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. 2014 Mar 13;370(11):1019-28.
doi: 10.1056/NEJMoa1310359. Epub 2014 Feb 26.

Constitutive activation of PKA catalytic subunit in adrenal Cushing's syndrome

Felix Beuschlein  1 , Martin Fassnacht, Guillaume Assié, Davide Calebiro, Constantine A Stratakis, Andrea Osswald, Cristina L Ronchi, Thomas Wieland, Silviu Sbiera, Fabio R Faucz, Katrin Schaak, Anett Schmittfull, Thomas Schwarzmayr, Olivia Barreau, Delphine Vezzosi, Marthe Rizk-Rabin, Ulrike Zabel, Eva Szarek, Paraskevi Salpea, Antonella Forlino, Annalisa Vetro, Orsetta Zuffardi, Caroline Kisker, Susanne Diener, Thomas Meitinger, Martin J Lohse, Martin Reincke, Jérome Bertherat, Tim M Strom, Bruno Allolio
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Constitutive activation of PKA catalytic subunit in adrenal Cushing's syndrome

Felix Beuschlein et al. N Engl J Med. .

Abstract

Background: Corticotropin-independent Cushing's syndrome is caused by tumors or hyperplasia of the adrenal cortex. The molecular pathogenesis of cortisol-producing adrenal adenomas is not well understood.

Methods: We performed exome sequencing of tumor-tissue specimens from 10 patients with cortisol-producing adrenal adenomas and evaluated recurrent mutations in candidate genes in an additional 171 patients with adrenocortical tumors. We also performed genomewide copy-number analysis in 35 patients with cortisol-secreting bilateral adrenal hyperplasias. We studied the effects of these genetic defects both clinically and in vitro.

Results: Exome sequencing revealed somatic mutations in PRKACA, which encodes the catalytic subunit of cyclic AMP-dependent protein kinase (protein kinase A [PKA]), in 8 of 10 adenomas (c.617A→C in 7 and c.595_596insCAC in 1). Overall, PRKACA somatic mutations were identified in 22 of 59 unilateral adenomas (37%) from patients with overt Cushing's syndrome; these mutations were not detectable in 40 patients with subclinical hypercortisolism or in 82 patients with other adrenal tumors. Among 35 patients with cortisol-producing hyperplasias, 5 (including 2 first-degree relatives) carried a germline copy-number gain (duplication) of the genomic region on chromosome 19 that includes PRKACA. In vitro studies showed impaired inhibition of both PKA catalytic subunit mutants by the PKA regulatory subunit, whereas cells from patients with germline chromosomal gains showed increased protein levels of the PKA catalytic subunit; in both instances, basal PKA activity was increased.

Conclusions: Genetic alterations of the catalytic subunit of PKA were found to be associated with human disease. Germline duplications of this gene resulted in bilateral adrenal hyperplasias, whereas somatic PRKACA mutations resulted in unilateral cortisol-producing adrenal adenomas. (Funded by the European Commission Seventh Framework Program and others.).

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Figures

Figure 1
Figure 1. Identification of Somatic PRKACA Mutation and Germline Genetic Duplications
Panel A shows a sequence chromatogram of normal adrenal tissue, and Panel B a chromatogram of a cortisol-producing adrenal adenoma. A somatic mutation in PRKACA (c.617A→C) was identified in the cortisol-producing adenoma, resulting in a Leu206Arg substitution that is not present in the adjacent normal tissue. Panel C shows an ideogram of chromosome 19 (top) with the genes included in the p13.2–p13.12 band (GRCh37/h19). The black blocks (bottom) represent the size and position of the duplications. The gray blocks represent the genes included only in the shared region of duplication. The red block represents PRKACA.
Figure 2
Figure 2. Functional Characterization of PRKACA Variants
Panel A shows the structure derived from the protein kinase A (PKA) tetramer, with the nonmutant catalytic subunit (Cα) depicted in green and the regulatory subunit (RIIβ) depicted in red. A zoomed view into the region of Leu206 in the Cα subunit is shown. Leu206 is depicted as a space-filling representation; the two residues in close proximity (Val115 and Tyr228) and additional residues from the inhibitory site (Arg111–Ser114, marked with an asterisk) of the regulatory subunit are depicted as sticks. Panel B shows the same region of the PKA tetramer, with Leu206 in the Cα subunit replaced by Arg206, also depicted as a space-filling representation. Panel C shows PKA activity of nonmutant and mutant PKA Cα subunits transfected in human embryonic kidney 293 cells, as determined by means of fluorescence resonance energy transfer (FRET) assay with a PKA reporter (for details, see Fig. S4 in the Supplementary Appendix). The results indicate that the mutant variants are constitutively active. Asterisks indicate P<0.05 for the comparison with Cαnonmutant + RIIβ. AKAR4-NES denotes a protein activity reporter 4 with a nuclear export signal. Panel D shows that high constitutive PKA activity was maintained when either mutant was cotransfected with an equal amount of non-mutant Cα subunit. Asterisks indicate P<0.05 for the comparison with Cαnonmutant + RIIβ. The data in Panels C and D were compared by means of a two-way analysis of variance followed by Bonferroni’s test. Panel E shows the quantification of enzymatic PKA activity; COS-7 cells were transfected with Cα (nonmutant or mutant) and RIIβ, with or without the addition of cyclic AMP (cAMP). Asterisks indicate P<0.05 for the comparison between samples with and those without the addition of cAMP. The hatch mark indicates P<0.05 for the comparison between samples transfected with nonmutant Cα subunit and those transfected with mutant Cα subunit without the addition of cAMP. In Panels C, D, and E, the I bars represent the standard error.
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