Discerning the genetic contributors to autonomous aldosterone production through whole genome sequencing

Malaysia, like the rest of the world, has a high prevalence of high blood pressure (hypertension), 32.7% for aged >18 years. Difficult to control hypertension can lead to co-morbidities and mortality as it is a major risk factor for stroke and heart diseases. Primary aldosteronism (PA), showing excess production of the hormone aldosterone from the adrenal glands, is probably the commonest curable cause of hypertension. While a recent report suggested that 60% of patients with hypertension have PA, a more conservative estimate, with concentration on curable patients, might suggest 5-10% of all patients, but in much higher proportions among those with treatment resistant hypertension, and ethnic groups with low-renin hypertension. In many patients, the discovery of a unilateral aldosterone-producing adenoma (APA) of the adrenal gland offers the opportunity for complete cure of hypertension. However developing country like Malaysia lacks the resources to identify these patients. Even making a reliable diagnosis of PA is currently fraught, requiring withdrawal of most first-line antihypertensive drugs from high-risk patients for the hormone (renin and aldosterone) measurements, often on more than one occasion if a confirmatory test (e.g saline or captopril suppression) is required. Hence, <1% of patients with PA are currently diagnosed. However, preventing excessive aldosterone is not only essential to attenuate high blood pressure which can lead to cardiovascular diseases, but also to prevent direct cardiac damage. We therefore aim in this proposal to identify the genetic contributors for autonomous aldosterone production, using the powerful whole genome sequencing technology, with the hope of developing a low-cost high-throughput peripheral blood assay that will allow mass diagnosis and targeted treatment of autonomous aldosterone production. Whole genome sequencing, a powerful technology to detect all the genetic sequences in an individual, will be able to identify heritable mutations associated with PA. Heredity is estimated to contribute 25-64% of inter-individual variation in blood pressure, yet currently genome-wide association studies have only identified ~3.5% of the genetic contribution with most hits occurring in the non-coding regions. This work will provide deeper genetic understanding of the disease development especially on the non gene-coding regulatory sequences and interplay between heritable germline mutations and somatic mutations that occur in the adrenal. Interestingly, one of the top hit GWAS polymorphism associated with PA in the recently released UK Biobank data is SMUG1, a DNA repair gene where the signature for its failure is C/T or G/A mutations. To note, almost half of aldosterone-producing cell clusters and micro-aldosterone-producing adenomas in PA patients have this signature. We hypothesize that WGS will discover informative, causal variants, e.g. in enhancer regions, which are rare (<1%) in control population databases, but >10-fold more frequent in documented PA with somatic mutation in aldosterone-driver genes. Such discovery may be rewarded by the potential for a clinically useful diagnostic test, a potential which is never realised by the common SNPs used in GWAS. We postulate that much of low-renin hypertension is due to undiagnosed autonomous aldosterone production and the low tech high throughput assay that can be developed from this study will cut cost of diagnosis and justify cheap preventive treatment despite spironolactone's known plethora of side effects. With the new information generated from this project, a simple genotyping test may be developed to identify hypertensive patients due to autonomous aldosterone production, which can then be treated by the cheap aldosterone receptor antagonist spironolactone, decreasing blood pressure and risk of cardiovascular diseases.

Malaysia, like the rest of the world, has a high prevalence of hypertension, 32.7% for aged >18 years. Persistent hypertension can lead to co-morbidities and mortality as it is a major risk factor for stroke and heart diseases. Primary aldosteronism (PA) is probably the commonest curable cause of hypertension. While a recent report suggested that 60% of patients with hypertension have PA, a more conservative estimate, with concentration on curable patients, might suggest 5-10% of all patients, but much higher proportions among those with treatment resistant hypertension, and ethnic groups with low-renin hypertension. Yet, sadly <1% of patients with PA are currently diagnosed due access and price of highly specialised screening/confirmatory tests. Early diagnosis and treatment of excessive aldosterone is not only essential to prevent high blood pressure, but also to prevent cardiac damage. We therefore aim in this proposal to perform whole genome sequencing on PA patients to elucidate genetic contributors to autonomous aldosterone production. Heredity contributes 25-64% of inter-individual variation in hypertension, yet genome-wide association studies have identified only ~3.5% of the genetic contribution with most hits occurring in the non-coding regions. WGS will provide deeper genetic understanding of the pathogenesis especially on the non-coding regulatory elements and interplay between germline mutation and somatic mutations in aldosterone driver-genes. The outcome of our proposal will facilitate the development of a low-cost high-throughput peripheral blood assay that will allow mass diagnosis of autonomous aldosterone production in Malaysia and justify targeted treatment with the cheap but non-selective aldosterone receptor antagonist spironolactone decreasing blood pressure and risk of cardiovascular diseases.

The prevalence of hypertension in Malaysia is 32.7% in adults, and causes fatal and non-fatal cardiovascular diseases. The adrenal hormone, aldosterone, stimulates salt retention, raises blood pressure and damages the heart. ~10% of hypertension is due to primary (=autonomous) aldosteronism (PA), but <1% of PA is diagnosed. Half of PA is due to aldosterone-producing adenomas (APAs) with gain-of-function somatic mutations. Since our description of a common sub-type of APA with Ca2+-channel (CACNA1D) mutations, >50 have been found in this gene, many also occurring in the microscopic aldosterone-producing cell clusters (APCCs) of apparently normal subjects. CACNA1D-mutant APAs are commonest in African-American patients, who have a high prevalence of low-renin (salt-dependent) hypertension.

We postulate that much of low-renin hypertension is due to undiagnosed CACNA1D-mutant APCCs, and that causative germline variants, e.g. in highly-expressed adrenal DNA repair or 'apolipoprotein RNA-editing' genes, increase susceptibility to somatic mutation.This study aims to identify germline genetic factors associated to autonomous aldosterone production through whole genome sequencing patients with CACNA1D somatic mutations. WGS can potentially identify causative rare and private variants in both coding genes and non-coding regulatory elements such as promoters and enhancers that are much more predictive than common 'GWAS polymorphisms'. By identifying genetic contributors for autonomous aldosterone production, we also hope to lay the groundwork of developing a low-cost high-throughput peripheral blood assay that will facilitate mass diagnosis and targeted treatment. In other words, a simple blood test to identify hypertensive patients due to autonomous aldosterone production, which can then be treated by the cheap aldosterone receptor antagonist spironolactone decreasing blood pressure and risk of cardiovascular diseases.

The main beneficiaries of the knowledge arising from the proposed research are:
i) Genetic and genomic scientists working on genetic contributors to human diseases. The scientific community is awaiting new analytical approaches and models to understand pathogenesis through whole genome sequencing. Our approaches to elucidate primary aldosteronism will contribute significantly to the endeavour especially on the interplay between germline and somatic mutations.
ii) Hypertensive patients who have primary aldosteronism. This not only includes those with an aldosterone-producing adenoma but also those with bilateral adrenal hyperplasia as predictive markers would be able to decrease unnecessary AVS and adrenalectomy.
iii) Endocrinologist and clinicians caring for primary aldosteronism. Through stratifying treatment according to who will benefit most from an adrenalectomy, endocrinologist and clinicians will use their time and resources most effectively.
iv) The national healthcare service/Ministry of health. An easier diagnosing method for identifying a curable cause of hypertension will increase diagnosing rate which will decrease other co-morbidities associated with hypertension such as cardiovascular diseases.
v) Pharmaceutical companies manufacturing antihypertensive medications. WGS of APAs may identify novel drug target to regulate aldosterone production.
vi) The Malaysian and UK Research Team. This UK-Malaysian collaboration will facilitate the transfer and exchange of research expertise between the countries. For example, it will help to build the capacity of analysing WGS data for medical use in the Malaysian counterpart.