肾单位肾痨新致病基因探索

Nephronophthisis (NPHP), a rare autosomal recessive monogenic inheritance disease caused by ciliary genetic defects, is the most common genetic causes of renal failure in children. To date, mutations of 19 genes (NPHP1–NPHP19) have been found to cause NPHP. However, more than 50 % of all cases have yet to find disease causing genes. We performed whole exome sequencing(WES) and bioinformatics analysis in 23 NPHP patients and found some patients carried recessive mutations (compound heterozygous or homozygous mutations) in ciliary genes, with the minor allele frequency(MAF)≤0.005 and predicted to be damaged. All these variants were confirmed in patients and their parents by Sanger sequencing. In addition, two of these genes were found in 2 patients respectively. These genes were predicted possible NPHP disease causing genes. In the present study, the expression of proteins encoded by target genes will further be explored in renal tissue of the patients. CRISPR/Cas9 technology will be applied to knockout target genes in vivo and in vitro. In 3 dimensions’ cultured MDCK cells, lumen and cyst formation will be observed. The relationship between target gene and the functional modules of NPHP and their intracellular signaling pathways will be analyzed to explore the possible pathogenesis of target gene. With positive results, target gene knockout zebrafish model and subsequently mouse model will be made to observe pronephric cyst formation in zebrafish, and the typical NPHP renal tubular interstitial changes in mice. These will help the confirmation of novel pathogenic gene of nephronophthisis and help the diagnosis of NPHP at an early stage and improve genetic counselling and enable prenatal testing.

肾单位肾痨（Nephronophthisis，NPHP）是一种罕见纤毛基因缺陷常染色体隐性单基因遗传病，是儿童肾衰竭最常见遗传因素。已知致病基因有19种，但仍有超过50%患者未明确致病基因。我们对23例患者进行全外显子测序分析，发现部分存在人群突变频率低(最小等位基因频率≤0.005）且蛋白功能预测有害的纤毛基因双重杂合或纯合突变（分别来自父母双方），其中两个基因分别在2个患者中同时检测到。这些基因有可能是NPHP新致病基因。本研究拟进一步检测靶基因编码蛋白在患者肾组织的表达；应用CRISPR/Cas9技术，先构建靶基因敲除MDCK细胞株，三维培养观察囊肿形成，并观察靶基因与NPHP相关纤毛功能复合体及其细胞内信号途径的关系，探讨其致病机制；再依次构建靶基因敲除斑马鱼和小鼠模型，观察斑马鱼前肾囊肿形成，小鼠肾脏特征性病理改变。以期发现NPHP新致病基因，为临床诊治、优生优育提供理论依据。

肾单位肾痨（Nephronophthisis，NPHP）是一种罕见纤毛基因缺陷常染色体隐性单基因遗传病，是儿童肾衰竭最常见遗传因素。已知致病基因有20种，但仍有超过50%患者未明确致病基因。目前我们一共收集到55例NPHP患者，通过生信分析筛选出部分可能致病基因，分别在MDCK细胞、小鼠和斑马鱼中构建疾病模型。本研究还发现致病基因CEP83新突变，除了引起肾小管囊肿外，还可引起显著肾小球囊肿和肾发育不良，CEP83可能为肾小球囊肿的新致病基因；相关论文已发表在Clinica Chimica Acta中（SCI论著，IF 2.79，小类二区）。进一步研究发现CEP83新突变位点可导致斑马鱼肾小球不发育或发育不良、伴心包水肿和脊索发育不良。另外，全外显子测序发现部分未检测到已知致病基因突变患儿存在其他纤毛基因突变，包括 ABHD12、CFAP77、DNAH9、DNAH10、DNAH14、PCNT共6个基因。此外，还发现纤毛基因IFT140纯合突变致Mainzer-Saldino综合征和SALL1新发突变致TBS综合征。同时，我们通过CRISPR/Cas9基因编辑技术首次成功构建具有肾脏表型（肾囊肿形成和肾间质纤维化）的NPHP1基因敲除小鼠模型。通过重组腺病毒转染在NPHP1小鼠模型中敲低ZONAB表达，可抑制NPHP小鼠肾囊肿形成，提示ZONAB可能是肾小管上皮细胞形成肾囊肿的关键转录调控因子。我们还初步研究了Hippo信号通路和TGF-β信号通路在NPHP1中的肾间质纤维化的发生作用机制。另外，通过腺相关病毒AAV-9型转染NPHP1大片段缺失小鼠模型使NPHP1重新表达，发现可有效改善了小鼠的肾内外表型。最后，我们对NPHP1小鼠的肾脏进行单细胞测序，与野生型小鼠对比分析后发现新的细胞亚群，推测是囊肿相关细胞。以上研究，为研究NPHP致病机制及治疗靶点提供了参考依据。