Abstract
Objectives
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is a common autosomal recessive disorder caused by defects in the CYP21A2 gene. We aimed to determine the prevalence of the most commonly reported mutations among 21-OHD Egyptian patients and correlate genotype with phenotype.
Methods
Molecular analysis of the CYP21A2 gene was performed for the detection of the six most common point mutations (p.P30L, p.I172N, p.V281L, p.Q318X, the splice site mutation Int2 [IVS2–13A/C>G], and the cluster of three mutations [p.I236N, p.V237E, and p.M239K] designed as CL6). Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method was performed on 47 unrelated Egyptian 21α-OH deficiency patients and their available parents to detect the presence of the six most common point mutations.
Results
Screening for the six most common point mutations in CYP21A2 gene, revealed mutations in 87.2% (82/94) of the studied alleles corresponding to 47 Egyptian patients. The most common mutation among the studied cases was IVS2-13C/A>G that was found to be presented in a frequency of 46.8% (44/94). The genotype/phenotype correlations related to null, A, and B groups were with PPV of 100, 55.5, and 83.3%, respectively.
Conclusions
The described method diagnosed CAH in 80.8% of the studied patients. Good correlation between genotype and phenotype in salt wasting and simple virilizing forms is determined, whereas little concordance is seen in nonclassical one. Furthermore, studying the carrier frequency of 21-OHD among the normal population is of great importance.
Funding source: National Research Centre
Award Identifier / Grant number: Molecular diagnostic markers for congenital adrena
Acknowledgments
The authors thank the patients and their families for accepting to participate in this study.
Reasearch funding: The research was funded by NRC (National Research Centre in Egypt) through the project entitled “Molecular diagnosis of 21 hydroxylase and 17-beta hydroxy steroid dehydrogenase-3 deficiency in congenital adrenal hyperplasia.”
Author contributions: All authors have read and approved the manuscript, and all authors contributed equally .
Competing interests: Nil.
Consent for publication: Not applicable.
Ethics approval and consent to participate: Written informed consents were taken from all subjects or from their guardians according to Medical Research Ethics Committee (MREC) of the NRC.
Availability of data and material: The data that support the findings of this study are available from the corresponding author upon reasonable request.
References
1. Riepe, FG, Sippell, WG. Recent advances in diagnosis, treatment, and outcome of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Rev Endocr Metab Disord 2007;8:349–63. https://doi.org/10.1007/s11154-007-9053-1.Search in Google Scholar
2. Trakakis, E, Basios, G, Trompoukis, P, Labos, G, Grammatikakis, I, Kassanos, D. An update to 21-hydroxylase deficient congenital adrenal hyperplasia. Gynecol Endocrinol 2010;26:63–71. https://doi.org/10.3109/09513590903015494.Search in Google Scholar
3. Tayel, SM, Ismael, H, Kandil, H, Abd Rabuh, AR, Sallam, H. Congenital adrenal hyperplasia in Alexandria, Egypt: a high prevalence justifying the need for a community-based newborn screening program. J Trop Pediatr 2010;57:232–4. https://doi.org/10.1093/tropej/fmq064.Search in Google Scholar
4. Mazen, I, Gad, Y, Khalil, A. Intersex disorders among Egyptian patients. J Arab Child 1996;7:607–25.Search in Google Scholar
5. El-Awady, M, Temtamy, S, Gad, Y. Adrenocortical androgen hypersecretion in hirsutism. Appl Endocrinol Egypt 1990;8:103.Search in Google Scholar
6. Essawi, M, Sharaf, S, Effat, L, Hafez, M, Amr, K. Molecular characterization of 21 hydroxylase deficiency congenital adrenal hyperplasia in Egyptian children: a pilot study. Med J Cairo Univ 2007;75:55.Search in Google Scholar
7. Gad, Y, Temtamy, S, El-Awady, M. Late onset 21-hydroxylase deficiency in childhood. Appl Endocrinol Egypt 1990;8:113–22.Search in Google Scholar
8. Trapp, CM, Levine, LS, Oberfield, SE. Congenital adrenal hyperplasia: pediatric endocrinology. Springer; 2018:311–34 pp.10.1007/978-3-319-73782-9_14Search in Google Scholar
9. Ghayee, HK, Auchus, RJ. Basic concepts and recent developments in human steroid hormone biosynthesis. Rev Endocr Metab Disord 2007;8:289–300. https://doi.org/10.1007/s11154-007-9052-2.Search in Google Scholar
10. Morel, Y, David, M, Forest, MG, Betuel, H, Hauptman, G, Andre, J, et al. Gene conversions and rearrangements cause discordance between inheritance of forms of 21-hydroxylase deficiency and HLA types. J Clin Endocrinol Metab 1989;68:592–9. https://doi.org/10.1210/jcem-68-3-592.Search in Google Scholar
11. Krone, N, Arlt, W. Genetics of congenital adrenal hyperplasia. Best Pract Res Clin Endocrinol Metab 2009;23:181–92. https://doi.org/10.1016/j.beem.2008.10.014.Search in Google Scholar
12. Witchel, SF, Azziz, R. Congenital adrenal hyperplasia. J Pediatr Adolesc Gynecol 2011;24:116–26. https://doi.org/10.1016/j.jpag.2010.10.001.Search in Google Scholar
13. 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. https://doi.org/10.1210/jcem.85.3.6441.Search in Google Scholar
14. Kharrat, M, Tardy, V, M’Rad, R, Maazoul, F, Jemaa, LB, Refai, 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. https://doi.org/10.1210/jc.2003-031056.Search in Google Scholar
15. Deneux, C, Tardy, V, Dib, A, Mornet, E, Billaud, L, Charron, D, et al. Phenotype-genotype correlation in 56 women with nonclassical congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Endocrinol Metab 2001;86:207–13. https://doi.org/10.1210/jcem.86.1.7131.Search in Google Scholar
16. 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. https://doi.org/10.1172/jci115897.Search in Google Scholar
17. Hughes, IA. Consequences of the Chicago DSD consensus: a personal perspective. Horm Metab Res 2015;47:394–400. https://doi.org/10.1055/s-0035-1545274.Search in Google Scholar
18. Bizzarri, C, Crea, F, Marini, R, Benevento, D, Porzio, O, Rava, L, et al. Clinical features suggestive of non-classical 21-hydroxylase deficiency in children presenting with precocious pubarche. J Pediatr Endocrinol Metab 2012;25:1059–64, https://doi.org/10.1515/jpem-2012-0241.Search in Google Scholar
19. Zerah, M, Ueshiba, H, Wood, E, Speiser, PW, Crawford, C, McDonald, T, et al. Prevalence of nonclassical steroid 21-hydroxylase deficiency based on a morning salivary 17-hydroxyprogesterone screening test: a small sample study. J Clin Endocrinol Metab 1990;70:1662–7. https://doi.org/10.1210/jcem-70-6-1662.Search in Google Scholar
20. Temtamy, S, Aglan, M. Consanguinity and genetic disorders in Egypt. Middle East J Med Genet 2012;1:12–7. https://doi.org/10.1097/01.mxe.0000407744.14663.d8.Search in Google Scholar
21. Ferenczi, A, Garami, M, Kiss, E, Pek, M, Sasvari-Szekely, M, Barta, C, et al. Screening for mutations of 21-hydroxylase gene in Hungarian patients with congenital adrenal hyperplasia. J Clin Endocrinol Metab 1999;84:2369–72. https://doi.org/10.1210/jc.84.7.2369.Search in Google Scholar
22. Grigorescu Sido, A, Weber, MM, Grigorescu Sido, P, Clausmeyer, S, Heinrich, U, Schulze, E. 21-Hydroxylase and 11β-hydroxylase mutations in Romanian patients with classic congenital adrenal hyperplasia. J Clin Endocrinol Metab 2005;90:5769–73. https://doi.org/10.1210/jc.2005-0379.Search in Google Scholar
23. Rabbani, B, Mahdieh, N, Sayarifar, F, Ashtiani, MT, New, M, Parsa, A, et al. A girl with 45,X/46,XX Turner syndrome and salt wasting form of congenital adrenal hyperplasia due to regulatory changes. Clin Lab 2012;58:1063–6. https://doi.org/10.7754/Clin.Lab.2011.110501.Search in Google Scholar
24. 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. https://doi.org/10.1016/j.ymgme.2006.12.005.Search in Google Scholar
25. Toraman, B, Okten, A, Kalay, E, Karaguzel, G, Dincer, T, Acikgoz, EG, et al. Investigation of CYP21A2 mutations in Turkish patients with 21-hydroxylase deficiency and a novel founder mutation. Gene 2013;513:202–8. https://doi.org/10.1016/j.gene.2012.10.059.Search in Google Scholar
26. Gomes, LG, Huang, N, Agrawal, V, Mendonca, BB, Bachega, TA, Miller, WL. Extraadrenal 21-hydroxylation by CYP2C19 and CYP3A4: effect on 21-hydroxylase deficiency. J Clin Endocrinol Metab 2009;94:89–95. https://doi.org/10.1210/jc.2008-1174.Search in Google Scholar
27. Kaupert, LC, Lemos-Marini, SH, De Mello, MP, Moreira, RP, Brito, VN, Jorge, AA, et al. The effect of fetal androgen metabolism-related gene variants on external genitalia virilization in congenital adrenal hyperplasia. Clin Genet 2013;84:482–8. https://doi.org/10.1111/cge.12016.Search in Google Scholar
28. Ezquieta, B, Cueva, E, Varela, J, Oliver, A, Fernandez, J, Jariego, C. Non-classical 21-hydroxylase deficiency in children: association of adrenocorticotropic hormone-stimulated 17-hydroxyprogesterone with the risk of compound heterozygosity with severe mutations. Acta Paediatr 2002;91:892–8, https://doi.org/10.1111/j.1651-2227.2002.tb02851.x.Search in Google Scholar
29. Friaes, A, Rego, AT, Aragues, JM, Moura, LF, Mirante, A, Mascarenhas, MR, et al. CYP21A2 mutations in Portuguese patients with congenital adrenal hyperplasia: identification of two novel mutations and characterization of four different partial gene conversions. Mol Genet Metab 2006;88:58–65. https://doi.org/10.1016/j.ymgme.2005.11.015.Search in Google Scholar
30. Balsamo, A, Cacciari, E, Baldazzi, L, Tartaglia, L, Cassio, A, Mantovani, V, et al. CYP21 analysis and phenotype/genotype relationship in the screened population of the Italian Emilia-Romagna region. Clin Endocrinol 2000;53:117–25. https://doi.org/10.1046/j.1365-2265.2000.01048.x.Search in Google Scholar
31. Tusie-Luna, MT, Traktman, P, White, PC. Determination of functional effects of mutations in the steroid 21-hydroxylase gene (CYP21) using recombinant vaccinia virus. J Biol Chem 1990;265:20916–22.10.1016/S0021-9258(17)45304-XSearch in Google Scholar
32. Goossens, K, Juniarto, AZ, Timmerman, MA, Faradz, SM, Wolffenbuttel, KP, Drop, SL, et al. Lack of correlation between phenotype and genotype in untreated 21-hydroxylase-deficient Indonesian patients. Clin Endocrinol 2009;71:628–35. https://doi.org/10.1111/j.1365-2265.2009.03550.x.Search in Google Scholar
33. Baş, 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. https://doi.org/10.4008/jcrpe.v1i3.49.Search in Google Scholar
34. Wedell, A, Thilen, A, Ritzen, EM, Stengler, B, Luthman, H. Mutational spectrum of the steroid 21-hydroxylase gene in Sweden: implications for genetic diagnosis and association with disease manifestation. J Clin Endocrinol Metab 1994;78:1145–52. https://doi.org/10.1210/jcem.78.5.8175971.Search in Google Scholar
35. 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 USA 2013;110:2611–6. https://doi.org/10.1073/pnas.1300057110.Search in Google Scholar
36. Levo, A, Partanen, J. Mutation-haplotype analysis of steroid 21-hydroxylase (CYP21) deficiency in Finland. Implications for the population history of defective alleles. Hum Genet 1997;99:488–97. https://doi.org/10.1007/s004390050394.Search in Google Scholar
37. Carvalho, TAAd, Souza, ICNd, Yoshioka, FKN, Caldato, MCF, Torres, NN, Garcia, LS, et al. CYP21 gene mutations in Brazilian patients with 21-hydroxylase deficiency from the Amazon region. Genet Mol Biol 2008;31:626–31. https://doi.org/10.1590/s1415-47572008000400004.Search in Google Scholar
38. Yadav, S, Birla, S, Marumudi, E, Sharma, A, Khadgawat, R, Khurana, ML et al. Clinical profile and inheritance pattern of CYP21A2 gene mutations in patients with classical congenital adrenal hyperplasia from 10 families. Indian J Endocrinol Metab 2015;19:644–8. https://doi.org/10.4103/2230-8210.163191.Search in Google Scholar
39. Podgorski, R, Aebisher, D, Stompor, M, Podgorska, D, Mazur, A. Congenital adrenal hyperplasia: clinical symptoms and diagnostic methods. Acta Biochim Pol 2018;65:25–33. https://doi.org/10.18388/abp.2017_2343.Search in Google Scholar
40. Torres, N, Mello, MP, Germano, CM, Elias, LL, Moreira, AC, Castro, M. Phenotype and genotype correlation of the microconversion from the CYP21A1P to the CYP21A2 gene in congenital adrenal hyperplasia. Braz J Med Biol Res 2003;36:1311–8. https://doi.org/10.1590/s0100-879x2003001000006.Search in Google Scholar
41. 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. https://doi.org/10.1210/jc.2002-021681.Search in Google Scholar
Supplementary Material
The online version of this article offers supplementary material (https://doi.org/10.1515/jpem-2019-0575).
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