The Clinical Research On Non-invasive Prenatal Test By Next-generation Sequencing (NGS)

Objective: The birth defect rate in China is 5.6%.Prenatal diagnosis plays important role in decreasing birth defect rate as secondary prevention measures,including ultrasound,maternal serum screening and invasive genetic testing of chromosomal disease.The false positive rate of maternal serum screening is up to 40%,so that many pregnant women are preformed on unnecessary invasive procedures which may cause miscarriage or intrauterine infection.It is imperative to develop a rapid and accurate method to screening fetal aneuploidy non-invasively in order to avoid above risks.The technology of non-invasive prenatal test(NIPT)is using next-generation sequencing(NGS)on cell free fetal DNA(cffDNA)in maternal plasma,which has been widely applied.However,majority of researches didn’t pay attention to the status of clinical application,but only focused on the accuracy of NIPT methods.Except common aneuploidy such as Down’s syndrome,there is another kind of chromosomal disease caused by copy number variations(CNVs).Most of CNVs in gene deserts are inherited or de novo,which cover 12 percents of human genome.CNVs is benign usually,but if the change of CNVs quantity affects the expression of dosage sensitive genes,a spectrum of phenotypes will be displayed,such as congenital malformation,intellectual disability and developmental delay.Common genomic diseases are including DiGeorge syndrome,Cri-du-chat syndrome and 1p36 deletion syndrome et al.Present technologies such as chromosomal microarray analysis(CMA)or copy number variations sequencing(CNV-seq)with higher resolution are widely applied to detect CNVs,which can reach 0.1Mb,and conventional karyotyping can detect CNVs>5Mb.The successful application of NIPT for detecting aneuploidy promotes extending NIPT for detecting fetal CNVs.Previous researches demonstrated NIPT showed high sensitivity of more than 99% for detecting CNVs>10Mb and the sensitivity of detecting CNVs<5Mb was needed to be improved.The limits of NIPT for detecting CNVs in the fetus with ultrasound anomalies were still remained.Firstly,our study will collect the clinic information of pregnancies who had received NIPT before in a large cohort.The statistic analysis will be preformed on the arrangedinformation in order to evaluating the status of clinical application.Then we will collect maternal peripheral blood sample,amniotic fluid and or fetal tissues after terminating pregnancy from the pregnancies with fetal ultrasound anomalies.The clinic information of these pregnancies will be collected meanwhile.The aim of this part is exploring the application indications of NIPT for detecting CNVs through discussing the factors related to NIPT accuracy.Methods: In partⅠ,we collected the clinic information of 12714 pregnancies who had received NIPT in the department of clinical genetics,Shengjing Hospital of China Medical University from Mar,2015 to Jun,2017.The statistic analysis was preformed on the groups of age,gestation week,indication,ultrasound findings in different gestation weeks,NIPT result,invasive prenatal diagnosis result and perinatal outcome.The status of clinical application was evaluated by the statistic results.In part Ⅱ,a total of 49 samples of maternal peripheral blood,47 samples of amniotic fluid and or fetal tissues after terminating pregnancy were collected,and the clinic information of these pregnancies were gathered meanwhile.The evaluation was preformed on the accuracy of NIPT for detecting CNVs,through the comparison between NIPT result,CNV-seq result and karyotype.Results: 1.The evaluation results of NIPT for detecting aneuploidy were as follows: the differences between the groups of high risk and low risk were statistically significant(P<0.05)by the groups of age and gestation week,respectively;the sensitivity of T21/18/13 and sex chromosomal abnormalities(SCAs)was 100%,and the specificity of T21/18/13 and SCAs was 99.94%,99.99%,99.95% and 99.79%,respectively,which were calculated by the standard of karyotype;the higher rate of NIPT positive results was in the group of ultrasound anomalies,especially in the group of increased NT;all of NIPT results in the group of non-structural soft markers were negative.2.The results of NIPT for detecting CNVs were as follows: there was no statistically significance(P>0.05)between the groups of CNVs>1Mb and no CNVs by the groups of age and gestation week,respectively;more CNVs were detected in the group of mutiple anomalies;the sensitivity and specificity of NIPT for detecting CNVs>1Mb were 83.33% and 97.3%;the accuracy of NIPT was independent of CNVs size.Conclusions: NIPT can screen aneuploidy instead of maternal serum screening,becauseof the effective reduction in unnecessary invasive procedures.When NIPT combines with ultrasound,the screening effectiveness of two methods will be improved significantly.NIPT can be applied for detecting CNVs in first trimester.Pregnant women with ultrasound anomalies should be recommended to receive invasive prenatal diagnosis sometimes,even if no CNVs is detected by NIPT.Pregnancy outcome must be determined by invasive prenatal diagnosis result.Karyotyping is still golden standard for detecting chromosomal abnormalities and can’t be completely replaced by higher resolution technologies.