Non-Invasive Prenatal Test Analysis Opens a Pandora's Box: Identification of Very Rare Cases of SRY-Positive Healthy Females, Segregating for Three Generations Thanks to Preferential Inactivation of the XqYp Translocated Chromosome

Abstract

The translocation of the testis-determining factor, the SRY gene, from the Y to the X chromosome is a rare event that causes abnormalities in gonadal development. In all cases of males and females carrying this translocation, disorder of sex development is reported. In our study, we described a peculiar pedigree with the first evidence of four healthy females from three generations who are carriers of the newly identified t(X;Y)(q28;p11.2)(SRY+) translocation with no evidence of ambiguous genitalia or other SRY-dependent alterations. Our study was a consequence of a Non-Invasive Prenatal Test (NIPT) showing a sexual chromosomal abnormality (XXY) followed by a chorionic villus analysis suggesting a normal karyotype 46,XX and t(X;Y) translocation detected by FISH. Here, we (i) demonstrated the inheritance of the translocation in the maternal lineage via karyotyping and FISH analysis; (ii) characterised the structural rearrangement via chromosomal microarray; and (iii) demonstrated, via Click-iT^® EdU Imaging assay, that there was an absolute preferential inactivation of the der(X) chromosome responsible for the lack of SRY expression. Overall, our study provides valuable genetic and molecular information that may lead personal and medical decisions.

Keywords: SRY-positive females; Y) unbalanced translocation; preferential X-inactivation; t(X.

Figure 1

Pedigree of the family. Arrow indicates the first patient enrolled in our study (proband (II,2) that carried the translocation under investigation. The females I,1, II,4 and III,1 are carriers of the same translocation.

Figure 2

(A) Karyotype test of II,2 (550 band resolution, magnification 100×) showing a sexual chromosomal anomaly reported as 46,X,der(X)?add(X)(q28), a pericentric inversion of chromosome 9 as chromosome heteromorphism was also detected (left); FISH analyses conducted on II,2 (right). Red signal refers to SRY region (spectrum orange LSI-SRY probe) while green signal refers to the centromere region of the X chromosome (spectrum green DXZ1 probe); (B) FISH analysis conducted on I,1 and II,4 (left and right, respectively) with the same probes mentioned for II,2. FISH analysis identified a derivative of an SRY-positive X chromosome arising from a translocation t(X;Y)(q28;p11.2) on all the investigated females.

Figure 3

CMA profile showing allele frequency and copy number change for DNA extracted from peripheral blood sample of II,2. (A) The red bar indicates the deleted region at terminal q-arm of the X chromosome (Xq28) and green bar represents duplicated probes mapping in shared pseudoautosomal region (PAR1) between Xp and Yp. The allelic profile based on the B allele frequency confirmed the copy number changes. (B) Green bar shows additional genomic material arising from Yp spanning from pseudoautosomal region (Yp11.32) to Y-specific region (Yp11.2) for about 9 Mb. (C) Ideogram demonstrates that the derivative X chromosome consists of Yp11.32p11.2 replacing the Xq28 deleted portion and includes the SRY gene, mapping in Yp11.2 (blue square).

Figure 4

der(X) inactivation test conducted by FISH analysis on fixed metaphases pre-treated with EdU fluorescent solution on II,2 (left) and II,4 (right) patients. Green signals indicate Xp and Yp subtelomeric regions and pink signal generated by conjugating with Alexa Fluor 647 identifies der(X) as inactive (strong pink coloration) in all analysed metaphases (>25 for each patient). In all FISH analyses, chromosomes were counterstained with DAPI (blue) and images were acquired at a magnification of 100× with a DMRA fluorescent microscope.