Research Of Noninvasive Prenatal Diagnosis Of Thalassemia Using Size-Fractionated Cell-Free Fetal DNA In Maternal Plasma

Background:Since the introduction of prenatal diagnosis to prevent birth defects,the risk of invasive prenatal diagnosis for both mother and foetus has been considered.Noninvasive prenatal diagnosis is one of the many long-sought goals in human genetics.The discovery of cell-free fetal DNA(cffDNA)in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis and monitoring.As a result of its development,fetal DNA in maternal plasma is considered as a hope for the noninvaSive prenatal diagnosis of single gene disorders such as thalassemia,which is one of the most common autosomal recessive single gene disorders and has markedly high incidence in Guangxi province.The development of noninvasive prenatal diagnosis has a significant contribution to women physical and mental health.In order to investigate the feasible noninvasive prenatal diagnosis of thalassemia,we established a whole set methodology for cffDNA enrichment and detection based upon its correlation characteristics.PartⅠDetection of Fetal SRY Gene Using Size-Fractionated Cell-Free Fetal DNA in Maternal PlasmaObject:To evaluate the feasibility of cffDNA-based noninvasive prenatal diagnosis,we developed a precise technique for fetal SRY gene detection using size-fractionated cell-free DNA in maternal plasma.Methods:Peripheral blood samples were collected form 157 pregnant women. CffDNA was extracted based on a column absorbent method and isolated by agarose gel electrophoresis.A dulex-polymerase chain reaction(PCR)was used to detected SRY gene and glycerol-dehyde-phosphate dehydrogenase(GAPDH) gene.Results:Both SRY and GAPDH gene were detected in 86 cffDNA samples form women bearing male fetuses.And only GAPDH gene was detected in 71 cffDNA samples from women bearing female fetuses.These results have a coincidence whit those of villus or amniotic fluid samples and postpartum follow-up.The specificity and sensitivity reached to 100%(157/157)and 100% (86/86),respectively.Conclusion:By agarose gel electrophoresis,re-extratedand and dulex PCR, size-fractionated cell-free fetal DNA in maternal plasma can be selective enriched and used to noninvasive prenatal diagnosis of sex-linked disorders and single gene disorders.PartⅡ.Detection of fetal STR genotypes Using Size-Fractionated Cell-Free Fetal DNA in Maternal PlasmaObject:To evaluate the reliable discrimination between the fetal and maternal DNA in maternal plasma,we analyzed 3 short tandem repeat(STR)genotypes (D5S818,D7S820 and D13S317)on basis of size-fractionated cell-free DNA in maternal plasma.Methods:CffDNA samples from 62 pregnant women were collected according to the method established in PartⅠ.Then foetus and its corresponding parents STR genotypes were analyzed by a mutilplex-PCR.Results:According to STR-PCR amplification,the fetal genotypes of 49/62 samples completely matched to those of corresponding parents.However,we observed maternal DNA contamination to D 13S317 amplification in 13 cases.Conclusion:The reliable discrimination between fetal and maternal DNA in maternal plasma has hitherto been a technical challenge.By selective enriching of cffDNA,we still can't completely exclude maternal DNA contamination,and such DNA background unavoidably interfered the conclusively identification. However,in our study,a specific amplification of maternal DNA was less likely based on the mutilplex-PCR technology.PartⅢ.Noninvasive prenatal diagnosis ofβ-thalassaemia using Size-Fractionated Cell-Free Fetal DNA in Maternal PlasmaObject:To investigated the clinical feasibility of cffDNA-based noninvasive prenatal diagnosis forβ-thalassaemia.Methods:According to commom mutational genotypes of 17 kinds ofβ-thalassaemia in Guangxi and Guangdong province,we designed 3 pairs of primers,biotin labeled.Then cffDNA was amplificated by a duplex PCR,and fetalβ-globin genotype was analyzed by revert dot-blot hybridization.The results were conclusively identified by traditional invasive methodology.Conclusion:Among 37 cffDNA samples,19 and 11 were identified asβ-thalassaemia major andβ-thalassaemia minor,respectively,and 7 were identified as normal.By comparing with that of invasive methods,3 cases were stated as misdiagnosis.The accurate rate reached to 91.9%(34/37).Conclusion:When fetal genotype is coincident with maternal,further analysis or re-examination is necessary for the unavoidable contamination of background DNA.Due to its facilely sampling,no risk for both of gravida and foetus,and no limited by duration of pregnancy,the cffDNA-based noninvasive prenatal screening forβ-thalassaemia has been expected clinical application.PartⅣ.Noninvasive prenatal diagnosis of Hb Bart's hydrops foetus using Size-Fractionated Cell-Free Fetal DNA in Maternal Plasma.Object:To explore a new noninvasive methodology for Hb Bart's hydrops foetus,we investigated PCR amplification efficiency discrimination between fetal cffDNA and maternal cfDNA.Methods:CffDNA samples form women bearing possible Hb Bart's hydrops foetus were collected.Fluorescence PCR and capillary electrophoresis(CE) were performed.And Hb Bart's hydrops foetus was conclusively identified because of different peak area ratio of products.Results:The peak area ratio of cffDNA sample from Hb Bart's hydrops foetus was significantly less than 1.However,the peak area ration of cffDNA sample from hydrops foetus due to another reason was approximately equal to 1.Conclusion:By fluorescence PCR and capillary electrophoresis,we developed a cffDNA-based prenatal screening for Hb Bart's hydrops foetus among large population.Additionally,traditional invasive methodology can provid sufficient foetus DNA samples to the PCR technology established in this study.As the result of these,a rapid screening for Hb Bart's hydrops foetus is under considered.