Routine cytogenetic analysis from cultured peripheral lymphocytes had been performed using a modified Edward-Troland method. After incubating for 72 hours in standard medium with phytohemaglutinin, mitotic arrest with Colcemid and harvesting procedure, the G banding technique was used. Metaphase spreads (500-band stage) were analyzed using VideoTest-Karyo software. The chromosome analysis revealed a 47, XYY karyotype.
The karyogram of the father was normal (46, XY). He refused further examination, thus, the reason for his mental disability remains unknown.
Fluorescence in situ hybridization (FISH) was performed on metaphase spreads using the standard procedure: pretreatment of the slide subsequently with pepsin, PBS and ethanol series in order to clean up the specimen. Denaturation was performed in both probe and slide (immersed in 70% formamid/2X SSC solution) at 72°C for 3 min. After placing the probe onto the slide, overnight hybridization was performed at 37°C in a dark chamber. Posthybridization washes were carried out the next day. A solution of 0.5 mg/mL DAPI in mounting medium was used as counter staining. Hybridization signals were detected using an Olympus fluorescence microscope (Olympus Optical, Hamburg, Germany) equipped with appropriate filters. The origin of the extra chromosome was confirmed using an α satellite centromere probe for the Y chromosome (Cytocell, Cat. No. LPEY cG; Cytocell Technologies, Cambridge, Cambridgeshire, UK). The presence of two Y chromosomes was confirmed in all cells, both metaphase spreads and interphase nuclei.