Glycine Receptor Autoantibodies Impair Receptor Function and Induce Motor Dysfunction.

OBJECTIVE: Impairment of glycinergic neurotransmission leads to complex movement and behavioral disorders. Patients harboring glycine receptor autoantibodies suffer from stiff-person syndrome or its severe variant progressive encephalomyelitis with rigidity and myoclonus. Enhanced receptor internalization was proposed as the common molecular mechanism upon autoantibody binding. Although functional impairment of glycine receptors following autoantibody binding has recently been investigated, it is still incompletely understood. METHODS: A cell-based assay was used for positive sample evaluation. Glycine receptor function was assessed by electrophysiological recordings and radioligand binding assays. The in vivo passive transfer of patient autoantibodies was done using the zebrafish animal model. RESULTS: Glycine receptor function as assessed by glycine dose-response curves showed significantly decreased glycine potency in the presence of patient sera. Upon binding of autoantibodies from 2 patients, a decreased fraction of desensitized receptors was observed, whereas closing of the ion channel remained fast. The glycine receptor N-terminal residues 29 A to 62 G were mapped as a common epitope of glycine receptor autoantibodies. An in vivo transfer into the zebrafish animal model generated a phenotype with disturbed escape behavior accompanied by a reduced number of glycine receptor clusters in the spinal cord of affected animals. INTERPRETATION: Autoantibodies against the extracellular domain mediate alterations of glycine receptor physiology. Moreover, our in vivo data demonstrate that the autoantibodies are a direct cause of the disease, because the transfer of human glycine receptor autoantibodies to zebrafish larvae generated impaired escape behavior in the animal model compatible with abnormal startle response in stiff-person syndrome or progressive encephalitis with rigidity and myoclonus patients. ANN NEUROL 2020;88:544-561.

Primary DQ effect in the association between HLA and neurological syndromes with anti-GAD65 antibodies.

The primary cause of neurological syndromes with antibodies against glutamic acid decarboxylase 65 (GAD65-Ab) is unknown, but genetic predisposition may exist as it is suggested by the co-occurrence in patients and their relatives of other organ-specific autoimmune diseases, notably type 1 diabetes mellitus (T1DM), and by the reports of a few familial cases. We analyzed the human leukocyte antigen (HLA) in 32 unrelated patients and compared them to an ethnically matched sample of 137 healthy controls. Four-digit resolution HLA alleles were imputed from available Genome Wide Association data, and full HLA next-generation sequencing-based typing was also performed. HLA DQA1*05:01-DQB1*02:01-DRB1*03:01 was the most frequent class II haplotype in patients (13/32, 41%). DQB1*02:01 was the only allele found to be significantly more common in patients than in controls (20/137, 15%, corrected p = 0.03, OR 3.96, 95% CI [1.54-10.09]). There was also a trend towards more frequent DQA1*05:01 among patients compared to controls (22/137, 16%; corrected p = 0.05, OR 3.54, 95% CI [1.40-8.91]) and towards a protective effect of DQB1*03:01 (2/32, 6% in patients vs. 42/137, 31% in control group; corrected p = 0.05, OR 0.15, 95% CI [0.02-0.65]). There was no significant demographic or clinical difference between DQ2 and non-DQ2 carriers (p > 0.05). Taken together, these findings suggest a primary DQ effect on GAD65-Ab neurological diseases, partially shared with other systemic organ-specific autoimmune diseases such as T1DM. However, it is likely that other non-HLA loci are involved in the genetic predisposition of GAD65-Ab neurological syndromes.

A Stiff Person Syndrome Misdiagnosed as a Psychiatric Illness: A Case Report.

BACKGROUND: Stiff Person Syndrome (SPS) is a rare autoimmune neurological disorder which is often misdiagnosed. We report here a case of SPS with a long diagnosis delay. CASE: A 36-year-old man presented with an 11-year history of progressive stiffness and painful spasms of his both legs, with recent worsening of his condition over the last year resulting in a considerable difficulty of standing up and walking. As the patient developed phobic symptoms, he was considered as having a psychiatric illness and treated with antianxiety and antidepressant drugs. As no real improvement was observed, the patient was referred to internal medicine. Neurological examination showed paraspinal, abdominal and lower limbs muscle contraction with lumbar rigidity. These symptoms were associated to adrenergic symptoms: profuse sweating, tachycardia and high bloodpressure. Initial routine investigations revealed high blood glucose level. Polygraphic electromyographic (EMG) evaluation from paraspinal and leg muscles showed continuous motor unit activity in agonist and antagonist muscle. Electroencephalography and brain magnetic resonance imaging were normal. Immunologic tests according to radio immune assay technique revealed high level of serum anti-glutamic acid decarboxylase (anti-GAD65) antibodies. Diagnosis of autoimmune SPS was retained based on clinical, electrophysiological, and immunological findings. Pregabalin at the dose of 150 mg, three times a day was prescribed with satisfying response. CONCLUSION: SPS is supported by an autoimmune pathogenesis and anti-GAD antibodies seems to be very helpful when SPS is clinically suspected. Treatment of SPS is a challenge, given the scarcity of the syndrome and the absence of established recommendations.

Disruption of a Structurally Important Extracellular Element in the Glycine Receptor Leads to Decreased Synaptic Integration and Signaling Resulting in Severe Startle Disease.

Functional impairments or trafficking defects of inhibitory glycine receptors (GlyRs) have been linked to human hyperekplexia/startle disease and autism spectrum disorders. We found that a lack of synaptic integration of GlyRs, together with disrupted receptor function, is responsible for a lethal startle phenotype in a novel spontaneous mouse mutant shaky, caused by a missense mutation, Q177K, located in the extracellular ß8-ß9 loop of the GlyR α1 subunit. Recently, structural data provided evidence that the flexibility of the ß8-ß9 loop is crucial for conformational transitions during opening and closing of the ion channel and represents a novel allosteric binding site in Cys-loop receptors. We identified the underlying neuropathological mechanisms in male and female shaky mice through a combination of protein biochemistry, immunocytochemistry, and both in vivo and in vitro electrophysiology. Increased expression of the mutant GlyR α1Q177K subunit in vivo was not sufficient to compensate for a decrease in synaptic integration of α1Q177Kß GlyRs. The remaining synaptic heteromeric α1Q177Kß GlyRs had decreased current amplitudes with significantly faster decay times. This functional disruption reveals an important role for the GlyR α1 subunit ß8-ß9 loop in initiating rearrangements within the extracellular-transmembrane GlyR interface and that this structural element is vital for inhibitory GlyR function, signaling, and synaptic clustering.SIGNIFICANCE STATEMENT GlyR dysfunction underlies neuromotor deficits in startle disease and autism spectrum disorders. We describe an extracellular GlyR α1 subunit mutation (Q177K) in a novel mouse startle disease mutant shaky Structural data suggest that during signal transduction, large transitions of the ß8-ß9 loop occur in response to neurotransmitter binding. Disruption of the ß8-ß9 loop by the Q177K mutation results in a disruption of hydrogen bonds between Q177 and the ligand-binding residue R65. Functionally, the Q177K change resulted in decreased current amplitudes, altered desensitization decay time constants, and reduced GlyR clustering and synaptic strength. The GlyR ß8-ß9 loop is therefore an essential regulator of conformational rearrangements during ion channel opening and closing.

Disturbed neuronal ER-Golgi sorting of unassembled glycine receptors suggests altered subcellular processing is a cause of human hyperekplexia.

Recent studies on the pathogenic mechanisms of recessive hyperekplexia indicate disturbances in glycine receptor (GlyR) α1 biogenesis. Here, we examine the properties of a range of novel glycine receptor mutants identified in human hyperekplexia patients using expression in transfected cell lines and primary neurons. All of the novel mutants localized in the large extracellular domain of the GlyR α1 have reduced cell surface expression with a high proportion of receptors being retained in the ER, although there is forward trafficking of glycosylated subpopulations into the ER-Golgi intermediate compartment and cis-Golgi compartment. CD spectroscopy revealed that the mutant receptors have proportions of secondary structural elements similar to wild-type receptors. Two mutants in loop B (G160R, T162M) were functional, but none of those in loop D/ß2-3 were. One nonfunctional truncated mutant (R316X) could be rescued by coexpression with the lacking C-terminal domain. We conclude that a proportion of GlyR α1 mutants can be transported to the plasma membrane but do not necessarily form functional ion channels. We suggest that loop D/ß2-3 is an important determinant for GlyR trafficking and functionality, whereas alterations to loop B alter agonist potencies, indicating that residues here are critical elements in ligand binding.

Molecular basis of the dominant negative effect of a glycine transporter 2 mutation associated with hyperekplexia.

Hyperekplexia or startle disease is a rare clinical syndrome characterized by an exaggerated startle in response to trivial tactile or acoustic stimuli. This neurological disorder can have serious consequences in neonates, provoking brain damage and/or sudden death due to apnea episodes and cardiorespiratory failure. Hyperekplexia is caused by defective inhibitory glycinergic neurotransmission. Mutations in the human SLC6A5 gene encoding the neuronal GlyT2 glycine transporter are responsible for the presynaptic form of the disease. GlyT2 mediates synaptic glycine recycling, which constitutes the main source of releasable transmitter at glycinergic synapses. Although the majority of GlyT2 mutations detected so far are recessive, a dominant negative mutant that affects GlyT2 trafficking does exist. In this study, we explore the properties and structural alterations of the S512R mutation in GlyT2. We analyze its dominant negative effect that retains wild-type GlyT2 in the endoplasmic reticulum (ER), preventing surface expression. We show that the presence of an arginine rather than serine 512 provoked transporter misfolding, enhanced association to the ER-chaperone calnexin, altered association with the coat-protein complex II component Sec24D, and thereby impeded ER exit. The S512R mutant formed oligomers with wild-type GlyT2 causing its retention in the ER. Overexpression of calnexin rescued wild-type GlyT2 from the dominant negative effect of the mutant, increasing the amount of transporter that reached the plasma membrane and dampening the interaction between the wild-type and mutant GlyT2. The ability of chemical chaperones to overcome the dominant negative effect of the disease mutation on the wild-type transporter was demonstrated in heterologous cells and primary neurons.

Stiff person-syndrome IgG affects presynaptic GABAergic release mechanisms.

The majority of patients with stiff person-syndrome (SPS) are characterized by autoantibodies to glutamate decarboxylase 65 (GAD65). In previous passive-transfer studies, SPS immunoglobulin G (IgG) induced SPS core symptoms. We here provide evidence that SPS-IgG causes a higher frequency of spontaneous vesicle fusions. Sustained GABAergic transmission and presynaptic GABAergic vesicle pool size remained unchanged. Since these findings cannot be attributed to anti-GAD65 autoantibodies alone, we propose that additional autoantibodies with so far undefined antigen specificity might affect presynaptic release mechanisms.

The GLRA1 missense mutation W170S associates lack of Zn2+ potentiation with human hyperekplexia.

Hyperekplexia is a neurological disorder associated primarily with mutations in the α1 subunit of glycine receptors (GlyRs) that lead to dysfunction of glycinergic inhibitory transmission. To date, most of the identified mutations result in disruption of surface expression or altered channel properties of α1-containing GlyRs. Little evidence has emerged to support an involvement of allosteric GlyR modulation in human hyperekplexia. Here, we report that recombinant human GlyRs containing α1 or α1ß subunits with a missense mutation in the α1 subunit (W170S), previously identified from familial hyperekplexia, caused remarkably reduced potentiation and enhanced inhibition by Zn(2+). Interestingly, mutant α1(W170S)ß GlyRs displayed no significant changes in potency or maximum response to glycine, taurine, or ß-alanine. By temporally separating the potentiating and the inhibitory effects of Zn(2+), we found that the enhancement of Zn(2+) inhibition resulted from a loss of Zn(2+)-mediated potentiation. The W170S mutation on the background of H107N, which was previously reported to selectively disrupt Zn(2+) inhibition, showed remarkable attenuation of Zn(2+)-mediated potentiation and thus indicated that W170 is an important residue for the Zn(2+)-mediated GlyR potentiation. Moreover, overexpressing the α1(W170S) subunit in cultured rat neurons confirmed the results from heterologous expression. Together, our results reveal a new zinc potentiation site on α1 GlyRs and a strong link between Zn(2+) modulation and human disease.

Analysis of hyperekplexia mutations identifies transmembrane domain rearrangements that mediate glycine receptor activation.

Pentameric ligand-gated ion channels (pLGICs) mediate numerous physiological processes and are therapeutic targets for a wide range of clinical indications. Elucidating the structural differences between their closed and open states may help in designing improved drugs that bias receptors toward the desired conformational state. We recently showed that two new hyperekplexia mutations, Q226E and V280M, induced spontaneous activity in α1 glycine receptors. Gln-226, located near the top of transmembrane (TM) 1, is closely apposed to Arg-271 at the top of TM2 in the neighboring subunit. Using mutant cycle analysis, we inferred that Q226E induces activation via an enhanced electrostatic attraction to Arg-271. This would tilt the top of TM2 toward TM1 and hence away from the pore axis to open the channel. We also concluded that the increased side chain volume of V280M, in the TM2-TM3 loop, exerts a steric repulsion against Ile-225 at the top of TM1 in the neighboring subunit. We infer that this steric repulsion would tilt the top of TM3 radially outwards against the stationary TM1 and thus provide space for TM2 to relax away from the pore axis to create an open channel. Because the transmembrane domain movements inferred from this functional analysis are consistent with the structural differences evident in the x-ray atomic structures of closed and open state bacterial pLGICs, we propyose that the model of pLGIC activation as outlined here may be broadly applicable across the eukaryotic pLGIC receptor family.

Propofol modulation of α1 glycine receptors does not require a structural transition at adjacent subunits that is crucial to agonist-induced activation.

Pentameric glycine receptors (GlyRs) couple agonist binding to activation of an intrinsic ion channel. Substitution of the R271 residue impairs agonist-induced activation and is associated with the human disease hyperekplexia. On the basis of a homology model of the α1 GlyR, we substituted residues in the vicinity of R271 with cysteines, generating R271C, Q226C, and D284C single-mutant GlyRs and R271C/Q226C and R271C/D284C double-mutant GlyRs. We then examined the impact of interactions between these positions on receptor activation by glycine and modulation by the anesthetic propofol, as measured by electrophysiological experiments. Upon expression in Xenopus laevis oocytes, D284C-containing receptors were nonfunctional, despite biochemical evidence of successful cell surface expression. At R271C/Q226C GlyRs, glycine-activated whole-cell currents were increased 3-fold in the presence of the thiol reductant dithiothreitol, whereas the ability of propofol to enhance glycine-activated currents was not affected by dithiothreitol. Biochemical experiments showed that mutant R271C/Q226C subunits form covalently linked pentamers, showing that intersubunit disulfide cross-links are formed. These data indicate that intersubunit disulfide links in the transmembrane domain prevent a structural transition that is crucial to agonist-induced activation of GlyRs but not to modulation by the anesthetic propofol and implicate D284 in the functional integrity of GlyRs.

Glycine receptor mouse mutants: model systems for human hyperekplexia.

Human hyperekplexia is a neuromotor disorder caused by disturbances in inhibitory glycine-mediated neurotransmission. Mutations in genes encoding for glycine receptor subunits or associated proteins, such as GLRA1, GLRB, GPHN and ARHGEF9, have been detected in patients suffering from hyperekplexia. Classical symptoms are exaggerated startle attacks upon unexpected acoustic or tactile stimuli, massive tremor, loss of postural control during startle and apnoea. Usually patients are treated with clonazepam, this helps to dampen the severe symptoms most probably by up-regulating GABAergic responses. However, the mechanism is not completely understood. Similar neuromotor phenotypes have been observed in mouse models that carry glycine receptor mutations. These mouse models serve as excellent tools for analysing the underlying pathomechanisms. Yet, studies in mutant mice looking for postsynaptic compensation of glycinergic dysfunction via an up-regulation in GABAA receptor numbers have failed, as expression levels were similar to those in wild-type mice. However, presynaptic adaptation mechanisms with an unusual switch from mixed GABA/glycinergic to GABAergic presynaptic terminals have been observed. Whether this presynaptic adaptation explains the improvement in symptoms or other compensation mechanisms exist is still under investigation. With the help of spontaneous glycine receptor mouse mutants, knock-in and knock-out studies, it is possible to associate behavioural changes with pharmacological differences in glycinergic inhibition. This review focuses on the structural and functional characteristics of the various mouse models used to elucidate the underlying signal transduction pathways and adaptation processes and describes a novel route that uses gene-therapeutic modulation of mutated receptors to overcome loss of function mutations.

Neuronal surface and glutamic acid decarboxylase autoantibodies in Nonparaneoplastic stiff person syndrome.

IMPORTANCE: High titers of autoantibodies to glutamic acid decarboxylase (GAD) are well documented in association with stiff person syndrome (SPS). Glutamic acid decarboxylase is the rate-limiting enzyme in the synthesis of γ-aminobutyric acid (GABA), and impaired function of GABAergic neurons has been implicated in the pathogenesis of SPS. Autoantibodies to GAD might be the causative agent or a disease marker. OBJECTIVE: To investigate the characteristics and potential pathogenicity of GAD autoantibodies in patients with SPS and related disorders. DESIGN: Retrospective cohort study and laboratory investigation. SETTING: Weatherall Institute of Molecular Medicine, University of Oxford. PARTICIPANTS: Twenty-five patients with SPS and related conditions identified from the Neuroimmunology Service. EXPOSURES: Neurological examination, serological characterization and experimental studies. MAIN OUTCOMES AND MEASURES: Characterization of serum GAD antibodies from patients with SPS and evidence for potential pathogenicity. RESULTS: We detected GAD autoantibodies at a very high titer (median, 7500 U/mL) in 19 patients (76%), including all 12 patients with classic SPS. The GAD autoantibodies were high affinity (antibody dissociation constant, 0.06-0.78 nmol) and predominantly IgG1 subclass. The patients' autoantibodies co-localized with GAD on immunohistochemistry and in permeabilized cultured cerebellar GABAergic neurons, as expected, but they also bound to the cell surface of unpermeabilized GABAergic neurons. Adsorption of the highest titer (700 000 U/mL) serum with recombinant GAD indicated that these neuronal surface antibodies were not directed against GAD itself. Although intraperitoneal injection of IgG purified from the 2 available GAD autoantibody­ositive purified IgG preparations did not produce clinical or pathological evidence of disease, SPS and control IgG were detected in specific regions of the mouse central nervous system, particularly around the lateral and fourth ventricles. CONCLUSIONS AND RELEVANCE: Autoantibodies to GAD are associated with antibodies that bind to the surface of GABAergic neurons and that could be pathogenic. Moreover, in mice, human IgG from the periphery gained access to relevant areas in the hippocampus and brainstem. Identification of the target of the non-GAD antibodies and peripheral and intrathecal transfer protocols, combined with adsorption studies, should be used to demonstrate the role of the non-GAD IgG in SPS.

Stiff-person syndrome (SPS) and anti-GAD-related CNS degenerations: protean additions to the autoimmune central neuropathies.

Stiff Person Syndrome (SPS) is a rare autoimmune neurological disease attributable to autoantibodies to glutamic acid decarboxylase (anti-GAD) more usually associated with the islet beta cell destruction of autoimmune type 1 diabetes (T1D). SPS is characterized by interference in neurons with the synthesis/activity of the inhibitory neurotransmitter gamma amino butyric acid (GABA) resulting in the prototypic progressive spasmodic muscular rigidity of SPS, or diverse neurological syndromes, cerebellar ataxia, intractable epilepsy, myoclonus and several others. Remarkably, a single autoantibody, anti-GAD, can be common to widely different disease expressions, i.e. T1D and SPS. One explanation for these data is the differences in epitope engagement between the anti-GAD reactivity in SPS and T1D: in both diseases, anti-GAD antibody reactivity is predominantly to a conformational epitope region in the PLP- and C-terminal domains of the 65 kDa isoform but, additionally in SPS, there is reactivity to conformational epitope(s) on GAD67, and short linear epitopes in the C-terminal region and at the N-terminus of GAD65. Another explanation for disease expressions in SPS includes ready access of anti-GAD to antigen sites due to immune responsiveness within the CNS itself according to intrathecal anti-GAD-specific B cells and autoantibody. Closer study of the mysterious stiff-person syndrome should enhance the understanding of this disease itself, and autoimmunity in general.

Human stiff-person syndrome IgG induces anxious behavior in rats.

BACKGROUND: Anxiety is a heterogeneous behavioral domain playing a role in a variety of neuropsychiatric diseases. While anxiety is the cardinal symptom in disorders such as panic disorder, co-morbid anxious behavior can occur in a variety of diseases. Stiff person syndrome (SPS) is a CNS disorder characterized by increased muscle tone and prominent agoraphobia and anxiety. Most patients have high-titer antibodies against glutamate decarboxylase (GAD) 65. The pathogenic role of these autoantibodies is unclear. METHODOLOGY/PRINCIPAL FINDINGS: We re-investigated a 53 year old woman with SPS and profound anxiety for GABA-A receptor binding in the amygdala with (11)C-flumazenil PET scan and studied the potential pathogenic role of purified IgG from her plasma filtrates containing high-titer antibodies against GAD 65. We passively transferred the IgG fraction intrathecally into rats and analyzed the effects using behavioral and in vivo electrophysiological methods. In cell culture, we measured the effect of patient IgG on GABA release from hippocampal neurons. Repetitive intrathecal application of purified patient IgG in rats resulted in an anxious phenotype resembling the core symptoms of the patient. Patient IgG selectively bound to rat amygdala, hippocampus, and frontal cortical areas. In cultured rat hippocampal neurons, patient IgG inhibited GABA release. In line with these experimental results, the GABA-A receptor binding potential was reduced in the patient's amygdala/hippocampus complex. No motor abnormalities were found in recipient rats. CONCLUSION/SIGNIFICANCE: The observations in rats after passive transfer lead us to propose that anxiety-like behavior can be induced in rats by passive transfer of IgG from a SPS patient positive for anti-GAD 65 antibodies. Anxiety, in this case, thus may be an antibody-mediated phenomenon with consecutive disturbance of GABAergic signaling in the amygdala region.

11C-flumazenil positron emission tomography demonstrates reduction of both global and local cerebral benzodiazepine receptor binding in a patient with Stiff Person Syndrome.

Stiff Person Syndrome (SPS) is a rare autoimmune disorder associated with antibodies against glutamic acid decarboxylase (GAD-Ab), the key enzyme in gamma-aminobutyric acid synthesis (GABA). In order to investigate the role of cerebral benzodiazepinereceptor binding in SPS, we performed [(11)C]flumazenil (FMZ) positron emission tomography (PET) in a female patient with SPS compared to nine healthy controls. FMZ is a radioligand to the postsynaptic central benzodiazepine receptor which is co-localized with the GABA-A receptor. In the SPS patient, we found a global reduction of cortical FMZ binding. In addition, distinct local clusters of reduced radiotracer binding were observed. These data provide first in vivo evidence for a reduced postsynaptic GABA-A receptor availability which may reflect the loss of GABAergic neuronal inhibition in SPS.

Immunobiology of stiff-person syndrome.

The two possibilities to explain the pathogenic basis of stiff-person syndrome (SPS) are intrathecal sensitization of GAD65-reactive CD4+T cells and synthesis of GAD65-specific autoantibodies within the CNS [Rakocevic et al., Arch. Neurol. 61: 902-904, 2004]; and peripheral antigen sensitization followed by CNS antigen recognition by autoantibodies that cross the blood-brain barrier. Antigen-specific CD4+ T cells are essential for the generation of high-affinity autoantibodies [Lanzavecchia, Nature 314: 537-539, 1985], but there is no evidence of cellular infiltration in the CNS of SPS patients [Warich-Kirches et al., Clin. Neuropathol. 16: 214-219, 1997; Ishizawa et al., Acta Neuropathol.(Berl) 97: 63-70, 1999]. This review discusses the possible role of autoantibodies and autoreactive T cells specific to neuronal antigens in SPS pathogenesis.

[Composition of the titin isoforms in muscles in pathology].

An electrophoretic study of changes in the composition of titin isoforms in skeletal and cardiac muscles of human and rat in pathology has been carried out. A more considerable decrease in the content of intact titin isoforms in comparison with the content of N2BA- and N2B-titins in hypertrophic heart of spontaneously hypertensive rats, N2A-titin in back muscle from patients with the "stiff-man syndrome", and in m. soleus of human and rats during the development of "muscle hypogravity syndrome" has been observed. In the last case, the relation between the reduction of titin content in m. soleus and the increase in the time the rats were in conditions of simulated microgravity has been revealed. On electrophoregrams of the left ventricle myocardium of patients with the terminal stage of dilated cardiomyopathy, intact titin and N2BA-titins were absent, and a considerable decrease in the content of N2B-titins has been observed, which could be a consequence of the terminal stage of pathology. Our results show that the development of the diseases is accompanied by a greater destruction of intact titin than of its other forms, which may be important for diagnostics of the pathological process.

The novel hyperekplexia allele GLRA1(S267N) affects the ethanol site of the glycine receptor.

Mutations in the GLRA1 gene, which encodes the alpha1-subunit of the inhibitory glycine receptor (GlyR), are the underlying causes in the majority of cases of hereditary startle disease (OMIM no. 149400). GlyRs are modulated by alcohols and volatile anesthetics, where a specific amino acid at position 267 has been implicated in receptor modulation. We describe a hyperekplexia family carrying the novel dominant missense allele GLRA1(S267N), that affects agonist responses and ethanol modulation of the mutant receptor. This study implies that a disease-related receptor allele carries the potential to alter drug responses in affected patients.

PET evidence of central GABAergic changes in stiff-person syndrome.

We measured expression of central nervous system GABA-A receptors with (11)C-flumazenil ((11)C-FMZ) and PET in two subjects with stiff person syndrome (SPS). We found reduced (11)C-FMZ binding potential (BP) in motor-premotor cortex, and increased (11)C-FMZ BP in the cerebellar nuclei. This is the first in vivo PET evidence of central GABA-A receptors dysfunction in SPS, possibly concurring to the motor symptoms.