Hyperekplexia is a human neurological disorder characterized by an excessive startle response and is typically caused by missense and nonsense mutations in the gene encoding the inhibitory glycine receptor (GlyR) α1 subunit (GLRA1)1-3. Genetic heterogeneity has been confirmed in isolated sporadic cases with mutations in other postsynaptic glycinergic proteins including the GlyR β subunit (GLRB)4, gephyrin (GPHN)5 and RhoGEF collybistin (ARHGEF9)6. However, many sporadic patients diagnosed with hyperekplexia do not carry mutations in these genes2-7. Here we reveal that missense, nonsense and frameshift mutations in the presynaptic glycine transporter 2 (GlyT2) gene (SLC6A5)8 also cause hyperekplexia. Patients harbouring mutations in SLC6A5 presented with hypertonia, an exaggerated startle response to tactile or acoustic stimuli, and life-threatening neonatal apnoea episodes. GlyT2 mutations result in defective subcellular localisation and/or decreased glycine uptake, with selected mutations affecting predicted glycine and Na+ binding sites. Our results demonstrate that SLC6A5 is a major gene for hyperekplexia and define the first neurological disorder linked to mutations in a Na+/Cl−-dependent transporter for a classical fast neurotransmitter. By analogy, we suggest that in other human disorders where defects in postsynaptic receptors have been identified, similar symptoms could result from defects in the cognate presynaptic neurotransmitter transporter.

Background

Inherited long QT syndrome (LQTS) is characterized by prolonged QT interval on the EKG, syncope and sudden death due to ventricular arrhythmia. Causative mutations occur mostly in cardiac potassium and sodium channel subunit genes. Confidence in mutation pathogenicity is usually reached through family genotype-phenotype tracking, control population studies, molecular modelling and phylogenetic alignments, however, biophysical testing offers a higher degree of validating evidence.

Methods and Results

By using in-vitro electrophysiological testing of transfected mutant and wild-type LQTS constructs into Chinese Hamster Ovary cells, we investigated the biophysical properties of 9 KCNQ1 missense mutations (A46T, T265I, F269S, A302V, G316E, F339S, R360G, H455Y, and S546L) identified in a New Zealand based LQTS screening programme. We demonstrate through electrophysiology and molecular modeling that seven of the missense mutations have profound pathological dominant negative loss-of-function properties confirming their likely disease-causing nature. This supports the use of these mutations in diagnostic family screening. Two mutations (A46T, T265I) show suggestive evidence of pathogenicity within the experimental limits of biophysical testing, indicating that these variants are disease-causing via delayed or fast activation kinetics. Further investigation of the A46T family has revealed an inconsistent co-segregation of the variant with the clinical phenotype.

Conclusions

Electrophysiological characterisation should be used to validate LQTS pathogenicity of novel missense channelopathies. When such results are inconclusive, great care should be taken with genetic counselling and screening of such families, and alternative disease causing mechanisms should be considered.

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.

Defects in mammalian glycinergic neurotransmission result in a complex motor disorder characterized by neonatal hypertonia and an exaggerated startle reflex, known as hyperekplexia (OMIM 149400). This affects newborn children and is characterized by noise or touch-induced seizures that result in muscle stiffness and breath-holding episodes. Although rare, this disorder can have serious consequences, including brain damage and/or sudden infant death. The primary cause of hyperekplexia is missense and non-sense mutations in the glycine receptor (GlyR) α1 subunit gene (GLRA1) on chromosome 5q33.1, although we have also discovered rare mutations in the genes encoding the GlyR β subunit (GLRB) and the GlyR clustering proteins gephyrin (GPNH) and collybistin (ARHGEF9). Recent studies of the Na+/Cl−-dependent glycine transporters GlyT1 and GlyT2 using mouse knockout models and human genetics have revealed that mutations in GlyT2 are a second major cause of hyperekplexia, while the phenotype of the GlyT1 knockout mouse resembles a devastating neurological disorder known as glycine encephalopathy (OMIM 605899). These findings highlight the importance of these transporters in regulating the levels of synaptic glycine.