LB-FP-3 EXOME SEQUENCING REVEALS FREQUENT SOMATIC MUTATIONS OF NA+/K+-ATPASE AND VOLTAGE-DEPENDENT CA2+ CHANNEL SUBUNITS IN A ZONA GLOMERULOSA-LIKE SUBTYPE OF ALDOSTERONE-PRODUCING ADENOMAS (APA)

Program: Late-Breaking Abstracts
Session: SAT-LB-Late-Breaking Poster Session 1
Bench to Bedside
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-LB-06
Elena AB Azizan1, Hanne Poulsen2, Junhua Zhou3, Michael Clausen2, Carmela Maniero3, Elena Bochukova3, Wanfeng Zhao4, Lalarukh Haris Shaikh3, Cheryl Brighton3, Tanja Dekkers5, Bas Tops6, Anthony P Davenport1, Benno Küsters6, Jiri Ceral7, Giles Yeo3, Ian McFarlane8, Miroslav Solar7, Jaap Deinum5, I Sadaf Farooqi3, Poul Nissen2 and Morris Jonathan Brown*1
1University of Cambridge, Cambridge, United Kingdom, 2Aarhus University, Denmark, 3University of Cambridge, United Kingdom, 4Addenbrookes Hospital, Cambridge, United Kingdom, 5Radboud University Nijmegen Medical Center, Nijmegen, Netherlands, 6Radboud University Nijmegen Medical Centre, Netherlands, 7Charles University - Faculty of Medicine in Hradec Kralove, Czech Republic, 8Addenbrooke’s Hospital, Cambridge, United Kingdom
Phenotypic differences between APAs with or without mutations of KCNJ5 prompted us to look for novel somatic mutations in a subgroup of ‘wild-type’ APAs. Ten were selected with histological features and gene expression profiles resembling normal adrenal zona glomerulosa (ZG) (1). APA and paired germline DNA were sent to BGI (Shenzen, China) for whole exome sequencing. Replication of novel mutations was tested in each of: 89 APAs of unknown genotype from two independent cohorts; 32 APAs of zona fasciculata (ZF)-like phenotype ± KCNJ5 mutations (n=20); and ten cortisol-secreting or non-secretory adenomas. APAs with novel mutations were compared by histology and immunohistochemistry (IHC) with KCNJ5-mutant APAs; and transcriptomes of the genotypically different APAs were compared by microarray both with each other and with adjacent ZG and ZF from which RNA had been separately collected by laser capture microdissection. The function of expressed mutants was studied in Xenopus oocytes and human adrenocortical (H295R) cells.

Exome sequencing showed nine of the 10 APAs to have a novel somatic mutation in one or other of two genes which encode either a Na+,K+-ATPase (n=4) or voltage-dependent Ca2+ channel (Cav) subunit (n=5). Two of the mutations occurred twice in the 10 samples, including the L104R mutation of ATP1A1 discovered independently, and replicated in 7/199 unselected APAs (2). This, and a 100-104del mutation spanning the same L104 residue, not only blocked Na+,K+ transport but caused a large inward leak of Na+ and H+ in oocytes; ATP1A1 L104R caused a 2-3 fold increase in aldosterone secretion and CYP11B2 expression in human adrenocortical cells. 5/39 APAs in a Czech cohort diagnosed in some cases by adrenal vein sampling (AVS) alone (i.e. with normal adrenal CT), had the L104R (n=4) or Cav mutations, compared to only 2/50 patients in a less selected Dutch cohort, and 0/42 of the controls. The function of the Cav mutations is not yet certain; but their clustering, conserved positions and recurrence in further APAs support a causal role. All APAs with the novel Na+,K+-ATPase or Cav mutations were <2 cms in diameter (eight were < 1cm), and had >40% compact cells. Unsupervised cluster analysis of the microarray data separated KCNJ5 mutant APAs from those with the new mutations; qPCR confirmed a cluster of genes which were more highly expressed both in normal ZG than ZF, and in APAs with the new mutations than the KCNJ5 mutants. IHC showed the Na+,K+-ATPase and Cavproteins themselves to be more abundant in ZG than ZF, and in the APAs of ZG-like phenotype.

The Na+,K+-ATPase and Cav mutations appear to define a subtype of APA, probably arising in ZG. The small size of the APAs (due partly to the more compact cells) and their higher prevalence in cohorts where absence of adenoma on CT or MRI did not preclude further investigation, suggest that ZG-like APAs are an easily overlooked, potentially curable cause of hypertension.

1. Azizan EA et al. J Clin Endocrinol Metab. 2012; 97: E819-29. 2. Beuschlein et al. Nat Genet. 2013; 45:440-4.

Nothing to Disclose: EAA, HP, JZ, MC, CM, EB, WZ, LH, CB, TD, BT, APD, BK, JC, GY, IM, MS, JD, ISF, PN, MJB

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm