Abstract
Objective
To screen for CLCN2 mutations in apparently sporadic cases of aldosterone-producing adenomas (APAs).
Description
Recently, CLCN2, encoding for the voltage-gated chloride channel protein 2 (ClC-2), was identified to be mutated in familial hyperaldosteronism II (FH II). So far, somatic mutations in CLCN2 have not been reported in sporadic cases of APAs. We screened 80 apparently sporadic APAs for mutations in CLCN2. One somatic mutation was identified at p.Gly24Asp in CLCN2. The male patient had a small adenoma in size but high aldosterone levels preoperatively. Postoperatively, the patient had normal aldosterone levels and was clinically cured.
Conclusion
In this study, we identified a CLCN2 mutation in a sporadic APA comprising about 1% of all APAs investigated. This mutation was complementary to mutations in other susceptibility genes for sporadic APAs and may thus be a driving mutation in APA formation.
Introduction
Dedicated cells of zona glomerulosa of adrenal cortex produce aldosterone in response to angiotensin II (1). The two most important physiological stimuli of aldosterone production are blood volume and level of serum potassium. Aldosterone maintains the blood pressure and blood volume by retaining salt and subsequently water follows. Excessive production of aldosterone is a pathological condition called primary aldosteronism (PA) and can be found in 5–10% of hypertensive patients (2). About 30% of patients with PA have inappropriately high levels of aldosterone due to aldosterone-producing adenomas (APAs), benign tumors that produce aldosterone, independent of the renin-angiotensin system resulting in high blood pressure that is resistant to conventional antihypertensive drugs.
The underlying genetic cause of 60–90% of APAs is known. Driving mutations are identified in the genes KCNJ5, ATP1A1, ATP2B3, CACNA1D and CTNNB1 (3, 4, 5, 6, 7, 8). The most frequent (40–50%) mutations are identified in KCNJ5 that encodes a K+ selective ion channel (Kir3.4). Mutations affect the selectivity filter of the ion channel which results in chronic depolarization of the cell membrane and opening of voltage-gated Ca2+ channels. Mutations in ATP1A1 and ATP2B3 have been identified in <10% each of APAs (3, 4, 5). Prevalence of CACNA1D mutation varies in different ethnic groups. Recently, targeted next-generation sequencing approach identified the higher percentage of CACNA1D mutation (>40%) in black population (8). All known mutations result in elevated cytoplasmic Ca2+ levels, and subsequently leads to increased aldosterone biosynthesis. In familial hyperaldosteronism (FH), the chimeric gene CYP11B1/CYP11B2 as well as germline mutations in KCNJ5, CACNA1H and CACNA1D have been identified in FH I, FH III, FH IV and primary aldosteronism, seizures, and neurologic abnormalities (PASNA) syndrome, respectively (6, 9, 10, 11). Of note, germline and de novo mutations in CLCN2, encoding the voltage-gated chloride channel ClC-2, were identified in familial hyperaldosteronism II (FH II) (12, 13). However, no somatic mutations in CLCN2 in sporadic APAs have been reported so far.
Patients and methods
Samples and nucleic acid isolation
This study encompasses 39 (primary cohort) unselected and consecutive patients with APAs diagnosed and treated in Norway, Sweden, and Germany (3). All patients had hypertension, variable hypokalemia and a high aldosterone-to-renin-ratio at the time of diagnosis. Following adrenal excision, the tumors were snap frozen at −80°C until analysis. The study was approved by the Local Ethical Committees in Linköping, Bergen and Essen and all patients gave informed consent. Regarding the primary cohort, tumor and blood DNA were available for 39 individuals. For only two APA samples, RNA was missing. The secondary cohort consisted of 41 APAs from Bergen, Norway.
Mutation analysis
The entire coding sequence of CLCN2, encoding the voltage-gated chloride channel ClC-2, was selected for Sanger sequencing on ABI 3500 Genetic Analyzer using Big Dye terminator v3.1 cycling kit for labeling of the samples. The primer sequences and PCR conditions are available on request.
Transcriptome analysis
The RNA samples of 37 APAs (primary cohort) were prepared and loaded on the Affymetrix Human Transcriptome Array 2.0 according to manufacturer’s protocol. The array data were normalized using RMA (Robust Multi-Array Average) sketch.
Results
Sequencing of the 39 APAs from the primary cohort revealed a somatic mutation (c.71G>A, p.Gly24Asp, rs1085307938) in the CLCN2 gene (Fig. 1A). The tumor from this patient did not have mutations in any other previously identified APA susceptibility gene (Fig. 1E). The mRNA expression of CLCN2 was observed at similar levels in wild-type and mutant CLCN2 APAs (Fig. 1F). The mutant tumor expressed high mRNA levels of CYP11B2 and the patient had high levels of plasma aldosterone despite a small sized APA ((Fig. 1G, H and I), Table 1). The male patient with this mutation was quite young (35 years) (Fig. 1J) and had unilateral disease as confirmed by preoperative venous catheterization. Postoperatively, the blood pressure as well as the aldosterone-to-renin-ratio was normalized. Pathology confirmed the presence of an adrenal adenoma.
CLCN2 mutations and comparison of pre-clinical and clinical variables. Sequencing chromatograms showing (A) the somatic mutation c.71G>A in a sporadic APA (top) and wild-type CLCN2 sequence in blood (bottom) and (B) the germline variant c.218G>A in both the APA (top) and blood (bottom). (C) Position of the identified somatic mutation (yellow dot) on the protein structure of the CIC-2 channel. (D) Amino acid sequence alignment of human CIC-2 channel with its orthologs. Arrow indicates the identified mutation in this study. (E) A somatic CLCN2 variant was found in 1 out of 39 sporadic APAs (primary cohort). This mutation is complementary to 20 other mutations in KCNJ5, ATP1A1, ATP2B3, CTNNB1 and CACNA1D. The remaining 18 APAs had no identified mutations in the listed susceptibility genes. (F) Log10 expression of CLCN2 after normalization of mRNA microarray data (unpublished data). (G) Relative mRNA levels of CYP11B2 in tumors with different mutations (real-time qPCR analysis of total RNA using HPRT1 as an internal control). (H) Plasma aldosterone levels in patients with different mutations (indicated on graph) and without mutation in the known susceptibility genes. (I) Age of the patients and (J) diameter of the tumors with regard to the identified mutation. neg, no mutation found.
Citation: European Journal of Endocrinology 181, 5; 10.1530/EJE-19-0377
CLCN2 mutations and comparison of pre-clinical and clinical variables. Sequencing chromatograms showing (A) the somatic mutation c.71G>A in a sporadic APA (top) and wild-type CLCN2 sequence in blood (bottom) and (B) the germline variant c.218G>A in both the APA (top) and blood (bottom). (C) Position of the identified somatic mutation (yellow dot) on the protein structure of the CIC-2 channel. (D) Amino acid sequence alignment of human CIC-2 channel with its orthologs. Arrow indicates the identified mutation in this study. (E) A somatic CLCN2 variant was found in 1 out of 39 sporadic APAs (primary cohort). This mutation is complementary to 20 other mutations in KCNJ5, ATP1A1, ATP2B3, CTNNB1 and CACNA1D. The remaining 18 APAs had no identified mutations in the listed susceptibility genes. (F) Log10 expression of CLCN2 after normalization of mRNA microarray data (unpublished data). (G) Relative mRNA levels of CYP11B2 in tumors with different mutations (real-time qPCR analysis of total RNA using HPRT1 as an internal control). (H) Plasma aldosterone levels in patients with different mutations (indicated on graph) and without mutation in the known susceptibility genes. (I) Age of the patients and (J) diameter of the tumors with regard to the identified mutation. neg, no mutation found.
Citation: European Journal of Endocrinology 181, 5; 10.1530/EJE-19-0377
CLCN2 mutations and comparison of pre-clinical and clinical variables. Sequencing chromatograms showing (A) the somatic mutation c.71G>A in a sporadic APA (top) and wild-type CLCN2 sequence in blood (bottom) and (B) the germline variant c.218G>A in both the APA (top) and blood (bottom). (C) Position of the identified somatic mutation (yellow dot) on the protein structure of the CIC-2 channel. (D) Amino acid sequence alignment of human CIC-2 channel with its orthologs. Arrow indicates the identified mutation in this study. (E) A somatic CLCN2 variant was found in 1 out of 39 sporadic APAs (primary cohort). This mutation is complementary to 20 other mutations in KCNJ5, ATP1A1, ATP2B3, CTNNB1 and CACNA1D. The remaining 18 APAs had no identified mutations in the listed susceptibility genes. (F) Log10 expression of CLCN2 after normalization of mRNA microarray data (unpublished data). (G) Relative mRNA levels of CYP11B2 in tumors with different mutations (real-time qPCR analysis of total RNA using HPRT1 as an internal control). (H) Plasma aldosterone levels in patients with different mutations (indicated on graph) and without mutation in the known susceptibility genes. (I) Age of the patients and (J) diameter of the tumors with regard to the identified mutation. neg, no mutation found.
Citation: European Journal of Endocrinology 181, 5; 10.1530/EJE-19-0377
Clinical characteristics of the patient with APA and the somatic CLCN2 mutation.
| Case ID | Gender | Age (years) | Aldosterone (ng/L) | Renin (ng/mL/h) | Tumor size (mm) | Gene | Mutation | Mutation | Protein alteration |
|---|---|---|---|---|---|---|---|---|---|
| B16 | M | 35.4 | 791 | 0.2 | 13 | CLCN2 | Somatic | c.71G>A | p.Gly24Asp |
Accession number for CLCN2, NM_004366.6.
APA, aldosterone-producing adenoma.
In addition to the somatic mutation, we found a germline variant (c.218G>A, p.Arg73His, rs144412275) in another patient (Fig. 1B). The variant p.Arg73His (rs144412275) is a rare variant with a minor allele frequency of 0.003 in Exome Aggregation Consortium (http://exac.broadinstitute.org/) and The Swedish Frequency resource for genomics (https://swegen-exac.nbis.se/) databases (14). We further sequenced the entire coding sequence of the CLCN2 gene in additional 41 sporadic APAs (secondary cohort) from Norway without finding any additional mutations.
Discussion
This is the first report of a somatic mutation in CLCN2 in a sporadic APA leading to an amino acid change at position 24 (p.Gly24Asp) of CIC-2. CIC-2 is a homo-dimeric chloride channel, open at hyperpolarized conditions and sensitive to intracellular Cl− concentrations (15). The amino acid region 21–46 is sensitive toward cell-swelling and extracellular pH (16, 17). A previous study reported that deletion of amino acids 21–46 and substitution mutations of several amino acids in this region leads to open channel (conductance) at resting condition (15). The heterozygous mutation p.Gly24Asp is located in a highly conserved amino acid region in the cytoplasmic domain of the CIC-2 channel (Fig. 1C and D). Recently, the mutation p.Gly24Asp was identified as a de novo mutation in FH II (12). The expression of mutant p.Gly24Asp was shown to drastically increase the current compared to wild-type channel at resting potential of −80 mV (12). The mutation p.Gly24Asp leads to depolarization of the adrenal cell membrane and activates the voltage-gated calcium channel that leads to Ca2+ influx, which in turn activates gene transcription (12, 13).
Regarding p.Arg73His, any functional effect is yet to be determined. A previous study reported that deletion of amino acids 66–76 had no effect on the channel activity (15). Polyphen2, SIFT, and MutationAssessor analysis predicted the single amino acid substitution as a benign alteration. A multi-species comparison of CLCN2 amino acid reveals the presence of histidine at position 73 in several vertebrate species, closely related to humans, including primate’s for example, orangutan (data not shown). If this variant would cause aldosterone-producing adenomas, such tumors should be prevalent among orangutans and selected against. Further, no familial history of hyperaldosteronism or hypertension is reported in the patient’s family and the patient has not developed any recurrence in 8 years. With these consideration, p.Arg73His seems to be a rare benign or neutral variant.
In conclusion, one somatic CLCN2 mutation was identified in about 1% of all sporadic APAs in our two cohorts combined (n = 80). The CLCN2 mutation is complementary to mutations in other sporadic APA susceptibility genes and may thus be a driving mutation in the formation of APAs.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this study.
Funding
This study was supported by ALF Grants, Region Östergötland, and by grants from LiU cancer to Oliver Gimm and Peter Söderkvist and from the Norwegian Cancer Society and the Western Norway Regional Health Authority (Helse Vest RHF) to T A.
Author contribution statement
R K D, O G, and P S were responsible for study design and manuscript writing. R K D designed and performed the experiments and analyzed the results of the study. O G, M W, P A, T A and A H provided the tumor tissue, blood and clinical data of the patients.
Acknowledgements
The authors thank Annette Molbaek and Åsa Schippert for technical assistant.
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