Mutation detection of CYP21A2 gene in nonclassical congenital adrenal hyperplasia patients with premature pubarche

Kolahdouz, Mahsa; Hashemipour, Mahin; Khanahmad, Hossein; Rabbani, Bahareh; Salehi, Mansoor; Rabbani, Ali; Naseri, Mona Mobalegh

Mutation detection of CYP21A2 gene in nonclassical congenital adrenal hyperplasia patients with premature pubarche

Authors

¹ Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

² Department of Pediatric Endocrinology, Isfahan Endocrine and Metabolism Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

³ Department of Biochemistry and Genetic Group, Faculty of Medical Sciences, Qazvin University of Medical Sciences, Qazvin, Iran

⁴ Growth and Development Research Center, Tehran University of Medical Sciences, Tehran, Iran

Abstract

Background: Congenital adrenal hyperplasia (CAH) due to mutations in the gene encoding 21-hydroxilase is one of common disease with an autosomal recessive form. In this study, our aim is to detect the prevalence of eight common mutations in nonclassical congenital adrenal hyperplasia (NCAH).
Materials and Methods: A total of 30 patients with clinical and laboratory evidence of NCAH was selected. Gene-specific polymerase chain reaction (PCR) without contamination of pseudogene was carried out, and PCR product of this step was used to amplification-refractory mutation system PCR on eight common mutations in CYP21A2 gene.

Results: Two heterozygote patients for I2G mutation and six heterozygote patients for Q318X mutation is reported in our study. These mutations associated with the classic form of CAH, and heterozygotes presented with NC symptom, including premature pubarche and hirsutism.
Conclusion: There are some data about the association of the mutation with the clinical form of CAH including classic (salt-wasting and simple virilizing) and NC form. I2G and Q318X mutations were reported in classic form in homozygote state, but the heterozygote form associated with NC form. CAH diagnosis with NC symptom and with measurement of 17-hydroxyprogestrone as NCAH is not a trusted assessment and require to molecular analysis for accurate diagnosis.

Keywords

References

1.	Campos VC, Pereira RM, Torres N, Castro Md, Aguiar-Oliveira MH. High frequency of Q318X mutation in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency in northeast Brazil. Arq Bras Endocrinol Metabol 2009;53:40-6.
2.	Stikkelbroeck NM, Hoefsloot LH, de Wijs IJ, Otten BJ, Hermus AR, Sistermans EA. CYP21 gene mutation analysis in 198 patients with 21-hydroxylase deficiency in The Netherlands: Six novel mutations and a specific cluster of four mutations. J Clin Endocrinol Metab 2003;88:3852-9.
3.	New MI, Wilson RC. Steroid disorders in children: Congenital adrenal hyperplasia and apparent mineralocorticoid excess. Proc Natl Acad Sci U S A 1999;96:12790-7.
4.	Witchel SF, Smith R, Suda-Hartman M. Identification of CYP21 mutations, one novel, by single strand conformational polymorphism (SSCP) analysis. Mutations in brief no 218. Online. Hum Mutat 1999;13:172.
5.	Cantürk C, Baade U, Salazar R, Storm N, Pörtner R, Höppner W. Sequence analysis of CYP21A1P in a German population to aid in the molecular biological diagnosis of congenital adrenal hyperplasia. Clin Chem 2011;57:511-7.
6.	Vrzalová Z, Hrubá Z, St'ahlová Hrabincová E, Pouchlá S, Votava F, Kolousková S, et al. Identification of CYP21A2 mutant alleles in Czech patients with 21-hydroxylase deficiency. Int J Mol Med 2010;26:595-603.
7.	Concolino P, Mello E, Minucci A, Giardina E, Zuppi C, Toscano V, et al. A new CYP21A1P/CYP21A2 chimeric gene identified in an Italian woman suffering from classical congenital adrenal hyperplasia form. BMC Med Genet 2009;10:72.
8.	Siegel SF, Hoffman EP, Trucco M. Molecular diagnosis of 21-hydroxylase deficiency: Detection of four mutations on a single gel. Biochem Med Metab Biol 1994;51:66-73.
9.	Rabbani B. Homozygous complete deletion of CYP21A2 causes a simple virilizing phenotype in an Azeri child. Asian Biomed 2011;5:889.
10.	Lako M, Ramsden S, Campbell RD, Strachan T. Mutation screening in British 21-hydroxylase deficiency families and development of novel microsatellite based approaches to prenatal diagnosis. J Med Genet 1999;36:119-24.
11.	Therrell BL. Newborn screening for congenital adrenal hyperplasia. Endocrinol Metab Clin North Am 2001;30:15-30.
12.	Speiser PW, White PC. Congenital adrenal hyperplasia. N Engl J Med 2003;349:776-88.
13.	Speiser PW, Dupont J, Zhu D, Serrat J, Buegeleisen M, Tusie-Luna MT, et al. Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Invest 1992;90:584-95.
14.	Bas F, Kayserili H, Darendeliler F, Uyguner O, Günöz H, Yüksel Apak M, et al. CYP21A2 gene mutations in congenital adrenal hyperplasia: Genotype-phenotype correlation in Turkish children. J Clin Res Pediatr Endocrinol 2009;1:116-28.
15.	Krone N, Roscher AA, Schwarz HP, Braun A. Comprehensive analytical strategy for mutation screening in 21-hydroxylase deficiency. Clin Chem 1998;44:2075-82.
16.	Speiser PW, Dupont B, Rubinstein P, Piazza A, Kastelan A, New MI. High frequency of nonclassical steroid 21-hydroxylase deficiency. Am J Hum Genet 1985;37:650-67.
17.	Wilson RC, Mercado AB, Cheng KC, New MI. Steroid 21-hydroxylase deficiency: Genotype may not predict phenotype. J Clin Endocrinol Metab 1995;80:2322-9.
18.	Xu Z, Chen W, Merke DP, McDonnell NB. Comprehensive mutation analysis of the CYP21A2 gene: An efficient multistep approach to the molecular diagnosis of congenital adrenal hyperplasia. J Mol Diagn 2013;15:745-53.
19.	Sharaf S, Hafez M, ElAbd D, Ismail A, Thabet G, Elsharkawy M. High frequency of splice site mutation in 21-hydroxylase deficiency children. J Endocrinol Invest 2014;38.5:505-511
20.	Haider S, Islam B, D'Atri V, Sgobba M, Poojari C, Sun L, et al. Structure-phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia. Proc Natl Acad Sci U S A 2013;110:2605-10.
21.	Krone N, Braun A, Roscher AA, Knorr D, Schwarz HP. Predicting phenotype in steroid 21-hydroxylase deficiency? Comprehensive genotyping in 155 unrelated, well defined patients from southern Germany. J Clin Endocrinol Metab 2000;85:1059-65.
22.	Anastasovska V, Kocova M. Intron 2 splice mutation at CYP21 gene in patients with congenital adrenal hyperplasia in the republic of Macedonia. Balkan Journal of Medical Genetics 2010;13:27-33.
23.	Ramazani A, Kahrizi K, Razaghiazar M, Mahdieh N, Koppens P. The frequency of eight common point mutations in CYP21 gene in Iranian patients with congenital adrenal hyperplasia. Iran Biomed J 2008;12:49-53.
24.	Lobato MN, Ordóñez-Sánchez ML, Tusié-Luna MT, Meseguer A. Mutation analysis in patients with congenital adrenal hyperplasia in the Spanish population: Identification of putative novel steroid 21-hydroxylase deficiency alleles associated with the classic form of the disease. Hum Hered 1999;49:169-75.
25.	Vakili R, Baradaran-Heravi A, Barid-Fatehi B, Gholamin M, Ghaemi N, Abbaszadegan MR. Molecular analysis of the CYP21 gene and prenatal diagnosis in families with 21-hydroxylase deficiency in northeastern Iran. Horm Res Paediatr 2005;63:119-24.
26.	Rabbani B, Mahdieh N, Ashtiani MT, Larijani B, Akbari MT, New M, et al. Mutation analysis of the CYP21A2 gene in the Iranian population. Genet Test Mol Biomarkers 2012;16:82-90.
27.	White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 2000;21:245-91.
28.	Wilson RC, Nimkarn S, Dumic M, Obeid J, Azar MR, Najmabadi H, et al. Ethnic-specific distribution of mutations in 716 patients with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. Mol Genet Metab 2007;90:414-21.
29.	Menabò S, Balsamo A, Baldazzi L, Barbaro M, Nicoletti A, Conti V, et al. A sequence variation in 3'UTR of CYP21A2 gene correlates with a mild form of congenital adrenal hyperplasia. J Endocrinol Invest 2012;35:298-305.
30.	Witchel SF, Bhamidipati DK, Hoffman EP, Cohen JB. Phenotypic heterogeneity associated with the splicing mutation in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Endocrinol Metab 1996;81:4081-8.
31.	Kharrat M, Tardy V, M'Rad R, Maazoul F, Jemaa LB, Refaï M, et al. Molecular genetic analysis of Tunisian patients with a classic form of 21-hydroxylase deficiency: Identification of four novel mutations and high prevalence of Q318X mutation. J Clin Endocrinol Metab 2004;89:368-74.
32.	Pinterova L, Garami M, Pribilincova Z, Behulova R, Mezenska R, Lukacova M, et al. PCR based diagnosis of 21-hydroxylase gene defects in Slovak patients with congenital adrenal hyperplasia. Endocr Regul 2000;34:65-72.
33.	New MI, Abraham M, Gonzalez B, Dumic M, Razzaghy-Azar M, Chitayat D, et al. Genotype-phenotype correlation in 1,507 families with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. Proc Natl Acad Sci U S A 2013;110:2611-6.

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