GENETIC TESTING Volume 6, Number 3, 2002 © Mary Ann Liebert, Inc. A PCR-RFLP Test for Simultaneous Detection of Two Single- Nucleotide Insertions in the Connexin-26 Gene Promoter MEHMET SIMSEK, 1 NADIA AL-WARDY, 1 AISHA AL-KHAYAT, 2 and MAZIN AL-KHABORY 3 ABSTRACT Comparisons of Connexin-26 (GJB2) gene sequences available in the GenBank data base indicate the pres- ence of a polymorphism in the promoter, but no easy method is available for the detection of this polymor- phism. We have developed a PCR-RFLP test for simultaneous detection of two single nucleotide insertions (G and A) in the GJB2 promoter. The test is based on amplification of a 146-bp DNA fragment, which was di- gested with Mae I to detect the G insertion in the promoter. A similar digestion with Hinf I detects the A in- sertion. The test was validated using direct DNA sequencing of amplified DNA from 33 samples. After vali- dation, we have used it to investigate DNA samples from 160 control subjects and 51 unrelated patients with nonsyndromic autosomal recessive deafness. All of the samples analyzed using the PCR test and DNA se- quencing were found to contain both the G and A insertions in the GJB2 gene promoter. This PCR test will be useful in studying the prevalence of these two insertions in other populations. 225 INTRODUCTION C ONGENITAL DEAFNESS is a common hearing impairment, with an incidenceof about1 in 1,000 live births.It is known that more than 50% of these cases are hereditary(Morton, 1991; Skvorak and Morton, 1999), of which approximately 80% are due to nonsyndromic autosomal recessive deafness (NARD). Recently, several mutations have been described in the coding region of the Connexin-26 (GJB2) gene. Some of these muta- tions have been detected in NARD patients at a relatively high frequency in different populations (Gasparini et al., 2000; Kelsell et al., 2001). However, mutations occurring in the pro- moter region of the GJB2 gene appear to be rare, and only a few cases have been described (Rabionet et al., 2000). Com- parisons of GJB2 gene sequences submitted to the GenBank in- dicate the presence of two single-base insertions (G and A), lo- cated at nucleotide positions (254) and (257), respectively, in relation to the initiation codon of the gene. Because the size of the Connexin-26 (GJB2) gene is rela- tively small (Zhang and Nicholson, 1989)—about 900 bp in length, including the promoter and coding region—direct DNA sequencing has been used to detect mutations in this gene (Kelsell et al., 1997; Estivill et al., 1998;Gasparini et al., 2000). Some laboratories have also used PCR-restriction fragment length polymorphism (RFLP) methods for detecting mutations in the coding region of the gene (Storm et al., 1999; Wilcox et al., 2000), but no PCR tests are available for the promoter mu- tations. Here, we describe a PCR test, that can detect the G and A insertions in the GJB2 gene promoter simultaneously. The test has been validatedusingdirectDNA sequencingon 33 sam- ples and then used to investigate DNA samples from 160 con- trol subjects and 51 NARD patients for the presence of these insertions in the GJB2 gene promoter. MATERIALS AND METHODS Genomic DNA was extracted from frozen whole blood of NARD patients and healthy subjects using a standard salting- out procedure (Miller et al., 1988). A 146-bp DNA fragment was amplified by PCR, and subsequentlytreated with Mae I or Hinf I. Primers used in the PCR were Cx112m (59-TCA GAG AAG TCT CCC TGT TCT GTC A TA-), and Cx258R (59-AAT GCT GGT GGA GTG TTT GTT CAC-). The underlined let- ter in the previous primer represents a mismatched base to in- activate an existing Mae I site. The PCR mixture (50 ml) con- 1 Biochemistry Department, Sultan Qaboos University, College of Medicine, Postal Code 123 Muscat, Sultanate of Oman. 2 Biology Department, Sultan Qaboos University, College of Medicine, Postal Code 123 Muscat, Sultanate of Oman. 3 Department of Otolaryngology, and Head and Neck Surgery, Al-Nahda Hospital, Postal Code 112 Muscat, Sultanate of Oman.