Role of two genes, CACNA1D and CADM1, with common or rare mutations in aldosterone producing adenomas of the adrenal.

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

Rare variants in single-minded 1 (SIM1) are associated with severe obesity

Single-minded 1 (SIM1) is a basic helix-loop-helix transcription factor involved in the development and function of the paraventricular nucleus of the hypothalamus. Obesity has been reported in Sim1 haploinsufficient mice and in a patient with a balanced translocation disrupting SIM1. We sequenced the coding region of SIM1 in 2,100 patients with severe, early onset obesity and in 1,680 controls. Thirteen different heterozygous variants in SIM1 were identified in 28 unrelated severely obese patients. Nine of the 13 variants significantly reduced the ability of SIM1 to activate a SIM1-responsive reporter gene when studied in stably transfected cells coexpressing the heterodimeric partners of SIM1 (ARNT or ARNT2). SIM1 variants with reduced activity cosegregated with obesity in extended family studies with variable penetrance. We studied the phenotype of patients carrying variants that exhibited reduced activity in vitro. Variant carriers exhibited increased ad libitum food intake at a test meal, normal basal metabolic rate, and evidence of autonomic dysfunction. Eleven of the 13 probands had evidence of a neurobehavioral phenotype. The phenotypic similarities between patients with SIM1 deficiency and melanocortin 4 receptor (MC4R) deficiency suggest that some of the effects of SIM1 deficiency on energy homeostasis are mediated by altered melanocortin signaling.Shwetha Ramachandrappa, Anne Raimondo, Anna M.G. Cali, Julia M. Keough, Elana Henning, Sadia Saeed, Amanda Thompson, Sumedha Garg, Elena G. Bochukova, Soren Brage, Victoria Trowse, Eleanor Wheeler, Adrienne E. Sullivan, Mehul Dattani, Peter E. Clayton, Vippan Datta, John B. Bruning, Nick J. Wareham, Stephen O'Rahilly, Daniel J. Peet, Ines Barroso, Murray L. Whielaw and I. Sadaf Farooq

KSR2 mutations are associated with obesity, insulin resistance, and impaired cellular fuel oxidation.

Kinase suppressor of Ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Targeted deletion of Ksr2 leads to obesity in mice, suggesting a role in energy homeostasis. We explored the role of KSR2 in humans by sequencing 2,101 individuals with severe early-onset obesity and 1,536 controls. We identified multiple rare variants in KSR2 that disrupt signaling through the Raf-MEKERK pathway and impair cellular fatty acid oxidation and glucose oxidation in transfected cells; effects that can be ameliorated by the commonly prescribed antidiabetic drug, metformin. Mutation carriers exhibit hyperphagia in childhood, low heart rate, reduced basal metabolic rate and severe insulin resistance. These data establish KSR2 as an important regulator of energy intake, energy expenditure, and substrate utilization in humans. Modulation of KSR2-mediated effects may represent a novel therapeutic strategy for obesity and type 2 diabetes.This work was supported by the Wellcome Trust (098497/Z/12/Z; 077016/Z/05/Z; 096106/Z/11/Z) (ISF and LRP), Medical Research Council (MC_U106179471) (NW), NIHR Cambridge Biomedical Research Centre (ISF, IB and SOR), and European Research Council (ISF). This study makes use of data generated by the UK10K Consortium (WT091310). A full list of the investigators who contributed to the generation of the data is available from http://www.UK10K.org.This is the final published version. It first appeared at http://www.cell.com/abstract/S0092-8674%2813%2901276-2

Rare Variant Analysis of Human and Rodent Obesity Genes in Individuals with Severe Childhood Obesity

A. Palotie on työryhmän UK10K Consortium jäsen.Obesity is a genetically heterogeneous disorder. Using targeted and whole-exome sequencing, we studied 32 human and 87 rodent obesity genes in 2,548 severely obese children and 1,117 controls. We identified 52 variants contributing to obesity in 2% of cases including multiple novel variants in GNAS, which were sometimes found with accelerated growth rather than short stature as described previously. Nominally significant associations were found for rare functional variants in BBS1, BBS9, GNAS, MKKS, CLOCK and ANGPTL6. The p.S284X variant in ANGPTL6 drives the association signal (rs201622589, MAF similar to 0.1%, odds ratio = 10.13, p-value = 0.042) and results in complete loss of secretion in cells. Further analysis including additional case-control studies and population controls (N = 260,642) did not support association of this variant with obesity (odds ratio = 2.34, p-value = 2.59 x 10(-3)), highlighting the challenges of testing rare variant associations and the need for very large sample sizes. Further validation in cohorts with severe obesity and engineering the variants in model organisms will be needed to explore whether human variants in ANGPTL6 and other genes that lead to obesity when deleted in mice, do contribute to obesity. Such studies may yield druggable targets for weight loss therapies.Peer reviewe

Radical housing and socially-engaged art – Reflections from a tenement town in Delhi’s extensive urbanisation

The relegation of workers housing to grey spaces or planned illegalities has ensured the availability of a permanently temporary migrant workforce to fuel the extensive urbanisation shaping around cities like Delhi. These grey spaces emerge as tenement towns in place of former agrarian villages, run and developed by agricultural landowners turned tenement entrepreneurs that exploit labour migrants as permanently temporary tenants. The challenges posed by the invisibilisation of such relations manifested sharply under the COVID-19 pandemic. A radical feminist politics has been emerging at the interstices as a counter against such violent dispossession employing creative narratives and ways of protest. This paper discusses the need for approaches that transcend research-activism boundaries while engaging with particularly marginalised communities. It discusses the potentials presented by socially engaged art in empowering radical politics through reflections from a long-term art-based inquiry conducted with women migrants in Kapashera, a tenement town located around Delhi

Pregnancy, Primary Aldosteronism, and Adrenal CTNNB1 Mutations.

This is a metadata record relating to an article that cannot be shared due to publisher copyright.Recent discoveries of somatic mutations permit the recognition of subtypes of aldosterone-producing adenomas with distinct clinical presentations and pathological features. Here we describe three women with hyperaldosteronism, two who presented in pregnancy and one who presented after menopause. Their aldosterone-producing adenomas harbored activating mutations of CTNNB1, encoding β-catenin in the Wnt cell-differentiation pathway, and expressed LHCGR and GNRHR, encoding gonadal receptors, at levels that were more than 100 times as high as the levels in other aldosterone-producing adenomas. The mutations stimulate Wnt activation and cause adrenocortical cells to de-differentiate toward their common adrenal-gonadal precursor cell type. (Funded by grants from the National Institute for Health Research Cambridge Biomedical Research Centre and others.).AEDT is supported by Agency for Science, Technology and Research (A*STAR) Singapore and the Wellcome Trust (085686/Z/08/A); SG and LHS are supported by the British Heart Foundation (FS/14/75/31134; FS/11/35/28871); EABA was supported by the Tunku Abdul Rahman Centenary Fund (St Catharine's College, Cambridge, UK) and the Austin Doyle Award (Servier Australia). JZ was supported by the Cambridge Overseas Trust Scholarship. MG is supported by the NIHR Cambridge Biomedical Research Centre (Metabolic), and MB is supported by the MRC (U105192713). The work was funded by the NIHR Cambridge Biomedical Research Centre (Cardiovascular) and an NIHR Senior Investigator award (NF-SI-0512-10052) to MJB