Patients. Ten families from the Republic of Macedonia were included in the analysis of the IT15 gene. Seven patients were referred for diagnostic testing due to the presence of clinical signs consistent with HD, while three individuals required predictive testing due to the presence of the disease in the family. The data on age, gender, family history and type of referral of the patients is summarized in Table 1.

      Methods. DNA was isolated from leukocytes using the proteinase K/SDS digestion-phenol/chloroform extraction-ethanol precipitation method, routinely used in our laboratory [9]. The conventional analyses consisted of polymerase chain reaction (PCR) analysis of exon 1 of the IT15 gene, denaturing polyacrylamide gel electrophoresis (PAGE) of the resulting fragments, transfer to a nylon membrane, hybridization with a digoxigenin labeled internal probe and visualization by autoradiography [10,11]. Polymerase chain reaction amplification was performed in a reaction volume of 25 mL using 50 ng of genomic DNA, 25 pmol of each primer (direct: 5'-ATG AAG GCC TTC GAG TCC CTC AAG TCC TTC-3' and reverse: 5'-AAA CTC ACG GTC GGT GCA GCG GCT CCT CAG-3'), 10 mM Tris (pH 8.3), 5 mM KCl, 2 mM MgCl₂, 200 mM (each) dNTPs, 10% dimethylsulfoxide and 2.5 U of Taq polymerase (AmpliTaq Gold; Applied BioSystems, Palo Alto, CA, USA). After initial heating at 94EC for 10 min, the reaction mix was subjected to 40 cycles of amplification, each consisting of incubation for 1 min at 94EC, 1 min at 60EC and 2 min at 72EC. Two mL of each PCR were diluted with two volumes of 95% formamide loading dye and heat denatured for 2 min at 95EC. The products were resolved on a 6% denaturing polyacrylamide gel (40 x 20 cm x 0.4 mm) containing 6 M urea and 32% formamide. The electrophoresis was run at 70 W constant power for 2 hours and the fragments were transferred to a positively charged nylon membrane (Hybond+; Amersham BioSciences UK Ltd., Little Chalfont, Buckinghamshire, England). The membranes were hybridized with a non radioactively labeled oligonucleotide probe (5’-CAG CAG CAG CAG CAG CG-3’) and detected by chemi­luminescence. Details for non radioactive labeling of oli­gonucleotide probes, allele specific hybridization and che­miluminiscent detection are as given previously [10,11]. A representative autoradioagram showing the results obtained using the PCR-PAGE method is given in Fig. 1. The results obtained with the non radioactive PCR-PAGE method were compared with the results obtained with automated fluorescent PCR/ capillary electrophoresis analysis using the ABI PRISM™ 310 Genetic Analyzer (Applied BioSystems). In this assay, PCR was performed under the same conditions as described above, the only difference being that the direct primer was labeled at the 5’-end with FAM fluorescent dye. Following amplification, 1-2 mL of the PCR reaction were mixed with 25 mL of deionized formamide and 1 mL of ROX-labeled size marker (Applied BioSystems), denatured for 3 min at 95EC and run on the ABI PRISM™ 310 Genetic Analyzer (Applied BioSystems). The results obtained were analyzed using the GeneScan Software II (Applied BioSystems). Electrophoretograms showing the detection of expanded CAG repeats in exon 1 of the HD gene in two HD patients are shown in Fig. 2.

D: diagnosis; P: predictive testing