中国当代儿科杂志2024，Vol.26Issue(2)：188-193,6.DOI:10.7499/j.issn.1008-8830.2309035
人苯丙氨酸羟化酶突变体p.R243Q、p.R241C和p.Y356X的体外表达及功能研究
In vitro expression and functional analyses of the mutants p.R243Q,p.R241C and p.Y356X of the human phenylalanine hydroxylase
摘要
Abstract
Objective To study the in vitro expression of three phenylalanine hydroxylase(PAH)mutants(p.R243Q,p.R241C,and p.Y356X)and determine their pathogenicity.Methods Bioinformatics techniques were used to predict the impact of PAH mutants on the structure and function of PAH protein.Corresponding mutant plasmids of PAH were constructed and expressed in HEK293T cells.Quantitative reverse transcription polymerase chain reaction was used to measure the mRNA expression levels of the three PAH mutants,and their protein levels were assessed using Western blot and enzyme-linked immunosorbent assay.Results Bioinformatics analysis predicted that all three mutants were pathogenic.The mRNA expression levels of the p.R243Q and p.R241C mutants in HEK293T cells were similar to the mRNA expression level of the wild-type control(P>0.05),while the mRNA expression level of the p.Y356X mutant significantly decreased(P<0.05).The PAH protein expression levels of all three mutants were significantly reduced compared to the wild-type control(P<0.05).The extracellular concentration of PAH protein was reduced in the p.R241C and p.Y356X mutants compared to the wild-type control(P<0.05),while there was no significant difference between the p.R243Q mutant and the wild type control(P>0.05).Conclusions p.R243Q,p.R241C and p.Y356X mutants lead to reduced expression levels of PAH protein in eukaryotic cells,with p.R241C and p.Y356X mutants also affecting the function of PAH protein.These three PAH mutants are to be pathogenic.[Chinese Journal of Contemporary Pediatrics,2024,26(2):188-193]关键词
苯丙氨酸羟化酶缺乏症/突变体/体外表达/细胞Key words
Phenylalanine hydroxylase deficiency/Mutant/In vitro expression/Cell引用本文复制引用
庞永红,陈桂荣,高翔羽,袁振亚,黄辉,王增芹,彭磊,李逸群,刘杰,刘冬..人苯丙氨酸羟化酶突变体p.R243Q、p.R241C和p.Y356X的体外表达及功能研究[J].中国当代儿科杂志,2024,26(2):188-193,6.基金项目
徐州市科学技术局社会发展项目(KC20081) （KC20081）
江苏省卫生健康委员会妇幼健康科研项目(F202058) （F202058）
江苏省妇幼保健协会科研课题(FYX201906) （FYX201906）
徐州医科大学科研课题(2018KJ19). （2018KJ19）
