Establishment of Clinical and Lab Algorithms for the Identification of Carriers of Mutations in CYP21A2 – A Study of 365 Children and Adolescents

Abstract

Background Mutations of CYP21A2 encoding 21-hydroxylase are the most frequent cause of congenital adrenal hyperplasia (CAH) and are associated either with elevated basal or ACTH-stimulated levels of 17-hydroxyprogesterone (17OHP) in blood.

Objective The study objective was to identify the most suitable of 12 different test algorithms and appropriate cut-off levels for that test to recognize patients with non-classical congenital adrenal hyperplasia (NCCAH) and carriers of clinically relevant mutations in CYP21A2.

Method and Patients Between July 2006 and July 2015 ACTH-tests were conducted in 365 children and adolescents (Age 1–20 y) suspected to have NCCAH. As a reference, results from subsequent gene sequencing of CYP21A2 was used. Inclusion criteria that were used were premature pubarche with accelerated bone age, hyperandrogenism, hirsutism, or menstrual irregularities. Receiver operating characteristics (ROC) were plotted. Evaluated test algorithms were composed around 17OHP measurements by radioimmunoassays. The most suitable test was identified by the greatest area under the curve (AUC).

Results Among the 12 tested algorithms, the sum of 30 min and 60 min stimulated 17OHP values (sum17OHPstim) showed the highest AUC of 0.774 for identifying heterozygous and bi-allelic mutations. A cut-off of 10.1 μg/l was advisable. Bi-allelic mutations only were best identified calculating the difference between 30 min and basal 17OHP values (Δ17OHP30). A cut-off of 9.4 μg/l was most effective.

Conclusion Alternatively to the above mentioned cut-offs the difference of 60 min after stimulation to basal 17OHP (Δ17OHP60) can be used for the benefit of a combined test to identify both heterozygotes and bi-allelic patients. There are minimal decreases in sensitivity and specificity compared to an approach that applies two tests. However, it denotes a simpler approach in the clinical routine.

Key words

CYP21A2 gene - ACTH-test - Congenital adrenal hyperplasia - 17OHP values

Publication History

Received: 12 March 2020
Received: 16 June 2020

Accepted: 08 July 2020

Article published online:
24 August 2020

• References

• 1 Speiser PW, Arlt W, Auchus RJ. et al. Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab 2018; 103: 4043-4088 DOI: 10.1210/jc.2018-01865.
• 2 Merke DP. Approach to the adult with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Endocrinol Metab 2008; 93: 653-660 DOI: 10.1210/jc.2007-2417
• 3 Gidlof S, Falhammar H, Thilen A. et al. One hundred years of congenital adrenal hyperplasia in Sweden: a retrospective, population-based cohort study. Lancet Diabetes Endocrinol 2013; 1: 35-42 DOI: 10.1016/S2213-8587(13)70007-X.
• 4 Gidlof S, Wedell A, Guthenberg C. et al. Nationwide neonatal screening for congenital adrenal hyperplasia in sweden: A 26-year longitudinal prospective population-based study. JAMA Pediatr 2014; 168: 567-574 DOI: 10.1001/jamapediatrics.2013.5321.
• 5 Fiet J, Gueux B, Gourmelen M. et al. Comparison of basal and adrenocorticotropin-stimulated plasma 21-deoxycortisol and 17-hydroxyprogesterone values as biological markers of late-onset adrenal hyperplasia. J Clin Endocrinol Metab 1988; 66: 659-667 DOI: 10.1210/jcem-66-4-659.
• 6 Witchel SF, Lee PA. Identification of heterozygotic carriers of 21-hydroxylase deficiency: Sensitivity of ACTH stimulation tests. Am J Med Genet 1998; 76: 337-342
• 7 Kreutzmann DJ, Cowell CT, Howard NJ. et al. Congenital adrenal hyperplasia family studies using the short ACTH test. Australian Paediatric Journal 1989; 25: 340-345
• 8 Bachega TA, Brenlha EM, Billerbeck AE. et al. Variable ACTH-stimulated 17-hydroxyprogesterone values in 21-hydroxylase deficiency carriers are not related to the different CYP21 gene mutations. J Clin Endocrinol Metab 2002; 87: 786-790 DOI: 10.1210/jcem.87.2.8247.
• 9 Knochenhauer ES, Cortet-Rudelli C, Cunnigham RD. et al. Carriers of 21-hydroxylase deficiency are not at increased risk for hyperandrogenism. J Clin Endocrinol Metab 1997; 82: 479-485 DOI: 10.1210/jcem.82.2.3759.
• 10 Dacou-Voutetakis C, Dracopoulou M. High incidence of molecular defects of the CYP21 gene in patients with premature adrenarche. J Clin Endocrinol Metab 1999; 84: 1570-1574 DOI: 10.1210/jcem.84.5.5683
• 11 Kulle AE, Riepe FG, Hedderich J. et al. LC-MS/MS based determination of basal- and ACTH-stimulated plasma concentrations of 11 steroid hormones: implications for detecting heterozygote CYP21A2 mutation carriers. Eur J Endocrinol 2015; 173: 517-524 DOI: 10.1530/EJE-14-1084.
• 12 Bidet M, Bellanne-Chantelot C, Galand-Portier MB. et al. Clinical and molecular characterization of a cohort of 161 unrelated women with nonclassical congenital adrenal hyperplasia due to 21-hydroxylase deficiency and 330 family members. J Clin Endocrinol Metab 2009; 94: 1570-1578 DOI: 10.1210/jc.2008-1582.
• 13 Tardy V, Forest M, De Rougemont A. et al. Detection of heterozygotes for 21-hydroxylase deficiency: validation of 21-deoxycortisol after ACTH test by sequencing of the entire CYP21 gene. Horm Res Paediatr 2005; 64: 41-42
• 14 Costa-Barbosa FA, Tonetto-Fernandes VF, Carvalho VM. et al. Superior discriminating value of ACTH-stimulated serum 21-deoxycortisol in identifying heterozygote carriers for 21-hydroxylase deficiency. Clin Endocrinol (Oxf) 2010; 73: 700-706 DOI: 10.1111/j.1365-2265.2010.03871.x.
• 15 Kulle AE, Riepe FG, Melchior D. et al. A novel ultrapressure liquid chromatography tandem mass spectrometry method for the simultaneous determination of androstenedione, testosterone, and dihydrotestosterone in pediatric blood samples: Age- and sex-specific reference data. J Clin Endocrinol Metab 2010; 95: 2399-2409
• 16 Escobar-Morreale HF, Sanchon R, San Millan JL. A prospective study of the prevalence of nonclassical congenital adrenal hyperplasia among women presenting with hyperandrogenic symptoms and signs. J Clin Endocrinol Metab 2008; 93: 527-533 DOI: 10.1210/jc.2007-2053
• 17 Nimkarn S, Gangishetti PK, Yau M. et al. 21-Hydroxylase-Deficient Congenital Adrenal Hyperplasia. In: Pagon RA, Adam MP, Ardinger HH, Eds. GeneReviews(R). Seattle (WA): University of Washington, Seattle University of Washington, Seattle. All rights reserved. 1993
• 18 Schenk D. Prämature Pubarche - ACTH-Test und Mutationsanalyse des 21-Hydroxylasegens; Dissertation, LMU München. 2005
• 19 Weil J, Bidlingmaier F, Sippell WG. et al. Comparison of two tests for heterozygosity in congenital adrenal hyperplasia (CAH). Acta endocrinologica 1979; 91: 109-121
• 20 New MI, Lorenzen F, Lerner AJ. et al. Genotyping steroid 21-hydroxylase deficiency: hormonal reference data. J Clin Endocrinol Metab 1983; 57: 320-326 DOI: 10.1210/jcem-57-2-320.
• 21 [Anonymous] In DIAsource ImmunoAssays S.A.
• 22 Gaedigk A, Ingelman-Sundberg M, Miller NA. et al. The Pharmacogene Variation (PharmVar) Consortium: Incorporation of the Human Cytochrome P450 (CYP) Allele Nomenclature Database. Clin Pharmacol Ther 2018; 103: 399-401 DOI: 10.1002/cpt.910.
• 23 Froud R, Abel G. Using ROC curves to choose minimally important change thresholds when sensitivity and specificity are valued equally: the forgotten lesson of pythagoras. theoretical considerations and an example application of change in health status. PLoS One 2014; 9: e114468 DOI: 10.1371/journal.pone.0114468
• 24 Moran C, Azziz R. 21-hydroxylase-deficient nonclassic adrenal hyperplasia: the great pretender. Seminars in reproductive medicine 2003; 21: 295-300 DOI: 10.1055/s-2003-43307
• 25 Livadas S, Dracopoulou M, Dastamani A. et al. The spectrum of clinical, hormonal and molecular findings in 280 individuals with nonclassical congenital adrenal hyperplasia caused by mutations of the CYP21A2 gene. Clin Endocrinol (Oxf) 2015; 82: 543-549 DOI: 10.1111/cen.12543.
• 26 Araujo RS, Mendonca BB, Barbosa AS. et al. Microconversion between CYP21A2 and CYP21A1P promoter regions causes the nonclassical form of 21-hydroxylase deficiency. J Clin Endocrinol Metab 2007; 92: 4028-4034 DOI: 10.1210/jc.2006-2163.
• 27 Chesover AD, Millar H, Sepiashvili L. et al. Screening for nonclassic congenital adrenal hyperplasia in the era of liquid chromatography-tandem mass spectrometry. J Endocr Soc 2020; 4: bvz030 DOI: 10.1210/jendso/bvz030.
• 28 Brandenberger G, Follenius M, Hietter B. Feedback from meal-related peaks determines diurnal changes in cortisol response to exercise. J Clin Endocrinol Metab 1982; 54: 592-596 DOI: 10.1210/jcem-54-3-592
• 29 Pasquali R, Biscotti D, Spinucci G. et al. Pulsatile secretion of ACTH and cortisol in premenopausal women: effect of obesity and body fat distribution. Clin Endocrinol (Oxf) 1998; 48: 603-612 DOI: 10.1046/j.1365-2265.1998.00458.x.
• 30 Quigley ME, Yen SS. A mid-day surge in cortisol levels. J Clin Endocrinol Metab 1979; 49: 945-947 DOI: 10.1210/jcem-49-6-945
• 31 Klose M, Lange M, Rasmussen AK. et al. Factors influencing the adrenocorticotropin test: role of contemporary cortisol assays, body composition, and oral contraceptive agents. J Clin Endocrinol Metab 2007; 92: 1326-1333 DOI: 10.1210/jc.2006-1791.
• 32 Krone N, Arlt W. Genetics of congenital adrenal hyperplasia. Best Pract Res Clin Endocrinol Metab 2009; 23: 181-192 DOI: 10.1016/j.beem.2008.10.014
• 33 Cinar N, Harmanci A, Aksoy DY. et al. Adrenocortical steroid response to ACTH in different phenotypes of non-obese polycystic ovary syndrome. J Ovarian Res 2012; 5: 42 DOI: 10.1186/1757-2215-5-42.
• 34 Luboshitzky R, Ishai A, Shen-Or Z. et al. Evaluation of the pituitary-adrenal axis in hyperandrogenic women with polycystic ovary syndrome. Neuro endocrinology letters 2003; 24: 249-254
• 35 Dorr HG, Binder G, Reisch N. et al. Experts’ Opinion on the Prenatal Therapy of Congenital Adrenal Hyperplasia (CAH) Due to 21-Hydroxylase Deficiency - Guideline of DGKED in cooperation with DGGG (S1-Level, AWMF Registry No. 174/013, July 2015). Geburtshilfe Frauenheilkd 2015; 75: 1232-1238 DOI: 10.1055/s-0041-109717.