Experimental Research Of DNA Copy Number Variation And DNA Methylation Change In Fetuses With Congenital Heart Defect

BackgroundCongenital heart defect, referred to as CHD for short, is the most common birthdefects and the main cause of fetal death in human. Causes of most CHDs have not yet beenknown. Only a few CHDs were caused by gene mutations and chromosome aberrations.The major causes of CHD resulted from interaction of multiple genes and environmentalfactors. To research associating genetic factors and epigenetics with CHD will help usfurther undestand CHD and early diagnose it, which could make more efficient screeningand treatment of fetal CHDs for the quality of the newborn.Copy number variation is common in submicroscopic genetic alterations.Chromosome submicroscopic deletions or amplifications will alter gene expression andimpair fetal development. Genes regulating cardiogenesis play an important role inproliferation, differentiation and functions of myocardial cells. Submicroscopicchromosomal imbalances involving cardiac-specific transcription factors will cause severeheart defects. Genome-wide copy number analysis can identify novel submicroscopicdeletions and amplifications associated with heart development. Researchers areincreasingly employing the microarray-based technique for genome-wide screening of copynumber variations which may help identify pathogenic DNA imbalance. Then geneticinformation will be more understanded.DNA methylation is an important modification of epigenetic regulation, which hassignificant functions in embryonic development. Abnormal DNA methylation may causediseases such as cardiovascular malformations and even fetal death. Daily exposure to toxicenvironmental contaminants or trophic factors can trigger genetic changes and influencecellur metabolism, which may disturb dynamic balance of DNA methylation patterns withserious consequences to fetus. To clarify the molecular mechanism of CHDs withtraditional knowledge is not easy, revealing epigenetic regulation of gene expression underlying CHDs may uncover environmental factors effects without changing DNAsequence. That will provide new scientific basis for effective prevention of fetalcardiovascular defects.ObjectiveThe aim of this study is to explore the molecular mechanism of CHDs and to find newmolecular biomarker of fetal cardiovascular malformations by analyses of genomic copynumber variation and DNA methylation changes associated with fetal CHDs. Next we mayprovide experimental evidence for early prenatal diagnosis of fetal CHDs.Methods1. Affymetrix SNP6.0array was used to analyse10samples for detection of genomiccopy number variations, including5CHD fetuses with normal karyotypes,2CHD fetuseswith abnormal chromosome and1normal fetus, compared with HapMap90Asianpopulation. Copy number variants with biological information were selected for furtheranalysis and verification.2. Fluorescence quantitative polymerase chain reaction (qPCR) assay was used toverified copy number deletions and amplifications in CHD fetal DNA, compared with itsparents, normal neonates and healthy volunteers, which contain genes associated withembryonic heart development and possibly related to cardiogenesis.3. Rhoche NimbleGene3×720K CpG Island Plus RefSeq Promoter array was used toanalyses Genome-wide DNA methylation between complex CHD fetuses and normalfetuses. We chose3cases with endocardial cushion defect, single atrioventricular valve andtransposition of the great arteries matched with3normal fetuses with same gestational ageand gender. Data of the microarray were processed using NimbleScan software. DNAmethylation changes with the genomic information can be viewed on sequence graph fileswith NimbleScan software between cases and controls.4. The same case samples and control samples were further tested by SequenomMassARRAY EpiTYPER DNA methylation platform. Quantitative methylationanalysis and validation were focus on the corresponding regions of DNA methylationdifferences between the cases and controls. Effects of epigenetic modifications on fetalcardiovascular malformations were noted in a preliminary analysis.Results1. Seven CHD fetuses total of10samples underwent genome-wide CNV analyses.Many CNVs arised, only some contained biological information. Some gene CNVs wereobserved that have an important role in heart development. The twin fetuses that one is TOFand the other is normal had no difference in copy number by subsequent verification. 2. We verified all CNVs possibly related to heart development by qPCR. In addition tothe18-trisomy with the copy number amplifications of GATA6related to heart defects, onecopy number deletion at14q23.1was validated which contains whole DAAM1andKIAA0666genes. The single-copy deletion existed in one normal karyotype fetus withcomplex CHDs. DAAM1is essential for early embryonic heart development and its copynumber deletion may be a potential cause of complex CHDs for fetuses.3. DNA methylation differences of3cases with complex CHDs when compared to3matched controls were found that Log2-ratio of RADIL, CHRNA10and RADIL genepromoter methylation were higher0.41~0.59than that of the controls, NXPH3, ZNF408and ABL1gene methylation level was lower0.34~0.62than controls.4. The same cases and controls’ umbilical cord blood DNA were further tested bySequenom MassARRAY system for verification of methylation differences. The screeningresults cannot to be validated by the another methylation analysis method.Conclusions1. More frequent copy number variants than chromose abberrations cause humangenetic diversity that also increase the risk of specific birth defects.2. Frequent CHDs in fetus with trisomy18were associated with overexpression ofHsa18genes especially GATA6, which disturbed cardiac differentiation and thedevelopment of the embryonic outflow tract.3. Copy number deletion on the locus of14q23.1will result in complex CHD, wherecontained DAAM1gene that is indispensable for heart development.4. Significant methylation differences were detected in3fetuses with complex CHDby genome-wide screening of methylation changes. But the methylation changes could notbe verified with subsequent mass spectrometry quantitative analysis of methylation withsame samples. Epigenetic mechanisms of DNA methylation in complex CHD may needadvanced study for more clinical samples.