| Down syndrome (DS) is the most frequent chromosomal disorder occurring in 1 800 to 1:600 of pregnant women, A majority of infants with DS have an IQ of less than 50, making this syndrome one of the leading causes of mental deficiency in the world, some survival have poor self-care ability, there is no effective treatments currently. Current means of prenatal diagnosis, such as amniocentesis and chorionic villus sampling, carry small but significant risks of miscarriage, although these methods are highly accurate, Currently no specific biomarkers exist for the screening of pregnancies at risk for DS. The genetic basis for DS is trisomy 21, one hypothesis is conferred by the increased expression of one or more of the district genes on the extra copy of chromosome 21.This DS chromosomal region (DCR) consists of 50-100 genes included SOD1, COL6A1, ETS2, CAF1A, CBS, DYRKA1, CRYA1, GART, IFNAR, APP, GLUR5, S100B, TAM, PFKL. Also the exact pathogenesis of DS is not fully understood, the genes in DCR are associated with the main features of DS. We speculate that there are any pathophysiological changes in serum of pregnant woman with DS because the over-expression genes, comparing with normal pregnant women.We analyzed quantitative proteomics characterization of the maternal serum samples of DS and normal pregnant women by iTRAQ technology coupled with MS/MS liquid chromatography, for determining different proteins. Bioinformatic analyses on the proteins identified were conducted deeply to obtain the information including molecular function, protein-protein interactions. We also investigated the differences between maternal serum metabolomics spectra of DS and normal pregnant women, then established prenatal screening models. Our study might provide the basis on new tests for improved DS screening.Section 1 Quantitative proteomics analysis of maternal serum of Down syndrome pregnancies using isobaric tagging reagent (iTRAQ)OBJECTIVE:To identify different proteins in maternal serum samples of DS compared with normal pregnant women using quantitative proteomics methods.METHODS:A nested, case-control study was performed using maternal serum from Down syndrome prenatal screening in Obstetrics and Gynecology Hospital affiliated Fudan University. Eighteen maternal serum samples of DS and gestational age-matched controls which consisted of 18 healthy pregnant women were investigated using isobaric tags for relative and absolute quantification (iTRAQ) technology coupled with MS/MS liquid chromatography. Vitamin D binding protein (VDBP) was vrified by western blotting.RESULTS:In our quantitative proteomics analysis, a total of 86 proteins were identified. We found 31 proteins differently expressed between the two groups using iTRAQ. Of these, involved four major biological process, that was cellular process (75.9%), biological regulation (72.4%), metabolic process (69%), response to stimulus (58.6%), and three major function which was protein binding (54.8%), transporter activity (38.7%), enzyme regulator activity(29%).CONCLUSION:Our study therefore indicates that the iTRAQ labelling approach may be indeed useful for the detection of novel biomarkers.Section 2 Bioinformatic analyses on the proteinsOBJECTIVE:To get the information including molecular function, protein-protein interactions, bioinformatic analyses on the proteins identified were conducted deeply.METHODS:Functional clustering were filterd using Database for Annotation, Visualization and Integrated Discovery (DAVID). Ingenuity Pathway Analysis system(IPA) was used to excavate the data from clustered proteins and the known screening markers for DS (AFP,HCG,PAPP-A,INHA) respectively. we also analysis the protein-protein interactions on DS related gene(SOD1, COL6A1, ETS2, CAF1A, CBS, DYRKA1. CRYA1. CART. IFNAR, APP. GLUR5, S100B.,TAM, PFKL).RESULTS:The cluster related DS is lipid transport, enzyme regulator activity, cell development and apoptosis. Three networks which were lipid metabalism,Cardiovascular System Development and Function. Nervous System Development and Function were obtained by IPA. The key node of those networks is ERK, Some key molecules (APOE,IGFBP3,AGT,KNG1,AFM) were related to ERK.CONCLUSION:Changes in ERK pathway may be the pathophysiology of nervous and cardiac developmental defects of Down syndrome fetus, APOE,IGFBP3,AGT KNG1,AFM may helpful for prenatal screening in future. Section 3 Application of metabolomics to establish prenatal screening for Down syndrome Model at the second trimesterOBJECTIVE:To establishment of prenatal screening models by analizing differences between maternal serum metabolomics spectra of DS and normal pregnant women,METHODS:A nested, case-control study was performed within a prospective cohort study of Down syndrome prenatal screening in Obstetrics and Gynecology Hospital affiliated Fudan University. The serum samples were obtained when they came for Down syndrome screening. We investigated nine maternal serum samples of DS and gestational age-matched controls which consisted of 44 healthy pregnant women. The serum metabolic profiles were obtained by High-performance liquid chromatography-time of flight mass spectrometry and the combination of the ESI+ and ESI-scans were used to provide more useful information about the samples. Principal components analysis(PCA) and Partial least squares discriminant analysis (PLS-DA) were used to compare such metabolic profiles. Support Vector Machines (SVM) was applied to build prenatal screening model.RESULTS:Case and control groups were clustered clearly by PCA and PLS-DA analysis. Precision of Down syndrome is 88.9%, while false positive rate is 2.3%. Ions were involved in forecasting from the ESI+ scans were 127.2Da,213.2Da,115.1 Da. 241.5Da,214.2Da,110Da,131.1 Da,143.2Da,459.5Da and ESI-scans was 470.8Da.CONCLUSION:Metabolomics Methods of prenatal screening model was better than the classical method. Further characterization and quantification of these markers in a larger cohort of subjects may provide the basis for new tests for improved DS screening.
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