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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 9 3626-3632
Copyright © 2006 by The Endocrine Society

17q22–24 Chromosomal Losses and Alterations of Protein Kinase A Subunit Expression and Activity in Adrenocorticotropin-Independent Macronodular Adrenal Hyperplasia

Isabelle Bourdeau, Ludmila Matyakhina, Sotirios G. Stergiopoulos, Fabiano Sandrini, Sosipatros Boikos and Constantine A. Stratakis

Division of Endocrinology, Department of Medicine (I.B.), Hôtel-Dieu du Centre Hospitalier de l’Université de Montréal, Montréal, Canada QC H2W 1T8; and Section on Endocrinology and Genetics (I.B., L.M., S.G.S., F.S., S.B., C.A.S.), Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1862

Address all correspondence and requests for reprints to: Constantine A. Stratakis, M.D., D.Sc., Chief, Section on Endocrinology and Genetics, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Room 1-3330, 10 Center Drive, MSC-1103, Bethesda, Maryland 20892. E-mail: stratakc{at}mail.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Primary adrenocortical hyperplasias leading to Cushing syndrome include primary pigmented nodular adrenocortical disease and ACTH-independent macronodular adrenal hyperplasia (AIMAH). Inactivating mutations of the 17q22–24-located PRKAR1A gene, coding for the type 1A regulatory subunit of protein kinase A (PKA), cause primary pigmented nodular adrenocortical disease and the multiple endocrine neoplasia syndrome Carney complex. PRKAR1A mutations and 17q22–24 chromosomal losses have been found in sporadic adrenal tumors and are associated with aberrant PKA signaling.

Objective: The objective of the study was to examine whether somatic 17q22–24 changes, PRKAR1A mutations, and/or PKA abnormalities are present in AIMAH.

Patients: We studied fourteen patients with Cushing syndrome due to AIMAH.

Methods: Fluorescent in situ hybridization with a PRKAR1A-specific probe was used for investigating chromosome 17 allelic losses. The PRKAR1A gene was sequenced in all samples, and tissue was studied for PKA activity, cAMP responsiveness, and PKA subunit expression.

Results: We found 17q22–24 allelic losses in 73% of the samples. There were no PRKAR1A-coding sequence mutations. The RIIß PKA subunit was overexpressed by mRNA, whereas the RI{alpha}, RIß, RII{alpha}, and C{alpha} PKA subunits were underexpressed. These findings were confirmed by immunohistochemistry. Total PKA activity and free PKA activity were higher in AIMAH than normal adrenal glands, consistent with the up-regulation of the RIIß PKA subunit.

Conclusions: PRKAR1A mutations are not found in AIMAH. Somatic losses of the 17q22–24 region and PKA subunit and enzymatic activity changes show that PKA signaling is altered in AIMAH in a way that is similar to that of other adrenal tumors with 17q losses or PRKAR1A mutations.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
ADRENOCORTICAL TUMORS, adenomas, carcinomas, or bilateral hyperplasias may produce cortisol and lead to ACTH-independent Cushing syndrome (CS). Although a number of genetic abnormalities have been detected in adrenocortical adenomas and carcinomas (1, 2), the genetic background of primary adrenal hyperplasias has been less well studied (3). ACTH-independent adrenal hyperplasias include primary pigmented nodular adrenocortical disease (PPNAD) and ACTH-independent macronodular adrenal hyperplasia (AIMAH) (3, 4). PPNAD may be associated with Carney complex (CNC) (5), which may be caused by inactivating mutations of PRKAR1A (CNC1) localized on the 17q22–24 region. PRKAR1A codes for the type 1A regulatory subunit of protein kinase A (PKA) (6, 7, 8). In CNC tumors, mutations in PRKAR1A (CNC1) lead to an increase of the cAMP-stimulated kinase activity (6). More recently, loss of heterozygosity of the 17q22–24 region has also been shown to occur in sporadic adrenocortical tumors, and somatic PRKAR1A mutations were found in sporadic adrenocortical adenomas (9). In addition, loss of heterozygosity of 2p16, the chromosomal region in which the other CNC locus (CNC2) has been mapped (10), has also been reported in adrenocortical tumors (1).

In the present study, we investigated the hypothesis that the PKA signaling pathway is altered in AIMAH. There is evidence that genes implicated in cyclic nucleotide-dependent signaling are involved in AIMAH tumorigenesis (11). In McCune-Albright syndrome, somatic {alpha}-subunit of the stimulatory G protein (GNAS) mutations induces constitutive cAMP production (12) that may lead to endocrine tumors including ACTH-independent bilateral nodular adrenal hyperplasia and CS (13, 14). Somatic activating mutations of GNAS gene have also been described in three adults with isolated AIMAH (15). In addition, in patients with AIMAH, the regulation of cortisol secretion is frequently mediated by aberrant expression of G protein-coupled receptors that involve the cAMP pathway (16, 17). For example, in one case of food-dependent CS, in vitro studies showed that gastric inhibitory polypeptide stimulated cAMP production, which implicated increased cAMP signaling in tumor cells (18).

In this study, we performed molecular cytogenetic analysis of adrenal samples of AIMAH to evaluate whether the 2p16 and 17q22–24 chromosomal regions may be altered as in PPNAD. We screened the PRKAR1A gene for mutations in AIMAH. Subsequently we examined the expression of the various PKA subunits using quantitative methods and immunohistochemistry; we also measured PKA activity in AIMAH tissues and control samples.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients and AIMAH samples

The institutional review boards of the participating institutions approved the collection of the adrenal samples from the patients for study, under National Institute of Child Health and Human Development, National Institutes of Health protocol 00CH160. Tissue samples from 14 patients with sporadic AIMAH were available for analysis. All patients were diagnosed with ACTH-independent CS by standard diagnostic testing. An in vivo aberrant cortisol secretion by membrane hormone receptors (17, 19) was identified in nine patients as described previously (Table 1Go). Adrenal tissues were obtained during surgery and were dissected; a part was frozen at –70 C until further use for genetic studies, and sections were fixed and paraffin-embedded for diagnostic and immunohistochemistry studies.


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  		TABLE 1. Description of clinical data and studies performed in patients with AIMAH

 
Fluorescent in situ hybridization (FISH) analysis for 17q allelic losses

Eleven adrenal AIMAH, three ACTH-dependent adrenal hyperplasia, and six normal adrenal gland samples were available for FISH analysis. Touch preparations of the tumors were made from frozen tumors. After fixation in methanol-acetic acid (3:1) for 20 min, they were air dried and equilibrated in 2x saline sodium citrate [0.3 mM NaCl, 30 mM sodium citrate (pH 7.0)] solution, followed by dehydration in ethanol series of 70, 80, 90, and 100%.

The probes used were bacterial artificial chromosomes (BACs) 400-P-14 and 514–0-11 from the 2p16 region (D2S2251-D2S2292) and CITB BAC 321-G-8 (7) containing PRKAR1A gene. All BACs were obtained from a commercially available library (Research Genetics). Control probes from other chromosomes were also used.

The BACs were grown and DNAs extracted as described elsewhere (20). DNAs were labeled by digoxigenin-11-deoxyuridine 5-triphosphate or biotin-11-deoxyuridine 5-triphosphate (Roche Molecular Biochemicals, Mannheim, Germany) by nick translation and hybridized to touch preparations of the tumors as previously described (21). After hybridization, cells were counterstained with 4',6'-diamidino-2-phenylindol-dihydrochloride. Hybridization signals were scored with the use of an epifluorescence microscope (Leica, Wetzlar, Germany), and fluorescence images were automatically captured on a cooled-charge-coupled device camera (Photometrics, Ltd., Tucson, AZ) using IP Lab Image software (Scanalytics, Inc., Fairfax, VA). At least 100 nonoverlapping cells with strong hybridization signals were scored per case. Presence of more than 20% cells with only one BAC signal was interpreted as an allelic loss. Normal adrenal tissues were used as control and showed 8 and 10% of cells with one signal of BACs 400-p-14 and 321-G-8, respectively. Control probes from other chromosomes were also hybridized to tumor cells and showed two expected signals in more than 90% of the cells. Chromosome 2- and 17-specific centromeric {alpha}-satellite probes (Vysis Inc., Downers Grove, IL) were used for the control of chromosomes 2 and 17 copy numbers in the tumor samples.

PRKAR1A mutation-screening; denaturing HPLC (DHPLC) analysis and sequencing

DNA was extracted from 14 AIMAH fresh-frozen tissues using standard proteinase K and phenol-chloroform methods. PCR was used to amplify exons 2–10 of the PRKAR1A gene, as previously described (6). Amplicons were screened using DHPLC instrument (HELIX; Varian, Inc., Woburn, MA) at column temperatures recommended by the DHPLC Melt program (http://insertion.stanford.edu/melt.html), as described elsewhere (22).

Heterozygote samples were sequenced using the BigDye Terminator kit (PerkinElmer, Norwalk, CT). The sequence traces were analyzed using Sequencher (Genecodes, Ann Arbor, MI).

Real-time PCR quantification of mRNA

Total RNA was extracted from 10 AIMAH frozen tissues using TRIZol reagent (Invitrogen, Carlsbad, CA) and further purified using RNeasy maxikits (QIAGEN, Inc., Valencia, CA) as described previously (23). The reference normal sample against which Taqman data were expressed was derived from total RNA extracted from normal adrenal glands of 62 Caucasian subjects (aged 15–61 yr) that is available commercially (CLONTECH Laboratories, Palo Alto, CA). We have demonstrated recently that this sample of pooled total RNA contains only a minimal amount of medulla (24).

Real-time (RT) PCR was performed using the RT-PCR system (PE Applied Biosystems, Foster City, CA). All reactions were performed according to the manufacturer’s recommendations. Sequences used for the PRKAR1A, PRKAR1B, PRKAR2A, PRKAR2B, and PRKACA probes were obtained from the genome databases. A melting curve and the cycle at detection were analyzed with the software of the apparatus. Data were expressed as CT values (the cycle number at which logarithmic PCR plots cross a calculated threshold line) and used to determine {Delta}CT values ({Delta}CT = CT of the normal adrenal glands minus CT of the targeted PKA subunit gene in AIMAH samples); positive {Delta}CT values represent overexpression and negative {Delta}CT low levels of respective PKA subunits expression, compared with the normal adrenal glands.

Statistical analysis was performed on mean {Delta}CT values of each subunit; all data are expressed as means ± SEM. Statistical analysis of comparisons between groups was undertaken using an all pairwise multiple comparison procedures (Tukey test) with the SigmaStat software package (Systat Software, Inc., Point Richmond, CA). The statistical analysis refers to the comparison among groups of the five subunits.

Immunohistochemistry

Sections from paraffin-embedded AIMAH samples were available for four patients. Sections were hybridized with monoclonal antibodies specific for RI{alpha}, and the other main PKA subunits (RII{alpha}, RIIß, and C{alpha}) (EMD Biosciences, San Diego, CA) as described previously (21, 25). Two blinded readers graded the specimens for staining and compared them with a normal adrenal gland. Staining was assigned one of four grades: 0 for nonstaining, 1 for weak staining, 2 for moderate staining, and 3 for strong staining.

PKA activity

PKA activity was measured in cell extracts from six AIMAH samples and three normal adrenal glands, as described previously (26). Kinase activity was measured, as described previously (6), in counts per minute (cpm) using {gamma}-32P-dATP (deoxyadenosine 5'-[{gamma}-32P]triphosphate, Amersham Pharmacia Biotech, Piscataway, NJ), in cell extracts from frozen tissues. PKA activity assays were performed twice for each sample. An average value was calculated for each experiment after correction for protein content. Total kinase activity represents enzymatic activity after stimulation with cAMP, whereas total PKA-specific activity is the difference between stimulated PKA activity with cAMP before and after the addition of protein kinase inhibitor (PKI) (6). Free PKA activity, which represents basal activity without stimulation of the cAMP, was calculated also as previously described (6). Both total and free PKA activities were expressed in units per milligram of protein.

Data from all samples were compared with the SigmaStat software package (Systat Software, Inc., Point Richmond, CA) using the t test for individual comparisons between the AIMAH samples and normal adrenal gland tissues. P < 0.05 was considered to indicate significance.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
FISH analysis: 2p16 and 17q allelic losses

Table 2Go summarizes results of FISH analysis on 20 human adrenal samples: 11 AIMAH tissues, three ACTH-dependent adrenal hyperplasia cases, and six normal adrenal glands. A dual-color FISH with the BACs 400-P-14 and 514–0-11 showed the allelic loss of the 2p16 region in 10 of 11 AIMAH (91%) in almost 100% of the cells. There were 2p16 deletions in less than 50% of the cells in three of three ACTH-dependent hyperplasia samples. There were no 2p16 deletions in six normal adrenal gland samples. A representative microphotograph illustrating loss of 2p16 is shown in Fig. 1AGo. Four AIMAH sample and one ACTH-dependent adrenal hyperplasia sample were studied by a dual-color FISH with a whole chromosome 2 painting probe and the BAC400-P-14. The results showed two copies of chromosome 2 in cells from the sample of patient 1, whereas only one copy of the chromosome was detected in cells from the sample of patient 2. Samples from patients 6 and 12 and a specimen from a patient with ACTH-dependent adrenal hyperplasia demonstrated that only part of the cells with deletions of the 2p16 region showed loss of chromosome 2 (Fig. 1BGo).


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  		TABLE 2. Summary of the results of the FISH study

 

Figure 1
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  		FIG. 1. Interphase FISH with BACs from 2p16 (A and B) and 17q22–24 (C) regions in AIMAH tissues. A, Dual-color FISH with BACs 400-P-14 (red) and 514-O-11 (green) from 2p16 region shows allelic losses of both BACs in adrenal tumor cells from patient 4. B, Adrenocortical tumor cells from patient 6 after hybridization with BAC 400-P-14 and {alpha}-satellite probe specific for chromosome 2 show an allelic deletion of BAC400-p-14 (one red signal). The presence of one green signal in two of three cells suggests that loss of large segments of chromosome 2 is also frequent. C, FISH with BAC321G-8 containing PRKARIA shows an allelic loss of 17q22–24 region in adrenal tumor cells of patient 2.

 
FISH analysis with the BAC 321-G-8 revealed allelic deletions of the 17q22–24 PRKAR1A-containing region in more than 90% of the cells from eight of 11 AIMAH samples (73%) (Fig. 1CGo). However, allelic deletions of the 17q22–24 region were found in less than 50% of cells in three of three ACTH-dependent adrenal hyperplasia samples. No significant 17q22–24 deletions were observed in six normal adrenal glands. Figure 1CGo shows an illustration of 17q22–24 allelic losses in AIMAH tissue. Five AIMAH samples and one ACTH-dependent hyperplasia sample were studied by a dual-color FISH with the BAC 321-G-8 and a chromosome 17-specific centromeric {alpha}-satellite probe or the BAC 426-B-2 that mapped to 17p (Table 2Go).

PRKAR1A gene screening

None of the 14 AIMAH samples analyzed showed PRKAR1A gene mutations. In one sample (patient 5), we found a single nucleotide polymorphism that has been previously reported in CNC patients and normal controls (7): it is located in intron 8 of the PRKAR1A gene (IVS-27G->A).

RT-PCR quantification of PKA mRNA

Results of RT-PCR on nine AIMAH samples are shown in Fig. 2AGo. Quantitative analysis demonstrated underexpression of the mRNAs of four of the main PKA subunits, compared with normal adrenal glands (RI{alpha}: {Delta}CT = –0.281 ± 0.3, RIß: {Delta}CT = –0.853 ± 0.3, RII{alpha}: {Delta}CT = –0.64 ± 0.3 and C{alpha}: {Delta}CT = –0.192 ± 0.2). The RIIß subunit (PRKAR2B) with a {Delta}CT = 0.924 ± 0.3 was overexpressed in eight of nine AIMAH samples studied, compared with normal adrenal glands.


Figure 2
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  		FIG. 2. A, Quantitative determination of the mRNA for the main PKA subunits by RT-PCR. This figure shows expression of the five PKA subunits (PRKAR1A, PRKAR1B, PRKAR2A, PRKAR2B, and PRKARCA) in AIMAH samples (n = 9), compared with normal adrenal glands (NA) (n = a pool of 62 normal adrenal glands). {Delta}CT values ({Delta}CT = CT of the normal adrenal glands minus CT of the target PKA subunit gene in AIMAH samples are provided on the y-axis. *, P ≤ 0.05. Statistical comparison among the five subunits using an all pairwise multiple comparison procedures (Tukey test) showed statistical difference expression among RIß, RII{alpha}, and RIIß groups. B, Total PKA activity (calculated as the difference between cAMP-stimulated PKA and PKI-inhibited PKA activities) and free PKA activity were higher in AIMAH (n = 6) than normal adrenal glands. Both total and free PKA activity are expressed in units per milligram of protein.

 
Comparison among groups of the five subunits using an all pairwise multiple comparison procedures (Tukey test) showed statistical difference among RIß, RII{alpha}, and RIIß groups as shown in Fig. 2AGo. There was overexpression of RIIß, compared with RIß (P = 0.002) and RII{alpha} (P = 0.007), and a trend for its overexpression, compared with RI{alpha} (P = 0.056) and C{alpha} (P = 0.09).

PKA activity

We measured PKA activity in six AIMAH tissues and three normal adrenal gland tissues. We compared the results of the normal adrenal glands with the six AIMAH samples. After exposure to cAMP, AIMAH samples had higher total kinase activity (10,794 ± 870 cpm/mg protein), compared with the normal adrenal glands 5,378 ± 2,277 cpm/mg protein (P = 0.028); all samples were done in duplicate, and the whole experiment was repeated for each sample at least twice. The activity was PKA specific because after inhibition by PKI, activity was 62,368 ± 4,150 cpm/mg protein for the AIMAH samples and 80,561 ± 37,416 cpm/mg for the normal adrenal samples, a difference that was not significant (P = 0.49), as expected. Accordingly, total PKA activity (10,605 ± 706 U/mg protein) was higher in AIMAH (n = 6) than normal adrenal glands (n = 3) (6 ± 3 U/mg protein; P < 0.001). Free PKA enzymatic activity (1835 ± 148 U/mg protein) was also higher in AIMAH (n = 6) than normal adrenal glands (n = 3) (0.4 ± 0.17 U/mg protein; P < 0.001).

Immunohistochemistry for PKA subunits

Sections were incubated with monoclonal antibodies specific for RI{alpha} and the other main PKA subunits (RII{alpha}, RIIß, and C{alpha}) (EMD Biosciences) as described previously (9). Staining for PRKAR1A (RI{alpha}) and the other main PKA subunits (RII{alpha}, RIIß, and C{alpha}) was compared with a normal adrenal gland. Overall, RI{alpha} staining in the AIMAH samples was decreased, compared with staining of the zona fasciculata/reticularis of normal adrenal gland in four of the four samples. RIIß staining was increased in two of four samples and RII{alpha} in all AIMAH samples. No significant changes occurred in the staining for C{alpha} (Table 3Go and Fig. 3Go).


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  		TABLE 3. Summary of the immunohistochemistry results of four AIMAH samples, compared with staining of the zona fasciculata/reticularis of a normal adrenal gland

 

Figure 3
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  		FIG. 3. Immunostaining of three human adrenal samples with monoclonal antibodies specific for total PKA, RI{alpha}, and the other main PKA subunits (RII{alpha}, RIIß, and C{alpha}). A, AIMAH (patient 5) showing increased staining for RIIß, compared with the other subunits. B, Staining is compared with the zona fasciculata/reticularis of normal adrenal gland. C, Tissue from PPNAD caused by a known PRKAR1A mutation.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The pathogenesis of AIMAH has not been extensively investigated (11). Although ectopic and abnormal expression of aberrant receptors regulates cortisol steroidogenesis in AIMAH, their role in the development of tumors remains to be elucidated (16). Several receptors found as aberrantly expressed are G protein-coupled-receptors and influence the cAMP signaling pathway. The molecular mechanisms responsible for the ectopic or abnormal expression and function of membrane receptors in adrenal CS have not been identified yet. In the present study, we investigated the hypothesis that the PKA system is altered in AIMAH, as is the case in PPNAD. We also investigated the possibility that the second PPNAD locus, 2p16 or CNC2 (10), is involved in the molecular pathogenesis of AIMAH.

The present study showed that 17q22–24 losses occur in 73% of AIMAH samples, but they are not associated with PRKAR1A mutations. Although the possibility that another gene at 17q22–24, not PRKAR1A, may be involved in AIMAH has not been excluded, loss of one allele of this gene leads to adrenocortical abnormalities as shown in both human (9) and mouse adrenocortical tissue (27). As in occasional cases of PPNAD and other adrenal tumors, in four of our specimens with AIMAH, there was more extensive loss of chromosome 17, indicating that additional genes on that chromosome participate in adrenocortical tumorigenesis.

Cytogenetic changes of the 2p16 chromosomal region that harbors the CNC2 locus were also frequently present in AIMAH tissues. The frequency is greater than in CNC tumors in which 2p16 deletions were demonstrated in only 32% of cases (28). In our previous study, 2p16 chromosomal region amplification was detected in 60% of the CNC tumors (28); however, in the AIMAH tissues, we did not find 2p16 amplification. Another study reported that the loss of genetic material of 2p16 was associated with adrenocortical carcinomas but not adenomas (29). Comparative genomic hybridization studies have also implicated chromosome 2p in adrenocortical tumorigenesis (1). Our present results support the notion that 2p16 is involved in the molecular pathogenesis of benign adrenocortical hyperplasias.

We found 2p16 and 17q22–24 deletions in both AIMAH and ACTH-dependent adrenal hyperplasia but not in normal adrenal glands. Whether these cytogenetic changes are a primary cause or secondary events in adrenocortical hyperplasias remains to be determined. These deletions may be secondary to various events in adrenocortical cells such as activation of PKA, ACTH stimulation, aberrantly expressed receptors, or other mechanisms yet to be identified.

PKA is an heterotetramer consisting of two regulatory (encoded by four genes: R1A, R1B, R2A, R2B) and two catalytic subunits. PRKAR1A encodes the regulatory subunit 1{alpha} (RI{alpha}) of PKA, which mediates cAMP signaling (30). Although no PRKAR1A mutations were found in our AIMAH cohort, decreased expression of the PRKARIA mRNA, compared with normal adrenal glands, was observed in two thirds of the AIMAH tissues. In addition, decreased expression of the protein was observed in the AIMAH samples. These observations may indicate a pathophysiological process that has similarities with that of PPNAD. The associated increase in expression of the RIIß subunit mRNA in 89% of the AIMAH samples also supports a role of aberrant PKA signaling in AIMAH. The RIIß subunit of the PKA tetramer has been found overexpressed in all mouse models of PRKAR1A down-regulation described to date (27, 31, 32) and is highly expressed in human PPNAD tissue (27), whereas it is not strongly expressed in normal adrenocortical tissue. Furthermore, Prkar2b–/– mouse demonstrates compensatory increases of RI{alpha} protein expression and altered cAMP-dependent total PKA activity (33). Thus, RIIß subunit overexpression may be associated with changes in cAMP-dependent total PKA activity: our findings indeed suggested that PKA activity is greater in AIMAH than normal adrenal glands (Fig. 2BGo).

In conclusion, this study demonstrated that inactivating mutations of PRKAR1A did not appear to be frequent in AIMAH. However, allelic losses of 17q22–24 (PRKAR1A-CNC1) and 2p16 (CNC2) were common in AIMAH. These chromosomal abnormalities were associated with altered PKA activity and PKA subunit expression. The RIIß PKA subunit in particular was overexpressed in AIMAH, as it was in PPNAD and mouse models of PRKAR1A down-regulation.


   Acknowledgments
 
The authors thank the physicians who referred the patients and the nurses for conducting the endocrine tests. We thank Dr. André Lacroix, Center Hospitalier de l’Université de Montréal (Montréal, Québec, Canada) for providing a number of AIMAH samples.


   Footnotes
 
This work was supported in part by intramural program Grant Z01 HD000642-04, National Institute of Child Health and Human Development, National Institutes of Health (principal investigator: Dr. C. A. Stratakis). I.B. was supported by a fellowship grant from the Fonds de la Recherche en Santé du Québec and intramural National Institutes of Health (NIH) funds.

First Published Online June 13, 2006

Abbreviations: AIMAH, ACTH-independent macronodular adrenal hyperplasia; BAC, bacterial artificial chromosome; CNC, Carney complex; CS, Cushing syndrome; CT, cycle threshold; DHPLC, denaturing HPLC; FISH, fluorescent in situ hybridization; PKA, protein kinase A; PKI, protein kinase inhibitor; PPNAD, primary pigmented nodular adrenocortical disease; RT, real time.

Received December 1, 2005.

Accepted June 1, 2006.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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C Vincent-Dejean, L Cazabat, L Groussin, K Perlemoine, G Fumey, F Tissier, X Bertagna, and J Bertherat
Identification of a clinically homogenous subgroup of benign cortisol-secreting adrenocortical tumors characterized by alterations of the protein kinase A (PKA) subunits and high PKA activity.
Eur. J. Endocrinol., June 1, 2008; 158(6): 829 - 839.
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