Introduction
The steroid hormone aldosterone is normally produced in the adrenal zona glomerulosa in response to either angiotensin II, which is produced in response to volume depletion, or hyperkalemia (high plasma K+ level). Both stimuli cause membrane depolarization, activating voltage-gated Ca2+ channels; increased intracellular Ca2+ provides the signal that triggers aldosterone production (Spät and Hunyady, 2004). In the setting of volume depletion, aldosterone signaling in renal and intestinal epithelia produces increased salt (re)absorption, promoting restoration of intravascular volume; in hyperkalemia, aldosterone promotes increased potassium secretion, restoring electrolyte balance.
Pathological secretion of aldosterone in the absence of normal physiological stimuli leads to primary aldosteronism (PA), producing increased salt (re)absorption and hypertension. Hypokalemia is a frequently associated finding, resulting from increased renal K+ elimination. PA is found in 10% of patients referred for evaluation of hypertension (Conn, 1955; Rossi et al., 2006). About half of these patients have adrenal aldosterone-producing adenomas (APAs). Germline mutations in three genes have been shown to cause rare Mendelian forms of early-onset PA. Gene fusions leading to constitutive expression of aldosterone synthase (encoded by CYP11B2), a rate-limiting enzyme in aldosterone biosynthesis, cause Glucocorticoid-Remediable Aldosteronism (GRA) (Lifton et al., 1992). Mutations in and near the selectively filter of the K+ channel encoded by KCNJ5 result in channels that conduct Na+, leading to adrenal glomerulosa cell depolarization and activation of Ca2+ channels, producing a Mendelian form of aldosteronism (Choi et al., 2011). Gain of function mutations in the calcium channel encoded by CACNA1D cause increased Ca2+ channel activity and another form of PA. These latter patients also have seizures, neurodevelopmental and neuromuscular abnormalities owing to gain of function effects of CACNA1D in the nervous system (Scholl et al., 2013). Families with GRA often have many affected subjects and were identified by linkage analysis in extended families (Lifton et al., 1992). Germline mutations in KCNJ5 are typically de novo or in small nuclear families; similarly, CACNA1D mutations to date are all de novo (Choi et al., 2011; Scholl et al., 2012, 2013). Germline mutations in KCNJ5 and CACNA1D were found following identification of the same or related somatic mutations as drivers of APAs (Choi et al., 2011; Scholl et al., 2012; Azizan et al., 2013; Scholl et al., 2013).
The causes of PA in many patients remain undetermined. Although Mendelian inheritance has been suggested by recurrence of PA in some kindreds without mutations in known genes (Stowasser et al., 1992; Torpy et al., 1998; Lafferty et al., 2000), traditional linkage analysis has failed to identify additional causative genes, likely due to a combination of factors including locus heterogeneity, high frequency of de novo mutations, reduced penetrance and/or variable expressivity. The advent of next-generation sequencing, allowing the search for recurrent mutations or greater burden of rare variants in individual genes than expected by chance, can permit identification of such loci in the absence of classical segregation patterns. Very rare phenotypes, such as childhood PA, are promising candidates for such traits.
Using exome sequencing, we here identify five independent occurrences of the identical mutation in CACNA1H among 40 subjects with unexplained PA in childhood. CACNA1H encodes a voltage-gated calcium channel that is expressed in adrenal glomerulosa. Electrophysiology demonstrates that this variant causes reduced inactivation and a shift of activation to more hyperpolarized potentials, effects inferred to produce increased calcium influx and PA.