Clinical Application Of Non-invasive Prenatal Testing Technology For Fetal Chromosome Copy Number Variation Detection

Objective: To evaluate the detection efficacy and clinical significance of non-invasive prenatal testing(NIPT)for fetal chromosome copy number variant(CNV)by combining copy number variation sequencing(CNV-seq)and karyotype analysis.Methods: A total of 22998 single pregnant women who underwent NIPT in the Second People’s Hospital of Nanning on January 1,2018 and August 31,2020 were selected.The pregnant women ranged in age from 18 to 48 years old,with an average of(32.0±5.5)years old.The gestational age ranged from 12 to 34 weeks,with an average of(17.4±3.1)weeks.For pregnant women with NIPT indicating high risk,deletion,duplication and sex chromosome abnormality,amniocentesis is recommended to extract amniotic fluid for chromosome karyotype analysis and Copy number variation sequencing(CNV-seq),and NIPT resμLts are verified.Chromosomal abnormalities were analyzed and the detection efficiency of NIPT for fetal chromosomal abnormalities was evaluated.NIPT recommends that low-risk women with no other abnormal outcomes continue their pregnancies and follow up.ResμLts:1.ResμLts of NIPT abnormalities: Among 22,998 pregnant women who underwent NIPT,318 cases of NIPT chromosomal abnormalities were detected,228 cases of non-CNV(76 cases with high-risk of trisomy 21,25 cases with highrisk of trisomy 18,23 cases with high-risk of trisomy 13,86 cases of sex chromosomal abnormalities,and 18 cases with high-risk of other chromosomes)and 90 cases of CNV.2.ResμLts of interventional prenatal diagnosis: 318 pregnant women with NIPT chromosomal abnormalities eventually underwent prenatal diagnosis by amniocentesis in 240 cases,180 of whom were non-CNV(69 with high risk of trisomy 21,18 with high risk of trisomy 18,22 with high risk of trisomy 13,62 with sex chromosomal abnormalities,and 9 with high risk of other chromosomes),and 60 with NIPT indicating CNV.The resμLts of CNV-seq and chromosome karyotype analysis showed that no pathogenic CNV or other chromosomal abnormalities were found in 180 non-CNV pregnant women.No pathogenic CNV or other chromosomal abnormalities were found in 31(51.67%)of the 60 pregnant women with CNV,while CNV-Seq resμLts showed that 28(46.67%)pregnant women showed fetal chromosomal abnormalities,of which 27 cases were consistent with the resμLts of NIPT and 1 case was inconsistent.In addition,1case only received chromosome karyotype analysis and refused CNV-Seq test.In the process of follow-up follow-up,it was found that among the 31 patients,1case of fetal growth restriction intrauterine stillbirth induction,1 case of fμLl-term cis polydactyl,and the rest of the newborns showed no abnormal phenotype at birth.3.Efficacy of NIPT in detecting fetal chromosome copy number variants.(1)The detection rate of CNV was 0.39%(90/22998),the sensitivity was 77.14%(27/35),the specificity was 99.85%(22663/22695),and the positive predictive value was 49.23%(32/65).The negative predictive value was 99.90%(22663/22680).(2)NIPT comparison with CNV-Seq resμLts: NIPT detection of different fragment sizes:(1)The CNV was detected as <5 MB copy number abnormality.NIPT detected 16 CNV abnormality,10 of which were confirmed by CNV-Seq by amniocentesis,and the remaining 6 CNV-Seq were not abnormal.The positive predictive value(PPV),sensitivity and specificity of NIPT were 66.67%,55.56%,and 99.97%,respectively.(2)for 5 Mb or greater CNV < 10 Mb.The PPV,sensitivity and specificity of NIPT were 50.00%,100% and 99.97%,respectively.The resμLts showed that CNV was abnormal in 13 cases,6 cases were confirmed by CNV-Seq and 6 cases were false positive.(3)For CNV fragment size ≥10Mb,NIPT detected 31 abnormal cases of CNV,and CNV-Seq confirmed that 11 cases were true positive,and 20 cases were false positive.The PPV,sensitivity and specificity of NIPT were 35.48%,100%,99.91%,respectively.NIPT showed no statistically significant difference in the specificity of CNV fragment sizes(P>0.05),but there were statistically significant differences in PPV and sensitivity(P <0.05).PPV of CNV< 5Mb was higher than 5 Mb≤CNV<10 Mb,while PPV of CNV≥ 10 Mb was lower.5.The sensitivity of CNV <10 Mb and CNV≥ 10 Mb was 100%,while the sensitivity of CNV< 5Mb was 55.56%,which was relatively low.(3)Among the resμLts of fetal chromosomal abnormalities suggested by NIPT,CNV-Seq confirmed that 5 cases were clinically major pathogenic CNV: 2cases of Angelman/Prader-Willi syndrome,1 case of Wolf-Hirschhorn syndrome,1 case of Cri du Chat syndrome,and 1 case of Di George syndrome).Among them,2 cases were investigated by the core family of the fetus’ parents,and one case of abnormal CNV was found to be a new mutation,and the other case of CNV was inherited from its parents.4.Follow-up pregnancy outcomes: All pregnant women were followed up,78 cases of NIPT high-risk pregnant women refused to interventional prenatal diagnosis and follow-up of pregnancy outcome: 4 cases were found in 30 cases of CNV has induced labor,1 case was born,1 case was lost to follow-up,the rest of the 24 patients health,48 cases of CNV in 10 cases,3 cases of natural labor termination of pregnancy was born,2 cases were lost to follow-up,the rest of the33 cases of health.Among the 22,680 NIPT low-risk women,9 fetal abnormalities were found during follow-up(1 intrauterine stillbirth,1 cleft lip and palate induced labor,1 meconium peritonitis induced labor,6 equinochus,hypospadias,natural heart disease,ductus arteriosus atresia,etc.).There were 8 cases of fetal abnormality found by B-μLtrasound,and CNV-Seq was finally confirmed as abnormal CNV by amniocentesis(1 case of growth restriction intrauterine stillbirth,1 case of live syndacta,and the rest were healthy).The other 1 case showed no abnormality in NIPT and CNV-Seq resμLts,but fetal abnormality was found by B-μLtrasound.The core family investigation of fμLl exon sequencing was conducted on her and her parents,and it was found that the two pathogenic gene mutation sites of heterozygous variation were from the parents of the fetus,and the pregnant woman selected the fetus for induction of labor after genetic counseling.Other newborns showed no abnormal phenotype at birth.Conclusion:NIPT technology is feasible for screening fetal CNV,but NIPT technology cannot replace CNV-Seq technology as a routine detection method for fetal CNV screening.While positive results requires interventional prenatal diagnosis,negative results need to contrast with enhanced-ultrasonography examination and follow-up examination,together with genetic counseling.