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Morris Brown and colleagues identify somatic mutations in ATP1A1 and CACNA1D in aldosterone-producing adenomas with features resembling zonaglomerulosa cells. They further show that the ATP1A1 mutations cause inward leak currents under physiological conditions, whereas the CACNA1D mutations induce a shift of voltage-dependent gating to more negative potentials and suppress channel inactivation. 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 1 , 2 . 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 3 . 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 Ca v 1.3. 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.

Somatic variants of the β-catenin gene were found in aldosterone-producing adenomas in two pregnant women. These adenomas expressed very high levels of two hormone receptors, which were presumably engaged by high levels of circulating cognate hormones. Systematic screening detects primary aldosteronism in 5 to 10% of all patients with hypertension and in approximately 20% of patients with treatment-resistant hypertension. 1 , 2 A unilateral aldosterone-producing adenoma is the most common potentially curable cause of hypertension in such cases. 2 Early detection of a unilateral aldosterone-producing adenoma is important both to maximize the likelihood of a complete cure of hypertension by means of adenoma removal and to prevent the onset of resistant hypertension and the risk of long-term cardiovascular complications. 3 The Wnt pathway, through β-catenin signaling, is critical for normal adrenocortical development and maintenance, in particular the zona glomerulosa of . . .

We aimed to assess the attitudes and perceptions of scholarly activity (SA) practices among emergency medicine (EM) physicians who are engaged in training residents. This study examined the belief and need for modern‐day SA, potential barriers, and department resources provided. We conducted a descriptive cross‐sectional survey study of EM physicians across the United States identified from the American College of Emergency Physicians and American College of Osteopathic Physicians directories. The survey consisted of 18 items regarding demographics, attitude toward SA, department support, and questions regarding residency programs. A total of 660 survey recipients completed the survey out of a possible pool of 4296 individuals (15% response rate), of which 530 (80%) indicated they were core faculty. Of core faculty, 428 (80.8%) were part of an allopathic program, whereas 102 (19.2%) were part of an osteopathic program. Department support was provided for protected time (385; 58.3%), research staff (346; 52.4%), Institutional Review Board preparation (240; 36.4%), and biostatistics (314; 47.6%). Of all the institutional roles, the largest percentage (82/125, 65.6%) of chair/vice chair/associate chairs strongly agreed or agreed (score of 5 or 4 of 5) with the statement, “Overall, I am satisfied with the scholarly support provided by my department.” There was no difference in agreement with this statement between respondents in an allopathic versus osteopathic program (210/428, 49.1% allopathic; 45/102, 44.1% osteopathic). There is a need for increased departmental support for SA. To optimally implement the Accreditation Council for Graduate Medical Education (ACGME) SA requirements into strategy and action, the ACGME should consider providing EM residency programs with an outline of best SA practices to foster a uniform consensus across academic institutions.

Aldosterone-producing adenomas (APAs) vary in phenotype and genotype. Zona glomerulosa (ZG)-like APAs frequently have mutations of an L-type calcium channel (LTCC) Ca V 1.3. Using a novel antagonist of Ca V 1.3, compound 8 , we investigated the role of Ca V 1.3 on steroidogenesis in the human adrenocortical cell line, H295R and in primary human adrenal cells. This investigational drug was compared with the common antihypertensive drug nifedipine, which has 4.5-fold selectivity for the vascular LTCC, Ca V 1.2, over Ca V 1.3. In H295R cells transfected with wild-type or mutant Ca V 1.3 channels, the latter produced more aldosterone than wild-type, which was ameliorated by 100 μM of compound 8 . In primary adrenal and non-transfected H295R cells, compound 8 decreased aldosterone production similar to high concentration of nifedipine (100 μM). Selective Ca V 1.3 blockade may offer a novel way of treating primary hyperaldosteronism, which avoids the vascular side effects of Ca V 1.2-blockade and provides targeted treatment for ZG-like APAs with mutations of Ca V 1.3.

Primary Aldosteronism (PA) is the commonest curable cause of hypertension. Whole exome sequencing (WES) in 2011 and 2013 identified common somatic mutations in genes regulating membrane polarisation in 60–80% of aldosterone-producing adenomas (APA). We undertook WES on 39 consecutive APAs in search of further variants. 1 APA revealed a somatic mutation (Val380Asp) within the single transmembrane domain of Cell Adhesion Molecule 1 (CADM1). An adjacent mutation (Gly379Asp) was discovered on WES from a PA patient in Munich. Both short and long isoforms (442 & 453 residues) of wild-type (WT) and both mutant CADM1 genes were cloned into lentivirus vectors and each transduced into adrenocortical (H295R) cells to assess its effect on aldosterone secretion and other parameters. Previous studies in pancreatic islet cells suggested a role of CADM1 in regulating gap junction (GJ) communication. To assess this we microinjected single WT or mutant H295R cells with the GJ permeable dye calceinAM and counted the dye-positive cells after 1 hour. The effect of inhibiting or silencing GJs in H295R cells using peptide gap27 or a Dharmacon smartpool was assessed. H295R cells were also co-transfected with WT or mutant CADM1 and the GJ protein CX43, tagged with the mApple fluorophore. These were mixed with cells transfected with CX43-Venus, allowing confocal visualisation of GJ formation. Protein modelling was undertaken to determine whether Asp in the intramembranous domain changes angulation of CADM1. All mutant isoforms had consistently different effects, shown as a range compared to WT. Cells transduced with mutant CADM1 showed 3-6-fold increase in aldosterone secretion (p<0.01) and 10-20-fold increase in CYP11B2 expression (p<0.001) compared to WT. Dye transfer assays showed paucity of dye transfer between neighbouring mutant CADM1 cells, while calcein passed easily through GJs in WT cells. CX43 inhibition increased aldosterone secretion 2-fold (p<0.01), and CYP11B2 expression 3 to 8-fold (<0.001). Knock-down of GJ proteins increased aldosterone secretion 1.5-fold (p<0.01) and CYP11B2 expression 1.7-fold (p<0.001). Protein modelling showed mutations to increase the angle of ectodomains to cell membrane, from 49o in WT cells, to 62o and 90o in Gly379Asp and Val380Asp respectively; increasing inter-cell distance from 21.2nm to 24.7 and 27.9nm. Mixing of Venus and mApple-tagged CX43 transfected cells showed fewer intact GJ channels in cells co-transfected with mutant compared to WT CADM1 [mutant 42/291 (14.4%) VS WT 68/212 (32.1%) p<0.001]. The CADM1 mutations shows the importance of membrane proteins in aldosterone regulation to extend beyond ion channels and transporters. A key role may be to bring opposing CX43 hemichannels close enough to form GJ channels, permitting the oscillating Ca2+ currents which regulate aldosterone in intact adrenal slices.

Sadaf Farooqi, Inês Barroso and colleagues report genome-wide SNP and CNV association analyses for severe obesity in children, selected at the extreme of the distribution for body mass index. They identify four loci newly associated with severe obesity, an enrichment of rare CNVs in severely obese cases and overlap in the loci associated with severe obesity in children and with BMI and obesity in the general population. Common and rare variants associated with body mass index (BMI) and obesity account for <5% of the variance in BMI. We performed SNP and copy number variation (CNV) association analyses in 1,509 children with obesity at the extreme tail (>3 s.d. from the mean) of the BMI distribution and 5,380 controls. Evaluation of 29 SNPs ( P < 1 × 10 −5 ) in an additional 971 severely obese children and 1,990 controls identified 4 new loci associated with severe obesity ( LEPR , PRKCH , PACS1 and RMST ). A previously reported 43-kb deletion at the NEGR1 locus was significantly associated with severe obesity ( P = 6.6 × 10 −7 ). However, this signal was entirely driven by a flanking 8-kb deletion; absence of this deletion increased risk for obesity ( P = 6.1 × 10 −11 ). We found a significant burden of rare, single CNVs in severely obese cases ( P < 0.0001). Integrative gene network pathway analysis of rare deletions indicated enrichment of genes affecting G protein–coupled receptors (GPCRs) involved in the neuronal regulation of energy homeostasis.

Most aldosterone-producing adenomas (APAs) have gain-of-function somatic mutations of ion channels or transporters. However, their frequency in aldosterone-producing cell clusters of normal adrenal gland suggests a requirement for codriver mutations in APAs. Here we identified gain-of-function mutations in both CTNNB1 and GNA11 by whole-exome sequencing of 3/41 APAs. Further sequencing of known CTNNB1 -mutant APAs led to a total of 16 of 27 (59%) with a somatic p.Gln209His, p.Gln209Pro or p.Gln209Leu mutation of GNA11 or GNAQ . Solitary GNA11 mutations were found in hyperplastic zona glomerulosa adjacent to double-mutant APAs. Nine of ten patients in our UK/Irish cohort presented in puberty, pregnancy or menopause. Among multiple transcripts upregulated more than tenfold in double-mutant APAs was LHCGR , the receptor for luteinizing or pregnancy hormone (human chorionic gonadotropin). Transfections of adrenocortical cells demonstrated additive effects of GNA11 and CTNNB1 mutations on aldosterone secretion and expression of genes upregulated in double-mutant APAs. In adrenal cortex, GNA11/Q mutations appear clinically silent without a codriver mutation of CTNNB1 . Sequence analysis identifies gain-of-function somatic mutations in GNA11 or GNAQ in CTNNB1 -mutant aldosterone-producing adenomas. Most patients with these mutations presented during puberty, pregnancy or menopause, with elevated LHCGR expression.

Aldosterone-producing adenomas (APAs) are the commonest curable cause of hypertension. Most have gain-of-function somatic mutations of ion channels or transporters. Herein we report the discovery, replication and phenotype of mutations in the neuronal cell adhesion gene CADM1 . Independent whole exome sequencing of 40 and 81 APAs found intramembranous p.Val380Asp or p.Gly379Asp variants in two patients whose hypertension and periodic primary aldosteronism were cured by adrenalectomy. Replication identified two more APAs with each variant (total, n  = 6). The most upregulated gene (10- to 25-fold) in human adrenocortical H295R cells transduced with the mutations (compared to wildtype) was CYP11B2 (aldosterone synthase), and biological rhythms were the most differentially expressed process. CADM1 knockdown or mutation inhibited gap junction (GJ)-permeable dye transfer. GJ blockade by Gap27 increased CYP11B2 similarly to CADM1 mutation. Human adrenal zona glomerulosa (ZG) expression of GJA1 (the main GJ protein) was patchy, and annular GJs (sequelae of GJ communication) were less prominent in CYP11B2-positive micronodules than adjacent ZG. Somatic mutations of CADM1 cause reversible hypertension and reveal a role for GJ communication in suppressing physiological aldosterone production. Recurrent somatic mutations altering residues in the transmembrane domain of CADM1 are identified in aldosterone-producing adenomas. Follow-up studies implicate a role of gap junction communication in regulating aldosterone production.

Melanocortin receptor accessory proteins (MRAPs) modulate signaling of melanocortin receptors in vitro. To investigate the physiological role of brain-expressed melanocortin 2 receptor accessory protein 2 (MRAP2), we characterized mice with whole-body and brain-specific targeted deletion of Mrap2, both of which develop severe obesity at a young age. Mrap2 interacts directly with melanocortin 4 receptor (Mc4r), a protein previously implicated in mammalian obesity, and it enhances Mc4r-mediated generation of the second messenger cyclic adenosine monophosphate, suggesting that alterations in Mc4r signaling may be one mechanism underlying the association between Mrap2 disruption and obesity. In a study of humans with severe, early-onset obesity, we found four rare, potentially pathogenic genetic variants in MRAP2, suggesting that the gene may also contribute to body weight regulation in humans.

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.