biostudies-literatureIshorst NElse Kröner-Fresenius-Stiftung (Else Kroner-Fresenius Foundation)Deutsche ForschungsgemeinschaftDeutsche Forschungsgemeinschaft (German Research Foundation)Else Kröner-Fresenius-StiftungDBT/Wellcome Trust India AllianceWellcome Trust595-606https://www.ebi.ac.uk/biostudies/studies/S-EPMC7617551biostudies-literatureUnknown33(5)Orofacial clefting (OFC) is a frequent congenital anomaly and can occur either in the context of underlying syndromes or in isolation (nonsyndromic). The two common OFC phenotypes are cleft lip with/without cleft palate (CL/P) and cleft palate only (CPO). In this study, we searched for penetrant CL/P genes, by evaluating de novo copy number variants (CNV) from an exome sequencing dataset of 50 nonsyndromic patient-parent trios. We detected a heterozygous 86 kb de novo deletion affecting exons 4-11 of ZFHX4, a gene previously associated with OFC. Genetic and phenotypic data from our in-house and the AGORA cohort (710 and 229 individuals with nonsyndromic CL/P) together with literature and database reviews demonstrate that ZFHX4 variants can lead to both nonsyndromic and syndromic forms not only of CL/P but also CPO. Expression analysis in published single-cell RNA-sequencing data (mouse embryo, zebrafish larva) at relevant time-points support an important role of Zfhx4/zfhx4 in craniofacial development. To characterize the role of zfhx4 in zebrafish craniofacial development, we knocked out/down the zebrafish orthologue. Cartilage staining of the zfhx4 CRISPR F0 knockout and morpholino knockdown at 4 days post-fertilization showed an underdeveloped and abnormally shaped ethmoid plate and cartilaginous jaw (resembling micrognathia). While there is evidence for the dominant inheritance of ZFHX4 variants in OFC, we here present a patient with a possible recessive inheritance. In conclusion, ZFHX4 has a highly heterogeneous phenotypic spectrum and variable mode of inheritance. Our data highlight that ZFHX4 should be considered in genetic testing in patients with nonsyndromic clefting.European journal of human genetics : EJHGRole of ZFHX4 in orofacial clefting based on human genetic data and zebrafish models.PMC7617551Q-614.1254MA 2546/5-1LU 1944/2-1IA/CRC/20/1/600002Henne SLudwig KUPande SKibris DGirisha KMIshorst NZametica BDrichel DMingardo ELambertz JHolzel SOngkosuwito ESiewert AOdermatt BChannab KNowak SGeyer MMangold EDegenhardt FDixon MGreve CYilmaz OCarels CLindenberg TKalanithy JCKruse Tvan Rooij IALMHagelueken GfalseRole of ZFHX4 in orofacial clefting based on human genetic data and zebrafish models.Orofacial clefting (OFC) is a frequent congenital anomaly and can occur either in the context of underlying syndromes or in isolation (nonsyndromic). The two common OFC phenotypes are cleft lip with/without cleft palate (CL/P) and cleft palate only (CPO). In this study, we searched for penetrant CL/P genes, by evaluating de novo copy number variants (CNV) from an exome sequencing dataset of 50 nonsyndromic patient-parent trios. We detected a heterozygous 86 kb de novo deletion affecting exons 4-11 of ZFHX4, a gene previously associated with OFC. Genetic and phenotypic data from our in-house and the AGORA cohort (710 and 229 individuals with nonsyndromic CL/P) together with literature and database reviews demonstrate that ZFHX4 variants can lead to both nonsyndromic and syndromic forms not only of CL/P but also CPO. Expression analysis in published single-cell RNA-sequencing data (mouse embryo, zebrafish larva) at relevant time-points support an important role of Zfhx4/zfhx4 in craniofacial development. To characterize the role of zfhx4 in zebrafish craniofacial development, we knocked out/down the zebrafish orthologue. Cartilage staining of the zfhx4 CRISPR F0 knockout and morpholino knockdown at 4 days post-fertilization showed an underdeveloped and abnormally shaped ethmoid plate and cartilaginous jaw (resembling micrognathia). While there is evidence for the dominant inheritance of ZFHX4 variants in OFC, we here present a patient with a possible recessive inheritance. In conclusion, ZFHX4 has a highly heterogeneous phenotypic spectrum and variable mode of inheritance. Our data highlight that ZFHX4 should be considered in genetic testing in patients with nonsyndromic clefting.2025-01-01T00:00:00Z2025 May2026-04-08T19:51:13.125Z2025-07-06T03:05:45.622ZS-EPMC76175513970259010.1038/s41431-024-01775-9