Clinical Phenotype and Mutation Spectrum of the CYP21A2 Gene in Patients with Steroid 21-Hydroxylase Deficiency

Abstract

Steroid 21-hydroxylase deficiency is caused by inactivating mutations in the CYP21A2 gene. This paper reports on the mutation spectrum and the genotype-phenotype correlation of 21-hydroxylase deficiency. 72 unrelated patients with congenital adrenal hyperplasia (CAH) were included. Molecular analysis of CYP21A2 was performed, via the multiplex ligation-dependent probe amplification (MLPA) analysis and sequence-specific differenzial PCR amplification of the CYP21A2 and CYP21A1P genes, using 4 pair-wise sequence-specific primers, followed by sequencing of the entire CYP21A2 gene. Large gene deletions were identified in 45 (31.3%) of the 144 unrelated CAH alleles, whereas the most frequent point mutations were intron 2 splice mutations (c.293-13A>G) (41/144, 28.5%). The MLPA analysis successfully identified 23 of 72 patients (31.9%) with single copy deletion in CYP21A2. This paper describes a rapid and accurate method for the molecular diagnosis of 21-hydroxylase deficiency, which relies on the identification of point mutations and structural rearrangements within the CYP21A2 gene.

Key words

Congenital adrenal ­hyperplasia - CYP21A2 - Steroid 21-hydroxylase deficiency

• References

• 1 Asanuma A, Ohura T, Ogawa E et al. Molecular analysis of Japanese patients with steroid 21-hydroxylase deficiency. J Hum Genet 1999; 44: 312-317
• 2 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: 164-170
• 3 Higashi Y, Tanae A, Inoue H et al. Evidence for frequent gene conversion in the steroid 21-hydroxylase P-450 (C21) gene: implications for steroid 21-hydroxylase deficiency. Am J Hum Genet 1988; 42: 17-25
• 4 Higashi Y, Yoshioka H, Yamada 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 USA 1986; 83: 2841-2845
• 5 Keen-Kim D, Redman JB, Alanes RU et al. Validation and clinical application of a locus-specific polymerase chain reaction- and minisequencing-based assay for congenital adrenal hyperplasia (21-hydroxylase deficiency). J Mol Diagn 2005; 7: 236-246
• 6 Koppens PF, Degenhart HJ. PCR-based detection of CYP21 deletions. Clin Chem 2003; 49: 1555-1556
• 7 Krone N, Arlt W. Genetics of congenital adrenal hyperplasia. Best Pract Res Clin Endocrinol Metab 2009; 23: 181-192
• 8 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: 1059-1065
• 9 Lee HH. CYP21 mutations and congenital adrenal hyperplasia. Clin Genet 2001; 59: 293-301
• 10 Lee HH, Chao HT, Ng HT et al. Direct molecular diagnosis of CYP21 mutations in congenital adrenal hyperplasia. J Med Genet 1996; 33: 371-375
• 11 Lee HH, Lee YJ, Chan P et al. Use of PCR-based amplification analysis as a substitute for the southern blot method for CYP21 deletion detection in congenital adrenal hyperplasia. Clin Chem 2004; 50: 1074-1076
• 12 Lee HH, Lee YJ, Wang YM et al. Low frequency of the CYP21A2 deletion in ethnic Chinese (Taiwanese) patients with 21-hydroxylase deficiency. Mol Genet Metab 2008; 93: 450-457
• 13 Lobato MN, Aledo R, Meseguer A. High variability of CYP21 gene rearrangements in Spanish patients with classic form of congenital adrenal hyperplasia. Hum Hered 1998; 48: 216-225
• 14 Merke DP, Bornstein ST. Congenital adrenal hyperplasia. Lancet 2005; 365: 2125-2136
• 15 Pang S, Murphey W, Levine LS et al. A pilot newborn screening for congenital adrenal hyperplasia in Alaska. J Clin Endocrinol Metab 1982; 55: 413-420
• 16 Pang SY, Wallace MA, Hofman L et al. Worldwide experience in newborn screening for classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Pediatrics 1988; 81: 866-874
• 17 Parajes S, Quinteiro C, Domínguez F et al. High frequency of copy number variations and sequence variants at CYP21A2 locus: implication for the genetic diagnosis of 21-hydroxylase deficiency. PLoS ONE 2008; 3: e2138
• 18 Schulze E, Scharer G, Rogatzki A et al. Divergence between genotype and phenotype in relatives of patients with the intron 2 mutation of steroid-21-hydroxylase. Endocr Res 1995; 21: 359-364
• 19 Speiser PW, Dupont J, Zhu D et al. Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Invest 1992; 90: 584-595
• 20 Torresani T, Biason-Lauber A. Congenital adrenal hyperplasia: diagnostic advances. J Inherit Metab Dis 2007; 30: 563-575
• 21 Tusié-Luna MT, White PC. Gene conversions and unequal crossovers between CYP21 (steroid 21-hydroxylase gene) and CYP21P involve different mechanisms. Proc Natl Acad Sci USA 1995; 92: 10796-10800
• 22 Wedell A, Thilen A, Ritzen EM et al. Mutational spectrum of the steroid 21-hydroxylase gene in Sweden: implications for genetic diagnosis and association with disease manifestation. J Clin Endocrinol Metab 1994; 78: 1145-1152
• 23 White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 2000; 21: 245-291
• 24 White PC, Tusie-Luna MT, New MI et al. Mutations in steroid 21-hydroxylase (CYP21). Hum Mut 1994; 3: 373-378