A CYP21A2 gene mutation in patients with congenital adrenal hyperplasia

Objectives: The aim of this study is to determine congenital adrenal hyperplasia (CAH) with the pattern of CYP21A2 gene-mutations in Saudi children. Methods: Between January 2011 and March 2014 at King Fahad Military Complex, Dhahran, Saudi Arabia, we thoroughly examined 11 patients with CAH and 2 asymptomatic individuals with a history of affected siblings. Additionally, we sequenced the full coding regions of the CYP21A2 gene and screened the gene for deletion(s)/duplication(s) using the multiplex ligation-dependent probe amplification (MLPA) technique. Results: Nine patients had classic CAH and presented with ambiguous genitalia and/or salt losing crisis. Two patients had the non-classic form of CAH and presented with precocious puberty. The remaining 2 subjects were asymptomatic. Screening the CYP21A2 gene, we detected p.Gln318X mutation in 4 patients, c.290 -13 C>G (IVS2-13C>G) in another 4, and a common deletion, involving exons 6 and 8 in 3 patients. Conclusion: Our strategy of Sanger sequencing followed by MLPA was very successful in detecting CYP21A2 mutations in all patients with CAH. Saudi Med J 2015; Vol. 36 (1): 113-116 doi: 10.15537/smj.2015.1.9697 C adrenal hyperplasia (CAH) is an autosomal recessive condition caused by a deficiency of one of 5 enzymes involved in the steroidogenesis pathway.1 The most common is 21-hydroxylase deficiency. This enzyme deficiency is caused by mutations in the CYP21A2 gene.2 The level of residual enzyme activity determines the clinical phenotype that ranges from mild virilization to salt losing life threatening crisis.1 Worldwide, the classic forms of 21-hydroxylase deficiency occur in one in 10,000 to 20,000 newborns.1,2 While the prevalence of the non-classical form of 21-hydroxylase deficiency is estimated to be one in 1,000 individuals.1 The recently introduced universal screening for CAH in Saudi Arabia reported an incidence of one in 6400 births.3 The prevalence of both classic and non-classic forms as well as their mutation pattern varies among different ethnic populations.4 Recent studies showed that an IVS2 AS -13 (A/C to G) mutation is prevalent in the Iranians while p.Q319X is common in Turkey, Tunisia, and East India.4-7 Different mutations result in variable deficiency of cortisol and aldosterone together with increased synthesis of androgen.8-10 The genotype/phenotype correlation of CAH has been reported in different populations and ethnic groups.4-7 Although the clinical presentations of CAH have been studied in Saudi children, literature review revealed no molecular report of 21-hydroxylase.3 Therefore, the aim of this study was to determine the pattern of CYP21A2 gene-mutations in Saudi children with CAH, and to describe the clinical phenotype of these patients. Methods. This study was conducted at King Fahad Military Complex, Dhahran, Saudi Arabia between January 2011 and March 2014. Children diagnosed with CAH during this period were recruited. Subjects were included if they presented with salt losing crisis, ambiguous genitalia, precocious puberty or virilization, and their 17 hydroxyprogestrone was above 50 nmol/l. Siblings who had previously documented high 17 hydroxyprogestrone were also included. Identified subjects were systematically reviewed with an emphasis on demographic features, family history, consanguinity, age, gender, and clinical presentation were documented using a data report sheet. This study was approved by the ethics committee at King Fahad Military Complex. It adhered to the tenets of the declaration of Helsinki and all participants-guardians consented to participate in this study. Our strategy was to first sequence the CYP21A2 gene by Sanger sequencing in order to test for single base subtitutions. If the patient was negative, we then proceed with the MLPA technique in order to search for deletetion(s) and/or duplication(s). Sample collection and DNA preparation. Blood samples (5 ml) were drawn in ethylenediaminetetraacetic acid (EDTA) tubes. Tubes were centrifuged at 5500 x g for 5 min and the Buffy layer was used for DNA extraction using the illustrated blood genomic Prep Mini Spin Kit (GE Healthcare, Buckinghamshire, UK) and stored at -20°C in aliquots until further use.