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

RESULTS AND DISCUSSION

The IVS-II mutation was detected in 17 (41.5%) of the CAH patients. Twelve (29.3%) were homozygous and five (12.2%) were heterozygous. All homozygous patients had severe classical CAH phenotype with 91.7% having the SW phenotype. Although this phenotype is usual in the homozygous state for this mutation [22], one of our homozygous patients displayed the SV phenotype. Three heterozygous patients with the SW phenotype, were compound heterozygotes having another of the tested mutations (p.Pro30Leu; 8 bp deletion in exon 3 (G110Δ8n)t; p.Ile172Asn; exon 6 cluster (p.Ile236Asn, p.Val237 Glu, p.Met239Lys); p.Phe306+t; p.Val281Leu; p.Gln 318X; p.Arg356- Trp). One of the compound heterozygotes had four different mutations: IVS-II, p.Val281Leu, p.Gln 318X and p.Arg356Trp. These complex alleles may have resulted from small or large gene conversions or from multiple mutation events. The second and the third compound heterozygote each had a mutation on another allele, p.Gln318X and p.Arg356Trp, respectively. The genotypes of the three compound heterozygotes and of their parents are presented in Table 2. In two other heterozygotes, one with SW and another with the LO phenotype, no second mutant allele was detected (Table 3). The frequency of the IVS-II mutation allele in our subjects is similar to that reported for other populations. This mutation has a high frequency in many parts of the world, except in some groups of patients in South Europe, and may be a hot-spot [23-25]. The IVS-II mutation was also found in 17 (30.9%) family members, of whom 10 (18.2%) were homozygotes and seven (12.7%) were heterozygotes. None had any clinical manifestation. The IVS-II mutation alters pre-mRNA splicing by activating another acceptor site for the splicing process and thus shifting the reading frame to create premature termination of translation [26]. Comparison of the phenotypic features with the IVS-II genotypes, shows phenotypic heterogeneity extending from classical SW CYP21 deficiency to asymptomatic carrier [26,27]. An unusually high frequency of “asymptomatic homozygotes” for a mutation expected to severely compromise CYP21 function has been described [28]. That an apparent homozygosity for the IVSII mutation may result from unequal amplification (“allele dropout”) of CYP21 alleles which leads to obscured normal alleles and represent a common diagnostic pitfall of methods employing PCR. For prenatal diagnosis, microsatellite typing could be used as a supplement to CYP21 genotyping so as to resolve ambiguities at nucleotide 656 on intron 2 [28,29]. However, asymptomatic homozygotes for the IVS-II mutation, may be polymorphic for unidentified splice regulatory factors such that the cryptic splice becomes ignored, permitting correct splicing of CYP21 pre-mRNA [28,30]. Since a severe IVS-II mutation almost totally abolishes CYP21 activity, homozygosity for splicing mutation in family members, could be attributed to the tendency of cyto chrome P450 enzymes to be “promiscuous” enzymes that bind many different substrates and catalyze a wide variety of hydroxylations, so that the expression of such an enzyme in the adrenal gland could account for the cryptic CYP21 activity [31]. The observed discordance between genotype and phenotype may result from either the postulated extra adrenal hydroxylase activity or from other factors that modify steroid CYP21 transcription, translation and action. Although some promoter elements important for a CYP21 gene transcription are known, and regulatory proteins that affect CYP21 expression have been proposed [32,33], much remains unexplained. Moreover, different receptor numbers or binding affinity for androgens, cortisol or aldo sterone may contribute to the phenotypic variability. Also, the activity of transcription factors and the expression of transport proteins may be individually regulated [34]. Strong genotype-phenotype correlation was observed in all Macedonian CAH patients with the detected IVS-II mutation. However, our results in the other family members confirm that the genotype cannot be completely predictive of phenotype [22,35].

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