Lawrence S. Kirschner

Mutations of the gene encoding the protein kinase A type I-α regulatory subunit in patients with the Carney complex

Carney complex (CNC) is a multiple neoplasia syndrome characterized by spotty skin pigmentation, cardiac and other myxomas, endocrine tumours and psammomatous melanotic schwannomas. CNC is inherited as an autosomal dominant trait and the genes responsible have been mapped to 2p16 and 17q22–24 (refs 6, 7). Because of its similarities to the McCune-Albright syndrome and other features, such as paradoxical responses to endocrine signals, genes implicated in cyclic nucleotide-dependent signalling have been considered candidates for causing CNC (ref. 10). In CNC families mapping to 17q, we detected loss of heterozygosity (LOH) in the vicinity of the gene (PRKAR1A) encoding protein kinase A regulatory subunit 1-α (RIα), including a polymorphic site within its 5′ region. We subsequently identified three unrelated kindreds with an identical mutation in the coding region of PRKAR1A. Analysis of additional cases revealed the same mutation in a sporadic case of CNC, and different mutations in three other families, including one with isolated inherited cardiac myxomas. Analysis of PKA activity in CNC tumours demonstrated a decreased basal activity, but an increase in cAMP-stimulated activity compared with non-CNC tumours. We conclude that germline mutations in PRKAR1A, an apparent tumour-suppressor gene, are responsible for the CNC phenotype in a subset of patients with this disease.

A genome-wide scan identifies mutations in the gene encoding phosphodiesterase 11A4 ( PDE11A ) in individuals with adrenocortical hyperplasia

Phosphodiesterases (PDEs) regulate cyclic nucleotide levels. Increased cyclic AMP (cAMP) signaling has been associated with PRKAR1A or GNAS mutations and leads to adrenocortical tumors and Cushing syndrome. We investigated the genetic source of Cushing syndrome in individuals with adrenocortical hyperplasia that was not caused by known defects. We performed genome-wide SNP genotyping, including the adrenocortical tumor DNA. The region with the highest probability to harbor a susceptibility gene by loss of heterozygosity (LOH) and other analyses was 2q31–2q35. We identified mutations disrupting the expression of the PDE11A isoform-4 gene (PDE11A) in three kindreds. Tumor tissues showed 2q31–2q35 LOH, decreased protein expression and high cyclic nucleotide levels and cAMP-responsive element binding protein (CREB) phosphorylation. PDE11A codes for a dual-specificity PDE that is expressed in adrenal cortex and is partially inhibited by tadalafil and other PDE inhibitors; its germline inactivation is associated with adrenocortical hyperplasia, suggesting another means by which dysregulation of cAMP signaling causes endocrine tumors.

Identification of a Novel Genetic Locus for Familial Cardiac Myxomas and Carney Complex

BACKGROUND Intracardiac myxomas are significant causes of cardiovascular morbidity and mortality through embolic stroke and heart failure. In the autosomal dominant syndrome Carney complex, intracardiac myxomas arise in the setting of lentiginosis and other lesions associated with cutaneous hyperpigmentation, extracardiac myxomas, and nonmyxomatous tumors. Genetic factors that regulate cardiac tumor growth remain unknown. METHODS AND RESULTS We used the molecular genetic techniques of linkage analysis to study 4 kindreds affected by Carney complex to determine the genetic basis of this syndrome. Our investigation confirmed genetic heterogeneity of Carney complex. Moreover, genetic linkage analysis with polymorphic short tandem repeats on the long arm of chromosome 17 revealed maximal pairwise LOD scores of 5.9, 1.5, 1.8, and 2.9 for families YA, YB, YC01, and YC11, respectively. Haplotype analysis excluded a founder effect at this locus. These data identify a major 17 cM locus on chromosome 17q2 that contains the Carney complex disease gene. CONCLUSIONS The ultimate identification and analysis of the Carney complex disease gene at this human chromosome 17q2 locus will facilitate diagnosis and treatment of cardiac myxomas and will foster new concepts in regulation of cardiac cell growth and differentiation.

Paradoxical Response to Dexamethasone in the Diagnosis of Primary Pigmented Nodular Adrenocortical Disease

Primary pigmented nodular adrenocortical disease is a bilateral adrenal disorder that leads to adrenocorticotropin (ACTH)-independent cases of the Cushing syndrome. In most cases, this disease occurs as part of the Carney complex, an autosomal dominant, multiple neoplasia syndrome that consists of skin lentigines, myxomas, and other nonendocrine and endocrine tumors (1). Cardiac myxoma, a tumor that may cause stroke and death, is among the most frequent and is often the first manifestation of the Carney complex (1, 2). The diagnosis of primary pigmented nodular adrenocortical disease should be followed by screening for the Carney complex and, in particular, its potentially fatal cardiac component (1, 2). Establishing the diagnosis of primary pigmented nodular adrenocortical disease can be difficult because the associated hypercortisolism usually develops slowly over several years and the clinical manifestations may be subtle (3, 4). Results of radiologic imaging can be normal or indistinguishable from those that indicate adrenal nodularity, which is frequently present in other primary forms of the Cushing syndrome and in normal elderly persons (5). In addition, plasma ACTH levels may not be fully suppressed, especially in mild or periodic cases of the Cushing syndrome (3, 4). Previous reports have mentioned a paradoxical increase in glucocorticoid level after administration of various doses of dexamethasone in patients with primary pigmented nodular adrenocortical disease (6-11), but this observation has not been systematically investigated. The Liddle test (12), the administration of low-dose and high-dose dexamethasone, is used to differentiate between pituitary-dependent and non-pituitary-dependent forms of the Cushing syndrome (12, 13). Suppression of urinary free cortisol greater than 90% and suppression of 17-hydroxycorticosteroid greater than 64% during the Liddle test identified all patients with pituitary-dependent cases of the Cushing syndrome in a large study of 118 patients (13). This test, however, has not been evaluated for its ability to differentiate between primary adrenocortical causes of the Cushing syndrome. We analyzed data from 21 patients with primary pigmented nodular adrenocortical disease who had been evaluated at the National Institutes of Health (NIH) over the past 30 years. Sixteen patients underwent the Liddle test in addition to other testing. We compared the usefulness of the Liddle test in diagnosing primary pigmented nodular adrenocortical disease and in differentiating this disease from other primary adrenocortical disorders. For this purpose, we investigated two control groups of patients with primary adrenocortical diseases that lead to ACTH-independent cases of the Cushing syndrome: patients with macronodular adrenocortical disease, a condition that is almost always bilateral (14-16), and patients with unilateral, single adrenocortical adenomas. Methods Patients We reviewed records of patients with ACTH-independent cases of the Cushing syndrome who were seen at the NIH clinical center over the past 30 years. The Carney complex was diagnosed on the basis of published criteria (2). Primary pigmented nodular adrenocortical disease, macronodular adrenocortical disease, and single adrenocortical adenoma were confirmed by histologic analysis after adrenalectomy or at autopsy, according to published criteria (1, 14-17). Macronodular disease, in particular, was diagnosed in the presence of multiple, bilateral, nonpigmented adrenocortical adenomas and a substantial increase in the weight of the adrenal glands (14-16). For some patients, little information was obtained about levels of plasma ACTH and urinary free cortisol because radioimmunoassays, which are now used to determine such variables, were not available until the late 1970s. The two control groups consisted only of patients for whom all data were available. For each patient, we analyzed plasma ACTH levels at 8:00 a.m.followed by ovine corticotropin-releasing hormone stimulationand diurnal plasma cortisol variation, as described elsewhere (13, 18). A 6-day Liddle test was conducted for each patient, as described elsewhere (12, 13): After 2 days of baseline measurement of urinary steroid excretion, dexamethasone, 0.5 mg, was given orally every 6 hours for 2 days starting at 6:00 a.m.; the dosage of dexamethasone was then increased to 2 mg every 6 hours for the last 2 days of the test. For children, the lowest dose of dexamethasone was adjusted to 7.2 g/kg of body weight and the highest dose was adjusted to 28.5 g/kg (3). In all patients, the 24-hour urinary free cortisol level was expressed per square meter of body surface area (g/m2); 17-hydroxycorticosteroid excretion was expressed per grams of creatinine excreted in 24 hours (mg/g). We also performed computed tomography of the adrenal glands, as described elsewhere (19). Hormone Assays Plasma ACTH and cortisol levels were measured as described elsewhere (13, 18). Urinary free cortisol excretion was measured by using direct radioimmunoassay (20, 21). The intra-assay coefficient of variation was 5%, and the interassay coefficient of variation was 10%. Urinary 17-hydroxycorticosteroid excretion was measured by using a modification of the colorimetric method described by Porter and Silber (20, 21). The intra-assay and interassay coefficients of variation were 6% and 11%, respectively. Statistical Analysis All data are expressed as the mean SE. For all statistical comparisons, a P value less than 0.05 was considered significant. Data were analyzed by using Statistica software (StatSoft, Inc., Tulsa, Oklahoma). Friedman analysis of variance was initially used within each group to determine differences in urinary free cortisol and 17-hydroxycorticosteroid levels in response to dexamethasone administration during the Liddle test. The Wilcoxon matched-pair test was used to determine which time points significantly differed from baseline. The Mann-Whitney U test was used to assess differences among groups. The specificity and sensitivity of the Liddle test were determined, and receiver-operating characteristic (ROC) curves were constructed, as described elsewhere (13, 18), to assess the usefulness of each method for differential diagnosis. Results Twenty-one patients (8 males and 13 females) with primary pigmented nodular adrenocortical disease were seen at the NIH clinical center over the past 30 years. Most patients were young: Age at diagnosis was 27.7 2.9 years. Four patients were children (2 boys and 2 girls; age at diagnosis, 10 1.8 years), and 17 patients were adults (6 men and 11 women; age at diagnosis, 31.9 2.6 years). No children were included in the two control groups. For 20 of 21 patients (95%), primary pigmented nodular adrenocortical disease occurred as a component of the Carney complex. Three of these patients had periodic cases of the Cushing syndrome (14%), which were characterized by periods of clinical symptoms and hypercortisolemia followed by periods of normalization of the body habitus and eucortisolemia, as described elsewhere (3, 4). The length of these periods varied considerably, from every 2 to 3 months (2 patients) to 1 to 2 weeks (1 patient). Four patients with primary pigmented nodular adrenocortical disease (19%) had subclinical cases of the Cushing syndrome, which were characterized by a relative paucity of clinical findings of the syndrome (with the exception of osteoporosis) and eucortisolemia with abnormal diurnal cortisol variation, as described elsewhere (4). Computed tomography revealed normal-sized adrenal glands in 19 patients with primary pigmented nodular adrenocortical disease (90%). Two patients had macronodules (nodules larger than 1 cm); one nodule had calcifications. An irregular contour of the adrenal glands, as described elsewhere (4, 5, 19), could be seen in 10 of the patients with primary pigmented nodular adrenocortical disease who had normal-sized adrenal glands (48%) (Figure 1). On computed tomography, all patients with macronodular adrenocortical disease had macronodules and all patients with single adenomas had a single mass. Figure 1. Macronodular appearance on computed tomography of the adrenal gland in a patient with primary pigmented adrenocortical disease ( left ) and the more typical bead-on-a-string appearance of an adrenal gland in a patient with primary pigmented nodular adrenocortical disease ( right ) Morning plasma ACTH levels were in the low end of the normal range but were still measurable in almost all of the patients in the three groups; however, no differences were seen in baseline ACTH values or responses of ACTH or cortisol to ACTH-releasing hormone stimulation (P>0.1 for all comparisons, Mann-Whitney U test) (data not shown). All patients demonstrated loss or reversal of the normal pattern of diurnal cortisol variation, but no differences were seen among groups (P>0.1 for all comparisons, Mann-Whitney U test) (data not shown). Data were available for 16 patients with primary pigmented nodular adrenocortical disease who had 17-hydroxycorticosteroid or urinary free cortisol responses to the Liddle test. Thirteen of these 16 patients had both glucocorticoids measured; for 3 patients only 17-hydroxycorticosteroid measurements were available. In these 16 patients, urinary excretion of both glucocorticoids increased gradually during the Liddle test (Figure 2). Friedman analysis of variance showed a P value less than 0.001 for 17-hydroxycorticosteroid and urinary free cortisol values in patients with primary pigmented nodular adrenocortical disease; the P value was greater than 0.2 for 17-hydroxycorticosteroid and urinary free cortisol values in patients with macronodular adrenocortical disease. For 17-hydroxycorticosteroid and urinary free cortisol values in patients with single adenomas, the P values were 0.05 and greater than 0.2, respectively. Figure 2. Mean percentage change in 24-hour excretion of 17-hyd

A Mouse Model for the Carney Complex Tumor Syndrome Develops Neoplasia in Cyclic AMP–Responsive Tissues

Carney complex is an autosomal dominant neoplasia syndrome characterized by spotty skin pigmentation, myxomatosis, endocrine tumors, and schwannomas. This condition may be caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A. To better understand the mechanism by which PRKAR1A mutations cause disease, we have developed conventional and conditional null alleles for Prkar1a in the mouse. Prkar1a(+/-) mice developed nonpigmented schwannomas and fibro-osseous bone lesions beginning at approximately 6 months of age. Although genotype-specific cardiac and adrenal lesions were not seen, benign and malignant thyroid neoplasias were observed in older mice. This spectrum of tumors overlaps that seen in Carney complex patients, confirming the validity of this mouse model. Genetic analysis indicated that allelic loss occurred in a subset of tumor cells, suggesting that complete loss of Prkar1a plays a key role in tumorigenesis. Similarly, tissue-specific ablation of Prkar1a from a subset of facial neural crest cells caused the formation of schwannomas with divergent differentiation. These observations confirm the identity of PRKAR1A as a tumor suppressor gene with specific importance to cyclic AMP-responsive tissues and suggest that these mice may be valuable tools not only for understanding endocrine tumorigenesis but also for understanding inherited predispositions for schwannoma formation.

Gene array analysis of macronodular adrenal hyperplasia confirms clinical heterogeneity and identifies several candidate genes as molecular mediators.

Corticotropin (ACTH)-independent macronodular adrenal hyperplasia (AIMAH) is a heterogeneous condition in which cortisol secretion may be mediated by gastrointestinal peptide (GIP), vasopressin, catecholamines and other hormones. We studied the expression profile of AIMAH by genomic cDNA microarray analysis. Total RNA was extracted from eight tissues (three GIP-dependent) and compared to total RNA obtained from adrenal glands from 62 normal subjects. Genes had to be altered in 75% of the patients, and be up- or downregulated at a cutoff ratio of at least 2.0; 82 and 31 genes were found to be consistently up- and downregulated, respectively. Among the former were regulators of transcription, chromatin remodeling, and cell cycle and adhesion. Downregulated sequences included genes involved in immune responses and insulin signaling. Hierarchical clustering correlated with the two main AIMAH diagnostic groups: GIP-dependent and non-GIP-dependent. The genes encoding the 7B2 protein (SGNE1) and WNT1-inducible signaling pathway protein 2 (WISP2) were specifically overexpressed in the GIP-dependent AIMAH. For these, and six more genes, the data were validated by semiquantitative amplification in samples from a total of 32 patients (the original eight, six more cases of AIMAH, and 18 other adrenocortical hyperplasias and tumors) and the H295R adrenocortical cancer cell line. In conclusion, our data confirmed AIMAHs clinical heterogeneity by identifying molecularly distinct diagnostic subgroups. Several candidate genes that may be responsible for AIMAH formation and/or progression were also identified, suggesting pathways that affect the cell cycle, adhesion and transcription as possible mediators of adrenocortical hyperplasia.

A transgenic mouse bearing an antisense construct of regulatory subunit type 1A of protein kinase A develops endocrine and other tumours: comparison with Carney complex and other PRKAR1A induced lesions

Background: Inactivation of the human type Iα regulatory subunit (RIα) of cyclic AMP dependent protein kinase (PKA) (PRKAR1A) leads to altered kinase activity, primary pigmented nodular adrenocortical disease (PPNAD), and sporadic adrenal and other tumours. Methods and results: A transgenic mouse carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline responsive promoter (the Tg(Prkar1a*x2as)1Stra, Tg(tTAhCMV)3Uh or tTA/X2AS line) developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia and other features reminiscent of PPNAD, including late onset weight gain, visceral adiposity, and non-dexamethasone suppressible hypercorticosteronaemia, with histiocytic, epithelial hyperplasias, lymphomas, and other mesenchymal tumours. These lesions were associated with allelic losses of the mouse chromosome 11 Prkar1a locus, an increase in total type II PKA activity, and higher RIIβ protein levels; the latter biochemical and protein changes were also documented in Carney complex tumours associated with PRKAR1A inactivating mutations and chromosome 17 PRKAR1A locus changes. Conclusion: We conclude that the tTA/X2AS mouse line with a downregulated Prkar1a gene replicates several of the findings in Carney complex patients and their affected tissues, supporting the role of RIα as a candidate tumour suppressor gene.

Clinical and Genetic Heterogeneity, Overlap with Other Tumor Syndromes, and Atypical Glucocorticoid Hormone Secretion in Adrenocorticotropin-Independent Macronodular Adrenal Hyperplasia Compared with Other Adrenocortical Tumors

OBJECTIVE ACTH-independent macronodular adrenal hyperplasia (AIMAH) is often associated with subclinical cortisol secretion or atypical Cushings syndrome (CS). We characterized a large series of patients of AIMAH and compared them with patients with other adrenocortical tumors. DESIGN AND PATIENTS We recruited 82 subjects with: 1) AIMAH (n = 16); 2) adrenocortical cortisol-producing adenoma with CS (n = 15); 3) aldosterone-producing adenoma (n = 19); and 4) single adenomas with clinically nonsignificant cortisol secretion (n = 32). METHODS Urinary free cortisol (UFC) and 17-hydroxycorticosteroid (17OHS) were collected at baseline and during dexamethasone testing; aberrant receptor responses was also sought by clinical testing and confirmed molecularly. Peripheral and/or tumor DNA was sequenced for candidate genes. RESULTS AIMAH patients had the highest 17OHS excretion, even when UFCs were within or close to the normal range. Aberrant receptor expression was highly prevalent. Histology showed at least two subtypes of AIMAH. For three patients with AIMAH, there was family history of CS; germline mutations were identified in three other patients in the genes for menin (one), fumarate hydratase (one), and adenomatosis polyposis coli (APC) (one); a PDE11A gene variant was found in another. One patient had a GNAS mutation in adrenal nodules only. There were no mutations in any of the tested genes in the patients of the other groups. CONCLUSIONS AIMAH is a clinically and genetically heterogeneous disorder that can be associated with various genetic defects and aberrant hormone receptors. It is frequently associated with atypical CS and increased 17OHS; UFCs and other measures of adrenocortical activity can be misleadingly normal.

Down-Regulation of Regulatory Subunit Type 1A of Protein Kinase A Leads to Endocrine and Other Tumors

Mutations of the human type Iα regulatory subunit (RIα) of cyclic AMP-dependent protein kinase (PKA; PRKAR1A) lead to altered kinase activity, primary pigmented nodular adrenocortical disease, and tumors of the thyroid and other tissues. To bypass the early embryonic lethality of Prkar1a−/− mice, we established transgenic mice carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline-responsive promoter. Down-regulation of Prkar1a by up to 70% was achieved in transgenic mouse tissues and embryonic fibroblasts, with concomitant changes in kinase activity and increased cell proliferation, respectively. Mice developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia, and other features reminiscent of primary pigmented nodular adrenocortical disease, histiocytic and epithelial hyperplasias, lymphomas, and other mesenchymal tumors. These were associated with allelic losses of the mouse chromosome 11 Prkar1a locus, an increase in total type II PKA activity, and higher RIIβ protein levels. This mouse provides a novel, useful tool for the investigation of cyclic AMP, RIα, and PKA functions and confirms the critical role of Prkar1a in tumorigenesis in endocrine and other tissues.

Chromosome 2 (2p16) abnormalities in Carney complex tumours

Carney complex (CNC) is an autosomal dominant multiple endocrine neoplasia and lentiginosis syndrome characterised by spotty skin pigmentation, cardiac, skin, and breast myxomas, and a variety of endocrine and other tumours. The disease is genetically heterogeneous; two loci have been mapped to chromosomes 17q22–24 (the CNC1 locus) and 2p16 (CNC2). Mutations in the PRKAR1A tumour suppressor gene were recently found in CNC1 mapping kindreds, while the CNC2 and perhaps other genes remain unidentified. Analysis of tumour chromosome rearrangements is a useful tool for uncovering genes with a role in tumorigenesis and/or tumour progression. CGH analysis showed a low level 2p amplification recurrently in four of eight CNC tumours; one tumour showed specific amplification of the 2p16-p23 region only. To define more precisely the 2p amplicon in these and other tumours, we completed the genomic mapping of the CNC2 region, and analysed 46 tumour samples from CNC patients with and without PRKAR1A mutations by fluorescence in situ hybridisation (FISH) using bacterial artificial chromosomes (BACs). Consistent cytogenetic changes of the region were detected in 40 (87%) of the samples analysed. Twenty-four samples (60%) showed amplification of the region represented as homogeneously stained regions (HSRs). The size of the amplicon varied from case to case, and frequently from cell to cell in the same tumour. Three tumours (8%) showed both amplification and deletion of the region in their cells. Thirteen tumours (32%) showed deletions only. These molecular cytogenetic changes included the region that is covered by BACs 400-P-14 and 514-O-11 and, in the genetic map, corresponds to an area flanked by polymorphic markers D2S2251 and D2S2292; other BACs on the centromeric and telomeric end of this region were included in varying degrees. We conclude that cytogenetic changes of the 2p16 chromosomal region that harbours the CNC2 locus are frequently observed in tumours from CNC patients, including those with germline, inactivating PRKAR1A mutations. These changes are mostly amplifications of the 2p16 region, that overlap with a previously identified amplicon in sporadic thyroid cancer, and an area often deleted in sporadic adrenal tumours. Both thyroid and adrenal tumours constitute part of CNC indicating that the responsible gene(s) in this area may indeed be involved in both inherited and sporadic endocrine tumour pathogenesis and/or progression.