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, 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.

The synthetic cannabinoid dehydroxylcannabidiol restores the function of a major GABAA receptor isoform in a cell model of hyperekplexia.

The functions of the glycine receptor (GlyR) and GABAA receptor (GABAAR) are both impaired in hyperekplexia, a neurological disorder usually caused by GlyR mutations. Although emerging evidence indicates that cannabinoids can directly restore normal GlyR function, whether they affect GABAAR in hyperekplexia remains unknown. Here we show that dehydroxylcannabidiol (DH-CBD), a synthetic nonpsychoactive cannabinoid, restores the GABA- and glycine-activated currents (IGABA and IGly , respectively) in HEK293 cells coexpressing a major GABAAR isoform (α1ß2γ2) and GlyRα1 carrying a human hyperekplexia-associated mutation (GlyRα1R271Q). Using coimmunoprecipitation and FRET assays, we found that DH-CBD disrupts the protein interaction between GABAAR and GlyRα1R271Q Furthermore, a point mutation of GlyRα1, changing Ser-296 to Ala-296, which is critical for cannabinoid binding on GlyR, significantly blocked DH-CBD-induced restoration of IGABA and IGly currents. This S296A substitution also considerably attenuated DH-CBD-induced disruption of the interaction between GlyRα1R271Q and GABAAR. These findings suggest that, because it restores the functions of both GlyRα1 and GABAAR, DH-CBD may represent a potentially valuable candidate drug to manage hyperekplexia.

Novel mutations in SLC6A5 with benign course in hyperekplexia.

Infants suffering from life-threatening apnea, stridor, cyanosis, and increased muscle tone may often be misdiagnosed with infantile seizures and inappropriately treated because of lack and delay in genetic diagnosis. Here, we report a patient with increased muscle tone after birth and hypertonic attacks with life-threatening apnea but no epileptiform patterns in EEG recordings. We identified novel compound heterozygous variants in SLC6A5 (NM_004211.4:c.[1429T > C];[1430delC]) by trio whole-exome sequencing, containing a base deletion inherited by the asymptomatic mother leading to a frameshift (c.1430delC, p.Ser477PhefsTer9) and a de novo base exchange leading to an amino acid change (c.1429T > C, p.Ser477Pro). To date, there are four known disease-associated genes for primary hyperekplexia, all of which are involved in the functioning of glycinergic synapses. SLC6A5 encodes the sodium- and chloride-dependent glycine transporter 2 (GlyT2), which recaptures glycine, a major inhibitory transmitter in the brainstem and spinal cord. The diagnosis altered the patient's medical care to his benefit because SLC6A5 mutations with rather benign courses of hyperekplexia may be spared of needless pharmacotherapy. Symptoms eventually decreased in frequency until about once in 2 mo at 2 yr age. We present the first report of halting hyperekplexia episodes by maternal soothing in multiple instances. We highlight the importance of clarifying the genetic diagnosis by rapid next-generation sequencing techniques in this group of infantile apneic attacks with hyperekplexia due to the broad differential diagnoses.

Hijacking of GABAA Receptors by Mutant Glycine Receptors.

Startle disease results from mutations in genes encoding inhibitory GlyR α1 and ß subunits or the presynaptic glycine transporter GlyT2. However, the most effective therapies are benzodiazepines that potentiate inhibitory GABAAR function. A recent publication by Zou et al. adds further complexity by suggesting that dominant GlyR α1 mutants assemble into pre- and extrasynaptic GABAARs.

The P429L loss of function mutation of the human glycine transporter 2 associated with hyperekplexia.

Glycine transporter 2 (GlyT2) mutations across the entire sequence have been shown to represent the presynaptic component of the neurological disease hyperekplexia. Dominant, recessive and compound heterozygous mutations have been identified, most of them leading to impaired glycine uptake. Here, we identified a novel loss of function mutation of the GlyT2 resulting from an amino acid exchange of proline 429 to leucine in a family with both parents being heterozygous carriers. A homozygous child suffered from severe neuromotor deficits. We characterised the GlyT2P429L variant at the molecular, cellular and protein level. Functionality was determined by glycine uptake assays. Homology modelling revealed that the mutation localises to α-helix 5, presumably disrupting the integrity of this α-helix. GlyT2P429L shows protein trafficking through various intracellular compartments to the cellular surface. However, the protein expression at the whole cell level was significantly reduced. Although present at the cellular surface, GlyT2P429L demonstrated a loss of protein function. Coexpression of the mutant with the wild-type protein, reflecting the situation in the parents, did not affect transporter function, thus explaining their non-symptomatic phenotype. Nevertheless, when the mutant was expressed in excess compared with the wild-type protein, glycine uptake was significantly reduced. Thus, these data demonstrate that the proline residue at position 429 is structurally important for the correct formation of α-helix 5. The failure in functionality of the mutated GlyT2 is most probably due to structural changes localised in close proximity to the sodium-binding site of the transporter.

Impaired glycinergic transmission in hyperekplexia: a model of parasomnia overlap disorder.

We report sleep phenotypes and polysomnographic findings in two siblings with a novel homozygous variant of the GLRA1 gene causing hereditary hyperekplexia (HH). Both sisters had startles during wakefulness and sleep, sleep terrors, and one had symptoms of REM sleep behavior disorder (RBD). Frequent startles were found in NREM sleep associated with NREM parasomnias in deep sleep. In REM sleep, both had motor behaviors and increased phasic/tonic muscle activities confirming RBD. Clonazepam improved startles, motor behaviors, and muscle activities in REM sleep. Impaired glycinergic transmission in human HH could be involved in the pathophysiology of RBD and NREM parasomnias.

Clinical features and genetic analysis of two siblings with startle disease in an Italian family: a case report.

BACKGROUND: Hyperekplexia also known as Startle disease is a rare neuromotor hereditary disorder characterized by exaggerated startle responses to unexpected auditory, tactile, and visual stimuli and generalized muscle stiffness, which both gradually subside during the first months of life. Although the diagnosis of Hyperekplexia is based on clinical findings, pathogenic variants in five genes have been reported to cause Hyperekplexia, of which GLRA1 accounts for about 80% of cases. Dominant and recessive mutations have been identified in GLRA1 gene as pathogenic variants in many individuals with the familial form of Hyperekplexia and occasionally in simplex cases. CASE PRESENTATION: In the present study, we describe clinical and genetic features of two Italian siblings, one with the major and one with the minor form of the disease. DNA samples from the probands and their parents were performed by NGS approach and validated by Sanger sequencing. The analysis of the GLRA1 gene revealed, in both probands, compound heterozygous mutations: c.895C > T or p.R299X inherited from the mother and c.587C > A or p.D98E inherited from the father. CONCLUSIONS: Until now, these two identified mutations in GLRA1 have not been reported before as compound mutations. What clearly emerges within our study is the clinical heterogeneity in the same family. In fact, even though in the same pedigree, the affected mother showed only mild startle responses to unexpected noise stimuli, which might be explained by variable expressivity, while the father, showed no clear signs of symptomatology, which might be explained by non-penetrance. Finally, the two brothers have different form of the disease, even if the compound heterozygous mutations in GLRA1 are the same, showing that the same mutation in GLRA1 could have different phenotypic expressions and suggesting an underling mechanism of variable expressivity.

Hyperekplexia-associated mutations in the neuronal glycine transporter 2.

Hyperekplexia or startle disease is a dysfunction of inhibitory glycinergic neurotransmission characterized by an exaggerated startle in response to trivial tactile or acoustic stimuli. Although rare, this disorder can have serious consequences, including sudden infant death. One of the most frequent causes of hyperekplexia are mutations in the SLC6A5 gene, encoding the neuronal glycine transporter 2 (GlyT2), a key component of inhibitory glycinergic presynapses involved in synaptic glycine recycling though sodium and chloride-dependent co-transport. Most GlyT2 mutations detected so far are recessive, but two dominant missense mutations have been described. The detailed analysis of these mutations has revealed structural cues on the quaternary structure of GlyT2, and opens the possibility that novel selective pharmacochaperones have potential therapeutic effects in hyperekplexia.

A novel nonsense autosomal dominant mutation in the GLRA1 gene causing hyperekplexia.

We present a family with two members affected by hyperekplexia and two unaffected members. All exons in the glycine receptor alpha 1 subunit gene (GLRA1) were sequenced in all four family members. Our index patient harbored a novel nonsense mutation (p.Trp314*; rs867618642) in the transmembrane domain three of the GLRA1 and a novel missense variant in the NH2-terminal part (p.Val67Met; rs142888296). After development of tolerance for the effective treatment with clobazam a drug holiday led to a sustained restoration of the treatment response.

Neonatal tremor episodes and hyperekplexia-like presentation at onset in a child with SCN8A developmental and epileptic encephalopathy.

SCN8A encephalopathy is a newly defined epileptic encephalopathy caused by de novo mutations of the SCN8A gene. We report herein a four-year-old boy presenting with severe non-epileptic abnormal movements, of possibly antenatal onset, progressively associated with pharmacoresistant epilepsy and regression, associated with a de novo heterozygous missense mutation of SCN8A. This case shows that paroxysmal non-epileptic episodes of severe tremor and hyperekplexia-like startles and a striking vegetative component can be the first early symptoms of severe SCN8A developmental and epileptic encephalopathy. Clinicians should be aware of these symptoms in order to avoid misdiagnosis and ensure early appropriate therapeutic management. [Published with video sequences on www.epilepticdisorders.com].