X连锁Alport综合征COL4A5剪接突变的致病性分析及剪接纠正  

Pathogenicity evaluation and splicing correction of COL4A5 splice mutation in X⁃linked Alportsyndrome

作　　者：周添荣 张壶涵 吴春燕[1] 李琪[2] 黄晟 段勇[1] Zhou Tianrong;Zhang Huhan;Wu Chunyan;Li Qi;Huang Sheng;Duan Yong(Department of Laboratory Medicine,the First Affiliated Hospital of Kunming Medical University,Kunming 650000,China;Pediatrics Department of the First Affiliated Hospital of Kunming Medical University,Kunming 650000,China;Emergency Department of the First Affiliated Hospital of Kunming Medical University,Kunming 650000,China)

机构地区：[1]昆明医科大学第一附属医院检验科,昆明650000 [2]昆明医科大学第一附属医院儿科,昆明650000 [3]昆明医科大学第一附属医院急诊科,昆明650000

出　　处：《中华肾脏病杂志》2021年第11期872-880,共9页Chinese Journal of Nephrology

摘　　要：目的分析一个X连锁显性遗传Alport综合征家系COL4A5基因的剪接突变位点,探索外显子特异性U1核小RNA(small nuclear RNA,snRNA)基因治疗的可能性。方法收集Alport综合征先证者及其家族成员临床资料,高通量测序技术检测先证者肾病系列基因全外显子组基因突变。用在线软件分析COL4A5 c.546+5G>A变异引起的剪接位点改变及其致病性。用minigene实验验证和分析Alport综合征家系先证者COL4A5基因突变位点c.546+5G>A及瞬时转染导入修饰过的U1 snRNA纠正剪接突变的效果。结果基因测序结果显示,先证者和其同母异父的兄长均存在COL4A5基因半合子变异,变异位点为c.546+5G>A。在线软件分析剪接变异致病性的结果显示,突变后原有的Donor剪切位点检测不到,提示影响剪切可能性很大。杂交小基因检测结果验证了COL4A5基因c.546+5G>A突变的异常剪接模式——外显子9缺失。经过修饰的U1 snRNA可部分纠正该异常的剪接突变,ExSpeU1(MT)、ExSpeU1(E9+1)、ExSpeU1(E9+9)、ExSpeU1(E9+11)对c.546+5G>A引发的外显子9缺失的纠正比例分别为0、43.81%、52.09%、48.12%。结论COL4A5新发剪接突变具有致病性,修饰过的U1 snRNA可部分纠正异常剪接。Objective To analyze the splicing mutation site of COL4A5 gene in a family with X⁃linked dominant Alport syndrome and explore the possibility of exon specific U1 small nuclear RNA(snRNA)gene therapy.Methods The clinical data of the proband and family members of Alport syndrome were collected,and the gene mutations in the whole exon of a series of nephropathy genes in the proband were detected by high⁃throughput sequencing.The splice site changes and pathogenicity caused by COL4A5 c.546+5G>A mutation were analyzed by online software.Minigene experiment was used to verify and analyze the effect of COL4A5 gene mutation site c.546+5G>A in the proband of Alport syndrome family,and transient transfection and introduction of modified U1 snRNA to correct splicing mutation.Results The results of gene sequencing showed that there was a hemizygous variation of COL4A5 gene in the proband and his half brother,and the variation site was c.546+5G>A The results of online software for analyzing the pathogenicity of splice variation showed that the original donor splicing site could not be detected after mutation,suggesting that there was a great possibility of affecting splicing.The abnormal splicing mode of COL4A5 gene with c.546+5G>A mutation—deletion of exon 9 was verified by hybridized small gene detection.The abnormal splicing mutation could be partially corrected by the modified U1 snRNA.The correction ratios of ExSpeU1(MT),ExSpeU1(E9+1),ExSpeU1(E9+9)and ExSpeU1(E9+11)to exon 9 deletion caused by c.546+5G>A were 0,43.81%,52.09%and 48.12%,respectively.Conclusions The pathogenicity of the new splicing mutation of COL4A5 is verified,and the modified U1 snRNA can partially correct the abnormal splicing.

关 键 词：肾炎 遗传性 DNA突变分析 基因治疗 ALPORT综合征 剪接突变 U1核小RNA 

分 类 号：R692.6[医药卫生—泌尿科学]