Novel Mutations in the SCNN1A Gene Causing
Pseudohypoaldosteronism Type 1
Jian Wang1,2., Tingting Yu1., Lei Yin3, Jing Li3, Li Yu2, Ye Shen3, Yongguo Yu1,3, Yongnian Shen3,
1Research Division of Birth Defects, Institute of Pediatric Translational Medicine, Shanghai Children’s Medical Center, Shanghai Jiaotong University School of Medicine,
Shanghai, P. R. China, 2Department of Laboratory Medicine, Boston Children’s Hospital, Boston, Massachusetts, United States of America, 3Department of Pediatrics,
Shanghai Children’s Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai, P. R. China
Pseudohypoaldosteronism type 1 (PHA1) is a rare inherited disease characterized by resistance to the actions of
aldosterone. Mutations in the subunit genes (SCNN1A, SCNN1B, SCNN1G) of the epithelial sodium channel (ENaC) and the
NR3C2 gene encoding the mineralocorticoid receptor, result in systemic PHA1 and renal PHA1 respectively. Common clinical
manifestations of PHA1 include salt wasting, hyperkalaemia, metabolic acidosis and elevated plasma aldosterone levels in
the neonatal period. In this study, we describe the clinical and biochemical manifestations in two Chinese patients with
systemic PHA1. Sequence analysis of the SCNN1A gene revealed a compound heterozygous mutation (c.1311delG and
c.1439+1G.C) in one patient and a homozygous mutation (c.814_815insG) in another patient, all three variants are novel.
Further analysis of the splicing pattern in a minigene construct showed that the c.1439+1G.C mutation can lead to the
retainment of intron 9 as the 59-donor splice site disappears during post-transcriptional processing of mRNA. In conclusion,
our study identified three novel SCNN1A gene mutations in two Chinese patients with systemic PHA1.
Citation: Wang J, Yu T, Yin L, Li J, Yu L, et al. (2013) Novel Mutations in the SCNN1A Gene Causing Pseudohypoaldosteronism Type 1. PLoS ONE 8(6): e65676.
Editor: Andreas R. Janecke, Innsbruck Medical University, Austria
Received November 22, 2012; Accepted April 27, 2013; Published June 6, 2013
Copyright: ? 2013 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by the National Natural Science Foundation of China (Grant No. 81201353) and Research Fund of Health Bureau of Shanghai
Municipality (Grant No. 20114y072). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Pseudohypoaldosteronism type 1 (PHA1) is a rare inherited
disease characterized by resistance to the actions of aldosterone. It
was first described in 1958 by Cheek and Perry , and common
clinical manifestations include salt wasting, hyperkalaemia, met-
abolic acidosis and elevated plasma aldosterone levels in the
neonatal period . According to the clinical manifestations and
Mendelian inheritance patterns, PHA1 can be classified as either
Renal PHA1 (autosomal dominant, OMIM177735) or the more
severe systemic PHA1 (autosomal recessive, OMIM264350) .
Renal PHA1 results from a defect in the tubular response to
aldosterone caused by inactivating mutations in the NR3C2 gene
(4q31) encoding the mineralocorticoid receptor . Systemic
PHA1 is caused by mutations in the genes encoding the subunits of
the epithelial sodium channel (ENaC): alpha subunit (SCNN1A;
12p13), beta subunit (SCNN1B; 16p12.2-p12.1), or gamma subunit
(SCNN1G; 16p12) . However, some special cases of PHA1 have
been reported. Hubert et al.  described a newborn with severe
recessive PHA1 caused by two heterozygous mutations in NR3C2.
Dirlewanger et al.  also reported that a homozygous missense
mutation in SCNN1A is responsible for a transient neonatal form of
In the 55 years since PHA was first known in the 1950s,
sufficient clinical descriptions have accumulated in the literatures.
Still, the symptoms of PHA1 are easily confused with that of other
endocrine disorders, especially if the pediatrician cannot get
specific biochemical findings . The symptoms of PHA1 are
easily confused with the symptoms of congenital adrenal hyper-
plasia (CAH) associated with 21-hydroxylase deficiency or 3-beta-
hydroxysteroid dehydrogenase deficiency; and the symptoms of
hypoaldosteronism (HA) due to aldosterone deficiency, antenatal
or infantile Bartter syndrome. In addition, respiratory tract
infections associated with PHA1 may be mistaken for symptoms
of cystic fibrosis . Such confusion delays diagnosis when prompt
treatment of PHA1 is essential, as death may occur as a result of
salt depletion and high blood potassium level .
In this study, two pediatric patients who suffer from PHA1 were
promptly diagnosed with molecular genetic tests and treated.
Using molecular genetic tests, we identified three novel mutations
in the SCNN1A gene. We further confirmed the pathogenicity of
the c.1439+1G.C mutation by analyzing its effect on splicing in a
Subjects and Methods
neonatal intensive care unit as a result of her refusal to be
breastfeed, poor response, weak cries and cold extremities.
Physical examination: Weight 3100 g (SDS=20.56), Height
49.5 cm (SDS=20.37), Heart rate 125/min, Respiratory rate
A 5-day-old female infant was admitted to the
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36/min, Blood pressure 71/32 mmHg, Rectal temperature
(37uC), SpO2 99%. Auscultation: no lung crackles and heart
murmur. Palpation: the liver edge about 2.5 cm below the costal
margin, spleen is not palpable. Skin color: mild jaundice. Genital
examination: female characteristics. Two days later, symptoms of
severe dehydration and vomiting appeared. Biochemical labora-
tory tests showed that she had severe hyperkalemia with
K+10.1 mmol/L, Na+105 mmol/L, Cl+83 mmol/L, PH 7.31,
BE(B) 210.8 mmol/L and the trans-tubular potassium gradient
(TTKG) is 4. Hormone indicator is as follows: aldosterone
1.08 nmol/L (range 0.17–0.47), plasma renin activity 14.0 ug/
L/h (range 0–15), ACTH 20.4 pmol/L, cortisol 687.2 nmol/L,
testosterone 0.94 nmol/L, and 17-OHP 25 nmol/L.
PHA1 was initially suspected on the basis of her clinical features,
and the infant was immediately given a supplement of 10%NaCl.
The results of hormone levels and TTKG value tests provided
Table 1. Primers for Amplify Exons and the Boundary Sequences of SCNN1A, SCNN1B, SCNN1G and NR3C2.
Primer Sequence (5’R3’)
5, 6gctctgaaaggcacaagtccctgctcctgaagacctccac 860
7, 8 ggtggctggaagcatgtattgggaaactgacagaggcaga723
3accttccgccatgattgtaa ccccagcgagactcaaatta 581
4 ttgagcatgtgtgagcatgaacaccgaggcacagaaaact 501
5actctcttcccctgctttcc ggtagcagccactcctcttg 434
6, 7 agtgggtagtggggtctcctaaagtgactggtcccacagg 813
11ccttcctcccctagaacagc cagtgacagagggaagcaca 474
12,13 ctgtttggaagggggatacactttggagagggcaccatac 849
2 ggggccgtaagagaagtaggggctgtttaccagcgttagc 751
3 ccacaggaagtcacacatgg aggggctagtggtcaaggag612
4 ccaacctgttcccctgagtaaccttttgctcccaagacct 413
6 gcagtgggagaggtggttta ggaccatgttcccttttgaa358
7 ccacagtaccaggcacctaa aactgcagaggactggaacc443
8 cataaggggcaggttcatgt atccaccgttcctacctcct 467
9,11tggtagaaagtgggaggagaaa gtggtgggaagagacagagg 650
2A tgttctgacatctcgacaagc aaacagacgggcttttctca784
7 ggcccagcagtattggtcta tgagtggttggatggatgaa569
Novel Mutations in SCNN1A Gene
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further evidence of PHA1 and the clinical diagnosis was confirmed
by molecular genetic testing. After increasing doses of oral
10%NaCl to 30 mEq/kg/d and doses of ion-exchange resins to
1g/kg/d, the serum potassium level was controlled at 4.5 to
5.5 mmol/L. Her development seemed normal at the last follow-
up visit (26 months). At that time, her plasma concentration of
aldosterone is 1.25 nmol/L and plasma renin activity is 1.67 ug/
A 2-months old female infant was referred to our
hospital with main complaint of electrolyte imbalance since she
was born. Physical examination: Weight 4750 g (SDS=24.13),
Height 51.1 cm (SDS=23.17), Heart rate 117/min, Respiratory
rate 33/min, Blood pressure 80/35 mmHg. Genital examination:
female characteristics. Other examination results showed that she
is without lung crackles or heart murmur, and has normal liver
and spleen size. This patient was born after 38 weeks of
pregnancy. She weighed 2900 g (SDS=20.65) at birth. At seven
days of age, She developed signs of hyponatremia, hyperkalaemia
and metabolic acidosis. She had initially been diagnosed with
CAH in another hospital on account of her clinical presentation.
However, 9a-fluorohydrocortisone (9a-FHC) supplement therapy
was ineffective. There was no history of parental consanguinity.
Laboratory test results are as follows: K+6.1 mmol/L, Na+125 m-
mol/L, Cl+92 mmol/L, PH 7.34, BE(B) 26.8 mmol/L and the
TTKG is 7. Hormone indicator is as follows: aldosterone
0.58 nmol/L (range 0.17–0.47), plasma renin activity 10.1 ug/
L/h (range 0–15), ACTH 4.5 pmol/L, cortisol 460.9 nmol/L,
testosterone 0.42 nmol/L, and 17-OHP 13 nmol/L.
After obtaining the molecular diagnostic result, the baby was
definitively diagnosed with PHA1. The serum potassium level was
controlled with an intake of 10%NaCl at 10 mEq/kg/d and ion-
exchange resins at 0.6 g/kg/d. At her last follow up at 12 months,
physical examination showed normal results and her serum
electrolytes are stable. Her weight was 9.5 kg (SDS=20.09),
and her height was 73 cm (SDS=20.21).
The study protocol was approved by the ethics committee of the
SCMC, and informed consent was obtained from the family
members and normal subjects.
Analysis of the NR3C2, SCNN1A, SCNN1B and SCNN1G
The genomic DNA of the patients and their parents was isolated
from whole blood samples by proteinase K digestion and phenol/
chloroform extraction. An additional group of 105 Chinese
subjects with no history of PHA1 were recruited as controls to
test whether the mutations found are common polymorphisms.
The study protocol was approved by the ethics committee of the
hospital, and informed consent was obtained from family members
and normal subjects.
All of the exons and exon-intron boundaries of the NR3C2
(GenBank NM_000901.2), SCNN1A (GenBank NM_001038.5),
SCNN1B (GenBank NM_000336.2) and SCNN1G (GenBank
NM_001039.3) gene were amplified by PCR (TaKaRa, Dalian,
China) using primers listed in Table 1. Primers were designed
using Primer 3 software (http://frodo.wi.mit.edu/primer3/). The
amplified PCR products were purified from agarose gel using
QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and
sequenced via the ABI3730XL sequencer (Applied Biosystems,
Minigene Construction and Transfection into HEK 293
The splice region (c.1439+1, intron 9) of SCNN1A gene was
amplified with specific primer pair (59-CCCaagcttGTAGA-
GAAAGCTGAGGTGCC-39, and 39-GACGCCGATTCAGA-
GAAAAAcctaggCTG-59) using PrimeSTAR HS DNA Polymer-
ase (TaKaRa, Dalian, China). The forward and reverse primer
Figure 1. Three novel mutations were identified in the SCNN1A gene. (A) Sequences showing a compound heterozygous mutations
(c.1311delG in exon 8 c.1439+1G.C in intron 9) in PHA1 patient of case 1. (B) Sequences showing a homozygous mutation (c.814_815insG in exon 4)
in PHA1 patient of case 2.
Novel Mutations in SCNN1A Gene
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sequences are located in intron 8 and in 39UTR, respectively. The
length of the PCR product is 1,863 bp. The restriction enzyme
sites HindIII and BamHI were inserted into the primer sequences to
enable directional cloning. DNA from a normal individual was
used as control. Minigene amplification products were purified,
digested, and ligated into the pcDNA3.1/Myc-His B vector
(Invitrogen, CA, USA) according to the protocol of the manufac-
turer. The constructions were used to transform E. coli DH5a
competent cells, and putative recombinant colonies were selected
to verify the presence of the correct mutation by sequencing
recovered plasmids. The recombinant plasmids with either wild-
type or mutant SCNN1A were transfected into HEK 293 cells.
Blank vector was used as a control.
RNA Extraction, cDNA Synthesis, and Splice Sites
Thirty-six hours after transfection, total RNA from the HEK
293 cells was extracted with an RNeasy Mini Kit (Qiagen, Hilden,
Germany). cDNA chains were obtained by reverse transcription
(TaKaRa, Dalian, China) and target fragment was amplified with
another primer pair (59-ttgacttctcctcagaccacc-39, and 59-agggtgac-
catcgtgacagag-39). The forward and reverse primer sequences are
located in exon 9 and at the junction between exon 10 and 11,
respectively. The expected length of the PCR product is 256 bp.
Then, PCR products were analyzed by running in 2% agarose gel
stained with ethidium bromide and sequenced.
In case 1, the patient and her parents were genotyped by direct
nucleotide sequencing. We found that the patient had compound
heterozygous mutations in the SCNN1A gene. One mutation was a
deletion of a single base ‘‘guanine’’ (c.1311delG) in exon 8, which
resulted in a frameshift leading to a premature stop codon
(p.Arg438GlyfsX43). The other mutation was a transversion of
guanine to cytosine (c.1439+1G.C) in the first base of the 59-
donor splice site of intron 9. This mutation occurs at a highly
conserved ‘‘AG-gt’’ sequence of the exon-intron boundary region.
The predicted effect of this G.C mutation is a disruption normal
splicing, possibly resulting in the retainment of intron 9 of the
SCNN1A gene. No other mutations were found in the entire exons
Figure 2. In vitro splicing assay of the c.1439+ +1G. .C mutation. (A) The splice region (c.1439+1, intron 9) of SCNN1A gene was amplified and
products were ligated into the pcDNA3.1/Myc-His B vector. (B) RT-PCR of HEK293 cells transfected with either wild-type or mutant SCNN1A. Minigenes
showed that the mutation c.1439+1G.C was sufficient to produce a longer band. (C) Lane 1: Empty pcDNA3.1 vector; Lane 2: wild-type SCNN1A
(256 bp); Lane 3: c.1439+1G.C mutant (361 bp); Lanes 4, 5, and 6: GAPDH used as control (245 bp).
Novel Mutations in SCNN1A Gene
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and the exon-intron boundaries of the patient’s NR3C2, SCNN1B
and SCNN1G gene. The patient’s mother was heterozygous for the
c.1311delG mutation, and her father was heterozygous for the
c.1439+1G.C mutation. No such mutations were observed in the
105 unrelated healthy individuals. Consequently, it was concluded
that the patient is a novel compound heterozygous individual who
inherited the maternal c.1311delG mutation and the paternal
c.1439+1G.C mutation (Fig. 1A).
In case 2, direct sequencing of the entire SCNN1A gene in the
patient revealed a homozygous insertion of one ‘‘G’’ in exon 4
(c.814_815insG). This frameshift mutation results in a premature
stop codon (p.Glu272GlyfsX38). The mutation was also found in
heterozygous state in both of her parents. DNA samples from 105
unrelated healthy individuals were subsequently tested for this
mutation, and it was not found in these individuals. Therefore, we
concluded that the patient is a novel homozygous individual who
inherited a copy of the c.814_815insG mutation from each of her
heterozygous parents (Fig. 1B).
In vitro Splicing Assays to the c.1439+1G.C Mutation
To assess the impact of the splice-site mutation, a minigene
construct was generated and tested in transiently transfected
cultured cells. HEK 293 cells were transfected with the mutant
SCNN1A, wild type SCNN1A, or empty pcDNA3.1 vector (negative
control). Three independent replicates were tested, and GAPDH was
used as a control for RNA quality and quantity. A difference in the
size of the cDNA fragment was observed between wild type and
mutated SCNN1A. The wild-type SCNN1A resulted in a single band
at 256 bp, whereas the mutated construct resulted in a longer band
at 361 bp. Sequencing confirmed that RNA resulted from mutant
SCNN1A construct contained the 105 nucleotides that constitute
intron 9. The loss of the 59-donor splice site at the exon-intron
boundary resulted in the retainment of intron 9 in the RNA (Fig. 2).
Pediatricians should consider the possibility of PHA1 when
encountering infants with unexplained hyponatremia, hyperkale-
mia and metabolic acidosis . PHA1 diagnosis is based on
elevated plasma aldosterone and renin levels, especially when
high-dose mineralocorticoid treatment cannot remedy the imbal-
ance of potassium and salt. Schweiger et al.  suggested that
TTKG index may be helpful in the acute setting to determine
whether mineralocorticoid deficiency or resistance is leading to
hyperkalemia. A typical TTKG value in a normal person on a
normal diet is 8–9 . During hyperkalemia or high potassium
intake, more potassium should be excreted in the urine and the
TTKG should be above 10. If the TTKG is low during
hyperkalemia, and this is also accompanied by hyponatremia
and increased urine sodium excretion, the presence of mineral-
ocorticoid deficiency or resistance should be considered. Belot
et al.  reported that there are two clinical manifestations in
patients with systemic PHA1, including life-threatening weight loss
and early cutaneous involvement. The dermatitis may be due to
high salt concentration in sweat. It is recommended for weight
changes to be monitored closely, and for sweat test (which
measures the concentration of chloride) to be administered for
early diagnosis . In addition, detailed family history is often
In our case, both patients had signs of salt loss soon after birth,
including hyponatremia, hyperkalemia, and metabolic acidosis.
We considered the possibility of ‘‘PHA1’’ in accordance with their
hormone levels and TTKG results. Molecular genetic tests were
performed on each patient and her family in order to confirm the
clinical diagnosis. All of the exons and exon-intron boundaries of
the NR3C2, SCNN1A, SCNN1B and SCNN1G genes were
sequenced by Sanger method. The results showed that the two
patients had novel mutations in the SCNN1A gene respectively.
Since the first identification of mutations in the Saudi and
Iranian Jewish kindred of PHA1 , nearly 25 kinds of mutations
have been identified in SCNN1A gene, including missense/
nonsense mutations, splicing mutations, small insertions/deletions,
and gross deletions [7,13–16]. However, no mutations have been
reported in the Chinese population. We have successfully
identified two novel frameshift mutations (c.1311delG and
c.814_815insG) and a novel splicing mutation (c.1439+1G.C)
in the SCNN1A gene in two Chinese patients with systemic PHA1.
The SCNN1A gene contains 13 exons and encodes the alpha
subunit of the ENaC, which is expressed in the distal nephron and
regulated by aldosterone . The ENaC is the rate-limiting step
for sodium reabsorption in the apical membrane of epithelia .
It plays a major role in the Na+- and K+-ion homeostasis, and has
a high affinity for the potassium-sparing diuretics amiloride and
triamterene. In our study, the frameshift mutations c.1311delG
and c.814_815insG induce premature termination of mRNA
translation and cause loss of the C-terminal domain (extracellular,
helical and cytoplasmic) of the protein. The splicing site mutation
(c.1439+1G.C) can lead to the retention of intron 9 as the 59-
donor splice site disappears during post-transcriptional processing
of mRNA. All of these novel mutations are thought to result in an
absence of a functional aENaC subunit, and constitute the
molecular basis for systemic PHA1 phenotype.
At present, the main treatment for PHA1 is the intake of high
doses of sodium together with ion exchange resins and dietary
manipulations to reduce potassium levels . During the course of
treatment, clinicians must gradually adjust the dose according to
the serum electrolytes and hormone levels [10,11]. When
treatment is effective, PHA1 patients will be able to maintain
the right balance of electrolytes and resume normal growth and
development; their plasma concentration of aldosterone and renin
activity will be reduced or return to normal . In order to reach
such successful treatment outcome, patients must take medicine on
time, maintain the right dosage, avoid high potassium foods and
closely observe signs and symptoms of PHA1 . In our case,
patient from case 1 was hospitalized again after four months due to
hyperkalemia caused by diarrhea. Because both patients suffer
from the systemic form of PHA1, both may need lifelong therapy.
Because PHA1 is rare, the relationship between genotype and
phenotype is not yet clear as a result of lack of sufficient cases.
However, our study has brought us a step closer to understanding
this relationship, and may ultimately contribute to the goal of
finding a simpler and more effective treatment for these patients.
We identified three novel SCNN1A gene mutations in two
Chinese patients with systemic PHA1. The c.1311delG mutation
and the c.814_815insG mutation are both frameshift mutations
that lead to premature stop codon. As demonstrated by the
functional analysis using an in vitro minigene system, the third
c.1439+1G.C mutation leads to aberrant splicing, in which
intron 9 is retains as the 59-donor splice site of intron 9
We thank Dr. Bai-lin Wu, Dr. Yiping Shen (Boston Children’s Hospital)
and Miss Sydney Song for their critical reading of the manuscript. We are
deeply grateful to the patients and their families for participating in this
Novel Mutations in SCNN1A Gene
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Conceived and designed the experiments: JW QF. Performed the
experiments: JW TY. Analyzed the data: JW L. Yu YY. Contributed
reagents/materials/analysis tools: JW L. Yu YY. Wrote the paper: JW QF.
Clinical diagnostic and therapy: L. Yin JL Ye Shen Yongnian Shen.
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