Materials. Two-hundred and twenty-eight unrelated patients from the Department of Pediatrics (n = 150) and the Institute for Respiratory Diseases (n = 78), Faculty of Medicine, Skopje, Republic of Macedonia, and numerous members of their families were referred for molecular CF analysis to the laboratory of the RCGEB, MASA, Skopje, Republic of Macedonia. Informed consent was obtained from all patients. The clinical diagnosis of CF was based on clinical features and positive sweat tests (Cl>60 mmol/L).

      Methods. DNA was isolated from leukocytes using the Proteinase K/SDS digestion-phenol/chloroform extraction-ethanol precipitation method, routinely used in our laboratory. The initial screening of all samples included polyacrylamide gel electrophoresis (PAGE) electrophoresis of PCR-amplified fragments for the detection of the DF508 mutation, as described previously [11]. In the first 3 years of the study other common mutations (G542X, W1282X, N1303K, R117H, 1717-1G®A, G85E, 444delA, G551D, R553X, R1162X, 3849G®A) were screened by PCR amplification, followed by allele specific oligonucleotide (ASO) hybridization and/or by restriction fragment length polymorphism (RFLP) analysis [12-15].

      Screening for unknown mutations was initiated in 1992, and it was initially performed by radioactive SSCP analysis [16] of exons 2, 3, 4, 5, 6a, 7, 8, 10, 11, 12, 17b, 18, 19, 20, 21 and 23. Since 1995, a DGGE analysis using primers and procedures described by Costes et al. [17] and Fanen et al. [18] has been used. Samples showing an abnormal pattern on SSCP or DGGE were further analyzed by direct sequence analysis of PCR amplified fragments using Sequence version 2.0 kit (Amersham Pharmacia Biotech UK Ltd., Little Chalfont, Buckinghamshire, UK) and 35SdATP [19] or fluorescent cycle sequencing on an ABI PRISM™ 310 Genetic Analyzer (Applied BioSystems, Foster City, CA, USA).

      Prenatal diagnosis was performed on DNA material isolated from chorionic villi, amniotic fluid and fetal blood using the procedure described above. Depending on the molecular defect, prenatal diagnosis was based on direct detection of the mutation and/or on haplotype analysis of polymorphic XV2c/TaqI and KM19/PstI markers [20].