| PART I NEWBORN SCREENING FOR G6PD DEFICIENCY IN CHINA AND DISTRIBUTION OF G6PD MUTANT GENESObjective: To investigate the Chinese neonatal screening population and provide information on the distribution of G6 PD gene mutations in patients with glucose-6-phosphate dehydrogenase deficiency in China.Methods: A total of 10,357 children with positive G6 PD enzyme activity tests from 29 newborn screening(NBS)centers in 12 provinces of China between 2013 to 2017 were included.The MMCA(Multicolor melting curve analysis)method was used to detect 16 hotspot mutation sites of the G6 PD gene in these children.DNA samples with negative MMCA test results were sequenced for the whole exon of G6 PD gene.Finally,the genotype distribution of G6 PD deficiency in NBS population in China was statistically calculated.Results: Of 10,357 samples included,9,950 were found the common mutations positive by MMCA test,accounting for 96.1%.A total of 13 mutation sites were detected(c.95A>G,c.383T>C,c.392G>T,c.487G>A,c.493A>G,c.517T>C,c.592C>T,c.871G>A,c.1004C>A,c.1024C>T,c.1360C>T,c.1376G>T,c.1388G>A).The samples of remaining 408 children was performed the whole exome sequencing,and 278 sampleswere successfully tested(68.1%).A total of 17 mutation sites were detected in the successfully sequenced samples,including 13 reported mutation sites(c.202G>A,c.593G>C,c.1003G>A,etc.)and 4 unreported mutation sites(c.152C>T,c.290A>T,c.697G>C,c.1285A>G).The most common G6 PD gene mutation sites in China were c.1376G>T,c.1388G>A,c.95A>G,c.1024C>T,c.871G>A.The distribution of G6 PD mutation sites were characterized with different regions,and the top five mutation sites in Sichuan,Chongqing,Guizhou,Hunan,Guangxi,Guangdong,and Hainan were: c.95A>G,c.871G>A,c.1024C>T,c.1376G>T,c.1388G>A,while in Shandong and Shaanxi provinces were: c.95A>G,c.487G>A,c.1024C>T,c.1376G>T,c.1388G>A.In addition,four novel missense mutation sites(c.152C>T,c.290A>T,c.697G>C,c.1285A>G)that had never been reported were detected in 7 male infants.Conclusion: A total of 26 known mutation sites have been detected in the G6 PD gene in Chinese neonates population,of which the most common mutation sites were c.1376G>T,c.1388G>A,c.95A>G,c.1024C>T,c.871G>A.There is a certain difference in the distribution of hotspot mutations between coastal cities and inland cities.In addition,our study first reported the presence of four novel mutation sites in the G6 PD gene in Chinese neonate population: c.152C>T,c.290A>T,c.697G>C,c.1285A>G,while their pathogenicity still needs to be clarified.PART II ANALYSIS OF THE PATHOGENICITY OF G6PD GENE c.697G>C MUTATIONObjective: To find the likely pathogenic mutation sites among these four novel mutation sites through overexpressing the wild-type and mutant vectors of G6 PD gene in HEK293 cells,combined with the clinical information,software prediction and protein structure analysis of the children.Methods: For the four novel mutation sites(c.152C>T,c.290A>T,c.697G>C,c.1285A>G)found by sequencing of the whole exon of G6 PD gene,pc DNA3.1 vector was used as the backbone,wild-type and mutant overexpression plasmids were constructed and transfected into HEK293 cells respectively.The relative expression of G6 PD gene in each group was detected by q RT-PCR,and the G6 PD enzyme activity level of each group was detected by the G6PD/6PGD enzyme activity detection method,then the possible pathogenic mutation sites were found by comparing the detection results of gene expression and enzyme activity,combining with the clinical information and prediction results of SIFT and Polyphen 2.Finally,the protein functional domains that mutation may affect were analyzed referring to literatures and Py MOL software modeling.Results: Four novel mutation sites(c.152C>T,c.290A>T,c.697G>C,c.1285A>G)and their corresponding wild-type pc DNA3.1-G6 PD overexpression plasmids were successfully constructed and transfected into HEK293 cells.The results showed that the expression level of G6 PD gene in the c.697G>C mutant group in HEK293 cells was 4 times higher than that in the WT control group,while the G6 PD enzyme activity was 30%lower than that in the WT group.The residual levels of G6 PD enzyme activity in the peripheral blood samples of three infants with c.697G>Cwere between 10% to 60%,which can be divided into WHO class III;the results predicted by SIFT and Polyphen 2 software both suggested the c.697G>C mutation may affect the function of G6 PD protein.Analysis of G6 PD protein structure using Py MOL software indicated that c.697G>C was located in the structural NADP+ binding domain of G6 PD protein,which may affect the thermal stability of G6 PD protein.Conclusion: G6 PD c.697G>C is a potential pathogenic mutation.According to the residual level of G6 PD enzyme activity in the neonatal peripheral blood samples carrying this mutation,it can be classified as a WHO III mutation.And it is likely to damage the function of G6 PD protein by affecting the binding of G6 PD to structural NADP+.PART III G6 PD c.697G>C MUTATION REDUCES G6PD ENZYME ACTIVITY IN CELLSObjective: To verify that the G6 PD gene c.697G>C mutation site leads to a decrease in G6 PD enzyme activity in cells.Methods: Sg RNAs and mutation homology arm sequences for G6 PD gene c.697G>C site were designed,CRISPR/Cas9 gene editing dual plasmid system was constructed,which was used to construct G6 PD gene c.697G>C mutated HEK293 cell line and erythroleukemia K562 cell line.Then q RT-PCR and Western-blot were used to detect the effect of c.697G>C mutation on G6 PD gene expression;CCK8 to detect the effect on cell proliferation;G6PD/6PGD enzyme activity detection method to detect the effect on G6 PD enzyme function;Crystal violet staining and Annexin V-APC/7-AAD to validate the tolerance of c.697G>C mutant HEK293 and K562 cell lines to the oxidative drug primaquine.Results: Three pairs of sg RNAs were designed for c.697G>C site and p X458-sg RNA plasmid was constructed.The p X458-Sg3 with the highest editing efficiency was selected to co-transfect HEK293 cells and K562 cells with the homology arm plasmid.The DNA sequencing results of the monoclonal cells verified the successful construction of HEK293 cell line and K562 cell line with G6 PD gene c.697G>C mutation.q RT-PCR and Western-blot detection revealed that the G6 PD gene c.697G>C mutation did not affect the expression of G6 PD gene in HEK293 and K562 cells;CCK8 test showed that the mutation of G6 PD c.697G>C significantly inhibited the cell proliferation of K562 cells,but had no effect on that of HEK293 cells;G6PD/6PGD enzyme activity test showed that G6 PD c.697G>C mutation resulted in a significant decrease of G6 PD enzyme activity in HEK293 cells and K562 cells by 20%-30%.The results ofcrystal violet staining and Annexin V-APC/7-AAD showed that the tolerance of G6 PD gene c.697G>C mutant HEK293 cells and K562 cells to primaquine decreased significantly at 200 and 250?M.Conclusion: The G6 PD c.697G>C mutation leads to a decrease in G6 PD enzyme activity in HEK293 and K562 cells,which is a pathogenic mutation. |
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