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Seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (SeSAME syndrome) caused by mutations in KCNJ10

Department of Genetics, The Howard Hughes Medical Institute, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 05/2009; 106(14):5842-5847. DOI: 10.1073/pnas.0901749106

ABSTRACT

We describe members of 4 kindreds with a previously unrecognized syndrome characterized by seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (hypokalemia, metabolic alkalosis, and hypomagnesemia). By analysis of linkage we localize the putative causative gene to a 2.5-Mb segment of chromosome 1q23.2-23.3. Direct DNA sequencing of KCNJ10, which encodes an inwardly rectifying K(+) channel, identifies previously unidentified missense or nonsense mutations on both alleles in all affected subjects. These mutations alter highly conserved amino acids and are absent among control chromosomes. Many of these mutations have been shown to cause loss of function in related K(+) channels. These findings demonstrate that loss-of-function mutations in KCNJ10 cause this syndrome, which we name SeSAME. KCNJ10 is expressed in glia in the brain and spinal cord, where it is believed to take up K(+) released by neuronal repolarization, in cochlea, where it is involved in the generation of endolymph, and on the basolateral membrane in the distal nephron. We propose that KCNJ10 is required in the kidney for normal salt reabsorption in the distal convoluted tubule because of the need for K(+) recycling across the basolateral membrane to enable normal activity of the Na(+)-K(+)-ATPase; loss of this function accounts for the observed electrolyte defects. Mice deficient for KCNJ10 show a related phenotype with seizures, ataxia, and hearing loss, further supporting KCNJ10's role in this syndrome. These findings define a unique human syndrome, and establish the essential role of basolateral K(+) channels in renal electrolyte homeostasis.

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Seizures, sensorineural deafness, ataxia, mental
retardation, and electrolyte imbalance (SeSAME
syndrome) caused by mutations in
KCNJ10
Ute I. Scholl
a
, Murim Choi
a
, Tiewen Liu
a
, Vincent T. Ramaekers
b
, Martin G. Ha
¨
usler
c
, Joanne Grimmer
d,1
,
Sheldon W. Tobe
e
, Anita Farhi
a
, Carol Nelson-Williams
a
, and Richard P. Lifton
a,2
a
Department of Genetics, The Howard Hughes Medical Institute, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510;
b
Department
of Paediatrics, Centre Hospitalier Universitaire, Domaine Universitaire du Sart Tilman, Baˆ timent B 35, B-4000 Lie` ge 1, Belgium;
c
Department of Paediatrics,
University Hospital, RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany;
d
Department of Nephrology, The Hospital for Sick Children, 555 University
Avenue, Toronto, ON, Canada M5G 1X8; and
e
Division of Nephrology, Sunnybrook Health Sciences Centre, 2075 Bayview Avenue, Toronto, ON, Canada
M4N 3M5
Contributed by Richard P. Lifton, February 18, 2009 (sent for review January 29, 2009)
We describe members of 4 kindreds with a previously unrecog-
nized syndrome characterized by seizures, sensorineural deafness,
ataxia, mental retardation, and electrolyte imbalance (hypokale-
mia, metabolic alkalosis, and hypomagnesemia). By analysis of
linkage we localize the putative causative gene to a 2.5-Mb
segment of chromosome 1q23.2–23.3. Direct DNA sequencing of
KCNJ10, which encodes an inwardly rectifying K
channel, identi-
fies previously unidentified missense or nonsense mutations on
both alleles in all affected subjects. These mutations alter highly
conserved amino acids and are absent among control chromo-
somes. Many of these mutations have been shown to cause loss of
function in related K
channels. These findings demonstrate that
loss-of-function mutations in KCNJ10 cause this syndrome, which
we name SeSAME. KCNJ10 is expressed in glia in the brain and
spinal cord, where it is believed to take up K
released by neuronal
repolarization, in cochlea, where it is involved in the generation of
endolymph, and on the basolateral membrane in the distal
nephron. We propose that KCNJ10 is required in the kidney for
normal salt reabsorption in the distal convoluted tubule because of
the need for K
recycling across the basolateral membrane to
enable normal activity of the Na
-K
-ATPase; loss of this function
accounts for the observed electrolyte defects. Mice deficient for
KCNJ10 show a related phenotype with seizures, ataxia, and
hearing loss, further supporting KCNJ10’s role in this syndrome.
These findings define a unique human syndrome, and establish the
essential role of basolateral K
channels in renal electrolyte
homeostasis.
Gitelman syndrome hypokalemia hypomagnesemia
inwardly rectifying K
channel renal salt wasting
T
ransmembrane ion flux via channels, transporters, and
pumps plays a critical role in diverse physiologic functions,
including neuronal signal transmission and electrolyte and vol-
ume homeost asis. In many cases, homologous electrolyte flux
processes in different tissues are mediated by the encoded
products of distinct genes, while in a few cases the identical gene
products are involved. Evidence of the latter comes from Men-
delian diseases in which mutation in a single gene produces
ef fects on both auditory and renal function. For example,
loss-of-function mutations in ATP6B1, which encodes a subunit
of the H
-ATPase, result in systemic acidosis because of a renal
defect in H
secretion and sensorineural hearing loss caused by
defective H
secretion into the c ochlear endoly mph, resulting in
impaired hair cell function and deafness (1). Similarly, mutations
in barttin, which encodes a subunit of the CLCNKA and
CLCNKB chloride channels, result in renal salt wasting and
deafness (2).
The genetic dissection of renal diseases featuring low serum
pot assium (hypok alemia) and metabolic alk alosis (high serum
pH) has identified many c omponents required for normal renal
electroly te homeostasis (2–7). In all cases, this syndrome has
resulted from increased activity of the epithelial Na
channel
(ENaC) on the apical membrane, which leads to increased
secretion of K
and H
because of the more negative luminal
potential. Hypokalemia with alk alosis can result either f rom
primary increases in ENaC activity because of mut ations in
ENaC itself (3), or f rom activation of ENaC by aldosterone in
response to reduced intravascular volume (7). Mutations that
cause impaired salt reabsorption in the thick ascending limb of
Henle or the dist al convoluted tubule cause salt wasting that
leads to secondary increases in ENaC activity and hypokalemic
alk alosis. Identified mutations in these diseases, referred to as
Bartter and Gitelman syndromes, are in genes including 2 apical
Na
-Cl
transporters that mediate Na-Cl entry into epithelia, 2
Cl
channel subunits that mediate exit of Cl
across the
basolateral membrane, and an apical K
channel (2, 4–7). These
syndromes are distinguished clinically by marked hypomag-
nesemia and low urinary calcium in Gitelman syndrome, while
hypercalciuria with normal or modest reductions in Mg
2
is
observed in Bartter syndrome.
Similarly, a number of Mendelian seizure disorders have been
described. Many of these result f rom mutations that depolarize
neurons, increasing neuronal excitability and reducing seizure
threshold. Examples include benign familial neonatal seizures
caused by mutations in the KCNQ2/3 K
channels (8, 9), benign
familial neonatal/infantile seizures caused by mutations in the
SCN2A gene encoding the alpha subunit of voltage gated Na
channels (10), and several idiopathic epilepsy syndromes caused
by mutations in the SCN1A sodium channel (11).
Considering the many similarities in the mechanisms govern-
ing renal electrolyte homeostasis and neuronal function, it is
surprising that relatively few single-gene disorders that have
ef fects on both have been identified. Here, we describe a
previously unrecognized complex syndrome featuring seizures,
sensorineural deafness, at axia, mental retardation and electro-
ly te imbalance (SeSAME), and demonstrate that it is caused
by mut ation in KCNJ10, which enc odes a K
channel ex pressed
in epithelia of the kidney and inner ear, as well as glial cells in
the CNS.
Author contributions: U.I.S., M.C., and R.P.L. designed research; U.I.S., M.C., T.L., V.T.R.,
M.G.H., J.G., S.W.T., A.F., C.N.-W., and R.P.L. performed research; U.I.S., M.C., T.L., and R.P.L.
analyzed data; and U.I.S., M.C., and R.P.L. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1
Present address: Department of Paediatrics, University of Western Ontario, 800 Commis-
sioners Road East, London, Ontario, Canada N6A 5W9.
2
To whom correspondence should be addressed. E-mail: richard.lifton@yale.edu.
5842–5847
PNAS
April 7, 2009
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no. 14 www.pnas.orgcgidoi10.1073pnas.0901749106
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Results
Case Report: Kindred 441. The index case, patient 441–1, is a
24-year-old female of Afghan ancestry who is the fifth of 6
of fspring of healthy first-cousins. Generalized seizures began at
3 months, oc curring several times daily. Seizures were controlled
in itially with phenobarbit al, and later diphenylhydantoin. Sitting
was first demonstrated at age 1 year, crawling at age 20 months.
At age 5 years 7 months, the patient presented for evaluation
of developmental delay. At this time, she was unable to walk or
speak. Physical examination was notable for atrophy of the lower
extremities without c ontractures. Motor strength was slightly
reduced in the upper, but markedly reduced in the lower
extremities. Reflexes were normal. There was marked ataxia.
Nerve conduction studies revealed reduced conduction velocit y
in the left peroneal and left tibial nerves (36 and 37 m/sec,
respectively; nl 4044 m/sec). A muscle and nerve biopsy showed
nor mal muscle other than fiber-type disproportion; there was
hypomyelination of the large myelinated nerve fibers in the sural
nerve, with moderate progressive axonal neuropathy. Brain MRI
showed normal myelination, and was normal with the exception
of slightly coarsened frontal sulci. EEG, abdominal ultrasound,
and karyotype analysis were normal.
L aboratory evaluation was remarkable for persistent hypoka-
lemia, metabolic alkalosis, and hypomagnesemia (Table 1).
Plasma renin activity (PRA) was elevated on repeated measures
(8.1 and 7.6 ng/ml per hour, nl 2.8 ng/ml per hour). Twenty-
four-hour urinary aldosterone level was elevated (31.3
g; nl
5.9–17.6) and the Ca
2
/creatin ine ratio was low (0.1 to 0.2
mmol/mmol). The patient was treated with oral potassium
replacement, and required 30 meq per day to maintain a K
level
in the normal range. At age 18, progressive hearing loss was
noted. Brainstem-evoked response audiometry and pure-tone
threshold audiometry were performed, and moderate-to-severe
sensorineural hearing loss was documented.
The patient’s family history is notable for 2 of 5 siblings with
a related disorder, and one spontaneous abortion. The first child,
a male, presented with seizures at age 4 months and was never
able to walk. He died at age 7 years during a diarrheal illness. The
sixth child, a male, presented with seizures and vomiting at age
5 months and was unable to walk until 16 months. He died at 18
months during an intercurrent infection. The other 3 children are
healthy.
Definition of a New Clinical Syndrome. In the review of 589 subjects
referred for evaluation of Gitelman and Bartter syndromes, we
rec ognized subjects f rom 3 additional kindreds with features
similar to patient 441–1. The shared features included early onset
seizures, mental retardation, ataxia, hypoton ia, and sensorineu-
ral hearing loss. Intention tremor was noted in several cases and
volume loss of the cerebellum in 2 cases (632–2 and 404–1).
Short stature was notable in 2 affected siblings (632–1 and
632–2), with a final height of 150 cm and 149 cm, respectively.
Electroly te abnormalities featured marked and persistent hy-
pok alemic metabolic alkalosis in the absence of hypertension
and striking hypomagnesemia that required electrolyte replace-
ment and, in many cases, use of phar macologic inhibitors of the
epithelial sodium channel or aldosterone antagonists to prevent
renal K
loss. Where available, 24-h urinary electrolyte mea-
surements revealed renal K
and Mg
2
wasting and high urinary
Na
levels. PRA and aldosterone levels, when measured, were
always elevated, and salt crav ing, enuresis, and polyuria/
polydipsia were reported. A summary of the patients’ clinical and
laboratory findings is prov ided in Table 1.
Mapping the Disease Locus. The recurrent clin ical features sug-
gested a previously unrecognized clinical syndrome. Moreover,
recurrence of a similar syndrome among siblings in 2 of these
k indreds, and its occurrence in the setting of parental c onsan-
guin ity in 2 kindreds, suggested autosomal recessive transmis-
sion. To attempt to map the underlying disease locus, we
performed genome-wide analysis of linkage in the 3 informative
k indreds (2 offspring of first-cousin marriage and 2 affected
siblings from unrelated parents). The results demonstrated
c omplete linkage of the putative trait locus to a single chromo-
some segment, 1q23.2-q23.3, with a lod sc ore of 3.0. The
maximum likelihood location is confined to a 2.5-Mb interval
extending from 158.1 M to 160.6 M base pairs (Fig. 1A). No other
chromosome segment showed linkage in all 3 kindreds. Signif-
icantly, the index case (441–1) did not show homozygosity at any
of the known loci for Bartter or Gitelman syndrome, and
sequencing of these genes revealed no evidence of pathogenic
mut ations.
Mutations in
KCNJ10
. The linked interval on chromosome 1
c ontains 70 well-defined and at least 6 hypothetical genes, none
of which has previously been implicated in human disease
phenot ypes that would explain the features found in these
patients. If the disease is caused by mutation in a single gene, we
anticipate that it would likely be expressed in the CNS, inner ear,
and k idney. We considered ion channels, transporters, and
Table 1. Clinical features of affected patients
Clinical feature
Patient number
327–1 404–1 441–1 632–1 632–2
Ancestry Great Britain Turkey Afghanistan Canada Canada
Consanguinity N/A Yes Yes No No
Seizures (age of onset in months) (N/A) (4) (3) (3) (3)
Ataxia ⫹⫹⫹⫹
Developmental delay ⫹⫹⫹⫹
Hearing loss ⫹⫹⫹⫹
K
(mmol/l) 2.9 3.15 3.1 3.1 2.9
Mg
2
(mmol/l) 0.55 0.62 0.54 0.56 0.6
HCO
3
(mmol/l) 28 30 29 31 33
U
Ca
/U
Cr
(mmol/mmol) 0.11 0.34 0.10 N/A 0.26
U
K
/U
Cr
(mmol/mmol) 38.18 24.28 21.67 N/A 16.18
U
Na
/U
Cr
(mmol/mmol) 19.09 25.86 32.50 N/A 23.86
K
, serum potassium, nl 3.5–5 mmol/l; Mg
2
, serum magnesium, nl 0.8 –1.2 mmol/l; HCO
3
, serum bicarbonate, nl 23–26 mmol/l;
U
Ca
/U
Cr
, urinary calcium/creatinine ratio, nl 0.4 mmol/mmol); U
K
/U
Cr
, urinary potasssium/creatinine ratio [nl 6 8 mmol/mmol, or 1–1.5
mmol/mmol in hypokalemia (36)]; U
Na
/U
Cr
, urinary sodium/creatinine ratio, values are elevated and in the range observed in patients with
Gitelman syndrome (30).
Scholl et al. PNAS
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regulators of their function to be leading candidates. We iden-
tified KCNJ10, which encodes the inwardly rectifying K
channel
K ir4.1 (also known as BIR10, BIRK1, K
AB
-2, Kir1.2), consisting
of 2 transmembrane segments and 1 pore, as a strong candidate
(Fig. 1B). KCNJ10 has been shown to be expressed in the CNS,
c ochlea, and distal nephron, and a mouse knockout has a
neurological phenotype with many features similar to those seen
in our patients (see Discussion).
We screened available members of the 4 kindreds with this
syndrome for KCNJ10 mutations by direct sequencing (Fig. 2).
We found homoz ygous missense mutations in the 2 consanguin-
eous kindreds, c ompound heterozygous missense mutations in 1
outbred kindred, and a compound missense/premature termi-
nation mutation in 1 kindred. In the compound heterozygous
patients, cloning of the coding region on single amplicons
c onfirmed that the 2 mutations identified are in trans. To assess
the significance of missense mut ations, we compared the
KCNJ10 amino acid sequence to orthologs in diverse vertebrate
species including mammals, Xenopus tropicalis, and zebrafish,
and closely related paralogs in chicken and Drosophila melano-
gaster (see Methods). These species split from a common ancestor
500 million years ago, and across these species, only 27% of the
amino acids were completely conserved. A ll of the identified
mut ations occurred at positions that were c ompletely conserved
among all vertebrate species and all but one occurred at positions
c ompletely conserved through Drosophila (see Fig. 2).
Patient 327–1 was compound heterozygous for a nonsense and
a missense mut ation (see Fig. 2 A). The nonsense mutation
introduces a premature termination codon at position 199 in the
c ytoplasmic C terminus. This deletes a PDZ-binding domain that
is known to be required for expression of Kir4.1 at the cell
surface (12). This patient also harbors an R65P substitution
immediately preceding the first transmembrane domain (Fig. 3).
This position is conserved in a related inward rectifier, Kir2.1,
and expression of Kir2.1 containing mutation at this position
abolished nearly all detectable whole-cell K
current when
ex pressed in Xenopus oocytes (13).
Af fected subject 404–1 was homozygous for a C140R missense
mut ation (see Fig. 2B). C140 is located in the P region near the
st art of the second transmembrane domain (see Fig. 3). This
position is conserved in the related channel ROMK (position
C153), and mutation of this residue to either alanine or serine
has been shown to abolish ROMK function (14).
A missense mut ation was found in kindred 441, resulting in a
T164I substitution (see Fig. 2C). The index case is homoz ygous
for the mutant allele, while both parents are heterozygotes and
Fig. 1. Mapping the disease locus. (A) An ideogram of chromosome 1 is
shown, with homozygous regions in patient 441–1 and 404 –1 indicated by
light gray boxes, and segments that are identical by descent (IBD) in the
siblings 632–1 and 632–2 marked by dark gray boxes. The overlap of these
intervals is marked, and represents the maximum likelihood location of the
disease locus on chromosome 1q23.2-q23.3, a 4-cM interval covering 2.5 Mb
from 158.1 M to 160.6 M base pairs. (B) The candidate interval contains KCNJ10
on chromosome 1q23.2. Neighbouring genes are represented by arrows in
their corresponding transcriptional orientations. KCNJ10 comprises 2 exons
indicated by boxes, with the coding sequence indicated in black.
Fig. 2. Mutations in KCNJ10 in affected patients. In each panel the DNA
sequences of the sense strand of wild-type subjects (Left) and affected subjects
(Right) are shown. The sequence of the encoded peptide is indicated in single
letter code. A ClustalW alignment of the Homo sapiens (H.s.) protein sequence
with orthologs and paralogs from Mus musculus (M.m.), Gallus gallus (G.g.),
Xenopus tropicalis (X.t.), Danio rerio (D.r.), and Drosophila melanogaster
(D.m.) is shown next to each mutation. The human sequence and residues
conserved in orthologs and paralogs are marked in yellow, and the mutant
residue is indicated. (A) Patient 327–1 is compound heterozygous for a mis-
sense and a nonsense mutation in KCNJ10.(B) Patient 404 –1 is homozygous
for a missense mutation, changing codon TGT (C140) to CGT (R140). (C)A
homozygous missense mutation was found in kindred 441, resulting in change
of codon ACC (T164) to ATC (I164). (D) In kindred 632, both affected siblings
are compound heterozygous for missense mutations: A167V and R297C.
Fig. 3. Location of KCNJ10 mutations in patients with SeSAME syndrome. A
schematic view of the protein is shown, with intracellular N- and C-termini, 2
transmembrane helices (plasma membrane shown in shaded gray), and 1
pore. This structure is characteristic of the inward rectifier family. Locations of
mutations are indicated by black circles, and the respective amino acid change
is noted.
5844
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Page 3
neither of 2 unaf fected siblings are homozygous, providing
further support for linkage (total lod score for link age after
inclusion of the 2 unaffected siblings increases to 3.25). Rapedius
et al. (15) have suggested that T164, which is located in the
sec ond transmembrane domain, forms an intra-subunit H-bond
with lysine 67 in the first transmembrane domain, and that this
interaction is critical for gating of the channel. Because this
lysine is predicted to form an H-bond not only with the backbone
carbonyl of Thr-164, but also with its side chain oxygen, mutation
to isoleucine would eliminate this interaction and potentially
af fect the gating properties of the channel in response to pH and
phosphatidylinositol 4,5-bisphosphate (PIP
2
) (see Discussion).
Finally, in kindred 632, both affected siblings are compound
heteroz ygotes for A167V and R297C mut ations (see Fig. 2D).
A167 is located at the end of the second transmembrane domain,
close to the constriction at the inner helix bundle that likely
c orresponds to the gate of the channel (16). R297 lies in a highly
c onserved segment in the C terminus of the protein (see Fig. 3).
Not ably, a mutation at the residue corresponding to R297 has
been found in ROMK (R292W), and was implicated as a loss of
function mutation in Bartter’s syndrome type II (17). Similarly,
mut ation of the conserved position in Kir2.1 (R312) to glu-
t amine g reatly reduces whole-cell currents and produces weak-
ened interaction with PIP
2
(see Discussion) (13).
None of the identified mutations are in the dbSNP database.
Resequencing of KCNJ10 in 103 unrelated Caucasian subjects
did not identif y any of these mutations and no missense variants
at c onserved residues were identified in any of the 206 alleles
studied.
Discussion
We have defined a previously unrecogn ized human syndrome
featuring prominent neurological and renal features and have
demonstrated that in all 4 kindreds studied the disease coseg-
regates w ith rare mutations in KCNJ10. The finding of 6 inde-
pendent rare KCNJ10 mutations in 4 families that significantly
c osegregate with the disease under a recessive model and which
show specificity for the disease provides genetic evidence that
these mutations are the cause of this syndrome. The fact that
many of the amino acids altered by mutations are conserved in
other members of the inward rectifier K
channel family and
have been shown to be essential for their normal function lends
strong support for the functional significance of these mutations.
The genetic and biochemical evidence support these mutations
being a genetic loss of function.
Several of the identified mut ations are likely to affect channel
activity v ia altered interaction with PIP
2
. Numerous functional
studies in closely related inward rectifier potassium channels
have underlined the crucial role of PIP
2
to sust ain activity of
these channels (13). PIP
2
is a membrane-delimited second
messenger, and binds proteins through electrostatic interactions
at basic amino acids. These sites have been defined in many
members of this gene family, and mutations at PIP
2
binding sites
have been implicated in other channelopathies, including Bart-
ter’s syndrome, caused by ROMK mutations, and Andersen’s
syndrome, caused by mutations in KCNJ2 encoding Kir2.1. A
similar mechanism likely ac counts for loss of function in at least
2 of the mutations identified here (R65P and R297C), which lie
at inferred PIP
2
binding sites. A third residue (T164) has been
implicated in an H-bond between the 2 transmembrane helices,
which again plays an important role in the channel’s gating in
response to PIP
2
and pH (15).
Sign ificant prior work has been done on KCNJ10; it appears
to function as a heteromultimer at least in some tissues. The
currents observed in native tissues have properties most similar
to those produced by coexpression of Kir4.1 and Kir5.1 in
heterologous systems, and immunofluorescence studies support
c olocalization of these gene products (18, 19). This observation
raises the question of whether a related syndrome might be
caused by mutation in Kir5.1.
In addition, mice with both constitutional and selective astro-
c yte knockout of KCNJ10 have been produced, with a resultant
phenot ype that is strikingly similar to the patients we describe
(20–22). The animals develop motor coordination deficits with
awkward and jerky movements and loss of balance, and drag the
hind limbs. They also suffer seizures and have sensorineural
hearing loss (20, 23). Additionally, mice with the constitutional
k nockout appear to have a salt-wasting phenotype; however, this
has not been well defined. These findings strongly support the
mut ations we identify as being loss of function.
In the brain, KCNJ10 appears to be primarily expressed in glial
cells (24), specifically in astrocytes surrounding synapses and
blood vessels, and oligodendroc yte cell bodies (20). Neuronal
repolarization after excitatory stimuli is achieved via efflux of
K
, and it has been proposed that KCNJ10 plays a role in
astroc yte clearance of this K
via ‘‘spatial buf fering.’’ If the
resting membrane potential is set by KCNJ10, a local increase in
extracellular K
c oncentration close to the synapse would favor
K
upt ake by astrocy tes, and efflux at remote positions that have
lower extracellular K
c oncentrations (i.e., the rise in extracel-
lular K
would cause the local glial E
K
to be less negative than
the agg regate cellular membrane potential). Loss of KCNJ10
would thus result in astrocyte depolarization [which is seen in
astroc ytes from KCNJ10-deficient mice (20)], loss of this K
clearance function, prolonged neuronal depolarization, and re-
duced seizure threshold. Similarly, astrocy te depolarization
would reduce clearance of the excitatory neurotransmitter glu-
t amate, which would also reduce seizure threshold [reduced
glut amate uptake is also seen in astroc ytes from KCNJ10-
deficient mice (20)]. While other mechan isms (activities of the
Na
-K
-ATPase or Na-K-Cl cotransporters) are also potentially
involved in the regulation of synaptic K
(25), the observed
seizure activity in humans deficient for KCNJ10 indicates an
import ant role of this channel in prevention of seizure activity.
Finally, it is of interest that c ommon variation in the KCNJ10
gene has been suggested to be associated with seizure suscep-
tibilit y (26), however, the functional significance of the impli-
cated variants and the replicability of this finding has not been
established.
K ir4.1 is expressed in inter mediate cells of the stria vascularis
(27), where it is believed to contribute to the generation of the
endoc ochlear potential, as demonstrated by hearing loss in the
KCNJ10-k nockout mouse (23) and the patients described herein.
Both mice and humans with KCNJ10 mutations have marked
at axia and there is also lower extremity weakness in the mouse
and some affected humans. Whether the ataxia is cerebellar in
origin or sensory (due to loss of proprioception) has not been
established and is hard to assess because of cognitive impairment
of affected subjects. Intention tremor and volume loss in the
cerebellum, as seen in some cases, suggest cerebellar involve-
ment. However, peripheral sensory neuropathy might also con-
tribute to the ataxia.
KCNJ10-deficient mice exhibit striking pathology of the spinal
c ord with dysmyelination, hypomyelination, and axonal degen-
eration along with massive spongiform vacuolation. MRI dem-
onstrates marked white-matter pathology in the spinal cord and
brainstem, while cerebellum, midbrain, and c ortical regions
seem unaffected at P12. It thus appears that Kir4.1 is required
for oligodendrocyte development, and at least spinal cord my-
elination (22). The observation that sural nerve biopsy in one of
our patients showed hypomyelination suggests a possible role for
KCNJ10 in the peripheral nervous system as well, and Kir4.1 has
been shown to be expressed in satellite cells (28).
The distinct electroly te abnormalities in our patients add
c onsiderable new insight into the role of KCNJ10 in renal
electroly te homeostasis. The KCNJ10 gene product has been
Scholl et al. PNAS
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MEDICAL SCIENCES
Page 4
immunolocalized in the kidney. In contrast to the apical K
channels (e.g., KCNJ1 enc oding ROMK and KCNMA1 encoding
Maxi-K) that mediate K
secretion in the distal nephron, Kir4.1
localizes to the basolateral membranes of epithelia of the distal
c onvoluted tubule, connecting tubule, and initial c ollecting
tubule (29). Weak immunoreactivity in the thick ascending limb
of Henle has also been described (12). Our patients with KCNJ10
deficienc y display hypokalemia, metabolic alk alosis, hypomag-
nesemia and, where studied, elevated levels of renin and aldo-
sterone. The high ren in and aldosterone, along with normal
blood pressure, hypokalemia, and metabolic alkalosis strongly
point to salt wasting as an incipient event in the renal features,
and the reports of salt craving, polyuria, and enuresis are
c onsistent with this. Moreover, these patients have elevated
urinary sodium/creatinine ratios in the range seen in patients
with Gitelman syndrome (see Table 1) (30). Loss of KCNJ10
function can result in salt wasting by impairing the activity of the
Na
-K
-ATPase. The Na
-K
-ATPase is on the basolateral
membrane, and its activity is required for Na
reabsorption,
pumping Na
out of epithelia and K
in against their electro-
chemical potentials. Because very large amounts of filtered Na
must be reabsorbed by renal epithelia, the K
that enters the
epithelial cell must be recycled to the interstitium by basolateral
K
channels to allow continued Na
reabsorption. Without this
mechan ism, the Na
-K
-ATPase could be inhibited and the
potential across the basolateral membrane diminished. This
dimin ished negative intracellular potential will also attenuate
the electrical gradient for the ef flux of Cl
(Fig. 4B). The
c ombined effects will produce impaired Na-Cl reabsorption.
Because much is k nown about the consequences of inhibition
of salt reabsorption in dif ferent nephron segments (31, 32), we
can make inferences about where the effects of KCNJ10 defi-
cienc y are impairing salt reabsorption. The only site at which
inhibition of salt reabsorption produces hypomagnesemia with
reduced urinary calcium is the distal c onvoluted tubule. In
c ontrast, loss of salt reabsorption in the thick ascending limb
produces marked hypercalciuria and little hypomagnesemia (5),
while loss of ENaC activit y in the collecting duct produces
hyperk alemia and acidosis rather than hypokalemia and alka-
losis (33). We consequently believe it is highly likely that
impaired salt reabsorption in the DCT plays a prominent role in
this syndrome. Because epithelial cells of the DCT have the
greatest per-cell Na
reabsorption and energy demand (34), it is
possible that sodium pump activity in other nephron segments is
also affected, but that the effect in the DCT predominates.
These considerations suggest an integ rated model in which
loss of Kir4.1 activity impairs salt reabsorption in the distal
c onvoluted tubule (see Fig. 4). Because salt reabsorption in the
DCT comprises 7% of the filtered load, loss of salt reabsorp-
tion here induces salt wasting, which activates the renin-
angiotensin system, increasing Na
reabsorption by the ENaC in
the connecting tubule and collecting duct. This increases the
electrical driving force for both K
and H
secretion, resulting
in hypokalemia and metabolic alkalosis. As described above, loss
of salt reabsorption in the DCT is also known to produce
hypocalciuria and hypomagnesemia. While these renal electro-
ly te defects seem relatively mild, it is noteworthy that 2 siblings
with this syndrome have died in the setting of diarrheal or other
intercurrent infections, suggesting impaired ability to defend
volume homeostasis under stress.
Little is known about the genes that underlie the most
prevalent forms of epilepsy. In the last decade, gene defects have
been identified that cause rare Mendelian for ms of idiopathic
epilepsy syndromes, and most of these genes encode ion chan-
nels, consistent with their role in maintaining membrane poten-
tial and regulating neuronal excitability (11). It will be interesting
to determine the prevalence of epilepsy caused by mutations in
the KCNJ10 gene. In addition to the characteristic neurological
features (developmental delay, ataxia, and hearing impair ment),
a simple blood test might help to screen for such patients, as all
patients in this report presented with significant hypokalemia
and hypomagnesemia.
These human findings raise the possibility that Kir4.1 could be
a useful target for pharmacologic manipulation. Similar to the
recently developed anticonvulsant drug retigabine, which opens
KCNQ2/3 channels (35), a Kir4.1 activator might have anticon-
vulsant effects; nonetheless, the expression of K ir4.1 in several
tissues raises the question of whether there might be pleiotropic
ef fects that could limit utility.
In summary, our data define a unique autosomal, recessive
syndrome characterized by seizures, sensorineural deafness,
at axia, mental retardation, and electrolyte imbalance (hypokale-
mic alkalosis and hypomagnesemia), and demonstrate that it is
caused by mutations in KCNJ10. We propose the acronym
SeSAME to refer to this disorder.
Materials and Methods
Patient Recruitment and DNA Preparation. The study protocol was approved by
the Yale Human Investigation Committee. Consent for participation was
obtained in accordance with Institutional Review Board standards. Patients
were referred for studies of hypokalemic salt-losing nephropathies, and kin-
dreds were chosen for further analysis by the presence of seizures, ataxia, and
hearing impairment. Genomic DNA was prepared from venous blood of
kindred members by standard procedures.
Genotyping. The samples were genotyped on the Illumina Human CNV370-
Duo (for 441–1) and Illumina Human 610-Quad (for 404–1, 632–1, and 632–2)
beadchips at the W.M. Keck Facility at Yale University. Sample processing and
labeling were performed using the manufacturer’s instructions. Mean call rate
of the 4 samples was 99.50%.
Mapping Homozygous and IBD Intervals. Because the genotype data from
441–1 and 404–1 were originated from different arrays, the data were com-
pared to generate a list of 346,073 shared SNPs to be subjected to homozy-
gosity mapping. Analysis of homozygous segments across 22 autosomes was
performed using the ‘‘Runs of homozygosity’’ tool implemented in PLINK
(v1.05). A fixed threshold of 200 consecutive SNPs and 2 Mb in length was
selected, and 1 heterozygous SNP within a segment was allowed.
To check the IBD shared regions between the affected siblings, the geno-
type data from 632–1 and 632–2 were directly compared. Missing calls were
Fig. 4. A model of impaired ion transport in the distal convoluted tubule
caused by mutations in the Kir4.1 inwardly rectifying potassium channel. (A)
Kir4.1/5.1 heteromultimers in the basolateral membrane of the distal convo-
luted tubule (DCT) recycle potassium entering the cell via the Na
-K
-ATPase
back into the interstitial space and contribute to the negative membrane
potential that promotes basolateral chloride exit. On the luminal surface,
sodium and potassium enter the cell via the thiazide sensitive cotransporter
NCCT, and Mg
2
enters via TrpM6, using the favorable electrical gradient. (B)
Disruption of Kir4.1 function inhibits the function of the Na
-K
-ATPase via
loss of potassium recycling, reduces basolateral chloride reabsorption by
rendering the membrane potential (E
m
) less negative, and thereby inhibits
both apical Na
and Cl
reabsorption by NCCT and Mg
2
reabsorption be-
cause of a less negative membrane potential. The resulting renal salt loss
activates the renin-angiotensin-aldosterone system. Increased amounts of
Na
and Cl
are delivered to the cortical collecting duct, where aldosterone
dependent Na
reabsoption via ENaC is coupled to K
and H
secretion (see
Discussion), thus accounting for the hypokalemic alkalosis observed.
5846
www.pnas.orgcgidoi10.1073pnas.0901749106 Scholl et al.
Page 5
discarded. Fixed thresholds of 200 consecutive SNPs and 1 Mb in length were
used to call IBD segments.
DNA Sequencing. A primer pair (KCNJ10F: 5-CATGGGGTGAGGGTTAGGAG-3
and KCNJ10R: 5-GGGAGTGGAGGATGGGTG-3) was used to amplify the
coding exon of KCNJ10 using as a template genomic DNA of disease family
members or controls. PCR generated a product with a size of 1,325 bp.
Products were analyzed via gel electrophoresis, and purified amplicons were
sequenced using the KCNJ10F, KCNJ10R, KCNJ10MF (5-CGGGCTGAGAC-
CATTCGTTTC-3) and KCNJ10MR (5-AGGCTTTTGCGCATATTGGAAC-3) prim-
ers. Disease-causing mutations were confirmed by at least 2 independent
sequences from different primers. In addition, in the 2 kindreds in which
affected subjects were apparent compound heterozygotes, the coding region
was amplified and cloned using the TOPO TA Cloning Kit (Invitrogen), and
independent clones from each patient were sequenced to determine whether
the identified mutations were in cis or trans.
Orthologs and Paralogs. Full-length orthologous and paralogous protein
sequences from vertebrate and invertebrate species (including rodents, bird,
fish, and fly) were extracted from GenBank. Orthologs were confirmed based
on database identity of annotation or in a BLAST of the protein sequence
against the human protein sequence, with the requirement that human
KCNJ10 be the top hit. If an ortholog could not be identified, a paralog was
studied. Protein sequences were aligned using the ClustalW algorithm. Gen-
Bank accession numbers were: NP002232.2 (human KCNJ10), NP001034573.1
(mouse KCNJ10), XP425554.2 (chicken paralog), NP 001072312.1 (frog
KCNJ10), XP001342993.1 (zebrafish ortholog), and NP001097884.1 (fly
paralog).
ACKNOWLEDGMENTS. We thank the members of the families studied and
their physicians for their invaluable contribution to this project. We thank Dr.
Sally-Anne Hulton (The Birmingham Children’s Hospital) and the Department
of Paediatrics, Klinikum Worms, Germany for referral of patients 327–1 and
404–1, respectively; Dr. Willem Proesmans (University Hospital Gasthuisberg,
Belgium) for advice in the evaluation of patient 441–1; Dr. Gerhard Giebisch
(Yale University School of Medicine), Dr. Stephen Waxman (Department of
Neurology, Yale University School of Medicine), and members of the Lifton,
State and Gu¨ nel laboratories (Department of Genetics, Yale University School
of Medicine) for helpful discussions. This study was supported by the Leducq
Transatlantic Network in Hypertension and the Yale O’Brien Center.
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5847
MEDICAL SCIENCES
Page 6
  • Source
    • "It should be noted that many features of SeSAME are recapitulated by rodent knockout of Kir4.1, including ataxia, stress-induced seizures, impaired hearing, altered ERG, and salt-wasting [15, 21, 50, 76, 117] . However , in contrast to Kir4.1 KO animals, patients afflicted with SeSAME/EAST do not exhibit any myelopathy [101]. A new zebrafish model of EAST syndrome using antisense oligonucleotide knockdown of KCNJ10 exhibited abnormal, ataxic movements and impaired renal function and may provide a useful screening tool for drugs targeting the channel, as these animals are easily manipulated and do not suffer from premature death [63]. "
    [Show abstract] [Hide abstract] ABSTRACT: Kir4.1 is an inwardly rectifying K+ channel expressed exclusively in glial cells in the central nervous system. In glia, Kir4.1 is implicated in several functions including extracellular K+ homeostasis, maintenance of astrocyte resting membrane potential, cell volume regulation, and facilitation of glutamate uptake. Knockout of Kir4.1 in rodent models leads to severe neurological deficits, including ataxia, seizures, sensorineural deafness, and early postnatal death. Accumulating evidence indicates that Kir4.1 plays an integral role in the central nervous system, prompting many laboratories to study the potential role that Kir4.1 plays in human disease. In this article, we review the growing evidence implicating Kir4.1 in a wide array of neurological disease. Recent literature suggests Kir4.1 dysfunction facilitates neuronal hyperexcitability and may contribute to epilepsy. Genetic screens demonstrate that mutations of KCNJ10, the gene encoding Kir4.1, causes SeSAME/EAST syndrome, which is characterized by early onset seizures, compromised verbal and motor skills, profound cognitive deficits, and salt-wasting. KCNJ10 has also been linked to developmental disorders including autism. Cerebral trauma, ischemia, and inflammation are all associated with decreased astrocytic Kir4.1 current amplitude and astrocytic dysfunction. Additionally, neurodegenerative diseases such as Alzheimer disease and amyotrophic lateral sclerosis demonstrate loss of Kir4.1. This is particularly exciting in the context of Huntington disease, another neurodegenerative disorder in which restoration of Kir4.1 ameliorated motor deficits, decreased medium spiny neuron hyperexcitability, and extended survival in mouse models. Understanding the expression and regulation of Kir4.1 will be critical in determining if this channel can be exploited for therapeutic benefit.
    Full-text · Article · Mar 2016 · Acta Neuropathologica
  • Source
    • "Эти опухоли вызывают повышение внутричерепного давления, метаболическую недостаточность, токсичность и вызывают гибель клеток [325] . Нарушение метаболизма ПА и мозгового кровообращения также происходит в процессе развития синдрома Дауна и синдрома Шнайдер–Робинсона [326][327][328][329], а также синдрома EAST/SeSAME с мутациями Kir4.1-каналов [307, 309, 330] , а астроциты не могут регулировать диаметр кровеносных капиля- ров [29, 293, 331] . Все эти состояния имеют исключительно глиальное происхождение. "
    Full-text · Article · Jan 2016 · Biologicheskie membrany
  • Source
    • "Kir4.1 is the major player for counteracting the extracellular accumulation of K + , which is achieved by K + influx into glia cells ( " spatial buffering " ) (Orkand et al., 1966). Mutations in human Kir4.1 lead to sensorineural deafness (Scholl et al., 2009). Whereas most publications concerning Kir4.1 in the auditory system address the cochlea, where the channel is involved in K + circulation (Hibino and Kurachi, 2006), no publication addresses the medullary auditory brainstem. "
    [Show abstract] [Hide abstract] ABSTRACT: In the mammalian auditory brainstem, the cochlear nuclear complex (CN) and the superior olivary complex (SOC) feature structural and functional specializations for ultrafast (<1 ms) and precise information processing. Their proteome, the basis for structure and function, has been rarely analyzed so far. Here we identified and quantified the protein profiles of three major auditory brainstem regions of adult rats, the CN, the SOC, and the inferior colliculus (IC). The rest of the brain served as a reference. Via label-free quantitative mass spectrometry and 2-D DIGE/MALDI-MS, we identified 584 and 297 proteins in the plasma membrane/synaptic vesicle proteome and the cytosolic proteome, respectively. 'Region-typical' proteins, i.e., those with higher abundance in one region than in the other three, were considered candidates for functional specializations. Key proteins were validated via Western blots and immunohistochemistry. Functional annotation clustering revealed an overrepresentation of neurofilament proteins among the CN+SOC-typical proteins. These are related to regulation of axon diameter and, thereby, conduction velocity. Interestingly, the sets of synapse-associated proteins differed between regions. For example, synaptotagmin-2 (Syt2), a Ca2+ sensor for fast exocytosis, was CN+SOC+IC-typical, whereas Syt1 was CN+SOC+IC-atypical. Together, our quantitative comparison of protein profiles has revealed several interesting candidate proteins for ultrafast and precise information processing.
    Full-text · Article · Jul 2015 · Molecular and Cellular Neuroscience
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