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1 Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS, Sophia Antipolis, France
2 Departments of Physiology
3 Biochemistry
8 INSERM UMR 546, Pitié-Salpêtrière Hospital, Paris, France
4 Department of Histology, Henri Mondor Hospital, Créteil, France
5 Explorations Fonctionnelles Neurologiques, Saint Brieuc Hospital, Saint Brieuc, France
6 Department of Neurology, Saint Joseph Hospital, Paris, France
7 Department of Neurology, Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT, USA
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Abstract |
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(Received 17 December 2004;
accepted after revision 13 April 2005;
first published online 14 April 2005)
Corresponding author S. Bendahhou: Université de Nice Sophia Antipolis, UMR 6097 CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, 660 Route des Lucioles, Sophia-Antipolis, 06560 Valbonne, France. Email: bendahhou{at}ipmc.cnrs.fr
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Introduction |
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Dysfunctions of Kir channels are linked to human diseases such as persistent hyperinsulinaemic hypoglycaemia of infancy (Thomas et al. 1995; Thomas et al. 1996), Bartter's syndrome (Simon et al. 1996), and Andersen's syndrome (AS) (Plaster et al. 2001). AS has been linked to the KCNJ2 gene that encodes for the Kir2.1 protein. Additional mutations on the same gene have been also associated with AS (Ai et al. 2002; Andelfinger et al. 2002; Tristani-Firouzi et al. 2002; Donaldson et al. 2003; Hosaka et al. 2003). Patients with AS mutations exhibit periodic paralysis (hypokalaemic, hyperkalaemic or normokalaemic), and cardiac manifestations (long QT, ventricular arrhythmia, bigeminy, torsade de pointes). It is interesting to note that AS mutations were found in patients with many dysmorphic features as has been noticed in the first report by Andersen et al. in 1971 (Andersen et al. 1971). These include low set ears, broad-base nose, hypertelorism, syndactyly, micrognathia, short stature, scoliosis and cleft palate. Functional analysis of AS mutations confirmed the role of Kir2.1 in skeletal muscle and cardiac tissues but did not elucidate its contribution to bone structuring. Many AS families do not carry any mutation in their KCNJ2 gene, suggesting that the disease must be genetically heterogeneous or other factors, such as partner or regulatory proteins, may alter Kir2.1 function.
In the present study, we report three families with the full clinical triad of AS phenotypes. Genetic screening identified an already reported mutation and two novel mutations in the KCNJ2 gene. In addition to clinical and electrophysiological studies on two of the probands, we carried out in vitro functional analysis of the two novel mutations in a mammalian cell background. To better understand the mechanism underlying AS, we further analysed the expression pattern of our two novel mutations as well as that of already described AS mutations in a muscle cell line.
Methods
Patients. Clinical studies were conducted after patients signed a consent form approved by the French and European Union bioethics law, and conformed with the Declaration of Helsinki. Patients were examined by one of the authors (G.B., A.F. or C.S.). Exams included ECG, EMG, CT-scan and clinical electrophysiology.
Electromyographic evaluations were performed using a protocol recently described (Fournier et al. 2004). Briefly, compound muscle action potentials (CMAPs) were recorded at the right and left abductor digiti minimi (ADM) muscles following supramaximal stimulation of the appropriate ulnar nerve at the wrist. A bandage around the hand prevented articulation displacements and changes in muscle volume during the exercise tests. Two to five supramaximal CMAPs were recorded at rest to ensure a stable baseline response. Two kinds of exercises were performed: (1) short exercise of the left ADM muscle lasting 10 s, with recording of the CMAPs immediately after exercise cessation and then every 10 s for 1 min; (2) long exercise of the right ADM muscle lasting 5 min, with brief (3–4 s) resting periods every 30–45 s to prevent ischaemia. CMAPs were recorded immediately after the 5 min exercise and every minute for 5 min, then every 5 min for 40 min. CMAP amplitude, duration and area were expressed as a percentage of the reference values measured before exercise. Changes in CMAP amplitude were compared with those observed in 30 healthy individuals. Myotonic discharges were also searched by needle EMG in the deltoid, extensor digitorum communis, first interosseus, vastus medialis and tibialis anterior muscles.
Molecular diagnosis and mutagenesis
DNA from patients was extracted from peripheral blood leucocytes and used as a template for subcloning. Whole KCNJ2 open reading frame was PCR-amplified using Pfu Taq DNA polymerase (Promega, USA). PCR product was cloned into the pGEM-T vector (Promega, USA) and the clones were sequenced. A mutation-containing clone was PCR-amplified with primers flanked with restriction enzyme sequences: BamHI and Not I for cloning into pXOOM vector, or EcoRI and SacII for cloning into pEGFP vector. The latter allowed fusion of the enhanced green fluorescent protein (EGFP) to the C-terminus of the Kir2.1 WT or mutant protein. The Kir2.1 WT protein (WT human cardiac KCNJ2 was obtained in the plasmid pBluescript KS(–) as a gift from Dr Vandenberg University of California, CA) was also fused to DsRed fluorescent protein using the same restriction enzymes. The mutated fusion proteins were PCR-amplified using primers flanked with the Not I sequence for subsequent cloning into the pBud vector (Invitrogen) under EF-1
promoter. Kir2.1-DsRed fusion was amplified with primers flanked with the Sal I site and subcloned into pBud vector under cytomegalovirus (CMV) promoter. Plasmid-containing mutant DNA was cut with Sal I restriction enzyme then Kir2.1-DsRed fusion was ligated into this site to generate a plasmid allowing simultaneous expression of WT and mutant protein. All PCR products and final constructs were sequenced to ensure fidelity.
Cell culture and in vitro electrophysiology
COS-7 cells, human embryonic kidney (HEK) 293 cells, and the C2C12 mouse cell line were maintained in Dulbecco's modified Eagle media supplemented with 10% fetal bovine serum and 100 U ml–1 streptomycin, 100 U ml–1 penicillin, at 37°C in a humidified 5% CO2 atmosphere. COS-7 cells were transiently transfected by the DEAE–dextran precipitate method using 1.5 µg Kir2.1WT or mutant DNA per 35-mm culture dish. Currents were recorded 48 h after transfection.
Recordings were conducted in the whole-cell configuration (Hamill et al. 1981) at room temperature (
22°C), using an EPC 10 amplifier (HEKA Electronic, Germany). The pipette solution contained (mM): 150 KCl, 0.5 MgCl2, 5 EGTA and 10 Hepes, pH 7.3. The bathing media was (mM): 150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2 and 10 Hepes, pH 7.3. Pipette resistance was 1.5–4 M
. Membrane currents were elicited by depolarizations ranging from –100 to +50 mV, from a holding potential of –80 mV. Only cells with series resistance less than 5 M were used for analysis. Data acquisition and analysis were performed using Pulse and Pulsefit (HEKA Electronic, Germany), and IgorPro (WaveMetrics Inc., OR, USA) software.
Confocal microscopy imaging
HEK293 cells were transiently transfected with DNA encoding for EGFP, DsRed, Kir2.1-EGFP, Kir2.1-DsRed, Kir2.1C154F-EGFP, Kir2.1T309I-EGFP, Kir2.1-DsRed/Kir2.1-EGFP, Kir2.1-DsRed/Kir2.1C154F-EGFP, and Kir2.1-DsRed/Kir2.1T309I-EGFP. HEK293 cells were cultured on a cover slip and transiently transfected with 1 µg of each DNA per 35-mm culture dish using the calcium phosphate precipitation technique (Graham & Van Der Eb, 1973). Mouse muscle C2C12 cells were cultured on a cover slip and transfected with a mixture of 2 µl Fugene 6 (Roche Diagnostics, USA) and 2 µg DNA of each clone, according to the manufacturer's procedure. Confocal microscopy (Leica) images were taken 24–48 h later with 100 x magnification lens.
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Results |
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In family A, a 27-year-old man (Fig. 1, patient A6), with no noticeable medical history, nor family history of muscular or cardiac disorders, presented with periodic paralysis. His first episode of muscle weakness occurred at the age of 14 following a long distance walk with spontaneous recovery. A second episode of regressive quadriplegia of unknown origin, as well as recurrent other episodes of muscle weakness of variable severity occurred once a year. One episode involved upper limbs with a prominent asymmetry. At the age of 25, investigations in our centre showed a small size (height 1.58 m), mild facial dysmorphism limited to a high broad, prominent forehead, and bone malformation, including abnormal radial curvature of the fifth digits and non-deviated short toes. Musculature was slightly hypertrophic with a permanent mild proximal muscle weakness of both lower limbs, and no clinical myotonia (no lid-lag). Facial, pharyngeal and respiratory muscle were not affected. Deep tendon reflexes (DTR) were weak but no other neurological abnormality was observed, kalaemia was normal at 4.3 mM at 12 h and 3.5 mM at 17 h, with no other biological abnormalities except for creatine kinase (CK) elevation at 689 IU l–1 (normal < 190 IU l–1). Thyroid function and serum lactate were normal. ECG, cardiac echography and spirometry showed no abnormality: QT interval was normal. However, a 24 h ECG monitoring revealed increased number of premature beat but no arrhythmic episode. During the recovery phase of an attack, kalaemia was low at 3.8 mM and rose to 5.5 mM 24 h later, strongly suggesting a hypokalaemic periodic paralysis. Bone radiographs revealed bilateral clinodactyly of the fifth fingers, phalangeal synostosis of the middle and distal phalanx of the fifth toes, no scoliosis, and no micrognathia. Deltoid muscle biopsy was normal (in particular no sarcoplasmic vacuolation was observed). The patient was not under acetazolamide medication.
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Clinical examination noticed dysmorphic features: short stature (1.56 m), low-set ears, broad forehead, and short philtrum. The patient had some cognitive impairment with learning difficulties at school. He suffered from palpitations. Cardiac examinations disclosed ventricular bigeminy, right bundle branch block, and short runs of ventricular tachycardia.
A proximal muscle weakness of the four limbs gradually appeared, that predominated in lower limbs including mild distal muscles involvement. Facial, respiratory and neck muscles were spared. CK level was as high as 1270 IU l–1 (normal is 0–200 IU l–1). Routine biological tests were normal. Serum K+ level was tested several times during attacks and remained in the normal range, but oral potassium ingestion triggered attack of paralysis.
In family C, the proband (Fig. 1, patient C3) is a 24-year-old woman. She was admitted at the hospital for the first time at age of 12. She has the particularity to be born with hip subluxation. Her father was known to suffer from hypokalaemic periodic paralysis attacks. The patient experienced her first attacks at age of 9: she described muscle discomfort associated with cramps and weakness, especially at rest after exercise. ECG showed ventricular extra-systolis with bigeminy. Cardiac examination showed ventricular tachycardia (150 min–1). The EMG, after an effort, was normal. CK level was slightly elevated at 127 IU l–1.
Electromyography
Electromyographic evaluations were performed in both A6 and B5 patients carrying mutations C154F and T309I, respectively (Fig. 1). No significant change of CMAPs was observed after a short exercise (Fig. 2A–C). Immediately after a long exercise, CMAPs exhibited an increase in duration in AS patients as well as in unaffected controls (Fig. 2D and E). This prolongation of the CMAPs may be explained by the well-known accumulation of K+ ions in the vicinity of T-tubules during sustained muscle activity, producing a sustained membrane depolarization that delays the normal repolarization process (Fournier et al. 2004). In AS patients, there was no significant change of CMAP amplitude during this first phase after long exercise (Fig. 2D–F). An abnormal decrease in CMAP amplitude appeared 5–10 min after exercise cessation, reaching its lowest point at 30 min (–65 and –56% in A6 and B5 patients, respectively). Duration and area of the CMAPs decreased over the same time, without any change of CMAP shape. Finally, needle EMG showed no myotonic discharges.
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AS has been linked to the KCNJ2 gene (Plaster et al. 2001). To date, about 20 missense mutations and 2 deletions have been described as causing the disease (Plaster et al. 2001; Andelfinger et al. 2002; Tristani-Firouzi et al. 2002; Donaldson et al. 2003; Hosaka et al. 2003). Since all AS mutations described to date have been identified in the same gene, we used KCNJ2 as a gene candidate for our screenings. All patients were also tested for mutations in channels that have been involved in periodic paralysis: CACNL1A3, SCN4A (exons 13, 22 and 24). No mutations were detected in these genes. Patient DNA was PCR-amplified with primer for KCNJ2 and directly sequenced. Sequence analysis revealed three distinct heterozygous mutations: G689T, G1127A and C1154T leading to amino acid changes C154F (patient A6), G300D (patient C3) and T309I (patient B5), respectively. C154F and T309I are novel mutations. However, mutation G300D has been reported earlier (Donaldson et al. 2003). Cysteine at position 154 is well conserved among all Kir family (Fig. 1D); however, threonine 309 is conserved only within the Kir2 and Kir6.1 subfamilies (Fig. 1D).
Loss-of-function mutants
Since G300D mutation has already been reported and studied, we focused this study on the two novel mutations C154F and T309I. KCNJ2 and mutant constructs were first cloned into the pXOOM vector and expressed under a CMV-driven promoter in COS-7 cells. EGFP was used in the pXOOM vector as a marker of cell transfection, not as a fusion protein. Only cells with green fluorescence were chosen for recordings. Kir2.1 WT showed a classical inwardly rectifying current (Fig. 3A). However, both of the mutants showed a complete lack of such a current (Fig. 3A).
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expressing promoter compared with the CMV promoter rather than to the type of fused protein. Similar to mutants expressed alone in the pXOOM vector, neither fused mutant proteins gave rise to any K+ current in COS-7 cells, confirming that both C154F and T309I mutations induced a loss of function of the channels.
AS mutations have been shown to cause the disease through a dominant-negative effect (Plaster et al. 2001; Ai et al. 2002; Andelfinger et al. 2002; Tristani-Firouzi et al. 2002; Bendahhou et al. 2003; Hosaka et al. 2003; Lange et al. 2003) or through a haplo-insufficiency mechanism (Bendahhou et al. 2003). In order to investigate the mechanism underlying the disease in these two families, we made up a construct in which Kir2.1-DsRed is expressed under the CMV promoter, and C154F-EGFP or T309I-EGFP under the EF-1 promoter in the same pBud vector. As shown in Fig. 3B, none of the constructs produced current, strongly suggesting that C154F and T309I acted in a dominant-negative fashion to abolish the inward rectifying K+ current. Random assembly of two equally expressed subunits into a tetramer would suggest that 1/16 of the tetrameric channels would be entirely WT, yet the currents appear to be smaller. This is probably due to differences in expression of EF-1 and CMV promoters.
Assembly and trafficking of AS-associated mutations
Confocal microscopy imaging was performed to monitor channel distribution in HEK293 cells on all DsRed- and EGFP-fused proteins. HEK293 cells (round shape) were preferred to COS-7 cells (flat) because they allow the cytoplasmic and membrane environments of the cell to be distinguished. Both single and double constructs were used. Figure 4A shows that all Kir constructs have a membrane distribution unlike DsRed and EGFP, which have a diffuse cytoplasmic localization. This indicates that C154F and T309I mutations do not affect signal trafficking to the membrane.
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AS mutants in a muscle cell line
Periodic paralysis is one feature of AS patients that has been reported in two-thirds of patients carrying a KCNJ2 mutation (Tristani-Firouzi et al. 2002). In order to monitor channel behaviour in a muscle cell line, both WT and mutant clones were introduced into C2C12, a mouse muscle cell line. Channel localization and distribution were monitored under a confocal microscope, taking advantage of the EGFP-tagged constructs. As shown in Fig. 4B, both WT and mutants (C154F and T309I) traffick to plasma membrane confirming our observations in HEK293 cells, and suggesting that both mutants act in a dominant-negative fashion to abolish Kir2.1 currents.
We have previously reported expression of many other AS mutants in either Xenopus oocytes or in HEK293 cells (Plaster et al. 2001; Tristani-Firouzi et al. 2002; Bendahhou et al. 2003). In order to monitor the behaviour of these mutants in a muscle cell line, D71V, 
95–98, G144S, R218Q, G300V, V302M and S314–Y315 were introduced into C2C12 cells. Figure 5 shows that all mutants have a behaviour similar to that seen in HEK293 cells. Mutations D71V, G144S, R218Q and G300V traffick properly to the plasma membrane. Deletions 95–98 and 314–315 showed a scattered cytoplasmic pattern reminiscent of degraded proteins or channel mistrafficking. However, we were not able to detect any fluorescence signal with substitution V302M after 24 h or even 48 h post-transfection, suggesting that these channels may have not been synthesized or may have been immediately degraded.
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Discussion |
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The new mutations introduced C154F and T309I amino acid changes, and are localized close to the pore region and to the C-terminal domain of the Kir2.1 channel protein, respectively. Cysteine 154 is a well-conserved residue among the inward rectifier K+ channel family, suggesting an important role in channel function. It resides 10 residues away from G144 that belongs to the K+ channel signature sequence GYG. A mutation of this residue has already been reported (G144S) to associate with AS. Residue T309 is only conserved within the Kir2 and Kir6.1 subfamilies, however.
We have introduced and monitored channel function, distribution and assembly of these two mutations in three cell lines. We have also validated already reported AS mutations in a murine C2C12 muscle cell line. Finally, in vivo functional data were reported for two patients.
Functional expression of the mutant channels has been carried out using the human cardiac inward rectifier Kir2.1 DNA alone in pXOOM expressing vector, or DNA fused to EGFP for mutants or to DsRed for WT in pBud vector. When expressed alone, only WT Kir2.1 construct gave rise to an inward rectifying current suggesting that the disorder in the two families is due to a loss of channel function. When the channels were fused to DsRed or EGFP, WT construct showed current properties that were indistinguishable from those of WT currents expressed without any tag. This finding demonstrates that neither EGFP nor DsRed affected Kir2.1 gating, assembly or trafficking. As seen with the constructs alone, the EGFP fused mutant proteins showed a loss of function. Kir2.1 channels form tetramers. To determine how mutant subunits affect the WT subunit in a whole channel complex, we carried out patch clamp experiments using the pBud vector, in which fused protein WT-DsRed was cloned under the control of the CMV promoter, and C154F-EGFP or T309I-EGFP were cloned in the same vector under the EF-1 promoter. We were unable to detect any substantial current under these conditions. Our data suggest that both of the mutations act in a dominant-negative fashion to abolish whole Kir2.1 current in mutation carriers in the two families.
AS is an autosomal dominant disorder. It has been reported that the majority of the mutations caused the disease through the same mechanism except one (Bendahhou et al. 2003). The V302M substitution was found in an AS family and was suggested to cause the disorder through a haplo-insufficiency mechanism.
We have also shown that AS mutations can assemble and traffick to the plasma membrane without producing any current. Deletion S314-Y315 that was associated with AS produced channels that did not traffick to the membrane. They co-assembled with WT channels and trapped them in the cytoplasm to abolish the K+ current in the membrane. In the present study, we used the same approach for C154F and T309I substitutions. Confocal microscopy images showed that both mutations trafficked properly to the plasma membrane and co-assembled with WT channels, and put the channel complexes in a silent mode.
Taken together, we suggest that the substitution C154F shuts down Kir2.1 current by altering channel gating. However, residue T309 lies close to the PIP2-binding site (R312) which has also been shown to cause AS (Plaster et al. 2001) and to destabilize PIP2 binding to Kir2.1 (Lopes et al. 2002). We hypothesize that an isoleucine at position 309 may destabilize PIP2 binding to the Kir2.1 protein leading to a channel shut down. However, adding PIP2 to the pipette did not rescue this mutation (data not shown). Mutations that belong to or are located close to a PIP2-binding site, and cause AS, account for 62% of the patients (Donaldson et al. 2003). PIP2 seems to be a major component in the modulation of the Kir family and may condition further channel regulation by other factors (Du et al. 2004).
Heterologous expression systems may lack factors necessary for protein processing, and data interpretation can be misleading. To address this problem, and to validate our previous and actual findings with AS mutations, we expressed most of the mutations in a mouse muscle cell line. Overall, there was no discrepancy between the expression of the channels in C2C12 cells or HEK293 cells. D71V, G144S, C154F, R218Q, G300V, E303K and T309I reached the plasma membrane, and S314-Y315 showed a scattered cytoplasmic pattern. The only difference seen was related to substitution V302M, which has the particularity to cause AS through a haplo-insufficiency mechanism. In HEK293 cells, we were able to see a faint green fluorescence (V302M-EGFP) in the cytoplasm, but we were not able to detect any protein product in western immunoblot from the same cells. In C2C12 cells, no fluorescence was detected 24 h or even 48 h post-transfection. Whatever the mechanism of degradation of the mutated channels is, our study of AS mutations in a muscle cell line validated the present and previously reported AS mutations in HEK293 cells.
We reported here clinical features of three families with periodic paralysis and cardiac arrhythmia. The mutations are sufficient to explain the manifestation of periodic paralysis in the affected family members, since kir2.1 channels may set rest membrane potential in myocytes. A loss of function may depolarize the cells, exacerbating Na+ channel activities that are in turn rapidly inactivated leading to muscle weakness. Kir2.1 channels are also expressed in the heart, and participate in Ik1 that are involved in the late-repolarization phase of the cardiac action potential. A decrease in Ik1 may delay cardiac repolarization and affect the maintenance of the resting membrane potential in cardiac myocytes. Such effects may underlie the LQT phenotype seen in AS patients. In a recent study on guinea pig cardiac myocytes, Miake and colleagues (Miake et al. 2003) have demonstrated that over-expression of Kir2.1 enhanced Ik1 by 100%, shortened action potential duration, accelerated phase 3 repolarization, and hyperpolarized resting membrane potential. In the same study, the authors showed that suppression of Ik1, by a dominant-negative Kir2.1 mutation variant, decelerated action potential repolarization, prolonged action potential duration, and depolarized resting membrane potential. These findings are consistent with our observations on AS patients, and corroborate all our findings on AS mutations in heterologous systems.
The loss-of-function hypothesis is further supported by EMG evaluations performed in vivo in two AS patients with C154F and T309I substitutions. In a recent study, it has been demonstrated that EMG can discriminate non-dystrophic myotonias and familial periodic paralyses in five main electrophysiological patterns (I to V) correlated with subgroups of ion channel mutations (Fournier et al. 2004). The two AS patients tested in the present study by following the same EMG protocol entered what has been described as pattern V. The latter is characterized by the absence of myotonia, no change of CMAPs after short exercise, but delayed decrease in CMAP amplitude during rest after long exercise. This late decline after long but not short exercise is a common feature that distinguishes patients with all types of periodic paralyses from normal subjects and from patients with myotonic syndromes (McManis et al. 1986; Kuntzer et al. 2000; Fournier et al. 2004). It attests to a decrease in muscle membrane excitability that accounts for exercise-induced episodes of weakness. In AS patients, the decrease in Ik1 may depolarize the cells, leading to membrane inexcitability. However, considering the involvement of the Kir2.1 channel in membrane repolarization, it is worth noting that in vivo evaluations of patients with AS failed to reveal any evidence of muscle membrane hyper-excitability, such as myotonic discharges or increase of CMAP amplitude following a short exercise. Finally, since pattern V is mainly shared by hypokalaemic periodic paralysis patients with a calcium channel mutation (Fournier et al. 2004), it is tempting to hypothesize that potassium and calcium channel mutations may lead to muscle membrane hypo-excitability via a common pathophysiological mechanism that remains to be determined.
Our study did not address the developmental consequences seen in affected members. Further studies on animal models are needed to determine the precise role of Kir2.1 channels in bone formation. Studying novel AS mutations in the KCNJ2 gene and the discovery of novel AS-associated genes in the remaining families with no KCNJ2 mutation may help to understand why bone disorders occur early and the skeletal and cardiac muscle abnormalities do not manifest until late childhood.
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: changes of CMAP amplitude in 30 control subjects (mean ±
S.E.M.). • and 
: changes of CMAP amplitude in patients A6 (C154F substitution) and B5 (T309I substitution), respectively.
