Miroslav Solar

Somatic mutations in ATP1A1 and CACNA1D underlie a common subtype of adrenal hypertension.

At least 5% of individuals with hypertension have adrenal aldosterone-producing adenomas (APAs). Gain-of-function mutations in KCNJ5 and apparent loss-of-function mutations in ATP1A1 and ATP2A3 were reported to occur in APAs. We find that KCNJ5 mutations are common in APAs resembling cortisol-secreting cells of the adrenal zona fasciculata but are absent in a subset of APAs resembling the aldosterone-secreting cells of the adrenal zona glomerulosa. We performed exome sequencing of ten zona glomerulosa–like APAs and identified nine with somatic mutations in either ATP1A1, encoding the Na+/K+ ATPase α1 subunit, or CACNA1D, encoding Cav1.3. The ATP1A1 mutations all caused inward leak currents under physiological conditions, and the CACNA1D mutations induced a shift of voltage-dependent gating to more negative voltages, suppressed inactivation or increased currents. Many APAs with these mutations were <1 cm in diameter and had been overlooked on conventional adrenal imaging. Recognition of the distinct genotype and phenotype for this subset of APAs could facilitate diagnosis.

Difficult-to-control arterial hypertension or uncooperative patients? The assessment of serum antihypertensive drug levels to differentiate non-responsiveness from non-adherence to recommended therapy

Difficult-to-control arterial hypertension is a common medical problem that may result from severe hypertensive disease or from poor adherence to the recommended medical treatment. The identification of non-adherent patients is challenging, especially when non-adherence is intentional. The current report describes the use of serum levels of prescribed antihypertensive drugs to evaluate the adherence in individuals with difficult-to-control arterial hypertension. Serum drug levels (SDLs) were evaluated by liquid chromatography with mass spectrometry. The chromatographic separation was performed on a reversed-phase column with a gradient flow of the mobile phase. The detection of analyzed substances was accomplished on a linear ion-trap mass spectrometer. The subjects were labeled as non-adherent when the serum level of at least one of the evaluated drugs was below the limit of quantification. The study used data from 84 patients with arterial hypertension who underwent SDL assessment to verify compliance with the recommended treatment. Patients who presented with uncontrolled blood pressure despite the recommended combination of at least three antihypertensives were enrolled in the analysis. Based on the evaluation of the SDLs, all of the evaluated drugs were found in the sera of 29 (34.5%) of the study patients. In the remaining 55 (65.5%) patients, non-adherence was diagnosed. None of the prescribed antihypertensive drugs was detected in the sera of the 29 (34.5%) patients. Our data suggest that an assessment of SDLs might be helpful before an extensive evaluation is initiated for difficult-to-control hypertension.

Adrenal venous sampling in primary aldosteronism: a low dilution of adrenal venous blood is crucial for a correct interpretation of the results

Objective In primary aldosteronism, adrenal venous sampling (AVS) is essential for subtype differentiation as it evaluates aldosterone secretion from both adrenals. Selectivity of adrenal sampling is assessed by the ratio of cortisol concentrations in adrenal venous blood and inferior vena cava blood (Cadrenal/Civc). Since the criteria for selective adrenal sampling differ among the reported literature, we performed a study to evaluate the influence of different selectivity criteria on AVS results. Design and methods Reports of AVS were screened retrospectively. All AVS were performed with cosyntrophin infusion. Reports containing samples with Cadrenal/Civc≥10 taken from both adrenals and at least one other adrenal sample characterised by Cadrenal/Civc≥1.1 were enrolled. For each individual, we chose reference samples that were defined by the highest Cadrenal/Civc achieved from each adrenal. The significance of the remaining samples with Cadrenal/Civc≥1.1 was analysed in regard to their respective reference samples. We assessed the impact of analysed samples on identification of lateralisation of aldosterone secretion that is crucial for decisions concerning adrenalectomy. Results AVS reports of 87 patients were enrolled. A total of 225 adrenal samples were analysed and divided into five groups according to Cadrenal/Civc:1.1–1.99, 2–2.99, 3–4.99, 5–9.99 and ≥10. By comparing reference with analysed samples, a concordant assessment with respect to lateralisation of aldosterone secretion was observed in 39, 52, 72, 85 and 94% of the respective groups of analysed samples. Conclusion AVS provides consistent information when adrenal samples with high cortisol concentrations are used.

Confirmatory testing in primary aldosteronism: extensive medication switching is not needed in all patients

Objective Confirmatory testing of suspected primary aldosteronism (PA) requires an extensive medication switch that can be difficult for patients with severe complicated hypertension and/or refractory hypokalemia. For this reason, we investigated the effect of chronic antihypertensive medication on confirmatory testing results. To allow the results to be interpreted, the reproducibility of confirmatory testing was also evaluated. Design and methods The study enrolled 114 individuals with suspected PA who underwent two confirmatory tests. The patients were divided into two groups. In Group A, both tests were performed on the guidelines-recommended therapy, i.e. not interfering with the renin–angiotensin–aldosterone system. In Group B, the first test was performed on chronic therapy with the exclusion of thiazides, loop diuretics, and aldosterone antagonists; and the second test was performed on guidelines-recommended therapy. Saline infusion, preceded by oral sodium loading, was used to suppress aldosterone secretion. Results Agreement in the interpretation of the two confirmatory tests was observed in 84 and 66% of patients in Groups A and B respectively. For all 20 individuals in Group A who ever had end-test serum aldosterone levels ≥240 pmol/l, aldosterone was concordantly nonsuppressible during the other test. Similarly, for all 16 individuals in Group B who had end-test serum aldosterone levels ≥240 pmol/l on modified chronic therapy, aldosterone remained nonsuppressible with guidelines-recommended therapy. Conclusion Confirmatory testing performed while the patient is on chronic therapy without diuretics and aldosterone antagonists can confirm the diagnosis of PA, provided serum aldosterone remains markedly elevated at the end of saline infusion.

Doxazosin: safety and efficacy in the treatment of resistant arterial hypertension

Five classes of antihypertensive drugs have proven efficacy in the prevention of cardiovascular morbidity and mortality. Among the remaining antihypertensives, the action of alpha‐1‐blockers is supported by most clinical evidence; however, in combination therapy, the published data concern their use as third‐line drugs at the most. The data from patients with drug‐resistant hypertension remain limited. The aim of our study was to evaluate the efficacy and safety of doxazosin in this clinical setting. Data from 97 patients with resistant hypertension treated by doxazosin were analysed retrospectively. Doxazosin was usually added as the fifth antihypertensive drug in individuals who were either unresponsive to or intolerant of the combination of other antihypertensives. The dose of doxazosin ranged from 2 to 16 mg/day. The mean duration of follow‐up was 21±17 months. Adverse events related to doxazosin treatment were rare and led to discontinuation of the therapy in only five patients (5.2%). Data from 34 patients were subjected to analysis of efficacy. In this subgroup, doxazosin therapy led to the reduction of blood pressure from 159±20/92±14 to 126±16/73±10 mmHg. We found that doxazosin is a well‐tolerated and effective drug for patients with resistant arterial hypertension who require a combination of multiple antihypertensive drugs.

Aldosterone-Producing Adenomas: Histopathology–Genotype Correlation and Identification of a Novel CACNA1D Mutation

Mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CTNNB1 are thought to cause the excessive autonomous aldosterone secretion of aldosterone-producing adenomas (APAs). The histopathology of KCNJ5 mutant APAs, the most common and largest, has been thoroughly investigated and shown to have a zona fasciculata–like composition. This study aims to characterize the histopathologic spectrum of the other genotypes and document the proliferation rate of the different sized APAs. Adrenals from 39 primary aldosteronism patients were immunohistochemically stained for CYP11B2 to confirm diagnosis of an APA. Twenty-eight adenomas had sufficient material for further analysis and were target sequenced at hot spots in the 5 causal genes. Ten adenomas had a KCNJ5 mutation (35.7%), 7 adenomas had an ATP1A1 mutation (25%), and 4 adenomas had a CACNA1D mutation (14.3%). One novel mutation in exon 28 of CACNA1D (V1153G) was identified. The mutation caused a hyperpolarizing shift of the voltage-dependent activation and inactivation and slowed the channel’s inactivation kinetics. Immunohistochemical stainings of CYP17A1 as a zona fasciculata cell marker and Ki67 as a proliferation marker were used. KCNJ5 mutant adenomas showed a strong expression of CYP17A1, whereas ATP1A1/CACNA1D mutant adenomas had a predominantly negative expression (P value =1.20×10−4). ATP1A1/CACNA1D mutant adenomas had twice the nuclei with intense staining of Ki67 than KCNJ5 mutant adenomas (0.7% [0.5%–1.9%] versus 0.4% [0.3%–0.7%]; P value =0.04). Further, 3 adenomas with either an ATP1A1 mutation or a CACNA1D mutation had >30% nuclei with moderate Ki67 staining. In summary, similar to KCNJ5 mutant APAs, ATP1A1 and CACNA1D mutant adenomas have a seemingly specific histopathologic phenotype.

Computed Tomography and Magnetic Resonance in the Imaging of the Aortic Valve and Ascending Aorta

Computed tomography and magnetic resonance imaging are modern tools that can be implemented in the diagnostic imaging of aortic insufficiency and aortic root disease. Both techniques are characterised by high reproducibility and the ability to perform the imaging in any plane. Compared to echocardiography, they are considered as alternatives in the evaluation of aortic regurgitation severity and left ventricular function, and superior in regard to the imaging of ascending aorta.

Aldosterone-Producing Adenomas

Mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CTNNB1 are thought to cause the excessive autonomous aldosterone secretion of aldosterone-producing adenomas (APAs). The histopathology of KCNJ5 mutant APAs, the most common and largest, has been thoroughly investigated and shown to have a zona fasciculata–like composition. This study aims to characterize the histopathologic spectrum of the other genotypes and document the proliferation rate of the different sized APAs. Adrenals from 39 primary aldosteronism patients were immunohistochemically stained for CYP11B2 to confirm diagnosis of an APA. Twenty-eight adenomas had sufficient material for further analysis and were target sequenced at hot spots in the 5 causal genes. Ten adenomas had a KCNJ5 mutation (35.7%), 7 adenomas had an ATP1A1 mutation (25%), and 4 adenomas had a CACNA1D mutation (14.3%). One novel mutation in exon 28 of CACNA1D (V1153G) was identified. The mutation caused a hyperpolarizing shift of the voltage-dependent activation and inactivation and slowed the channel’s inactivation kinetics. Immunohistochemical stainings of CYP17A1 as a zona fasciculata cell marker and Ki67 as a proliferation marker were used. KCNJ5 mutant adenomas showed a strong expression of CYP17A1, whereas ATP1A1/CACNA1D mutant adenomas had a predominantly negative expression (P value =1.20×10−4). ATP1A1/CACNA1D mutant adenomas had twice the nuclei with intense staining of Ki67 than KCNJ5 mutant adenomas (0.7% [0.5%–1.9%] versus 0.4% [0.3%–0.7%]; P value =0.04). Further, 3 adenomas with either an ATP1A1 mutation or a CACNA1D mutation had >30% nuclei with moderate Ki67 staining. In summary, similar to KCNJ5 mutant APAs, ATP1A1 and CACNA1D mutant adenomas have a seemingly specific histopathologic phenotype.

Aldosterone-Producing AdenomasNovelty and Significance: Histopathology–Genotype Correlation and Identification of a Novel CACNA1D Mutation

Mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CTNNB1 are thought to cause the excessive autonomous aldosterone secretion of aldosterone-producing adenomas (APAs). The histopathology of KCNJ5 mutant APAs, the most common and largest, has been thoroughly investigated and shown to have a zona fasciculata–like composition. This study aims to characterize the histopathologic spectrum of the other genotypes and document the proliferation rate of the different sized APAs. Adrenals from 39 primary aldosteronism patients were immunohistochemically stained for CYP11B2 to confirm diagnosis of an APA. Twenty-eight adenomas had sufficient material for further analysis and were target sequenced at hot spots in the 5 causal genes. Ten adenomas had a KCNJ5 mutation (35.7%), 7 adenomas had an ATP1A1 mutation (25%), and 4 adenomas had a CACNA1D mutation (14.3%). One novel mutation in exon 28 of CACNA1D (V1153G) was identified. The mutation caused a hyperpolarizing shift of the voltage-dependent activation and inactivation and slowed the channel’s inactivation kinetics. Immunohistochemical stainings of CYP17A1 as a zona fasciculata cell marker and Ki67 as a proliferation marker were used. KCNJ5 mutant adenomas showed a strong expression of CYP17A1, whereas ATP1A1/CACNA1D mutant adenomas had a predominantly negative expression (P value =1.20×10−4). ATP1A1/CACNA1D mutant adenomas had twice the nuclei with intense staining of Ki67 than KCNJ5 mutant adenomas (0.7% [0.5%–1.9%] versus 0.4% [0.3%–0.7%]; P value =0.04). Further, 3 adenomas with either an ATP1A1 mutation or a CACNA1D mutation had >30% nuclei with moderate Ki67 staining. In summary, similar to KCNJ5 mutant APAs, ATP1A1 and CACNA1D mutant adenomas have a seemingly specific histopathologic phenotype.

LBOS 02-06 HISTOPATHOLOGICAL-SPECIFIC MUTATION SPECTRUM OF ALDOSTERONE-PRODUCING ADENOMAS.

Objective: Aldosterone-producing adenoma (APA) is a common curable cause of hypertension. Somatic mutations in five genes (KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CTNNB1) have been found to cause the excess aldosterone production of two thirds of APAs [1–4]. KCNJ5 mutant APAs, the most common and largest, had explicit genotype-phenotype relationship - a low protein expression of KCNJ5 relative to their peritumoural zona glomerulosa (ZG) and a zona fasciculata-like composition [5–6]. Conversely for the other genes, controversy arises on whether they have the opposite cell phenotype [4,7–8]. This prospective study aim to to characterize the histopathological-specific mutation spectrum of APAs. Design and method: Targeted sequencing of ‘hotspots’ for mutations associated with primary aldosteronism were performed on all APAs. Diagnosis of APA was confirmed through CYP11B2 expression as measured by immunohistochemistry. Immunohistochemical staining using KCNJ5 as a ZG cell marker and CYP17A1 as a ZF cell marker was used to characterize the APAs in addition to hematoxylin and eosin staining. The relationship between genotype and phenotype was then compared. Results: APAs with intense staining of KCNJ5 frequently had a mutation in ATP1A1 or CACNA1D. APAs with intense staining of CYP17A1 frequently had a mutation in KCNJ5. In addition to previously reported mutations, a new causal mutation in exon 28 of CACNA1D was identified. Conclusions: ATP1A1 and CACNA1D mutant APAs comprise of ZG-like cell composition with intense staining of KCNJ5 whereas KCNJ5 mutant APAs comprise of ZF-like cell composition with intense staining of CYP17A1. As ZG-like APAs have been reported to be small [4], diagnosis of an APA in these patients could be made easier through identification of the causal mutation through genotyping the free-circulating ZG-like APA DNA pre-adrenalectomy.