Sir, We read with great interest, the article by Barel et al. (2017) presenting the first cases of disease causing mutations in the trafficking protein, kinesin binding 1 gene (TRAK1). They described six patients from three unrelated consanguineous families of Arab descent, who suffered from a fatal encephalopathy and carried the same homozygous truncating mutation c.287-2A>C in TRAK1. TRAK1 is a kinesin adaptor protein that has several putative roles. Gilbert et al. (2006) described a hypertonic mouse model with mutated Trak1. These mice had lower GABAA receptors in their CNS, especially in lower motor neurons, thus implicating Trak1 as a crucial regulator of GABAA receptor homeostasis. Webber et al. (2008) found that TRAK1 interacts with Hrs (hepatocyte-growth-factor-regulated tyrosine kinase substrate) and regulates endosome to lysosome trafficking. A possible role in neuronal inhibition was suggested following the identification of a susceptibility locus for childhood absence epilepsy in the 3p23-p14 locus that includes TRAK1 in a genome wide linkage analysis (Chioza et al., 2009). The most studied role of TRAK1 is in mitochondrial trafficking, as TRAK1 links mitochondria to kinesin motor proteins together with the Rho GTPase, Miro. A recent study suggested that TRAK1 dependent mitochondrial trafficking differs between young and mature neurons. In hippocampal and cortical neurons TRAK1 traffics mitochondria mainly in axons, but in young neurons it does so both in axons and dendrites (Loss and Stephenson, 2017). Another role of TRAK1 is in mitochondrial fusion via interaction with mitofusins. Depletion of TRAK1 was found to cause mitochondrial fragmentation (Lee et al., 2017). Barel et al. (2017) demonstrated that the mutation in TRAK1, damaged mitochondrial distribution, membrane potential and motility. A study on genetic causes for intellectual disability (Anazi et al., 2017), found mutations in TRAK1 in two families. The patients presented with: neonatal respiratory distress, seizures, delayed myelination and brain atrophy. In this letter, we describe the identification of a homozygous missense variant in the TRAK1 gene in two siblings of unrelated Ashkenazi-Jewish parents who presented with hyperekplexia and fatal refractory status epilepticus. The full clinical presentation of this new syndrome was described in 2004 (Lerman-Sagie et al., 2004). The study was approved by the Helsinki committee of the Israeli Health Department. Written informed consents were obtained from all subjects and their respective guardians. The patients, a girl (Patient 1) and a boy (Patient 2), were born 4 years apart after a normal pregnancy and delivery to healthy parents. On their first day of life they presented with increased muscle tone and an exaggerated startle response to tactile stimulation. Motor development was mildly delayed but social, fine motor, and language development were normal. The mother described a tendency to ‘startle much too often’ since birth; the babies would become suddenly rigid, particularly during baths (Supplementary Videos 1 and 2 of both patients). Exam during the first year of life revealed hypotonia, hyperekplexia, and hyperreflexia, with no dysmorphic features. The boy was treated with clonazepam since birth to ameliorate the hyperekplexia and prevent seizures. Following a febrile illness (the girl at the age of 18 months, and the boy at 12 months) they experienced a tonic-clonic seizure that progressed into status epilepticus. Seizures were not controlled despite adequate anti-epileptic treatment and trials of steroids and intravenous immunoglobulin (IVIG). The patients expired following refractory status epilepticus after 17 and 12 days, respectively. An evaluation before seizure onset, revealed normal MRI, EEG, and metabolic testing. In Patient 1, a muscle biopsy was normal; a brain MRI during the status epilepticus demonstrated a hyperintense lesion in the right thalamus and bilateral tissue loss in the Sylvian fissure; and a mtDNA duplication was identified by Southern blot analysis. An autopsy revealed normal muscle and mitochondria, and brain tissue showed increased microglia in the cortex and hippocampus, mild astrocytic proliferation in the hippocampal region, and perivascular mononuclear infiltrates. Mutations in genes known to be associated with hyperekplexia or related to glycine or GABA receptors [GLRA1, GLRB, GPHN, ARHGEF9, GlyT1 (SLC6A9), GlyT2 (SLC6A5), GABARAP and GABARAPL1] were sequenced and excluded in this family thus it was suggested that these patients had a new autosomal recessive syndrome. We sought to identify the disease-causing variants by homozygosity mapping (the parents’ ancestors were both from the same region in Romania) combined with whole exome sequencing. Whole genome single nucleotide polymorphism (SNP) analysis revealed few regions of shared homozygosity; the largest regions were Ch.3:36.624.933–42.503.035 and Ch.10:92.280.193–96.115.639. Exome sequencing was performed on Patient 1; variants were filtered based on an allele frequency <0.01 according to online databases: dbSNP, 1000G, ExAC, gnomAD. Likely pathogenicity was assessed if the variant was truncating (splicing or non-sense), missense or an in-frame indel. Missense and in-frame indels were considered if they were predicted to be pathogenic by online prediction tools: PolyPhen-2, SIFT and MutationTaster. We found two non-synonymous coding SNPs in the homozygous region in chromosome 3 predicting the amino acid exchanges p.L329P in TRAK1, and p.R798Q in the Villin-like protein (VILL). In the chromosome 10 region, there was no unreported SNP. Apart from these variants no additional homozygous or compound heterozygous unreported variants were identified. We analysed the variants in both genes in all family members and found that the two variants segregated with the disease (Fig. 1A). Confirmation and familial segregation were performed using direct Sanger sequencing (3100 Genetic Analyzer Applied Biosystems). The affected children were found to be homozygous and the parents and one healthy child heterozygous (Fig. 1A and B) for the c.986T>C mutation in the TRAK1 gene (NM_001042646) and for the c.2393G>A in the VILL gene (NM_015873). SIFT and PolyPhen-2 predicted the p.L329P variant in TRAK1 to be probably damaging, and the p.R798Q variant in VILL to be tolerable or benign in the main transcripts of the gene. In the Genome Aggregation Database (http://gnomad.broadinstitute.org), the heterozygous allele count is 7/244 764 alleles for the p.L329P in the TRAK1 gene and 70/274 990 alleles for the p.R798Q variant in the VILL gene, with zero homozygous cases for both. The seven variants present in the gnomAD database for TRAK1 were only detected in the Ashkenazi-Jewish population (7/9808 alleles). Further TRAK1 variants were not found in seven additional patients with hyperekplexia and epilepsy belonging to a large cohort of patients with hyperekplexia. Figure 1 View largeDownload slide Family pedigree, identified mutations and evolutionary conservation of the altered amino acid in the TRAK1 gene. (A) Pedigree of the family, the last child was born after identification of the mutation. (B) Sanger confirmation analysis of the missense variant L329P in the TRAK1 gene (NM_001042646 c.986 T>C) Carrier heterozygosity in the parents and homozygosity in the affected individual were confirmed. (C) Evolutionary conservation of the TRAK1 gene. Figure 1 View largeDownload slide Family pedigree, identified mutations and evolutionary conservation of the altered amino acid in the TRAK1 gene. (A) Pedigree of the family, the last child was born after identification of the mutation. (B) Sanger confirmation analysis of the missense variant L329P in the TRAK1 gene (NM_001042646 c.986 T>C) Carrier heterozygosity in the parents and homozygosity in the affected individual were confirmed. (C) Evolutionary conservation of the TRAK1 gene. The TRAK1 and not the VILL variant was considered responsible for the patient’s severe neurological involvement for the following reasons. The Trak1 mouse model manifests a similar phenotype to our patients with hypertonia and jerky movements (Gilbert et al., 2006), and demonstrates the involvement of TRAK1 in GABAA receptor trafficking. In addition, the TRAK1 protein is expressed in the CNS (the human protein atlas https://www.proteinatlas.org). The p.L329P variant, affecting a highly conserved amino acid (Fig. 1C), was predicted to probably damage the protein structure and is very rare in the Genome Aggregation data. In contrast, a mouse model, the oligotriche mouse (olt/olt homozygous mutant) with a 234 kbp deletion including the VILL gene orthologue in mice, Vill (villin like), and additional genes, does not have a neurological phenotype. These mice have alopecia and male infertility, attributed to the Plcd1 gene included in the deleted region (Runkel et al., 2008). The VILL gene, part of the villin/gelsolin family, which is suggested to participate in actin bundling, has not been described as disease causing and is transcribed and expressed mainly in the gut and not in the CNS. The variant found in our patients was not predicted to damage the protein and is 10 times more prevalent than the variant found in the TRAK1 gene. We functionally tested the p.L329P variant in TRAK1 using a Xenopus laevis oocyte expression system. Co-expression of the TRAK1 wild-type or mutant proteins with the most abundant GABAA receptor comprised α1β2γ2 subunits revealed no difference in the recorded GABA-evoked responses using two-microelectrode voltage clamping (data not shown). This suggests that the interaction of wild-type or mutant TRAK1 protein with GABAA receptors does not differentially affect trafficking, function or stability of these GABAA complexes in Xenopus oocytes. Other functional assays, exploring the effects of this variant on GABAA receptors and mitochondrial function, will therefore be necessary to understand the disease mechanism. Hyperekplexia is a rare neurogenetic disorder characterized by neonatal hypertonia and exaggerated startle reflex after sudden tactile, auditory or visual stimuli accompanied by temporary generalized stiffness and falls. It is mostly dominantly inherited and caused by mutations in the glycine receptors α and β subunit genes (GLRA1 and GLRB) that facilitate fast-response, inhibitory neurotransmission in the spinal cord and brainstem. Additional mutations were found in other postsynaptic proteins of glycinergic synapses as in GPHN, collybistin and ARHGEF9 (Harvey et al., 2008). In contrast to most patients with hyperekplexia, our patients exhibited hypertonicity only during the neonatal period, with decreased tone in infancy. Seizures are infrequently described in patients with hyperekplexia (Harvey, 2004; Zeydan et al., 2017); however, refractory status epilepticus has not been described. Our patients’ clinical presentation is different from that described by Barel et al. since the most dominant feature was congenital hyperekplexia. In addition, their development was only mildly delayed, and they did not have epilepsy until they succumbed to refractory status epilepticus, at the age of 12 and 18 months. In contrast, the patients reported by Barel et al. (2017) presented during infancy (age 1 month to 19 months) with myoclonus, they had global developmental delay, epilepsy started in the first 2 years of life, developmental regression occurred after the onset of epilepsy, and in addition they all developed spasticity. Hyperekplexia was only described in one of the patients from the Barel series and another was described as having an exaggerated startle response. All other patients were described with early onset myoclonus. The Trak1 mouse manifested ‘jerky movements’, which could be the equivalent of hyperekplexia or myoclonus in mice. Barel et al. (2017) demonstrated that TRAK1-deficient fibroblasts showed irregular distribution, altered motility, reduced membrane potential, and diminished respiration of mitochondria. At the molecular level, the variant resulted in the formation of an early termination codon and nonsense mediated decay, suggesting that TRAK1 interaction with trafficking or mitochondrial transport would be lacking in the homozygous carriers. Conversely, the variant in our patients (p.L329P) is located close to the Hrs interaction site involved in endosome to lysosome trafficking and far from the part of the protein responsible for generation of mitochondrial complexes (Stephenson, 2014). Therefore, we suggest that the pathomechanism involved in our patients, producing a different phenotype, may not be related to mitochondrial dysfunction but rather to dysregulation of endosome to lysosome trafficking of neuronal inhibitory proteins. Further research is needed to elucidate the exact mechanism. Acknowledgements This paper is in memory of Dr Esther Leshinsky-Silver who found the gene in 2011 and enabled the birth of a healthy child in this family. Funding The generous support of Rabbi Bochner of the Bonei Olam organization is acknowledged. Supplementary material Supplementary material is available at Brain online. References Anazi S , Maddirevula S , Salpietro V , Asi YT , Alsahli S , Alhashem A , et al. Expanding the genetic heterogeneity of intellectual disability . Hum Genet 2017 ; 136 : 1419 – 29 . Google Scholar CrossRef Search ADS PubMed Barel O , Malicdan MCV , Ben-Zeev B , Kandel J , Pri-Chen H , Stephen J , et al. Deleterious variants in TRAK1 disrupt mitochondrial movement and cause fatal encephalopathy . Brain 2017 ; 140 : 568 – 81 . Google Scholar CrossRef Search ADS PubMed Chioza BA , Aicardi J , Aschauer H , Brouwer O , Callenbach P , Covanis A , et al. Genome wide high density SNP-based linkage analysis of childhood absence epilepsy identifies a susceptibility locus on chromosome 3p23-p14 . Epilepsy Res 2009 ; 87 : 247 – 55 . Google Scholar CrossRef Search ADS PubMed Gilbert SL , Zhang L , Forster ML , Iwase T , Soliven B , Donahue LR , et al. Trak1 mutation disrupts GABAA receptor homeostasis in hypertonic mice . Nat Genet 2006 ; 38 : 245 – 50 . Google Scholar CrossRef Search ADS PubMed Harvey K . The GDP-GTP exchange factor collybistin: an essential determinant of neuronal gephyrin clustering . J Neurosci 2004 ; 24 : 5816 – 26 . Google Scholar CrossRef Search ADS PubMed Harvey RJ , Topf M , Harvey K , Rees MI . The genetics of hyperekplexia: more than startle! Trends Genet 2008 ; 24 : 439 – 47 . Google Scholar CrossRef Search ADS PubMed Lee CA , Chin LS , Li L . Hypertonia-linked protein Trak1 functions with mitofusins to promote mitochondrial tethering and fusion . Protein Cell 2017 , in press . doi: 10.1007/s13238-017-0469-4 . Google Scholar CrossRef Search ADS Lerman-Sagie T , Watemberg N , Vinkler C , Fishhof J , Leshinsky-Silver E , Lev D . Familial hyperekplexia and refractory status epilepticus: a new autosomal recessive syndrome . J Child Neurol 2004 ; 19 : 522 – 5 . Google Scholar CrossRef Search ADS PubMed Loss O , Stephenson FA . Developmental changes in trak-mediated mitochondrial transport in neurons . Mol Cell Neurosci 2017 ; 80 : 134 – 47 . Google Scholar CrossRef Search ADS PubMed Runkel F , Aubin I , Simon-Chazottes D , Büssow H , Stingl R , Miething A , et al. Alopecia and male infertility in oligotriche mutant mice are caused by a deletion on distal chromosome 9 . Mamm Genome 2008 ; 19 : 691 – 702 . Google Scholar CrossRef Search ADS PubMed Stephenson FA . Revisiting the TRAK family of proteins as mediators of GABAA receptor trafficking . Neurochem Res 2014 ; 39 : 992 – 6 . Google Scholar CrossRef Search ADS PubMed Webber E , Li L , Chin LS . Hypertonia-associated protein Trak1 is a novel regulator of endosome-to-lysosome trafficking . J Mol Biol 2008 ; 382 : 638 – 51 . Google Scholar CrossRef Search ADS PubMed Zeydan B , Gunduz A , Demirbilek V , Dervent A . Visually evoked startle response in a patient with epilepsy: a case report and review of the literature . Neurocase 2017 ; 23 : 79 – 81 . Google Scholar CrossRef Search ADS PubMed © The Author(s) (2018). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: email@example.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
Brain – Oxford University Press
Published: May 26, 2018
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