There are approximately 6,000 persons with hearing loss or hearing impairment in Macedonia. Patients with hearing impairment have been ascer­tained through two regional otorhinolaryngology centers: Audiology Center, Clinic for Othorinolar-yngology, Medical faculty, Skopje and Association of Deaf and Hard of Hearing, Skopje, R. Mace­donia. We performed molecular analyses of the GJB2 gene on DNA samples from 80 individuals with non syndromic hearing loss (NSHL) belong­ing to 33 affected, unrelated families. Twenty-three families were of Macedonian, six of Albanian, one Turkish and three of Gypsy origin. After informed consent was obtained and examination of complete medical histories was made, the family inheritance and audiological status were ascertained. DNA was isolated from peripheral white blood cells using a phenol/chloroform extraction, ethanol precipitation method [8].

The 35delG mutation was identified by the method described by Storm etal. [9], in which poly­merase chain reaction (PCR) amplifications were performed with two sets of primers: GJB1: 5'-GTG AGG TTG TGT AAG AGT TG-3'; GJB2: 5'-CTG GTG GAG TGT TTG TTC CCA-3'; and as con­trol sequence: GJB7 5'-CCA GGC TGC AAG AAC GTG TGC-3' and GJB9: 5'-CTC ATG TCT CCG GTA GGC CAC-3' under standard PCR conditions. The PCR products were digested with BszYI (Boeh-ringer, Mannheim, Germany) and analyzed by 2% agarose gel electrophoresis (Figure 1A).

To identify the nucleotide substitutions respon­sible for altered the electrophoretic mobility de­tected by SSCP analysis, the PCR fragments were sequenced by BigDye sequencing kit v3.1 (PE Ap­plied Biosystems, Foster City, CA, USA), accord­ing to the manufacturer's instructions and separated on an Applied Biosystems 310 Genetic analyzer.

The presence of the deletion delD13S1830 en­compassing part of the GJB6 gene was analyzed by use of three primers to simultaneously amplify the breakpoint-containing fragment and the normal GJB2 allele [10]. The primers used were: GJB6-1F 5'-AGT GAT CCA TCT GCC TCA GC-3'; GJB6-2RN 5'-GTC TGT GCT CTC TCT TTG ATC TC-3' and GJB6-3RD 5'-GGA AGG TGT GGA TCA CAG TC-3', under cycling conditions of initial denaturation of 10 min. at 95°C, followed by 30 cycles of 1 min. at 94°C, 1 min. at 60°C and 1 min. at 72°C in the Applied Biosystems 2720 thermal-cycler. The PCR fragments were analyzed by 1.5% agarose gel electrophoresis. The presence of a 478 bp fragment indicates the presence of the 342 kb deletion (Primers GJB6-1F and GJB6-3RD), while the presence of a 650 bp fragment indicates the nor­mal allele (primers GJB6-1F and GJB6-2RN).