GlyT1 encephalopathy: Characterization of presumably disease causing GlyT1 mutations
Introduction
Glycine is an important inhibitory neurotransmitter predominantly in caudal regions of the central nervous system (CNS). Additionally, glycine acts as a coagonist to glutamate at N-methyl-D-aspartate receptors (NMDAR). After its presynaptic release at inhibitory synapses, it binds to glycine receptors opening an intrinsic ion channel and facilitating the chloride conductance of the postsynaptic cell (Lynch, 2004). To allow for neurotransmission to proceed with high spatial and temporal resolution, the released neurotransmitter has to be rapidly removed from the synaptic cleft and (finally) shuttled back into the presynaptic terminal. The key players that are assumed to contribute crucially to both, regulation of the extracellular glycine concentrations, as well as the efficient recycling, are the high affinity glycine transporters GlyT1 and GlyT2, which belong to the large family of SLC6A transporters (Gomeza et al., 2006). GlyT2 is expressed exclusively by glycinergic neurons and is responsible for the reuptake of glycine into the presynapse (Gomeza et al., 2003b). In contrast GlyT1, that is expressed by major glial cell populations and a subset of glutamatergic neurons, facilitates the rapid glycine clearance from the synaptic cleft and additionally regulates NMDAR function by controlling the extracellular glycine concentration at a subset of excitatory synapses (Gomeza et al., 2003a; Zafra et al., 1995). Pharmacological inhibition of GlyT1 through irreversible inhibitors results in a concentration dependent elevation of the extracellular glycine level only in the CSF but not in other body fluids and without influencing other amino acid concentrations (Martina et al., 2004).
Mice lacking GlyT1 expression are not viable and die within a few hours after birth most likely due to respiratory deficiencies. Here, insufficient synaptic clearance was shown to lead to elevated extracellular glycine concentrations and thus inducing GlyR hyperactivity, which results in glycine mediated overinhibition (Gomeza et al., 2003a). This phenotype was caused by glial GlyT1 expression, since glia specific GlyT1 gene inactivation caused a phenocopy of the full GlyT1 knock out at least at neonatal stages (Eulenburg et al., 2010). In contrast inactivation of neuronal GlyT1 expression caused significant changes in the rodent behaviour suggesting altered glutamatergic signalling via NMDARs (Eulenburg et al., 2010; Martina et al., 2005).
Taken together these findings corroborate the hypothesis that the phenotype seen in GlyT1 KO mice results from overactive glycinergic inhibition caused by accumulation of glycine in the extracellular space.
Previous work suggested that mutations within the coding regions of the SLC6A9 gene might be causal for glycine transporter 1 encephalopathy in humans (Alfadhel et al., 2016; Kurolap et al., 2016), a disease displaying many but not all facets of nonketotic hyperglycinemia, previously associated with loss of function mutations of the mitochondrial glycine cleavage system (Applegarth and Toone, 2001).
Up to now three different families with different mutations and in total 6 individuals carrying GlyT1 mutations homozygously and displaying the disease phenotype have been identified (Alfadhel et al., 2016; Kurolap et al., 2016). Symptoms described in all patients include arthrogryposis and increased nuchal translucency in ultrasound scans during pregnancy. Lifeborn infants showed severe respiratory failure requiring persistent ventilation, encephalopathy, hypotonia progressing to limp hypertonicity in response to loud sounds and tactile stimulation, global developmental delay and dysmorphic features. Further symptoms were also muscular abnormalities, including clubfeet, hyperextension of the knees and joint laxity (Alfallaj and Alfadhel, 2019). In all patients a mildly elevated glycine concentration in the CSF with no changes in serum glycine was observed, which might be a good diagnostic marker for GlyT1 encephalopathy in the future (Kurolap et al., 2016).
Three different mutations in the SLC6A9 gene have been reported in the context of GlyT1 encephalopathy in three different families: a homozygous missense mutation c.1219 A>G (p.Ser407Gly), a homozygous small deletion c.928_932 delAAGTC (p.Lys310Phe+fs*31), and a homozygous nonsense mutation c.1717 C>T (p.Gln573*) (Alfadhel et al., 2016; Kurolap et al., 2016) due to consanguinity an autosomal recessive inheritance pattern is suggested (Alfallaj and Alfadhel, 2019).
Although the correlation of homozygous mutations and the disease phenotype suggests a direct link, the consequence of the mutation on GlyT1 functions is unclear at present. In this study we now present a functional characterization of the GlyT1 mutations found in GlyT1 encephalopathy patients including one previously unpublished mutation.
Section snippets
Results
A novel mutation within the SLC6A9 gene was identified in two fetuses in subsequent pregnancies of the same parents that showed increased nuchal translucency and severe arthrogryposis in ultrasound scans. Based on these findings both pregnancies were terminated. The observed malformations were very similar to previously described cases of GlyT1 encephalopathy (Alfadhel et al., 2016; Kurolap et al., 2016). Indeed, a retrospective genomic analysis of both fetuses by NGS revealed a homozygous
Discussion
In this study, we show that all hGlyT1 mutations previously identified in patients with GlyT1 encephalopathy, as well as a novel mutation identified in two fetuses with abnormalities consistent with GlyT1 encephalopathy, result in severe transporter dysfunction. These findings support the hypothesis that the identified GlyT1 mutations are causative for the phenotype of GlyT1 encephalopathy. The GlyT1 mutations, hGlyT1Q573* and hGlyT1K310F+fs*31 identified by Kurolap et al. (2016) have been
Genetic analysis
The parents of the two fetuses received genetic counselling and agreed to the genomic analysis and the subsequent analysis of the identified mutation. For the genetic analysis DNA was extracted from cord blood of the fetus, and conducted whole exome sequencing on a NovaSeq6000 platform (Illumina, San Diego, USA) after enrichment with the SureSelectXT Human All Exon v6 kit (Agilent, Santa Clara, USA). Sequencing reads were mapped to the Genome Reference Consortium Human Genome Build 37 (GRCh37)
Author’s contributions
Katharina Hauf: Investigation, methodology, formal analysis, writing-original draft, visualization; Lukas Barsch, Investigation, formal analysis; Daniel Bauer: Investigation, formal analysis; Rebecca Buchert: Investigation; Anja Armbruster: Investigation; Leonie Frauenfeld: Investigation; Ute Grasshoff: Investigation; Volker Eulenburg: Conceptualization, Investigation, Supervision, Project administration, writing -original draft. All authors have read and approved the final draft of the
Declaration of competing interest
The authors have nothing to declare.
Acknowledgements
We would like to thank Ina Bosse and Konstanze Büttner, for excellent technical assistance. This work was supported by fundings from the Leipzig University and a Grant from the Deutsche Forschungs Gemeinschaft to V.E. (DFG, EU110/6-1). Calculations for this research were conducted on the Lichtenberg high performance computer of the TU Darmstadt.
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