Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism and accounts for 90–95% of CAH cases. In the present work, we analyzed the functional consequence of four novel previously reported point CYP21A2 mutations -p.R132C, p.R149C, p.M283V, p.E431K- found in Argentinean 21-hydroxylase deficient patients. In addition, we report an acceptor splice site novel point mutation, c.652-2A>G, found in a classical patient in compound heterozygosity with the rare p.R483Q mutation. We performed bioinformatic and functional assays to evaluate the biological implication of the novel mutation. Our analyses revealed that the residual enzymatic activity of the isolated mutants coding for CYP21A2 aminoacidic substitutions was reduced to a lesser than 50% of the wild type with both progesterone and 17-OH progesterone as substrates. Accordingly, all the variants would predict mild non-classical alleles. In one non-classical patient, the p.E431K mutation was found in cis with the p.D322G one. The highest decrease in enzyme activity was obtained when both mutations were assayed in the same construction, with a residual activity most likely related to the simple virilizing form of the disease. For the c.652-2A>G mutation, bioinformatic tools predicted the putative use of two different cryptic splicing sites. Nevertheless, functional analyses revealed the use of only one cryptic splice acceptor site located within exon 6, leading to the appearance of an mRNA with a 16 nt deletion. A severe allele is strongly suggested due to the presence of a premature stop codon in the protein only 12 nt downstream.
References
[1]
New MI, White PC, Pang S, Dupont B, Speiser PW (1989) The Metabolic Basis of Inherited Disease: The adrenal hyperplasias New York Press.1881–1917 pp.
[2]
Miller WL (1994) Genetics, diagnosis and management of 21-hydroxylase deficiency. J Clin Endocrinol Metab 78: 241–246. doi: 10.1210/jcem.78.2.8106606
[3]
Pang S, Shook MK (1997) Current status of neonatal screening for congenital adrenal hyperplasia. Curr Opin Pediatr 9: 419–423. doi: 10.1097/00008480-199708000-00018
[4]
Therell BL (2001) Newborn screening for congenital adrenal hyperplasia. Endocrinol Metab Clin North Am 30: 15–30.
[5]
Van der Kamp HJ, Wit JM (2004) Neonatal screening for congenital adrenal hyperplasia. Eur J Endocrinol 151: U71–U75. doi: 10.1530/eje.0.151u071
Speiser PW, Dupont B, Rubinstein P, Piazza A, Kastelan A, et al. (1985) High frequency of nonclassical steroid 21-hydroxylase deficiency. Am J HumGenet 37: 650–667. doi: 10.1097/00006254-198604000-00017
[8]
Donohoue PA, van Dop C, McLean RH, White PC, Jospe N, et al. (1986) Gene conversion in salt-losing congenital adrenal hyperplasia with absent complement C4B protein. J Clin Endocrinol Metab 62: 995–1002. doi: 10.1210/jcem-62-5-995
[9]
Higashi Y, Tanae A, Inohue H, Fujii-Kuriyama Y (1988) Evidence for frequent gene conversions in the steroid 21-hydroxylase (P-450c21) gene: implications for 21-hydroxylase deficiency. Am J Hum Genet 42: 17–25.
[10]
White PC, Speiser PW (2000) Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 21: 245–291. doi: 10.1210/edrv.21.3.0398
[11]
Robins T, Carlsson J, Sunnerhagen M, Wedell A, Persson B (2006) Molecular Model of Human CYP21 Based on Mammalian CYP2C5: Structural Features Correlate with Clinical Severity of Mutations Causing Congenital Adrenal Hyperplasia. Mol Endocrinol 20: 2946–2964. doi: 10.1210/me.2006-0172
[12]
Minutolo C, Nadra A, Fernández C, Taboas M, Buzzalino N, et al. (2011) Structure-based analysis of five disease causing-mutation in 21-Hydroxylase deficient patients. PLoS ONE doi:10.1371/journal.pone.0015899.
[13]
Haider S, Islam B, D’Atri V, Sgobba M, Poojari C, et al. (2013) Structure–phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia. Proc Natl Acad Sci USA 110: 2605–2610. doi: 10.1073/pnas.1221133110
[14]
den Dunnen JT, Antonarakis SE (2000) Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat 15: 7–12. doi: 10.1002/(sici)1098-1004(200001)15:1<7::aid-humu4>3.0.co;2-n
[15]
White PC, New MI, Dupont B (1986) Structure of the human steroid 21- hydroxylase genes. Proc Natl Acad Sci USA 83: 5111–5115. doi: 10.1073/pnas.83.14.5111
[16]
Dain LB, Buzzalino ND, Oneto A, Belli S, Stivel M, et al. (2002) Classical and nonclassical 21-hydroxylase deficiency: A molecular study of Argentine patients. Clin Endocrinol 56: 239–245. doi: 10.1046/j.0300-0664.2001.01419.x
[17]
Pasqualini T, Alonso G, Tomasini R, Galich AM, Buzzalino N, et al. (2007) Congenital adrenal hyperplasia: clinical characteristics and genotype in newborn, childhood and adolescence. Medicina 67: 253–261.
[18]
Owerbach D, Ballard L, Draznin MB (1992) Salt-wasting congenital adrenal hyperplasia: detection and characterization of mutations in the steroid 21- hydroxylase gene, CYP21, using the polymerase chain reaction. J Clin Endocrinol Metab 74: 553–558. doi: 10.1210/jcem.74.3.1740489
[19]
Wedell A, Luthman H (1993) Steroid 21-hydroxylase deficiency: two additional mutations in salt-wasting disease and rapid screening of disease-causing mutations. Hum Molec Genet 2: 499–504. doi: 10.1093/hmg/2.5.499
[20]
Desmet FO, Hamroun D, Lalande M, Collod-Béroud G, Claustres M, et al. (2009) Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res e67 doi:10.1093/nar/gkp215.
[21]
Krone N, Riepe FG, Gr？tzinger J, Partsch CJ, Sippell WG (2005) Functional Characterization of Two Novel Point Mutations in the CYP21 Gene Causing Simple Virilizing Forms of Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency. J Clin Endocrinol Metab 90: 445–454. doi: 10.1210/jc.2004-0813
[22]
Bleicken C, Loidi L, Dhir V, Parajes S, Quinteiro C, et al. (2009) Functional characterization of three CYP21A2 sequence variants (p.A265V, p.W302S, p.D322G) employing a yeast co-expression system. Hum Mutat 30: 443–450. doi: 10.1002/humu.20926
[23]
Tusie-Luna MT, Traktman P, White PC (1990) Determination of functional effects of mutations in the steroid 21-hydroxylase gene (CYP21) using recombinant vaccinia virus. J Biol Chem 265: 20916–20922.
[24]
Fernández Solari JJ, Pozzi AG, Ceballos NR (2002) Seasonal changes in the activity of the cytochrome P450c17 from the testis of Bufo arenarum. J Comp Physiol 172: 685–690. doi: 10.1007/s00360-002-0296-6
[25]
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye banding. Anal Biochem 72: 248–254. doi: 10.1006/abio.1976.9999
[26]
Ghayee HK, Rege J, Watumull LM, Nwariaku FE, Carrick KS, et al. (2011) Clinical, Biochemical, and Molecular Characterization of Macronodular Adrenocortical Hyperplasia of the Zona Reticularis: A New Syndrome. J Clin Endocrinol Metab 96: 243–250. doi: 10.1210/jc.2010-1222
[27]
Muro AF, Caputi M, Pariyarath R, Pagani F, Buratti E, et al. (1999) Regulation of fibronectin EDA exon alternative splicing: possible role of RNA secondary structure for enhancer display. Mol Cell Biol 19: 2657–2671.
[28]
Chu X, Ding H, Cui G, Xu Y, Wang DW, et al. (2013) Functional consequences of a novel point mutation in the CYP21A2 gene identified in a Chinese Han patient with nonclassic 21-hydroxylase deficiency. Clin Endocrinol doi:10.1111/cen.12309.
[29]
Nikoshkov A, Lajic S, Vlamis-Gardikas A, Tranebjaerg L, Holst M, et al. (1998) Naturally occurring mutants of human steroid 21-hydroxylase (P450c21) pinpoint residues important for enzyme activity and stability. J Biol Chem 273: 6163–6165. doi: 10.1074/jbc.273.11.6163
[30]
Nikoshkov A, Lajic S, Holst M, Wedell A, Luthman H, et al. (1997) Synergistic effect of partially inactivating mutations in steroid 21-hydroxylase deficiency. J Clin Endocrinol Metab 82: 194–199. doi: 10.1210/jcem.82.1.3678
[31]
Menassa R, Tardy F, Despert F, Bouvattier-Morel C, Brossier JP, et al. (2008) p.H62L a rare mutation of the CYP21 gene identified in two form of 21-hydroxylase deficiency. J Clin Endocrinol Metab 93: 1901–1908. doi: 10.1210/jc.2007-2701
[32]
Soardi FC, Barbaro M, Lau IF, Lemos-Marini SH, Baptista MT, et al. (2008) Inhibition of CYP21A2 enzyme activity caused by novel missense mutations identified in Brazilian and Scandinavian patients. J Clin Endocrinol Metab 93: 2416–2420. doi: 10.1210/jc.2007-2594
[33]
Tardy V, Menassa R, Sulmont V, Lienhardt-Roussie A, Lecointre C, et al. (2010) 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 95: 1288–1300. doi: 10.1210/jc.2009-1202
[34]
Wilson RS, Mercado AB, Cheng KC, New MI (1995) Steroid 21-hydroylase deficiency: genotype may not predict phenotype. J Clin Endocrinol Metab 80: 2322–2329. doi: 10.1210/jcem.80.8.7629224
[35]
Jaaskelainen J, Levo A, Voutilainen R, Partanen J (1997) Population-wide evaluation of disease manifestation in relation to molecular genotype in steroid 21-hydroxylase (CYP21) deficiency: good correlation in a well defined population. J Clin Endocrinol Metab 82: 3293–3297. doi: 10.1210/jcem.82.10.4271
[36]
Nimkarn S, Cerame BI, Wei JQ, Dumic M, Zunec R, et al. (1999) Congenital adrenal hyperplasia (21-hydroxylase deficiency) without demonstrable genetic mutations. J Clin Endocrinol Metab 84: 378–381. doi: 10.1210/jcem.84.1.5554
[37]
Krone N, Braun A, Roscher AA, Knorr D, Schwartz HP (2000) Predicting phenotype in steroid 21-hydroxylase deficiency? Comprehensive genotyping in 155 unrelated, well defined patients from southern Germany. J Clin Endocrinol Metab 85: 1059–1065. doi: 10.1210/jcem.85.3.6441
[38]
Ezquieta B, Cueva E, Varela J, Oliver A, Fernández J, et al. (2002) 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 91: 892–898. doi: 10.1111/j.1651-2227.2002.tb02851.x
[39]
Dolzan V, Sólyom J, Fekete G, Kovács J, Rakosnikova V, et al. (2005) Mutational spectrum of steroid 21-hydroxylase and the genotype-phenotype association in Middle European patients with congenital adrenal hyperplasia. Eur J Endocrinol 153: 99–106. doi: 10.1530/eje.1.01944
[40]
Gomes LG, Huang N, Agrawal V, Mendon？a BB, Bachega TASS, et al. (2008) The Common P450 Oxidoreductase Variant A503V is Not a Modifier Gene for 21- Hydroxylase Deficiency. J Clin Endocrinol Metab 93: 2913–2916. doi: 10.1210/jc.2008-0304
[41]
Dolzan V, Prezelj J, Vidan-Jeras B, Breskvar K (1999) 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: 132–139. doi: 10.1530/eje.0.1410132
[42]
Admoni O, Israel S, Lavi I, Gur M, Tenenbaum-Rakover Y (2006) Hyperandrogenism in carriers of CYP21 mutations: the role of genotype. Clin Endocrinol 64: 645–651. doi: 10.1111/j.1365-2265.2006.02521.x
[43]
Speiser PW, Azziz R, Baskin LS, Ghizzoni L, Hensle TW, et al. (2010) Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 95: 4133–4160. doi: 10.1210/jc.2009-2631
[44]
New MI, Abraham M, Gonzalez B, Dumic M, Razzaghy-Azar M, et al. (2013) Genotype-phenotype correlation in 1,507 families with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. Proc Natl Acad Sci USA 110: 2611–2616. doi: 10.1073/pnas.1300057110
[45]
Lajic S, Wedell A (1996) An intron 1 splice mutation and a nonsense mutation (W23X) in CYP21 causing severe congenital adrenal hyperplasia. Hum Genet 98: 182–184. doi: 10.1007/s004390050186
[46]
Ordo？ez-Sánchez ML, Ramírez-Jiménez S, López-Gutierrez AU, Riba L, Gamboa-Cardiel S, et al. (1998) Molecular genetic analysis of patients carrying steroid 21-hydroxylase deficiency in the Mexican population: identification of possible new mutations and high prevalence of apparent germ-line mutations. Hum Genet 102: 170–177. doi: 10.1007/s004390050672
[47]
Billerbeck AE, Mendonca BB, Pinto EM, Madureira G, Arnhold IJ, et al. (2002) Three novel mutations in CYP21 gene in Brazilian patients with the classical form of 21-hydroxylase deficiency due to a founder effect. J Clin Endocrinol Metab 87: 4314–4317. doi: 10.1210/jc.2001-011939
[48]
Lee HH, Chao HT, Lee YJ, Shu SG, Chao MC, et al. (1998) Identification of four novel mutations in the CYP21 gene in congenital adrenal hyperplasia in the Chinese. Hum Genet 103: 304–310. doi: 10.1007/s004390050821
[49]
Lee HH, Chang SF (2001) Multiple transcripts of the CYP21 gene are generated by the mutation of the splicing donor site in intron 2 from GT to AT in 21-hydroxylase deficiency. J Endocrinol 171: 397–402. doi: 10.1677/joe.0.1710397
[50]
Robins T, Bellanne-Chantelot C, Barbaro M, Cabrol S, Wedell A, et al. (2007) Characterization of novel missense mutations in CYP21 causing congenital adrenal hyperplasia. J Mol Med 85: 247–255. doi: 10.1007/s00109-006-0121-x
[51]
Ono M, Kashimada K, Miyai K, Onishi T, Takagi M, et al. (2008) In Vitro Enzyme Assay of CYP21A2 Mutation (R483Q) by A Novel Method Using Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS). Clin Pediat Endocrinol 17: 49–56. doi: 10.1297/cpe.17.49
[52]
Stikkelbroeck NM, Hoefsloot LH, de Wijs IJ, Otten BJ, Hermus AR, et al. (2003) 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 88: 3852–3859. doi: 10.1210/jc.2002-021681
