A total of 72 subjects were initially enrolled in the study, which were generally categorized into three groups: autosomal aneuploidy (n=22, 30.55%); sex chromosome aneuploidy (n=40, 55.56%); submicroscopic CNVs (n=10, 13.89%). But five samples were excluded due to lack of enough DNA after CMA testing (Table S1). Therefore, 67 samples (40 sex chromosome aneuploidy, 22 autosomal aneuploidy and 5 submicroscopic CNVs) were eventually analyzed (Fig. 1).
Diagnostic concordance of CNV-seq and CMA vs karyotyping
Positive result of CMA was obtained on 63.08 % (41/65) samples which showed a mosaic pattern identified by karyotyping, of which, 37 samples showed a mosaic pattern with the level as low as 20% (Fig. 1). Besides, CMA detected additional mosaic trisomy 8 and mosaic partial trisomy 8 which was not detected by karyotyping in the cultured AF sample. In comparison, CNV-Seq not only identified all 43 chromosomal aneuploidies or submicroscopic CNVs detected by CMA, but also provided a 34.88% (15/43) increased yield compared with CMA(Figure 1). Besides, the level of mosaicism defined by CNV-seq range from 6% to 92%. The chromosomal map intervals, size, and copy number of the reportable mosaicisms detected by both DNA-based techniques were almost identical. In addition, there was a 100% negative concordance of CNV-Seq for the 9 remaining samples diagnosed as normal by CMA.
Chromosomal mosaicism for autosomal aneuploidy
As showed in Figure 1, a total of 22 subjects were enrolled in the group, which involving 21 cases identified by karyotyping and 1 case identified by CMA. High-risk noninvasive prenatal screening (16/22) were the most prevalent diagnosis indications (Table 1). The relative frequencies of mosaic aneuploidies showed 54.54 % (12/22) for trisomy 21, 9.09 % (2/22) for trisomy 18, 9.09 % (2/22) for trisomy 15 and 27.27 % (6/22) for other autosomal aneuploidies.
13 of 22 cases were confirmed by CMA with the level as low as 20%, while consistent CNV-seq and chromosome results were noted in 19 cases with the identified mosaicism at levels as low as 5 % (Fig. 2). Of the 19 cases confirmed by CNV-seq, the percentages of cells for trisomy 21, 18 and 13 were in good agreement when CNV-seq and cytogenetics were compared. While in Case 16, Case 17, Case 18, Case 19, Case 22, the proportion of abnormal chromosome was much lower in culture samples compared with uncultured. Notably, in Case 18, the mosaic trisomy 8 was not detected in the cultured AF sample by metaphase analysis of 100 G-banded cells, while CMA and CNV-seq showed 24 % and 18 % trisomy 8 mosaicism, respectively (Supplemental Fig. S1). Of the remaining three cases (case 11, 20, 21), both CNV-seq and CMA showed a normal result in uncultured amniotic fluid cells but karyotype showed a mosaic pattern of trisomy 21, trisomy 9 and trisomy 20 in cultured amniotic fluid cells, respectively.
Chromsomal mosaicism for sex chromosome aneuploidy
The 40 cases with sex chromosome aneuploidies accounted for 59.70 % (40/67) of total cases with a mosaic pattern identified by karyotyping. Clinical indications for these cases included abnormal ultrasound structure (aUS, n=6), advanced maternal age (AMA, n=11), high-risk of maternal serum screening (hMSS, n=12) or NIPS (n=33), and poor maternal history (n=2). Details of the chromosomes involved and the clinical course of the 40 pregnancies are presented in Table 1.The mosaic findings including twenty-five cases for monosomy X(45, X/46, XX), seven cases for monosomy X and trisomy X(45, X /47, XXX) or monosomy X and disomy X(45, X /47, XXY),five cases for disomy X(47, XXY /46, XY), one case for disomy Y(47, XYY /46, XY), one case for trisomy X(47, XXX /46, XX), and one case for both monosomy X, disomy X, and trisomy X(45, X /46, XX/47, XXX) are listed in Table 1.
Based on the karyotyping data, the percentage of monosomic or trisomic cells varied from 3.8 % to 92 % (Table 1). Of the 40 sex chromosome aneuploidies identified by karyotyping, 25 cases including mosaic pattern in 23 cases were confirmed by CMA with the mosaicism level as low as 20 %, while consistent CNV-seq and chromosome results were noted in 34 cases with the level as low as 8 %. Namely, the incremental yield of mosaicism less than 20% achieved by CNV-Seq was 22.5% (9/40). There was a 100% positive concordance between CMA and CNV-Seq for 23 samples. It should be noted that the proportion of monosomy X or disomy Y was more than 30% differences in culture samples compared with uncultured samples in 5 samples (Case 38, Case 47, Case 50, Case 54 and Case 55).
In addition, for the 9 incremental cases of mosaicism identified by CNV-seq, CNV-Seq showed the level of mosaicism was range from 8 % to 23 %. Besides, for Case 36 and Case 41, which showed 1.92 and 1.90 haploid equivalents of chromosome X for the amniotic fluid sample, CNV-seq of induced fetal placenta confirmed the placental mosaicism with chromosome X of 1.17-1.87 and 1.3-1.85 haploid equivalents (Supplemental Fig.S2).
In Case 23, 26, 27, 30, 32 and 61, both CNV-seq and CMA showed a normal result in uncultured amniotic fluid cells but karyotype showed a mosaic pattern of monosomy X or disomy X in cultured amniotic fluid cells. Among these cases, karyotype detected a mosaic pattern of monosomy X or disomy X less than 10% in 5 cases. The negative results of CMA and CNV-seq were most likely due to technical limitations.
Chromsomal mosaicism for submicroscopic CNVs
A total of 5 cases with a mosaic submicroscopic CNVs pattern were enrolled in the group. 4 out of 5 had a mosaic pattern with small supernumerary marker chromosome (sSMC) or unclarified derived chromosome identified by karyotyping were clarified the character, origin and pathogenicity of sSMC with the help of CMA (Number 64, 65, 66, 67)(Supplemental Fig. S3-S7). Details of the chromosomes involved and the clinical course of the 5 pregnancies are presented in Table 1.