Mutations in SZT2 Result in Early‐Onset Epileptic Encephalopathy

Page 1of13 between this versionandtheVersionofrecord. Pleasecitethis articleasdoi:10.1002/ajmg.a.38717. through thecopyediting, typesetting, paginationandproofreadingprocess, whichmayleadtodifferences This istheauthormanuscript acceptedforpublicationandhasundergone full peerreviewbuthasnotbeen Pizzino et al.Pizzino et Amy Pizzino Leukoencephalopathy and Epileptic Encephalopathy Early-Onset In Result SZT2 in Mutations

To whom correspondence should be addressed be addressed should correspondence whom To Adeline Vanderver Adeline Miriam Bloom Miriam 6. 1. 7. 8. 9. 10. 2. 3. 4. 5. Laura A. Adang: [email protected] Adang: Laura A. Washington, DC, USA USA DC, Washington, Center, Medical National Children’s Imaging and Radiology, Diagnostic Department of Department of Neurology, Children’s National Medical Center, Washington, DC, USA USA DC, Medical Center, Washington, National Children’s Neurology, Department of Illumina, Inc., San Diego, CA, USA USA CA, Inc., Diego, San Illumina, DC, USA USA DC, Washington, Medical Center, National esearch, Children's R Medicine Center for Genetic DC, USA USA DC, Washington University, Washington George Health Sciences, of and Medicine School Division of Neurology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA USA PA, Philadelphia, of Philadelphia, Hospital Children’s of Neurology, Division Columbia. Columbia. of District Washington, of Medicine, University School Washington The George (NHGRI), Bethesda, MD USA USA MD Bethesda, (NHGRI), Institute Genome Research Human National at the Program Disease Undiagnosed Australia Australia Lucia, Queensland, Queensland, St. of University Bioscience, Institute for Molecular Department of Pediatrics, Children’s National Medical Center, Washington, DC, USA USA DC, Center, Washington, Medical National Children’s Department of Pediatrics, 1 , Matthew Whitehead , Matthew

Author Manuscript5 , Sarah H. Evans , Sarah H.

1 , John G. Murnick, , John 2,6 , Parisa Sabet Rasekh Sabet , Parisa

10 10

1 6 Joan Conry, Joan 1 , Jennifer Murphy , Jennifer SZT2 and Leukoencephalopathy and 1 Ryan J. Taft Ryan J. 3 , Guy Helman Guy , 4,7 , Cas Simons , Cas

4 , 4 ,

SZT2 sequencing exome Whole Myelination Leukoencephalopathy encephalopathy epileptic Early onset Keywords leukoencephalopathies. and for EOEE investigation Pizzino et al.Pizzino et understanding of SZT2 the understanding our clinical case This expands of central myelination. loss findingnovel of progressive mutations in SZT2 in mutations heterozygous compound with regression and developmental EOEE, macrocephaly, with an individual present EOEE. We cause of a identified newly gene, are regulator threshold seizure severe epilepsies often associated with psychomotor regression. Mutations in SZT2 in Mutations regression. psychomotor with associated often epilepsies severe of collection genetically a heterogeneous are (EOEE) encephalopathies epileptic Early onset

Author Manuscript

as identified by whole exome sequencing. Serial imaging characterized the the characterized imaging Serial sequencing. as identifiedexome by whole This article isprotected by copyright. All rights reserved. -phenotype and emphasizes the role of this gene in diagnosticthe in gene role the of this emphasizes and-phenotype American JournalofMedicalGenetics:PartA

John Wiley&Sons, Inc. ABSTRACT ABSTRACT

2 SZT2 and Leukoencephalopathy and , a known , a known

Page 2of13

Page 3of13 literature and present the case of an affected child with compound heterozygous variants in in heterozygous variants compound with child ancase affected of the literature and present review we existing the will context, In this et al., 2016). Venkatesan et al., 2017; Tshuchida et al., 2017; Nakamura Falcone et al., 2013; et al., 2013; (Basel-Vanagaite (Table 1) callosum of corpus the and atrophy dysgenesis findings include imaging Reported disability. intellectual Pizzino et al.Pizzino et (Baldassari et al., 2016; Peng et al., 2017; Wolfson et al., 2017). et al., 2017). Wolfson al., 2017; Peng et et(Baldassari al., 2016; suppression the of mTOR inhibits Szt2 dysfunctional that It theorized is et al., 2010). Toutzaris Li, & 2017; Yin, Peng, & 2009; O'Brien, Beyer, Yang, Frankel, Mahaffey, 2016; Pippucci, & Bisulli, Licchetta, Tinuper, (Baldassari, models and animal humans in epileptogenesis and (mTOR) target of rapamycin mammalian of regulation the with associated is system, SZT2 central nervous the in Highly expressed 2016). & Millichap, Angle, Venkatesan, al., 2017; et Tshuchida 2017; et al., Nakamura 2013; et al., Falcone et al., 2013; EOEE (Basel-Vanagaite at 20 months of age. Her neurologic examination was notable for macrocephaly (head (head macrocephaly was for notable examination neurologic of age. Her months at 20 in described, mutations recently As psychomotor regression. with are associated typically children and in epilepsies (Table 1). (Table 1). myelination central ofnovel finding of loss the with encephalopathy epileptic and early onset Prior clinical reports have described the role of role the of described reports have clinical Prior

Author seizures with attention medical to presented who Salvador El girl from aThe proband is Manuscript heterogeneous genetically group a of are (EOEE) encephalopathies epileptic Early onset SZT2 This article isprotected by copyright. All rights reserved. , a known seizure threshold regulator gene, are a rare cause of cause a of rare gene, are regulator threshold seizure , a known American JournalofMedicalGenetics:PartA CLINICAL REPORT REPORT CLINICAL INTRODUCTION INTRODUCTION John Wiley&Sons, Inc.

3 SZT2 in refractory epilepsy and refractory in epilepsy SZT2 and Leukoencephalopathy and

SZT2

by the age of 7, she had lost the ability to communicate, purposefully use her limbs, and was limbs, her use purposefully communicate, ability the to had lost ageby of she 7, the proton density (< 1 year) or T2 FLAIR images (>1 year) were also obtained in accordance with with accordance in year) obtained also were year) (>1 or FLAIR T2 images 1 density (< proton phenobarbital, topiramate, rufinamide, midazolam, and the ketogenic diet. and diet. ketogenic the midazolam, rufinamide, topiramate, phenobarbital, age. Post contrast coronal and axial T1WI were performed in 3 exams after intravenous injection injection intravenous after exams 3 T1WI in performed were and axial coronal contrast age. Post Pizzino et al.Pizzino et developmental delay of unknown etiology, although neither parent was affected. was affected. parent neither etiology, although unknown delay of developmental with relatives for maternal significant pedigree was A three-generation and sepsis. chronic pancreatitis, (SIADH), hormone antidiuretic of inappropriate syndrome by complicated course, years hospital a of prolonged age at 8 died following She and areflexia. choreoathetosis, and rashes, alopecia for were diffuse notable examinations Her later dependent. tube gastrostomy and, was progressive Her encephalopathy were limited. language skills receptive andwords, her never She produced specific months. 40 at a stand to and pulled months at 15 independently she sat Developmentally, reflexes. tendon deep global hypotonia, and intact joints), extensible and hyper- fingers, tapered bridge, long depressed nasal microophthalmia, hypertelorism, bossing, (frontal features norm), dysmorphic above standard deviations >2 51cm, circumference or axial gradient echo, and axial diffusion weighted images were performed in all cases. Axial cases. all Axial in performed were weighted images diffusion axial and gradient echo, or axial T2WI, coronal coronal T2WI, T1WI, axial Sagittal WI). Milwaukee, (General Electric, magnet refractory to multiple medications, including lamotrigine, levetiracetam, phenytoin, phenytoin, levetiracetam, including lamotrigine, medications, refractory multiple to Her was epilepsy bilateral seizures. into convulsive evolution intermittent with hemispheres from both arising consciousness impaired with seizures focal motor frequent had discharges. She Her electroencephalogram (EEG) was characterized by multi-focal epileptiform epileptiform by multi-focal characterized (EEG) was Her electroencephalogram

Author a or 3T 1.5T either on were performed exams imaging (MRI) resonance Brain magnetic Manuscript This article isprotected by copyright. All rights reserved. American JournalofMedicalGenetics:PartA NEUROIMAGING NEUROIMAGING John Wiley&Sons, Inc.

SZT2 and Leukoencephalopathy and

Page 4of13

Page 5of13 progressive diffuse brain volume loss with eventual development of abnormal signal in the signal the in of abnormal eventual development with loss volume brain progressive diffuse Pizzino et al.Pizzino et (Fig 1.2c). (Fig 1.2c). creatine low spectroscopy revealed MR and b). However, (Fig 1.2a of life MRI months at 21 and brain a follow-up exam between this interim the in change occurred significant No time. at this appropriate was age1.1a). Myelination (Fig dysgenesis callosal with consistent rostrum, of the and blunting intermedia massa of thalamic the absence included anomalies Midline andage b). (Fig forcorpus 1.1a callosum diameter and thickened fronto-occipital increased (TR 2000 msec, TE 288 msec). msec). msec, TE288 2000 (TR exams 3 in ganglia of voxel basal left cm 2 2 over interest the x a x 2 with was performed (MRS) spectroscopy magnetic resonance voxel Single dimeglumine. gadopentetate mmol/kg of 0.1 Genomics using the Illumina Nextera Rapid Capture kit, and captured libraries were sequenced sequenced were libraries and captured kit, Capture Rapid Illumina Nextera using the Genomics for Medical at the Queensland Centre was performed of DNA (WES) sequencingexome whole Triplet Medical Center. at Children's National Leukoencephalopathies of Unclassified (Fig images 1.6d). orbital on noted is microphthalmia symmetric mild and 1.6a). Hypertelorism age (Fig 7 at MRI follow-up on signal pituitary posterior of normal and loss increasing size with (Fig 1.5a), enlarged mildly is adenohypophysis the 4, By age (Figand 1.6). splenium 1.5 and of myelination furtheryears regression 1.6) show 7 (Fig and years(Fig 1.5), 1.4), 4 years at 3 (Fig exams Follow-up (Fig 1.3d). pronounced was less depression 1.3a-c). Creatine By 31 months, mild diffuse brain volume loss and hypomyelination had developed (Fig had developed hypomyelination and loss diffuse brain volume mild By months, 31 The initial brain MRI at 11 months of age demonstrated megalencephaly with an with megalencephaly of age demonstrated months at 11 brain MRI The initial

Author studyProject Disorders Bioregistry Myelin the in enrolled was prospectively The patient Manuscript This article isprotected by copyright. All rights reserved. American JournalofMedicalGenetics:PartA MOLECULAR ANALYSES ANALYSES MOLECULAR John Wiley&Sons, Inc.

5 SZT2 and Leukoencephalopathy and

performed using the Real Time Genomics integrated analysis tool rtgFamily v3.2 (Cleary et al., (Cleary v3.2 rtgFamily tool analysis integrated Genomics Time Real the performed using predicted to be “probably damaging” by PolyPhen-2 (score: 0.995) and “deleterious” by SIFT by “deleterious” and 0.995) (score: PolyPhen-2 by damaging” “probably bepredicted to at NM_015284.3:c.6916G>A substitution inherited maternally a and truncation protein premature in result to predicted (p.Phe1834Serfs*47) NM_015284.3:c.5499delC pair deletion Pizzino et al.Pizzino et candidate variants. candidate variants. verified Sanger sequencing information. and pedigree variant data annotated the incorporating workflow an in-house with performed was of candidate variants and identification analysis Subsequent et al., 2012). (Cingolani v2.9 and dbNSFP database, dbSNP147, GRCh37.75 SnpEff from the data and filtered using v4.2 usingannotated SnpEff were variants All 2014). was calling variant informed and pedigree (GRCh37) genome reference human aligned the to 92%). were Reads (average of >20x depth a read to were bases sequenced of 90% targeted that such depth a was minimum to sequenced Each 100nt). exome (2 x Illumina 2000 anon HiSeq mutations in the the in mutations novel identified WES whom for myelination, of central loss epilepsy, and regression, refractory GnomAD or LOVD. LOVD. or GnomAD in has been reported FigureNeither variant 1B. in as shown linages, vertebrate all conserved in is residue Theal., 2014). p.Gly2306 (Kircher et score CADD of 31 a significant havevariants Both & Kumar, 2009). Ng, Henikoff, 2012; et al., Cingolani al., 2010; et (Adzhubei (score: 0.01) is variant missense The p.Gly2306Arg et al., 2016). (Vanderver 1) (Table (p.Gly2306Arg) children with mild-to-moderate intellectual disability without seizures and with normal brain normal with and seizures without disability intellectual mild-to-moderate children with Two candidate compound heterozygous mutations were identified: a were identified: heterozygous mutations compound candidateTwo Author delayand globalyoung developmental girl profound with a weIn describe report, this Manuscript SZT2

gene (Table 1). Falcone et al reported a family with three macrocephalic threewith macrocephalic family reported a et al gene Falcone (Table 1). This article isprotected by copyright. All rights reserved. American JournalofMedicalGenetics:PartA John Wiley&Sons, Inc. DISCUSSION DISCUSSION

6 SZT2 and Leukoencephalopathy and de novo single base single base

Page 6of13

Page 7of13 patient in this report is similar to previous reports of SZT2 the reports previous to similar report is this in patient phenotype associated with mutations in in mutations with associated phenotype studies could be used to explore the role the of explore be to used could studies rather than a direct effect from effect a direct rather than epilepsy refractory the to atrophy secondary progressive the is that It possible Kleefield, is 1995). & Moses, Evagelopoulou, Mantzoros, (Papapostolou, SIADH with clinically associated can patient, be our in as found pituitary posterior signal, typical of The loss neuroimaging. on signal weighted T2/T1 of normal loss a slow yearsage, by followed of 2 approximately until myelination typical demonstrates our patient although yet be elucidated, to is myelination myelination and worsening global atrophy (Figure 1A). The connection between between connection 1A).(Figure The global atrophy and worsening myelination of development of typical loss a progressive evolving into myelination normal revealed our patient for serial obtained images The Table(Figure 1). 1A, imaging findings unique have diminished reflexes as well (Tshuchida et al., 2017). 2017). et al., (Tshuchida as well reflexes have diminished to was noted patient reported One previously (Dali et al., 2015). leukodystrophies among several feature a common neuropathy, a peripheral with be consistent could our patient in of reflexes cases, including our own patient (Table 1) (Unterberger, Bauer, Walser, & Bauer, 2016). The Bauer, The 2016). & Bauer, Walser, (Unterberger, (Table patient 1) our own cases, including Pizzino et al.Pizzino et syndromes and disorders of brain development and were identified in most of the reported of reported the most in were and identified of brain development and disorders syndromes genetic epilepsy many to common changes callosum are Corpus 2016). et al., Venkatesan 2017; et al., Tshuchida et al., 2017; Nakamura et al., 2013; (Basel-Vanagaite (Table 1) abnormalities et al., 2016). Venkatesan et al., 2017; Tshuchida et al., 2017; Nakamura 2013; imaging (Falcone et al., 2013). Several additional case reports describe a more severe clinical clinical severe a more describe caseadditional reports Several et al., 2013). imaging (Falcone

Author Manuscript migrational and corpus callosum thick a short, changes include MRI reported Previously This article isprotected by copyright. All rights reserved. American JournalofMedicalGenetics:PartA SZT2 . In the future, electromyography and nerve conduction conduction and nerve In electromyography . future, the John Wiley&Sons, Inc. SZT2 SZT2

, similar to our patient (Basel-Vanagaite et al., , (Basel-Vanagaite our patient similar to 7 in peripheral demyelination as well, as the loss as as loss the well, peripheral in demyelination SZT2 -related EOEE, but demonstrates demonstrates but -related EOEE, and Leukoencephalopathy and SZT2 and

SZT2

Pizzino et al.Pizzino et SZT2 SZT2 SZT2 with associated variability the broad phenotypic underscore These cases et al., 2016). Venkatesan et al., 2017; Tshuchida et al., 2017; Penget al., 2009; Frankel et al., 2013; Falcone2013; (NIDDK). Diseases Kidney and Digestive Diabetes and Institute of National the and (NINDS), and Stroke Disorders Institute of Neurological National the between NCATS, collaboration funded through is consortium This and NCATS. (ORDR), Research Rare Diseases of Office the of initiative an (RDCRN), Network Research Clinical of Rare the Diseases a part is (U54NS065768) Network Lysosomal Disease The Disease Network. Lysosomal grantand the CURE from the grants by LA: Award. Supported Research Discovery Early Career Research Council by an Australian RJT Education, Neurology Fund for Pediatric Delman bysupported the Shire. R.J.T. is an employee of Illumina. an employeeIllumina. is of R.J.T. Shire. report. this in as demonstrated defects including myelination imaging abnormalities, of potential as breadth the as well encephalopathy, demonstrate the emerging role of role of emerging the demonstrate these reports Collectively, neuroimaging. and on atrophy of myelination development who presented with early onset epileptic encephalopathy and progressive loss of typical loss and progressive encephalopathy early epileptic presented who onset with In this case study, we describe an affected child with compound heterozygous variants in in variants heterozygous compound with child affected an we describe In study, case this GH, AP, and AV are supported by the Myelin Disorders Bioregistry Project. GH is GH is Bioregistry Project. Disorders Myelin byare the and supported AVGH, AP, A.V. receives funding and research support from Illumina, Gilead Sciences, Eli Lilly,and Eli Gilead Sciences, Illumina, from research support and A.V. funding receives

Author Manuscriptmutations

ranging from mild intellectual disability to severe early onset epileptic epileptic onset early disability severe to , ranging intellectual from mild This article isprotected by copyright. All rights reserved. American JournalofMedicalGenetics:PartA SZT2 ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS John Wiley&Sons, Inc. in epilepsy and cerebral function (Basel-Vanagaite et al., (Basel-Vanagaite function cerebral epilepsy in and DISCLOSURE DISCLOSURE

8 SZT2 and Leukoencephalopathy and

Page 8of13

Page 9of13 Nakamura, Y., Togawa, Y., Okuno, Y., Muramatsu, H., Nakabayashi, K., Kuroki, Y., . . . Saitoh, Y., .Saitoh, Kuroki, K., H., Nakabayashi, Y., Muramatsu, Y., Okuno, Y., Togawa, Nakamura, Papapostolou, C., Mantzoros, C. S., Evagelopoulou, C., Moses, A. C., & Kleefield, J. (1995). Kleefield, J. & A. C., Moses, C., Evagelopoulou, S., C. Mantzoros, C., Papapostolou, Kircher, M., Witten, D. M., Jain, P., O'Roak, B. J., Cooper, G. M., & Shendure, J. (2014). A J. & O'Roak, G. M., Cooper, Shendure, P., B. J., Jain, M., D. Kircher, Witten, M., Kumar, P., Henikoff, S., & Ng, P. C. (2009). Predicting the effects of coding non-synonymous non-synonymous of coding effects the (2009). Predicting & C. P. Ng, Henikoff, S., Kumar, P., Toutzaris, D., Lewerenz, J., Albrecht, P., Jensen, L. T., Letz, J., Geerts, A., .Methner, A. Letz,L. J., T., Albrecht, P., Jensen, Lewerenz, J., D., Toutzaris, Peng, M., Yin, N., & Li, M. O. (2017). SZT2 dictates GATOR control of mTORC1 signalling. mTORC1 of control GATOR dictates O. (2017). SZT2 M. Li,& Peng, N., Yin, M., Frankel, W. N., Yang, Y., Mahaffey, C. L., Beyer, B. J., & O'Brien, T. P. (2009). Szt2, a novel Szt2, (2009). &L., T.P. B. O'Brien, J., Beyer, C. Mahaffey, N.,Yang, Y., W. Frankel, Cingolani, P., Platts, A., Wang le, L., Coon, M., Nguyen, T., Wang, L., . . . Ruden, D. M. (2012). D. M. .Ruden, L., . Nguyen, T.,Wang, L., M., A., Wang Coon, le, Platts, P., Cingolani, Cleary, J. G., Braithwaite, R., Gaastra, K., Hilbush, B. S., Inglis, S., Irvine, S. A., . . . De La A., .DeIrvine, S. Inglis, S., B. S., Hilbush, Gaastra, R., G., K., Braithwaite, Cleary, J. Baldassari, S., Licchetta, L., Tinuper, P., Bisulli, F., & Pippucci, T. (2016). GATOR1 complex: complex: GATOR1 T.(2016). & F., Pippucci, Bisulli, Licchetta, P., L., Tinuper, S., Baldassari, Pizzino et al.Pizzino et Adzhubei, I. A., Schmidt, S., Peshkin, L., Ramensky, V. E., Gerasimova, A., Bork, . P., A., Gerasimova, L., V. E., Peshkin, I. S., Ramensky, Adzhubei, A., Schmidt, Dali, C. I., Barton, N. W., Farah, M. H., Moldovan, M., Mansson, J. E., Nair, N., . . . Krarup, C. C. E.,Nair, N.,.Krarup, J. Mansson, M., I., H., Farah, Moldovan, M. N.W., Barton, C. Dali, Falcone, M., Yariz, K. O., Ross, D. B., Foster, J., 2nd, Menendez, I., & Tekin, M. (2013). An M. I.,& 2nd, Tekin, Menendez, D. B., J., Foster, Ross, K. O., Yariz, M., Falcone, Basel-Vanagaite, L., Hershkovitz, T., Heyman, E., Raspall-Chaure, M., Kakar, N., Smirin-Yosef, N.,Smirin-Yosef, Kakar, T.,Heyman, Raspall-Chaure, M., E., L., Hershkovitz, Basel-Vanagaite, (3), 310-315. doi:10.1038/ng.2892 doi:10.1038/ng.2892 Genet,(3), 46 310-315. Nat Intern Med,Intern 237 Nature, 543 Nature, (6), 811-820. doi:10.1016/j.freeradbiomed.2009.12.023 doi:10.1016/j.freeradbiomed.2009.12.023 Med,(6), 48 811-820. Biol Radic Nat Methods, 7 Methods, Nat doi:10.1016/j.braindev.2017.08.003 doi:10.1016/j.braindev.2017.08.003 doi:10.1111/j.1601-183X.2009.00509.x doi:10.1111/j.1601-183X.2009.00509.x Imaging of the sella in the syndrome of inappropriate secretion of antidiuretic hormone. hormone. of antidiuretic secretion of inappropriate syndrome the in sella the Imaging of general framework for estimating the relative pathogenicity of human genetic variants. of human relative the pathogenicity estimating for framework general (7), 1073-1081. 4(7), 1073-1081. Protoc, Nat algorithm. using SIFT the function protein on variants (2010). A novel giant peroxisomal superoxide dismutase motif-containing protein. protein. dismutase superoxide motif-containing giant peroxisomal (2010). A novel doi:10.1038/nprot.2009.86 doi:10.1038/nprot.2009.86 developmental delay, macrocephaly and a dysmorphic corpus callosum. corpus callosum. and a dysmorphic delay, macrocephaly developmental epilepsy, entity with a SZT2 discernible in cause clinical (2017). Biallelic mutations S. One, 8 disability. non-syndromicwith intellectual a family in inSZT2 acid deletion amino gene for seizure threshold in mice. mice. in threshold gene for seizure epilepsy and dysmorphic corpus callosum. corpus callosum. and dysmorphic epilepsy with encephalopathy cause infantile mutations Biallelic SZT2 (2013). .Borck, P., G. doi:10.1016/j.ajhg.2013.07.005 doi:10.1016/j.ajhg.2013.07.005 (Austin), 6 iso-3. iso-2; w1118; strain melanogaster of genome the Drosophila in SNPs SnpEff: of single polymorphisms, nucleotide effects the and predicting annotating A program for high-throughput sequencing data. sequencing high-throughput from pedigrees on identification variant mutation and de novo F. (2014). Joint Vega, M. the common genetic actor in focal in epilepsies. genetic actor common the doi:10.1089/cmb.2014.0029 doi:10.1089/cmb.2014.0029 Sunyaev, S. R. (2010). A method and server for predicting damaging missense mutations. mutations. missense damaging for and server predicting method A (2010). Sunyaev, R. S. Ann Clin Transl Neurol, 2 Neurol, Transl Clin leukodystrophy. Ann metachromatic neuropathy in of severity with correlate levels (2015). Sulfatide doi:10.1136/jmedgenet-2016-103883 doi:10.1136/jmedgenet-2016-103883

Author Manuscript doi:10.1371/journal.pone.0082810 e82810. (12),

(2), 80-92. doi:10.4161/fly.19695 (2), doi:10.4161/fly.19695 80-92. (7645), 433-437. doi:10.1038/nature21378 doi:10.1038/nature21378 (7645), 433-437.

(4), 248-249. doi:10.1038/nmeth0410-248 doi:10.1038/nmeth0410-248 (4), 248-249. (2), 181-185. 181-185. (2), This article isprotected by copyright. All rights reserved. American JournalofMedicalGenetics:PartA John Wiley&Sons, Inc. REFERENCES REFERENCES (6), 21 405-419. Biol, J Comput (5), 568-576. 8(5), 568-576. Behav, Genes Brain

(5), 518-533. doi:10.1002/acn3.193 (5), doi:10.1002/acn3.193 518-533. Am J Hum Genet,Hum 93 JAm (8), 503-510. 503-510. Genet,(8), 53 J Med SZT2 and Leukoencephalopathy and (3), 524-529. (3), 524-529. Brain Dev Brain

. Free PLoS PLoS Fly Fly J

Wolfson, R. L., Chantranupong, L., Wyant, G. A., Gu, X., Orozco, J. M., Shen, K., . . . Sabatini, Shen, M., K., .Sabatini, J. Gu, X., Orozco, L., G. A., L., Wyant, R. Wolfson, Chantranupong, Venkatesan, C., Angle, B., & Millichap, J. J. (2016). Early-life epileptic encephalopathy encephalopathy epileptic (2016). Early-life J. J. & B., Millichap, Venkatesan, Angle, C., Unterberger, I., Bauer, R., Walser, G., & Bauer, G. (2016). Corpus callosum and epilepsies. and epilepsies. callosum (2016). Corpus Bauer, G. G., & Walser, I.,Bauer, R., Unterberger, Pizzino et al.Pizzino et Vanderver, A., Simons, C., Helman, G., Crawford, J., Wolf, N. I., Bernard, G., . . . Taft, R. J. I., J. G., .Taft, R. N. Bernard, Wolf, J., G., Helman, Crawford, C., Vanderver, A., Simons, Tshuchida, N., Nakashima, M., Miyauchi, A., Yoshitomi, S., Kimizu, T.,Ganesan, Kimizu, S., V., . A., Yoshitomi, Miyauchi, M., N.,Nakashima, Tshuchida, Neurol, 79 Neurol, doi:10.1684/epd.2016.0828 doi:10.1684/epd.2016.0828 to regulate mTORC1. regulateto mTORC1. nutrients for necessary lysosome and the is to recruits GATOR1 (2017). KICSTOR D. M. secondary to SZT2 pathogenic recessive variants. variants. recessive pathogenic secondary SZT2 to epileptic encephalopathy. encephalopathy. epileptic threeearly-onset in cases of SZT2 mutations biallelic N.(2017). Novel Matsumoto, Seizure, 37 Seizure,

Author Manuscript abnormalities. white matter with patients in sequencing exome (2016). Whole

(6), 1031-1037. doi:10.1002/ana.24650 doi:10.1002/ana.24650 (6), 1031-1037. , 55-60. doi:10.1016/j.seizure.2016.02.012 doi:10.1016/j.seizure.2016.02.012 , 55-60. This article isprotected by copyright. All rights reserved. American JournalofMedicalGenetics:PartA Nature, 543 Nature, Clin Genet Clin John Wiley&Sons, Inc. (7645), 438-442. doi:10.1038/nature21423 doi:10.1038/nature21423 (7645), 438-442. . doi:10.1111/cge.13061 . doi:10.1111/cge.13061

10 (2), 18 195-200. Disord, Epileptic SZT2 and Leukoencephalopathy and Ann Ann

Page 10of13

Page 11of13 Table 1. Clinical and radiographic features of of features radiographic and Clinical 1. Table

patient) (current (current 11 2017) al., et (Nakamura 10 2017) al., et (Tshuchida 9 2017) al., et (Tshuchida 8 2017) al., et (Tshuchida 7 2013) al., et (Falcone (siblings) 4-6 2016) al., et (Venkatesan 3 2013) al., et Vanagaite (Basel- 2 2013) al., et Vanagaite (Basel- 1 Individual

Author Manuscript

p.Tyr2866Leufs p.Leu939Aspfs*19 p.Glu1317Glyfs*4 p.Gly1829Valfs*52 p.Asn1234Alafs*35 p.Phe1401del p.Thr1170ArgfsTer22 p.Phe1834Serfs*47 c.8596dup: c.8596dup: p.Ser2721Cys c.8162C>G: p.Arg2435Trp c.7303C>T: c.2929+1G>A: c.3947dup: c.5482del: c.3700_3716del: c.4202_4204delTTC: p.Arg3235* C>T: c.9703 c.3509_3512delCAGA: c.2092C>T:p.Gln698* p.Ser499Ile c.1496G>T: p.Arg25* c.73C>T: c.6916G>A: p.Gly2306Arg c.6916G>A: c.5499delC: 3delinsCTCGTG c.2930-17_2930- and p.Pro1394Leu) c.4181C>T: SZT2 Gene : Protein : Gene This article isprotected by copyright. All rights reserved. Mutation Mutation American JournalofMedicalGenetics:PartA

∗ 42

John Wiley&Sons, Inc.

Homozygous heterozygous Compound heterozygous Compound Homozygous heterozygous Compound heterozygous Compound heterozygous Compound heterozygous Compound heterozygous Compound Mutation Mutation type

pathogenic variants variants SZT2 pathogenic

bridge, hypertelorism) bridge, nasal flattened bossing, macrocephaly ID, Mild-moderate fissures) palpebral slanted down- (macrocephaly, features dysmorphic EOEE, fissures) palpebral downslanting ptosis, forehead, (high features dysmorphic EOEE, fissures) palpebral downslanting ptosis, forehead, (high features dysmorphic EOEE, (macrocephaly with frontal frontal with (macrocephaly features dysmorphic EOEE, hypertelorism forehead, high macrocephaly, ID, moderate EOEE, microcephaly palate, high-arched anomaly, Duane ID, severe EOEE, forehead high ID, severe EOEE, eyebrows) arched ptosis, forehead, (high features dysmorphic ID, severe EOEE, Clinical phenotype Clinical

Normal progressive atrophy progressive Migrational abnormalities Migrational pellucidum septum cavum persistent CCdysgenesis, pellucidum septum cavum peristent CCdysgenesis, myelination and and myelination of loss CCdysgenesis, CCdysgenesis ventricles enlarged CCatrophy, ventricles enlarged pellucidum, septum cavus persistent CCatrophy, CCdysgenesis Imaging findings by MRI by findings Imaging

Author Manuscript disability intellectual = ID callosum; corpus = CC codon; stop * = frameshift, fs = encephalopathy; epileptic earlyonset EOEE= deletion; = del This article isprotected by copyright. All rights reserved. American JournalofMedicalGenetics:PartA John Wiley&Sons, Inc. Page 12of13

Page 13of myelination has regressed. myelinationinterval has ganglia shows Single over basal voxel the MRS left normalization Cr, of and ventricles. intermedia thalamic and The mass is Myelin (whiteabsent progression3a). asterisk, isthan less 3. Images at 31 months show at 31months Images 3. interval mild of onset brain volume loss withdiffuse mild sulci of prominence the left basal ganglia shows intervalganglialeft shows the basal mild an remainswhichmildly Cr increase in diminished with persistent 2. Imaging at 21months is at Imaging with 2. persistent unchanged megalencephaly, thickening callosum,corpus the of expectedwith delayedconsistent myelination.spectroscopy for MR of age, Singleover or regression voxel callosum. The blunted The callosalMyelination callosum. rostrumis consistent1a). is with mild dysgenesis arrow, (white arrow, 5c). Myelination continues to regress.Myelination to 5c). adenohypophysis continues arrow, The of is newlywith maintenance enlarged 4. Images at depict years 3 Images 4. interval further diffuse volume loss with mild brain sulci of prominence (white ventricles.decreasedissplenium abnormally now The further and in has size 5a), (asterisk, bright (white overganglialeftreveals the basal elevationdiminished with consequent Cholineto of (Cho) (Cr) creatine bluntingand rostrum, the myelination. normal of Single voxelTE 1500, TR spectroscopy288) MR (MRS; 1. Imaging at 11months demonstrates at Imaging 1. macroencephaly with mild corpus the generalized of thickening 5. Images at 4 years show interval at show years 4 Images 5. further diffuse brainvolumesulci of loss withand mild prominence arrow, 4b) and ventricles 4b) arrow, and (white and isgenu the 4b). The smallersplenium asterisk, in than caliber

AuthorSerial resonance magnetic patient A. a of imaging mutations. with(MRI) SZT2 Manuscript

creatine and N-acetylaspartate (NAA) to N-acetylaspartate creatine(NAA) creatineratios and forage. This article isprotected by copyright. All rights reserved. elevation of Cho:Cr and NAA:Cr and elevationCho:Cr of ratiosforage. American JournalofMedicalGenetics:PartA NAA:Cr, and ratiosfor and Cho:Cr NAA:Cr, age. John Wiley&Sons, Inc. normal. normal.

B. Alignment of the the Alignment of protein B. r SZT2 sequence mutated the surrounding homologous SZT2 protein homologousSZT2 sequences representativefrom vertebrate species. proteinSZT2 sequences were ventricles, decreased splenium, and regression and ventricles,myelination. of increased decreased The further splenium, has adenohypophysis 6. Images at demonstrate years 7 6. interval Images further diffusebrain volume sulci lossof with prominence and in size with new loss of normal posteriorin sizeimaging The shows with(white pituitaryloss normal new 6a). of arrow, signal Author Manuscriptretrievedfull-length proteinuniprot were and alignments generatedOmega. from with Clustal

hypertelorismwith mild microphthalmia (6d). symmetric This article isprotected by copyright. All rights reserved. American JournalofMedicalGenetics:PartA normal posteriornormal signal. pituitary 165x167mm(300 300DPI) x John Wiley&Sons, Inc.

esiduep.Gly2306arrow)by to (shown Page 14of13