Maternal Plasma DNA Analysis With Next-generation Sequencing For Noninvasive Prenatal Diagnosis Of Trisomy21、18

ObjectiveTo prospectively study the sensitivity and specificity of next-generation sequencing technology to detect chromosome aneuploid using cell-free fetal DNA in maternal plasma. To compare the screening efficiency between traditional serum screening and non-invasive screening in the second trimester pregnancy. To evaluate the screening efficiency and clinical feasibility of next-generation sequencing technology to detect chromosome aneuploid using cell-free fetal DNA in maternal plasma.Materials and MethodsBlood samples were collected in a prospective,blinded study from Peking Union Medical College Hospital and obstetric clinics of Haidian Women and Children hospital from April2011to October2011.We study all singleton pregnancies who agreed to accept non-invasive detection. Eligibility criteria included pregnant women with more than11weeks of gestation who have no chromosomal abnormalities and invasive procedures;they did not receive allogeneic blood transfusion, transplant surgery, maternal peripheral blood stem cell therapy experience. Blood samples were used to traditional serum screening and non-invasive screening which were dectected by12-plexes the flux Hiseq2000platform. Pregnancies with screening positive,non-invasive positive or advanced maternal age should be offered definitive diagnosis through amniocentesis or CVS. Follow up after birth,if the result of serum screening or cffDNA detection is normal.Results1.2236pregnant women were enrolled and provided blood, all singleton pregnancies,138cases (6.2%)were lost; cffDNA testing failed with84cases (3.8%), of which74cases have follow-up data. There were2024samples eligible for selection.2.1741cases of second trimester screening pregnancies were collected, the positive rate of them were14.3%(249/1741), in which249cases are with trisomy21(Down’s Syndrome, DS) high-risk,3cases with trisomy18.3. Within an analysis non-invasive test samples of2,236,successfully reported2,152 cases, failed84cases (3.8%). Non-invasive test detected22cases of chromosome aneuploid:one case of T13,6cases of T18,12cases of T21,2cases of the XO, one case of XXY, positive rate of1.02%(22/2152)4. Follow-up rate of93.8%.432gravidas accepted invasive prenatal diagnosis.29abnormal choromosome karyotypes were diagnosed,as13fetals with DS (incidence rate0.54%),1fetal with13-trisome,.5fetals with18-trisome,5fetals with45,X(one of them is mosaicism),1fetal with47, XXY.3case initiatively do induction of labor;19fetals with multiple fetal malformations,the majority involved urinary system, cardiovascular system;10cases with abnormal pregnancy outcomes, because of fetal death, the late inevitable abortion.and so on.5.1fetal with T18and1fetal with47, XXY were detected by non-invasive test,but karyotypes were normal;1fetal with T21,2fetals with45,X and1fetal with47, XXY were detected by invasive prenatal diagnosis,but non-invasive test were normal.6. With detection.of21,18,13chromosome by non-invasive test and traditional serological screening,the false positive rate(FPR) of them were0.05%,14.05%, the positive predictive value (PPV) of them were94.74%,2.41%, the detection rate(DR) of them were94.74%,54.55%.7. With detection.of21,18.13.X,Y chromosome by non-invasive test and traditional serological screening,the false positive rate(FPR) of them were0.10%、14.08%, the positive predictive value (PPV) of them were90.91%、2.41%, the detection rate(DR) of them were83.33%、40%.8. Detection of DS by non-invasive test, sensitivity was92.3%and specificity was100%; detection of18,13trisomy,the sensitivity of them were100%,100%and specificity of them were99.95%,100%detection of XO, XXY with lower sensitivity, specificity were100%,99.95%.9.18cases of IVF pregnant women (all singleton pregnancies) accounted for0.9%(18/2236).1case with46, XY,-21,+21rob (21;21), inv (9) by invasive prenatal diagnosis, non-invasive test result was T21.The remaining17cases were normal.Conclusions1. The FPR of next-generation sequencing technology to detect T21、T18using cell-free fetal DNA in maternal plasma is lower,DR and PPV is higher, which initially prove the clinical feasibility of next-generation sequencing technology to detect chromosome aneuploid as screening protocol2. The screening efficiency of invasive screening is superior to traditional serum screening.3. It can detect the chromosomal aneuploid of chromosome13and sex chromosome which serum screening can not detect.4. The test is suitable for IVF donor pregnancies.