Screening Of Novel Biomarkers In Maternal Blood For Prenatal Screening Of Down’s Syndrome And Construction Of Detection Methods For Biomarkers

Down’s syndrome(DS) is one of the most common geneogenous physical and intellectual developmental deficiencies caused by chromosomal abnormalities in humans, with a prevalence rate of 0.1 to 0.2% for newborn infants. Owing to the increasing work pressure and maternal age, as well as the application of assisted reproductive technologies, the majority of DS has been rising. However, there is no effective method to cure the disease, and DS-affected patients are usually deprived of self-care agency, which may result in much trouble for the patients, affected families and society. Therefore, a large number of prenatal screening and prenatal diagnosis models are introduced to DS-affected pregnancies, which is very important and valuable in achieving a final diagnosis of DS and may provide more choices for DS-affected pregnant women.Chromosome analysis of fetus cast-off cells obtained by invasive procedures is the gold standard for prenatal diagnosis of Down syndrome(DS), whereas the invasiveness of amniocentesis or chorionic villus sampling may result in some abortions with a ratio of 1%, and warrants the discovery of novel noninvasive predictive biomarkers. Thus, the prenatal screening tests have been applied to the risk evaluation in DS-affected pregnancies in the majority of countries. There are various voluntary screening tests used for the prenatal screening tests of DS-affected pregnancies. There screening tests based on their pregnancy gestation, age, and weight present many combinations of biomarkers, which include alpha fetoprotein(AFP), total human chorionic gonadotropin(h CG), free beta subunit of h CG(β-h CG), unconjugated estriol(u E3), pregnancy-associated plasma protein A(PAPP-A), proform of eosinophil major basic protein(Pro MBP), inhibin-A, placental growth factor(PGF), and so on. In this study, we utilized the meta-analysis method to systematically evaluate the screening performance of the serum triple screening test(STS) consisting of alpha fetoprotein(AFP), unconjugated estriol(u E3), and human chorionic gonadotropin(h CG) measurements and the integrated screening test(INS) composed of theultrasonographic nuchal translucency(NT) measurement and serum screening tests, and compare the discrepancy between the screening performance of the two tests. The results revealed that the INS presented better screening performance than the STS, and could be recommended as the first-choice screening test for DS, whereas more research would be performed so as to search for other biomarkers with higher specificities and more predictive power, which would help to improve prenatal screening tests for DS-affected pregnancies.Therefore, on the basis of earlier studies, the preliminary investigation into metabonomics and micro-RNomics in maternal serum or plasma of DS-affected pregnancies were also performed to screen for other kinds of potential biomarkers for DS-affected pregnancies. Then, the western blot test was conducted to verify the four proteins including complement factor H-related protein 1 precursor(CFHR1), d GTPase, kininogen 1 isoform 2(KNG1), and β2-glycoprotein I(β2-GPI), and the time-resolved fluoroimmunoassay(TRFIA) methods were established to detect the concentrations of these proteins in maternal serum.Objectives: 1.Evaluate the screening performance of the STS and INS; 2.Screen for the potential metabolic markers in maternal serum of DS-affected pregnancies and explore the pathological mechanism of DS which is associated with the metabolic pathways. 3.Screen for the potential micro RNA markers in maternal plasma of DS-affected pregnancies and explore the pathogenesis of DS which is associated with the gene expression. 4.Verify the level changes of CFHR1, d GTPase, KNG1, and β2-GPI in maternal serum and establish the TRFIA detection methods.Methods: 1.A systematic literature search was conducted in Pub Med, the ISI Science Citation Index, EMBASE, China Biology Medicine disc(CBM disc), China National Knowledge Infrastructure, CNKI), and VIP resource integration service platform to acquire the most comprehensive publications. Based on the inclusion criteria and exclusion criteria,several studies were determined and evaluated by the pooled sensitivity, specificity, positive likelihood ratio(PLR), negative likelihood ratio(NLR), diagnostic odds ratio(DOR), fitting summary receiver operating characteristic(SROC) curves, and the area under the curve(AUC), and the screening performance of the STS and INS was assessed, to provide the theoretical basis for subsequent research. 2.The liquid chromatography-tandem mass spectrometry(LC-MS/MS) analysis and the gas chromatography-mass spectroscopy(GC-MS) analysis were employed, and combined with the results of bioinformatics studies, to set up the analysis model of metabonomics in maternal serum from UP and DP. Then, the principal component analysis(PCA), cluster analysis, and random forest(RF) analysis were performed, and the Welch t-test was used to screened for the metabolic molecules whose levels differed significantly between UP and DP. 3.Total RNA was extracted with TRIzol LS and purified using RNeasy mini kit, and RNA quality and quantity was measured using Nano Drop-1000 spectrophotometer. After that, the mi RCURYTM Array Power Labeling kit was used for RNA labeling and array hybridization, and the slides were scanned using the Axon Gene Pix 4000 B microarray scanner, and the raw intensity of the image was read and analyzed using Gene Pix pro V6.0. to set up the analysis model of micro-RNomics in maternal plasma from UP and DP. Then, the cluster analysis, and RF analysis were performed, and the unpaired t-test was used to screened for the micro RNA molecules whose levels differed significantly between UP and DP. 4.The western blot test was performed to verify the four proteins including CFHR1, d GTPase, KNG1, and β2-GPI, and the TRFIA methods were established to detect the concentrations of d GTPase, KNG1, and β2-GPI in maternal serum. Then, the methodological evaluation, including the linear detection ranges, the sensitivity exploration, the precision experiments, the specificity experiments, and so on, was conducted, and the concentration ranges of d GTPase, KNG1, and β2-GPI were preliminarily investigated in serum of DS-unaffected pregnant women(UP) and DS-affected pregnant women(DP).Results: 1.(1) After the systematic search and selection, eighteen publications with 183,998samples met the inclusion criteria and were determined for this meta-analysis, which included thirteen studies for STS and six for INS.(2) The results of INS revealed that the pooled sensitivity was 0.77 with the 95% confidence interval(CI) of 0.73-0.81, the pooled specificity was 0.94(95% CI = 0.94-0.94), the PLR and NLR were 9.78(95% CI = 6.87-13.93) and 0.26(95% CI = 0.22-0.31); the pooled DOR was 44.72(95% CI = 30.77-65.01), the Q value of the SROC curve was 0.8381, and the AUC was 0.9064, respectively.(3) The results of INS showed that the pooled sensitivity presented 0.93(95% CI = 0.90-0.95), the pooled specificity presented 0.93(95% CI = 0.93-0.93), the PLR and NLR were 22.38(95% CI = 12.47-40.14) and 0.08(95% CI = 0.05-0.11); the pooled DOR was 289.81(95% CI = 169.08-496.76), and the Q value and AUC for the SROC curve were 0.9337 and 0.9781, respectively.(4) It was apparent that the INS curve was above the STS curve in one figure, and the z test indicated that both the AUC and Q value were remarkably greater for the INS than the STS(z statistic = 3.957, p < 0.01; z statistic = 4.613, p < 0.01, respectively). 2.(1) The results of PCA for metabonomics in serum showed that the contribution ratios of comp 1, comp 2, and comp 3 were 34.86%, 10.46% and 5.99%, suggesting the stability for the analysis model of metabonomics.(2) The RF analysis provided a forecast accuracy of 97%, and revealed that the pathogenesis of DS might be related with carbohydrate metabolism, amino acid metabolism, heme metabolism, energy metabolism, lipid metabolism, nucleotide metabolism.(3) A total of 193 metabolic compounds expressed significantly differentially were screened out, including the increased levels of 16 molecules and decreased levels of 177 molecules. 3.(1) Results of the cluster analysis for micro RNA in plasma showed that the levels of micro RNA between six samples within a group were homogeneous, presenting a good discrimination between UP and DP.(2) The scan results of micro RNA chips revealed that gene loci were clarity, the background of image was distinct, and the colour and luster were uniform.(3) A total of 135 micro RNA molecules expressed significantly differentially werescreened out, including the increased levels of 95 molecules and decreased levels of 40molecules. 4.(1) Western blot tests revealed that levels of CFHR1, d GTPase, and KNG1 in DP were increased significantly, and β2-GPI was decreased remarkably, compared with UP.(2) The TRFIA detection results of d GTPase showed that the suitable concentrations of the fixed antibody, free antibody, and Eu3+- labelled antibody were 25 μg/ml, 10 μg/ml and 10 μg/ml respectively, and the equation of standard curve was “lg(y) = 0.867lg(x)+lg1789”(R2 = 0.992), the sensitivity was 3.06 ng/ml.(3) The TRFIA detection results of KNG1 showed that the suitable concentrations of the fixed antibody, free antibody, and Eu3+- labelled antibody were 25 μg/ml, 2.5 μg/ml and 5 μg/ml respectively, and the equation of standard curve was “lg(y) = 0.982lg(x)+lg1579”(R2 = 0.994), the sensitivity was 2.16 ng/ml.(3) The TRFIA detection results of β2-GPI showed that the suitable concentrations of the fixed antibody, free antibody, and Eu3+- labelled antibody were 10 μg/ml, 5 μg/ml and 5 μg/ml respectively, and the equation of standard curve was “lg(y) = 1.009lg(x)+lg1164”(R2 = 0.991), the sensitivity was 3.91 ng/ml. All results of the methodological evaluation for three proteins, including the precision experiments, the specificity experiments, the recovery experiments, and so on, met the research requirements.(4) The preliminary concentration ranges of d GTPase, KNG1, and β2-GPI in UP, DS-unaffected high-risk pregnant women group, and DP were 40.9±1.7 μg/ml, 43.2±2.6 μg/ml and 95.6±4.4 μg/ml, 84.3±1.3 μg/ml, 98.2±4.8 μg/ml, and 231.9±8.2 μg/ml, 215.9±13.6 μg/ml, 203.4±11.1 μg/ml, and 103.8±1.4 μg/ml, respectively. There were significant differences in the levels of d GTPase, KNG1, and β2-GPI between UP and DP, as well as between DS-unaffected high-risk pregnant women group and DP, while no significant difference existed between DS-unaffected high-risk pregnant women group and UP.Conclusions: 1.Both STS and INS presented relatively low specificities and screening performance. Therefore, more research would be performed so as to search for other novel biomarkers with higher specificities and more predictive power. 2.In metabonomic study, a total of 193 metabolic compounds whose levels differedsignificantly between UP and DP were discovered, which might provide the foundation for the screening of potential metabolic markers for DS-affected pregnancies. 3.The exploration of the metabolic pathways might provide some clues for the exploration of the pathological mechanism of DS. 4.In micro RNA study, a total of 135 micro RNA molecules, such as hsa-mi R-483-3p, hsa-mi R-3667-5p, hsa-mi R-16-2-3p, hsa-mi R-576-5p, and so on, whose levels differed significantly between UP and DP, were discovered. The research might provide the foundation for the screening of potential micro RNA markers for DS-affected pregnancies, 5.The exploration of micro RNA markers and the gene expression might provide some clues for the exploration of the pathogenesis of this disease. 6.The results of western blot tests suggested that CFHR1, d GTPase, KNG1, and β2-GPI might be potential novel protein markers for DS. 7.The TRFIA methods for d GTPase, KNG1, and β2-GPI in maternal serum were established, the relevant methodological evaluation was conducted, and the concentration ranges of three proteins were preliminarily investigated, which provided some foundations for the exploration of novel prenatal screening strategies for DS-affected pregnancies.