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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

Division of Pediatric Endocrinology, Department of Pediatrics, Christian Albrechts University of Kiel (M.V., M.P., W.G.S.), D-24105 Kiel, Germany; SANITAS Ostseeklinik Boltenhagen (M.P.), D-23946 Boltenhagen, Germany; Children’s Hospital of Málaga (J.P.L.-S.), E-20911 Málaga, Spain; and Children’s Hospital of Bremen-Nord (G.S.-S.), D-28755 Bremen, Germany

Address all correspondence and requests for reprints to: Prof. W. G. Sippell, M.D., Division of Pediatric Endocrinology, Department of Pediatrics, Schwanenweg 20, Universitäts Kinderklinik, D-24105 Kiel, Germany. E-mail: sippell{at}pediatrics.uni-kiel.de

	Abstract

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Pseudohypoaldosteronism type 1 (PHA1) is characterized by neonatal salt wasting resistant to mineralocorticoids. There are 2 forms of PHA1: the autosomal recessive form with symptoms persisting into adulthood, caused by mutations in the amiloride-sensitive luminal sodium channel, and the autosomal dominant or sporadic form, which shows milder symptoms that remit with age. Mutations in the gene encoding the human mineralocorticoid receptor (hMR) are, at least in some patients, responsible for the latter form of PHA1. We here report the results of a genetic study in a sporadic case and in 5 affected patients from 2 families with autosomal dominant PHA1. In the sporadic case we identified a new frameshift mutation, Ins2871C, in exon 9 of the hMR gene. Family members were asymptomatic and had no mutation. This mutation is the first described in exon 9 and impairs the last 27 amino acids of the hormone-binding domain. In 2 kindreds with autosomal dominant PHA1 we found no mutation of the hMR gene. Our results confirm the hypothesis that autosomal dominant or sporadic PHA1 is a genetically heterogeneous disease involving other, as yet unidentified, genes.

	Introduction

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PSEUDOHYPOALDOSTERONISM type 1 (PHA1) is a rare hereditary salt-wasting disorder first described by Cheek and Perry in 1958 (1). At least two forms of PHA1 have been defined since then (2, 3, 4, 5, 6, 7). The autosomal recessive inherited form of PHA1 is characterized by severe multiple target organ resistance to aldosterone, including the kidneys, colon, and sweat and salivary glands, and usually persists into adulthood (6, 8). The autosomal recessive form of PHA1 is caused by mutations in one of the three subunits of the amiloride-sensitive luminal sodium channel (ENaC) that is responsible for sodium reabsorption (9). The autosomal dominant inherited or sporadic form of PHA1, predominantly with a renal resistance to aldosterone, is characterized by renal salt loss, hyperkalemia, metabolic acidosis, failure to thrive, and elevated PRA and aldosterone levels in infancy. Clinical symptoms usually remit with age, whereas aldosterone and PRA remain high. Exogenous mineralocorticoids have no effect, but patients respond well to occasional excessive salt supplementation (10). Some patients are clinically asymptomatic, but have elevated PRA and aldosterone levels (6).

Recently, Geller et al. identified 4 mutations in the human mineralocorticoid receptor gene (hMR) responsible for the autosomal dominant and sporadic form of PHA1, i.e. 2 frameshift mutations and 1 stop mutation in exon 2 and an intron 5 splice mutation (11). One missense mutation in exon 8 was found by Tajima et al. (12). The hMR gene cloned by Arizza et al. consists of 9 exons, with a coding region spanning from exons 2–9, encoding for 984 amino acids (13). Other researchers found no mutations of the hMR gene in 1 autosomal dominant (14) and in 2 sporadic cases of PHA1 (15, 16). Cases with no mutation found in the hMR gene are more likely to be autosomal recessive forms of PHA1 in view of severity of the disease and the consanguinity of the parents (15, 17).

The aim of the present report on a new hMR gene mutation in a sporadic case and five cases with autosomal dominant PHA1 without mutations is to provide further evidence for the marked genetic heterogeneity of PHA1, suggesting the involvement of other unidentified genes in the etiology of this life-threatening neonatal disease.

	Materials and Methods

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Analyses of plasma steroids and ACTH levels and PRA were performed using standard RIA procedures of comparable quality criteria.

Blood samples for molecular genetic studies were taken after informed consent was obtained from the patients and/or their parents. Genomic DNA was extracted from peripheral blood leukocytes, and the hMR gene was amplified using 19 pairs of primers as described by Geller et al. (11). PCR was performed for 35 amplification cycles (denaturation at 94 C for 40 s, annealing at 58 or 65 C for 40 s, extension at 72 C for 40 s, last extension at 72 C for 5 min) using PCR Super Mix high fidelity (Life Technologies, Inc., Gaithersburg, MD). Before sequencing, PCR products were purified using the QIAquick-spin PCR purification kit (QIAGEN, Bothell, WA). The nucleotide sequences of both strands of the PCR products were directly determined using an automated fluorescent sequencer (ABI Prism 310 Genetic Analyzer, Perkin-Elmer Corp., Wellesley, MA). Sequencing included all translated exons (2, 3, 4, 5, 6, 7, 8, 9) of the hMR gene and the exon/intron boundaries.

	Results

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Patients

Family 1. Patient 1 is a girl, now 16 yr old, who presented at 2 weeks after birth with failure to thrive, vomiting, diarrhea, and mild dehydration. Her parents were nonconsanguineous, and pregnancy and delivery were uncomplicated. Birth weight was 3150 g. She was breast fed and received no medication until admission. On admission, she had hyponatremia, hyperkalemia, and elevated aldosterone (Table 1), but no metabolic acidosis. The urinary Na/K ratio was elevated (1.4; normal range for healthy infants between 8 days and 6 months, 0.3–0.4). Other parameters, including plasma creatinine, ACTH, cortisol, and 17-hydroxyprogesterone were normal. Intravenous urography was normal, ruling out obstructive uropathy mimicking transient PHA (18).

Family 2. Patient 2 is a girl, now 5 yr old, who presented at age 2 weeks with the same clinical signs as patient 1. She also had elevated aldosterone levels (Table 1) and an inappropriately elevated urinary Na/K ratio of 2.6. Other parameters, including plasma creatinine, ACTH, cortisol, and 17-hydroxyprogesterone, were normal, as was ultrasound of the kidneys. Parents are nonconsanguineous, pregnancy and delivery were normal, and birth weight was 3140 g. The infant was breast fed and received no medication before admission. Mineralocorticoid administration had no effect. With NaCl supplementation given for 14 months she had no symptoms and showed marked catch-up growth (length, -1.2 to 0.7 SD), but aldosterone levels remained elevated. Her mother, patient 3, never had symptoms of salt loss, but also had highly elevated plasma aldosterone levels. The mother of patient 3 probably had a salt-loosing crisis during infancy, but detailed clinical or laboratory data are no longer available.

Family 3. Patient 4, a girl, was diagnosed at age 2 yr with failure to thrive. She never had an overt salt-loosing crisis. Sodium levels at time of diagnosis were low normal, but never decreased; however, plasma aldosterone was always high (Table 1). Patient 5, her brother, was diagnosed in infancy with failure to thrive. He suffered from hyponatremia during an episode of obstructive bronchitis. Plasma aldosterone levels were high and remain so to date (Table 1). Both siblings demonstrated catch-up growth with oral NaCl supplementation (height: patient 4, -3.4 to -1.6 SD; patient 5, -2.1 to -1.8 SD), whereas without this therapy, growth rate decreased. Urinary Na/K ratios during sodium supplementation were still slightly elevated (1.3 and 1.1, respectively; normal range for 6 months to 2 yr, 0.5–0.8). Patient 6, a cousin, was diagnosed as having PHA1 at 3 days of age with hyponatremia, severe dehydration, convulsions, and elevated plasma aldosterone levels (Table 1). In all patients other routine laboratory parameters, including creatinine, were normal, as was kidney ultrasound. Mineralocorticoids were of no benefit in terms of weight gain or hyponatremia. In the parental generation, no family member had symptoms suggesting PHA1; however, clinical analysis revealed that both mothers, their sister, and the grandmother were all affected (19, 20), proving an autosomal dominant trait.

Kindreds of the families and main quantitative clinical data are shown in Fig. 1 and Table 1.

View larger version (11K): [in this window] [in a new window]   Figure 1. Pedigrees of the three families with PHA1. Sporadic case: A, patient 1 (II,2); autosomal dominant cases: B, patients 2 (III,1) and 3 (II,1); I,1 had suspected salt loss in infancy; C, patients 4 (III,2), 5 (III,5), and 6 (III,6)). C, Asymptomatic patients, as described previously (19 20 ).

Direct sequencing of PCR products of the hMR gene from the sporadic case of PHA1 (patient 1, family 1) showed a heterozygous insertion of a cytosine at position 2871 in exon 9, according to the numbering of nucleotides in the publication of the DNA structure of the hMR gene (Fig. 2). This mutation leads to a frame shift, resulting in a nonsense protein from codon 958 and a first stop codon at position 1012. Both parents and the three brothers of the patient had no mutation in the entire coding sequence of the hMR gene.

View larger version (31K): [in this window] [in a new window]   Figure 2. Heterozygous mutation Ins2871C in exon 9 of the hMR gene in patient 1 (A) and the wild-type sequence of his father’s DNA (B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We here report on a patient presenting with the sporadic form of PHA1. This patient had a heterozygous mutation in exon 9 of the hMR gene (Ins2871C; Fig. 2), causing a frame shift with a nonsense amino acid sequence from codon 958 of the hMR and a first stop codon at codon 1012. This mutation lies within the end of the hormone-binding domain of the hMR gene (Fig. 3) (21) and underlines the physiological importance of the last 27 amino acids of the hMR. Whether heterozygous mutations of the hMR impair biological function because of haploinsufficiency, as suggested by Geller et al. (11), or a dominant negative effect needs further clarification. To our knowledge, this is the third report of a mutation in the hMR gene in patients with the sporadic form of PHA1 (11, 12). Tajima et al. (12) described a missense mutation in a highly conserved region of the hormone-binding domain, whereas Geller et al. (11) described mutations involving the complete DNA- and hormone-binding domain.

View larger version (28K): [in this window] [in a new window]   Figure 3. Exons of the hMR gene with mutations described to date. Top, Mutations found by Geller et al. (11 ) and Tajima et al. (12 ); bottom, mutation of codon 958 found in patient 1 (present study). Nucleotide numbering is according to Arizza et al. (13 ) (GenBank Accession No. M16801, beginning at translation start).

In family 3, no mutations in the ENaC gene were found (22), consistent with the observation that mutations in the ENaC gene are associated with autosomal recessive inheritance causing severe salt loss (9).

These findings support the hypothesis that PHA1 is a genetically markedly heterogeneous disorder in which other, as yet unidentified and probably regulatory, genes must be involved (23). Moreover, in patients with sporadic PHA1, Arai et al. (24) recently reported a significantly increased concurrence of hMR and ENaC gene polymorphisms, either of which alone does not cause PHA1. These researchers found a significantly increased concurrence of hMR and ENaC gene polymorphisms in sporadic PHA1 patients. The concordance of these polymorphisms, the hMR gene mutations reported here and by other researchers (11, 12), and the lack of obvious mutation in any of the known genes involved in salt homeostasis in other patients with PHA1 can be best explained by multigenic expression and heredity in PHA1.

	Acknowledgments

 
We thank Mrs. Gisela Hohmann for her expert technical assistance with molecular biology techniques, and we are grateful to Mrs. Joanna Voerste for linguistic editing of the manuscript.

Received July 17, 2000.

Revised January 12, 2001.

Accepted January 18, 2001.

	References

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