Patients. Case 1 was referred for cytogenetic investigation at 11 years of age, when she started developing as a boy, instead of a girl as her parents had expected. At birth, she was of normal weight and length, but had an obvious genital ambiguity, so that she underwent a clitoro­plasty when she was 3 years old.

      Physical examination revealed short stature for her age, abnormal body proportions with short extremities and well developed muscles. Mild somatic features of Turner syndrome (short stature, webbed neck, cubitus valgus) were noticed. The external genitalia consisted of a small phallus with hypertrophy of labia majora, two perineal openings without palpable gonads on labioscrotal structures or in the inguinal region. The internal genitalia consisted of an intra-abdominal gonad on the right side, a fibrous structure on the left side, and a small uterus. She was referred for surgical removal of the right gonad. Macroscopic and microscopic examination of this gonad revealed a dysgenetic testis.

      Case 2 was 5 years old when she was admitted for karyotype investigation, because her parents requested a second opinion concerning the previous finding of a mosaic karyotype with Y chromosome in her peripheral blood lymphocytes. Ambiguous genitalia were obvious at birth (peniform clitoris, vaginal vestibule with two perineal openings, labioscrotal structure with palpable gonad on the right side which seemed to be a testis on ultrasonographic examination). Vaginography revealed the presence of a vagina and pelvic ultrasonography showed a normal uter­us. At 2 years of age a laparotomy was performed, and the right gonad was removed. On the left side, the presence of a tube and of a gonad with macroscopic appearance of an ovary led to the conservation of that gonad. Macroscopic and microscopic examination of the removed gonad revealed a dysgenetic testis. At 5 years of age, the child showed mild growth delay.

      Classical Cytogenetics. Written and informed consent of the patient’s parents was obtained. Chromosomal studies were performed from peripheral blood samples, for both subjects and the parents of Case 1. Cell cycle synchronization, GTG- and CBG-banding techniques were applied as described [15]. Karyotype description followed the International System for Human Cytogenetic Nomenclature reccomendations [16]. Microscopic analysis was performed on a NIKON E800 microscope and CCD camera (NIKON DS-2MBWc; Nikon Corporation, Tokyo, Japan), using LUCIA karyotyping software (Laboratory Imaging, Praque, Czech Republic).

      Fluorescence In Situ Hybridization (FISH). In this study, FISH was carried out with commercial probes according to the manufacturer’s instructions. These included: Y-specific painting probe (XCP-Y; MetaSystems GmbH, Altlussheim, Germany); centromeric probe for Y (CEN DYZ3); Yq12(DYZ1) (Vysis Inc., Downers Grove, IL, USA); subtelomeric probes for short arm X and Y locus DXYS130; subtelomeric probes for long arm of X and Y, locus DXYS224 (Q-Biogene Inc., Irvine, CA, USA); locus specific Yp11.3 SRY (Vysis Inc.); DAPI counterstaining. Bacterial artificial chromosomes (BAC) clones RP11-400O10 and RP11-140H23 were used in Case 2.

      Selection and Isolation of BAC DNA For Fluorescence In Situ Hybridization Analysis. Two BAC clones from RPCI-11 human libraries were selected from NCBI (http://www.ncbi.nlm.nih.gov) databases. The RP11-400O10 clone is localized on Yp11.3 [position 2,709,521-2,838,553 (http://www.ncbi.nlm.nih.gov/entrez/viewer. fogi?list_uids=10801470] and covers the entire SRY gene. The RP11-140H23 clone is localized on Yq11.2 (position 23,691,183-23,794,414). The BAC clones were supplied by the Wellcome Trust Sanger Institute (Hinxton, Cambridgeshire, UK). The FISH probes were directly labeled with Spectrum Green-dUTP (RP11-140H23) and Rho­damine (RP11-400O10) using commercially available kits (Vysis Inc.; and Roche Diagnostics GmbH, Mannheim, Germany). The FISH analysis was performed on an epi­fluorescence microscope (NIKON E800; Nikon) equipped with a CCD camera (NIKON DS-2MBWc; Nikon) and dedicated software (NIS BR Elements - LUCIA; Laboratory Imaging). A minimum of five metaphases containing the structurally abnormal Y chromosome were examined with each probe.

      Polymerase Chain Reaction (PCR). Molecular studies were performed on genomic DNA that was extracted by a salting out method from fresh peripheral blood collected on EDTA [17]. Two DNA markers were amplified by PCR: i) a short DNA sequence from the first intron of Amelogenin gene which is localized on X chromosome (Xp22.31-p22.1 region) is 106 bp long, and Y chromosome (Yp11.2 band) is 112 bp long. ii) The DYS392 STR marker which is localized on the long arm of the Y chromosome (Yq11.2, position 21,043,146-21,043,399) and has the [TAT]_n repeat. The primer pair used in this study (STRBase) amplified a 93-125 bp DNA sequence, depending on the existent allele [18].

      The amplified DNA sequence, two PCR products should be obtained in the case of male individuals. The PCR mixture consisted of 1X PCR buffer (10X PCR Buf­fer: 200 mM Tris-HCl, pH 8,4, 500 mM KCl), 1,5 mM MgCl₂, 200 mM dNTPs, 200 mM of each primer, 1 unit Taq-DNA polymerase, 1 mg/mL bovine serum albumin (BSA) and sterile water up to 20 mL.           

  After an initial DNA denaturation step at 94°C for 2 min., 35 cycles were performed, each consisting of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C. The reaction was terminated with a 7 min. extension at 72°C. The PCR reactions were performed in a UNO II Biometra® Thermo­cycler (Biometra Biomedizinische Analytik GmbH, Goet­tingen, Germany). The PCR products were demonstrated by electrophoresis on 8% PAA (polyacrylamide) gel and silver-stained [19]. All PCR assays were carried out in duplicate, and control reactions were also performed with DNA extracted from a normal male, from a normal female, and a negative control (with water).