A Preliminary Study On Genomic Instability And Methylation Status Of Imprinted Genes In Neural Tube Defects

Birth defects are structural or functional abnormalities during fetal development and the leading cause of death for foetus, physical or mental disability for infants and adults. Neural tube defects (NTDs), including anencephaly and spina bifida, are among the commonest human birth defects, affecting around1in every1000births throughout the world. Infants with anencephaly are stillborn or die shortly after birth, whereas infants with spina bifida can survive but are likely to have severe lifelong disabilities, which greatly affect their quality of life. Studies on NTDs have been conducted for many years and mainly include two aspects:first, looking for candidate genes for human NTDs; second, investigating the role of environmental factors in human NTDs. Researchers have been searching for candidate genes for NTDs. Biochemical and developmental pathways, mouse models and positional evidences have provided more than100candidate genes for the study of human neural tube defects. However, data from case-control or family-based association studies in human populations have indicated: NTDs can not be explained by dysfunction of single gene. The environmental factors involved in NTDs include maternal nutritional factors, infection as well as some physical and chemical factors. Among environmental factors, maternal folate status appears to be a key determinant of NTDs susceptibility. Low maternal vitamin B12and folic acid status may be risk factors for human NTDs. Preconceptional folic acid supplementation can effectively prevent the majority of NTDs (50%to75%). Folic acid plays a important role in the occurrence of human NTDs. Folic acid participates in DNA synthesis, DNA methylation and other important physiological processes as a methyl donor. Folate deficiency increased uracil misincorporation in DNA in mice that may lead to genomic instability; in addition, folate deficiency caused global DNA hypomethylation.It is possible that genomic instability and change of DNA methylation status play important roles in NTDs. Nevertheless, few are reports of NTDs associated with genomic stability and methylation status of imprinted genes. Microsatellite instability (MSI) is the one signal of genomic instability. The major mechanism of MSI is that strand slippage during DNA synthesis results in the addition or deletion of repeat units within the microsatellite and these DNA changes escape from DNA mismatch repair system (MMR) correction. Therefore, on one hand, MSI suggests the dysfunction of mismatch repairs genes; on the other hand, microsatellites can be important regulators of gene expression. Changes in microsatellite sequence length may affect related gene expression such as transcription rate, RNA stability, splicing efficiency and RNA-protein interactions.Epigenetics is the study of mitotically and/or meiotically heritable changes in gene function that can not be explained by changes in DNA sequence. DNA methylation is an important component of epigenetics that can regulate gene expression. Hypermethylation of some gene promoters could lead to their inactivation; DMRs(differentially methylated regions) were key regulators of the imprinted gene expression, methylation status of DMRs of the imprinted gene were associated with many congenital growth disorders.Despite these accomplishments, the mechanism of NTDs remains unclear. Therefore, it is conducive to explore further mechanism of NTDs from the genomic instability and epigenetic alterations.Objective:1.To investigate genomic instability in NTDs by MSI experiments.2. To investigate hMLHl and hMSH2promoter methylation patterns in NTDs.3. To investigate H19DMR1and IGF2DMRO methylation status in NTDs.4. To explore the connection between the genetic stability, abnormal methylation of imprinted genes and NTDs.Methods:1. PCR-urea denaturing polyacrylamide gel electrophoresis and Genescan were used to analyze MSI in NTDs. Six microsatellite loci Bat25, Bat26, Bat34C4, D2S123, D2S119and D3S1611were chosen as detection targets for MSI.2. Methyl-specific PCR (MSP) was used to analyze methylation status of hMLH1and hMSH2promoters in NTDs.3. Bisulfite sequencing PCR, subcloning and sequencing were used to analyze H19DMR1, IGF2MR0methylation status and hMLH1, hMSH2promoter methylation patterns.Results: 1. MSI exists in23of50NTDs by PCR-urea denaturing polyacrylamide gel electrophoresis and Genescan, total positive rate of MSI in NTDs is46%.2. hMLH1and hMSH2promoter sequences are unmethyled in NTDs by MSP; hMLH1and hMSH2promoter methylation patterns are detected by BSP, subcloning and sequencing, these results show that hMSH2promoter sequence is unmethyled as that of MSP, and that hMLH1promoter shows specific methylation pattern in4,5CG sites, which is no significant difference between NTDs and control groups.3. H19DMR1hypermethylation exists in14of21NTDs samples (66.7%) and in2of7control samples (28.6%) respectively, the hypermethylation rate of NTDs group is2.33times higher than that of control group.4. IGF2DMRO hypomethylation is detected in NTDs and control groups. There is no significant difference between NTDs and control groups.Conclusion:MSI and H19DMR1hypermethylation occur in NTDs. They maybe are associated with the occurrence of NTDs. IGF2TMR0is hypomethylation in the embryonic brain tissue studied, which indicates that IGF2DMRO methylation status does not associated with H19DMR1methylation status.