Author response: Recurrent gain of function mutation in calcium channel CACNA1H causes early-onset hypertension with primary aldosteronism (2015)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract 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. https://doi.org/10.7554/eLife.06315.001 eLife digest The consequence of mutations to the large majority of human genes is unknown. Most mutations that are currently known were discovered by tracing their effects through families. This allows the locations of mutations to be pinpointed on chromosomes—the structures that genetic material is packaged into. Other mutations are harder to trace because individuals with these mutations may develop very different signs and symptoms, or not develop clinical abnormalities at all. Alternatively, a trait may appear sporadically in a family because the mutation arises anew in the affected subject. Recently developed technologies that allow scientists to rapidly sequence all the gene-encoding regions of an individual's DNA—their genome—offer a new way to identify harmful genetic variants. Comparing the genomes of individuals with rare disorders can reveal if the individuals share any genetic mutations in common that could cause their symptoms. Scholl et al. used this strategy to sequence the genomes of 40 individuals with a rare type of hypertension—a condition that causes high blood pressure, and increases the risk of strokes, kidney failure and heart attacks—that develops early in childhood. In this form of the disease, high blood pressure is caused by the adrenal glands above the kidneys producing too much of a hormone called aldosterone. Some genetic causes of this form of the disease have already been identified. Now, Scholl et al. have found a new genetic mutation present in five families with this condition. Two of the individuals were the first in their families to develop this mutation, while three others inherited it. Some of the family members with this mutation had hypertension and some did not. The mutation is in a gene that encodes a type of calcium channel—a protein found in the membrane that surrounds cells, and which can open and close to control the amount of calcium in the cell. This particular calcium channel is abundant in the cells of the adrenal gland. Scholl et al. found that the mutation causes the calcium channels to be more likely to open and take longer to close. This increases the number of calcium ions that move into the cell, which causes the adrenal gland to produce more aldosterone. These new insights have provided a new way of diagnosing early-onset hypertension, and suggest that targeting calcium channels could help to develop new treatments for this disease. https://doi.org/10.7554/eLife.06315.002 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. Results Whole-exome sequencing of 40 subjects with PA From a cohort of more than 1500 unrelated subjects referred for evaluation of genetic forms of hypertension, we identified 40 subjects diagnosed with hypertension and PA by age 10 years in whom disease-causing mutations in CYP11B2, KCNJ5, and CACNA1D (Lifton et al., 1992; Choi et al., 2011; Scholl et al., 2013) were excluded. Clinical details are shown in Supplementary file 1A. All subjects had hypertension with elevated aldosterone levels despite low plasma renin activity (PRA). None of the subjects studied were the offspring of consanguineous union. DNA from peripheral blood was subjected to exome capture and sequencing; mean coverage was 73 independent reads per targeted base (Supplementary file 1B). Variants were called as described in ‘Materials and methods’ (Lemaire et al., 2013). We performed three analyses tailored to the expectation of a rare genetic disease (‘Materials and methods’). We sought previously unreported (absent in dbSNP, NHLBI, 1000Genomes and Yale exome databases) protein-altering variants that occurred in more than one subject (Supplementary file 1C); we performed gene burden analyses to search for previously unreported or rare (minor allele frequency [MAF] < 0.01%) heterozygous variants that collectively occurred in any gene more often than expected by chance (Supplementary file 1D); we searched for rare (MAF < 0.1%) homozygous and potential compound heterozygous variants that collectively occurred in any genes more often than expected by chance (Supplementary file 1E). Identification of a recurrent novel variant in CACNA1H There was only one result that surpassed genome-level significance: we found five apparently unrelated subjects with the identical previously unreported heterozygous A > G variant, resulting in a p.Met1549Val substitution in CACNA1H, which encodes the pore-forming alpha subunit of a T-type, low voltage-activated calcium channel (aka CaV3.2) (Figure 1, Table 1, Supplementary file 1F) (Perez-Reyes, 2003). This variant is absent among more than 129,000 alleles sequenced from diverse populations in the Exome Aggregation Consortium (Exome Aggregation Consortium), and Yale databases. No other CACNA1H alleles with allele frequencies <0.01% were found among our cohort. Like other Ca2+ channel alpha subunits, CACNA1H contains four homologous repeats (I–IV), each with six transmembrane segments (S1–S6). The CACNA1HM1549V variant lies in the S6 segment of repeat III (Marksteiner et al., 2001). Sanger sequencing in each case confirmed the heterozygous variant (Figure 1A). Figure 1 with 1 supplement see all Download asset Open asset Kindreds with hypertension and primary aldosteronism (PA) with CACNA1HM1549V mutation at conserved position of S6 domain. (A) Pedigrees of kindreds with CACNA1HM1549V mutation are shown. Studied subjects with early-onset hypertension are shown as black filled symbols, and subjects with early-onset hypertension by family history (K333) or low renin with normal blood pressure (K1393) are shown as grey filled symbols. Genotypes are indicated below each symbol (+/+ denotes wild type sequence; +/M denotes heterozygosity for CACNA1HM1549V variant). Corresponding Sanger sequencing results for selected subjects are depicted to the right. (B) Transmembrane structure of CaV3.2 (encoded by CACNA1H), the pore-forming subunit of a voltage-gated Ca2+ channel, is shown. These channels have four internal homologous repeats (I–IV), each with six transmembrane segments (S1–S6) and a membrane-associated loop between the pore-forming S5 and S6 segments. The p.Met1549Val mutation is located in S6 of repeat III. (C) Conservation of CACNA1HM1549 in CACNA1H orthologs and paralogs. The amino acid sequences of the S6 segment of domain III of CACNA1H, orthologs and paralogs are shown. The S6 segment, including Met1549, is virtually completely conserved (highlighted in yellow) among orthologs and all paralogs that are activated by small changes in membrane potential (l, low voltage-activated) but not those activated by large changes (h, high voltage-activated). M1549 is part of the Met-Phe-Val sequence that is implicated in rapid channel inactivation (Marksteiner et al., 2001). https://doi.org/10.7554/eLife.06315.003 Figure 1—source data 1 Source data corresponding to Figure 1. https://doi.org/10.7554/eLife.06315.004 Download elife-06315-fig1-data1-v1.xlsx Table 1 Clinical features of index cases with CACNA1HM1549V https://doi.org/10.7554/eLife.06315.006 Subject IDGenderAge dxBP (%ile)Aldo (ng/dl)PRA (ng/ml/hr)ARR (ng/dl: ng/ml/hr)1347-1M3 yrs160/105 (>99th)20<0.1>2001390-1F7 yrs150/90 (>99th)660.23301368-1M8 yrs140/90 (>99th)20<0.2>100333-2M9 yrs192/144 (>99th)40<0.7>571393-1M2 mos170/110 (>99th)87<0.6>145 M, male; F, female; age dx, age at diagnosis of hypertension; yrs, years; mos, months; BP, blood pressure; (%ile), percentile adjusted for age and gender; Aldo, serum aldosterone; PRA, plasma renin activity; ARR, aldosterone:renin ratio, values >20 with aldosterone level >15 are considered indicative of primary aldosteronism (PA). Three index cases were of European ancestry, one Hispanic, and one African American by self-report and principal component analysis (Figure 1—figure supplement 1). Members of the extended families were recruited, and sequencing of these subjects demonstrated that CACNA1HM1549V was a de novo mutation (absent in the biological parents) in both the index case of kindred 1347, and in the affected mother of the index case in kindred 1390 (Figure 1). Analysis of highly polymorphic markers confirmed paternity and maternity in both kindreds (Supplementary file 1G). This establishes independent occurrences of CACNA1HM1549V in these two kindreds. In the remaining three kindreds, the variant was transmitted to the index case from a parent, and samples from grandparents were not available for further analysis of transmission (Figure 1). Analysis of kinship coefficients using SNP genotypes of affected subjects from Illumina Human 1M-Quad beadchips and the KING algorithm (Manichaikul et al., 2010) provided no evidence that these three kindreds shared recent common ancestry (Supplementary file 1H, ‘Materials and methods’). Further, haplotypes flanking the CACNA1H mutation were phased using the BEAGLE program, revealing that the maximum shared haplotype flanking the CACNA1H mutation among these three kindreds was only 53.6 kb (87.0 kb for the two European kindreds, Figure 2). From this data, the maximum likelihood estimate of the number of generations since the last shared common ancestor among these subjects is estimated to be 714 generations 95% CI 290-1268 (Genin et al., 2004). A more conservative analysis identifying homozygous discordant SNPs (eliminating inference of haplotypes by phasing) still limited the shared haplotype to less than 127.1 kb, consistent with the results of phasing using BEAGLE. These findings indicate that the CACNA1HM1549V mutation in these three kindreds has not been inherited from a recent common ancestor, and has either arisen independently or has been inherited from an extremely remote common ancestor. The latter possibility is extremely unlikely given the absence of this mutation in more than 129,000 alleles studied to date. Figure 2 Download asset Open asset Shared haplotypes in subjects with inherited CACNA1HM1549V variant. Haplotypes of three affected individuals from kindreds without proven de novo occurrence of CACNA1HM1549V variant were phased using BEAGLE (‘Materials and methods’) (Browning and Browning, 2007). This analysis identified a very small maximum interval shared among all three individuals (∼53.6 kb, green box) flanked by rs1075789 and rs3760122. If only homozygous discordant calls (*) were considered in the absence of phasing, the maximum interval shared by all three subjects would be 127.1 kb and the longest pairwise shared haplotype would be 200.0 kb between 1393-1 and 333-1. https://doi.org/10.7554/eLife.06315.007 The probability of finding any recurrent protein-altering de novo mutation among 40 kindreds is ∼4.0 × 10−6 (see ‘Materials and methods’). Even with a conservative estimate of the allele frequency of CACNA1HM1549V of 0.00001 in the general population (‘conservative’ because it has never been seen among >129,000 alleles in subjects not selected for early PA), the probability of finding three additional instances of this mutation in 38 unrelated subjects is ∼8.4 × 10−12. Combined, the probability of finding these five instances of the identical variant by chance is conservatively estimated to be 3.4 × 10−17, providing extremely strong statistical support for the role of this mutation in PA. CACNA1H is expressed in human adrenal glomerulosa If the CACNA1HM1549V mutation causes early-onset PA, CACNA1H (CaV3.2) should be expressed in human adrenal glomerulosa. CACNA1H transcripts have previously been reported in human kidney, liver, heart and brain (Cribbs et al., 1998), and our prior analysis of gene expression of human adrenal cortex showed that CACNA1H was the second most highly expressed calcium channel alpha subunit, after CACNA1D (Scholl et al., 2013). We performed immunohistochemistry with two different antibodies specific for the encoded channel protein (CaV3.2), demonstrating strong staining of human adrenal glomerulosa; this staining was abolished after preincubation with immunogenic peptide (Figure 3). These results are consistent with prior in situ hybridization and electrophysiological studies of rodent and bovine glomerulosa (Schrier et al., 2001; Hu et al., 2012) as well as a recent study of human adrenal gland (Felizola et al., 2014). Figure 3 Download asset Open asset Immunohistochemistry of CaV3.2 in normal human adrenal gland. Sections of normal human adrenal are shown. C denotes adrenal capsule; G, glomerulosa; F, fasciculata. (A) Normal adrenal gland stained with hematoxylin and an antibody to CaV3.2 (Alomone). (B) Higher power image of adrenal in panel (A). (C, D) Absence of staining after preincubation of the antibody with the antigenic peptide, demonstrating specificity. (E) A second normal human adrenal gland stained for CACNA1H as in (A, B). (F) Gland from (A–D) stained with a second α-CACNA1H antibody (Santa Cruz). Scale bars, 100 μm (A, C); 50 μm (B, D, E, F). The results demonstrate expression of CaV3.2 in the normal zona glomerulosa, which is only several cells in depth. https://doi.org/10.7554/eLife.06315.008 Clinical features of subjects with CACNA1HM1549V variant The clinical features of the index cases harboring the CACNA1HM1549V variant were uniform. All presented with hypertension by age 10 and had persistent inappropriate elevation of serum aldosterone with suppressed PRA and high aldosterone:PRA ratio, indicative of autonomous adrenal aldosterone production (Table 1). Adrenal imaging by computed tomography, magnetic resonance or ultrasound showed no evidence of mass or hyperplasia at the time of presentation. There were no other recurrent or distinctive features of the index cases, specifically no history of seizures, neurologic or neuromuscular disorders as found in patients with CACNA1D mutations (Scholl et al., 2013). Additional details are presented in Appendix 1. By direct Sanger sequencing, we identified five additional CACNA1HM1549V mutation carriers among family members, including four parents and one uncle of an index case (Figure 1). Of these five, three were diagnosed with early severe hypertension while two were not, and in fact were normotensive as adults (Appendix 1 and Supplementary file 1I). For example, subject 1390-2 was diagnosed with severe hypertension and PA at age 17; her hypertension was difficult to control, leading to unilateral adrenalectomy at age 29. Her hypertension nonetheless recurred, requiring reinstitution of treatment. Interestingly, the histology of her adrenal gland showed striking microscopic hyperplasia. While the normal adrenal glomerulosa comprises only a few cell layers and is about 70 μm in depth, the glomerulosa of subject 1390-2 was ∼30 cell layers and ∼300 μm in depth (Figure 4A–C). CACNA1H was expressed in the hyperplastic glomerulosa layer (Figure 4, Appendix 1). In this kindred, the CACNA1HM1549V variant arose concordantly and segregated precisely with PA and early hypertension (Table 2). Two mutation carriers (1368-2 and 1393-3) were normotensive as adults and had not been diagnosed as hypertensive in childhood; in subject 1393-3, PRA was at the lower limit of normal with normal aldosterone level, while PRA and aldosterone levels were normal in 1368-2 (Supplementary file 1I). Figure 4 Download asset Open asset Glomerulosa hyperplasia in adrenal gland of subject 1390-2 with CACNA1HM1549V mutation. C denotes adrenal capsule; G, glomerulosa; F, fasciculata; R, reticularis; M, medulla. (A) Low power image stained with hematoxylin and eosin. Scale bar 1000 μm. (B, C) Higher power images of adrenal from panel (A), scale bars 400 μm (B) or 100 μm (C). The mutant adrenal shows marked zona glomerulosa hyperplasia, with micronodular invasion of the capsule (denoted by *). (D) Same adrenal gland stained with hematoxylin and antibody to CaV3.2 (Santa Cruz), demonstrating specific staining of zona glomerulosa. Scale bar, 400 μm. (E, F), higher power images stained with second antibody to CaV3.2 (Alomone). Scale bars 250 μm (E) or 100 μm (F). CaV3.2 is expressed in the hyperplastic zona glomerulosa. https://doi.org/10.7554/eLife.06315.009 Table 2 Laboratory values of carriers and of CACNA1HM1549V in kindred 1390 Subject IDGenderAge (ng/dl)PRA (ng/ml/hr)ARR renin M, male; F, female; in years was serum potassium Aldo, serum aldosterone; direct renin is indicative of hypertension; ARR, aldosterone:renin ratio, values >20 or values of renin with aldosterone level greater than are considered indicative of PA. samples were on the same and values were in the same for in whom values are at age Figure 1 for and are not in Figure 1, and are of of CACNA1HM1549V variant for early-onset PA the of this mutation for early-onset PA, we performed targeted Sanger sequencing for the CACNA1HM1549V variant in DNA of additional unrelated subjects with PA diagnosed after age 10 with hypertension and adrenal hyperplasia, and referred for potential genetic causes of hypertension without evidence of PA. We also sequenced DNA of including 40 that did not have mutations in previously implicated genes and et al., 2011; Azizan et al., 2013; et al., 2013; Scholl et al., No additional CACNA1HM1549V mutations were demonstrating striking for early-onset PA. 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