0000001892

0000003310

Enokizono, Takashi

FUKUSHIMA, Hiroko

Kosaki, Kenjiro

OHTO, Tatsuyuki

Suzuki, Hisato

Takada, Hidetoshi

Takenouchi, Toshiki

Takeshita, Yohei

Tanaka, Mai

Uehara, Tomoko

Human Genome Variation

English

Tsukuba Repository  (Tulips-R)

Takeshita et al. Human Genome Variation             (2020) 7:9 https://doi.org/10.1038/s41439-020-0094-2 Human Genome VariationDATA REPORT Open Ac ce s sNovel ARX mutation identified in infantile spasmsyndrome patientYohei Takeshita1, Tatsuyuki Ohto 2,3, Takashi Enokizono2, Mai Tanaka2, Hisato Suzuki 4, Hiroko Fukushima2,3,Tomoko Uehara4, Toshiki Takenouchi 4, Kenjiro Kosaki4 and Hidetoshi Takada2,3AbstractWe report a 7-year-old boy with infantile spasms caused by a novel mutation in the Aristaless-related homeobox (ARX)gene. He showed infantile spasms and hypsarrhythmia on electroencephalogram from early infancy. Brain MRI did notreveal severe malformation of the brain except mild hypoplasia of the corpus callosum. Two-fold adrenocorticotropichormone (ACTH) therapy failed to control the seizures, and ketogenic diet therapy and multi-antiepileptic drugtherapy were required as he showed intractable daily tonic-clonic seizures. Exome sequencing identified a hemizygousmutation in the ARX gene, NG_008281.1(ARX_v001):c.1448+ 1 G > A, chrX: 25025227 C > T (GRCh37). To ourknowledge, this mutation has not been reported previously.X-linked infantile spasm syndrome-1 (ISSX1), alsoknown as early infantile epileptic encephalopathy-1(EIEE1; MIM 308350, X-linked recessive), is character-ized clinically by infantile spasms, hypsarrhythmia andsevere psychomotor retardation. ISSX1 is caused bymissense mutations or expansion mutations in the poly-alanine tract in the Aristaless-related homeobox (ARX)gene, as well as intellectual disabilities and epilepsywithout severe brain malformation, including X-linkedintellectual disability with or without dystonia andOhtahara syndrome1. Here, we present a 7-year-old boywith infantile spasm syndrome caused by a novel muta-tion of the ARX gene, as confirmed by exome sequencing.The 7-year-old patient was the second child born tohealthy nonconsanguineous Japanese parents. The familyhistory was unremarkable. The boy was born at 39 weeksgestation without asphyxia. The birth weight was 2844 g.Tonic spasms involving flexion of the upper limbs wereobserved from 1 month of age, and he was referred to ourhospital with involuntary movement at 3 months of age.He showed no visual response or head control, and thegeneral muscle tone was poor. Brain MRI did not revealsevere malformation of the brain except mild hypoplasiaof the corpus callosum. Various blood tests, includingamino acid analysis, lactate and pyruvate, cerebrospinalfluid test, urine organic acid analysis, and ophthalmologicexamination, were within the normal limits. Karyotypeanalysis showed a normal male karyotype of 46,XY. As theelectroencephalogram revealed hypsarrhythmia, it wasdiagnosed as infantile spasm syndrome. Adrenocortico-tropic hormone (ACTH) injection therapy (0.015 mg/kg/day) was started at 4 months of age. Seizure and hypsar-rhythmia, however, did not disappear after 2 weeks oftherapy, and the dose of ACTH was increased to0.025 mg/kg/day from the third week. Four weeks afterstarting therapy, it was stopped due to elevation of liverenzymes (aspartate aminotransferase (AST) 162 IU/L,alanine aminotransferase (ALT) 215 IU/L, gamma-glutamyl transferase (GGT) 123 IU/L). High-dose phe-nobarbital therapy in combination with potassium bro-mide, zonisamide, and benzodiazepines was ineffective.The second ACTH therapy was performed from10 months of age, but the convulsions continued, and thepatient started to have a daily tonic-clonic seizure. Keto-genic diet therapy and multi-antiepileptic drug therapy© The Author(s) 2020OpenAccessThis article is licensedunder aCreativeCommonsAttribution 4.0 International License,whichpermits use, sharing, adaptation, distribution and reproductionin any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate ifchangesweremade. The images or other third partymaterial in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to thematerial. Ifmaterial is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.Correspondence: Tatsuyuki Ohto (tohto@md.tsukuba.ac.jp)1Department of Pediatrics, Ibaraki Seinan Medical Center Hospital, Sakai-machi,Japan2Department of Pediatrics, University of Tsukuba Hospital, Tsukuba, JapanFull list of author information is available at the end of the articleOfficial journal of the Japan Society of Human Genetics1234567890():,;1234567890():,;1234567890():,;1234567890():,;relieved the seizures, which remained intractable. Heshowed profound intellectual disability and spastic tetra-plegia and was tube-fed via gastrostoma. An exome ana-lysis was performed at the age of 7.Prior to an exome analysis, informed consent from theparents and approval from the University of TsukubaHospital review board, based on the tenets of theDeclaration of Helsinki, were obtained for the molecularstudies. Written consent was obtained from the patient’sparents. Genomic DNA was extracted from peripheralblood leukocytes of the patient and his parents. An exomeanalysis of the trio was performed as described pre-viously2. Briefly, all the exons were captured using theSureSelect All Exon V6 Kit (Agilent Technologies, SantaClara, CA); then, exome analyses were performed usingthe HiSeq2500 platform (Illumina, San Diego, CA).Exome sequencing showed a hemizygous mutation in theARX gene, NG_008281.1(ARX_v001):c.1448+ 1G>A,chrX: 25025227C >T (GRCh37), which was not identified inhis parents, resulting in truncation of the ARX proteinwithout an aristaless domain (Figs. 1 and 2). To ourknowledge, this mutation has never been reported previously,and no healthy person with this mutation was found in theGenome Aggregation Database (gnomAD), the HumanGenetic Variation Database (HGVD) or the Tohoku MedicalMegabank Organization (ToMMo) database.The Aristaless-related homeobox (ARX) protein, the ver-tebrate homolog of Drosophila Aristaless, belongs to theAristaless-related subset of the paired (Prd) class of home-odomain proteins3. It is located in Xp21.3 and has a centralhomeodomain (HD) and a conserved C-terminal aristalessdomain (AD). It also has three polyalanine (polyA) tracts N-terminal to the HD and a fourth polyA tract between the HDand AD4,5. In situ hybridization of ARX in human fetal brainsections at various developmental stages showed the highestexpression in neuronal precursors in the germinal matrix ofthe ganglionic eminence and in the ventricular zone of thetelencephalon. Expression was also observed in the hippo-campus, cingulate, subventricular zone, cortical plate, cau-date nucleus, and putamen. These findings suggest that ARXplays crucial roles in the differentiation and maintenance ofspecific neuronal cell types in the human brain6.Mutations in ARX cause a broad variety of neurologicsymptoms, ranging from severe brain malformation tostructurally normal brains having intellectual disabilities andepilepsy7,8. In addition, there is a reasonably close phenotype−genotype correlation in the ARX gene. In a review of29 males with ARX mutations, Kato et al. found that thosewith premature termination or nonsense mutations hadbrain malformation syndromes, including LISX2 and Proudsyndrome, whereas those with missense and expansion of thepolyA tract had epileptic encephalopathy or intellectual dis-ability without brain malformations1,9.Our patient’s mutation (ARX:c.1448+ 1G>A) is thoughtto cause failure to splice intron 4, as the splice donor site atthe 5′ end of intron 4 would not be recognized. Predictedpre-messenger RNA should have information of intron 4with stop codon (TAA) near the 3′ end of exon 4, resulting inthe truncation of ARX protein between the fourth polyAtract and the AD (Fig. 2). A mutation in the splice acceptorsite at the 3′ end of intron 4, ARX:c.1449-1G >C, has beenreported in a boy with early-onset developmental and epi-leptic encephalopathy, and this mutation was also predictedto abolish the splice acceptor site, retaining intron 4 andleading to a premature termination codon immediately afterexon 410. As exon 5 is the last exon of the ARX gene, it wouldbe predicted to escape nonsense-mediated mRNA decay(NMD), and analysis of cDNA from the lymphoblastoid cellsof this patient confirmed retention of intron 4 and loss ofdetectable expression of ARX mRNA across exon 4 to exon5. Although mRNA analysis was not performed in our case,there is a possibility that our mutation also causes the loss ofARX mRNA expression after exon 4, escaping NMD,because it results in the same intron 4 splicing failure. TheFig. 1 Sanger sequencing results of the ARX gene in the patient and his parents and predicted amino acid sequences. The patient had anARX:c.1448+ 1 G > A de novo mutation (red arrow), which caused the termination of the ARX protein. The double line indicates the splice donor siteat the 5′ end of intron 4.Takeshita et al. Human Genome Variation             (2020) 7:9 Page 2 of 3Official journal of the Japan Society of Human Geneticspredicted ARX protein in our case has no AD, and thephenotype and genotype were similar to those previouslyreported as intellectual disability and infantile onset devel-opmental and epileptic encephalopathies with deficit ofAD10,11, suggesting that our patient’s mutation should bepathological.Genotype−phenotype correlation in animal models withARX mutation has been reported as compatible with that inhumans. In Arx-deficient male mice, aberrant migration anddifferentiation of interneurons containing gamma-aminobutyric acid (GABAergic interneurons) in the gang-lionic eminence and neocortex resembled some of the clin-ical features of X-linked lissencephaly with abnormalgenitalia (XLAG) in humans12. On the other hand, a mousemodel expressing the human ARX c.428_451dup24 dupli-cation mutation, the most frequent human mutation inpolyA tract 2, presented with hyperactivity, enhanced ste-reotypes and alerted contextual fear memory13. These animalmodels are relevant for the investigation of the pathophy-siological effects of these mutations and for therapeuticapproaches because of their similarities with human patients.In conclusion, we identified a novel ARX mutation(c.1448+ 1 G > A) in a Japanese boy who presented withinfantile spasm syndrome without severe brainmalformation.HGV databaseThe relevant data from this Data Report are hosted at the Human GenomeVariation Database at https://doi.org/10.6084/m9.figshare.hgv.2826.AcknowledgementsThis research is funded by the Japan Agency for Medical Research andDevelopment (AMED).Author details1Department of Pediatrics, Ibaraki Seinan Medical Center Hospital, Sakai-machi,Japan. 2Department of Pediatrics, University of Tsukuba Hospital, Tsukuba,Japan. 3Department of Child Health, Faculty of Medicine, University of Tsukuba,Tsukuba, Japan. 4Center for Medical Genetics, Keio University School ofMedicine, Tokyo, JapanConflict of interestThe authors declare that they have no conflict of interest.Publisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Received: 19 January 2020 Revised: 25 February 2020 Accepted: 26February 2020References1. Kato, M. Genotype−phenotype correlation in neuronal migration disordersand cortical dysplasias. Front. Neurosci. 9, 181 (2015).2. Takenouchi, T. et al. Novel overgrowth syndrome phenotype due to recurrentde novo PDGFRB mutation. J. Pediatr. 166, 483–486 (2015).3. Bienvenu, T. et al. ARX, a novel Prd-class-homeobox gene highly expressed inthe telencephalon, is mutated in X-linked mental retardation. Hum. Mol. Genet.11, 981–991 (2002).4. Strømme, P. et al. Mutations in the human ortholog of Aristaless cause X-linked mental retardation and epilepsy. Nat. Genet. 30, 441–445 (2002).5. Nasrallah, I. M., Minarcik, J. C. & Golden, J. A. A polyalanine tract expansion inArx forms intranuclear inclusions and results in increased cell death. J. Cell Biol.167, 411–416 (2004).6. Ohira, R. et al. Human ARX gene: genomic characterization and expression.Mol. Genet. Metab. 77, 179–188 (2002).7. Friocourt, G. & Parnavelas, J. G. Mutations in ARX result in several defectsinvolving GABAergic neurons. Front. Cell Neurosci. 4, 4 (2010).8. Olivetti, P. R. & Noebels, J. L. Interneuron interrupted: molecular pathogenesisof ARX mutations and X-linked infantile spasms. Curr. Opin. Neurobiol. 22,859–865 (2012).9. Kato, M. et al. Mutations of ARX are associated with striking pleiotropyand consistent genotype-phenotype correlation. Hum. Mutat. 23,147–159 (2004).10. Shoubridge, C. et al. Splice variant in ARX leading to loss of C-terminal regionin a boy with intellectual disability and infantile onset developmental andepileptic encephalopathy. Am. J. Med. Genet. A 179, 1483–1490 (2019).11. Kato, M., Koyama, N., Ohta, M., Miura, K. & Hayasaka, K. Frameshiftmutations of the ARX gene in familial Ohtahara syndrome. Epilepsia51, 1679–1684 (2010).12. Kitamura, K. et al. Mutation of ARX causes abnormal development of forebrainand testes in mice and X-linked lissencephaly with abnormal genitalia inhumans. Nat. Genet. 32, 359–369 (2002).13. Dubos, A. et al. A new mouse model of ARX dup24 recapitulates the patients’behavioral and fine motor alterations. Hum. Mol. Genet. 27, 2138–2153 (2018).14. Gage, B. K. et al. The role of ARX in human pancreatic endocrine specification.PLoS ONE 10, e0144100 (2015).Fig. 2 Schematic representation of the ARX gene and predicted protein. Genomic structure of the ARX gene and approximate protein domainsand important functional regions of ARX, including the octapeptide domain (OP), polyalanine tract (polyA), acidic domain (Acidic), prd-likehomeodomain (prd-like HD), and aristaless domain (AD). The number above the exons indicates the sizes in base pairs (bp). The predicted ARXprotein in the patient is truncated and has no aristaless domain. Modified from Gage et al.14 and Ohira et al.6.Takeshita et al. Human Genome Variation             (2020) 7:9 Page 3 of 3Official journal of the Japan Society of Human Genetics

Novel ARX mutation identified in infantile spasm syndrome patient

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Novel ARX mutation identified in infantile spasm syndrome patient

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