Abstract
The aim of the current study was to search for the presence of genetic variants in the CYP21A2 Z promoter regulatory region in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Screening of the 10 most frequent pseudogene-derived mutations was followed by direct sequencing of the entire coding sequence, the proximal promoter, and a distal regulatory region in DNA samples from patients with at least one non-determined allele. We report three non-classical patients that presented a novel genetic variant—g.15626A>G—within the Z promoter regulatory region. In all the patients, the novel variant was found in cis with the mild, less frequent, p.P482S mutation located in the exon 10 of the CYP21A2 gene. The putative pathogenic implication of the novel variant was assessed by in silico analyses and in vitro assays. Topological analyses showed differences in the curvature and bendability of the DNA region bearing the novel variant. By performing functional studies, a significantly decreased activity of a reporter gene placed downstream from the regulatory region was found by the G transition. Our results may suggest that the activity of an allele bearing the p.P482S mutation may be influenced by the misregulated CYP21A2 transcriptional activity exerted by the Z promoter A>G variation.
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References
M.I. New, P.C. White, S. Pang, B. Dupont, P.W. Speiser, The Adrenal Hyperplasias, in The Metabolic Basis of Inheritated Disease, 6th edn., ed. by C. Scriver, A.L. Beaudet, S. Sly, D. Valle (McGraw-Hill, New York, 1989), pp. 1881–1917
Y. Higashi, A. Tanae, H. Inoue, Y. Fujii-Kuriyama, Evidence for frequent gene conversion in the steroid 21-hydroxylase P-450(C21) gene: implications for steroid 21-hydroxylase deficiency. Am. J. Hum. Genet. 42(1), 17–25 (1988)
S.F. Chang, B.C. Chung, Difference in transcriptional activity of two homologous CYP21A genes. Mol. Endocrinol. 9(10), 1330–1336 (1995)
M.K. Tee, G.O. Babalola, P. Aza-Blanc, M. Speek, S.E. Gitelman, W.L. Miller, A promoter within intron 35 of the human C4A gene initiates abundant adrenal-specific transcription of a 1 kb RNA: location of a cryptic CYP21 promoter element? Hum. Mol. Genet. 4(11), 2109–2116 (1995)
S.D. Wijesuriya, G. Zhang, A. Dardis, W.L. Miller, Transcriptional regulatory elements of the human gene for cytochrome P450c21 (steroid 21-hydroxylase) lie within intron 35 of the linked C4B gene. J. Biol. Chem. 274(53), 38097–38106 (1999)
R.S. Araujo, B.B. Mendonca, A.S. Barbosa, C.J. Lin, J.A. Marcondes, A.E. Billerbeck, T.A. Bachega, Microconversion between CYP21A2 and CYP21A1P promoter regions causes the nonclassical form of 21-hydroxylase deficiency. J. Clin. Endocrinol. Metab. 92(10), 4028–4034 (2007)
H.J. Zhang, J. Yang, M.N. Zhang, W. Zhang, J.M. Liu, W.Q. Wang, G. Ning, X.Y. Li, Variations in the promoter of CYP21A2 gene identified in a Chinese patient with simple virilizing form of 21-hydroxylase deficiency. Clin. Endocrinol. (Oxf) 70(2), 201–207 (2009)
C. Minutolo, A.D. Nadra, C. Fernandez, M. Taboas, N. Buzzalino, B. Casali, S. Belli, E.H. Charreau, L. Alba, L. Dain, Structure-based analysis of five novel disease-causing mutations in 21-hydroxylase-deficient patients. PLoS One 6(1), e15899 (2011)
M. Taboas, C. Fernandez, S. Belli, N. Buzzalino, L. Alba, L. Dain, Isolated p.H62L mutation in the CYP21A2 gene in a simple virilizing 21-hydroxylase deficient patient. Case Rep. Genet. 2013, 143781 (2013)
X. Chu, H. Ding, G. Cui, Y. Xu, D.W. Wang, Y. He, Functional consequences of a novel point mutation in the CYP21A2 gene identified in a Chinese Han patient with nonclassic 21-hydroxylase deficiency. Clin. Endocrinol. (Oxf) 80(6), 927–928 (2014)
L.B. Dain, N.D. Buzzalino, A. Oneto, S. Belli, M. Stivel, T. Pasqualini, C. Minutolo, E.H. Charreau, L.G. Alba, Classical and nonclassical 21-hydroxylase deficiency: a molecular study of Argentine patients. Clin. Endocrinol. (Oxf) 56(2), 239–245 (2002)
T. Pasqualini, G. Alonso, R. Tomasini, A.M. Galich, N. Buzzalino, C. Fernandez, C. Minutolo, L. Alba, L. Dain, Congenital adrenal hyperplasia clinical characteristics and genotype in newborn, childhood and adolescence. Medicina (B Aires) 67(3), 253–261 (2007)
P.C. White, M.I. New, B. Dupont, Structure of human steroid 21-hydroxylase genes. Proc. Natl. Acad. Sci. USA 83(14), 5111–5115 (1986)
D. Ulgiati, D.C. Townend, F.T. Christiansen, R.L. Dawkins, L.J. Abraham, Complete sequence of the complement C4 gene from the HLA-A1, B8, C4AQ0, C4B1, DR3 haplotype. Immunogenetics 43(4), 250–252 (1996)
A. Wedell, H. Luthman, Steroid 21-hydroxylase deficiency: two additional mutations in salt-wasting disease and rapid screening of disease-causing mutations. Hum. Mol. Genet. 2(5), 499–504 (1993)
K. Vlahovicek, L. Kajan, S. Pongor, DNA analysis servers: plot.it, bend.it, model.it and IS. Nucleic Acids Res. 31(13), 3686–3687 (2003)
I. Brukner, R. Sanchez, D. Suck, S. Pongor, Trinucleotide models for DNA bending propensity: comparison of models based on DNaseI digestion and nucleosome packaging data. J. Biomol. Struct. Dyn. 13(2), 309–317 (1995)
M.G. Munteanu, K. Vlahovicek, S. Parthasarathy, I. Simon, S. Pongor, Rod models of DNA: sequence-dependent anisotropic elastic modelling of local bending phenomena. Trends Biochem. Sci. 23(9), 341–347 (1998)
A. Dorn, C. Benoist, D. Mathis, New B-lymphocyte-specific enhancer-binding protein. Mol. Cell. Biol. 9(1), 312–320 (1989)
M. Barbaro, S. Lajic, L. Baldazzi, A. Balsamo, P. Pirazzoli, A. Cicognani, A. Wedell, E. Cacciari, Functional analysis of two recurrent amino acid substitutions in the CYP21 gene from Italian patients with congenital adrenal hyperplasia. J. Clin. Endocrinol. Metab. 89(5), 2402–2407 (2004)
M. Barbaro, F.C. Soardi, L.J. Ostberg, B. Persson, M.P. de Mello, A. Wedell, S. Lajic, In vitro functional studies of rare CYP21A2 mutations and establishment of an activity gradient for nonclassic mutations improve phenotype predictions in congenital adrenal hyperplasia. Clin. Endocrinol. (Oxf) 82(1), 37–44 (2015)
A. Balsamo, E. Cacciari, L. Baldazzi, L. Tartaglia, A. Cassio, V. Mantovani, S. Piazzi, A. Cicognani, P. Pirazzoli, B. Mainetti, F. Zappulla, CYP21 analysis and phenotype/genotype relationship in the screened population of the Italian Emilia-Romagna region. Clin. Endocrinol. (Oxf) 53(1), 117–125 (2000)
G. Di Pasquale, M. Wasniewska, M. Caruso, G. Salzano, M. Coco, F. Lombardo, F. De Luca, Salt wasting phenotype in a compound heterozygous girl with P482S mutation associated with anovel mutation of CYP21 gene (Q481P). J. Endocrinol. Invest. 28(11), 1038–1039 (2005)
P. Concolino, C. Carrozza, A. Minucci, C. Santonocito, F. Ameglio, C. Zuppi, E. Capoluongo, A case of patient affected by hirsutism carrying the P482S CYP21 gene mutation associated with loss of heterozygosity (LOH). Clin. Chim. Acta 370(1–2), 201–202 (2006)
M.I. New, M. Abraham, B. Gonzalez, M. Dumic, M. Razzaghy-Azar, D. Chitayat, L. Sun, M. Zaidi, R.C. Wilson, T. Yuen, Genotype-phenotype correlation in 1,507 families with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. Proc. Natl. Acad. Sci. USA 110(7), 2611–2616 (2013)
C. Bleicken, L. Loidi, V. Dhir, S. Parajes, C. Quinteiro, F. Dominguez, J. Grotzinger, W.G. Sippell, F.G. Riepe, W. Arlt, N. Krone, Functional characterization of three CYP21A2 sequence variants (p. A265V, p.W302S, p.D322G) employing a yeast co-expression system. Hum. Mutat. 30(2), E443–E450 (2009)
V. Dolzan, J. Prezelj, B. Vidan-Jeras, K. Breskvar, Adrenal 21-hydroxylase gene mutations in Slovenian hyperandrogenic women: evaluation of corticotrophin stimulation and HLA polymorphisms in screening for carrier status. Eur. J. Endocrinol. 141(2), 132–139 (1999)
O. Admoni, S. Israel, I. Lavi, M. Gur, Y. Tenenbaum-Rakover, Hyperandrogenism in carriers of CYP21 mutations: the role of genotype. Clin. Endocrinol. (Oxf) 64(6), 645–651 (2006)
Acknowledgments
The authors would like to thank Dr. W. L. Miller (University of San Francisco, CA, USA) for kindly providing the pC21/−0.3 Kb/Luc and.pC21/−5.6 Kb/Luc plasmids used in this work and Msc. F. Pisciottano for the helpful discussion of sequence conservation data.
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The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
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All the procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written informed consent was obtained from all patients and parents involved in this work. The study was approved by the ethics committee of the Instituto de Biología y Medicina Experimental, and the Centro Nacional de Genética Médica, Buenos Aires, Argentina.
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Fernández, C.S., Bruque, C.D., Taboas, M. et al. Misregulation effect of a novel allelic variant in the Z promoter region found in cis with the CYP21A2 p.P482S mutation: implications for 21-hydroxylase deficiency. Endocrine 50, 72–78 (2015). https://doi.org/10.1007/s12020-015-0680-0
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DOI: https://doi.org/10.1007/s12020-015-0680-0