| Objectives:Cleft lip（CL）is a common congenital deformity with 1‰-4‰morbidityis.In China,its morbidity at birth is about 1.84‰.The category of CL includes simple cleft lip,simple palate cleft and cleft palate and lip.According to whether it is accompanied with abnormality of other tissues,it can be categorized as syndromic cleft lip with or without cleft palate（SCLP）and nonsyndromic cleft lip with or without cleft palate（NSCLP）,which takes up to 30%and 70%respectively of the total amounts of CL/P.The pathogenicity of cleft palate and lip is very complicated since it includes the interaction of environment and genetics and also is regulated by many factors.If the pathogenic locus or susceptibility genes of CL could be found,it will be very helpful to the early diagnosis,risk forecasting,prevention and intervention of nonsyndromic cleft lip（NSCL）.DNA methylation is one of the epihenetic modifications found in genome.DNA methylation can suppress gene expressions normally,whereas demethylation can induce the re-activations and expressions of genes.Till now,technologyies used in DNA methylation inspection included Bisulfite Sequencing PCR（BSP）method,Methylation-Specific PCR（MSP）method,MethyLight method and ChIP onchip,etc.among them,CHIP onchip has become an important platform for candidate genes screening of multe-genes complicated genetic diseases.In the study,we applied the methylation chip technology to screen the abnormal methylation locus in partial lesion tissues from NSCL fetus and the candidate locus were preliminarily validated through pyrosequencing among enlarged samples.Functions of candidate genes were studied in primary rat myoblast.It is expected to confirm the candidate genes of NSCL for future prenatal diagnosis.Methods:1.Samples Collection.（1）NSCL Group:16 lesion issues from odinopoeia NSCL fetus diagnosed by Prenatal Diagnosis Center of 2nd Affiliated Hospital of CMU before 2012 Dec,whose gestational weeks were from 22 to 30.（2）Normal control Group:15 lip tissues from non-CL odinopoeia fetus with matched gestational weeks.2.Screening of the abnormal methylation lotus using chip technology.3 cases were selected（2 female fetuses cases,1 male fetus case）from NSCLgroup and control group respectively.DNA was extracted from cleft tissues and performed methylation chip.The Illumina 450K Infinium Methylation BeadChip was utilized in the experiment.3.Bioinformatics analysis of methylation chip results.MINI-R software,GO annotation,Pathway metabolic pathway annotation,cluster analysis of multi-samples expression modes and other bioinformatics were applied for functional analysis on candidate genes.4.Detection of candidate genes expressions in tissues samples by Real-Time PCR.We identified the candidate genes based on two criteria.First,the genes that have ever been reported to be related with CL morbidity and the ones which showed significantly different methylation level in our chip results with strong significant difference per statistical analysis（p<0.05,|beta.difference|>0.14）.Second,those that play important role in cell pathway.In our experiment,we used RealTime-PCR method to detect the expression of candidate genes in 16 cases of NSCL group and 15 cases of control group.5.Validation of abnormal methylation genes using pyrosequencing.DNA was extracted and purified by methylation conversion Kit.PCR was run with conversed DNA samples as templates:10μL of PCR products were analyzed on the PyroMark Q96 real-time quantitate pyrophosphoric acid series analysis meter.6.Candidate gene proteins were detected with Western Blot and immunohistochemistry in the NSCL and control groups.Protein levels from NSCL group and control group were validated by Western Blot technology.At the same time,lesion tissues were utilized using immunohistochemistry.7.Functional studies of candidate genes in primary rat myoblast.Dab1 gene expression in primary rat myoblast was silenced by SiRNA l.Transfection efficiency at different time was measured by Real-Time PCR and Western Blot method.Cell migration capability was detected by cell wound scratch assay and transwell method.Cell apoptosis level was detected by flow cytometry.Cell proliferation capability was detected by MTT method.Results:1.The screening of methylation discrepancy genes in the lesion tissue samples by methylation chips.（1）There existed lots of abnormal methylation genes in the NSCL odinopoeia fetus comparing with the normal tissues,among which there were 3661 high methylation gene locus and 1218 low methylation gene locus.These loci distributed in2849 genes and spread widely in chromosomes.Most abnormal methylation genes were distributed in the gene noumenon region and CpG islands.There were also some abnormal methylation level genes located in TSS1500 and island shore.（2）Several genes that showed abnormal methylation level（p<0.05）included MSX1,BMP7,EGFR,PAX9,GLI2 and IRF6 have been reported to be related to CLP morbidity.（3）Two key transcription factors in REELIN cell pathway,both DAB1 and FYN,showed high methylation levels.In view of REELIN being the key member of the pathway,we will regard REELIN,DAB1 and FYN all as research objects in the following research.2.The mRNA expression of candidate gene was detected in tissue lesions by Real-Time PCR.We used real-time PCR to detect the expression of 9 candidate genes in tissue lesions.The result showed that DAB1,FYN and REELIN genes expression were decreased in NSCL group（p<0.05）.MSX1,GLI2 and IRF6 gene expression levels were increased in NSCL group（p<0.05）.BMP7,while no difference was found fir EGFR and PAX9 genes.3.The chip results were validated by pyrosequencing.We enlarged the samples numbers and pyrosequenced DAB1 and FYN which showed strongly significant decrease in NSCL samples.The result showed that there really existed significant methylation level discrepancy（p<0.05）for DAB1 gene.The correlation analysis result showed that the methylation degree of one of the two loci presented negative relationship with the DAB1gene’s expression level（P<0.01,r=-0.6076）.Namely,there existed high methylation phenomenon in NSCL group compared with control group,while there was no statistical significance for FYN.4.DAB1 and FYN proteins in the lesion tissues were detected using Western Blot and immunohistochemistry.In our study,we detected the protein level of DAB1 and FYN in tissues using Western Blot and immunohistochemical,and the protein showed significantly decreased in NSCL tissues（P<0.001）.5.The silence efficiency of siRNA-Dab1 was measured in primary rat myoblast.The Dab1 levels were detected 12 hrs,24 hrs and 48 hrs after SiRNA-Dab1 transfection.The RNA expression level reached the lowest level at 24 hrs after transfection.Dab1 protein expression is mostly inhibited at 48 hrs after transfection.6.The measurement of cell transfer capability and apoptosis status of primary rat myoblast after DAB1 gene was inhibited.The cell wound scratch assay and Transwell assay results indicated that the cell transfer level was suppressed after the DAB1 gene was silenced.The apoptosis detection result by streaming cell meter showed no change in apoptosis level.The result of cell proliferation inspected by MTT method indicated that there was neither difference in proliferation.So it can be assumed that the high methylation of DAB1 gene may affect its expression level,which could influence cellular transfer capability and result in the tissue fusion abnormality during facial formation.Conclusion:1.There were plenty of abnormal methylation loci widely spread in genome in NSCL lesion tissues.The abnormal methylation loci mainly distributed in gene noumenon region and CpG Island,few was found in TSS1500 and island shore.2.The abnormal gene expression of REELIN signals pathway is related with NSCL morbidity.3.The low DAB1 expression resulted from its abnormal high methylation in NSCL lesion tissue is one of the factors related to the occurrence of NSCL.4.In primary rat myoblast,decrease of Dab1 gene expression can result in the significant reduction of cell migration capability,but it has no impact on apoptosis and proliferation. |
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