The Prevalence of 21-Hydroxylase Deficiency in Adrenal Incidentalomas - Hormonal and Mutation Screening
Authors
H. Wagnerova1, I. Lazúrová1, V. Habalová2, D. Dudášová1, A. Vrzgula3
Affiliations
1 1st Department of Internal Medicine, Medical Faculty, University Košice, Slovakia
2 Department of Medical Biology, Medical Faculty, University Košice, Slovakia
3 1st Department of Surgery, Medical Faculty, University Košice, Slovakia
Key words
· adrenal incidentalomas · 21-hydroxylase deficiency · 17-OH progesterone
Abstract
&
The aim of the present study was to evaluate and compare the response of 17 OHP to ACTH stimu- lation in patients with various types of adrenal incidentalomas and to examine the occurence of germline CYP21 mutation in these patients. Subjects and Methods: 40 patients (27 females, 13 males) with unilateral and bilat- eral masses were screened for five most com- mon mutations of the CYP21 in peripheral blood DNA samples. A hormonal evaluation, i.e. baseline plasma values of 17OHP, DHEAS as well as plasma 17OHP and DHEA after ACTH stimulation, was performed in all patients. 21 of them had unilateral adrenal adenoma, 13 patients had adrenal hyperplasia (six of them unilateral) and 6 patients had CT characteristics of other tumors (myelolipomas, cysts, adreno- cortical carcinoma).
Results: There were no significant differ- ences in plasma 17OHP, DHEAS and plasma cortisol between all three groups. Stimulated plasma values of DHEA and 17OHP after ACTH administration were significantly higher in patients with adenomas (p<0.05 and p<0.01) and with hyperplasia (p<0.05 and p<0.05) compared with those with other tumors. An exaggerated response of 17 OHP was found in 5 (12%) patients. However, mutation screening in peripheral blood samples revealed no CYP21 mutation in all examined groups.
Summary: Although 12% of patients with adre- nal incidentalomas had an exaggerated response of 17 OHP after ACTH administration indicating a possible 21-hydroxylase deficiency, these find- ings are not associated with CYP21 mutation estimated in peripheral blood samples. There was found no germline CYP21 mutation in all patients with various adrenal incidentalomas.
| received | 09.10.2007 |
| first decision | 30.10.2007 |
| accepted | 03.12.2007 |
Bibliography DOI 10.1055/s-2007-1004551 Published online: March 25, 2008 Exp Clin Endocrinol Diabetes 2008; 116: 272-275 @J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart . New York ISSN 0947-7349
Correspondence H. Wagnerova, MD 1st Department of Internal Medicine Medical Faculty University Košice Tr.SNP 1 040 66 Slovak Republic
Tel .: +42/190/533 01 24 wagnerh2002@yahoo.com
Introduction &
Clinically inapparent adrenal massess, often ref- fered to as adrenal incidentalomas, are one of the most prevalent of all human tumors. Their inci- dence increases not only because of wide use of imaging techniques, but also because of aging of the population.
At autopsy series the prevalence of previously undiagnosed adrenal massess ranges between 1.5 upto 8.7% (Devenyi et al., 1967, Hedeland et al., 1968) and large CT studies indicate a prevalence between 2-4% (Abecassis et al., 1985). The major- ity of adrenal incidentalomas are benign and nonfunctioning adrenal adenomas, however recent studies demonstrate a higher occurrence of subclinical hypercortisolism (Rossi et al., 2000, Mantero et al., 2000).
Despite of the rarity of androgen overproduction, some authors found an exaggerated response of 17 OH progesterone after ACTH administration in large number of patients with adrenal incidenta- lomas (25-59%), more frequently seen in those with bilateral lesions. Study of Jaresch and col- leagues showed that adrenal adenomas are more frequent in homozygous patients (82%) in com- parison with the heterozygous carriers for con- genital 21 hydroxylase deficiency (Jaresch et al., 1992).
The aim of this study was to evaluate a response of 17 OH progesterone to ACTH administration, to compare the response to ACTH between groups with unilateral and bilateral lesions and to exam- ine the occurrence of CYP21 mutations in patients with unilateral and bilateral massess.
Patients and Methods
&
40 patients (27 females and 13 males) were included in this study. 33 of them had unilateral lesions, while 7 had bilateral adrenal masses or hyperplasia. Size of adrenal masses ranged from 0.5 up to 10 cm. None of these patients had clinical symp- toms of endocrine activity including those due to adrenal hyper- androgenism.
In all patients, a careful hormonal evaluation excluded hyper- cortisolism, primary hyperaldosteronism, pheochromocytoma and hyperandrogenism.
Based on CT characteristics, 21 patients had adrenal adenoma and 13 patients had unilateral (6) and bilateral (7) hyperplasia. In other 6 patients a CT scan suggested cysts (3 cases), myelo- lipoma (2 cases) and adrenocortical carcinoma (1 case), respec- tively.
11 patients underwent adrenal surgery. Histological evaluation confirmed carcinoma in one patient, myelolipoma in 2 patients, cyst in one and adenomas in 7 patients, respectively.
Endocrine evaluation
An overnight 1 mg Dexametasona supression test and plasma adrenocorticotrophic hormone (ACTH) were used for a detection of hypercortisolism, plasma free metanephrines were estimated to rule out pheochromocytoma and aldosterone-renin ratio (ARR) was calculated to rule out primary aldosteronism.
Plasma ACTH, plasma renin activity (PRA), plasma aldosterone, 17OH progesterone (17 OHP), dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) were measured radioimmunologically using RIA kits Immunotech (France), free plasma metanephrines (metanephrin and normetanephrin) were evaluated radioimmunologically using RIA kits Labor Diag- nostika Nord (Germany).
18 patients underwent an ACTH simulating test using 0,25 mg of synthetic analogue of ACTH - tetracosactide hexaacetate (Syn- acthen, Novartis, Switzerland). ACTH was administered intrave- nously at 7.00 o’clock and blood samples for investigation of 17 OHP and DHEA were collected in 0, 30 and 60 minutes. In women
| Primer | Sequences |
|---|---|
| CYP1 | 5'-TTCAGGCGATTCAGGAAGGC-3' |
| CYP48 | 5'-CAGAGCAGGGAGTAGTCTC-3' |
| P3 | 5'-TTGTCCTTGGGAGACTACTCC-3' |
| P4 | 5'-ACCTCTCGCACCCCAGTATGACT-3' |
| P5 | 5'-GCTCCGGAGCCTCCACCTCG-3' |
| P6 | 5'-TTGGAGTTCAGCACCAC-3' |
| P7 | 5'-GTGGTGCTGAACTCCAA-3' |
| P8 | 5'-ACACCAGCTTGTCTGCAGGAGGCG-3' |
| P16 | 5'-TGTGGTGGAGGCTGGTCCCCG-3' |
| P17 | 5'-AGGGCAGGGCGTCCCCGGAGC-3' |
| Mutation | Primers | PCR product (bp) | Restriction enzyme | Fragment size (bp) | |
|---|---|---|---|---|---|
| Wild allele | Mutated allele | ||||
| Pro-30-Leu | P5P6 | 249 | Hha I | 228.21 | 249 |
| Intron 2 (A,C->G) | P7P8 | 378 | Hha I | 378 | 364.24 |
| Val-281-Leu + | P3P4 | 2219 | Apal I | 990.853.376 | 1366.853 |
| Arg -339-His | 1229.990 | ||||
| Pro-453-Ser | P16P17 | 223 | Hha I | 147.51.23.2 | 147.74.2 |
with normal menstrual cycle the test was performed on 5th day of the cycle.
An increase in plasma 17 OHP more than 12 mmol/l (7) or its plasma value after ACTH administration higher than 30 nmol/l (8) suggested a 21 hydroxylase deficiency. Plasma DHEA value higher than 20 ng/ml was considered to indicate the 3 beta HSD deficiency (9).
Mutation analysis CYP21 gene
DNA was isolated from peripheral blood using Wizard Genomic DNA Isolation kit (Promega, Co, Ltd, USA). DNA samples of patients were analyzed for the presence of five most common mutations in gene for 21-hydroxylase. CYP 21 gene was ampli- fied in two parts, from the beginning of the gene to exon 3 (1129bp) and from the 8bp deletion in exon 3 to exon 10 (2219bp). Primers CYP1 and CYP 48 (Wedell and Luthman, 1993) were used for the amplification of the first part and P3, P4 (Oriola et al., 1997) for the second part of gene. The amplicon from each first PCR was subjected to the subsequent PCR-RFLP or RFLP analyses under similar conditions as described in paper Oriola et al. (Oriola et al., 1997). Appropriate primers are listed in Table 1. Using fragment 1 as template for second PCR-RFLP analysis we detected Pro-30-Leu and intron 2 (A, C++G) mutations. Fragment 2 was used for detection of Val281-Leu +Arg339His mutations and served as template for PCR-RFLP detection of Pro-453-Ser mutation. Basic characteristics of mutational analysis of the CYP 21 gene are described in Table 2.
Statistical analysis
Mean values of the investigating parameters are expressed as mean ±SEM. Comparison of the parameters between groups was performed using an unpaired T test. A relationship between parameters was evaluated using linear regression analysis.
Results
&
The clinical data of patients with unilateral and bilateral adrenal massess are shown in Table 3.
A diagnosis of adrenal adenoma was postulated in 21 patients, hyperplasia in 13 and other tumors in 6 patients, respectively. There were no significant differences in mean age between the patients with adenomas, hyperplasia and those with other tumors. Size of adrenal masses ranged from 0.5 up to 10.0 cm.
There was a higher prevalence of female gender in adrenal ade- nomas. The prevalence of unilateral and bilateral hyperplasia were similar, while adenomas and other tumors were predomi- nantly unilateral.
Baseline plasma concentrations of 17 OHP, DHEA, DHEAS and plasma cortisol are shown in Table 4.
There were no significant differences in plasma 17 OHP, DHEAS, DHEA and plasma cortisol between all three groups, although
| Parameter | N | Female/Male | Age Mean Range | Uni/Bilat | Size (cm) |
|---|---|---|---|---|---|
| adenoma | 21 | 15/6 | 56.5 (39-71) | 20/1 | 1.5-7.9 |
| hyperplasia | 13 | 9/4 | 51.5 (36-72) | 6/7 | 0.5-1.7 |
| other | 6 | 3/3 | 50.4 (35-68) | 6/1 | 1.8-10.0 |
| Parameter | All group X± SEM | Adenomas X + SEM | Hyperplasia X ± SEM | Other X + SEM |
|---|---|---|---|---|
| 17-OHP (nmol/l) | 2.84±0.39 | 3.01 ±0.27 | 3.19±0.76 | 1.76±0.41 |
| DHEA (ng/ml) | 1.88±0.25 | 1.61 ±0.28 | 2.12±0.41 | 2.5± 1.23 |
| DHEAS (ng/l) | 63.81 ± 12.8 | 55.02 ± 10.1 | 82.2± 37.5 | 58.17±0.45 |
| plasma cortisol (umol/l) | 0.37 ±0.02 | 0.389 ± 0.02 | 0.4±0.05 | 0.288 ±0.04 |
| Parameter | All group X ± SEM | Adenomas X ± SEM | Hyperplasia X ± SEM | Other |
|---|---|---|---|---|
| X ± SEM | ||||
| 17-OHP (nmol/l) | 9.75±1.78 | 12.1 ±3.6* | 9.59 ±0.99* | 5.27 ± 1.25 |
| DHEA (ng/ml) | 3.71 ±0.9 | 4.53 ±0.7* | 8.26±1.8 | 2.6±0.14 |
| plasma cortisol (umol/l) | 0.63±0.05 | 0.66± 0.03 | 0.68 ±0.05 | 0.59 ± 0.04 |
p<0.05
p<0.01
| Parameter | Adenomas vs hyper- plasia | Adeno- mas vs others | Hyper- plasia vs others |
|---|---|---|---|
| 17-OHP basal (nmol/l) | ns | ns | ns |
| 17-OHP stimulated (nmol/l) | ns | p<0.05 | p<0.05 |
| DHEA basal (ng/ml) | ns | ns | ns |
| DHEA stimulated (ng/ml) | ns | p<0.05 | p<0.01 |
| plasma cortisol basal (umol/l) | ns | ns | ns |
| plasma cortisol stimulated (umol/I) | ns | ns | ns |
there was a tendency to higher values of 17 OHP and plasma cortisol in adenomas and hyperplasia.
Table 5 shows plasma values of 17 OHP, DHEA and plasma corti- sol after ACTH administration in all group of patients and groups with adenomas, hyperplasia and other tumors.
Stimulated plasma values of DHEA after ACTH administration were significantly higher in patients with adenomas (p<0.05) and adrenal hyperplasia (p<0.01) compared with those with other adrenal massess. Additionally stimulated plasma 17 OHP was significantly higher in patients with adenomas (p<0.05) and hyperplasia (p<0.05) than those with other tumors (Table 6).
There was found a significant negative correlation between ACTH-stimulated DHEA values and size of the tumor (r= - 0.56, p<0.01) (· Fig. 1).
An exaggerated response of 17-OHP (more thad 12 nmol/l) was found in 5(12%) patients (4 female, 1 male). In these patients the CT scan revealed an adrenal adenoma in 3 cases (23%) and bilat- eral hyperplasia in 2 cases (28%), respectively. Nobody of the patients had an exaggerated response of DHEA after ACTH stim- ulation.
8
6
Size (cm)
4
2
0
0
1
5
9
13
17
DHEA (ng/ml)
Mutation screening in peripheral blood samples of patients with adrenal incidentalomas revealed no mutation of Pro30Leu, Val- 281Leu and no mutations of Arg339His, Pro453Ser as well as intron 2 (A/C-G) in all examined groups.
Discussion
&
In the present study we performed a hormonal evaluation and mutation analysis to detect a 21-hydroxylase deficiency in patients with adrenal incidentalomas.
An exaggerated response of 17 OHP after ACTH administration represents a frequent endocrine abnormality ranging between 17 upto 71% of patients with adrenal incidentalomas (Mantero
and Arnaldi, 1999). Some authors found a positive relationship between the severity of 21 hydroxylase deficiency and the prev- alence of adrenal massess (45% of heterozygous and 82% of homozygous) (Jaresch et al., 1992).
In our group of patients we found a significantly higher ACTH- stimulated 17OHP as well as DHEA values in patients with adre- nal adenomas and adrenal hyperplasia than those with other lesions (cysts, myelolipomas and carcinoma). Baseline levels were mildly but not significantly higher. Unfortunately we were not able to demonstrate a significant difference between unilat- eral and bilateral lesions, although ACTH-stimulated values of DHEA were nonsignificantly higher in group with hyperplasia. It has been demonstrated that patients with bilateral adrenal incidentalomas have significantly increased plasma 17 OHP lev- els in response to ACTH stimulation compared with those with unilateral tumors. On the other hand, an exaggerated response of 17OHP to ACTH more frequently occurs in patients with bilat- eral than in unilateral massess (Bernini et al., 1998). In a recent study Patócs et al. found a higher ACTH-stimulated plasma 17OHP concentrations in patients with bilateral adrenal massess, although the difference between bilateral and unilateral tumors was not significant (Patócs et al., 2002).
Mancini et al found an increased response of 17 OHP (more than 30 nmol/l) in approximately 25% of patients with adrenal tumors. However, they suppose that this exaggerated response of 17OHP is not specifi c for 21-hydroxylase defi ciency and it can reflect an alteration of intratumoral steroidogenesis (Mancini et al., 2003).
Based on the response of 17OHP after ACTH administration we supposed a 21-OH deficiency in 5 patients with adrenal masses - three adenomas and two cases of bilateral hyperplasia. The prevalence of this exaggerated response indicating a 21 hydroxy- lase deficiency was 12%. However we did not detect a CYP21 mutation in these patients. Additionaly, none of the patients with adrenal massess had mutation of all evaluated alels.
The difference in stimulated values of 17 OHP between adrenal adenomas, hyperplasia in comparison with other tumors have not been described in previous literature. Although the group of other tumors was very heterogenous, a lower values of stimu- lated 17 OH progesterone may indicate that some of these tumors were histologically not fully of adrenocortical origin (cysts, myelolipomas).
The prevalence of 21 hydroxylase deficiency in Caucasian popu- lation has been estimated as one of 1 000, but selective groups may have a higher prevalence. In study of Patócs, the detection of one homozygous and three heterozygous mutations in 19 patients with bilateral (21%) and five heterozygous germline mutations in 31 patients with unilateral tumors (16%) suggests a higher frequency of mutations in both groups of adrenal tumors (Patócs et al., 2002). However our results do not support this finding. Our finding can be supported by others who did not confirmed a 21-hydroxylase mutation in patients with adrenal incidentalomas (Beuschlein et al., 1998) indicating that 21 hydroxylase deficiency is not involved into the pathogenesis of adrenal massess,.
In study of Reincke et al. authors demostrated that patients with adrenal incidentalomas have an increased response of mineralo- corticoid and glucocorticoid precursors after ACTH administra- tion. It is possible that mechanism other than impairment of 21-hydroxylase activity should be considered since decreased
activity of the 11beta-hydroxylase enzyme has already been documented (Reincke et al., 1997). On the other hand, Mancini and colleagues demonstrated an improvement of the exagger- ated response of 17OHP to ACTH stimulation after adrenalec- tomy and suppose that this endocrine abnormality is related to an enzymatic defect inside the tumor mass (Mancini et al., 2003). Because there was no case of germline CYP21 mutation in our group of patients we may support the hypothesis of intratu- moral enzymatic defect in unilateral or bilateral adrenal mass. Second possible explanation is that other genes or other effects involved into the metabolism of adrenal steroids may influence the clinical and/or hormonal abnormalities presenting in these patients.
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