Mutations in the gene encoding GlyT2 (SLC6A5) define a presynaptic component of human startle disease

Abstract

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][2][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 genes 2-7 . 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 Correspondence and requests for materials (subject to a Material Transfer Agreement) should be addressed to R.J.H. (robert.harvey@pharmacy.ac.uk) or M. I.R. (m.i.rees@swansea.ac.uk).. † these authors contributed equally to this work. COMPETING INTERESTS STATEMENT:The authors declare that they have no competing financial interests. Europe PMC Funders GroupAuthor Manuscript Nat Genet. Author manuscript; available in PMC 2011 October 31. Glycine transporters (GlyTs) are members of the Na + /Cl − -dependent neurotransmitter transporter superfamily 9,10 , integral membrane proteins that utilise electrochemical gradients to control the concentration of neurotransmitters at central synapses. This superfamily also includes transporters for GABA, biogenic amines (norepinephrine, dopamine, serotonin, proline), betaine, taurine and creatine. GlyTs have dual functions at both inhibitory and excitatory synapses, resulting from the differential localisation of two distinct transporters 9,10 , GlyT1 and GlyT2. GlyT1 is predominantly expressed in glial cells 9,10 , exhibits a 2 Na + /1 Cl − /1 glycine stoichiometry and bi-directional glycine transport 11 . These properties are appropriate for the control of extracellular glycine concentrations in the submicromolar range for modulation of N-methyl-D-aspartate receptors 12 , and also for lowering extracellular glycine levels at inhibitory glycinergic synapses 13,14 . By contrast, GlyT2 is found in glycinergic axons, exhibits a 3 Na + /1 Cl − /1 glycine stoichiometry and does not display reverse uptake 11 , reflecting an essential role for GlyT2 in maintaining a high presynaptic pool of neurotransmitter at glycinergic synapses 15 . Na + /Cl − -dependent tr...

Mutations in the GlyT2 Gene (SLC6A5) Are a Second Major Cause of Startle Disease

“…Mutation Analysis of SLC6A5 in Hyperekplexia-A total of 93 index cases were screened for genetic variation in SLC6A5 coding exons and donor and acceptor splice junctions by Sanger DNA sequencing (17). Sequence variants were assigned as potential disease-causing mutations after exclusion from a panel of 400 human controls and cross-referencing with common SLC6A5 polymorphisms (17) and those found in dbSNP (supplemental Table S1).…”

“…Sequence variants were assigned as potential disease-causing mutations after exclusion from a panel of 400 human controls and cross-referencing with common SLC6A5 polymorphisms (17) and those found in dbSNP (supplemental Table S1). This revealed 20 sequence variants that were exclusive to 17 index cases, of which the vast majority (14 of 17) showed homozygous or compound heterozygous recessive inheritance ( Fig.…”

“…Molecular Genetics-Exons and intron-exon boundaries of SLC6A5 were amplified from genomic DNA isolated from peripheral blood using established primers sets (17). To avoid allelic dropout, all of the primers were placed in intronic regions that were devoid of known single-nucleotide polymoryphisms found in the National Center for Biotechnology Information data base dbSNP.…”

“…In the absence of glycine, Because low levels of glycine uptake are found in HEK293 cells (17), [ 3 H]glycine uptake was calculated as nmol/min/mg of protein and then expressed as a percentage of the empty expression vector transfected control. Only wildtype GlyT2 and mutant A275T result in any detectable uptake above control values.…”

“…This led to the identification of missense, nonsense, and frameshift mutations in the GlyT2 gene (SLC6A5), encoding a Na ϩ /Cl Ϫ -dependent neurotransmitter transporter that maintains a high presynaptic pool of glycine at glycinergic synapses (17,18). Using detailed structure-function analyses, we demonstrated that GlyT2 mutations disrupted transporter membrane trafficking, Na ϩ , or glycine-binding sites (17). Subsequently, unique GlyT2 mutations were discovered in cattle and dogs, causing startle disorders with early neonatal lethality (19,20).…”