PMC5470200_01
Cytogenetics and FISH characterization of marker. a QFQ banding of the normal chromosome X (right) and the marker (left). b RBA banding shows the inactivation of the marker (arrow) c FISH analysis with the specific probe for Xp11.1-q11.1 alpha-satellite (DXZ1, red signals) on the normal chromosome X and the marker (arrow). d FISH analysis with b167P23 BAC probe (Xp11.22, red signals) on the normal chromosome X and the marker (arrow). e FISH analysis with b217H1 BAC probe (Xq13.2-21.1, red signals) on the normal chromosome X and the marker (arrow). f FISH analysis with the specific probe for Xp/Yp telomere (DXYS129, red signals) on the normal chromosome X and the marker (arrow). g FISH analysis with the probe for common telomeric sequences (TTAGGG, red signals) on all chromosomes. The arrow indicates the marker. h Schematic representation of marker chromosome structure
13039_2017_323_Fig1_HTML.jpg
PMC5470200_01_13039_2017_323_Fig1_HTML.jpg
Fig1
It was possible to recognize the marker morphology as a double apparently complete chromosome X, probably an isochromosome, sized more than chromosome 1 (Fig. 1a).  Xp arms, where usually the telomeric regions are located. X inactivation study by means of RBA banding evidenced that the marker was inactive in all analysed cells (Fig. 1b). The centromeric X alpha-satellite probe (DXZ1) showed an intense positive signal in the middle of the chromosome (new AC) and other two positive regions equally distant from the new centromere in the canonical position (Fig. 1c). Using BAC probes mapped in Xp11.22 (b167P23) and Xq13.2-21.1 (b217H1) an Xp paracentric inversion involving the entire p arm was identified (Fig. 1d, e, and h). In fact, the two hybridization signals for the 167P23 BAC probe were located at both sites of the AC instead near the ICs (Fig. 1d, h). The specific Xp telomere probe (DXYS129) showed two hybridization signals localized near the two ICs (Fig. 1f) confirming the Xp inversion. The common telomeric sequences (TTAGGG) were evidenced in 4 different positions: 2 in correspondence of Xq telomeres and 2 next to the ICs (Fig. 1g, h).
FISH characterization of deleted marker. a FISH analysis with the specific probe for Xp11.1-q11.1 alpha-satellite (DXZ1, red signals) on the normal chromosome X and the deleted marker (arrow). b FISH analysis with the probe for common telomeric sequences (TTAGGG, red signals) on all chromosomes. The grey and the white arrows, indicate the deleted marker and the acentric fragment respectively. c FISH analysis with the specific probe for Xp/Yp telomere (DXYS129, red signals) on the normal chromosome X and the entire marker. The arrow indicates the possible breaking point. d FISH analysis with the specific probe for Xp/Yp telomere (DXYS129, red signals) on the normal chromosome X and the deleted marker (arrow). e Schematic representation of deleted marker structure
13039_2017_323_Fig2_HTML.jpg
PMC5470200_01_13039_2017_323_Fig2_HTML.jpg
Fig2
The breakpoint was identified between the Xp TTAGGG sequences and the IC, as shown in Fig. 2a where the third centromeric signal of DXZ1 probe is not visible, probably retained by the lost fragment, while TTAGGG sequence was maintained on the deleted psu itrc(X) (Fig. 2b). A schematic representation clarify the mechanism (Fig. 2e). 
Hypothetical mechanism of marker formation. a Normal chromosome X. b Crossing-over mechanism between Xp telomere and the centromeric telomere-like sequences. c Paracentric inversion involving the Xp arm that results in a shift of the p end of centromere. d Duplication of the entire X chromosome, that gives rise to an isochromosome X
13039_2017_323_Fig3_HTML.jpg
PMC5470200_01_13039_2017_323_Fig3_HTML.jpg
Fig3