Balkan Journal of Medical Genetics

INTRON 2 SPLICE MUTATION AT CYP21 GENE IN PATIENTS WITH CONGENITAL ADRENAL HYPERPLASIA IN THE REPUBLIC OF MACEDONIA
Anastasovska V, Kocova M
*Corresponding Author: Mirjana Kocova, Department of Endocrinology and Genetics, University Children’s Clinic, Vodnjanska 17, 1000 Skopje, Republic of Macedonia; Tel.: +389-70-242-694; Fax: +389-23-129-027; E-mail: mirjanakocova@yahoo.com
page: 27

MATERIALS AND METHODS

We studied 41 Macedonian patients (27 girls and 14 boys) with clinical and laboratory signs of CAH evaluated at the Department of Endocrinology and Genetics, University Children’s Clinic, Skopje, Republic of Macedonia, and 55 of their healthy parents and siblings, belonging to 37 unrelated families. There was one patient in 33 families and two patients in four families. Informed consent for the genetic study was obtained from each family. The CAH patients were of mixed ethnicity (28 of Macedonian ethnicity, nine of Albanian ethnicity and four Gypsies). All patients had elevated plasma 17-hydroxyprogesterone (17-OHP) and were classified according to standard criteria [2]. Of the 41 patients, 19 (46.3%) had the SW form characterized by extremely elevated 17-OHP levels (up to 75 nmol/L) with onset of dehydration and/or shock associated with hyperkalemia and hyponatremia. Females had ambiguous genitalia. The diagnosis was made within 2 months after birth. Four patients (9.8%) with SV form at the age 2-14 years were diagnosed due to signs of androgen excess, after corticotropin stimulation. Eighteen patients (43.9%) had the LO form characterized in girls by normal external genitalia and in both sexes by precocious pubarche and elevated 17-OHP levels, 60 min. after stimulation [2]. The diagnoisis was made at age 4-17 years. Molecular Analysis. DNA samples of all subjects were obtained from peripheral blood lymphocytes by the standard proteinase K-phenolchloroform method [20]. Direct molecular detection of the IVS-II-656 mutation was performed by the polymerase chain reaction/amplification created restriction site (PCR/ACRS) method, followed by restriction enzyme digestion [21]. Eight other common pseudogene-derived point mutations: p.Pro30Leu, 8 bp deletion in exon 3 (G110Δ8nt), p.Ile172Asn, exon 6 cluster (p.Ile236Asn, p.Val237Glu, p.Met239Lys), p.Phe306+t, p.Val281Leu, p.Gln318X and p.Arg356Trp were also tested in all subjects (Table 1). The primary differential PCR amplification of the active CYP21 gene, without contamination from the highly homologous pseudogene sequence, was performed with 20 pmol of each CYP21 specific primer (21BF: 5’-TCG GTG GGA GGG TAC CTG AAG-3’ and 21BR: 5’-AAT TAA GCC TCA ATC CTC TGC AGC G-3’) under the PCR conditions described in Gene Amp XL (Extra Long) PCR Kit (Applied Biosystems, Branchburg, NJ, USA). The EcoRI digestion of the 3.2 kb PCR product from the CYP21 gene results in two fragments (1.0 and 2.2 kb), whereas the pseudogene digested similarly results in three fragments (0.5, 0.6, and 2.2 kb). This ensured that only the active gene sequence had been amplified and analyzed (Figure1). The primary PCR product was used as a template for a secondary PCR amplification using the ACRS method with primers specific for detection of the IVS-II mutation (C3B: 5’-TTC ATC AGT TCC CAC CC TCC AGC CCC gA-3’ and C4A: 5’-CTT CTT GTG GGC TTT CCA GAG CAG GtA-3’). The secondary ACRS PCR was carried out in a final volume of 50 μL containing primary PCR product, 50 pmol of each primer, 200 μM of each dNTP, 1,5 mM MgCl2 and 1.25 U Taq DNA polymerase (Ampli Taq Gold; Applied Biosystems) for 30 cycles at 95°C for 1 min., 62°C for 1 min. and 72°C for 2.5 min. A 115 bp PCR product was obtained (Figure 2). The amplified fragments were digested with 5-10 U of a SacI restriction enzyme at 37°C overnight and the digestion products analyzed on 2% high resolution agarose gel detected by ethidium bromide staining on UV radiation. The mutant allele provides a recognition site for SacI, so that digestion with this enzyme produces two fragments of 85 and 30bp. The normal allele contains no restriction site for SacI (Figure 2). The digestion with the restriction enzyme allowed not only the mutation detection but also determination of the zygosity of the analyzed mutation in an individual.

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