Genomic DNA was extracted from the peripheral blood leukocytes using a standard phenol-chloroform extraction method (3). The splice junctions and all coding regions of the ABCD1 gene of the proband were amplified by polymerase chain reaction (PCR) using primers ALDe6-F (5’-CGC TCT CTG GCG TCA GCG-3’) and ALDe6-R (5’-TGG TGT TGG TCC TCC CTG-3’). The conditions for PCR were as follows: initial denaturation for 15 seconds at 96°C, followed by 25 cycles of denaturation for 15 seconds at 96°C, annealing for 15 seconds at 55°C and a final extension for 4 min. at 60°C. The reaction mixture at final volume (100 µL) contained 1X PCR buff er II (PE Applied BioSystems, Foster City, CA, USA), 1.5 mM MgCl2, 0.5 M betaine (Sigma, St. Louis, MO, USA), 200 µM dNTP, 1 µM of each primer, two units Taq polymerase and 100 ng of DNA template. The PCR products were sequenced directly using the DyeTM Terminator cycle sequencing kit (PE Applied BioSystems). Restriction enzyme digestions were performed using 40 µL of the initial PCR products of exon 6 using primers ALDe- -F and ALDe6-R, and were incubated at 37°C overnight with five units of HhaI restriction enzyme. The restriction fragments were separated by 3% agarose gel electrophoresis under 100 V for 4 hours.