Application For Prenatal Diagnosis By Comparative Genomic Hybridization And Fetal DNA In Maternal Plasma

ⅠApplication for noninvasive prenatal diagnosis by fetal DNA in maternal plasmaBackgrounds: The traditional samplings for prenatal diagnosis are invasive. Those methods include chorionic villus sampling, amniocentesis and cordocentesis, and so on. All of them are dangerous to the fetus in some extent and can induce many complications, such as abortion, intrauterine infection and fetal phocomely. For noninvasive prenatal diagnosis fetal cells in maternal blood are good source. But there are few fetal cells in maternal blood and it is difficult to separate and purify them efficiently. Moreover the reagent necessary for experiment is expensive. Recently, with the discovery of free fetal DNA in maternal plasma, it becomes possible for noninvasive prenatal diagnosis. Recently, free fetal DNA has been applied in prenatal investigation of sex-linked diseases, fetal RhD status, pregnancy-associated disorders, and fetal chromosomal abnormalities. However, the conclusion about the correlation of free fetal DNA in maternal plasma and familiar chromosomal disorder such as Down's syndrome (DS) wasn't certain and we don't know the tissue origin and released mechanism of free fetal DNA in maternal plasma until now. In addition, there have been some reports on noninvasive prenatal diagnosis of Rh blood system, but we don't find related reports about ABO blood system. In China ABO maternal-fetal incompatibility incidence is relatively higher and it is the primary reason of HDN. One section of our study is qualitative analysis. We detected fetal ABO genotype for noninvasive prenatal diagnosis using fetal DNA in maternal plasma of blood group O mothers. The aim of the study is to investigate the accuracy and feasibility of this noninvasive prenatal diagnosis of fetal blood type. Another section is quantification analysis. To provide a new marker for noninvasive prenatal screening Down's syndrome, the level of maternal free fetal DNA was detected quantitatively using TagMan probe real-time quantitative PCR among the pregnancies with DS fetus and DS high-risk pregnancies. And we explored the tissue origin and released mechanism of free fetal DNA in maternal plasma in this study. Section IUse of maternal plasma for noninvasive prenatal diagnosis of fetal ABO genotypesBackground and objective: ABO maternal-fetal incompatibility is very common and it can induce various kinds of gestational final results such as stillborn and HDN. Prenatal diagnosis fetal ABO genotyping can provide basis for prevention and therapy of maternal-fetal incompatibility. Noninvasive diagnosis is a trend of research at present. Use of free fetal DNA in maternal plasma opened a door for noninvasive prenatal diagnosis. There have been some reports on noninvasive prenatal diagnosis of Rh blood system. As far as ABO blood system, we don't find related reports. So we identified fetal ABO genotypes using fetal DNA in plasma of blood group O pregnant women. The aim of the study is to investigate the accuracy and feasibility of this method.Methods: A total of 105 blood group O women in middle or late pregnancy were enrolled. Fetal DNA in maternal plasma samples and genomic DNA in newborns' umbilical blood were extracted with QIAamp DNA Blood Kit. DNA was amplified to identify ABO genotypes by polymerase chain reaction with sequence-specific primers (PCR-SSP). The results of genotypes that were evaluated by the serologic tests for phenotypes were not known until the reports of fetal ABO genotypes by using of free fetal DNA in maternal plasmaResults: (1) Using DNA from umbilical blood, ABO genotyping of 105 newborns was all identified successfully with PCR-SSP. (2) Using fetal DNA from maternal plasma, 88.6%(93/105) fetal ABO genotyping was correct; twelve false fetal genotypes were from 66 pregnant women with fetuses of type non-O. The accuracy in middle pregnancy was lower than that in late pregnancy although there was no statistical difference (0.050.05). (3) The mean level ofβ-HCG was 96.8±42.9 IU/ml for pregnant women carrying a trisomy 21 fetus, 58.1±25.3 IU/ml for DS high-risk pregnant women carrying a male fetus, and 38.1±19.4 IU/ml for normal pregnant women carrying a male fetus in maternal plasma. The remarkable difference between each two groups was statistically significance by t-test (p<0.01). (4)β-HCG is a sensitive marker for DS screening. (5) The level of free fetal DNA in maternal plasma was obviously relative toβ-HCG concentration in maternal serum (Pearson correlation coefficient r=0.86,P< 0.0001).Conclusions: (1) Real-time PCR possesses many merit, such as sensitive, specific, high-speed, convenient operation, high volume, etc. These advantages are suited to the large-scale prenatal screening and diagnosis of clinical diseases. Non-invasive prenatal sex diagnosis is very accurate by using of Real-time PCR. (2) Fetal DNA in maternal plasma might be a new marker for prenatal screening DS. (3) The other markers were needed for free fetal DNA to be an effective measure for prenatal DS screening. (4) There was strong relevance between the level of free fetal DNA andβ-HCG concentration. This is the first time to put forward that syncytial trophoblast maybe the predominant tissue origin of free fetal DNA in maternal plasma, and the abnormally released mechanism of fetal DNA in maternal plasma for pregnant women carrying trisomy 21 fetus is the destroy and apoptosis of syncytial trophoblast. ⅡThe improvement and application of comparative genomichybridization in prenatal diagnosis of fetalchromosomal abnormalitiesBackground and objective: Currently, conventional cytogenetic analysis of metaphase chromosomes and fluorescence in situ hybridization (FISH) are the most common methods for screening of abnormalities, but these methods have significant limitations. Standard cytogenetic may cause errors due to external contamination, culture failure and selective growth of maternal cells. The probes used in FISH technology are not designed for prenatal detection of all unbalanced chromosomal abnormalities. Only a few regions of the genome can be explored in one preparation. As a new chromosome analysis technique, CGH made quite a progress in cytogenetic approaches. The CGH technique has many advantages, since tissue that is fresh, frozen, formalin-fixed, or paraffin-embedded can be used for the analysis and the tissue or cells needed are much less. Comparative genomic hybridization is a technique that offers a molecular approach to cytogenetic analysis and allows the entire genotype to be screened in a single hybridization without the need of specific probe and prior knowledge of the location of chromosomal abberations. CGH has been applied primarily in cancer genetics, but is considered useful for clinical cytogenetics as well, including the application in the research of malignant hematological diseases, cell lines, diagnosis of hereditary diseases and embryo development research. Now the application of CGH in prenatal diagnosis has been the studying central point. The aim of this study is to create a useful CGH for prenatal diagnosis through the improvement. Comparing with conventional cytogenetic analyses, we evaluate the feasibility and reliability of CGH in the detection of chromosomal abnormalities in malformed fetuses or high-risk cases.Methods: Fetuses were chosen from 5 pregnancies with abnormal cytogenetic analyses results and we examined 12 cases of malformed infants or fetal death. Genomic DNA was extracted from amniotic fluid or umbilical cord blood samples using a Tiangen DNA extraction kit. Sample DNA and control DNA were labeled with SpectrumGreen dUTP and SpectrumRed dUTP respectively using the Vysis nick translation kit. Probes were prepared by mixing 200ng of SpectrumGreen dUTP labeled sample DNA, 200ng SpectrumRed labeled male human genomic DNA and 10μg of unlabeled Human Cot-1 DNA. The probe mixture was precipitated and then resuspended in CGH hybridization buffer. The probe mix was added to the hybridization site on the normal male metaphase target slide and covered with a sealing film. The slide and probe mix were co-denatured at 73℃and placed in a moist chamber at 37℃for 48-72 h. Following hybridization slides were washed and then counterstained with DAPIⅡ. Simultaneously, we designed fluorochrome-exchanged CGH: Sample DNA was labeled with SpectrumRed dUTP and genomic DNA was labeled with SpectrumGreen. CGH slides were analyzed on an epifluorescence microscope equipped using specific filter sets for DAPI, SpectrumGreen and SpectrumRed signals. A sequence of blue, green and red digital images was acquired under VideoTesT CGH software control. Karyotyping was performed based on DAPI banding pattern. A fluorescence intensity ratio (FR) profile was calculated after background correction and normalization of the green to red ratio for each metaphase to 1.0. Mean ratio profiles for each chromosome were determined after data from all analyzed metaphases were combined. Trisomies or partial chromosome gains were defined as FR>1.25. Monosomies or partial chromosome losses were defined as FR<0.75.The results of conventional cytogenetic analyses were not known until the finish of CGH analysis.Results: (1) Our CGH method was confirmed perfect for clear background, and good fluorescence hybridization. What's important, each of the 17 samples was analyzed successfully for chromosomal abnormalities by CGH. (2) Using fluorochrome-exchanged CGH, 3 pregnancies with trisomy 21 fetus, 1 case of trisomy 18 and 1 case of 45, XO were all detected correctedly comparing with conventional cytogenetic analyses. Two fetuses, one of which was Dandy-Walker syndrome were identified normal as well as cytogenetic method. (3)The karyotype of a stillborn fetus was 46, XY, Del 4q32-qter by CGH, while only 4q distal losses was identified by conventional cytogenetic analyses. (4) Among the 17 pregnancies, cytogenetic analyses had no results in 9 cases, but chromosomal abnormalities were identified by CGH in three cases and were verified by fluorochrome-exchanged CGH. For case 12, trisomy 21 was detected by CGH (when sample DNA was labeled green). For case 16, deletion 2p24-pter and duplication 12p13 were identified by CGH and verified by fluorochrome-exchanged CGH. For case 17, the unbalanced karyotype of del 1p33-pter and del 22q11-12 was identified, although 22p had to be excluded from analysis because of heterochromatic DNA.Conclusions: (1) Exchanging the labeled fluorochrome can reduce inconsistencies in the results caused by deviations in the process of DNA labeling and hybridization, and can increase the accuracy and reliability of analysis. This is the first report about fluorochrome-exchanged CGH. (2) The CGH is more sensitive than conventional cytogenetic analysis, and can be a useful and necessary supplement for the latter. Fluorochrome-exchanged CGH can provide a safe, accurate and useful alternative to traditional banding analysis especially in cases where conventional cytogenetic analysis is unavailable. (3) Chromosomal abnormalities are a significant component of the etiology of fetal malformations, but not attributed to some of malformations such as Dandy-Walker.