C. L. Hammond

Pathophysiological Mechanisms of Dominant and Recessive GLRA1 Mutations in Hyperekplexia

Hyperekplexia is a rare, but potentially fatal, neuromotor disorder characterized by exaggerated startle reflexes and hypertonia in response to sudden, unexpected auditory or tactile stimuli. This disorder is primarily caused by inherited mutations in the genes encoding the glycine receptor (GlyR) α1 subunit (GLRA1) and the presynaptic glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of GLRA1 in 88 new unrelated human hyperekplexia patients revealed 19 sequence variants in 30 index cases, of which 21 cases were inherited in recessive or compound heterozygote modes. This indicates that recessive hyperekplexia is far more prevalent than previous estimates. From the 19 GLRA1 sequence variants, we have investigated the functional effects of 11 novel and 2 recurrent mutations. The expression levels and functional properties of these hyperekplexia mutants were analyzed using a high-content imaging system and patch-clamp electrophysiology. When expressed in HEK293 cells, either as homomeric α1 or heteromeric α1β GlyRs, subcellular localization defects were the major mechanism underlying recessive mutations. However, mutants without trafficking defects typically showed alterations in the glycine sensitivity suggestive of disrupted receptor function. This study also reports the first hyperekplexia mutation associated with a GlyR leak conductance, suggesting tonic channel opening as a new mechanism in neuronal ligand-gated ion channels.

The Glycinergic System in Human Startle Disease: A Genetic Screening Approach

Human startle disease, also known as hyperekplexia (OMIM 149400), is a paroxysmal neurological disorder caused by defects in glycinergic neurotransmission. Hyperekplexia is characterised by an exaggerated startle reflex in response to tactile or acoustic stimuli which first presents as neonatal hypertonia, followed in some with episodes of life-threatening infantile apnoea. Genetic screening studies have demonstrated that hyperekplexia is genetically heterogeneous with several missense and nonsense mutations in the postsynaptic glycine receptor (GlyR) α1 subunit gene (GLRA1) as the primary cause. More recently, missense, nonsense and frameshift mutations have also been identified in the glycine transporter GlyT2 gene, SLC6A5, demonstrating a presynaptic component to this disease. Further mutations, albeit rare, have been identified in the genes encoding the GlyR β subunit (GLRB), collybistin (ARHGEF9) and gephyrin (GPHN) – all of which are postsynaptic proteins involved in orchestrating glycinergic neurotransmission. In this review, we describe the clinical ascertainment aspects, phenotypic considerations and the downstream molecular genetic tools utilised to analyse both presynaptic and postsynaptic components of this heterogeneous human neurological disorder. Moreover, we will describe how the ancient startle response is the preserve of glycinergic neurotransmission and how animal models and human hyperekplexia patients have provided synergistic evidence that implicates this inhibitory system in the control of startle reflexes.

A novel GABRG2 mutation, p.R136*, in a family with GEFS+ and extended phenotypes.

Genetic mutations in voltage-gated and ligand-gated ion channel genes have been identified in a small number of Mendelian families with genetic generalised epilepsies (GGEs). They are commonly associated with febrile seizures (FS), childhood absence epilepsy (CAE) and particularly with generalised or genetic epilepsy with febrile seizures plus (GEFS+). In clinical practice, despite efforts to categorise epilepsy and epilepsy families into syndromic diagnoses, many generalised epilepsies remain unclassified with a presumed genetic basis. During the systematic collection of epilepsy families, we assembled a cohort of families with evidence of GEFS+ and screened for variations in the γ2 subunit of the γ-aminobutyric acid (GABA) type A receptor gene (GABRG2). We detected a novel GABRG2(p.R136*) premature translation termination codon in one index-case from a two-generation nuclear family, presenting with an unclassified GGE, a borderline GEFS+ phenotype with learning difficulties and extended behavioural presentation. The GABRG2(p.R136*) mutation segregates with the febrile seizure component of this familys GGE and is absent in 190 healthy control samples. In vitro expression assays demonstrated that γ2(p.R136*) subunits were produced, but had reduced cell-surface and total expression. When γ2(p.R136*) subunits were co-expressed with α1 and β2 subunits in HEK 293T cells, GABA-evoked currents were reduced. Furthermore, γ2(p.R136*) subunits were highly-expressed in intracellular aggregations surrounding the nucleus and endoplasmic reticulum (ER), suggesting compromised receptor trafficking. A novel GABRG2(p.R136*) mutation extends the spectrum of GABRG2 mutations identified in GEFS+ and GGE phenotypes, causes GABAA receptor dysfunction, and represents a putative epilepsy mechanism.

Genetic epilepsy with febrile seizures plus: definite and borderline phenotypes

Generalised epilepsy with febrile seizures plus (GEFS+) is the most studied familial epilepsy syndrome. However, characteristics of UK families have not previously been reported. Among the first 80 families recruited to our families study, four broad subphenotypes were identified: families with classical GEFS+; families with borderline GEFS+; families with unclassified epilepsy; and families with an alternative syndromal diagnosis. Borderline GEFS+ families shared many characteristics of classical GEFS+ families—such as prominent febrile seizures plus and early onset febrile seizures—but included more adults with focal epilepsies (rather than the idiopathic generalised epilepsies predominating in GEFS+) and double the prevalence of migraine. Thus the authors believe that a novel and robust familial epilepsy phenotype has been identified. Subcategorising families with epilepsy is helpful in targeting both clinical and research resources. Most families with GEFS+ have no identified causal mutation, and so predicting genetic homogeneity by identifying endophenotypes becomes more important.

Ethnicity can predict GLRA1 genotypes in hyperekplexia

Objectives Hyperekplexia is predominantly caused by mutations in the α-1 subunit of the inhibitory glycine receptor (GLRA1). Three quarters of cases show autosomal-recessive inheritance. Methods We carefully ascertained reports of ethnicity from our hyperekplexia research cohort. These were compared with all published cases of hyperekplexia with an identified genetic cause. Ethnicities were subgrouped as Caucasian, Asian, Arabic, Turkish, Jewish or Afro-American. Results We report the ethnicity of 90 cases: 56 cases from our service augmented by 34 cases from the literature. Homozygous deletions of exons 1 to 7 are predominantly seen in people with Turkish backgrounds (n=16/17, p<0.001). In contrast, the dominant point mutation R271 is seen in people of Asian, Caucasian and African-American heritage (n=19) but not in people with Arab or Turkish ethnicities (p<0.001). Conclusions Self-declared ethnicity can predict gene-screening outcomes. Cultural practices influence the inheritance patterns and a Caucasian founder is postulated for R271 mutations.

Implications for Families of Advances in Understanding the Genetic Basis of Epilepsy

Investigations into families with a large number of individuals with epilepsy have led to the discovery of epilepsy-causing (or epilepsy associated) gene mutations. These discoveries offer advantages and insights for the patient, family, healthcare professionals and biomedical scientists. Despite these benefits, there is little evidence about the impact of participation in genetic research for families with epilepsy. Here we report on the reflections of individuals who have participated in epilepsy genetic research through the Wales Epilepsy Research Network (WERN). Undergoing genetic investigation for inherited epilepsy has extensive emotive impact, both positive and negative, on individuals and families. Recognising these impacts is imperative to researchers working with families; having implications for study design, research consent and the provision of appropriate support.

PATH40 Spectrum of clinical disease in hyperekplexia: the genotypes and phenotypes of 48 cases

Hyperekplexia is a rare, potentially treatable neurogenetic condition: two major genes of effect are described—one postsynaptic (GLRA1) the other presynaptic (SLC6A5). It is currently not known whether a genetic diagnosis can predict clinical presentation. Sixty individuals were identified from a genetic database and the referring clinicians were contacted using a standard questionnaire; replies for 48 (80%) were received. 37 people (from 29 families) had mutations in GLRA1 and 11 (10 families) in SLC6A5. They all exhibited classical symptoms of hyperekplexia and clonazepam is the treatment of choice (92% had a sustained benefit). We found no genetic evidence for “major” or “minor” forms of hyperekplexia. Apnoea attacks are common (40%) and delayed milestones, learning difficulties or speech and language impairment was seen in 44%. Patients with SLC6A5 mutations were significantly more likely than those with GLRA1 mutations: to have had serious infantile apnoeas (p<0.005); and to have both apnoeas and developmental difficulties (p<0.01). The developmental delay demonstrated in some patients may represent failure of neurogenesis in a glycine-poor environment. There is a compelling argument for early genetic testing for symptomatic neonates and preconception counselling for parents who carry SLC6A5 mutations. Ninety per cent of neonates with hyperekplexia due to SLC6A5 mutations have multiple serious apnoeas and cognitive development is impaired in two thirds.

Bench-to-bedside: first molecular epilepsy outcomes from the Wales epilepsy research network: a novel GABRG2 mutation in an epilepsy family

A case of acute onset neuropsychological deficit and vertical gaze palsy, a 61-year-old woman with paroxysmal atrial fibrillation, hypertension and migraine presented with sudden onset of flashing lights, severe headache, double vision, and subsequently became unresponsive requiring intubation and intensive care unit admission. Initial brain CT to exclude subarachnoid haemorrhage was normal, but a repeat scan 24 h later demonstrated ischemic lesions in right thalamus and internal capsule. Three days later, she was extubated but noted to be confused. She was drowsy, sleepy and amnesic. Eye examination revealed impairment of vertical eyes movements. The rest of cranial nerves were normal. She had normal upper limbs examination. In the lower limbs she had brisk reflexes, mild gait ataxia and up going plantars. Blood tests and CSF examination were normal. Brain (MRI) demonstrated bilateral paramedian thalamic infarction. CT-angiography showed normal carotid arteries. She was eventually transferred to neurorehabilitation unit, where she continued to suffer from hypersomnolense and vertical gaze palsy. A repeat brain MRI did not show significant changes. Detailed neuropsychological assessment demonstrated profound cognitive impairment. In conclusion, it is important to consider this type of ischemic infarction in the differential diagnosis of patients with recurrent episodes of unresponsiveness, cognitive impairment and gaze palsy. Email: odaijumma@yahoo.co.uk

PO054 Ten years of studying familial epilepsy in wales

Aim We have been recruiting multiplex families into the ‘genetics of familial epilepsy’ study since 2006. Method Clinicians referred patients with a familial history (typically three or more relatives with epilepsy). We conducted telephone interviews, performed home visits, collected DNA samples, reviewed medical notes, constructed pedigrees and performed epilepsy phenotyping. We directly sequenced candidate genes, performed exome sequencing and functional laboratory experiments where possible. Results We recruited 110 families and collected DNA samples from 251 affected and 479 unaffected individuals. 36 families have predominantly generalised epilepsy, 27 predominantly focal epilepsy, 23 families have both focal and generalised epilepsy, and 24 families have unclassifiable epilepsy. We identified several specific familial phenotypes including: genetic epilepsy with febrile seizures plus (GEFS+) n=16; familial temporal lobe epilepsy n=9; benign familial neonatal seizures n=2; autosomal dominant lateral temporal lobe epilepsy n=1 and familial cavernomas n=1. Causative mutations were found in genes including GABRG2, KCNQ2, KRIT1, LGI1 and SLC2A1. Conclusion We identified a monogenetic cause for the epilepsy in 5% of our families. 20% of our unresolved families have individuals with generalised and individuals with focal epilepsy, a finding which has been replicated in other recent studies, suggesting that common genetic mechanisms can cause ‘mixed’ phenotypes.

SUDEP and familial epilepsy [Conference Abstract]

Intra-rectal Diazepam (DZ) is the first rescue medication for acute prolonged convulsive seizures in children in many countries. In this study, we aimed at assessing the experience of the families of patients presenting Dravet Syndrome (DS) with respect to the use of intra-rectal DZ.