Mutations in the GABA Transporter SLC6A1 Cause Epilepsy with Myoclonic-Atonic Seizures
2015; Elsevier BV; Volume: 96; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2015.02.016
ISSN1537-6605
AutoresGemma L. Carvill, Jacinta M. McMahon, Amy L. Schneider, Matthew Zemel, Candace T. Myers, Julia Saykally, John Nguyen, Angela Robbiano, Federico Zara, Nicola Specchio, Oriano Mecarelli, Robert L. Smith, Richard J. Leventer, Rikke S. Møller, Marina Nikanorova, Petia Dimova, Albena Jordanova, Steven Petrou, Ingo Helbig, Pasquale Striano, Sarah Weckhuysen, Samuel F. Berkovic, Ingrid E. Scheffer, Heather C. Mefford,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoGAT-1, encoded by SLC6A1, is one of the major gamma-aminobutyric acid (GABA) transporters in the brain and is responsible for re-uptake of GABA from the synapse. In this study, targeted resequencing of 644 individuals with epileptic encephalopathies led to the identification of six SLC6A1 mutations in seven individuals, all of whom have epilepsy with myoclonic-atonic seizures (MAE). We describe two truncations and four missense alterations, all of which most likely lead to loss of function of GAT-1 and thus reduced GABA re-uptake from the synapse. These individuals share many of the electrophysiological properties of Gat1-deficient mice, including spontaneous spike-wave discharges. Overall, pathogenic mutations occurred in 6/160 individuals with MAE, accounting for ∼4% of unsolved MAE cases. GAT-1, encoded by SLC6A1, is one of the major gamma-aminobutyric acid (GABA) transporters in the brain and is responsible for re-uptake of GABA from the synapse. In this study, targeted resequencing of 644 individuals with epileptic encephalopathies led to the identification of six SLC6A1 mutations in seven individuals, all of whom have epilepsy with myoclonic-atonic seizures (MAE). We describe two truncations and four missense alterations, all of which most likely lead to loss of function of GAT-1 and thus reduced GABA re-uptake from the synapse. These individuals share many of the electrophysiological properties of Gat1-deficient mice, including spontaneous spike-wave discharges. Overall, pathogenic mutations occurred in 6/160 individuals with MAE, accounting for ∼4% of unsolved MAE cases. SLC6A1 (MIM 137165) encodes GAT-1, a voltage-dependent gamma-aminobutyric acid (GABA) transporter that is responsible for the re-uptake of GABA from the synapse. GABA is the principal inhibitory neurotransmitter that counterbalances neuronal excitation in the brain and disruption of this inhibitory balance can result in seizures. To date, mutations in SLC6A1 have not been shown to cause epilepsy in humans, although mutations in other genes that cause altered GABA signaling have been reported. Overlapping 3p25.3 microdeletions have been reported in individuals with a wide spectrum of neurodevelopmental disorders.1Dikow N. Maas B. Karch S. Granzow M. Janssen J.W. Jauch A. Hinderhofer K. Sutter C. Schubert-Bast S. Anderlid B.M. et al.3p25.3 microdeletion of GABA transporters SLC6A1 and SLC6A11 results in intellectual disability, epilepsy and stereotypic behavior.Am. J. Med. Genet. A. 2014; 164A: 3061-3068Crossref PubMed Scopus (21) Google Scholar Here, we describe a de novo 3p25.3 deletion in an individual with myoclonic-atonic epilepsy (MAE; also called myoclonic-astatic epilepsy or Doose syndrome; Table 1). This 315.6-kb deletion refines the critical interval to just two genes, SLC6A1 and SLC6A11 (Figure S1). In addition, two single de novo SLC6A1 mutations in a cohort of individuals with intellectual disability and autism were reported by two independent, large exome sequencing studies.2Sanders S.J. Murtha M.T. Gupta A.R. Murdoch J.D. Raubeson M.J. Willsey A.J. Ercan-Sencicek A.G. DiLullo N.M. Parikshak N.N. Stein J.L. et al.De novo mutations revealed by whole-exome sequencing are strongly associated with autism.Nature. 2012; 485: 237-241Crossref PubMed Scopus (1455) Google Scholar, 3Rauch A. Wieczorek D. Graf E. Wieland T. Endele S. Schwarzmayr T. Albrecht B. Bartholdi D. Beygo J. Di Donato N. et al.Range of genetic mutations associated with severe non-syndromic sporadic intellectual disability: an exome sequencing study.Lancet. 2012; 380: 1674-1682Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar These molecular genetics studies, as well as the function of GAT-1 at the synapse, suggest that SLC6A1 is an excellent candidate gene for epileptogenesis.Table 1Clinical and Molecular Findings in Individuals with Pathogenic SLC6A1 MutationsIndividualAge and SexEpilepsy SyndromecDNA Change, Protein Change, and InheritanceGERP, CADD, PolyPhen-2, Grantham, and SIFT scoresFamily HistoryDevelopment prior to Seizure OnsetAge at Seizure OnsetSeizure Type at OnsetDevelopment after Seizure OnsetOther Seizure TypesAge at Seizure OffsetEEGNeuroimagingOther FeaturesMedicationsOriginal Cohort of 569 Individuals with Epileptic Encephalopathy18 years, FMAEc.131G>A (p.Arg44Gln), de novo4.37, 35, 0.99 (damaging), 43, 1 (tolerated)negativedelayed30 monthsatonic drop attacksplateaued, mild IDatypical absences (onset 32 months) with blinking, myoclonic seizures (onset 2.5 years),4 yearsposterior predominant 3.5–4 Hz GSW, bilateral occipital spike-wave on eye closure, no PPR,delayed myelinationManual stereotypies, autistic features, hypertelorism, broad short nasal tipCZP and VPA stopped drop attacks, VPA ceased at 5 years of age216 years, FMAEc.889G>A (p.Gly297Arg), de novo4.8, 27.6, 0.37 (benign), 125, 0 (damaging)father’s first cousin has absence seizuresisolated speech delay31 monthsatonic drop attacksregression at 4 years, severe IDabsences with eyelid myoclonias, myoclonic status, nonconvulsive status epilepticusongoing3 Hz GSW, GPSW, PPRnormalAutistic features, moderately severe tremor, reluctant to use hands at 14 years, aggression, thoracic scoliosis∗VPA, LTG, ∗CLB, LEV, TPM, ∗ETX; 3.5 years seizure free on CLB before seizure recurrence310 years, FMAEc.1000G>C (p.Ala334Pro), maternally inh (9% mosaic)5.37, 34, 1.00 (damaging), 27, 0.04 (damaging)maternal great aunt with visual auras, paternal great uncle with GTCS, bilateral family history of speech disordersdelayed12 monthsdrop attacksmoderate IDabsences with eyelid myoclonias, atonic drop attacks preceded by eyelid flutter, GTCS (onset 9 years)ongoing; GTCS2.5–3 Hz GSW, no PPRnormalhyperlaxity, lumbar lordosisVPA, LTG, ∗LEV, ∗CZP, and ∗ETX stopped drop attacks; KD was effective but did not completely abolish seizures410 years, MMAE at 4 years, evolving to aBECTsc.1369_1370 delGG (p.Gly457Hisfs∗10), de novoNAnegativedelayed3 yearsmyoclonic-atonic, atonic seizuresIDabsence, myoclonic seizures6 years2.5–3 Hz GSW, right centro-temporal region; CSWS on initial EEG, resolvednormalautistic features, attention deficit hyperactivityVPA and LEV, since 2012 only ∗LEVValidation Cohort of 75 Individuals with MAE512 years, FMAEc.578G>A (p.Trp193∗), de novo4.86, 38, NA, NA, NAnegativedelayed38 monthsmyoclonic-atonic seizuresmild IDabsence, myoclonic seizures3 years and 7 monthsGSW, GPSW, PPRnormalautistic features (mild)VPA, ETX, ∗CLZ622 years, FMAEc.863C>T (p.Ala288Val), inh from affected mother4.98, 29.7, 1.00 (damaging), 64, 0 (damaging)mother has MAEdelayed14 monthsmyoclonic-atonic drop attacksregression from 2 years, moderate IDabsences, absences with eyelid myoclonias, GTCS rareongoing; catamenial GTCS, daily absences, myoclonic-atonic seizures.5–4 Hz GSW, PSW, atypical absences on IPS with PPR and on HV; slow background with excessive beta (drug-related)normalautistic features, pyramidal signs, ataxia, tremor, dyslalia, dysarthriaVPA, CBZ, LTG, CZP, CLB, LEV, TPM, ETX; benzodiazepines indispensable; CZP and CLB7 (mother)44 years, FMAEc.863C>T, (p.Ala288Val), de novoas abovenegativedelayed12 monthsone febrile seizure, myoclonic-atonic seizuresregression at puberty, moderate IDabsences, absences with eyelid myoclonias, GTCS (increase with age)ongoing; catamenial GTCS, daily absences, myoclonic-atonic seizures2.5–4.5 Hz GSW, PSW, IPS with PPR; HV provoked subclinical paroxysmsnormaloppositional behaviors (mild)VPA, LTG, LEV, CZP, TPMNovel 3p25.3 Microdeletion3p25.3 deletion7 years, FMAEdeletion includes SLC6A11 and exon 1 of SLC6A1, de novoNAnegativedelayed3 yearsatonic drop attacksmoderate IDabsences with eyelid myocloniaongoingGSW, bilateral, posterior high-voltage activitynormalhypotonia, autistic traits, absent speechVPAPreviously Published Whole-Exome Sequencing Study CasesRauch, 2012 (ZH50743)12 years, FNA (cohort of individuals with ID)c.452 delT (p.Leu151Argfs∗35), de novoNAnegative, Italian origindelayed speech (48 months) and walking (26 months)5.5 yearsmyoclonic-astatic seizuresmoderate ID (IQ < 50)NANANAMRI at 5 years showed mild cerebellar atrophyautistic features, repetitive behavior, aggression, short attention span, flat and long face, large upper incisors, prognathism,NASanders, 2012 (13832.p1)NA, MNA (cohort of individuals with autism spectrum disorders)c. 863C>T (p.Ala288Val), de novo4.55, NA, 1.00 (probably damaging), 64, 0.02 (damaging)NANA1.5 yearspetit mal (absence)delayed speech, then regression with loss of speechNAongoingabnormal at 2 yearsMRI normal at 3 yearsautism, attention deficit disorderADD medications, AEDs, mood stabilizersMutation coordinates based on SLC6A1: NM_003042.3 and protein NP_003033.3Genome evolutionary rate profiling (GERP) scores range from least (−12.3) to most highly (6.17) conserved residues. Combined annotation dependent depletion (CADD) Phred-scaled scores range 0–99. All PolyPhen-2 scores were calculated under the HumVar model for Mendelian disorders and ranged from 0–1, where 1 is most likely to be damaging. Grantham scores ranged from 0–215 where 215 is predicted to be most damaging. Sorting intolerant from tolerant (SIFT) scores ranged from 0–1, where 0 is predicted to be most damaging. Abbreviations are as follows: inh, inherited; F, female; M, male; aBECTS, atypical benign epilepsy with centro-temporal spikes; CSWS, continuous spike-wave discharges during slow sleep; MAE, myoclonic-atonic epilepsy; ID, intellectual disability; IPS, intermittent photic stimulation; ADD, attention deficit disorder; GTCS, generalized tonic-clonic seizures; AED, anti-epileptic drug; GSW, generalized spike wave; PSW, polyspike wave; PPR, photo-paroxysmal response; HV, hyperventilation; NA, not available; VPA, sodium valproate; LTG, lamotrigine; CLB, clobazam; CBZ, carbamazepine; LEV, levetiracetam; TPM, topiramate; ETX, ethosuxamide; KD, ketogenic diet; CZP, clonazepam. ∗current medication Open table in a new tab Mutation coordinates based on SLC6A1: NM_003042.3 and protein NP_003033.3 Genome evolutionary rate profiling (GERP) scores range from least (−12.3) to most highly (6.17) conserved residues. Combined annotation dependent depletion (CADD) Phred-scaled scores range 0–99. All PolyPhen-2 scores were calculated under the HumVar model for Mendelian disorders and ranged from 0–1, where 1 is most likely to be damaging. Grantham scores ranged from 0–215 where 215 is predicted to be most damaging. Sorting intolerant from tolerant (SIFT) scores ranged from 0–1, where 0 is predicted to be most damaging. Abbreviations are as follows: inh, inherited; F, female; M, male; aBECTS, atypical benign epilepsy with centro-temporal spikes; CSWS, continuous spike-wave discharges during slow sleep; MAE, myoclonic-atonic epilepsy; ID, intellectual disability; IPS, intermittent photic stimulation; ADD, attention deficit disorder; GTCS, generalized tonic-clonic seizures; AED, anti-epileptic drug; GSW, generalized spike wave; PSW, polyspike wave; PPR, photo-paroxysmal response; HV, hyperventilation; NA, not available; VPA, sodium valproate; LTG, lamotrigine; CLB, clobazam; CBZ, carbamazepine; LEV, levetiracetam; TPM, topiramate; ETX, ethosuxamide; KD, ketogenic diet; CZP, clonazepam. ∗current medication To investigate the role of SLC6A1 in the etiology of the severe infantile and childhood epilepsies, we performed targeted resequencing in 569 individuals with a range of epileptic encephalopathies. Epileptic encephalopathies are a group of infantile- and childhood-onset epilepsies characterized by multiple seizure types and developmental delay or regression; they are associated with abundant epileptiform activity, which contributes to cognitive impairment.4Berg A.T. Berkovic S.F. Brodie M.J. Buchhalter J. Cross J.H. van Emde Boas W. Engel J. French J. Glauser T.A. Mathern G.W. et al.Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005-2009.Epilepsia. 2010; 51: 676-685Crossref PubMed Scopus (3259) Google Scholar All individuals or their parents or legal guardians gave informed consent to participate in the study and the institutional review boards of the University of Washington, and the University of Melbourne approved this study. We captured all 14 coding SLC6A1 exons and at least five base pairs of flanking intronic sequences by using molecular inversion probes (MIPs); next-generation sequencing, data analysis, and variant calling were performed as described previously.5Carvill G.L. Heavin S.B. Yendle S.C. McMahon J.M. O’Roak B.J. Cook J. Khan A. Dorschner M.O. Weaver M. Calvert S. et al.Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1.Nat. Genet. 2013; 45: 825-830Crossref PubMed Scopus (473) Google Scholar In brief, we used MIPs and 100 ng of each proband’s DNA to capture all target DNA and performed PCR with universal primers that contained a unique 8-bp barcode on the reverse primer. Amplified PCR products from all individuals were pooled and sequenced on an Illumina Hiseq according to a 101-bp paired-end protocol. We mapped raw reads to the genome (UCSC Genome Browser hg19) by using the Burrows-Wheeler Aligner (BWA) and performed variant calling by using the Genome Analysis Toolkit (GATK). Variants that did not adhere to the following criteria were excluded from further analysis: allele balance > 0.75, quality (QUAL) < 30, quality by depth (QD) < 5, coverage < 50×, and presence in homopolymer runs ≥4 bp. A threshold of 50× coverage was used for this methodology given that variants below this cut-off have a high false-positive rate; this is in contrast to other technologies, such as exome sequencing, which have a threshold of ∼20×. Variants were annotated with SeattleSeq (see Web Resources), and the exome aggregation consortium (ExAC) dataset (see Web Resources) was used for assessments of variant frequency in the control population. Overall, we sequenced 90% of SLC6A1 to a depth of at least 50× and at an average coverage of 652× across all samples (Figure S2). We performed segregation analysis in parental DNA samples for all nonsynonymous, frameshift, and splice-site variants that were not present in the ExAC set of ∼61,000 exomes (see Web Resources). We performed segregation analysis on the proband and parental DNA by using Sanger sequencing with primers designed to flank the variant of interest. Maternity and paternity were confirmed with the PowerPlex S5 system (Promega) for all de novo mutations. We identified four likely pathogenic SLC6A1 mutations in a cohort of 569 individuals with epileptic encephalopathies (Table 1, Table S1, and Figure 1). All mutations adhered to the aforementioned criteria and occurred at a highly conserved nucleotide; none were present in ExAC, and each of the positions at which these mutations occurred was covered at a sequence depth of 20× or greater in the controls. In addition, amino acid changes were predicted to be damaging by one or more of the prediction tools (Polyphen2, Grantham, and SIFT; see Web Resources)6Grantham R. Amino acid difference formula to help explain protein evolution.Science. 1974; 185: 862-864Crossref PubMed Scopus (1686) Google Scholar that we used (Table 1). Moreover, we considered these variants to be likely pathogenic on the basis that they occurred de novo in the affected individual in three cases, and in one individual the variant was inherited from an unaffected mother who was a somatic mosaic for this mutation. By using a single molecular MIP (smMIP) that targeted this mutation as described previously7Hiatt J.B. Pritchard C.C. Salipante S.J. O’Roak B.J. Shendure J. Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation.Genome Res. 2013; 23: 843-854Crossref PubMed Scopus (227) Google Scholar in the unaffected mother, we detected four alleles with the mutant C allele and 43 alleles with the reference allele. This suggests that approximately 18% of the mother’s white blood cells carry the mutant allele. Strikingly, all individuals with SLC6A1 mutations showed phenotypic homogeneity4Berg A.T. Berkovic S.F. Brodie M.J. Buchhalter J. Cross J.H. van Emde Boas W. Engel J. French J. Glauser T.A. Mathern G.W. et al.Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005-2009.Epilepsia. 2010; 51: 676-685Crossref PubMed Scopus (3259) Google Scholar (Table 1) in that MAE is characterized by the onset of myoclonic, myoclonic-atonic, and atonic seizures between 7 months and 6 years of age and the presence of generalized spike-wave or polyspike-wave discharges. Development prior to seizures is usually normal.8Oguni H. Fukuyama Y. Tanaka T. Hayashi K. Funatsuka M. Sakauchi M. Shirakawa S. Osawa M. Myoclonic-astatic epilepsy of early childhood—clinical and EEG analysis of myoclonic-astatic seizures, and discussions on the nosology of the syndrome.Brain Dev. 2001; 23: 757-764Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar In our cohort, 85/569 individuals had a diagnosis of MAE for which no molecular cause had been previously identified.5Carvill G.L. Heavin S.B. Yendle S.C. McMahon J.M. O’Roak B.J. Cook J. Khan A. Dorschner M.O. Weaver M. Calvert S. et al.Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1.Nat. Genet. 2013; 45: 825-830Crossref PubMed Scopus (473) Google Scholar This statistically significant enrichment of SLC6A1 mutations in MAE-affected probands (4/85) as compared to those with other epileptic encephalopathy phenotypes (0/484; p value 0.0005, Fisher’s two-tailed test) prompted us to use the same methodology to screen this gene in an additional cohort of 75 individuals with MAE. In this validation cohort, we identified two additional SLC6A1 mutations (Table 1 and Figure 1). The c.578G>A (p.Trp193∗) mutation arose de novo, whereas the c.863C>T (p.Ala288Val) mutation was inherited from a mother who also had MAE. We performed further segregation analysis in the maternal grandparents and showed that the c.863C>T (p.Ala288Val) mutation arose de novo in the mother, who passed this mutation on to her affected daughter. Overall, in a cohort of 160 probands with MAE, we identified six SLC6A1 point mutations, accounting for ∼4% of previously unsolved MAE cases. SLC6A1 is widely expressed throughout developing and mature human, mouse, and rat brains, and its expression follows that of the GABAergic pathways.9Scimemi A. Structure, function, and plasticity of GABA transporters.Front. Cell. Neurosci. 2014; 8: 161PubMed Google Scholar, 10Miller J.A. Ding S.L. Sunkin S.M. Smith K.A. Ng L. Szafer A. Ebbert A. Riley Z.L. Royall J.J. Aiona K. et al.Transcriptional landscape of the prenatal human brain.Nature. 2014; 508: 199-206Crossref PubMed Scopus (753) Google Scholar GAT-1 is primarily located in the axon and nerve terminals of GABAergic interneurons, whereas GAT-3 is more abundant in astrocytes.11Conti F. Melone M. De Biasi S. Minelli A. Brecha N.C. Ducati A. Neuronal and glial localization of GAT-1, a high-affinity gamma-aminobutyric acid plasma membrane transporter, in human cerebral cortex: with a note on its distribution in monkey cortex.J. Comp. Neurol. 1998; 396: 51-63Crossref PubMed Scopus (117) Google Scholar, 12Minelli A. DeBiasi S. Brecha N.C. Zuccarello L.V. Conti F. GAT-3, a high-affinity GABA plasma membrane transporter, is localized to astrocytic processes, and it is not confined to the vicinity of GABAergic synapses in the cerebral cortex.J. Neurosci. 1996; 16: 6255-6264Crossref PubMed Google Scholar, 13Chiu C.S. Jensen K. Sokolova I. Wang D. Li M. Deshpande P. Davidson N. Mody I. Quick M.W. Quake S.R. Lester H.A. Number, density, and surface/cytoplasmic distribution of GABA transporters at presynaptic structures of knock-in mice carrying GABA transporter subtype 1-green fluorescent protein fusions.J. Neurosci. 2002; 22: 10251-10266Crossref PubMed Google Scholar At the pre-synaptic terminal, GAT-1 is responsible for the re-uptake of GABA from the synaptic cleft. This voltage-dependent transport requires the exchange of two sodium ions and one chloride ion for each GABA molecule.14Radian R. Kanner B.I. Stoichiometry of sodium- and chloride-coupled gamma-aminobutyric acid transport by synaptic plasma membrane vesicles isolated from rat brain.Biochemistry. 1983; 22: 1236-1241Crossref PubMed Scopus (102) Google Scholar In Gat1-deficient mice, GABA uptake is impaired, resulting in both increased ambient GABA levels and spontaneous spike-wave discharges.15Jensen K. Chiu C.S. Sokolova I. Lester H.A. Mody I. GABA transporter-1 (GAT1)-deficient mice: differential tonic activation of GABAA versus GABAB receptors in the hippocampus.J. Neurophysiol. 2003; 90: 2690-2701Crossref PubMed Scopus (186) Google Scholar In this study, we identified two truncating alterations (c.1369_1370delGG [p.Gly457Hisfs∗10] and c.578G>A [p.Trp193∗]) and one partial gene deletion that most likely lead to loss of GAT-1 function. Importantly, no truncating alterations have been identified in the ∼61,000 ExAC exomes, providing further evidence that loss-of-function protein changes are likely pathogenic. Mutagenesis and functional experiments at the sites of the four missense substitutions (c.131G>A [p.Arg44Gln], c.889G>A [p.Gly297Arg], c.1000G>C [p.Ala344Pro], and c.863C>T [p.Ala288Val]) suggest that they lead to a loss of GAT-1 function. Substitution at the Arg44 position has been shown to result in approximately 98% (p.Arg44Ser) and 70% (p.Arg44Lys) decreases in GABA transport activity.16Ben-Yona A. Kanner B.I. Functional defects in the external and internal thin gates of the γ-aminobutyric acid (GABA) transporter GAT-1 can compensate each other.J. Biol. Chem. 2013; 288: 4549-4556Crossref PubMed Scopus (20) Google Scholar We anticipate that the p.Arg44Gln substitution described here will have a similar negative effect on GABA uptake. Similarly, a p.Ala288Cys substitution reduced GABA transport activity to 5%–7% of the activity seen in wild-type-like GAT-1.17Rosenberg A. Kanner B.I. The substrates of the gamma-aminobutyric acid transporter GAT-1 induce structural rearrangements around the interface of transmembrane domains 1 and 6.J. Biol. Chem. 2008; 283: 14376-14383Crossref PubMed Scopus (27) Google Scholar The p.Ala288Val substitution described here had the most damaging scores possible according to PolyPhen2 and SIFT and most likely results in loss of GAT-1 function. Although no mutagenesis data exist for the p.Gly297Arg alteration, Gly297, along with Ala61, Leu300, and Trp400, forms the GABA binding site.18Wein T. Wanner K.T. Generation of a 3D model for human GABA transporter hGAT-1 using molecular modeling and investigation of the binding of GABA.J. Mol. Model. 2010; 16: 155-161Crossref PubMed Scopus (28) Google Scholar Replacement of this small amino acid with a large positively charged residue is likely to occlude the GABA binding pocket. Finally, the p.Ala334Pro substitution occurs in transmembrane domain 7, and the presence of a large aromatic residue is likely to alter the conformation of this domain and disrupt function. In summary, although we have not performed mutagenesis studies for these specific substitutions, the evidence suggests that all six alterations could lead to a loss of function and reduced GABA uptake from the synapse, in a manner similar to that seen in Gat1-knockout mice. Given that GABA is the major inhibitory neurotransmitter in the brain, it seems paradoxical that increased GABA levels would cause seizures with hypersynchronous epileptiform neuronal activity. However, an elevation in ambient and synaptic GABA, due to decreased clearance, has the capacity to enhance both phasic and tonic inhibition. Increases in either of these two modes of inhibition have been associated with the appearance of spike-wave discharges.19Cope D.W. Di Giovanni G. Fyson S.J. Orbán G. Errington A.C. Lorincz M.L. Gould T.M. Carter D.A. Crunelli V. Enhanced tonic GABAA inhibition in typical absence epilepsy.Nat. Med. 2009; 15: 1392-1398Crossref PubMed Scopus (319) Google Scholar, 20Hosford D.A. Wang Y. Cao Z. Differential effects mediated by GABAA receptors in thalamic nuclei in lh/lh model of absence seizures.Epilepsy Res. 1997; 27: 55-65Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar Moreover, Gat1-knockout mice, as well as mice administered a GAT-1 inhibitor, show spontaneous spike-wave discharges typical of absence seizures,19Cope D.W. Di Giovanni G. Fyson S.J. Orbán G. Errington A.C. Lorincz M.L. Gould T.M. Carter D.A. Crunelli V. Enhanced tonic GABAA inhibition in typical absence epilepsy.Nat. Med. 2009; 15: 1392-1398Crossref PubMed Scopus (319) Google Scholar a seizure type seen in all individuals with SLC6A1 mutations. Finally, tiagabine, an anti-epileptic drug that is effective in treating focal seizures and that blocks GAT-1 can cause both absence status epilepticus and myoclonic seizures in human subjects.21Koepp M.J. Edwards M. Collins J. Farrel F. Smith S. Status epilepticus and tiagabine therapy revisited.Epilepsia. 2005; 46: 1625-1632Crossref PubMed Scopus (48) Google Scholar, 22Schousboe A. Madsen K.K. Barker-Haliski M.L. White H.S. The GABA synapse as a target for antiepileptic drugs: a historical overview focused on GABA transporters.Neurochem. Res. 2014; 39: 1980-1987Crossref PubMed Scopus (50) Google Scholar These studies suggest that GABA function may extend beyond inhibition. Overall, we identified six likely pathogenic SLC6A1 mutations in seven individuals, including an affected mother and daughter, and an additional eighth individual with a deletion disrupting SLC6A1; all eight individuals have MAE (Table 1). The median age of seizure onset was 30.5 months (mean = 26.1 months; range = 12–38 months). All of these individuals had absence seizures, notably including eyelid myoclonia in four cases. All individuals also had drop attacks, which were myoclonic atonic in four individuals and atonic in the other four. Recording myoclonic-atonic seizures in a child with MAE can be challenging because formal video-EEG monitoring has shown that myoclonic, atonic, and myoclonic-atonic seizures can all occur in a single individual and can be hard to differentiate.8Oguni H. Fukuyama Y. Tanaka T. Hayashi K. Funatsuka M. Sakauchi M. Shirakawa S. Osawa M. Myoclonic-astatic epilepsy of early childhood—clinical and EEG analysis of myoclonic-astatic seizures, and discussions on the nosology of the syndrome.Brain Dev. 2001; 23: 757-764Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar All individuals had generalized spike-waves >2.5 Hz on their EEGs, and four had a photoparoxysmal response. Seizures settled in three of the individuals, all children between the ages of 3 and 8 years; the remaining five individuals, aged 7 to 44 years, had ongoing seizures. Although the overall electroclinical pattern was consistent with MAE, atypical features were noted. Specifically, preceding developmental delay, which can occur in a minority of individuals with MAE, occurred in all eight individuals here. Developmental slowing or regression occurred in four individuals. All individuals had intellectual disabilities that ranged from mild to severe. Six individuals had autistic features. Tremors were marked in two individuals, one of whom also had ataxia. Another individual had prominent manual stereotypies. Also, generalized tonic-clonic seizures are frequently observed in MAE,23Kilaru S. Bergqvist A.G. Current treatment of myoclonic astatic epilepsy: clinical experience at the Children’s Hospital of Philadelphia.Epilepsia. 2007; 48: 1703-1707Crossref PubMed Scopus (82) Google Scholar, 24Kaminska A. Ickowicz A. Plouin P. Bru M.F. Dellatolas G. Dulac O. Delineation of cryptogenic Lennox-Gastaut syndrome and myoclonic astatic epilepsy using multiple correspondence analysis.Epilepsy Res. 1999; 36: 15-29Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 25Nabbout R. Kozlovski A. Gennaro E. Bahi-Buisson N. Zara F. Chiron C. Bianchi A. Brice A. Leguern E. Dulac O. Absence of mutations in major GEFS+ genes in myoclonic astatic epilepsy.Epilepsy Res. 2003; 56: 127-133Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar but only three of our individuals had this seizure type; of these three, two were the mother and daughter, who had catamenial generalized tonic-clonic seizures. Seven of the eight affected individuals, including the affected mother, were female, but this might simply reflect the relatively small cohort rather than true biological significance. Interestingly, in a large exome sequencing study that focused on gene discovery under a de novo mutation model, the c.863C>T [p.Ala288Val] substitution was identified in an individual with autism spectrum disorder (13832.p1).2Sanders S.J. Murtha M.T. Gupta A.R. Murdoch J.D. Raubeson M.J. Willsey A.J. Ercan-Sencicek A.G. DiLullo N.M. Parikshak N.N. Stein J.L. et al.De novo mutations revealed by whole-exome sequencing are strongly associated with autism.Nature. 2012; 485: 237-241Crossref PubMed Scopus (1455) Google Scholar The clinical features of individual 13832.p1, as noted in the Simons Foundation Autism Research Initiative (SFARI; Table 1), indicate that this individual had absence seizures, regression, and autism, perhaps reflecting an overlapping phenotype with MAE. Interestingly, the median seizure onset for the three individuals with the p.Ala288Val substitution was 14 months, which is much earlier than that of the other six individuals described here, for whom the median onset was 33.5 months. Further studies are needed to determine whether this difference correlates with an underlying biological mechanism. We identified pathogenic SLC6A1 mutations in 6/160 probands with MAE, suggesting that mutations in this gene account for ∼4% of individuals with this severe epilepsy syndrome and are more likely in individuals with pre-existing developmental delay. We also describe a de novo deletion in one individual, whose phenotype was strikingly similar to that observed in individuals with SLC6A1 point mutations, despite the inclusion of the adjacent gene, SLC6A11 (MIM 607592), in the deletion. A genetic etiology for MAE has been proposed since its initial description by Doose and is supported by family studies.26Doose H. Gerken H. Leonhardt R. 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Genet. 2001; 68: 859-865Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar Glucose transporter 1 deficiency has also been implicated in MAE, and SLC2A1 (MIM 138140) mutations have been found in a small subset (4/84) of MAE-affected individuals with both inherited and de novo mutations.35Mullen S.A. Marini C. Suls A. Mei D. Della Giustina E. Buti D. Arsov T. Damiano J. Lawrence K. De Jonghe P. et al.Glucose transporter 1 deficiency as a treatable cause of myoclonic astatic epilepsy.Arch. Neurol. 2011; 68: 1152-1155Crossref PubMed Scopus (100) Google Scholar More recently, the role of de novo mutations in MAE has expanded given that mutations in GABRG2 (MIM 137164) have been identified in a single individual and mutations in CHD2 (MIM 602119) have been identified in two individuals.5Carvill G.L. Heavin S.B. Yendle S.C. McMahon J.M. O’Roak B.J. Cook J. Khan A. Dorschner M.O. Weaver M. Calvert S. et al.Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1.Nat. Genet. 2013; 45: 825-830Crossref PubMed Scopus (473) Google Scholar Each of these genes contributes slightly to the etiology of MAE, but they are also associated with a wide spectrum of epilepsy phenotypes ranging from benign to severe. Mutations in SLC6A1 seem to occur specifically in individuals presenting with MAE. Although this observation requires further validation, it is supported by the lack of mutations in the remaining 484 individuals who had other epileptic encephalopathies and were sequenced in this study, as well as in 264 probands with infantile spasms or Lennox-Gastaut syndrome.36Allen A.S. Berkovic S.F. Cossette P. Delanty N. Dlugos D. Eichler E.E. Epstein M.P. Glauser T. Goldstein D.B. Han Y. et al.Epi4K ConsortiumEpilepsy Phenome/Genome ProjectDe novo mutations in epileptic encephalopathies.Nature. 2013; 501: 217-221Crossref PubMed Scopus (1086) Google Scholar Collectively, these findings suggest that SLC6A1 mutations might cause a specific epilepsy syndrome: MAE that occurs in the context of abnormal early development. These early abnormalities might be due to the specific function of GAT-1 and GABA transport in the developing human brain. The members of the EuroEPINOMICS Rare Epilepsy Syndrome Myoclonic-Astatic Epilepsy & Dravet working group are Albena Jordanova, Sarah von Spiczak, Hiltrud Muhle, Hande Caglayan, Katalin Sterbova, Dana Craiu, Dorota Hoffman, Anna-Elina Lehesjoki, Kaja Selmer, Christel Depienne, Johannes Lemke, Carla Marini, Renzo Guerrini, Bernd Neubauer, Tiina Talvik, Peter De Jonghe, Arvid Suls, and Eric Leguern. G.L.C. and I.H. are members of the scientific advisory board of Ambry Genetics. We are grateful to Angelika Ackerhans and Kerstin Wuhlbrandt (Department of Neuropediatrics, University of Kiel) for database and sample management. We appreciate access to phenotypic data on SFARI Base, and we are grateful to all of the families at the participating Simons Simplex Collection (SSC) sites, as well as the principal investigators. This research was supported by the NIH (NINDS 1R01NS069605 to H.C.M.; 1K99NS089858 to G.L.C.), the American Epilepsy Society and the Lennox and Lombroso Fund (G.L.C.), the National Health and Medical Research Council of Australia (S.F.B. and I.E.S.), the Bulgarian Ministry of Education and Science National Science Fund (grant DTK02/67), the Research Fund of the University of Antwerp (grant TOP-BOF-29069 to A.J.). Intramural funds from the University of Kiel and the German Research Foundation (HE5415/5-1 and HE5415/6-1) further supported I.H. Additional funding sources are listed in the Supplemental Data. Download .pdf (.29 MB) Help with pdf files Document S1. Figures S1 and S2 and Table S1 The URLs for data presented herein are as follows:Burrows-Wheeler Aligner, http://bio-bwa.sourceforge.net/CADD, http://cadd.gs.washington.edu/ExAC Browser, http://exac.broadinstitute.org/PolyPhen-2,http://www.genetics.bwh.harvard.edu/pph2/GATK, http://www.broadinstitute.org/gatk/OMIM, http://www.omim.org/SFARI, https://base.sfari.orgSeattleSeq Annotation 138, http://snp.gs.washington.edu/SeattleSeqAnnotation138/SIFT, http://sift.bii.a-star.edu.sg/SSC population dataset described in this study, https://ordering.base.sfari.org/sfari-download-prepared-datasets.htmlUCSC Genome Browser, http://genome.ucsc.edu
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