Pitfalls in molecular diagnosis of 21-hydroxylase deficiency in congenital adrenal hyperplasia

Kolahdouz, Mahsa; Mohammadi, Zahra; Kolahdouz, Parisa; Tajamolian, Masoud; Khanahmad, Hossein

Pitfalls in molecular diagnosis of 21-hydroxylase deficiency in congenital adrenal hyperplasia

Authors

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

² Department of Genetics and Molecular Biology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

Abstract

Congenital adrenal hyperplasia (CAH) is a putative error of metabolism with autosomal recessive heredity pattern. The main manifestations of classic form of CAH are salt-wasting, dehydration and simple virilization in both sexes and ambiguous genitalia in female gender. 21-hyroxylase (CYP21A2) impairment with prevalence value of 1 in 10,000–15,000 live births is the most common etiology of CAH. Because of consanguineous marriages, the frequency of the CAH in Iran is very high. A wide range of mutations diversity exists in CYP21A2 gene and a large number of these mutations derived from a highly homologous pseudogene, CYP21A1P, through gene conversion. In addition, new mutations such as small and large deletion and point mutations can also result in enzyme deficiency. Various methods for mutation detection were performed. The main obstacle in molecular diagnosis of CAH is amplification of pseudogene during polymerase chain reaction of CYP21A2. All attempts focus on discrimination of pseudogene from gene; that is why, there is the majority of mutations on pseudogene, and if we have contamination with the pseudogene, the result will be unreliable. Here, we discuss this methods and advantage and disadvantage of those.

Keywords

References

1.	López-Gutiérrez AU, Riba L, Ordoñez-Sánchez ML, Ramírez-Jiménez S, Cerrillo-Hinojosa M, Tusié-Luna MT. Uniparental disomy for chromosome 6 results in steroid 21-hydroxylase deficiency: Evidence of different genetic mechanisms involved in the production of the disease. J Med Genet 1998;35:1014-9.
2.	Keegan CE, Killeen AA. An overview of molecular diagnosis of steroid 21-hydroxylase deficiency. J Mol Diagn 2001;3:49-54.
3.	Speiser PW, White PC. Congenital adrenal hyperplasia. N Engl J Med 2003;349:776-88.
4.	White PC, Grossberger D, Onufer BJ, Chaplin DD, New MI, Dupont B, et al. Two genes encoding steroid 21-hydroxylase are located near the genes encoding the fourth component of complement in man. Proc Natl Acad Sci U S A 1985;82:1089-93.
5.	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 U S A 1986;83:2841-5.
6.	Lee HH, Chao HT, Ng HT, Choo KB. Direct molecular diagnosis of CYP21 mutations in congenital adrenal hyperplasia. J Med Genet 1996;33:371-5.
7.	Higashi Y, Hiromasa T, Tanae A, Miki T, Nakura J, Kondo T, et al. Effects of individual mutations in the P-450(C21) pseudogene on the P-450(C21) activity and their distribution in the patient genomes of congenital steroid 21-hydroxylase deficiency. J Biochem 1991;109:638-44.
8.	Mornet E, Crété P, Kuttenn F, Raux-Demay MC, Boué J, White PC, et al. Distribution of deletions and seven point mutations on CYP21B genes in three clinical forms of steroid 21-hydroxylase deficiency. Am J Hum Genet 1991;48:79-88.
9.	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.
10.	Dupont B, Oberfield SE, Smithwick EM, Lee TD, Levine LS. Close genetic linkage between HLA and congenital adrenal hyperplasia (21-hydroxylase deficiency). Lancet 1977;2:1309-12.
11.	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.
12.	Fitness J, Dixit N, Webster D, Torresani T, Pergolizzi R, Speiser PW, et al. Genotyping of CYP21, linked chromosome 6p markers, and a sex-specific gene in neonatal screening for congenital adrenal hyperplasia. J Clin Endocrinol Metab 1999;84:960-6.
13.	Anastasovska V, Kocova E, Kocova M. A p. P30 L Mutation at the CYP21A2 gene in macedonian patients with Nonclassical Congenital Adrenal Hyperplasia. Balkan J Med Genet 2010;13:17-21.
14.	Wilson RC, Wei JQ, Cheng KC, Mercado AB, New MI. Rapid deoxyribonucleic acid analysis by allele-specific polymerase chain reaction for detection of mutations in the steroid 21-hydroxylase gene. J Clin Endocrinol Metab 1995;80:1635-40.
15.	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.
16.	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.
17.	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.
18.	Dolzan V, Stopar-Obreza M, Zerjav-Tansek M, Breskvar K, Krzisnik C, Battelino T. Mutational spectrum of congenital adrenal hyperplasia in Slovenian patients: A novel Ala15Thr mutation and Pro30Leu within a larger gene conversion associated with a severe form of the disease. Eur J Endocrinol 2003;149:137-44.
19.	Ferenczi A, Garami M, Kiss E, Pék M, Sasvári-Székely 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.
20.	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.
21.	Coeli-Lacchini FB, Turatti W, Elias PC, Elias LL, Martinelli CE Jr, Moreira AC, et al. A rational, non-radioactive strategy for the molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Gene 2013;526:239-45.
22.	Mao R, Nelson L, Kates R, Miller CE, Donaldson DL, Tang W, et al. Prenatal diagnosis of 21-hydroxylase deficiency caused by gene conversion and rearrangements: Pitfalls and molecular diagnostic solutions. Prenat Diagn 2002;22:1171-6.
23.	Tusie-Luna MT, Speiser PW, Dumic M, New MI, White PC. A mutation (Pro-30 to Leu) in CYP21 represents a potential nonclassic steroid 21-hydroxylase deficiency allele. Mol Endocrinol 1991;5:685-92.
24.	Lee HH, Lee YJ, Lin CY. PCR-based detection of the CYP21 deletion and TNXA/TNXB hybrid in the RCCX module. Genomics 2004;83:944-50.
25.	Anastasovska V, Kocova M. Intron 2 splice mutation at CYP21 gene in patients with congenital adrenal hyperplasia in the republic of macedonia. BJMG 2010;13: 3-10.
26.	Day DJ, Speiser PW, White PC, Barany F. Detection of steroid 21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection reaction. Genomics 1995;29:152-62.
27.	Chin D, Speiser PW, Imperato-McGinley J, Dixit N, Uli N, David R, et al. Study of a kindred with classic congenital adrenal hyperplasia: Diagnostic challenge due to phenotypic variance. J Clin Endocrinol Metab 1998;83:1940-5.
28.	Krone N, Braun A, Weinert S, Peter M, Roscher AA, Partsch CJ, et al. Multiplex minisequencing of the 21-hydroxylase gene as a rapid strategy to confirm congenital adrenal hyperplasia. Clin Chem 2002;48:818-25.
29.	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.
30.	Witchel SF, Lee PA, Suda-Hartman M, Trucco M, Hoffman EP. Evidence for a heterozygote advantage in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Endocrinol Metab 1997;82:2097-101.
31.	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.
32.	Witchel SS, Lee PA, Trucco M. Who is a carrier? Detection of unsuspected mutations in 21-hydroxylase deficiency. Am J Med Genet 1996;61:2-9.
33.	Bobba A, Iolascon A, Giannattasio S, Albrizio M, Sinisi A, Prisco F, et al. Characterisation of CAH alleles with non-radioactive DNA single strand conformation polymorphism analysis of the CYP21 gene. J Med Genet 1997;34:223-8.
34.	Yang YP, Corley N, Garcia-Heras J. Reverse dot-blot hybridization as an improved tool for the molecular diagnosis of point mutations in congenital adrenal hyperplasia caused by 21-hydroxylase deficiency. Mol Diagn 2001;6:193-9.
35.	Rabbani B. Homozygous complete deletion of CYP21A2 causes a simple virilizing phenotype in an Azeri child. Asian Biomed 2011;5:889.
36.	Krone N, Roscher AA, Schwarz HP, Braun A. Comprehensive analytical strategy for mutation screening in 21-hydroxylase deficiency. Clin Chem 1998;44:2075-82.
37.	Concolino P, Mello E, Toscano V, Ameglio F, Zuppi C, Capoluongo E. 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;402:164-70.
38.	White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 2000;21:245-91.
39.	New MI. Steroid 21-hydroxylase deficiency (congenital adrenal hyperplasia). Am J Med 1995;98:2S-8.

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