This Article Abstract Full Text (PDF) All Versions of this Article: 91/9/3671    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fernandes-Rosa, F. L. Articles by Antonini, S. R. Search for Related Content PubMed PubMed Citation Articles by Fernandes-Rosa, F. L. Articles by Antonini, S. R. Related Collections Adrenal and Hypertension Cardiovascular Endocrinology

Recurrence of the R947X Mutation in Unrelated Families with Autosomal Dominant Pseudohypoaldosteronism Type 1: Evidence for a Mutational Hot Spot in the Mineralocorticoid Receptor Gene

Division of Pediatric Endocrinology (F.L.F.-R., M.d.C., A.C.L., S.R.A.), Department of Pediatrics, School of Medicine of Ribeirao Preto, University of Sao Paulo, 14049-900 Sao Paulo, Brazil; and Division of Pediatric Endocrinology (W.G.S., F.G.R.), Department of Pediatrics, University Hospital Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany

Address all correspondence and requests for reprints to: Sonir R. Antonini, M.D., Ph.D., Department of Pediatrics, School of Medicine of Ribeirão Preto, Avenida Bandeirantes, 3900-Ribeirão Preto, 14049-900 Sao Paulo, Brazil. E-mail: s.antonini{at}hcrp.fmrp.usp.br.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Background: The renal form of pseudohypoaldosteronism type 1 (PHA1) is a rare disease characterized by congenital mineralocorticoid resistance of the kidney. Twenty-two different loss-of-function mutations in the mineralocorticoid receptor gene have been described in families with PHA1. These mutations were not recurrent and resulted in a large phenotypic variability.

Objective: The objective of this study is to analyze the recurrence of an inactivating mutation in the mineralocorticoid receptor gene in unrelated families with autosomal dominant PHA1.

Patients: Seventeen members from three unrelated families with autosomal dominant PHA1 were studied, including 11 affected patients with variable clinical manifestations. Fifty healthy subjects were used as controls.

Methods: Genomic DNA was extracted, and the entire coding region of the mineralocorticoid receptor gene was submitted to automatic sequencing. Four dinucleotide microsatellite markers spanning a region of 3.2 cM in the human mineralocorticoid receptor gene locus, and two intragenic polymorphisms were used for haplotype analysis.

Results: A heterozygous point mutation at codon 947 (c.2839C>T) changing arginine to stop codon (R947X) was found in the three families. Different haplotypes segregated with the R947X mutation in each family, demonstrating the absence of a founder effect for this mutation.

Conclusion: Codon 947 of the mineralocorticoid receptor is the first mutational hot spot for autosomal dominant PHA1.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
PSEUDOHYPOALDOSTERONISM type 1 (PHA1) is characterized by congenital mineralocorticoid resistance of the kidney or other mineralocorticoid target tissues (1). The affected subjects present with renal salt loss, dehydration, and failure to thrive associated with hyponatremia, hyperkalemia, and metabolic acidosis, with high plasma renin and aldosterone levels and no response to the administration of exogenous mineralocorticoids (2, 3). Two models of inheritance of PHA1, autosomal recessive (systemic form) and autosomal dominant or sporadic (renal form), have been described (4, 5).

In the renal form of PHA1, the mineralocorticoid resistance is restricted to the kidney. Clinical symptoms usually abate with age, whereas aldosterone and plasma renin activity (PRA) may remain high. Patients respond well to salt supplementation, but exogenous mineralocorticoids have no effect (6). Most of the autosomal and sporadic PHA1 cases are caused by loss-of-function mutations in the human mineralocorticoid receptor (hMR) gene (NR3C2).

The hMR gene has an amino-terminal region that harbors a ligand-independent transactivation function (AF1) coded by exon 2, a conserved DNA binding domain coded by exons 3 and 4, and a C-terminal domain responsible for ligand binding and ligand-dependent transactivation coded by exons 5–9 (7). To date, 22 mutations in the three domains of the hMR have been described in PHA1 patients (2, 8, 9, 10, 11, 12, 13, 14, 15). There was no recurrence of these mutations among the affected families.

The R947X mutation of the hMR gene was previously described in a Turkish family with PHA1 (13). In this study, we report for the first time the recurrence of this mutation in two other unrelated families with PHA1. Therefore, the purpose of this study was to ascertain a common haplotype in the hMR gene in the three families with the R947X or whether codon 947 of hMR is a mutational hot spot.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
The local Ethics Committees approved this study, and informed consent was obtained from the individuals and/or their parents.

Family 1

The index male patient, the first child of nonrelated Brazilian parents, was born at term after an uneventful pregnancy, with a birth weight of 3250 g. He was admitted to hospital at 2 months of age with insufficient weight gain, vomiting, dehydration, and poor sucking from the age of 2 wk. Hyponatremia (126 mmol/liter), hyperkalemia (6.3 mmol/liter), elevated urinary Na (42 mmol/liter), and normal sweat Na (23.8 mmol/liter; normal range: 15–80 mmol/liter) were noted. Plasma aldosterone was elevated (296 ng/dl or 8.19 nmol/liter) as was PRA (7.3 ng/ml·h or 1460 fmol/liter·sec). Plasma 17-hydroxyprogesterone, ACTH, and cortisol were normal. Fludrocortisone administration had no effect on hyponatremia, weight gain, or PRA; however, the infant showed marked catch-up in growth and normalization of the electrolytes with oral NaCl supplementation (2 g/d). Plasma aldosterone normalized (66 ng/dl or 1.82 nmol/liter) but PRA remained elevated (12 ng/ml·h or 2400 fmol/liter·sec). Aldosterone levels increased (100 ng/dl or 2.77 nmol/liter) and PRA remained elevated (8.5 ng/ml·h or 1700 fmol/liter·sec) after cessation of NaCl supplementation at 10 months old. The patient has grown normally and his electrolytes remain normal to the present time. The mother had slightly elevated plasma aldosterone (38 ng/dl or 1.05 nmol/liter) and PRA (3.9 ng/ml·h or 780 fmol/liter·sec) with mild symptoms during infancy (vomiting and poor sucking), but she recovered without treatment. The father had no symptoms and normal plasma aldosterone and PRA. Five maternal uncles, two maternal cousins, and the maternal grandmother were investigated.

Family 2

The index male patient, the first child of nonrelated parents of Turkish origin, was born at term after an uneventful pregnancy with a birth weight of 3600 g. He was admitted to hospital with insufficient weight gain and renal salt loss at 2 wk old. Hyponatremia (129 mmol/liter), hyperkalemia (5.8 mmol/liter), and elevated urinary Na (33 mmol/liter) were noted. Plasma aldosterone was elevated (212 ng/dl or 5.87 nmol/liter) as was plasma renin (8676 ng/liter or 205 pmol/liter). Plasma 17-hydroxyprogesterone and cortisol were normal. The neonate presented weight gain and normalization of electrolytes with oral NaCl supplementation (4 mmol/kg·d). Both parents are clinically free of symptoms and have normal plasma electrolytes and aldosterone levels.

Family 3

This Turkish family has been previously described (13). The symptomatic proband and his healthy father are carriers of the R947X mutation in the hMR gene.

Clinical and laboratory testing

PRA was determined using a commercial kit (GammaCoat RIA kit, Clinical Assays; INCSTAR, Stillwater, MN). Assay sensitivity was 0.2 ng/ml·h, and intraassay and interassay coefficients of variation were 2.8 and 7.6%, respectively. Reference values for PRA were 1.24 ± 1.09 ng/ml·h or 248 ± 218 fmol/liter·sec. Serum 17-hydroxyprogesterone and cortisol were measured by RIA, and electrolytes were measured by the ion-selective electrode method (16). Plasma aldosterone levels were measured by RIA after extraction in family 1 (16) and in families 2 and 3 (IBL, Hamburg, Germany). Plasma aldosterone levels above 28 ng/dl or 0.77 nmol/liter in adults and 90 ng/dl or 2.49 nmol/liter in infants were considered elevated (17).

Genomic DNA isolation and DNA sequencing

Genomic DNA was extracted from peripheral blood leukocytes using the QIAmp kit (QIAGEN Inc., Valencia, CA). All translated exons (2, 3, 4, 5, 6, 7, 8, 9) of the mineralocorticoid receptor gene and the exon/intron boundaries were sequenced as previously described (2, 12). PCR products were sequenced using a commercial kit (ABI Prism Big Dye; PE Applied Biosystems, Foster City, CA) and analyzed with an ABI Prism 310 Genetic Analyzer (PE Applied Biosystems).

Microsatellite analysis

The D4S1586, D4S3031, D4S3014, and D4S3008 microsatellite markers, spanning a region of 3.2 cM around the hMR gene (Fig. 1), were amplified by PCR. The primer sequences were obtained from Ensembl (www.ensembl.org), and the sense primers were 5'-end labeled with fluorescence (6-FAM; PE Applied Biosystems). PCR was performed under the following conditions: denaturation at 94 C for 5 min; 30 cycles of denaturation at 94 C for 30 sec, annealing at 55 C or 56 C, and extension at 72 C for 2 min; final extension at 72 C for 10–15 min. Alleles were submitted to electrophoresis using an ABI 310 apparatus with ROX-500 (PE Applied Biosystems) as size standard. Genotype analysis was automated using GENESCAN 3.0 and GENOTYPER 2.5 (PE Applied Biosystems). Haplotype analysis was performed in 11 members of family 1 (seven affected), three members of family 2 (two affected), and three members of family 3 (two affected). The microsatellite D4S3014 was also studied in 50 healthy Brazilians.

View larger version (8K): [in this window] [in a new window]   FIG. 1. Schematic representation of the hMR gene locus on chromosome 4 showing the distance in centimorgans of microsatellite markers D4S1586, D4S3031, D4S3014, and D4S3008 in relation to the hMR gene. The location of the intragenic polymorphisms I180V and A241V and the R947X mutation are also shown.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

PCR direct sequencing of exon 9 of the hMR gene in the proband of family 1 revealed the heterozygous point mutation at codon 947 (c.2839C>T), changing arginine (CGA) to stop codon (TGA) (R947X). His mother, his grandmother, two uncles, and two cousins also presented this mutation (Fig. 2). The same heterozygous mutation was found in the proband of family 2 and his father, and was previously described in the proband of family 3 and his father (13).

View larger version (34K): [in this window] [in a new window]   FIG. 2. Pedigree structure and haplotypes for families 1 (A), 2 (B), and 3 (C). Females are represented by circles and males are represented by squares. Affected individuals are indicated by black symbols. Genotypes for markers D4S1586, D4S3031, D4S3014, and D4S3008, hMR gene polymorphisms I180V and A241V, and the R947X mutation are displayed in relation to the hMR gene. The disease-associated haplotype is indicated by black bars.

Haplotype analysis

To investigate whether a common ancestral chromosome accounted for the recurrence of the same mutation in these three unrelated families, we constructed mutation-associated haplotypes by genotyping all family members for four dinucleotide repeat microsatellites in addition to the two intragenic polymorphisms described in the DNA sequencing section (Figs. 1 and 2) found in the Brazilian family. The D4S3031 marker and the A241V polymorphism were not informative, because all subjects studied were homozygous for the two markers. The 248-bp allele of the D4S3014 marker was found in all carriers of the R947X mutation in the three families. However, this is the most common allele (63 of 100 alleles) found in healthy Brazilians. The D4S1586 and D4S3008 marker alleles were shared by the affected individuals in all three families, but they were different in each family. The I180V polymorphism was found in the heterozygous state only in affected subjects in families 1 and 3, but not in family 2. Taken together, these results indicate that the cosegregating haplotype was different in each of these families.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
In this study, we describe for the first time the occurrence of the R947X mutation in the hMR in three unrelated families with the autosomal dominant form of PHA1. The R947X mutation is located in the C-terminal ligand binding domain of the hMR. Deletions at the C-terminal end of the hMR greatly reduced aldosterone binding capacity, whereas the loss of the last four amino acids completely abolished the aldosterone binding, as previously demonstrated (18). The mutant receptor R947X impairs the translation of the last 37 amino acids of the hMR and leads to the loss of -helices H11 and H12. These -helices are responsible for folding the receptor into a ligand-binding competent state and for stabilizing the active receptor conformation (19). Therefore, it is expected that the R947X mutation reduces the ligand-binding capacity, explaining the PHA1 phenotype in the affected patients.

Two of the families with the R947X mutation are from Turkey, whereas one is from Brazil. The Brazilian population comprises different ethnic backgrounds, including immigrants from the Middle East, a region colonized by Turkey, and therefore, a common founder effect of the R947X mutation could not be ruled out. To verify this hypothesis, we performed haplotype analysis using four dinucleotide microsatellite markers spanning a region of 3.2 cM in the hMR gene locus on chromosome 4 and two intragenic polymorphisms. We found three different haplotypes segregating with the mutation R947X in each family. The possibility of recombination between these markers is improbable due to the distance from the hMR gene; therefore, our data demonstrate the absence of a founder effect for the R947X mutation in these three families. On the other hand, these data indicate that codon 947 of the hMR is a hot spot for loss-of-function mutations, the first described in this gene. The nucleotide region that coded for codon 947 harbors a CpG dinucleotide (CGA) and is located within a CG-rich region in the 3' region of the hMR gene. The recurrent mutation in this nucleotide region could suggest deamination-induced mutation of the cytosine at the CpG site, which results in thymine. Deamination of 5-methylcytosine to thymine at CpG sites is probably the most important cause of point mutations in humans, accounting for more than 20% of all base substitutions that give rise to genetic disease (20).

In conclusion, we report for the first time the occurrence of the R947X mutation in the hMR in three unrelated families with PHA1. Our results suggest that this mutation has arisen independently in these families; therefore, codon 947 of the hMR might be considered to be a hot spot for loss-of-function mutations in PHA1.

	Acknowledgments

 
We gratefully acknowledge Dr. David S. Geller for the MR gene primer sequences and the technical assistance of Emilia M. Pinto and Sandra S. Santos.

	Footnotes

 
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo 04/15362-6, Brazil.

First Published Online June 6, 2006

Abbreviations: hMR, Human mineralocorticoid receptor; PHA1, pseudohypoaldosteronism type 1; PRA, plasma renin activity.

Received March 21, 2006.

Accepted May 31, 2006.

	References

Top Abstract Introduction Patients and Methods Results Discussion References

 

1. Cheek DB, Perry JW 1958 A salt wasting syndrome in infancy. Arch Dis Child 33:252–256[Medline]
2. Geller DS, Rodriguez-Soriano J, Vallo Boado A, Schifter S, Bayer M, Chang SS, Lifton RP 1998 Mutations in the mineralocorticoid receptor gene cause autosomal dominant pseudohypoaldosteronism type I. Nat Genet 19:279–281[CrossRef][Medline]
3. Zennaro MC, Lombes M 2004 Mineralocorticoid resistance. Trends Endocrinol Metab 15:264–270[CrossRef][Medline]
4. Kuhnle U, Nielsen MD, Tietze HU, Schroeter CH, Schlamp D, Bosson D, Knorr D, Armanini D 1990 Pseudohypoaldosteronism in eight families: different forms of inheritance are evidence for various genetic defects. J Clin Endocrinol Metab 70:638–641[Abstract]
5. Hanukoglu A 1991 Type I pseudohypoaldosteronism includes two clinically and genetically distinct entities with either renal or multiple target organ defects. J Clin Endocrinol Metab 73:936–944[Abstract]
6. Oberfield SE, Levine LS, Carey RM, Bejar R, New MI 1979 Pseudohypoaldosteronism: multiple target organ unresponsiveness to mineralocorticoid hormones. J Clin Endocrinol Metab 48:228–234[Abstract]
7. Zennaro MC, Keightley MC, Kotelevtsev Y, Conway GS, Soubrier F, Fuller PJ 1995 Human mineralocorticoid receptor genomic structure and identification of expressed isoforms. J Biol Chem 270:21016–21020[Abstract/Free Full Text]
8. Sartorato P, Lapeyraque AL, Armanini D, Kuhnle U, Khaldi Y, Salomon R, Abadie V, Di Battista E, Naselli A, Racine A, Bosio M, Caprio M, Poulet-Young V, Chabrolle JP, Niaudet P, De Gennes C, Lecornec MH, Poisson E, Fusco AM, Loli P, Lombes M, Zennaro MC 2003 Different inactivating mutations of the mineralocorticoid receptor in fourteen families affected by type I pseudohypoaldosteronism. J Clin Endocrinol Metab 88:2508–2517[Abstract/Free Full Text]
9. Tajima T, Kitagawa H, Yokoya S, Tachibana K, Adachi M, Nakae J, Suwa S, Katoh S, Fujieda K 2000 A novel missense mutation of mineralocorticoid receptor gene in one Japanese family with a renal form of pseudohypoaldosteronism type 1. J Clin Endocrinol Metab 85:4690–4694[Abstract/Free Full Text]
10. Viemann M, Peter M, Lopez-Siguero JP, Simic-Schleicher G, Sippell WG 2001 Evidence for genetic heterogeneity of pseudohypoaldosteronism type 1: identification of a novel mutation in the human mineralocorticoid receptor in one sporadic case and no mutations in two autosomal dominant kindreds. J Clin Endocrinol Metab 86:2056–2059[Abstract/Free Full Text]
11. Riepe FG, Krone N, Morlot M, Ludwig M, Sippell WG, Partsch CJ 2003 Identification of a novel mutation in the human mineralocorticoid receptor gene in a German family with autosomal-dominant pseudohypoaldosteronism type 1: further evidence for marked interindividual clinical heterogeneity. J Clin Endocrinol Metab 88:1683–1686[Abstract/Free Full Text]
12. Nystrom AM, Bondeson ML, Skanke N, Martensson J, Stromberg B, Gustafsson J, Anneren G 2004 A novel nonsense mutation of the mineralocorticoid receptor gene in a Swedish family with pseudohypoaldosteronism type I (PHA1). J Clin Endocrinol Metab 89:227–231[Abstract/Free Full Text]
13. Riepe FG, Krone N, Morlot M, Peter M, Sippell WG, Partsch CJ 2004 Autosomal-dominant pseudohypoaldosteronism type 1 in a Turkish family is associated with a novel nonsense mutation in the human mineralocorticoid receptor gene. J Clin Endocrinol Metab 89:2150–2152[Abstract/Free Full Text]
14. Sartorato P, Khaldi Y, Lapeyraque AL, Armanini D, Kuhnle U, Salomon R, Caprio M, Viengchareun S, Lombes M, Zennaro MC 2004 Inactivating mutations of the mineralocorticoid receptor in type I pseudohypoaldosteronism. Mol Cell Endocrinol 217:119–125[CrossRef][Medline]
15. Geller DS, Zhang J, Zennaro MC, Vallo-Boado A, Rodriguez-Soriano J, Furu L, Haws R, Metzger D, Botelho B, Karaviti L, Haqq AM, Corey H, Janssens S, Corvol P, Lifton RP 2006 Autosomal dominant pseudohypoaldosteronism type 1: mechanisms, evidence for neonatal lethality, and phenotypic expression in adults. J Am Soc Nephrol 17:1429–1436[Abstract/Free Full Text]
16. Germano CM, de Castro M, Crescencio JC, Gallo Jr L, Antunes-Rodrigues J, Moreira AC, Elias LL 2005 The interaction of plasma renin activity and plasma atrial natriuretic peptide in 21-hydroxylase deficiency patients. J Endocrinol Invest 28:300–304[Medline]
17. Kuhnle U, Lewicka S 2003 Diagnostic procedures in endocrine disorders of sodium regulation. In: Ranke MB, ed. Diagnostics of endocrine function in children and adolescents. Basel, Switzerland: Karger; 224–239
18. Couette B, Jalaguier S, Hellal-Levy C, Lupo B, Fagart J, Auzou G, Rafestin-Oblin ME 1998 Folding requirements of the ligand-binding domain of the human mineralocorticoid receptor. Mol Endocrinol 12:855–863[Abstract/Free Full Text]
19. Hellal-Levy C, Fagart J, Souque A, Wurtz JM, Moras D, Rafestin-Oblin ME 2000 Crucial role of the H11–H12 loop in stabilizing the active conformation of the human mineralocorticoid receptor. Mol Endocrinol 14:1210–1221[Abstract/Free Full Text]
20. Krawczak M, Ball EV, Cooper DN 1998 Neighboring-nucleotide effects on the rates of germ-line single-base-pair substitution in human genes. Am J Hum Genet 63:474–488[CrossRef][Medline]

This article has been cited by other articles:

				A. Balsamo, A. Cicognani, M. Gennari, W. G Sippell, S. Menabo, F. Baronio, and F. G Riepe Functional characterization of naturally occurring NR3C2 gene mutations in Italian patients suffering from pseudohypoaldosteronism type 1 Eur. J. Endocrinol., February 1, 2007; 156(2): 249 - 256. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 91/9/3671    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fernandes-Rosa, F. L. Articles by Antonini, S. R. Search for Related Content PubMed PubMed Citation Articles by Fernandes-Rosa, F. L. Articles by Antonini, S. R. Related Collections Adrenal and Hypertension Cardiovascular Endocrinology