Sporadic forms of PA represent the majority of cases and are due either to bilateral adrenal hyperplasia or APA. APA are found in approximately one- third of patients, although frequency may vary depending on diagnostic criteria and thresholds used for subtype identification. By applying whole exome sequencing to paired tumour and germline DNA samples, it has been possible to identify recurrent somatic mutations in different genes coding for ion channels (KCNJ5 and CACNA1D) and ATPases (ATP1A1 and ATP2B3) regulating intracellular ionic homeostasis and cell membrane potential. Mutations of KCNJ5 and CACNA1D are similar to those identified in FH- III and PASNA and lead to increased intracellular calcium concentrations, which activate calcium signalling and aldosterone biosynthesis. Mutations in ATP1A1, coding for the α1 subunit of the Na+, K+- ATPase, lead to a loss of pump activity and inward proton or sodium leak, which increase aldosterone production through cell membrane depolarization and increased calcium influx or increased intracellular acidification. ATP2B3 codes for the plasma membrane calcium- transporting ATPase 3 (PMCA3). Mutations lead to loss of the physiological pump function and reduced Ca2+ export; they also lead to severely depolarized membrane potential, which is explained by a pathological sodium leak associated with or con ducted by the mutant PMCA3. Finally, somatic mutations in CTNNB1, coding for β- catenin which plays an important role in adrenal cortex development and tumorigenesis, are found in 2– 5% of APA, and mutations in PRKACA (coding for the cAMP- dependent protein kinase catalytic subunit alpha) in rare cases. Mutations in those genes are also found in cortisol- producing adenoma and adrenocortical cancer; the mechanisms whereby the same mutations lead to different hormonal phenotypes remain yet to be established.
KCNJ5 mutations are the most prevalent mutations found in ≈40% of APA, with a higher prevalence in Asian populations. Carriers of KCNJ5 mutations are younger and more often women. Some studies also report larger tumour size, a ZF- like cellular phenotype, and higher plasma aldosterone levels when compared to non- carriers, but these results, as well as other genotype- phenotype correlations, are not replicated in other studies. ATPase mutations were reported to be more frequent in men and CACNA1D mutations are associated mainly with smaller tumours. CTNNB1 mutations seem to be more prevalent in women.
Recent data indicate that the frequency of somatic mutations in APA might be much higher than previously described, due to tumour heterogeneity and the presence of multiple nodules, which do not all express aldosterone synthase. Indeed, aldosterone synthase immunohistochemistry- guided targeted sequencing on DNA extracted from formalin- fixed paraffin embedded adrenal samples revealed an overall frequency of somatic mutations of 88%, with a similar mutational spectrum then in previous studies.
Genetic testing in patients with APA prior to surgery is challenged by the difficulty of detecting somatic mutations from peripheral blood. This could be circumvented by identifying surrogate biomarkers of the mutation status. Genotype- specific steroid profiles have been recently associated with APA, in particular a 7- steroid fingerprint able to correctly classify 92% of the APA according to genotype. Future use of this type of screening could allow to select patients for adrenal vein sampling and to apply targeted treatments, once these approaches have been prospectively validated. Indeed, mutated KCNJ5 channels have peculiar pharmacological characteristics and are blocked by calcium- channel blockers, such as verapamil, and Na- channel blockers, such as amiloride at high therapeutic doses. Macrolide antibiotics, including roxithromycin, potently inhibit mutant channels as well as CYP11B2 expression and aldosterone production in adrenocortical cells and in primary cell lines from APA. Current studies are ongoing to test whether these compounds can be used to identify and treat patients with PA due to APA with KCNJ5 mutations.
