Functional Study On EIF2B5 Gene Mutations In Vanishing White Matter Disease

Functional study on EIF2B5 gene mutations in Vanlshing White Matter diseaseLeukoencephalopathy with Vanishing white matter(VWM,OMIM#603896) is one of the most prevalent inherited white matter disorders in childhood,and so far its specificness lies in that the disease results from direct defects in protein translation initiation.Typical clinical manifestation of this disease is the slow progress of the nervous system degeneration and motor function is more severe than cognitive function.The features of cranial magnetic resonance imaging(MRI) are unique, which shows specificity of abnormal signal of all or almost all cerebral white matter and its replacement by cerebrospinal fluid(CSF)。This change is best shown by fluid-attenuated inversion recovery(FLAIR) images,in which abnormal white matter just have the similar signal to CSF。Since 2001-2002,people have found that VWM is caused by mutation in any of the five genes(EIF2B1,EIF2B2,EIF2B3,EIF2B4, EIF2B5)which encoding the subunits of eukaryotic translation initiation factor eIF2B(eIF2Bα,eIF2Bβ,eIF2Bγ,eIF2Bδ,eIF2Bε).Up to date,functional study about EIF2B1-5 proteins is under of start stage.There was no reported from Chinese VWM patients.The pathogenesis of this disease is in-depth study by which we will further understands of protein translation initiation of this important and complex physiological processes as well as abnormal protein translation of the common occurrence of disease mechanisms.Objective:To analyze EIF2B1-5 gene mutations in four Chinese patients with VWM diagnoses clinically.To understand effect of EIF2B5 gene mutations in different regions of eIF2Bε,functional study about EIF2B5 mutagenesis from Chinese VWM patients was performed.This would be useful to speculate and further study the distributions of the difference EIF2B5 domains,and provide the clue for exploring pathogenesis of VWM.Method:Four VWM patients diagnoses clinically were enrolled from Peking Univesity First Hospital in 2007-2008.All exons and exon-intron boundaries of EIF2B1-5 genes were amplified by polymerase chain reaction(PCR) and followed by direct DNA sequencing.Five mutant p.CMV-EIF2B5 for novel mutations found from Chinese VWM patients,c.185 A＞T(p.D62V),c.805C＞T(P.R269X),c.1004 G＞C (p.C335S),c.1126A＞C(p.N376D),c.l827₁838del(p.S610_D613del),were constructed by site-directed mutagenesis assays.HEK293 cells were used for transfection.Expression of EIF2B5 protein from wildtype and mutant types were detected using Western Blot methods.Result:Sequencing results of EIF2B1-5 for four VWM patients shows:Two heterozygous mutations of EIF2B5 were identified c.337 C＞T(p.R113C) in exon3 and c.806G＞A(p.R269Q) in exon6 from patient 1.One homozygous novel mutation of EIF2B3 were identified c.1037 T＞C(p.I346T) in exon9 from patient 2.Their parents were found the heterozygous variation in those sites.The other two patients didn't find any changes from EIF2B1-5.Expression results of EIF2B5 protein by Western Blot methods demonstrated that there were decreased from mutants c.805C＞T (P.R269X) and c.1126A＞C(p.N376D) compared with wildtype.With the statistical significance,this two kinds of mutant EIF2B5 have significant change in protein expression(P＜0.05).The protein of mutants c.805C＞T(P.R269X) has more significant decrease.Conclusion:Through analysis of EIF2B1-5 for four VWM patients diagnoses clinically,c.337 C＞T(p.R113C),c.806G＞A(p.R269Q) and c.1037 T＞C(p.I346T) were found from two patients.The first two mutations were reported and the latter is novel mutation not reported around world yet.Expression of EIF2B5 protein decreased from mutant c.805C＞T(P.R269X) and c.1126A＞C(p.N376D) which demonstrated that there are different effects in different regions of EIF2B5 gene mutations.Thus there may have different mechanisms of EIF2B5 protein function by different domains.This is the first report about functional study of ELF2B5 mutations from VWM patients in China.