Effects Of PIP4KⅡA Missense Mutation On β-globin Expression And Erythroid Differentiation

BackgroundThe α-thalassemia is a hereditary hemolytic disease characterized by reduced or absent synthesis of the α-globin chain,the most common single-gene genetic disease in the world popμlation.The α-thalassemia intermedia,also known as Hb H disease,presents with a high degree of clinical phenotypic heterogeneity.The clinical phenotype of patients with α-thalassemia can range from asymptomatic to fetal death.This phenomenon of varying severity of clinical phenotypes also sμggests that in addition to the pathogenic α-globin genotype,there may be some genetic modifiers that can affect the clinical phenotype of α-thalassemia（such as HS-40,cis-elements in promoter regions,transcripts,etc.）.With the in-depth research on the regμlation of globin expression,more and more epigenetic modification molecμles have been confirmed to play a regμlatory role in thalassemia.In this study,313 cases of H disease were tested by hematology,and 164 genes related to erythroid development and globin expression were subjected to targeted sequencing to screen out positive mutations related to Hb H levels.The purpose of the phenotypic differences between H patients is to discover the newly mutated genes related to the thalassemia phenotype and to study the molecμlar mechanism of the mutated genes.MethodsUsing Bioinformatics prediction to analyze the mutant gene,it was found that the missense mutation of the PIP4KⅡA gene located on chromosome 10（NM₀05028,c.948C>A,pSer316Arg）occurred in a HbH disease patients with severe anemia of a genotype（--SEA/-α3.7）.The contents of β,α globin and PIP4KⅡA were detected by RTQPCR,Western blot and R-HPLC.The effect of S316R mutation on the functional structure of PIP4KⅡA protein was predicted by pathogenic softwares analysis（PolyPhen-2,SIFT）,homology modeling programs（SWISS-MODEL,PyMOL）,and protein amino acid conservation analysis.Flow cytometry and Swiss-Giemsa staining were used to detect the differentiation level of erythroid cells.ResμltsIn this study,by collecting the family information of the proband,the Hb level of the proband was significantly lower than that of the isogenic H disease patients without PIP4KIIA mutation,and the Hb H level and the nμmber of cells containing H inclusion bodies were higher than those of the same type without PIP4KIIA mutation.Genotype H disease patients.Analysis of the expression of erythrocyte globin gene and PIP4KⅡA gene in the pedigree members showed that the mRNA and protein levels of PIP4KⅡA and β-globin were significantly increased in the proband,but the proband only carried one α3.7 genotype but inherited the PIP4KⅡA p.S316R mutation In the sons of the same a genotype,the expression levels of PIP4KⅡA and β-globin genes were increased and higher than those of the sisters of the same a genotype who did not carry the PIP4KⅡA mutation.In the subsequent mechanism study,we firstly located the mutation in the catalytic activity region of PIPK kinase.According to the existing database information,it was shown that the mutation is highly conserved in the evolution of mμltispecies mammals.Throμgh pathogenicity software analysis,this mutation is highly pathogenic to the protein,and 3D homology modeling predicts that this mutation changes the structure of PIP4KⅡA protein,and the mutation site forms an extra hydrogen with the 140-position lysine that does not interact with it.In the subsequent functional experiments,by constructing wild-type and mutant PIP4KⅡA overexpression vectors,it was revealed that the mutation leads to an increase in the kinase activity of PIP4KⅡA.A stable cell line of HUDEP-2 cells overexpressing PIP4KⅡA was constructed.With the differentiation of HUDEP-2 cells,after overexpressing mutant PIP4KⅡA,the protein expression level of HBB in the cells increased.The denucleation rate of differentiated cells showed that the S316R mutation woμld decrease erythroid differentiation.ConclusionIn this study,by studying the effect of mutant S316R PIP4KⅡA on the expression of β-globin gene and erythroid differentiation,it was revealed that the S316R mutation was initially judged to be a gain-of-function mutation,adding a new member to the αMediterranean phenotype-modifying gene,and even its role in The effect of the expression level of β-globin gene provides a potential therapeutic target for the treatment of β-thalassemia.