Pathogenicity evaluation and splicing correction of COL4A5 splice mutation in X-linked Alport syndrome
10.3760/cma.j.cn441217-20201123-00133
- VernacularTitle:X连锁Alport综合征 COL4A5剪接突变的致病性分析及剪接纠正
- Author:
Tianrong ZHOU
1
;
Huhan ZHANG
;
Chunyan WU
;
Qi LI
;
Sheng HUANG
;
Yong DUAN
Author Information
1. 昆明医科大学第一附属医院检验科，昆明 650000
- Keywords:
Nephritis, hereditary;
DNA mutational analysis;
Genetic therapy;
Alport syndrome;
Splicing mutation;
U1 snRNA
- From:
Chinese Journal of Nephrology
2021;37(11):872-880
- CountryChina
- Language:Chinese
-
Abstract:
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.
