CTNNB1-Mutant Aldosterone-Producing Adenomas With Somatic Mutations of GNA11/GNAQ Have Distinct Phenotype and Genotype
Authors: Zhou Junhua , Boulkroun Sheerazed , Cabrera Claudia P , Azizan Elena A B , Fernandes-Rosa Fabio , Cottrell Emily , Argentesi Giulia , Wu Xilin , O’Toole Sam , Marker Alison , Jordan Suzanne , Berney Daniel M , Lines Kate , Metherell Louise , Teo Ada , Thakker Rajesh V , Drake William , Wozniak Eva , Mein Charles A , Storr Helen L , Zennaro Maria-Christina , Brown Morris J 
Published: May 2021

Background: We report (this meeting) somatic mutation of GNA11/Q in CTNNB1-mutant APAs. The recurrent co-driver mutation causes reversible hypertension in puberty, pregnancy, or menopause. We have investigated the molecular mechanism of this association. Methods: Gene expression profiles in 3 double mutant APAs were studied by unsupervised hierarchical clustering analysis and enrichment analysis of 362 differentially expressed genes and validated by qPCR, IFC and IHC in 10 double mutant APAs or transfected primary adrenal cells. Multiple region biopsies were performed in 7 adrenals adjacent to double-mutant APAs and 4 APAs with KCNJ5 or CACNA1D mutations. The findings of APA mutations in adjacent adrenals were replicated in each case by ddPCR ± NGS. Results: Unsupervised hierarchical clustering analysis showed clustering of the double-mutant APAs, and a high proportion of genes were many-fold upregulated compared to other APAs. LHCGR, TMEM132E, DKK1, C9orf84, FAP, GNRHR and MPP3 are among the genes with high expression. A small number of genes are down-regulated in the double-mutant APAs, including CYP11B1. qPCR confirmed an average of ~10 to 1000-fold higher expression of the hallmark genes in double-mutants. Enrichment analysis showed significant enrichment of features or terms concerned with cell junction and cell adhesion (P&lt;10–8). IFC confirmed LHCGR intensity was 31–144 fold higher in primary adrenal cells with GNA11-p.Gln209Pro transfection and high CTNNB1 intensity. LHCGR intensity was qualitatively and quantitatively associated with immunofluorescence for CTNNB1. IHC of double-mutant APAs showed absent CYP11B1 but strong staining of CYP11B2. qPCR confirmed a lower CYP11B1/CYP11B2 ratio and a higher LHCGR expression (P&lt;10–3, both). IHC confirmed LHCGR positivity in double-mutant APAs but distribution varied both within and between cells. Adjacent ZG was hyperplastic, with absence of both CYP11B1 and CYP11B2 staining, but weak/moderate staining for LHCGR. The same GNA11 ± CTNNB1 somatic mutations were detected in multiple regions of the adjacent adrenals to 3 double mutant APAs. qPCR of hallmark APA genes differed from the APAs. High concordance between ddPCR, NGS and Sanger sequencing of the findings of APA mutations in adjacent adrenals when analysed in the same sample. No mutations were found in 4 adrenals adjacent to APAs with KCNJ5 or CACNA1D mutations, nor in other 4 adrenals adjacent to double-mutant APAs. Conclusions: Patients harboring APAs with somatic mutations in both GNA11/GNAQ Q209 and CTNNB1 have distinct phenotype in both the APAs and their adjacent adrenals. Same GNA11 ± CTNNB1 somatic mutations were found in the adjacent adrenals to double mutant APAs. We infer that a double-hit within related pathways is more likely than a single-hit to cause large increases in expression of LHCGR, and of other genes which may influence clinical presentation.