Screening And Verification For Genes Associated With Neural Tube Defects By Methylome Analysis

Objective: Neural tube defects are a group of complex congenital malformations of the central nervous system,affecting 0.5-2 per 1000 pregnancies worldwide.In China,the incidence of NTDs is very high,especially in Shanxi Province.NTDs can lead to spontaneous abortion,infant death,and multiple handicaps,the latter of which can be a dual pressure on the spirit and finances of surviving children and their families.Therefore,it is of great significance to study the mechanism of NTDs for finding out the scientific prevention and treatment methods.The etiologies of NTDs are complicated,with both genetic and environmental factors implicated.Researches showed that aberrant methylation could play some causal roles in increasing the risk of failed neural tube closure.A previous study demonstrated that abnormal global DNA methylation due to the 677C>T variant in 5,10-methylene tetrahydrofolate reductase gene(MTHFR)significantly increased the risk of human NTDs.DNA methylation levels of long interspersed nucleotide elements reduced significantly in the brain tissue of NTD samples.Chen X et al.reported the association between global DNA hypomethylation and NTD-affected pregnancy in fetal brain tissue.In addition,hypermethylation or hypomethylation of specific genes associated with DNA repair,folate receptor,imprinting,transposon have also been revealed to play vital roles in NTDs.The above experiments were performed mostly by using the brain tissues from NTD animal models.However,due to the lesion of the spina bifida is mainly located in the spinal cord,and the pattern of DNA methylation showed tissue specific,it is more appropriate to study spina bifida by using spinal cord tissues.Unfortunately,there was only one study which focus on the DNA methylation pattern of spinal cord in NTD and showed no aberrant methylation in specific genes in spinal cord and brain in NTD cases.It is reasonable to explore the underlying mechanism of NTD by using spinal cord tissue at both of the genetic and epigenetic levels.Therefore,in present study we explored the novel aberrant DNA methylation in genome-wide level in fetal spinal cord tissues of NTDs using the Infinium Human Methylation450 BeadChip Array(TM)(Infinium 450K)and further examined the expression levels of candidate genes.And we explored the candidate genes on different embryonic days in the animal model,in order to further explore the pathogenesis of NTDs,provide scientific basis for preventio and treatment.Methods: DNA from spinal cord tissues from both NTD group(3 cases)and control group(3 cases)were used for methylation array assay.Online SAS analysis system was used for GO annotation,Pathway metabolic pathway annotation,clustering analysis and other bioinformatics methods for the functional analysis of differentially expressed proteins.14 cases of spinal cord tissue and 14 cases of fetal spinal cord tissue of control group were collected respectively.Real-time PCR was used to detect the mRNA levels of candidate genes.Pyrosequencing or bisulfite sequencing(BSP)methods were performed for identifying the methylation level of different transcription regulatory regions in candidate genes.To investigate whether SNP contributes to the altered expression of candidate gene in children with NTDs,in addition to DNA methylation,we analyzed genomic DNA sequencing result of TRIM4 in 100 NTD patients,which were independent of these 14.Western blot analysis was performed to further verify the change of TRIM4 protein expression level in NTDs compared with controls.An animal model of spina bifida was established to detect the mRNA expression of candidate genes in different embryonic stages and the mRNA levels in animal models were changed.The methylation level of candidate genes was detected by BSP method.Results Statistical Real-time PCR data were expressed as 2-??ct values.Western blot analysis was expressed as relative density and the data were shown as mean ± SEM.The Graphpad Prism6 statistical analysis software performs statistical analysis on experimental data.For data subject to normal distribution,the paired t test or one-way analysis of variance is used for comparison between the experimental group and the control group.For the data that does not obey the normal distribution,the experimental group and the control Wilcoxon paired sign rank sum test was used for comparison between groups,p<0.05 considered statistically significant.Results: A total of 4648 methylated sites were screened out,among which 1987 sites were highly methylated and 2661 were highly methylated.There were 2217 genes corresponding to these methylation loci.We performed GO analysis on 2217 DNA methylation differential genes and found that these genes involved a total of 14 molecular functions,11 cell components,and 17 biological processes.The interest gene mRNA levels were measured.In the abnormal group mRNA expression upregulated and have a statistically significant difference between the gene STAT1,STT3 A,SEC61G,EIF2S1,ApoA1,APOA1 BP,TRIM4,tlr1,PTRF,NFIX,CD40;expression was downregulated and have a statistically significant difference between the gene SLC7A11,MAP2K2(P < 0.05).Therefore,these genes are candidate genes for preliminary screening.The expression level of pyrosequencing of the differential gene of mRNA,found that there are differences of methylation levels of CD40,TRIM4 and STT3A gene.In CD40 the overall methylation level of these regions of the tube was hypomethylated,deformity group in neural p=0.0002,the difference was statistically significant.In TRIM4 the overall methylation level of the two regions is significantly lower in the NTD group,p=0.0003,with significant difference.We apply the BSP method to verify the STT3 A gene differences between CpG sites methylation level.The results showed that compared with the normal group,neural tube malformation group CpG sites 3 methylation level and 5 CpG sites in the overall level of methylation was increased,there was statistically significant difference.The expression of TRIM4 in fetal spinal cord tissues was significantly higher in the NTD group than the control group,as measured by Western blotting.To investigate whether SNP contributes to the increased expression of TRIM4 gene in children with NTDs,in addition to DNA methylation,next-sequencing and Sanger sequencing was used.Putting together our mRNA expression data,DNA methylation data and the sequencing data we suggested that genetic variants in TRIM4 genes have only faint contribution to pathogenesis of human NTD.The main factor affecting the mRNA level of TRIM4 in NTDs is the change of DNA methylation.We detected the mRNA expression of candidate genes in different embryo stages in a rat embryonic spina bifida model.Both the literature and our microarray results show that the methylation level of MGMT in human neural tube defects is different from that in the control group and not in animal spina bifida model.Therefore,we also use the MGMT gene as a candidate.One of the genes.Since the TRIM4 gene was not studied in rats,mRNA sequences could not be found so that primers could not be designed for PCR.We only detected mRNA expression levels of CD40,STT3 A,and MGMT.We found that CD40,STT3 A,and MGMT were all highly expressed during the early embryo stage,and the expression was lowest after E14,and CD40 and STT3 A were expressed in the neural tube malformation group and the normal control group.No difference.The expression of E14 and E18 decreased,and the difference was statistically significant.We used BSP method to detect the methylation level of MGMT promoter,and found that the mean methylation level of MGMT was not statistically different from controls.Conclusion: A total 4648 differential methylation locis were screened out by Illumina 450 K methylation chip in neural tube defects,among which 1987 sites were highly methylated and 2661 were highly methylated.There were 2217 genes corresponding to these methylation loci.The expression and methylation level of STT3 A,CD40 and TRIM4 genes between NTD and control groups has significant differences,they are epigenetic neural tube defects marker.TRIM4 protein is highly expressed in the NTD group and is associated with neural tube defects.Genetic variants in TRIM4 genes have only faint contribution to pathogenesis of human NTD.The main factor affecting the mRNA level of TRIM4 in NTDs is the change of DNA methylation The expression of mRNA of MGMT was down regulated in the rat spina bifida model induced by retinoic acid,but there was no significant difference in the methylation level.DNA damage and DNA repair response were involved in SBA rats.