Study On Pathogenicity Of Novel Exon And Intron Double Mutations In A Wiskott-aldrich Syndrome Family In Vitro

Objective: Wiskott-Aldrich syndrome(WAS)is a rare X-linked recessive primary immunodeficiency disease,which is caused by WASP gene mutation and abnormal expression of Wiskott-Aldrich syndrome protein(WASp).WASP gene mutation along with WASp expression level are closely related to clinical phenotypes of WAS.In this study,in order to explore the exon splicing effect of novel intron mutation and the correlation between the genotype and clinical phenotype,the theory of minigene technology was used to conduct the study in vitro on novel mutations in the WAS patients.Methods:1.Collect the clinical data,family history,protein expression and sequencing results of the patients and family members;2.Analyze the pathogenicity of novel mutations by online bio-informatics tools(The Mutation Taster and Polyphen-2);3.Construct four groups for wild-type(mini-WASP-WT),exon mutant(mini-WASP-Mut1),intron mutant(mini-WASP-Mut2)and combined mutant(mini-WASP-Mut3)recombinant plasmids in vitro and transfect them into COS-7 cells;4.Detect the relative expression level of empty plasmid,wild-type and three groups of mutant plasmids in vitro by reverse transcription real-time quantitative PCR(RT-q PCR);5.Direct sequence for the c DNA products and perform comparison analysis.Results:1.Both the patient and his cousin had thrombocytopenia with small platelets,infection and bleeding tendency with a family history of thrombocytopenia,and the clinical scores were,respectively,4 and 5.Both patients had novel c.158T>C mutation on exon 2 of WASP gene,resulting in p.L53 P.In addition,the cousin had a second new intron mutation of IVS2+14C>T.The results of the protein expression by flow cytometry and western blot were significantly reduced in the patient;2.Both bio-informatics prediction tools showed that the predicted pathogenicity of novel c.158T>C mutation was possibly to be pathogenic;3.Recombinant plamids expressing part of genomic gene sequences in wild-type and 3 mutant groups were successfully constructed in vitro and verified by sequencing.The results showed c.158T>C in mini-WASP-Mut1,IVS2+14C>T in mini-WASP-Mut2,two mutations in mini-WASP-Mut3 and normal sequence in mini-WASP-WT.The consistent results were acquired by sequencing for verifying successful transfection into COS-7 cells;4.The results of RT-q PCR showed that the relative expression of the mini-WASP-WT was 1681.849;mini-WASP-Mut1 was 2498.951;mini-WASP-Mut2 was 2055.928 and the mini-WASP-Mut3 was 881.948;5.The sequencing of c DNA in vitro showed that the mini-WASP-Mut2 and mini-WASP-Mut3 carrying intron mutation contained 360 bp of the first three exons without addition and deletion of gene sequence.The mini-WASP-Mut2 and mini-WASP-WT showed normal gene sequences;The mini-WASP-Mut1 and mini-WASP-Mut3 revealed a 360 bp sequence that only contained the mutation c.158T>C.Conclusions: This study confirmed that novel intron mutation did not affect the splicing of exons,and excluded the influence of the mutation on the severe clinical manifestations of the cousin at the transcription level.The study in vitro using minigene provided a new sight for exploring the pathogenesis of novel mutations in WAS.