case_images: PMC11788280_01
case_images: Sex chromosomes plot for different fetal fraction ranges. The horizontal axis represents the normalized chromosome value for the X chromosome (NCVx), and the vertical axis represents the normalized chromosome value for the Y chromosome (NCVy). The dotted lines indicate the normal range for female and male euploid samples across different fetal fraction (FF) ranges () females with FFs between 2% and 12%; () males with FFs between 2.0% and 3.0% () males with FFs between 5.0% and 6.0%; () males with FFs between 6.0% and 10.0%; () males with FFs between 10.0% and 12.0%. The reported cases are shown as gray and black diamonds for the first and second sequencing of case A, respectively, and as a black square for case B. As observed in both cases, the NCVy:NCVx ratio deviates significantly from that expected for male fetuses with FFs in the 5% and 10% ranges, respectively.
case_images: fgene-15-1502287-g001.jpg
case_images: PMC11788280_01_fgene-15-1502287-g001.jpg
case_images: F1
case_images: Given that these values were significantly lower than that expected for a male fetus with a 5.0% FFi (Figure 1), the possibility of underlying clinical factors interfering with the result was discussed with the physician.
case_images: Analysis of the overrepresented genetic material belonging to the Y-chromosome. Case (A). (A) Left: Graphical plot of 5-Mb bin Z-scores across the X chromosome. Right: Graphical plot of Chr. Y 5-Mb z-scores across the Y chromosome. In all cases, the bin Z-score values have been calculated with regard to a female reference of normalcy (zscore_fem), or with regard to a male reference of normalcy (zscore_male). Between positions 5-10 Mb, a bin with a Z-score of approximately 100 is observed, a value 10 times higher than expected for a male fetus with a native fetal fraction (FFi) of around 5%. (B) Graphical representation of the ratio of Y chromosome reads to total reads within the genomic region of the Y chromosome spanning positions ChrY: 6,400,000-7400,000 (GRCh37/hg19) for (from left to right): CASE A, a set of 50 samples from pregnant patients with female fetuses (FFs between 5% and 12%), and a set of 50 samples from pregnant patients with male fetuses (FFs between 5% and 12%). In case A, the ratio exceeds by more than ten-fold the number of reads aligned to the Y chromosome in samples from patients with fetuses classified by the analysis algorithm as XY. (C) PCR amplification of the amelogenin (green peak) containing regions and fragment analysis of PCR products by capillary electrophoresis on AB3500 (ThermoFisher) and GeneMapper (rr). Top: patient; Middle: female reference; Bottom: male reference. The 119 bp peak represents AMELX, and the 124 bp peak represents AMELY. The marker under each allele peak represents the number of repeats.
case_images: fgene-15-1502287-g002.jpg
case_images: PMC11788280_01_fgene-15-1502287-g002.jpg
case_images: F2
case_images: An analysis of the distribution of DNA fragments along the Y chromosome revealed that DNA from this chromosome was not homogeneously present; Y-DNA fragments were concentrated solely to the cytogenetic region Yp11,2 covering the genomic coordinates ChrY:6,400,000_6,900,000 (GRCh37/hg19) (Figure 2A). In contrast, fetuses classified as male exhibited a ratio of aligned Y-DNA fragments to total reads much lower than that found in the case described in this report (8 fold lower in the general male fetus sample set vs. case A) (Figure 2B). To elucidate whether the overrepresentation of fragments was caused by an insertion of Y-chromosome genetic material into the maternal genome, fluorescent PCR was performed on white blood cell DNA using specifically designed primers. The amplification results revealed the presence of the expected female variant AMELX and the unexpected male variant AMELY in the maternal genome (Figure 2C).
case_images: PMC11788280_02
case_images: Analysis of the overrepresented genetic material belonging to the Y-chromosome. Case (B). (A) Left: Graphical plot of 5-Mb bin Z-scores across the X chromosome; right: Graphical plot of Chr. Y-5 Mb Z-scores across the Y chromosome. In all cases, the bin Z-score values have been calculated with regard to a female reference of normalcy (zscore_fem), or with regard to a male reference of normalcy (zscore_male). Between positions 5-10 Mb, a bin with a Z-score of approximately 125 is observed, a value 10 times higher than expected for a male fetus with a native fetal fraction (FFi) of around 11%. (B) Graphical representation of the ratio of Y chromosome reads to total reads within the genomic region of the Y chromosome spanning positions ChrY: 6,400,000-7400,000 (GRCh37/hg19) for (from left to right): CASE B, a set of 50 samples from pregnant patients with female fetuses (FFs between 5% and 12%), and a set of 50 samples from pregnant patients with male fetuses (FFs between 5% and 12%). In case B, the ratio exceeds by more than ten-fold the number of reads aligned to the Y chromosome in samples from patients with fetuses classified by the analysis algorithm as XY. (C) PCR amplification of amelogenin and PRKY and the Y:7,350,250-7,350,345 containing regions and fragment analysis of PCR products by capillary electrophoresis on AB3500 (ThermoFisher) and GeneMapper (rr): C_1 and C_4: patient; C_2 and C_5: female reference; C_3 and C_6: male reference. Green peak: The 119 bp peak represents AMELX, and the 124 bp peak represents AMELY. The blue peak at 208 bp represents PRKY and the blue peak at 96 bp is a region in chromosome Y at Y:7,350,241-7,350,341.
case_images: fgene-15-1502287-g003.jpg
case_images: PMC11788280_02_fgene-15-1502287-g003.jpg
case_images: F3
case_images: The Z-score value for the Y chromosome was 40.02 with a SeqFF-based estimated FF of 8.8% and an FFy of 3.5%; however, the Y-DNA fragment distribution analysis revealed alignment with the cytogenetic region Yp11.2, specifically between genomic coordinates ChrY: 7,000,000-7,500,000 (GRCh37/hg19) (Figure 3A). Similarly, the ratio of the number of Y-DNA readings to total readings in this region was significantly higher than that identified in a set of 50 samples classified as male fetuses (4.5 fold lower in the general male fetus sample set vs. case B) (Figure 3B). Fluorescent PCR on white blood cell DNA confirmed the insertion of the PRKY-including region into the patient's genome (Figure 3C).