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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
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{at}mm21.ukl.uni-freiburg.de
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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-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 aldosterone-producing adenoma.
The P450c21 gene expression correlated 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. |
Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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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, 6, 7). Despite considerable experimental and clinical efforts, the tumorigenesis of adrenocortical adenomas has not yet been elucidated in the majority of cases. Recently, Jaresch et al. (8) detected clinically silent macronodular adrenal hyperplasia and adrenocortical adenomas in 80% of patients with homozygous congenital adrenal hyperplasia using abdominal computed tomography. These authors also showed that 45% of heterozygous carriers in families with CAH had uni- or bilateral 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, 11, 12, 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 undisputed, because hypersecretion of multiple precursors of the glucocorticoid and mineralocorticoid pathway after ACTH/CRH stimulation have been documented in patients with adrenal adenomas (14, 15). Moreover, in vitro analysis of steroidogenic enzyme expression in adrenal tumors indicates a complex pattern of disorganized expression of these enzymes, arguing against a specific enzyme 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.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Nineteen patients with adrenocortical tumors were studied: two patients had nonfunctional, incidentally detected adenomas; seven had cortisol-producing adenomas; six had aldosterone-producing adenomas; 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, nucleotide position 1638 and CYP6, 5'-AGCAGGGAGTAGTCTCCCAAG, position 2400 for fragment I (763 bp); CYP9, 5'-TCCTTGGGAGACTACTCCCTG, position 2378 and CYP10, 5'-TGCTCAGAGCTGAGTGAGGGT, position 3535 for fragment II (1158 bp); CYP11, 5'-CTTGGGAGACTACTCCCTGCT, 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. Sequencing reactions were performed with the Thermo SequenAse cycle sequencing kit (Amersham, Braunschweig, Germany) with IRD41-labeled 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 unspecific 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
formaldehyde as denaturing agents, directly transferred to a nylon
membrane by vacuum, and immobilized by ultraviolet cross-linking.
Plasmids containing side- chain cleavage enzyme (P450scc),
17--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
[32P]deoxycytidine triphosphate (Random Primed
Labeling Kit, Boehringer, Mannheim, Germany), consecutive hybridization
steps followed by blot stripping were performed. The blot was washed
under high-stringency conditions and exposed for autoradiography 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
concentrations are expressed as % ± SEM of normal
adrenals (100%).
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Genomic DNA from leukocytes and adrenal tumors was used for
specific amplification of the CYP21B gene. A heterozygous 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
carcinoma (Table 1
). In these two
patients, identical mutations were detected in the tumor tissue. The
Val281Leu mutation is a relatively mild mutation of the CYP21B gene,
and homozygous patients have about 50% of wild-type enzymatic activity
for conversion of 17-hydroxyprogesterone to 11-deoxycortisol (22, 23).
In one patient with an aldosterone-producing adenoma, a tumor-specific
deletion of the 8-nucleotide sequence in exon 3, which is specific for
the CYP21B gene, was detected. In the other samples, only wild-type
CYP21B sequences were found.
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Dot blots of tumor mRNA after hybridization with a P450c21 probe
are shown in Fig. 1a. The results after
quantitation of the P450c21 mRNA expression are summarized in Fig. 1b.
Cortisol-producing adenomas, cortisol-producing carcinomas, and
aldosterone-producing adenomas had similar P450c21 mRNA steady state
levels compared with normal adrenals (n = 4; 100 ± 10%). In
contrast, nonfunctional adenomas had very low steady state mRNA
expression of 21-hydroxylase.
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). 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.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The concept that 21-OHD may be involved in adrenal tumorigenesis was renewed by the recent finding of exaggerated responses of 17-hydroxyprogesterone after ACTH 1–24 stimulation in patients with adrenal incidentalomas (10, 11, 12, 13). This response is characteristic for heterozygous carriers 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-heterozygous 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 tumorigenesis. 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 carcinoma. 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 evidence 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 mutations in tumors most likely to harbor defects in 21-hydroxylase activity (nonfunctional or virilizing tumors), our data argue against the hypothesis that somatic mutations within the CYP21B gene are responsible for the clinical and biochemical 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 carcinomas 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 downregulation in nonfunctional
adenomas indicating a complex regulatory process in these tumor
tissues. However, the mechanisms responsible for P450 enzyme expression
remains to be elucidated.
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Footnotes |
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Received December 19, 1997.
Revised April 3, 1998.
Accepted April 10, 1998.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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