Hyperekplexia: a treatable neurogenetic disease

Brain & Development 24 (2002) 669–674 www.elsevier.com/locate/braindev

Review article

Hyperekplexia: a treatable neurogenetic disease Lan Zhou, Kipp L. Chillag, Michael A. Nigro* Children’s Hospital of Michigan, Wayne State University School of Medicine, 3901 Beaubien Boulevard, Detroit, MI 48201, USA Received 23 April 2002; accepted 29 May 2002

Abstract Hyperekplexia is primarily an autosomal dominant disease characterized by exaggerated startle reflex and neonatal hypertonia. It can be associated with, if untreated, sudden infant death from apnea or aspiration pneumonia and serious injuries and loss of ambulation from frequent falls. Different mutations in the a1 subunit of inhibitory glycine receptor (GLRA1) gene have been identified in many affected families. The most common mutation is Arg271 reported in at least 12 independent families. These mutations uncouple the ligand binding and chloride channel function of inhibitory glycine receptor and result in increased excitability in pontomedullary reticular neurons and abnormal spinal reciprocal inhibition. Three mouse models from spontaneous mutations in GLRA1 and b subunit of inhibitory glycine receptor (GLRB) genes and two transgenic mouse models are valuable for the study of the pathophysiology and the genotype–phenotype correlation of the disease. The disease caused by mutation in GLRB in mice supports the notion that human hyperekplexia with no detectable mutations in GLRA1 may harbor mutations in GLRB. Clonazepam, a gamma aminobutyric acid (GABA) receptor agonist, is highly effective and is the drug of choice. It enhances the GABA-gated chloride channel function and presumably compensates for the defective glycinegated chloride channel in hyperekplexia. Recognition of the disease will lead to appropriate treatment and genetic counseling. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Hyperekplexia; Inhibitory glycine receptor; Gene mutation; Mouse model; Clonazepam

1. Introduction Hyperekplexia, also known as hereditary startle disease, is a rare neurogenetic disorder characterized by exaggerated startle response and neonatal hypertonia [1,2]. It was first reported in 1958 by Kirstein and Silfverskiold as ‘emotionally precipitated drop seizure’ [3]. It was subsequently reported by Suhren et al. in 1966 using the Greek term ‘hyperexplexia’ [4] and was corrected to ‘hyperekplexia’ a year later by Gastaut and Villeneuve [5]. The other terms used in the past to report the same disease included ‘congenital stiff-man syndrome’ [6] and ‘hereditary stiff-baby syndrome’ [7]. In this article, we review the clinical features, genetic causes, animal models, pathophysiology, and treatment of hyperekplexia.

2. Clinical features Hyperekplexia is predominantly an autosomal dominant disease with much fewer autosomal recessive and sporadic cases reported. The disease is rare and the prevalence * Corresponding author. Tel.: 11-248-553-0010; fax: 11-248-553-5957. E-mail address: [email protected] (M.A. Nigro).

remains unknown. It mainly affects northern European descendants, although two Japanese families have been reported as well [8]. Newborns with hyperekplexia manifest diffuse hypertonia, hyperreflexia, and exaggerated startle response to noise and handling shortly after birth [3,9]. The startle attack can be easily elicited by nose tapping that persists with repetitive stimuli and constitutes sudden head retraction and body tonic flexion. The tactile stimuli from feeding in newborns and infants can induce oropharyngeal incoordination and poor air exchange that often result in apnea or aspiration pneumonia and consequently sudden infant death [9–11]. The recurrent abdominal muscle contraction from exaggerated startle response can increase the abdominal pressure and cause a high incidence of inguinal hernia. The hypertonicity and hyperreflexia are transient and usually diminish spontaneously after the first year of life, suggesting maturation of a compensatory mechanism. The acquisition of early motor milestones is often delayed due to hypertonicity, but catch-up occurs as the muscle stiffness diminishes. The patients may refuse to ambulate in their early childhood because of the fear of frequent falls from exaggerated startle reflex that usually persists into adult life [1]. In the pedigree we studied, the young children had the peculiar feature of

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ambulating on their knees prior to attaining the usual bipedal gait. Children with hyperekplexia often develop episodic nocturnal clonic limb jerking around age 5. Each episode lasts for several minutes. The lower extremities are more severely involved than upper extremities. This nocturnal clonic jerking is not epileptic in nature and does not respond to antiepiletic drugs [1]. Untreated adults with hyperekplexia can be severely debilitated and eventually wheelchair-bound to avoid severe injury from frequent falls with excessive startle response [1,4]. The abnormal startle response is usually triggered by loud or unexpected noises and constitutes sudden stiffness and fall to the ground with arms by two sides and without loss of consciousness. Patients can suffer severe facial laceration, skull or limb fractures from the attacks. They usually have uncertain gait with the fear of falling [1,4]. When Suhren et al. [4] reported 25 members with hyperekplexia in a Dutch family of five generations in 1966, they described two forms: the minor form with only inconstant excessive startle response while the major form with additional clinical features as mentioned above. However, the minor form lacks the genetic mutation and the typical electrophysiological abnormalities as detected in the major form [12–14], suggesting that it could just represent a normal variant. The intelligence of patients with hyperekplexia is usually normal or mildly impaired. Profound mental retardation or seizure is not a feature. The sporadic cases often display these atypical features and do not harbor common genetic mutations of the disease [15]. The presence of atypical features in these cases suggests different genetic or nongenetic etiologies, such as brainstem tumor, infarction, or infection [16–19]. The diagnosis of hyperekplexia is not difficult as long as one is aware of this disease. However, it is often misdiagnosed as spastic quadriplegia, epilepsy, or cerebellar disorder due to infantile hypertonicity, tonic spasm during the attack, nocturnal clonic limb jerking, and unsteady gait [1]. Therefore, nose tapping should be included in the routine examination when patients present neonatal or infantile hypertonicity, hyperreflexia, episodic tonic flexion, or apnea [9]. The genetic testing of hyperekplexia is currently available only in research laboratories.

3. Genetic causes In 1992, the hyperekplexia gene was linked to the long arm of chromosome 5 (5q33-35) by a linkage study in a large kindred with autosomal dominant hyperekplexia [20]. This locus contains several neurotransmitter receptor genes, including two GABA receptor subunit (GABRA1 and GABRG2) genes, a glutamate receptor gene and an aadrenergic receptor gene. Radiation hybrid mapping in four

unrelated hyperekplexia families, including one family we had been following, further localized the gene to the area between the markers CSF1R and D5S379 [21], where the a1 subunit of the inhibitory glycine receptor (GLRA1) gene was subsequently mapped to [22]. GLRA1 was proved to be the defective gene in hyperekplexia based on two findings. First, the plant alkaloid strychnine, a GLRA1 antagonist, caused exaggerated startle response and hypertonia in mouse, resembling human hyperekplexia [23]. Second, mutations in GLRA1 gene were detected in many affected families [24]. The inhibitory glycine receptor is a member of neurotransmitter-gated ion channel superfamily that includes GABA, glutamate, and nicotinic acetylcholine receptors. It is a ligand-gated chloride channel provoking postsynaptic hyperpolarization which mediates synaptic inhibition in brainstem and spinal cord where it is primarily expressed. Human inhibitory glycine receptor has three well-characterized subunits, a1, a2 (GLRA2), and b that assemble into a pentameric complex mediating chloride channel function [25]. Two inhibitory glycine receptor isoforms have been identified in mammals. The neonatal isoform is a homopentamer of a2 subunits, while the adult isoform is a hetero-pentamer comprising three a1 subunits and two b subunits [26]. Human GLRA2 gene has been mapped to chromosome Xp [27] and GLRB gene to chromosome 4q [28]. GLRA1 gene has nine exons encoding a transmembrane protein with four membrane-spanning domains, M1–M4 [24,29]. It has a short intracellular loop between M1 and M2, a short extracellular loop between M2 and M3, and a long intracellular loop between M3 and M4. The long Nterminal domain containing the ligand binding sites and a short C-terminal domain are extracellularly located. Upon ligand binding, the M2 domains undergo conformational change for chloride entry and the M1–M2 and M2–M3 loops facilitate this change [30]. The M2–M3 loop contains positively charged amino acids, including Arg271, and functions as the mouth of the chloride channel to allow chloride binding and entry [24]. Several missense mutations of GLRA1 gene have been identified in families with autosomal dominant hyperekplexia. These missense mutations result in amino acid changes, including Pro250Thr [31], Val260Met [32], Gln266His [33], Arg271Leu and Arg271Gln [12,15,24,34,41,42], Lys276Glu [35], and Tyr279Cys [15]. These mutations are mainly located in M1–M2 and M2–M3 loops, exerting dominant negative effects by uncoupling ligand binding and chloride channel function [30,36–40]. The most common mutations are G1192T and G1192A in exon 6 that result in the substitution of uncharged amino acids (leucine and glutamine, respectively) for Arg271, a highly conserved amino acid among different species. The Arg271 mutations have been reported in at least 12 independent families from Netherlands, US, UK, and Switzerland [12,15,24,34,41,42]. Arg271Leu and Arg271Gln have

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been demonstrated to reduce the ligand sensitivity of the receptor [30]. Three reported autosomal recessive hyperekplexia cases are also caused by mutations in GLRA1 gene. All these patients were born to consanguineous parents. One is caused by Ile244Asn mutation in M1 domain [34]. The other two are caused by homozygous null mutations with a large deletion of exons 1–6 in one patient [44] and Y202Stop missense mutation in the other [43]. Ile244Asn probably renders GLRA1 unable to integrate into the glycine receptor complex and thus causes recessive disease [34]. Most cases of the sporadic hyperekplexia do not carry mutations in GLRA1 gene, although compound heterozygous mutations in GLRA1 gene have been described in two reports. One report described two siblings inheriting Arg252His from their father and Arg392His from their mother [45]. The other report presented a patient carrying a paternally derived Met147Val and a maternally derived 1 base pair deletion (delC 601-605) [43]. The disease in these cases is caused by the combination of two different loss of function mutations in GLRA1 alleles. This mechanism may be responsible for more sporadic cases if more frequently searched for. Since GLRB is part of the adult form of inhibitory glycine receptor complex, it is tempting to speculate that mutations in GLRB gene might contribute to some cases of hyperekplexia with no detectable mutations in GLRA1 gene. However, no such cases have been reported yet. Gephyrin, a 93 kDa protein, has recently been demonstrated of playing an essential role in clustering and anchoring inhibitory glycine receptor complex [46]. Gephyrin deficient mice display some features of hyperekplexia such as hypertonicity and exaggerated startle response [47]. Therefore, it is possible that mutations in Gephyrin gene may account for some atypical hyperekplexia.

4. Animal models Three hyperekplexia mouse models have been identified resulting from spontaneous mutations. Unlike human hyperekplexia that mainly is an autosomal dominant disease, all three mouse models display autosomal recessive inheritance [21,48,49], indicating that they harbor loss of function mutations. Spasmoid mouse (spd) harbors Ala52Ser mutation at Nterminal of GLRA1, the gene being mapped to mouse chromosome 11 [21]. The mechanism by which spd mutation affects the receptor is unknown. The mutation does not affect ligand binding, receptor assembly, receptor gating, or GLRA1 expression. It might affect the interaction of GLRA1 with other components of the receptor complex. Spastic mouse (spa) bears an insertion of a 7.1 kb retrotransposon LINE-1 element within intron 6 of GLRB gene located in mouse chromosome 3 [48]. This mutation causes down-regulation of GLRB mRNA with no evidence of abnormal splicing or premature termination.

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Spd and spa are phenotypically identical and bear striking similarities to human hyperekplexia, including exaggerated startle reflex, muscle rigidity, and frequent falls [21,48]. This supports the hypothesis that some human hyperekplexia cases without mutations in the coding region of GLRA1 gene might harbor mutations in the non-coding region of GLRA1 gene or in GLRB gene. Despite phenotypes similar to human hyperekplexia, spd and spa usually exhibit the symptoms 2 weeks after birth corresponding to a rapid transition from neonatal inhibitory glycine receptor isoform to the adult one at this developmental stage. However, human patients with hyperekplexia usually express the phenotype right after birth and the developmental regulation of the isoform transition in human has not been well studied. It most likely takes place at prenatal stage. The third mouse model with spontaneous mutation, oscillator, is caused by a microdeletion in GLRA1 [49]. This seven base pair deletion causes a frameshift resulting in loss of highly conserved M3–M4 loop and M4 transmembrane domain. Like spd and spa, homozygous oscillator mice appear normal during the first 2 weeks of postnatal life. But they subsequently develop a rapid progression of rigidity and tremor, spastic gait, exaggerated startle response, and usually die within 10 days. Human GLRA1 null mutation, however, has mild phenotype and is not neonatal lethal, indicating a better compensatory mechanism for the loss of GLRA1 in human [34,43,44]. These mouse models are valuable for the study of pathophysiology in hyperekplexia. Two transgenic mouse models of hyperekplexia have been generated recently [50,51]. Expression of wild-type GLRB in spa reduced hyperekplexia phenotype in a dosedependent manner and more closely resembled human hyperekplexia than spa [50]. Exogenous expression of mutant GLRA1 with Arg271Gln, the most common mutation in human hyperekplexia, caused phenotype similar to the human disease [51]. This finding confirms the dominant negative role of Arg271Gln mutation. It will be interesting to see whether the knock-in of mutant GLRA1 with Arg271Gln would cause hyperekplexia with autosomal dominant inheritance.

5. Pathophysiology Hyperekplexia does not appear to have gross or microscopic pathology in the nervous system. No histopathological abnormalities have been identified in hyperekplexia mouse model [48]. Likewise, head computerized tomography (CT) of human patients is unremarkable [1]. Extensive electrophysiological studies have been performed in patients and in the mouse models to characterize the physiological abnormalities. Electromyographic (EMG) reflex studies, recording the response of head and limb muscles to acoustic and tactile stimuli, have demon-

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strated higher sensitivity, shorter latency, higher muscle response amplitude, and much less habituation in hyperekplexia as compared to normal controls [13,52–55]. The earliest responder to the stimuli is the sternocleidomastoid muscle, suggesting a brainstem origin of the reflex. There is no aberrant electrophysiological response other than exaggerated normal startle response. The abnormality is unlikely to be located in auditory or somatic sensory pathways because brainstem auditory evoked responses (BAER) and somatic sensory evoked potential (SSEP) are essentially unremarkable [54]. These findings strongly support the notion that the primary physiological abnormality in hereditary hyperekplexia is a lowered threshold and increased excitability in pontomedullary reticular neurons that result in a widespread elevated gain of vestigial withdrawal reflexes [54]. Floeter et al. [56] have studied the spinal inhibitory pathways in patients with hyperekplexia. Inhibitory glycine receptor is highly expressed in the spinal cord by inhibitory interneurons, including Renshaw cells, Ia, and some Ib interneurons, mediating several types of inhibition of stretch reflexes. Patients with hyperekplexia have abnormal reciprocal inhibition and normal recurrent inhibition, indicating that dysynaptic reciprocal inhibition is primarily mediated through glycinergic interneurons while monosynaptic recurrent inhibition has a non-glycinergic contribution, presumably from GABA-gated chloride channel. However, Arg271Gln caused reduction of both glycine receptor and GABA receptor mediated inhibitory transmission in spinal cord ventral horn of the transgenic mice [51]. The abnormal reciprocal inhibition may contribute to muscle stiffness in hyperekplexia. The other electrophysiological studies are unremarkable, including normal or non-specific spikes and waves during tonic spasm on electroencephalography (EEG), normal nerve conduction studies, and normal needle EMG [1,9,54]. There are a few reports of large SSEP and long-loop reflexes (‘C-responses’) in hyperekplexia patients that may indicate increased cortical neuronal excitability [57,58]. In favor of this notion, hyperekplexia patients display striking tolerability to high dose of clonazepam, a GABARA1 agonist. This issue has been addressed by magnetic resonance spectroscopy (MRS) study in four unrelated patients and 20 healthy controls [59]. The ratio of Nacetylaspartate (NAA)/choline (Cho) 1 creatine (Cr) was reduced in frontal and central areas of patients, suggesting the existence of frontal neuronal dysfunction in hyperekplexia. However, these findings could also be epiphenomena of brainstem abnormalities. A large-scale study is needed to obtain more conclusive results.

6. Treatment Fortunately, hyperekplexia is a highly treatable disease as opposed to the majority of neurogenetic disorders. Clona-

zepam is the drug of choice that dramatically diminishes exaggerated startle response and consequently reduces morbidities and mortalities associated with the disease. However, it does not reduce infantile hypertonicity to the same degree. Patients usually require high doses (0.1– 0.2 mg/kg/day) of clonazepam and tolerate it very well without losing effectiveness with time [1,2,9]. Clonazepam is a GABARA1 agonist, enhancing GABA-gated chloride channel function and presumably compensating for the defective glycine-gated chloride channel function in hyperekplexia. The association of apnea and sudden infant death requires immediate monitoring of at-risk infants and initiation of clonazepam as emphasized in our previous report [9]. Newborns with hyperekplexia should be discharged home with apnea monitoring after clinical improvement achieved with clonazepam. Hyperekplexia should be considered in the evaluation of neonates and infants with diffuse muscular rigidity, episodic tonic spasm, apnea, aspiration pneumonia, and near-miss sudden infant death syndrome. Hyperekplexic startle response to nose tapping should be included in the routine examination of all newborns. If the disease is misdiagnosed as seizure, treatment will be futile as the commonly used anti-convulsants are not benzodiazepines and are ineffective [1]. Several other drugs have been tried but their effectiveness has not been well established. Vigabatrin, a GABA transaminase inhibitor, fails to improve the startle activity in a double-blind and placebo-controlled study in four patients [60]. The effectiveness of valproic acid [61], clobazam [62], and fluoxitin [63] has been reported in a few sporadic cases that may have different genetic or non-genetic etiologies. 7. Conclusions Hyperekplexia is a treatable neurogenetic disease and clonazepam is the treatment of choice. It is characterized by exaggerated startle reflex and infantile hypertonicity due to increased excitability in pontomedullary reticular neurons and abnormal spinal reciprocal inhibition. This disease is caused by mutations in GLRA1. It can also be caused by mutation in GLRB in the mouse model. Recognition of the disease is essential for appropriate treatment and genetic counseling. References [1] Andermann F, Keene DL, Andermann E, Quesney LF. Startle disease or hyperekplexia: future delineation of the syndrome. Brain 1980;103:985–997. [2] Praveen V, Patole SK, Whitehall JS. Hyperekplexia in neonates. Postgrad Med J 2001;77:570–572. [3] Kirstein L, Silfverskiold BP. A family with emotionally precipitated ‘drop seizure’. Acta Psychiatr Neurol Scand 1958;33:471–476. [4] Suhren O, Bruyn GW, Tuynman JA. Hyperexplexia. A hereditary startle syndrome. J Neurol Sci 1966;3:577–605.

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