Effect On Gene Expression Profiles Caused By A39S Mutated DJ-1 Protein And The Research On Detection Of DJ-1 Mutations And Serum DJ-1 Protein Concentration

PartⅠThe effect on gene expression profiles caused by A39S mutation of DJ-1BackgroundDJ-1 protein is a multifunction protein encoded by DJ-1 gene,one of the virulent genes of AREP.It participates in many physiological processes such as cell cycle modulation,transcriptional regulation,RNA stability,signal transduction in the androgen receptor pathway, oncogenesis,oxidative stress and mitochondrial function.Also,it could serve as a transcriptional co-activator playing a role in the dopamine synthesis,oxidative tress and apoptosis through regulating the transcription activities of PSF,TH,AR,Nrf2 and p53 genes.The concrete mechanism of DJ-1 mutation in PD is still unknown,traditional methods, instead of detecting all of the proteins or genes that could be associated with DJ-1,were limited to one or few proteins or genes that could probably interact with DJ-1.DNA microarray retrieves this defect.ObjectiveTo identify whether the A39S mutation could change the gene expression profiles of DJ-1 and contribute to onset of PD.MethodsGenerate HEK293 monoclonal cell lines which stably express Flag-tagged pCMV-Tag2A empty vector,pCMV-Tag2A-DJ-1 and pCMV-Tag2A-DJ-1-A39S vectors respectively.DNA microarray assays were used in these monoclonal cell lines to detect genes whose expressions are abnormally regulated by A39S mutated DJ-1 protein.ResultsCompared with the cell line expressing empty vector,we found expression levels of 14 genes upregulated and 28 genes downregulated in expressing wild-type DJ-1 protein cells;expression levels of 6 genes upregulated and 2 genes downregulated in expressing A39S mutated DJ-1 protein cells respectively.Compared with the cell line expressing wild-type DJ-1 protein,we found the expression level of UGT2B7 gene upregulated in expressing A39S mutated DJ-1 protein cell.These genes with differential expression were classified to several groups such as signal transduction,cell adhesion,transcription regulation,cell cycle regulation,protein modification,apoptosis,oxidative stress and neurotoxicity.It suggested that A39S mutated DJ-1 protein probably affects the normal function of these pathways via changing these associated gene expression levels to participate the pathogenesis of PD.ConclusionsA39S mutation could disturb the transcriptional activities of DJ-1 protein. PartⅡScreening for DJ-1 gene Mutation in patients of EOP by real-time PCR and direct sequencingBackgroundEarly-onset parkinsonism(EOP) is a group of syndromes carrying the same characteristics with the idiopathic PD with onset age younger than 50 years old.Besides the common features,EOP also has its own characteristics such as being lighten at morning and serious at dusk, dysmyotonia relieved after rest,dyskinesis emerging at early stage of dopamine treatment,hyperreflexia et al.EOP usually inherits at the mode of recessive inheritance.The mutations of gene Parkin,DJ-1,PINK1 and ATP13A2 contribute to the onset of EOP.DJ-1 is one of these pathogenic genes of EOP with a low mutation rate at 1～2%or below.To date,more than 20 mutations of DJ-1 including exon rearrangement mutation have been found.The exon rearrangements may present in the heterozygous state and thus escape detection by DNA direct sequencing because there is still a normal copy of the exon.Sequencing combined with quantitative polymerase chain reaction has been universally used to detect DJ-1 gene mutations.ObjectiveTo investigate the mutation frequency and clinical characteristics of DJ-1 gene. MethodsDuplications or deletions of DJ-1 gene were detected using real-time fluorescent quantitative PCR;point mutations and small deletion/duplication mutations were detected by direct sequencing.ResultsIn this research we screened for mutation of DJ-1 in 160 sporadic EOP patients by real-time PCR combined with DNA direct sequencing and identified a novel pathogenic mutation -the heterozygous deletion of exon 2 in 4 patiens with a mutation rate at 2.5%.Also in the same patient sample,we detected four known polymorphisms IVS4+30 T→G, IVS4+45 G→A,IVS4+46 G→A,IVS5+31G→A and a new polymorphism IVS6+52C→T.No DJ-1 clinical specificity was found in this study.Conclusions1.A novel pathogenic mutation -the heterozygous deletion of exon 2 was found in Chinese sporadic EOP patients using real-time fluorescent quantitative PCR;2.The rate of DJ-1 gene exon2 arrangement was 2.5%in this study, indicating that the exon rearrangement mutation may play important roles in Chinese Sporadic EOP patiens.3.Four known SNPs and a new SNP IVS6+52C→T were found in Chinese sporadic EOP patients using direct sequencing. PartⅢThe relationship of serum DJ-1 protein's concentration and PDBackgroundThe major pathological changes of PD is the selective loss of dopaminergic neurons from the substantia nigra pars compacta and the presence of Lewy bodies and these changes present far more than the appearance of clinical symptoms.It is hard to differentiate PD from other diseases with similar symptoms such as essential tremor,the parkinsonian plus syndromes,multiple system atrophy,progressive supranuclear palsy and corticobasal degeneration just by symptoms criteria.Also,after the establishment of diagnosis,there are still problems on the assessment of severity and progressing rate.In addition,the evaluation of neuroprotective drugs' effect calls a direct and reliable criteria.The search for biomarker of PD is the result of this need.For PD,the utilization of biomarker is focused on the following three aspects:first the right diagnosis at the early stage,second the assessment of severity and progressing rate and last the evaluation of drugs' effect. DJ-1 is generally distributed in all of the body and could be detected in the cerebrospinal fluid(CSF),plasma and serum.It was proved that the concentration of DJ-1 protein could act as the biomarker of some tumors, cerebrovascular disorders or neurodegenerative disease.ObjectiveTo identify the level of serum DJ-1 protein in Chinese sporadic PD patients and justify whether the serum DJ-1 protein concentration could serve as the biomarker of PD.MethodsDetect the concentration of serum DJ-1 in 120 sporadic PD patients and 126 age,gender matched controls with DJ-1-ELISA Kit.Statistic methods were used to analysis the difference between these two groups.ResultsThe difference of serum DJ-1 concentration between PD group(no staging) and control is not statistically significant(p=0.113);The difference of serum DJ-1 concentration between PD groups(different Hoehn-Yahr stage) and control is not statistically significant(p=0.441); The difference of serum DJ-1 concentration between PD groups(different course of disease) and control is not statistically significant(p=0.145); The difference of serum DJ-1 concentration between PD different age groups is not statistically significant(p=0.834);The difference of serum DJ-1 concentration between PD different age groups is not statistically significant(p=0.059);ConclusionsThe first research in China to detect the Serum DJ-1 protein concentration of sporadic PD patients and controls;there was no apparent difference between these two groups and it was not associated with the disease severity or course of disease.So there was no sufficient proof to justify the serum DJ-1 concentration as a PD biomarker.