The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 5 2150-2152
Copyright © 2004 by The Endocrine Society
Autosomal-Dominant Pseudohypoaldosteronism Type 1 in a Turkish Family Is Associated with a Novel Nonsense Mutation in the Human Mineralocorticoid Receptor Gene
Felix G. Riepe,
Nils Krone,
Michel Morlot,
Michael Peter,
Wolfgang G. Sippell and
Carl-Joachim Partsch
Division of Pediatric Endocrinology, Department of Pediatrics (F.G.R., N.K., W.G.S., C.-J.P.), Christian-Albrechts-University Kiel, D-24105 Kiel, Germany; Kinderkrankenhaus auf der Bult (M.M.), D-30173 Hannover, Germany; and Sanitas Ostseeklinik Boltenhagen (M.P.), D-23946 Boltenhagen, Germany
Address all correspondence and requests for reprints to: Dr. Felix G. Riepe, Department of Pediatrics, Christian-Albrechts-University of Kiel, Schwanenweg 20, D-24105 Kiel, Germany. E-mail: friepe{at}pediatrics.uni-kiel.de.
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Abstract
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Pseudohypoaldosteronism type 1 (PHA1) is a rare congenital disease inherited in either an autosomal-recessive or an autosomal-dominant trait. The autosomal-dominant form manifests with renal salt loss in infancy and a gradual improvement with advancing age. PHA1 presents with potential life-threatening salt wasting and failure to thrive in early infancy. Autosomal-dominant forms of PHA1 are often caused by heterozygous mutations of the MR gene coding for the mineralocorticoid receptor. Whether heterozygous mutations of the MR gene impair biological function as a result of haplo-insufficiency or due to a dominant-negative effect needs further clarification. We report a case of a renal form of PHA1 in a Turkish family. A heterozygous nonsense mutation c3055C>T (R947X) in exon 9 of the MR gene leading to a premature stop codon was identified in the index patient. The truncated receptor is free of aldosterone binding. The segregation analysis revealed the identical mutation in the patient’s father, who never showed any symptoms of PHA. This shows the incomplete penetrance of the phenotype, although a mild salt loss might have been overlooked in the father’s childhood.
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Introduction
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ALDOSTERONE IS THE main mineralocorticoid in humans. The absence of aldosterone, or an insufficiency of aldosterone-mediated signal transduction due to an impairment of the mineralocorticoid receptor (MR), leads to excessive renal salt loss. Aldosterone acts through MR, a member of the nuclear receptor superfamily. Nuclear receptors have structurally and functionally related regions assigned to distinct domains (1, 2, 3). The amino terminal domain (N-ter) is necessary for a ligand-independent transactivation. The highly conserved central DNA-binding domain consists of two zinc finger motives involved in DNA binding and receptor dimerization. The C-terminal ligand-binding domain (LBD) consists of approximately 250 amino acids with a complex tertiary structure and is responsible for ligand binding, interaction with heat-shock proteins, dimerization, nuclear targeting, and hormone-dependent transactivation. MR induces or represses several aldosterone-regulated genes by binding to cis-acting DNA elements in the regulatory region of the target genes (4). Possible targets for MR-mediated aldosterone-dependent genes are N-Myc downstream-regulated gene 2, K-Ras2, c-Myc, c-Jun, c-Fos, and Fra-2(5, 6). However, the role of these gene products for water and salt homeostasis is so far unknown.
Pseudohypoaldosteronism type 1 (PHA1) is a rare, congenital disease characterized by renal salt loss resistant to mineralocorticoids with highly elevated plasma renin activity (PRA) and plasma aldosterone levels. Cheek and Perry (7) were the first to describe the disease as early as 1958. The autosomal-dominant form shows a renal resistance to aldosterone, characterized by renal salt loss, hyperkalemia, metabolic acidosis, failure to thrive, elevated PRA, and elevated aldosterone levels in infancy. Patients can be treated with oral salt supplementation (8). Patients with the autosomal-dominant form of PHA1 typically show a gradual clinical improvement with regard to renal salt loss during childhood. Some individuals are clinically asymptomatic but may have elevated PRA and aldosterone levels (9).
In some patients, the autosomal-dominant form is caused by mutations of the human mineralocorticoid receptor gene (MR). The human MR gene consists of 10 exons. Translation starts in exon 2 coding for N-ter. Exons 3–5 code the DNA-binding domain, and exons 5–9 code for the LBD (4). To date, 13 mutations associated with PHA1 have been described (10, 11, 12, 13, 14). Geller et al. (10) identified four mutations in the human MR gene, two frameshift mutations, one nonsense mutation in exon 2, and an intron 5 splice mutation. One missense mutation in exon 8 was found by Tajima et al. (11). Our group recently described a frameshift mutation in exon 9 and a nonsense mutation in exon 2 of the MR gene (12, 13). Sartorato et al. (14) described two frameshift mutations in exon 2, one nonsense mutation in exon 4, and three missense mutations in exons 3, 5, and 9 in a group of 14 families.
We present a novel MR gene mutation in a Turkish family with autosomal-dominant PHA1, showing different penetrance within the affected family. We provide additional evidence for a high interindividual clinical heterogeneity of PHA1, suggesting the involvement of unknown coregulators in the etiology of this disease.
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Subjects and Methods
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The male index patient was born of consanguineous parents (first-degree cousins) after an uneventful pregnancy by cesarean section at term with a birth weight of 3310 g (–0.76 SD). Inpatient treatment was necessary because of recurrent vomiting and failure to thrive at 4 wk of age. He presented with a weight of 3210 g, body mass index of 11 kg/m2 (–3.3 SD), and a length of 54 cm (–7.6 SD). On admission, he had hyponatremia (129 mmol/liter) and hyperkalemia (6.4 mmol/liter), but no metabolic acidosis. Plasma aldosterone (2554 pg/ml: normal reference range for age, 150-1050 pg/ml) and PRA were elevated (>40 ng/ml·h, normal reference range for age 1.5–10.2 ng/ml·h). 17-Hydroxyprogesterone, ACTH, and cortisol were normal. Plasma creatinine and sweat electrolytes were in the normal range. After acute therapy with iv saline substitution, the infant showed marked catch-up growth with oral NaCl supplementation of 3–8 mmol/kg·d [after 19 months: length, 79 cm (–1.6 SD); weight, 12 kg; and body mass index, 19.2 kg/m2 (+1.5 SD)]. Plasma electrolytes remained normal under oral NaCl therapy. NaCl treatment was stopped within 24 months. Plasma sodium remained unchanged (135 mmol/liter), but plasma aldosterone was still elevated [1395 pg/ml (3870 nmol/liter): normal reference range for age, 100–880 pg/ml (277–2441 nmol/liter)]. Both parents were clinically free of symptoms. The father’s serum renin and aldosterone levels were slightly elevated [renin, 43.2 mU/liter; normal reference range, 3.3–41 mU/liter; and aldosterone, 379 pg/ml (1051 nmol/liter), normal reference range for age 40–310 pg/ml (111–560 nmol/liter), respectively], whereas sodium and potassium were in the normal range. The excretion of the typical aldosterone metabolites (tetrahydroaldosterone and 18-hydroxy-tetrahydroaldosterone) in a 24-h urinary sample was normal. No events of prior electrolyte disturbance or inpatient treatment were recorded or recollected by the parents.
Analysis of plasma steroids was performed by RIA (IBL, Hamburg, Germany). ACTH levels and PRA/renin were measured using standard procedures (Lumitest ACTH, Brahms Diagnostics, Berlin, Germany; PRA RIA, IBL, Hamburg, Germany; Renin CLIA, Nichols Institute Diagnostics, Bad Vilbel, Germany). Urinary steroid analysis was performed by gas chromatography/mass spectrometry.
Blood samples for molecular genetic analysis were taken after obtaining informed consent. Genomic DNA was extracted from peripheral blood leukocytes, and the mineralocorticoid receptor gene (MR) was amplified using 19 primer pairs with PCR conditions described previously (10, 13). The nucleotide sequences of the PCR products were directly determined using an automated fluorescent sequencer (ABI Prism 310 Genetic Analyzer, PerkinElmer Corp., Wellesley, MA). MR sequencing included all translated exons (2, 3, 4, 5, 6, 7, 8, 9) of the MR gene and the exon/intron boundaries. PCR-restriction analysis of a 258-bp fragment of exon 9 of the MR gene including the mutation was carried out with DdeI (Roche, Mannheim, Germany) at 37 C for 2 h to verify the mutant MR allele.
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Results
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Direct sequencing of PCR products of the MR gene in the patient revealed a heterozygous nucleotide substitution c3055C>T (R947X) in exon 9 (Fig. 1
), according to the nucleotide numbering of Arriza et al. (4). This mutation leads to a premature stop codon, resulting in a truncated protein. The father showed the same heterozygous mutation of the MR gene as the index patient. The mutant allele was detected by restriction enzyme digestion of a 258-bp PCR fragment with DdeI (Fig. 1). The DdeI restriction site was introduced in the mutant allele. The mutant MR PCR fragment was digested to 174- and 84-bp fragments. The analysis of the maternal MR gene revealed a wild-type allele on both chromosomes. No DdeI restriction site is seen in the wild-type allele.
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FIG. 1. A, Family pedigree showing autosomal-dominant segregation of R947X. Solid symbols indicate the R947X mutation. B, Restriction analysis of a 258-bp PCR fragment of exon 9 of the MR gene digested with DdeI, showing a 258-, 174-, and an 84-bp PCR fragment in the heterozygous state for R947X and a single 258-bp fragment in the wild-type sequence. MK, Marker. C, Mutational analysis of MR showing heterozygous substitution of Stop (TGA) for Arg (CGA) in the index patient (II.1) and his father (I.1). The homozygous wild-type allele (CGA) was found in the mother (I.2).
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Discussion
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We identified a novel nonsense mutation (R947X) in the MR gene in a Turkish infant with a renal form of PHA1. The nonsense mutation is localized in the LBD of the MR. The carboxy terminus of the receptor is truncated by 37 amino acids. It is known that artificial MR mutants lacking the last 20 or 40 amino acids show no ligand binding at all (15). Even without the last four amino acids, there is no aldosterone binding. Couette et al. (15) demonstrated that the reason for the impaired ligand binding is due to receptor misfolding. Due to these findings, we can postulate that the mutant R947X-MR protein will have no ligand-binding capacity.
The segregation analysis of the allele carrying the mutation R947X revealed the father as heterozygous for the identical nonsense mutation. However, he was clinically free of symptoms and had normal plasma electrolytes, normal aldosterone urinary metabolites, and only a slightly elevated plasma renin and aldosterone level. No occasion of salt loss, in particular during infancy and childhood, was recorded, and he had never received inpatient treatment. The MR truncation resulting from R947X will lead to a missing binding of cortisol (15). Therefore, a local increase in cortisol concentration at the MR receptor, for example, due to decreased 11ß-HSD type II activity, cannot be responsible for the restoration of the salt homeostasis in this family. We have to speculate that other rescue mechanisms must be involved in this case.
In conclusion, our findings support the finding that PHA1 due to MR gene mutations can show incomplete phenotypical penetrance. The discovery of novel MR mutations in PHA1 may lead to a better understanding of the physiology of renal salt conservation and may disclose alternative regulating mechanisms in sodium homeostasis.
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Acknowledgments
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We are grateful to Gisela Hohmann and Brigitte Andresen for their expert technical assistance. We also thank S. Wudy, Department of Pediatrics, University of Giessen, for the measurement of urinary steroids. In addition, we thank Joanna Voerste for linguistic help with the manuscript.
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Footnotes
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Abbreviations: LBD, Ligand-binding domain; MR, mineralocorticoid receptor; PHA1, pseudohypoaldosteronism type 1; PRA, plasma renin activity.
Received September 5, 2003.
Accepted February 3, 2004.
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Abstract
Introduction
