Audrey Robinson-White

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.

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.

Large Deletions of the PRKAR1A Gene in Carney Complex

Purpose: Since the identification of PRKAR1A mutations in Carney complex, substitutions and small insertions/deletions have been found in ∼70% of the patients. To date, no germ-line PRKAR1A deletion and/or insertion exceeded a few base pairs (up to 15). Although a few families map to chromosome 2, it is possible that current sequencing techniques do not detect larger gene changes in PRKAR1A–mutation-negative individuals with Carney complex. Experimental Design: To screen for gross alterations of the PRKAR1A gene, we applied Southern hybridization analysis on 36 unrelated Carney complex patients who did not have small intragenic mutations or large aberrations in PRKAR1A, including the probands from two kindreds mapping to chromosome 2. Results: We found large PRKAR1A deletions in the germ-line of two patients with Carney complex, both sporadic cases; no changes were identified in the remaining patients, including the two chromosome-2-mapping families. In the first patient, the deletion is expected to lead to decreased PRKAR1A mRNA levels but no other effects on the protein; the molecular phenotype is predicted to be PRKAR1A haploinsufficiency, consistent with the majority of PRKAR1A mutations causing Carney complex. In the second patient, the deletion led to in-frame elimination of exon 3 and the expression of a shorter protein, lacking the primary site for interaction with the catalytic protein kinase A subunit. In vitro transfection studies of the mutant PRKAR1A showed impaired ability to bind cyclic AMP and activation of the protein kinase A enzyme. The patient bearing this mutation had a more-severe-than-average Carney complex phenotype that included the relatively rare psammomatous melanotic schwannoma. Conclusions: Large PRKAR1A deletions may be responsible for Carney complex in patients that do not have PRKAR1A gene defects identifiable by sequencing. Preliminary data indicate that these patients may have a different phenotype especially if their defect results in an expressed, abnormal version of the PRKAR1A protein.

A cAMP-specific phosphodiesterase ( PDE8B ) that is mutated in adrenal hyperplasia is expressed widely in human and mouse tissues: a novel PDE8B isoform in human adrenal cortex

Bilateral adrenocortical hyperplasia (BAH) is the second most common cause of corticotropin-independent Cushing syndrome (CS). Genetic forms of BAH have been associated with complex syndromes such as Carney Complex and McCune–Albright syndrome or may present as isolated micronodular adrenocortical disease (iMAD) usually in children and young adults with CS. A genome-wide association study identified inactivating phosphodiesterase (PDE) 11A (PDE11A)-sequencing defects as low-penetrance predisposing factors for iMAD and related abnormalities; we also described a mutation (c.914A>C/H305P) in cyclic AMP (cAMP)-specific PDE8B, in a patient with iMAD. In this study we further characterize this mutation; we also found a novel PDE8B isoform that is highly expressed in the adrenal gland. This mutation is shown to significantly affect the ability of the protein to degrade cAMP in vitro. Tumor tissues from patients with iMAD and no mutations in the coding PDE8B sequence or any other related genes (PRKAR1A, PDE11A) showed downregulated PDE8B expression (compared to normal adrenal cortex). Pde8b is detectable in the adrenal gland of newborn mice and is widely expressed in other mouse tissues. We conclude that PDE8B is another PDE gene linked to iMAD; it is a candidate causative gene for other adrenocortical lesions linked to the cAMP signaling pathway and possibly for tumors in other tissues.

The demonstration of histamine release in clinical conditions: A review of past and present assay procedures

SummaryTopics related to the measurement of histamine in human plasma and other body fluids are reviewed. These include (1) an overview of the data obtained by the biological, fluorometric and radioenzymatic assays over the past 45 years; (2) the various modifications of the radioenzymatic isotopic assay of histamine and the development of a single extraction step assay; (3) a compilation of values obtained in our laboratory by the radioenzymatic assay of histamine levels in various body fluids in disease states associated with abnormal histamine production or release; and (4) factors that affect histamine levels in plasma and some experimental considerations for monitoring changes in free histamine levels. The last topic includes a discussion of the halflife of histamine in the circulation, its clearance across various vascular beds, and the fact that capillary endothelial cells are one site of inactivation of circulating histamine.ZusammenfassungÜber neueste Entwicklungen in der Messung von Histamin im Plasma und anderen Körperflüssigkeiten beim Menschen wird referiert. Diese schließen ein (1) eine Überblick über Daten, die mit biologischen fluorometrischen und radioenzymatischen Methoden in den letzten 45 Jahren erhalten wurden; (2) die verschiedenen Modifikationen der radioenzymatischen Isotopenmethode für Histamin und die Entwicklung einer Einmalextraktionsschritt-Methode; (3) eine Zusammenstellung von mit der radioenzymatischen Methode erhaltenen Histaminwerten aus unserem Labor in verschiedenen Körperflüssigkeiten bei Krankheitszuständen, die mit abnormaler Histaminbildung oder-freisetzung einhergehen; und (4) Faktoren, die den Histaminwert im Plasma und einige experimentelle Überlegungen beeinflussen zwecks Überwachung von Veränderungen von freiem Histamin. — Der letzte Topic schließt eine Diskussion der Halbwertszeit von Histamin im Kreislauf ein, seine Clearance entlang verschiedener Gefäßabschnitte und das Faktum, daß kapilläre Endothelzellen ein Ort für die Inaktivierung von zirkulierendem Histamin sind.

Protein Kinase A Signaling

Abstract: Protein kinase A (PKA) signaling, in “classic” endocrine cell functioning, is known to mediate cAMP effects, generated through adenylate cyclase as a response to the activation of G protein‐coupled receptors (GPCRs). This signaling system is highly versatile; its flexibility is supported by a number of adenylate cyclases, four PKA regulatory and three catalytic subunits, and several phosphodiesterases that close the negative feedback loop of cAMP generation, most molecules that are expressed in a tissue‐specific manner. A central question, however, remains: how do the hundreds of GPCRs mediate their specific effects? Tissue specificity of the expression of the various components of the PKA system, albeit necessary, cannot be the only answer. It helps more to view PKA as a central hub that interacts with a variety of other signaling pathways in endocrine cells, not only mediating but also communicating cAMP effects to the mitogen‐activated protein kinase (MAPK), protein kinase C and B (PKC and PKB/Akt, respectively). The net result of these complex interactions, evidence for which is reviewed in this chapter, is what we know as “cAMP effects.” It is, perhaps, because of this complexity that investigations of PKA signaling in vivo and in vitro often give contradictory results and are difficult to interpret.

Protein kinase A effects of an expressed PRKAR1A mutation associated with aggressive tumors.

Most PRKAR1A tumorigenic mutations lead to nonsense mRNA that is decayed; tumor formation has been associated with an increase in type II protein kinase A (PKA) subunits. The IVS6+1G>T PRKAR1A mutation leads to a protein lacking exon 6 sequences [R1 alpha Delta 184-236 (R1 alpha Delta 6)]. We compared in vitro R1 alpha Delta 6 with wild-type (wt) R1 alpha. We assessed PKA activity and subunit expression, phosphorylation of target molecules, and properties of wt-R1 alpha and mutant (mt) R1 alpha; we observed by confocal microscopy R1 alpha tagged with green fluorescent protein and its interactions with Cerulean-tagged catalytic subunit (C alpha). Introduction of the R1 alpha Delta 6 led to aberrant cellular morphology and higher PKA activity but no increase in type II PKA subunits. There was diffuse, cytoplasmic localization of R1 alpha protein in wt-R1 alpha- and R1 alpha Delta 6-transfected cells but the former also exhibited discrete aggregates of R1 alpha that bound C alpha; these were absent in R1 alpha Delta 6-transfected cells and did not bind C alpha at baseline or in response to cyclic AMP. Other changes induced by R1 alpha Delta 6 included decreased nuclear C alpha. We conclude that R1 alpha Delta 6 leads to increased PKA activity through the mt-R1 alpha decreased binding to C alpha and does not involve changes in other PKA subunits, suggesting that a switch to type II PKA activity is not necessary for increased kinase activity or tumorigenesis.

PRKAR1A Inactivation Leads to Increased Proliferation and Decreased Apoptosis in Human B Lymphocytes

The multiple neoplasia syndrome Carney complex (CNC) is caused by heterozygote mutations in the gene, which codes for the RIalpha regulatory subunit (PRKAR1A) of protein kinase A. Inactivation of PRKAR1A and the additional loss of the normal allele lead to tumors in CNC patients and increased cyclic AMP signaling in their cells, but the oncogenetic mechanisms in affected tissues remain unknown. Previous studies suggested that PRKAR1A down-regulation may lead to increased mitogen-activated protein kinase (MAPK) signaling. Here, we show that, in lymphocytes with PRKAR1A-inactivating mutations, there is increased extracellular signal-regulated kinase (ERK) 1/2 and B-raf phosphorylation and MAPK/ERK kinase 1/2 and c-Myc activation, whereas c-Raf-1 is inhibited. These changes are accompanied by increased cell cycle rates and decreased apoptosis that result in an overall net gain in proliferation and survival. In conclusion, inactivation of PRKAR1A leads to widespread changes in molecular pathways that control cell cycle and apoptosis. This is the first study to show that human cells with partially inactivated RIalpha levels have increased proliferation and survival, suggesting that loss of the normal allele in these cells is not necessary for these changes to occur.

A mouse model for Carney complex.

Mice with complete inactivation of the type Iα regulatory subunit (RIα) of cyclic (c) AMP‐dependent protein kinase (PKA) (coded by the Prkar1a gene) die early in embryonic life. 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. Mice developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia, and other features reminiscent of PPNAD, and histiocytic and epithelial hyperplasias, lymphomas, and other mesenchymal tumors. This mouse provides a useful tool for the investigation of cAMP, RIα, and PKA functions and confirms Prkar1as critical role in tumorigenesis in endocrine and other tissues.