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A Novel Missense Mutation of Mineralocorticoid Receptor Gene in One Japanese Family with a Renal Form of Pseudohypoaldosteronism Type 1

Department of Pediatrics (T.T., J.N., K.F.), Hokkaido University School of Medicine, Sapporo 060-8638; Institute for Molecular and Cellular Bioscience (H.K., S.K.), The University of Tokyo, Tokyou 113-0032; Department of Endocrinology and Metabolism (S.Y., K.T., M.A., S.S.), Kanagawa Children’s Medical Center, Yokohama 232-8555; and Department of Pediatrics, Toranomon Hospital (S.Y.), Tokyo 105-8470, Japan

Address correspondence and requests for reprints to: Kenji Fujieda, M.D., Ph.D., Department of Pediatrics, Hokkaido University School of Medicine, N15, W7, Kita-ku, Sapporo 060-8638, Japan. E-mail: Ken-fuiji{at}med.hokudai.ac.jp

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Pseudohypoaldosteronism type 1 (PHA1) is a rare condition characterized by neonatal salt loss with dehydration, hypotension, hyperkalemia, and metabolic acidosis, despite elevated plasma aldosterone levels and PRA. Two modes of inheritance of PHA1 have been described: an autosomal dominant form and an autosomal recessive form. An autosomal recessive form manifests severe life-long salt wasting resulting from multiple mineralocorticoid target tissue such as sweat, salivary glands, the colonic epithelium, and lung. Contrary, an autosomal dominant PHA1 manifests milder salt wasting that gradually improves with advancing age. Recently, in one sporadic and four dominant cases, four different mutations including two frame shift mutations, two premature termination codons, and one splice site mutation in the mineralocorticoid receptor (MR) gene were identified.

We studied the molecular mechanisms of one Japanese family with a renal form of PHA1. PCR and direct sequencing of the MR gene identified a heterozygous point mutation changing codon 924 Leu (CTG) to CCG (Pro) (L924P) in all affected members. COS-1 cells were transfected with expression vectors for either wild type or the mutant MR-L924P receptors, together with the reporter plasmid (glucocorticoid response element tk-CAT). Aldosterone increased CAT activity in cells expressing wild-type receptor, but had no effect in cells expressing the mutant receptors. These results suggest that mineralocorticoid resistance in this family is due to a missense mutation in the MR gene. To our knowledge, this is the first case of the missense mutation of the MR gene in renal PHA1.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
PSEUDOHYPOALDOSTERONISM type 1 (PHA1) is characterized by congenital resistance of the kidney and/or other mineralocorticoid target tissues to aldosterone, resulting in excessive salt wasting. Two modes of inheritance of PHA1 have been described: an autosomal dominant form and an autosomal recessive form. An autosomal recessive form manifests severe life-long salt wasting resulting from multiple mineralocorticoid target tissue such as sweat, salivary glands, the colonic epithelium, and lung (1, 2, 3, 4, 5, 6). On the other hand, an autosomal dominant PHA1 manifests milder salt wasting that gradually improves with advancing age (3, 7, 8). In autosomal dominant form the target organ defect is confined to renal tubules only.

The loss of function mutations of the amiloride-sensitive epithelial sodium channel were identified in the autosomal recessive familial PHA1 (6, 9, 10). On the other hand, a recent report has identified the nonsense and frameshift mutations of the mineralocorticoid receptor (MR) gene in several patients with a renal form of PHA1 (Fig. 1) (8). These findings indicate that at least some cases of renal PHA1 are a disease of the mineralocorticoid resistance due to the dysfunction of the MR (8). MRs belonging to the steroid hormone receptor family are conditional transcriptional factors that play important roles by controlling specific genes (11, 12, 13, 14). Members of the steroid hormone receptor family are structurally characterized by three distinct domains: an N-terminal transcriptional activation domain, a central DNA binding domain (DBD), and C-terminal ligand-binding domain (LBD). This LBD exerts multifunctional actions such as dimerization and ligand-dependent transactivation function (11, 12, 13, 14).

View larger version (11K): [in this window] [in a new window]   Figure 1. Schematic representation of the MR gene. The boxes denote the exons, and the MR gene had 10 exons (from exon 1b to exon 9). A vertical line above the MR gene points to the mutation detected in this study, and the arrows below the schema indicate the mutation previously described (8 ). The black boxes (exons 3 and 4) encode the DNA-binding domain, and the regions of exons 5, 6, 7, 8, and 9 (shaded boxes) encode the hormone binding domain.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Pedigree and endocrine findings in the family are shown in Fig. 2A and Table 1. The proband (II-1) was born by spontaneous vaginal delivery at full term. There was no consanguinity. At 2 weeks of age she presented with poor weight gain and failure to thrive. Initial laboratory examinations showed hyponatremia (129 mmol/L), hyperkalemia (6.8 mmol/L), an extremely elevated plasma aldosterone concentration (24,880.2 pmol/L), and PRA (>25 µg/L/h) (Table 1). Urinary Na and K levels were 41 mmol/L and 21 mmol/L, respectively, and a urinary Na value was inappropriately high in the face of hyponatremia (Table 1). Adrenal and renal functions were normal. The electrolyte disturbance quickly resolved with salt supplementation (1 g/day), and weight gain was restored to normal. At 10 months of age salt supplement was withheld, and she remains well and is growing normally since then. Her mother (I-2) demonstrated elevated plasma aldosterone concentration (1,658.8 pmol/L) (Table 1), and she reported the episode of poor weight gain until 8 months of age. The younger brother of the proband (II-2) was also hospitalized for poor weight gain and failure to thrive at the age of 7 days. His serum sodium was 132 mmol/L; potassium, 4.9 mmol/L; plasma aldosterone, 15,040.6 pmol/L; and PRA, >25 µg/L/h (Table 2). Urinary Na and K levels were 36 mmol/L and 29 mmol/L, respectively. Salivary Na and Cl were 13 mmol/L and 9 mmol/L, respectively, within normal range (Table 1). He was also treated with salt supplementation (1 g/day). Serum sodium returned to normal values, and he has gradually improved over time with diminishing needs for salt supplementation. Sodium supplementation was discontinued at age 11 months.

View larger version (54K): [in this window] [in a new window]   Figure 2. A, Family pedigree showing autosomal dominant segregation with PHA type 1. The proband is II-1. Arrows indicate the affected members. Solid symbol indicates the L924P mutation. B, The result from the proband (II-1). The T residue was changed to C residue, accompanying with the substitution of Pro (CCG) for Leu (CTG) at codon 924 in exon 8 denoted by an arrowhead. The determination of whole sequence confirmed the mutation was on only one allele. C, His brother (II-2) shared the same mutation as heterozygous. D, While their father (I-1) had only wild-type alleles, their mother (II-2) (E), with a renal form of PHA1, had the wild and mutant alleles.

Methods

This study was approved by the internal review board, and informed consent for DNA analysis was obtained from their parents.

MR gene analysis

To analyze the MR gene from this family, DNA was prepared from white blood cells using standard techniques. The primers of PCR were selected in the intron-exon boundaries of the MR gene according to published sequences of the MR gene (8). All PCR was conducted with primer pairs, consisting of 5 min 94 C, followed by 30 cycles of 40 s at 94 C, 40 s at 57 C, and 1 min at 72 C. If nonspecific bands were present, the expected PCR product was purified by 2% NuSieve (FMC Bioproducts, Rockland, ME) gel electrophoresis. Direct sequence of these PCR products was performed using the ABI PRISM Dye Terminator Cycle Sequencing Kit and automated fluorescent sequencer ABI 373A (PE Applied Biosystems, Foster City, CA), according to a previous method (15).

Ligand-induced receptor function of wild type and the mutant MR

The technique of site-directed mutagenesis by overextension PCR was used to replace the cytosine (C) residue at position of the normal human MR cDNA with a thymidine (T) residue, using the MR cDNA. The recombinant plasmid was designated MR-L924P. COS-1 cells were cultured, and cotransfections with 1 µg of either the wild-type MR or the mutant MR-L924P and 3 µg reporter plasmid of glucocorticoid response element (GRE)-tk-CAT, reporter plasmid, in which GREs are coupled to the CAT gene, were carried out in the presence of lipofectin reagent (Life Technologies, Gaithersburg, MD) (16). Cells were incubated with 10 nM aldosterone. CAT assay was performed as described previously (16).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
MR gene analysis

Sequencing of exon 8 of the MR gene in the proband (I-1) revealed the heterozygous point mutation changing codon 924 Leu (CTG) to CCG (Pro) (L924P) (Fig. 2B). Her younger brother (II-2) shared this heterozygous mutation (Fig. 2C). Although their father (I-1) did not have any mutation of the MR gene (Fig. 2D), their mother (I-2) had also both of the normal and mutant alleles (Fig. 2E). To determine whether this mutation was not merely polymorphism, we sequenced the MR gene from 50 normal Japanese individuals by PCR-direct sequencing, and none had this nucleotide transition. These results strongly indicate that this mutation is responsible for the development of this disorder, and that the inheritance might be autosomal dominant.

Functional analysis

After transfection, we determined mRNA levels by Northern blot analysis, and there was no difference in the wild and mutant receptor (data not shown). To determine whether the identified base substitution is responsible for mineralocorticoid resistance, COS-1 cells were transfected with the wild-type or the mutant MR-L924P plasmid, together with GRE-tk-CAT. In cells transfected with the wild-type MR, aldosterone induced a 7-fold increase in CAT activity (Fig. 3). However, there was no stimulation of CAT activity by aldosterone in cells, which had been transfected with the mutant MR-L924P plasmid (Fig. 3), indicating that this mutant receptor can not transduce ligand-dependent transactivation.

View larger version (12K): [in this window] [in a new window]   Figure 3. Ligand-induced receptor function of wild type and the mutant MR. Transcriptional activities of wild-type MR and the mutant MR in COS-1 cells. Each of receptor expression vectors (1 µg) was transfected together with 3 µg GRE-tk-CAT reporter plasmid in the absence (-) and presence (+) of 10 nM of aldosterone, and CAT-assay was performed. Each value represents mean + SE of three individual transfections and is shown as fold induction from background activity of reporter plasmid.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

As stated above, there are only five cases (case 1, 2-1, 3-1, 4-1, and 5-1) of renal PHA1, in whom MR mutations were identified (Table 2). All these patients showed renal salt wasting with hyperkalaemic acidosis despite high aldosterone levels, and improved with age and were asymptomatic without treatment (8). MR mutations of these patients would lead to a truncated protein, thus resulting in a complete loss of MR function (Table 2) (8). Our missense mutation, L924P, affects a highly conserved residue and results in complete absence of MR function. Thus, all mutations of MR have equally severe functional consequences in vitro. Accordingly, this severe heterozygous loss of function mutations would develop clinically evident disease. However, regarding with serum Na, K, and plasma aldosterone in patients and affected family members, their ranges are variable despite of severe mutations of the MR gene (Table 2). It is also reported that many adult gene carriers have elevated aldosterone concentrations but no history of clinical manifestations (8). Moreover, several dominant and many sporadic kindreds do not have MR gene mutation (8). Taken together, it is possible that not only MR mutation, but also either mutations in additional genes or nongenetic factor may contribute to clinical and biochemical phenotypes. To address these issues, it is necessary to analyze more patients with a renal form of PHA1.

In conclusion, we identified a novel missense mutation of the MR gene in a Japanese family with a renal form of PHA1. To our knowledge, this is the first report that the naturally occurring mutant MR can not activate ligand-dependent transactivation. This will give new insights for understandings the pathophysiology of this disorder.

	Footnotes

 
¹ These authors contributed equally to this work.

Received April 19, 2000.

Revised August 2, 2000.

Accepted August 30, 2000.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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