Frequent protein kinase A regulatory subunit A1 mutations but no GNAS mutations as potential driver in sporadic cardiac myxomas.

PURPOSE: Cardiac myxomas (CMs) are the second most common benign primary cardiac tumors, mainly originating within the left atrium. Approximately 5% of CM cases are associated with Carney Complex (CNC), an autosomal dominant multiple neoplasia syndrome often caused by germline mutations in the protein kinase A regulatory subunit 1A (PRKAR1A). Data concerning PRKAR1A alterations in sporadic myxomas are variable and sparse, with PRKAR1A mutations reported to range from 0% to 87%. Therefore, we investigated the frequency of PRKAR1A mutations in sporadic CM using next-generation sequencing (NGS). Additionally, we explored mutations in the catalytic domain of the Protein Kinase A complex (PRKACA) and examined the presence of GNAS mutations as another potential driver. METHODS AND RESULTS: This study retrospectively collected histological and clinical data from 27 patients with CM. First, we ruled out the possibility of underlying CNC through clinical evaluations and standardized interviews for each patient. Second, we performed PRKAR1A immunohistochemistry (IHC) analysis and graded the reactivity of myxoma cells semi-quantitatively. NGS was then applied to analyze the coding regions of PRKAR1A, PRKACA, and GNAS in all 27 cases. Of the 27 sporadic CM cases, 13 (48%) harbored mutations in PRKAR1A. Among these 13 mutant cases, six displayed more than one mutation in PRKAR1A. Most of the identified mutations resulted in premature stop codons or affected splicing. In PRKAR1A mutant CM cases, the loss of PRKAR1A protein expression was significantly more common. In two cases with missense mutations, protein expression remained preserved. Furthermore, a single mutation was detected in the catalytic domain of the protein kinase A complex, while no GNAS mutations were found. CONCLUSION: We identified a relatively high frequency of PRKAR1A mutations in sporadic CM. These PRKAR1A mutations may also represent an important oncogenic mechanism in sporadic myxomas, as already known in CM cases associated with CNC.

Superficial Angiomyxomas Frequently Demonstrate Loss of Protein Kinase A Regulatory Subunit 1 Alpha Expression: Immunohistochemical Analysis of 29 Cases and Cutaneous Myxoid Neoplasms With Histopathologic Overlap.

Superficial angiomyxomas (SAMs) are benign cutaneous tumors that arise de novo and in the setting of the Carney complex (CC), an autosomal dominant disease with several cutaneous manifestations including lentigines and pigmented epithelioid melanocytomas. Although most SAM do not pose a diagnostic challenge, a subset can demonstrate histopathologic overlap with other myxoid tumors that arise in the skin and subcutis. Traditional immunohistochemical markers are of limited utility when discriminating SAM from histopathologic mimics. Since protein kinase A regulatory subunit 1 alpha (PRKAR1A) genetic alterations underlie most CC cases, we investigated whether SAM demonstrate loss of PRKAR1A protein expression by immunohistochemistry. In our series, 29 SAM, 26 myxofibrosarcoma, 5 myxoid dermatofibrosarcoma protuberans, 11 superficial acral fibromyxomas, and 18 digital mucous cysts were characterized. Of the 29 SAM examined in this study, 1 was associated with documented CC in a 5-year-old girl. SAM tended to arise in adults (mean 49.7 y; range: 5 to 87 y). Loss of PRKAR1A was seen in 55.2% of cases (16/29) and had a male predilection (87.5%, 12/16). PRKAR1A-inactivated SAM demonstrated significant nuclear enlargement (100%, 16/16 vs. 23.1%, 3/13), multinucleation (81.3%, 13/16 vs. 23.1%, 3/13), and presence of neutrophils (43.8%, 7/16 vs. 0%, 0/13). In contrast, PRKAR1A was retained in all cases of myxofibrosarcoma (100%, 26/26), myxoid dermatofibrosarcoma protuberans (100%, 5/5), superficial acral fibromyxomas (100%, 11/11), and digital mucous cyst (100%, 18/18). Taken together, PRKAR1A loss by immunohistochemistry can be used as an adjunctive assay to support the diagnosis of SAM given the high specificity of this staining pattern compared with histopathologic mimics.

Noncanonical protein kinase A activation by oligomerization of regulatory subunits as revealed by inherited Carney complex mutations.

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function.

The regulatory 1α subunit of protein kinase A modulates renal cystogenesis.

The failure of the polycystins (PCs) to function in primary cilia is thought to be responsible for autosomal dominant polycystic kidney disease (ADPKD). Primary cilia integrate multiple cellular signaling pathways, including calcium, cAMP, Wnt, and Hedgehog, which control cell proliferation and differentiation. It has been proposed that mutated PCs result in reduced intracellular calcium, which in turn upregulates cAMP, protein kinase A (PKA) signaling, and subsequently other proliferative signaling pathways. However, the role of PKA in ADPKD has not been directly ascertained in vivo, although the expression of the main regulatory subunit of PKA in cilia and other compartments (PKA-RIα, encoded by PRKAR1A) is increased in a mouse model orthologous to ADPKD. Therefore, we generated a kidney-specific knockout of Prkar1a to examine the consequences of constitutive upregulation of PKA on wild-type and Pkd1 hypomorphic (Pkd1RC) backgrounds. Kidney-specific loss of Prkar1a induced renal cystic disease and markedly aggravated cystogenesis in the Pkd1RC models. In both settings, it was accompanied by upregulation of Src, Ras, MAPK/ERK, mTOR, CREB, STAT3, Pax2 and Wnt signaling. On the other hand, Gli3 repressor activity was enhanced, possibly contributing to hydronephrosis and impaired glomerulogenesis in some animals. To assess the relevance of these observations in humans we looked for and found evidence for kidney and liver cystic phenotypes in the Carney complex, a tumoral syndrome caused by mutations in PRKAR1A These observations expand our understanding of the pathogenesis of ADPKD and demonstrate the importance of PRKAR1A highlighting PKA as a therapeutic target in ADPKD.

Functional Characterization of PRKAR1A Mutations Reveals a Unique Molecular Mechanism Causing Acrodysostosis but Multiple Mechanisms Causing Carney Complex.

The main target of cAMP is PKA, the main regulatory subunit of which (PRKAR1A) presents mutations in two genetic disorders: acrodysostosis and Carney complex. In addition to the initial recurrent mutation (R368X) of the PRKAR1A gene, several missense and nonsense mutations have been observed recently in acrodysostosis with hormonal resistance. These mutations are located in one of the two cAMP-binding domains of the protein, and their functional characterization is presented here. Expression of each of the PRKAR1A mutants results in a reduction of forskolin-induced PKA activation (measured by a reporter assay) and an impaired ability of cAMP to dissociate PRKAR1A from the catalytic PKA subunits by BRET assay. Modeling studies and sensitivity to cAMP analogs specific for domain A (8-piperidinoadenosine 3',5'-cyclic monophosphate) or domain B (8-(6-aminohexyl)aminoadenosine-3',5'-cyclic monophosphate) indicate that the mutations impair cAMP binding locally in the domain containing the mutation. Interestingly, two of these mutations affect amino acids for which alternative amino acid substitutions have been reported to cause the Carney complex phenotype. To decipher the molecular mechanism through which homologous substitutions can produce such strikingly different clinical phenotypes, we studied these mutations using the same approaches. Interestingly, the Carney mutants also demonstrated resistance to cAMP, but they expressed additional functional defects, including accelerated PRKAR1A protein degradation. These data demonstrate that a cAMP binding defect is the common molecular mechanism for resistance of PKA activation in acrodysosotosis and that several distinct mechanisms lead to constitutive PKA activation in Carney complex.

Defects of the Carney complex gene (PRKAR1A) in odontogenic tumors.

The surgical treatment of some odontogenic tumors often leads to tooth and maxillary bone loss as well as to facial deformity. Therefore, the identification of genes involved in the pathogenesis of odontogenic tumors may result in alternative molecular therapies. The PRKAR1A gene displays a loss of protein expression as well as somatic mutations in odontogenic myxomas, an odontogenic ectomesenchymal neoplasm. We used a combination of quantitative RT-PCR (qRT-PCR), immunohistochemistry, loss of heterozygosity (LOH) analysis, and direct sequencing of all PRKAR1A exons to assess if this gene is altered in mixed odontogenic tumors. Thirteen tumors were included in the study: six ameloblastic fibromas, four ameloblastic fibro-odontomas, one ameloblastic fibrodentinoma, and two ameloblastic fibrosarcomas. The epithelial components of the tumors were separated from the mesenchymal by laser microdissection in most of the cases. We also searched for odontogenic pathology in Prkar1a(+) (/) (-) mice. PRKAR1A mRNA/protein expression was decreased in the benign mixed odontogenic tumors in association with LOH at markers around the PRKAR1A gene. We also detected a missense and two synonymous mutations along with two 5'-UTR and four intronic mutations in mixed odontogenic tumors. Prkar1a(+) (/) (-) mice did not show evidence of odontogenic tumor formation, which indicates that additional genes may be involved in the pathogenesis of such tumors, at least in rodents. We conclude that the PRKAR1A gene and its locus are altered in mixed odontogenic tumors. PRKAR1A expression is decreased in a subset of tumors but not in all, and Prkar1a(+) (/) (-) mice do not show abnormalities, which indicates that additional genes play a role in this tumor's pathogenesis.

Sporadic Carney complex without PRKAR1A mutation in a young patient with ischemic stroke.

We describe a 29-year-old male, with a previous history of testicular tumor, who presented with a posterior circulation ischemic stroke associated to an atrial myxoma. Dermatologic observation disclosed spotty skin and mucosal pigmentation (lentigines), and a cutaneous myxoma was histopathologically confirmed. Although there was no family history of any of the Carney complex (CNC) features and no mutations in the PRKAR1A gene were found, these findings lead to the diagnosis of CNC. We emphasize the importance of recognizing this entity in young patients with stroke.

Differential role of PKA catalytic subunits in mediating phenotypes caused by knockout of the Carney complex gene Prkar1a.

The Carney complex is an inherited tumor predisposition caused by activation of the cAMP-dependent protein kinase [protein kinase A (PKA)] resulting from mutation of the PKA-regulatory subunit gene PRKAR1A. Myxomas and tumors in cAMP-responsive tissues are cardinal features of this syndrome, which is unsurprising given the important role played by PKA in modulating cell growth and function. Previous studies demonstrated that cardiac-specific knockout of Prkar1a causes embryonic heart failure and myxomatous degeneration in the heart, whereas limited Schwann cell-specific knockout of the gene causes schwannoma formation. In this study, we sought to determine the role of PKA activation in this phenotype by using genetic means to reduce PKA enzymatic activity. To accomplish this goal, we introduced null alleles of the PKA catalytic subunits Prkaca (Ca) or Prkacb (Cb) into the Prkar1a-cardiac knockout (R1a-CKO) or limited Schwann cell knockout (R1a-TEC3KO) line. Heterozygosity for Prkaca rescued the embryonic lethality of the R1a-CKO, although mice had a shorter than normal lifespan and died from cardiac failure with atrial thrombosis. In contrast, heterozygosity for Prkacb only enabled the mice to survive 1 extra day during embryogenesis. Biochemical analysis indicated that reduction of Ca markedly reduced PKA activity in embryonic hearts, whereas reduction of Cb had minimal effects. In R1a-TEC3KO mice, tumorigenesis was completely suppressed by a heterozygosity for Prkaca, and by more than 80% by heterozygosity for Prkacb. These data suggest that both developmental and tumor phenotypes caused by Prkar1a mutation result from excess PKA activity due to PKA-Ca.

Clinical and molecular genetics of Carney complex.

Carney complex (CNC) is a rare multiple familial neoplasia syndrome that is characterized by multiple types of skin tumors and pigmented lesions, endocrine neoplasms, myxomas and schwannomas and is inherited in an autosomal dominant manner. Clinical and pathologic diagnostic criteria are well established. Over 100 pathogenic variants in the regulatory subunit type 1A (RI-A) of the cAMP-dependent protein kinase (PRKAR1A) have been detected in approximately 60% of CNC patients, most leading to R1A haploinsufficiency. Other CNC-causing genes remain to be identified. Recent studies provided some genotype-phenotype correlations in CNC patients carrying PRKAR1A-inactivating mutations, which provide useful information for genetic counseling and/or prognosis; however, CNC remains a disease with significant clinical heterogeneity. Recent mouse and in vitro studies have shed light into how R1A haploinsufficiency causes tumors. PRKAR1A defects appear to be weak tumorigenic signals for most tissues; Wnt signaling activation and cell cycle dysregulation appear to be important mediators of the tumorigenic effect of a defective R1A.

Mutations and polymorphisms in the gene encoding regulatory subunit type 1-alpha of protein kinase A (PRKAR1A): an update.

PRKAR1A encodes the regulatory subunit type 1-alpha (RIalpha) of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA). Inactivating PRKAR1A mutations are known to be responsible for the multiple neoplasia and lentiginosis syndrome Carney complex (CNC). To date, at least 117 pathogenic variants in PRKAR1A have been identified (online database: http://prkar1a.nichd.nih.gov). The majority are subject to nonsense mediated mRNA decay (NMD), leading to RIalpha haploinsufficiency and, as a result, activated cAMP signaling. Recently, it became apparent that CNC may be caused not only by RIalpha haploinsufficiency, but also by the expression of altered RIalpha protein, as proven by analysis of expressed mutations in the gene, consisting of amino acid substitutions and in-frame genetic alterations. In addition, a new subgroup of mutations that potentially escape NMD and result in CNC through altered (rather than missing) protein has been analyzed-these are frame-shifts in the 3' end of the coding sequence that shift the stop codon downstream of the normal one. The mutation detection rate in CNC patients is recently estimated at above 60%; PRKAR1A mutation-negative CNC patients are characterized by significant phenotypic heterogeneity. In this report, we present a comprehensive analysis of all presently known PRKAR1A sequence variations and discuss their molecular context and clinical phenotype.