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Why are somatic mutations in the adrenal gland a common cause of human hypertension? Supervisor: Professor Morris Brown, Clinical Pharmacology Unit. Project code for applications: SCM041 Background: Hypertension is frequently due to aldosterone-producing adenomas (APA) of the adrenal. Most APAs have appeared to arise not in the physiological site of aldosterone production, the zona glomerulosa (ZG), but in the adjacent zona fasciculata (ZF). This paradox was resolved with our discovery that APAs of the ZG do indeed occur, but are usually much smaller than ZF-APAs, and easily missed on CT scans.1 However, our finding of frequent, specific somatic mutations in ZG, distinct from those causing ZF-APAs, raises the further puzzle of how mutation can commonly arise in a non-proliferative tissue, and be selected in small, non-proliferative tumours.2 We have found a probable solution in the transcriptome of the human adrenal, together with immunohistochemical and functional analysis of key genes, upregulated in ZG vs ZF. Among these were the stem-cell marker and Wnt-signalling gene, LGR5 (>50-fold), DACH1 (23-fold), and several other Wnt signalling genes, including the LGR5-ligand, RSPO3. Transfection and silencing of LGR5 or DACH1, in human adrenal cells, and responses to RSPO3, show each of these to regulate aldosterone production, in part through regulation of cell turnover. ZG of human adrenal shows significant positivity for markers of apoptosis and proliferation. These findings are all unique to human adrenal. Hypothesis: Because [i] deletion of the gene encoding aldosterone synthesis (CYP11B2) in mice, or treatment of other mammals with a CYP11B2 inhibitor, leads to proliferation, migration and apoptosis of ZG cells, and [ii] salt excess suppresses aldosterone production, we have developed the hypothesis that: ZG cells are driven by Wnt to switch between synthetic and ‘proliferative’ modes; that, high salt intake by humans generally suppresses aldosterone production, hence favouring proliferation etc; and the proliferation permits frequent mutations, with those leading to constitutive aldosterone production being selected because protective against apoptosis. The hypothesis would explain why human ZG looks so much thinner, with only patchy CYP11B2 expression, compared to other species; and why several genes which are upregulated many-fold in normal ZG, but not in ZG-like APAs, have a role in suppressing aldosterone production, in part by directing the cells into either apoptosis or migration to the inner adrenal zones. The hypothesis would also explain why ZG-like APAs are so small, namely the cells have a survival advantage not through proliferation, but by switching on genes that maintain constitutive aldosterone production. Project and techniques: The PhD student will be part of a vibrant group studying genes expressed in the human adrenal, which are of interest either because frequently mutated, or because upregulated in ZG. Most of the upregulated genes have yet to be studied. The aim is to test our hypothesis above, and to explain why APAs are one of the commonest tumours in Medicine. Several of the findings are being introduced into the routine diagnosis of APAs at an earlier stage e.g. through detection of somatic mutations in adrenal vein blood of patients with an adrenal adenoma on CT or MR scan, or of upregulated genes in ultrasound-guided aspirations from adenomas. The student will have the opportunity to be part of this clinical innovation, while learning techniques that include: highthroughput DNA sequencing, RNA analyses, immunohistochemistry, 2- and 3-D cell culture, transfection, silencing RNA, radioimmunoassay. Elena Azizan undertook the studies cited below during her PhD, and won Young Investigator prizes at the International Society of Hypertension, and American Endocrine Society. 1. Azizan EA, Lam BY, Newhouse SJ, et al. Microarray, qPCR, and KCNJ5 sequencing of aldosterone-producing adenomas reveal differences in genotype and phenotype between zona glomerulosa- and zona fasciculata-like tumors. J Clin Endocrinol Metab. 2012; 97(5): E819-29. 2. Azizan EA, Poulsen H, Tuluc P, et al. Somatic mutations in ATP1A1 and CACNA1D underlie a common subtype of adrenal hypertension. Nat Genet. 2013; 45(9): 1055-60.