Functional analysis of TMEM53, the disease gene for a novel type of sclerosing bone disorder

Purpose: The goal of this study is to explore the molecular mechanism of the phenotypes found in the TMEM53 mutant human and mouse. Methods: Calvarial mRNA profiles of 3-day-old Tmem53+/- and Tmem53-/- mice were generated by sequencing, in triplicate, using NovaSeq 6000 sequencer. The reads from the RNA-seq were aligned to the mouse genome (NCBI build37.2) using Tophat. Aligned reads were quantified using Cufflinks. Differential gene expression analysis was performed with Cuffdiff. Gene ontology (GO) term enrichment analysis was performed by DAVID 6.8. Results:The gene expression profiling displayed a total of 14,836 genes, including 1,278 differentially expressed genes (DEGs). These DEGs were enriched in some GO terms, and the most enriched term is "ossification," which is considered to be closely associated with the thickened calvaria in the Tmem53-/- mice. The known marker genes of osteogenic precursors, osteoblasts and osteocytes (Runx2, Sp7, Alpl, Bglap and Ibsp) were up-regulated in Tmem53-/- mice according to the RNA-seq dataset. The DEGs were also enriched in the reported target gene set of Bmp-Smad pathway. The representative downstream transcriptional targets of Bmp signaling, Id1, Dlx3 and Dlx5, were up-regulated in the calvaria of the Tmem53-/- mice. Conclusions: Our results shows enhanced osteoblastogenesis in the calvaria of the Tmem53-/- mice, which would be attributed to the over-activated Bmp-Smad pathway caused by the Tmem53 deficiency. Calvarial mRNA profiles of 3-day-old Tmem53+/- (n=3) and Tmem53-/- mice (n=3)一、研究目的：本研究旨在探究TMEM53突变人类与小鼠所呈现表型的分子机制。 二、研究方法：采用NovaSeq 6000测序仪对3日龄Tmem53杂合子（Tmem53+/-）及纯合敲除（Tmem53-/-）小鼠的颅盖骨mRNA进行转录组测序，每组设置3次生物学重复。使用Tophat将RNA-seq原始读段比对至小鼠基因组（NCBI build37.2），通过Cufflinks对比对上的读段进行定量分析，采用Cuffdiff完成差异基因表达分析，借助DAVID 6.8完成基因本体（Gene Ontology, GO）功能富集分析。 三、研究结果：本次基因表达谱共检测到14836个基因，其中差异表达基因（differentially expressed genes, DEGs）共计1278个。上述DEGs显著富集于多项GO功能条目，富集程度最高的条目为“骨化（ossification）”，该条目被认为与Tmem53-/-小鼠增厚的颅盖骨密切相关。RNA-seq数据集显示，成骨前体细胞、成骨细胞及骨细胞的已知标记基因Runx2、Sp7、Alpl、Bglap及Ibsp在Tmem53-/-小鼠中均呈上调表达。DEGs同时显著富集于已报道的骨形态发生蛋白-Smad（Bmp-Smad pathway）信号通路靶基因集。Bmp信号通路的典型下游转录靶标Id1、Dlx3及Dlx5在Tmem53-/-小鼠的颅盖骨中均呈上调表达。 四、研究结论：本研究结果表明，Tmem53-/-小鼠颅盖骨中成骨过程显著增强，该现象可归因于Tmem53缺失导致的Bmp-Smad信号通路过度激活。本研究使用的测序数据为3日龄Tmem53+/-小鼠（n=3）及Tmem53-/-小鼠（n=3）的颅盖骨mRNA表达谱。