Patients. Fifty unrelated patients with hemophilia A, and members of their families, were included in this study. Severe, moderate and mild phenotypes were observed in 29, 10 and 11 of the patients, respectively. The clinical severity of the disease was classified according to standard criteria, based on the remaining fVIII activity [8].

DNA was isolated from peripheral white blood cells using the phenol/chloroform extraction and the ethanol precipitation method, routinely in use in our laboratory [9,10].

Southern Blotting. The intron 22 inversion of the fVIII gene was detected by Southern blotting [5]. Namely, 10 mg of genomic DNA was digested with BclI at 50°C, separated on 0.7% agarose gels, transferred to nylon mem­branes (Hybond N+; Amersham Pharmacia Biotech, Frei­burg, Germany) and hybridized at 65°C with a 0.9 kb EcoRI/SstI fragment of plasmid p482.6 (ATCC), contain­ing a part of the intron 22 homologous sequence. The probe was non radioactively labeled using the Fluorescein Gene Images Labeling and Detection System (Amersham Pharmacia Biotech).

The same Southern blot method was used for the determination of gross gene deletions or insertions in the fVIII gene. Genomic DNA was digested with the TaqI restriction enzyme, while the digested DNA was hybrid­ized with probes A and B representing cDNA of the fVIII gene from exons 1-14 and 14-26, respectively [11].

Polymerase Chain Reaction (PCR), Single Strand Conformation Polymorphism (SSCP) and Denatur­ation Gradient Gel Electrophoresis (DGGE). Patients in whom an inversion in intron 22 was not found as a cause of hemophilia A, were further analyzed for the presence of single nucleotide changes or small deletions/insertions in the coding regions of the fVIII gene. All exons, except exon 14, were separately amplified from genomic DNA by PCR using oligonucleotide primers and cycling conditions as described by David et al. [12] for SSCP or Diamond et al. [13] for DGGE.

Generally, 400 ng of genomic DNA, 50 pM/mL of each oligonucleotide, and 1 U Taq DNA polymerase were added to a final volume of 50 mL of 1X reaction buffer, 2 mmol/L MgCl₂, 0.2 mmol/L dNTP each [14]. Most of the PC reactions were performed under the conditions of initial denaturation of 5 min. at 95°C, followed by 32 cycles of denaturation at 94°C for 1 min., annealing at 56°C for 1 min. and elongation at 72°C for 1 min., with a final extension at 72°C for 10 min.

The SSCP was performed on the DeCode System (Bio-Rad Laboratories, Hercules, CA, USA). The PCR products were loaded onto a non denaturing 12% acryl­amide/Bisacrylamide (39:1) gel. Electrophoresis was per­formed at a constant power of 25W, at 4°C for about 20 hours.

DGGE analysis for exons 4 and 11, 5’ of exon 14, 16, 22, 23, and 26, were performed on the same DeCode Sys­tem (Bio-Rad Laboratories). Heteroduplexes were pre­pared by mixing PCR products of the same region from a patient and normal DNA. The mixture was denatured for 5 min. at 95°C and allowed to reanneal slowly for 30 min. to 37°C. The PCR fragments were loaded onto an 8% polyacrylamide gel with a linear gradient of denaturing agents (urea and formamide) in a concentration of 10-60%. PCR fragments were visualized with silver staining of the gel.

DNA Sequencing. To identify the nucleotide substitu­tions responsible for altered electrophoretic mobility detected by SSCP analysis, or heteroduplex formation by DGGE analysis, each of the PCR fragments was sequenced, either manually using the Sequenase Version 2.0 sequencing kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) [15], or by the ABI PRISMO 377 automated sequencer (DNA Sequencing Kit; PE Applied BioSystems, Foster City, CA, USA).