Test with 5 mcg ACTH for Diagnosis of Non-classic Congenital Adrenal Hyperplasia

Background: Short test with 250 mcg corticotropin (Tetracosactide acetate, a substance of first 24 amino acids from 39-acid chain of endogenous ACTH) is a diagnostic standard of non-classic form of congenital adrenal hyperplasia (CAH). However, it is well known that morning ACTH levels in healthy people fluctuate between 10 and 60 pg/ml, and peak cortisol levels can be achieved with 1-24 ACTH dose of 1 mcg or 5 mcg. Objective: To study the sensitivity and specificity of the test with 5 mcg ACTH to diagnose non-classic form of congenital adrenal hyperplasia (NCAN). Materials and Methods: During from 2006 to 2011, we screened 435 women aged from 16 to 35 (25 (21; 29) years) for CAH after exclusion of neoplastic hyperandrogenism. Protocol of low-dose (5 mcg) and standard (250 mcg) 1-24 ACTH tests: Blood sampling for basal 17-ОНР and cortisol levels; blood sampling for 17-ОНР and cortisol at 30 and 60 minutes after 5 mcg or 250 mcg 1-24 ACTH stimulation. Molecular genetic analysis for most prevalent CYP21 mutations was performed using allele specific polymerase chain reaction. Results: Diagnosis of non-classic CAH with 21-hydroxylase deficiency was proved in 5.3% (23/435) of patients. Conclusion: Low dose test with Tetracosactide 5 mcg can identify 21-hydroxylase deficiency in patients with stimulated 17-ОНР concentrations over 14 ng/ml. Test with Tetracosactide 5 mcg had sensitivity of 72.7% and specificity of 100% with positive prognostic value (+PV) of 100%, and negative prognostic value (-PV) of 89.3%.


Introduction
Short test with 250 mcg corticotropin (Tetracosactide acetate, a substance of first 24 amino acids from 39-acid chain of endogenous adrenocorticotropic hormone (ACTH)) is a diagnostic standard of non-classic form of congenital adrenal hyperplasia (CAH). This ACTH dose results in serum ACTH concentrations of up to 2000 pg/ml. However, it is well known that morning ACTH levels in healthy people fluctuate between 10 and 60 pg/ml, and peak cortisol levels can be achieved with 1-24 ACTH dose of 1 mcg or 5 mcg [1]. Thus, standard ACTH tests may result in false-positive results due to over stimulation of adrenal glands.
Study inclusion criteria were following: 1. Dermatological or biochemical signs of hyperandrogenism in women with normal gonadotropins anovulatory and ovulatory cycle.
First group of patients comprises of 234 patient with clinically and/or biochemically established non-neoplastic hyperandrogenism; 201 women without hyperandrogenism were included in the second group. Ovulation absented in 75.2% (176/234) of hyperandrogenic women and in 85.6% (172/201) of patients without hyperandrogenism. In our study we also included some women with recurrent pregnancy loss (12 in first group and 29 in second group).
For differential diagnostic of non-classic CAH with 21-hydroxylase deficiency we performed low-dose (5 mcg) 1-24 ACTH test with Tetracosactide acetate solution (250 mcg per 1 ml, NOVARTIS, Switzerland). Immediately before the test, 1 ml Tetracosactide acetate solution (250 mcg) was diluted by 49 ml of 0.9% NaCl solution to get concentration of 1-24 ACTH of 5 mcg per 1 ml. For intravenous bolus 1 ml of the final solution (5 mcg 1-24 ACTH) injected with 4 ml of 0.9% NaCl solution. In 20 women, standard test with 250 mcg 1-24 ACTH was additionally needed for further diagnosis.
Molecular genetic analysis (MGA) for most prevalent CYP21 mutations was performed using allele specific polymerase chain reaction [3].
For sensitivity and specificity analysis of low-dose ACTH test we compared results of low-dose (5 mcg) and standard (250 mcg) Tetracosactide tests with data of molecular genetic analysis for CYP21.
Statistical data analysis was run using software STATISTICA 6.0 and MedCalc Version 7.4.2.0. We used Shapiro-Wilk test to assess normality of distribution, and relevant t-test for comparison of two depending or non-depending samples. Descriptive non-parametrical statistics included Median (Me), Mean (M), standard deviation (SD), quartiles [25; 75] and Min-Max range. To compare quantitative parameters of non-related samples, Mann-Whitney (U) test was applied. Wilcoxon test (W) and Friedman variance analysis (F) was used when comparing two or more related samples. Kendall's coefficient of concordance (nonparametric correlation coefficient between two variables) was used to assess the relationship between multidimensional variables. Chi-squared test (χ ² ) was done to analyze difference in distribution of categorical parameters in two groups.
Correlation between numerical parameters was tested by Spearman (r) and Kendall's Tau criteria. Sensitivity, specificity and accuracy of 5 mcg 1-24 ACTH test were calculated using standard formulas. Significance level was chosen at p<0.05.

Results
Anovulation in women from both groups was diagnosed as abnormally prolonged menstrual cycle (over 35 days up to amenorrhea, Me=52 days) and/or negative ovulation tests in at least 5 cycles. In group 1 hyperandrogenism was proven clinically (at least 8 scores of Ferriman-Gallwey scale in 62.4% (146/234), acne in 29.5% (69/234) of patients) and biochemically (Table 1).
According to New et al. [4] and Moran et al. [5], non-classical deficiency of 21-hydroxylase should be considered in patients with elevated 17-ОНР levels (basal as well as ACTH stimulated) >10 ng/ml.
2) To evaluate the relevance of absolute values of stimulated 17-ОНР levels.

Non-classic CAH is unlikely
To evaluate relevance of increase ratio BUT NOT absolute stimulated levels of 17-ОНР for the diagnostics of 21-hydroxylase deficiency, we compared increase rate of 17-ОНР and cortisol concentrations. Such approach prevents underestimation of maximal 17-ОНР concentrations <10-14 ng/ml due to «low dose» of ACTH.

Test results are ambiguous and further standard test with 250 mcg of ACTH is needed for differential diagnosis of CAH
Further search for 21-hydroxilase deficiency was suggested in 48 patients with basal 17-ОНР level over 1.7 ng/ml (recommended basal 17-ОНР level for the diagnosis of 21-hydroxilase deficiency [5]) and documented >3-fold increase of 17-ОНР.
There was a subgroup of patients (6/48 (12.5%)) with dose dependent adrenal response to ACTH who needed further search for non-classical ACH. All these 6 women had maximal stimulated 17-ОНР concentrations after 5 mcg Tetracosactide test ≥ 6.7 ng/ml and dermatological signs of hyperandrogenism (hirsutism).

Molecular genetic analysis of CYP21 gene
Twenty-seven patients had basal or stimulated 17-ОНР concentrations over 10 ng/ml, and 17 of them had molecular genetic analysis of CYP21 gene. Patients with stimulated 17-ОНР levels between 10 ng/ml and 14 ng/ml were found to be heterozygous carriers of most common CYP21 mutations. Women with maximal 17-ОНР levels ≥ 14 ng/ml had homozygous mutations or combinations of different heterozygous mutations (Table 3) (Figures 1 and 2). Molecular genetic analysis in 14 patients with maximal 17-ОНР concentrations between 4 and 6.2 ng/ml revealed no CYP21 mutations using allele-specific polymerase chain reaction (n=10) or direct CYP21 gene sequencing. Thus, 21-hydroxylase deficiency was excluded in these women. 334 women with non-significant 17-ОНР rise (<6.2 ng/ml) did not need further investigation for the non-classical CAH and were excluded from the study. In other 320 patients 21hydroxylase deficiency could be surely excluded only after either standard test with 250 mcg ACTH or direct CYP21 gene sequencing. We found no CYP21 mutations in 21 patients with dose independent adrenal response to 5 mcg tetracosactide. There were 13 women with maximal 17-ОНР concentrations >6.7 ng/ml. However, non-classical CAH was not proved in them during standard test and allele specific PCR of CYP21 gene. Molecular-genetic analysis in 6 women with dose-dependent response to Tetracosactide 5 mcg and Tetracosactide 250 mcg revealed 2 cases of homozygous CYP21 mutations: V281L/V281L [6], V237E/ V237E and one case of chimera gene CYP21P/CYP21 [7] (Figure 3). Non-classical CAH was proved only in women with maximal 17-ОНР concentration ≥ 6.7 ng/ml after stimulation with 5 mcg Tetracosactide and >14 ng/ml after stimulation with 250 mcg tetracosactide.
Three patients with maximal 17-ОНР levels under 6.0 ng/ml after stimulation with 5 mcg Tetracosactide and under 10 ng/ml after 250 mcg Tetracosactide, had heterozygous carrier state of V281L mutation.
We evaluated sensitivity and specificity of the main hyperandrogenism symptoms, such as hirsutism and anovulation to define those women who need examination for 21-hydroxylase deficiency.
Hirsutism seems to be the main symptom which disturbs women with NC-CAH. However regular and ovalutary menstrual circle cannot rule out NC-CAH.   Sensitivity and specificity analysis of low-dose test with 1-24 ACTH-5 mcg included data from 36 patients who run through lowdose and standard ACTH test as well as genetic analysis of CYP21.
These were patients with 17-ОНР concentration over 14 ng/ml and homozygous or mixed heterozygous mutations of CYP21 gene. Other patients with heterozygous mutations and 17-ОНР under 14 ng/ml (n=10), and 2 patients with dose-independent reaction to ACTH, max 17-ОНР level >7 ng/ml and no proven mutations of CYP21 gene (PCR) were excluded from this analysis. Unfortunately, 10 clinically hyperandrogenic patients and high Tetracosactide 5 mcg-stimulated 17-ОНР levels (over 14 ng/ml) withdrew from the study. This fact may have influenced the results of sensitivity analysis.
According to the results of the test with 5 mcg Tetracosactide, absolute stimulated 17-ОНР levels but not grades of its increase are relevant for the diagnostics of 21-hydroxilase. Steroidal response to Tetracosactide may be dose dependent in 12.5% of patients, and they have higher peak 17-ОНР levels after stimulation with Tetracosactide-250 mcg. Lack of low-dose 1-24 ACTH stimulation can be probably explained by an adaptation of hyhperplase adrenal glands to grater pituitary stimuli.

Conclusion
1. Low dose test with Tetracosactide 5 mcg can identify 21hydroxylase deficiency in patients with stimulated 17-ОНР concentrations over 14 ng/ml.
2. Standard stimulation test with Tetracosactide 250 mcg and MGA of CYP21 gene is indicated for patients with dermatological signs of hyperandrogenism and maximal 17-ОНР levels ≥ 6.7 ng/ml after stimulation with Tetracosactide 5 mcg.
It should be noted, that clinical relevance of mild non-classic CAH which cannot be detected with low-dose but not with standard-dose Tetracosactide test is still uncertain. It is also known, that most women with heterozygous mutation of CYP21 gene are symptoms free and have normal fertility. Further studies of the cased with discordant results of tests with Tetracosactide 5 mcg and 250 mcg may bring useful information.