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A ?-subunit mutation in the acetylcholine receptor channel gate causes severe slow-channel syndrome

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A ?-subunit mutation in the acetylcholine receptor channel gate causes severe slow-channel syndrome
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  A p-Subunit Mutation in the Acetylcholine Receptor Channel Gate Causes Severe Slow-Channel Syndrome Christopher M. Gomez, MD, PhD,* Ricardo Maselli, MD,? Jason Gammack, BS,* Jose Lasalde, PhD, Shiori Tamamizu, l’hD, David R. Cornblath, MD,5 Mohamed Lehar, MD, Mark McNamee, PhD, and Ralph W. Kuncl, MD, PhDg ~ ~~~ Point mutations in the genes encoding the acetylcholine receptor AChR) subunits have been recognized in some patients with slow-channel congenital myasthenic syndromes CMS). Clinical, electrophysiological, and pathological differences between these patients may be due to the distinct effects of individual mutations. We report that a spontaneous mutation of the p subunit that interrupts the leucine ring of the AChR channel gate causes an eightfold increase in channel open time nd a severe CMS characterized by severe endplate myopathy and extensive remodeling of the postsynaptic mem- brane. The pronounced abnormalities in neuromuscular synaptic architecture and function, muscle fiber damage nd weakness, resulting from a single point mutation are a dramatic example of a mutation having a dominant gain of function and of hereditary excitotoxicity. Gomez CM, Maselli R, Gainmack J, Lasalde J, Tamamizu S, Cornblath DR, Lehar M, McNamee M, Kuncl RW. A P-subunit mutation in the acetylcholine receptor channel gate causes severe slow-channel syndrome. Ann Neurol 1996;39:712-723 The congenital myasthenic syndromes are a family of disorders of neuromuscular transmission in which the physiological and sometimes molecular defects can be predicted because of the accessibility of the neuromus- cular junction (NMJ) to electrophysiological, immu- nological, and morphological analysis [ 11. Electrophys- iological studies have indicated that a subgroup of these disorders, the slow-channel congenital myas- thenic syndromes (SCCMSs) are due to inherited de- fects in the function of the nicotinic acetylcholine re- ceptor (AChR) of the NMJ [l-31, and the mutations responsible for some of these cases have been identified. The 290-kd AChR is a ligand-gated ion channel consisting of five similar subunits (a2p6&), ach en- coded by a separate gene [4]. The channel is formed by homologous transmembrane domains (M2) of the five subunits, each with an a-helical conformation [5]: Sequence comparison, mutational analysis, and 9 A structural studies have suggested that the ion-channel gate consists of a hydrophobic ring of leucine residues at homologous positions in each subunit that opens due to conformational changes upon acetylcholine (ACh) binding [6, 71. Differences in the clinical, electrophysiological, and pathological features between patients with SCCMS suggests that there is heterogeneity in the effects of the mutations responsible for these syndromes [ 11. Cases of SCCMS have been attributed to missense mutations within the &-subunit M2 domain [%lo], the a- subunit ACh-binding domain [I ll, and to nonsense mutations encoding truncated AChR E subunits [12]. We report a case of SCCMS with a severe endplate myopathy and evidence of extensive remodeling of the postsynaptic membrane due to a mutation in the puta- tive gate of the AChR channel. Materials and Methods Clinical Data The patient, presently 32 years of age, presented with poor head control and a weak suck after a normal birth. He sat at 30 months and walked at 5 years of age. Ophthalmopa- resis was noted at 8, and he developed fatigability at age 10 years. By age 13 he had knee and hip contractures and was wheelchair bound. Edrophonium test was positive. Antibody titer to AChR was negative. Prednisone produced subjective improvement at age 14. From age 15 to 16 he was treated From the ‘Department of Neurology, University of Minnesota, Minneapolis, MN; Sections of ?Neuroscience and $Molecular and Cellular Biology, University of California-Davis, Davis, CA; and %Department of Neurology, Johns HoPkins Hospital, Balcimore, MD. Received Sep 19, 1995, and in revised form Dec 15. Accepted for publication Dec 15, 1995. Address correspondence D~ G~~~~, ~~~~~~~~~ f ~~~ Universiry of Minnesota, 420 Delaware Sr SE, Minneapolis, MN 55455. 712 Copyright 996 by the American Neurological Association  with pyridostigmine, guanidine, and thymectomy without improvement. Neither of the parents nor any of his 4 iblings have any symptoms of the disease. There was no consanguin- ity and no question on nonpaternity. The father died at age 55 of multiple myeloma. On examination, the patient was a thin male with short stature, a long, thin face, high-arched palate, and high-pitched voice. He had near complete oph- thalmoparesis, fatigable ptosis, and severe atrophy and weak- ness of all the limb muscles. Motor point muscle biopsies and venous blood samples from the patient (deltoid age 25, anconeus age 27), and blood samples from his 4 asymptomatic siblings and his mother were obtained in accordance with the guidelines of and with the approval of the respective institutional human studies review boards. Genomic DNA from his deceased father was extracted from slides of his bone marrow bi- opsy (Gentra Systems, Minneapolis, MN). Normal human genomic DNA samples from the Centre d'Etude du Poly- morphisme Humain (CEPH) panel were used as control DNA. used for on-line capture of MEPPs. The kinetics of extracel- Mar recorded endplate noise and MEPPs are independent of the cable properties of the muscle membrane. Under these conditions the mean channel open time can be obtained from the corner frequency of the power spectrum of noise using the expression: z = 1/27~& where F = corner fre- quency. In addition, the decays of extracellularly recorded MEPPs are identical to the decays of intracellularly recorded miniature endplate currents (MEPCs) under voltage-clamp conditions. Exponential fits to the MEPC decays were per- formed using a nonlinear least-square fitting routine. The mean channel open time was deduced from the time con- stants of MEI'C decays. The resolution of these techniques is insufficient to identify distinct channel populations and, therefore, provides an estimate of the average open time of a presumably mixed population of AChR channels. Pathological Studies, Morphometry, and a-Bungarotoxin Binding Motor point biopsies were obtained from deltoid and anco- neus muscles. Portions of muscle were prepared routinely for frozen-section histochemistry and resin-section light micros- copy. Other portions of the biopsy were prepared for special testing as follows: Motor nerve terminals and NMJs were simultaneously visualized with bromoindoxyl acetate for cho- linesterase and with silver-gold impregnation for nerve ter- minals [ 151. For electron microscopy, glutaraldehyde (2- 4%)-fixed tissue was stained for cholinesterase, dissected into regions containing NMJs, and embedded in Araldite for ultrathin sectioning. For niorphometric studies electron micrographs (EMS) of endplate sections were enlarged uni- formly to 18,000X final magnification. Length and areas were measured using a Houston Instruments Hi-Pad digitiz- ing tablet interfaced with a computer and Bioquant RMS software (R and M Biometrics, Nashville, TN). The analysis included nerve terminal area, vesicle number, pre- and post- synaptic membrane lengths, and postsynaptic membrane as previously established [ 161. From these primary data, vesicle density, postsynaptic membrane density, and the postsynap- tidpresynaptic ratios of areas and lengths were calculated. A separate portion of muscle was incubated for 3 hours in '2'I-a-bungarotoxin ('*'IaBT), washed [ 171, and then fixed in 2% paraformaldehydel100 mM lysindl0 mM so- dium periodate/560 mM sucrose for 1 hour. This specimen was submitted for EM autoradiography and quantitative junctional AChR measurement [ 171. The specific binding of 'L51aBT o NMJs was calculated using at least 100 to 200 muscle fibers 1171. Electrophysiological Studies Clinical electromyographic recording was performed ac- cording to standard methods. For in vitro microelectrode studies, part of the anconeus muscle was surgically removed using a previously described technique [ 131. Recordings of miniature endplate potentials (MEPPs) and endplate potcn- tials (EPPs) were performed using previously described elec- trophysiologic techniques [ 141. During the recordings, the preparation was continuously superfused with Tyrode's aer- ated with a 95% 02 %) COz gas mixture. Recordings were conducted at room temperature (26°C) and the pH was maintained between 7.3 and 7.4. Neurally evoked EPPs were obtained by stimulating fine intramuscular nerve branches with a concentric bipolar electrode. Spontaneous MEPPs and neurally evoked EPPs were recorded using a WPI-750 elec- trometer (WPI Inc, Hamden, CT). The output of the recording instrument was amplified, filtered, and sampled at 10 kHz by a 12-bit analog/digital (ND) converter (Data Translation 28 18, Marlboro, MA). All electrical signals were acquired on-line and stored on an IBM-AT computer for subsequent analysis. To minimize the differences in MEPP and EPP amplitudes resulting from dif- ferences in the resting membrane potential (RMP), correc- tion of MEPP and EPP amplitudes for RMP was performed according to the following equation: MEPP,,,,,,,,,, = MEPP X (-77/RMP). The quanta1 content of EPPs (m) was auto- matically calculated from EPP files using the indirect vari- ance method: m = mean EPI' amolitude/(SD). In vivo extracellular recording of endplate noise and MEPPs was performed in the left supinator muscle (R. Ma- selli, unpublished). All recordings were obtained using a monopolar needle that was connected to the preamplifier of a TECA TE4 electromyograph (TECA Corp, Pleasantville, NY . The limb temperature was maintained at 33°C. The analog output of this instrument was simultaneously con- nected to the AID board of two separate computers. One computer was equipped with Electrophysiological Data Anal- ysis software (Dagan), a program that enabled it to perform spectral analysis of endplate noise. The other computer was Genetic Analysis of the M2 Domains Polymerase chain reaction (PCR) and single-strand confor- mation polymorphism (SSCP) analyses used to amplify and screen the M2 domains of the four adult AChR subunits were performed as described [ 101 (Robocycler 40, Strata- gene). The primer set for the M2 domain of the a subunit corresponds to the intron sequences flanking exon 7 (181, and amplifies a 280-bp fragment. The primer sets for the M2 domains of the p and 6 subunits correspond to sequences in intron (as determined by nucleotide sequence analysis) and Gomez et al: Slow-Channel Syndrome 713  the 3’ extreme of exon 8 [19, 201, as predicted [21, 221, and amplify 300- and 2 10-bp fragments, respectively. The primer set for the &-subunit M2 domain corresponds to sequences in exon 7 immediately upstream of intron 7 and the 3’ ex- treme of exon 8 [231, as predicted from the mouse E genomic structure [22]. The 220-bp fragment amplified includes the entire E M2 coding sequence along with E intron 7 - 100 bp). For allele-specific analysis, distinct SSCP conformers were excised and eluted directly from gels, and reamplified using the same primers. Nucleotide sequence analysis of M2 domains amplified from genomic DNA or from SSCP conformers was performed manually (Sequenase, DeltaTaq, USB) In Xtro Expression Studies Site-directed mutagenesis of mouse P-subunit cDNA to gen- erate the homologous L263M mutation was performed using single-stranded plasmid generated from the vector pSelect (Promega). The mutation was confirmed by dideoxy nucleo- tide sequence determination. Mutant and wild-type AChR subunit mRNAs [24-261 were transcribed in vitro from lin- earized plasmids using T7 polymerase. The oocyte vitelline membrane was removed manually after incubation in hypertonic solution composed of 150 mM NaC1, 2 mM KC1, 3% sucrose, and 5 mM HEPES (pH 7.6). The oocytes were placed in a recording chamber containing bath solution (100 mM KCI, 1 mM MgCI2, and 10 mM HEPES, pH 7.2) at 20 to 22°C. The patch pipettes were made of thick-walled borosilicate glass (Sutter Instru- ments, Novato, CA) exhibiting resistances of 8 to 12 MQ. The pipette solution contained 100 mM KCI, 10 mM HEPES, 10 mM EGTA, pH 7.2, and 4 pM ACh. All experi- ments were performed in a cell-attached patch configuration. Single-channel currents were recorded using a Dagan 3900 amplifier (Dagan, Minneapolis, MN), filtered at 5 kHz (Fre- quency Devices Inc, Haverhill, MA), and stored on VHS tapes using a digital data recorder (VR-IOB, Instrutech Corp, Mineola, NY). The data traces were played back into an IBM-compatible computer through a IXgiData 1200 inter- face (Axon Instruments, Foster City, CA) and digitized at 50 psec. Single-channel currents were detected with a half- amplitude crossing algorithm (IPROC) and data analysis was performed using pCLAMP (Axon Instruments). Results Electropbyjiological Data Clinical electromyographic studies provided evidence for both prolonged responses to ACh and severe im- pairment of the safety factor of neuromuscular trans- mission. A single supramaximal stimulus of the ulnar nerve elicited at least three well-defined repetitive re- sponses after the initial compound muscle action po- tential (CMAP) in the adductor digiti minimi. In Fig- ure 1A, the repetitive responses can be seen after the first three stimuli. Repetitive stimulation at 0.5 to 20 Hz elicited decremend responses (see Fig 1A) between the first and fourth response of up to 74%, with the greatest decrement occurring at 3 Hz. After edropho- nium, the amplitude of the first response increased by A B C L Fig 1. Repetitive compound action potentials, decremental responses, and markedly prolonged endplate potential (EPP) decay phases. (A) Repetitive compound muscle action poten- tials (CMAPs) recorded over the adductor digiti minimi dur- ing repetitive nerve stimulation at I Hz show repetitive CMAPs in the first three responses and decremental response of the prima y action potential. (B and C) Exumples of EPPs recordedfrom the patient (B) andfiom an age-matched con- trol (C). In the patient, EPP decays are extremely prolonged. Calibration: (A) horizontal bar, 3 msec; vertical bur, 1 ml/: (B and C) Horizontal bar, 20 msec; vertical bar, 2 mV in B and 5 mV in C. 150°/o, but there was no change in the decrement at the fourth response. Electromyography revealed normal insertional activity and no spontaneous activity. Volun- tary motor unit potentials had short durations and low amplitudes with an early recruitment pattern. Motor and sensory nerve conduction studies were normal aside from reduced motor-evoked amplitudes. The duration of the EPPs recorded from anconeus muscle fibers was greatly increased as a result of ex- treme prolongation of the EPP decay phases (Fig 1B). The mean half-decay time of the patient EPPs (38.62 I _ 3.2 msec, n = 14) was more than 10 times longer than the mean half-decay time of EPPs recorded from control patients (Fig 1C) (3.43 0.23 msec, n = 7, p < 0.001). ‘The mean amplitude of the patient EPPs (1.08 0.21 mV, n = 14) was profoundly reduced in comparison with the mean amplitude of EPPs recorded from control patients (15.78 2.45 mV, n = 16, p < 0.001). In addition, MEPP amplitudes were extremely small or undetectable from the background noise at most of the recorded endplates. The mean MEPP am- plitude estimated from those detected above background was 0.15 - 0.07 mV, n = 3, versus 0.81 t 0.08 714 Annals of Neurology Vol 39 No 6 June 1996  mV, n = 70. The mean EPP quanta1 content was also significantly reduced (17.72 3.4, n = 16, versus 32.7 2 3.8, n = 14 in control patients, p < 0.01). The mean channel open time of the patient deduced from the power spectrum of endplate noise (6.55 ? 1.77 msec, n = 4) and the MEPC time constants (7.47 - 0.60 msec, n = 8) were markedly prolonged in comparison with the channel open time of control pa- tients (1.28 0.14 msec, n = 16, and 1.53 _ 0.11 msec, n = 30, respectively, p < 0.001) (Fig 2). This value indicates the presence of a population of channels with severely impaired hnetics, although a mixed pop- ulation of AChR channels may be present. 10 7 Pathological Data Sections of the deltoid muscle showed increased vari- ability in fiber size, occasional split fibers, and type 1 fiber predominance (not shown). In addition, in the anconeus, autophagic lysosomal vacuoles were prevalent within the majority of myofibers (Fig 3). By contrast, necrosis was nearly absent. Autophagic vacuoles were frequently present near NMJs under the sarcolemma and in the long central axis of myofibers. The same autophagic contents, lined by remnants of basement membrane, filled the extracellular space, separating in- dividual muscle fibers and associated with some fi- brosis. Silver cholinesterase stains of the deltoid motor point biopsy revealed elongated, multisegmented end- plates with multiple terminal zonal branches innervat- ing single muscle fibers (not shown). Acetylcholinester- ase stains were consistently positive. Ultrastructural study of 10 endplate regions in del- toid and five endplates in anconeus revealed regions of endplate myopathy alongside areas of marked remodel- ing of the postsynaptic membrane. The features of end- plate myopathy were as follows. Junctional sarcoplasm contained autophagic vacuoles and myeloid structures (Fig 4A, B, D, F-H) and areas of myofibrillar disrup- tion (Fig 4C). Empty vesicles were rare. In areas where postsynaptic folds were focally simplified (Fig 4A, D-G), the junctional basal lamina was highly re- duplicated, the primary and secondary synaptic clefts focally widened, and the space filled with extruded ve- sicular autophagic debris. Adjacent to the degenerated postsynaptic folds, the postsynaptic membrane was also focally enlarged and remodeled. In some junctions, postsynaptic folds were excessively complex and elongated within enlarged postsynaptic areas (see Fig 4B-D, H). There were nu- merous endocytotic vesicles in the junctional folds (see Fig 4, all). In some fibers, folds that resembled postsyn- aptic membrane because of their finger-like projections and endocytotic vesicles extended over 100 pM from the endplate region proper (see Fig 4B). Minor presyn- + - 10 9 L- 0 1 02 Hz B 10 102 Hz ig 2. Power spectral analysis of endplate noise in vivo pre- dicts acetylcholine receptors with prolonged burst durations. Examples of power spectra of endplate noise recorded fiom the patient (A) andfiom an age-matched control (B). In both cases the spectrum was best$tted with a single Lorenzian finction. In the patient? curve there is a marked shtfi- offie- quencies toward the low end of the spectrum. The mean chan- nel open time was 655 msec in the patient and 1.05 msec in the subject control. Gomez et al: Slow-Channel Syndrome 715  Fig 3. Light microscopy of anconeus muscle. The myopathic changes of increased variabilip in fiber size, fiber qlitting, andjbrosis are more severe than in the deltoid. Autophagic lysosomal uucuoles are present in the majority of myofbers but are especially prominent in the large jiber (upper right) at the region bearing a neuromuscular unction (arrow). Bar = SO pm. aptic abnormalities were also present. Occasional junc- tions were denuded of their nerve terminals or con- tained an unmyelinated sprout (see Fig 411). Nerve terminals were slightly small but otherwise normal in vesicle diameter and density (see Fig 4A, -H). We applied standard rnorphometric techniques to compare quantitatively the dimensions of the patient’s deltoid NMJ to normal intercostal (Table). As with other SCCMSs, both the nerve terminal area and length were mildly reduced. On the other hand, the postsynaptic membrane area and length were increased compared with published controls. As a result, the cal- culated ratios of postsynaptic/presynaptic membrane area and length were greater than those in normal end- plates, compared with the same ratios in other SCCMS patients, which were less than normal [2, 3, 271. In addition, the “normal” overall postsynaptic membrane density measurement obscures the combination of focal postsynaptic membrane elaboration and degeneration (see Fig 4D). e-Bungarotoxin Binding Studies In ‘”IaBT-reacted autoradiography, silver grains were present over motor endplate regions as well as selec- tively over both intracellular autophagolysosomes and extracellular autophagic debris (data not shown). The number of AChRs per endplate, as estimated by 1251~BT inding was sixfold greater in the patient’s deltoid fibers (12.0 X 10’ AChRdendplate) and 45- fold greater in the anconeus muscle fibers (95 X 10’ AChRdendplate) compared with normal and disease control deltoid fibers (2.1 _t 0.2 X 10’; 95% confi- dence interval, 1.5-3.9 X 10’ AChRdendplate) [17]. Genetic Analysis of M2 Domains We screened the M2 domains of the AChR a-, J3- I-, and &subunit genes amplified from the patient’s genomic DNA using the PCR and SSCP analysis. An abnormal conformer was identified in the sequences amplified from the P-subunit M2 region of the patient genomic DNA, present in equal abundance with the normal conformer (Fig 5, lane 3). This abnormal con- former was absent from P-subunit M2 sequences am- plified from the genomic DNA of his clinically normal parents and his 4 normal siblings (Fig 5, lanes 1, 2, 4-7). The abnormal conformer was also not present in 100 normal control samples. We obtained template for allele-specific sequence analysis by isolating and reamplifying the SSCP con- formers directly from the SSCP gel using the srcinal P-subunit M2 primer set. The abnormal SSCP con- former contained a C-to-A transversion within codon 263, coding for a leucine-to-methionine substitution (L2”M) in the ninth residue of the P-subunit M2 do- main (Fig 6a, arrow). This mutation was not present in the sequence amplified from the normal conformer (Fig Gb, arrow). The P-subunit M2 DNA amplified from the patient’s genomic DNA contained both the wild-type and mutant sequences (Fig 4C, arrow), while the J3-subunit M2 regions amplified from genomic DNA of both the patient‘s parents (Fig 4D and E) and from his 4 siblings (not shown) contained only the ~ ~~ ig 4. Electron microscopic features of endplate rnyopathy and postsynaptic remodeling. Selected deltoid neurornuscular junc- tions A-H) illustrate focal degeneration of postsynaptic folds (A, D, E; curved arrows) with widening of pvima y and sec- ondary synaptic clefis F, G; stars); complex remodeling of postsynaptic membrane, which is increased in length and urea (B, C, D, H), even adjdcent to the focal degeneration (D); postsynaptic folds that extend fir beyond the endplute (B, F; long arrowheads); autophagic vacuoles or nyelin figures in junctional sarcoplasm (B, D, F, G, H; arge white arrows), which sometimes engulf perijunctional nuclei (A); mtophagic debris in synaptic clefis all junctions; asterisks); focul myo- Jbrillm disruption C; small white arrows); and overabun- dant endorytotic vesicles in postsynaptic membrane (all junctions, eg black arrowheads in A, B, F, H). Occasional junctions contain an unmyelinated sprout (D, E). Nerve ter- minah are otherwise normal, though slightly small. Bar I prn. (Fig 4 continues on page 718.) 716 Annals of Neurology Vol 35 No 6 June 1996

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