OBJECTIVE:Hypertension is a major cardiovascular risk factor affecting about 1 in 3 adults. Although the majority of hypertension cases (∼90%) are classified as "primary hypertension" (PHT), endocrine hypertension (EHT) accounts for ∼10% of cases and is caused by underlying conditions such as primary aldosteronism (PA), Cushing's syndrome (CS), pheochromocytoma or paraganglioma (PPGL). EHT is often misdiagnosed as PHT leading to delays in treatment for the underlying condition, reduced quality of life and costly, often ineffective, antihypertensive treatment. MicroRNA (miRNA) circulating in the plasma is emerging as an attractive potential biomarker for various clinical conditions due to its ease of sampling, the accuracy of its measurement and the correlation of particular disease states with circulating levels of specific miRNAs. METHODS:This study systematically presents the most discriminating circulating miRNA features responsible for classifying and distinguishing EHT and its subtypes (PA, PPGL, and CS) from PHT using 8 different supervised machine learning (ML) methods for the prediction. RESULTS:The trained models successfully classified PPGL, CS, and EHT from PHT with area under the curve (AUC) of 0.9 and PA from PHT with AUC 0.8 from the test set. The most prominent circulating miRNA features for hypertension identification of different disease combinations were hsa-miR-15a-5p and hsa-miR-32-5p. CONCLUSIONS:This study confirms the potential of circulating miRNAs to serve as diagnostic biomarkers for EHT and the viability of ML as a tool for identifying the most informative miRNA species.

Abstract Disclosure: L.A. Birch: None. J.L. Fullerton: None. S.M. MacKenzie: None. L.M. Work: None. E. Davies: None. Vascular cognitive impairment (VCI) encompasses all disorders of vascular origin that result in cognitive deficits. Stroke is a leading cause of death, and doubles risk of VCI, in turn those with VCI have an increased risk of stroke. Hypertension is the most significant modifiable risk factor for both stroke and VCI. The mineralocorticoid aldosterone is an essential regulator of blood pressure, acting via the mineralocorticoid receptor (MR), and hyperaldosteronism increases risk of cerebrovascular disease including stroke and VCI. In addition to its epithelial role, MR is distributed throughout the vasculature and brain and has potential as a twofold target for VCI given that MR antagonism has proved beneficial in both stroke and VCI. Expression of the MR gene, NR3C2, can be repressed by microRNAs post-transcriptionally binding NR3C2 mRNA at its 3’ untranslated region (3’UTR). MicroRNAs circulate within extracellular vesicles (EVs), which have shown promise in therapeutic delivery and can cross the blood-brain barrier. This project aims to target MR expression by EV-mediated delivery of miRNAs targeting the NR3C2-3’UTR In silico analysis identified 74 microRNAs as potentially binding the NR3C2-3’UTR. Several were also highlighted in a recent review of circulating microRNAs implicated in stroke, including the brain-specific microRNA-124-3p (miR-124-3p). Pathway analysis also revealed additional mRNA targets of miR-124-3p relevant to VCI and aldosterone signalling, including serum/glucocorticoid regulated kinase 1 (SGK1), a protein kinase downstream of MR that mediates aldosterone action. We confirmed binding of miR-124-3p to the NR3C2-3’UTR in vitro by dual luciferase assay, with significantly decreased reporter gene expression observed after treatment with miR-124-3p precursor. Following induction of ischaemic stroke in stroke-prone spontaneously hypertensive rats by the transient middle cerebral artery occlusion model, brain miR-124-3p levels were significantly altered, with reduced expression confined to the infarct region. EVs were then isolated from rat plasma using size exclusion chromatography and loaded with miR-124-3p mimic by electroporation. When incubated with B50 rat neuronal cells, these EVs resulted in a significant 2-fold increase in miR-124-3p level, indicating successful delivery. No change in NR3C2 or SGK1 mRNA level was observed. Similarly, following oxygen-glucose deprivation, loaded EV treatment increased miR-124-3p levels but NR3C2 and SGK1 mRNA levels were unchanged. MR protein levels are still to be quantified. In conclusion, miRNA-124-3p is a promising therapeutic mediator of NR3C2 expression. Ongoing studies will include further in vitro characterization of its effects involving additional cell types of the neurovascular unit and exploration of neuroprotective effects in an in vivo bilateral carotid artery stenosis model of VCI. Presentation: 6/3/2024

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Aldosterone is a cardiovascular hormone with a key role in blood pressure regulation, among other processes, mediated through its targeting of the mineralocorticoid receptor in the renal tubule and selected other tissues. Its secretion from the adrenal gland is a highly controlled process subject to regulatory influence from the renin-angiotensin system and the hypothalamic-pituitary-adrenal axis. MicroRNAs are small endogenous non-coding RNA molecules capable of regulating gene expression post-transcriptionally through stimulation of mRNA degradation or suppression of translation. Several studies have now identified that microRNA levels are changed in cases of aldosterone dysregulation and that microRNAs are capable of regulating the expression of various genes involved in aldosterone production and action. In this article we summarise the major studies concerning this topic. We also discuss the potential role for circulating microRNAs as diagnostic biomarkers for primary aldosteronism, a highly treatable form of secondary hypertension, which would be highly desirable given the current underdiagnosis of this condition.

Abstract Disclosure: S. Lamprou: None. S.M. MacKenzie: None. J.D. McClure: None. S. Robertson: None. A. Riddell: None. J.C. van Kralingen: None. A. Gimenez-Roqueplo: None. P. Reel: None. S. Reel: None. G. Assié: None. A. Laurence: None. F. Beuschlein: None. G. Rossi: None. J. Deinum: None. M. Reincke: None. G. Eisenhofer: None. E. Jefferson: None. M. Zennaro: None. E. Davies: None. Primary Aldosteronism (PA) is the most common endocrine hypertension subtype, caused by autonomous aldosterone secretion. While readily treatable, accurate diagnosis of PA – particularly its differentiation from primary hypertension (PHT) – is a complex and protracted process that can entail delays in effective treatment and lead to more severe comorbidities. Given the established role of microRNAs (miRNA) in regulating aldosterone biosynthesis, we hypothesize that circulating miRNAs are potentially viable biomarkers for PA that could inform more accurate and rapid diagnosis. To detect changes in circulating miRNA profiles specific to PA, we analyzed plasma levels of 172 selected microRNAs in a retrospectively-generated cohort of PHT (n=111) and PA patients (n=109). The dataset consisted of ΔCt values for each miRNA (normalized to multiple endogenous miRNAs), as determined by real time RT-qPCR assay. Two-sample t-tests were used to assess whether the means of the two disease groups are equal, corrected using False Discovery Rate (FDR; significance level q < 0.05). Ingenuity Pathway Analysis software (QIAGEN) was employed to analyze the miRNAs bioinformatically and identify mRNA targets involved in blood pressure regulation. Pathway analysis was performed to discover associations between PA and differentially-expressed miRNAs. We identified 83 circulating microRNAs that showed significant differential expression: 27 were upregulated and 56 downregulated in PA relative to PHT. The upregulated miRNA demonstrating the greatest fold change was hsa-miR-451a (FC:1.8, q=0.001), while the most downregulated was hsa-miR-409-3p (FC:0.5, q=0.001). Bioinformatics analysis predicted several dysregulated miRNAs to target mRNAs in PA-related pathways. For example, the most upregulated miRNA, hsa-miR-451a, is predicted to target mRNAs in biochemical pathways relevant to PA pathophysiology, including SGK1 and MAP3K1. SGK1 contributes to the signaling cascade induced by the mineralocorticoid receptor-aldosterone complex that stimulates Na+ reabsorption in the kidney, while MAP3K1 participates in the Renin-Angiotensin system, a key regulator of aldosterone secretion, as part of a signaling cascade that leads to the activation of the AP1 complex in the nucleus. In addition to hsa-miR-451a, other differentially-expressed miRNAs were also revealed to target multiple mRNAs with direct and indirect connections to PA. In summary, analysis has identified miRNAs that are significantly differentially expressed in the plasma of PA patients compared to PHT patients, indicating promise for a biomarker-based approach to PA diagnosis utilizing distinctive circulating microRNA signatures. Further analysis is now being conducted to validate our findings in an independent population of hypertensive patients. Presentation: Friday, June 16, 2023

Background Arterial hypertension represents a worldwide health burden and a major risk factor for cardiovascular morbidity and mortality. Hypertension can be primary (primary hypertension, PHT), or secondary to endocrine disorders (endocrine hypertension, EHT), such as Cushing's syndrome (CS), primary aldosteronism (PA), and pheochromocytoma/paraganglioma (PPGL). Diagnosis of EHT is currently based on hormone assays. Efficient detection remains challenging, but is crucial to properly orientate patients for diagnostic confirmation and specific treatment. More accurate biomarkers would help in the diagnostic pathway. We hypothesized that each type of endocrine hypertension could be associated with a specific blood DNA methylation signature, which could be used for disease discrimination. To identify such markers, we aimed at exploring the methylome profiles in a cohort of 255 patients with hypertension, either PHT ( n = 42) or EHT ( n = 213), and at identifying specific discriminating signatures using machine learning approaches. Results Unsupervised classification of samples showed discrimination of PHT from EHT. CS patients clustered separately from all other patients, whereas PA and PPGL showed an overall overlap. Global methylation was decreased in the CS group compared to PHT. Supervised comparison with PHT identified differentially methylated CpG sites for each type of endocrine hypertension, showing a diffuse genomic location. Among the most differentially methylated genes, FKBP5 was identified in the CS group. Using four different machine learning methods—Lasso (Least Absolute Shrinkage and Selection Operator), Logistic Regression, Random Forest, and Support Vector Machine—predictive models for each type of endocrine hypertension were built on training cohorts (80% of samples for each hypertension type) and estimated on validation cohorts (20% of samples for each hypertension type). Balanced accuracies ranged from 0.55 to 0.74 for predicting EHT, 0.85 to 0.95 for predicting CS, 0.66 to 0.88 for predicting PA, and 0.70 to 0.83 for predicting PPGL. Conclusions The blood DNA methylome can discriminate endocrine hypertension, with methylation signatures for each type of endocrine disorder.

BackgroundArterial hypertension is a major cardiovascular risk factor. Identification of secondary hypertension in its various forms is key to preventing and targeting treatment of cardiovascular complications. Simplified diagnostic tests are urgently required to distinguish primary and secondary hypertension to address the current underdiagnosis of the latter.MethodsThis study uses Machine Learning (ML) to classify subtypes of endocrine hypertension (EHT) in a large cohort of hypertensive patients using multidimensional omics analysis of plasma and urine samples. We measured 409 multi-omics (MOmics) features including plasma miRNAs (PmiRNA: 173), plasma catechol O-methylated metabolites (PMetas: 4), plasma steroids (PSteroids: 16), urinary steroid metabolites (USteroids: 27), and plasma small metabolites (PSmallMB: 189) in primary hypertension (PHT) patients, EHT patients with either primary aldosteronism (PA), pheochromocytoma/functional paraganglioma (PPGL) or Cushing syndrome (CS) and normotensive volunteers (NV). Biomarker discovery involved selection of disease combination, outlier handling, feature reduction, 8 ML classifiers, class balancing and consideration of different age- and sex-based scenarios. Classifications were evaluated using balanced accuracy, sensitivity, specificity, AUC, F1, and Kappa score.FindingsComplete clinical and biological datasets were generated from 307 subjects (PA=113, PPGL=88, CS=41 and PHT=112). The random forest classifier provided ∼92% balanced accuracy (∼11% improvement on the best mono-omics classifier), with 96% specificity and 0.95 AUC to distinguish one of the four conditions in multi-class ALL-ALL comparisons (PPGL vs PA vs CS vs PHT) on an unseen test set, using 57 MOmics features. For discrimination of EHT (PA + PPGL + CS) vs PHT, the simple logistic classifier achieved 0.96 AUC with 90% sensitivity, and ∼86% specificity, using 37 MOmics features. One PmiRNA (hsa-miR-15a-5p) and two PSmallMB (C9 and PC ae C38:1) features were found to be most discriminating for all disease combinations. Overall, the MOmics-based classifiers were able to provide better classification performance in comparison to mono-omics classifiers.InterpretationWe have developed a ML pipeline to distinguish different EHT subtypes from PHT using multi-omics data. This innovative approach to stratification is an advancement towards the development of a diagnostic tool for EHT patients, significantly increasing testing throughput and accelerating administration of appropriate treatment.FundingEuropean Union's Horizon 2020 Research and Innovation Programme under Grant Agreement No. 633983, Clinical Research Priority Program of the University of Zurich for the CRPP HYRENE (to Z.E. and F.B.), and Deutsche Forschungsgemeinschaft (CRC/Transregio 205/1).

Extra-mammary Paget's disease (EMPD) is a chronic erythematous dermatological condition which can be classified as either primary or secondary. Primary EMPD is thought to arise from skin adnexal apocrine glands, whereas, secondary tends to arise from visceral carcinomas, such as a colorectal or gynaecological source.1Heymann W.R. Extramammary Paget's disease.Clin Dermatol. 1993; 11: 83-87Abstract Full Text PDF PubMed Scopus (76) Google Scholar,2Shepherd V. Davidson E.J. Davies-Humphreys J. Extramammary Paget's disease.BJOG. 2005; 112: 273-279Crossref PubMed Scopus (168) Google Scholar EMPD developing from a non-invasive colorectal adenoma is incredibly rare, with very few documented cases in the current literature. As such, there is very little guidance on treatment and prognosis, although reports suggest these patients may have better outcomes.3Hutchings D. Windon A. Assarzadegan N. Salimian K.J. Voltaggio L. Mongtomery E.A. Perianal Paget’s disease as spread from non-invasive colorectal adenomas.Histopathology. 2021; 78: 276-280Crossref PubMed Scopus (5) Google Scholar Thus, it is critical for a clinical pathological consensus to avoid over treatment. This case report presents an incidental finding of EMPD in a patient with a rectal tubulo-villous adenoma (TVA). A 70-year-old woman presented for assessment of mild rectal bleeding in the context of known sigmoid diverticular disease. She had no history of malignancy and her only past history was a previous hysterectomy performed decades prior. She had a family history of colorectal cancer (first degree relative). Her laboratory investigations including full blood count and iron studies were within normal limits. Her index colonoscopy revealed a 6 mm pedunculated polyp at the dentate line that was resected with cold snare polypectomy. Histology revealed a TVA with low grade dysplasia. A surveillance procedure performed one year later revealed tumour recurrence at the site of previous resection and a new 12 mm lesion (Fig. 1A). This, and surrounding tissue were removed with combined cold and hot snare polypectomy. Histology revealed the same findings as the original polyp. A second surveillance procedure six months later revealed ongoing recurrence with an 8 mm pedunculated lesion at the dentate line. Cold snare polypectomy was again performed. Given the ongoing recurrence, submucosal fibrosis, and the difficult endoscopic location, she was referred for an endoscopic mucosal resection. A 15 mm polypoid lesion was completely resected using endoscopic submucosal dissection. Histology revealed a TVA with a focal area of high grade dysplasia that was clear of the inked margin (Fig. 2A–C). Adjacent to the adenoma, there was a small fragment of attached squamous mucosa that showed large atypical cells in a pagetoid spread, containing intracytoplasmic mucin. Immunohistochemistry showed that both the adjacent adenoma and these cells were positive for cytokeratin (CK)7, CK20, CDX-2 and SATB2, indicating an intestinal phenotype (Fig. 2D–F). Gross cystic disease fluid protein (GCDFP)-15, oestrogen receptor, GATA-3 and mammaglobin were negative. She was referred for gynaecological and colorectal evaluation. Speculum examination of the vaginal vault did not reveal evidence of anterior extension of disease. Serum tumour markers were negative and contrast enhanced computed tomography of the chest, abdomen and pelvis did not reveal a distant primary tumour. There was no evidence of local invasion seen on pelvic magnetic resonance imaging. Examination under anaesthesia (EUA) revealed subtle macroscopic changes including mild induration and oedema from 7 o'clock to 1 o'clock which were consistent with Paget's (Fig. 1B). The biopsies showed extensive disease, confined to the epithelium, with the same intestinal phenotype as the original case. The final pathological diagnosis was EMPD arising from a rectal adenoma. The patient has been reviewed by a multi-disciplinary team and planned for regular surveillance with repeat EUA and biopsy. Secondary EMPD usually arises when there is intraepithelial invasion (pagetoid spread) of an underlying or contiguous visceral carcinoma. The most common sites are the lower gastro-intestinal or urogenital tract. Differential diagnoses for the microscopic appearance in these areas include melanoma and pagetoid Bowen's disease;2Shepherd V. Davidson E.J. Davies-Humphreys J. Extramammary Paget's disease.BJOG. 2005; 112: 273-279Crossref PubMed Scopus (168) Google Scholar however, these can readily be differentiated with an appropriate immunohistochemical (IHC) panel. Primary perianal Paget's is rare, whilst secondary Paget's disease makes up to 60% of perianal cases.4Lam C. Funaro D. Extramammary Paget’s disease: summary of current knowledge.Dermatol Clin. 2010; 28: 807-826Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar Presentations include itch and irritation of the peri-anal area, as well as ulceration and a palpable mass.5Liao X. Liu X. Fan X. Lai J. Zhang D. Perianal Paget’s disease: a clinicopathological and immunohistochemical study of 13 cases.Diagn Pathol. 2020; 15: 1-7Crossref PubMed Scopus (11) Google Scholar Distinguishing primary from secondary EMPD involves both immunohistochemistry and exclusion of an underlying carcinoma. Immunohistochemically, primary tumours arising from adnexal structures tend to have the following IHC markers: CK7+, CK20−, GCDFP+6De Nisi M.C. D’Amuri A. Toscano M. Lalinga A.V. Pirtoli L. Miracco C. Usefulness of CDX2 in the diagnosis of extramammary Paget disease associated with malignancies of intestinal type.Br J Dermatol. 2005; 153: 677-679Crossref PubMed Scopus (28) Google Scholar and may also express HER2 and androgen receptors. In contrast, secondary EMPD of anorectal origin tends to be CK7−, although this is not specific, and CK20+7Ohnishi T. Watanabe S. The use of cytokeratins 7 and 20 in the diagnosis of primary and secondary extramammary Paget’s disease.Br J Dermatol. 2000; 142: 243-247Crossref PubMed Scopus (172) Google Scholar,8Battles O.E. Page D.L. Johnson J.E. Cytokeratins, CEA, and mucin histochemistry in the diagnosis and characterization of extramammary Paget’s disease.Am J Clin Pathol. 1997; 108: 6-12Crossref PubMed Scopus (70) Google Scholar can also express a range of antigens dependent on the underlying tumour. Of note CDX2 is a specific lower gastrointestinal adenocarcinoma associated EMPD supportive marker.6De Nisi M.C. D’Amuri A. Toscano M. Lalinga A.V. Pirtoli L. Miracco C. Usefulness of CDX2 in the diagnosis of extramammary Paget disease associated with malignancies of intestinal type.Br J Dermatol. 2005; 153: 677-679Crossref PubMed Scopus (28) Google Scholar However, not all secondary EMPDs follow these patterns of expression, such as certain gynaecological and breast carcinomas. Hence, the diagnosis needs to be made in conjunction with the clinical findings and exclusion of other causes. Unlike Paget's disease of the nipple associated with ductal carcinoma in situ, EMPD arising from a non-invasive colorectal adenoma is unusual. Very few cases have been reported in the current literature. In a series of 11 cases of perianal Paget's disease, five of the patients had documented synchronous rectal adenocarcinoma and they were CK7+, CK20+, and GCDFP−. Of the six patients who did not have rectal adenocarcinoma, four were CK7+, CK20−, and GCDFP+. The other two cases were CK7+, CK20+ and GCDFP−, similar to our case. In both cases there was evidence of recurrence after wide local excision, and one was subsequently treated with radiotherapy.9Goldblum J.R. Hart W.R. Perianal Paget's disease: a histologic and immunohistochemical study of 11 cases with and without associated rectal adenocarcinoma.Am J Surg Pathol. 1998; 22: 170-179Crossref PubMed Scopus (200) Google Scholar In their case series of 13 patients, Liao and colleagues also found that GCDFP was only positive in patients with primary peri-anal disease, and CDX2 was only positive in secondary disease, whilst CK20 was present in both primary and secondary disease. Eight of the 13 cases also had a synchronous carcinoma, whilst one had an adenoma with high grade dysplasia.5Liao X. Liu X. Fan X. Lai J. Zhang D. Perianal Paget’s disease: a clinicopathological and immunohistochemical study of 13 cases.Diagn Pathol. 2020; 15: 1-7Crossref PubMed Scopus (11) Google Scholar The largest case series to date of EMPD associated with colorectal adenomas published by Hutchings et al. included four cases. Similar to our case, all four had evidence of high grade dysplasia with no evidence of invasion or an invasive adenocarcinoma elsewhere and IHC markers were CK7+, CK20+, GCDFP− and CDX2 +, where available. Only one patient required adjuvant treatment after excision. One had recurrence at 8 months and another was found to have a mucinous adenocarcinoma 36 months after presentation.3Hutchings D. Windon A. Assarzadegan N. Salimian K.J. Voltaggio L. Mongtomery E.A. Perianal Paget’s disease as spread from non-invasive colorectal adenomas.Histopathology. 2021; 78: 276-280Crossref PubMed Scopus (5) Google Scholar In summary, secondary EMPD is a rare condition, especially arising from non-invasive rectal adenomas such as in our case. As such, it is important that pathologists are aware of this condition. Thorough clinical assessment in addition to IHC markers are essential to determine (1) whether these are invasive or non-invasive lesions, and (2) whether these are primary or secondary lesions. Due to the limited number of cases, the optimal treatment strategy following excision of the primary lesion remains uncertain. In any case, after careful assessment and exclusion of a malignant primary, regular surveillance appears to be a reasonable follow-up strategy in order to avoid undue harm from over treatment. However, clinicians need to be aware that recurrence and metachronous malignancy may still occur in these patients. The authors state that there are no conflicts of interest to disclose.

Additional file 5: Table S5. Sample characteristics.

RATIONALE:Major depressive disorder (MDD) is a leading cause of disability worldwide but currently prescribed treatments do not adequately ameliorate the disorder in a significant portion of patients. Hence, a better appreciation of its aetiology may lead to the development of novel therapies.OBJECTIVES:In the present study, we have built on our previous findings indicating a role for protease-activated receptor-2 (PAR2) in sickness behaviour to determine whether the PAR2 activator, AC264613, induces behavioural changes similar to those observed in depression-like behaviour.METHODS:AC264613-induced behavioural changes were examined using the open field test (OFT), sucrose preference test (SPT), elevated plus maze (EPM), and novel object recognition test (NOR). Whole-cell patch clamping was used to investigate the effects of PAR2 activation in the lateral habenula with peripheral and central cytokine levels determined using ELISA and quantitative PCR.RESULTS:Using a blood-brain barrier (BBB) permeable PAR2 activator, we reveal that AC-264613 (AC) injection leads to reduced locomotor activity and sucrose preference in mice but is without effect in anxiety and memory-related tasks. In addition, we show that AC injection leads to elevated blood sera IL-6 levels and altered cytokine mRNA expression within the brain. However, neither microglia nor peripheral lymphocytes are the source of these altered cytokine profiles.CONCLUSIONS:These data reveal that PAR2 activation results in behavioural changes often associated with depression-like behaviour and an inflammatory profile that resembles that seen in patients with MDD and therefore PAR2 may be a target for novel antidepressant therapies.

Primary aldosteronism (PA) is a common and highly treatable condition, usually resulting from adrenocortical tumorous growth or hyperplasia. PA is currently underdiagnosed owing to its complex and protracted diagnostic procedures. A simplified biomarker-based test would be highly valuable in reducing cardiovascular morbidity and mortality. Circulating microRNAs are emerging as potential biomarkers for a number of conditions due to their stability and accessibility. PA is known to alter microRNA expression in adrenocortical tissue; if these changes or their effects are mirrored in the circulating miRNA profile, then this could be exploited by a diagnostic test. However, the reproducibility of studies to identify biomarker-circulating microRNAs has proved difficult for other conditions due to a series of technical challenges. Therefore, any studies seeking to definitively identify circulating microRNA biomarkers of PA must address this in their design. To this end, we are currently conducting the circulating microRNA arm of the ongoing ENS@T-HT study. In this review article, we present evidence to support the utility of circulating microRNAs as PA biomarkers, describe the practical challenges to this approach and, using ENS@T-HT as an example, discuss how these might be overcome.

Abstract Primary aldosteronism (PA), also known as Conn’s syndrome, is a common curable cause of hypertension. Family studies of essential hypertensive patients suggest that heritable genetic factors play a role in blood pressure regulation1. Interestingly, single nucleotide polymorphisms (SNP) in genes encoding enzymes involved with adrenal steroidogenesis, CYP11B2, CYP11B1 and CYP17A1, associate with increased risk of hypertension2. Therefore, we analysed whether selected SNPs in these genes are associated with PA. We performed an association study using genotype imputation for selected SNPs of the steroidogenic enzyme genes CYP11B2 (rs4546, rs1799998, rs13268025), CYP11B1 (rs6410, rs149845727), and CYP17A1 (rs1004467, rs138009835, rs2150927) from a pilot genome wide association study of Malaysian PA patients and healthy controls which was merged with the Singapore Genome Variation Project (SGVP) population dataset3. Genotype imputation for minor and major alleles was validated using PCR sequencing (n>10 for each genotype). Further, one SNP from each steroidogenic enzyme (CYP11B2:rs1799998, CYP11B1:rs6410 and CYP17A1:rs1004467) was validated using commercial TaqMan genotyping assays on the ABI 7000 Sequence Detection System which was performed on 149 PA patients and 78 non-hypertensive healthy individuals. Case-control genetic association analysis was performed at http://www.oege.org/software/orcalc.html and the association between genotypes and phenotypes was done using the independent-samples Kruskal-Wallis test on SPSS (version 25). The Minor Allele Frequencies (MAFs) for rs1004467, rs6410 and rs1799998 were similar to East Asian populations but differed significantly different from European, African, American and South Asian populations (rs1004467 MAF: C=0.258/298, rs6410 MAF: A=0.265/298, rs1799998 MAF: C=0.225/298). In Chinese patients matched by gender, heterozygotes for rs6410 had significantly increased risk of PA compared to common homozygotes (OR: 3.15, 95% CI: 1.01–9.8, p=0.04). Across patients of different ethnicity, the distribution of aldosterone levels was significantly different (p=0.039). In summary, only SNP rs6410 in Chinese patients matched by gender showed association with PA in our South East Asian cohort. More functional experiments need to be done to find out whether this is causal for PA or whether the SNP is in linkage disequilibrium with the actual functional causative SNPs. Once the functional SNP is known, identification of these germline variants in asymptomatic family members would allow early screening of PA to be offered and potentially provide novel drug targets to treat the disease. References: 1Timberlake et al., Curr Opin Nephrol Hypertens. 2001 Jan;10(1):71-9. 2MacKenzie et al., Int J Mol Sci. 2017 Mar 7;18(3). pii: E579. 3Teo et al., Genome Res. 2009 Nov;19(11):2154-62.

The secretion of aldosterone by the adrenal cortex is a tightly regulated process. Loss of this control can result in severe hypertension and end-organ damage, so detailed understanding of the various mechanisms by which the body regulates aldosterone biosynthesis is key. The emergence of microRNAs (miRNAs) as negative regulators of numerous physiological processes has naturally led to their study in the context of aldosterone production. We summarise several studies that have demonstrated a significant role for microRNAs in aldosterone biosynthesis and action, thereby presenting a possible therapeutic role in the treatment of common forms of hypertension such as primary aldosteronism. Furthermore, the presence of microRNAs in the circulation offers the prospect of accessible and informative biomarkers that may simplify the currently protracted and technically difficult diagnosis of such conditions.

Context and objective Common genetic variants in CYP17A1 associate with higher blood pressure, putatively from impaired 17α-hydroxylase activity and mineralocorticoid excess. However, the same variants protect against obesity and insulin resistance. We tested whether CYP17A1 variants that enhance 17α-hydroxylase activity cause ‘relative corticosterone deficiency’. Since corticosterone is thought to contribute disproportionately to negative feedback in the hypothalamic-pituitary-adrenal axis, we also tested whether lower corticosterone associates with higher cortisol and hence with metabolic syndrome. Design Cross-sectional studies within the population-based Orkney Complex Disease Study (ORCADES; n=2018), VIKING Health Study Shetland (VIKING; n=2098), East Hertfordshire study (EHERTS; n=279), Edinburgh Type 2 Diabetes Study (ET2DS; n=903), and the Swiss Kidney Project on Genes in Hypertension (SKIPOGH; n=888). Outcome measures Cortisol and corticosterone in morning plasma samples in ORCADES, VIKING and ET2DS, and in EHERTS in plasma following overnight dexamethasone suppression (0.25mg) and 30 mins after ACTH1-24 (1µg); cortisol and corticosterone metabolites in day and night urine samples in SKIPOGH. Features of the metabolic syndrome including body mass index, systolic blood pressure, lipid profile, fasting glucose, fasting insulin and HOMA-IR. Results In ORCADES, ET2DS and SKIPOGH, CYP17A1 variants were associated with corticosterone:cortisol ratio. In ORCADES, VIKING and ET2DS there were consistent associations of morning plasma cortisol and corticosterone with BMI, blood pressure, lipid profile, fasting glucose and HOMA-IR. In EHERTS, however, after dexamethasone suppression and ACTH1-24 stimulation, impaired glucose tolerance and insulin sensitivity were associated with higher cortisol but lower corticosterone. Similarly, in SKIPOGH, low corticosterone:cortisol metabolite ratios were associated with high BMI and dyslipidemia. Conclusions ‘Relative corticosterone deficiency’, due to a primary alteration in adrenal steroidogenesis favouring cortisol over corticosterone, may mediate the associations of genetic variation in CYP17A1 with metabolic syndrome. However, additional determinants of variation in plasma corticosterone are likely to explain its generally positive associations with features of metabolic syndrome.

Secretion of the major mineralocorticoid aldosterone from the adrenal cortex is a tightly-regulated process enabling this hormone to regulate sodium homeostasis and thereby contribute to blood pressure control. The circulating level of aldosterone is the result of various regulatory mechanisms, the most significant being those controlled by the renin-angiotensin system and plasma potassium levels. The importance of maintaining tight control over aldosterone secretion is demonstrated by cases of dysregulation, which can result in severe hypertension and significantly increased cardiovascular risk. In this article we summarize current knowledge of the major regulatory mechanisms, focusing particularly on the systems operating within the adrenocortical zona glomerulosa cells; we also describe some of the other factors that influence aldosterone production to a lesser but still significant extent. Finally, we discuss the influence of common genetic polymorphisms on aldosterone secretion in large sections of the population and also the emerging role of microRNA as significant regulators of this system.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Background: Myocardial infarction (MI) is a leading cause of heart failure and death worldwide. Preservation of contractile function and protection against adverse changes in ventricular architecture (cardiac remodeling) are key factors to limiting progression of this condition to heart failure. Consequently, new therapeutic targets are urgently required to achieve this aim. Expression of the Runx1 transcription factor is increased in adult cardiomyocytes after MI; however, the functional role of Runx1 in the heart is unknown. Methods: To address this question, we have generated a novel tamoxifen-inducible cardiomyocyte-specific Runx1 -deficient mouse. Mice were subjected to MI by means of coronary artery ligation. Cardiac remodeling and contractile function were assessed extensively at the whole-heart, cardiomyocyte, and molecular levels. Results: Runx1 -deficient mice were protected against adverse cardiac remodeling after MI, maintaining ventricular wall thickness and contractile function. Furthermore, these mice lacked eccentric hypertrophy, and their cardiomyocytes exhibited markedly improved calcium handling. At the mechanistic level, these effects were achieved through increased phosphorylation of phospholamban by protein kinase A and relief of sarco/endoplasmic reticulum Ca 2+ -ATPase inhibition. Enhanced sarco/endoplasmic reticulum Ca 2+ -ATPase activity in Runx1-deficient mice increased sarcoplasmic reticulum calcium content and sarcoplasmic reticulum–mediated calcium release, preserving cardiomyocyte contraction after MI. Conclusions: Our data identified Runx1 as a novel therapeutic target with translational potential to counteract the effects of adverse cardiac remodeling, thereby improving survival and quality of life among patients with MI.

The aldosterone synthase (CYP11B2) and 11β-hydroxylase (CYP11B1) enzymes are known to be important players in the development of hypertension. Sequencing of the CYP11B2 and CYP11B1 genes and quantification of their respective mRNAs is greatly complicated by their high degree of sequence similarity. The need to ensure gene specificity during such analysis has required the development of particular methods for the detection of key polymorphisms at these loci, which are detailed in this chapter.

The majority of genes contributing to the heritable component of blood pressure remain unidentified, but there is substantial evidence to suggest that common polymorphisms at loci involved in the biosynthesis of the corticosteroids aldosterone and cortisol are important. This view is supported by data from genome-wide association studies that consistently link the CYP17A1 locus to blood pressure. In this review article, we describe common polymorphisms at three steroidogenic loci (CYP11B2, CYP11B1 and CYP17A1) that alter gene transcription efficiency and levels of key steroids, including aldosterone. However, the mechanism by which this occurs remains unclear. While the renin angiotensin system is rightly regarded as the major driver of aldosterone secretion, there is increasing evidence that the contribution of corticotropin (ACTH) is also significant. In light of this, we propose that the differential response of variant CYP11B2, CYP11B1 and CYP17A1 genes to ACTH is an important determinant of blood pressure, tending to predispose individuals with an unfavourable genotype to hypertension.