PRDM1 DNA-binding zinc finger domain is required for normal limb development and is disrupted in Split Hand/Foot Malformation

Split Hand/Foot Malformation (SHFM) is a rare limb abnormality with clefting of the fingers and/or toes. For many patients, the genetic etiology is unknown. Through whole exome and targeted sequencing, we detected three novel variants in a transcription factor, PRDM1 that arose de novo in families with SHFM or segregated with the phenotype. PRDM1 has been shown to be required for limb development; however, its role is not well understood, and it is unclear how the PRDM1 variants affect protein function. Using transient and stable overexpression rescue experiments in zebrafish, we show that the variants have reduced function compared to wildtype PRDM1 likely due to disruption of the proline/serine-rich and DNA-binding zinc finger domains. Through expression assays, RNA-seq, and CUT&RUN in isolated pectoral fin cells, we demonstrate that Prdm1a directly binds to and regulates genes required for limb induction, outgrowth, and anterior/posterior patterning, such as fgfr1a, dlx5a, dlx6a, and smo. Together, these results improve our understanding of the role of PRDM1 in the limb gene regulatory network and help predict the pathogenicity of PRDM1 variants in humans Overall design: RNA-seq: Comparative gene expression analysis from isolated pectoral fins of Tg(Mmu:Prx1-EGFP) wildtype and Tg(Mmu:Prx1-EGFP);prdm1am805/m805 mutant zebrafish embryos at 48 hours post fertilization (hpf). CUT&RUN: Cleavage Under Targets and Release Using Nuclease (CUT&RUN) for IgG, H3K27Ac, and Prdm1a in isolated pectoral fin cells of wildtype and prdm1am805/m805 zebrafish embryos at 24 hpf contributor: University of Washington Center for Mendelian Genomics