CLCN2 clicks with aldosterone-producing adenomas, too!

In primary aldosteronism, inappropriately elevated aldosterone levels cause hypertension and – as an optional finding – hypokalemia. Contrary to historic estimates, primary aldosteronism is now considered the commonest cause of secondary hypertension, present in >5% of hypertensive patients. Patients with primary aldosteronism typically either have an aldosterone-producing adenoma (a benign tumor of the adrenal cortex) or bilateral adrenal hyperplasia. Rarely, familial aggregation is observed (‘familial hyperaldosteronism’) (1). Genetic studies over the last decade have provided insight into the molecular pathogenesis of primary aldosteronism, identifying new somatic (tumor-specific) mutations in aldosterone-producing adenomas and germline (inherited or de novo) mutations in familial hyperaldosteronism. The two genes that are most frequently mutated in aldosterone-producing adenomas are KCNJ5, encoding for a potassium channel, and CACNA1D, encoding for a calcium channel. Interestingly, germline mutations in the same genes can also cause Mendelian forms of primary aldosteronism (2, 3). Contrary, mutations in some genes, such as ATP1A1 and ATP2B3 encoding ATPases (4), are only found in aldosterone-producing adenomas, but not in familial hyperaldosteronism, likely because germline mutations would be lethal. Mutations in other genes, namely CYP11B2 (aldosterone synthase) and CACNA1H (encoding for another calcium channel), have so far only been identified in familial hyperaldosteronism (5), raising the question whether do not provide sufficient proliferative stimulus for adenoma formation.

Most recently, two groups reported germline mutations in the CLCN2 gene in familial hyperaldosteronism. The report by Scholl et al. described mutations in eight families (two de novo, 17 individuals total with CLCN2 mutations) (6) and dubbed the associated syndrome familial hyperaldosteronism type II; the study by Fernandes-Rosa et al. identified one de novo case (7). CLCN2 encodes for the voltage-gated chloride channel ClC-2, the first anion channel implicated in primary aldosteronism and hypertension. In both studies, electrophysiology demonstrated that ClC-2 mutations in familial hyperaldosteronism cause increased chloride permeability. The ensuing cellular depolarization leads to activation of voltage-gated calcium channels, influx of calcium and increased aldosterone production.

In a landmark paper in this issue of the European Journal of Endocrinology, Dutta et al. add an important piece of the primary aldosteronism genetics puzzle. They Sanger sequenced the CLCN2 coding regions in 80 aldosterone-producing adenomas from Norway, Sweden and Germany (8). One of these tumors carried a somatic CLCN2 mutation. Remarkably, this somatic mutation (p.Gly24Asp) was identical with the de novo germline mutation reported in the patient described by Fernandes-Rosa et al. (7). The tumor with CLCN2 mutation was 13 mm in size (thus, rather small) and was found in a 35-year-old man with elevated aldosterone levels. Preoperative adrenal venous sampling demonstrated lateralization of aldosterone production. After surgical tumor removal, both blood pressure and aldosterone:renin ratio normalized. RNA expression levels of CYP11B2 (aldosterone synthase) in the tumor were high, providing further evidence that the lesion was the cause of the patient’s primary aldosteronism.

The p.Gly24Asp mutation changes a conserved residue in the N-terminus of the ClC-2 channel. Its impact on channel function and aldosterone production has been well studied in vitro. Mutant channels show higher current amplitudes and altered voltage-dependent gating. In a cellular model of glomerulosa function, transfection of channels carrying the mutation significantly raises aldosterone production compared to wildtype channels (7).

Collectively, there is no doubt that the somatic CLCN2 mutation reported by Dutta et al. accounts for increased aldosterone production in the tumor. Some open questions remain: Do CLCN2 mutations cause sufficient proliferation to cause tumor formation (as mentioned, the tumor reported by Dutta et al. was quite small) or do tumors with CLCN2 mutations carry additional somatic mutations that account for proliferation? How frequent are somatic CLCN2 mutations in aldosterone-producing adenomas? Are CYP11B2 or CACNA1H mutations also rare causes of aldosterone-producing adenomas? What is the phenotype associated with CLCN2 mutations (e.g. tumors with KCNJ5 mutations are typically large and associated with female gender)? Exome sequencing of the tumor with CLCN2 mutation and normal tissue, as well as targeted sequencing of large cohorts could provide additional insights. In any case, the study by Dutta et al. is a milestone toward the identification of additional rare somatic mutations in aldosterone-producing adenomas, further emphasizing the long-neglected role of anion channels in zona glomerulosa function and primary aldosteronism. Lastly, it provides yet another example of the fruitful interactions between the studies of rare Mendelian diseases (such as familial hyperaldosteronism) and more common sporadic disorders (such as aldosterone-producing adenomas): The identification of germline mutations in the KCNJ5 and the CACNA1D genes in familial hyperaldosteronism was inspired by the discovery of corresponding somatic mutations in aldosterone-producing adenomas (2, 3). Dutta et al. turn the story around, expanding the disease spectrum associated with gain-of-function mutations in CLCN2 from familial hyperaldosteronism to aldosterone-producing adenomas.