Elsevier

Gene

Volume 507, Issue 1, 1 October 2012, Pages 20-26
Gene

Steroid 21-hydroxylase gene mutational spectrum in 50 Tunisian patients: Characterization of three novel polymorphisms

https://doi.org/10.1016/j.gene.2012.07.027Get rights and content

Abstract

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease of steroid biosynthesis in humans. More than 90% of all CAH cases are caused by mutations of the 21-hydroxylase gene (CYP21A2), and approximately 75% of the defective CYP21A2 genes are generated through an intergenic recombination with the neighboring CYP21A1P pseudogene. In this study, the CYP21A2 gene was genotyped in 50 patients in Tunisia with the clinical diagnosis of 21-hydroxylase deficiency. CYP21A2 mutations were identified in 87% of the alleles. The most common point mutation in our population was the pseudogene specific variant p.Q318X (26%). Three novel single nucleotide polymorphism (SNP) loci were identified in the CYP21A2 gene which seems to be specific for the Tunisian population. The overall concordance between genotype and phenotype was 98%. With this study the molecular basis of CAH has been characterized, providing useful results for clinicians in terms of prediction of disease severity, genetic and prenatal counseling.

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

References (42)

  • S.M. Baumgartner-Parzer et al.

    Carrier frequency of congenital adrenal hyperplasia (21-hydroxylase deficiency) in a middle European population

    J. Clin. Endocrinol. Metab.

    (2005)
  • R.G. Cotton et al.

    Proof of “disease causing” mutation

    Hum. Mutat.

    (1998)
  • V. Dolzan

    Mutational spectrum of steroid 21-hydroxylase and the genotype–phenotype association in Middle European patients with congenital adrenal hyperplasia

    Eur. J. Endocrinol.

    (2005)
  • M. Dracopoulou-Vabouli et al.

    The spectrum of molecular defects of the CYP21 gene in the Hellenic population: variable concordance between genotype and phenotype in the different forms of congenital adrenal hyperplasia

    J. Clin. Endocrinol. Metab.

    (2001)
  • M.G. Forest

    Recent advances in the diagnosis and management of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

    Hum. Reprod. Update

    (2004)
  • H. Globerman et al.

    Nonsense mutation causing steroid 21-hydroxylase deficiency

    J. Clin. Invest.

    (1988)
  • J. Jaaskelainen et al.

    Population-wide evaluation of disease manifestation in relation to molecular genotype in steroid 21-hydroxylase (CYP21) deficiency: good correlation in a well defined population

    J. Clin. Endocrinol. Metab.

    (1997)
  • M. Kharrat

    Molecular genetic analysis of Tunisian patients with a classic form of 21-hydroxylase deficiency: identification of four novel mutations and high prevalence of Q318X mutation

    J. Clin. Endocrinol. Metab.

    (2004)
  • M. Kharrat et al.

    A novel 13-bp deletion in exon 1 of CYP21 gene causing severe congenital adrenal hyperplasia

    Diagn. Mol. Pathol.

    (2005)
  • M. Kharrat et al.

    Detection of a frequent duplicated CYP21A2 gene carrying a Q318X mutation in a general population with quantitative PCR methods

    Diagn. Mol. Pathol.

    (2011)
  • N. Krone

    Multiplex minisequencing of the 21-hydroxylase gene as a rapid strategy to confirm congenital adrenal hyperplasia

    Clin. Chem.

    (2002)
  • View full text