Prenatal Genotyping Of RHD From Cell-free Fetal DNA In RhD-negative Maternal Plasma

In this prospective study,we assessed the feasibility of fetal RHD genotyping by analysis of DNA extracted from plasma samples of RhD-negative pregnant women using a polymerase chain reaction with sequence-specific primers(PCR-SSP) and real-time PCR to diagnosis and prevent hemolytic disease of newborn.The fetal RhD phenotype may be accurately predicted from RhD-negative maternal plasma in Chinese subjects.These methods could be spread in clinical application.This study is designed to cover following two aspects.Part 1 Detection of fetal RHD genotyping via PCR-SSP using free DNA in RhD-negative maternal plasmaAs reported that the Rh blood group has been mapped to chromosome 1 (1p34.3-36.1) and contain two homologous genes of RHD and RHCE.One gene encodes for the D antigen,which is present on the erythrocyte membrane of Rh-positive individuals.The other gene encodes for the C/c and E/e polypeptide present on red blood cells of all individuals.Persons are clinically classified as Rh-positive and Rh-negative depending on the presence or absence of the D antigen on the red cell surface.Antisera to five Rh antigens(D,C,c,E and e) are available and are used to type individuals serologically.Hemolytic disease of the newborn(HDN) is caused by maternal anti-D(IgG) antibody crossing the placenta and binding to red blood cells, followed by their destruction,which results in anemia. Majority of RhD-negative individuals are associated with the deletion of the RHD gene between the upstream and downstream Rhesus boxes in Caucasian.38%of Chinese RhD-negative individuals had various RHD alleles,80%in RhD-negative Black-persons,however,66%of these cases carry RHD_ψallele.In china,currently prenatal testing of fetal RhD status is achieved through amniocentesis or chorionic villus sampling,an invasive procedure that usually is performed late in pregnancy and carries risks to the fetal and mother.More recently,it has become clear that invasive sampling can be avoided by the use of fetal cells or cell-free fetal DNA present in the maternal circulation,leading to a virtually risk-free means of prenatal RhD blood group testing in women negative for the target antigen.Studies of noninvasive determination of the fetal RhD blood type using free fetal DNA in Caucasians have provided considerable information,and these techniques have begun to achieve widespread acceptance in Europe.On the other hand,relatively few studies of noninvasive fetal RHD genotyping have been performed on Asian populations including Chinese RhD-negative pregnant women.Genetics influence the incidence of the RhD-negative phenotype according to ethnic origin.Interestingly,several studies,however,have revealed that RhD-negative individuals may have a partial or intact RHD gene,and this genotyping occurs at different frequencies depending on the ethnic population.The most frequent RHD allele among Asian individuals is the Del variant RHD1227A, which serves as an important genetic marker in RhDel persons.The Del phenotype has been detected in 30%of apparently RhD-negative Chinese individuals,and the RHD1227A allele was detected in all the Del samples in this population.In fact,the detection of RHD1227A and hybrid alleles has been shown to be predictive of Rh phenotypes in the Chinese population.In this study,we investigated the occurrence of RhD-negative alleles in the southeastern region of China.Using PCR-SSP of fetal DNA in maternal plasma,we developed an improved fetal RHD genotyping strategy for RhD-negative pregnant women in Chinese populations.We explore a method for pretanal diagnosis of the fetal RHD genotyping from RhD-negative pregnant wome to diagnosis and prevent hemolytic disease of the newborn.It could be spread in clinical application.Methods1.Screening for pregnant women with single fetus by B ultrasonic at gestational stage ranging from 11 to 40 weeks.2.Select RhD-negative pregnant women through the conventional serological analysis.3.DNA was extracted using QIAamp DNA blood Mini Kit(Qiagen,Germany).4.PCR-SSP was established in our laboratory.5.Theβ-globin(GLO) control gene was amplication and served as a control to confirm the presence and quality of gDNA in each sample.6.The SRY gene amplification confirmed the presence of fetal DNA in maternal plasma sample.7.Specific primes were designed according to the difference between RHD and RHCE.8.Specific primes were achieved in Chinese RhDel for the detection of RHD1227A, which is a useful genetic marker for RhDel identification.9.Observed fetal sex and confirmed accurately Rh phenotype through serological measure of fetal umbilic blood after delivery.10.Adsorption and elution tests were performed on the samples with RHD1227A.Results1.To select 58 cases in RhD-negative pregnant women.2.Amplication products of GLO and SRY were positive in the 32 samples.3.Amplication products intron 4,exon5,exon7 and exon10 were positive in 24 samples.4.Amplication products intron 4,exon5,exon7 and exon10 were negative in 6 samples. 5.Amplication products exon7 and exon10 were positive in the only one sample.6.Amplication product exon10 was positive in the only one sample.7.Genotyping RhD-positive samples in these PCR-SSP tests were further examined for the RHD1227A allele.In these samples analyzed,RHD1227A allele was available only 2 of the 24 cases.8.Among the 32 specimens,genotyping results of 28 cases were in complete concordance with phenotyping results of serological measure of fetal umbilic blood after delivery.9.2 cases of RhDel phenotype were assessed by detecting RHD1227A allele gene. The final accuracy rate of 93.75%can be obtained.ConclusionsThe method of using fetal DNA in RhD-negative maternal plasma for noninvasive prenatal diagnosis of fetal RhD blood type is a simple quick and specificity non-invasive prenatal gene diagnosis to diagnosis and prevent hemolytic disease of newborn,could be spread in clinical application.Part 2 Quantitative analysis of fetal RHD genotyping from fetal DNA in RhD-negative maternal plasmaIn the preliminary study,we used the PCR-SSP method to forecast male fetal RhD blood type.Female fetuses were not detected.In order to obtain a complete research data,to rule out interference caused by some technical factors,to increase sensitivity and specificity of detection,we have adopted a real-time PCR technology.Fetal RhD blood type was predicted successfully.A feasible method was established completely in the prenatal gene diagnosis of fetal RhD blood type. Methods1.Screening for pregnant women with single fetus by B ultrasonic at gestational stage ranging from 14 to 41 weeks.2.To select RhD-negative pregnant women through the conventional serological analysis.3.DNA was extracted using QIAamp DNA blood Mini Kit(Qiagen,Germany).4.DNA was extracted using DNA extraction kit(PEL-FREEZ,Beijing) for buffy coat from pregnant women.5.Theβ-globin(GLO) control gene was amplificated and served as a control to confirm the presence and quality of gDNA in each sample.6.The SRY gene amplification confirmed the presence of fetal DNA in maternal plasma sample.7.DNA samples of the negative results of amplification SRY were further analysis STR polymorphism with huffy coat from pregnant women.The father-derived gene fragment in fetal DNA would be found.8.Specific primes were designed according to the difference between RHD and RHCE.9.To establish standard curve by standard-specific gene copy number.10.Real-time PCR was established in our laboratory.11.Specific primes were achieved in Chinese RhDel for the detection of RHD1227A, which is a useful genetic marker for RhDel identification.12.To screen Rh negative pregnant women and observ newborn gender and collect cord blood for phenotyping identification of Rh blood group after delivery.13.The amplified products of RHD127A allele were purified and further directed sequencing.Results1.To select 78 cases of RhD negative pregnant women through serological measurement. 2.41 cases of positive results by SRY and GLO amplification in the 78 samples.3.37 samples of negative results by SRY amplification were further examinated the STR polyrnorphism with buffy coat from pregnant women.Father-derived gene fragments were achieved.The presence of fetal DNA was confirmed.4.65 cases of all positive results through intron4,exon 5,7,10 amplification.5.10 cases of all negative results through intron4,exon5,7,10 amplification.6.Only one case of positive results of exon7,10 through intron4,exon5,7,10 amplification.7.Two case of positive results of exon10 through intron4,exon5,7,10 amplification.8.65 samples of all the positive results through all replicates intron 4,exon5,7,10 amplification were further amplified RHD1227A alleles in five cases of positive results.9.Genotyping results were consistent with serology phenotyping results in 70 of 78 cases,the rate was 90%(70/78).10.Five cases of RHD1227A allele were directly sequencing.There was a G＞A base mutation in the 1227 position between exon 9 and intron 9.11.Five casese were identified as RhDel by detecting RHD1227A allel.The final accuracy rate of 94.9%(74/78) can be obtained in the fetal RHD genotyping.12.The detection rate was 89.7%(70/78).From 5 cases false positive,4 cases RhDel phenotype were assessed by detecting RHD1227A allele gene.ConclusionsNon-invasive prenatal diagnosis of fetal RhD blood type using real-time PCR is conducive to the standardization of technology.Test results were quantified.This method raise the specificity and sensitivity of detection,is a feasible method of accurate predicting of fetal RhD blood type...