Chromosome disease is one of the most serious birth defects in newborns. At present, there is no effective treatment, prenatal screening is a more effective way to reduce birth defects. NIPT is an important means of prenatal screening. In 2012, (ISPD) of the International Institute of Prenatal diagnosis and (ACOG) of the American College of Obstetrics and Gynaecology also recommended NIPT as a detection method for high-risk groups of chromosome aneuploidy[7,8].Through the screening of NIPT in 25502 pregnant women, the screening efficiency of NIPT in fetal autosomal, sex chromosome, and other chromosome aneuploidy and CNV was analyzed, and its clinical value was explored. In this study, NIPT suggested that there were 48 cases (0.19%) of high-risk T21, 15 cases (0.06%) of T18, and 8 cases (0.04%) of T13. The PPV of T21, T18, and T13 is 90.90%, 45.45%, and 33.33%, respectively. It is consistent with the literature report[9]. The decrease of PPV, in turn, depends not only on the sensitivity of NIPT but also on the decrease of incidence of T21, T18, and T13. At the same time, due to the absence of chromosome karyotype examination in the tissues of induced labor or spontaneous abortion, it may contain some true positive samples of NIPT, which also affects PPV. This study shows that NIPT has high accuracy and reliability in screening T21, T18, and T13, especially for T21[10].
Besides, 13 cases of T21mos, T18mos, and T13mos were detected in this study, and 1 case of true positive (T21mos) sample number was 20AH00515, 30 years old, 20 weeks pregnant for 4 weeks. The results of Down's screening were as follows: T21,1/620, chromosome karyotype indicated that 21q21.1 repetition was 1.76Mb, involving one OMIM gene, and there was no report of repetitive pathogenicity of CLINGEN. The clinical significance of the repetition was unknown. After genetic counseling, we chose to continue pregnancy and now delivered, and the baby was born as a normal girl. The detection efficiency of NIPT for trisomy 21, 18, and 13 was significantly lower than that of NIPT for T21, T18, and T13. The analysis of the reason may be due to the non-separation of mitosis in the early embryo or the delayed division in the later stage, in which the multicellular system is composed of normal karyotype and aberrant karyotype makes it difficult to detect NIPT. The free DNA detected by NIPT comes from placental trophoblast cells, not a fetus. The inconsistency of chimerism in different regions of placental tissue leads to more false-positive NIPT detection[11], especially in distinguishing true chimerism from false chimerism or low proportion chimerism. Therefore, NIPT is not recommended for screening chimeric chromosomal abnormalities.
In 72 cases of a sex chromosome aneuploidy, the detection rate of SCA was 0.28%. The detection rate of PPV for sex chromosome aneuploidy was 40.07%. Among them, 3, 6, 8 and 6 cases were true positive cases for 45,X0, 47,XXY, 47,XXX, 47 ,XYY, respectively. The detection efficiency of NIPT for sex chromosome aneuploidy was 12.00%, 50.00%, 72.73%, and 75.0%, respectively, which was not as effective as that for autosomal aneuploidy, and its efficiency for autosomal aneuploidy was lower than that for autosomal aneuploidy. The detection efficiency was the lowest (PPV 12.00%) for 45,XO, but moderate for 47,XXY, 47XXX, and 47,XYY (PPV 64.52%), which was consistent with that reported in the literature[12]. Because X and Y chromosomes are highly homologous, it is not easy to detect, and many fragments of the Y chromosome are similar to other chromosomes, so the accuracy of sequencing is reduced. Factors such as the deviation of cytosine and thymine content on the X chromosome, random inactivation of the X chromosome, restricted placental chimerism (CPM), and maternal SCA can also reduce the specificity of NIPT and detection[13]. Although the equipment, Z value, and concentration settings used in different experiments are different, which may have a certain impact on the detection results, NIPT still has certain clinical applicability for the detection of sex chromosome triploid, while the detection efficiency for 45,XO is not stable enough. In some cases of SCA, there is no obvious clinical phenotype and ultrasound abnormality in the fetus. Phenotypic abnormality can not be found in the fetal and infantile follow-up. After puberty, it will have a serious impact on physiology and psychology. Therefore, prenatal screening, genetic counseling, and long-term follow-up of SCA are particularly important[14].
This study shows that the efficiency of NIPT in the detection of aneuploidy of other chromosomes is low and has obvious limitations. NIPT revealed chromosome abnormalities in 34 cases, true positive in 3 cases, false positive in 21 cases, and no further verification in 10 cases. T7 was the most detected, followed by T8, T10, and T16, which was similar to that reported in the literature[15]. Its PPV is 12.50%, which is significantly lower than autosomal aneuploidy and sex chromosome aneuploidy. The reason for the higher false positive may be that the incidence of trisomy of these rare chromosomes is very low. and related factors such as "trisomy self-rescue" in the process of embryogenesis, placental chimerism caused by mitotic errors of diploid embryos, maternal cell contamination, maternal tumor, and so on[16,17]. The true positives of the 3 cases were T2, T9, and T16 respectively. T2 was the sample number of AB198R51420,43 years old, 20-5 weeks of gestation. The karyotype of amniotic fluid was 46 XN chromosome 2 trisomy 30% verified by CMA. Fetal ultrasound examination was normal. According to the database query, some abnormal phenotypes were reported, such as oligohydramnios, abnormal face, congenital heart abnormalities, etc. and there were also reports that some fetuses had no obvious abnormal pregnancy outcome. After genetic counseling, we chose to continue the pregnancy, and the phenotype of the fetus was normal after birth. The sample number of T16 was AB19HG04493,33 years old, and the gestation was 15 to 5 weeks. The result of CNV-Seq verification was sep[hg19] dup (16) (p13.3q24.3) repetitive 90.35Mb. There was no abnormality in B-ultrasound. After database inquiry, trisomy 16 showed fetal intrauterine limitation and a congenital heart defect. After genetic counseling, we chose to continue the pregnancy,at present, the fetus has been born.T9 is the sample number AB19HG06905,28 years old, 16 weeks pregnant and 4 weeks pregnant. Verified by the karyotype of amniotic fluid cells + CMA:(46,XX [31]/47,XX [59]); CMA results are 60~70% trisomy of chromosome 9. After database query: its phenotypic growth retardation, cognitive impairment, microphthalmia, heart abnormality, skeletal development abnormality, etc., may cause disease after genetic counseling, and choose to directly induce labor. Therefore, NIPT should combine the routine examination of pregnant women and fetal B-ultrasound screening for other chromosome aneuploidy results, and provide information helpful to the judgment of results, genetic counseling, and clinical decision-making as far as possible, to reduce maternal anxiety and unnecessary pregnancy termination. Liang et al[18] reported nearly 100000 samples and found that the detection rate of aneuploidy of other chromosomes was relatively low by expansive NIPT screening, with a PPV of 28%. The authors believe that it may also be related to the low total incidence of chromosomal abnormalities mentioned above. CPM. (ACMG) of the American Society of Medical Genetics and Genomics pointed out in 2016 that NIPT is not suitable for the detection of autosomal aneuploidy other than T21, T18, and T13[19]. Therefore, NIPT as a fetal aneuploid detection of other chromosomes, the accuracy is insufficient, the use of NIPT to detect other chromosome aneuploidy should fully inform pregnant women of its limitations, the results need more verification.
In the detection of chromosome MMS by NIPT, CNV samples were detected in 104 cases, 18 cases were true positive, PPV was 32.14%, and 38 cases were false positive. NIPT has a certain detection efficiency for CNV, but there are still many false positives, and the results still need to be further verified by chromosome karyotype analysis or CMA detection[20,21], which is especially important for pathogenic or possibly pathogenic CNV. Although there are many false positives of MMS detected by NIPT, it is difficult to find that MMS, NIPT below 10Mb can make up for the deficiency of chromosome karyotype analysis and reduce the missed diagnosis caused by artificial visual judgment. The results of this study suggest that the conventional sequencing depth of NIPT is less effective for CNV detection, and the reason may be related to the refusal of further CMA verification in some samples, and the interference of low fetal free DNA level restricted placental chimerism and abnormal maternal chromosome copy number[22,23], which may also be the common reason for the low efficiency of multiple high-risk samples (Table 1). Therefore, when the fetus is CNV detected by NIPT, we should further understand the medical history of pregnant women, combined with the results of prenatal ultrasound diagnosis, consider the need for interventional prenatal diagnosis, and combine chromosome karyotype analysis and CMA detection techniques to further improve the detection rate of abnormal chromosomes[24,25,26], and select fetal parents' chromosomes for control analysis if necessary. Among the true positive samples of CNV in this study (Table 5), there were 5 cases of pathogenicity, 3 cases of possible pathogenicity, and 10 cases of unknown clinical significance. Sample 19AH05194:NIPT reported that there were 2.5Mb duplicates in the 3p26.3 region, and the chip results showed that arr[HG19]3p26.3(285856-2499708)×3 involved three OMIM genes, including CHL (607416), CNYN (607220), and CNTN (607280). Some patients with autism, cognitive impairment, and epilepsy were found in PUBMED to repeat with this area, and the repetition was possible to cause disease. After genetic counseling, choose to continue the pregnancy, the fetus has been born, normal growth and development, parents refused to do carrier testing. Sample 20AH01147: the chip result showed that the 2q13 deletion was 2.12Mb, which was possible pathogenicity, which was consistent with its mother's CMA test sample (serial number CMA20200277).It was caused by maternal inheritance and chose to continue the pregnancy. the fetus has been born and its growth and development are normal. Sample 20AH00928: no karyotype was performed. B-ultrasound showed that fetal left hydronephrosis with left ureter dilatation, permanent left superior vena cava, and widened coronary sinus. Chip results: there were multiple abnormalities on chromosome 8, 8p23.1p12 region deletion involved 8p23.1 deletion syndrome, and the characteristic phenotypes were congenital heart abnormality, renal abnormality, growth retardation, and mental retardation. 8p12q24.3 region repetition involves trisomy 8, which is characterized by mental retardation, a special face, heart abnormality, and so on. After genetic counseling for pathogenic CNV, pregnant women chose to induce labor. Sample 20AH02846: the karyotype of amniotic fluid was 46del (5)(p14). The result of microarray showed that there was a 5p15.33-p14.3 deletion of 22.5 Mb, involving 56 OMIM genes. The deletion in this region involved Cri-du-chat syndrome. The main phenotypes included mental retardation, language disorder, cat-like crying in infancy, and so on. The deletion was not abnormal in peripheral blood karyotype of pathogenic CNV, parents, and induced labor after genetic counseling. The sample AB198R02189: chip result is 1q21.1q21.2.71MB, involving 13 OMIM genes. The repetition in this region is related to 1q21.1 microrepetition syndrome, and the main phenotypes are mild to moderate mental retardation, autism, hyperactivity disorder, tycoon, etc,the syndrome is not complete, and the penetrance rate is 29.1%. The repetition is pathogenic CNV, after genetic counseling, choose to terminate pregnancy. Therefore, from the above sample analysis, it can be seen that the detection of CNV with clear pathogenicity by NIPT is of clinical significance and can provide an important basis for subsequent karyotype analysis or CMA detection[27]. In recent years, scalable NIPT checking has helped to further improve the performance of MMS detection, but the 2015 ISPD update guidelines still limit NIPT to the scope of MMS that has been studied[28].
In summary, as an important method of prenatal screening, NIPT has high accuracy in the screening of trisomy 21, 18, and 13 with a higher incidence, especially trisomy 21, which is very effective for SCA. And MMS testing also has a certain reference value. But it is not recommended for the detection of other chromosomal aneuploidies. We believe that in the future, by upgrading the sequencing technology process, improving the bioinformatics analysis algorithm and library construction, high-precision collection of free fetal DNA, and increasing the coverage of the whole genome,it may further improve the detection efficiency of NIPT, combined with maternal medical history examination, serology, and Ultra-acoustic auxiliary inspection, chromosome karyotype analysis, fluorescence in situ hybridization technology, CMA detection technology, etc. can better utilize the advantages of NIPT technology.