Download PDF
Horm Metab Res 2014; 46(07): 515-520
DOI: 10.1055/s-0034-1371864
Endocrine Care
© Georg Thieme Verlag KG Stuttgart · New York

Functional and Structural Analysis of Four Novel Mutations of CYP21A2 Gene in Italian Patients with 21-Hydroxylase Deficiency

A. Massimi
1   Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy
,
M. Malaponti
1   Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy
,
L. Federici
2   Department of Experimental and Clinical Sciences, Ce.S.I. Center of ­Excellence on Aging, University of Chieti G. d’Annunzio, Chieti, Italy
,
D. Vinciguerra
1   Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy
,
M. L. Manca Bitti
3   Pediatric Diabetology and Endocrinology Unit, Policlinico Tor Vergata, Rome, Italy
,
A. Vottero
4   Departments of Pediatrics, University of Parma, Parma, Italy
,
L. Ghizzoni
5   Division of Endocrinology, Diabetology, and Metabolism, Department of Internal Medicine, University of Turin, Turin, Italy
,
M. Maccarrone
6   Center of Integrated Research, Campus Bio-Medico University of Rome, Rome, Italy
,
M. Cappa
7   Endocrinology and Diabetology Unit and Research Laboratory, Bambino Gesù Childrenʼs Hospital, Rome, Italy
,
S. Bernardini
1   Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy
,
O. Porzio
1   Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy
› Author Affiliations
Further Information

Publication History

received 14 November 2013

accepted 06 March 2014

Publication Date:
05 May 2014 (online)

Also available at
    Permissions and Reprints

Abstract

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by defects in the 21-hydroxylase gene (CYP21A2), coding for the enzyme 21-hydroxylase (21-OH). About 95% of the mutations arise from gene conversion between CYP21A2 and the inactive pseudogene CYP21A1P: only 5% are novel CYP21A2 mutations, in which functional analysis of mutant enzymes has been helpful to correlate genotype-phenotype. In the present study, we describe 3 novel point mutations (p.L122P, p.Q481X, and p.E161X) in 3 Italian patients with CAH: the fourth mutation (p.M150R) was found in the carrier state. Molecular modeling suggests a major impact on 21-hydroxylase activity, and functional analysis after expression in COS-7 cells confirms reduced enzymatic activity of the mutant enzymes. Only the p.M150R mutation affected the activity to a minor extent, associated with NC CAH. CYP21A2 genotyping and functional characterization of each disease-causing mutation has relevance both for treatment and genetic counseling to the patients.

Key words

congenital adrenal ­hyperplasia (CAH) - CYP21A2 - 21-hydroxylase - genotype-phenotype - molecular modeling
 

  • References

  • 1 White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 2000; 21: 245-291
    CrossrefPubMedGoogle Scholar
  • 2 Higashi Y, Yoshioka H, Yamane M, Gotoh O, Fujii-Kuriyama Y. 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
    CrossrefPubMedGoogle Scholar
  • 3 White PC, Grossberger D, Onufer BJ, Chaplin DD, New MI, Dupont B, Strominger JL. Two genes encoding steroid 21-hydroxylase are located near the genes encoding the fourth component of complement in man. Proc Natl Acad Sci USA 1985; 82: 1089-1093
    CrossrefPubMedGoogle Scholar
  • 4 Riepe FG, Hiort O, Grötzinger J, Sippell WG, Krone N, Holterhus PM. Functional and structural consequences of a novel point mutation in the CYP21A2 gene causing congenital adrenal hyperplasia: potential relevance of helix C for P450 oxidoreductase-21-hydroxylase interaction. J Clin Endocrinol Metab 2008; 93: 2891-2895
    CrossrefPubMedGoogle Scholar
  • 5 Barbaro M, Baldazzi L, Balsamo A, Lajic S, Robins T, Barp L, Pirazzoli P, Cacciarie E, Cicognani A, Wedell A. Functional studies of two novel and two rare mutations in the 21-hydroxylase gene. J Mol Med 2006; 84: 521-528
    CrossrefPubMedGoogle Scholar
  • 6 Robins T, Bellanne-Chantelot C, Barbaro M, Cabrol S, Wedell A, Lajic S. Characterization of novel missense mutations in CYP21 causing congenital adrenal hyperplasia. J Mol Med 2007; 85: 247-255
    CrossrefPubMedGoogle Scholar
  • 7 Concolino P, Mello E, Patrosso MC, Penco S, Zuppi C, Capoluongo E. pH282N and p.Y191H: 2 novel CYP21A2 mutations in Italian congenital adrenal hyperplasia patients. Metabolism 2012; 61: 519-524
    CrossrefPubMedGoogle Scholar
  • 8 Tardy V, Menassa R, Sulmont V, Lienhardt-Roussie A, Lecointre C, Brauner R, David M, Morel Y. Phenotype-genotype correlations of 13 rare CYP21A2 mutations detected in 46 patients affected with 21-hydroxylase deficiency and in one carrier. J Clin Endocrinol Metab 2010; 95: 1288-1300
    CrossrefPubMedGoogle Scholar
  • 9 Marino R, Ramirez P, Galeano J, Perez Garrido N, Rocco C, Ciaccio M, Warman DM, Guercio G, Chaler E, Maceiras M, Bergadà I, Gryngarten M, Balbi V, Pardes E, Rivarola MA, Belgorosky A. Steroid 21-hydroxylase gene mutational spectrum in 454 Argentinean patients: genotype-phenotype correlation in a large cohort of patients with congenital adrenal hyperplasia. Clin Endocrinol (Oxf) 2011; 75: 427-435
    CrossrefPubMedGoogle Scholar
  • 10 Finkielstain GP, Chen W, Mehta SP, Fujimura FK, Hanna RM, Van Ryzin C, McDonnell NB, Merke DP. Comprehensive genetic analysis of 182 unrelated families with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Endocrinol Metab 2011; 96: E161-E172
    CrossrefPubMedGoogle Scholar
  • 11 New MI, Abraham M, Gonzalez B, Dumic M, Razzaghy-Azar M, Chitayat D, Sun L, Zaidi M, Wilson RC, Yuen T. Genotype-phenotype correlation in 1,507 families with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. Proc Natl Acad Sci USA 2013; 110: 2611-2616
    CrossrefPubMedGoogle Scholar
  • 12 Concolino P, Vendittelli F, Mello E, Minucci A, Carrozza C, Rossodivita A, Giardina B, Zuppi C, Capoluongo E. Functional analysis of two rare CYP21A2 mutations detected in Italian patients with a mildest form of congenital adrenal hyperplasia. Clin Endocrinol (Oxf) 2009; 71: 470-476
    CrossrefPubMedGoogle Scholar
  • 13 Porzio O, Cunsolo V, Malaponti M, De Nisco E, Acquafredda A, Cavallo L, Andreani M, Giardina E, Testi M, Cappa M, Federici G. Divergent Phenotype of Two Siblings Human Leukocyte Antigen Identical, Affected by Nonclassical and Classical Congenital Adrenal Hyperplasia Caused by 21-Hydroxylase Deficiency. J Clin Endocrinol Metab 2006; 91: 4510-4513
    CrossrefPubMedGoogle Scholar
  • 14 Ghizzoni L, Cappa M, Vottero A, Ubertini G, Carta D, Di Iorgi N, Gasco V, Marchesi M, Raggi V, Ibba A, Napoli F, Massimi A, Maghnie M, Loche S, Porzio O. Relationship of CYP21A2 genotype and serum 17-hydroxyprogesterone and cortisol levels in a large cohort of Italian children with premature pubarche. Eur J Endocrinol 2011; 165: 307-314
    CrossrefPubMedGoogle Scholar
  • 15 Jang JH, Jin DK, Kim JH, Tan HK, Kim JW, Lee SY, Ki CS, Park HD. Multiplex ligation-dependent probe amplification assay for diagnosis of congenital adrenal hyperplasia. Ann Clin Lab Sci 2011; 41: 44-47
    PubMedGoogle Scholar
  • 16 Winqvist O, Karlsson FA, Kampe O. 21-Hydroxylase, a major autoantigen in idiopathic Addison’s disease. Lancet 1992; 339: 1559-1562
    CrossrefPubMedGoogle Scholar
  • 17 Thompson JD, Higgins DG, Gibson TJ, Clustal W. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994; 2: 4673-4680
    PubMedGoogle Scholar
  • 18 Wester MR, Johnson EF, Marques-Soares C, Dansette PM, Mansuy D, Stout CD. Structure of a substrate complex of mammalian cytochrome P450 2C5 at 2.3 A resolution: evidence for multiple substrate binding modes. Biochemistry 2003; 42: 6370-6379
    CrossrefPubMedGoogle Scholar
  • 19 Rai BK, Madrid-Aliste CJ, Fajardo JE, Fiser A. MMM: a sequence-to-structure alignment protocol. Bioinformatics 2006; 22: 2691-2692
    CrossrefPubMedGoogle Scholar
  • 20 Sali A, Blundell TL. Comparative protein modeling by satisfaction of spatial restraints. J Mol Biol 1993; 234: 779-815
    CrossrefPubMedGoogle Scholar
  • 21 Laskowski RA, Moss DS, Thornton JM. Main-chain bond lengths and bond angles in protein structures. J Mol Biol 1993; 231: 1049-1067
    CrossrefPubMedGoogle Scholar
  • 22 White PC. Neonatal screening for congenital adrenal hyperplasia. Nat Rev Endocrinol 2009; 5: 490-498
    CrossrefPubMedGoogle Scholar
  • 23 Wedell A. Molecular genetics of 21-hydroxylase deficiency. Endocr Dev 2011; 20: 80-87
    PubMedGoogle Scholar
  • 24 Nimkarn S, New MI. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency: a paradigm for prenatal diagnosis and treatment. Ann N Y Acad Sci 2011; 1192: 5-11
    PubMedGoogle Scholar
  • 25 Haider S, Islam BM, Poojari C, Sun L, Yuen T, Zaidi M, New MI. Structure-phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia. Proc Natl Acad Sci USA 2013; 110: 2605-2610
    CrossrefPubMedGoogle Scholar
  • 26 Robins T, Carlsson J, Sunnerhagen M, Wedell A, Persson B. Molecular model of human CYP21 based on mammalian CYP2C5: structural features correlate with clinical severity of mutations causing congenital adrenal hyperplasia. Mol Endocrinol 2006; 20: 2946-2964
    CrossrefPubMedGoogle Scholar
  • 27 Baradaran-Heravi A, Vakili R, Robins T, Carlsson J, Ghaemi N, A’rabi A, Abbaszadegan MR. Three novel CYP21A2 mutations and their protein modelling in patients with classical 21-hydroxylase deficiency from northeastern Iran. Clin Endocrinol (Oxf) 2007; 67: 335-341
    CrossrefPubMedGoogle Scholar
  • 28 Minutolo C, Nadra AD, Fernández C, Taboas M, Buzzalino N, Casali B, Belli S, Charreau EH, Alba L, Dain L. Structure-based analysis of five novel disease-causing mutations in 21-hydroxylase-deficient patients. PLoS One 2011; 6: e15899
    CrossrefPubMedGoogle Scholar
  • 29 Di Pasquale G, Wasniewska M, Caruso M, Salzano G, Coco M, Lombardo F, De Luca F. Salt wasting phenotype in a compound heterozygous girl with P482S mutation associated with a novel mutation of CYP21 gene (Q481P). J Endocrinol Invest 2005; 28: 1038-1039
    CrossrefPubMedGoogle Scholar