Background: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease caused by mutations in the CYP21A2 gene, which codes for steroid 21-hydroxylase. More than 90% of patients with CAH have mutations in CYP21A2 or have large deletions in the RCCX module on chromosome 6p21.3, which also includes the pseudogene CYP21A1P. Genotyping of CYP21A2 is required for diagnosis of CAH, but current genotyping methods, such as direct sequencing, allele-specific PCR amplification, or PCR amplification and restriction fragment length polymorphism (PCR-RFLP) still need further improvements to reduce test time and cost.
Methods: We developed a novel CAH mutation screening method based on allele-specific primer extension (ASPE), followed by bead-array hybridization, for the ten major point mutation sites and the 8 bp deletion in CYP21A2, and a long PCR assay to detect large deletions between CYP21A1P and CYP21A2. After the first long PCR amplification, a second short PCR amplification was adapted to increase the ASPE efficiency. The total genotyping procedure takes approximately 8 hours.
Results: Eighteen CAH patients and two controls were tested using the bead-array method. Homozygous or heterozygous large gene deletions and three point mutation sites were detected by this method, and most of the results were consistent with sequencing or PCR-RFLP analysis. Nine of the 18 patients had a large deletion in the RCCX module, which was not easily detected using the conventional genotyping method.
Conclusion: A novel CAH mutation screening method has been developed to detect ten point mutations and the 8 bp deletion in CYP21A2, as well as large deletions between CYP21A1P and CYP21A2. This novel genotyping strategy is superior to PCR-RFLP-based methods and equally as accurate as sequencing.
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Drs Yongtaek Oh and Sung Won Park contributed equally to this work.
This work was supported by grant no. 2007-S1025909 from the Small and Medium Business Administration of Korea (Daejeon, Korea). Drs Oh, Han, and Chun are employees of the diagnostics company YeBT (Seoul, Korea). Dr Han owns stock options in YeBT. Dr Lim is an employee of Ubioslab Co. (Seoul, Korea) and has a patent pending (#2007-0056018).
White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 2000; 21: 245–91PubMedCrossRefGoogle Scholar
Krone N, Braun A, Roscher AA, et al. Predicting phenotype in steroid 21-hydroxylase deficiency? Comprehensive genotyping in 155 unrelated, well defined patients from southern Germany. J Clin Endocrinol Metab 2000; 85: 1059–65PubMedCrossRefGoogle Scholar
Liivak K, Tobi S, Schlecht H, et al. Incidence of classical 21-hydroxylase deficiency and distribution of CYP21A2 mutations in Estonia. Horm Res 2008; 69: 227–32PubMedCrossRefGoogle Scholar
Speiser PW, Dupont B, Rubinstein P, et al. High frequency of nonclassical steroid 21-hydroxylase deficiency. Am J Hum Genet 1985; 37: 650–67PubMedGoogle Scholar
White PC, New MI, Dupont B. Structure of human steroid 21-hydroxylase genes. Proc Natl Acad Sci U S A 1986; 83: 5111–5PubMedCrossRefGoogle Scholar
Higashi Y, Yoshioka H, Yamane M, et al. Complete nucleotide sequence of two steroid 21-hydroxylase genes tandemly arranged in human chromosome: a pseudogene and a genuine gene. Proc Natl Acad Sci U S A 1986; 83: 2841–5PubMedCrossRefGoogle Scholar
Grigorescu-Sido A, Schulze E, Grigorescu-Sido P, et al. Mutational analysis and genotype-phenotype correlation in patients with classic 21-hydroxylase deficiency from Transylvania (north-west Romania). J Pediatr Endocrinol Metab 2002; 15: 1505–14PubMedCrossRefGoogle Scholar
Groschl M, Rauh M, Dorr HG. Cortisol and 17-hydroxyprogesterone kinetics in saliva after oral administration of hydrocortisone in children and young adolescents with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Endocrinol Metab 2002; 87: 1200–4PubMedCrossRefGoogle Scholar
Kosel S, Burggraf S, Fingerhut R, et al. Rapid second-tier molecular genetic analysis for congenital adrenal hyperplasia attributable to steroid 21-hydroxylase deficiency. Clin Chem 2005; 51: 298–304PubMedCrossRefGoogle Scholar
Huynh T, McGown I, Cowley D, et al. The clinical and biochemical spectrum of congenital adrenal hyperplasia secondary to 21-hydroxylase deficiency. Clin Biochem Rev 2009; 30(2): 75–86PubMedGoogle Scholar
Lacey JM, Minutti CZ, Magera MJ, et al. Improved specificity of newborn screening for congenital adrenal hyperplasia by second-tier steroid profiling using tandem mass spectrometry. Clin Chem 2004; 50(3): 621–5PubMedCrossRefGoogle Scholar
Speiser PW, White PC, Dupont J, et al. Molecular genetic prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency by allele-specific hybridization. Recent Prog Horm Res 1994; 49: 367–71PubMedGoogle Scholar
Mao R, Nelson L, Kates R, et al. Prenatal diagnosis of 21-hydroxylase deficiency caused by gene conversion and rearrangements: pitfalls and molecular diagnostic solutions. Prenat Diagn 2002; 22: 1171–6PubMedCrossRefGoogle Scholar
Asanuma A, Ohura T, Ogawa E, et al. Molecular analysis of Japanese patients with steroid 21-hydroxylase deficiency. J Hum Genet 1999; 44: 312–7PubMedCrossRefGoogle Scholar
Hayashi Z, Orimo H, Araki T, et al. Prenatal diagnosis of steroid 21-hydroxylase deficiency by analysis of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) profiles. Prenat Diagn 1997; 17: 435–42PubMedCrossRefGoogle Scholar
Day DJ, Speiser PW, White PC, et al. Detection of steroid 21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection reaction. Genomics 1995; 29: 152–62PubMedCrossRefGoogle Scholar
Higashi Y, Tanae A, Inoue H, et al. Molecular genetic analysis of steroid 21-hydroxylase [P-450(C21)] deficiency. Acta Paediatr Jpn 1998; 30 Suppl.: 105–10Google Scholar
Krone N, Braun A, Weinert S, et al. Multiplex minisequencing of the 21-hydroxylase gene as a rapid strategy to confirm congenital adrenal hyperplasia. Clin Chem 2002; 48: 818–25PubMedGoogle Scholar
Parajes S, Quinteiro C, Domínguez F, et al. A simple and robust quantitative PCR assay to determine CYP21A2 gene dose in the diagnosis of 21-hydroxylase deficiency. Clin Chem 2007; 53: 1577–84PubMedCrossRefGoogle Scholar
Concolino P, Mello E, Toscano V, et al. Multiplex ligation-dependent probe amplification (MLPA) assay for the detection of CYP21A2 gene deletions/ duplications in congenital adrenal hyperplasia: first technical report. Clin Chim Acta 2009; 402: 164–70PubMedCrossRefGoogle Scholar
Lee YH, Park ES, Kang SH, et al. Characterization of a novel DNA polymorphism in the human CYP21 gene and application for DNA diagnosis of congenital adrenal hyperplasia. Clin Endocrinol (Oxf) 2000; 53: 419–22CrossRefGoogle Scholar
Bortolin S, Black M, Modi H, et al. Analytical validation of the Tag-It high-throughput microsphere-based universal array genotyping platform: application to the multiplex detection of a panel of thrombophilia-associated single-nucleotide polymorphisms. Clin Chem 2004; 50: 2028–36PubMedCrossRefGoogle Scholar
Wilson RC, Nimkarn S, Dumic M, et al. Ethnic-specific distribution of mutations in 716 patients with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. Mol Genet Metab 2007; 90(4): 414–21PubMedCrossRefGoogle Scholar
Jin DK, Kim JS, Song SM, et al. A study on the relationship between genotype and phenotype in Korean patients with congenital adrenogenital syndrome caused by 21-hydroxylase deficiency. J Korean Soc Endocrinol 2000; 15(2): 237–47Google Scholar
White PC, Vitek A, Dupont B, et al. Characterization of frequent deletions causing steroid 21-hydroxylase deficiency. Proc Natl Acad Sci U S A 1988; 85: 4436–40PubMedCrossRefGoogle Scholar
Neocleous V, Ioannou YS, Bartsota M, et al. Rare mutations in the CYP21A2 gene detected in congenital adrenal hyperplasia. Clin Biochem 2009; 42: 1363–7PubMedCrossRefGoogle Scholar