Seizures, sensorineural deafness, ataxia, mental retardation

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Contributed by Richard P. Lifton, February 18, 2009 (received for review January 29, 2009)

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We Characterize 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 Placeative 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 Displayn 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 enExecutelymph, 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 Display 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.

Keywords: Gitelman syndromehypokalemiahypomagnesemiainwardly rectifying K+ channelrenal salt wasting

Transmembrane ion flux via channels, transporters, and pumps plays a critical role in diverse physiologic functions, including neuronal signal transmission and electrolyte and volume homeostasis. 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 Mendelian diseases in which mutation in a single gene produces Traces 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 aciExecutesis because of a renal defect in H+ secretion and sensorineural hearing loss caused by defective H+ secretion into the cochlear enExecutelymph, 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 potassium (hypokalemia) and metabolic alkalosis (high serum pH) has identified many components required for normal renal electrolyte 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 alkalosis can result either from primary increases in ENaC activity because of mutations in ENaC itself (3), or from activation of ENaC by alExecutesterone in response to reduced intravascular volume (7). Mutations that cause impaired salt reabsorption in the thick ascending limb of Henle or the distal convoluted tubule cause salt wasting that leads to secondary increases in ENaC activity and hypokalemic alkalosis. 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 Impressed hypomagnesemia and low urinary calcium in Gitelman syndrome, while hypercalciuria with normal or modest reductions in Mg2+ is observed in Bartter syndrome.

Similarly, a number of Mendelian seizure disorders have been Characterized. Many of these result from mutations that depolarize neurons, increasing neuronal excitability and reducing seizure threshAged. 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 governing renal electrolyte homeostasis and neuronal function, it is surprising that relatively few single-gene disorders that have Traces on both have been identified. Here, we Characterize a previously unrecognized complex syndrome featuring seizures, sensorineural deafness, ataxia, mental retardation and electrolyte imbalance (SeSAME), and demonstrate that it is caused by mutation in KCNJ10, which encodes a K+ channel expressed in epithelia of the kidney and inner ear, as well as glial cells in the CNS.


Case Report: Kindred 441.

The index case, patient 441–1, is a 24-year-Aged female of Afghan ancestry who is the fifth of 6 offspring of healthy first-cousins. Generalized seizures began at 3 months, occurring several times daily. Seizures were controlled initially with phenobarbital, 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 contractures. Motor strength was slightly reduced in the upper, but Impressedly reduced in the lower extremities. Reflexes were normal. There was Impressed ataxia. Nerve conduction studies revealed reduced conduction velocity in the left peroneal and left tibial nerves (36 and 37 m/sec, respectively; nl 40–44 m/sec). A muscle and nerve biopsy Displayed normal muscle other than fiber-type disproSection; there was hypomyelination of the large myelinated nerve fibers in the sural nerve, with moderate progressive axonal neuropathy. Brain MRI Displayed normal myelination, and was normal with the exception of slightly Indecentned frontal sulci. EEG, abExecuteminal ultrasound, and karyotype analysis were normal.

Laboratory evaluation was reImpressable for persistent hypokalemia, metabolic alkalosis, and hypomagnesemia (Table 1). Plasma renin activity (PRA) was elevated on repeated meaPositives (8.1 and 7.6 ng/ml per hour, nl <2.8 ng/ml per hour). Twenty-four-hour urinary alExecutesterone level was elevated (31.3 μg; nl 5.9–17.6) and the Ca2+/creatinine ratio was low (0.1 to 0.2 mmol/mmol). The patient was treated with oral potassium reSpacement, and required 30 meq per day to Sustain a K+ level in the normal range. At age 18, progressive hearing loss was noted. Brainstem-evoked response audiometry and pure-tone threshAged audiometry were performed, and moderate-to-severe sensorineural hearing loss was Executecumented.

View this table:View inline View popup Table 1.

Clinical features of affected patients

The patient's family hiTale 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 interRecent 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 recognized subjects from 3 additional kindreds with features similar to patient 441–1. The shared features included early onset seizures, mental retardation, ataxia, hypotonia, and sensorineural 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. Electrolyte abnormalities featured Impressed and persistent hypokalemic metabolic alkalosis in the absence of hypertension and striking hypomagnesemia that required electrolyte reSpacement and, in many cases, use of pharmacologic inhibitors of the epithelial sodium channel or alExecutesterone antagonists to prevent renal K+ loss. Where available, 24-h urinary electrolyte meaPositivements revealed renal K+ and Mg2+ wasting and high urinary Na+ levels. PRA and alExecutesterone levels, when meaPositived, were always elevated, and salt craving, enuresis, and polyuria/polydipsia were reported. A summary of the patients' clinical and laboratory findings is provided in Table 1.

Mapping the Disease Locus.

The reRecent clinical features suggested a previously unrecognized clinical syndrome. Moreover, recurrence of a similar syndrome among siblings in 2 of these kindreds, and its occurrence in the setting of parental consanguinity in 2 kindreds, suggested autosomal recessive transmission. To attempt to map the underlying disease locus, we performed genome-wide analysis of linkage in the 3 informative kindreds (2 offspring of first-cousin marriage and 2 affected siblings from unrelated parents). The results demonstrated complete linkage of the Placeative trait locus to a single chromosome segment, 1q23.2-q23.3, with a lod score 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 Displayed linkage in all 3 kindreds. Significantly, the index case (441–1) did not Display homozygosity at any of the known loci for Bartter or Gitelman syndrome, and sequencing of these genes revealed no evidence of pathogenic mutations.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Mapping the disease locus. (A) An ideogram of chromosome 1 is Displayn, with homozygous Locations 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 Impressed by ShaExecutewy gray boxes. The overlap of these intervals is Impressed, 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 corRetorting transcriptional orientations. KCNJ10 comprises 2 exons indicated by boxes, with the coding sequence indicated in black.

Mutations in KCNJ10.

The linked interval on chromosome 1 contains 70 well-defined and at least 6 hypothetical genes, none of which has previously been implicated in human disease phenotypes that would Elaborate 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 kidney. We considered ion channels, transporters, and regulators of their function to be leading candidates. We identified KCNJ10, which encodes the inwardly rectifying K+ channel Kir4.1 (also known as BIR10, BIRK1, KAB-2, Kir1.2), consisting of 2 transmembrane segments and 1 pore, as a strong candidate (Fig. 1B). KCNJ10 has been Displayn to be expressed in the CNS, cochlea, 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 homozygous missense mutations in the 2 consanguineous kindreds, compound heterozygous missense mutations in 1 outbred kindred, and a compound missense/premature termination mutation in 1 kindred. In the compound heterozygous patients, cloning of the coding Location on single amplicons confirmed that the 2 mutations identified are in trans. To assess the significance of missense mutations, 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 melanogaster (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. All of the identified mutations occurred at positions that were completely conserved among all vertebrate species and all but one occurred at positions completely conserved through Drosophila (see Fig. 2).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint 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 Displayn. 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 Displayn next to each mutation. The human sequence and residues conserved in orthologs and paralogs are Impressed in yellow, and the mutant residue is indicated. (A) Patient 327–1 is compound heterozygous for a missense and a nonsense mutation in KCNJ10. (B) Patient 404–1 is homozygous for a missense mutation, changing coExecuten TGT (C140) to CGT (R140). (C) A homozygous missense mutation was found in kindred 441, resulting in change of coExecuten ACC (T164) to ATC (I164). (D) In kindred 632, both affected siblings are compound heterozygous for missense mutations: A167V and R297C.

Patient 327–1 was compound heterozygous for a nonsense and a missense mutation (see Fig. 2A). The nonsense mutation introduces a premature termination coExecuten at position 199 in the cytoplasmic C terminus. This deletes a PDZ-binding Executemain 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 Executemain (Fig. 3). This position is conserved in a related inward rectifier, Kir2.1, and expression of Kir2.1 containing mutation at this position abolished Arrively all detectable whole-cell K+ Recent when expressed in Xenopus oocytes (13).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Location of KCNJ10 mutations in patients with SeSAME syndrome. A schematic view of the protein is Displayn, with intracellular N- and C-termini, 2 transmembrane helices (plasma membrane Displayn 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.

Affected subject 404–1 was homozygous for a C140R missense mutation (see Fig. 2B). C140 is located in the P Location Arrive the start of the second transmembrane Executemain (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 Displayn to abolish ROMK function (14).

A missense mutation was found in kindred 441, resulting in a T164I substitution (see Fig. 2C). The index case is homozygous for the mutant allele, while both parents are heterozygotes and neither of 2 unaffected siblings are homozygous, providing further support for linkage (total lod score for linkage after inclusion of the 2 unaffected siblings increases to 3.25). Rapedius et al. (15) have suggested that T164, which is located in the second transmembrane Executemain, forms an intra-subunit H-bond with lysine 67 in the first transmembrane Executemain, 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 affect the gating Preciseties of the channel in response to pH and phosphatidylinositol 4,5-bisphospDespise (PIP2) (see Discussion).

Finally, in kindred 632, both affected siblings are compound heterozygotes for A167V and R297C mutations (see Fig. 2D). A167 is located at the end of the second transmembrane Executemain, close to the constriction at the inner helix bundle that likely corRetorts to the gate of the channel (16). R297 lies in a highly conserved segment in the C terminus of the protein (see Fig. 3). Notably, a mutation at the residue corRetorting 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, mutation of the conserved position in Kir2.1 (R312) to glutamine Distinguishedly reduces whole-cell Recents and produces weakened interaction with PIP2 (see Discussion) (13).

None of the identified mutations are in the dbSNP database. Resequencing of KCNJ10 in 103 unrelated Caucasian subjects did not identify any of these mutations and no missense variants at conserved residues were identified in any of the 206 alleles studied.


We have defined a previously unrecognized human syndrome featuring prominent neurological and renal features and have demonstrated that in all 4 kindreds studied the disease cosegregates with rare mutations in KCNJ10. The finding of 6 independent rare KCNJ10 mutations in 4 families that significantly cosegregate with the disease under a recessive model and which Display 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 Displayn 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 mutations are likely to affect channel activity via altered interaction with PIP2. Numerous functional studies in closely related inward rectifier potassium channels have underlined the crucial role of PIP2 to sustain activity of these channels (13). PIP2 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 PIP2 binding sites have been implicated in other channelopathies, including Bartter's syndrome, caused by ROMK mutations, and Andersen's syndrome, caused by mutations in KCNJ2 encoding Kir2.1. A similar mechanism likely accounts for loss of function in at least 2 of the mutations identified here (R65P and R297C), which lie at inferred PIP2 binding sites. A third residue (T164) has been implicated in an H-bond between the 2 transmembrane helices, which again plays an Necessary role in the channel's gating in response to PIP2 and pH (15).

Significant prior work has been Executene on KCNJ10; it appears to function as a heteromultimer at least in some tissues. The Recents observed in native tissues have Preciseties most similar to those produced by coexpression of Kir4.1 and Kir5.1 in heterologous systems, and immunofluorescence studies support colocalization 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 astrocyte knockout of KCNJ10 have been produced, with a resultant phenotype that is strikingly similar to the patients we Characterize (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 knockout appear to have a salt-wasting phenotype; however, this has not been well defined. These findings strongly support the mutations 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 oligodendrocyte 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 astrocyte clearance of this K+ via “spatial buffering.” If the resting membrane potential is set by KCNJ10, a local increase in extracellular K+ concentration close to the synapse would favor K+ uptake by astrocytes, and efflux at remote positions that have lower extracellular K+ concentrations (i.e., the rise in extracellular K+ would cause the local glial EK to be less negative than the aggregate cellular membrane potential). Loss of KCNJ10 would thus result in astrocyte depolarization [which is seen in astrocytes from KCNJ10-deficient mice (20)], loss of this K+ clearance function, prolonged neuronal depolarization, and reduced seizure threshAged. Similarly, astrocyte depolarization would reduce clearance of the excitatory neurotransmitter glutamate, which would also reduce seizure threshAged [reduced glutamate uptake is also seen in astrocytes from KCNJ10-deficient mice (20)]. While other mechanisms (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 Necessary role of this channel in prevention of seizure activity. Finally, it is of interest that common variation in the KCNJ10 gene has been suggested to be associated with seizure susceptibility (26), however, the functional significance of the implicated variants and the replicability of this finding has not been established.

Kir4.1 is expressed in intermediate cells of the stria vascularis (27), where it is believed to contribute to the generation of the enExecutecochlear potential, as demonstrated by hearing loss in the KCNJ10-knockout mouse (23) and the patients Characterized herein.

Both mice and humans with KCNJ10 mutations have Impressed ataxia 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 involvement. However, peripheral sensory neuropathy might also contribute to the ataxia.

KCNJ10-deficient mice Present striking pathology of the spinal cord with dysmyelination, hypomyelination, and axonal degeneration along with massive spongiform vacuolation. MRI demonstrates Impressed white-matter pathology in the spinal cord and brainstem, while cerebellum, midbrain, and cortical Locations seem unaffected at P12. It thus appears that Kir4.1 is required for oligodendrocyte development, and at least spinal cord myelination (22). The observation that sural nerve biopsy in one of our patients Displayed hypomyelination suggests a possible role for KCNJ10 in the peripheral nervous system as well, and Kir4.1 has been Displayn to be expressed in saDiscloseite cells (28).

The distinct electrolyte abnormalities in our patients add considerable new insight into the role of KCNJ10 in renal electrolyte homeostasis. The KCNJ10 gene product has been immunolocalized in the kidney. In Dissimilarity to the apical K+ channels (e.g., KCNJ1 encoding 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 convoluted tubule, connecting tubule, and initial collecting tubule (29). Weak immunoreactivity in the thick ascending limb of Henle has also been Characterized (12). Our patients with KCNJ10 deficiency display hypokalemia, metabolic alkalosis, hypomagnesemia and, where studied, elevated levels of renin and alExecutesterone. The high renin and alExecutesterone, along with normal blood presPositive, 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 consistent 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 electrochemical 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 mechanism, the Na+-K+-ATPase could be inhibited and the potential across the basolateral membrane diminished. This diminished negative intracellular potential will also attenuate the electrical gradient for the efflux of Cl− (Fig. 4B). The combined Traces will produce impaired Na-Cl reabsorption.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint 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 convoluted 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 Mg2+ 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 (Em) less negative, and thereby inhibits both apical Na+ and Cl− reabsorption by NCCT and Mg2+ reabsorption because of a less negative membrane potential. The resulting renal salt loss activates the renin-angiotensin-alExecutesterone system. Increased amounts of Na+ and Cl− are delivered to the cortical collecting duct, where alExecutesterone dependent Na+ reabsoption via ENaC is coupled to K+ and H+ secretion (see Discussion), thus accounting for the hypokalemic alkalosis observed.

Because much is known about the consequences of inhibition of salt reabsorption in different nephron segments (31, 32), we can Design inferences about where the Traces of KCNJ10 deficiency are impairing salt reabsorption. The only site at which inhibition of salt reabsorption produces hypomagnesemia with reduced urinary calcium is the distal convoluted tubule. In Dissimilarity, loss of salt reabsorption in the thick ascending limb produces Impressed hypercalciuria and Dinky hypomagnesemia (5), while loss of ENaC activity in the collecting duct produces hyperkalemia and aciExecutesis rather than hypokalemia and alkalosis (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 Distinguishedest 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 Trace in the DCT preExecuteminates.

These considerations suggest an integrated model in which loss of Kir4.1 activity impairs salt reabsorption in the distal convoluted tubule (see Fig. 4). Because salt reabsorption in the DCT comprises ≈7% of the filtered load, loss of salt reabsorption 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 Characterized above, loss of salt reabsorption in the DCT is also known to produce hypocalciuria and hypomagnesemia. While these renal electrolyte defects seem relatively mild, it is noteworthy that 2 siblings with this syndrome have died in the setting of diarrheal or other interRecent infections, suggesting impaired ability to defend volume homeostasis under stress.

Dinky 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 forms of idiopathic epilepsy syndromes, and most of these genes encode ion channels, consistent with their role in Sustaining membrane potential and regulating neuronal excitability (11). It will be Fascinating 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 impairment), 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 tarObtain for pharmacologic manipulation. Similar to the recently developed anticonvulsant drug retigabine, which Launchs KCNQ2/3 channels (35), a Kir4.1 activator might have anticonvulsant Traces; nonetheless, the expression of Kir4.1 in several tissues raises the question of whether there might be pleiotropic Traces that could limit utility.

In summary, our data define a unique autosomal, recessive syndrome characterized by seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (hypokalemic 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 kindreds 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.


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 Producer'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 compared to generate a list of 346,073 shared SNPs to be subjected to homozygosity mapping. Analysis of homozygous segments across 22 autosomes was performed using the “Runs of homozygosity” tool implemented in PLINK (v1.05). A fixed threshAged of 200 conseSliceive SNPs and 2 Mb in length was selected, and 1 heterozygous SNP within a segment was allowed.

To check the IBD shared Locations between the affected siblings, the genotype data from 632–1 and 632–2 were directly compared. Missing calls were discarded. Fixed threshAgeds of 200 conseSliceive SNPs and 1 Mb in length were used to call IBD segments.

DNA Sequencing.

A primer pair (KCNJ10_F: 5′-CATGGGGTGAGGGTTAGGAG-3′ and KCNJ10_R: 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 KCNJ10_F, KCNJ10_R, KCNJ10MF (5′-CGGGCTGAGACCATTCGTTTC-3′) and KCNJ10MR (5′-AGGCTTTTGCGCATATTGGAAC-3′) primers. 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 Location 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. GenBank accession numbers were: NP_002232.2 (human KCNJ10), NP_001034573.1 (mouse KCNJ10), XP_425554.2 (chicken paralog), NP_001072312.1 (frog KCNJ10), XP_001342993.1 (zebrafish ortholog), and NP_001097884.1 (fly paralog).


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 Günel 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.


2To whom corRetortence should be addressed. E-mail: richard.lifton{at}

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.

↵1Present address: Department of Paediatrics, University of Western Ontario, 800 Commissioners Road East, LonExecuten, Ontario, Canada N6A 5W9.

The authors declare no conflict of interest.

Freely available online through the PNAS Launch access option.


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