PRKAR1A mutations in primary pigmented nodular adrenocortical disease

Primary Pigmented Nodular Adrenocortical Disease (PPNAD) is a rare primary bilateral adrenal defect causing corticotropin-independent Cushing’s syndrome. It occurs mainly in children and young adults. Macroscopic appearance of the adrenals is characteristic with small pigmented micronodules observed in the cortex. PPNAD is most often diagnosed in patients with Carney complex (CNC), but it can also be observed in patients without other manifestations or familial history (isolated PPNAD). The CNC is an autosomal dominant multiple neoplasia syndrome characterized by the association of myxoma, spotty skin pigmentation and endocrine overactivity. One of the putative CNC genes has been identified as the gene of the regulatory R1A subunit of protein kinase A (PRKAR1A), located at 17q22-24. Germline heterozygous inactivating mutations of PRKAR1A have been reported in about 45% of patients with CNC, and up to 80% of CNC patients with Cushing’s syndrome due to PPNAD. Interestingly, such inactivating germline PRKAR1A mutations have also been found in patients with isolated PPNAD. The hot spot PRKAR1A mutation termed c.709[-7-2]del6 predisposes mostly to isolated PPNAD, and is the first clear genotype/phenotype correlation described for this gene. Somatic inactivating mutations of PRKAR1A have been observed in macronodules of PPNAD and in sporadic cortisol secreting adrenal adenomas. Isolated PPNAD is a genetic heterogenous disease, and recently inactivating mutations of the gene of the phosphodiesterase 11A4 (PDE11A4) located at 2q31–2q35 have been identified in patients without PRKAR1A mutations. Interestingly, both PRKAR1A and PDE11A gene products control the cAMP signaling pathway, which can be altered at various levels in endocrine tumors.     

Frequent mutations of beta-catenin gene in sporadic secreting adrenocortical adenomas

Objective Molecular alterations remain largely unknown in most sporadic adrenocortical tumours and hyperplasias. In our previous work, we demonstrated the differential expression of several Wnt/β‐catenin signalling‐related genes implicated in ACTH‐independent macronodular adrenal hyperplasias (AIMAH). To better understand the role of Wnt/β‐catenin signalling in adrenocortical tumours, we performed mutational analysis of the β‐catenin gene. Methods We studied 53 human adrenocortical samples (33 adenomas, 4 carcinomas, 13 AIMAH, 3 ACTH‐dependent adrenal hyperplasias) and the human adrenocortical cancer cell line NCI‐H295R. All samples were screened for somatic mutations in exons 3 and 5 of the β‐catenin gene. Eleven and six samples were analysed for β‐catenin protein expression by Western blotting and immunohistochemistry, respectively. Results No mutations were detected in adrenocortical carcinomas, AIMAH and ACTH‐dependent hyperplasias. Genetic alterations were found in 5 (15%) out of 33 adenomas: three cortisol‐secreting adenomas, one aldosterone‐secreting adenoma and one nonfunctional adenoma. Two‐point mutations occurred at serine residues of codons 37 and 45 (S37C and S45F). The remaining three tumours contained deletions of 6, 55 and 271 bp. H295R cells carry an activating S45P mutation. Western blot analysis of samples with 55‐ and 271‐bp deletions showed an additional shorter and more intense band representing an accumulation of the mutated form of β‐catenin protein. In addition, cytoplasmic and/or nuclear accumulation of β‐catenin was observed in mutated adenomas by immunohistochemistry. Conclusions Activating mutations of exon 3 of the β‐catenin gene are frequent in adrenocortical adenomas, and further characterization of the Wnt/β‐catenin signalling pathway should lead to a better understanding of adrenal tumourigenesis.     

New genes and/or molecular pathways associated with adrenal hyperplasias and related adrenocortical tumors

Over the course of the last 10 years, we have studied the genetic and molecular mechanisms leading to disorders that affect the adrenal cortex, with emphasis on those that are developmental, hereditary and associated with adrenal hypoplasia or hyperplasia, multiple tumors and abnormalities in other endocrine glands. On the basis of this work, we propose an hypothesis on how adrenocortical tumors form and the importance of the cyclic AMP-dependent signaling pathway in this process. The regulatory subunit type 1-α (RIα) of protein kinase A (PKA) (the PRKAR1A gene) is mutated in most patients with Carney complex and primary pigmented nodular adrenocortical disease (PPNAD). Phosphodiesterase-11A (the PDE11A gene) and -8B (the PDE8B gene) mutations were found in patients with isolated adrenal hyperplasia and Cushing syndrome, as well in patients with PPNAD. PKA effects on tumor suppression and/or development and the cell cycle are becoming clear: PKA and/or cAMP act as a coordinator of growth and proliferation in the adrenal cortex. Mouse models in which the respective genes have been knocked out see m to support this notion. Genome-wide searches for other genes responsible for adrenal tumors and related diseases are ongoing; recent evidece of the involvement of the mitochondrial oxidation pathway in adrenocortical tumorigenesis is derived from our study of rare associations such as those of disorders predisposing to adrenomedullary and related tumors (Carney triad, the dyad of paragangliomas and gastric stromal sarcomas or Carney–Stratakis syndrome, hereditary leiomyomatosis and renal cancer syndrome) which appear to be associated with adrenocortical lesions.     

How does cAMP/protein kinase A signaling lead to tumors in the adrenal cortex and other tissues?

The overwhelming majority of benign lesions of the adrenal cortex leading to Cushing syndrome are linked to one or another abnormality of the cAMP signaling pathway. A small number of both massive macronodular adrenocortical disease and cortisol-producing adenomas harbor somatic GNAS mutations. Micronodular adrenocortical hyperplasias are either pigmented (the classic form being that of primary pigmented nodular adrenocortical disease) or non-pigmented; micronodular adrenocortical hyperplasias can be seen in the context of other conditions or isolated; for example, primary pigmented nodular adrenocortical disease usually occurs in the context of Carney complex, but isolated primary pigmented nodular adrenocortical disease has also been described. Both Carney complex and isolated primary pigmented nodular adrenocortical disease are caused by germline PRKAR1A mutations; somatic mutations of this gene that regulates cAMP-dependent protein kinase are also found in cortisol-producing adenomas, and abnormalities of PKA are present in most cases of massive macronodular adrenocortical disease. Micronodular adrenocortical hyperplasias and some cortisol-producing adenomas are associated with phosphodiesterase 11A and 8B defects, coded, respectively, by the PDE11A and PDE8B genes. Mouse models of Prkar1a deficiency also show that increased cAMP signaling leads to tumors in adrenal cortex and other tissues. In this review, we summarize all recent data from ours and other laboratories, supporting the view that Wnt-signaling acts as an important mediator of tumorigenicity induced by abnormal PRKAR1A function and aberrant cAMP signaling.     

Mutational analysis of PRKAR1A and Gs(alpha) in sporadic adrenocortical tumors.

Little is known about the pathogenesis of adrenocortical tumors. The cAMP-dependent pathway is physiologically activated by ACTH in adrenocortical cells and different components of this cascade may be altered in some functioning adrenocortical tumors. Recently, mutations of the gene encoding the PKA type 1 A regulatory subunit (R1 A), PRKAR1A, associated with loss of heterozygosity (LOH) at PRKAR1A locus, have been demonstrated in primary pigmented nodular adrenocortical disease (PPNAD), either isolated or associated with Carney complex. Moreover, activating mutations of the Gs(alpha) gene (the gsp oncogene) have also been found in a small number of adrenocortical cortisol-secreting adenomas. Aim of this study was to investigate the presence of such genetic alterations on a series of 10 ACTH-independent Cushing syndrome due to non-PPNAD adrenocortical adenomas. The coding sequence of PRKAR1A, evaluated by PCR and direct sequencing analysis, revealed the absence of mutations while heterozygosity for at least 1 polymorphism excluded LOH in most tumors. In one single adenoma gsp mutation was detected. In conclusion, we provide additional evidence that the only mutational changes able to activate the cAMP pathway so far identified, i.e. PRKAR1A mutations and gsp oncogene, are a rare event in adrenocortical tumors.     

Hypermethylation of adenomatous polyposis coli gene promoter is associated with novel Wnt signaling pathway in gastric adenomas

Background and Aim: Gastric adenomas (GAs) are considered as premalignant lesions of gastric adenocarcinoma. The role of Wnt signaling pathway in GAs is rarely identified. In the present study, we aimed to determine whether Wnt signaling plays a role in the pathogenesis of GAs, and to clarify the mechanism of Wnt signaling in GAs. Methods: The study investigated the relationship between clinicopathological characteristics, Helicobacter pylori (Hp) infection, adenomatous polyposis coli (APC) promoter methylation, APC and β‐catenin immunohistochemistry expression and mutation status, compared with 38 gastric adenoma and periadenomatous tissues (PTs). Results: The abnormal expression of β‐catenin in PTs, low‐grade adenomas (LGAs) and high‐grade adenomas (HGAs) was 0%, 9.09% and 81.25%. For APC, immunoreactive score (IRS) was 5.50 ± 0.5 in PTs, 3.59 ± 1.4 in LGAs and 1.8 ± 2.0 in HGAs. The scores in LGAs and HGAs were significantly lower than those in PTs (P = 0.000). IRS reflected significantly reduced expression of APC in HGAs (P = 0.002). The absent expression of APC had a correlation with the expression of β‐catenin (P = 0.000). Four LGAs (18.18%) and nine HGAs (56.25%) had methylation of APC. APC promoter methylation correlated with the grade (P = 0.014) and the expression of β‐catenin and APC (P = 0.000). Genes mutation was detected in only two adenomas (5.3%). The presence of Hp in HGAs (43.8%) was significantly higher than in LGAs (13.6%) (P = 0.038). But there was no statistical correlation to growth pattern, size, APC hypermethylation and gene mutation. Conclusion: Hypermethylation of APC promoter, instead of mutations involving APC and β‐catenin, may play a role in the development and progression of GAs contributing to moderate activation of Wnt signaling. Helicobacter pylori may accelerate the progress of gastric adenoma, but the pathogenesis needs further research.     

Carney complex and other conditions associated with micronodular adrenal hyperplasias

Carney complex (CNC) is a multiple neoplasia syndrome that is inherited in an autosomal dominant manner and is characterized by skin tumors and pigmented lesions, myxomas, schwannomas, and various endocrine tumors. Inactivating mutations of the PRKAR1A gene coding for the regulatory type I-α (RIα) subunit of protein kinase A (PKA) are responsible for the disease in most CNC patients. The overall penetrance of CNC among PRKAR1A mutation carriers is near 98%. Most PRKAR1A mutations result in premature stop codon generation and lead to nonsense-mediated mRNA decay. CNC is genetically and clinically heterogeneous, with specific mutations providing some genotype-phenotype correlation. Phosphodiesterase-11A (the PDE11A gene) and -8B (the PDE8B gene) mutations were found in patients with isolated adrenal hyperplasia and Cushing syndrome, as well in patients with PPNAD. Recent evidences demonstrated that dysregulation of cAMP/PKA pathway can modulate other signaling pathways and contributes to adrenocortical tumorigenesis.     

Molecular genetics of adrenocortical tumours, from familial to sporadic diseases

Adrenal masses can be detected in up to 4% of the population, and are mostly of adrenocortical origin. Adrenocortical tumours (ACTs) may be responsible for excess steroid production and, in the case of adrenocortical cancers, for morbidity or mortality due to tumour growth. Our understanding of the pathogenesis of ACTs is more limited than that for other tumours. However, studies of the genetics of ACTs have led to major advances in this field in the last decade. The identification of germline molecular defects in the hereditary syndrome responsible for ACTs has facilitated progress. Indeed, similar molecular defects have since been identified as somatic alterations in sporadic tumours. The familial diseases concerned are Li-Fraumeni syndrome, which may be due to germline mutation of the tumour-suppressor gene TP53 and Beckwith-Wiedemann syndrome, which is caused by dys-regulation of the imprinted IGF-II locus at 11p15. ACTs also occur in type 1 multiple endocrine neoplasia (MEN 1), which is characterized by a germline mutation of the menin gene. Cushing's syndrome due to primary pigmented nodular adrenocortical disease (PPNAD) has been observed in Carney complex patients presenting inactivating germline PRKAR1A mutations. Interestingly, allelic losses at 17p13 and 11p15 have been demonstrated in sporadic adrenocortical cancer and somatic PRKAR1A mutations have been found in secreting adrenocortical adenomas. More rarely, mutations in Gs protein (gsp) and the gene for ACTH receptor have been observed in ACTs. The genetics of another group of adrenal diseases that can lead to adrenal nodular hyperplasia -- congenital adrenal hyperplasia (CAH) and glucocorticoid-remediable aldosteronism (GRA) -- have also been studied extensively. This review summarizes recent advances in the genetics of ACTs, highlighting both improvements in our understanding of the pathophysiology and the diagnosis of these tumours.     

Primary pigmented nodular adrenocortical disease reveals insulin-like growth factor binding protein-2 regulation by protein kinase A.

OBJECTIVE: Primary pigmented nodular adrenocortical disease (PPNAD) can occur as an isolated trait or part of Carney complex, a familial lentiginosis-multiple endocrine neoplasia syndrome frequently caused by mutations in PRKAR1A, which encodes the 1alpha regulatory subunit of protein kinase A (PKA). Because alterations in the insulin-like growth factor (IGF) axis, particularly IGF-II and IGF binding protein (IGFBP)-2 overexpression, have been implicated in sporadic adrenocortical tumors, we sought to examine the IGF axis in PPNAD. DESIGN: RNA samples and paraffin-embedded sections were procured from adrenalectomy specimens of patients with PPNAD. Changes in expression of IGF axis components were evaluated by real-time quantitative RT-PCR and immunohistochemistry. NCI-H295R cells were used to study PKA and IGF axis signaling in adrenocortical cells in vitro. RESULTS: IGFBP-2 mRNA level distinguished between the two genetic subtypes of this disease; increased IGFBP-2 expression in PRKAR1A mutation-positive PPNAD tissues was also confirmed by immunohistochemistry. Moreover, PKA inhibitors increased IGFBP-2 expression in NCI-H295R adrenocortical cells, and anti-IGFBP-2 antibody reduced their proliferation. CONCLUSIONS: IGFBP-2 expression is increased in PPNAD caused by PRKAR1A mutations, and in adrenocortical cancer cells. This is the first evidence for PKA-dependent regulation of IGFBP-2 expression in adrenocortical cells.     

Carney Complex Complicated with Primary Pigmented Nodular Adrenocortical Disease without Cushing's Syndrome Recurrence for Five Years after Unilateral Adrenalectomy

We herein report a case of Carney complex (CNC) complicated with primary pigmented nodular adrenocortical disease (PPNAD) after unilateral adrenalectomy. A 44-year-old woman was admitted to our hospital for PPNAD surgery. She had previously undergone surgery for cardiac myxoma and had a PRKAR1A mutation with no family history of CNC. She had Cushing''s signs, but her metabolic abnormalities were mild. Adrenal insufficiency due to poor medication adherence was a concern, so she underwent unilateral adrenalectomy. Cushing''s signs improved postoperatively and without recurrence for five years. Treatment plans for PPNAD should be determined based on the patient''s condition, medication adherence, and wishes.     

Mutations of the PRKAR1A gene in Cushing's syndrome due to sporadic primary pigmented nodular adrenocortical disease

Primary pigmented nodular adrenocortical disease (PPNAD) is a cause of ACTH-independent Cushing's syndrome. This condition can be difficult to diagnose because hypercortisolism may be periodic and adrenal imaging may not demonstrate an adrenal tumor. PPNAD can be part of the Carney complex (CNC), an autosomal dominant multiple neoplasia syndrome. Germline mutations of the regulatory subunit R1A of PKA (PRKAR1A) have been observed in about 45% of CNC kindreds. To improve our understanding of sporadic PPNAD and develop a potential diagnostic tool, we investigated the genetics of patients with sporadic and isolated PPNAD. Patients undergoing surgery for bilateral ACTH-independent Cushing's syndrome in whom pathological examination revealed PPNAD were subjected to endocrinological investigations and a systematic search for other manifestations of CNC. The PRKAR1A gene was sequenced using DNA from frozen adrenal tissues and leukocytes from three patients with sporadic isolated PPNAD and using leukocyte DNA from two additional patients. Different inactivating germline mutations of the PRKAR1A gene were found in the five patients. For three cases, study of the parents' DNA demonstrated a de novo mutation. One patient presented with an unusual 2.5-cm macronodule of the right adrenal mimicking an adrenal adenoma. A somatic 16-bp deletion of PRKAR1A gene was also found in this macronodule. Inactivating germline mutations of PRKAR1A are frequent in sporadic and isolated cases of PPNAD. The wild-type allele can be inactivated by somatic mutations, consistent with the hypothesis of the gene being a tumor suppressor gene. Thus, genetic analysis can be of help to the clinician in the diagnosis of this difficult form of adrenal Cushing's syndrome.     

Genetic alterations of Wnt signaling pathway-associated genes in hepatocellular carcinoma

Background and Aim: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Recently, abnormal activation of the Wnt pathway has been found to be involved in the carcinogenesis of HCC. However, the relationship between genetic changes in the Wnt pathway–associated genes and its protein expression has not been studied in patients with HCC and cirrhotic nodules. The purpose of this study is to explore the contribution of inappropriate activation of the Wnt pathway in liver carcinogenesis. Methods: Somatic mutation in exons 3–5 of AXIN1 and exon 3 of β‐catenin were analyzed by direct sequencing and expression of axin and β‐catenin proteins by immunohistochemistry in a series of 36 patients with HCC and cirrhosis. Results: The AXIN1 and β‐catenin gene mutations were observed in 25% (9/36) and 2.8% (1/36) of HCCs, respectively. All mutations detected in AXIN1 and β‐catenin genes were missense point mutations. Abnormal nuclear expression of β‐catenin was observed in 11 of 36 cases of HCCs (30.6%), but not in cirrhotic nodules. Reduced or absent expression of axin was seen in 24 of 36 HCCs (66.7%). The abnormal expression of β‐catenin and axin proteins was closely correlated with mutations of AXIN1 and β‐catenin (P < 0.0001 and P = 0.008, respectively). Conclusions: These data suggest that mutation of AXIN1 gene is a frequent and late event for HCC associated with cirrhosis, and is correlated significantly with abnormal expression of axin and β‐catenin. Therefore, activation of Wnt signaling through AXIN1 rather than β‐catenin mutation might play an important role in liver carcinogenesis.     

Cyclical Cushing syndrome presenting in infancy: an early form of primary pigmented nodular adrenocortical disease, or a new entity?

Cushing syndrome is uncommon in childhood and rare in infancy. We report the case of a 3-yr-old child who presented with symptoms of Cushing syndrome beginning shortly after birth. Her hypercortisolemia was cyclical, causing relapsing and remitting symptoms, which eventually led to suspicions of possible Munchausen syndrome by proxy. Investigation at the National Institutes of Health excluded exogenous administration of glucocorticoids and indicated ACTH-independent Cushing syndrome. Paradoxical response to dexamethasone stimulation (Liddle's test) suggested a diagnosis of primary pigmented nodular adrenocortical disease (PPNAD).After bilateral adrenalectomy, both glands showed micronodular adrenocortical hyperplasia, but histology was not consistent with typical PPNAD. DNA analysis of the coding sequences of the PRKAR1A gene (associated with PPNAD and Carney complex) and the GNAS gene (associated with McCune-Albright syndrome) showed no mutations.We conclude that hypercortisolemia in infancy may be caused by micronodular adrenocortical hyperplasia, which can be cyclical and confused with exogenous Cushing syndrome. A paradoxical rise of glucocorticoid excretion during Liddle's test may delineate these patients. Infantile micronodular disease has some features of PPNAD and may represent its early form; however, at least in the case of the patient reported here, micronodular hyperplasia was not caused by coding mutations of the PRKAR1A or GNAS genes or associated with typical histology or any other features of Carney complex or McCune-Albright syndrome and may represent a distinct entity.     

A novel PRKAR1A mutation associated with hepatocellular carcinoma in a young patient and a variable Carney complex phenotype in affected subjects in older generations

Context Carney complex (CNC) is an autosomal dominant multiple endocrine neoplasia syndrome (OMIM 160980). About 70% of cases are familiar; most have mutations of the PRKAR1A gene on chromosome 17q22–24. There is little phenotype–genotype correlation known to date. Objective To study the genotype–phenotype correlation in a family with newly diagnosed CNC and three generations of subjects bearing the same PRKAR1A mutation. The proband was diagnosed with hepatocellular carcinoma, a tumour that appears to be associated with CNC. Design The study consisted of clinical and genetic analysis of a total of 10 individuals belonging to a large Italian family. Patients The index case was referred for PRKAR1A gene mutation analysis because he met the diagnostic criteria for a clinical diagnosis of CNC. Results The PRKAR1A‐inactivating mutation c.502 +1G > A in the intron 5 splice‐donor site was detected after bidirectional sequencing of germline DNA. The mutation causes a frameshift in the transcribed sequence and a nonsense mRNA that was shown to be degraded; this leads to PRKAR1A haploinsufficiency in all tissues. All available relatives were screened first by DNA testing and, if the latter was positive, by clinical, biochemical and imaging means. Conclusions A novel PRKAR1A mutation with an apparently low penetrance and variable expression is reported; the same mutation is also associated with a hepatocellular carcinoma. This is the first time a PRKAR1A mutation is reported in individuals who were diagnosed with CNC after retrospective family screening and following the identification of a proband; the finding has implications for genetic counselling on PRKAR1A and/or CNC.     

Primary pigmented nodular adrenocortical disease: paradoxical responses of cortisol secretion to dexamethasone occur in vitro and are associated with increased expression of the glucocorticoid receptor

Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent adrenal Cushing's syndrome (CS), which is often associated with Carney complex (CNC). We have recently described a paradoxical increase in cortisol excretion after dexamethasone administration in most patients with PPNAD. In the present study we investigated the hypothesis that this phenomenon is due to a primary abnormality of the tissues affected by PPNAD, rather than a defect of the patients' hypothalamic-pituitary-adrenal axis; as such it should be replicated in vitro by adrenal slices exposed directly to dexamethasone. We were able to study adrenal tissues from eight patients with CS caused by PPNAD; two patients were also studied in vivo according to a protocol first described in ACTH-independent macronodular adrenal hyperplasia (AIMAH) for the clinical detection of aberrant hormone receptor expression. Their DNA has been previously screened for inactivating mutations of the PRKAR1A gene, the most frequent molecular defect leading to PPNAD and/or CNC. We also investigated whether glucocorticoid receptor (GR) expression underlies paradoxical dexamethasone responses in PPNAD by immunohistochemistry and semiquantitative PCR, and we correlated GR expression with that of other markers for PPNAD (e.g. synaptophysin). Indeed, we demonstrated that dexamethasone induced cortisol secretion in vitro in five of these tumors; no such increase was seen in adenomatous or AIMAH tissues that were treated in the same manner. GR mRNA was expressed, and GR immunoreactivity was detected in PPNAD nodular cells. Staining for GR was not seen in surrounding cortical cells, and hence, it correlated with synaptophysin, which also stains PPNAD in a similar manner. In normal adrenal tissue, GR was detected mostly in medullary areas, whereas GR immunoreactivity was weak in adenomatous and AIMAH tissues. We conclude that 1) dexamethasone produces an increase in glucocorticoid synthesis by PPNAD adrenal slices in vitro, suggesting a direct effect on adrenocortical tissue, and 2) this phenomenon is accompanied by increased expression of the GR in PPNAD nodules. PPNAD and/or CNC patients with and without mutations leading to protein kinase A activation demonstrated in vitro and/or in vivo paradoxical dexamethasone responses and GR expression, indicating that PRKAR1A alterations are not necessary for these phenomena.