| Monozygotic twins(MZ)are formed by the development and division of the same fertilized egg.They have the same set of gene sequences and should theoretically have the same phenotype.However,one of the twins often has abnormal development or malformation in clinic,and the cause of the disease is not clear.Epigenetics is a newly emerging genetic research in recent years.It means that the expression and function of genes change without changing DNA sequence.Epigenetics may be an important pathogenesis of dysplasia in monozygotic twins.This dissertation intends to use DNA methylation analysis method to explore the etiology and to analyze the correlation between fetal diseases and epigenetics,which has important clinical significance for early prevention and treatment.Objective: To investigate the role of DNA methylation in the development of one of the monozygotic twins by analyzing the genome-wide DNA methylation levels and disease-related gene differentially methylated regions(DMRs)of monozygotic twins with different phenotypes.Materials and Methods:From May 2016 to February 2019,patients with dysplasia of one twin were selected in our hospital.First,the identical/heterogeneous twins were identified by Quantitative Fluorescent Polymerase Chain Reaction(QF-PCR),and the cases identified as monozygotic twins were included in the study.Whole genome DNA methylation sequencing(WGBS)was performed after excluding copy number variations(CNVs)and single-nuceotide polymorphisms(SNPs)abnormalities and disease-related SNPs sequence differences among monozygotic twins by Chromosome microarray analysis(CMA)and whole exome sequencing(WES).DNA methylation levels among monozygotic twins were analyzed;monozygotic twins with similar phenotypes were grouped and DMRs among monozygotic twins in the same group were analyzed.Realtime quantitative fluorescent polymerase chain reaction(q PCR)and bisulfite sequencing polymerase chain reaction(BSP)was used to verify the detection of disease-related target gene DMRs.Results:(1)Twelve monozygotic twins were analyzed by WGBS.According to clinical phenotype,there are 7 pairs of monozygotic twins with congenital heart diseases(CHD)(14 cases),2 pairs of monozygotic twins with cleft lip and palate(4 cases),1 pair of monozygotic twin with multiple malformations(2 cases),1 pair of monozygotic twin with diaphragmatic hernia(2 cases),and 1 pair of monozygotic twin with exomphalos(2cases).According to the type of specimen,there were 2 pairs of amniotic fluid(4 cases),5 pairs of prenatal umbilical cord blood(10 cases),3 pairs of postnatal umbilical cord blood(6 cases),and 2 pairs of peripheral blood(4 cases).(2)There were significant differences in DNA methylation levels between monozygontic twins with different phenotypes(P<2.2e-16).(3)The level of DNA methylation in amniotic fluid samples was lower than that in cord blood and peripheral blood,and there were also statistical differences in DNA methylation levels between prenatal umbilical cord blood,postnatal umbilical cord blood and peripheral blood at different developmental stage(P<0.014～0.006).(4)Seven DMRs related to cleft lip and palate were found in the cleft lip and palate group of monozygotic twins,of which COL9A1 and YAP1 genes had significant methylation differences.25 DMRs related to cardiac dysplasia were found in the CHD group of monozygotic twins,among which the methylation of EHMT1 gene was more significant;and no DMRs related to disease gene was found in other groups.(5)A family with cleft lip and palate was verified by BSP and q PCR.The results showed that the expression of m RNA in genes of COL9A1 and YAP1 was high in patients with cleft lip and palate,which coincided with the downregulation of gene methylation.(6)Six pairs of twins in one of the monozygotic twins with cardiac dysplasia were tested for EHMT1 gene by BSP technique(One pair of twins failed to carry out validation experiments due to insufficient sample size).It was found that the DNA methylation level of EHMT1 gene in the affected group was significantly higher than that in the normal group.Conclusion:(1)There are obvious differences in DNA methylation levels among phenotypically discordant monozygotic twins.(2)DNA methylation levels were significantly different in amniotic fluid and blood samples from different tissue sources,and DNA methylation levels were also different in blood samples from different developmental stages.(3)The methylation levels of COL9A1 and YAP1 genes were significantly down-regulated in cleft lip and palate patients and affected the expression of these two genes.Considering that the methylation of COL9A1 and YAP1 genes was related to cleft lip and palate deformity in one of the monozygotic twins.(4)DNA methylation of EHMT1 gene was up-regulated in one of the identical twins with cardiac dysplasia.Considering that DNA methylation of EHMT1 gene was related to the occurrence of cardiac dysplasia in one of the monozygotic twins. |
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