Ute I. Scholl

Recurrent gain of function mutation in calcium channel CACNA1H causes early-onset hypertension with primary aldosteronism

Many Mendelian traits are likely unrecognized owing to absence of traditional segregation patterns in families due to causation by de novo mutations, incomplete penetrance, and/or variable expressivity. Genome-level sequencing can overcome these complications. Extreme childhood phenotypes are promising candidates for new Mendelian traits. One example is early onset hypertension, a rare form of a global cause of morbidity and mortality. We performed exome sequencing of 40 unrelated subjects with hypertension due to primary aldosteronism by age 10. Five subjects (12.5%) shared the identical, previously unidentified, heterozygous CACNA1HM1549V mutation. Two mutations were demonstrated to be de novo events, and all mutations occurred independently. CACNA1H encodes a voltage-gated calcium channel (CaV3.2) expressed in adrenal glomerulosa. CACNA1HM1549V showed drastically impaired channel inactivation and activation at more hyperpolarized potentials, producing increased intracellular Ca2+, the signal for aldosterone production. This mutation explains disease pathogenesis and provides new insight into mechanisms mediating aldosterone production and hypertension. DOI: http://dx.doi.org/10.7554/eLife.06315.001

Novel somatic mutations in primary hyperaldosteronism are related to the clinical, radiological and pathological phenotype.

Aldosterone‐producing adenomas (APAs) and bilateral adrenal hyperplasia are important causes of secondary hypertension. Somatic mutations in KCNJ5, CACNA1D, ATP1A1, ATP2B3 and CTNNB1 have been described in APAs.

Whole-Exome Sequencing Characterizes the Landscape of Somatic Mutations and Copy Number Alterations in Adrenocortical Carcinoma

CONTEXTnAdrenocortical carcinoma (ACC) is a rare and lethal malignancy with a poorly defined etiology, and the molecular genetics of ACC are incompletely understood.nnnOBJECTIVEnTo utilize whole-exome sequencing for genetic characterization of the underlying somatic mutations and copy number alterations present in ACC.nnnDESIGNnScreening for somatic mutation events and copy number alterations (CNAs) was performed by comparative analysis of tumors and matched normal samples from 41 patients with ACC.nnnRESULTSnIn total, 966 nonsynonymous somatic mutations were detected, including 40 tumors with a mean of 16 mutations per sample and one tumor with 314 mutations. Somatic mutations in ACC-associated genes included TP53 (8/41 tumors, 19.5%) and CTNNB1 (4/41, 9.8%). Genes with potential disease-causing mutations included GNAS, NF2, and RB1, and recurrently mutated genes with unknown roles in tumorigenesis comprised CDC27, SCN7A, and SDK1. Recurrent CNAs included amplification at 5p15.33 including TERT (6/41, 14.6%) and homozygous deletion at 22q12.1 including the Wnt repressors ZNRF3 and KREMEN1 (4/41 9.8% and 3/41, 7.3%, respectively). Somatic mutations in ACC-established genes and recurrent ZNRF3 and TERT loci CNAs were mutually exclusive in the majority of cases. Moreover, gene ontology identified Wnt signaling as the most frequently mutated pathway in ACCs.nnnCONCLUSIONSnThese findings highlight the importance of Wnt pathway dysregulation in ACC and corroborate the finding of homozygous deletion of Wnt repressors ZNRF3 and KREMEN1. Overall, mutations in either TP53 or CTNNB1 as well as focal CNAs at the ZNRF3 or TERT loci denote mutually exclusive events, suggesting separate mechanisms underlying the development of these tumors.

PRKACA mutations in cortisol-producing adenomas and adrenal hyperplasia - a single-center study of 60 cases

OBJECTIVEnCortisol excess due to adrenal adenomas or hyperplasia causes Cushings syndrome. Recent genetic studies have identified a somatic PRKACA(L206R) mutation as a cause of cortisol-producing adenomas. We aimed to compare the clinical features of PRKACA-mutant lesions with those of CTNNB1 mutations, and to search for similar mutations in unilateral hyperplasia or tumors co-secreting aldosterone.nnnDESIGN, PATIENTS, AND METHODSnIn this study, 60 patients with cortisol excess who had adrenalectomies at our institution between 1992 and 2013 were assessed, and somatic mutations were determined by Sanger sequencing. A total of 36 patients had overt Cushings syndrome, the remainder were subclinical: 59 cases were adenomas (three bilateral) and one was classified as hyperplasia. Four tumors had proven co-secretion of aldosterone.nnnRESULTSnAmong cortisol-secreting unilateral lesions without evidence of co-secretion (n=52), we identified somatic mutations in PRKACA (L206R) in 23.1%, CTNNB1 (S45P, S45F) in 23.1%, GNAS (R201C) in 5.8%, and CTNNB1+GNAS (S45P, R201H) in 1.9%. PRKACA and GNAS mutations were mutually exclusive. Of the co-secreting tumors, two (50%) had mutations in KCNJ5 (G151R and L168R). The hyperplastic gland showed a PRKACA(L206R) mutation, while patients with bilateral adenomas did not have known somatic mutations. PRKACA-mutant lesions were associated with younger age, overt Cushings syndrome, and higher cortisol levels vs non-PRKACA-mutant or CTNNB1-mutant lesions. CTNNB1 mutations were more significantly associated with right than left lesions.nnnCONCLUSIONSnPRKACA(L206R) is present not only in adenomas, but also in unilateral hyperplasia and is associated with more severe autonomous cortisol secretion. Bilateral adenomas may be caused by yet-unknown germline mutations.

An Update on Familial Hyperaldosteronism.

Familial forms of primary aldosteronism have been suggested to account for up to 6% of cases in referral centers. For many years, the genetics of familial hyperaldosteronism remained unknown, with the notable exception of glucocorticoid-remediable aldosteronism, due to unequal crossing over and formation of a chimeric 11β-hydroxylase/aldosterone synthase gene. Over the past 5 years, mutations in 3 additional genes have been shown to cause familial forms of primary aldosteronism. Gain-of-function heterozygous germline mutations in KCNJ5, which encodes an inward rectifier potassium channel, cause autosomal dominant syndromes of PA and hypertension with or without adrenal hyperplasia. Germline mutations in CACNA1D, which codes for an L-type calcium channel, have so far only been found in 2 cases with a syndrome of primary aldosteronism, seizures, and neurologic abnormalities. Both KCNJ5 and CACNA1D mutations in familial hyperaldosteronism were only discovered following identification of similar or identical somatic mutations in aldosterone-producing adenomas. In contrast, a recent exome sequencing study identified germline mutations in CACNA1H (a T-type calcium channel), previously undescribed in adenomas, in 5 unrelated families with early-onset primary aldosteronism and hypertension, without any additional shared symptoms. Future exome or genome sequencing studies are expected to shed light on the genetic basis of many cases of familial hyperaldosteronism that remain unexplained.

CACNA1HM1549V Mutant Calcium Channel Causes Autonomous Aldosterone Production in HAC15 Cells and Is Inhibited by Mibefradil

We recently demonstrated that a recurrent gain-of-function mutation in a T-type calcium channel, CACNA1H(M1549V), causes a novel Mendelian disorder featuring early-onset primary aldosteronism and hypertension. This variant was found independently in five families. CACNA1H(M1549V) leads to impaired channel inactivation and activation at more hyperpolarized potentials, inferred to cause increased calcium entry. We here aimed to study the effect of this variant on aldosterone production. We heterologously expressed empty vector, CACNA1H(WT) and CACNA1H(M1549V) in the aldosterone-producing adrenocortical cancer cell line H295R and its subclone HAC15. Transfection rates, expression levels, and subcellular distribution of the channel were similar between CACNA1H(WT) and CACNA1H(M1549V). We measured aldosterone production by an ELISA and CYP11B2 (aldosterone synthase) expression by real-time PCR. In unstimulated cells, transfection of CACNA1H(WT) led to a 2-fold increase in aldosterone levels compared with vector-transfected cells. Expression of CACNA1H(M1549V) caused a 7-fold increase in aldosterone levels. Treatment with angiotensin II or increased extracellular potassium levels further stimulated aldosterone production in both CACNA1H(WT)- and CACNA1H(M1549V)-transfected cells. Similar results were obtained for CYP11B2 expression. Inhibition of CACNA1H channels with the T-type calcium channel blocker Mibefradil completely abrogated the effects of CACNA1H(WT) and CACNA1H(M1549V) on CYP11B2 expression. These results directly link CACNA1H(M1549V) to increased aldosterone production. They suggest that calcium channel blockers may be beneficial in the treatment of a subset of patients with primary aldosteronism. Such blockers could target CACNA1H or both CACNA1H and the L-type calcium channel CACNA1D that is also expressed in the adrenal gland and mutated in patients with primary aldosteronism.

Src-family protein tyrosine kinase phosphorylates WNK4 and modulates its inhibitory effect on KCNJ1 (ROMK)

Significance Genetic, biochemical, and animal model studies have implicated with-no-lysine kinase 4 (WNK4) in regulation of the balance between renal salt reabsorption and K secretion. It has been proposed that serum/glucocorticoid-induced kinase 1 (SGK1)-induced phosphorylation of WNK4 plays an important role in switching from inhibiting KCNJ1 (ROMK) channels during volume depletion to promoting K secretion during increasing K intake. However, it is not understood why a high SGK1 level fails to stimulate ROMK channels during the volume depletion, whereas it increases ROMK channel activity and K secretion during high K intake. The present study provides insights into the mechanism illustrating how SFK-induced phosphorylation of WNK4 regulates ROMK to distinguish the physiologic response to hyperkalemia and volume depletion. With-no-lysine kinase 4 (WNK4) inhibits the activity of the potassium channel KCNJ1 (ROMK) in the distal nephron, thereby contributing to the maintenance of potassium homeostasis. This effect is inhibited via phosphorylation at Ser1196 by serum/glucocorticoid-induced kinase 1 (SGK1), and this inhibition is attenuated by the Src-family protein tyrosine kinase (SFK). Using Western blot and mass spectrometry, we now identify three sites in WNK4 that are phosphorylated by c-Src: Tyr1092, Tyr1094, and Tyr1143, and show that both c-Src and protein tyrosine phosphatase type 1D (PTP-1D) coimmunoprecipitate with WNK4. Mutation of Tyr1092 or Tyr1143 to phenylalanine decreased the association of c-Src or PTP-1D with WNK4, respectively. Moreover, the Tyr1092Phe mutation markedly reduced ROMK inhibition by WNK4; this inhibition was completely absent in the double mutant WNK4Y1092/1094F. Similarly, c-Src prevented SGK1-induced phosphorylation of WNK4 at Ser1196, an effect that was abrogated in the double mutant. WNK4Y1143F inhibited ROMK activity as potently as wild-type (WT) WNK4, but unlike WT, the inhibitory effect of WNK4Y1143F could not be reversed by SGK1. The failure to reverse WNK4Y1143F-induced inhibition of ROMK by SGK1 was possibly due to enhancing endogenous SFK effect on WNK4 by decreasing the WNK4–PTP-1D association because inhibition of SFK enabled SGK1 to reverse WNK4Y1143F-induced inhibition of ROMK. We conclude that WNK4 is a substrate of SFKs and that the association of c-Src and PTP-1D with WNK4 at Tyr1092 and Tyr1143 plays an important role in modulating the inhibitory effect of WNK4 on ROMK.

Intracellular Molecular Differences in Aldosterone- Compared to Cortisol-Secreting Adrenal Cortical Adenomas.

The adrenal cortex is a major site of steroid hormone production. Two hormones are of particular importance: aldosterone, which is produced in the zona glomerulosa in response to volume depletion and hyperkalemia, and cortisol, which is produced in the zona fasciculata in response to stress. In both cases, acute stimulation leads to increased hormone production, and chronic stimulation causes hyperplasia of the respective zone. Aldosterone- and cortisol-producing adenomas (APAs and CPAs) are benign tumors of the adrenal cortex that cause excess hormone production, leading to primary aldosteronism and Cushing’s syndrome, respectively. About 40% of the APAs carry somatic heterozygous gain-of-function mutations in the K+ channel KCNJ5. These mutations lead to sodium permeability, depolarization, activation of voltage-gated Ca2+ channels, and Ca2+ influx. Mutations in the Na+/K+-ATPase subunit ATP1A1 and the plasma membrane Ca2+-ATPase ATP2B3 similarly cause Na+ or H+ permeability and depolarization, whereas mutations in the Ca2+ channel CACNA1D directly lead to increased calcium influx. One in three CPAs carries a recurrent gain-of-function mutation (L206R) in the PRKACA gene, encoding the catalytic subunit of PKA. This mutation causes constitutive PKA activity by abolishing the binding of the inhibitory regulatory subunit to the catalytic subunit. These mutations activate pathways that are relatively specific to the respective cell type (glomerulosa versus fasciculata), and there is little overlap in mutation spectrum between APAs and CPAs, but co-secretion of both hormones can occur. Mutations in CTNNB1 (beta-catenin) and GNAS (Gsα) are exceptions, as they can cause both APAs and CPAs through pathways that are incompletely understood.

A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis

BackgroundDysregulated WNT signaling dominates adrenocortical malignancies. This study investigates whether silencing of the WNT negative regulator DKK3 (Dickkopf-related protein 3), an implicated adrenocortical differentiation marker and an established tumor suppressor in multiple cancers, allows dedifferentiation of the adrenal cortex.MethodsWe analyzed the expression and regulation of DKK3 in human adrenocortical carcinoma (ACC) by qRT-PCR, immunofluorescence, promoter methylation assay, and copy number analysis. We also conducted functional studies on ACC cell lines, NCI-H295R and SW-13, using siRNAs and enforced DKK3 expression to test DKK3’s role in blocking dedifferentiation of adrenal cortex.ResultsWhile robust expression was observed in normal adrenal cortex, DKK3 was down-regulated in the majority (>75%) of adrenocortical carcinomas (ACC) tested. Both genetic (gene copy loss) and epigenetic (promoter methylation) events were found to play significant roles in DKK3 down-regulation in ACCs. While NCI-H295R cells harboring β-catenin activating mutations failed to respond to DKK3 silencing, SW-13 cells showed increased motility and reduced clonal growth. Conversely, exogenously added DKK3 also increased motility of SW-13 cells without influencing their growth. Enforced over-expression of DKK3 in SW-13 cells resulted in slower cell growth by an extension of G1 phase, promoted survival of microcolonies, and resulted in significant impairment of migratory and invasive behaviors, largely attributable to modified cell adhesions and adhesion kinetics. DKK3-over-expressing cells also showed increased expression of Forkhead Box Protein O1 (FOXO1) transcription factor, RNAi silencing of which partially restored the migratory proficiency of cells without interfering with their viability.ConclusionsDKK3 suppression observed in ACCs and the effects of manipulation of DKK3 expression in ACC cell lines suggest a FOXO1-mediated differentiation-promoting role for DKK3 in the adrenal cortex, silencing of which may allow adrenocortical dedifferentiation and malignancy.

Genetic mechanisms of human hypertension and their implications for blood pressure physiology

Hypertension, or elevated blood pressure, constitutes a major public health burden that affects more than 1 billion people worldwide and contributes to ~9 million deaths annually. Hereditary factors are thought to contribute to up to 50% of interindividual blood pressure variability. Blood pressure in the general population approximately shows a normal distribution and is thought to be a polygenic trait. In rare cases, early-onset hypertension or hypotension are inherited as Mendelian traits. The identification of the underlying Mendelian genes and variants has contributed to our understanding of the physiology of blood pressure regulation, emphasizing renal salt handling and the renin angiotensin aldosterone system as players in the determination of blood pressure. Genome-wide association studies (GWAS) have revealed more than 100 variants that are associated with blood pressure, typically with small effect sizes, which cumulatively explain ~3.5% of blood pressure trait variability. Several GWAS associations point to a role of the vasculature in the pathogenesis of hypertension. Despite these advances, the majority of the genetic contributors to blood pressure regulation are currently unknown; whether large-scale exome or genome sequencing studies will unravel these factors remains to be determined.