Patients. We studied nine patients after allogeneic BMT, five male children and one adult female, by FISH with alternatively labeled X and Y probes, after transplantation from different sex donors. Three male-to-male donor-recipient pairs were studied by genotyping of STR and VNTR markers. The clinical data of the patients are shown in Table 1.
Fluorescent In Situ Hybridization. We used alternatively labeled green and orange centromere probes for the X- and Y-chromosome (Cat. # 32-132023 and Cat.# 32-130024; Vysis, Downers Grove, IL, USA). Lymphocytes were isolated using Limphoprep (Nycomed Pharma AS, Oslo, Norway) from peripheral blood. The FISH procedure was carried out as described in “CEP procedure” (Vysis). Fluorescence signals were detected using a fluorescence microscope with respective filters (Olympus BX60; Olympus Optical Co., Tokyo, Japan) And a software program (ISIS; MetaSystems Altlussheim, Germany). The percentage of chimerism was determined from the analysis of at least 200 successfully hybridized cells (Fig. 1).
Genotyping of Microsattelite Loci. Three male-to-male donor-recipient pairs were studied by genotyping of six STR markers (ACPP, D3S1282, D3S1509, SST, RHO, D3S1212) and two VNTR markers (SERT and DAT1). DNA from whole blood was obtained by protein­ase K digestion, phenol/chloroform extraction and subsequent ethanol precipitation. Primers available in GenBank Database were used.
For the STR markers, PCR was performed in 15 mL containing 200 ng genomic DNA, 1 x PCR buffer, 9 pmol (12.5 pmol for ACPP) of each primer, 200 mM dNTPs, 0.4U Taq DNA polymerase (GIBCO BRL, Gaithersburg, MA, USA), 1.5 mM MgCl2. The reaction was carried out on an OmniGene thermal cycler (Hybaid Ltd., Ashford, Middx, UK). The amplification conditions were: 94°C (5 min.), followed by 30 cycles at 94°C (40 seconds), 1 min. annealing step at the temperature corresponding to the annealing temperature of primer pairs, 72°C (1 min.) and a final extension at 72°C (7 min.). Separation and detection of the amplified STR fragments were by 4 or 6% high-resolution vertical denaturing polyacrylamide gel electrophoresis (PAGE) for 2.5 hours at 60 W and silver staining, respectively. The size of each allele was calculated to 100 bp DNA ladder (GIBCO BRL).
For the VNTR markers, PCR was performed in a volume of 12 mL that included 20 ng genomic DNA, reaction buffer, 5 pmol of each primer, 200 mM dNTPs, 1 unit of TaqDNA polymerase, 1.5 mM MgCl2. Amplification consisted of 94°C for 3 min., touchdown PCR conditions using primer annealing temperatures of 66°C and 64°C at two cycles each, followed by 30 cycles of 94°C for 30 seconds, 62°C for 45 seconds and 72°C for 45 seconds, and a final extension step at 72°C for 10 min. The amplification products were resolved on 1% agarose/1.5% Meta­phore gels and visualized by ethidium bromide transillum­mination.