Screening Of Genes Associated With Early Onset Parkinson’s Disease And The Role Of Ddx56 In The Disease

Parkinson’s disease(PD)is a common neurodegenerative disease,mostly in the middle and old age,with a complex genetic background.Researches has found that the occurrence of aging and senile diseases is mostly based on the materials at early stage.The disease sensitivity caused by gene variation,plays a function in the whole stage of life,affects the course of the disease,and even brings forward the onset of the disease.This phenomenon is especially obvious in the early-onset cases.We identified new,PD related genes by using SNP microarray,linkage analysis,and whole exon sequencing(WES)in a multi-generation member of an early-onset PD genetic family.In this study,17 family members were genotyped in a family consisting of multiple members with early-onset PD(N = 8,5 of 8 were early-onset).Three chromosome segments(154.0-155.2 c M on chromosome 2,0-5.5 c M on chromosome 14,and13.2-15.0 c M on chromosome 16)were found to be related to pathogenic genes by linkage analysis.A total of 186 mutant locus were screened from linked regions.Combined with WES and coexpression analysis,24 PD related genes were screened out,and these loci showed family specificity.Then,we conducted first-generation sequencing to verify the DNA of individuals in the family,and Spearman rank correlation analysis showed that five locus genotypes were relate to PD,which Spearman coefficient R>0.6and P<0.05.We collected DNA samples from 28 sporadic PD patients and 30 normal people in Fuzhou,Sanming and other areas,conducted out-of-family verification of the above sites by sequencing,and screened out mutation sites with high risk for subsequent in vivo and in vitro functional studies.Through the construction of regression equation and risk calculation,the mutation of Ddx56 gene(OR= 10.923,P=0.041<0.05)and Aspn gene(OR=8.198,P=0.007<0.05)had significant influence on the outcome of PD.The probability of PD was 10.923 and 8.198 times,respectively.Subsequently,we conducted in vivo functional analysis of the above selected target genes.Firstly,four genes with high conserved(overall >60%)were selected through amino acid sequence conserved analysis.Tg(Vmat2: GFP)neuron fluorescent zebrafish strain were selected to perform knockdown of four target genes,and the dopamine nerve cells were observed.It was found that Galnt13 and Ddx56 gene knockdown significantly decreased the fluorescence area of dopamine neurons(P<0.05),but the changes of Aspn and Pdxdc1 were not significant.Then,gene expression pattern detection was performed on AB strain zebrafish.The results of overall in situ hybridization showed that Ddx56 was a maternal-expressed gene,which was widely expressed in early embryo.After 48 hours post fertilization(48 hpf),Ddx56 was mainly expressed in brain,spine and pancreas.Galnt13 was widely and strongly expressed in the early embryo,mainly in the midbrain region,but no obvious signal was seen in the spinal region and other organs.And quantitative PCR(QPCR)analysis showed that Galnt13 was highly expressed in brain and eye,and Ddx56 was highly expressed in brain and testis,both of which were correlated with the distribution and developmental phenotype of brain neurons.We also examined the expression of dopamine-related and PD-related genes,and the expression of dopamine-related genes in both neurons was altered.Compared with the control group,the expressions of PD-related Sncgb,Syn2 b and Lrrk2 genes were significantly different when the Galnt13 knocked down(P<0.05).When Ddx56 knockdown,the gene expressions of PD-related genes Sncga,Sncgb,Polg,Pink,Parkin,Lrrk2,Gba and Sncb were significantly changed(P<0.05).The function of Ddx56 gene was significant,which was included in the next study.We performed an informatic analysis of Ddx56 mutation on protein structure.The serine at position 302 of the protein is mutated into lysine,which has some difference in properties.The mutation site was located in a segment of alpha helix,located at the C-terminal of helicase,which may be closely related to the basic function of this gene.Then,the Ddx56 gene knockout zebrafish was constructed by CRISPR/Cas9 system.The sequencing was performed on the F1 generation and homozygous lethal detection on the F2 generation.And the behavior trajectory experiment of 5dpf juvenile fishes was conducted.It was found that Ddx56(-/-)zebrafish died 4-10 days post fertilization(4-10 dpf)during development.At the same time,the percentage of Ddx56 knockout in the moving defect group was significantly higher than that in the moving normal group at each time point.The results showed that the gene mutation had an effect on the mobility of zebrafish in the early development stage,similar to the stride and gait disorders in the early and pre-onset PD.Immunofluorescence assay showed that the expression of tyrosine hydroxylase(TH1)protein in brain region of F2 Ddx56(+/-)、Ddx56(-/-)juvenile fish was significantly decreased(P<0.05).AO staining showed that the apoptosis of Ddx56(+/-)、Ddx56(-/-)brain regions was significantly increased(P<0.05).Preferential behavior analysis and T-maze behavior detection found that the part of cognitive function of Ddx56 knocked out fish decreased significantly,similar to the cognitive impairment of PD.After the in vivo experiment,the function of Ddx56 gene was studied in vitro.The lentiviral vector of Ddx56 interference(Ddx56-Sh)/overexpression(Ddx56-OE)was constructed and transfected into SH-SY5 Y human neuroma blasts.The results of QPCR showed that Ddx56 gene had 14.69 times overexpression and 0.28 times interference expression.The protein overexpression level of WB was 1.31 times and the knockout expression level was 0.71 times.Flow cytometry showed that the percentage of apoptosis in Ddx56-Sh group was significantly increased(P<0.05).There was no significant difference in apoptosis ratio in Ddx56-OE group.KEGG functional clustering analysis of RNA-seq data showed that the differential gene changes caused by Ddx56 knockout were mainly concentrated in the immune system,energy metabolism,protein translation,signal transduction and other aspects,especially in the ribosome and metabolic pathways,with more than 20 differentially expressed genes in each pathway.Meanwhile,differentially expressed genes are significantly associated with neurodegenerative diseases such as PD,Huntington’s disease,and Alzheimer’s disease,and change the level of oxidative phosphorylation.In this study,through sequencing analysis of PD genetic families and sporadic patient samples,the mutation sites that may be associated with the disease were screened out.Through multi-level informatics analysis and in vitro and in vivo experiments,the basic expression pattern of Ddx56 gene in early development was identified,and it was found that Ddx56 could induce neuron apoptosis and behavioral disorders in zebrafish.The association between the mutation of this gene and the apoptosis of dopamine neurons was established.The effect of gene knockdown on neuron survival was verified by in vitro experiments,and its close association with neurodegenerative diseases was confirmed at the transcriptome level.The action of this gene in the early stage may affect the probability of individual disease occurrence and the course of disease progression,and its molecular mechanism needs to be further studied.This study lays a foundation for early molecular diagnosis,prediction and evaluation of PD.