Primary aldosteronism (PA) accounts for 5-10% of all hypertension. One of the major causes of PA is sporadic formation of aldosterone-producing adenomas (APAs). These benign tumours develop in the cortical region of adrenal glands and autonomously secrete excessive amounts of aldosterone. This hormone increases sodium retention and water reabsorption by the kidneys, leading to high blood pressure. Landmark discoveries of somatic mutations in APAs led to better understanding of molecular mechanisms causing autonomous aldosterone secretion. The first mutations were found in KCNJ5, followed by ATP1A1, ATP2B3 and CACNA1D, all encoding cation-channels or transporters. Several in vitro studies showed disruption of cellular ion-balance leading to the phenotype of hyper-aldosterone secretion from APAs. Following our lab's discovery of initial four somatic mutations by whole exome sequencing, over 30 single-base change mutations have been reported in the CACNA1D gene, which encodes the a1 subunit of an L-type Ca2+ channel (LTCC), CaV1.3. Initial and several subsequent mutations cause electrophysiological gain-of-function with increased activation and/or slowed inactivation of CaV1.3. Prior to the discovery of these mutations, L-type Ca2+ channels were not considered important in regulation of aldosterone production. In the first part of my thesis, I investigated two of the mutations and showed that the gain-of-function results in increased aldosterone secretion from an adrenocortical carcinoma cell line, H295R, when transiently transfected with the mutants. I also showed that CaV1.3 can play a role in physiological aldosterone secretion, finding that CYP11B2 expression is reduced by 50% in the adrenals of CaV1.3 knockout mice. The discovery of mutations in CACNA1D led to a drug discovery challenge award from a pharmaceutical company in which high-throughput screening of CaV1.3-expressing cells was undertaken against the company's 1.8M compound library. I identified the adrenal isoforms of the channel's alpha and beta subunits (CACNA1D and CACNB2), and helped development of the stable HEK293 cell line used for screening. This led to 3 tool compounds (A, B & C) that were selective antagonists for CaV1.3 over another family member of the ion channels in high-throughput electrophysiological experiments using IonWorks Barracuda and QPatch platforms. I showed compound B to effectively inhibit aldosterone secretion in both H295R and primary adrenal cells isolated from a normal adrenal. This finding is a significant step in developing compound B further into a CaV1.3-selective drug for treating PA patients without cardiovascular side effects as in the case of existing dihydropyridine class of Ca2+ channel blockers. The second part of my thesis focused on genotyping and whole exome sequencing of 59 APAs from 52 patients, in order to identify further genes underlying primary aldosteronism. Mutations in previously reported genes were identified in 34 of the APAs (57.6%). CACNA1D was the most commonly mutated gene (20.3%) in this cohort, but not KCNJ5 (16.9%) as previously reported. This variation in the frequencies observed is perhaps due to the different methods used for screening PA. For example, many of our patients were detected by renin measurement in resistant hypertension, and their APA identified by a unique PET-CT (using C11 metomidate), in place of adrenal vein sampling. In addition to this, novel somatic mutation was found in a gene not encoding an ion channel, however, this protein was previously linked to cell-cell adhesion and tumour suppression. The gene identified is CADM1, a cell adhesion molecule 1, and the mutation found leads to substitution of uncharged by negatively charged amino acid in the single transmembrane domain of this cell surface protein. The likely significance of this discovery was greatly enhanced when we ascertained that one of the 'private' somatic mutations found on whole exome sequencing of APAs in Munich was in fact a similar substitution in the adjacent amino acid of the membrane-spanning domain. High expression of CADM1 in zona glomerulosa (ZG) was found, the site of aldosterone synthesis in the adrenal cortex and in the APAs, as well as the aldosterone producing cell clusters (APCCs) within the ZG. In vitro experiments using H295R cells showed both mutations in CADM1 lead to 10-20 fold upregulation of CYP11B2 transcription, on qPCR, resulting in 2-4-fold increase of aldosterone secretion, compared to the wild-type CADM1. Despite the introduction of a negative charge into the transmembrane domain, both mutants could translocate to the cell surface. The evidence to date, points to the loss of cell-cell adhesion in the presence of mutant CADM1 as the cause of uncontrolled aldosterone synthesis. Silencing of CADM1 in H295R cells revealed downregulation of aldosterone synthesis and secretion. Transcriptome analysis by RNAseq, of H295R cells expressing wild-type or mutant CADM1 or silenced CADM1 showed a large number of differentially expressed genes. Mutant CADM1 upregulated genes involved in steroidogenesis and ACTH response pathways. A possible role of CADM1 was found to be in the regulation of inter-cell communication via gap junction protein, connexin-43 (Cx43). This was upregulated with higher expression on plasma membrane in the CADM1 silenced cells. TSG101, a protein involved in lysosomal degradation of Cx43 was downregulated in the absence of CADM1 and possibly the mechanism for increased Cx43 expression. Also, immunostaining of adrenal sections showed internalised para-nuclear staining localisation of Cx43 in the ZG, APAs and APCCs, regions with high CADM1 expression compared to membranous localisation of Cx43 in ZF. In contrast to the common and numerous mutations in CACNA1D, mutations in CADM1 are rare. Nonetheless, they may enhance our understanding of the functional significance of glomerular structure of the outer zone of adrenal cortex, where cell-cell adhesion and intercellular communication appear critical for the regulation of aldosterone secretion.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:767736 |
| Date | January 2019 |
| Creators | Garg, Sumedha |
| Contributors | Gurnell, Mark |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/289390 |
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