Autosomal dominant pseudohypoaldosteronism type 1 with a novel splice site mutation in MRgene

Pseudohypoaldosteronism type 1 (PHA1) is a rare inherited condition that is characterized by renal insensitivity to the action of mineralcorticoids (aldosterone) [1]. At least two forms of PHA1, autosomal dominant and recessive of the disease have been described. However, patients with autosomal dominant PHA1 generally have much milder symptoms of having renal PHA than those with autosomal recessive PHA1 which shows multiple target organ unresponsiveness to mineralcorticoids such as sweat and salivary glands, colon and kidney [2]. Aldosterone plays a crucial role in the regulation of blood pressure and potassium homeostasis. It binds to the mineralcorticoid receptor (MR) in the distal nephron which triggers increased sodium reabsorption via the epithelial sodium channel (ENaC) to restore intravascular volume. Recently, it became clear that mutations in the MR gene (MR or NR3C2) leads to autosomal dominant renal PHA1 and mutations in the ENaC alpha, beta and gamma subunit genes (SCNN1A, SCNN1B and SCNN1G) leads to autosomal recessive severe systemic PHA1 [3, 4].

Patients with autosomal dominant renal PHA1 generally require oral salt supplementation, but typically show a gradual clinical improvement in renal salt loss during childhood. Some individuals are clinically asymptomatic but may have elevated PRA and aldosterone levels [2]. In most cases, renal PHA1 is the result of mutations of the gene MR, which consists of 10 exons [5]. The MR gene contains an amino-terminal region that harbors a ligand-independent transactivation function coded by exon 2 and a conserved DNA binding domain encoded by exon 3-4 and a ligand binding domain responsible for ligand binding and ligand-dependent transactivation encoded by exons 5-9 [6]. To date more than 50 different PHA1-causing mutations in the MR gene have been identified, including nonsense, frameshift, missense, and splice site mutations distributed throughout the gene [7–15].

This report concerns a patient with PHA1 with a novel splice acceptor site mutation which leads to exon skipping and frame shift result in premature termination at the transcript level. The mRNA from peripheral blood lymphocytes and urinary sediments showed evidence of wild type and exon-skipped RT-PCR products.

In this study we identified a novel mutation c.2511-2A>C in intron 6 in the MR gene and this mutation was strongly suspected of eliminating a consensus splice acceptor site, resulting in exon 7 skipping in the mRNA. Although no clear evidence has been shown, it is believed that this mutation in the MR gene reduces the ligand-binding capacity of MR [6]. This same mutation was found in the patient's mother.

Both mother and daughter had a very mild phenotype in comparison with typical patients with PHA1. For this reason, our patient needed only a small amount of sodium replacement (0.5 g/day) and this supplement could be terminated early, at the age of 7 months. Her mother was clinically free of symptoms, had normal plasma electrolytes, and showed only elevated PRA and aldosterone level. No occasion of salt loss, not even during infancy or childhood, was recorded, and she had never undergone treatment as an inpatient. Viemann at al reported that the patients with typical PHA1 need a much larger amount of salt replacement than did our patient, 4 g/day to 12 g/day for therapy [7], and it is reported the patients needed the supplement for a relatively longer time, 1 to 3 years [16]. However, the finding that some patients carrying the MR mutation have no symptomatic or very mild symptomatic manifestations can be found in some reports of PHA1 [7, 17]. Moreover, distinct differences of the disease severity between various affected members within one family or between unrelated individuals carrying the same mutation could be observed [7, 14]. We should consider that salt intake in the diet may modulate the phenotype and that some patients may compensate their phenotype without treatment just by a high salt diet. However, there is another possibility that the phenotype of PHA1 may not originate from the genotype of only the MR mutation. As postulated by Geller [5] and Tajima [8], it is possible that either mutations in additional genes or non-genetic factors may contribute to the clinical and biochemical phenotypes of the carriers of MR mutations. However, several attempts to pin down these additional genes or factors have been without success [17, 18].

About genotype-phenotype correlations, Riepe describes in his recent review article that no association could be drawn from the available clinical data and MR gene mutations [19]. This fact may also suggest the existence of other factors.

Many human diseases that are caused by loss-of-function mutations in nuclear receptors have been reported, but the mechanism of the disease differs for each receptor. Moreover, whether MR mutations cause mineralcorticoid resistance via haploinsufficiency or dominant negative mechanism remains controversial. To test the pathogenic mechanism, Sartorato et al showed in co-transfection experiments that the two mutated receptors they identified, Q776R and L979P, exerted a dominant negative effect on the wild type receptors, and stated that this dominant negative effect is necessary to give rise to autosomal dominant PHA1 [7]. However, Riepe et al and Balsamo et al performed the same co-transfection experiments and showed that the mutated receptors they identified, C818L, A1012X, Y134X and L772X, exhibited no dominant negative effect on the wild-type MR proteins. Therefore, Riepe et al and Balsamo et al claimed that haploinsufficiency of the MR locus is sufficient to generate PHA1 in their patients [16, 17].

Geller et al had access to RNA analysis to test for nonsense-mediated mRNA decay (NMD) in a patient with a nonsense mutation. They found wild type but not mutant MR mRNA in peripheral blood lymphocytes of a PHA1 patient with a nonsense mutation in exon 3 (A590X). The absence of a mutant RNA was probably due to NMD, indicating the patient possessed the onset mechanism of haploinsufficiency because there was no mutant mRNA due to NMD [20].

With genetic analysis using RT-PCR, the problem is sometimes encountered that NMD interferes with the detection of nonsense mutations. The NMD pathway is an mRNA surveillance system that typically degrades transcripts containing premature termination codons (PTCs) to prevent translation of unnecessary or aberrant transcripts. Failure to eliminate these mRNAs with PTCs may result in the synthesis of abnormal proteins that can be toxic to cells through dominant negative or gain-of-function effects. Geller et al suggested that haploinsufficiency is sufficient to generate the PHA1 phenotype because of this mechanism [14]. However, we confirmed by means of RT-PCR that transcripts with exon 7 skipped remain in mRNA from not only peripheral blood lymphocytes but also urinary sediments. Exon 7 of the MR gene contains 131 bp and this exon skipping creates an out-of-frame mutation and leads to PTC in exon 8 in mRNA. However, we could identify mutant RT-PCR products in our patient, which means NMD does not always affect mutations with PTCs. On the other hand, it is possible that the pathogenic mechanism of PHA1 in patients with MR mutations depends on the mutation position. PHA1 is such a rare inherited disease that only about 40 mutations have been described by now, so that mRNA analytical studies of this disease are quite rare and additional studies are necessary to identify the mechanism of the phenotype of PHA1. It should be noted that the molar abundance of the smaller mRNA appears to be lower than the wild type transcript, that is possibly indicating that there is incomplete NMD of the mutant transcript in our study. Moreover, this study includes two limitations. First, we have not conducted quantitative PCR because we could not amplify clear PCR products with the one-step PCR, therefore, conducted nested PCR. Second, we have not determined the mutant peptide expression analysis because kidney tissues were unavailable in this disease. We must also hypothesize that, although not mediated by NMD, haploinsufficiency given to the loss of major functional domains of the receptor, may be the pathogenic mechanism of PHA1 in this patient. However, we can say, at least, some amount of mutant transcripts are expressed in the kidney tissues and the onset mechanism of haploinsufficiency because of complete NMD was denied in our patient.

By using mRNA extracted from urinary sediments, we could detect transcript abnormality noninvasively. Igarashi et al. were the first to identify transcript abnormalities by extracting mRNA from urinary sediment cells of patients with Dent disease [21]. Following their report, our group used this method to detect splicing abnormalities in mRNA from kidney cells [22–24]. Since urinary sediments contain cells derived from the kidney, genetic analysis of those cells constitutes an entirely noninvasive, simple method for the diagnosis of inherited kidney diseases, which can also be used for diagnosis of all other inherited kidney diseases including renal tubular disorders and glomerular diseases. Our study is the first to use this method for PHA1.

Our report describes a novel MR mutation in a patient with PHA, and this mutation was found to eliminate a splice acceptor site, which results in exon 7 skipping and subsequently in premature termination in exon 8. This was demonstrated in mRNA from both peripheral blood leukocytes and urine sediments.

Our patient and her mother showed very mild symptoms in spite of a robust mutation of PHA1, which raises the possibility that MR mutation may not be involved in the severity of the symptoms of PHA1.

In this novel mutation, RT-PCR products of mRNA extracted from peripheral blood lymphocytes and urinary sediments showed evidence of the presence of wild-type and mutated abnormal mRNA. In our study we could provide clear evidence that NMD does not operate in mRNA. We could thus prove that NMD does not always observed in patients with MR mutations with PTCs. Additional studies including mRNA analysis will be needed to identify the exact mechanism of the phenotype of PHA.