A loss-of-function variant in canine GLRA1 associates with a neurological disorder resembling human hyperekplexia.

Hereditary hyperekplexia is a rare neuronal disorder characterized by an exaggerated startle response to sudden tactile or acoustic stimuli. In this study, we present a Miniature Australian Shepherd family showing clinical signs, which have genetic and phenotypic similarities with human hereditary hyperekplexia: episodes of muscle stiffness that could occasionally be triggered by acoustic stimuli. Whole genome sequence data analysis of two affected dogs revealed a 36-bp deletion spanning the exon-intron boundary in the glycine receptor alpha 1 (GLRA1) gene. Further validation in pedigree samples and an additional cohort of 127 Miniature Australian Shepherds, 45 Miniature American Shepherds and 74 Australian Shepherds demonstrated complete segregation of the variant with the disease, according to an autosomal recessive inheritance pattern. The protein encoded by GLRA1 is a subunit of the glycine receptor, which mediates postsynaptic inhibition in the brain stem and spinal cord. The canine GLRA1 deletion is located in the signal peptide and is predicted to cause exon skipping and subsequent premature stop codon resulting in a significant defect in glycine signaling. Variants in GLRA1 are known to cause hereditary hyperekplexia in humans; however, this is the first study to associate a variant in canine GLRA1 with the disorder, establishing a spontaneous large animal disease model for the human condition.

Clinical and Neurophysiologic Phenotypes in Neonates With BRAT1 Encephalopathy.

BACKGROUND AND OBJECTIVES: BRAT1 encephalopathy is an ultra-rare autosomal recessive neonatal encephalopathy. We delineate the neonatal electroclinical phenotype at presentation and provide insights for early diagnosis. METHODS: Through a multinational collaborative, we studied a cohort of neonates with encephalopathy associated with biallelic pathogenic variants in BRAT1 for whom detailed clinical, neurophysiologic, and neuroimaging information was available from the onset of symptoms. Neuropathologic changes were also analyzed. RESULTS: We included 19 neonates. Most neonates were born at term (16/19) from nonconsanguineous parents. 15/19 (79%) were admitted soon after birth to a neonatal intensive care unit, exhibiting multifocal myoclonus, both spontaneous and exacerbated by stimulation. 7/19 (37%) had arthrogryposis at birth, and all except 1 progressively developed hypertonia in the first week of life. Multifocal myoclonus, which was present in all but 1 infant, was the most prominent manifestation and did not show any EEG correlate in 16/19 (84%). Video-EEG at onset was unremarkable in 14/19 (74%) infants, and 6 (33%) had initially been misdiagnosed with hyperekplexia. Multifocal seizures were observed at a median age of 14 days (range: 1-29). During the first months of life, all infants developed progressive encephalopathy, acquired microcephaly, prolonged bouts of apnea, and bradycardia, leading to cardiac arrest and death at a median age of 3.5 months (range: 20 days to 30 months). Only 7 infants (37%) received a definite diagnosis before death, at a median age of 34 days (range: 25-126), and almost two-thirds (12/19, 63%) were diagnosed 8 days to 12 years postmortem (median: 6.5 years). Neuropathology examination, performed in 3 patients, revealed severely delayed myelination and diffuse astrogliosis, sparing the upper cortical layers. DISCUSSION: BRAT1 encephalopathy is a neonatal-onset, rapidly progressive neurologic disorder. Neonates are often misdiagnosed as having hyperekplexia, and many die undiagnosed. The key phenotypic features are multifocal myoclonus, an organized EEG, progressive, persistent, and diffuse hypertonia, and an evolution into refractory multifocal seizures, prolonged bouts of apnea, bradycardia, and early death. Early recognition of BRAT1 encephalopathy allows for prompt workup, appropriate management, and genetic counseling.

Four Turkish families with hyperekplexia: A missense mutation and the exon 1-7 deletion in the GLRA1 gene.

BACKGROUND: Hyperekplexia is a disease that progresses with excessive startle attacks and is included in the differential diagnosis of epilepsy and many movement disorders. METHODS: The WES results were validated in available family members by Sanger sequencing, or in the case of deletion, PCR followed by agarose gel electrophoresis was performed. RESULTS: WES analysis revealed the previously reported homozygous c.277C>T p.Arg93Trp variant in the GLRA1 gene (ENST00000455880.2) in Family 1. In all other three families, the previously reported homozygous deletion of exons 1-7 of the GLRA1 gene was identified using CNV analysis based on the WES data. CONCLUSIONS: The homozygous exon1-7 deletion has been described several times in different populations and may be a founder mutation in the Kurdish people in Turkey. The family with Arg93Trp variant stems from the Black Sea region of Turkey where close consanguinity is common. These analyses are important to provide genetic counseling to families and for a better understanding of the pathophysiology of the disease.

Clinical, genetic, and functional characterization of the glycine receptor ß-subunit A455P variant in a family affected by hyperekplexia syndrome.

Hyperekplexia is a rare neurological disorder characterized by exaggerated startle responses affecting newborns with the hallmark characteristics of hypertonia, apnea, and noise or touch-induced nonepileptic seizures. The genetic causes of the disease can vary, and several associated genes and mutations have been reported to affect glycine receptors (GlyRs); however, the mechanistic links between GlyRs and hyperekplexia are not yet understood. Here, we describe a patient with hyperekplexia from a consanguineous family. Extensive genetic screening using exome sequencing coupled with autozygome analysis and iterative filtering supplemented by in silico prediction identified that the patient carries the homozygous missense mutation A455P in GLRB, which encodes the GlyR ß-subunit. To unravel the physiological and molecular effects of A455P on GlyRs, we used electrophysiology in a heterologous system as well as immunocytochemistry, confocal microscopy, and cellular biochemistry. We found a reduction in glycine-evoked currents in N2A cells expressing the mutation compared to WT cells. Western blot analysis also revealed a reduced amount of GlyR ß protein both in cell lysates and isolated membrane fractions. In line with the above observations, coimmunoprecipitation assays suggested that the GlyR α1-subunit retained coassembly with ßA455P to form membrane-bound heteromeric receptors. Finally, structural modeling showed that the A455P mutation affected the interaction between the GlyR ß-subunit transmembrane domain 4 and the other helices of the subunit. Taken together, our study identifies and validates a novel loss-of-function mutation in GlyRs whose pathogenicity is likely to cause hyperekplexia in the affected individual.

Hyperekplexia: A Frequent Near Miss in Infants and Young Children.

Hyperekplexia, an underdiagnosed motor paroxysm of infancy, mimics epilepsy closely. It is hallmarked by episodic and excessive startle response, brief episodes of intense, generalized hypertonia, or stiffness in response to unexpected auditory and/or tactile stimuli right from birth. Though a seemingly benign entity with an excellent prognosis, hyperekplexia has been occasionally associated with recurrent apneas, feeding difficulties, and sudden infant death syndrome (SIDS). We describe three unrelated children with hyperekplexia (two SLC6A5; one GLRA1). All three children had the onset of motor paroxysms from the neonatal period and were initially labeled as drug-resistant epilepsy leading to a variable diagnostic delay, the longest being 2.5 years. An excellent response to oral clonazepam with a good neurodevelopmental outcome was observed. The lack of habituation on the nose-tapping test is a simple clinical clue to the diagnosis. Early differentiation from epilepsy minimizes treatment cost, allays caregiver anxiety, and empowers them with abortive measures.

Anesthetic Management of an Adult With Hyperekplexia Undergoing a Laparoscopic Colectomy: A Case Report.

Hyperekplexia is a rare genetic disorder characterized by an exaggerated startle response to innocuous stimuli. There are several case reports documenting the administration of general anesthesia to infants and children with hyperekplexia and 1 case report documenting the use of a labor epidural in a parturient. These cases suggest a possible resistance to depolarizing neuromuscular blocking agents and increased risk of malignant hyperthermia. There are no case reports of adults with hyperekplexia receiving general anesthesia. We report the case of a 20-year-old woman with hyperekplexia who safely received general anesthesia without neuromuscular blockade for a laparoscopic colectomy.

Advances in hyperekplexia and other startle syndromes.

Startle, a basic alerting reaction common to all mammals, is described as a sudden involuntary movement of the body evoked by all kinds of sudden and unexpected stimulus. Startle syndromes are heterogeneous groups of disorders with abnormal and exaggerated responses to startling events, including hyperekplexia, stimulus-induced disorders, and neuropsychiatric startle syndromes. Hyperekplexia can be attributed to a genetic, idiopathic, or symptomatic cause. Excluding secondary factors, hereditary hyperekplexia, a rare neurogenetic disorder with highly genetic heterogeneity, is characterized by neonatal hypertonia, exaggerated startle response provoked by the sudden external stimuli, and followed by a short period of general stiffness. It mainly arises from defects of inhibitory glycinergic neurotransmission. GLRA1 is the major pathogenic gene of hereditary hyperekplexia, along with many other genes involved in the function of glycinergic inhibitory synapses. While about 40% of patients remain negative genetic findings. Clonazepam, which can specifically upgrade the GABARA1 chloride channels, is the main and most effective administration for hereditary hyperekplexia patients. In this review, with the aim at enhancing the recognition and prompting potential treatment for hyperekplexia, we focused on discussing the advances in hereditary hyperekplexia genetics and the expound progress in pathogenic mechanisms of the glycinergic-synapse-related pathway and then followed by a brief overview of other common startle syndromes.

Teaching Video NeuroImage: Hereditary Hyperekplexia Mimicking Tonic Seizures in an Infant.

Hereditary hyperekplexia is a rare neurologic disorder characterized by an exaggerated startle response with profound muscle stiffness.1,2 Given the nature of the spells, this condition is often misdiagnosed as epilepsy. Mutations in glycine receptors and transporters are the primary cause of this syndrome.1 We present an example of stimulus-induced hyperekplexia captured on video EEG in a 7-week-old girl with compound heterozygous variants in the presynaptic glycine transporter gene SLC6A5.

Rescue of two trafficking-defective variants of the neuronal glycine transporter GlyT2 associated to hyperekplexia.

Hyperekplexia is a rare sensorimotor syndrome characterized by pathological startle reflex in response to unexpected trivial stimuli for which there is no specific treatment. Neonates suffer from hypertonia and are at high risk of sudden death due to apnea episodes. Mutations in the human SLC6A5 gene encoding the neuronal glycine transporter GlyT2 may disrupt the inhibitory glycinergic neurotransmission and cause a presynaptic form of the disease. The phenotype of missense mutations giving rise to protein misfolding but maintaining residual activity could be rescued by facilitating folding or intracellular trafficking. In this report, we characterized the trafficking properties of two mutants associated with hyperekplexia (A277T and Y707C, rat numbering). Transporter molecules were partially retained in the endoplasmic reticulum showing increased interaction with the endoplasmic reticulum chaperone calnexin. One transporter variant had export difficulties and increased ubiquitination levels, suggestive of enhanced endoplasmic reticulum-associated degradation. However, the two mutant transporters were amenable to correction by calnexin overexpression. Within the search for compounds capable of rescuing mutant phenotypes, we found that the arachidonic acid derivative N-arachidonoyl glycine can rescue the trafficking defects of the two variants in heterologous cells and rat brain cortical neurons. N-arachidonoyl glycine improves the endoplasmic reticulum output by reducing the interaction transporter/calnexin, increasing membrane expression and improving transport activity in a comparable way as the well-established chemical chaperone 4-phenyl-butyrate. This work identifies N-arachidonoyl glycine as a promising compound with potential for hyperekplexia therapy.

A proline-rich motif in the large intracellular loop of the glycine receptor α1 subunit interacts with the Pleckstrin homology domain of collybistin.

Introduction: The inhibitory glycine receptor (GlyR), a mediator of fast synaptic inhibition, is located and held at neuronal synapses through the anchoring proteins gephyrin and collybistin. Stable localization of neurotransmitter receptors is essential for synaptic function. In case of GlyRs, only beta subunits were known until now to mediate synaptic anchoring. Objectives: We identified a poly-proline II helix (PPII) in position 365-373 of the intra-cellular TM3-4 loop of the human GlyRα1 subunit as a novel potential synaptic anchoring site. The potential role of the PPII helix as synaptic anchoring site was tested. Methods: Glycine receptors and collybistin variants were generated and recombinantly expressed in HEK293 cells and cultured neurons. Receptor function was assessed using patch-clamp electrophysiology, protein-protein interaction was studied using co-immuno-precipitation and pulldown experiments. Results: Recombinantly expressed collybistin bound to isolated GlyRα1 TM3-4 loops in GST-pulldown assays. When the five proline residues P365A, P366A, P367A, P369A, P373A (GlyRα1P1-5A) located in the GlyRα1-PPII helix were replaced by alanines, the PPII secondary structure was disrupted. Recombinant GlyRα1P1-5A mutant subunits displayed normal cell surface expression and wildtype-like ion channel function, but binding to collybistin was abolished. The GlyRα1-collybistin interaction was independently confirmed by o-immunoprecipitation assays using full-length GlyRα1 subunits. Surprisingly, the interaction was not mediated by the SH3 domain of collybistin, but by its Pleckstrin homology (PH) domain. The mutation GlyRα1P366L, identified in a hyperekplexia patient, is also disrupting the PPII helix, and caused reduced collybistin binding. Conclusion: Our data suggest a novel interaction between α1 GlyR subunits and collybistin, which is physiologically relevant in vitro and in vivo and may contribute to postsynaptic anchoring of glycine receptors.

Abnormal neurodevelopment outcome in case of neonatal hyperekplexia secondary to missense mutation in GLRB gene.

Hyperekplexia is an exaggerated startle to external stimuli associated with a generalised increase in tone seen in neonates with both sporadic and genetic predisposition. This is an uncommon neurological entity that is misdiagnosed as seizure. A 28-days-old infant was admitted to us with characteristic intermittent generalised tonic spasm being treated as a seizure disorder. The infant had characteristic stiffening episode, exaggerated startle and non-habituation on tapping the nose. Hyperekplexia was suspected and confirmed by genetic testing (mutation in the ß subunit of glycine was found). Initial improvement was seen with the use of clonazepam, which was not sustained. At the age of 4.5 years, the child is still having neurobehavioural issues like hyperactivity and sensory hyper-responsiveness. Usually, hyperekplexia is benign in nature. We report a case of hyperekplexia with non-sense mutation in the ß subunit of GlyR gene having abnormal neurodevelopmental findings at 4.5 years.

Charged residues at the pore extracellular half of the glycine receptor facilitate channel gating: a potential role played by electrostatic repulsion.

KEY POINTS: The Arg271Gln mutation of the glycine receptor (GlyR) causes hereditary hyperekplexia. This mutation dramatically compromises GlyR function; however, the underlying mechanism is not yet known. This study, by employing function and computation methods, proposes that charged residues (including the Arg residue) at the pore extracellular half from each of the five subunits of the homomeric α1 GlyR, create an electrostatic repulsive potential to widen the pore, thereby facilitating channel opening. This mechanism explains how the Arg271Gln mutation, in which the positively charged Arg residue is substituted by the neutral Gln residue, compromises GlyR function. This study furthers our understanding of the biophysical mechanism underlying the Arg271Gln mutation compromising GlyR function. ABSTRACT: The R271(19')Q mutation in the α1 subunit of the glycine receptor (GlyR) chloride channel causes hereditary hyperekplexia. This mutation dramatically compromises channel function; however, the underlying mechanism is not yet known. The R271 residue is located at the extracellular half of the channel pore. In this study, an Arg-scanning mutagenesis was performed at the pore extracellular half from the 262(10') to the 272(20') position on the background of the α1 GlyR carrying the hyperekplexia-causing mutation R271(19')Q. It was found that the placement of the Arg residue rescued channel function to an extent inversely correlated with the distance between the residue and the pore central axis (perpendicular to the plane of the lipid bilayer). Accordingly, it was hypothesized that the placed Arg residues from each of the five subunits of the homomeric α1 GlyR create an electrostatic repulsive potential to widen the pore, thereby facilitating channel opening. This hypothesis was quantitatively verified by theoretical computation via exploiting basic laws of electrostatics and thermodynamics, and further supported by more experimental findings that the placement of another positively charged Lys residue or even a negatively charged Asp residue also rescued channel function in the same manner. This study provides a novel mechanism via which charged residues in the pore region facilitate channel gating, not only for the disease-causing 19'R residue in the GlyR, but also potentially for charged residues in the same region of other ion channels.

Hyperekplexia and other startle syndromes.

Abnormal startle syndromes are classified into hyperekplexia, stimulus-induced, and neuropsychiatric startle syndromes. Hyperekplexia is attributed to a genetic, idiopathic, or symptomatic cause. Hereditary hyperekplexia is a treatable neurogenetic disorder. In patients with a hyperactive startle response, the first step is to characterize the extent and associations of 'response.' Secondary or symptomatic causes are particularly important in children, as they provide useful clinical clues to an underlying neurodevelopmental or neurodegenerative disorders. Particular attention should be given to any neonate or infant with generalized or episodic stiffness, drug-refractory seizures, recurrent apnea, stimulus-sensitive behavioral states, or sudden infant death syndrome. Eliciting a non-habituating head-retraction reflex to repeated nose tapping should be a part of routine examination of all new-borns. Vigevano maneuver should be taught to all families and health-care workers as an emergency rescue measure. The onset of excessive startle after infancy should direct investigations for an acquired cause such as brainstem injury, antibodies against glycine receptors, and neurodegeneration. Finally, one should not forget to evaluate unexplained cases of abnormal gait and frequent falls in adults for underlying undiagnosed startle syndromes. Oral clonazepam is an effective therapy besides behavioral and safety interventions for hereditary cases. The outcomes in genetic cases are good overall.

Top of Basilar Syndrome Presenting With Hyperekplexia Initially Diagnosed as a Convulsive Status Epilepticus.

BACKGROUND: Hyperekplexia is a rare neurologic disorder characterized by pronounced startle responses to tactile or acoustic stimuli and increase tone. Acquired hyperekplexia is usually seen in brainstem pathologies and when it develops acutely it can be easily misdiagnosed as a convulsive seizure. CASE REPORT: A 38-year-old man presented with acute onset generalized brief involuntary jerky movements and a decreased level of consciousness. He was initially diagnosed with convulsive status epilepticus for which he received multiple antiseizure medications without any improvement. Further investigations revealed abnormal oculocephalic reflex response and that his movements were in fact hyperkeplexia caused by brainstem infarction with basilar artery thrombus secondary to right vertebral artery dissection. Emergent thrombectomy was performed and he was eventually discharged to a rehabilitation facility. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Emergency physicians should be aware of hyperekplexia and how to differentiate it from convulsive stats epilepticus because the pathology and the emergent treatment of these 2 serious conditions are different. An underlying acquired brainstem pathology (especially basilar artery thromboembolism) should be suspected in any patient with untypical convulsive like movements along with focal neurologic signs compatible with brain stem pathology even when computed tomography imaging is normal. © 2020 Elsevier Inc.

The startle disease mutation α1S270T predicts shortening of glycinergic synaptic currents.

KEY POINTS: Loss-of-function mutations in proteins found at glycinergic synapses, most commonly in the α1 subunit of the glycine receptor (GlyR), cause the startle disease/hyperekplexia channelopathy in man. It was recently proposed that the receptors responsible are presynaptic homomeric GlyRs, rather than postsynaptic heteromeric GlyRs (which mediate glycinergic synaptic transmission), because heteromeric GlyRs are less affected by many startle mutations than homomers. We examined the α1 startle mutation S270T, at the extracellular end of the M2 transmembrane helix. Recombinant heteromeric GlyRs were less impaired than homomers by this mutation when we measured their response to equilibrium applications of glycine. However, currents elicited by synaptic-like millisecond applications of glycine to outside-out patches were much shorter (7- to 10-fold) in all mutant receptors, both homomeric and heteromeric. Thus, the synaptic function of heteromeric receptors is likely to be impaired by the mutation. ABSTRACT: Human startle disease is caused by mutations in glycine receptor (GlyR) subunits or in other proteins associated with glycinergic synapses. Many startle mutations are known, but it is hard to correlate the degree of impairment at molecular level with the severity of symptoms in patients. It was recently proposed that the disease is caused by disruption in the function of presynaptic homomeric GlyRs (rather than postsynaptic heteromeric GlyRs), because homomeric GlyRs are more sensitive to loss-of-function mutations than heteromers. Our patch-clamp recordings from heterologously expressed GlyRs characterised in detail the functional consequences of the α1S270T startle mutation, which is located at the extracellular end of the pore lining M2 transmembrane segment (18'). This mutation profoundly decreased the maximum single-channel open probability of homomeric GlyRs (to 0.16; cf. 0.99 for wild type) but reduced only marginally that of heteromeric GlyRs (0.96; cf. 0.99 for wild type). However, both heteromeric and homomeric mutant GlyRs became less sensitive to the neurotransmitter glycine. Responses evoked by brief, quasi-synaptic pulses of glycine onto outside-out patches were impaired in mutant receptors, as deactivation was approximately 10- and 7-fold faster for homomeric and heteromeric GlyRs, respectively. Our data suggest that the α1S270T mutation is likely to affect the opening step in GlyR activation. The faster decay of synaptic currents mediated by mutant heteromeric GlyRs is expected to reduce charge transfer at the synapse, despite the high equilibrium open probability of these mutant channels.

Approach to exaggerated startle reflex: a case of hyperekplexia minor.

A broad set of conditions may present with an exaggerated startle reflex in clinics. This, combined with the overall rarity of these disorders, may pose diagnostic uncertainty in the mind of the treating physician. Herein, we report a case of a patient who presented to us with the complaint of exaggerated startle reflex and outline a simple approach towards characterisation of these disorders.