The Improvement And Application In The Techniques Of The Non-invasive Prenatal Genetic Diagnosis

IntroductionPrenatal diagnosis is an effective method to avoid birth defects. Genetic amniocentesis, chorion villus sampling are invasive diagnostic methods , and many of these women are reluctant to expose the fetus to the risk associated with an invasive prenatal diagnostic procedure. Because of this, there is a great interest in risk - free prenatal diagnostic techniques . Identifying fetal cells from maternal blood was first reported by Walknows-ka et al. who, in 1969, found XY metaphases in maternal blood of pregnant women carrying male fetuses. Among various fetal cell types isolated from maternal blood, nucleated red blood cells ( NRBCs ) have emerged as the best candidates for non - invasive prenatal diagnosis, because they have a short lifespan, are present in significant numbers in the peripheral blood of early fetuses and have a full complement of nucleogenes.Hemoglobin production involves two developmental switches: from embryonic (ζ₂ ε₂ ) to fetal hemoglobin (α₂ γ₂ ) commencing at 6 - 7 weeks of gestation, and from fetal to adult hemoglobin (α₂ β₂) at birth. Our previous studies showed the possibility of identifying erythroblasts enriched from maternal blood by using the fetal γ - globin chain as a marker. However, a very small amount of fetal hemoglobin is also expressed in adults and pregnancy itself causes an increase in maternal cells producing fetal hemoglobin. In contrast, embryonic globin chains are unique to fetal erythroblasts and they are therefore potentially the best cell markers to beused in identification of enriched fetal cells from iriaternal blood. The embryonic hemoglobin e - chain has the potential for clinical practice to be used in the future as a unique fetal cell marker in the first trimester of pregnancy.But the antibody marking is high - cost and the procedure is complex. A rapid, simple and direct chemical staining method adapted from the classical Kleihauer test was developed to select fetal cells. Precise differentiation between fetal and maternal erythroblasts is based on the constitutional difference between fetal and adult haemoglobin. The fetal cells appear with an intense pink cytoplasmic staining while maternal cells with adult haemoglobin are colourless.After the isolation of positive fetal cells with micromanipulation, the whole genome amplification is required. Genomic DNA should be evenly amplified with complete coverage and consistent representation of all genes. The usefulness of a WGA method depends on its ability to represent the entire genome with minimal amplification bias. Much progress has been made over the past 12 years to amplify genomic DNA ( gDNA). Several successful amplification strategies such as PEP and DOP have been developed. However, PCR - based WGA methods may generate nonspecific amplification artifacts , give incomplete coverage of loci, and generate DNA less than 1 kb long that cannot be used in many applications. Recently, A newer isothermal method called multiple displacement amplification ( MDA) , based on a rolling circle amplification , provides a highly uniform representation across the genome, representing the entire genome with minimal amplification bias. We use this method to amplify the whole genome of fetal cells, to discuse its feasibility in the application of the non - invasive prenatal genetic diagnosis.Materials and MethodsWe obtained maternal blood samples from 20 pregnancies, at 7 ²5 weeks'gestation, 12 of these pregnancies were at high risk for DMD and 5 for PKU . They were followed up with molecular genetic analysis of amniotic fluids. Blood from 2 normal non - pregnant women was used as negative control and 1 fetal umbilical cord blood was used as positive control.NRBCs were separated with percoll using a discontinuous density gradient method ,and then smeared on microscope slides using cytocentifugation. Some slides were antibodies against the -y or e - chain of fetal hemoglobin. The others were stained with Kleihauer test . Then the positive NRBCs were collected by micromanipulator under microscopic observation, and then amplified them by MDA. Sex and STR analysis were determind from a small aliquot of the reaction. This can further verify the origin of NRBCs and make prenatal diagnosis of genetic disease at the same time.ResultsNRBCs stained with e - chain of fetal hemoglobin were found earliest at 7 week of gestation. And the optimum week in which to perform a reliable non -invasive prenatal diagnosis is around 7 15 week. While NRBCs stained with Kleihauer test were found earliest at 8 week of gestation. And the optimum week is around 10 22 week. The number of detected NRBCs from 10ml of maternal blood ranged from 4 to 19 in each case and average was 9. 5 in 10ml. The product length of MDA is > 15kb. We finished genetic diagnosis of 12 fetus at high risk of DMD ,4 were diagnosed male DMD patient, and 4 were normal male fetus , 1 sample were diagnosed female carrier, and 3 were normal female fetus. 5 fetus at high risk of PKU,2 were diagnosed PKU patient,2 were normal fetus, and 1 sample wasnt detected for nonsensical information at STR loci.ConclusionsWe used the embryonic hemoglobin e - chain as a unique fetal cell marker in the first trimester of pregnancy. We developed a rapid, simple and direct chemical staining method called Kleihauer test to mark the fetal cell. In addi-tion,we founded MDA method to amplify the whole genome of fetal cells. Combind with sex detection and STR linkage analysis, we further verified the origin of NRBC, and completed the prenatal diagnosis of genetic disease at the same time.