Human Medical Genetics & Genomics Graduate Program, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado, USA.
Department of Craniofacial Development, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado, USA.
Birth Defects Res. 2024 Mar;116(3):e2327. doi: 10.1002/bdr2.2327.
Split hand/foot malformation (SHFM) is a congenital limb disorder presenting with limb anomalies, such as missing, hypoplastic, or fused digits, and often craniofacial defects, including a cleft lip/palate, microdontia, micrognathia, or maxillary hypoplasia. We previously identified three novel variants in the transcription factor, PRDM1, that are associated with SHFM phenotypes. One individual also presented with a high arch palate. Studies in vertebrates indicate that PRDM1 is important for development of the skull; however, prior to our study, human variants in PRDM1 had not been associated with craniofacial anomalies.
Using transient mRNA overexpression assays in prdm1a mutant zebrafish, we tested whether the PRDM1 SHFM variants were functional and could lead to a rescue of the craniofacial defects observed in prdm1a mutants. We also mined previously published CUT&RUN and RNA-seq datasets that sorted EGFP-positive cells from a Tg(Mmu:Prx1-EGFP) transgenic line that labels the pectoral fin, pharyngeal arches, and dorsal part of the head to examine Prdm1a binding and the effect of Prdm1a loss on craniofacial genes.
The prdm1a mutants exhibit craniofacial defects including a hypoplastic neurocranium, a loss of posterior ceratobranchial arches, a shorter palatoquadrate, and an inverted ceratohyal. Injection of wildtype (WT) hPRDM1 in prdm1a mutants partially rescues the palatoquadrate phenotype. However, injection of each of the three SHFM variants fails to rescue this skeletal defect. Loss of prdm1a leads to a decreased expression of important craniofacial genes by RNA-seq, including emilin3a, confirmed by hybridization chain reaction expression. Other genes including dlx5a/dlx6a, hand2, sox9b, col2a1a, and hoxb genes are also reduced. Validation by real-time quantitative PCR in the anterior half of zebrafish embryos failed to confirm the expression changes suggesting that the differences are enriched in prx1 expressing cells.
These data suggest that the three SHFM variants are likely not functional and may be associated with the craniofacial defects observed in the humans. Finally, they demonstrate how Prdm1a can directly bind and regulate genes involved in craniofacial development.
分裂手/足畸形（SHFM）是一种先天性肢体疾病，表现为肢体异常，如缺失、发育不良或融合的手指，以及经常出现颅面缺陷，包括唇裂/腭裂、小牙症、小颌畸形或上颌骨发育不全。我们之前在转录因子 PRDM1 中发现了三个与 SHFM 表型相关的新变体。一个个体还表现出高拱形腭。脊椎动物的研究表明，PRDM1 对颅骨发育很重要；然而，在我们的研究之前，人类 PRDM1 中的变体尚未与颅面异常相关。
使用瞬时 mRNA 过表达试验在 prdm1a 突变的斑马鱼中，我们测试了 PRDM1 SHFM 变体是否具有功能，并且是否可以挽救 prdm1a 突变体中观察到的颅面缺陷。我们还挖掘了以前发表的 CUT&RUN 和 RNA-seq 数据集，这些数据集从 Tg（Mmu：Prx1-EGFP）转基因系中分离出 EGFP 阳性细胞，该系标记胸鳍、咽弓和头部的背部分，以检查 Prdm1a 结合以及 Prdm1a 缺失对颅面基因的影响。
prdm1a 突变体表现出颅面缺陷，包括发育不全的神经颅、后鳃弓弓缺失、短腭方骨和倒置的半椎骨。在 prdm1a 突变体中注射野生型（WT）hPRDM1 部分挽救了腭方骨表型。然而，注射三种 SHFM 变体中的每一种都未能挽救这种骨骼缺陷。通过 RNA-seq 证实，prdm1a 的缺失导致重要颅面基因的表达减少，包括 emilin3a，通过杂交链反应表达验证。其他基因，包括 dlx5a/dlx6a、hand2、sox9b、col2a1a 和 hoxb 基因也减少。在斑马鱼胚胎的前半部分进行实时定量 PCR 验证未能证实表达变化，这表明差异在表达 prx1 的细胞中富集。
这些数据表明，这三种 SHFM 变体可能没有功能，可能与人类观察到的颅面缺陷有关。最后，它们展示了 Prdm1a 如何直接结合并调节参与颅面发育的基因。
