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BRIEF REPORT |
Second Department of Medicine (J.M., A.P., K.B., M.T., P.G., A.S., A.M., K.R.), School of PhD Studies (G.B.), and Third Department of Medicine (P.P., Z.P.), Faculty of Medicine, Semmelweis University, H-1088 Budapest, Hungary
Address all correspondence and requests for reprints to: Károly Rácz, M.D., D.Sc., 2nd Department of Medicine, Semmelweis University, Szentkiralyi 46, H-1088 Budapest, Hungary. E-mail: racz{at}bel2.sote.hu.
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Abstract |
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Objective: The objective of the study was to examine whether N363S and ER22/23K variants of the GR gene may be associated with the development of adrenal incidentalomas and whether these variants may contribute to metabolic abnormalities frequently present in these patients.
Design, Setting, and Patients: The study included 99 patients with unilateral and 44 patients with bilateral adrenal incidentalomas, 102 population-matched control subjects, and 100 patients with type 2 diabetes mellitus.
Main Outcome Measures: Metabolic and hormonal parameters and GR gene variants were determined.
Results: When compared with control subjects, the carrier frequency for the N363S variant was markedly and significantly higher in patients with bilateral (7.8 vs. 20.5%, P < 0.05) but not in those with unilateral incidentalomas (7.1%) or in patients with type 2 diabetes (13.0%). Type 2 diabetes occurred more frequently in patients with bilateral, compared with those with unilateral incidentalomas (40.9 vs. 22.2%, P < 0.05). In patients with bilateral incidentalomas, a significant association of the N363S variant with impaired glucose homeostasis but not with body mass index, hypertension, hyperlipidemia, or history of coronary artery disease was found. The carrier frequency of the ER22/23EK variant was similar in all groups, and this variant failed to show any association with metabolic abnormalities.
Conclusion: These results suggest that the N363S variant of the GR gene may play a role in the pathogenesis of bilateral adrenal incidentalomas, although the mechanism still remains to be investigated.
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Introduction |
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It has been shown that 8.9–15% of all adrenal incidentalomas are bilateral (2, 5), and a few studies indicated an increased prevalence of mild hormonal abnormalities in patients with bilateral adrenal incidentalomas, compared with those with unilateral tumors (5, 6). The development of bilateral incidentalomas strongly supports the role of possible systemic pathogenetic effect(s), although the presence of systemic factors has been found in only a relatively few patients (7, 8). It seems also important that bilateral incidentalomas occur more frequently than expected from the coincidence of two unilateral incidentalomas. If incidentalomas in the left and right adrenals of a single patient develop independently with a given probability (q), the statistical probability of finding bilateral incidentalomas (q2) among all patients with incidentalomas [q(2-q)] predicts a low occurrence of bilateral tumors (0.09–1.11%), which is in contrast with their observed frequency of 8.9–15% among all incidentalomas.
Of the numerous single-nucleotide polymorphisms of the glucocorticoid receptor (GR) gene, the N363S sequence variant has been detected with an allele frequency of 2.3–8% in the general European population (9, 10, 11, 12, 13, 14) and has been associated with an increased sensitivity to glucocorticoids and with several metabolic abnormalities (9, 10, 11, 12, 13, 14, 15, 16, 17). Another rare polymorphism of the GR gene is the ER22/23EK variant, which was shown to occur with an allele frequency of 1.8–4.5% in the Caucasian population (13, 18) and was found to be associated with decreased glucocorticoid sensitivity and better metabolic profile (13, 16, 17, 18).
We performed the present study with the following two aims: 1) to examine whether the N363S and ER22/23EK variants of the GR gene may be associated with the development of unilateral and/or bilateral adrenal incidentalomas and 2) whether the presence of these gene variants may be linked to metabolic or hormonal abnormalities frequently found in patients with adrenal incidentalomas.
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Patients and Methods |
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The study included 143 patients with adrenal incidentalomas. Of the 143 patients, 99 had unilateral tumors [75 women and 24 men; mean age at diagnosis 53.8 yr, range 22–78 yr; body mass index (BMI) 29.7 kg/m2, range 17.2–43.7 kg/m2], and 44 had bilateral tumors (30 women and 14 men; mean age at diagnosis 56.9 yr, range 34–74 yr; BMI 30.8 kg/m2, range 19.8–46.1 kg/m2). In addition, 102 population-matched control subjects (65 women and 37 men; mean age 47.1 yr, range 28–79 yr; BMI 25.17 kg/m2, range 17.67–37.98 kg/m2), and 100 patients with type 2 diabetes mellitus (42 women and 58 men; mean age at diagnosis 60 yr, range 28–82 yr; BMI 29.25 kg/m2, range 19.4–43.5 kg/m2) were tested. Control subjects and patients with type 2 diabetes had no clinical signs of endocrine malfunction. The study was approved by the local Ethical Committee of Semmelweis University. Informed consent was obtained from all individuals who participated in the study.
Adrenal incidentalomas were discovered by abdominal ultrasonography or CT, performed for the evaluation of unrelated diseases. The tumors were documented by CT as homogeneous adrenal lesions with densities of 10 Hounsfield units or less on thin-section unenhanced scans. All patients with adrenal incidentalomas underwent a detailed clinical and hormonal evaluation that excluded Cushing’s syndrome, primary aldosteronism, pheochromocytoma, and hyperandrogenism.
Clinical assessment
On physical examination none of the patients or control subjects showed signs of an overt endocrine dysfunction. Blood pressure was measured three times using a standardized automated sphygmomanometer. Plasma glucose, cholesterol, triglyceride, and other standard blood analytes were determined by routine methods.
In patients with adrenal incidentalomas, blood samples were collected for hormone measurements at midnight, after an overnight fast between 0800 and 0900 h and after an overnight 1-mg dexamethasone test. Plasma cortisol and dehydroepiandrosterone sulfate concentrations were measured by RIA (19). Plasma ACTH was determined by an immunochemiluminometric assay (Nichols Institute Ltd., San Juan, CA).
Diabetes mellitus and impaired glucose tolerance (IGT) were diagnosed according to the recommendations of the American Diabetes Association, with a cut-off value of 7.0 mmol/liter fasting plasma glucose level and/or 11.1 mmol/liter 2-h postload plasma glucose during an oral glucose tolerance test for diabetes and with a cut-off value of 5.6 mmol/liter fasting plasma glucose level and/or 7.8 mmol/liter 2-h postload plasma glucose during an oral glucose tolerance test for IGT. Hypertension was diagnosed according to the World Health Organization guidelines, with cut-off values of 140 mm Hg systolic and/or 90 mm Hg diastolic blood pressures. Increased blood lipid levels were diagnosed when serum cholesterol and/or triglyceride levels were above 5.1 and 2.3 mmol/liter, respectively.
Genetic analysis
DNA was isolated from peripheral blood leukocytes. For screening the N363S variant, a new allele-specific PCR method was used (20). The ER22/23EK variant was screened by restriction fragment length polymorphism, using a modified method described by Koper et al. (15). Primer sequences and PCR conditions are available on request. The results obtained by allele-specific PCR and restriction fragment length polymorphism were confirmed by direct sequencing using an automated sequencer 310 genetic analyzer from Applied Biosystems (Foster City, CA).
Statistical analysis
Data were analyzed using the SPSS 12.0 software (SPSS Inc., Chicago, IL). Differences between proportions were tested by 
2 test or Fischer’s exact test. Differences between continuous variables were tested with Student’s t test or Mann-Whitney U test. Levels of statistical significance were set at P < 0.05. Hardy-Weinberg equilibrium was tested with the Monte Carlo method.
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Results |
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Clinical and demographic data of patients with unilateral and bilateral adrenal incidentalomas are shown in Table 1
. The mean age, sex ratio, and BMI were similar in both groups. The prevalence of type 2 diabetes mellitus in patients with bilateral incidentalomas (40.9%) was significantly higher, compared with that found in patients with unilateral adrenal tumors (23.2%), but hypertension, hyperlipidemia, and history of coronary artery disease occurred with a similar frequency in both groups. Plasma hormone concentrations failed to show significant differences between the two groups.
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Among the 102 population-matched control subjects analyzed, eight were heterozygous for the N363S variant (carrier frequency, 7.8%), whereas seven heterozygous patients were identified among the 99 patients with unilateral adrenal incidentalomas (carrier frequency, 7.1%). Of the 100 patients with type 2 diabetes, 13 heterozygous patients corresponding to a carrier frequency of 13% were detected, but this moderately increased carrier frequency was not statistically different from those found in control subjects or patients with unilateral incidentalomas. By contrast, the finding of nine heterozygous patients among the 44 patients with bilateral incidentalomas (carrier frequency, 20.5%) indicated a significant overrepresentation of the N363S variant, compared with control subjects or patients with unilateral adrenal incidentalomas (P < 0.05) (Fig. 1A). This carrier frequency indicated a relative risk of 3.02 (95% confidence interval 1.04–9.45) for detecting bilateral incidentalomas in N363S carriers vs. noncarriers. Genotypic distribution was in accordance with Hardy-Weinberg equilibrium in all groups.
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None of the patients or controls had both N363S and ER22/23EK alleles. Heterozygous ER22/23EK variant was detected in five of the 102 population-matched control subjects (carrier frequency 4.9%), six of the 99 patients with unilateral adrenal incidentalomas (carrier frequency 6.2%), and two of the 44 patients with bilateral adrenal tumors (carrier frequency 4.5%). Of the 100 patients with type 2 diabetes mellitus, five patients were heterozygous carriers and one patient was a homozygous carrier of the ER22/23EK variant (carrier frequency 6%). There were no statistically significant differences in carrier frequencies among the different groups (Fig. 1A
). Genotypic distribution was again in accordance with Hardy-Weinberg equilibrium in all groups.
Associations between the two gene variants and hormonal or metabolic characteristics
Among all patients with adrenal incidentalomas, impaired glucose homeostasis was significantly (P < 0.05) associated with the presence of the N363S variant (53.5 and 93.8% in noncarriers and carriers, respectively). When analyzed separately in the two groups of patients, impaired glucose homeostasis in patients with bilateral tumors was present in 57.1% of noncarriers and 100% of carriers (P < 0.05), but in patients with unilateral tumors, the occurrence of impaired glucose homeostasis was not statistically different between noncarriers and carriers (52.2 vs. 85.7%) (Fig. 1B). The N363S carrier status, analyzed in either all patients or the two groups of patients with adrenal tumors, failed to show associations with hormonal findings, tumor mass, body weight, BMI, hypertension, increased blood lipid levels, or a history of coronary artery disease.
In patients with type 2 diabetes, associations between the N363S allele and body weight, BMI, hypertension, or need for insulin therapy were absent.
Comparison of clinical and biochemical findings in ER22/23EK carriers and noncarriers indicated no significant differences in any groups of patients or controls.
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Discussion |
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Although the relatively small number of patients included in the study may limit the results, our findings suggest a role for the N363S variant in the development of bilateral adrenal incidentalomas. The N363S variant has been shown to be associated with increased glucocorticoid sensitivity in vivo (9), presumably due to an increased transactivating capacity of the GR (17). In addition, earlier studies suggested an association of the N363S variant with several metabolic abnormalities including impairment of glucose homeostasis, although variable results have been reported. It has been proposed that the pathophysiological mechanism underlying metabolic abnormalities in N363S carriers may be a relative hypersensitivity for insulin elicited by changes in cortisol levels (11) and that the N363S variant may play a role in type 2 diabetes. However, other studies including ours failed to document association between the N363S variant and diabetes or glucose metabolism in healthy, random, or diabetic subjects (10, 12, 13, 14). Nevertheless, it cannot be entirely ruled out that the increased transactivating capacity of the N363S variant may enhance transcription of target genes involved in both the formation of bilateral adrenal incidentalomas and the frequent occurrence of type 2 diabetes in these patients.
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Acknowledgments |
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Footnotes |
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Disclosure statement: The authors have nothing to declare.
First Published Online April 24, 2006
Abbreviations: BMI, Body mass index; CT, computed tomography; GR, glucocorticoid receptor; IGT, impaired glucose tolerance.
Received January 12, 2006.
Accepted April 17, 2006.
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References |
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