An inducible mouse model of OI type V reveals aberrant osteogenesis caused by Ifitm5 c.-14C>T mutation

Osteogenesis imperfecta (OI) Type V is typically characterized by radial head dislocation, calcification of interosseous membrane and post-fracture hyperplastic callus. It is caused by the c.-14C>T mutation in the 5UTR of the IFITM5 gene, adding five amino acids (MALEP) to the N-terminal of IFITM5 protein. Previous studies have suggested a neomorphic function of the MALEP-IFITM5 protein. However, the underlying mechanisms remain unclear due to embryonic lethality in previous models. Therefore, we developed an inducible animal model of Ifitm5flox c.-14C>T that could be induced by Cre expressing at different developmental stages to explore the pathogenic effects of the neomorphic MALEP-IFITM5. Specifically, Prx1-Cre; Ifitm5flox c.-14C>T mutant mice were born with fractures in all limbs, showing deficient ossification and enhanced chondrogenesis associated with increased SOX9 abundance. We isolated skeletal cells from tibia of Ifitm5 mutant mouse at P6 stage and conducted single RNA sequencing (10X Genomics). The data were compared to the RNA-seq data generated from the tibia of control mouse at the same stage (GSE159544). The protocol to isolate the mutant skeletal cells for single cell RNA sequencing was the same as the control sample (GSE159544). Right and left tibia from Prx1-Cre; Ifitm5flox c.-14C>T mutant mouse at P6 stage were dissected with soft tissues removed. Bone tissue was cut into small pieces and blood cells were removed with TrypLE Express digession for 10 minutes at 37 degree. The remaining bone tissue was digested with type II collagenase (0.25%) and Dispase (0.25%) in HBSS for 1.5 hours. Cells were collected every half an hour, and digestion medium was changed each time. Red blood cells were lysised and dead cells were removed using kits. The remaining cells were tested for cell viability (>80%). Single cells were encapsulated for library preparation and sequencing using the Chromium single cell platform (10X Genomics Inc)according to the manufacturers protocol. Library size and concentration were determined by Qubit, quantitative PCR, and Bioanalyzer assays. A raw total input of 15000 cells was estimated for each sample. Library preparation and Illumina (NovaSeq 6000) sequencing (151 bp paired end) were performed at HKU CPOS, at 150 Gbp throughput per sample.成骨不全V型（Osteogenesis imperfecta, OI）的典型特征为桡骨头脱位、骨间膜钙化及骨折后增生性骨痂形成。该疾病由IFITM5基因5'非翻译区（5'UTR）的c.-14C>T突变所致，该突变会在IFITM5蛋白的N端添加5个氨基酸（MALEP）。既往研究提示，携带该突变的MALEP-IFITM5蛋白存在新功能型活性。然而，由于既往模型存在胚胎致死性，其潜在致病机制仍未明确。为此，本研究构建了可被不同发育阶段表达的Cre重组酶诱导的Ifitm5flox c.-14C>T诱导型动物模型，以探究MALEP-IFITM5新功能型的致病效应。具体而言，Prx1-Cre; Ifitm5flox c.-14C>T突变小鼠出生时即出现四肢全段骨折，表现为骨化不全与软骨生成增强，且伴随SOX9蛋白表达水平升高。研究人员于出生后第6日（P6）从Ifitm5突变小鼠的胫骨中分离骨骼细胞，并利用10X Genomics平台开展单细胞RNA测序（single RNA sequencing）。将该测序数据与同阶段对照小鼠胫骨的RNA测序数据（GSE159544）进行比对。突变骨骼细胞的单细胞RNA测序分离流程与对照样本（GSE159544）完全一致：摘取Prx1-Cre; Ifitm5flox c.-14C>T突变小鼠出生后第6日的左右胫骨，去除软组织；将骨组织剪为碎块，于37℃下用TrypLE Express消化10分钟以去除血细胞；剩余骨组织置于汉克平衡盐溶液（HBSS）中，用0.25% II型胶原酶与0.25%分散酶（Dispase）消化1.5小时，每半小时收集一次细胞并更换消化液。使用试剂盒裂解红细胞并去除死细胞，剩余细胞的活率需大于80%。随后采用Chromium单细胞平台（10X Genomics Inc.）按照制造商的操作流程进行单细胞包裹以制备文库并测序。文库的片段大小与浓度通过Qubit、定量PCR及生物分析仪（Bioanalyzer）进行检测。每个样本的初始细胞投入量预计为15000个。文库制备与Illumina NovaSeq 6000测序（151 bp双端测序）均在香港大学临床前研究中心（HKU CPOS）完成，单样本测序通量达150 Gbp。