The role of 21-hydroxylase in the pathogenesis of adrenal masses: Review of the literature and focus on our own experience
L. Barzon1, P. Maffei2, N. Sonino3, C. Pilon2, L. Baldazzi4, A. Balsamo4, O. Del Maschio5, G. Masi1, M. Trevisan1, M. Pacenti1, and F. Fallo2
1Department of Histology, Microbiology and Medical Biotechnologies; 2Department of Medical and Surgical Sciences; 3Department of Statistical Sciences, University of Padua; 4Department of Pediatrics, University of Bologna; 5Anatomical Pathology Unit, City Hospital of Mestre, Mestre, Italy
ABSTRACT. An exaggerated response of 17- hydroxyprogesterone (17-OHP) to exogenous ACTH stimulation has been found in 30 to 70% of patients with incidentally discovered adrenal tu- mors, supporting the concept that congenital 21- hydroxylase deficiency may be a predisposing fac- tor for adrenocortical tumorigenesis. Decreased expression of 21-hydroxylase gene has been ob- served in sporadic non-functioning adrenocorti- cal adenomas and adrenocortical carcinomas, in agreement with the reduced steroidogenic ac- tivity found in these types of tumors. Screening studies for the presence of mutations in CYP21A2 gene, encoding 21-hydroxylase, in patients with
sporadic adrenocortical tumors yielded discordant results. Overall, a higher frequency of germline 21-hydroxylase mutation carriers has been found among patients with adrenal tumors, including incidentalomas, than in the general population. However, the presence of mutations did not cor- relate with endocrine test results and tumor mass features, suggesting that 21-hydroxylase defi- ciency does not represent a relevant mechanism in adrenal tumorigenesis. Mechanisms leading to reduced 21-hydroxylase expression and activity are still unknown.
(J. Endocrinol. Invest. 30: 615-623, 2007) @2007, Editrice Kurtis
INTRODUCTION
Adrenal masses are common findings in clinical prac- tice, affecting 1-4% of the population (1). The preva- lence of such masses increases with the patients’ age, being 0.2% in young subjects as compared with 6.9% in subjects older than 70 yr of age (2). Most are benign non-functioning adrenocortical adenomas and, less fre- quently, pheochromocytomas, whereas adrenocortical carcinomas and malignant pheochromocytomas are rare. The pathogenesis of sporadic adrenal tumors is poorly understood, whereas progress has been made in the identification of the molecular basis of inherited syndromes associated with the risk of development of
adrenal masses, such as congenital adrenal hyperplasia (CAH) due to impaired activity of steroid 21-hydroxylase enzyme (3, 4). In this syndrome, decreased production of cortisol leads to increased ACTH secretion, resulting in overproduction of adrenal androgens and adrenal hyperplasia. The mildest forms of 21-hydroxylase defi- ciency are probably underdiagnosed due to inconspic- uous symptoms and the first clinical manifestation may be the incidental discovery of adrenal enlargement (5-7). On the other hand, an exaggerated response of 17-hydroxyprogesterone (17-OHP) to exogenous ACTH stimulation (the hallmark of 21-hydroxylase defi- ciency) is frequently found in patients with incidentally discovered adrenal tumors (1), supporting the concept that congenital 21-hydroxylase deficiency may be a predisposing factor for adrenocortical tumorigenesis. However, the impact of 21-hydroxylase deficiency in the pathogenesis of adrenal masses is a field not fully understood. The aim of this review article is to discuss this issue on the basis of the published literature and our own experience.
Correspondence: F. Fallo, MD, Department of Medical and Surgical Sci- ences, University of Padua, Via Ospedale 105, 35128 Padova, Italy. E-mail: francesco.fallo@unipd.it
MOLECULAR GENETICS OF 21-HYDROXYLASE
Steroid 21-hydroxylase is a microsomal cytochrome P450 enzyme involved in the conversion of 17-OHP to 11-deoxycortisol and of progesterone to de- oxycorticosterone (DOC) (8). It is encoded by the CYP21A2 gene, which is located in the HLA major histocompatibility complex on chromosome 6p21.3, approximately 30 kb apart from its inactive pseu- dogene CYP21A1P. The nucleotide sequences of CYP21A2 and CYP21A1P are 98% identical in exons and approximately 96% identical in introns.
The 21-hydroxylase gene is exclusively expressed in the adrenal cortex and, within the adrenal cor- tex, in all three zones. At lower levels than in the fe- tal adrenal gland, CYP21A2 is present in other fetal tissues, such as placenta, ovary, testis, aorta, liver, thymus, and brain (9). Expression of 21-hydroxy- lase in the zona fasciculata is mainly regulated by ACTH, which induces its transcription (10). In the H295R human adrenocortical carcinoma cell line and in primary cultures of human adrenocortical cells, CYP21A2 expression is also induced by cAMP (the second messenger for ACTH), angiotensin II, insulin, and IGF-I (11, 12). The promoter region of the CYP21A2 gene contains binding sites for sev- eral transcription regulators, such as the nuclear factor-granulocyte macrophage b NF-GMb com- plex (13), which is involved in cell proliferation and differentiation, and steroidogenic factor-1 (SF-1) (14), an “orphan” nuclear hormone receptor that is required for expression of most steroidogenic enzymes. Adrenal-specific expression of 21-hy- droxylase is hypothesized to be mediated by an enhancer sequence that is able to influence expres- sion of several adjacent genes within the human leukocyte antigen (HLA) major histocompatibility complex locus (15). Liver receptor homologue- 1 (LRH-1), an orphan nuclear hormone receptor that is closely related to SF-1 and is expressed in most steroidogenic tissues (16), has been demon- strated to regulate expression of several steroid- metabolizing enzymes, even though no data are available for CYP21A2. It is however conceivable that orphan nuclear receptors, such as the general activator LRH-1 and the repressors DAX-1, small heterodimer partner (SHP), and testicular recep- tor 4 (TR4), modulate CYP21A2 expression through interaction with other coactivators or corepressors, such as SF-1 (16-18). Expression of steroidogenic enzymes is also regulated by DNA methylation and, in particular, inhibition of DNA methyla- tion has been demonstrated to reduce basal and cAMP-induced expression of steroidogenic acute regulatory (StAR) protein and of most steroidog- enic enzymes, including CYP21A2 (19).
EXPRESSION AND ACTIVITY OF 21-HYDROXYLASE IN NORMAL ADRENAL CORTEX AND IN SPORADIC ADRENOCORTICAL TUMORS
In the fetal adrenal cortex, expression of CYP21A2 is detectable by histochemical analysis in the fetal zone and transitional zone between 14 weeks and term and in the definitive zone starting at 24 weeks and continuing to term (20, 21). Expression of CYP21A2 during human fetal development differs from the expression observed in the adult adrenal gland, where intense expression was observed in the zona glomerulosa and zona fasciculata, with little or no expression in the zona reticularis (20).
Expression of 21-hydroxylase transcripts and protein as well as 21-hydroxylase activity have been investi- gated by several studies. Decreased expression of CYP21A2 mRNA and 21-hydroxylase activity have been documented in sporadic adrenocortical car- cinomas (22, 23). Low CYP21A2 mRNA levels have been demonstrated also in non-functioning adreno- cortical adenomas, as compared with the normal ad- renal cortex (24, 25). These adenomas also showed normal aldosterone and cortisol levels, but signifi- cantly increased 17-OHP content. At variance, func- tioning tumors showed normal levels of CYP21A2 mRNA (24, 25).
Gene expression profiling by microarray analysis and real-time quantitative RT-PCR and serial analy- sis of gene expression have been employed to in- vestigate expression of steroidogenic enzymes in aldosterone-producing adenomas and in cortisol- producing adenomas. Microarray analysis demon- strated that aldosterone-producing adenomas pro- duced higher levels of CYP21A2 mRNA, together with increased HSD3B2 and CYP11B2 (aldosterone synthase) mRNA compared with normal adrenal, whereas cortisol-producing adenomas had higher levels of CYP11A (cholesterol side-chain cleavage), CYP17 (17a-hydroxylase/17-20 lyase), HSD3B2, and CYP11B1 (11ß-hydroxylase) mRNA (26). Overexpres- sion of CYP21A2 and CYP11B2 mRNA in aldoster- one-producing adenomas has also been shown by serial analysis of gene expression (27). In our series of adrenocortical tumors, CYP21A2 mRNA expres- sion, as measured by quantitative real-time RT-PCR, was significantly higher in aldosterone-producing adenomas than in the normal adrenal cortex, but significantly lower in non-functioning adrenocortical adenomas, thus confirming the results reported in the literature (Fig. 1).
21-HYDROXYLASE DEFICIENCY: CLINICAL ASPECTS
Congenital adrenal hyperplasia is a group of auto- somal recessive disorders characterized by impaired
300000
CYP21A2 mRNA (copies/mg total RNA)
250000
200000
150000
*
100000
50000
*
0
Normal
APA
CPA
NFA
ACC
cortisol secretion from the adrenal gland. More than 95% of CAH cases are caused by 21-hydroxylase en- zyme deficiency. Biochemically, this inherited disor- der is characterized by adrenal overproduction of sex hormone precursors, that do not require 21-hydroxy- lation for their synthesis. Clinical expression of the syndrome is related to the degree of deficiency of 21-hydroxylase activity, varying from conditions with inadequate aldosterone production to maintain sodi- um balance and risk of life-threatening hyponatremic dehydration (“salt wasters”), to those with isolated prenatal virilization of girls and rapid somatic growth with early epiphyseal fusion in both sexes (“simple virilizers”), to a mild “non-classic form” of the disor- der in which affected females have little or no viriliza- tion at birth. Most mutations causing 21-hydroxylase deficiency are the result of recombinations between CYP21A2 and the CYP21A1P pseudogene. Two mechanisms of recombination have been described: unequal crossing over during meiosis, resulting in a net deletion of CYP21A2, or gene conversion that transfers deleterious mutations normally present in CYP21A1P to CYP21A2 (4). Thus, a limited number of mutations are responsible for the majority of cas- es of 21-hydroxylase deficiency. A good agreement between clinical presentation and type of mutations has been observed (4, 28).
ADRENAL TUMORS IN CONGENITAL ADRENAL HY- PERPLASIA DUE TO 21-HYDROXYLASE DEFICIENCY
In a prospective study, Jaresch et al. (29) reported a high prevalence of adrenal masses, nearly 82% in homozygous and 45% in heterozygous patients with
CAH, including deficiencies of 21-hydroxylase, 11ß- hydroxylase, and 3ß-hydroxysteroid dehydrogenase. The risk of developing adrenal masses seems to be higher in patients who take inadequate glucocorti- coid therapy (30, 31). Histological types of adrenal tumor include adenoma, myelolipoma, and heman- gioma (32-34). Steroid-responsive hyperplastic adre- nal nodules can present in previously undiagnosed patients late in life and can potentially be confused with virilizing adrenal adenomas (35-37). Rarely, viriliz- ing adrenal carcinoma has been found in 21-hydrox- ylase deficiency patients (38, 39), but most adrenal masses in children with 21-hydroxylase deficiency are benign (40). We reported one case of congenital 21- hydroxylase deficiency out of a series of 202 patients with adrenal incidentaloma (7). The case was a male patient, bearing bilateral adrenal hyperplasia with a monolateral 1.5 cm-macronodule. He had markedly elevated plasma 17-OHP levels both before and after ACTH stimulation, and increased ACTH and DHEAS levels. Classic virilizing 21-hydroxylase deficiency was diagnosed by genetic testing. The mass remained unchanged at follow-up.
The presence of undiagnosed mild CAH due to 21- hydroxylase deficiency has been investigated in pa- tients with incidentally discovered adrenal masses (incidentalomas). Indeed, 30-60% of patients with adrenal incidentalomas have been reported to have an exaggerated 17-OHP response to ACTH stimu- lation and the frequency of abnormal responses is even higher in patients with bilateral adrenal masses (1, 41-44), thus supporting the concept that congeni- tal 21-hydroxylase deficiency may be a predisposing factor for adrenocortical tumorigenesis.
Discordant findings have been reported by stud- ies investigating the presence of CYP21A2 muta- tions in patients with adrenocortical tumors. Re- sults from these reports are summarized in Table 1. Methodological aspects could account for the different prevalence of mutations among studies. In fact, analysis of point mutations in the CYP21A2 gene by sequencing or allele-specific PCR may miss large gene conversions and deletions, which can be detected by Southern blot analysis. Search for germline and somatic CYP21A2 mutations by sequence analysis in a group of sporadic adreno- cortical tumors, including 6 aldosterone-producing adenomas, 7 cortisol-producing adenomas, 2 non- functioning adenomas, and 4 adrenocortical carci- nomas demonstrated the presence of heterozygous germline mutations (V281L) in 2 patients, one with
a cortisol-producing adenoma and the other with an androgen-secreting adrenocortical carcinoma, and a somatic, heterozygous microdeletion in ex- on 3 of one aldosterone-producing adenoma (45) (Table 1). In another study (44), allele-specific PCR analysis of 4 aldosterone-producing adenomas, 4 cortisol-producing adenomas, 6 non-functioning adenomas, and 13 adrenocortical carcinomas, in- cluding both non-functioning and functioning can- cers, demonstrated CYP21A2 mutations only in a vi- rilizing adrenocortical carcinoma, which carried two somatic heterozygous mutations (Table 1). Patocs et al. performed mutation screening of the 9 most frequent pseudogene-derived point mutations by allele-specific PCR analysis in a series of 50 adrenal incidentalomas, including 19 bilateral and 31 unilat- eral masses (46) (Table 1). They demonstrated that
| Reference | Method of mutation screening | CYP21A2 mutations in adrenal tumors | |||
|---|---|---|---|---|---|
| Aldosterone- producing adenoma | Cortisol-producing adenoma/ hyperplasia | Non-functioning adenoma | Adrenocortical carcinoma | ||
| Beuschlein et al., 1998 (25) | PCR-sequencing | 1/6 (somatic micro- deletion in exon 3) | 1/7 (heterozygous V281L) | 0/2 | 1/4 (heterozygous V281L) |
| Kjellman et al., 1999 (45) | allele-specific PCR | 0/4 | 0/4 | 0/6 | 1/13 (somatic heterozygous V281L and L307insT) |
| Patocs et al., 2002 (46) | allele-specific PCR | – | – | 5/31 unilateral masses (3 heterozygous I172N; 2 heterozygous V281L); 4/19 bilateral masses (1 homozygous V281L; 2 heterozygous V281L; 1 heterozygous R356W) | – – |
| Baumgartner-Parzer et al., 2002 (47) | PCR-sequencing and Southern-blotting | – | – | 9/48 unilateral masses (1 compound heterozygous deletion and gene conversion; 5 heterozygous large gene conversions; 2 heterozygous Q318X; 1 heterozygous int2 mutation); 0/2 bilateral masses | |
one patient (2% of cases) with bilateral incidentalo- ma had the non-classical form of 21-hydroxylase de- ficiency due to germline homozygous V281L muta- tion and that a relatively high proportion of patients carried germline heterozygous mutations. The fre- quency of mutations was similar in patients with bi- lateral and unilateral adrenal incidentalomas (21.1% and 16.1%, respectively). No further mutations were identified in tumor samples. Endocrine evaluation demonstrated a considerable degree of divergence between the mutational spectrum of the CYP21A2 gene and ACTH-stimulated plasma 17-OHP levels, which varied greatly both in patients with mutations and in those without mutations.
Screening for germline CYP21A2 mutations in a group of 50 patients with adrenal incidentalomas was also performed by Baumgartner-Parzer et al. (47) who, at variance, employed both PCR-sequencing and Southern-blot analysis, a method which allows the identification of large gene deletion/conversion events. They diagnosed 21-hydroxylase deficiency in one (2%) 70-yr-old patient who had a combination of a large gene deletion and a conversion event in the other allele consistent with a severe impairment of 21-hydroxylase enzymatic activity. Eight other patients (16%) had classic 21-hydroxylase muta- tions (5 had heterozygous large gene conversions and 3 had heterozygous single base substitutions). No significant differences in clinical signs, tumor size, or presence of bilateral masses was observed between patients with mutations and those with- out mutations. Overall, in this study too there was a higher frequency of classic 21-hydroxylase mutation carriers (2% homozygous and 16% heterozygous) among patients with adrenal incidentalomas than in the general population (carrier prevalence for classic 21-hydroxylase deficiency: 1-2%; disease frequency: one in 15,000 live births) (4).
The prevalence of 17-OHP hyper-response to ACTH stimulation in patients with non-functioning adrenal adenomas is higher than the prevalence of CYP21A2 mutations. Moreover, such patients of- ten show hyperresponse of 11-deoxycortisol and DOC, in addition to 17-OHP, to ACTH stimulation (48, 49), thus suggesting the presence of a general impairment of steroidogenesis in non-functioning adrenal adenomas. Moreover, the finding of a re- lationship between tumor size and 17-OHP-stimu- lated secretion reported in patients with adrenal incidentalomas not associated with 21-hydroxyla- se deficiency (50-52) indicates that such endocrine abnormalities may simply parallel the increased volume of adrenal tissue or intratumoral functional impairment of enzyme activity, rather than a true enzymatic defect.
MECHANISM OF ADRENAL TUMORIGENESIS IN 21- HYDROXYLASE DEFICIENCY ACTH
In patients with 21-hydroxylase deficiency the de- creased production of cortisol leads to persistent or intermittent ACTH hypersecretion, which has been suggested to have a main role in the formation of tumors within the hyperplastic adrenal cortex. The effect of ACTH is mediated by binding to its cog- nate receptor [melanocortin 2 receptor (MC2-R)], fol- lowed by activation of several intracellular pathways, which have been implicated not only in the regula- tion of steroidogenesis, but also in the regulation of adrenocortical growth, differentiation and tumori- genesis. MC2-R has been suggested to have a tumor suppressor function, since loss of heterozygosity of the MC2-R gene, resulting in reduced expression of MC2-R mRNA, has been found in advanced tumor stages (53). In vitro studies in human primary cell cultures have demonstrated that ACTH has an anti- proliferative effect (54), although ACTH treatment in- creases DNA synthesis and cell growth and induces expression of potent mitogens, such as jun, fos, and myc (55). In an in vivo model, physiological doses of ACTH, acting through the MC2-R, have been dem- onstrated to have antiproliferative effects and to in- hibit adrenal tumor growth (56). The observation is apparently in disagreement with that observed in pa- tients with isolated familial glucocorticoid deficiency due to inactivating mutations of the MC2-R gene, who show ACTH insensitivity and adrenal hypoplasia (57). Other proopiomelanocortin-derived peptides have been shown to have effects on adrenocortical growth, such as fragments of melanocyte-stimulat- ing hormone (MSH) (58).
CRH
Increased production of CRH might contribute to adrenal enlargement. In fact, CRH exherts a direct activity on the adrenal cortex through its type 1 and type 2 receptors, which have been found to be up- regulated in adrenocortical tumors (59). CRH directly stimulates DHEA secretion by adrenocortical cell in vitro (59) and, in vivo, in hypophysectomized rats, high doses of CRH have been demonstrated to re- duce adrenocortical atrophy (60).
Adrenal sex steroid hormones
Patients with homozygous and heterozygous 21- hydroxylase deficiency carriers have increased ad- renal androgens and testosterone concentrations compared to healthy individuals (61). It cannot be excluded that the mechanism leading to adrenal en- largement is a result of estrogens and/or androgens stimulation. We recently demonstrated that estro-
gen receptors (ER), androgen receptors (AR), as well as aromatase are expressed in the human adrenal cortex and in adrenocortical tumors and that adreno- cortical carcinomas are characterized by increased ERa/ERB ratio, variable AR expression, and overex- pression of aromatase (unpublished observations). Thus, adrenal androgens could be aromatized to es- trogens within the adrenal gland and stimulate cell proliferation in an autocrine/paracrine loop, as we demonstrated with the human adrenocortical carci- noma cell line H295R (62).
GLUCOCORTICOID THERAPY FOR ADRENAL IN- CIDENTALOMAS WITH HIGH 17-OHP LEVELS: A PUZZLING CASE
It is unclear whether glucocorticoid treatment of pa- tients with 21-hydroxylase deficiency reduces the risk of developing adrenal masses. It is also unknown whether ACTH suppression by glucocorticoids could reduce tumor mass size in patients with adrenal inci- dentalomas and 17-OHP hypersecretion. We recent- ly used a very low dose of dexamethasone to treat a 58-yr-old woman with an incidentally discovered non-functioning mass of 5 cm in size at the right ad- renal, discovered 18 yr after left adrenalectomy for a 1.5-cm adrenal incidentaloma (micronodular adreno- cortical hyperplasia at histological analysis) (Fig. 2A). On admission, the patient was obese [body mass index (BMI) 32 kg/m2], without hirsutism or cushin- goid features; her blood pressure was normal with the anti-hypertensive therapy and endocrine evalua-
tion demonstrated normal adrenal function, with the exception of high 17-OHP levels (4.8 nmol/l, normal range in menopausal condition 0.3-1.5 nmol/l). At ACTH stimulation test (Synacthen 0.250 mg iv bolus) with plasma cortisol and 17-OHP measurements at 0 and 60 min, cortisol rose from 386 (normal values at 08:00 h, 198-695 nmol/l) to 534 nmol/l and 17- OHP from 3.3 to 7.2 mmol/l. The ACTH-stimulated 17-OHP level was compatible with 21-hydroxylase deficiency heterozygous condition carrier. An ab- dominal computed tomography (CT) scan confirmed the presence of a 5.2x4 cm inhomogeneous mass with clear margins, characterized by the presence of a cystic area with liquid content. The unenhanced attenuation value was less than 10 Hounsfield Units (Fig. 2B). Radioiodine (131I)-metaiodobenzylguanidine (MIBG) scintigraphy showed no abnormal uptake by the mass and a positron emission tomography with 18F-fluorodeoxyglucose, to exclude malignancy, de- tected no abnormal metabolic activity at adrenal level or at other sites. Treatment with low-dose dexametha- sone (0.25 mg/day) was started, while awaiting the re- sults of germline molecular analysis of CYP21A2 (28), which revealed no gene mutations. After 6 months of dexamethasone treatment, 17-OHP and cortisol levels were suppressed. At control CT, the adrenal mass showed a slight modification due to an increase of liquid content but no change in size and in attenu- ation value (Fig. 2C). Since there was no change in size of the adrenal mass, dexamethasone therapy was continued at a lower dose (0.125 mg/day), scheduling adrenal hormone measurements and CT scan after
A
10-Feb-2004
H-SP-CR
B
17:52:57.67
4 IMA 22
SPI 4
SP-202.5
R
10cm
kV, 120
et mAs 145
mA 362
1105
C
201-Jun-1940
Volume Zoom VA47C
16-Nov.2005
WATC
H-SP-CR
D
27-Sep-2004
H-SP-CR
18:02-09.71
18:39:53,07
SIMA 18
4 IMA 16
SPF4
SPI 4
SP -64.0
SP -245.5
R
10cm
10cm
V 120
off mAs 100
MA 306
₩ 110
TI 0 5
MAS 169
108
OT 0.0
TOO
14.0/25/5
15.000
12
E
1 yr. Intermediate (at 3 months interval) abdominal ultrasound scans were also performed to check ad- renal morphology. At 20 months of dexamethasone treatment, the endocrine profile was unchanged and the adrenal mass showed only a minimal increase in size (Fig. 2D). Dexamethasone was maintained at the same daily dose of 0.125 mg. The mechanism of tum- origenesis in this case of bilateral incidentaloma is un- known. Germline CYP21A2 gene mutations were ex- cluded by sequence analysis, and adrenal tissue was not available to exclude somatic CYP21A2 mutations. A paracrine ACTH-dependent mechanism of adre- nal cell growth stimulation, as recently documented in bilateral macronodular adrenal hyperplasia (63), could be hypothesized, although circulating ACTH was normal (6.2 pmol/l, normal range 2.2-11 pmol/ I) in our patient. During long-term treatment by low- dose dexamethasone, which led to inhibition of the pituitary-adrenal axis, there was a change in adrenal tumor content from solid to liquid with only a minimal increment in mass size. Up to now, close observation with endocrine work-up and ultrasound/CT scan has allowed delaying the decision about surgery on the solitary mass. However, as recommended for all pa- tients bearing large adrenal tumors, a cautionary ap- proach will be maintained. The risk of the appearance of a mass in the contralateral gland is in fact not neg- ligible in patients with adrenal incidentalomas, i.e., 3% in our series experience (64), and could be higher after unilateral adrenalectomy, as in our patient.
CONCLUSIONS
An exaggerated response of 17-OHP to exogenous ACTH stimulation has been found in 30 to 70% of patients with incidentally discovered adrenal tumors, supporting the concept that congenital 21-hydroxy- lase deficiency may be a predisposing factor for adrenocortical tumorigenesis. Decreased expres- sion of 21-hydroxylase gene has been observed in sporadic non-functioning adrenocortical adenomas and adrenocortical carcinomas, in agreement with the reduced steroidogenic activity found in these types of tumors. In spite of a higher prevalence of germline 21-hydroxylase mutation carriers among patients with adrenocortical tumors, including inci- dentalomas, this condition does not seem to repre- sent a relevant mechanism in adrenal tumorigenesis. Mechanisms leading to reduced 21-hydroxylase ex- pression and activity are still unknown.
ACKNOWLEDGMENTS
G. Masi is a recipient of a fellowship from IRCCS-IOV (Istituto On- cologico Veneto-Padova, Italy).
REFERENCES
1. Barzon L, Sonino N, Fallo F, Palù G, Boscaro M. Prevalence and natural history of adrenal incidentalomas. Eur J Endo- crinol 2003, 149: 273-85.
2. Kloos RT, Gross MD, Francis IR, Korobkin M, Shapiro B. Inciden- tally discovered adrenal masses. Endocr Rev 1995, 16: 460-84.
3. Merke DP, Bornstein SR. Congenital adrenal hyperplasia. Lancet 2005, 365: 2125-36.
4. White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 2000, 21: 245-91.
5. Ravichandran R, Lafferty F, McGinniss MJ, Taylor HC. Con- genital adrenal hyperplasia presenting as massive adrenal incidentalomas in the sixth decade of life: report of two patients with 21-hydroxylase deficiency. J Clin Endocrinol Metab 1996, 81: 1776-9.
6. Nagasaka S, Kubota K, Motegi T, et al. A case of silent 21- hydroxylase deficiency with persistent adrenal insufficiency after removal of an adrenal incidentaloma. Clin Endocrinol (Oxf) 1996, 44: 111-6.
7. Barzon L, Scaroni C, Sonino N, et al. Incidentally discovered adrenal tumors: endocrine and scintigraphic correlates. J Clin Endocrinol Metab 1998, 83: 55-62.
8. Paine AH, Hales DB. Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr Rev 2004, 25: 947-70.
9. Pezzi V, Mathis JM, Rainey WE, Carr BR. Profiling transcript levels for steroidogenic enzymes in fetal tissues. J Steroid Biochem Mol Biol 2003, 87: 181-9.
10. John ME, John MC, Boggaram V, Simpson ER, Waterman MR. Transcriptional regulation of steroid hydroxylase genes by corticotropin. Proc Natl Acad Sci USA 1986, 83: 4715-9.
11. Bird IM, Mason JI, Rainey WE. Protein kinase A, protein ki- nase C, and Ca2+-regulated expression of 21-hydroxylase cytochrome P450 in H295R human adrenocortical cells. J Clin Endocrinol Metab 1998, 83: 1592-7.
12. Endoh A, Yang L, Hornsby PJ. CYP21 pseudogene tran- scripts are much less abundant than those from the active gene in normal human adrenocortical cells under various conditions in culture. Mol Cell Endocrinol 1998, 137: 13-9.
13. Shannon MF, Pell LM, Lenardo MJ, et al. A novel tumor necrosis factor-responsive transcription factor which rec- ognizes a regulatory element in hemopoetic growth factor genes. Mol Cell Biol 1990, 10: 2950-9.
14. Wijesuriya SD, Zhang G, Dardis A, Miller WL. Transcription- al regulatory elements of the human gene for cytochrome P450c21 (steroid 21-hydroxylase) lie within intron 35 of the linked C4B gene. J Biol Chem 1999, 274: 38097-106.
15. Tee MK, Babalola GO, Aza-Blanc P, Speek M, Gitelman SE, Miller WL. A promoter within intron 35 of the human C4A gene initiates abundant adrenal-specific transcription of a 1 kb RNA: location of a cryptic CYP21 promoter element? Hum Mol Genet 1995, 4: 2109-16.
16. Sirianni R, Seely JB, Attia G, et al. Liver receptor homologue- 1 is expressed in human steroidogenic tissues and activates transcription of genes encoding steroidogenic enzymes. J Endocrinol 2002, 174: R13-7.
17. Bavner A, Sanyal S, Gustafsson JA, Treuter E. Transcriptional corepression by SHP: molecular mechanisms and physio- logical consequences. Trends Endocrinol Metab 2005, 16: 478-88.
18. Lee HJ, Lee YF, Chang C. TR4 orphan receptor represses the human steroid 21-hydroxylase gene expression through the monomeric AGGTCA motif. Biochem Biophys Res Comm 2001, 285: 1361-8.
19. Liu J, Li XD, Vaheri A, Voutilainen R. DNA methylation af- fects cell proliferation, cortisol secretion and steroidogenic gene expression in human adrenocortical NCI-H295R cells. J Mol Endocrinol 2004, 33: 651-62.
20. Coulter CL, Jaffe RB. Functional maturation of the primate fetal adrenal in vivo: 3. Specific zonal localization and devel- opmental regulation of CYP21A2 (P450c21) and CYP11B1/ CYP11B2 (P450c11/aldosterone synthase) lead to inte- grated concept of zonal and temporal steroid biosynthesis. Endocrinology 1998, 139: 5144-50.
21. Narasaka T, Suzuki T, Moriya T, Sasano H. Temporal and spa- tial distribution of corticosteroidogenic enzymes immunore- activity in developing human adrenal. Mol Cell Endocrinol 2001, 174: 111-20.
22. Sasano H, Suzuki T, Nagura H, Nishikawa T. Steroidogen- esis in human adrenocortical carcinoma: biochemical ac- tivities, immunohistochemistry, and in situ hybridization of steroidogenic enzymes and histopathologic study in nine cases. Hum Pathol 1993, 24: 397- 404.
23. Ogo A, Haji M, Yanase T, Kato K, Nawata H. The modifica- tion and expression of 21-hydroxylase gene in normal hu- man adrenal gland and adrenal cancer. J Endocrinol Invest 1991, 14: 831-7.
24. Rácz K, Pinet F, Marton T, Szende B, Gláz E, Corvol P. Expres- sion of steroidogenic enzyme messenger ribonucleic acids and corticosteroid production in aldosterone-producing and “nonfunctioning” adrenal adenomas. J Clin Endocrinol Metab 1993, 77: 677-82.
25. Beuschlein F, Schulze E, Mora P, et al. Steroid 21-hydroxy- lase mutations and 21-hydroxylase messenger ribonucleic acid expression in human adrenocortical tumors. J Clin En- docrinol Metab 1998, 83: 2585-8.
26. Bassett MH, Mayhew B, Rehman K, et al. Expression profiles for steroidogenic enzymes in adrenocortical disease. J Clin Endocrinol Metab 2005, 90: 5446-55.
27. Assié G, Auzan C, Gasc JM, et al. Steroidogenesis in al- dosterone-producing adenoma revisited by transcriptome analysis. J Clin Endocrinol Metab 2005, 90: 6638-49.
28. Balsamo A, Cacciari E, Baldazzi L, et al. CYP21 analysis and phenotype/genotype relationship in the screened popula- tion of the Italian Emilia-Romagna region. Clin Endocrinol (Oxf) 2000, 53: 117-25.
29. Jaresch S, Kornely E, Kley HK, Schlaghecke R. Adrenal inci- dentaloma and patients with homozygous or heterozygous congenital adrenal hyperplasia. J Clin Endocrinol Metab 1992, 74: 685-9.
30. Wang J, Bissada MA, Williamson HO, Yakout H, Bissada NK. Adrenal tumors associated with inadequately treated con- genital adrenal hyperplasia. Can J Urol 2002, 9: 1563-4.
31. Kurtoglu S, Atabek ME, Keskin M, Patiroglu TE. Adreno- cortical adenoma associated with inadequately treated congenital adrenal hyperplasia. J Pediatr Endocrinol Metab 2003, 16: 1311-4.
32. Ravichandran R, Lafferty F, McGinniss MJ, Taylor HC. Con- genital adrenal hyperplasia presenting as massive adrenal incidentalomas in the sixth decade of life: report of two patients with 21-hydroxylase deficiency. J Clin Endocrinol Metab 1996, 81: 1776-9.
33. Umpierrez MB, Fackler S, Umpierrez GE, Rubin J. Adrenal myelolipoma associated with endocrine dysfunction: review of the literature. Am J Med Sci 1997, 314: 338-41.
34. Pignatelli D, Vendeira P, Cabral AC. Adrenal incidentalomas: adrenal hemangioma in a patient with congenital adrenal hyperplasia. South Med J 1998, 91: 775-9.
35. Mokshagundam S, Surks MI. Congenital adrenal hyperpla- sia diagnosed in a man during workup for bilateral adrenal masses. Arch Intern Med 1993, 153: 1389-91.
36. Norris AM, O’Driscoll JB, Mamtora H. Macronodular con- genital adrenal hyperplasia in an adult with female pseudo- hermaphroditism. Eur Radiol 1996, 6: 470-2.
37. Falhammar H, Thoren M. An 88-year-old woman with adre- nal tumor and congenital adrenal hyperplasia: connection or coincidence? J Endocrinol Invest 2005, 28: 449-53.
38. Dubey GK, Dotiwalla HH, Choubey BS, Kher A. A case re- port of virilising adrenal cortical carcinoma. J Assoc Physi- cians India 1981, 29: 491-3.
39. Bauman A, Bauman CG. Virilizing adrenocortical carcinoma. Development in a patient with salt-losing congenital adre- nal hyperplasia. JAMA 1982, 248: 3140-1.
40. Lightner ES, Levine LS. The adrenal incidentaloma. A pedi- atric perspective. Am J Dis Child 1993, 147: 1274-6.
41. Seppel T, Schlaghecke R. Augmented 17 alpha-hydroxy- progesterone response to ACTH stimulation as evidence of decreased 21-hydroxylase activity in patients with inci- dentally discovered adrenal tumours (‘incidentalomas’). Clin Endocrinol (Oxf) 1994, 41: 445-51.
42. Ambrosi B, Peverelli S, Passini E, et al. Abnormalities of en- docrine function in patients with clinically “silent” adrenal masses. Eur J Endocrinol 1995, 132: 422-8.
43. Terzolo M, Osella G, Ali A, et al. Different patterns of steroid secretion in patients with adrenal incidentaloma. J Clin En- docrinol Met 1996, 81: 740-4.
44. Bernini GP, Brogi G, Vivaldi MS, et al. 17-Hydroxyprogester- one response to ACTH in bilateral and monolateral adrenal incidentalomas. J Endocrinol Invest 1996, 19: 745-52.
45. Kjellman M, Holst M, Backdahl M, Larsson C, Farnebo LO, Wedell A. No overrepresentation of congenital adrenal hy- perplasia in patients with adrenocortical tumours. Clin En- docrinol (Oxf) 1999, 50: 343-6.
46. Patócs A, Tóth M, Barta C, et al. Hormonal evaluation and mutation screening for steroid 21-hydroxylase deficiency in patients with unilateral and bilateral adrenal incidentalo- mas. Eur J Endocrinol 2002, 147: 349-55.
47. Baumgartner-Parzer SM, Pauschenwein S, Waldhäusl W, Pöl- zler K, Nowotny P, Vierhapper H. Increased prevalence of het-
erozygous 21-OH germline mutations in patients with adrenal incidentalomas. Clin Endocrinol (Oxf) 2002, 56: 811-6.
48. Barzon L, Boscaro M. Diagnosis and management of adre- nal incidentalomas. J Urol 2000, 163: 398-407.
49. Reincke M, Peter M, Sippell WG, Allolio B. Impairment of 11ß-hydroxylase but not 21-hydroxylase in adrenal ‘inciden- talomas’. Eur J Endocrinol 1997, 136: 196-200.
50. Dall’Asta C, Barbetta L, Libe R, Passini E, Ambrosi B. Co- existence of 21-hydroxylase and 11 beta-hydroxylase de- ficiency in adrenal incidentalomas and in subclinical Cush- ing’s syndrome. Horm Res 2002, 57: 192-6.
51. Toth M, Racz K, Adleff V, et al. Comparative analysis of plasma 17-hydroxyprogesterone and cortisol responses to ACTH in patients with various adrenal tumors before and after unilat- eral adrenalectomy. J Endocrinol Invest 2000, 23: 287-94.
52. Sadoul JL, Kézachian B, Altare S, Hadjali Y, Canivet B. Ap- parent activities of 21-hydroxylase, 17alpha-hydroxylase and 17,20-lyase are impaired in adrenal incidentalomas. Eur J Endocrinol 1999, 141: 238-45.
53. Reincke M, Mora P, Beuschlein F, Arlt W, Chrousos GP, Al- lolio B. Deletion of the adrenocorticotropin receptor gene in human adrenocortical tumors: implications for tumorigen- esis. J Clin Endocrinol Metab 1997, 82: 3054-8.
54. Simonian MH, Gill GN. Regulation of the fetal human ad- renal cortex: effects of adrenocorticotropin on growth and function of monolayer cultures of fetal and definitive zone cells. Endocrinology 1981, 108: 1769-79.
55. Kimura E, Sonobe MH, Armelin MC, Armelin HA. Induction of FOS and JUN proteins by adrenocorticotropin and phor- bol ester but not by 3’,5’-cyclic adenosine monophosphate derivatives. Mol Endocrinol 1993, 7: 1463-71.
56. Zwermann O, Schulte DM, Reincke M, Beuschlein F. ACTH 1-24 inhibits proliferation of adrenocortical tumors in vivo. Eur J Endocrinol 2005, 153: 435-44.
57. Clark AJ, Weber A. Adrenocorticotropin insensitivity syn- dromes. Endocr Rev 1998, 19: 828-43.
58. Lowry PJ, Silas L, McLean C, Linton EA, Estivariz FE. Pro-gamma- melanocyte-stimulating hormone cleavage in adrenal gland un- dergoing compensatory growth. Nature 1983, 306: 70-3.
59. Willenberg HS, Haase M, Papewalis C, Schott M, Scherbaum WA, Bornstein SR. Corticotropin-releasing hormone recep- tor expression on normal and tumorous human adrenocorti- cal cells. Neuroendocrinology 2005, 82: 274-81.
60. Bornstein SR, Ehrhart M, Scherbaum WA, Pfeiffer EF. Adrenocortical atrophy of hypophysectomized rats can be reduced by corticotropin-releasing hormone (CRH). Cell Tis- sue Res 1990, 260: 161-6.
61. Knochenhauer ES, Cortet-Rudelli C, Cunnigham RD, Con- way-Myers BA, Dewailly D, Azziz R. Carriers of 21-hydroxy- lase deficiency are not at increased risk for hyperandrogen- ism. J Clin Endocrinol Metab 1997, 82: 479-85.
62. Montanaro D, Maggiolini M, Recchia AG, et al. Antiestro- gens upregulate estrogen receptor ß expression and inhibit human adrenocortical cell proliferation. J Mol Endocrinol 2005, 35: 245-56.
63. Lefebvre H, Duparc C, Chartrel N, et al. Intraadrenal adreno- corticotropin production in a case of bilateral macronodular adrenal hyperplasia causing Cushing’s syndrome. J Clin En- docrinol Metab 2003, 88: 3035-42.
64. Barzon L, Scaroni C, Sonino N, Fallo F, Paoletta A, Boscaro M. Risk factors and long-term follow-up of adrenal inciden- talomas. J Clin Endocrinol Metab 1999, 84: 520-6.