ANXA11 Mutations Prevail in Chinese ALS Patients with and Without Cognitive Dementia E237

Volume 4, Number 3, June 2018 Neurology.org/NG

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ARTICLE ANXA11 mutations prevail in Chinese ALS patients with and without cognitive dementia e237

ARTICLE Determining the incidence of familiality in ALS: A study of temporal trends in Ireland from 1994 to 2016 e239

ARTICLE Rare variants and de novo variants in mesial temporal lobe epilepsy with hippocampal sclerosis e245

CLINICAL/SCIENTIFIC NOTE Brain copper storage aft er genetic long-term correction in a mouse model of Wilson disease e243 Academy Officers Neurology® is a registered trademark of the American Academy of Neurology (registration valid in the United States). Ralph L. Sacco, MD, MS, FAAN, President Neurology® Genetics (eISSN 2376-7839) is an open access journal published James C. Stevens, MD, FAAN, President Elect online for the American Academy of Neurology, 201 Chicago Avenue, Ann H. Tilton, MD, FAAN, Vice President Minneapolis, MN 55415, by Wolters Kluwer Health, Inc. at 14700 Citicorp Drive, Bldg. 3, Hagerstown, MD 21742. Business offices are located at Two Carlayne E. Jackson, MD, FAAN, Secretary Commerce Square, 2001 Market Street, Philadelphia, PA 19103. Production offices are located at 351 West Camden Street, Baltimore, MD 21201-2436. Janis M. Miyasaki, MD, MEd, FRCPC, FAAN, Treasurer © 2018 American Academy of Neurology. Terrence L. Cascino, MD, FAAN, Past President Neurology® Genetics is an official journal of the American Academy of Neurology. Journal website: Neurology.org/ng, AAN website: AAN.com Executive Office, American Academy of Neurology Copyright and Permission Information: Please go to the journal website (www.neurology.org/ng) and click the Permissions tab for the relevant Catherine M. Rydell, CAE, Executive Director/CEO article. Alternatively, send an email to [email protected]. 20l Chicago Ave General information about permissions can be found here: https://shop.lww.com/ journal-permission. Minneapolis, MN 55415 Disclaimer: Opinions expressed by the authors and advertisers are not Tel: 612-928-6000 necessarily those of the American Academy of Neurology, its affiliates, or of the Publisher. The American Academy of Neurology, its affiliates, and the Publisher disclaim any liability to any party for the accuracy, completeness, Editorial Office efficacy, or availability of the material contained in this publication (including drug dosages) or for any damages arising out of the use Patricia K. Baskin, MS, Executive Editor or non-use of any of the material contained in this publication. Kathleen M. Pieper, Senior Managing Editor, Neurology Advertising Sales Representatives: Wolters Kluwer, 333 Seventh Avenue, Lee Ann Kleffman, Managing Editor, Neurology: Genetics New York, NY 10001. Contacts: Eileen Henry, tel: 732-778-2261, fax: 973-215- 2485, [email protected] and in Europe: Avia Potashnik, Wolters Sharon L. Quimby, Managing Editor, Neurology® Clinical Practice Kluwer, tel: +44 207 981 0722; +44 7919 397 933 or e-mail: avia.potashnik@ Morgan S. Sorenson, Managing Editor, Neurology® Neuroimmunology & Neuroinflammation wolterskluwer.com. Careers & Events: Monique McLaughlin, Wolters Kluwer, Two Commerce Cynthia S. Abair, MA, Senior Graphics Editor Square, 2001 Market Street, Philadelphia, PA 19103, tel: 215-521-8468, fax: 215- Andrea R. Rahkola, Production Editor, Neurology 521-8801; [email protected]. Robert J. Witherow, Senior Editorial Associate Reprints: Meredith Edelman, Commercial Reprint Sales, Wolters Kluwer, Two Commerce Square, 2001 Market Street, Philadelphia, PA 19103, tel: 215-356-2721; Karen Skaja, Senior Editorial Associate [email protected]; [email protected]. Kaitlyn Aman Ramm, Editorial Assistant Special projects: US & Canada: Alan Moore, Wolters Kluwer, Two Kristen Swendsrud, Editorial Assistant Commerce Square, 2001 Market Street, Philadelphia, PA 19103, tel: 215-521-8638, [email protected]. International: Andrew Andrea Willgohs, Editorial Assistant Wible, Senior Manager, Rights, Licensing, and Partnerships, Wolters Kluwer; [email protected]. Publisher Wolters Kluwer Baltimore, MD

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Scientiﬁc Integrity Advisor Robert B. Daroﬀ, MD, FAAN TABLE OF CONTENTS Volume 4, Number 3, June 2018 Neurology.org/NG

e239 Determining the incidence of familiality in ALS: A study of temporal trends in Ireland from 1994 to 2016 M. Ryan, M. Heverin, M.A. Doherty, N. Davis, E.M. Corr, A. Vajda, N. Pender, R. McLaughlin, and O. Hardiman Open Access

e242 Chorea-acanthocytosis: Homozygous 1-kb deletion in VPS13A detected by whole-genome sequencing S. Walker, R. Dad, B. Thiruvahindrapuram, M.I. Ullah, A. Ahmad, M.J. Hassan, S.W. Scherer, and B.A. Minassian Open Access

e240 Neurodegeneration as the presenting symptom in 2 adults with xeroderma pigmentosum complementation group F N.M. Shanbhag, M.D. Geschwind, J.J. DiGiovanna, C. Groden, R. Godfrey, M.J. Yousefzadeh, E.A. Wade, L.J. Niedernhofer, M.C.V. Malicdan, K.H. Kraemer, W.A. Gahl, and C. Toro Open Access

e244 Absence of NEFL in patient-specific neurons in early-onset Charcot-Marie-Tooth neuropathy M.T. Sainio, E. Ylikallio, L. Mäenpää, J. Lahtela, P. Mattila, M. Auranen, J. Palmio, and H. Tyynismaa Open Access

Clinical/Scientific Notes

e232 Expanding the global prevalence of spinocerebellar ataxia type 42 K. Ngo, M. Aker, L.E. Petty, J. Chen, F. Cavalcanti, A.B. Nelson, S. Hassin-Baer, M.D. Geschwind, S. Perlman, D. Italiano, A. Lagan`a, S. Cavallaro, G. Coppola, J.E. Below, and B.L. Fogel Open Access

e243 Brain copper storage after genetic long-term correction in Editorial a mouse model of Wilson disease R. Uerlings, D. Moreno, O. Murillo, C. Gazquez, R. Hern´andez-Alcoceba, e241 Whole-exome sequencing to disentangle the complex G. Gonz´alez-Aseguinolaza, and R. Weiskirchen genetics of hippocampal sclerosis–temporal lobe epilepsy Open Access P. Striano and C. Nobile Open Access Companion article, e245 Correction

Articles e238 Expanding the global prevalence of spinocerebellar ataxia type 42 e245 Rare variants and de novo variants in mesial temporal lobe epilepsy with hippocampal sclerosis J.K.L. Wong, H. Gui, M. Kwok, P.W. Ng, C.H.T. Lui, L. Baum, P.C. Sham, P. Kwan, and S.S. Cherny Open Access Editorial, e241 e236 Somatic GNAQ mutation in the forme fruste of Sturge-Weber syndrome M.S. Hildebrand, A.S. Harvey, S. Malone, J.A. Damiano, H. Do, Z. Ye, L. McQuillan, W. Maixner, R. Kalnins, B. Nolan, M. Wood, E. Ozturk, N.C. Jones, G. Gillies, K. Pope, P.J. Lockhart, A. Dobrovic, R.J. Leventer, I.E. Scheffer, and S.F. Berkovic Open Access Cover image e237 ANXA11 mutations prevail in Chinese ALS patients with Immunocytochemical analysis of MAP2 (green) and TUBB3 (red) and without cognitive dementia proteins in iPSC-derived neurons of a patient with early-onset Charcot- “ ﬁ K. Zhang, Q. Liu, K. Liu, D. Shen, H. Tai, S. Shu, Q. Ding, H. Fu, S. Liu, Marie-Tooth neuropathy. See Absence of NEFL in patient-speci c Z. Wang, X. Li, M. Liu, X. Zhang, and L. Cui neurons in early-onset Charcot-Marie-Tooth neuropathy.” Open Access See e244 EDITORIAL OPEN ACCESS Whole-exome sequencing to disentangle the complex genetics of hippocampal sclerosis–temporal lobe epilepsy

Pasquale Striano, MD, PhD, and Carlo Nobile, PhD Correspondence Dr. Striano Neurol Genet 2018;4:e241. doi:10.1212/NXG.0000000000000241 [email protected]

Mesial temporal lobe epilepsy with Mesial temporal lobe epilepsy with hippocampal sclerosis RELATED ARTICLE (MTLE-HS) is a common epilepsy syndrome accounting for approximately 20% of people with epilepsy.1 It typically shows electroclinical features indicative of seizure onset in the mesial or Rare variants and de novo limbic structures of the temporal lobe, i.e., epigatric/visceral, autonomic, psycho-affective, and variants in mesial temporal sensorial symptoms, including déjàvu.1 Awareness is generally preserved at onset, but loss of lobe epilepsy with consciousness may also occur, with motionless stare and oro-alimentary, vocal, or gestural hippocampal sclerosis automatisms, eventually followed by a convulsive seizure. EEGs show anterior or mid-temporal Page e245 epileptic abnormalities combined with focal slowing. Hippocampal sclerosis (HS) is de- monstrable on coronal MRI sequences by a unilateral (or asymmetrical) decrease in hippocampal volume and an increase in signal on T2-weighted sequences. The neuropathologic hallmark of HS is a combination of atrophy and astrogliosis of the amygdala, hippocampus, uncus, para- hippocampal gyrus, and the entorhinal cortex.1 The diagnosis of MTLE-HS is crucial because it is often uncontrolled by antiseizure drugs but typically responsive to resective surgery.2,3

The etiology of MTLE-HS remains largely elusive. Although generally perceived as an acquired – disorder, a few familial cases have been reported,4 6 suggesting complex inheritance, similar to that widely accepted for genetic generalized epilepsies. Hitherto, this condition is an appropriate target for contemporary approaches to complex disorders, such as genome-wide association studies for common genetic variants or deep sequencing for rare variants. A relatively recent association study, including ;1,000 patients with MTLE-HS compared with ;7,500 controls, identiﬁed a robust association with a polymorphic marker in the sodium channel gene SCN1A.7

In this issue of Neurology Genetics®, Wong et al.8 investigated the role of rare and de novo genetic variants in MTLE-HS by whole-exome sequencing (WES) performed in a small well- characterized cohort of Han Chinese patients from Hong Kong clinically homogeneous as to seizure semiology, ictal/interictal EEG recordings, and high-deﬁnition brain MRI studies. Age at onset of epilepsy was ≥2 years. As control, the authors used WES data from 692 Hong Kong Han Chinese participants with no history of developmental or neuropsychiatric disorders. Association studies of rare variants (frequency of minor allele <1% in population databases) were performed at gene or gene-set levels by comparing the total amounts of variation found in individual genes or in groups of clinically or functionally connected genes, respectively, in cases and controls. Moreover, in a subgroup of patients, of whom both parents were available, rare variant analysis of parent-proband trios was performed to uncover de novo variants not transmitted by either parent and potentially recessive mutations inherited by both parents.

Overall, WES data from 47 patients (26 females), including 23 trios, led Wong et al.8 to identify rare and de novo variants in a number of genes. Notably, compared to population controls, signiﬁcant enrichment of rare variants was observed in SEC24B, a gene involved in vesicle traﬃcking and development, whereas gene-set association analysis showed variant enrichment

From the Pediatric Neurology and Muscular Diseases Unit (P.S.), Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, “G. Gaslini” Institute, Genova; and CNR-Neuroscience Institute and Department of Biomedical Sciences (C.N.), University of Padua, Italy.

Funding information and disclosures are provided at the end of the article. Full disclosure form information provided by the authors is available with the full text of this article at Neurology.org/NG. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 in the fragile X mental retardation protein (FMRP)-related preliminary findings emerging from this WES study. In a re- group of genes, which comprises hundreds of genes regulated cent study, the group of E. Aronica examined the pathologic by the FMRP protein, including the mammalian target of cellular pathways involved in different phases of epilepto- rapamycin (mTOR) pathway. In addition, analysis of trios genesis in human and animal hippocampus.10 This analysis revealed 21 de novo variants, many of which are known to be revealed involvement of several key pathogenic pathways associated with different neuropsychiatric disorders. These underlying epileptogenesis, including inflammation, gliosis results, however, while providing useful hints for future and deregulation of the extracellular matrix. Better un- studies, should be considered with caution on a clinical/ derstanding of gene expression and regulation during the diagnostic ground because of the low statistical power of the course of epileptogenesis in MTLE may eventually produce cohort investigated. Indeed, the enrichment of variants significant advances for the development of preventive revealed in SEC24B, based on 3/47 variants identified in the treatment for this common chronic neurologic disease. patients vs 1/652 in the controls, only indicates a statistical trend to be confirmed in studied of larger patient cohorts; Study funding variant enrichment in the FMRP gene set might reflect the No targeted funding reported. higher number of genes making up this gene group as compared to the other gene sets investigated. Also, the conclusion Disclosure that FMRP targets by its putative interaction with the mTOR P. Striano has served on the scientific advisory board of the pathway may play a pathogenic role in MTLE-HS sounds Italian Agency of the Drug (AIFA) and received honoraria attractive, but it is speculative. However, the frequency of de from Kolfarma s.r.l., UCB pharma, and Eisai Inc., and research novo mutations detected in the patients (21/23 trios) does support from the Italian Ministry of Health. C. Nobile re- not exceed that expected in the general population (approx- ceived research support from the Telethon Foundation imately 1 de novo variant per individual). Some of them affect (Grant no. GGP15229) and the Genetics Commission of the genes such as ROBO4, NLGN3, and CEP170B, which have Italian League Against Epilepsy. Full disclosure form in- been found to harbor variants in autism spectrum disorder, formation provided by the authors is available with the full but their involvement in epilepsy awaits confirmatory studies. text of this article at Neurology.org/NG.

Overall, no major hit emerged for MTLE-HS from this study, References supporting the view that the genetic architecture underlying 1. Blümcke I, Aronica E, Miyata H, et al. International recommendation for a comprehensive neuropathologic workup of epilepsy surgery brain tissue: a consensus task MTHE-HS is complex and that MTLE-HS and other neu- force report from the ILAE Commission on Diagnostic Methods. Epilepsia 2016;57: ropsychiatric disorders may have shared biology. The major 348–358. 2. Androsova G, Krause R, Borghei M, et al. Comparative effectiveness of antiepileptic limitation of this study is the relatively small cohort of in- drugs in patients with mesial temporal lobe epilepsy with hippocampal sclerosis. vestigated patients, particularly the small number of analyzed Epilepsia 2017;58:1734–1741. 3. Wiebe S, Blume WT, Girvin JP, et al. Effectiveness and efficiency of surgery for trios, which is unlikely to produce positive results, especially if temporal lobe epilepsy study group. N Engl J Med 2001;345:311–318. considering the likely polygenic nature of the disease. Genetic 4. Briellmann RS, Torn-Broers Y, Jackson GD, Berkovic SF. Seizures in family members of patients with hippocampal sclerosis. Neurology 2001;57:1800–1804. epilepsies include over 30% of all epilepsy syndromes. Next- ’ ff 5. Kobayashi E, D Agostino MD, Lopes-Cendes I, et al. Hippocampal atrophy and T2- generation sequencing has proven to be e ective in identify- weighted signal changes in familial mesial temporal lobe epilepsy. Neurology 2003;60: ing mutations for mendelian, single-gene disorders.9 By 405–409. 6. Striano P, Gambardella A, Coppola A, et al. Familial mesial temporal lobe epilepsy contrast, this technique have showed so far limited success in (FMTLE): a clinical and genetic study of 15 Italian families. J Neurol 2008;255: the identification of variants causing more complex pheno- 16–23. types where the phenotypes are more heterogeneous, and it is 7. Kasperaviciute D, Catarino CB, Matarin M, et al. Epilepsy, hippocampal sclerosis and febrile seizures linked by common genetic variation around SCN1A. Brain 2013;136: unclear whether they result from the action of a single gene, 3140–3150. multiple genes, or a complex interaction between the genetic 8. Wong JKL, Hongsheng G, Maxwell K, et al. Rare variants and de novo variants in and environmental factors. mesial temporal lobe epilepsy with hippocampal sclerosis. Neurol Genet 2018;4: e245. 9. Orsini A, Zara F, Striano P. Recent advances in epilepsy genetics. Neurosci Lett 2018; The increasing development of experimental tools and bio- 667:4–9. 10. Korotkov A, Mills JD, Gorter JA, van Vliet EA, Aronica E. Systematic review and meta- informatics analysis for a large-scale evaluation of gene ex- analysis of differentially expressed miRNAs in experimental and human temporal lobe pression may help overcome this limitation to confirm the epilepsy. Sci Rep 2017;7:11592.

2 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG ARTICLE OPEN ACCESS Rare variants and de novo variants in mesial temporal lobe epilepsy with hippocampal sclerosis

John K.L. Wong, PhD, Hongsheng Gui, PhD, Maxwell Kwok, BSc, Ping Wing Ng, MD, Colin H.T. Lui, MD, Correspondence Larry Baum, PhD, Pak Chung Sham, MD, PhD, Patrick Kwan, MD, PhD, and Stacey S. Cherny, PhD Dr. Cherny [email protected] or Neurol Genet 2018;4:e245. doi:10.1212/NXG.0000000000000245 Dr. Kwan [email protected] or Dr. Sham [email protected]

Abstract RELATED ARTICLE Objective Editorial We investigated the role of rare genetic variants and of de novo variants in the pathogenesis of Whole-exome sequencing mesial temporal lobe epilepsy related to hippocampal sclerosis (MTLE-HS). to disentangle the complex genetics of hippocampal – Methods sclerosis temporal lobe Whole-exome sequencing (WES) was performed in patients with MTLE-HS and their un- epilepsy aﬀected parents (trios). Genes or gene sets that were enriched with predicted damaging rare Page e241 variants in the patients as compared to population controls were identiﬁed. Patients and their parents were compared to identify whether the variants were de novo or inherited.

Results After quality control, WES data from 47 patients (26 female), including 23 complete trios, were available for analysis. Compared with population controls, significant enrichment of rare variants was observed in SEC24B. Integration of gene set data describing neuronal functions and psychiatric disorders showed enrichment signal on fragile X mental retardation protein (FMRP) targets. Twenty-one de novo variants were identified, with many known to cause neuropsychiatric disorders. The FMRP-targeted genes also carried more de novo variants. Inherited compound heterozygous and homozygous variants were identified.

Conclusions The genetic architecture underlying MTHE-HS is complex. Multiple genes carrying de novo variants and rare variants among FMRP targets were identiﬁed, suggesting a pathogenic role. MTLE-HS and other neuropsychiatric disorders may have shared biology.

From the Centre for Genomic Sciences and Department of Psychiatry (J.K.L.W., H.G., L.B., P.C.S., S.S.C.), Li Ka Shing Faculty of Medicine, The University of Hong Kong; Department of Medicine and Therapeutics (M.K., P.K.), The Chinese University of Hong Kong; Department of Medicine (P.W.N.), United Christian Hospital; Department of Medicine (C.H.T.L.), Queen Elizabeth Hospital, Hong Kong, China; Departments of Medicine and Neurology (P.K.), The University of Melbourne, Royal Melbourne Hospital, Australia; Department of Epide- miology and Preventive Medicine (S.S.C.) and Department of Anatomy and Anthropology (S.S.C.), Sackler Faculty of Medicine, Tel Aviv University, Israel; and The State Key Laboratory of Brain and Cognitive Sciences (P.C.S., S.S.C.).

The Article Processing Charge was funded by the University of Hong Kong. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary ASD = autism spectrum disorder; ExAC = Exome Aggregation Consortium; FMRP = fragile X mental retardation protein; GWAS = genome-wide association study; HS = hippocampal sclerosis; IL = interleukin; MAF = minor allele frequency; MGI = Mouse Genome Informatics; mGluR = metabotropic glutamate receptor; MTLE-HS = mesial temporal lobe epilepsy related to hippocampal sclerosis; NMDAR = NMDA receptor; SNP = single nucleotide polymorphism; WES = whole-exome sequencing.

Mesial temporal lobe epilepsy related to hippocampal sclerosis HS with concordant findings from seizure semiology, EEG (MTLE-HS) is among the most drug-resistant types of focal (interictal and ictal recording during prolonged video EEG epilepsy.1 Histologically, MTLE-HS is characterized by atrophy monitoring), MRI (1.5T or 3T) findings characteristic of hip- and astrogliosis of the amygdala, hippocampus, para- pocampal sclerosis (HS), and histologic confirmation of HS in hippocampal gyrus, and the entorhinal cortex.2 In drug- patients who had undergone resective epilepsy surgery.11 All resistant patients, resection of the sclerotic hippocampus and patients were ethnic Han Chinese with an age at onset of epi- the surrounding mesial temporal structures can be an effective lepsy of ≥2years.Patientswereexcludediftherewasevidenceof treatment3; hence, MTLE-HS remains one of the most com- extratemporal lobe seizures, they had no history of seizure, had mon indications for epilepsy surgery.4 Understanding the psychogenic nonepileptic seizures, or had other epileptogenic molecular basis of MTLE-HS may lead to identification of lesions identified on MRI. Parents of probands were eligible for novel drug targets and alleviate the need for invasive treatment. inclusion if they did not have epilepsy or history of febrile seizure. Each participant provided either venous blood or saliva The pathogenesis of MTLE-HS is unknown. Several studies samples from which DNA was extracted for sequencing using have investigated the role of common susceptibility variants in standard protocols. A total of 48 patients with MTLE-HS were the pathogenesis of MTLE-HS.5 Early studies reported asso- enrolled. Both parents were recruited for 23 patients, forming ciations between single nucleotide polymorphisms (SNPs) in complete trios. The study was approved by the ethics com- interleukin (IL)-1, PDYN, GABBR1, and PRNP and mesial mittees of the participating hospitals. All participants or their temporal lobe epilepsy, but the results have been controversial.6 legal guardians provided written informed consent. A recent genome-wide association study (GWAS) identified an association with SCN1A polymorphisms.7 However, a large Population controls heritability study of focal epilepsy suggested that common For the purpose of quality assessment and association testing, variants explained only 3% of heritability.8 WES data from 692 Hong Kong Han Chinese participants (298 men and 394 women) were added to the calling set Recently, whole-exome sequencing (WES) of 356 trios dis- (Supplemental materials, links.lww.com/NXG/A55) (mean – covered 429 de novo variants in patients with epileptic age: 41.1 years, range: 15 55 years). They were participants in encephalopathies, with recurrent mutations in 19 genes.5 We a population-based study investigating lumbar disc de- hypothesized that, similar to other focal epilepsy syndromes,9 generation and did not have a history of developmental or 12 both rare and de novo variants may underlie the unexplained neuropsychiatric disorders. genetic susceptibility to MTLE-HS. WES was applied in Standard protocol approvals, registrations, a recent study of patients with a variety of common general- 10 and patient consents ized and focal epilepsy syndromes, but trios were not examined. We performed WES on patients with MTLE-HS and The study was approved by the Joint Chinese University of some unaffected parents in this study. Hong Kong-New Territories East Cluster Clinical Research Ethics Committee (ref. No. 2004.068 and 2004.268). All participants or their legal guardians provided written informed consent. Methods Whole-exome sequencing and Study design bioinformatics pipeline We performed WES on patients with MTLE-HS and their Detailed methodology for WES and the bioinformatics unaffected parents. This enabled us to examine the effects of pipeline including quality control, variant calling, and in silico rare variants among all the patients by comparing with pop- analysis, is provided in supplementary materials. ulation controls and to identify genes containing de novo and inherited variants in trio-based analysis. Association analysis

Participants Gene-based association tests Patients with MTLE-HS (probands) and their parents were Case-control analyses of rare variants (minor allele frequency recruited from 3 regional hospitals (Prince of Wales Hospital, [MAF] <1% in the 1000 Genomes Project Phase III, Exome United Christian Hospital, and Queen Elizabeth Hospital) in Aggregation Consortium (ExAC), and dbSNP137 databases) Hong Kong. Inclusion criteria of probands were “pure” MTLE- included all the patients with MTLE-HS and population

2 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG controls. Principle component analysis on the patient group variant. In addition to the quality control described above, (N = 47) and control group (N = 692) revealed no systematic each de novo variant required a read depth ≥8 to be called. bias between the 2 data sets (figure e-1, links.lww.com/NXG/ Mutations found in any of the 3 population databases A55). Burden tests on rare damaging variants were performed (dbSNP137, ExAC, or 1000 Genome phase III) were ex- per gene from the human RefGene database. Three criteria cluded. All de novo variants were validated by Sanger se- were adopted to select rare damaging variants within genes for quencing. The nonsynonymous to synonymous ratio was the test: (1) single nucleotide variants with MAF >1% in the compared with other exome sequencing studies. The can- 1000 Genomes Project Phase III, ExAC, and dbSNP137 didate focal epilepsy gene list (table e-1, links.lww.com/ databases were excluded; (2) missense and nonsense variants NXG/A55) has been compared with our de novo variant that passed quality control were used in the tests; and (3) only gene list for overlaps. The 25 gene sets tested for rare variant genes carrying 3 or more variants were included in the gene- association were also tested for enrichment of de novo var- based association tests. A burden-style test from PLINK/SEQ iants by hypergeometric test, so as to assess whether the (https://atgu.mgh.harvard.edu/plinkseq) was chosen for the same biological network was disrupted among the de novo purpose. The burden test was performed using 1,000,000 genes. rounds of adaptive permutations. Multiple testing corrections were applied for the number of tested genes using the false Inherited variant analysis: homozygotes and discovery rate.13 Gene annotation and damaging variant compound heterozygotes ff prediction was performed using KGGSeq, which combines To investigate the e ect of heterozygous and homozygous ≥ multiple prediction methods.14 (double hit) events, genes carrying 2 mutations in heterozygous and homozygous configurations were summa- Set-based association tests rized. At least 1 hit inherited from each unaffected parent is For set-based association tests, we considered groups of genes required to fit the sporadic nature of the recruited samples. with similar biological functions as the unit of testing (gene Rare variants at MAF ≤ 1% (in the 1000 Genomes Project set). We first included a set of focal epilepsy candidate genes15 Phase III, ExAC, and dbSNP137 databases) were considered (table e-1, links.lww.com/NXG/A55). In addition, given the in the analysis. We considered the following scenarios of high prevalence of psychiatric disorders among patients genes carrying double hit variants, which are rare/absent in with focal epilepsy,16 we curated additional gene sets by the population: (1) candidate genes suggested by functional considering those of psychiatric disorders and of important databases (Phenolyzer and SynaptomeDB) and with ad- neuronal functions (table e-2, links.lww.com/NXG/A55). justed p values <0.05; (2) both contributing variants are Gene sets were obtained from the “Genebook” websi- nonsense and not found among phased controls (Genome of te(atgu.mgh.harvard.edu/;spurcell/genebook/genebook.cgi) the Netherlands project suggested double knockout by Loss- 22 previously used for a large-scale exome sequencing study of of-Function variants are very rare) ; and/or (3) genes with schizophrenia.17 The primary and secondary gene sets on the de novo variants carried by the patients with MTLE-HS. website cover 2,546 gene candidates involved in important Details of each approach are described in Supplemental systems of neuronal functions and previous studies of psychi- materials, links.lww.com/NXG/A55. The recurrences in the atric disorders, including intellectual disability, schizophrenia, subsequent gene list were further investigated for their rel- fragile X syndrome, and autism spectrum disorders (ASD). In evance to MTLE-HS. particular, rare variants of fragile X mental retardation protein (FMRP)-targeted genes have been found to be enriched in Integrative genomic annotation multiple psychiatric disorders (fragile X, ASD, and schizo- To investigate whether the gene sets suggested by the asso- phrenia).18 FMRP is encoded by the gene FMR1, an RNA- ciation tests are enriched at the transcriptome level, we binding protein that regulates translation of synaptic genes for reviewed published expression studies on hippocampal tissues normal neurogenesis.19 We combined the 2 previously repor- of patients with MTLE-HS to derive an intersected list of ted FMRP sets20,21 to better evaluate the importance of FMRP dysregulated genes (table e-3, links.lww.com/NXG/A55). fi targets in MTLE-HS. The same criteria for variant selection Seven expression studies were identi ed after excluding and statistical tests as used for gene-based association were used studies on other types of epilepsies. Genes reported by at least for gene set–based association. 2 studies were included in the gene list. The genes were tested for enrichment with the 25 candidate gene sets derived from Trio-based analysis the “Genebook” website. Hypergeometric tests were per- To identify de novo and inherited variants attributed to formed, where p values were corrected for the number of tests MTLE-HS, trio-based analyses were performed in the sub- (25 sets × 7 studies). group of MTLE-HS patients with complete trios. To investigate the functional relevance of the candidate genes De novo variants and gene set enrichment analysis carrying de novo variants, we queried the gene list against KGGSeq was used for the discovery and annotation of de Mouse Genome Informatics (MGI) and ClinVar.23 Genes novo variants in the trios. The predicted damaging effects and reported to cause any neural abnormalities in knockout variation intolerance scores were annotated per gene and per mouse models or by human genetic studies were tabulated.

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 3 Results reported in patients with other neuropsychiatric disorders, including intellectual disability (5.6:1), ASD (3.1:1), and Participants schizophrenia (5.1:1)24 (table e-4, links.lww.com/NXG/ A total of 48 patients with MTLE-HS were enrolled. Sequence A55). Two of the patients with de novo variants have a family data from 1 patient did not pass quality control and was history of epilepsy, implying that, as expected, not all of the de excluded (supplemental data, links.lww.com/NXG/A55), novo variants cause epilepsy. leaving 47 patients (26 female) for further analysis. The characteristics of the patients are shown in table 1. The me- The list of the 18 genes carrying the nonsynonymous de novo dian age at onset of epilepsy was 17.7 years (range 1.5–43 variants was compared with 3 studies investigating de novo years), and the median age of recruitment was 39.5 years variants in different neuropsychiatric disorders (epileptic (range 13–57.4 years). There were equal numbers of patients encephalopathies,25 ASD,26 and schizophrenia24). We found with left and right HS (21 patients each); 5 patients had that 5 of 18 genes were reported by one of the studies, and 3 bilateral disease. Thirty patients with unilateral drug-resistant genes (ROBO4, NLGN3, and CEP170B) were found to overlap MTLE-HS had undergone epilepsy surgery (anterior tem- with the ASD study (table 4). However, none of the 18 genes poral lobectomy and amygdalohippocampectomy). Seizure affected by nonsynonymous de novo variants was found in the onset was confirmed on ictal video EEG recording in all focal epilepsy gene set (table e-1, links.lww.com/NXG/A55). patients before surgery. Histology confirmed HS in the resected hippocampus in all patients. Nineteen were seizure- Gene set enrichment analysis for de novo variants free after surgery. Both parents were recruited for 23 patients, The enrichment test suggests that the FMRP-targeted genes forming complete trios. None of the parents had a history of also carried more de novo variants in the patients with MTLE- epilepsy or febrile seizure. All probands and parents were of HS, which is in agreement with the association results Han Chinese descent. mentioned above (table e-5, links.lww.com/NXG/A55). Al- though testing of the 2 FMRP-targeted gene sets20,21 sepa- Association analysis rately were not significant, the merged FMRP set achieved fi Gene-based and set-based association tests a signi cant p value (p < 0.0013). Gene-based p values are shown in table 2. The SEC24B gene remained significant after correction for multiple testing (q < Inherited variants: homozygotes and compound heterozygotes 0.041, 5,154 genes). The association was contributed by 4 We assessed the patients for inherited damaging variants predicted damaging variants, 3 of which were found in the acting in a recessive manner, either as compound hetero- patients and 1 in the population controls. zygotes or homozygotes. Genes fulfilling such criteria were considered to be carrying “double-hit” variants. For loss-of- Table 3 shows results of the set-based tests using 25 gene sets. function variants, we considered all rare events (MAF < 1%). After correction for multiple comparisons, significant en- Only 1 gene, P2RX7, was identified, harboring a homozygous richment was observed in the FMRP-related gene sets. The nonsense variant (NM_002562:c.1591G>T:p.E531*) in bigger FMRP set20 achieved a higher significance level, in- − a single patient. The mutation was validated by Sanger cluding all rare variants in the comparison (p < 3.88 × 10 4). sequencing. When restricting to the rare predicted damaging variants, the unified set of 2 FMRP gene sets20,21 also achieved statistical − For missense variants, we considered genes carrying double significance (p < 2.89 × 10 4). There was no significant en- hit variants, which are recurrent in MTLE-HS or present in richment for rare variants in the candidate focal epilepsy the de novo gene list. In addition to restricting MAF to 1% in gene set. population databases, we used 3 criteria described in the Trio-based analysis Methods section to identify 3 genes carrying missense variants as either homozygous or compound heterozygous: CEP170B, De novo variants UBR4, and CALHM1. In total, 27 de novo variants were identified. One variant was excluded from validation because of technical difficulties. We found 2 patients carrying homozygous or compound Among the remaining 26 variants, 21 were validated by Sanger heterozygous rare variants in CEP170B. Among 4 of the sequencing. Therefore, our analysis pipeline achieved a true contributed variants, one of them is a de novo variant. The 2 positive rate of 81% (21/26). There were no recurrent de compound heterozygous variants were validated. One of the novo mutated genes. The validated de novo mutations were probands carrying a compound heterozygous variant in found in 13 patients (8 had 1, 2 had 2, and 3 had 3 mutations). CEP170B had a reported family history of epilepsy (sibling Of the 21 validated de novo mutations, 18 were non- and son) (table e-6, links.lww.com/NXG/A55). synonymous and 3 were synonymous (table 4). The nonsynonymous to synonymous variant ratio of all de novo Integrative genomic annotation variants was 6:1 (p < 0.22), which is higher than the neutral Comparison between the 25 gene sets used in the association rate reported in other studies (2.8:1)24 and is higher than that tests and published transcriptomic studies of MTLE-HS

Table 1 Clinical characteristics of patients with mesial temporal lobe epilepsy related to hippocampal sclerosis and their corresponding genes reported

Trios group (included in association analysis and trio-based analysis)

Participant Age at Age at Duration of History of Epilepsy Surgical Family history of Inheritance Gene no. Sex recruitment onset epilepsy FS1 surgery outcomea Lateralization epilepsy or FS modelb name

5 Female 34.3 6 28.3 Yes No — L Paternal granduncles —— (epilepsy)

8 Male 57.4 29 28.4 No No — LN ——

9 Female 52.8 34 18.8 No No — R N De novo FAM65A

CH CALHM1

Rare burden SEC24B

12 Male 42 13 29 No No — B N De novo CEP170B

De novo FGB

De novo MASTL

15 Female 25.5 12 13.5 No Yes P L N Homozygous P2RX7

17 Male 39.7 27 12.7 No Yes G R N —

19 Male 50.3 14 36.3 Yes No — L N De novo GRASP

De novo SLC5A12

De novo CDC42EP1

21 Female 40.9 17 23.9 No No — BN ——

22 Female 33.7 17 16.7 Yes Yes G R N ——

24 Male 31.1 23 7.9 Yes No — L N De novo NBEAL1

CH UBR4

25 Male 35.2 31 3.7 No Yes G R N Homozygous CEP170B

26 Female 25.6 2.3 23.3 Yes Yes P L N De novo NPC1L1

Rare burden SEC24B

28 Female 21.9 13 8.6 No Yes G R N ——

30 Female 41.1 12 29.3 Yes Yes G B N De novo ANXA6

33 Male 17.6 6 11.3 No Yes G R N CH CALHM1

34 Female 31.3 17 14.4 No Yes G L N De novo NLGN3

Continued 5 6 erlg:Gntc oue4 ubr3|Jn 08Neurology.org/NG 2018 June | 3 Number 4, Volume | Genetics Neurology:

Table 1 Clinical characteristics of patients with mesial temporal lobe epilepsy related to hippocampal sclerosis and their corresponding genes reported (continued)

Trios group (included in association analysis and trio-based analysis)

Participant Age at Age at Duration of History of Epilepsy Surgical Family history of Inheritance Gene no. Sex recruitment onset epilepsy FS1 surgery outcomea Lateralization epilepsy or FS modelb name

37 Female 31.5 15 16.6 Yes No — R N De novo SBSPON

39 Female 46.4 7 39.6 Yes No — L N De novo BAIAP2

De novo RASEF

40 Female 47 36 10.9 No No — R Elder brother (epilepsy) De novo ROBO4 and son (febrile convulsion)

CH CEP170B

41 Male 25.9 6 19.8 Yes No — L Elder sister (febrile —— convulsion)

43 Male 53.6 10 43.8 Yes No — R Younger sister De novo PLEC (epilepsy)

De novo TACC2

44 Male 42.9 14 28.9 No No — R N De novo BHLHE40

45 Male 49.3 21 27.9 No No — LN ——

Cases-only group (included in association analysis)

Participant Age at Age at Duration of History of Epilepsy Surgical Family history of Inheritance Gene no. Sex recruitment onset epilepsy FS surgery outcomea Lateralization epilepsy or FS modelb name

1 Female 42.4 29 13.4 No Yes P B N ——

2 Female 25.7 19 6.7 No Yes G R N ——

3 Female 56.2 43 13.2 No Yes P R Younger sister —— (epilepsy)

4 Female 38.7 9 29.7 Yes Yes P R N ——

6 Female 35.1 6 29.1 No Yes G L N ——

7 Female 49 6 43 No Yes G L N ——

10 Female 52.2 30 22.2 No No — LN ——

11 Female 39.5 10 29.5 Yes Yes G R N ——

13 Male 46.3 33 13.3 No Yes P L N ——

Continued erlg.r/GNuooy eeis|Vlm ,Nme ue2018 June | 3 Number 4, Volume | Genetics Neurology: Neurology.org/NG

Table 1 Clinical characteristics of patients with mesial temporal lobe epilepsy related to hippocampal sclerosis and their corresponding genes reported (continued)

Cases-only group (included in association analysis)

Participant Age at Age at Duration of History of Epilepsy Surgical Family history of Inheritance Gene no. Sex recruitment onset epilepsy FS surgery outcomea Lateralization epilepsy or FS modelb name

14 Male 42.7 16 26.7 Yes Yes G L N ——

16 Male 33 17 16 No Yes G R N ——

18 Female 36.1 24 12.1 Yes Yes P L N ——

20 Female 23.3 13 10.3 No Yes G L N ——

23 Male 51.6 30 21.7 No Yes G R N ——

27 Female 30.7 23 7.9 Yes No — B Maternal aunt —— (epilepsy)

29 Female 53.6 7 46.4 No Yes G L N ——

31 Female 15 1 13.5 No Yes G R N Rare burden SEC24B

32 Male 13 9 3.5 No Yes — RN ——

35 Male 55.1 41 14.6 No Yes P L N ——

38 Male 39.3 6 33.3 No Yes G R N ——

42 Female 37.5 7 30.9 No No — RN ——

46 Male 42.3 35 7 Yes No — LN ——

47 Male 28 NA NA Yes Yes G L N CH UBR4

48 Male 43.6 NA NA No Yes P R N ——

Abbreviations: — = not applicable; B = bilateral; CH = compound heterozygous; FS = febrile seizure; L = left; R = right. a G, good outcome with complete seizure freedom after surgery (except on drug withdrawal) and/or complete seizure freedom for 5 years or more at the last follow-up; P, poor outcome with ongoing drug-resistant seizures after surgery. All patients had at least 2 years of follow-up after surgery. b The 3 models of inheritance are de novo, CH and homozygous (homozygous for the same variant), and rare burden (carrying a rare risk variant reported in the gene-based association test). 7 MTOR gene was reported to be one of the top 5 FMRP- Table 2 Results of the gene-based association test regulated targets; for example, the expression of FMR1 was 20 Tested Corrected shown to reduce the mTOR protein level by 30% in vitro. Gene variants p Value p value The mTOR pathway was found to be inactive in sclerotic 29 ﬂ −6 hippocampus and is believed to induce in ammatory reac- SEC24B 48.00×100.041 tions in neurons through the PI3K/Akt/mTOR signaling − SUCO 52.70×105 0.139 pathway in patients with MTLE-HS.30 Our case-control rare − MAN1C1 46.15×105 0.317 variant association study suggested that FMRP targets are ﬁ −4 − signi cantly associated with MTLE-HS (p < 3.88 × 10 ). It is TRDMT1 33.83×104 1 plausible that the dysregulation of mTOR could be caused by − KIF3A 36.12×104 1 gene mutations of the FMRP pathway. The enrichment test of

− SS18L1 56.19×104 1 de novo variants among MTLE-HS cases also suggests that mutations could be introduced to FMRP targets by sponta- −4 QSER1 56.49×101 neous mutations. Both transcriptome and genomic analysis − BBS5 39.33×104 1 highlighted the importance of FMRP targets in MTLE-HS

− pathogenesis. However, the size of the FMRP gene set is CLCN1 79.73×104 1 a potential bias. For the candidate focal epilepsy gene set test, ACMSD 3 0.0010 1 we observed no signiﬁcant enrichment, but the candidate GALNTL5 3 0.0010 1 gene list in table-e1 (links.lww.com/NXG/A55) may be incomprehensive. NCKAP5 6 0.0011 1

Genes carrying 3 or more variants were included in the gene-based asso- Expression data further revealed the complexity of dysregu- ciation tests (5,154 genes tested). The gene-based burden test results of 47 lated pathways related to neural functions in MTLE-HS. In mesial temporal lobe epilepsy related to hippocampal sclerosis cases vs 692 controls. addition to FMRP targets, the results also suggested that mGluR5, PSD, and NMDAR-associated gene sets may play roles in the pathogenesis of MTLE-HS. These gene sets are important for dendritic development and function; hence, showed that the NMDA receptor (NMDAR), PSD, FMRP, they could also be candidate genes of MTLE-HS. and metabotropic glutamate receptor (mGluR) 5 gene sets are significantly enriched in the differentially expressed genes The case-control gene-based association analysis identified (table e-3, links.lww.com/NXG/A55). SEC24B as a potential candidate gene for MTLE-HS. Little is known about the function of this gene, although mutations of Candidate genes in the de novo list were queried against the SEC24B have been reported to cause neural tube de- ClinVar database, and none was known to be associated with velopment defects in humans and knockout mice.31 How this epilepsy. The MGI database showed a myriad of genes asso- relates to the pathogenesis of MTLE-HS is unknown. Tar- ciated with the abnormal nervous system phenotype in geted studies on our reported candidate genes from the as- knockout mice. Six of the 18 genes in our de novo list were sociation analysis could be performed to confirm the also listed by MGI, suggesting that their knockout mice model association signal. Animal models may be used to verify the might produce aberrant nervous system phenotypes effect of the observed variants on neural development. Apart (BHLHE40, TACC2, ROBO4, GRASP, BAIAP2, and from the investigation of well-defined epilepsy subgroups, NLGN3); keywords such as “abnormal nervous system de- meta-analysis by aggregating next-generation sequencing velopment” and “disrupted synaptic transmission” were fre- studies in consortium settings could be pursued in the light of quently observed in the list. gaining discovery power. The approach was demonstrated by an International League Against Epilepsy Consortium5 Discussion GWAS studies, which reported associations with genes such as SCN1A. However, we did not detect any association signal In this study, we have identified de novo variants and rare and de novo variants in the coding region of SCN1A. This variants possibly involved in the genetic risks of MTLE-HS. could be due to the risk that SNPs of MTLE-HS are more Consideration of rare variants in our WES and that of differ- frequently found within the promoter region of SCN1A.A entially expressed genes in resected hippocampal tissues both common variant association study on focal epilepsy also suggest that FMRP targets play a potential role in the patho- suggested the risk that SNPs of SCN1A could act through its genesis of MTLE-HS. In addition, we have identified rare var- expression modulation.7 iants in SEC24B, which might be associated with the disease.27 By screening candidate genes carrying de novo variants, we FMRP, encoded by the FMR1 gene, regulates a number of identified another 2 patients carrying compound heterozygous genes, many of which are expressed in the brain and are or homozygous rare variants in the gene CEP170B. A total of 3 implicated in psychiatric disorders.28 It is important that the patients carried either inherited or de novo CEP170B variants.

8 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG brain. The function of CEP170B is not well characterized, but Table 3 Results of the gene set burden test the deletion of its paralog CEP170 is associated with seizures, 33 Damaging microcephaly, and corpus callosum abnormalities. Given the Seta Genes p valueb All p valuec number of recurrences and the extensive expression of

24 23 CEP170B in the brain, it is plausible that it may play a role in FMRP-Ascano + FMRP-Darnell 1,557 2.89 × 10 2.15 × 10 MTLE-HS pathogenesis. − 2 FMRP-Ascano 939 3.15 × 10 3 3.88 × 10 4 scz-denovo-lof 87 0.013 0.027 We found a relatively high ratio of nonsynonymous to synonymous de novo variants, suggesting that de novo variants ARC complex 28 0.025 0.077 may be a contributing factor in the pathogenesis of MTLE- calcium-channel 26 0.033 0.013 HS. Notably, a number of the de novo variants identiﬁed

FMRP-Darnell 788 0.040 0.450 overlap with those reported in previous family-based studies of neuropsychiatric disorders. In particular, among the 18 PSD-95 65 0.040 0.167 genes carrying the nonsynonymous de novo variants, 3 have PSD 685 0.059 0.303 been reported in ASD. These include the gain-of-function p.R451C mutation in the esterase domain of NLGN3 kirov-denovo-cnv 234 0.118 0.048 (Neuroligin 3).34 p.R451C mutant mice showed increased mGluR5 39 0.220 0.360 AMPA receptor-mediated excitatory synaptic transmission in ID-candidates 196 0.289 0.200 the hippocampus, raising the amount of NMDA receptors by twofold.35 In our patient (no. 34), the de novo variant is also ASD-candidates 112 0.357 0.538 found within the esterase domain of NLGN3 (p.S499L). This FMRP-ASD-overlap-Ascano 93 0.538 1.000 adds further support to the hypothesis of shared biology be- 27 psych-cnv 346 0.538 0.833 tween epilepsy and ASD.

ASD-49-gene-network 49 0.583 0.833 One of the patients was found to have a de novo mutation in scz-denovo-nonsyn 611 0.583 0.120 GRASP. Relevant knockout rat models showed reduced dendritic outgrowth in immature hippocampal neurons.36 ASD-74-gene-network 74 0.600 0.282 The p.N162S mutation carried by our patient maps to the ID-denovo-nonsyn 132 0.667 0.833 PDZ domain of the GRASP protein, where it binds to

ASD-denovo-nonsyn 743 0.714 1.000 mGluRs and gamma-aminobutyric acid B receptor 2. Hence, altered GRASP function might affect the development of the CHD8-network 6 0.714 0.018 hippocampus. miR-137 446 0.714 0.450 scz-gwas 479 0.714 0.370 Our study has limitations. The sample size is relatively small, particularly the number of trios. Trios studies are challenging NMDAR network 61 0.833 0.833 to perform in adults because of logistic reasons (e.g., parents ASD-denovo-lof 128 1.000 0.833 often do not live with probands or unable to participate because of ill health). The possibility of additional extratemporal ID-denovo-lof 30 1.000 0.833 lobe seizures cannot be completely ruled out in all the Abbreviations: ARC = activity-regulated cytoskeleton-associated protein; patients. However, strict criteria were used to define MTLE- ASD = autism spectrum disorder; FMRP = fragile X mental retardation pro- HS, and nearly two-thirds of patients analyzed had histologic tein; ID = intellectual disability; mGluR = metabotropic glutamate receptor; NMDAR = NMDA receptor; PSD = postsynaptic density. confirmation of HS. Parents or controls were not specifically Bold: significant after multiple testing correction for 48 sets. The gene set burden test results of 47 mesial temporal lobe epilepsy related screened for HS by MRI. However, parents or controls with to hippocampal sclerosis cases vs 692 controls. epilepsy were excluded, and the prevalence of HS in people a For detail of gene sets, see table e-2, links.lww.com/NXG/A55. b Test on rare and predicted damaging variants. without epilepsy is rare. In a study of 207 patients who un- c Test on all rare variants. derwent high-resolution MRI for nonepilepsy indication (hearing loss), HS was found in 2, both had history of seizures.37 Hence the prevalence of HS in people without epilepsy Notably, 1 patient who carried compound heterozygous var- is estimated to be less than 0.5%. iants on CEP170B also showed a family history of MTLE-HS. We could not confirm whether the affected family members This is a study specifically investigating rare variants and de also carry 1 or more copies of these CEP170B variants, as they novo variants associated with MTLE-HS, revealing complex have not been tested. The remaining 2 cases affected by re- genetic architecture. The findings provide further support to cessive CEP170B variants appear to be sporadic. De novo the involvement of the PI3K/Akt/mTOR pathway in the variants in CEP170B have also been reported in patients with pathogenesis of MLTE, potentially via FMRP regulation, and ASD. According to ProteomicsDB,32 protein expression of shared pathobiology between epilepsy and other neuropsy- CEP170B has been detected mostly in the fetal brain and adult chiatric disorders. Collaboration effort to increase discovery

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 9 Table 4 Genes carrying nonsynonymous de novo variants in probands of trios

Participant Variant ExAC no. Gene type Chr Position Change frequency Mouse phenotype

− 9 FAM65A M 16 67574094 g.595C>T:p.R199C 8.3 × 10 6 NA

12 FGB M 4 155491720 g.1394T>C:p.V465A 0 NA

12 MASTL M 10 27459014 g.1126A>T:p.S376C 0 Abnormal embryonic neuroepithelium morphology

− 12 CEP170B M 14 105353332 g.2756C>T:p.T919M 1.7 × 10 5 NA

19 SLC5A12 M 11 26743102 g.160G>C:p.G54R 0 Abnormal neuron differentiation, abnormal excitatory postsynaptic potential, reduced long term potentiation

19 GRASP M 12 52407501 g.56A>G:p.N19S 0 Abnormal neuron differentiation, abnormal excitatory postsynaptic potential, reduced long term potentiation

19 CDC42EP1 F 22 37962638 g.283delG:p.P95fs 0 NA

24 NBEAL1 M 2 203921179 g.335C>G:p.T112S 0 NA

− 26 NPC1L1 M 7 44578845 g.1151C>T:p.S384L 4.9 × 10 5 NA

30 ANXA6 M 5 150481051 g.1980C>G:p.D660E 0 Increased ventricle muscle contractility

34 NLGN3 M X 70387443 g.1496C>T:p.S499L 0 Abnormal CNS synaptic transmission, decreased brain size, abnormal nervous system development

37 SBSPON M 8 74005154 g.149G>T:p.C50F 0 NA

39 RASEF M 9 85615372 g.1551G>T:p.K517N 0 NA

39 BAIAP2 M 17 79090095 g.1649C>T:p.A550V 0 Abnormal CNS synaptic transmission

− 40 ROBO4 S 11 124763780 g.1480C>T:p.R494* 5.3 × 10 5 Abnormal telencephalon development

43 PLEC1 M 8 144998224 g.5954C>T:p.T1985M 0.0004 Decreased nerve conduction velocity

− 43 TACC2 M 10 123842395 g.380C>T:p.A127V 1.6 × 10 5 NA

44 BHLHE40 M 3 5024745 g.607G>A:p.E203K 0 Increased susceptibility to pharmacologically induced seizures

Abbreviations: ExAC = Exome Aggregation Consortium; F = frameshift; M = missense; NA = not available; S = stop-gain. Bold: the same gene reported by other de novo variants studies. 1: epileptic encephalopathies, 2: autism spectrum disorder, and 3: schizophrenia.

power and different types of genetic abnormalities (copy Acknowledgment number variations [CNVs], methylation) and analysis of the The authors thank all patients, their families, and all healthy resected hippocampal tissues to identify somatic mutations controls for their participation. may further improve our understanding of the pathogenesis of this genetically heterogeneous disorder, potentially leading to Study funding novel therapeutic targets. This research was supported by the Hong Kong University Grants Council General Research Fund Grant HKU 7630/ Author contributions 12M (PI: SSC). J.K.L. Wong and H. Gui performed data analysis and prepared the manuscript. M. Kwok performed laboratory work Disclosures and collected data. P.W. Ng and C.H.T. Lui collected data J.K.L. Wong, H. Gui, and M. Kwok report no disclosures. P.W. and provided and cared for study patients. L. Baum advised Ng serves or has served on the editorial board of Cerebro- on data analysis and prepared the manuscript. P.C. Sham vascular Diseases, Journal of Neurologic Sciences, and Hong Kong served as a scientific advisor. P. Kwan advised on data Medical Journal. C.H.T. Lui reports no disclosures. L. Baum analysis, prepared the manuscript, and served as a scientific has received research support from the Chinese University of advisor. S.S. Cherny supervised the project, advised on data Hong Kong, St. Paul Co-educational College, and University analysis, prepared the manuscript, and served as a scientific of Macau; and holds a patent regarding nanoparticles for advisor. Alzheimer’s disease screening by MRI. P.C. Sham serves or

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Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 11 ARTICLE OPEN ACCESS Somatic GNAQ mutation in the forme fruste of Sturge-Weber syndrome

Michael S. Hildebrand, PhD,* A. Simon Harvey, MD,* Stephen Malone, MBBS,* John A. Damiano, BSc, Correspondence Hongdo Do, PhD, Zimeng Ye, MSc, Lara McQuillan, BBiomed, Wirginia Maixner, MBBS, Renate Kalnins, MBBS, Dr. Berkovic [email protected] Bernadette Nolan, MBBS, Martin Wood, MBBS, Ezgi Ozturk, PhD, Nigel C. Jones, PhD, Greta Gillies, MSc, or Dr. Hildebrand Kate Pope, BSc, Paul J. Lockhart, PhD, Alexander Dobrovic, PhD, Richard J. Leventer, MBBS, PhD, [email protected] Ingrid E. Scheffer, MBBS, PhD,* and Samuel F. Berkovic, MD*

Neurol Genet 2018;4:e236. doi:10.1212/NXG.0000000000000236 Abstract Objective To determine whether the GNAQ R183Q mutation is present in the forme fruste cases of Sturge- Weber syndrome (SWS) to establish a deﬁnitive molecular diagnosis.

Methods We used sensitive droplet digital PCR (ddPCR) to detect and quantify the GNAQ mutation in tissues from epilepsy surgery in 4 patients with leptomeningeal angiomatosis; none had ocular or cutaneous manifestations.

Results Low levels of the GNAQ mutation were detected in the brain tissue of all 4 cases—ranging from 0.42% to 7.1% frequency—but not in blood-derived DNA. Molecular evaluation conﬁrmed the diagnosis in 1 case in which the radiologic and pathologic data were equivocal.

Conclusions We detected the mutation at low levels, consistent with mosaicism in the brain or skin (1.0%–18.1%) of classic cases. Our data conﬁrm that the forme fruste is part of the spectrum of SWS, with the same molecular mechanism as the classic disease and that ddPCR is helpful where conventional diagnosis is uncertain.

*These authors contributed equally to this work.

From the Department of Medicine (Austin Hospital) (M.S.H., J.A.D., Z.Y., L.M., I.E.S., S.F.B.), University of Melbourne, Heidelberg, Victoria, Australia; Murdoch Childrens Research Institute (M.S.H., A.S.H., G.G., K.P., P.J.L., R.J.L.), Parkville, Victoria, Australia; Department of Paediatrics (Royal Children’s Hospital) (A.S.H., G.G., K.P., P.J.L., R.J.L., I.E.S.), Department of Pathology (H.D., R.K., A.D), and Department of Medicine (Royal Melbourne Hospital) (E.O., N.C.J.), University of Melbourne, Parkville, Victoria, Australia; Department of Neurology (A.S. H., R.J.L., I.E.S.) and Department of Neurosurgery (W.M.), Royal Children’s Hospital, Parkville, Victoria, Australia; Department of Neurosciences (S.M., B.N.) and Neurosurgical Department (M.W.), Lady Cilento Children’s Hospital, Brisbane, Queensland, Australia; Translational Genomics and Epigenomics Laboratory (H.D., A.D.), Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia; School of Cancer Medicine (H.D., A.D.), La Trobe University, Bundoora, Victoria, Australia; Anatomical Pathology (R.K.), Austin Health, Heidelberg, Victoria, Australia; Department of Neuroscience (N.C.J.), Central Clinical School, Monash University, Victoria, Australia; and Department of Neurology (N.C.J.), The Alfred Hospital, Melbourne, Victoria, Australia.

Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary ddPCR = droplet digital PCR; LMA = leptomeningeal angiomatosis; SWS = Sturge-Weber syndrome.

Sturge-Weber syndrome (SWS) is a rare, sporadic neuro- Standard protocol approvals, registrations, cutaneous disorder that occurs in 1 in 20,000 newborns, and patient consents typically characterized by brain pathology— The Human Research Ethics Committees of The Royal leptomeningeal angiomatosis (LMA), cortical atrophy and Children’s Hospital, Melbourne, Australia (project no. calcification, and layer 1 fusion—port-wine stain, and 29077F), and Austin Health, Melbourne, Australia (project vascular glaucoma.1 Clinical manifestations and severity no. H2007/02961), approved this study. Informed consent are heterogeneous with drug-resistant epilepsy, hemi- was obtained from the patients, or their parents in the case of paresis and cognitive impairment the most common neu- minors, for participation in the study. rologic features, glaucoma the most frequent ocular presentation, and port-wine stain the predominant der- Droplet digital PCR matological feature.1 Sometimes, the characteristic men- We used a commercially available ddPCR Mutation De- ingeal lesions of SWS are seen without skin or ocular tection Assay (ID: 10049047; Bio-Rad, Hercules, CA) to features2,3—this is referred to as forme fruste of SWS, or detect the GNAQ c.548G>A (p.R183Q) mutation and sometimes type III SWS, and diagnosis can be challenging. wild-type allele. Briefly, the ddPCR reaction mixture was assembled from a 2× ddPCR Supermix for Probes (No A somatic mosaic mutation (c.548G>A; p.R183Q) of the dUTP; Bio-Rad), 20× ddPCR Mutation Detection Assay, GNAQ gene that disrupts the activity of the encoded gua- and 10 ng of DNA sample to a final volume of 23 μL. Twenty nosine triphosphatase is present in classic SWS and also in microliters of each reaction mixture was then loaded into the patients who only have a port-wine stain.4 This mutation sample well of an 8-channel droplet generator cartridge was found in studies from different populations to be (Bio-Rad), and droplets were generated with 70 μLof present in the brain or skin of more than 80% of patients.4,5 droplet generation oil (Bio-Rad) using the manual QX200 Enrichment of this mutation in endothelial cells of both Droplet Generator. Following droplet generation, samples SWS skin and brain specimens,6,7 and SWS brain paren- were manually transferred to a 96-well PCR plate, heat- chyma not affected by LMA,6 has also recently been sealed, and amplified on a C1000 Touch thermal cycler using reported. the following cycling conditions: 95°C for 10 minutes for 1 cycle, followed by 40 cycles at 94°C for 30 seconds and 55°C Droplet digital PCR (ddPCR) is an ultra-sensitive technique for60seconds,1cycleat98°Cfor10minutesand12°Cfor recently reported for detection of the SWS mutation.5,7 It infinite. Post-PCR products were read on the QX200 droplet uses microfluidics and surfactant chemistries to emulsify reader (Bio-Rad) and analyzed using QuantaSoft software. input DNA into thousands of uniformly sized droplets and We established the detection limit of the ddPCR assay by then to amplify them with fluorescently labeled TaqMan serially diluting mutant samples with wild-type DNA to probes before measuring fluorescence on a droplet reader, as obtain different mutant/(mutant + wild-type) ratios: 5%, we and others have previously described.8,9 Based on fluo- 1%, 0.5%, 0.25%, and 0.1%. These mixed DNA samples were rescence intensity, the number of mutation-positive and subjected to ddPCR as described above. wild-type templates is quantified to calculate the frequency of a mutant allele. Here, we used this approach to screen 4 patients with forme fruste SWS including 1 in which the di- Results agnosis was equivocal. Clinical report Four patients presented during childhood with forme fruste or Methods SWS type III with drug-resistant epilepsy (table 1) and LMA on MRI and histopathology (figures 1, A–C and 2, A–C, Patients figure e-1, links.lww.com/NXG/A48, table 1), without port- We ascertained 4 patients with forme fruste SWS through our wine stains. Fresh-frozen (cases 1, 2, and 4) or formalin-fixed epilepsy surgery programs at Austin Health, Royal Children’s paraffin-embedded (case 3) brain tissue was available fol- Hospital, Melbourne, and the Lady Cilento Children’s Hos- lowing epilepsy surgery. The diagnoses of SWS type III for pital, Queensland, Australia. Genomic DNA was extracted cases 1, 2, and 4 were definitive based on imaging and path- from the brain using the DNA Genotek PrepIt 2CD Kit ologic data (figure 1, A–C, figure e-1, links.lww.com/NXG/ (Ontario, Canada) or Qiagen AllPrep DNA/RNA Kit and A48, table 1). In case 3, the diagnosis was less certain, as CT peripheral blood using the Macherey-Nagel NucleoBond CB and MRI showed calcification in the left occipital region 100 Kit (Duren, Germany) or Qiagen QIAamp DNA Maxi posteroinferiorly without convincing focal atrophy (figure 2, Kit (Hilden, Germany). A and B, table 1). Pathologically, in the subarachnoid plane,

2 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG Table 1 Clinical characteristics of forme fruste cases of Sturge-Weber syndrome

Seizure Age at Leptomeningeal onset surgery GNAQ R183Q, frequency Case angiomatosisa (mo) Seizure types Surgery (y) (%) of the mosaic mutation

1 Definite 10 Focal impaired awareness seizures; Right temporo-parieto- 57.1 left hemiclonic seizures occipital disconnection

2 Definite 9 Right hemiclonic; focal impaired Left temporo-parieto- 25.8 awareness seizures; myoclonic, occipital disconnection atonic

3 Subtle 20 Focal impaired awareness seizures; Left occipital 21 2.1 tonic-clonic lesionectomy

4 Definite 12 Focal impaired awareness seizures; Left functional 7 0.42 tonic-clonic hemispherotomy a Based on imaging and histopathologic analyses. a small vascular malformation was seen with some arterial frequency, comparable with a previously reported limit features, coupled with underlying parenchymal calcification (0.1%) for a similar assay.5 and cortical dyslamination (figure 2C, table 1). Genomic DNA isolated from the resected brain tissue and Mutation detection in the brain-derived that from the peripheral blood were analyzed using ddPCR. genomic DNA by ddPCR The GNAQ p.R183Q mutant allele was detected only in ge- We established the detection limit for the GNAQ mutation nomic DNA extracted from the brain tissue (7.1% frequency detection ddPCR by assaying serially mixed mutant and wild- in case 1, 5.8% in case 2, 2.1% in case 3, and 0.42% in case 4) type samples in triplicate. The mutant allele at a frequency but not in genomic blood-derived DNA from 3 patients ≥0.25% was consistently detected, while detection of the (figures 1, D, E and 2, D, E, figures e-1, e-3 to e-7, links.lww. mutant allele at 0.1% was only achieved in 2 of the 3 wells com/NXG/A48). Blood-derived DNA was not available from (figure e-2, links.lww.com/NXG/A48, table e-1). Thus, the case 3. It should be noted that although very low, the 0.42% detection limit in our hands was 0.25% mutant allele mutant allele frequency of case 4 was above our established

Figure 1 Imaging, histopathology, and molecular evaluation of case 1 with definite leptomeningeal angiomatosis

(A) Precontrast T1-weighted axial MRI scan showing right temporal and occipital atrophy and right occipital cortical calcification. (B) Postcontrast T1-weighted axial MRI scan showing leptomeningeal enhancement. (C) Hematoxylin and eosin–stained image of the neocortex showing a small area of densely clustered leptomeningeal vessels. (D) Identification of the wild-type GNAQ allele in green (present in the brain and blood) by digital PCR. (E) Identification of the mutant GNAQ R183Q allele (in blue) in the brain-derived but not blood-derived DNA—rare blue dots in blood are signal from droplets containing multiple DNA templates (supplemental data, links.lww.com/NXG/A48). Droplets without DNA templates are gray. Y- axis, amplitude of fluorescent signal. WT = wild-type GNAQ probe; MUT = mutant GNAQ R183Q probe.

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 3 Figure 2 Imaging, histopathology, and molecular evaluation of case 3 with subtler MRI findings

(A) Precontrast T2-weighted coronal MRI scan showing subtle signal change and calcification in the left occipital region (arrow) posteroinferiorly involving the occipital cortex or leptomeninges. Calcifica- tion was confirmed on CT (not shown). (B) Postcontrast T1-weighted coronal MRI scan showing leptomeningeal enhancement in the same region. Enhancement in the right occipital region (asterisk) is due to the normal transverse sinus. (C) Hematoxylin and eosin stained image showing subarachnoid angiomatosis (starred) between adjacent cerebral gyrae with cortical calcification (arrow). (D) Identification of the wild-type GNAQ allele (in green) in the brain by digital PCR. (E) Identification of the mutant GNAQ R183Q allele (in blue) in the brain. Droplets without genomic DNA templates are gray. Y- axis, amplitude of fluorescent signal. WT = wild-type GNAQ probe; MUT = mutant GNAQ R183Q probe.

detection limit (figures e-2, e-3, and e-7, links.lww.com/ terms of percentage mosaicism in the tissue tested, suggesting NXG/A48, table e-1). that there are other, as yet unidientified, influences on genotype-phenotype correlation. For case 1, fluorescent droplets were observed in the blood- derived genomic DNA below the expected amplitude, but In formalin-fixed paraffin-embedded samples, low-level so- these did not overlap with the true positive signal in the brain- matic mosaic mutations are challenging to detect because the derived genomic DNA when fluorescence intensity was DNA is of low quality and often has impurities. Despite these viewed on 2D plots (figure e-3, links.lww.com/NXG/A48). challenges, we were able to identify the somatic mutation in Instead, this is fluorescent signal from droplets containing case 3 from a 3-year-old pathologic specimen. This and other multiple genomic templates, a phenomenon not infrequently sensitive mutation detection technologies are showing in- observed when running ddPCR assays. creasing utility in elucidating the role of somatic mosaicism in brain-specific neurologic disorders, as shown recently for tu- berous sclerosis,12 in addition to SWS. Discussion The important discovery of a recurrent, somatic GNAQ mu- Author contributions tation provided the first insights into the molecular biology of M.S.H., I.E.S., and S.F.B. initiated and directed the project. – SWS. Initial reports focused on classic SWS,4 6,10,11 and here, M.S.H., J.A.D., H.D., Z.Y., L.M., E.O., and G.G. performed we extend these findings to forme fruste cases, a far more molecular genetics experiments. A.S.H., S.M., W.M., B.N., subtle, sometimes unrecognized, form of SWS. Our findings M.W., K.P., R.J.L., I.E.S., and S.F.B. conducted clinical phe- confirm that forme fruste cases are caused by the somatic notyping. R.K. performed histopathologic analyses. M.S.H., GNAQ p.R183Q mutation present at low to very low levels in N.C.J., P.J.L., A.D., and S.F.B. provided equipment and brain tissue due to mosaicism, consistent with a few reported reagents. M.S.H., A.S.H., S.M., I.E.S., and S.F.B. wrote the cases.4,11 It is intriguing that the mutation was only present in paper. All authors discussed the results and commented on the brain tissue of these forme fruste cases, and not in blood (of the manuscript. 3 cases), suggesting that the mutation may have arisen later during development than for classic cases, although we did Acknowledgment not have other tissues available from our cases to confirm this. The authors thank the patients and their families for their As MRI and even pathologic diagnosis can be equivocal for participation in this study. subtle LMA lesions, as for case 3 (figure 2, table 1), molecular evaluation may have specific diagnostic value. The relatively Study funding low level of the GNAQ mutation in the brain tissue of case 3 is This study was supported by the National Health and Medical consistent with the milder imaging and pathologic manifes- Research Council Program Grant (1091593) to I.E.S. and S.F. tations; however, case 4 had an even lower mutant load in B., a Project Grant (1129054) to S.F.B., a Project Grant

4 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG (1079058) to M.S.H., a Practitioner Fellowship (1006110) to GW Pharmaceuticals; has received research support from the I.E.S., and a R.D Wright Career Development Fellowship National Health and Medical Research Foundation, National (1063799) to M.S.H. R.J.L. is supported by a Melbourne Institute of Health, University of Melbourne School of Health Children’s Clinician Scientist Fellowship. Sciences, March of Dimes Foundation, Queensland Emer- gency Medicine Research Foundation, Health Research Disclosure Council New Zealand, Medical Research Future Fund, and M.S. Hildebrand has received research support from the Rebecca L Cooper Medical Research Foundation; and National Health and Medical Research Council. A.S. Harvey receives royalty payments for Diagnostic and Therapeutic reports no disclosures. S. Malone has served on the scientific Methods for EFMR (Epilepsy and Mental Retardation Lim- advisory boards of UCB Pharma and BioMarin; has received ited to Females). S.F. Berkovic has served on the scientific travel funding/speaker honoraria from UCB Pharma and advisory boards of UCB Pharma and Eisai Australia; has BioMarin; and has received research support from UCB served on the editorial boards of Brain, Epileptic Disorders, and Pharma. J.A. Damiano reports no disclosures. H. Do has given Lancet Neurology; has received speaker honoraria from UCB lectures and educational presentations for Bio-Rad. Z. Ye and Pharma, Novartis Pharmaceuticals, Sanofi-Aventis, and Jan- L. McQuillan report no disclosures. W. Maixner receives sen Cilag; holds a patent for SCN1A testing held by Bi- publishing royalties from Springer. R. Kalnins and B. Nolan onomics Inc and licensed to various diagnostic companies; is report no disclosures. M. Wood has been a consultant to one of the inventors listed on a patent held by Bionomics Inc Medtronic. E. Ozturk and N.C. Jones report no disclosures. G. on Diagnostic testing of using the SCN1A gene; is one of the Gillies and K. Pope have received research support from the inventors on pending patent WO61/010176: Therapeutic National Health and Medical Research Council. P.J. Lockhart compound that relates to discovery of PCDH19 gene as the has served on the editorial boards of Genetics Research In- cause of familial epilepsy with mental retardation limited to ternational, Open Access (OA) Genetics and the Journal of females; and has received research support from the National Neurochemistry and has received research support from the Health and Medical Research Council. Full disclosure form National Health and Medical Research Council. A. Dobrovic information provided by the authors is available with the full has served on the editorial boards of BMC Cancer and Mo- text of this article at Neurology.org/NG. lecular Cancer; has been a consultant to Astra Zeneca; has given lectures and educational presentations for Bio-Rad; and Received February 6, 2018. Accepted in final form March 26, 2018. has received research support from the National Health and References Medical Research Council. R.J. Leventer has received research 1. Comi AM. Sturge-Weber syndrome. Handb Clin Neurol 2015;132:157–168. support from the National Health and Medical Research 2. Crosley CJ, Binet EF. Sturge-Weber syndrome: presentation as a focal seizure dis- ff order without nevus flammeus. Clin Pediatr (Phila) 1978;17:606–609. Council and Campbell Edwards Trust. I.E. Sche er has re- 3. Rasmussen T, Mathieson G, Le Blanc F. Surgical therapy of typical and a forme fruste ceived speaker honoraria from UCB Pharma, Eisai, GSK, variety of the Sturge-Weber syndrome. Schweiz Arch Neurol Neurochir Psychiatr Athena Diagnostics, and Transgenomics; has served on the 1972;111:393–409. 4. Shirley MD, Tang H, Gallione CJ, et al. Sturge-Weber syndrome and port-wine scientific advisory boards of GSK, UCB Pharma, BioMarin, stains caused by somatic mutation in GNAQ. N Engl J Med 2013;368: Ramaciotti Foundation, and Nutricia; received the 2014 1971–1979. 5. Uchiyama Y, Nakashima M, Watanabe S, et al. Ultra-sensitive droplet digital PCR for Prime Ministers Prize For Science; has served on the editorial detecting a low-prevalence somatic GNAQ mutation in Sturge-Weber syndrome. Sci boards of Neurology, Epilepsy Currents, Annals of Neurology, Rep 2016;6:22985. 6. Sundaram SK, Michelhaugh SK, Klinger NV, et al. GNAQ mutation in the venous Epileptic Disorders, Progress in Epileptic Disorders series, and vascular malformation and underlying brain tissue in Sturge-Weber syndrome. Virtual Neuro Centre; holds patents/pending patents for Neuropediatrics 2017;48:385–389. 7. Huang L, Couto JA, Pinto A, et al. Somatic GNAQ mutation is enriched in brain SCN1A testing held by Bionomics Inc licensed to various endothelial cells in Sturge-Weber syndrome. Pediatr Neurol 2017;67:59–63. diagnostic companies, Diagnostic and therapeutic methods 8. Oxnard GR, Paweletz CP, Kuang Y, et al. Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyp- for EFMR, A diagnostic method for epilepsy, A diagnostic ing of cell-free plasma DNA. Clin Cancer Res 2014;20:1698–1705. method for epilepsy (also published as Methods for the di- 9. Tsao SC, Weiss J, Hudson C, et al. 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Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 5 ARTICLE OPEN ACCESS ANXA11 mutations prevail in Chinese ALS patients with and without cognitive dementia

Kang Zhang, PhD,* Qing Liu, MD, PhD,* Keqiang Liu, PhD, Dongchao Shen, PhD, Hongfei Tai, PhD, Shi Shu, MD, Correspondence PhD, Qingyun Ding, MD, Hanhui Fu, PhD, Shuangwu Liu, PhD, Zhili Wang, PhD, Xiaoguang Li, MD, Dr. X. Zhang [email protected] or Dr. Cui Mingsheng Liu, MD, Xue Zhang, MD, PhD, and Liying Cui, MD, PhD [email protected] Neurol Genet 2018;4:e237. doi:10.1212/NXG.0000000000000237 Abstract Objective To investigate the genetic contribution of ANXA11, a gene associated with amyotrophic lateral sclerosis (ALS), in Chinese ALS patients with and without cognitive dementia.

Methods Sequencing all the coding exons of ANXA11 and intron-exon boundaries in 18 familial amyotrophic lateral sclerosis (FALS), 353 unrelated sporadic amyotrophic lateral sclerosis (SALS), and 12 Chinese patients with ALS-frontotemporal lobar dementia (ALS-FTD). The transcripts in peripheral blood generated from a splicing mutation were examined by reverse transcriptase PCR.

Results We identiﬁed 6 nonsynonymous heterozygous mutations (5 novel and 1 recurrent), 1 splice site mutation, and 1 deletion of 10 amino acids (not accounted in the mutant frequency) in 11 unrelated patients, accounting for a mutant frequency of 5.6% (1/18) in FALS, 2.3% (8/353) in SALS, and 8.3% (1/12) in ALS-FTD. The deletion of 10 amino acids was detected in 1 clinically undetermined male with an ALS family history who had atrophy in hand muscles and myotonic discharges revealed by EMG. The novel p. P36R mutation was identiﬁed in 1 FALS index, 1 patient with SALS, and 1 ALS-FTD. The splicing mutation (c.174-2A>G) caused in-frame skipping of the entire exon 6. The rest missense mutations including p.D40G, p.V128M, p.S229R, p.R302C and p.G491R were found in 6 unrelated patients with SALS.

Conclusions The ANXA11 gene is one of the most frequently mutated genes in Chinese patients with SALS. A canonical splice site mutation leading to skipping of the entire exon 6 further supports the loss-of-function mechanism. In addition, the study ﬁndings further expand the ANXA11 phenotype, ﬁrst highlighting its pathogenic role in ALS-FTD.

*These authors contributed equally to the manuscript. From the Department of Neurology and Laboratory of Clinical Genetics, Peking Union Medical College Hospital (K.Z., Q.L., D.S., H.T., Q.D., H.F., S.L., Z.W., X.L., M.L., X.Z., L.C.) and McKusick-Zhang Center for Genetic Medicine (K.L., S.S., X.Z.), State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College; and Neuroscience Center (K.Z., Q.L., K.L., D.S., H.T., S.S., Q.D., H.F., S.L., Z.W., X.L., M.L., X.Z., L.C.), Chinese Academy of Medical Sciences, Beijing, China.

The Article Processing Charge was funded by the authors. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary ALS = amyotrophic lateral sclerosis; ExAC = Exome Aggregation Consortium; FALS = familial amyotrophic lateral sclerosis; FTD = frontotemporal lobar dementia; MAF = minor allele frequency; PUMCH = Peking Union Medical College Hospital; SALS = sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurologic disease C9ORF72, ATXN2, AR, DCTN1, NEFH, PRPH, DAO, TFG, characterized by progressive paralysis and ultimately respira- TAF15, and GRN) using a massive parallel sequencing gene tory failure within 5 years of symptom onset.1 Approximately panel by the Ion Torrent PGM system as described before8 5%–10% of ALS cases exhibit familial amyotrophic lateral scle- and Sanger sequencing of genes including CHCHD10, TBK1, rosis (FALS) inheritance, and causative gene mutations can be CCNF, and GLE1. C9orf72 expansion was also examined found in 60% of patients with FALS. The remaining 90%–95% using repeat-primed PCR. of ALS cases exhibit sporadic amyotrophic lateral sclerosis(SALS), and mutations in the same genes are responsible Transcripts investigation of the for 10% of patients with SALS.2 To date, rare variants in more splicing mutation than 30 genes have been reported to cause or be associated with Total RNA was extracted using TRIzol (Invitrogen) from ALS.3 Recently, mutations of ANXA11 have been identified in peripheral white blood cells. Then, 2.5 μg of RNA was used to patients with ALS of European ancestry, but pathogenicity of perform reverse transcriptase PCR according to the Reverse ANXA11 in other ALS cohorts remained unproved.4 In the Transcription System (Promega, Madison, WI) instructions. current study, we investigated ANXA11 mutations in Chinese cDNA was amplified using the primer pair ANXA11-RT-F ALS patients with or without cognitive decline. (59-CCATGAGCTACCCTGGCTAT-39)andANXA11-RT-R (59-GACTCCCCAGGCAGTCAAT-39) located at ANXA11 exon 4 and exon 8 (shown in figure 3), respectively. The PCR products were isolated on a 1.5% agarose gel. DNA fragments of Methods interest were gel purified and sequenced. Study population A total of 383 Chinese patients with ALS were recruited at the Standard protocol approvals, registrations, ALS clinic of Neurology Department, Peking Union Medical and patient consents College Hospital (PUMCH) from January 2016 to August The study was approved by the Ethical Review Board of 2017. Patients were diagnosed according to the established PUMCH. After an informed consent form was obtained from – clinical criteria and standard protocol by specialists in ALS.5 7 the participant or his family, the blood specimen of the participant was collected. Mutation screening Genomic DNA was extracted from peripheral blood leukocytes using a QIAamp DNA Blood Midi Kit (Qiagen, Valencia, CA) Results according to the manufacturer’s instructions. All coding The current patient cohort included 18 FALS index patients exons and at least 100 bp of flanking introns of ANXA11 and 365 unrelated patients with SALS. Twelve patients with (NM_145869) were amplified and put to Sanger Sequencing SALS had concomitant frontotemporal lobar dementia (ALS- 4 with published primer sequences using an ABI 3730 automated FTD). Of the 12 patients with ALS-FTD, 11met the probable DNA sequencing system (Applied Biosystems). Sequence behavioral variant FTD (bvFTD) diagnosis following the alignment were performed against human genome (UCSC hg Rascovsky criteria,7 and the remaining one was diagnosed as 19) using CodonCode Aligner. Each identified mutation was semantic dementia. The demographic features of the current reamplified and resequenced from both ends with the same cohort are presented in table 1. primer pairs. Rare variants (minor allele frequency [MAF] < 0.1%) with high conservation were further assessed for patho- Mutations in AXAN11 and updated mutation genicity using online prediction software, SIFT (sift.bii.a-star.- spectrum of ALS causal genes in Chinese edu.sg), and PolyPhen-2 (genetics.bwh.harvard.edu/pph2/). patients with ALS Bioinformatics analysis software MutationTaster (muta- In total, we identified 6 nonsynonymous heterozygous mutationtaster.org), ESEfinder 3.0 (krainer01.cshl.edu/cgi-bin/tools/ tions (5 novel and 1 recurrent), 1 splice site mutation, and 1 ESE3/esefinder.cgi?process=home), and Human Splicing Finder deletion of 10 amino acids (not accounted in the mutant fre- 3.0 (umd.be/HSF3/index.html) were applied to predict the ef- quency) in 11 unrelated patients, accounting for a mutant fect of mutation on mRNA splicing. frequency of 5.6% (1/18) in FALS, 2.3% (8/353) in SALS, and 8.3% (1/12) in ALS-FTD (figures 1 and 2 and figure e-1, The current cohort of patients had been screened for known links.lww.com/NXG/A57). These mutations were all absent in ALS genes (SOD1, ALS2, SETX, FUS, VAPB, ANG, TARDBP, 384 healthy controls. No additional mutations were detected FIG4, OPTN, VCP, UBQLN2, SIGMAR1, CHMP2B, PFN1, in other known ALS genes. A missense variant we identified

2 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG was also detected in 1 clinically undetermined male patient Table 1 Demographics of the study population whose father was diagnosed as having ALS and deceased at

Variable FALS SALS ALS-FTD the age of 70 years before DNA was obtainable. The son had mild atrophy in the left hand, which was noticed at the age of N 18 353 12 31 years; myotonic discharges in bilateral limbs and thoracic Mean onset age (y) 44.71 ± 12.54 51.92 ± 11.56 58.60 ± 9.812 paraspinal muscles were revealed by EMG (ﬁgure e-1).

Male, n (%) 10 (55.6) 197 (55.8) 8 (66.7) Effect of the c.174-2A>G variation on ANXA11 Site of onset, bulbar (%) 2 (11.1) 48 (13.6) 2 (16.7) mRNA splicing The c.174-2A>G variation was predicted to affect splicing by Abbreviations: ALS = amyotrophic lateral sclerosis; FALS = familial amyotrophic lateral sclerosis; FTD = frontotemporal lobe dementia; SALS = spo- multiple bioinformatics analysis. RNA extracted from the radic amyotrophic lateral sclerosis. patient’s peripheral blood showed the alternatively spliced transcript lacking the entire coding part of exon 6 (p.A58_Q187del). Electrophoresis on a 1.5% agarose gel revealed 1 fragment (c.119A>G, p.D40G) was already reported in European encompassing exons 5–7 in the healthy control. Of interest, 3 patients with ALS. Three novel mutations (c.174-2A>G, distinct fragments were observed in the mutant patient. Sanger p.A58_Q187del; c.382G>A, p.V128M; and c.687T>A, sequencing for those 3 fragments confirmed that an approximate p.S229R) were identified, which had not been documented in 750-bp transcript was a normal spliced fragment encompassing online databases of human polymorphisms including exons 5–7, an approximate 250-bp transcript was an aberrant dbSNP147, 1000 Genomes, and Exome Aggregation Consor- one resulting from exon 6 skipping, and an approximate 500-bp tium (ExAC). The remaining 3 coding variants (c.107C>G, transcript was a heterodimer consisting a normal transcript and p.P36R; c.904C>T, p.R302C; and c.1471G>A, p.G491R) an abnormal transcript. Therefore, the c.174-2A>G mutation had an allele frequency of less than 0.005% in the ExAC affects exon 6 causing 58–187 amino acid deletions. The sche- database and were predicted to be damaging by bio- matic diagram of gel fractionation and sequence traces of reverse informatics analysis. Of note, the p.P36R mutation was transcriptase PCR products are shown in figure 3. detected both in patients with ALS and in patients with ALS- FTD. Detailed information concerning the ANXA11 muta- Clinical characteristics of patients with the tions and mutation carriers identified in the current cohort are ANXA11 mutation presented in table e-1 (links.lww.com/NXG/A56). In addi- All10patients(7menand3women)carryingANXA11variant tion, a deletion mutation (c.1146_1175del, p.L383_V392del) came from Chinese mainland, and the age of symptom onset

Figure 1 Mutations identified in ANXA11 in ALS and ALS-FTD patients

Mutations found in the previous study and in the present study are marked in black and in red, respectively. A deletion mutation associated to a clinically undetermined patient is marked in blue. Conservation of amino acid across species is shown at the bottom. ALS = amyotrophic lateral sclerosis; FTD = frontotemporal lobe dementia.

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 3 Figure 2 Updated mutation spectrum of FALS and SALS in Chinese populations

SOD1 is the most common mutant gene in FALS, and ANXA11 in SALS. FALS = familial amyotrophic lateral sclerosis; SALS = sporadic amyotrophic lateral sclerosis.

ranged from 37 to 71 years. Four patients initially exhibited causative genes, including MATR3, TUBA4A, CHCHD10, – dysarthria and dysphagia, and 6 patients exhibited limb weakness GLE1, TBK1, NEK1,andCCNF3,9 16,wereconfirmed to be as the first symptom. Two patients (case 534, p.P36R, and case related to ALS. However, the frequency of mutations in these 395, p.P36R) died, survived 28 and 24 months, respectively. The genes is very low in Chinese patients with ALS. Lately, 6 rest of the patients are still alive, and 2 individuals (case 479, ANXA11 heterozygous missense mutations were reported in p.G491R, and case 545, p.A58_Q187del) survived for over 5 European patients with ALS.4 In the present study, we screened years. All patients denied sarcoidosis. There was a patient (case the ANXA11 gene mutation in a large ALS and ALS-FTD 534, p.P36R) with a positive family history of ALS whose older cohort of Chinese mainland for the first time. We found 7 brother had similar symptoms including dysarthria and dyspha- potentially pathogenic mutations in a total of 10 unrelated gia and died of respiratory failure 2 years after disease onset. The cases. The ANXA11 mutation frequency in our current cohort p.P36R mutation carrier (case 474) initially presented slurred is 2.6% (10/383), with 5.6% (1/18) in FALS, 2.3% (8/353) in speech at age 70 years. A few months later, his symptom quickly SALS, and 8.3% (1/12) in ALS-FTD, which is higher than the extended to his both legs with muscle weakness and atrophy. frequency in a European cohort (1% in FALS and 1.7% in About a year and a half later, His family members reported that SALS).4 A recent meta-analysis regarding Asian ALS pop- he became easily irritable, aggressive and inappropriate behaviors, ulations revealed that the most frequent mutations were SOD1 such as laughing at inappropriate occasions. Over the subsequent (SALS 1.5%, FALS 30.0%), followed by FUS (SALS 0.9%, 1 year, his both arms showed weakness. EMG demonstrated FALS 6.4%), C9orf72 (SALS 0.3%, FALS 2.3%), and TARDBP chronic and acute denervation changes in the cervical, thoracic, (SALS 0.2%, FALS 1.5%) mutations.17 Moreover, we pre- and lumbar segments. MRI showed bilateral temporal lobe at- viously reported the prevalence of FUS, SOD1, and OPTN rophy and moderate frontal atrophy. 18F-fluorodeoxyglucose- responsible for 1.7%, 1.5%, and 1.3% of Chinese patients with PET imaging showed bilateral frontotemporal hypometabolism. SALS respectively, whereas the mutation frequency in Chinese His Mini-Mental State Examination score was 20/30, and the FALS was 30.6% for SOD1 mutations, 5.6% for FUS mutations, Montreal Cognitive Assessment(MoCA)testscorewas15/30. and 5% for OPTN mutations.8 Considering those studies to- He was diagnosed as having ALS with bvFTD. In addition, case gether, among Chinese patients with ALS, ANXA11 is the 479 (p.G491R) was accompanied by cognitive impairment. leading gene in SALS, and SOD1 is the most frequent causal The detailed clinical information is summarized in table e-1 gene in FALS (figure 2), which indicate a different genetic (links.lww.com/NXG/A56). architecture between Caucasians and Chinese. Therefore, the sequencing analysis of SOD1, ANXA11,andFUS is preferen- Discussion tially recommended in Chinese patients with ALS. With evolving technologies for gene sequencing, a large num- All variants identified except for p.D40G are first reported. ber of ALS causal genes were found. In the past 3 years, 7 The p.D40G mutation was previously reported in European

4 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG Figure 3 Transcripts of the splicing mutation (c.174-2A>G) detected by reverse transcription PCR analysis

A 1.5% agarose gel fractionation of RT-PCR products of blood RNA shows the distinct fragments of cDNA obtained by specific primers. Lane WT represents a normal transcript in a healthy control. Lane MU represents an aberrant transcript in a patient carrying the c.174-2A>G mutation, which contains 3 different bans. Sequencing results show that the band located at 750 bp implies a normal transcript; the band located at 250 bp implies an aberrant transcript, which wascaused by exon 6 skipping; and the band located at 500 bp is confirmed to be a result of formation of heterodimer consisting of 1 normal transcript and 1 aberrant transcript. ALS = amyotrophic lateral sclerosis; cDNA = complementary DNA; FTD = frontotemporal lobe dementia; RT-PCR = reverse transcription PCR. patients with ALS and had a common European founder.4 was different from other patients. These variable manifestations Among these mutations, p.P36R, p.R302C, and p.G491R imply apparent clinical heterozygous among the specific had a very low allele frequency (<0.005%) in the ExAC ANXA11 mutation carriers. Case 660 is a p.S229R carrier of database. Nevertheless, the pathogenicity of the 3 muta- Han ethnicity, and she also harbors c.688C>T (p.R230C). The tional sites is supported by the following evidences. First, the common single nucleotide polymorphism (rs1049550, C>T, 3 mutations were absent from our healthy controls. Second, p.R230C, MAF 0.44) in ANXA11 was associated with the in- the 3 mutant positions are all highly conserved across spe- creased risk of sarcoidosis.19 Actually, the ANXA11 rs1049550 cies, implicating that those amino acids are of functional T allele is a protective factor for affecting sarcoidosis in the importance. Third, different in silico prediction algorithms, Chinese Han population.20 It is notable that nobody had all demonstrated the 3 sites pathogenic. We should recog- a family or personal history of sarcoidosis in our cohort. Of nize that many definite pathogenic variants of disease- interest, the p.L383_V392del mutation carrier (case 399) did causing genes, such as cancer gene and cardiomyopathy not fulfill the diagnosis of ALS, although his father died of the gene, may have an allele frequency of less than 0.01% in the disease. This may further suggest the varied phenotype caused ExAC database.18 In addition, the p.V128M variant was by the ANXA11 mutation and would require long-term follow- predicted to be benign by bioinformatics software, but it was up of the patient. not observed in population-based databases and our controls. No mutations of other ALS causal genes were dis- In the current study, 2 unrelated patients with ALS (case 534 covered in the patient carrying p.V128M. The p.V128M and case 395) harboring the p.P36R mutation had no cog- mutation may play a detrimental role in ALS, and further nitive impairment. Case 534 has a positive family history. functional experiment and additional cohorts screening will Unfortunately, unavailability of DNA samples from his draw firm conclusions. family members precluded us from confirming whether the mutation segregates with the disease. The same genetic It is difficult to pinpoint dominant features of ANXA11- change was also detected in an individual (case 474) having mutated patients. Patients carrying the same variant may have ALS-FTD and had classic upper and lower motor nervous different clinical presentations. For instance, in contrast to our system damage combined with bilateral frontotemporal at- 1 patient (case 506) harboring the p.D40G variant initially rophy. To our knowledge, it is the first time for us to find the presented left arm weakness at his 59 years, European p.D40G ANXA11 mutation in the patient with ALS-FTD, which variant carriers exhibited late disease onset (average, 67 years) provides further genetic support for ALS and FTD as a dis- and mostly bulbar onset reported by the original ariticle.4 ease continuum. Indeed, the number of causal ALS-FTD Similarly, the cognitive function varied among patients har- genes increased rapidly in the past few years. Mutations in boring the identical p.P36R variant, with normal cognitive C9orf72, TARDBP,andTBK1 are the major genetic causes in ability and concomitant FTD observed in the present study. ALS-FTD, and TDP-43 inclusions as a common pathologic – Moreover, we found that a splice site mutation c.174-2A>G character are resulted from each of these variants.1,21 25 (p.A58_Q187del) and the living affected carrier (case 545) Pathogenic mutations in other genes including FUS, showed slow progression and >8 years disease duration, which CHCHD10, CCNF, UBQLN2, SQSTM1,andVCP have also

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 5 been found in patients with ALS-FTD.26 However, the ge- Acknowledgment netic etiology of quite a few patients with ALS-FTD remains The authors thank all the patients and their families for their unclear. cooperation in this study.

ANXA11 is located on human chromosome 10q22.3 and Study funding encodes the 505 amino acid annexin A11 protein that This study was funded by the National Key Research and belongs to a group of calcium-dependent phospholipid- Development Program of China (grant nos. 2016YFC0905100 binding proteins. Annexin A11 is a member of the annexin and 2016YFC0905103); the Chinese Academy of Medical protein family. Four conserved annexin domains (annexin Sciences (CAMS) Innovation Fund for Medical Sciences 1, annexin 2, annexin 3, and annexin 4) constitute its (CIFMS) (grant nos. 2016-I2M-1-002 and 2016-I2M-1-004); 27 conserved C terminus. Unlike other annexins, it has the National Natural Science Foundation of China (NSFC) a unique long N-terminal domain containing the binding (grant no. 81230015); and the Beijing Municipal Science and – 28 site of calcyclin (residues 50 62). Calcyclin can mediate Technology Commission (grant no. Z151100003915078). the ubiquitination and proteasomal degradation of many target proteins.29 Functional data showed that p.D40G and Disclosure p.G38R, which both located in proximity to the calcyclin The authors report no disclosures. Full disclosure form in- binding region, could result in abnormal binding of calcy- formation provided by the authors is available with the full clin.4 That may be the major cause of formation of annexin text of this article at Neurology.org/NG. A11–positive inclusions in postmortem nervous tissue observed from ANXA11 mutant carriers. Of note, the exon- Received January 8, 2018. Accepted in final form March 22, 2018. skipping mutation c.174-2A>G (p.A58_Q187del) result- – ing in 58 187 amino acid deletions directly impaired the References functional domain, undoubtedly disrupted calcyclin bind- 1. Taylor JP, Brown RH Jr, Cleveland DW. Decoding ALS: from genes to mechanism. ing. Furthermore, the p. P36R mutation close to the resi- Nature 2016;539:197–206. 2. Andersen PM, Al-Chalabi A. Clinical genetics of amyotrophic lateral sclerosis: what dues 50–62 might affect normal calcyclin binding as well, do we really know? Nat Rev Neurol 2011;7:603–615. although there is no functional study to support this pos- 3. Brown RH, Al-Chalabi A. Amyotrophic lateral sclerosis. N Engl J Med 2017;377: 162–172. sibility. Combined with original article’sexperimental 4. Smith BN, Topp SD, Fallini C, et al. Mutations in the vesicular trafficking protein data,4 13 of the 23 ANXA11 mutations (p.P36R, p.G38R, annexin A11 are associated with amyotrophic lateral sclerosis. Sci Transl Med 2017;9: eaad9157. p.D40G, and p.A58_Q187del) are around the binding site 5. Brooks BR, Miller RG, Swash M, Munsat TL; World Federation of Neurology Re- of calcyclin. We speculate that this region is a hot spot of search Group on Motor Neuron Diseases. El Escorial revisited: revised criteria for the fi diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor mutation and has signi cant biological functions. Those Neuron Disord 2000;1:293–299. variants disrupt the normal physiologic function of annexin 6. Neary D, Snowden JS, Gustafson L, et al. Frontotemporal lobar degeneration: a consensus on clinical diagnostic criteria. Neurology 1998;51:1546–1554. A11, suggesting that the pathogenesis of ANXA11 muta- 7. Rascovsky K, Hodges JR, Knopman D, et al. Sensitivity of revised diagnostic tions is mediated by a loss-of-function mechanism. How- criteria for the behavioural variant of frontotemporal dementia. Brain 2011;134: 2456–2477. ever, the pathogenetic mechanism of the remaining 8. Liu Q, Liu F, Cui B, et al. Mutation spectrum of Chinese patients with familial and ANXA11 mutations is unclear, and further functional sporadic amyotrophic lateral sclerosis. J Neurol Neurosurg Psychiatry 2016;87: 1272–1274. experiments are needed to shed light on this. 9. Johnson JO, Pioro EP, Boehringer A, et al. Mutations in the Matrin 3 gene cause familial amyotrophic lateral sclerosis. Nat Neurosci 2014;17:664–666. 10. Smith BN, Ticozzi N, Fallini C, et al. Exome-wide rare variant analysis identifies Our result showed that the ANXA11 gene is one of the most TUBA4A mutations associated with familial ALS. Neuron 2014;84:324–331. frequently mutated genes, which runs just next to SOD1 in 11. Bannwarth S, Ait-El-Mkadem S, Chaussenot A, et al. A mitochondrial origin for Chinese patients with ALS. Patients in our cohort with frontotemporal dementia and amyotrophic lateral sclerosis through CHCHD10 involvement. Brain 2014;137:2329–2345. ANXA11 mutations showed varied disease characteristics, but 12. Kaneb HM, Folkmann AW, Belzil VV, et al. Deleterious mutations in the essential no phenotype-genotype correlations were observed. A ca- mRNA metabolism factor, hGle1, in amyotrophic lateral sclerosis. Hum Mol Genet 2015;24:1363–1373. nonical splice site mutation leading to skipping of the entire 13. Cirulli ET, Lasseigne BN, Petrovski S, et al. Exome sequencing in amyotrophic lateral exon 6 further supports the loss-of-function mechanism. In sclerosis identifies risk genes and pathways. Science 2015;347:1436–1441. 14. Freischmidt A, Wieland T, Richter B, et al. Haploinsufficiency of TBK1 causes familial addition, our findings further expanded the ANXA11 phe- ALS and fronto-temporal dementia. Nat Neurosci 2015;18:631–636. notype, first highlighting its pathogenic role in ALS-FTD. 15. Brenner D, Muller K, Wieland T, et al. NEK1 mutations in familial amyotrophic lateral sclerosis. Brain 2016;139:e28. 16. Williams KL, Topp S, Yang S, et al. CCNF mutations in amyotrophic lateral sclerosis Author contributions and frontotemporal dementia. Nat Commun 2016;7:11253. 17. Zou ZY, Zhou ZR, Che CH, Liu CY, He RL, Huang HP. Genetic epidemiology of Kang Zhang, Qing Liu, Xue Zhang, and Liying Cui designed amyotrophic lateral sclerosis: a systematic review and meta-analysis. J Neurol Neu- the experiments. Kang Zhang, Qing Liu, Keqiang Liu, Dong- rosurg Psychiatry 2017;88:540–549. 18. Kobayashi Y, Yang S, Nykamp K, Garcia J, Lincoln SE, Topper SE. Pathogenic variant chao Shen, Hongfei Tai, Shi Shu, Hanhui Fu, Shuangwu Liu, burden in the ExAC database: an empirical approach to evaluating population data for and Zhili Wang performed the experiments. Qing Liu, Qingyun clinical variant interpretation. Genome Med 2017;9:13. 19. Hofmann S, Franke A, Fischer A, et al. Genome-wide association study identifies Ding, Xiaoguang Li, Mingsheng Liu, and Liying Cui recruited ANXA11 as a new susceptibility locus for sarcoidosis. Nat Genet 2008;40: and evaluated the patients. Kang Zhang, Qing Liu, Keqiang Liu, 1103–1106. 20. Feng X, Zang S, Yang Y, et al. 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6 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG 21. van der Zee J, Gijselinck I, Van Mossevelde S, et al. TBK1 mutation spectrum in an 25. Sreedharan J, Blair IP, Tripathi VB, et al. TDP-43 mutations in familial and sporadic extended european patient cohort with frontotemporal dementia and amyotrophic amyotrophic lateral sclerosis. Science 2008;319:1668–1672. lateral sclerosis. Hum Mutat 2017;38:297–309. 26. Crook A, Williams K, Adams L, Blair I, Rowe DB. Predictive genetic testing for 22. DeJesus-Hernandez M, Mackenzie IR, Boeve BF, et al. Expanded GGGGCC hex- amyotrophic lateral sclerosis and frontotemporal dementia: genetic counselling anucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked considerations. Amyotroph Lateral Scler Frontotemporal Degener 2017;18:475–485. FTD and ALS. Neuron 2011;72:245–256. 27. Wang J, Guo C, Liu S, et al. Annexin A11 in disease. Clin Chim Acta 2014;431: 23. Renton AE, Majounie E, Waite A, et al. A hexanucleotide repeat expansion in 164–168. C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 2011;72: 28. Lesniak W, Slomnicki LP, Filipek A. S100A6-new facts and features. Biochem Biophys 257–268. Res Commun 2009;390:1087–1092. 24. Kabashi E, Valdmanis PN, Dion P, et al. TARDBP mutations in individuals 29. Fukushima T, Zapata JM, Singha NC, et al. Critical function for SIP, a ubiquitin E3 with sporadic and familial amyotrophic lateral sclerosis. Nat Genet 2008;40: ligase component of the beta-catenin degradation pathway, for thymocyte de- 572–574. velopment and G1 checkpoint. Immunity 2006;24:29–39.

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 7 ARTICLE OPEN ACCESS Determining the incidence of familiality in ALS A study of temporal trends in Ireland from 1994 to 2016

Marie Ryan, MRCPI, Mark Heverin, MSc, Mark A. Doherty, BSc, Nicola Davis, MRes, Emma M. Corr, PhD, Correspondence Alice Vajda, PhD, Niall Pender, PhD, Russell McLaughlin, PhD, and Orla Hardiman, MD Marie Ryan [email protected] Neurol Genet 2018;4:e239. doi:10.1212/NXG.0000000000000239 Abstract Objective To assess temporal trends in familial amyotrophic lateral sclerosis (FALS) incidence rates in an Irish population and to determine factors inﬂuencing FALS ascertainment.

Methods Population-based data collected over 23 years, using the Irish amyotrophic lateral sclerosis (ALS) register and DNA biobank, were analyzed and age-standardized rates of FALS and associated familial neuropsychiatric endophenotypes were identiﬁed.

Results Between 1994 and 2016, 269 patients with a family history of ALS from 197 unique families were included on the register. Using stringent diagnostic criteria for FALS, the mean age- standardized FALS incidence rate for the study period was 11.1% (95% conﬁdence interval [CI], 8.8–13.4). The FALS incidence rate increased steadily from 5.2% in 1994 to 19.1% in 2016, an annual increase of 0.7% (95% CI, 0.5–0.9, p < 0.0001). Inclusion of the presence of neuropsychiatric endophenotypes within kindreds increased the FALS incidence rate to 30%. The incidence of FALS in newly diagnosed individuals from known families increased significantly with time, accounting for 50% of all FALS diagnoses by 2016. The mean annual rate of recategorization from “sporadic ALS” to “FALS” was 3% (95% CI, 2.6–3.8).

Conclusions The true population-based rate of FALS is at least 20%. Inclusion of extended endophenotypes within kindreds increases the rate of FALS to 30%. Cross-sectional analysis of clinic-based cohorts and stringent deﬁnitions of FALS underestimate the true rate of familial disease. This has implications for genetic counseling and in the recognition of presymptomatic stages of ALS.

From the Academic Unit of Neurology (M.R., M.H., N.D., E.M.C., A.V., O.H.), Trinity College; Department of Genetics (M.A.D., R.M.), Trinity College; and Department of Psychology (N.P.), Beaumont Hospital, Dublin, Ireland.

The Article Processing Charge was funded by Research Motor Neurone. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary ALS = amyotrophic lateral sclerosis; CI = conﬁdence interval; FALS = familial ALS; FTD = frontotemporal lobar dementia; SALS = sporadic ALS.

Amyotrophic lateral sclerosis (ALS) is recognized as a pri- with FTD. For those identified posthumously, a direct chart mary motor system degeneration of complex genetic origin. review and interview with a family member is conducted where The condition is usually distinguished into “familial ALS” possible. The ALS DNA biobank has been used to identify (FALS), in which other family members are reported to have established pathogenic variants in genes implicated in ALS had ALS, and “sporadic ALS” (SALS), in which there is no pathogenesis.16 These DNA samples are stored to allow for discernible family history. At least 30 mendelian-inherited retrospective assessment of new genes. genes have been implicated in the familial form of ALS.1 However, variants in the majority of mendelian-inherited Standard protocol approvals, registrations, – genes have also been identified in apparently sporadic cases.2 5 and patient consents All patients included in the Irish ALS register between January Recent modeling suggests that the interaction between the 1, 1994, and December 31, 2016, were invited to participate in genetic and environmental risk factors varies depending on the study. Informed written consent for the study was the specific genetic variants involved, implying that most ge- obtained from all participants. This study was approved by the netic causes of ALS are likely to be incompletely penetrant, Beaumont Hospital Research Ethics Committee (15/40). and that most apparently, SALS occurs in the context of variable genomic risk.6,7 Moreover, higher rates of neuro- Diagnostic criteria for FALS fi psychiatric conditions have been described among first- and The presence and classi cation of FALS was determined us- fi fi 17 second-degree relatives of ALS probands,8,9 and 14% poly- ing our previously de ned criteria ( gure 1). genic overlap has been reported between ALS and schizo- Analysis of register data phrenia.10 Whether the presence of these neuropsychiatric Data from the Irish ALS register from 1994 to 2016 were phenotypes within relatives should be considered in the def- interrogated. All cases reporting a history of suspected or inition of familial disease remains to be determined. confirmed ALS or FTD in at least 1 relative were collated. Where necessary, genealogical records were reviewed. The In this study, we have used previously proposed criteria with DNA database was cross-referenced with the clinical database, varying levels of stringency to assess temporal trends in FALS and the genetic status was determined in all cases for whom incidence rates in an Irish population over a 23-year period. DNA was available. Additional individuals with a known gene We have taken into account the evolution of thinking around variant, not previously identified by a family history of ALS or the definition of FALS and have sought to evaluate the impact FTD, were collated. Cases with a diagnosis of Kennedy dis- of recently identified genetic and phenotypic representations ease were excluded. Individuals who had a family history of ALS and related conditions on how we define FALS. suspicious for ALS (e.g., relative died of “muscle wasting disease”), in whom we could not confirm the diagnosis, were excluded. Similarly, individuals with a relative with dementia, Methods in whom the nature of the dementia could not be accurately Data collection determined, were excluded. An Irish population-based register for patients with ALS has fi been in operation since 1994 with an associated DNA bank Our previously reported criteria were applied to all identi ed – operating since 1999.11 13 Details of case ascertainment and FALS cases. Annual age-standardized incidence rates for FALS validation methodologies have been described extensively elsewhere.11,12 Briefly, individuals confirmed to have possible, probable, or definite ALS according to the El Escorial criteria14 fi Figure 1 Byrne criteria for familial amyotrophic lateral are enrolled on the register. Following con rmation of di- sclerosis (FALS) agnosis, a semistructured telephone interview is conducted using a standardized questionnaire. All informants are asked specifically about the occurrence of any neurodegenerative or neuropsychiatric conditions among their first- and second- degree relatives. A diagnosis of frontotemporal lobar dementia (FTD) in a relative is accepted if (1) they were diagnosed by a physician with expertise in cognitive disorders or (2) the description of the relative was deemed to meet the Neary criteria15 by a neurologist experienced in diagnosing patients

2 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG and FALS subclassifications were calculated as a proportion of reference population data sets were filtered. Variants that were the total number of ALS cases diagnosed annually, using the present in the ALSonline genetics database (ALSoD)19 or the pooled Irish ALS register population from 1994 to 2016 and Human Gene Mutation Database V.2017.220 and are reported considering the following age bands: ≤39, 40–49, 50–59, in the literature as being familial or highly penetrant were 60–69, 70–79 and 80+ years. considered to be mendelian causes of ALS. Patients for whom DNA was not available and without confirmed family history of Where multiple members of the same kindred were identified, ALS or FTD were categorized as nonfamilial cases. All cases pedigrees were constructed to include first-, second-, and with an established mendelian-inherited ALS gene variant were third-degree relatives where possible. All newly diagnosed identified, and the crude incidence rate of mendelian-inherited individuals from previously identified kindreds were identi- ALS was calculated by dividing by the number of patients di- fied. Crude incidence rates of newly diagnosed individuals agnosed with ALS annually. from known families were calculated as a proportion of total number of ALS families presenting annually. Dates of di- Statistical analysis agnosis of FALS for all cases were obtained from the ALS We fitted linear regression models to estimate the annual register and cross-referenced against medical records where mean change in incidence rates with calendar year as the applicable. All cases were grouped by whether they were predictor variable and dependent variables: total FALS, defi- recategorized from SALS to FALS or identified as FALS at the nite FALS, probable FALS, possible FALS, FALS from time of diagnosis. Annual rates of recategorization were cal- previously identified families, recategorized FALS, mendelian- culated by dividing the number of recategorized FALS indi- inherited ALS and probands with a positive family history of viduals by the number of individuals diagnosed with ALS FTD, all-type dementia, and schizophrenia/psychosis, re- annually. spectively. To determine whether increasing rates of FALS were a function of higher rates of “possible FALS” diagnoses, Analysis by additional phenotype we tested the hypothesis that the total FALS (b1) and com- and endophenotype bined “definite” and “probable FALS” (b2) beta coefficients All cases with a confirmed family history of FTD were identi- were not statistically different from each other. A simple linear fied. Secondary analysis identified all probands with a confirmed regression model with total FALS and combined “definite” family history of all-type dementia and/or schizophrenia/ and “probable FALS” as main effects with joint interaction psychosis. The crude incidence rates of probands with a con- term (b1*b2) was fitted via bias corrected bootstrap (1,000 firmed family history of FTD, all-type dementia, and possible resamples). SPSS Statistics Version 24 was used to identify familial endophenotypes (e.g., schizophrenia/psychosis13)were and estimate the parameters of the linear models and to test calculated by dividing by the number of patients diagnosed with for statistical significance. ALS annually. Genetic screening and analysis Results All patients were tested for established high-penetrance ALS- associated variants. Patients were screened for the presence of Demographics the pathogenic GGGGCC hexanucleotide repeat expansion in A total of 2173 individuals, diagnosed with ALS between 1994 C9orf72 by repeat-primed PCR as described previously.18 This and 2016, were recorded on the Irish ALS register. Of these, methodology has previously been validated with positive and 313 individuals had potential FALS based on a family history negative controls using reverse transcriptase PCR and Southern of suspected or confirmed ALS or FTD in at least 1 relative or blots. Amplified fragments were measured by capillary elec- the presence of the mendelian-inherited ALS gene mutation trophoresis on an Applied Biosystems 3500 Series Genetic in the proband (figure 2A). Thirty-five individuals with a Analyzer and visualized using Gene Mapper v.4.0. Patients with family history suspicious for ALS or FTD in whom it was not 30 hexanucleotide repeats or above were deemed positive for possible to confirm the relatives’ diagnoses and 9 individuals the expansion. Next-generation DNA sequencing was per- with proven Kennedy disease were excluded. Two hundred formed through Illumina paired-end, PCR-free, whole-genome sixty-nine registered patients with ALS comprising 197 sequencing and Illumina paired-end, target-enriched sequenc- unique families were included in the final analysis (figure 2B). ing. Sequence data were generated for 676 patients and 446 Ninety-four individuals carried a known ALS-causative gene population-matched controls. Participants were screened for mutation (C9orf72 [89], TARDBP [1], FUS [2], SOD1 [1], exonic and splice-site variants in the exons of 30 genes and SQSTM1 [1]). Fifty-one patients carrying the C9orf72 considered to be linked to ALS (ALS2, ANG, CHCHD10, variant reported a family history of FTD (figure e1, links. CHMP2B, DAO, DCTN1, ELP3, ERBB4, FIG4, FUS, lww.com/NXG/A49). hnRNPA1, LMNB1, MATR3, NEFH, OPTN, PFN1, PRPH, SETX, SIGMAR1, SOD1, SPAST, SPG11, SQSTM1, TAF15, Secondary analysis of the Irish ALS register identified 392 TARDBP, TBK1, UBQLN2, UNC13A, VAPB, and VCP). To patients with a confirmed relative with dementia (reported as screen for high penetrance, variants that were present in any Alzheimer disease [131], FTD [51], and unspecified [210]) and controls or at a maximum allele frequency exceeding 0.05 in 57 patients with a confirmed family history of schizophrenia.

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 3 FALS from previously identified families and Figure 2 Flow chart of patients who met the inclusion/ex- recategorized FALS clusion criteria for the study A mean of 2.9% (95% CI, 1.8–4.1) of individuals diagnosed with ALS each year were from known FALS families, increasing by 0.3% annually (95% CI, 0.2–0.4, p < 0.0001). The relative contribution of newly diagnosed individuals from known families increased annually (p = 0.001), accounting for 50% of all FALS diagnoses in 2016. To prevent an un- derestimation of effect size due to unrecognized FALS in SALS individuals (i.e., those in whom a second family member has not yet been affected), the final 3 years of data collection were excluded. For the remaining years, the overall mean rate of recategorization from sporadic to FALS was 3% (95% CI, 2.6–3.8) annually. This did not change with time (p = 0.177).

Mendelian-inherited ALS From 1999 to 2016, the mean crude incidence rate for known mendelian-inherited ALS was 4.49% (95% CI, 2.8–6.2) annually. A temporal increase of 0.4% (95% CI, 0.2–0.6) annually (p = 0.02) was observed, driven by C9orf72-positive ALS patients, who accounted for 89 of 94 known mendelian- inherited forms. The other cases, all of which were sporadic, were associated with mutations in TARDBP (1), FUS (2), SQSTM1 (1), and a previously recognized rare SOD1 variant.

Impact of phenotype From 1994 to 2016, a mean of 1.9% (95% CI, 1.1–2.9) of (A) All patients with ALS registered with the Irish ALS register from 1994 ﬁ to 2016 who reported a history of suspected or confirmed ALS or FTD in at individuals diagnosed with ALS annually had a con rmed least 1 relative were identified. All patients with an established, highly positive family history of FTD, increasing by 0.2% (95% CI, penetrant ALS variant (C9orf72 89, TARDBP 1, FUS 2, SOD1 1, and SQSTM11) were identified from the DNA database. (B) Thirty-five patients with a family 0.01–0.39; p = 0.001) per year. The mean crude incidence rates history suspicious for ALS or FTD in whom it was not possible to confirm the ﬁ relatives’ diagnoses and 9 patients with Kennedy disease were excluded. of probands with a con rmed family history of any form of Five C9orf72-positive patients with a family history suspicious for ALS or FTD dementia or schizophrenia were 14.87% (95%% CI, 9.3–20.5) in whom it was not possible to confirm the relatives’ diagnoses were reca- – tegorized into gene-positive only category. ALS = amyotrophic lateral scle- and 2.2% (95% CI, 1.4 3.0), respectively. Both demonstrated rosis; FTD = frontotemporal lobar dementia. annual increases of 1.8% (95% CI, 1.4–2.2; p = 0.0001) and 0.2% (95% CI, 0.18–0.22; p = 0.0001), respectively.

Discussion Incidence of FALS The mean annual crude FALS incidence rate was 11.1% (95% The Irish ALS population-based register is flexibly designed to confidence interval [CI], 8.9–13.3) for the study period, and the allow for both the categorization of FALS and the application of corresponding mean age-standardized FALS incidence rate was different levels of stringency of the definition of FALS. The 11.1% (95% CI, 8.8–13.4). However, the age-standardized FALS availability of data from the register, combined with island status, incidence rate increased steadily from 5.2% in 1994 to 19.1% low immigration rates, and comparatively large family sizes in 2016, representing an overall increase of 0.7% (95% CI, makes Ireland an ideal place to study FALS. By applying our 0.5–0.9, p < 0.0001) per annum (figure 3A). Using our previously previously reported criteria to the Irish ALS population-based published criteria for “definite FALS,” the mean age-standardized register, we have shown that the mean annual age-standardized incidence rate was 4% (95% CI, 2.9–5.0) for the entire study incidence rate for any definition of FALS over a 23-year period period. However, between 1994 and 2016, this increased at a rate was 11.1%. This is consistent with the commonly quoted figure of 0.2% (95% CI, 0.01–0.3, p = 0.007) annually (figure 3B). For of 10% for the population rate of FALS observed in other “probable FALS,” the age-standardized incidence rate was 3.1% populations of European ancestry.21,22 However, we have also (95% CI, 2.3–3.9) but did not increase with time (p = 0.318) identified an increasing trend in FALS ascertainment, ranging (figure 3C). Conversely, the age-standardized incidence for from 5.2% in 1994 to 19.1% in 2016, with over 50% of newly “possible FALS” increased by 0.4% (95% CI, 0.2–0.5, p < 0.0001) diagnosed FALS cases in 2016 coming from second gen- annually, with an overall mean rate of 4.6% (95% CI, 3.1–6.1) erations within known FALS families. This in turn may drive (figure 3D). There was no difference between the total FALS the observed increasing trend in “definite” FALS as with in- (b = 0.007) and combined “definite” and “probable FALS” (b = creasing awareness of the prevalent FALS families, certainty of 0.003) beta coefficients (p = 0.671) (figure 3E). FALS classification increases.

4 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG Figure 3 Temporal trends in FALS incidence

Temporal trends in age-standardized FALS incidence for total FALS (A), definite FALS (B), probable ALS (C), and possible FALS (D). Temporal trends in age-standardized FALS incidence for total FALS compared with definite and probable FALS (E). ALS = amyotrophic lateral sclerosis; FALS = familial ALS.

The mean rate of recategorization from “SALS” to “FALS” the 35 C9orf72-positive ALS patients without a confirmed was 3% annually. This is higher than previously reported family history of ALS or FTD, 9 patients were unable to crude incidence rates of ALS in relatives of patients with SALS provide complete information on their relatives. The of 1.2%.23 The difference is most likely a function of differences remaining patients reported at least 1 first- or second-degree in classification criteria used. Indeed, there are numerous rea- relative with unspecified dementia, a neuropsychiatric disor- sons why some FALS individuals are misclassified as SALS, der or unconfirmed ALS (figure e-2, links.lww.com/NXG/ including incomplete ascertainment of extended kindreds, and A49). Our findings are thus in keeping with our previous work small family size.17 Less frequently, SALS may be misclassified suggesting that truly “sporadic” ALS associated with C9orf72 as FALS.24 The methodological approach used in this study is repeat expansions are rare and that the majority of those consistent with standard approaches used in a clinical envi- carrying this variant have a family history of either neurode- ronment with only ALS cases with confirmed ALS and/or FTD generative or neuropsychiatric disease. cases in relatives included in our analysis. Overall, as the op- portunities to misclassify FALS as SALS are greater than vice We have also demonstrated an increased trend in incidence of versa,17 the possibility of underestimating the true FALS rate probands with confirmed positive family histories of FTD, all- within this study far exceeds the possibility an overestimation. type dementia, and schizophrenia reflecting the recognition of the importance of the extended phenotype within ALS kin- We have also found a mean incidence rate of known dreds. Our findings also demonstrate a systematic difference mendelian-inherited genes associated with ALS of 4.49% in the clinical phenotype between historical cases and those (95% CI, 2.8–6.2), increasing annually. This figure is driven recently enrolled consistent with information creep, a com- by C9orf72-positive ALS, which accounts for the majority of mon finding in longer standing registers.25 Our observations known mendelian-inherited ALS in Ireland. In our data set, of strongly suggest that FALS criteria should be expanded to

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 5 incorporate the presence of these extended phenotypes cluster analysis of ALS kindreds with extended neuropsychi- within ALS kindreds. We have previously shown that the atric endophenotypes.9 Conversely, assuming a lifetime risk of likelihood of FALS increases based on the number of patients developing dementia in those over 65 of approximately 1 in with ALS within a kindred and the size of the extended kin- 5,28 the presence of dementia in 2 relatives within an ALS dred.17 Using the same analyses, we have now calculated that kindred is insufficient to make a diagnosis of likely FALS, a kindred of 38 will introduce a 5% probability of having irrespective of the family size (figure 4B). a relative with FTD26 by chance alone, but that the presence of 2 or more relatives with FTD is sufficiently unlikely by Using stringent diagnostic criteria and excluding the presence chance (p = 0.025) to provide a credible criterion for FALS. of a neuropsychiatric endophenotype within kindreds, our Similarly,27 we have calculated a 5% probability of having 1 data suggest that at least 20% of ALS cases in Ireland have relative with schizophrenia in the presence of at least 5 un- FALS. Of these, over 40% of Irish ALS families carry the affected relatives. However, there is a diminishing probability C9orf72 repeat expansion. However, by including a wider of having 2 or more first- or second-degree relatives with neuropsychiatric endophenotype among relatives as an ad- schizophrenia within extended kindreds, rendering the pres- ditional criterion, the rate of FALS is likely to be in the region ence of 3 or more family members with schizophrenia, of 30%, although definitive estimates cannot yet be generated a credible criterion by which to extend the definition of FALS using our population-based register data, as historical ascer- (figure 4A). This calculation is consistent with our recent tainment is incomplete.

Figure 4 Binomial probability distribution

For the number of relatives with schizophrenia (A) or dementia (B) against the kindred size. p < 0.05 is highlighted in blue.

6 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG This study has limitations. The presence of the C9orf72 repeat from Janssen Cilag, Biogen Idec, Sanofi Aventis, Novartis, expansion was determined by repeat-primed PCR plus and Merck-Serono; has been a member of advisory panels amplicon length analysis in blood samples. Although confir- for Biogen Idec, Allergan, Ono Pharmaceuticals, Novartis, mation of each repeat expansion length using Southern Cytokinetics, Treeway, Wave, NINDS CDE Team for ALS/ blotting is recommended, and although there is acknowl- MND, and Sanofi Aventis; serves as Editor-in-Chief of edged variation in amplicon length across the tissues, the Amyotrophic Lateral Sclerosis and Frontotemporal Dementia; approach used in this study was validated using positive and serves on the editorial board of the Journal of Neurology negative controls confirmed using Southern blot and is con- Neurosurgery; coholds patents for Treatment of Central sistent with that adopted in the setting of diagnostic screening. Nervous System Injury Inventors (RCSI); consults for Biogen Idec and Cytokinetics; and has received research The definition and utility of the concept of FALS remains support from Science Foundation Ireland. Full disclosure a matter of debate. Our data demonstrate that the estimated form information provided by the authors is available with frequency of FALS within a population can be biased by both the full text of this article at Neurology.org/NG. ascertainment methods, the level of stringency applied to the definition, and the inclusion or exclusion of extended phe- Received February 6, 2018. Accepted in final form April 6, 2018. notypes and endophenotypes that are biologically associated References with ALS. Our population-based longitudinal data indicate 1. Al-Chalabi A, van den Berg LH, Veldink J. Gene discovery in amyotrophic lateral that at least 20% of ALS is familial using stringent criteria. sclerosis: implications for clinical management. Nat Rev Neurol 2017;13:96–104. 2. Jackson M, Al-Chalabi A, Enayat ZE, Chioza B, Leigh PN, Morrison KE. Copper/ However, our data also suggest that a wider diagnostic cate- zinc superoxide dismutase 1 and sporadic amyotrophic lateral sclerosis: analysis of gorization, to include FTD and neuropsychiatric conditions, 155 cases and identification of a novel insertion mutation. Ann Neurol 1997;42: is warranted. 803–807. 3. Kabashi E, Valdmanis PN, Dion P, et al. TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet 2008;40:572–574. Author contributions 4. Hubers A, Just W, Rosenbohm A, et al. De novo FUS mutations are the most frequent genetic cause in early-onset German ALS patients. Neurobiol Aging 2015;36:3117. M. Ryan: study concept and design, analysis and interpretation e1–3117.e6. of data, and manuscript composition. M. Heverin: study 5. Gibson SB, Downie JM, Tsetsou S, et al. The evolving genetic risk for sporadic ALS. Neurology 2017;89:226–233. concept and design, analysis and interpretation of data, 6. Al-Chalabi A, Calvo A, Chio A, et al. Analysis of amyotrophic lateral sclerosis as and revision of the manuscript for intellectual content. a multistep process: a population-based modelling study. Lancet Neurol 2014;13: 1108–1113. M.A. Doherty: acquisition of data, analysis and interpretation 7. Chio A, Calvo A, Mazzini L, et al. Genetic mutations shorten the multistep process in of data, and revision of the manuscript for intellectual content. ALS. Amyotroph Lateral Scler Frontotemporal Degener 2017;18:1–73. 8. Byrne S, Heverin M, Elamin M, et al. Aggregation of neurologic and neuropsychiatric N. Davis and E.M. Corr: acquisition of data. A. Vajda and disease in amyotrophic lateral sclerosis kindreds: a population-based case-control N. Pender: study concept and design. R. McLaughlin: analysis cohort study of familial and sporadic amyotrophic lateral sclerosis. Ann Neurol 2013; 74:699–708. and interpretation of data and revision of the manuscript for 9. O’Brien M, Burke T, Heverin M, et al. Clustering of neuropsychiatric disease in first- intellectual content. O. Hardiman: study concept and design, degree and second-degree relatives of patients with amyotrophic lateral sclerosis. analysis and interpretation of data, and revision of the manu- JAMA Neurol 2017;74:1425–1430. 10. McLaughlin RL, Schijven D, van Rheenen W, et al. Genetic correlation between script for intellectual content. amyotrophic lateral sclerosis and schizophrenia. Nat Commun 2017;8:14774. 11. Traynor BJ, Codd MB, Corr B, Forde C, Frost E, Hardiman O. Incidence and prevalence of ALS in Ireland, 1995–1997: a population-based study. Neurology 1999; Study funding 52:504–509. The project is supported through the funding provided by 12. O’Toole O, Traynor BJ, Brennan P, et al. Epidemiology and clinical features of amyotrophic lateral sclerosis in Ireland between 1995 and 2004. J Neurol Neurosurg Science Foundation Ireland (15/SPP/3244 and 16/ERCD/ Psychiatry 2008;79:30–32. 3854). O.H. receives support from the Health Research Board 13. Byrne S, Elamin M, Bede P, et al. Cognitive and clinical characteristics of patients with amyotrophic lateral sclerosis carrying a C9orf72 repeat expansion: a population-based including Joint Programme in Neurodegeneration, Research cohort study. Lancet Neurol 2012;11:232–240. Motor Neurone, and Science Foundation Ireland (Futur- 14. Brooks BR, Miller RG, Swash M, Munsat TL. El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor eNeuro Centre). R.L.M. receives support from the Motor Neuron Disord 2000;1:293–299. Neurone Disease Association (MNDA). 15. Neary D, Snowden JS, Gustafson L, et al. Frontotemporal lobar degeneration: a consensus on clinical diagnostic criteria. Neurology 1998;51:1546–1554. 16. Kenna KP, McLaughlin RL, Byrne S, et al. Delineating the genetic heterogeneity of Disclosure ALS using targeted high-throughput sequencing. J Med Genet 2013;50:776–783. M. Ryan has received research support from Science Foun- 17. Byrne S, Bede P, Elamin M, et al. Proposed criteria for familial amyotrophic lateral sclerosis Amyotroph Lateral scler 2011;12:157–159. dation Ireland. M. Heverin, M.A. Doherty, N. Davis, and 18. Renton AE, Majounie E, Waite A, et al. A hexanucleotide repeat expansion in E.M. Corr report no disclosures. A. Vajda has received re- C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 2011;72: 257–268. search support from the Irish Health Research Board. 19. Abel O, Shatunov A, Jones AR, Andersen PM, Powell JF, Al-Chalabi A. Development N. Pender has received a speaker honorarium from Biogen; of a smartphone app for a genetics website: the amyotrophic lateral sclerosis online genetics database (ALSoD). JMIR Mhealth and Uhealth 2013;1:e18. has served on the editorial board of the International Journal 20. Stenson PD, Mort M, Ball EV, et al. The Human Gene Mutation Database: towards of Neuroscience; and has received research support from the a comprehensive repository of inherited mutation data for medical research, genetic diagnosis and next-generation sequencing studies. Hum Genet 2017;136:665–677. Monkstown Hospital Foundation. R. McLaughlin has re- 21. Byrne S, Walsh C, Lynch C, et al. Rate of familial amyotrophic lateral sclerosis: ceived research support from the Motor Neurone Disease a systematic review and meta-analysis. J Neurol Neurosurg Psychiatry 2011;82: 623–627. Association of England and Science Foundation Ireland. 22. Andersen PM. Genetic factors in the early diagnosis of ALS. Amyotroph Lateral Scler O. Hardiman has received speaker honoraria/travel funding Other Motor Neuron Disord 2000;1(suppl 1):S31–S42.

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 7 23. Hanby MF, Scott KM, Scotton W, et al. The risk to relatives of patients with sporadic 26. Coyle-Gilchrist IT, Dick KM, Patterson K, et al. Prevalence, characteristics, and amyotrophic lateral sclerosis. Brain 2011;134:3454–3457. survival of frontotemporal lobar degeneration syndromes. Neurology 2016;86: 24. Rabe M, Felbecker A, Waibel S, et al. The epidemiology of CuZn-SOD mutations in 1736–1743. Germany: a study of 217 families. J Neurol 2010;257:1298–1302. 27. Saha S, Chant D, Welham J, McGrath J. A systematic review of the prevalence of 25. Rooney JPK, Brayne C, Tobin K, Logroscino G, Glymour MM, Hardiman O. Beneﬁts, schizophrenia. PLoS Med 2005;2:e141. pitfalls, and future design of population-based registers in neurodegenerative disease. 28. Seshadri S, Wolf PA. Lifetime risk of stroke and dementia: current concepts, and Neurology 2017;88:23212329. estimates from the Framingham Study. Lancet Neurol 2007;6:1106–1114.

8 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG ARTICLE OPEN ACCESS Chorea-acanthocytosis Homozygous 1-kb deletion in VPS13A detected by whole-genome sequencing

Susan Walker, PhD, Rubina Dad, MPhil, Bhooma Thiruvahindrapuram, MSc, Muhammed Ikram Ullah, PhD, Correspondence Arsalan Ahmad, MD, Muhammad Jawad Hassan, PhD, Stephen W. Scherer, PhD, and Berge A. Minassian, MD Dr. Minassian Berge.Minassian@ Neurol Genet 2018;4:e242. doi:10.1212/NXG.0000000000000242 UTSouthwestern.edu Abstract Objective To determine a molecular diagnosis for a large multigenerational family of South Asian ancestry with seizures, hyperactivity, and episodes of tongue biting.

Methods Two aﬀected individuals from the family were analyzed by whole-genome sequencing on the Illumina HiSeq X platform, and rare variants were prioritized for interpretation with respect to the phenotype.

Results A previously undescribed, 1-kb homozygous deletion was identiﬁed in both individuals sequenced, which spanned 2 exons of the VPS13A gene, and was found to segregate in other family members.

Conclusions VPS13A is associated with autosomal recessive chorea-acanthocytosis, a diagnosis consistent with the phenotype observed in this family. Whole-genome sequencing presents a comprehensive and agnostic approach for detecting diagnostic mutations in families with rare neurologic disorders.

From the Centre for Applied Genomics (S.W., B.T., S.W.S.), The Hospital for Sick Children; Program in Genetics and Genome Biology (S.W., R.D., B.T., S.W.S., B.A.M.), The Hospital for Sick Children, Toronto, Ontario, Canada; Atta-ur Rahman School of Applied Biosciences (R.D., M.J.H.), National University of Sciences and Technology (NUST), Islamabad; Department of Biochemistry (I.M.U.), University of Health Sciences, Lahore; Division of Neurology (A.A.), Shifa International Hospital, Shifa Tameer e Millat University, Islamabad, Pakistan; Department of Molecular Genetics (S.W.S.), University of Toronto; McLaughlin Centre (S.W.S.), University of Toronto, Canada; and Department of Pediatrics (B.A.M.), University of Texas Southwestern, Dallas.

Chorea-acanthocytosis (CHAC; MIM number 200150) is an [figure]) were suspected to have a clinically separate neuro- autosomal recessive neurodegenerative disorder characterized logic disorder with symptoms including intellectual disability, by chorea and blood cells with abnormal morphology seizures, migraine, depression, and osteoarthritis. (acanthocytosis). Additional common features include dystonia, seizures, tics, and uncontrollable tongue biting. CHAC is caused by homozygous or compound heterozygous muta- Methods tions in VPS13A,1 which encodes chorein, a protein of un- ff known function. Here, we report a family with previously DNA from 22 family members, including the 5 a ected unexplained epilepsy found by whole-genome sequencing individuals, was extracted from whole blood using a standard (WGS) to carry a novel mutation in VPS13A, leading to phenol-chloroform method. Participants III-1 and IV-16 a diagnosis of CHAC. (index case) were selected for analysis by WGS. WGS was performed at The Centre for Applied Genomics (Toronto, Canada) with total genomic DNA following standard proto- Clinical report cols. Library preparation was performed from 100 ng of DNA using the Illumina TruSeq Nano DNA Library Prep Kit fol- A large multigeneration Pakistani family was recruited from lowing the manufacturer’s recommended protocol. Libraries the province of Khyber Pakhtunkhwa, Pakistan, presenting were pooled in equimolar quantities and sequenced on an with a history of seizures, episodes of hyperactive and psy- Illumina HiSeq X platform following Illumina’s recom- chiatric problems and headache, nausea, sleep problems, and mended protocol to generate paired-end reads of 150 bases in persistent tics with onset beginning late in the third decade of length. Base calling and data analysis were performed using life. Within the family, there are 4 affected individuals across 3 Illumina HiSeq Analysis Software version 2-2.5.55.1311. generations and an additional younger participant suspected Reads were mapped to the hg19 reference sequence using to be in the early stages of developing the same disorder. Isaac alignment software (Isaac alignment software: SAAC00776.15.01.27), and SNV and small indel variants Clinical details of index case IV:16 were called using the Isaac variant caller (Isaac Variant Caller The index patient, a 37-year-old woman, first visited the [Starling]: 2.1.4.2). Rare variants were defined as those with – neurologist at Shifa International Hospital, Islamabad, pre- less than 1% frequency in population databases.2 4 CNVs senting with hyperactive behavior beginning 1 year prior, with were detected using the read depth methods as previously episodes of tongue biting commencing 6 months later and 3 described.5 Rare CNVs were defined as those less than 1% generalized tonic-clonic seizures during the month before the frequency among unrelated, unaffected individuals sequenced consultation. She had been diagnosed with epilepsy and mi- using the same technology.6 Participants were screened for graine 3 years earlier but since developed hyperactivity and regions of homozygosity using PLINK (v1.90) “Runs of difficulty in concentrating in daily activities. She also suffered homozygosity” implementation. frequent tongue bites while speaking. On examination, she was found to have bilateral ptosis, but other cranial nerves Standard protocol approvals, registrations and were normal. Tone and power in all 4 limbs were normal, patient consents as were plantar responses. Higher mental function and speech Informed consent was obtained from all participants, and this were also typical. She was diagnosed as having a congenital study was approved by The Hospital for Sick Children syndrome with epilepsy, Tourette syndrome, and bilateral Research Ethics Board, Toronto, Canada. ptosis and prescribed escitalopram, procyclidine, carbamaze- pine, and later, haloperidol. One genetic test was sent for, namely Huntington disease, for which CAG repeats at the HD Results locus were normal (27 repeats). Red blood cell morphology was studied and reported normal. Through analysis of rare CNVs detected from WGS data, a 1168-bp deletion was detected, spanning exons 8 and 9 Her parents were reportedly nonrelated, and the symptoms of VPS13A (chr9:79827422-79828590); c.556_696del; were also present in 3 other family members: participants III: p.(Thr186_Leu232del). The deletion was homozygous in 1, IV:13, and V:8 in the figure. Individual V:6 was also sus- both individuals sequenced and would likely result in an in- pected of being in the early stages of developing the same frame deletion of 47 amino acids. PCR amplification and disorder. The family history and clinical features of affected Sanger sequencing confirmed the deletion and were used to individuals are described in table 1. Six additional family test the pattern of segregation among all family members, members (individuals III:16, III:21, V:2, V:4, V:9, and V:13 where DNA was available (figure). Homozygosity mapping

2 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG Figure Pedigree of the family

“−” indicates the presence of VPS13A p.(Thr186_Leu232del). “+” indicates no deletion. Individuals without genotypes shown were not available for analysis. Black shapes indicate individuals affected with the primary disorder, and gray shapes indicate individuals affected with the second, separate phenotype. Individual V:7 died at the age of 10 years before the typical age at onset; thus, the affection status could not be determined. Numbers inside circles indicate more than 1 individual in the family. The index case is denoted by an arrow.

from the WGS data for participants III-1 and IV-16 showed kinase–associated neurodegeneration) with similar but distinct that the deletion resides in an extended region of homozy- phenotypic features, and the clinical presentation of CHAC gosity in both individuals (chr9:78,311,259-98,376,367 in III- also overlaps with other disorders, presenting a diagnostic 1 and chr9:79,451,734-97,016,027 in IV-16). Individuals IV: challenge.7,8 The presence of acanthocytes can be helpful to 13, V:8, and V:6 were found to be homozygous for the de- suggest a diagnosis of CHAC, but hematology results are letion. Family relationships were confirmed by the analysis of inconsistent,7,8 as is the case in this family. With WGS, short tandem repeats. Analysis of single nucleotide variants a precise diagnosis could be rapidly achieved without the and small insertion/deletions did not yield any pathogenic or requirement of any prior awareness of the disorder by the likely pathogenic variants (all homozygous variants in coding referring clinician. Achieving a correct diagnosis now allows fi regions identi ed in either individual are shown in table e-1, accurate anticipation of the course of the disorder (particu- links.lww.com/NXG/A50). On peripheral blood smear, larly in the case of the youngest, mildly affected individual) acanthocytes were detected in 3 of the 4 individuals with the and facilitates appropriate treatment.9 The diagnosis also primary condition (III:1, IV:13, and V:8) and in V:6, but not prevents further unnecessary testing for possible nutritional in the index case IV:16. or infectious etiologies and helps to remove any stigma associated with behaviors in the family.

Discussion The nature of the mutation in this family further demonstrates fi The phenotype of the affected individuals is in keeping with the the bene t of using WGS compared with other nontargeted known CHAC phenotype. All 4 experienced choreoathetosis, technologies. Such a small deletion would be too small to seizures, and cognitive/psychiatric symptoms. Four members detect by microarray analysis and challenging to identify using of the family were found to have acanthocytes on peripheral exome sequencing. It may also not have been detected using blood smear, supporting the diagnosis of CHAC. The molec- a targeted gene or gene panel test, depending on the specific ular findings and hematology testing also confirmed the di- methodologies used. Small CNVs affecting VPS13A, such as agnosis of the youngest individual, V:6, presenting with the the one identified in this family, have been found previously in early stages of the condition. None of the 6 individuals with the cases with CHAC, although the deletion we describe had not second phenotype was homozygous for the deletion in been reported to date.10 VPS13A (3 did not carry the variant, and 3 were heterozygous), confirming that they likely have a second, distinct disorder. Here, we present a large multigenerational family from Pakistan with chorea-acanthocytosis attributable to a 1168-bp, 2-exon There are 4 neuroacanthocytosis syndromes (CHAC, McLeod deletion in VPS13A. This family showcases the potential for syndrome, Huntington disease-like 2, and pantothenate WGS as a first-tier diagnostic test in neurologic disorders.

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 3 Table 1 Clinical features of affected individuals

III:1 IV:13 IV:16 V:8 V:6

Age at onset 35 30 27 26

Present age 18

Choreoathetosis ++++

Orofacial dyskinesia ++++

Hyporeflexia +

Dysarthria ++++

Seizures +++++

Tics ++++

Dystonia ++

Parkinsonism ++

Caudate atrophy + No MRI No MRI No MRI

Putamen atrophy + No MRI No MRI No MRI

Limb muscular atrophy +

Limb muscle weakness +

Dementia +

Personality changes +++++

Mood changes ++++

Anxiety +++

Psychosis + One episode

Aggressiveness ++

Dysphagia +++

Drooling ++

Self-mutilation of the tongue +++

Hematology Acanthocytes Acanthocytes Acanthocytes Acanthocytes

Author contributions and the Canada Foundation for Innovation. R.D. gratefully S. Walker: acquisition of data and analysis and in- acknowledges her funding by the Higher Education terpretation of data. R. Dad and B. Thiruvahindrapuram: Commission of Pakistan under the International Research analysis and interpretation of data. I.M. Ullah: sample col- Support Initiative Program (HEC-IRSIP). S.W.S. is funded lection. A. Ahmad: acquisition of data and analysis and by the GlaxoSmithKline-CIHR Chair in Genome Sciences at interpretation of data. M.J. Hassan, S.W. Scherer, and the University of Toronto and The Hospital for Sick B.A. Minassian: critical revision of the manuscript and study Children. B.A.M holds the University of Toronto Michael supervision. Bahen Chair in Epilepsy Research and the University of Texas Southwestern Jimmy Elizabeth Westcott Distin- Acknowledgment guished Chair in Pediatric Neurology. The authors thank the family for their participation in the study and The Centre for Applied Genomics for their Study funding analytical and technical support. This work was supported by This work was supported by The Centre for Applied Genomics, The Centre for Applied Genomics, the University of Toronto the University of Toronto McLaughlin Centre, Genome McLaughlin Centre, Genome Canada/Ontario Genomics Canada/Ontario Genomics Institute, the Canadian Institutes of Institute, the Canadian Institutes of Health Research Health Research (CIHR), the Canadian Institute for Advanced (CIHR), the Canadian Institute for Advanced Research, Research, and the Canada Foundation for Innovation.

4 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG Disclosure MECP2,andVMA21. Full disclosure form information pro- S. Walker, R. Dad, B. Thiruvahindrapuram, M.I. Ullah, vided by the authors is available with the full text of this article A. Ahmad, and M.J. Hassan report no disclosures. S.W. Scherer at Neurology.org/NG. holds the GlaxoSmithKline-CIHR Chair in Genome Scien- fi ces at the University of Toronto and The Hospital for Received January 10, 2018. Accepted in nal form April 5, 2018. Sick Children; is on the scientific advisory board of Deep fi References Genomics; has served on the scienti c advisory board of 1. Rampoldi L, Dobson-Stone C, Rubio JP, et al. A conserved sorting-associated protein Population Diagnostics; has served on the editorial boards is mutant in chorea-acanthocytosis. Nat Genet 2001;28:119–120. 2. Lek M, Karczewski KJ, Minikel EV, et al. Analysis of protein-coding genetic variation of Genomic Medicine, Genes, Genomes, Genetics, the Journal of in 60,706 humans. Nature 2016;536:285–291. Personalized Medicine, the Open Genomics Journal, the Hugo 3. 1000 Genomes Project Consortium. A global reference for human genetic variation. Nature 2015;526:68–74. Journal, Genome Medicine, the Journal of Neurodevelopmental 4. Fu W, O’Connor TD, Jun G, et al. Analysis of 6,515 exomes reveals the recent origin Disorders, Autism Research, PathoGenetics, Comparative and of most human protein-coding variants. Nature 2013;493:216–220. Functional Genomics, BMC Medical Genomics,andCytoge- 5. Trost B, Walker S, Wang Z, et al. A comprehensive workflow for read depth-based identification of copy-number variation from whole-genome sequence data. Am J netics and Genome Research; and has received research sup- Hum Genet 2018;102:142–155. port from the Genome Canada/Ontario Genomics Institute, 6. Yuen RKC, Merico D, Bookman M, et al. Whole genome sequencing resource identifies 18 new candidate genes for autism spectrum disorder. Nat Neurosci 2017; Canadian Institutes of Health Research, Canadian Institute 20:602–611. for Advanced Research, McLaughlin Centre, Canada Foun- 7. Walterfang M, Evans A, Looi JCL, et al. The neuropsychiatry of neuroacanthocytosis syndromes. Neurosci Biobehav Rev 2011;35:1275–1283. dation for Innovation, Government of Ontario, and NIH. 8. Walker RH. Untangling the thorns: advances in the neuroacanthocytosis syndromes. B.A. Minassian holds patents for diagnostic testing of the J Mov Disord 2015;8:41–54. 9. Benninger F, Afawi Z, Korczyn AD, et al. Seizures as presenting and prominent following genes: EPM2A, EPM2B, MECP2,andVMA21;has symptom in choreaacanthocytosis with c.2343del VPS13A gene mutation. Epilepsia received research support from the NIH; and has received 2016;57:549–556. 10. Tomiyasu A, Nakamura M, Ichiba M, et al. Novel pathogenic mutations and copy license fee payments/royalty payments from patents for di- number variations in the VPS13A gene in patients with chorea-acanthocytosis. Am J agnostic testing of the following genes: EPM2A, EPM2B, Med Genet B Neuropsychiatr Genet 2011;156B:620–631.

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 5 ARTICLE OPEN ACCESS Neurodegeneration as the presenting symptom in 2 adults with xeroderma pigmentosum complementation group F

Niraj M. Shanbhag, MD, PhD, Michael D. Geschwind, MD, PhD, John J. DiGiovanna, MD, Correspondence Catherine Groden, MS, CRNP, Rena Godfrey, PA, Matthew J. Yousefzadeh, PhD, Erin A. Wade, BA, Dr. Toro [email protected] Laura J. Niedernhofer, MD, PhD, May Christine V. Malicdan, MD, PhD, Kenneth H. Kraemer, MD, William A. Gahl, MD, PhD, and Camilo Toro, MD

Neurol Genet 2018;4:e240. doi:10.1212/NXG.0000000000000240 Abstract Objective To describe the features of 2 unrelated adults with xeroderma pigmentosum complementation group F (XP-F) ascertained in a neurology care setting.

Methods We report the clinical, imaging, molecular, and nucleotide excision repair (NER) capacity of 2 middle-aged women with progressive neurodegeneration ultimately diagnosed with XP-F.

Results Both patients presented with adult-onset progressive neurologic deterioration involving chorea, ataxia, hearing loss, cognitive deficits, profound brain atrophy, and a history of skin photosensitivity, skin freckling, and/or skin neoplasms. We identified compound heterozygous pathogenic mutations in ERCC4 and confirmed deficient NER capacity in skin fibroblasts from both patients.

Conclusions These cases illustrate the role of NER dysfunction in neurodegeneration and how adult-onset neurodegeneration could be the major symptom bringing XP-F patients to clinical attention. XP-F should be considered by neurologists in the diﬀerential diagnosis of patients with adult- onset progressive neurodegeneration accompanied by global brain atrophy and a history of heightened sun sensitivity, excessive freckling, and skin malignancies.

From the Department of Neurology (N.M.S., M.D.G.), University of California San Francisco, CA; Laboratory of Cancer Biology and Genetics (J.J.D., K.H.K.), Center for Cancer Research, National Cancer Institute, National Institutes of Health; NIH Undiagnosed Diseases Program (C.G., R.G., M.C.V.M., W.A.G., C.T.), National Human Genome Research Institute, National Institutes of Health, Bethesda, MD; and Department of Molecular Medicine (M.J.Y., E.A.W., L.J.N.), Center on Aging, The Scripps Research Institute, Jupiter, FL.

The Article Processing Charge was funded by the NIH. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary CS = Cockayne syndrome; MoCA = Montreal Cognitive Assessment; NER = Nucleotide excision repair; UDP = Undiagnosed Diseases Program; UDS = unscheduled DNA synthesis; UV = ultraviolet; XP = xeroderma pigmentosum; XP-F =XP complementation group F.

Patients with genome instability caused by defects in the DNA common inherited adult-onset neurodegenerative disorders. damage repair response illustrate the detrimental effect of Additional clinical details are summarized in the table. cumulative DNA damage. This includes heightened cancer risk, accelerated aging, and neurodegeneration. A broad Patient 1 spectrum of neurologic manifestations has been associated A now 60-year-old woman of Northern European descent with with disorders of DNA repair, including xeroderma pigmen- a history of infertility and skin photosensitivity but no skin tosum (XP), Cockayne syndrome (CS), ataxia telangiectasia, neoplasms sought medical attention at the age 46 years with 1 trichothiodystrophy, and Nijmegen breakage syndrome.1 year of progressive memory and balance difficulties. Her skin examination revealed moderate freckling of sun-exposed XP is a rare autosomal recessive disorder arising from deficient regions (figure 1A). On neurologic examination, she demon- DNA repair. XP is caused primarily by mutations in genes strated mild chorea of her extremities and face, limb ataxia, encoding components of the nucleotide excision repair (NER) absent ankle reflexes, and impaired gait. She had 1 sibling with DNA repair pathway. NER recognizes and repairs several MS but no other similarly affected relatives (figure 1B). Brain types of DNA lesions, including those due to ultraviolet (UV) MRI showed severe global atrophy out of proportion to her radiation, which results in bulky dimers at dipyrimidine sites.2 recent symptom onset (figure 2, A–D). EMG and nerve con- The typical result is sun damage to exposed skin and eyes. duction studies indicated diffuse axonal sensorimotor poly- Patients with XP have a high frequency of skin cancer in re- neuropathy. A sural nerve biopsy showed decreased myelinated sponse to genomic damage caused by UV radiation. Approx- fibers with clusters of regenerating fibers (figure 1D). Neuro- imately one-fourth of patients with XP experience neurologic logic progression was gradual but relentless over the next 11 deterioration, and when present, shortens life expectancy.3 years; the patient became dependent on a walker for ambulation Cumulative toxicity by non–UV-mediated unrepaired geno- and deteriorated in essentially all cognitive domains, with an 4 mic lesions in nonreplicating tissue explains neuronal injury. early Mini-Mental State Examination score of 26/30, a Mon- treal Cognitive Assessment (MoCA)5 score of 18/30 at 6 years, We report 2 middle-aged Caucasian women with adult-onset and an MoCA score of 16/30 at 8 years after her initial visit. chorea, ataxia, dystonia, neuropathy, and later progressive cognitive impairment, but with life-long acute skin sun- Patient 2 burning on minimal sun exposure, freckle-like skin lesions on A 52-year-old woman of Ashkenazi Jewish descent presented sun-exposed skin, and early onset of skin cancer in 1 case. with a 20-year history of progressive dystonia, gait ataxia, hearing Both were found to have compound heterozygous mutations loss, and worsening cognition. Her past medical history included in ERCC4 and significantly reduced NER capacity, confirming photosensitivity with blistering skin lesions after limited sun the diagnosis of XP complementation group F (XP-F). Their exposure resulting in multiple facial lentigines and basal cell presentations highlight the importance of adult neurologists carcinomas as a teenager. Because of aggressive photo- considering XP-F when evaluating patients with atypical protection, she had minimal freckling but multiple scars from fi neurodegeneration. basal cell carcinoma resections ( gure 1E). There was a maternal history of mild postural tremor but no similarly affected relatives (figure 1F). On neurologic examination, the patient Methods was disoriented, partly amnestic, and struggled to follow simple commands. She had dystonic and choreiform movements of the Standard protocol approvals, registrations, head and neck, a coarse, irregular appendicular tremor, ataxia, and patient consents and spasticity and was using a wheelchair. She had brisk reflexes Patients were referred by their neurologists to participate in and upgoing toes. Brain MRI demonstrated global cerebral at- the NIH Undiagnosed Diseases Program (UDP). They en- rophy and inner table hyperostosis (figure 2, E–H). There was rolled in protocol 76-HG-0238 (ClinicalTrials.gov Identifier: no electrophysiologic evidence of polyneuropathy. The patient NCT00369421) approved by the National Human Genome died at age 54 years from secondary medical complications. Research Institute (NHGRI) Institutional Review Board. Written informed consent was obtained according to protocol guidelines. Both patients had completed extensive clinical Results evaluations over the preceding decades, including investigations for toxic, metabolic, infectious, autoimmune, and Following singleton whole-exome sequencing, variants were inflammatory processes and had undergone genetic testing for prioritized based on known disease association, population

2 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG Table XP-F patients’ molecular, clinical, and neurologic findings

Case 1 (UDP_3675) Case 2 (UDP_7356)

ERCC4 (NM_005236.2) mutations c.2395C>T, p.Arg799Trp/c.1765C>T, p.Arg589Trp c.2395C>T, p.Arg799Trp/c.1376C>A, p.Ser459*

Age (y)

At NIH evaluation 53 54

Current 60 Deceased

Ethnicity Northern European Ashkenazi Jewish

History of acute skin sun-burning on minimal Yes Yes exposure

Freckle-like skin lesions Moderate Mild

Skin cancer No ≥20 basal cell carcinomas (first by age 16 y)

Symptom prompting first neurologic Cognitive impairment; 46 Chorea and ataxia; 34 evaluation; age (y)

First subjective neurologic symptom; age “Twitchy, affected dexterity”; mid 20s “Clumsy”; late teens

Sequence of neurologic symptoms and signs Chorea/ataxia/neuropathy, cognitive impairment, Chorea/ataxia, dystonia, spasticity, dementia and dementia

Height (cm); weight (kg); BMI 162.3; 60.2; 22.9 147; 49; 22.7

Developmental milestones Normal Normal

Education College classes College degree

Speech Ataxic dysarthria (mild) Ataxic dysarthria (mild)

Swallowing Mild dysphagia with tongue weakness and protrusion Normal swallow study

Hearing No subjective complaint of hearing loss. Audiometry Neurosensory hearing loss (onset mid 30’s) not available

Activities of daily living Needs some assistance Fully dependent

Gait; assisting device Shuffling and ataxic; walker Ataxic and spastic; wheelchair

DTRs and plantar responses Absent ankle jerks, extensor Brisk throughout, extensor

Urinary incontinence Yes Yes

Polyneuropathy Diffuse axonal sensorimotor polyneuropathy No

Other neoplasm history No No

Keratopathy No No

Optic discs; retina Normal; normal Pale; Normal

Brain imaging Severe global atrophy Severe global atrophy and hyperostosis of the inner table

DNA repair (UDS) 26.0% ± 4.8% of normal 21.3% ± 5.1% of normal

Abbreviations: BMI = body mass index; DTR = deep tendon reflex; UDS = unscheduled DNA synthesis.

frequency, and various in-silico pathogenicity models. Both NER capacity was investigated by measuring unscheduled patients had compound heterozygous mutations in ERCC4 DNA synthesis (UDS) determining the incorporation of (table). Patient 1 (figure 1C) carried 2 known ERCC4 nucleotides into the nuclear genome of nonreplicating, UV-C mutations associated with XP-F; NM_005236.2: c.1765C>T; irradiated patient-derived fibroblasts (see figure 1 legend for p.Arg589Trp and c.2395C>T,p.Arg799Trp.6,7 Patient 2 details). The percent repair was obtained by comparing irra- (figure 1G) had the p.Arg799Trp allele and an ultra-rare diated and unirradiated patient cells to reference standards ERCC4 truncating mutation; NM_005236.2:c.1376C>A, NER-proficient human fibroblasts (C5RO) and NER- p.Ser459*. Segregation analysis of the families confirmed deficient human fibroblasts (XP51RO). XP51RO cells were compound heterozygous allele pairs in both. from a patient with a homozygous p.Arg153Pro ERCC4

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 3 Figure 1 Two cases of XP-F with adult-onset neurologic deterioration

Patient 1 (top): panels (A–D) and patient 2 (bottom): panels (E–G). (A and E) Face photographs of patients 1 and 2 demonstrating prominent skin freckling (patient 1) or scarring (open arrows) at the sites of prior basal cell carcinoma resections (patient 2). (B and F) Nuclear family pedigrees for patient 1 (UDP_3675) and patient 2 (UDP_7356). (C and G) Sanger chromatogram for patient 1 and patient 2 demonstrating relevant mutations in both ERCC4 alleles (arrows). (D) Representative image of sural nerve biopsy (embedded in Epon and stained with toluidine blue) from patient 1 demonstrating a decrease in the number of myelinated fibers with evidence of degeneration/regeneration clusters (×400). (H) Measurement of UDS in patient fibroblasts. Values are normalized to those obtained for NER-proficient (C5RO) and NER-deficient (XP51RO) control fibroblasts.8 Cells were irradiated with 24 J/m2 UV and then incubated in the presence of the thymidine analog EdU for 2.5 hours to allow DNA repair. AlexaFluor647 was conjugated to EdU by Click-iT chemistry before fixation, stained with DAPI, and quantification of the fluorescence signal in G1 cells measured by flow cytometry. UDS was measured in duplicate for each cell line in 3 independent experiments. Values represent mean ± SD, ****p < 0.0001 by 1-way ANOVA.

mutation associated with a phenotype of XFE progeroid past the 4th decade.10 Neurodegeneration is largely reported syndrome and severely reduced (<3%) UDS.8 UDS of in younger patients in complementation groups XP-A, XP-B, fibroblasts from patients 1 and 2 had 26.0% ± 4.8% and 21.3% XP-D, and XP-G but not in XP-C or XP-E and is often her- ± 5.1% of the expected NER capacity, respectively, indicating alded by progressive neurosensory hearing loss.3 Neurologic significant NER impairment (figure 1H). features reported in XP-F include gait disturbances, ataxia, neuropathy, chorea, sensorineural hearing loss, cognitive de- Discussion cline, and cerebral and cerebellar atrophy.6,7,10 Our observations support reports suggesting that ERCC4: p.Arg799Trp The prevalence of XP in the general population approximates 1 might be a common allele among XP-F patients with adult- in 1 million, being higher in Japan because of a founder mu- onset neurodegeneration.6 Besides XP-F, ERCC4 mutations tation in XPA. One-fourth of individuals with XP exhibit are associated with other diseases, including XFE progeria with neurologic manifestations including acquired microcephaly, very low UDS, Fanconi Anemia, and XP/CS.3,8 neuropathy, sensorineural hearing loss, and progressive cognitive impairment. In a long-term follow-up study of 89 patients Like other reported XP-F cases with neurodegeneration, our with XP in the United Kingdom, three patients were diagnosed patients manifested disparity between severity of brain atrophy with XP-F, one of whom demonstrated neurologic features by MRI at the time of first imaging and relatively modest early similar to our patients.9 Up to 10% of all XP is XP-F.3,10 XP-F is cognitive deficits, consistent with compensation of a very slow caused by mutations in the ERCC4 gene. The encoded neurodegenerative process over decades. This contrasts with ERCC4/XPF protein forms a tight complex with the ERCC1 other neurodegenerative disorders, such as Alzheimer disease, protein. This heterodimer provides 59-nuclease activity for in which profound impairment usually accompanies such se- a crucial excision step of NER.2 vere degree of brain atrophy.

Compared with other XP subtypes, patients with XP-F have These cases elicit consideration of several points. First, dys- less propensity for skin neoplasms and present later, often function of the NER pathway can be associated with profound

4 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG Figure 2 Representative axial and sagittal sections of T1-weighted brain MRIs

Patient 1 (top, A–D) and patient 2 (bottom, E–H) demonstrate prominent and diffuse atrophy in the supra- and infra-tentorial compartments.

neurodegeneration. Second, there is a causal role of cumula- Acknowledgment tive unrepaired DNA damage in neurodegenerative disorders The authors thank their patients, their families, and referring in general. The precise type of damage leading to neuro- physicians. Dr. Dennis Landis contributed to the clinical degeneration is unclear but may relate to stable oxidative evaluations of these patients at the NIH. genomic lesions. Finally, the diagnosis of XP, and particularly XP-F, should be considered in adult patients with unexplained Study funding neurodegeneration associated with global brain atrophy, es- Intramural Research Program of the National Institutes of pecially when accompanied by a history of photosensitivity, Health (NIH) NHGRI and the NCI, Common Fund, Office of skin malignancies, and/or excessive freckling. the Director; the NIH/NHLBI grant (HHSN268201400058); the NIH/NIA grant (P01AG043376): M.J.Y., E.A.W. and Author contributions L.J.N.; and the Michael J. Homer Family Fund: M.D.G. N.M. Shanbhag: wrote the initial draft of the manuscript. M.D. Geschwind, J.J. DiGiovanna, C. Groden, and R. God- Disclosure frey: clinical consult and contributed to manuscript writing. Dr. Shanbhag has received research support from the NIH M.J. Yousefzadeh: measured NER in fibroblasts, in- NINDS and Alzheimer’s Association. Dr. Geschwind has re- terpretation of data, and contributed to manuscript writing. ceived funding for travel and/or speaker honoraria from E.A. Wade and L.J. Niedernhofer: measured NER in fibro- Oakstone Publishing, Inc; has served on the editorial board of blasts, interpretation of data, data analysis, and contributed to Dementia & Neuropsychologia; serves or has served as a con- manuscript writing. M.C.V. Malicdan: interpretation of data, sultant to Advanced Medical Inc, Best Doctors Inc, Grand data analysis, sample logistics, and contributed to manuscript Rounds, Gerson Lehrman Group Inc, Guidepoint Global, writing. K.H. Kraemer: study design, interpretation of data, MEDACorp, LCN Consulting, Optio Biopharma Solutions, and contributed to manuscript writing. W.A. Gahl: study various medical-legal consulting, Biohaven Pharmaceuticals design, contributed to manuscript writing, and funding ac- Inc, Teva Pharmaceuticals, and Quest Diagnostics; and quisition. C. Toro: study design, clinical consult, in- receives or has received research support from the NIH/NIA terpretation of data, data analysis, and wrote the initial draft of (R01 AG031189) PI 2013-2018, Alliance Biosecure, Michael the manuscript. J. Homer Family Fund, CurePSP, and Tau Consortium. Dr.

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 5 DiGiovanna serves or has served on scientific sdvisory boards ManNAc; and receives research support from the NIH. Dr. of the Medical and Scientific Advisory Board of the Foun- Toro is a full-time employee of the NIH and receives funding dation for Ichthyosis and Related Skin Types; holds patents from the NIH intramural program. Full disclosure form in- with regard to the use of synthetic peptides to disrupt the formation provided by the authors is available with the full cytoskeleton and therapeutic uses of hmgn1 and hmgn2; text of this article at Neurology.org/NG. consults or has consulted for the US Food and Drug Ad- ministration; and is employed by the National Cancer Received January 22, 2018. Accepted in final form April 23, 2018. Institute and NIH. Dr. Groden, Dr. Godfrey, and Dr. You- References sefzadeh report no disclosures. Dr. Wade is or has been 1. McKinnon PJ. DNA repair deficiency and neurological disease. Nat Rev Neurosci employed by The Scripps Research Institute and The Na- 2009;10:100–112. 2. Sijbers AM, de Laat WL, Ariza RR, et al. Xeroderma pigmentosum group F tional Institute on Aging. Dr. Niedernhofer holds patents with caused by a defect in a structure-specific DNA repair endonuclease. Cell 1996; regard to a rapid test to measure DNA repair capacity of an 86:811–822. individual; has received research support from the NIH/ 3. Kraemer KH, DiGiovanna JJ. Xeroderma pigmentosum. In: Adam MP, Ardinger HH, Pagon RA, et al, editors. GeneReviews((R)). Seattle: University of Washington; NHLBI, NIH/NIA, and Glenn Award for Aging Research. 1993–2016. Dr. Malicdan serves or has served on the editorial board of 4. Folstein MF, Folstein SE, McHugh PR. “Mini-mental state.” A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res 1975;12: BMC Musculoskeletal Disorders; holds patents with regard to 189–198. therapeutic pharmaceutical agent for diseases associated with 5. Nasreddine ZS, Phillips NA, Bedirian V, et al. The Montreal Cognitive Assessment, MoCA: a brief screening tool for mild cognitive impairment. J Am Geriatr Soc 2005; decrease in function of gne protein, food composition, and 53:695–699. food additive, Gne-/-hGNED176VTg as a mouse model for 6. Gregg SQ, Robinson AR, Niedernhofer LJ. Physiological consequences of defects in ERCC1-XPF DNA repair endonuclease. DNA Repair (Amst) 2011;10:781–791. DMRV/HIBM and CANNABINOID RECEPTOR MEDI- 7. Moriwaki S, Nishigori C, Imamura S, et al. A case of xeroderma pigmentosum ATING COMPOUNDS; and is employed by the NIH complementation group F with neurological abnormalities. Br J Dermatol 1993;128: 91–94. Intramural Research Program. Dr. Kraemer serves or has 8. Niedernhofer LJ, Garinis GA, Raams A, et al. A new progeroid syndrome reveals that served on the editorial board of Photodermatology and Pho- genotoxic stress suppresses the somatotroph axis. Nature 2006;444:1038–1043. 9. Fassihi H, Sethi M, Fawcett H, et al. Deep phenotyping of 89 xeroderma pigmen- toimmunology. Dr. Gahl has received funding for travel and/or tosum patients reveals unexpected heterogeneity dependent on the precise molecular speaker honoraria from Cystinosis Research Network; serves defect. Proc Natl Acad Sci USA 2016;113:E1236–E1245. 10. Tofuku Y, Nobeyama Y, Kamide R, Moriwaki S, Nakagawa H. Xeroderma pigmen- or has served on the editorial board of Molecular Genetics and tosum complementation group F: report of a case and review of Japanese patients. Metabolism; receives or has received licensing royalties from J Dermatol 2015;42:897–899.

6 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG ARTICLE OPEN ACCESS Absence of NEFL in patient-speciﬁc neurons in early-onset Charcot-Marie-Tooth neuropathy

Markus T. Sainio, MSc, Emil Ylikallio, MD, PhD, Laura Mäenpää, MSc, Jenni Lahtela, PhD, Pirkko Mattila, PhD, Correspondence Mari Auranen, MD, PhD, Johanna Palmio, MD, PhD, and Henna Tyynismaa, PhD Dr. Tyynismaa [email protected] Neurol Genet 2018;4:e244. doi:10.1212/NXG.0000000000000244 Abstract Objective We used patient-specific neuronal cultures to characterize the molecular genetic mechanism of recessive nonsense mutations in neurofilament light (NEFL) underlying early-onset Charcot- Marie-Tooth (CMT) disease.

Methods Motor neurons were diﬀerentiated from induced pluripotent stem cells of a patient with early- onset CMT carrying a novel homozygous nonsense mutation in NEFL. Quantitative PCR, protein analytics, immunocytochemistry, electron microscopy, and single-cell transcriptomics were used to investigate patient and control neurons.

Results We show that the recessive nonsense mutation causes a nearly total loss of NEFL messenger RNA (mRNA), leading to the complete absence of NEFL protein in patient’s cultured neurons. Yet the cultured neurons were able to differentiate and form neuronal networks and neurofilaments. Single-neuron gene expression fingerprinting pinpointed NEFL as the most downregulated gene in the patient neurons and provided data of intermediate filament transcript abundancy and dynamics in cultured neurons. Blocking of nonsense-mediated decay partially rescued the loss of NEFL mRNA.

Conclusions The strict neuronal specificity of neurofilament has hindered the mechanistic studies of recessive NEFL nonsense mutations. Here, we show that such mutation leads to the absence of NEFL, causing childhood-onset neuropathy through a loss-of-function mechanism. We pro- pose that the neurofilament accumulation, a common feature of many neurodegenerative diseases, mimics the absence of NEFL seen in recessive CMT if aggregation prevents the proper localization of wild-type NEFL in neurons. Our results suggest that the removal of NEFL as a proposed treatment option is harmful in humans.

From the Research Programs Unit (M.T.S., E.Y., L.M., M.A., H.T.), Molecular Neurology, University of Helsinki; Clinical Neurosciences, Neurology (E.Y., M.A.), University of Helsinki and Helsinki University Hospital; Institute for Molecular Medicine Finland (FIMM) (J.L., P.M.), University of Helsinki; Neuromuscular Research Center (J.P.), Tampere University Hospital and University of Tampere; and Department of Medical and Clinical Genetics (H.T.), University of Helsinki, Finland.

Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary CMT = Charcot-Marie-Tooth; INA = internexin; iPSC = induced pluripotent stem cell; mRNA = messenger RNA; NCV = nerve conduction velocity; NEFH = neurofilament heavy; NEFL = neurofilament light; NEFM = neurofilament medium; NMD = nonsense-mediated mRNA decay; PRPH = peripherin; qPCR = quantitative PCR; UMI = unique molecular identifier.

Neurofilaments are 10-nm-wide intermediate filaments ex- details are described in the e-Methods (appendix e-1, link- clusive to neurons and crucial for the maintenance of neu- s.lww.com/NXG/A54). rite structure and integrity.1 Neurofilament light chain (NEFL) is among the core subunits of neurofilament, usually forming heterodimers with medium (NEFM) and Results heavy chains (NEFH), sometimes supplemented with either α-internexin (INA) or peripherin (PRPH). The toxic Clinical findings accumulation of neurofilament is a hallmark of many neu- P1 was first examined as an infant because of nystagmus and – rodegenerative disorders,2 4 and thus, the removal of NEFL tremor. Her feet were deformed resembling clubfoot, and has been investigated as a treatment option.5 In addition, her movements were clumsy. She learned to walk at the age the potential of NEFL as a serum biomarker of neuronal of 20 months. Electrophysiology showed strongly de- injury in a number of neurologic disorders is currently creased nerve conduction velocities (NCVs) corresponding – investigated.6 9 to demyelinating sensorimotor neuropathy. At the age of 8 years, there was no response from the sensory median Gene mutations in NEFL have been found to underlie Charcot- nerve. Ulnar sensory NCVs from the wrist to finger and the Marie-Tooth disease (CMT), either the demyelinating elbow to wrist were 9 and 19 m/s, respectively. Median and CMT1F4 or axonal CMT2E10 form.10,11 Most disease-causing ulnar motor NCVs from the elbow to wrist were 21 and 16 NEFL mutations are dominantly inherited missense variants m/s, respectively. The only response in the lower leg was functioning through a gain-of-function mechanism,12,13 in recorded from tibialis anterior. Four years later, sensory which the missense mutant NEFL protein disrupts neurofila- NCVs were absent in all upper limb nerves (ie, radial, ulnar, ment assembly and organelle transport in the axons by forming and median). Median motor response was also absent, and aggregates.14,15 All reported recessive NEFL mutations have ulnar NCVs were markedly reduced (ie, 11 m/s from the been homozygous nonsense variants.13,16,17 Elucidation of the elbow to wrist, no response from the wrist to finger). disease mechanism of the recessive variants in humans has been Needle examination showed denervation. There were un- complicated by the restricted neuronal expression of NEFL.18 specific mild white matter lesions on her brain MRI that Thus, it is not known if the homozygous nonsense variants were not progressive. Her younger brother, P2, had similar cause disease through aggregation effects mediated by a trun- symptoms and electrophysiologic findings, although less cated protein or by the loss of NEFL protein through severe, and his brain MRI was normal. During their school nonsense-mediated messenger RNA (mRNA) decay (NMD). years, the patients were estimated to be approximately 2 Here, we used patient-specific induced pluripotent stem cell years behind their peers in cognitive development. How- (iPSC)-derived neuronal cultures and report that the complete ever, both managed to finish supported elementary school absence of NEFL in patients with a homozygous NEFL non- despite difficulties. sense mutation causes early-onset CMT. As neuropathy slowly progressed, distal muscle weakness became apparent in upper and lower extremities. Muscle atrophy was evident in the legs and intrinsic hand muscles. Both Methods patients underwent orthopedic surgery for feet deformities Standard protocol approvals, registrations, and tight Achilles tendon. Distal weakness and waddling gait and patient consents were observed. P2, aged 27 years, can still walk 50 m without Patient and control samples were taken according to the aid, whereas P1 lost ambulation at the age of 25 years. For Declaration of Helsinki, with informed consent. The study both patients, grip strength was reduced, pinching impossible, was approved by the Institutional Review Board of the Hel- and fine motor skills decreased. Weakness in finger extension sinki University Hospital. and flexion was severe (1-2/5 Medical Research Council [MRC] scale). All movements were essentially lost in ankle Patients plantar and dorsiflexion. There was also proximal muscle Two siblings, patients P1 and P2, now aged 30 and 27 years, weakness in the upper and lower limbs but to a lesser extent respectively, who were born to healthy unrelated parents of (3-4/5 MRC). Tendon reflexes were absent. No clear sensory Finnish origin, were investigated for motor and de- disturbances were found. In both patients, articulation was velopmental delay in early childhood. The skin fibroblasts slow, but there were no dysarthria, facial weakness, or other used in the study were derived from patient P1. Experimental bulbar symptoms.

2 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG P1 was also diagnosed with ventricle septum defect in child- Absence of NEFL in patient-specific hood and later aortic valve stenosis and regurgitation, sleep cultured neurons apnea, and severe obesity. The parents were healthy; both To investigate the consequences of the NEFL nonsense var- underwent electrophysiologic studies as part of their child- iant, we differentiated patient-specific iPSC, reprogrammed ren’s investigations with normal NCVs. from P1 skin fibroblasts, into neurons. Two iPSC clones were used from P1, and 1 clone each from 3 unrelated control Genetic findings iPSCs. We used the motor neuron differentiation protocol, Targeted next-generation sequencing panel for known modified from reference 21 as summarized in figure 2A. We CMT disease genes19 for the DNA sample of P1 revealed verified the neuronal differentiation by quantitative PCR a novel homozygous c.1099C>T (g.24811765C>T) variant (qPCR) of microtubule-associated protein 2 (MAP2) and in exon 2 of the NEFL gene, predicting a nonsense change βIII-tubulin (TUBB3) mRNA expression (figure 2B) and by p.Arg367*. The patients were homozygous for the variant, immunocytochemistry with TUBB3 (TUJ1) and MAP2 and parents were heterozygous carriers (figure 1A). The antibodies (figure 2C). Approximately 90% of DAPI-positive GnomAD20 database (277,044 alleles) lists 15 heterozygous cells were also MAP2 and/or TUJ1 positive in each imaged carriers of the variant in Finland, indicating an enrichment of frame. To validate the motor neuronal identity of the differ- the variant with a carrier frequency of 0.00058. The locali- entiated neurons, we analyzed the expression levels of ISL zation of the variant together with previously reported LIM homeobox 1 (ISL1), motor neuron and pancreas disease-causing variants in NEFL domains is summarized in homeobox 1 (MNX1), and acetylcholine transferase (CHAT) figure 1B. by qPCR (figure 2D). Although some variation was detected

Figure 1 Dominant and recessive missense and nonsense variants in neurofilament light (NEFL)

(A) Sequencing traces of the c.1099C>T variant in the family members show that both parents of the patients are heterozygous carriers of the mutation. (B) NEFL protein domains are depicted, and the localization of the reported missense and nonsense variants is indicated (modified from references 17 and 25). The nonsense variant A367* identified in this study is shown in red.

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 3 Figure 2 Neuron differentiation and validation

(A) Work-flow of fibroblast-derived induced pluripotent stem cell (iPSC) differentiation into motor neurons; Wnt signaling pathway activator (WNT act), retinoic acid (RA), Sonic hedgehog (SHH), growth factors (GF; BDNF, IGF-1 and CNTF), Poly-D-lysine (PDL). (B) Validation of the expression of neural transcripts MAP2 and TUBB3 against GAPDH by quantitative PCR (qPCR) in total culture RNA of patient 1 clones 1 (Pt C1) and 2 (Pt C2) and controls 1-3 (ctr 1-3) after motor neuron differentiation. (C) Immunocytochemical analysis of MAP2 (green) and TUBB3 (red) proteins in patient 1 and control neuronal cultures. (D) Validation of the expression of motor neural transcripts ISL1, MNX1, and CHAT by qPCR as in B. (E) Immunocytochemical analysis of ISL1 (red) and NEFM (green) protein in patient 1 and control neuronal cultures. ISL1-positive neurons are shown in larger cell clusters in the final differentiation stage (day 14 of in PDL + laminin- coated plates). (F) Expression of intermediate filament subunits neurofilament medium (NEFM), neurofilament heavy (NEFH), and neurofilament light (NEFL) by qPCR as in B. The bars in each graph represent mean levels ± SD, n = 3 for each cell line. All scale bars 50 μm. 49,6-diamidino-2-phenylindole (DAPI) indicates nuclear staining.

4 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG between the different clones for the motor neuron markers, neurons did not show any staining with NEFL antibody, no decrease in differentiation potential was observed in pa- confirming that they were devoid of NEFL. However, they tient lines in comparison with control lines. ISL1 expression were still able to form as long, branching projections as the was confirmed by immunocytochemistry in the neuronal control neurons, suggesting that the intermediate filament cultures (figure 2E). network in the absence of NEFL was sufficient for axonal maintenance in culture. We then analyzed the mRNA levels of NEFL, NEFM, and NEFH and observed that the patient neuronal cultures had Intermediate filament transcript dynamics in a markedly decreased NEFL mRNA, with a residual level of cultured neurons about 5% in patient vs control samples, whereas patient To examine the gene expression fingerprints in single cultured NEFM and NEFH mRNA levels were comparable with con- neurons, we used the differentiated neuronal cultures of P1 trol levels (figure 2F). This suggested that the nonsense clone 1 and control 1 for single-cell transcriptomics by the mutant NEFL transcript was degraded by NMD. We next Macosko22 method with 10X Genomics Single Cell Plat- investigated whether NEFL protein could be detected in pa- form.23 After quality control, 1,336 cells could be profiled tient neurons by immunoblotting or immunocytochemistry. from P1 clone 1 and 418 cells from control 1. The expression Relatively, even neuronal differentiation of lysed samples was of 17,318 genes could be reliably detected in these cells. validated by immunoblotting for TUJ1, NEFM, and CHAT Clustering of the individual cells based on their transcriptome proteins (figure 3A). The NEFL nonsense variant had pre- profiles resulted in 5 clusters. Neuronal cells clustered dis- dicted a potential C-terminally truncated protein of 366 tinctly from other cells, driven by the expression of neuron- amino acids (approximately 45 kDa). However, the Western specific transcripts (figure 4A). Clustering revealed that 26.1% blots of patient neuron lysates showed no full-length NEFL of the captured cells from P1 clone 1 (349 of 1,336 cells) and polypeptide (68 kDa) or signs of a truncated NEFL protein 23.0% from control 1 (96 of 418 cells) had a neural identity, using either an N-terminal monoclonal (recognizing residues depicted in t-SNE projections colored by the expression of 6–25) or a polyclonal pan-NEFL antibody (figure 3A), in- MAP2, microtubule-associated protein tau (MAPT), growth- dicating that patient neurons were absent of NEFL. Using associated protein 43 (GAP43), and synaptophysin (SYP) immunocytochemistry, highly similar neurite structures and (figure 4B). The captured neurons also expressed motor neuronal networks were seen in patient and control motor neuronal markers as depicted in figure 4C in which CHAT, neurons by NEFM immunostaining (figure 3B). The patient SLC18A3, ISL1, MNX1, LHX1, LHX3, DCC, ONECUT1, and

Figure 3 Complete loss of neurofilament light (NEFL) protein in cultured patient neurons

(A) Immunoblotting of whole cell lysates of patient 1 clones 1 and 2 (Pt C1 and C2) and controls 1-3 (ctr 1-3) after motor neuronal differentiation with anN- terminal monoclonal or a polyclonal pan-NEFL antibody. Protein levels of neuronal markers ChAT, TUJ1, and neurofilament medium (NEFM) and the loading control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as well as the stain-free blot are shown. (B) Immunocytochemical analysis of NEFM (green) and NEFL (orange) of neurite architecture in patient 1 and control neurons after motor neural differentiation. 49,6-diamidino-2-phenylindole (DAPI) indicates nuclear staining. Scale bars 50 μm.

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 5 Figure 4 Transcriptome dynamics of patient and control neurons

(A) Clustering of the single cells derived from patient 1 and control iPSCs in the tSNE-plot based on their gene expression fingerprints. Different clusters are color coded. In the combined single-cell sequencing data, patient cells are shown as filled dots and control cells as diamonds. Neurons are clustered as a clearly separate group of cells (cluster 1, red). (B) MAP2, MAPT, SYP, and GAP43 expression is high in the neuronal cluster cells. Red indicates high expression, and gray indicates low. (C) Cells in the neural cluster express motor neuron lineage-specific transcripts, CHAT, SLC18A3, ISL1, MNX1, LHX1, LHX3, DCC, ONECUT1,andONECUT2, summed in the tSNE-plot. Purple indicates high expression, and gray indicates low. (D) The most significantly upregulated and downregulated transcripts (adjusted p <0.001 and absolute fold change ≥1.5) in the neural cluster between patient and control cells. Neurofilament light (NEFL) is the most downregulated transcript in the patient neurons. (E) In the violin plots, each individual cell is shown with its specific transcript level, depicting the most downregulated transcript NEFL in patient neurons, and evenly expressed intermediate filament subunit transcripts INA, NEFM, NEFH, PRPH,andVIM. Expression refers to normalized log(e) expression scale. (F) Expression of NEFL against GAPDH by qPCR from total culture RNA of patient clones 1 (Pt C1) and 2 (Pt C2) and controls 1-3 (Ctr 1-3) after motor neuronal differentiation, nontreated (NT) or treated with 200 μg/mL cycloheximide (CHX) for 18 hours. The comparisons were made individually between each cell line with and without CHX treatment, n = 3 for each cell line and treatment (unpaired 2-tailed t test, **p <0.001,*p < 0.01). Bars represent mean levels ± SD.

6 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG ONECUT2 are summed, indicating that these neurons are 2(UTS2), a small cyclic peptide shown previously to regulate part of the motor neuronal lineage. The neuronal differ- intracellular calcium in rat spinal cord neurons,24 was the most entiation efficiency in cultures was much greater than the upregulated gene in patient vs control neurons, but its ex- percentage of captured neurons, which reflects the difficulty pression profile showed high variation within the individual to capture neuronal cells with long extensions in contrast to patient neurons (figure e-2, links.lww.com/NXG/A52). morphologically more favorable cell types. However, large Therefore, its specific role in association with NEFL loss is not numbers of neurons were successfully captured from pa- clear. tient and control cultures, and the proportion of neuronal cells of all captured cells was comparable in patient and Neurite architecture control data. Because NEFL is believed to be a fundamental building block in the intermediate filament network of axons and dendrites,1 we To gain an overall appreciation of the abundancy of the cap- examined neurites in our cultures. By immunocytochemistry, tured mRNAs per gene in the cultured neurons and of the we did not detect defects in neurite morphology in the patient’s ratios of the different intermediate filament transcripts, we cultured neurons in comparison with control neurites. The analyzed the mean unique molecular identifier (UMI) counts neurite signals for TUJ1, MAP2, and NEFM were not reduced for each gene in the control neuronal cell cluster. The most in patient cell lines (figure e-3A, links.lww.com/NXG/A53). highly expressed gene was MALAT1 (metastasis associated We also performed electron microscopic analysis to further lung adenocarcinoma transcript 1), followed by a number of examine neurite structure. Neurite areas varied in cross sections, 2 genes for cytoskeletal proteins such as tubulin and actin, ri- from 7,000 to 170,000 nm , but did not significantly differ bosome subunits, and mitochondrial-DNA encoded oxidative between control and patient samples (figure e-3B). Un- phosphorylation complex subunits (figure e-1, links.lww.com/ expectedly, cross sections of patient neurites showed in addition NXG/A51). NEFM was the most highly captured intermediate to microtubules a clear presence of intermediate filaments filament transcript (46th in abundance), closely followed by (figure 5). We counted the percentages of neurite cross sections NEFL (66th), whereas INA (238), VIM (2,728), PRPH that contained microtubules or filament bundles and observed (2,991), and NEFH (3,456) were much less frequent (figure similar numbers in control and patient samples (figure e-3C). e-1). The UMI counts per gene thus indicated that both NEFL Furthermore, the longitudinal sections of patient neurites dis- and NEFM were very highly expressed transcripts in control played no signs of neurofilament accumulation or abnormalities cultured neurons. indicating dysregulation in the microtubule network or axonal transport. Collectively, these results showed that cultured hu- At a single-cell level, when comparing the autosomal tran- man neurons can form neurofilaments and maintain axonal script levels in the patient neurons with control neurons structure in the absence of NEFL. (adjusted p < 0.001 and absolute fold change ≥1.5), NEFL was the most significantly downregulated transcript (10-fold) in Discussion the patient neurons (figure 4D). Violin plots in figure 4E demonstrate the reduced level of NEFL transcripts in in- We describe here CMT1F patients with a novel homozygous dividual neurons of patient identity in comparison to control nonsense mutation in NEFL, and demonstrate that the mu- identity, whereas other intermediate filaments were not sig- tation leads to the absence of NEFL in patient-derived cul- nificantly altered, indicating no transcriptional compensation. tured neurons. Both patients had the disease onset at infancy Although we found no evidence of stable NEFL protein in and presented with severely reduced NCVs and slowly pro- cultured patient neurons, the violin plot in figure 4E shows gressive distal muscle weakness in lower and upper extremi- that some neurons still expressed a low level of NEFL mRNA. ties. The low NCVs suggested that myelin was lost in the To study NEFL transcript dynamics, we blocked NMD by peripheral neurons, but nerve biopsies were not available from treating the neuronal cultures with cycloheximide (CHX), an the patients to investigate whether the reduced NCVs were inhibitor of protein synthesis. CHX significantly increased the due to the dramatic loss of axonal caliber in the absence of amount of nonsense NEFL mRNA in patient neuronal cul- NEFL or the loss of myelin. Intermediate to severe reduction tures, but at the same time, CHX dramatically decreased the in NCVs has been previously reported in association with amount of wild-type NEFL mRNA in control neurons (figure certain NEFL mutations.4,11 In addition to peripheral nerve 4F). These results indicated that NMD machinery was re- involvement, both of our patients had mild intellectual dis- sponsible for the nonsense mRNA degradation but could not ability possibly also resulting from the NEFL defect, since completely abolish it because of its high abundancy. In ad- abnormalities in cognitive development have been previously dition, the NEFL mRNA levels appear to be tightly regulated reported in a few patients with dominant or recessive NEFL in relation to protein synthesis. mutations.13,25

In this study, we did not investigate in detail the potential Recessively inherited NEFL nonsense mutations typically other transcriptional alterations that were associated with cause an early-onset CMT.13,16,17 Homozygous p.Glu140* NEFL loss in patient’s cultured neurons, as these ﬁndings mutation was described in 1 patient with gait disturbance and require substantial additional studies. For example, urotensin progressive muscle weakness since school age,16 p.Glu210* in

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 7 Figure 5 Neurite structure is not disrupted by the lack of neurofilament light (NEFL)

Representative electron microscopy images of neurite architecture in patient 1 and control neurons. Intermediate filaments (outlined arrow) and microtubules (filled arrow) are indicated in cross sections. Normal neurofilament network is seen in longitudinal sections of patient neurites. Scale bars 500 nm.

4 siblings with slowly progressive distal muscle weakness and neuronal injury.30,31 In the cultured neurons of this study, we atrophy starting at approximately 1.5 years,13 and p.Glu163* found the intermediate filaments expressed in the following or- in an adolescent girl with muscle weakness and gait disturbances der of abundance: NEFM>NEFL>INA>VIM>NEFH>PRPH. during the first decade.17 Although neurofilament aggregation is In the patient neurons lacking NEFL, we found no indication of well documented for dominant NEFL mutations,12,13,26 as well transcriptional compensation of other neurofilament poly- as in other neurodegenerative disorders,2,3 the molecular con- peptides, although we could detect neurofilaments in the neurites sequences of recessive nonsense mutations in NEFL have not by electron microscopy. This suggests that the intermediate fil- been fully investigated. Neuronal specificity of NEFL has pre- ament formation in cultured neurons does not require NEFL. viously prevented studying the nonsense mutations in detail, and However, a recent study reported that a CMT patient with especially in cells with endogenous levels of mutant NEFL recessive NEFL nonsense mutations had no neurofilament in mRNA. Using neurons differentiated from patient-specificiPSC, axons in a nerve biopsy as detected by electron microscopy.17 we unexpectedly observed that the recessive NEFL nonsense Combined with our demonstration of NEFL nonsense muta- mutation led to a complete absence of NEFL protein, through tions leading to NEFL absence, their result indicates that in NMD of the nonsense mutant mRNA. human peripheral axons, the lack of NEFL protein indeed leads to neurofilament loss. It is possible that the transport of neuro- In this study, we demonstrate the loss of NEFL mRNA and filaments to the long distal sural nerve may be impaired in protein in human neurons. In the literature, NEFL is largely patients, and this cannot be reproduced by the current in vitro considered as an essential component of neurofilament in mature model. It is important that the attempts to remove NEFL as neurons together with NEFM and NEFH.15 The composition of a therapeutic intervention to its toxic accumulation5 should take neurofilaments is also dependent on the neuronal type and de- into account that its loss is equally harmful to peripheral neurons velopmental stage.15 Our single-neuron transcriptomics showed and caused a severe early-onset disease in our patients. It is also that NEFL and NEFM were highly abundant transcripts in the noteworthy that the full Nefl mouse knockout only displayed cultured neurons, whereas NEFH was not. Low NEFH transcript a phenotype following nerve injury,32 suggesting major differ- capture is consistent with its expression increasing only as a result ences in the neurofilament biology between humans and mice, of axonal maturation concomitant with myelination.27 INA and which may be connected to axon length. PRPH may also contribute to neurofilament formation, but are mostly expressed during early embryonic neuronal differentia- Previous study of iPSC-derived neurons from CMT indi- tion or in early postnatal brain, respectively,28,29 or following viduals carrying a NEFL missense variant found NEFL

8 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG aggregate retention in the perikarya of neurons, possibly European Research Council. Full disclosure form information disrupting the neurofilament network and axonal mainte- provided by the authors is available with the full text of this nance.33 Our results indicate that CMT can be caused by article at Neurology.org/NG. both the loss of NEFL and its toxic accumulation.12 We therefore speculate that in the cases of NEFL accumulation, Received January 17, 2018. Accepted in final form April 19, 2018. the toxicity is at least partly caused by the aggregates pre- References venting the proper localization and function of wild-type 1. Brown HG, Troncoso JC, Hoh JH. Neurofilament-L homopolymers are less NEFL, as well as disrupting the maintenance and turnover of mechanically stable than native neurofilaments. J Microsc 1998;191:229–237. fi 2. Hirano A, Nakano I, Kurland LT, Mulder DW, Holley PW, Saccomanno G. Fine intermediate laments in the axon. This could result in structural study of neurofibrillary changes in a family with amyotrophic lateral scle- NEFL loss in critical parts of the axons, similar to the situ- rosis. J Neuropathol Exp Neurol 1984;43:471–480. ationinpatientswithrecessiveNEFL nonsense mutations. 3. Israeli E, Dryanovski DI, Schumacker PT, et al. Intermediate filament aggregates cause mitochondrial dysmotility and increase energy demands in giant axonal neuropathy. Indeed, reduced neurofilament has been detected in cuta- Hum Mol Genet 2016;25:2143–2157. neous nerve fibers of patients with dominant CMT2E, 4. Jordanova A, De Jonghe P, Boerkoel CF, et al. Mutations in the neurofilament light chain gene (NEFL) cause early onset severe Charcot-Marie-Tooth disease. Brain suggesting that aggregates in cell bodies led to neurofilament 2003;126:590–597. disruption distally.34 5. Yadav P, Selvaraj BT, Bender FL, et al. Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling. Acta Neuropathol 2016; 132:93–110. Here, we demonstrated that the absence of NEFL in human 6. Meeter LH, Dopper EG, Jiskoot LC, et al. Neurofilament light chain: a biomarker for genetic frontotemporal dementia. Ann Clin Transl Neurol 2016;3:623–636. neurons causes early-onset CMT. As a limitation of our study, 7. Disanto G, Barro C, Benkert P, et al. Serum neurofilament light: a biomarker of skin fibroblasts of only 1 patient from the family were available neuronal damage in multiple sclerosis. Ann Neurol 2017;81:857–870. 8. Weydt P, Oeckl P, Huss A, et al. Neurofilament levels as biomarkers in asymptomatic for iPSC generation. The lack of an obvious defect in neu- and symptomatic familial amyotrophic lateral sclerosis. Ann Neurol 2016;79: rofilament formation in cultured patient-specific neurons 152–158. 9. Byrne LM, Rodrigues FB, Blennow K, et al. Neurofilament light protein in blood as challenges the use of the current model system in studies of a potential biomarker of neurodegeneration in huntington’s disease: a retrospective pathogenic mechanisms. In addition, we presented a case in cohort analysis. Lancet Neurol 2017;16:601–609. 10. Mersiyanova IV, Perepelov AV, Polyakov AV, et al. A new variant of Charcot-Marie- which single-neuron transcriptomics could be used to identify Tooth disease type 2 is probably the result of a mutation in the neurofilament-light the genetic defect based on the consequent gene expression gene. Am J Hum Genet 2000;67:37–46. 11. De Jonghe P, Mersivanova I, Nelis E, et al. Further evidence that neurofilament light alteration. chain gene mutations can cause Charcot-Marie-Tooth disease type 2E. Ann Neurol 2001;49:245–249. Author contributions 12. Sasaki T, Gotow T, Shiozaki M, et al. Aggregate formation and phosphorylation of neurofilament-L Pro22 Charcot-Marie-Tooth disease mutants. Hum Mol Genet All authors acquired and analyzed data and contributed to the 2006;15:943–952. writing of the manuscript. M.T. Sainio, E. Ylikallio, J. Lahtela, 13. Yum SW, Zhang J, Mo K, Li J, Scherer SS. A novel recessive nefl mutation causes a severe, early-onset axonal neuropathy. Ann Neurol 2009;66:759–770. P. Mattila, M. Auranen, and H. Tyynismaa designed the 14. Gentil BJ, Minotti S, Beange M, Baloh RH, Julien JP, Durham HD. Normal role of the experiments. L. Mäenpää performed bioinformatic analysis. low-molecular-weight neurofilament protein in mitochondrial dynamics and disruption in Charcot-Marie-Tooth disease. FASEB J 2012;26:1194–1203. J. Palmio performed clinical investigations. E. Ylikallio and 15. Gentil BJ, Tibshirani M, Durham HD. Neurofilament dynamics and involvement in H. Tyynismaa supervised the study. neurological disorders. Cell Tissue Res 2015;360:609–620. 16. Abe A, Numakura C, Saito K, et al. Neurofilament light chain polypeptide gene mutations in Charcot-Marie-Tooth disease: nonsense mutation probably causes Acknowledgment a recessive phenotype. J Hum Genet 2009;54:94–97. 17. Fu J, Yuan Y. A novel homozygous nonsense mutation in NEFL causes autosomal The authors thank Riitta Lehtinen for technical help. They recessive Charcot-Marie-Tooth disease. Neuromuscul Disord 2018;28:44–47. acknowledge the Electron Microscopy Unit of the Institute of 18. Shy ME, Patzko A. Axonal Charcot-Marie-Tooth disease. Curr Opin Neurol 2011;24: 475–483. Biotechnology, University of Helsinki, for providing labora- 19. Ylikallio E, Johari M, Konovalova S, et al. Targeted next-generation sequencing reveals tory facilities and electron microscopy-sample preparation, further genetic heterogeneity in axonal Charcot-Marie-Tooth neuropathy and a mutation in HSPB1. Eur J Hum Genet 2014;22:522–527. and the Biomedicum Stem Cell Center, University of 20. Lek M, Karczewski KJ, Minikel EV, et al. Analysis of protein-coding genetic variation Helsinki, for iPSC generation and technical help. in 60,706 humans. Nature 2016;536:285–291. 21. Du ZW, Chen H, Liu H, et al. Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells. Nat Commun 2015;6:6626. Study funding 22. Macosko EZ, Basu A, Satija R, et al. Highly parallel genome-wide expression profiling This work was supported by the Academy of Finland, Sigrid of individual cells using nanoliter droplets. Cell 2015;161:1202–1214. 23. Zheng GX, Terry JM, Belgrader P, et al. Massively parallel digital transcriptional Juselius Foundation, University of Helsinki, Helsinki Uni- profiling of single cells. Nat Commun 2017;8:14049. versity Hospital, Doctoral Programme in Biomedicine, and 24. Filipeanu CM, Brailoiu E, Le Dun S, Dun NJ. Urotensin-II regulates intracellular calcium in dissociated rat spinal cord neurons. J Neurochem 2002; Finska Läkaresällskapet. 83:879–884. 25. Horga A, Laura M, Jaunmuktane Z, et al. Genetic and clinical characteristics of NEFL- related Charcot-Marie-Tooth disease. J Neurol Neurosurg Psychiatry 2017;88: Disclosure 575–585. Markus T. Sainio reports no disclosures. Emil Ylikallio has 26. Leung CL, Nagan N, Graham TH, Liem RK. A novel duplication/insertion mutation of NEFL in a patient with Charcot-Marie-Tooth disease. Am J Med Genet A 2006; received research support from the Academy of Finland, 140:1021–1025. University of Helsinki, and Emil Aaltonen Foundation. Laura 27. Haynes RL, Borenstein NS, Desilva TM, et al. Axonal development in the cerebral white matter of the human fetus and infant. J Comp Neurol 2005;484: Mäenpää, Jenni Lahtela, Pirkko Mattila. Mari Auranen, and 156–167. Johanna Palmio report no disclosures. Henna Tyynismaa has 28. Escurat M, Djabali K, Gumpel M, Gros F, Portier MM. Differential expression of two fi neuronal intermediate-filament proteins, peripherin and the low-molecular-mass served on the editorial board of Scienti c Reports and has neurofilament protein (NF-L), during the development of the rat. J Neurosci 1990; received research support from the Academy of Finland and 10:764–784.

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 9 29. Kaplan MP, Chin SS, Fliegner KH, Liem RK. Alpha-internexin, a novel neuronal 32. Zhu Q, Couillard-Despres S, Julien JP. Delayed maturation of regenerating intermediate filament protein, precedes the low molecular weight neurofilament myelinated axons in mice lacking neurofilaments. Exp Neurol 1997;148: protein (NF-L) in the developing rat brain. J Neurosci 1990;10:2735–2748. 299–316. 30. Beaulieu JM, Kriz J, Julien JP. Induction of peripherin expression in subsets of brain 33. Saporta MA, Dang V, Volfson D, et al. Axonal Charcot-Marie-Tooth disease patient- neurons after lesion injury or cerebral ischemia. Brain Res 2002;946:153–161. derived motor neurons demonstrate disease-specific phenotypes including abnormal 31. Troy CM, Muma NA, Greene LA, Price DL, Shelanski ML. Regulation of peripherin electrophysiological properties. Exp Neurol 2015;263:190–199. and neurofilament expression in regenerating rat motor neurons. Brain Res 1990;529: 34. Pisciotta C, Bai Y, Brennan KM, et al. Reduced neurofilament expression in cutaneous 232–238. nerve fibers of patients with CMT2E. Neurology 2015;85:228–234.

10 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG CLINICAL/SCIENTIFIC NOTES OPEN ACCESS Expanding the global prevalence of spinocerebellar ataxia type 42

Kathie Ngo, BS, Mamdouh Aker, Lauren E. Petty, MS, Jason Chen, PhD, Francesca Cavalcanti, MD, Correspondence Alexandra B. Nelson, MD, Sharon Hassin-Baer, MD, Michael D. Geschwind, MD, PhD, Susan Perlman, MD, Dr. Fogel [email protected] Domenico Italiano, MD, PhD, Angelina Lagan`a, MD, Sebastiano Cavallaro, MD, PhD, Giovanni Coppola, MD, Jennifer E. Below, PhD, and Brent L. Fogel, MD, PhD

Neurol Genet 2018;4:e232. doi:10.1212/NXG.0000000000000232

Spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders that involve the degeneration of the cerebellum and brainstem.1 These genetic diseases are characterized by autosomal dominant inheritance with approximately 44 known subtypes. Recently, dominant mutations in the CACNA1G gene, encoding the voltage-gated calcium channel 2 3,4 CaV3.1, have been linked to SCA42 in French and Japanese families. SCA42 prevalence elsewhere in the world has yet to be documented. Through a combination of whole-exome sequencing (WES) and linkage analysis, we have identiﬁed an SCA42 mutation in patients from 3 additional countries, expanding the worldwide prevalence of this disease.

Methods All study methods were approved by the Institutional Review Board of UCLA. All patients received a comprehensive clinical evaluation for acquired causes of ataxia.5 All patients provided written informed consent for collection of DNA. Only patients who tested negative for common genetic ataxias (SCA1, SCA2, SCA3, SCA6, SCA7, and Friedreich ataxia) were enrolled.5 WES was performed for 8 members of 3 families (ﬁgure, A and B). Genomic DNA (gDNA) libraries were prepared using the Nextera Rapid Capture Exome kit (Illumina, San Diego, CA). Sequencing for these libraries was performed with 107-bp paired-end reads on a HiSeq 2500 sequencer in the rapid- run mode platform (Illumina, San Diego, CA). The gDNA library for B-III-3 was prepared using the SureSelect Human All Exon V4 Capture kit (Agilent Technologies, Santa Clara, CA), and 101-bp paired-end reads were sequenced on the Illumina HiSeq 4000 platform (Illumina, San Diego, CA). Sequencing data were processed as described.5 Linkage analysis was conducted with ALLEGRO on 190,569 single nucleotide polymorphisms (SNPs) (average distance between SNPs ≈ 0.015 Mb) under a dominant model (f0/f1/f2 = 0/1/1) with estimated minor allele frequencies drawn from HapMap3 project6 Centre d'Etude du Polymorphism Humain - Utah population data, comprised Utah residents with Northern and Western European ancestry.7 Self-reported ancestry was con- ﬁrmed via principal component analysis (PCA) of WES variants with a minor allele frequency of >5% and 1000 Genomes Project Phase 38 reference data. Segregation analysis for family A was performed by PCR, followed by Sanger sequencing.

Results Family A was clinically evaluated in Italy, their country of origin, and PCA conﬁrmed European ancestry. Aﬀected members exhibited pure cerebellar ataxia with onset between ages 22 and 58

From the Department of Neurology (K.J.N., M.A., S.P., G.C., B.L.F.), Program in Neurogenetics, David Geffen School of Medicine, University of California, Los Angeles; Department of Medical Genetics (L.E.P., J.E.B.), Vanderbilt University Medical Center, Nashville, TN; Department of Psychiatry and Biobehavioral Sciences (J.A.C., G.C.), University of California, Los Angeles, CA; Institute of Neurological Sciences (F.C., S.C.), Italian National Research Council, Mangone, Italy; Department of Neurology (A.B.N., M.D.G.), UCSF Memory and Aging Center, University of California, San Francisco; Sackler Faculty of Medicine (S.H.), Tel-Aviv University, Israel; Italian College of General Practitioners and Primary Care (D.I.), Department of Clinical and Experimental Medicine (A.L.), University of Messina, Italy; and the Department of Human Genetics (B.L.F.), David Geffen School of Medicine, University of California, Los Angeles.

(A) Pedigree of family A. (B) Pedigree of family B. Probands are indicated by an arrow. Shaded symbols represent affected individuals. Individuals who had WES are indicated by an asterisk. Genotypes of the c.5144 position in CACNA1G are shown under each patient sequenced. G is the reference, and A is the p. R1715H mutation. Individuals who were unavailable for genotyping are represented in gray with dashed lines. Shaded gray individuals were reportedas affected. SCA = spinocerebellar ataxia.

years (mean 37.5 years). All affected members initially expe- with a sense of imbalance and intermittent jerking movements rienced a subjective sense of leg weakness and imbalance that of his neck that slowly progressed to a tremor with cervical slowly progressed to gait and limb ataxia with dysarthria. dystonia manifesting as a rightward tilt. His imbalance pro- There was no nystagmus, abnormal saccadic pursuit, motor gressed to a mild gait and lower limb ataxia. The remainder of deficits, pyramidal or extrapyramidal signs, abnormal sensa- his neurologic examination was normal. MRI of the brain tion, or cognitive decline noted in any patients. MRI of the showed mild cerebellar atrophy, primarily of the vermis. His brain demonstrated cerebellar atrophy in all patients. WES parents and 6 younger siblings were all reported as asymp- identified a known pathogenic variant in the CACNA1G gene tomatic; however, none were available for examination or in all 3 affected family members (A-II-8, A-III-10, and A-III-7) genetic testing. Although the 1000 Genomes Project does not tested. All other dominant SCAs were excluded by linkage include Yemeni samples, this sample clustered with admixed analysis and/or by the absence of rare missense variation.5 American samples, indicating primarily European ancestry The CACNA1G variant (hg19:chr17:48694921G>A, with small amounts of Asian and African admixture, consis- p.Arg1715His) was previously observed in patients from France tent with expectations for a Middle Eastern population. – and Japan.2 4 This variant was not present in the ExAC (exac. broadinstitute.org) or gnomAD (gnomad.broadinstitute.org) We next questioned whether CACNA1G variants were com- databases of human variation. The variant segregated with dis- mon in neurologic disease in the US population, so we ease and was located within a linkage peak, consistent with reviewed a recent analysis of 3,040 clinical WES cases,9 com- being pathogenic. prising 1,082 patients with involvement of the CNS, including cerebellar ataxia, and noted that it did not identify any patients In a second family with pure cerebellar ataxia, family B of with the p.Arg1715His variant we detected and found only 1 Eastern European ancestry living in the United States, the patient with a novel variant in CACNA1G deemed pathogenic, proband (B-III-1) developed, at age 67 years, a sense of suggesting that the mutation of this gene may be rare in the US imbalance that slowly progressed to a gait and limb ataxia with population. Next, to assess the prevalence of this disease within dysarthria. His first cousin once removed (B-IV-1) was also a specific US ataxia population, we reviewed WES data from affected, with an age at onset of 39 years, initially also involving 225 sporadic or familial cases seen at our tertiary referral center his balance and speech and slowly progressing to a gait and limb at UCLA. Aside from family B, we identified no additional ataxia with dysarthria. MRI of the brain showed cerebellar at- cases. Therefore, we estimate the frequency in our ataxia rophy. PCA verified European ancestry. The above CACNA1G cohort to be 1 of 225 (0.4%) undiagnosed families. variant was identified in both patients by WES. The proband’s sister (B-III-3) also carried the variant but reported herself as asymptomatic and was unavailable for neurologic examination. Discussion Lastly, WES identified the same variant in a man from Yemen, In this report, we describe 3 additional SCA42 families orig- clinically evaluated in Israel, who, at age 25 years, presented inating from Italy, the United States (via Europe), and Yemen,

2 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG respectively, expanding the worldwide prevalence of the dis- Global, MEDACorp, LCN Consulting, Optio Biopharma order. In addition, we report a case with the new finding of Solutions, various medical-legal consulting, Biohaven Phar- cervical dystonia, along with tremor, previously reported in maceuticals Inc, Teva Pharmaceuticals, and Quest Diag- another family.3 Although previously reported in French and nostics; and has received research support from the NIH, the – Japanese families,2 4 none of the families in this report had Michael J. Homer Family Fund, CurePSP, and the Tau clear evidence of French or Japanese ancestry to the best of Consortium. S. Perlman has received research support from our ability to ascertain. Although we find the disorder to be EryDel, Reata, Viropharma/Shire, Retrotope, Edison, Teva, rare in our center’s US ataxia population (0.4%, N = 225), we Pfizer, Biohaven, and the National Ataxia Foundation. D. recommend that clinicians broadly consider the possibility of Italiano and A. Lagana report no disclosures. S. Cavallaro has SCA42 in undiagnosed patients with autosomal dominant served on the editorial boards of the ARC Journal of Neuro- ataxia, without regard to country of origin. science, Current Medicinal Chemistry, Genes and Genome Jour- nal, ISRN Genomics, Jacobs Journal of Biomarkers, Journal of Biological Informatics & Biodiversity, Journal of Genomes and Author contributions Proteomes, Letters in Drug Design & Discovery, Medicinal F.C., G.C., J.E.B., and B.L.F. contributed to the conception Chemistry, The Open Biochemistry Journal, and Source Journal and design of the research project and K.J.N., M.A., L.E.P., of Genomics. G. Coppola receives publishing royalties from and J.A.C. were responsible for its execution. A.B.N., M.D.G., Oxford University Press and has received research support S.H.-B., S.P., D.I., A.L., S.C., and B.L.F. supervised collection from Takeda Pharmaceutical Company, the NIH, the Adel- of the clinical data and samples and/or managed patient care. son Medical Research Foundation, the Tau Consortium, the K.J.N., L.E.P., J.E.B., and B.L.F. conducted all bioinformatics CHDI, Takeda Pharmaceutical Company, the John Douglas analysis. M.A., K.J.N., and B.L.F. wrote the manuscript, and all French Alzheimer’s Foundation, and the FARA. J.E. Below authors were responsible for its review and critique. has received research support from the Sanofi Innovation Awards Program, the Tulane National Primate Research Study funding Center, the NIH/Wayne State University Genetic Study of This work was supported in part by the National Institute for Stuttering, the NIH/Johns Hopkins University Baylor-Johns Neurological Disorders and Stroke (Grant R01NS082094 to Hopkins Center for Mendelian Genetics, and the NIH/Broad Dr. Fogel) and the National Ataxia Foundation (Young In- Institute of MIT. B.L. Fogel has received travel funding/ vestigator Award to Dr. Fogel). The research described was speaker honoraria from the American Academy of Neurology, supported by the NIH/National Center for Advancing Trans- the American Physician Institute for Advanced Professional lational Science UCLA Clinical and Translational Science In- Studies, and the National Ataxia Foundation; has served on stitute grant UL1TR000124. Dr. Fogel acknowledges the the editorial boards of Neurology: Genetics and Neurology support through donations to the University of California by the Today; and has received research support from the NIH/ Rochester Ataxia Foundation. MDG was funded by the Michael J. NINDS and the National Ataxia Foundation. Full disclosure Homer Family Fund. ABN is supported by the Richard and form information provided by the authors is available with the Shirley Cahill Endowed Chair in Parkinson’s Disease Research. full text of this article at Neurology.org/NG. The authors acknowledge the support of the NINDS Informatics Center for Neurogenetics and Neurogenomics (P30 NS062691). Received November 27, 2017. Accepted in final form February 21, 2018.

Disclosure References K.J. Ngo, M. Aker, and L.E. Petty report no disclosures. J.A. Chen 1. Shakkottai VG, Fogel BL. Clinical neurogenetics: autosomal dominant spinocer- is a cofounder/advisor of Verge Genomics; has received re- ebellar ataxia. Neurol Clin 2013;31:987–1007. 2. Coutelier M, Blesneac I, Monteil A, et al. A recurrent mutation in CACNA1G alters search support from the NIH/NINDS; and holds stock/stock cav3.1 T-type calcium-channel conduction and causes autosomal-dominant cerebellar options in Verge Genomics. F. Cavalcanti reports no disclosures. ataxia. Am J Hum Genet 2015;97:726–737. 3. Morino H, Matsuda Y, Muguruma K, et al. A mutation in the low voltage-gated A.B. Nelson has received research support from the NIH/ calcium channel CACNA1G alters the physiological properties of the channel, causing NINDS, the Parkinson Disease Foundation, the Dystonia spinocerebellar ataxia. Mol Brain 2015;8:89. 4. Kimura M, Yabe I, Hama Y, et al. SCA42 mutation analysis in a case series of Japanese Medical Research Foundation, the Brain Research Foun- patients with spinocerebellar ataxia. J Hum Genet 2017;62:857–859. dation, and the Richard and Shirley Cahill Foundation. 5. Fogel BL, Lee H, Deignan JL, et al. Exome sequencing in the clinical diagnosis of sporadic or familial cerebellar ataxia. JAMA Neurol 2014;71:1237–1246. S. Hassin-Baer has received consultancy fees from AbbVie 6. International HapMap C; Altshuler DM, Gibbs RA, et al. Integrating common and and Medtronic. M.D. Geschwind has received speaker hon- rare genetic variation in diverse human populations. Nature 2010;467:52–58. 7. Gudbjartsson DF, Jonasson K, Frigge ML, Kong A. Allegro, a new computer program oraria from Oakstone Publishing, Inc; has served on the for multipoint linkage analysis. Nat Genet 2000;25:12–13. editorial board of Dementia & Neuropsychologia; has been 8. Genomes Project C, Auton A, Brooks LD, et al. A global reference for human genetic variation. Nature 2015;526:68–74. a consultant of Advanced Medical Inc, Best Doctors Inc, 9. Retterer K, Juusola J, Cho MT, et al. Clinical application of whole-exome sequencing Grand Rounds, Gerson Lehrman Group Inc, Guidepoint across clinical indications. Genet Med 2016;18:696–704.

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 3 CLINICAL/SCIENTIFIC NOTES OPEN ACCESS Brain copper storage after genetic long-term correction in a mouse model of Wilson disease

Ricarda Uerlings, BSc, Daniel Moreno, PhD, Oihana Murillo, PhD, Cristina Gazquez, PhD, Rubén Hernández- Correspondence ´ Alcoceba, MD, PhD, Gloria González-Aseguinolaza, PhD, and Ralf Weiskirchen, PhD Dr. Gonzalez-Aseguinolaza [email protected] or Neurol Genet 2018;4:e243. doi:10.1212/NXG.0000000000000243 Dr. Weiskirchen [email protected]

Wilson disease is a rare autosomal recessive condition caused by mutations in the copper- transporting ATPase ATP7B gene (OMIM: 606882) provoking loss of function and resulting in variable hepatic and neurologic symptoms. Currently, the treatment of Wilson disease focuses on achieving a negative copper balance either with chelators (e.g., D-penicillamine, trientine, and tetrathiomolybdate) or zinc, which reduces copper absorption, or a combination thereof.1 However, these lifelong treatment regimens often cause side eﬀects and do not restore normal copper metabolism.

Recently, the construction and characterization of an AAV8 vector system in which the human ATP7B cDNA is placed under the control of the liver-specific human α1-antitrypsin (AAT) promoter has been described.2 This targeted therapy showed clear and robust long-term benefit − − in the Atp7b / mouse model, representing a well-established model for therapeutic inter- ventions such as drug, gene, and cell therapy.3 In particular, the AAV8 vector induced a dose- dependent therapeutic effect as assessed by reduction in serum transaminases and urinary copper excretion, normalization of serum holo-ceruloplasmin, and restoration of physiologic biliary copper excretion in response to copper overload without any side effects.2 Although the findings of this study are overall promising, the therapeutic effects from reducing cerebral copper have not been tested yet.

− − We have previously analyzed in detail the age-dependent accumulation of copper in the Atp7b / mouse model in which a portion of the Atp7b exon 2 is replaced by a disruption cassette incorporating an early termination codon and a frameshift mutation giving rise to a shortened mRNA, which is not capable to produce detectable levels of ATP7B protein.4 This former analysis in respective mice revealed about a twofold stable increase in copper throughout the brain parenchyma, whereas in periventricular regions, copper was decreased by a factor of up to 3.5, especially in the fourth ventricle where lumen was systematically discernable in null but not in wild-type animals.5 It is known that these impairments of homeostatic mechanisms in brain copper metabolism are connected with distinct cognitive alterations, neurodegeneration, and morphologic changes of normal astrocyte architecture, which are the consequences of varying regional susceptibility to copper within the brain.5 Therefore, for the successful development of future adeno-associated virus (AAV)-based gene therapy as a novel option in the management of human Wilson disease, it will be of fundamental importance to investigate the eﬀect of the transgene on cerebral copper concentration and distribution.

To test whether the AAV8-based therapy allows for the correction of cerebral copper overload in the Atp7b null mice, we extended the previous study and performed laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) on brain tissue sections of male homozygous

From the Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (R.U., R.W.), RWTH University Hospital Aachen, Aachen, Germany; Gene Therapy and Regulation of Gene Expression Program (D.M., O.M., C.G., R.H.-A., G.G.-A.), CIMA, Foundation for Applied Medical Research, University of Navarra; Instituto de Investigacion Sanitaria de Navarra (IDISNA) (D.M., O.M., C.G., R.H-.A., G.G.-A.); and Vivet-Therapeutics (G.G.-A.), Pamplona, Spain.

The Article Processing Charge was funded by the authors.

Comment on animal manipulation: Animal experimentation was approved by the Ethical Committee for Animal Testing of the University of Navarra.

Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 − − Atp7b null mice. To do so, 6-week-old Atp7b / mice (n = 6) capability and allows for the analysis of a large variety of received an injection of AAV8 expressing a codon optimized biological materials with high spatial resolution.6 To visualize (co)versionofanATP7B mini gene (miATP7B) engineered the concentrations of individual elements in the analyzed to allow optimize expression in mouse. The construct was sections, we transmitted the measurements obtained in our directed under transcriptional control of the human AAT LA-ICP-MS line by line scans into an Excel spreadsheet and promoter (AAV-AAT-co-miATP7B) at a dose of 1 × 1011 vg/ generated parametric images using open source ELAI soft- − − mouse, whereas another group of Atp7b / mice (n = 5) of the ware recently developed by us.7 At the end, the results are same age was left untreated. Moreover, we have incorporated processed into 2-dimensional images, which can be trans- − a group of age-matched Atp7b+/ littermates (n = 6) taken as formed into common file formats such as TIFF or JPG while a healthy control group because these heterozygous animals retaining exact proportion of their X/Y dimensions. show no alterations of copper metabolism and expression of the ATP7B protein compared with wild-type controls.3 In our analysis, we found that the brains of animals receiving the transgene had overall lower concentrations of total cere- Animals were sacrificed 14 weeks later, brains were harvested, bral copper (figure 1), most prominently noticeable in the and 30-μm thick tissue cryosections were prepared for LA- cerebellum, cerebellar white matter, corpus callosum, 3rd and ICP-MS metal imaging. This technique has multielement 4th ventricles, and surrounding tissue, and a slight decrease in

Figure 1 Metal bioimaging in 30-μm thick cryosections taken from the brains of untreated and AAV8-AAT-co-miATP7B-treated Atp7b null mouse

The contents of copper (Cu), iron (Fe), zinc (Zn), manganese (Mn), sodium (Na), magnesium (Mg), potassium (K), calcium (Ca), phosphorus (P), sulfur (S), carbon (C), chromium (Cr), nickel (Ni), and lead (Pb) in each section are shown. Individual images of elements were done with the ELAI software tool.7 Light − microscopic (LM) images of cryosections and pictures of brains analyzed are shown for orientation in the left margin. In this analysis, Atp7b+/ mice served as a further control. Please note that the content of C serving as reference is given in %, whereas concentrations of all other elements are given in μg/g liver tissue. Details about animal manipulation are given elsewhere.2

2 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG the basal ganglia. While the mean cerebral copper content was no neurologic alterations have been reported. Only the 3.80 ± 0.2 μg/g brain tissue in the untreated group, the de- progeny of homozygous mutant females demonstrate neuro- livery of the transgene reduced the copper content to a mean logic symptoms at young age.3 However, when these neurologic − concentration of 3.05 ± 0.17 μg/g. The content in the Atp7b+/ symptoms are apparent, respective animals do not survive be- control mice that show no alterations in copper metabolisms yond 2 weeks of age.9 Thereafter, alterations in behavior pro- was determined to be 2.34 ± 0.09 μg/g. The coefficient of voked by cerebral copper overload are negligible. As such, the variation in these measurements was calculated to be 5.1% in lack of severe neurologic alterations is a limitation of the current the nontreated group, 5.7% in the treated group, and 4.0% in model when comparing with human Wilson disease. the control group. The concentrations of other metals including iron, zinc, manganese, sodium, magnesium, potas- We hope that our note will further encourage clinical studies sium, calcium, phosphorus, chromium, nickel, and lead were aiming to use AAV therapeutic gene transfer in patients with unaffected. These findings reveal that the delivery of AAV8- Wilson disease. The ability to elicit robust and long-term AAT-co-miATP7B is capable of reducing the overall cerebral ATP7B gene expression in vivo with AAV vectors might be- copper content without affecting other metals. Moreover, the come an attractive therapy to complement the mainstay single IV administration with AAV8-AAT-co-miATP7B pro- therapy for Wilson disease of relying on chelating agents and voked reduced urinary copper excretion, increased cerulo- medications that block excess copper absorption. plasmin activity in blood, and reduced liver damage as indicated by lower activities in alanine transaminase (figure 2, Author contributions A-C). This therapeutic effect on liver metal content was also R. Uerlings: performance and evaluation of LA-ICP-MS recently demonstrated by metal bioimaging showing the measurements. D. Moreno, C. Gazquez, and O. Murillo: ani- overall reduction of hepatic copper throughout the tissue.8 mal experimentation. R. Hernández-Alcoceba, G. González- Therefore, it is most likely that the reduction of cerebral Aseguinolaza, and R. Weiskirchen: design of the study and copper in our liver-directed therapy is secondary and induced writing of the manuscript. by hepatic ATP7B expression lowering systemic copper concentration. All these changes demonstrate that AAV- Acknowledgment treated animals recovered from copper overload. The authors thank Astrid Küppers (Zentralinstitut für Engi- neering, Elektronik und Analytik, Forschungszentrum Jülich, Under therapy, we noticed no alterations in neurologic Jülich, Germany) for excellent technical assistance in LA-ICP- functions. However, this is not surprising because the most MS measurements. They acknowledge Drs Nick Weber and striking phenotype of this experimental Wilson disease model Bérnard Benichou for critical comments and careful review and is the formation of gross anatomical liver abnormalities, and Dr. Andreas Matusch (Division of Molecular Neuroimaging,

Figure 2 Analysis of copper in urine and ceruloplasmin and ALT in control, untreated, and AAV8-AAT-co-miATP7B-treated Atp7b null mouse

(A) Copper in 24 hour urine was analyzed 4, 9, and 14 weeks after the administration of the vector. (B) Ceruloplasmin levels in serum were determined 4 weeks after vector administration. (C) Alanine transaminase (ALT) levels indicating liver damage were determined 14 weeks after the administration of the vector. p values for significance are **p < 0.01.

Neurology.org/NG Neurology: Genetics | Volume 4, Number 3 | June 2018 3 Institute of Neuroscience and Medicine-2, Forschungszentrum Clinical Microbiology and Antimicrobials, EC Gastroenterology and Jülich, Germany) for help in preparing murine brains. Digestive System, Journal of Pharmacology and Pharmaceutical Research, Stem Cells and Regenerative Medicine, American Journal Study funding of Family Medicine, American Journal of Liver & Clinical Research, R. Weiskirchen is supported by grants from the Deutsche Journal of Clinical Pharmacology and Research, Annals of Proteo- Forschungsgemeinschaft (SFB/TRR 57 projects P13 and mics and Bioinformatics, and International Journal of Experimental Q3) and from the IZKF Aachen (Project O3-1). Biology and applied for a patent regarding pharmaceutical composition targeting the hepatic content of amyloid beta (Aβ)to Disclosure treat liver fibrosis/cirrhosis. Full disclosure form information R. Uerlings, D. Moreno, O. Murillo, and C. Gazquez report no provided by the authors is available with the full text of this disclosures. R. Hernández-Alcoceba has served on the editorial article at Neurology.org/NG. board of Human Gene Therapy; has participated in a project fi- nanced by Vivet Therapeutics; and has received research support Received December 7, 2017. Accepted in final form April 11, 2018. from the Foundation for Applied Medical Research (FIMA). G. González-Aseguinolaza is affiliated with Vivet Therapeutics, References a company focused on optimizing gene therapy approaches; 1. Patil M, Sheth KA, Krishnamurthy AC, Devarbhavi H. A review and current per- spective on Wilson disease. J Clin Exp Hepatol 2013;3:321–336. holds a patent regarding AAV vector expressing ATP7B for the 2. Murillo O, Luqui DM, Gazquez C, et al. Long-term metabolic correction of Wilson’s treatment of Wilson’s disease; and has received research support disease in a murine model by gene therapy. J Hepatol 2016;64:419–426. 3. Buiakova OI, Xu J, Lutsenko S, et al. Null mutation of the murine ATP7B (Wilson from the Spanish Ministry of Economy and Competitiveness disease) gene results in intracellular copper accumulation and late-onset hepatic and FundacióPerAmoral’Art. Ralf Weiskirchen has served on nodular transformation. Hum Mol Genet 1999;8:1665–1671. 4. Boaru SG, Merle U, Uerlings R, et al. Simultaneous monitoring of cerebral metal the editorial boards of Faculty 1000 in Medicine, Journal of accumulation in an experimental model of Wilson’s disease by laser ablation in- Cellular Biochemistry, Hepatitis Monthly, Frontiers in Gastrointes- ductively coupled plasma mass spectrometry. BMC Neurosci 2014;15:98. fi 5. Scheiber IF, Mercer JF, Dringen R. Metabolism and functions of copper in brain. Prog tinal Sciences (Frontiers in Physiology), The Scienti c World Journal Neurobiol 2014;116:33–57. (Section Hepatology), Laboratory Animals, Frontiers in Physiology, 6. Weiskirchen R, Uerlings R. Laser ablation inductively coupled plasma mass spectrometry in biomedicine and clinical diagnosis. Cell Mol Med 2015;19:806–814. Hepatobiliary Surgery and Nutrition, Frontiers in Pharmacology, 7. Uerlings R, Matusch A, Weiskirchen R. Reconstruction of laser ablation inductively Gastrointestinal Sciences Frontiers in Physiology, American Journal coupled plasma mass spectrometry (LA-ICP-MS) spatial distribution images in Microsoft Excel 2007. Int J Mass Spectrom 2016;395:27–35. of Biochemistry and Molecular Biology, World J Biological Chem- 8. Moreno D, Murillo O, Gazquez C, et al. Visualization of the therapeutic efficacy of istry, EC Orthopaedics, Scientific Journal of Biology, Journal of a gene correction approach in Wilson’s disease by laser-ablation inductively coupled mass spectrometry. J Hepatol 2018;68:199–201. Development Biology and Regenerative Medicine, SF Journal 9. Lutsenko S. Atp7b-/- mice as a model for studies of Wilson’s disease. Biochem Soc of Biotechnology and Biomedical Engineering, American Journal of Trans 2008;36:1233–1238.

4 Neurology: Genetics | Volume 4, Number 3 | June 2018 Neurology.org/NG CORRECTION Expanding the global prevalence of spinocerebellar ataxia type 42 Neurol Genet 2018;4:e238. doi:10.1212/NXG.0000000000000238

In the Clinical/Scientiﬁc Note “Expanding the global prevalence of spinocerebellar ataxia type 42” by Ngo et al.,1 the ﬁrst and fourth authors’ names are missing their middle initials, which should read Kathie J. Ngo and Jason A. Chen, respectively. The publisher regrets the omission. There is also a small typo in the Methods section, which should read, “…with estimated minor allele frequencies drawn from HapMap3 project Centre d’Etude du Polymorphisme Humain—Utah population data.” The authors regret the error.

Reference 1. Ngo K, Aker M, Petty LE, et al. Expanding the global prevalence of spinocerebellar ataxia type 42. Neurol Genet 2018;4:e232.