COMMENTS
Steroid 21-Hydroxylase Mutations and 21-Hydroxylase Messenger Ribonucleic Acid Expression in Human Adrenocortical Tumors*
FELIX BEUSCHLEIN, EGBERT SCHULZE, PATRICIA MORA,
HANS-PETER GENSHEIMER, CHRISTIANE MASER-GLUTH, BRUNO ALLOLIO, AND MARTIN REINCKE
Schwerpunkt Endokrinologie, Abt. Innere Medizin II, Klinikum der Albert-Ludwigs-Universität Freiburg (F.B., P.M. M.R.); Schwerpunkt Endokrinologie, Medizinische Universitätsklinik Würzburg (F.B., B.A.); and Institut für Pharmakologie, Universität Heidelberg (E.S., H .- P.G., C.M .- G.), Heidelberg, Germany
ABSTRACT
Twenty-one hydroxylase (P450c21) is a key enzyme essential for normal zona glomerulosa and fasciculata function. Recently, 21-hy- droxylase deficiency has been implicated in the pathogenesis of ad- renocortical tumors. Therefore, we investigated the mutational spec- trum of the CYP21B gene and the messenger RNA expression of P450c21 in six aldosterone-producing adenomas, seven cortisol-pro- ducing adenomas, two nonfunctional incidentally detected adenomas, and four adrenal carcinomas. DNA from leukocytes and tumors was amplified by PCR using primers specific for the CYP21B gene. The 10 exons, intron 2, intron 7, all other exon/intron junctions, and 380 bp of the promoter region of CYP21B were automatically sequenced. Poly(A) RNA was extracted from tumor tissue, dot blotted on a nylon membrane, and hybridized with 32P-labeled P450 side-chain cleav- age, P450 17-a-hydroxylase, and P450c21 complementary DNA probes. We detected heterozygous germline mutations (exon 7, Val 281Leu) in two patients, one with a cortisol-producing adenoma and
the other with an androgen-secreting adrenocortical carcinoma. A somatic, heterozygous microdeletion was found in exon 3 of one al- dosterone-producing adenoma. The P450c21 gene expression corre- lated with the clinical phenotype of the tumor, with low P450c21 messenger RNA expression in nonfunctional adenomas (18.8%, 1.5%) compared with high P450c21 expression in aldosterone- and cortisol- producing adenomas (84 ± 8% and 101 ± 4%, respectively, vs. normal adrenals, 100 ± 10%). In conclusion, the prevalence of heterozygous germline mutations in the CYP21B gene was higher in patients with adrenocortical tumors (11%; 95% confidence interval, 1-34%) than in the general European population (2%; 95% confidence interval, 1.93- 2.06%), but this difference is questionable because of the low number of subjects in our series. The pathophysiological significance of this finding in the presence of one normal CYP21B gene seems to be low, suggesting that 21-hydroxylase deficiency is not a major predisposing factor for adrenal tumor formation. (J Clin Endocrinol Metab 83: 2585-2588, 1998)
C ONGENITAL adrenal hyperplasia (CAH) caused by a deficiency of adrenal 21-hydroxylase (P450c21) is the most frequent inherited disorder of steroid metabolism (1, 2). The prevalence of this disorder in central Europe is about 1/7000 with an estimated population frequency of heterozy- gosity of about 1/50 (3). Molecular analysis of DNA from affected patients has demonstrated deletions and point mu- tations within the CYP21B gene impairing 21-hydroxylase function (1, 2).
The wide application of abdominal imaging procedures has led to the detection of a high number of adrenal masses (4). The majority of this so-called incidentalomas is benign and hormonally silent (5-7). Despite considerable exper- imental and clinical efforts, the tumorigenesis of adreno- cortical adenomas has not yet been elucidated in the ma-
Received December 19, 1997. Revision received April 3, 1998. Ac- cepted April 10, 1998.
Address all correspondence and requests for reprints to: M. Reincke Abt. Innere Medizin II, Klinikum der Universität, Hugstetter Strasse 55 79106 Freiburg, Germany. E-mail: reincke@mm21.ukl.uni-freiburg.de.
* This work was supported by the Wilhelm-Sander-Stiftung, München.
jority of cases. Recently, Jaresch et al. (8) detected clinically silent macronodular adrenal hyperplasia and adrenocor- tical adenomas in 80% of patients with homozygous con- genital adrenal hyperplasia using abdominal computed tomography. These authors also showed that 45% of het- erozygous carriers in families with CAH had uni- or bi- lateral adrenal nodules on computed tomography scans. In addition, several cases of functional adrenocortical tumors have been described in CAH patients (for review, see Ref. 9). An exaggerated response of 17-hydroxyprogesterone after ACTH stimulation was found in 30-70% of patients with incidentally detected adrenal tumors (10-13), further supporting the concept that 21-hydroxylase deficiency (21- OHD) could be a predisposing factor for adrenocortical tumor formation. However, this concept is not undis- puted, because hypersecretion of multiple precursors of the glucocorticoid and mineralocorticoid pathway after ACTH/CRH stimulation have been documented in pa- tients with adrenal adenomas (14, 15). Moreover, in vitro analysis of steroidogenic enzyme expression in adrenal tumors indicates a complex pattern of disorganized ex- pression of these enzymes, arguing against a specific en-
zyme defect in the tumor tissues (16). To clarify this issue, we investigated the prevalence of germline and somatic CYP21B mutations, as well as the relative messenger RNA (mRNA) expression of P450c21 in a variety of functional and nonfunctional adrenocortical tumors.
Patients and Methods
Patients
Nineteen patients with adrenocortical tumors were studied: two pa- tients had nonfunctional, incidentally detected adenomas; seven had cortisol-producing adenomas; six had aldosterone-producing adeno- mas; and four had adrenocortical carcinomas producing cortisol (n = 2) or androgens (n = 2). The clinical and pathological diagnosis was made according to established criteria (17). All patients gave written informed consent, and the study protocol was approved by the ethics committee of the University of Würzburg.
Tissues
Normal adult adrenals were obtained after organs were removed from brain-dead patients for transplantation. Tumor tissue was collected at adrenalectomy, snap-frozen in liquid nitrogen, and immediately stored at -80 C until analyzed.
Mutational analysis of CYP21B
Genomic DNA was extracted from EDTA blood and tumor tissue of all patients using the Blood & Cell Culture DNA Kit (Qiagen, Hilden, Germany). Four fragments (I-IV) were specifically amplified by PCR using oligonucleotides CYP5, 5’-AGCTATAAGTGGCACCTCAGG, nu- cleotide position 1638 and CYP6, 5’-AGCAGGGAGTAGTCTCCCAAG, position 2400 for fragment I (763 bp); CYP9, 5’-TCCTTGGGAGAC- TACTCCCTG, position 2378 and CYP10, 5’-TGCTCAGAGCTGAGT- GAGGGT, position 3535 for fragment II (1158 bp); CYP11, 5’-CTTGG- GAGACTACTCCCTGCT, position 2380 and CYP12, 5’-GTTCGT- ACGGGAGCAATAAAG, position 4444 for fragment III (2065 bp); and CYP-Pro1,5’-GCAGGGACTGCCATTTTCTCT, position 1289 and CYP6 for fragment IV (1112 bp). Base numbering is identical to that reported in White et al. (18). PCR reactions were performed as described in Schulze et al. (19) and Day et al. (20). The PCR fragments I-IV were used for direct sequencing of all exons, the exon/intron junctions, intron 2, intron 7, and 380 bp of the promoter region of the CYP21B gene. Se- quencing reactions were performed with the Thermo SequenAse cycle sequencing kit (Amersham, Braunschweig, Germany) with IRD41-la- beled oligonucleotides (MWG-Biotech, Ebersberg, Germany) on a Licor 4000L automatic DNA sequencer (Licor, Lincoln, NE). Large deletions of the CYP21 gene locus on chromosome 6 and deletions leading to loss of a 8-nucleotide sequence in exon 3 of CYP21B gene were screened and quantified by polyacrylamide electrophoresis of IRD41-labeled unspe- cific PCR products of exon 3 according to Rumsby et al. (21) using the oligonucleotides CYP19, 5’-ACAAGCTGGTGTCTAAGAAC, position 2346 and CYP20, 5’-TCACAGAACTCCTGGGTCAG, position 2480 as PCR primers.
Dot blot
Poly(A)-RNA was isolated from tumor tissue using the Oligotex Direct mRNA Kit (Qiagen). For dot blotting, 1.5 µg mRNA was dried, redissolved in 10 µL blotting buffer containing formamid and formal- dehyde as denaturing agents, directly transferred to a nylon membrane by vacuum, and immobilized by ultraviolet cross-linking. Plasmids con- taining side- chain cleavage enzyme (P450scc), 17-a-hydroxylase (P450c17), and P450c21 complementary DNAs (kindly provided by Dr. W.L. Miller, University of California, San Francisco) and a mouse ß-actin probe (Stratagene, Heidelberg, Germany), respectively, were digested with EcoRI and separated by agarose gel electrophoresis. After labeling the probes with [a32P]deoxycytidine triphosphate (Random Primed La- beling Kit, Boehringer, Mannheim, Germany), consecutive hybridiza- tion steps followed by blot stripping were performed. The blot was washed under high-stringency conditions and exposed for autoradiog- raphy at -80 C with intensifying screens. For normalization the blot was
stripped again and rehybridized with the ß-actin probe. Resulting dots were quantified by scanning densitometry (IMAGE program, National Institutes of Health, Bethesda, MD). The steady state mRNA concen- trations are expressed as % ± SEM of normal adrenals (100%).
Results
CYP21B mutations
Genomic DNA from leukocytes and adrenal tumors was used for specific amplification of the CYP21B gene. A het- erozygous missense mutation (Val281Leu) in exon 7 was found in the genomic DNA of one patient with a cortisol- secreting adenoma and in one androgen-producing carci- noma (Table 1). In these two patients, identical mutations were detected in the tumor tissue. The Val281Leu mutation
| Tissue | n | DNA leukocytes | DNA tumor |
|---|---|---|---|
| Nonfunctional adenomas | 2 | No mutations | No mutations |
| Cortisol-producing adenoma | 7 | 1 hetero | 1 hetero |
| Val281Leu | Val281Leu | ||
| Aldosterone-producing adenoma | 6 | No mutations | 1 hetero |
| Del (Exon 3) | |||
| Adrenal carcinoma | |||
| Cortisol producing | 2 | No mutations | No mutations |
| Androgen producing | 2 | 1 hetero | 1 hetero |
| Val281Leu | Val281Leu |
A
APA
CPA
NFA
APC
CPA
APA
P450c21
B-actin
B
21-hydroxylase mRNA
mRNA expression (% of normal adrenal)
120
OOOH
100
8
0
₹
80
0
0
O
60
40
20
0
0
0
NFA (n=2)
CPA (n=7)
APA (n=6)
CPC (n=2)
APC (n=2)
| Tissue | n | mRNA | ||
|---|---|---|---|---|
| P450scc | P450c17 | P450c21 | ||
| Nonfunctional adenomas | 2 | 51%, 53% | 0%, 5% | 19%, 2% |
| Cortisol-producing adenoma | 7 | 96 ± 9% | 144 ± 19% | 101 ± 4% |
| Aldosterone-producing adenoma | 6 | 83 ± 4% | 108 ± 14% | 84 ± 8% |
| Cortisol-producing carcinoma | 2 | 50%, 82% | 58%, 66% | 102%, 71% |
| Androgen-producing carcinoma | 2 | 74%, 82% | 42%, 80% | 3%, 1% |
is a relatively mild mutation of the CYP21B gene, and ho- mozygous patients have about 50% of wild-type enzymatic activity for conversion of 17-hydroxyprogesterone to 11-de- oxycortisol (22, 23). In one patient with an aldosterone-pro- ducing adenoma, a tumor-specific deletion of the 8-nucleo- tide sequence in exon 3, which is specific for the CYP21B gene, was detected. In the other samples, only wild-type CYP21B sequences were found.
P450c21 expression
Dot blots of tumor mRNA after hybridization with a P450c21 probe are shown in Fig. 1a. The results after quan- titation of the P450c21 mRNA expression are summarized in Fig. 1b. Cortisol-producing adenomas, cortisol-producing carcinomas, and aldosterone-producing adenomas had sim- ilar P450c21 mRNA steady state levels compared with nor- mal adrenals (n = 4; 100 ± 10%). In contrast, nonfunctional adenomas had very low steady state mRNA expression of 21-hydroxylase.
The expression of P450scc and P450c17 mRNA was similar to that of P450c21 in cortisol and aldosterone-producing tu- mors (Table 2). Nonfunctional adenomas expressed P450scc but did not express P450c17 mRNA. Androgen-secreting carcinomas expressed P450c17 mRNA, which is required for androgen synthesis, but did not express P450c21 mRNA required for glucocorticoid synthesis.
Discussion
Classical CAH has been shown to be associated with the formation of symptomatic and asymptomatic adrenocortical tumors (9). It is assumed, that chronic ACTH excess in CAH patients results in diffuse or nodular adrenocortical hyper- plasia, which later may become autonomous because of on- cogenic mutations in the tissue.
The concept that 21-OHD may be involved in adrenal tumorigenesis was renewed by the recent finding of exag- gerated responses of 17-hydroxyprogesterone after ACTH 1-24 stimulation in patients with adrenal incidentalomas (10-13). This response is characteristic for heterozygous car- riers or for homozygous late-onset 21-OHD (24). However, the interpretation of ACTH stimulation tests in these patients has been controversial (10, 15, 25).
Molecular analysis of the gene locus of CYP21B revealed that 21-OHD is caused by homozygous or compound-het- erozygous mutations within the CYP21B gene (1, 2, 26). The majority of mutations that cause 21-OHD appear to result from recombinations between the CYP21B gene and its closely homologous pseudogene CYP21A. These are either deletions caused by unequal crossing over during meiosis or apparent transfers of deleterious sequences from CYP21A to
CYP21B. In this study we analyzed whether mutations in the CYP21B gene are a predisposing factor for adrenal tumori- genesis. We detected two heterozygous germline mutations in 19 patients (prevalence: 11%, 95% confidence interval 1-34%), one in a patient with a cortisol-producing adenoma and the other in a patient with an androgen-secreting car- cinoma. The prevalence of heterozygous CYP21B mutations in our patients was, therefore, higher than the prevalence of carriers of 21-OHD in the general European population (2%; 95% confidence interval 1.93-2.06%) (3). The significance of this difference, however, seems to be questionable because of the small cohort size resulting in an substantial overlap of the confidence intervals. In addition, the majority of patients, including two incidentaloma patients, did not have CYP21B mutations, suggesting that CAH is not a major predisposing factor for adrenal tumorigenesis. Also, we did not find ev- idence for a correlation between the clinical phenotype of the patients and the mutational spectrum of CYP21B, because the Val281Leu mutation was found in a virilized patient with an androgen-secreting carcinoma as well as in a patient with a cortisol-producing adenoma causing Cushing’s syndrome.
In one patient with an aldosterone-producing adenoma a heterozygous somatic mutation (deletion of 8 bp in exon 3) was detected. Evidently, the phenotype of mineralocorticoid hypertension in this patient cannot be explained by the CYP21B mutation. Because we did not detect somatic mu- tations in tumors most likely to harbor defects in 21-hydrox- ylase activity (nonfunctional or virilizing tumors), our data argue against the hypothesis that somatic mutations within the CYP21B gene are responsible for the clinical and bio- chemical phenotype of adrenocortical tumors or are per se oncogenic.
Dot blot analysis of P450c21 mRNA showed that P450c21 expression correlated well with the clinical and biochemical phenotype of the patients (Fig. 1, a and b and Table 2). Nonfunctional adenomas and androgen-secreting carcino- mas did not express P450c21 mRNA in significant amounts. Glucocorticoid and mineralocorticoid-producing tumors had P450c21 levels similar to tissue from adrenals of brain- dead patients. Similar data could be observed with respect to P450scc and P450c17 mRNA expression in the present tumor series as well as by others (27, 28), with upregulation in cortisol- and aldosterone-producing adenomas and down- regulation in nonfunctional adenomas indicating a complex regulatory process in these tumor tissues. However, the mechanisms responsible for P450 enzyme expression re- mains to be elucidated.
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