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Original Studies |
Department of Physiology, Nippon Medical School (K.A., T.S.), 1–1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan; and the Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health (K.Z., G.P.C.), Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Keiko Arai, M.D., Department of Physiology, Nippon Medical School, 1–1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. E-mail: arai_keiko/phys2{at}nms.ac.jp
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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, ß, and 
complementary DNAs
(cDNAs) in a series of five sporadic cases of PHA, whose MR cDNA
contained nonconservative homozygous
(C944
T944,
Ala241Val241) and/or a conservative
heterozygous substitutions (A760G760,
Ileu180Val180), which, however, were also
present at high frequencies in a control population with apparently
normal salt conservation. We found a nonconservative substitution
(A2086G2086,
Thr663Ala663) in the ENaC in all five of
our patients, two of whom were homozygous and three of whom were
heterozygous for this alteration, which was also present in the
homozygous and heterozygous form in 31% and 64% of control subjects,
respectively. We also found a nonconservative homozygous substitution
(C1006G1006,
Pro336Ara336) in the ßENaC and three
nonconservative and conservative homozygous substitutions
(T554A554,
Trp178Arg178;
C1526G1526,
Pro501Ala501;
T1862G1862,
Ser614Ala614) in the ENaC of all five of
our patients and in a substantial proportion of control subjects.
Interestingly, when the patient group was compared to controls, a
significantly increased concurrence of the MR and ENaC polymorphisms
was found in the patients (P < 0.025). We conclude
that the changes identified in the cDNA of the three ENaC subunits in
the patients with sporadic PHA are polymorphisms, which on their own
have no apparent pathophysiological significance. We hypothesize,
however, that these polymorphisms might influence salt conservation
negatively if they are present concurrently with other genetic defects
of the MR or other proteins that participate in sodium homeostasis. The
latter would be compatible with a sporadic presentation and digenic or
multigenic expression and heredity in PHA. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The rat and human ENaC consist of three subunits, , ß, and ,
whose cDNAs were recently cloned (28, 29, 30, 31). These three subunits form a
channel located at the apical site of renal epithelial cells of the
distal convoluted tubule that allows passive transport of sodium into
the cell in response to aldosterone. Whereas most of sodium transport
functions lie within the -subunit, the ß- and -subunits play
synergistic roles and are required for full function (29, 31).
Recently, several studies demonstrated abnormalities of the ß- and
-subunits in patients with Liddle’s syndrome, a rare condition
whose pathophysiological mechanism is excessive reabsorption of sodium
by the distal convoluted tubule, i.e. the mirror image of
PHA (32, 33, 34). These reports suggested that the ENaC might be the
postreceptor locus of the defect in many patients with PHA;
accordingly, mutations of the- and ß-subunits of the ENaC were
reported in PHA patients from autosomal recessive kindreds (35, 36). In
this study, we examined the cDNA structure of the three subunits of
ENaC cDNAs in our five sporadic patients with PHA (23, 26).
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We examined the cDNAs and genomic DNA of the 3 subunits of ENaC
in 5 previously reported patients with PHA and 42 unrelated control
subjects with apparently normal salt conservation (23, 26). We
summarize the clinical, biochemical, and hormonal characteristics of
the patients in Table 1
.
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The proband, a 17-yr-old white male, was born by spontaneous vaginal delivery at full term. In the first week of life, he developed severe dehydration, hyponatremia, hyperkalemia, and acidosis, associated with profound urinary salt loss. After a stormy course, he had an extensive evaluation at the Pediatric Clinical Research Unit of New York Hospital-Cornell Medical Center, which led to the diagnosis of pseudohypoaldosteronism and confirmed the presence of multiple organ resistance to aldosterone (8). He was maintained on high doses of salt and up to the age of 7 yr required frequent hospital admissions for iv hydration. On this first admission to the NIH at the age of 16 yr, he was placed on carbenoxolone therapy, which allowed him to grow and progress to puberty.
This patient was the only clinically affected member of his family. His father was healthy and had normal serum electrolytes and plasma aldosterone as well as normal 24-h urinary aldosterone excretion. Of the patient’s three sisters, one had died at the age of 4 days from "cerebral hemorrhage," whereas his other two sisters were reportedly healthy.
Case 2
The proband, a 13-yr-old black female, was born by spontaneous vaginal delivery without complication at full term. She presented at 10 days of age with shock and severe hyponatremia [sodium (Na), 110 mmol/L] and hyperkalemia [potassium (K), 8.7 mmol/L]. The diagnosis of PHA was made at 11 months of age, when she was evaluated at New York Hospital (8). During the evaluation, serum sodium was 131 mmol/L, and potassium was 6.5 mmol/L, whereas her urinary sodium was 150 mmol/24 h, despite taking 3 mmol/kg·day sodium. Her plasma aldosterone level and PRA were markedly elevated (9.1 nmol/L and 1.29–28.07 ng/mL·h, respectively). Salivary and sweat sodium were also elevated at 144 and 148 mmol/L, respectively. Her renal and adrenal functions were normal. The family history was notable for hypertension, and the pedigree did not reveal consanguinity.
Case 3
The proband, a 3-yr-old Portuguese female, was born at full term by spontaneous vaginal delivery without complication. At 7 days of age, she presented at St. Barnabas Hospital in New Jersey in a state of shock with severe hyponatremia (Na, 109 mmol/L) and hyperkalemia (K, 8.9 mmol/L). The diagnosis of pseudohypoaldosteronism was based on her extremely elevated plasma aldosterone levels (8.48–142.9 nmol/L) and PRA (5054–8049 ng/mL·h). Her sweat sodium level was higher than normal (67.3 mmol/L; normal, 15.9–45.9). Her parents were third cousins and had normal plasma aldosterone concentrations and PRA.
Case 4
The proband, a 5-week-old Moroccan male, was born by spontaneous vaginal delivery without complications at full term. He presented at 5 weeks of age at the Hospital Universitaire des Enfants Reine Fabiola in Brussels with frequent vomiting and severe dehydration, associated with marked hyponatremia (Na, 115 mmol/L) and hyperkalemia (K, 8.8 mmol/L). His plasma aldosterone concentration was extremely elevated (36 nmol/L), as was his PRA (38.9 ng/mL·h), which led to the diagnosis of pseudohypoaldosteronism. His saliva sodium concentration was 134 mmol/L. His parents were first cousins. There was no family history of hypertension.
Case 5
The proband, a 7-month-old white male, was born by spontaneous vaginal delivery at full term, with the only complication being mild maternal preeclampsia. At 1 month of age, he presented with poor weight gain and failure to thrive. Initial evaluation revealed hyponatremia (Na, 126 mmol/L) and hyperkalemia (K, 5.6 mmol/L). At 2 months of age, he was referred to the Department of Pediatrics, West Virginia University Charleston Division, for further evaluation. This revealed an extremely elevated plasma aldosterone concentration (46.8 nmol/L; normal, 0.22–0.43) and PRA (>998.9 ng/mL·h; normal, 1.1–4.1). There was no history of consanguinity.
Methods
Establishment of permanent cell lines. Epstein-Barr virus-transformed lymphoblast lines were established from all patients and/or their parents, as previously described (37). Cells were harvested in RPMI 1640 medium with 10% FBS and 2 mmol/L glutamine.
Sequencing of genomic DNA. Genomic DNA was isolated from
Epstein-Barr virus-transformed lymphoblast lines, as previously
described (29). The genomic DNA fragments were amplified by PCR. The
sets of primers used for amplifying -, ß-, and ENaC were
described by Chang et al. (35). The genomic DNA fragments of
ENaC were sequenced directly by the dideoxynucleotide chain
termination method, as previously described (32), whereas those of
ßENaC and ENaC were sequenced directly by Big Dye terminator cycle
sequencing (PE Biosystems, Foster City, CA) with an ABI 377 automated
DNA sequencer (PE Biosystems, Foster City, CA).
Population study of identified mutations in ENaC. An
identified base substitution in ENaC created a new recognition site
for Aci 1 (C/CGC) at position 2086 of the ENaC cDNA, whereas the
wild-type allele was not cut by this enzyme. We amplified the 440-bp
PCR fragment of genomic DNA (cDNA position 1820–2260) of the
ENaC cDNA from the 5 patients and 42 unrelated control subjects with
apparently normal salt conservation. Fifty microliters of the PCR
products were digested with 100 U Aci 1 (New England Biolab, Beverly,
MA) at 37 C for 16 h and separated on a 2% agarose gel. The
normal PCR fragment was cut into 3 fragments (226, 163, and 51 bp) by
Aci 1, whereas the PCR fragment containing the mutant allele was cut
into 4 fragments (226, 43, 123, and 51 bp).
Patient and control population study of identified amino acid
substitutions in ß- and ENaC. We studied the frequency of the
amino acid substitutions identified in ßENaC and ENaC of our
patients in 25 of our control subjects by directly sequencing their
genomic DNA.
Statistical analysis. The 
2 test was employed
to examine the difference between the patient group and normal subjects
in having concurrent polymorphisms of the ENaC cDNA and the MR
cDNA.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. A nonconservative amino acid
substitution (A2086G2086,
Thr663Ala663) was identified in the ENaC
of the 5 patients, 2 in the homozygous and 3 in the heterozygous form,
respectively (Fig. 1). This amino acid substitution was also found in a
high proportion of 42 unrelated control subjects. The homozygosity and
heterozygosity frequencies of the ENaC Ala663 were 31%
and 64%, respectively.
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G1006,
Pro336Ara336) in the ßENaC and 3
homozygous amino acid substitutions
(T554A554,
Trp178Arg178;
C1526G1526,
Pro501Ala501;
T1862G1862,
Ser614Ala614) in the ENaC were identified
in all 5 of our patients and in all 25 control subjects examined.
Three of the four (75%) patients with multiple tissue resistance
to aldosterone had both ENaC (heterozygous or homozygous) and MR
(homozygous) mutations, whereas only 7% of our controls with
apparently normal salt conservation had the same concurrent
abnormalities (Table 2
; P < 0.025).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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ENaC and a
missense mutation of ßENaC resulted in loss of amiloride-sensitive
sodium channel activity in PHA patients from autosomal recessive
kindreds (35, 36). However, we found none of these mutations in our
five sporadic cases of PHA. Instead, we identified a polymorphism
located at a position close to the C-terminal of the ENaC, which was
also present at high frequency in apparently normal controls. A
previous report also showed that this polymorphism was present in 49%
of a control population in the heterozygous form (38). The proline-rich
region of the C-terminal of the ENaC that we identified is important
for binding to -spectrin and for stabilization of the sodium channel
in the membrane, suggesting that the above polymorphism might have
functional significance (39). We identified two different amino acid substitutions at the immunogenic domain of the MR in our PHA patients with multiple end-organ resistance to aldosterone, whereas we found no abnormalities in the DNA- or ligand-binding domains; one of these amino acid substitutions was nonconservative (23, 27). The base changes that led to these amino acid substitutions, however, were also present in a substantial proportion of apparently normal individuals and, therefore, should not be sufficient by themselves to cause PHA.
Finally, we identified four amino acid substitutions in the ß- and
ENaC subunits in all patients and control subjects examined,
suggesting that they might have been wrongly sequenced originally or
that they are extremely common polymorphisms in the general
population.
We have to assume that the ENaC amino acid substitutions
identified in our patients and significant proportion of control
subjects are pathophysiologically insignificant polymorphisms by
themselves. Warnock et al. previously described a ßENaC
polymorphism that might influence sodium transport (40). We cannot rule
out, however, the possibility that they may confer vulnerability in
salt conservation, which might be expressed fully only when
concurrently present with other genetic defects of the MR or other
proteins that participate in sodium homeostasis, such as Nedd 4
(41).
Indeed, we did find an increased co-occurrence of ENaC and MR
polymorphisms in our patients. Our hypothesis, if true, would be
compatible with a sporadic presentation or a digenic or multigenic
expression and heredity, as previously described in retinitis
pigmentosa (42). In this case, hereditary transmission might be complex
and appear as either a dominant and/or a recessive trait with variable
penetrance.
We conclude that the amino acid substitutions of the ENaC identified
in our patients are polymorphisms that do not cause the disease by
themselves. We hypothesize, however, that the disease might result from
a combination of abnormalities in the ENaC, MR, and other molecules
important for sodium transport and conservation (31, 32).
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Acknowledgments |
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Footnotes |
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Received October 28, 1998.
Revised March 31, 1999.
Accepted April 7, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunits of the human
epithelial sodium channel. Am J Physiol. 268:C1157–C116.
subunit: genetic heterogeneity of Liddle syndrome. Nat Genet. 11:76–82.[CrossRef][Medline]
subunit of the epithelial
sodium channel gene in three pseudohypoaldosteronism type 1 families. Nat Genet. 13:248–250.[CrossRef][Medline]
ENaC)
mediates its localization at the apical membrane. EMBO J. 13:4440–4450.[Medline]
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