of-Function Variant in KHDC3L Gene Causes a Range of Adverse Pregnancy Outcomes: A Case Report.","date":"2025","source":"Molecular genetics & genomic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39763182","citation_count":5,"is_preprint":false},{"pmid":"37918946","id":"PMC_37918946","title":"Familial recurrent molar pregnancy: positive for KHDC3L gene mutation.","date":"2023","source":"BMJ case reports","url":"https://pubmed.ncbi.nlm.nih.gov/37918946","citation_count":4,"is_preprint":false},{"pmid":"35361411","id":"PMC_35361411","title":"A familial case of recurrent hydatidiform mole with p.Asp108Ilefs∗30 causing mutation in KHDC3L: A genetic and clinical report.","date":"2022","source":"Taiwanese journal of obstetrics & gynecology","url":"https://pubmed.ncbi.nlm.nih.gov/35361411","citation_count":0,"is_preprint":false},{"pmid":"40908712","id":"PMC_40908712","title":"The retrospective data analysis of NLRP7 and KHDC3L mutations in Turkish patients with recurrent hydatidiform mole.","date":"2025","source":"Turkish journal of obstetrics and gynecology","url":"https://pubmed.ncbi.nlm.nih.gov/40908712","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.02.20.639225","title":"An  <i>Nlrp5</i>  -null mutation leads to attenuated  <i>de novo</i>  methylation in oocytes, accompanied by a significant reduction in DNMT3L","date":"2025-02-23","source":"bioRxiv","url":"https://doi.org/10.1101/2025.02.20.639225","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14095,"output_tokens":2545,"usd":0.04023,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9683,"output_tokens":2803,"usd":0.059245,"stage2_stop_reason":"end_turn"},"total_usd":0.099475,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"Biallelic loss-of-function mutations in C6orf221 (KHDC3L) cause familial biparental hydatidiform mole (FBHM), implicating KHDC3L as a regulator of genomic imprinting in the human oocyte; the gene family's biological properties suggest KHDC3L and NLRP7 may interact as components of an oocyte complex required for determination of epigenetic status on the oocyte genome.\",\n      \"method\": \"Sanger sequencing of affected families; identification of biallelic mutations segregating with FBHM phenotype (genome-wide failure to specify/maintain maternal epigenotype at imprinted loci)\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via biallelic mutations with defined phenotype, but molecular interaction with NLRP7 proposed rather than directly demonstrated\",\n      \"pmids\": [\"21885028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ECAT1 (KHDC3L) belongs to a eutherian-specific gene family characterized by an atypical KH RNA-binding domain; the protein is specifically expressed in oocytes and/or embryonic stem cells, absent in fish, bird, and marsupial genomes, and encoded in a syntenic genomic region that has undergone rapid evolution in eutherians.\",\n      \"method\": \"Comparative genomics, phylogenetic analysis, domain structure analysis, RT-PCR expression profiling across species\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — multiple orthogonal approaches (comparative genomics, domain analysis, expression) but no direct functional assay of the KH domain\",\n      \"pmids\": [\"17913455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"KHDC3L protein displays a juxta-perinuclear signal and co-localizes with NLRP7 in lymphoblastoid cell lines from normal subjects; protein-truncating mutations in KHDC3L do not alter this subcellular localization in hematopoietic cells.\",\n      \"method\": \"Immunofluorescence microscopy in lymphoblastoid cell lines from normal subjects and KHDC3L-mutant patients\",\n      \"journal\": \"European journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct immunofluorescence localization with functional context (mutant vs. wild-type), single lab, single method\",\n      \"pmids\": [\"23232697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NLRP7 and KHDC3L co-localize to the oocyte cytoskeleton and cortical region in growing human oocytes; after first cell division, both proteins become asymmetrically confined to the outer cortical region and excluded from the cell-to-cell contact region until the blastocyst stage, at which point KHDC3L redistributes to the nucleus.\",\n      \"method\": \"High-resolution confocal immunofluorescence and electron microscopy on 164 human oocytes and preimplantation embryos\",\n      \"journal\": \"Human reproduction (Oxford, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization with functional context, high-resolution confocal + electron microscopy, large sample size (164 oocytes/embryos), single lab with multiple orthogonal imaging methods\",\n      \"pmids\": [\"25358348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"KHDC3L is a component of the subcortical maternal complex (SCMC) whose integrity is essential for de novo DNA methylation genome-wide in the female germline; oocytes from a patient homozygous for an inactivating KHDC3L mutation show genome-wide deficit of DNA methylation including at germline differentially methylated regions (gDMRs) of imprinted genes, with no selectivity toward imprinted genes. Post-fertilization, methylation defects at imprinted genes persist while most non-imprinted regions recover near-normal methylation.\",\n      \"method\": \"Single-cell bisulphite sequencing (scBS-seq) of oocytes from a KHDC3L c.1A>G homozygous patient; genome-wide methylation analysis of preimplantation embryo and molar tissue from the same patient\",\n      \"journal\": \"Genome medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-coverage genome-wide single-cell methylation sequencing in patient-derived oocytes with matched embryo and molar tissue; multiple genomic features assessed; inactivating mutation with clear functional readout\",\n      \"pmids\": [\"31847873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"KHDC3L is expressed in human epiblast cells and ensures genome stability and viability; KHDC3L binds to PARP1 and stimulates its enzymatic activity. In response to DNA damage, KHDC3L localizes to DNA damage sites and facilitates homologous recombination (HR)-mediated DNA repair. ATM kinase phosphorylates KHDC3L at Thr145 and Thr156, which is essential for KHDC3L's functions. RPL-associated deletions p.E150_V160del and p.E150_V172del both lack Thr156 and are impaired in PARP1 activation and HR repair.\",\n      \"method\": \"Co-immunoprecipitation (KHDC3L–PARP1 binding), in vitro PARP1 activity assays, DNA damage localization by immunofluorescence, HR repair assays, site-directed mutagenesis of Thr145/Thr156, analysis of patient-derived deletion mutants\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, enzymatic assay, mutagenesis, DNA repair assay, patient mutation analysis), direct mechanistic readouts; single lab but comprehensive experimental coverage\",\n      \"pmids\": [\"31609975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ECAT1 (KHDC3L) is a subunit of the subcortical maternal complex required for spindle assembly in human oocytes; RNAi-mediated knockdown of ECAT1 impairs spindle assembly, reduces oocyte maturation and fertilization rates, and decreases cleavage rate of resulting zygotes. ECAT1 is localized to the cytoplasm with enrichment in the cortical region of oocytes and preimplantation embryos.\",\n      \"method\": \"RNA interference (RNAi) in human oocytes; immunofluorescence for spindle assembly and protein localization; RT-PCR and quantitative expression analysis across developmental stages\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function with direct cellular phenotype (spindle assembly defect, maturation rate, fertilization rate, cleavage rate) combined with localization in human oocytes; multiple functional readouts in the relevant cell type\",\n      \"pmids\": [\"27917907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"KHDC3L is essential for integrity of the subcortical maternal complex (SCMC) in human oocytes; loss of KHDC3L function causes a genome-wide failure of de novo DNA methylation in oocytes, establishing SCMC integrity as a prerequisite for normal oocyte epigenome establishment.\",\n      \"method\": \"Single-cell bisulphite sequencing of patient oocytes; genome-wide methylation profiling\",\n      \"journal\": \"Genome medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — genome-wide single-cell functional assay in patient-derived material with inactivating mutation; mechanistic conclusion directly supported by data\",\n      \"pmids\": [\"31847873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Bovine Ecat1 (ortholog of KHDC3L) protein localizes to the cytoplasm of oocytes and its transcript peaks at the 8- to 16-cell embryo stage, consistent with a maternal-to-embryonic transition role.\",\n      \"method\": \"Immunofluorescence, RT-PCR, quantitative real-time PCR across bovine oocyte and embryo developmental stages; 3' RACE-PCR\",\n      \"journal\": \"Reproduction in domestic animals\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — expression and localization data in a non-human model (bovine), single lab, no functional perturbation\",\n      \"pmids\": [\"25366560\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"KHDC3L (also known as ECAT1/C6orf221) is a eutherian oocyte-specific protein with an atypical KH RNA-binding domain that functions as a component of the subcortical maternal complex (SCMC): it co-localizes with NLRP7 at the oocyte cytoskeleton and cortical region, is essential for de novo genome-wide DNA methylation in the oocyte (required for genomic imprinting), supports spindle assembly and meiotic progression, and in epiblast cells binds and activates PARP1 while facilitating ATM-dependent (Thr145/Thr156 phosphorylation) homologous recombination repair of DNA damage; loss-of-function mutations cause familial biparental hydatidiform mole and recurrent pregnancy loss.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"KHDC3L (ECAT1/C6orf221) is an oocyte- and embryo-expressed protein that functions as a subunit of the subcortical maternal complex (SCMC), where it governs establishment of the maternal epigenome and meiotic competence [#4, #6]. In growing human oocytes it co-localizes with NLRP7 at the cytoskeleton and cortical region, becomes asymmetrically confined to the outer cortex after the first cell division, and redistributes to the nucleus by the blastocyst stage [#3]. SCMC integrity dependent on KHDC3L is a prerequisite for genome-wide de novo DNA methylation in the female germline: oocytes from a patient with an inactivating KHDC3L mutation show a non-selective, genome-wide methylation deficit, and after fertilization the defect persists specifically at imprinted germline differentially methylated regions while most non-imprinted regions recover [#4, #7]. KHDC3L is also required for spindle assembly, oocyte maturation, fertilization, and early cleavage, consistent with a cytoplasmic cortical role in meiotic progression [#6]. Beyond the oocyte, KHDC3L is expressed in epiblast cells where it binds and stimulates PARP1, localizes to sites of DNA damage, and facilitates homologous-recombination repair; ATM-dependent phosphorylation at Thr145 and Thr156 is essential for these activities, and recurrent-pregnancy-loss deletions that remove Thr156 abolish PARP1 activation and HR repair [#5]. Biallelic loss-of-function mutations in KHDC3L cause familial biparental hydatidiform mole [#0].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established KHDC3L (ECAT1) as a member of a eutherian-specific gene family with an atypical KH RNA-binding domain restricted in expression to oocytes and embryonic stem cells, framing it as a candidate germline-specific regulator.\",\n      \"evidence\": \"Comparative genomics, phylogenetic and domain analysis, and cross-species RT-PCR expression profiling\",\n      \"pmids\": [\"17913455\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional assay of the KH domain or demonstration of RNA binding\", \"Molecular role in oocytes not addressed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified KHDC3L as a disease gene by showing biallelic loss-of-function mutations cause familial biparental hydatidiform mole, linking it to maternal imprinting establishment and predicting a functional partnership with NLRP7.\",\n      \"evidence\": \"Sanger sequencing of affected families with mutation segregation against the FBHM imprinting phenotype\",\n      \"pmids\": [\"21885028\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"NLRP7 interaction proposed but not directly demonstrated\", \"Molecular mechanism connecting mutation to imprinting failure unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Provided first direct evidence that KHDC3L co-localizes with NLRP7 (juxta-perinuclear signal) and showed truncating mutations do not relocalize the protein in hematopoietic cells.\",\n      \"evidence\": \"Immunofluorescence in patient and control lymphoblastoid cell lines\",\n      \"pmids\": [\"23232697\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Co-localization in a non-germline surrogate cell type\", \"No co-immunoprecipitation confirming physical interaction\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Resolved the dynamic localization of KHDC3L in the relevant cell type, showing cortical/cytoskeletal co-localization with NLRP7 in oocytes, asymmetric cortical confinement after first division, and nuclear redistribution at blastocyst stage.\",\n      \"evidence\": \"High-resolution confocal and electron microscopy on 164 human oocytes and preimplantation embryos\",\n      \"pmids\": [\"25358348\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of stage-specific redistribution not tested\", \"Mechanism driving nuclear relocalization unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated a causal cellular function: KHDC3L as an SCMC subunit is required for spindle assembly, oocyte maturation, fertilization, and early cleavage.\",\n      \"evidence\": \"RNAi knockdown in human oocytes with immunofluorescence spindle/localization readouts and developmental rate analysis\",\n      \"pmids\": [\"27917907\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link between KHDC3L and spindle machinery not defined\", \"How cortical SCMC controls a spindle process is unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established SCMC integrity dependent on KHDC3L as a prerequisite for genome-wide de novo oocyte DNA methylation, showing a non-selective methylation deficit that resolves to an imprinted-locus-specific defect after fertilization.\",\n      \"evidence\": \"Single-cell bisulphite sequencing of patient oocytes with matched embryo and molar tissue methylation profiling\",\n      \"pmids\": [\"31847873\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which a cortical complex controls nuclear DNA methylation machinery is unknown\", \"Why imprinted loci selectively fail to recover post-fertilization unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined a distinct DNA-repair function in epiblast cells: KHDC3L binds and stimulates PARP1, localizes to damage sites, and promotes homologous recombination, with ATM phosphorylation at Thr145/Thr156 essential and RPL deletions removing Thr156 abolishing these activities.\",\n      \"evidence\": \"Co-IP, in vitro PARP1 activity assays, DNA-damage immunofluorescence, HR repair assays, site-directed mutagenesis, and patient deletion-mutant analysis\",\n      \"pmids\": [\"31609975\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this DNA-repair role operates in oocytes or only epiblast unclear\", \"Relationship between SCMC function and PARP1/HR activity not integrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a cortically localized SCMC subunit mechanistically directs nuclear processes — de novo DNA methylation and HR repair — and whether its KH domain binds RNA in vivo remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No demonstrated RNA target despite the predicted KH domain\", \"No structural model of KHDC3L within the SCMC\", \"Mechanistic connection between cortical SCMC and nuclear methylation/repair machinery undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 6]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [4, 7]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [\n      \"subcortical maternal complex (SCMC)\"\n    ],\n    \"partners\": [\n      \"NLRP7\",\n      \"PARP1\",\n      \"ATM\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}