Steroid 21-hydroxylase gene mutational spectrum in 50 Tunisian patients: Characterization of three novel polymorphisms
Highlights
► Molecular analysis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency in Tunisian patients was realized. ► Three novel variants in CYP21A2 gene were identified. ► Results were important in terms of prediction of disease severity, genetic and prenatal counseling.
Introduction
Congenital adrenal hyperplasia (CAH, OMIM#201910) due to steroid 21-hydroxylase deficiency (21-OHD) is one of the most common inborn endocrine disorders and is inherited as an autosomal recessive disease (Lee et al.). Molecular abnormalities of the 21-hydroxylase gene (CYP21A2) lead to various degrees of impaired cortisol and aldosterone biosynthesis, and to androgen excess. There are three major phenotypes depending on severity of the enzymatic defect: the classical salt-wasting form (SW), classical simple virilizing form (SV), both revealed at birth, and the non classical form (NC) with late-onset symptoms and diagnosis in childhood or after puberty (Speiser et al., 1992). The incidence is estimated at 1/15000 in live births for classical SW/SV CAH (Tian et al.) and 1/1000 for NC-CAH (Witchel and Azziz, 2010). The CYP21A2 gene is located on chromosome 6p21.3 in close proximity to a highly homologous inactive pseudogene CYP21A1P. CYP21A2 and CYP21A1P contain 10 exons and 9 introns, spaced over 3.4 kb (Vrzalova et al.). Their nucleotide sequences are approximately 98% identical in exons and 96% identical in introns (Trakakis et al.). To date, complete gene deletions or large gene conversions as well as point mutations have been reported as the cause of the disease, and more than 150 alleles have been identified in patients with CAH (Human Gene Mutation Database: http://www.hgmd.cf.ac.uk/ac/index.php) (Urabe et al., 1990, Wilson et al., 1995). Most of these point mutations arise from micro-conversion of the CYP21A1P with the CYP21A2 (Lee et al., 2000, Tusie-Luna and White, 1995). Molecular genetic testing of the CYP21A2 gene for a panel of the ten most common mutations and gene deletions detects about 90–95% of disease-causing alleles, whereas complete sequencing is needed to detect rarer alleles (White and Speiser, 2000). The incidence of the genetic defects of 21-OH has been extensively studied and ethnic-specific distribution of mutations has been reported (Wilson et al., 2007).
In this paper, the genetic analysis of CYP21A2 in 50 Tunisian patients is presented. As far as we know, this is the first report about distribution of mutations causing the classical and non classical forms of 21-OHD in the Tunisian population to be published. The analysis includes the complete CYP21A2 sequence in order to detect common and rare mutated alleles in all patients and MLPA, when needed. Allele variability and the distribution of mutations among the patients were estimated and compared with other populations. Three new CYP21A2 variants were identified in introns 4, 5 and 7 respectively. In addition, genotype–phenotype correlation was studied to provide more evidence for using genotype as a reliable predictive tool in clinical practice.
Section snippets
Patients
We studied 50 patients with CAH presumably due to 21-hydroxylase deficiency, referred from all over the country to the laboratory of Human Cytogenetics, Molecular Genetics and Reproductive Biology, Farhat Hached University Hospital, Sousse (Tunisia). The patients (21 males and 29 females) came from 44 unrelated families. In five families, there was more than one patient with 21-OHD. Consanguinity was documented in 23 families (52.27%), absent in 16 families, and unknown in five families.
Distribution of disease-causing alleles
In the present study we systematically genotyped 50 Tunisian patients clinically diagnosed as having a CAH due to 21-hydroxylase deficiency from 44 families. The clinical characteristics and genotyping results of the probands are shown in Table 1. Among the 50 patients, twenty two presented with SW-CAH (9 girls and 13 boys), fourteen had SV-CAH (3 boys and 11 girls) and fourteen had the NC-CAH (5 boys and 9 girls). The overall frequency of the molecular defects detected in all patients is
Discussion
Congenital adrenal hyperplasia (CAH) is comprised of a family of autosomal recessive disorders leading to the defect in cortisol biosynthesis. The 21-hydroxylase deficiency (21-OHD) is the most common cause of CAH which accounts for 90% of the CAH patients. There are various phenotypes due to combination of different mutations. Ten common mutations in CYP21A2 gene, located on 6q21.3, have been reported in many populations around the world.
In this study, we described the spectrum and frequency
Conclusion
In conclusion, our genotyping approach allowed accurate and sensitive identification of CYP21A2 gene mutations in CAH patients and their families and offered reliable information needed for diagnostics and for adequate genetic counseling in Tunisia. In general, there are significant relationships between phenotype and genotype, where the clinical manifestations of CAH, according to its autosomal recessive inheritance, reflect the allele bearing the mutation that predicts the least impairment of
Acknowledgments
We wish to express our appreciation for the cooperation and generosity of all contributed families. We are also grateful to doctors: Rached Jomaa, Nejla Salem, Samia Tilouche, Hédia Houissa Sliman, Asma Bouaziz Abed, Faiza Chtiwi Lamine, and Hela Marmouche for providing clinical information and blood samples. We appreciate the expert technical assistance of Ahlem Msakni, Sihem Sassi, Gisela Hohmann, Tanja Dahm and Michèle Nigou. This work was supported by the European Society for Pediatric
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