Following prenatal diagnosis of 5,367 pregnancies with karyotyping and CMA, 72 fetuses were identified with mosaic results, including 22 with autosomal aneuploidy (30%), 40 with sex chromosome aneuploidy (n = 56%) and 10 with large cryptic genomic rearrangements (14%). Five samples with large cryptic genomic rearrangements were excluded from the analysis due to a lack of DNA following CMA testing (Additional file 1: Table S1). Therefore, 67 samples (40 sex chromosome aneuploidies, 22 autosomal aneuploidies and 5 large cryptic genomic rearrangements) were eventually analyzed by both CMA and CNV-seq (Fig. 1).
Diagnostic concordance of CNV-seq and CMA versus karyotyping
For the 65 of 67 samples identified as mosaic by karyotyping, 41(63%) were also confirmed by CMA (Fig. 1). Of the 41 positives, 37 showed low levels of mosaicism around 20%. Two samples normal by karyotyping, we revealed as mosaic trisomy 8 and mosaic partial trisomy 8 by CMA. In comparison, CNV-Seq not only identified all 43 mosaics detected by CMA, but also identified an additional 15 mosaic samples, increasing the yield of mosaic detection by 35% over CMA. The levels of mosaicism defined by CNV-seq ranged from 6 to 92%. Further, the chromosomal map intervals, size, and copy number of the reportable mosaicisms detected by both DNA-based techniques were almost identical. Nine samples diagnosed as normal by CMA were also confirmed by CNV-seq.
Chromosomal mosaicism for autosomal aneuploidy
For mosaic autosomal aneuploidy, there were 21 cases identified by karyotyping and 1 case by CMA. The majority of mosaic cases (16 of 22) were from high-risk noninvasive prenatal screening group (Table 1). The mosaics identified involved trisomy 21 (12, 55%), trisomy 18 (2, 9%), trisomy 15 (2, 9%) and other autosomal trisomies (6, 27%).
CMA analysis identified 13 of 22 cases with mosaicism levels as low as 20% whereas CNV-seq identified 19 of the 22 cases with mosaicism at levels down to 5% (Fig. 2). For all 19 cases confirmed by CNV-seq, the percentages of trisomic cells for trisomy 21, 18 and 13 were in good close agreement with karyotyping results. However, for case 16, 17, 18, 19 and 22, the proportion of aneuploidy was much lower in culture samples compared with uncultured. Notably, for case 18, the mosaic trisomy 8 was not detected in the cultured AF sample by metaphase analysis of 100 G-banded cells whereas CMA and CNV-seq showed 24% and 18% trisomy 8 mosaicism, respectively (Additional file 1: Fig. S1). For the remaining three cases 11, 20 and 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.
Chromosomal mosaicism for sex chromosome aneuploidy
There were 40 cases with mosaic sex chromosome aneuploidies accounting for 60% of all cases identified by karyotyping. Clinical indications for these cases were broader including aUS (n = 6), AMA (n = 11), hMSS (n = 12), NIPS (n = 33) and poor fertility history (n = 2). Details of the sex chromosomes involved and the clinical course of the 40 pregnancies are presented in Table 1. The mosaic findings including 25 cases for monosomy X (45, X/46, XX), 7 cases for monosomy X and trisomy X (45, X /47, XXX) or monosomy X and disomy X (45, X /47, XXY), 5 cases for disomy X (47, XXY /46, XY), 1 case for disomy Y (47, XYY /46, XY), 1 case for trisomy X (47, XXX /46, XX) and 1 case for both monosomy X, disomy X, and trisomy X (45, X /46, XX/47, XXX).
Based on the karyotyping data, the percentage of monosomic or trisomic cells varied from as low as 4% to as high as 92% (Table 1). Of the 40 sex chromosome mosaics identified by karyotyping, CMA identified 23 cases of mosaicism (levels as low as 20%), 2 cases of whole sex chromosome aneuploidy and 15 normals. In contrast, CNV-seq identified 34 cases of mosaicism (level as low as 8%) with an incremental yield of mosaicism of 22.5% over CMA. There was a 100% positive concordance between CMA and CNV-Seq for 23 mosaic samples. It should be noted that in case samples 38, 47, 50, 54 and 55, the proportion of monosomy X or disomy Y varied by more than 30% in cultured samples compared with uncultured samples.
For the 9 additional cases of mosaicism identified by CNV-seq, the measured levels of mosaicism were low, ranging from 8 to 23%. Further, for cases 36 and 41, there were 1.92 and 1.90 haploid equivalents of chromosome X in the amniotic fluid samples, respectively, whereas CNV-seq analysis of available fetal placenta confirmed placental mosaicism with chromosome X of 1.17–1.87 and 1.3–1.85 haploid equivalents (Additional file 3: Fig.S2).
For cases 23, 26, 27, 30, 32 and 61, both CNV-seq and CMA showed a normal result in uncultured amniotic fluid cells, but karyotyping showed a mosaic pattern of monosomy X or disomy X in cultured amniotic fluid cells. Among these cases, karyotyping detected a mosaic pattern of monosomy X or disomy X of less than 10% in 5 of the 6 cases. The negative results of CMA and CNV-seq may have been due to technical limitations or culture artifacts by karyotyping.
Chromosomal mosaicism for large cryptic genomic rearrangements
A total of 5 cases with large cryptic genomic rearrangements were identified by karyotyping. Of these, 4 (cases 64, 65, 66, 67) had a mosaic pattern involving a small supernumerary marker chromosome (sSMC) or unclarified derived chromosome. By CMA, the character, origin and pathogenicity of these sSMC was further clarified (Additional file 4: Fig. S3–Additional file 8: Fig. S7). Details of the chromosomes involved and the clinical course of the 5 pregnancies are presented in Table 1.