背景:手/足分裂畸形(SHFM)是一种先天性肢体疾病,表现为肢体异常,如失踪,发育不良,或融合的数字,通常是颅面缺陷,包括唇腭裂,microdontia,小颌畸形,或上颌骨发育不全。我们先前在转录因子中鉴定了三个新的变体,与SHFM表型相关的PRDM1。一个人还表现出高弓腭。脊椎动物的研究表明,PRDM1对颅骨的发育很重要;然而,在我们研究之前,PRDM1的人类变异与颅面异常无关.
方法:使用prdm1a-/-突变斑马鱼的瞬时mRNA过表达测定,我们测试了PRDM1SHFM变异体是否具有功能性,是否可以导致prdm1α-/-突变体中观察到的颅面缺损的抢救.我们还挖掘了以前发表的CUT&RUN和RNA-seq数据集,这些数据集从标记胸鳍的Tg(Mmu:Prx1-EGFP)转基因品系中分选EGFP阳性细胞,咽弓,和头部的背侧部分来检查Prdm1a结合以及Prdm1a丢失对颅面基因的影响。
结果:prdm1a-/-突变体表现出颅面缺陷,包括发育不良的神经颅骨,后颈支气管弓的丧失,一个较短的掌方,和一个倒置的头皮。在prdm1a-/-突变体中注射野生型(WT)hPRDM1部分挽救了palatquirate表型。然而,三种SHFM变体中的每一种的注射都无法挽救这种骨骼缺陷。prdm1a的缺失导致RNA-seq引起的重要颅面基因的表达减少,包括Emilin3a,通过杂交链反应表达证实。其他基因包括dlx5a/dlx6a,hand2,sox9b,col2a1a,和hoxb基因也减少。通过实时定量PCR在斑马鱼胚胎的前半部分中进行的验证未能证实表达变化,表明差异在prx1表达细胞中富集。
结论:这些数据表明三种SHFM变体可能没有功能,并且可能与人类观察到的颅面缺陷有关。最后,他们展示了Prdm1a如何直接结合和调节与颅面发育有关的基因。
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.