P4HB基因新突变导致成骨不全Cole-Carpenter综合征的分子机制研究

Osteogenesis imperfect (OI) is a hereditary bone disease of a single gene mutation characterized by increased bone fragility. Nowadays, most virulence genes are discovered related to the synthesis of collagen type I, osteoblast differentiation and bone mineralization. The exact molecular mechanism of the recent discovery of the specific type OI called cole-carpenter syndrome (CCS) which is caused by the P4HB gene mutation is still unclear. In our previous study on the OI patients by using second-generation sequencing, we detected that 1 CCS patients of P4HB new heterozygous mutation of p.C400R. The loci is highly conservative in various species. The protein structure shows that the site is located in the center of the reaction of the gene encoding protein disulfide isomerase. We hypothesized that the apoptosis induced by endoplasmic reticulum stress may be the key to the P4HB mutation resulted in CCS. Our research group intends to establish a P4HB-C400R knock-in mouse model to investigate the phenotype and function of skeletal resulting from this mutation. At the same time, skin fibroblasts of the patient would be cultured to study the changes of the function of collagen type I. The somatic cells would be reprogrammed to induced pluripotent stem (iPS) cells and would be induced to differentiate into osteoblasts and bone tissue. We would observe the differentiation process and the expression of stress pathway protein in endoplasmic reticulum of osteoblasts and bone tissue to verify the exact molecular mechanism of P4HB mutation. This study will provide a new target for the development of effective drugs for OI.

成骨不全（OI）是一种以骨脆性增加为主要特征的单基因遗传骨病。目前发现的致病基因多与Ⅰ型胶原的合成、成骨细胞分化及骨矿化相关。最近发现的P4HB基因突变导致的OI特殊类型Cole-carpenter综合症（CCS），其确切分子机制不清。我们前期对OI患者二代测序发现1例P4HB基因新杂合突变p.C400R导致的CCS。该位点高度保守，位于基因所编码的二硫键异构酶蛋白的反应中心。我们推测内质网应激诱导的成骨细胞凋亡可能是P4HB突变致CCS的关键。本课题拟构建P4HB基因C400R敲入小鼠模型，研究该突变导致的骨骼表型和功能，同时培养患者皮肤成纤维细胞，研究Ⅰ型胶原功能改变；重编辑体细胞为诱导多能干（iPS）细胞，诱导其分化为成骨细胞和骨组织，通过观察其分化及内质网应激通路蛋白的表达，以证实P4HB突变之确切分子机制。本研究将为研发治疗OI的有效药物提供新靶点。

背景：近年发现P4HB基因突变导致成骨不全症(OI)的特殊类型Cole-carpenter综合症（CCS），其确切分子机制不清。.方向：本课题拟构建P4HB基因C402R敲入小鼠模型（相当于人P4HB基因C400R突变），并转染P4HBC400R质粒到人源间充质干细胞（MSC）观察其骨形成能力及内质网应激情况，以证实P4HB突变之确切分子机制。.主要内容：利用CRISPR/Cas9技术成功构建P4HBC402R/+小鼠，对突变小鼠表型分析；观察已转染P4HBC400R人源MSC成骨及软骨分化后骨形成能力以及Ⅰ型胶原表达情况。分析突变小鼠成骨细胞及软骨祖细胞内质网的应激指标，并测定内质网应激PERK通路蛋白。临床继续收集95例OI先证者及其家系，进行相应的基因谱及表型谱研究。.重要结果:我们观察到P4HBC402R突变小鼠无脆性骨折及颅面部畸形特征，却有长骨明显缩短的表型。P4HBC402R突变是通过损害小鼠生长板发育影响长骨长度，该突变影响小鼠软骨细胞增殖、分化和成熟，但不影响其凋亡。P4HBC400R人源MSC提示该突变在翻译后水平影响成骨分化和胶原分泌。内质网应激在P4HBC402R/+小鼠软骨细胞及MSC中被触发，激活了未折叠蛋白反应（UPR）中PERK通路。泛素化异常、脯氨酸羟化酶活性缺陷和二硫键异构酶(PDI)活性异常导致细胞内PDI的积累可能是内质网应激增强和UPR的原因。在临床研究中，我们不仅总结了国人1型胶原基因突变导致OI的表型谱及基因突变谱的特征，还探索了其新发和遗传性突变的差异，及其他罕见致病基因导致OI的特征。.科学意义：我们探索了P4HBC402R突变在软骨发生中的重要作用，以及导致CCS生长迟缓的潜在致病机制，为研发治疗OI的有效药物提供了重要的理论和实验基础。对OI患者的临床研究丰富了国人OI的致病基因谱及表型谱，对临床早期诊断治疗OI具有重要指导意义。

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