PHEX基因突变致X-连锁低血磷性佝偻病分子机制及治疗策略研究

The X-linked hypophosphatemic rickets (XLHR) is a common metabolic bone disease caused by mutations in the PHEX gene. We have carried PHEX genotype analysis in 28 children with XLHR and discovered 10 novel PHEX mutations. This study will continue to collect XLHR cases. Sequencing analysis of all the exons and their adjacent sequence and promoter region will be performed to clarify the gene variation of XLHR patients in China. Genotype- phenotype correlation study will be carried on the basis of genotype results. Missense and nonsense mutations are the two major types of mutations caused XLHR in PHEX gene. In this study, we will take function analysis of the detected missense mutations and classify them into endoplasmic reticulum retention and enzyme activity defective types. We next want to discuss the corrective effect of chemical chaperones drugs Orkambi and 4-PBA on endoplasmic reticulum retention mutant protein in vitro and vivo. In recent years，read-through drugs are playing increasing prominent roles in the researches related to genetic diseases caused by nonsense mutations．We will study whether the read-through drugs NB54, NB84, PTC124 can inhibit the role of PHEX gene nonsense mutation; promote complete PHEX protein synthesis; recovery of endopeptidase activity. Our study will provide a new experimental basis for the clinical treatment of XLHR.

X-连锁低血磷性佝偻病(XLHR)是儿童常见的致畸/致残性遗传病，由PHEX基因突变所致。申请者前期对28例XLHR患儿进行了PHEX基因型分析，发现10种新突变。本研究将进一步扩大收集XLHR病例，对其进行PHEX基因外显子及其邻近序列、启动子区序列进行测序以明确我国XLHR病人基因变异特征，并对XLHR的临床表型与基因突变类型进行关联研究。PHEX基因错义和无义突变是致XLHR的两大突变类型。本研究将对已检测到的PHEX基因错义突变进行功能研究，将错义突变进行内质网滞留型和酶活缺失型分类，进而探讨化学分子伴侣类药物Orkambi和4-PBA对内质网滞留型突变蛋白的纠正作用；同时研究促通读药物NB54、NB84、PTC124是否能够抑制PHEX基因无义突变的作用，促进完整PHEX蛋白合成、恢复内肽酶活性，为XLHR病人的治疗提供新思路。

背景：XLHR的临床表型多样，且严重程度差异很大，既往研究关于基因型与表型相关性一直存在争议。为了研究我们在XLHR患者中基因型-表型无相关性的研究结论，我们分析了不同类型PHEX基因突变的生物学特性，包括PHEX蛋白突变体的蛋白表达、亚细胞定位和内肽酶酶活性改变。.方法：首先构建了PHEX野生型和10种突变型重组表达载体，将质粒转染HEK293细胞，Western blotting检测突变对PHEX蛋白表达水平的影响，免疫荧光实验观察PHEX突变体亚细胞定位，利用糖苷内切酶Endo H检测PHEX蛋白糖基化状态。构建分泌型PHEX重组表达载体，转染细胞后，富集细胞上清蛋白，利用荧光多肽底物酶水解反应检测PHEX内肽酶活性。.结果：野生型和突变型PHEX在HEK293细胞中表达水平检测结果显示，9种PHEX突变体（p.Cys77Tyr，p.Cys85Ser，p.Ile281Lys，p.Ile333del，p.Ala514Pro，p.Gly572Ser，p.Gln714*，p.Arg567*和p.Arg747*）蛋白表达水平较野生型显著下降，糖苷内切酶Endo H检测结果显示这9种PHEX突变体蛋白均表达为不成熟的糖基化形式。免疫荧光实验观察到9种突变体不能锚定到细胞膜上，均滞留在细胞基质内。仅p.Gly553Glu突变型与野生型一样，大部分表达为成熟的糖基化蛋白，且主要表达在细胞膜上。分泌型PHEX质粒转染与上清蛋白检测结果显示， 细胞基质滞留型PHEX突变体均不能分泌到胞外。仅p.Gly553Glu突变型分泌到细胞上清，荧光多肽底物酶水解反应检测到p.Gly553Glu突变型PHEX内肽酶活性为野生型的13%。.结论：XLHR患者中发现的PHEX非截短突变型突变体的主要改变是蛋白表达水平降低，糖基化不完全和细胞膜定位缺陷，这些生物学特性与截短突变型一致。本研究的XLHR患者临床回顾性分析和PHEX突变生物学功能分析结果均显示XLHR临床表现严重程度与PHEX基因突变类型之间无相关性。