Table of Contents Neurology.Org/Ng Online ISSN: 2376-7839 Volume 3, Number 3, June 2017

Table of Contents Neurology.org/ng Online ISSN: 2376-7839 Volume 3, Number 3, June 2017

THE HELIX e151 HSP and deafness: Neurocristopathy caused by e157 Collaboration, workshops, and symposia a novel mosaic SOX10 mutation S.M. Pulst S. Donkervoort, D. Bharucha-Goebel, P. Yun, Y. Hu, P. Mohassel, A. Hoke, W.M. Zein, D. Ezzo, A.M. Atherton, A.C. Modrcin, M. Dasouki, A.R. Foley, and C.G. Bönnemann EDITORIAL e159 ARHGEF9 mutations cause a speciﬁc recognizable X-linked intellectual disability e155 Genetic analysis of age at onset variation in syndrome spinocerebellar ataxia type 2 P. Striano and F. Zara K.P. Figueroa, H. Coon, N. Santos, L. Velazquez, Companion article, e148 L.A. Mederos, and S.M. Pulst

ARTICLES e158 Previously unrecognized behavioral phenotype in e148 ARHGEF9 disease: Phenotype clariﬁcation and Gaucher disease type 3 genotype-phenotype correlation M. Abdelwahab, M. Potegal, E.G. Shapiro, and M.Alber,V.M.Kalscheuer,E.Marco,E.Sherr, I. Nestrasil G. Lesca, M. Till, G. Gradek, A. Wiesener, C. Korenke, S. Mercier, F. Becker, T. Yamamoto, S.W. Scherer, C.R. Marshall, S. Walker, U.R. Dutta, A.B. Dalal, e160 Intramyocellular lipid excess in the mitochondrial V. Suckow, P. Jamali, K. Kahrizi, H. Najmabadi, and disorder MELAS: MRS determination at 7T B.A. Minassian S. Golla, J. Ren, C.R. Malloy, and J.M. Pascual Editorial, e159 e149 Clinicopathologic and molecular spectrum of CLINICAL/SCIENTIFIC NOTES RNASEH1-related mitochondrial disease e147 Camptocormia and shufﬂing gait due to a novel E. Bugiardini, O.V. Poole, A. Manole, A.M. Pittman, MT-TV mutation: Diagnostic pitfalls A. Horga, I. Hargreaves, C.E. Woodward, J. Reimann, D. Lehmann, S.A. Hardy, G. Falkous, M.G. Sweeney, J.L. Holton, J.-W. Taanman, G.T. Plant, C.V.Y. Knowles, R.L. Jones, W.S. Kunz, R.W. Taylor, and J. Poulton, M. Zeviani, D. Ghezzi, J. Taylor, C. Smith, C. Kornblum C. Fratter, M.A. Kanikannan, A. Paramasivam, K. Thangaraj, A. Spinazzola, I.J. Holt, H. Houlden, M.G. Hanna, and R.D.S. Pitceathly e150 SCA8 should not be tested in isolation for ataxia R.H. Roda, A.B. Schindler, and C. Blackstone e152 Intragenic DOK7 deletion detected by whole- genome sequencing in congenital myasthenic e153 Compound heterozygous mutations in MASP1 in syndromes a deaf child with absent cochlear nerves Y. Azuma, A. Töpf, T. Evangelista, P.J. Lorenzoni, E. Kari, I. Schrauwen, L. Llaci, L.M. Fisher, J.L. Go, A. Roos, P. Viana, H. Inagaki, H. Kurahashi, and M. Naymik, J.A. Knowles, M.J. Huentelman, and H. Lochmüller R.A. Friedman Table of Contents continued e154 Biallelic TOR1A variants in an infant with severe e156 Febrile ataxia and myokymia broaden the SPG26 arthrogryposis hereditary spastic paraplegia phenotype S.C. Reichert, P. Gonzalez-Alegre, and R. Dad, S. Walker, S.W. Scherer, M.J. Hassan, G.H. Scharer M.D. Alghamdi, B.A. Minassian, and R.A. Alkhater

Podcast Video LOE classiﬁcation LOE recommendation

tinyurl.com/NeurologyNG twitter.com/GreenJournal

Cover image: Ultrastructural examination of skeletal muscle showing increased numbers of mitochondria, many of which are structurally abnormal. Stylized by Kaitlyn Aman Ramm, Neurology Editorial Assistant. See “Clinicopathologic and molecular spectrum of RNASEH1-related mitochondrial disease.” Neurology.org/ng Online ISSN: 2376-7839 Volume 3, Number 3, June 2017

fi VISION: Neurology® Genetics will be the premier peer-reviewed Executive Of ce, American Academy of Neurology fi Catherine M. Rydell, CAE, Executive Director/CEO journal in the eld of neurogenetics. 201 Chicago Ave Minneapolis, MN 55415 MISSION: Neurology: Genetics will provide neurologists with Tel: 612-928-6100 outstanding original contributions that elucidate the role of genetic and epigenetic variations in diseases and biological traits Editorial Office of the central and peripheral nervous systems. Patricia K. Baskin, MS, Executive Editor Kathleen M. Pieper, Senior Managing Editor, Neurology Editor Lee Ann Kleffman, Managing Editor, Neurology: Genetics Neurology: Genetics Sharon L. Quimby, Managing Editor, Neurology® Clinical Practice Stefan M. Pulst, MD, Dr med, FAAN Morgan S. Sorenson, Managing Editor, Neurology® Neuroimmunology & fl Professor and Chair, Department of Neurology Neuroin ammation University of Utah Cynthia S. Abair, MA, Senior Graphics Editor Salt Lake City, UT Andrea R. Rahkola, Production Editor, Neurology [email protected] Robert J. Witherow, Senior Editorial Associate Specialties: Genetics, movement disorders Karen Skaja, Senior Editorial Associate Kaitlyn Aman Ramm, Editorial Assistant Editor-in-Chief Kristen Swendsrud, Editorial Assistant Neurology® Andrea Willgohs, Editorial Assistant Robert A. Gross, MD, PhD, FAAN Professor of Neurology and of Pharmacology and Physiology Strong Epilepsy Center Publisher University of Rochester Medical Center Wolters Kluwer Rochester, NY Baltimore, MD [email protected]

Editorial Inquiries Publishing Staff Tel: 612-928-6400 Kim Jansen, Executive Publisher Toll-free: 800-957-3182 (US) Alexandra Lazerow, Production Team Leader, AAN Journals Fax: 612-454-2748 Steve Rose, Editorial Assistant [email protected] Stacy Drossner, Production Associate

Academy Ofﬁcers Ralph L. Sacco, MD, MS, FAAN, President James C. Stevens, MD, FAAN, President Elect Ann H. Tilton, MD, FAAN, Vice President Carlayne E. Jackson, MD, FAAN, Secretary Janis M. Miyasaki, MD, MEd, FRCPC, FAAN, Treasurer Terrence L. Cascino, MD, FAAN, Past President Neurology.org/ng Online ISSN: 2376-7839 Volume 3, Number 3, June 2017

Editor Biostatistics Neurology® Genetics Richard J. Kryscio, PhD Stefan M. Pulst, MD, Dr med, FAAN University of Kentucky Professor and Chair, Department of Neurology Lexington, KY University of Utah Christopher A. Beck, PhD Salt Lake City, UT University of Rochester [email protected] Rochester, NY Specialties: Genetics, movement disorders Sue Leurgans, PhD Editor-in-Chief Rush University Medical Center Neurology® Chicago, IL Robert A. Gross, MD, PhD, FAAN Level of Evidence Evaluations Professor of Neurology and of Pharmacology and Physiology Gary S. Gronseth, MD, FAAN Strong Epilepsy Center University of Kansas University of Rochester Medical Center Kansas City, KS Rochester, NY [email protected] Podcasts Ted M. Burns, MD Deputy Editors University of Virginia Neurology Charlottesville, VA Bradford B. Worrall, MD, MSc, FAAN Andrew M. Southerland, MD, MSc, Deputy Podcast Editor Professor of Neurology and Public Health Sciences University of Virginia University of Virginia Charlottesville, VA Charlottesville, VA Ombudsman Neurology: Genetics David S. Knopman, MD, FAAN Nicholas Elwood Johnson, MD Mayo Clinic University of Utah Rochester, MN Salt Lake City, UT Specialties: Muscle diseases, patient-centered outcomes Scientiﬁc Integrity Advisor Robert B. Daroff, MD, FAAN Associate Editors Case Western Reserve University Neurology: Genetics Cleveland, OH Alexandra Durr, MD, PhD Editorial Board Hôpital de la Salpêtrière Hilary Coon, PhD, University of Utah, Salt Lake City, UT Paris, France Giovanni Coppola, MD, UCLA, Los Angeles, CA Specialties: Spastic paraplegia, Huntington disease, ataxia Chantal Depondt, MD, PhD, Université Libre de Bruxelles, Brussels, Belgium Massimo Pandolfo, MD, FAAN Brent L. Fogel, MD, PhD, FAAN, UCLA, Los Angeles, CA Hôpital Erasme Anthony J. Griswold, PhD, University of Miami, Miami, FL Université Libre de Bruxelles Orhun H. Kantarci, MD, Mayo Clinic, Rochester, MN Brussels, Belgium Julie R. Korenberg, PhD, MD, University of Utah, Salt Lake City, UT Specialties: Epilepsy, Friedreich ataxia, stem cells Margherita Milone, MD, PhD, Mayo Clinic, Rochester, MN Davide Pareyson, MD, C. Besta Neurological Institute IRCCS Foundation, Raymond P. Roos, MD, FAAN Milan, Italy University of Chicago Medical Center Shoji Tsuji, MD, PhD, University of Tokyo Graduate School of Medicine, Chicago, IL Tokyo, Japan Specialties: Motor neuron disease, prion disease, neurovirology/neuroimmunology Dineke S. Verbeek, PhD, University Medical Center Groningen, Groningen, The Netherlands Jeffery M. Vance, MD, PhD David Viskochil, MD, PhD, University of Utah, Salt Lake City, UT University of Miami Juliane Winkelmann, MD, Technische Universität München, Helmholtz Miami, FL Zentrum Münche, Munich, Germany Specialties: Parkinson, Alzheimer disease, inherited neuropathies Juan I. Young, PhD, University of Miami, Miami, FL Level of Evidence Review Team Melissa J. Armstrong, MD, Gainesville, FL Richard L. Barbano, MD, PhD, FAAN, Rochester, NY Richard M. Dubinsky, MD, MPH, FAAN, Kansas City, KS Jeffrey J. Fletcher, MD, MSc, Ann Arbor, MI Gary M. Franklin, MD, MPH, FAAN, Seattle, WA David S. Gloss II, MD, MPH&TM, Charleston, WV John J. Halperin, MD, FAAN, Summit, NJ Jason Lazarou, MSc, MD, Toronto, Ontario, Canada Steven R. Messé, MD, FAAN, Philadelphia, PA Pushpa Narayanaswami, MBBS, DM, FAAN, Boston, MA Alex Rae-Grant, MD, Cleveland, OH Neurology.org/ng Online ISSN: 2376-7839 Volume 3, Number 3, June 2017

Publication Information Neurology® is a registered trademark of the American Academy of Neurology (registration valid in the United States). Neurology® Genetics (eISSN 2376-7839) is an open access journal published online for the American Academy of Neurology, 201 Chicago Avenue, Minneapolis, MN 55415, by Wolters Kluwer Health, Inc. at 14700 Citicorp Drive, Bldg. 3, Hagerstown, MD 21742. Business offices are located at Two Commerce Square, 2001 Market Street, Philadelphia, PA 19103. Production offices are located at 351 West Camden Street, Baltimore, MD 21201-2436. © 2017 American Academy of Neurology. Neurology® Genetics is an official journal of the American Academy of Neurology. Journal website: Neurology.org/ng, AAN website: AAN.com Copyright and Permission Information: Please go to the journal website (www.neurology.org/ng) and click the “©Request Permissions” icon for the relevant article. Alternatively, send an email to [email protected]. General information about permissions can be found here: https://www.lww.com/journal-permission. Disclaimer: Opinions expressed by the authors and advertisers are not 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, efficacy, or availability of the material contained in this publication (including drug dosages) or for any damages arising out of the use or non-use of any of the material contained in this publication. Advertising Sales Representatives: Wolters Kluwer, 333 Seventh Avenue, New York, NY 10001. Contacts: Eileen Henry, tel: 732-778-2261, fax: 973-215-2485, [email protected] and Elizabeth S. Hall, tel: 267-804-8123, [email protected]. In Europe: Avia Potashnik, Wolters Kluwer, tel: 144 207 981 0722; 144 7919 397 933 or e-mail: [email protected]. Careers & Events: Monique McLaughlin, Wolters Kluwer, Two Commerce Square, 2001 Market Street, Philadelphia, PA 19103, tel: 215-521- 8468, fax: 215-521-8801; [email protected]. Reprints: Meredith Edelman, Commercial Reprint Sales, Wolters Kluwer, Two Commerce Square, 2001 Market Street, Philadelphia, PA 19103, tel: 215-555-1212 (office), 215-356-2721 (mobile); [email protected]; [email protected]. Special projects: US & Canada: Alan Moore, Wolters Kluwer, Two Commerce Square, 2001 Market Street, Philadelphia, PA 19103, tel: 215-521-8638, [email protected]. International: Andrew Wible, Senior Manager, Rights, Licensing, and Partnerships, Wolters Kluwer; [email protected]. THE HELIX Collaboration, workshops, and symposia

Stefan M. Pulst, MD, Readers may have noticed the presence of a March will be mutually beneficial and provide platforms Dr med issue of Neurology® Genetics that added to the cus- for the rapid dissemination of new research findings. tomary bimonthly publication schedule. The March issue is a supplement focused on research presented STUDY FUNDING Correspondence to by the International Stroke Genetics Consortium No targeted funding reported. Dr. Pulst: [email protected] (ISGC). DISCLOSURE Genetics of stroke is paradigmatic for the chal- S.M. Pulst serves on the editorial boards of Journal of Cerebellum, Neuro- Neurol Genet lenges of analyzing polygenic risk in highly complex Molecular Medicine, CONTINUUM, Experimental Neurology, Neurogenetics, 2017;3:e157; doi: 10.1212/ disease phenotypes with significant environmental and Nature Clinical Practice Neurology; receives research support from NIH, NXG.0000000000000157 Target ALS, National Ataxia Foundation, and ISIS Pharmaceuticals; has and lifestyle confounders. The consortium brings consulted for Ataxion Therapeutics; served on a speakers’ bureau for Athena together stroke neurologists and geneticists that col- Diagnostics, Inc.; is a stockholder of Progenitor Life Sciences; has received laborate to achieve sample sizes and diverse cohorts license fee payments from Cedars-Sinai Medical Center; holds patents for to define genetic risk for a number of stroke subtypes. Nucleic acids encoding ataxin-2 binding proteins, Nucleic acid encoding Schwannomin-binding proteins, Transgenic mouse expressing a polynucleo- The supplementary issue published proceedings of tide encoding a human ataxin-2 polypeptide, Methods of detecting spinocer- – the 19th and 20th ISGC workshops.1 3 The ISGC ebellar ataxia-2 nucleic acids, Nucleic acid encoding spinocerebellar ataxia-2 workshops have been held on a semiannual basis since and products related thereto, Schwannomin-binding proteins, and Compo- the first meeting in 2007. The Department of sitions and methods for spinocerebellar ataxia; and receives an honorarium from the AAN as the Editor of Neurology: Genetics. Go to Neurology.org/ng Neurology at the University of Utah sponsored for full disclosure forms. a workshop in 2014, and I could personally witness that members of ISGC are highly interactive and REFERENCES committed to sharing of data. In addition to the 1. Anderson CD, Boncoraglio G, Falcone G. Proceedings of excitement for discovery, the group also shared in the 19th and 20th International Stroke Genetics Consor- a desire to sample the local surroundings, including tium Workshops. Neurol Genet 2017;3:S1. doi: 10.1212/ regional cuisine, hiking, and skiing. NXG.0000000000000137. 2. Debette S, Saba Y, Vojinovic D, et al. 19th Workshop of Neurology: Genetics recently published the pro- the International Stroke Genetics Consortium, April 28–29, 4 ceedings from another group of geneticists. This 2016, Boston, Massachusetts, USA. Neurol Genet 2017;3: research conference dealt with the phenotypic diver- S2–S11. doi: 10.1212/NXG.0000000000000100. sity associated with mutations in the ATP1A3 gene. 3. Woo D, Debette S, Anderson C. 20th Workshop of the The ATP1A3-related disease conference presented International Stroke Genetics Consortium, November 3–4, – a summary of findings, whereas the ISGC proceed- 2016, Milan, Italy. Neurol Genet 2017;3:S12 S18. doi: 10. 1212/NXG.0000000000000136. ings will allow the reader to access individual 4. Rosewich H, Sweney MT, DeBrosse S, et al. Research con- abstracts. ference summary from the 2014 International Task Force It is the hope of the editors that these types of on ATP1A3-Related Disorders. Neurol Genet 2017;3:e139. collaborations of the journal with genetics consortia doi: 10.1212/NXG.0000000000000139.

From the Department of Neurology, University of Utah, Salt Lake City. 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.

Neurology.org/ng Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 EDITORIAL ARHGEF9 mutations cause a specific recognizable X-linked intellectual disability syndrome

Pasquale Striano, MD, Mutations in more than 100 genes have been re- including tonic-clonic seizures in 9 subjects, focal in PhD ported to cause X-linked intellectual disability 9, tonic in 2, and myoclonic in 1 individual. EEG Federico Zara, PhD (XLID), mainly in males. By contrast, the identified recordings revealed generalized, focal, or even multi- X-linked genes in which de novo mutations specifi- focal epileptic abnormalities. All patients with epi- cally cause ID in females are still limited so far.1 lepsy were treated with antiseizure drugs, usually Correspondence to Next-generation sequencing has nowadays enabled a polytherapy, and seizures were drug resistant in 4 Dr. Striano: [email protected] the screening for a causative mutation in XLID using cases. Minor abnormalities on neuroimaging were targeted panel, X-exome, and whole-exome sequenc- observed in 8 subjects and included nonspecific T2 Neurol Genet ing methods.2 Mutations in the X-linked cell division hyperintensities/enlarged perivascular spaces in 3 2017;3:e159; doi: 10.1212/ cycle 42 guanine nucleotide exchange factor (GEF)-9 subjects and hippocampal sclerosis, hypoplastic fron- NXG.0000000000000159 gene (ARHGEF9) have been recently associated with tal lobe, brain atrophy, polymicrogyria, and delayed a wide phenotypic spectrum, including ID, behavior myelination in 1 each. Eight subjects had de novo disorders, autism spectrum disorder, hyperekplexia, hemizygous single nucleotide mutations, 3 had and infantile epileptic encephalopathy.3–5 A number maternally inherited mutations, and 7 carried chro- of patients with ARHGEF9 mutations, encompassing mosomal disruptions. All female patients had strongly missense and nonsense mutations, deletions, and skewed X-inactivation. However, the most important complex rearrangements, have been reported. How- merit of this article is to provide quite accurate ever, so far, the clinical features of ARHGEF9 disease genotype-phenotype correlations. Indeed, ID was are nonspecific, with extreme phenotypic and genetic moderate in individuals carrying with less severe mu- heterogeneity. tations. Moreover, male patients were most severely In this issue of Neurology® Genetics, Alber et al.6 affected with the overall neurologic syndrome (i.e., report 18 patients (including 5 females) identified most severe ID and seizures) and showed distinctive through clinical practice (8 previously unpublished) and consistent facial features, including enlarged, fle- and from a review of the literature. Detailed medical shy ear lobes, and sunken appearance of the middle history and examination findings were obtained for face (i.e., midface hypoplasia) in combination with each patient via a standardized questionnaire. Pa- prognathism. Finally, all the 3 male patients harbor- tients’ age widely ranged from 4 to 57 years, and ing mutations in exon 9 that do affect a specific pleck- age at onset of the symptoms varied from day 1 to strin homology (PH) domain of the protein did not age 7 years, with a mean age of 15 months. Presenting develop epilepsy. symptoms included seizures (5 patients), develop- ARHGEF9 encodes collybistin, a protein which is mental delay in combination with seizures (4 sub- highly expressed in multiple regions of the brain dur- jects), and developmental delay/ID alone (6 ing development and structured by an N-terminal patients). Most patients (13 subjects) showed delayed SRC homology 3 (SH3) domain followed by tandem early developmental milestones, and all individuals DBL homology (DH), PH domains, and a C-terminal had ID ranging from mild-moderate to severe degree, proline-rich sequence.7 This protein structure iden- with associated autistic features or hyperactivity/ tifies ARHGEF9 as a member of the Rho GTPase aggressive behavior in some cases. Thirteen patients activator family, responsible for the activation of had epilepsy with onset at a mean age of 20 months Rho-family GTPases and involved in a number of (range: 1 week–7 years). Seizure types were variable, cell signaling transduction pathways.7 In addition,

See article From the Laboratory of Neurogenetics (P.S.), Istituto “Giannina Gaslini,” Genova, Italy; and Pediatric Neurology and Muscular Diseases Unit (F.Z.), Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, “G. Gaslini” Institute, Italy. Funding information and disclosures are provided at the end of the editorial. Go to Neurology.org/ng for full disclosure forms. The Article Processing Charge for this editorial was waived at the discretion of the Editor. 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.

Neurology.org/ng Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 collybistin is essential for the clustering of the scaf- warranted in the future to clarify the full spectrum folding protein gephyrin and of GABAA receptors at of ARHGEF9 disease and to shed light on the the postsynaptic membrane, 2 proteins associated accumulating evidence implicating neuronal synaptic with ID and epilepsy.8 The reduction of inhibitory gene products as key molecular factors underlying the receptor clusters in the brain has been proposed as etiologies of a diverse range of neurodevelopmental a plausible underlying pathophysiologic mechanism. conditions. In fact, collybistin-deficient male mice show reduced exploratory behavior, impaired spatial learning, STUDY FUNDING increased anxiety scores, and reduction of gephyrin- No targeted funding reported. dependent GABA receptor clusters in amygdala and DISCLOSURE hippocampus.7 P. Striano received honoraria from FB Health, Kolfarma s.r.l., UCB The reasons behind the clinical variability of pharma, and Eisai Inc. and research support from the Italian Ministry ARHGEF9 mutations remain unknown and could of Health and the Telethon Foundation. F. Zara received research be to be linked to several factors. First, the clinical support from the Italian Ministry of Health, the European Community features observed in mutated females may depend on Sixth, the Italian Ministry of Health, the Telethon Foundation, and the Italian League Against Epilepsy. Go to Neurology.org/ng for full the degree of X-inactivation skewing. Thus, while disclosure forms. males have more pronounced ID and dysmorphic features, females may show variable symptoms with REFERENCES a phenotype similar to that of males when displaying 1. Tarpey PS, Smith R, Pleasance E, et al. A systematic, large- the 100% X-inactivation.9 However, X-inactivation scale resequencing screen of X-chromosome coding exons and expression in blood might not necessarily reflect in mental retardation. Nat Genet 2009;41:535–543. what is occurring in the brain, and other factors may 2. de Ligt J, Willemsen MH, van Bon BW, et al. Diagnostic exome sequencing in persons with severe intellectual dis- modify expression, such as variants in regulatory se- ability. N Engl J Med 2012;367:1921–1929. quences of ARHGEF9 and related genes. Second, 3. Lesca G, Till M, Labalme A, et al. De novo Xq11.11 certain ARHGEF9 mutations (e.g., C-terminal trun- microdeletion including ARHGEF9 in a boy with mental cations) clearly cause dominant-negative effects on retardation, epilepsy, macrosomia, and dysmorphic fea- gephyrin and GABAA receptor clustering in neuronal tures. Am J Med Genet A 2011;155A:1706–1711. systems.10 Moreover, functional studies of ARHGEF9 4. Kalscheuer VM, Musante L, Fang C, et al. A balanced chromosomal translocation disrupting ARHGEF9 is asso- missense mutations affecting the PH domain struc- ciated with epilepsy, anxiety, aggression, and mental retar- ture that binds phosphatidylinositol-3-phosphate dation. Hum Mutat 2009;30:61–68. show consequent loss in the ability of collybistin to 5. Long P, May MM, James VM, et al. Missense mutation mediate gephyrin clustering.10 Nevertheless, given the R338W in ARHGEF9 in a family with X-linked intellec- variability in clinical phenotypes associated with tual disability with variable macrocephaly and macro-or- ARHGEF9 mutations, it is evident that clinical obser- chidism. Front Mol Neurosci 2016;8:83. vation, flanked by molecular genetics, is the best 6. Alber M, Kalscheuer V, Marco E, et al. ARHGEF9 disease: phenotype clarification and genotype-phenotype correla- approach for a correct approach to patients with ID tion. Neurol Genet 2017;3:e148. doi: 10.1212/NXG. and other neuropsychiatric features. In fact, patients 0000000000000148. with ID often undergo a diagnostic odyssey before 7. Mayer S, Kumar R, Jaiswal M, et al. Collybistin activation receiving the correct diagnosis, mainly because of by GTP-TC10 enhances postsynaptic gephyrin clustering clinical heterogeneity, unusual presentations, and lack and hippocampal GABAergic neurotransmission. Proc – of specific genotype-phenotype correlations. On the Natl Acad Sci USA 2013;110:20795 20800. 8. Saiepour L, Fuchs C, Patrizi A, et al. Complex role of other hand, the identification of the disease-related collybistin and gephyrin in GABAA receptor clustering. mutation is of paramount importance to patients J Biol Chem 2010;285:29623–29631. and their families, as awareness of the causing condi- 9. Shimojima K, Sugawara M, Shichiji M, et al. Loss-of- tion relieves uncertainty, improves communication function mutation of collybistin is responsible for X-linked with professionals, and provides prognostic informa- mental retardation associated with epilepsy. J Hum Genet – tion and enhances social support. Alber et al.6 provide 2011;56:561 565. 10. Papadopoulos T, Schemm R, Grubmüller H, Brose N. an excellent clinical genetic summary of the Lipid binding defects and perturbed synaptogenic ARHGEF9 mutations, providing great aid in the activity of a Collybistin R290H mutant that causes clinical dissection of ARHGEF9 variants. Further epilepsy and intellectual disability. J Biol Chem 2015; observations and longitudinal follow-up studies are 290:8256–8270.

2 Neurology: Genetics ARHGEF9 disease Phenotype clarification and genotype-phenotype correlation

Michael Alber, MD ABSTRACT Vera M. Kalscheuer, PhD Objective: We aimed to generate a review and description of the phenotypic and genotypic spec- Elysa Marco, MD tra of ARHGEF9 mutations. Elliott Sherr, MD, PhD Methods: Patients with mutations or chromosomal disruptions affecting ARHGEF9 were identi- Gaetan Lesca, MD fied through our clinics and review of the literature. Detailed medical history and examination Marianne Till, MD findings were obtained via a standardized questionnaire, or if this was not possible by reviewing Gyri Gradek, MD the published phenotypic features. Antje Wiesener, MD Christoph Korenke, MD, Results: A total of 18 patients (including 5 females) were identified. Six had de novo, 5 had mater- PhD nally inherited mutations, and 7 had chromosomal disruptions. All females had strongly skewed Sandra Mercier, MD, X-inactivation in favor of the abnormal X-chromosome. Symptoms presented in early childhood PhD with delayed motor development alone or in combination with seizures. Intellectual disability Felicitas Becker, MD was severe in most and moderate in patients with milder mutations. Males with severe intellectual Toshiyuki Yamamoto, disability had severe, often intractable, epilepsy and exhibited a particular facial dysmorphism. ’ MD, PhD Patients with mutations in exon 9 affecting the protein s PH domain did not develop epilepsy. Stephen W. Scherer, PhD Conclusions: ARHGEF9 encodes a crucial neuronal synaptic protein; loss of function of which Christian R. Marshall, results in severe intellectual disability, epilepsy, and a particular facial dysmorphism. Loss of only PhD the protein’s PH domain function is associated with the absence of epilepsy. Neurol Genet 2017;3: Susan Walker, PhD e148; doi: 10.1212/NXG.0000000000000148 Usha R. Dutta, PhD Ashwin B. Dalal, MD GLOSSARY Vanessa Suckow AED 5 antiepileptic drug; Cb 5 collybistin; NGS 5 next-generation sequencing; XLID 5 X-linked intellectual disability. Payman Jamali, MD Kimia Kahrizi, MD X-chromosomal mutations are a common cause of intellectual disability (X-linked intellec- Hossein Najmabadi, PhD tual disability [XLID]) in males accounting for 10%–12% of ID.1,2 XLID is highly hetero- Berge A. Minassian, MD geneous and is usually divided into syndromic and nonsyndromic forms, depending on the association with particular clinical findings.1,2 Epileptic seizures accompany XLID in almost half the disorders, in some beginning in infancy prior to the developmental delay being Correspondence to Dr. Alber: evident. Recent next-generation sequencing (NGS) approaches have expedited the identifi- – [email protected] cation of XLID genes, including those associated with epilepsy,1 3 many of which encode proteins involved in synaptic function.4 ARHGEF9 is one such gene,5–9 encoding collybistin (Cb), a brain-specific guanine nucleotide exchange factor with an essential role in inhibitory synaptic transmission.5 Cb interacts with the inhibitory receptor–anchoring protein gephyrin and is required for the formation of gephyrin and gephyrin-dependent

See editorial From the Department of Paediatrics (Neurology) (M.A., B.A.M.), Program in Genetics and Genome Biology (S.W.S., C.R.M., S.W., B.A.M.), The Hospital for Sick Children and University of Toronto, Canada; Research Group Development and Disease (V.M.K., V.S.), Max Plank Institute for Molecular Genetics, Berlin, Germany; Department of Neurology (E.M., E.S.), University of California, San Francisco; Service de Cytogenetique Constitutionnelle (G.L., M.T.), Hospice Civils de Lyon, France; Center for Medical Genetics and Molecular Medicine (G.G.), Haukeland University Hospital, Bergen, Norway; Humangenetisches Institut (A.W.), Universitaetsklinikum Erlangen, Germany; Zentrum für Kinder- und Jugendmedizin (C.K.), Elisabeth Kinderkrankenhaus, Oldenburg, Germany; Service de Genetique Medicale (S.M.), CHU Hotel Dieu, Nantes, France; Neurologische Universitätsklinik (F.B.), Tübingen, Germany; Institute of Medical Genetics (T.Y.), Tokyo Women’s Medical University, Japan; Diagnostics Division (U.R.D., A.B.D.), Center for DNA Fingerprinting and Diagnostics, Telangana, India; Shahrood Welfare Organization (P.J.), Shahrood, Iran; and Genetics Research Center (K.K., H.N.), University of Social Welfare and Rehabilitation Sciences, Tehran, Iran. Funding information and disclosures are provided at the end of the article. Go to Neurology.org/ng for full disclosure forms. The Article Processing Charge was paid 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.

Neurology.org/ng Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 from patients for research purposes. Written consent was ob- GABAA clusters in the postsynaptic mem- ’ brane (figure 1).5,8,10 Cb knockout mice tained from patients parents for the publication of recognizable photographs. exhibit increased anxiety, impaired spatial learning, and convulsions.10,11 X-chromosome inactivation analysis. In the unpublished cases (patients P1, P3, and P4) X-chromosome inactivation anal- Over 10 patients with ARHGEF9 muta- ysis was performed with the human androgen receptor assay: – – tions have been reported,5 9,12 15 indicating amplification of the CAG repeat in exon 1 of the androgen recep- that this will likely be one of the more com- tor gene was performed by PCR with fluorescence-tagged pri- mon XLIDs. Descriptions of the published mers. Subsequently, digestion with the methylation-sensitive enzyme HpaII was performed, and fragments were analyzed with cases vary from scant (in large NGS projects) an automated capillary sequencer (ABI 3100; Applied Bio- to case reports with substantial detail. In the systems, Foster City, CA). Genescan and Genotyper Software present study, we combine detailed descrip- (Applied Biosystems) were used to determine fragment sizes and intensities, and the degree of X-inactivation was calculated. tions of the phenotypes of 18 patients with ARFGH9 mutations. In 4 cases, adequate RESULTS Genetic findings. Tables 1 and 2 summa- detail was present at first published descrip- rize the genetic findings of the 18 patients. Eleven had tion. In 6 cases, we went back to the patients hemizygous ARHGEF9 mutations, including prema- mentioned in tables of NGS results and ture stop codon, splicing, and point mutations. Six worked with their physicians to obtain clin- were de novo mutations; the 2 above brothers (P10 ical details. The remaining are new unpub- and 11) and patients P8, P15, and P18 showed lished patients. a maternal inheritance, the latter with 3 affected brothers with a similar phenotype. Seven had chromosomal disruptions. The 11 mutations were in METHODS Two brothers (patients P10 and P11) with ID and epilepsy and unremarkable MRIs were tested by clinical whole- exons 8 (3), 9 (2), 1 (1), 3 (1), 4 (1), 5 (1), and 7 exome sequencing and found to have mutations in the ARH- (1), and in intron 9 (1) (exon numbering based on the GEF9 gene. Further 16 patients (including 5 females) with longest ARHGEF9 transcript NM_015185.2). The mutations or chromosomal disruptions affecting ARHGEF9 were patients with chromosomal disruptions had deletions identified through a literature review and by contacting genetic (4), translocations (2), and a paracentric inversion (1). centers in North America, Asia, and Europe. The 5 female patients all showed skewed X-inactivation Standard protocol approvals, registrations, and patient of the normal chromosome. consents. A detailed developmental epilepsy and general medical Clinical features. Tables 1 and 2 summarize the clinical history together with examination findings were obtained for each patient via a standardized questionnaire filled by their treating features of the 18 patients. physician, or if this was not possible by a review of the described Demographics. Current age or age at last examination phenotypic features in publications. No samples were obtained ranged from 4 to 57 years. Presenting symptoms: onset

Figure 1 Interaction of collybistin in the postsynaptic membrane

Schematic representation of the interactions of collybistin in the formation of gephyrin and gephyrin-dependent GABAA clusters in the postsynaptic membrane.

2 Neurology: Genetics Table 1 Genetic findings and phenotypic features of patients with ARHGEF9 mutations or disruptions (females)

Genetic findings and clinical features (reference) P1 P28 P3 P4 P59

Chromosomal disruption Balanced Balanced translocation (46, 27 kb Xq11.1 deletion: 7.5 kb Xq11.1 deletion: Paracentric inversion (46, translocation (46,XX, t X, t(X;18)(q11.1;q11.21)) arrXq11.1 arrXq11.1 (62,854,862– X,inv(X)(q11.1q27.3)) (X;20)(q12;P13)) (62,838,630– 62,862,403)x1 62,865,334)

Inheritance De novo De novo De novo De novo De novo

X-inactivation (testing) Skewed 100 (RT-PCR Skewed 100 (RT-PCR with Skewed 95:5 (RT-PCR Skewed 90:10 (Mercier) Skewed 100 (replication with ARHGEF9 ARHGEF9 primers vs with ARHGEF9 primers studies on primers vs controls) controls) vs controls) phytohemagglutinin- stimulated T lymphocytes after BrdU incorporation)

Functional study N/A Loss of gephyrin 1 GABAAR N/A N/A N/A clusters

Current age/age of 10 15 (AE) 9 4 25 examination (AE)

Presenting age and 10 mo; SZ, DD 7 mo; DD 12 mo; DD 31 mo; SZ, DD 18 mo; HA, DD symptoms

Dysmorphic features CD Facial Facial Facial Facial

Somatic parameter, C N/A; H ,3; W 3 C ,3; H ,3; W ,3 C 50; H 3; W 90 C 90; H 90; W 90 C N/A; H 65; W 60 percentile

Developmental S: 12; W: 16 S: 21 S: 15; W: 22 S: 11; W: 30; Wo: 42 W: 13; Wo: 20 milestones, mo

Intellectual disability Severe Severe Moderate Moderate Moderate (48) (IQ testing)

Autistic features Yes NR No No No

Hyperactivity Yes Yes Yes No No

Hyperekplexia No No No No No

Hyperarousal No No No No Yes

MRI Normal Normal Normal N/A UF

Epilepsy Yes Yes No Yes No

Seizure types GTC GTC — F, GTC —

Seizure onset 10 mo 7 y — 31 mo —

EEG findings Gen. SW Slow BG — Normal —

AEDs VPA, PHT, PGB VPA, CLB — None —

Response to AED ,50% red NR — Seizure free —

Other features Nonverbal, Nonverbal (auto-) Hypotonia, pes PE, enhanced tendon Sensory hyperarousal hyperpigmentation aggressive behavior, sleep adductus, sleep reflexes, mild joint problems, OSA, insensitivity problems hypermobility to thermal pain

Abbreviations: AED 5 antiepileptic drug; BG 5 background; BrdU 5 bromodeoxyuridine; C 5 head circumference; CD 5 clinodactyly; CLB 5 clobazam; DD 5 developmental delay; GTC 5 generalized tonic-clonic seizures; H 5 height; HA 5 hyperammonemia; N/A 5 not applicable; NR 5 not reported; OSA 5 obstructive sleep apnea; PE 5 pectus excavatum; PGB 5 pregabalin; PHT 5 phenytoin; RT 5 real time; S 5 sitting; SW 5 spike and wave; SZ 5 seizures; UF 5 unspecific findings; VPA 5 valproate; W 5 body weight; W 5 independent walking.

of symptoms occurred at a mean age of 15 months (7) to severe (10). Of the 10 patients with severe (median 9 months, range 1 day–7 years). Present- ID, 7 were nonverbal. ing symptoms varied from seizures (5), develop- Neuropsychiatric features. Autistic features were re- mental delay in combination with seizures (4), ported in 4 patients. Five patients had hyperactivity. and developmental delay/intellectual disability Dysmorphic features. Photographs for review of dys- alone (6). One patient presented with hyperek- morphic features were available for 9 of the 18 pa- plexia and seizures shortly after birth and 1 patient tients. Eight patients showed mild facial dysmorphic with hyperarousal and developmental delay (with features. Out of this group, it appears that the male no seizures). patients who are most severely affected with the over- Intellectual disability. The majority of patients (13) all neurologic syndrome (i.e., most severe ID and seiz- showed delayed early developmental milestones. All ures) show more distinctive and consistent facial patients had ID ranging from mild (1) to moderate features, including enlarged, fleshy earlobes, a sunken

Neurology: Genetics 3 4

Table 2 Genetic findings and phenotypic features of patients with ARHGEF9 mutations or disruptions (males)

Genetic findings and clinical features (reference) P67 P76 P86 P95 P10 P11 P12

Chromosomal disruption/ 1.29 Mb Xq11.11 737 kb Xq11.11 NM_001173479 NM_015185.2 NM_015185.2 NM_015185.2 NM_015185.2 NM number deletion: arrXq11.1 deletion: arrXq11.1 (61,848,414– (62,321,746– 63,138,698) [Hg18]x0 63,058,548) x.dn

Complementary DNA level N/A N/A c.4C.T c.164G.A c.950C.G c.950C.G c.530T.C

a erlg:Genetics Neurology: Protein level N/A N/A p.Q2 p.G55A p.S317W p.S317W p.L177P

Genomic DNA N/A N/A 63005022G.A 62944437C.T 62885872G.C 62885872G.C 62917036A.G

Exon N/A N/A 1 3 8 8 5

Domain N/A N/A None SH3 None None DH

Functional study N/A N/A N/A Loss of gephyrin 1 N/A N/A N/A GABAAR clusters

Inheritance De novo De novo Maternally De novo Maternally Maternally De novo

Current age/age of 11 5 (AE) 5 (AE) D (4) 23 31 4 examination (AE)

Presenting age and 5 mo; SZ 9 mo; DD 6 mo; DD Birth; HE; SZ 13 mo; SZ 30 mo; SZ 4 mo; SZ symptoms

Dysmorphic features Facial No No NR Facial Facial No

Somatic parameter, C 97; H 97; W 97 C 90–97; H 75–90; W C75–90; H 50–75; W NR C 50; H 25; W 10–25 C P50; H 97; W .97 C 25–50; H 90–97; W percentile .97 10–25 50–75

Developmental S: 12; W: 23 S: 12; W: no S: 12; W: 24 NR S: 12; W: 18 S: 6; W: 15 W: 20 milestones, mo

Intellectual disability (IQ Severe Severe Severe Severe Severe Severe Severe testing)

Autistic features No NR NR NR Yes No Yes

Hyperactivity YesNRNRNRNoNoNo

HE No NR NR Yes No No No

Hyperarousal No NR NR NR No No No

MRI Normal HFL PMG BA UF Normal Normal

Epilepsy Yes Yes Yes Yes Yes Yes Yes

Seizure types FD, GTC FD GTC T (startle) GTC F, FD, GTC FC, FD, GTC

Seizure onset 5 mo 24 mo 20 mo Birth 13 mo 30 mo 4 mo

EEG findings Normal Bilateral temporal SW CSWS NR Gen. 2 Hz SW, slow BG Gen. 2 Hz SW, slow BG Slow BG, 4 Hz rhythm

AEDs CBZ, PB, TPM, LEV, VPA Multiple AEDs PB, LTG VPA, CBZ, CLB CBZ, CLB VPA, LEV, LTG OXC

Continued Table 2 Continued

Genetic findings and clinical features (reference) P67 P76 P86 P95 P10 P11 P12

Response to AED 50% red Seizure free Refractory Refractory 50% red 50% red 50% red

Other features Nonverbal, PE, Nonverbal, trigono- Nonverbal, ataxic gait HE — Nonverbal Nonverbal, hypospadia aggressive behavior cephalic head nevus flammeus, sleep problems

Genetic findings and clinical features (reference) P13 P1415 P1512,13 P1614 P1714 P18

Chromosomal disruption/NM NM_015185.2 NM_015185.2 NM_015185.2 NM_015185.2 NM_015185.2 NM_015185.2 number

Complementary DNA level c.311G.A c.869G.A c.1012C.T c.1198G.A c.130012T.C c.1067G.A

Protein level p.R104Q p.R290H p.R338W p.E400K p. ? Exon skipping p.R356Q

Genomic DNA 62926208C.T 62893973C.T 62885810G.A 62875476C.T 62875372A.G 62875607C.T

Exon 4 7 8 9 Intron 9 9

Domain DH DH PH PH None PH

Functional study N/A Reduced PI3P-binding 1 Reduced PI3P-binding 1 N/A N/A N/A loss of gephyrin clusters loss of gephyrin clusters

Inheritance De novo De novo Maternally De novo De novo Maternally

Current age/age of 15 57 26 (AE) 2 (AE) 3 (AE) 28 examination (AE)

Presenting age and symptoms 12 mo; SZ, DD 6 mo; SZ, DD N/A 6 mo; DD 2 mo; SZ 7 y; ID

Dysmorphic features No No Facial Facial Facial, CD No

Somatic parameter, C 60; H ,3; W ,3 N/A C 90; H 70 C 10–25; H 25–50; W C 97; H 50; W N/A C 97; H 75; W 75 percentile 10–25

Developmental milestones, S: 18; W: no N/A NR S: 12; W: 29 S:12; W: no S: 6; W: 12; Wo: 12 mo

Intellectual disability (IQ Severe Moderate (40–50) Moderate (43) Moderate Moderate Mild (50–70) testing)

Autistic features Yes No No NR NR No erlg:Genetics Neurology: Hyperactivity YesNoNRNRNRNo

HE No No NR NR NR No

Hyperarousal No No NR NR NR No

MRI UF HS N/A Normal MD N/A

Epilepsy Yes Yes Yes No No No

Seizure types F, GTC, M, T FD, GTC NR ———

Seizure onset 12 mo 6 mo NR ———

EEG findings MISF, 4 Hz rhythm, slow BG Temporal SW NR ———

Continued 5 appearance of the middle face (midface hypoplasia) in combination with a protruding of the jaw (prognathism) (figure 2). Two patients showed 5-digit primidone;

5 clinodactyly. Neuroimaging findings. MRI data were available in

independent walking; 15 patients. Findings were none (7), hippocampal continuous spikes and 5 myoclonic seizures; MD generalized tonic-clonic 5 sclerosis (1), hypoplastic frontal lobe (1), brain atro- 5 5 P18 — phy (1), polymicrogyria (1), delayed myelination (1), or nonspecific (T2 hyperintensities, accentuated polymicrogyria; PRM

5 perivascular spaces) (3). body weight; W

lamotrigine; M Seizure/epilepsy details. Thirteen patients had epi- 5 clobazam; CSWS 5

5 lepsy with onset at a mean age of 20 months (median 12 months, range 1 week–7 years). Seizure types were variable, including generalized tonic-clonic (9), focal 14 valproate; W dyscognitive (5), focal (4), myoclonic (1), and tonic 5 P17 Fetal finger pads, short fifth finger

focal dyscognitive seizures; GTC (2) seizures. EEG findings were available for 11 of the pectus excavatum; PMG 5 levetiracetam; LTG clinodactyly; CLB

5 13 patients and showed generalized (3), bilateral (1), 5 5 multifocal (1), and focal (1) epileptiform discharges and were normal in 2 patients. One patient with vigabatrin; VPA

5 polymicrogyria showed continuous spike-and-wave discharges in slow-wave sleep. Two patients had on- phenobarbital; PE lacosamide; LEV

14 ly moderate background slowing. 5 5 carbamazepine; CD P16 ——— ——— sleep problems Epilepsy treatment. Four patients had medically focal clonic seizures; FD 5

5 refractory epilepsy, 4 showed 50% seizure reduction with antiepileptic drugs (AEDs), 1 showed seizure reduction of less than 50%, and 2 became completely unspecific findings; VGB seizure free without further AED treatment. AEDs 5 oxcarbazepine; PB ketogenic diet; LCS

5 consisted mainly of combination therapy (10); only 5

febrile seizures; FC 1 patient was on monotherapy (1). 5 12,13

head circumference; CBZ Additional features included sleep disorder (4), 5 P15 NR Macro-orchidism Fetal finger and toe pads, ataxic gait (1), aggressive behavior (2), fetal finger topiramate; UF

5 and toe pads (2), pigmentation abnormalities (2),

not reported; OXC pectus excavatum (2), hyperreflexia (1), tachypnea 5 bromide; C (1), macro-orchidism (1), and insensitivity to thermal 5 eslicarbazepine; F

5 pain (1). hippocampal sclerosis; KD 5

DISCUSSION

tonic seizures; TPM We describe and review the pheno- 15

5 types of 18 patients with ARHGEF9 mutations and not applicable; NR VPA, TPM, LEV, PRM,ESL LCS, — background; Br 5 5 chromosomal disruptions. Symptoms usually present in early childhood with delayed motor development

seizures; T alone or in combination with seizures. ID later on is 5

developmental delay; ESL usually moderate to severe with the majority of severe

5 cases being nonverbal. hypoplastic frontal lobe; HS brain atrophy; BG 5 Epilepsy is common (13/18), starting usually at an 5 early age (median 12 months). Seizure semiology is variable with a majority of patients showing general- spike and wave; SZ deceased; DD CBZ, VPA, TPM, LEV, LTG,Br, VGB, STM, CLB, LCS, KD Refractory Refractory NR Nonverbal, episodic tachypnea 5

5 ized tonic-clonic seizures and many showing multiple seizure types. Most of the epilepsies were difficult to multiple independent spike foci; N/A hyperekplexia; HFL 5 treat with 10 patients on combination AED therapy. 5

antiepileptic drug; BA Facial dysmorphism, in particular, enlarged ear- 5

sulthiame; SW lobes, midface hypoplasia, and prognathism appear 5 Continued to be a constant feature of the neurologically severely height; HE

5 affected male patients. The overall picture, therefore, is one of a moder- first words. sitting; STM 5 ate-to-severe XLID (with severe cases nonverbal), Genetic findings and clinical features (reference) P13 P14 AEDs Response to AED Other features Table 2 myelination delay; MISF 5 Lifted over from Hg18 to Hg19. seizures; H 5 waves during slow-wave sleep; D S Abbreviations: AED a Wo moderate-to-severe epilepsy (with severe cases poorly

6 Neurology: Genetics mutation. To clarify this, we briefly review some Figure 2 Facial dysmorphism of 2 severely affected male patients aspects of Cb structure and function. Inhibitory

neurotransmitter receptor channels (GABAA and glycine) imbedded in the postsynaptic membrane are anchored and clustered therein by a submem- brane lattice composed of the gephyrin protein. The gephyrin lattice itself binds the plasma membrane via intermediary proteins critical among which is Cb. Cb binds gephyrin through its DH domain and attaches to the membrane through both its ter- mini as follows: the N-terminal SH3 domain binds the membrane-integral neuroligin-2 protein, and the C-terminal PH domain binds the phosphoino-

sitol 3-phosphate (PI3P) constituent of the plasma membrane (figure 1).5,8,10 In male patients P6 and P7, the entire ARHGEF9 gene is deleted, and in patient P8, there is a premature stop in the second codon (table 1). These 3 patients therefore lack Cb altogether, and their phenotypes would be represen- tative of the complete loss of Cb function. Table 2 shows that all 3 have severe ID and epilepsy. Pa- tients 9 through 15 have various missense mutations affecting different regions of the protein, but

not the C-terminal PI3P-interacting PH domain. All have epilepsy. Where functional experiments were performed (patients 9, 14, and 15), it was shown that the mutations result in loss of gephyrin clusters.5,12,16 The 3 remaining patients (16 through 18) all have mutations that affect the PH domain. None of these patients have epilepsy (and all have mild-to-moderate ID). Therefore, tentatively, mu-

tationsaffectingonlythePI3P-interacting C-terminus of the protein are not associated with epilepsy. Future functional experiments for these and subsequent PH domain mutations may uncover differ- Patients P6 (A) and P11 (B) from tables 1 and 2. Note enlarged fleshy earlobes, midface ences from the effects of the epileptogenic hypoplasia, and prognathism. mutations and a possible understanding of epilepto- genesisinthisdisease. responsive or intractable to AEDs), and a facial dys- An additional important observation gleaned from morphism in the severe cases. our summary tables is comparing male patients P8 Mothers of the various male patients in our study and P9 (table 2). Patient P8 has a stop mutation, do not have XLID, indicating that random skewing of p.Q2*, in the very first exon, and his symptom con- X-inactivation protects female carriers of ARHGEF9 stellation is very severe and similar to patients P6 and mutations. We did, however, find 5 female patients. P7 in whom the entire gene is deleted. This first exon X-inactivation studies in all 5 showed skewing in is coding, and therefore mutation p.Q2* truncating, favor of the abnormal X-chromosome (table 1), ren- in a shorter transcript (NM_001173479) that skips dering them equivalent to male counterparts with over the next 2 exons utilized by the longest transcript hemizygous mutations. The female phenotype would (NM_015185.2) of the gene. The shorter transcript, invariably be affected by remaining expression from NM_001173479, was confirmed to be highly ex- the normal allele in cells in which it is active, and by pressed in both the developing and adult human differences in the extent of X-inactivation skewing in brain and may be the dominant transcript.6 Patient the brain compared to leucocytes. P9 is the very first ARHGEF9 patient described, and Closeanalysisofmalesinthegenotypeandphe- a striking feature of his presentation was hyperekplex- notype tables (1 and 2, respectively) suggests a tenta- ia. His mutation is in exon 2, of the longer transcript, tive genotype—epilepsy phenotype correlation which is not utilized by the shorter transcript. Hyper- related to the region of the protein affected by the ekplexia is not found in any of the patients described

Neurology: Genetics 7 after that first patient (tables 1 and 2), which suggests LaFollette, Johnson; Huff Powell & Bailey; and Bell, Roper and that this symptom may result from a particular func- Kohlmyer; and has served on the Board of Directors of the National Organization of Disorders of the Corpus Callosum. Dr. Lesca, Dr. Till, tion of the slightly longer protein encoded by the Dr. Gradek, Dr. Wiesener, Dr. Korenke, Dr. Mercier, and Dr. Becker longer transcript. report no disclosures. Dr. Yamamoto has received research support from As in so many other neurogenetic disorders, our the Japan Ministry of Education, Science, Sports and Culture. Dr. Scher- er has served on the scientific advisory board of Population Diagnostics; review reveals that the ARHGEF9 disease is not uni- has served on the editorial boards of Genomic Medicine; Genes, Genomes, form and varies with the gene mutation and likely Genetics; Journal of Personalized Medicine; The Open Genomics Journal; other genetic and extragenetic factors. Therefore, The Hugo Journal; Genome Medicine; the Journal of Neurodevelopmental the diagnostic approach in candidate patients still Disorders; Autism Research; PathoGenetics; Comparative and Functional Genomics; BMC Medical Genomics; and Cytogenetics and Genome Research; requires chromosomal microarray testing as the and has received research support from Genome Canada/Ontario Ge- first-line genetic testing because of the substantial nomics Institute, Canadian Institutes of Health Research, Canadian Insti- diagnostic yield and low relative cost, followed by tute for Advanced Research, McLaughlin Centre, Canada Foundation for a gene panel or whole-exome sequencing approach Innovation, the government of Ontario, Autism Speaks, and SickKids Foundation. Dr. Marshall has received travel funding from Affymetrix as second tier. and Life Technologies and serves on the editorial board of Genes, Ge- There is, however, a core phenotype of moderate- nomes, Genetics. Dr. Walker reports no disclosures. Dr. Dutta has to-severe ID and epilepsy with a facial dysmorphism received research support from the Department of Biotechnology—India (University of Mysore) and SERB. Dr. Dalal has served on the editorial (large earlobes, midface hypoplasia, and prognathism) board of Official Publication of Society for Indian Academy of Medical in patients with complete loss of the gene’s function. Genetics and has received research support from the Department of Sci- This phenotype is moderated with lesser mutations— ence and Technology (Govt. of India) and the Indian Council of Medical less severe intellectual disability and seizures, and Research (Govt. of India). Dr. Suckow, Dr. Jamali, Dr. Kahrizi, and Dr. Najmabadi report no disclosures. Dr. Minassian holds patents for diag- absence of facial dysmorphism. As mentioned, mu- nostic testing of the following genes: EPM2A, EPM2B, MECP2, tations affecting the last exon alone appear to be VMA21; has received research support from the National Institute of nonepiletogenic. Neurological Disorders and Stroke of the NIH under award number P01 NS097197; and receives license fee payments/royalty payments from AUTHOR CONTRIBUTIONS patents for diagnostic testing of the following genes: EPM2A, EPM2B, MECP2, VMA21. Go to Neurology.org/ng for full disclosure forms. Michael Alber: acquisition of data, analysis and interpretation of data, and study supervision. Vera M. Kalscheuer, Elysa Marco, Elliott Received November 17, 2016. Accepted in final form March 14, 2017. Sherr, Gaetan Lesca, Marianne Till, Gyri Gradek, Antje Wiesener, Christoph Korenke, Sandra Mercier, Felicitas Becker, Toshiyuki REFERENCES Yamamoto, Stephen W. Scherer, and Christian R. Marshall: acquisition of data and critical revision of manuscript for intellectual content. 1. Ropers HH Genetics of early onset cognitive impair- – Susan Walker: acquisition of data, analysis of data, and critical revi- ment. Annu Rev Genomics Hum Genet 2010;11:167 sion of manuscript for intellectual content. Usha R. Dutta, Ashwin 187. B. Dalal, Vanessa Suckow, Payman Jamali, Kimia Kahrizi, and Hossein 2. Stevenson RE, Charles E, Schwartz R, Rogers RC. Atlas of Najmabadi: acquisition of data and critical revision of manuscript X-linked Intellectual Disability Syndromes, 2nd ed. New for intellectual content. Berge A. Minassian: acquisition of data, anal- York: Oxford University Press; 2012. ysis and interpretation of data, and critical revision of manuscript for 3. Hu H, Haas SA, Chelly J, et al. X-exome sequencing of intellectual content. 405 unresolved families identifies seven novel intellectual disability genes. Mol Psychiatry 2016;21:133–148. ACKNOWLEDGMENT 4. Humeau Y, Gambino F, Chelly J, Vitale N. X-linked The authors thank the patients and their families. They thank Ms. Wendy mental retardation: focus on synaptic function and plas- Gu (University Toronto) for generating the graphic of figure 1. ticity. J Neurochem 2009;109:1–14. 5. Harvey K, Duguid IC, Alldred MJ, et al. The GDP-GTP STUDY FUNDING exchange factor collybistin: an essential determinant of Study funded by Genome Canada and the Ontario Brain Institute. This neuronal gephyrin clustering. J Neurosci 2004;24:5816– work was supported by the Ontario Brain Institute, Genome Canada, The McLaughlin Foundation, The Centre for Applied Genomics and 5826. the EU FP7 project GENCODYS, grant number 241995 (to V.M.K., 6. Shimojima K, Sugawara M, Shichiji M, et al. Loss-of- H.N., and K.K.). B.A.M. holds the University of Toronto Michael function mutation of collybistin is responsible for X-linked Bahen chair in Epilepsy Research. mental retardation associated with epilepsy. J Hum Genet 2011;56:561–565. DISCLOSURE 7. Lesca G, Till M, Labalme A, et al. De novo Xq11.11 Dr. Alber has received travel funding/speaker honoraria from AES Fel- microdeletion including ARHGEF9 in a boy with mental lows Program 2015. Dr. Kalscheuer reports no disclosures. Dr. Marco retardation, epilepsy, macrosomia, and dysmorphic fea- has been a consultant for Grand Rounds Second Opinions (Clinical opin- tures. Am J Med Genet A 2011;155A:1706–1711. ions through UCSF) and has received research support from the Wallace 8. Kalscheuer VM, Musante L, Fang C, et al. A balanced Research Foundation. Dr. Sherr has served on the scientific advisory chromosomal translocation disrupting ARHGEF9 is asso- board of InVitae; holds a patent for Blood-based diagnostic for autism; ciated with epilepsy, anxiety, aggression, and mental retar- has been a consultant for Personalis; has received research support from dation. Hum Mutat 2009;30:61–68. National Institute of Neurological Disorders and Stroke, Simons Founda- tion, CURE Foundation, and the John and Marsha Goldman Founda- 9. Marco EJ, Abidi FE, Bristow J, et al. ARHGEF9 disruption; holds stock/stock options in InVitae and ChemoCentryx; has tion in a female patient is associated with X linked mental worked with the following law firms: Sheuerman, Martini & Tabari; retardation and sensory hyperarousal. J Med Genet 2008; Galloway and Lucchese; Zuger Kirmis & Smith; Donahoe & Kearney; 45:100–105.

8 Neurology: Genetics 10. Papadopoulos T, Soykan T. The role of collybistin in and linkage to Xq12-q21. J Med Genet 1998;35:1026– gephyrin clustering at inhibitory synapses: facts and open 1030. questions. Front Cell Neurosci 2011;24:5–11. 14. de Ligt J, Willemsen MH, van Bon BW, et al. Diagnostic 11. Papadopoulos T, Korte M, Eulenburg V, et al. Impaired exome sequencing in persons with severe intellectual dis- GABAergic transmission and altered hippocampal synaptic ability. N Engl J Med 2012;367:1921–1929. plasticity in collybistin-deficient mice. EMBO J 2007;26: 15. Lemke JR, Riesch E, Scheurenbrand T, et al. Targeted 3888–3899. next generation sequencing as a diagnostic tool in epileptic 12. Long P, May MM, James VM, et al. Missense mutation disorders. Epilepsia 2012;53:1387–1398. R338W in ARHGEF9 in a family with X-linked intellec- 16. Papadopoulos T, Schemm R, Grubmüller H, Brose N. tual disability with variable macrocephaly and macro-or- Lipid binding defects and perturbed synaptogenic activ- chidism. Front Mol Neurosci 2016;8:83. ity of a collybistin R290H mutant that causes epilepsy 13. Johnson JP, Nelson R, Schwartz CE. A family with mental and intellectual disability. J Biol Chem 2015;290:8256– retardation, variable macrocephaly and macro-orchidism, 8270.

Neurology: Genetics 9 Clinicopathologic and molecular spectrum of RNASEH1-related mitochondrial disease

Enrico Bugiardini, MD ABSTRACT Olivia V. Poole, MRCP Objective: Pathologic ribonuclease H1 (RNase H1) causes aberrant mitochondrial DNA (mtDNA) Andreea Manole, BSc segregation and is associated with multiple mtDNA deletions. We aimed to determine the preva- Alan M. Pittman, PhD lence of RNase H1 gene (RNASEH1) mutations among patients with mitochondrial disease and Alejandro Horga, MD establish clinically meaningful genotype-phenotype correlations. Iain Hargreaves, PhD Methods: RNASEH1 was analyzed in patients with (1) multiple deletions/depletion of muscle Cathy E. Woodward, BSc mtDNA and (2) mendelian progressive external ophthalmoplegia (PEO) with neuropathologic evi- Mary G. Sweeney, BSc dence of mitochondrial dysfunction, but no detectable multiple deletions/depletion of muscle Janice L. Holton, mtDNA. Clinicopathologic and molecular evaluation of the newly identified and previously re- FRCPath, PhD ported patients harboring RNASEH1 mutations was subsequently undertaken. Jan-Willem Taanman, . PhD Results: Pathogenic c.424G A p.Val142Ile RNASEH1 mutations were detected in 3 pedigrees Gordon T. Plant, among the 74 probands screened. Given that all 3 families had Indian ancestry, RNASEH1 FRCOphth, MD genetic analysis was undertaken in 50 additional Indian probands with variable clinical presenta- Joanna Poulton, tions associated with multiple mtDNA deletions, but no further RNASEH1 mutations were con- FRCPCH, DM firmed. RNASEH1-related mitochondrial disease was characterized by PEO (100%), cerebellar Massimo Zeviani, MD, ataxia (57%), and dysphagia (50%). The ataxia neuropathy spectrum phenotype was observed in . PhD 1 patient. Although the c.424G A p.Val142Ile mutation underpins all reported RNASEH1- Daniele Ghezzi, PhD related mitochondrial disease, haplotype analysis suggested an independent origin, rather than John Taylor, PhD a founder event, for the variant in our families. Conrad Smith, PhD Conclusions: In our cohort, RNASEH1 mutations represent the fourth most common cause of Carl Fratter, MPhil adult mendelian PEO associated with multiple mtDNA deletions, following mutations in POLG, Meena A. Kanikannan, RRM2B,andTWNK. RNASEH1 genetic analysis should also be considered in all patients with MD POLG-negative ataxia neuropathy spectrum. The pathophysiologic mechanisms by which the Arumugam Paramasivam, c.424G.A p.Val142Ile mutation impairs human RNase H1 warrant further investigation. PhD Neurol Genet 2017;3:e149; doi: 10.1212/NXG.0000000000000149 Kumarasamy Thangaraj, PhD GLOSSARY Antonella Spinazzola, MD COX 5 cytochrome c oxidase; IBD 5 identity by descent; mtDNA 5 mitochondrial DNA; NCS 5 nerve conduction studies; PCA 5 principal component analysis; PEO 5 progressive external ophthalmoplegia; RNase H1 5 ribonuclease H1; RRF 5 Ian J. Holt, PhD ragged red fiber. Henry Houlden, FRCP, PhD Mitochondrial diseases are commonly inherited disorders caused by mutations in nuclear Michael G. Hanna, and mitochondrial DNA (mtDNA). Dysfunction in a subset of nuclear genes involved with FRCP, MD mtDNA maintenance results in the secondary accumulation of multiple deletions and/or Robert D.S. Pitceathly, depletion of muscle mtDNA.1 The clinical spectrum of this group of disorders is broad, MRCP, PhD

From the MRC Centre for Neuromuscular Diseases (E.B., O.V.P., A.M., A.H., J.L.H., H.H., M.G.H., R.D.S.P.), UCL Institute of Neurology and National Hospital for Neurology and Neurosurgery; Department of Molecular Neuroscience (A.M., A.M.P., J.L.H., H.H., M.G.H.), Division of Correspondence to Neuropathology (J.L.H.), Department of Clinical Neuroscience (J.-W.T., A.S., I.J.H.), UCL Institute of Neurology; Neurometabolic Unit (I.H.), Dr. Pitceathly: Neurogenetics Unit (C.E.W., M.G.S.), Department of Neuro-ophthalmology (G.T.P.), National Hospital for Neurology and Neurosurgery, [email protected] London; Nuffield Department of Obstetrics and Gynaecology (J.P.), University of Oxford; MRC-Mitochondrial Biology Unit (M.Z.), Cambridge, UK; Unit of Molecular Neurogenetics (D.G.), Fondazione IRCCS Istituto Neurologico “Carlo Besta,” Milan, Italy; Oxford Medical Genetics Laboratories (J.T., C.S., C.F.), Oxford University Hospitals NHS Foundation Trust, Churchill Hospital, UK; Department of Neurology (M.A.K.), Nizam’s Institute of Medical Sciences; CSIR-Centre for Cellular and Molecular Biology (A.P., K.T.), Hyderabad, Telangana, India; MRC Mill Hill Laboratory (I.J.H.), London, UK; Biodonostia Research Institute (I.J.H.), San Sebastián, Spain; and Department of Basic and Clinical Neuroscience ’ Supplemental data (R.D.S.P.), Institute of Psychiatry, Psychology and Neuroscience, King s College London, UK. at Neurology.org/ng Funding information and disclosures are provided at the end of the article. Go to Neurology.org/ng for full disclosure forms. The Article Processing Charge was funded by the Medical Research Council. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Neurology.org/ng Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 ranging from severe infantile hepatocerebral Genetic studies. Whole-exome and Sanger sequencing was used syndromes to benign, late-onset progressive to analyze the 8 coding exons and intron-exon boundaries of RNASEH1 (primer sequences available on request). external ophthalmoplegia (PEO). Mutations in the mtDNA maintenance gene RNASEH1, Clinicopathologic and molecular evaluation of patients. Patients harboring RNASEH1 mutations were assessed clinically encoding ribonuclease H1 (RNase H1), have by the authors. Medical records and muscle tissue histopathologic recently been linked to adult mitochondrial and electron microscopy findings were reviewed. Real-time disease presenting with CNS and neuromuscu- quantitative PCR of DNA extracted from muscle was under- lar involvement.2 RNase H1 degrades RNA taken in all patients harboring RNASEH1 mutations and multiple mtDNA deletions to exclude coexisting mtDNA depletion. 3 hybridized to DNA, and knockout mice Previously published RNASEH1 variants (pathogenic and of suffer embryonic lethality owing to mtDNA unknown significance) were evaluated2,7 and clinicopathologic depletion,4 attributable to persistent mtDNA and genotypic data extracted and combined with the London- RNA/DNA hybrids.5 However, humans with Oxford cohort to accurately define the clinical genetic spectrum of all known RNASEH1-mediated mitochondrial disease. RNASEH1 mutations develop a relatively mild clinical syndrome, comprising adult- Principal component analysis, identity by descent, and haplotyping. Principal component analysis (PCA), using GCTA onset PEO associated with multiple mtDNA software (version 1.26.0), was performed relative to the HapMap deletions secondary to the impaired physical project populations (CEU, JPT, and YRI) to study the geograph- segregation of mtDNA molecules.6 The prev- ical origin of 2 Indian families where exome-sequencing data were available (families A and B). Identity by descent (IBD) analysis alence and clinical spectrum of RNASEH1- was utilized in plink software (v1.90 b1g) to explore the possi- related mitochondrial disease is currently bility of a common lineage for the pedigrees. Haplotyping was unknown. adopted to investigate the possibility of a common origin for the . We screened 74 unrelated probands with c.424G A p.Val142Ile mutation. Six single nucleotide variants extracted from available exome data were used to construct genetically undetermined mitochondrial haplotypes around the mutation site, and Sanger sequencing of disease for RNASEH1 mutations. The pre- the variants in affected individuals from the London-Oxford viously reported pathogenic missense transi- cohort and in 2 previously reported families was undertaken.6 tion c.424G.A p.Val142Ile was detected in Haplotypes were phased around homozygous variant sites only, given parental samples were unavailable. 3 families with Indian ancestry: homozygous mutations were confirmed in 2 pedigrees Standard protocol approvals, registrations, and patient consents. The study was approved and performed under the eth- and compound heterozygous mutations ical guidelines issued by our institutions for clinical studies, with in combination with a novel c.442T.C written informed consent obtained from all participants for p.Cys148Arg variant in a third. Detailed genetic studies. clinicopathologic and molecular profiling of RESULTS RNASEH1 sequence data analysis. Seventy- these newly identified families harboring four unrelated probands were recruited from the RNASEH1 mutations, and evaluation of all London and Oxford NHS England nationally com- previously reported cases, was subsequently missioned service for mitochondrial diseases and undertaken to determine the phenotypic spec- categorized as follows: multiple deletions (n 5 33) trum of RNASEH1-mediated mitochondrial and depletion (n 5 21) of muscle mtDNA and disease and establish clinically meaningful mendelian PEO with neuropathologic evidence genotype-phenotype correlations. Finally, of mitochondrial dysfunction, but no detectable the ancestral origins of the “common” multiple deletions/depletion of muscle mtDNA RNASEH1 c.424G.A p.Val142Ile mutation (n 5 20). Homozygous RNASEH1 c.424G.A were investigated. p.Val142Ile mutations were identified in 2, apparently unrelated, nonconsanguineous families (family A: A-III.8, A-III.9, A-III.10, and A-III.11; METHODS Participants. Patients referred to the London and Oxford NHS England nationally commissioned service for and family B: B-II.1 and B-II.8), and a third singleton mitochondrial diseases were recruited to the study according to case (family C: C-II.1) was compound heterozygous the following criteria: (1) confirmed multiple deletions/depletion with the novel missense mutation c.442T.C of muscle mtDNA and (2) mendelian PEO with pathologic evi- p.Cys148Arg (figure 1A). Parental segregation was dence of mitochondrial dysfunction, including ragged red fibers confirmed. All 3 families had Indian ancestry. (RRFs) and/or cytochrome c oxidase (COX)-negative muscle fi- Affected individuals harbored multiple deletions of bers, without detectable multiple deletions or depletion of muscle mtDNA. RNASEH1 genetic analysis was also undertaken in muscle mtDNA. Fifty additional unrelated Indian a cohort of Indian patients with evidence of multiple mtDNA probands with multiple deletions of muscle mtDNA deletions detectable in muscle. deletions were screened for RNASEH1 mutations

2 Neurology: Genetics Figure 1 Pedigrees and histopathologic findings of patients harboring RNASEH1 c.424G>A p.Val142Ile mutations

(A) Pedigrees of families harboring RNASEH1 mutations. Filled blue symbols represent affected individuals. Arrows indicate probands. Long range PCR (LPCR) and Southern blot (SB) demonstrate multiple deletions of muscle mitochondrial DNA (B-II.8) in patient (P) when compared with control muscle (C). (B) Muscle biopsy histology (A-III.8) demonstrating ragged red fibers (modified Gomori trichrome, GT), ragged blue fibers (succinate dehydrogenase, SDH), and several muscle fibers deficient in cytochrome c oxidase (COX), arrows. Scale bar represents 50 mm in GT and 200 mm in SDH and COX. (C) Ultrastructural examination (A-III.8) showing increased numbers of mitochondria, many of which are structurally abnormal, including the presence of paracrystalline inclusions, arrows. Scale bar represents 1 mm.

(see table e-1 at Neurology.org/ng, for clinicopatho- proportion of patients (57%) exhibited cerebellar dys- logic spectrum), but no known or novel RNASEH1 function. Additional clinical features (summarized in variants were detected. table 1) included dysphagia (50%), proximal muscle weakness (36%), peripheral neuropathy (36%), and Clinicopathologic and molecular features in RNASEH1 pyramidal signs (14%). NCS and EMG were consis- mutations. Mean age of symptom onset in the tent with involvement of motor and/or sensory nerve London-Oxford cohort was 29 years (range, 13–36 fibers with demyelinating, axonal, or ganglionic fea- years). All patients presented with either ptosis or tures. RRFs and/or COX-deficient fibers and multi- imbalance. The clinical phenotype was characterized ple mtDNA deletions were reported in all cases when by PEO, proximal muscle weakness, and cerebellar muscle tissue was available. ataxia. One patient had a sensory ataxic neuropathy, The c.424G.A p.Val142Ile mutation was de- with nerve conduction studies (NCS) and EMG tected in all 7 families (both newly reported and pre- indicative of a nonlength-dependent, predominantly viously published). Three were homozygous and 4 sensory, axonal neuropathy. Brain MRI demon- were compound heterozygous with the following mu- strated moderate generalized parenchymal volume tations: c.442T.C p.Cys148Arg (n 5 2), c.469C.T loss (n 5 2). Histopathologic examination of muscle p.Arg157*,andc.554C.T p.Ala185Val. revealed RRFs and COX-negative fibers in all cases examined, while ultrastructural examination showed PCA, IBD, and haplotyping. PCA revealed that fami- increased and abnormal mitochondria, many with lies A and B clustered to the same ethnic group paracrystalline inclusions (figure 1, B and C). There (figure 2A, top panel), while IBD analysis implied was no evidence of a coexisting reduction in muscle a distant relationship to approximately second mtDNA copy number (B-II.8 and C-II.1). cousins (mean PI_HAT 0.049 6 0.013). Haplo- Three families (6 affected individuals) with con- typing of families A and B suggested one shared firmed RNASEH1 mutations have previously been founder haplotype of 3.67 Mb which was also pres- reported.2 An additional case was identified from var- ent in family C. However, given that the iants of unknown significance published in exome- c.424G.A p.Val142Ile variant was heterozygous, sequencing studies.7 These data were combined with it was not possible to decipher whether this repre- the London-Oxford cohort and confirmed that PEO sented a population-specific or an ancestral muta- was a universal feature in patients with RNASEH1- tion (figure 2A, bottom panel). Analysis of the same related mitochondrial disease and that a substantial haplotype marker set in 2 previously reported pedigrees

Neurology: Genetics 3 4

Table 1 Clinicopathologic, biochemical, and genetic features of newly reported and previously published adults with RNASEH1-related mitochondrial disease

Multiple Age at Skeletal muscle mtDNA RNASEH1 cDNA and amino Patient Clinical features onset, y Brain CT/MRI NCS/EMG histochemistry RCEA deletions acid change

. erlg:Genetics Neurology: A-III.8 PEO, ptosis, facial weakness, proximal muscle 33 Normal Myopathy RRFs, COX-deficient ND Y c.424G A p.Val142Ile; weakness fibers c.424G.A p.Val142Ile

A-III.9 PEO, ptosis 32 ND ND RRFs ND ND c.424G.A p.Val142Ile; c.424G.A p.Val142Ile

A-III.10 PEO, ptosis ND ND ND ND ND ND c.424G.A p.Val142Ile; c.424G.A p.Val142Ile

A-III.11 PEO, ptosis ND ND ND ND ND ND c.424G.A p.Val142Ile; c.424G.A p.Val142Ile

B-II.1 PEO, ptosis, ataxia, facial and proximal muscle 36 Cerebral/cerebellar atrophy ND SDH-positive/COX- ND Y c.424G.A p.Val142Ile; weakness deficient fibers c.424G.A p.Val142Ile

B-II.8 PEO, ptosis, dysarthria, ataxia, facial and 33 Cerebral/cerebellar atrophy Sensory . motor neuronopathy RRFs, SDH-positive/ Normal Y c.424G.A p.Val142Ile; proximal muscle weakness and myopathy COX-deficient fibers, c.424G.A p.Val142Ile neurogenic changes

C-II.1 PEO, ptosis, proximal muscle weakness, ataxia, 13 ND Sensory neuropathy RRFs ND Y c.424G.A p.Val142Ile; diabetes c.442T.C p.Cys148Arg

S12 PEO, ptosis, dysphagia, muscle pain, exercise 20 Cerebellar/brain stem atrophy Mild demyelinating motor RRFs, COX-deficient Low I/IV Y c.424G.A p.Val142Ile; intolerance, respiratory and lower limb neuropathy and myopathy fibers c.469C.T p.Arg157* weakness, ataxia

S22 PEO, ptosis, limb and axial weakness, head 23 ND Mild neurogenic features RRFs, COX-deficient Low I/IV Y c.554C.T p.Ala185Val; drop, pyramidal features dysphagia, reduced fibers c.424G.A p.Val142Ile visual acuity, cerebellar signs

S32 PEO, dysphagia, respiratory impairment ND ND ND ND ND Y c.424G.A p.Val142Ile; c.424G.A p.Val142Ile

S42 PEO, dysphagia, respiratory impairment ND ND ND ND ND Y c.424G.A p.Val142Ile; c.424G.A p.Val142Ile

S52 PEO, ptosis, dysphonia, dysphagia, pyramidal 45 Cerebellar/cortical atrophy, Neurogenic features RRFs, COX-deficient ND ND c.424G.A p.Val142Ile; signs, cerebellar signs, cognitive impairment deep periventricular white fibers c.424G.A p.Val142Ile matter hyperintensities

S62 PEO, gait instability, severe dysphagia, 40 ND ND RRFs, COX-deficient ND Y c.424G.A p.Val142Ile; respiratory impairment fibers c.424G.A p.Val142Ile

10197 Bilateral ptosis, PEO, ataxia, fatigue, dysphagia 44 ND ND RRFs, COX-deficient ND ND c.424G.A p.Val142Ile; fibers c.442T.C p.Cys148Arg

Abbreviations: COX 5 cytochrome c oxidase; mtDNA 5 mitochondrial DNA; NCS 5 nerve conduction studies; ND 5 not determined; PEO 5 progressive external ophthalmoplegia; RCEA 5 respiratory chain enzyme analysis; RRF 5 ragged red fiber; SDH 5 succinate dehydrogenase. Families A, B, and C reported in the current study; S1–62 and 10197 are previously published patients. nationally commissioned service for mitochondrial Figure 2 Principal component and haplotype analysis of families harboring c.424G>A p.Val142Ile variant and schematic illustrating the newly diseases, RNASEH1 mutations represent the fourth identified and previously reported RNASEH1 mutations to date most common cause of mendelian PEO associated with multiple mtDNA deletions in adults (2.7%, 3/ 109), following mutations in POLG (24.7%, 27/ 109), RRM2B (16.5%, 18/109), and TWNK (also known as C10orf2, PEO1, or Twinkle, 16.5%, 18/ 109). Additional, but less frequent, causes of adult mendelian PEO associated with multiple mtDNA deletions in our cohort include SLC25A4 (ANT1), OPA1, MFN2, TYMP, TK2, and SPG7 (1/109, 0.9%), while AFG3L2, DNA2, MGME1, POLG2, DGUOK, and MPV17 were not detected. Two families, in whom affected individuals harbored multiple mtDNA deletions, were homozygous for the previously reported c.424G.A p.Val142Ile mutation, thus confirming its pathogenicity and the importance of RNase H1 in mtDNA maintenance. A third proband was compound heterozygous with a novel missense mutation c.442T.C p.Cys148Arg in a highly conserved region of the enzyme, resulting in a hydrophobic amino acid being replaced by a pos- itively charged residue within the RNase H1 active site. This mutation was previously reported as a variant of unknown significance in a patient with PEO harboring the POLG2 variant c.1105A.G p.Arg369Gly, which was favored as being causative.7 However, a minor allele frequency of 0.003 suggests that the POLG2 variant c.1105A.G p.Arg369Gly is a benign polymorphism and unlikely to be deleterious.8 Furthermore, the similarity in clinical phenotypes and association with identical mutations in our cohort supports the pathogenic basis of RNASEH1 c.442T.C p.Cys148Arg when a second mutant

(A) Principal component analysis (top panel). X-axis represents component 1; Y-axis repre- allele is present. sents component 2. Families A and B cluster to the same ethnic group. Haplotype analysis Clinically, RNASEH1-linked mitochondrial disease of individuals harboring the RNASEH1 mutation c.424G.A p.Val142Ile (bottom panel). “1” is relatively homogenous, comprising PEO, cerebellar “ ” “ ” indicates the presence of the marker, 0 indicates the absence of the marker, and 0/1 is ataxia, dysphagia, and proximal muscle weakness. This used when haplotype could not be phased. Haplotypes reported are for A-III.8, B-II.8, C-II.1, S-1, and S-3. Green 5 c.424G.A mutation; gray 5 markers differing from reference hap- could reflect similar structural and functional conse- lotype. Families A and B shared a haplotype of at least 3.57 Mb. However, 2 additional quences of the reported RNASEH1 mutations. Indeed, haplotypes were different, suggesting distant recombination events or that the haplotypes all patients are homozygous or heterozygous for arose independently. (B) The RNASEH1 gene has 8 exons encoding 4 protein domains as . follows: mitochondrial targeting sequencing domain (M), hybrid-binding domain (HBD), con- c.424G A p.Val142Ile, and remaining variants all nection domain (CD), and ribonuclease H domain (H-domain). The latter conducts all catalytic occur within in the same catalytic domain (figure activity. Both herein newly identified and previously reported mutations are illustrated in the 2B). Additional deleterious mutations associated with “ ” . schematic. To date, all affected individuals harbor the common c.424G A p.Val142Ile more severe, early-onset clinical phenotypes might mutation either in homozygous or heterozygous states. Brackets 5 number of families with each mutation. Red 5 newly reported variant. exist. However, we did not identify any cases in our mtDNA depletion cohort. Furthermore, it is possible with European ancestry (S1 and S3) revealed a differ- that very detrimental mutations are embryonic lethal ent haplotype at single nucleotide polymorphism as is seen in knockout mice which are completely 4 rs10186193, 74 base pairs away from the c.424G.A deficient in RNase H1. Finally, sensory ataxic neu- p.Val142Ile mutation. These data are consistent with ropathy, previously unreported with RNASEH1 mu- an independent origin for the c.424G.A p.Val142Ile tations, expands the recognized clinical phenotype to mutation in the patients analyzed. include ataxia neuropathy spectrum, most commonly associated with recessive POLG mutations.9 DISCUSSION Based on data obtained from patients IBD analysis confirmed that families A and B referred to the London and Oxford NHS England were distantly related. However, their haplotype

Neurology: Genetics 5 differed from 2 previously reported European pedi- Institute of Neurology sequencing facility, which received a propor- ’ grees.2 These data suggest that the c.424G.A tion of funding from the Department of Health s National Institute for Health Research Biomedical Research Centres funding scheme. p.Val142Ile mutation has arisen independently in R.D.S.P. is funded by the National Institute for Health Research. the lineages analyzed. European admixing of the O.V.P. has received funding from the Lily Foundation. J.P. receives London-Oxford Indian families harboring support from the Lily Foundation, NewLife (SG/14-15/11), the Medical Research Council (MR/J010448/1), and the Wellcome Trust c.424G.A p.Val142Ile could account for the lack (0948685/Z/10/Z). K.T. is supported by the Department of Biotech- of RNASEH1 mutations in the 50 additional Indian nology and the Council of Scientific and Industrial Research (BioAge: probands screened. Furthermore, the close proximity BSC0118), Government of India. A.P. is supported by the Science of all 3 families to the CEU (European) HapMap pro- and Engineering Research Board (PDF/2016/000881), Government of India. A.S. is supported by the Medical Research Council Senior ject populations (figure 2A) suggests that they belong Non-Clinical Fellowship, MC_PC_13029. to the Ancestral North Indian gene pool, which shares up to 70% genetic affinities with Europeans.10 DISCLOSURE Our data confirm that RNASEH1 mutations are Enrico Bugiardini, Olivia V. Poole, Andreea Manole, Alan M. Pittman, an important cause of mitochondrial disease result- Alejandro Horga, Iain Hargreaves, Cathy E. Woodward, and Mary G. Sweeney report no disclosures. Janice L. Holton has received travel funding from the secondary accumulation of multiple ing from Merck-Serono; has served on the editorial board of Neuropa- mtDNA deletions and that the phenotypic spec- thology and Applied Neurobiology; has been an employee of University trum in adults is relatively benign. Nevertheless, College London; and has received research support from Alzheimer’s RNASEH1 genetic analysis should be considered Research Trust, The Margaret Watson Memorial Trust Grant from The Sarah Matheson Trust, Action Medical Research, Brain Net Europe: in all patients presenting with ataxia neuropathy Support for the Queen Square Brain Bank for Neurological Disorders, spectrum once POLG mutations have been The Sarah Matheson Trust, Myositis Support Group, The Multiple excluded. Finally, although the commonly occur- System Atrophy Trust, the Michael J Fox Foundation for Parkinson’s Research, Alzheimer’s Research UK, MSA Coalition, and the King Bau- ring c.424G.A p.Val142Ile RNASEH1 variant douin Foundation Sophia Fund. Jan-Willem Taanman has served on the could indicate a mutation hotspot within the gene, scientific advisory board of Novintum Bioscience Ltd.; has received it might instead reflect an inability of the mutant research support from Royal Free Charity; and receives royalty payments enzyme to bind with a partner protein, thereby from the University of Oregon. Gordon T. Plant has served on the editorial board of Neuro-Ophthalmology. Joanna Poulton and Massimo allowing Val142Ile RNase H1 to attack its substrates Zeviani report no disclosures. Daniele Ghezzi has served on the editorial 6 indiscriminately. As such, loss-of-function mutations board of Orphanet Journal of Rare Diseases and has received research could confer different clinical phenotypes. support from the Italian Ministry of Health, European Communities, Foundation Telethon, CARIPLO Foundation Italy, and the Pierfranco and Luisa Mariani Foundation of Italy. John Taylor, Conrad Smith, Carl AUTHOR CONTRIBUTIONS Fratter, and Meena A. Kanikannan report no disclosures. Arumugam Enrico Bugiardini: study design, analysis and interpretation of data, and Paramasivam has received research support from DST-SERB National drafting the manuscript. Olivia V. Poole: analysis and interpretation of Post Doctoral Fellowship No.: PDF/2016/000881. Kumarasamy Thangaraj data and revising the manuscript. Andreea Manole: acquisition, analysis has served on the editorial boards of Mitochondrion, PLoS One, BMC and interpretation of data, and revising the manuscript. Alan M. Pittman, Medical Genetics, Scientific Reports,andClinical Genetics. Antonella Alejandro Horga, Iain Hargreaves, Cathy E. Woodward, Mary G. Sweeney, Spinazzola reports no disclosures. Ian J. Holt has received research support Janice L. Holton, Jan-Willem Taanman, and Gordon T. Plant: acquisition, from Medical Research Council UK. Henry Houlden has received research analysis and interpretation of data, and revising the manuscript. Joanna support from The Medical Research Council (MRC) UK, The BRT, The Poulton: study concept, acquisition of data, and revising the manuscript. MDA USA, Muscular Dystrophy UK, Ataxia UK, Muscular Dystrophy Massimo Zeviani, Daniele Ghezzi, John Taylor, and Conrad Smith: acquisi- UK, Rosetrees Trust, The Wellcome Trust, and the National Institute for tion and interpretation of data and revising the manuscript. Carl Fratter: study Health (NIHR). Michael G. Hanna has been a consultant for Novartis and design, acquisition and interpretation of data, and revising the manuscript. has received research support from an MRC Centre Grant and the Myositis Meena A. Kanikannan and Arumugam Paramasivam: acquisition and inter- Support Group. Robert D.S. Pitceathly reports no disclosures. Go to Neu- pretation of data and revising the manuscript. Kumarasamy Thangaraj: study rology.org/ng for full disclosure forms. concept, acquisition and interpretation of data, and revising the manuscript. Antonella Spinazzola, Ian J. Holt, and Henry Houlden: study concept Received December 19, 2016. Accepted in final form March 13, 2017. and revising the manuscript. Michael G. Hanna: study concept, interpretation of data, and revising the manuscript. Robert D.S. Pitceathly: study design, analysis and interpretation of data, and drafting the manuscript. REFERENCES 1. DiMauro S, Schon EA, Carelli V, Hirano M. The clinical maze of mitochondrial neurology. Nat Rev Neurol 2013; ACKNOWLEDGMENT 9:429–444. The authors acknowledge the Telethon-Italy Network of Genetic Bio- 2. Reyes A, Melchionda L, Nasca A, et al. RNASEH1 muta- banks (GTB12001J) for DNA supply. tions impair mtDNA replication and cause adult-onset mitochondrial encephalomyopathy. Am J Hum Genet STUDY FUNDING 2015;97:186–193. The research leading to these results has received funding from the European 3. Keller W, Crouch R. Degradation of DNA RNA hybrids Community’s Seventh Framework Programme (FP7/2007-2013) under by ribonuclease H and DNA polymerases of cellular and grant agreement no. 2012-305121 “Integrated European—omics research viral origin. Proc Natl Acad Sci USA 1972;69:3360–3364. project for diagnosis and therapy in rare neuromuscular and neurodegenerative diseases (NEUROMICS).” This work is also supported by 4. Cerritelli SM, Frolova EG, Feng C, Grinberg A, Love PE, a Medical Research Council Centre grant (G0601943) and Wellcome Crouch RJ. Failure to produce mitochondrial DNA results Trust grant on Synaptopathies. Part of this work was undertaken in in embryonic lethality in Rnaseh1 null mice. Mol Cell the University College London Hospitals/University College London 2003;11:807–815.

6 Neurology: Genetics 5. Holmes JB, Akman G, Wood SR, et al. Primer retention 8. Lek M, Karczewski KJ, Minikel EV, et al. Analysis of owing to the absence of RNase H1 is catastrophic for mito- protein-coding genetic variation in 60,706 humans. chondrial DNA replication. Proc Natl Acad Sci USA 2015; Nature 2016;536:285–291. 112:9334–9339. 9. Lax NZ, Whittaker RG, Hepplewhite PD, et al. Sen- 6. Akman G, Desai R, Bailey LJ, et al. Pathological ribonu- sory neuronopathy in patients harbouring reces- clease H1 causes R-loop depletion and aberrant DNA sive polymerase gamma mutations. Brain 2012;135: segregation in mitochondria. Proc Natl Acad Sci USA 62–71. 2016;113:E4276–E4285. 10. Metspalu M, Romero IG, Yunusbayev B, et al. Shared 7. Lieber DS, Calvo SE, Shanahan K, et al. Targeted exome and unique components of human population structure sequencing of suspected mitochondrial disorders. Neurol- and genome-wide signals of positive selection in South ogy 2013;80:1762–1770. Asia. Am J Hum Genet 2011;89:731–744.

Neurology: Genetics 7 Intragenic DOK7 deletion detected by whole-genome sequencing in congenital myasthenic syndromes

Yoshiteru Azuma, MD, ABSTRACT PhD Objective: To identify the genetic cause in a patient affected by ptosis and exercise-induced mus- Ana Töpf, PhD cle weakness and diagnosed with congenital myasthenic syndromes (CMS) using whole-genome Teresinha Evangelista, sequencing (WGS). MD Methods: Candidate gene screening and WGS analysis were performed in the case. Allele-specific Paulo José Lorenzoni, PCR was subsequently performed to confirm the copy number variation (CNV) that was suspected MD, PhD from the WGS results. Andreas Roos, PhD Pedro Viana, MD Results: In addition to the previously reported frameshift mutation c.1124_1127dup, an intra- 9 Hidehito Inagaki, PhD genic 6,261 bp deletion spanning from the 5 untranslatedregiontointron2oftheDOK7 gene Hiroki Kurahashi, MD, was identified by WGS in the patient with CMS. The heterozygous deletion was suspected based PhD on reduced coverage on WGS and confirmed by allele-specific PCR. The breakpoints had micro- Hanns Lochmüller, MD homology and an inverted repeat, which may have led to the development of the deletion during DNA replication. Conclusions: We report a CMS case with identification of the breakpoints of the intragenic Correspondence to DOK7 deletion using WGS analysis. This case illustrates that CNVs undetected by Sanger Dr. Lochmüller: [email protected]. sequencing may be identified by WGS and highlights their relevance in the molecular diagnosis uk of a treatable neurologic condition such as CMS. Neurol Genet 2017;3:e152; doi: 10.1212/ NXG.0000000000000152

GLOSSARY aCGH 5 array comparative genomic hybridization; AChE 5 acetylcholinesterase; CMS 5 congenital myasthenic syndromes; CNV 5 copy number variation; MLPA 5 multiplex ligation–dependent probe amplification; MuSK 5 muscle-specific tyrosine kinase; NMJ 5 neuromuscular junction; WES 5 whole-exome sequencing; WGS 5 whole-genome sequencing.

Congenital myasthenic syndromes (CMS) are inherited disorders characterized by fatigable muscle weakness with or without other associated signs or symptoms.1 They are caused by mutations in genes expressed at the neuromuscular junction (NMJ). DOK7 is one of the components of the NMJ and an activator of the muscle-specific tyrosine kinase (MuSK).2 Recessive mutations in DOK7 cause approximately 10% of the genetically diagnosed CMS cases.1 CMS are heterogeneous diseases, and to date, more than 25 genes have been reported to be causative. Consecutive single-gene screening has been routinely used as a diagnostic tool; however, next-generation sequencing allows the analysis of all these genes simultaneously to identify the causative variant and obtain a genetic diagnosis. The efficacy of whole-exome sequencing (WES) for the diagnosis of CMS cases has been reported,3,4 as well as its ability to identify new causal genes.5,6 However, the limitation is that WES is designed to detect only protein- coding regions and exon-intron boundaries of the genome.

Supplemental data at Neurology.org/ng From the Institute of Genetic Medicine (Y.A., A.T., T.E., P.J.L., A.R., H.L.), Newcastle University, UK; Division of Neurology (P.J.L.), Federal University of Parana, Brazil; Leibniz-Institut für Analytische Wissenschaften ISAS e.V. (A.R.), Germany; Department of Neurosciences and Mental Health (P.V.), University of Lisbon, Portugal; and Division of Molecular Genetics (H.I., H.K.), Fujita Health University, Japan. Funding information and disclosures are provided at the end of the article. Go to Neurology.org/ng for full disclosure forms. The Article Processing Charge was funded by the Medical Research Council. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Neurology.org/ng Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 On the other hand, whole-genome changes on facial muscles. Repetitive nerve stimula- sequencing (WGS) allows the analysis of deep tion showed a remarkable decremental response of intronic, intergenic, and other noncoding re- 76% in proximal muscles. Both antiacetylcholine- gions. Furthermore, WGS allows to detect receptor and anti-MuSK antibodies were negative, and immunosuppressive treatment was unsuccessful. copy number variations (CNVs), as coverage Acetylcholinesterase (AChE) inhibitor of pyr- is more homogeneous than that of WES.7 idostigmine up to 360 mg/d for 10 years had little We present a CMS case in which a large effect and was discontinued without clinical deterio- intragenic DOK7 deletion was identified by ration after the trial of oral administration of salbu- WGS compound heterozygous to a known tamol which effected significantly. He has not exonic mutation. experienced severe muscle weakness for 5 years since salbutamol was started. METHODS DOK7 screening. DNA from the patient was extracted from whole blood by standard methods. Screening of DOK7 screening. Based on the limb-girdle clinical hot-spot mutations was performed by Sanger sequencing, en- presentation of the patient, a hot-spot region of compassing a region of ;600 bp covering the previously reported DOK7 was investigated as a first screening step. 2 European founder mutation c.1124_1127dup. Subsequently, Sanger sequencing revealed that the patient carried full screening of coding regions and exon-intron boundaries of the heterozygous c.1124_1127dup reported as the DOK7 gene was performed. Primer sequences are listed in 2 table e-1 at Neurology.org/ng. Annotation of the human DOK7 a founder mutation in European CMS patients. This cDNA is according to the GenBank accession number NM_ mutation was not present in the mother (DNA from 173660. the father was unavailable). However, this single Mutation analysis by WGS. WGS was performed by the Tru- heterozygous mutation does not explain DOK7- Seq PCR–free library preparation kit and HiSeqX v2 SBS kit CMS, which invariably shows autosomal recessive (Illumina, San Diego, CA) for 303 mean coverage on a HiSeqX inheritance. To identify a second heteroallelic DOK7 sequencer. Reads were mapped against hg19 reference genome variant, the whole coding region and exon-intron 8 using the Burrows-Wheeler transform, and duplicates were boundaries of the DOK7 gene were Sanger removed using Picard tools.9 sequenced, but no potentially pathogenic exonic or Sequence variants were called using the Genome Analysis Toolkit.10 WGS data were then analyzed using deCODE’s plat- splice site variants were found. The sample was form (Clinical Sequence Miner; WuXi NextCODE, Cambridge, therefore subjected to WGS to try to identify other MA). Rare variants were filtered by threshold of coverage ($8), mutations within the DOK7 gene or elsewhere in the variant call ($2), and ratio of variant ($0.2) and allele frequency genome. of 1% in 1000 Genomes database.11 WGS analysis. As expected, applying a standard pipe- Sanger sequencing of large deletion. We amplified line for variant filtering (minor allele frequency 1% in DNA samples to identify the suspected intragenic deletion with primers 59-CCCAGATGGTGCGCTTGCTCC-39and 59- coding region), the heterozygous c.1124_1127dup in GCCCACCCCCTCACGCTCAG-39. The PCR protocol com- DOK7 was detected in the WGS data. This filtering prised 35 cycles and annealing temperature of 68°C using did not identify any other coding variants in known HotStarTaq DNA polymerase with Q-Solution for the GC rich CMS causal genes. region (QIAGEN, Düsseldorf, Germany). However, visual inspection of the sequencing Standard protocol approvals, registrations, and patient reads of the DOK7 gene for this patient revealed that consents. All human studies including genetic analysis were the read depth for exons 1 and 2 was lower than that approved by institutional review boards, and appropriate written of neighboring regions and other control samples informed consent was obtained from all the patients and family (figure 1A). Furthermore, there were no heterozy- members. gous variants within this region, indicating a run of RESULTS Clinical findings. The patient is a 39-year- homozygosity or hemizygosity suggesting a single old Portuguese man who presented with bilateral copy region. Close inspection of the boundaries of ptosis and exercise-induced muscle weakness. He had this region showed that in some instances, sections no family history of muscle disease, and his motor of the sequencing reads did not match the reference milestones in childhood were normal. He showed sequence. These reads were considered chimeric or mild ptosis from infancy and noticed mild lower limb split reads, as the unmatched sequences did align to weakness at 13 years of age. He was admitted to a different region of the genome. Split reads are hospital for a month because of sudden severe gen- indicative of structural variation. In fact, the 39 sec- eralized muscle weakness and worsening ptosis at 15 tion of the split reads of the proximal boundary years of age. He has bilateral facial weakness and aligns to the 39end of the distal boundary, and vice winged scapula, and the clinical diagnosis of a neu- versa (figure 1B, red underline and red box). The romuscular transmission defect was confirmed by proximal and distal breakpoints lie approximately neurophysiologic studies. EMG showed myopathic 6 kb away. These findings suggested that this patient

2 Neurology: Genetics Figure 1 Whole-genome sequencing analysis and allele-specific PCR

(A) Both index case and his mother show reduced read depth (coverage) from exon 1 to deep intron 2 of the DOK7 gene (red arrow). Controls 1–4correspondto samples sequenced and analyzed through the same pipeline and without the diagnosis of congenital myasthenic syndromes. (B) Split reads were observedat both presumed breakpoints. Nucleotides matching the reference sequence of DOK7 are highlighted in orange/blue. Single unmatched nucleotides are highlighted in yellow, and further unmatched sequences are not highlighted. The unmatched sequence (indicated with red/green underline) of the split reads of the proximal breakpoint aligns to the reference sequence (indicated in green/red boxes) at the distal breakpoint, and vice versa. (C) The expected products amplified by allele-specific PCR were identified in the index case and the mother. (D) The junction of the breakpoint in the allele with the intragenic deletion was confirmed by Sanger sequencing of the PCR product. Coverage and reads were drawn by the graphical user interface of Sequence Miner 5.21.1 (WuXi NextCODE).

has a heterozygous 6-kb deletion in DOK7 encom- designed around 250 bp away from the presumed passing exons 1 and 2. breakpoints of the deletion, between the 59 untranslated region and intron 2. The expected Identification and analysis of the intragenic DOK7 product of 488 bp was amplified in the DNA deletion. We performed PCR using a pair of primers samples of the patient, but not in control DNA

Neurology: Genetics 3 (figure 1C). The junction of the 2 breakpoints was in the mother, who did not carry the identified by Sanger sequencing of the PCR prod- c.1124_1127dup mutation. We therefore con- uct (figure 1D). The exact size of the deletion is cluded that the CMS in the patient is caused by 6,261 bp. The deletion was also detected by PCR the compound heterozygous mutations in DOK7.

Figure 2 Analysis of the breakpoints of the intragenic 6-kb deletion

(A) University of California Santa Cruz genome browser (genome.ucsc.edu/) view of the deleted region showing the Simple Tandem Repeats track (based on Tandem Repeats Finder, TRF18) and the Repeating Elements track (based on Repeat- Masker19). GT-rich repeat regions (green box) are seen around the distal breakpoint, and a G-rich region (green arrow) is located near the proximal breakpoint. (B) The secondary DNA structure with the lowest delta G value was predicted by the mfold tool (unafold.rna.albany.edu/?q5mfold) for the 800 and 200 bp regions around the proximal breakpoint. An enlarged view of the breakpoint area highlighting the complementary nucleotides is also shown. The proximal breakpoint (indicated by the red arrows) is at the boundary of a loop and a 12-bp inverted repeat that may cause stalling of DNA replication. It is possible that deletion/duplication can occur if stalled replication resumes using an alternate location on the same chromosome. Red/blue/green bars represent hydrogen bonds between G-C/T-A/G-T.

4 Neurology: Genetics The 2 breakpoints of the deletion have a C-triplet of CNVs by read depth less reliable. This can be homology region, and the deleted region contains overcome by the homogenous coverage of WGS, al- a G-rich region and GT-rich repeat region (figure 2A). lowing both the detection of single nucleotide as well In silico secondary structure analysis using the prediction as CNV. program mfold12 showed that the proximal breakpoint is WGS analysis is still more expensive than WES at the boundary of a loop and a 12-bp inverted repeat and Sanger sequencing. In addition, computational (figure 2B). This may cause stalling of DNA replication tools need further improvement in sensitivity and and subsequently result in chromosomal structural specificity to detect CNVs exhaustively.15 Taken changes including deletions, if replication resumes using together, we believe that WGS is advantageous and an alternate chromosomal location. will become the method of choice for genetic diagnosis in rare, heterogeneous conditions such as CMS. Screening of the intragenic deletion in a CMS cohort. To We suggest that previously unsolved cases or the identify carriers of single heterozygous mutations in carriers of a single mutation in a causal gene are DOK7 (i.e., without a second rare variant within cod- especially suitable cases of CMS for WGS analysis. ing regions and exon-intron boundaries), we inter- The 6-kb deletion was not identified in other cases rogated our database of clinically diagnosed CMS tested by PCR, although it is inherited from the – cases referred to us in the years 1996 2015. The total mother, suggesting this is likely a private mutation. number of patients with CMS was 577, of which 7 However, it is possible that other CNVs in DOK7 genetically unsolved cases had single frameshift mu- underlie in CMS cases. tations in DOK7 (c.1124_1127dup in 6 cases and We also determined the breakpoints of the 6-kb c.1378dup in 1 case). These samples were amplified deletion, and analysis of the sequence and secondary using the deletion-specific pair of primers used to structure suggested that long inverted repeats might detect the 6-kb deletion of the index family. All 7 cause the development of the deletion due to a stall samples were negative using this PCR method. This of replication, and microhomology might have played does not exclude that they carry CNVs in DOK7 a role in the repair process.16 Further documentation different from the one described in this study. of breakpoints and sequences would help understand the mechanism for the development of CNVs. DISCUSSION We identified an intragenic DOK7 Obtaining genetic diagnosis of CMS is very deletion in a patient with clinically diagnosed CMS. important because the therapy varies depending on Patients lacking a second heteroallelic mutation in the affected gene. Poor response to AChE inhibitors DOK7 were reported in a previous study.2 Moreover, is often observed in patients affected by limb-girdle multiexon genomic deletions of RAPSN13 and CMS due to DOK7 mutations. Salbutamol therapy COLQ14 have also been identified as causative of has now been started for the patient described in this CMS. It is therefore conceivable that CNVs in study, which has been reported of good response in DOK7 may explain a proportion of cases assessed as DOK7-CMS.17 negative or inconclusive by conventional sequencing analysis. AUTHOR CONTRIBUTIONS Yoshiteru Azuma: drafting the manuscript, acquisition of data, and Our study shows the advantage of WGS analysis analysis and interpretation. Ana Töpf: analysis and interpretation and and detailed interrogation for detecting CNVs, using critical revision of the manuscript. Teresinha Evangelista and Paulo José coverage and visual analysis of split reads. Tradition- Lorenzoni: acquisition of data. Andreas Roos: analysis and interpreta- ally, multiplex ligation–dependent probe amplification and study supervision. Pedro Viana: acquisition of data. Hidehito Inagaki and Hiroki Kurahashi: analysis and interpretation. Hanns tion (MLPA) is considered the method of choice to Lochmüller: study concept and design and study supervision. detect previously described CNVs, where kits are available commercially. To identify new CNVs, STUDY FUNDING however, specific MLPA primers for each gene need Study funded by European Commission’s Seventh Framework Pro- gramme (FP7/2007-2013) under grant agreement no. 2012-305121 to be designed, rendering it expensive and time (NEUROMICS). Hanns Lochmüller—funding from the Medical consuming for testing a genetically heterogeneous Research Council as part of the MRC Centre for Neuromuscular Dis- syndrome such as CMS. Array comparative genomic eases (reference G1002274, grant ID 98482) and by the European Union hybridization (aCGH) is also a valuable method for Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 305444 (RD-Connect). CNVs analysis; nevertheless, deletions/duplications are not detectable by aCGH if they are shorter than DISCLOSURE the spacing of the hybridization probes. In addition, Yoshiteru Azuma, Ana Töpf, and Teresinha Evangelista report no disclo- neither MLPA nor aCGH can detect single nucleo- sures. Paulo José Lorenzoni has received research support from CNPq tide variants. Despite WES being widely used for (Brazil). Andreas Roos, Pedro Viana, Hidehito Inagaki, and Hiroki Kurahashi report no disclosures. Hanns Lochmüller has served on the clinical sequencing, the library preparation step re- scientific advisory boards of German Duchenne parents project, IRDiRC sults in uneven coverage, which makes the estimation Interdisciplinary Scientific Committee, German Muscular Dystrophy

Neurology: Genetics 5 Network, Myotubular Trust Patient Registry, Action Duchenne Patient technologies for the complete capture of protein-coding Registry, German Patient Registries on DMD, and SMA; has received regions. Hum Mutat 2015;36:815–822. travel funding/speaker honoraria from PTC Therapeutics Inc. and Ultra- 8. Li H, Durbin R. Fast and accurate long-read alignment genyx Pharmaceuticals Inc.; serves on the editorial boards of the Journal with Burrows-Wheeler transform. Bioinformatics 2010; of Neuromuscular Diseases and the Journal of Neurology; has been a con- 26:589–595. sultant for Roche Pharmaceuticals, ASD Therapeutics Partners LLC, IOS 9. Picard. Available at: broadinstitute.github.io/picard/. Press, Alexion Pharmaceuticals Inc., Ultragenyx Pharmaceutical Inc., and Fondazione Cariplo (funding from each paid to Newcastle University); Accessed March 23, 2017. and has received research support from Marigold Foundation Ltd., Ultra- 10. McKenna A, Hanna M, Banks E, et al. The Genome genyx Pharmaceutical Inc., PTC Therapeutics Inc., Eli Lilly and Co., Analysis Toolkit: a MapReduce framework for analyzing Action Benni & Co., GSK (GlaxoSmithKline), Trophos SA, European next-generation DNA sequencing data. Genome Res Commission (RD-Connect), European Commission (OPTIMISTIC), 2010;20:1297–1303. European Commission (NeurOmics), Medical Research Council (MRC), 11. Auton A, Brooks LD, Durbin RM, et al. A global National Institute for Health Research (NIHR), Action Duchenne, Asso- reference for human genetic variation. Nature 2015; ciation Francaise Contre les Myopathies, British Heart Foundation, Mus- 526:68–74. cular Dystrophy UK, National Cancer Institute, Spinal Muscular Atrophy 12. Zuker M. Mfold web server for nucleic acid folding and Support UK, Wellcome Trust, Jennifer Trust, and Duchenne Parent hybridization prediction. Nucleic Acids Res 2003;31: Project. Go to Neurology.org/ng for full disclosure forms. 3406–3415. 13. Gaudon K, Penisson-Besnier I, Chabrol B, et al. Mul- Received January 19, 2017. Accepted in final form March 14, 2017. tiexon deletions account for 15% of congenital myasthenic syndromes with RAPSN mutations after REFERENCES negative DNA sequencing. J Med Genet 2010;47: 1. Engel AG, Shen XM, Selcen D, Sine SM. Congenital 795–796. myasthenic syndromes: pathogenesis, diagnosis, and treat- 14. Wang W, Wu Y, Wang C, Jiao J, Klein CJ. Copy number ment. Lancet Neurol 2015;14:420–434. analysis reveals a novel multiexon deletion of the COLQ 2. Beeson D, Higuchi O, Palace J, et al. Dok-7 mutations gene in congenital myasthenia. Neurol Genet 2016;2: underlie a neuromuscular junction synaptopathy. Science e117. doi: 10.1212/NXG.0000000000000117. 2006;313:1975–1978. 15. Pirooznia M, Goes FS, Zandi PP. Whole-genome CNV 3. Das AS, Agamanolis DP, Cohen BH. Use of next- analysis: advances in computational approaches. Front generation sequencing as a diagnostic tool for congenital Genet 2015;6:138. myasthenic syndrome. Pediatr Neurol 2014;51:717–720. 16. Hastings PJ, Ira G, Lupski JR. A microhomology- 4. Garg N, Yiannikas C, Hardy TA, et al. Late presentations mediated break-induced replication model for the origin of congenital myasthenic syndromes: how many do we of human copy number variation. PLoS Genet 2009;5: miss? Muscle Nerve 2016;54:721–727. e1000327. 5. Bauche S, O’Regan S, Azuma Y, et al. Impaired presyn- 17. Lorenzoni PJ, Scola RH, Kay CS, et al. Salbutamol ther- aptic high-affinity choline transporter causes a congenital apy in congenital myasthenic syndrome due to DOK7 myasthenic syndrome with episodic apnea. Am J Hum mutation. J Neurol Sci 2013;331:155–157. Genet 2016;99:753–761. 18. Benson G. Tandem repeats finder: a program to 6. O’Connor E, Topf A, Muller JS, et al. Identification of analyze DNA sequences. Nucleic Acids Res 1999;27: mutations in the MYO9A gene in patients with congenital 573–580. myasthenic syndrome. Brain 2016;139:2143–2153. 19. Smit A, Hubley R, Green P. RepeatMasker Open-3.0. 7. Lelieveld SH, Spielmann M, Mundlos S, Veltman JA, 1996–2010. Available at: repeatmasker.org. Accessed Gilissen C. Comparison of exome and genome sequencing March 23, 2017.

6 Neurology: Genetics HSP and deafness Neurocristopathy caused by a novel mosaic SOX10 mutation

Sandra Donkervoort, MS, ABSTRACT CGC Objective: To identify the underlying genetic cause in 2 sisters affected with progressive lower Diana Bharucha-Goebel, extremity spasticity, neuropathy, and early-onset deafness. MD Methods: Whole-exome sequencing was performed, and segregation testing of variants was Pomi Yun, MPH investigated using targeted Sanger sequencing. An inherited paternal mosaic mutation was fur- Ying Hu, MS ther evaluated through quantitative analysis of the ratio of mutant vs wild-type allele in genomic Payam Mohassel, MD DNA from various tissues, including blood, dermal fibroblasts, and saliva. Ahmet Hoke, MD, PhD . Wadih M. Zein, MD Results: A novel heterozygous nonsense mutation (c.1140C A; p.Y380X) in SOX10 was identified Daniel Ezzo, BS in the affected sisters. Paternal mosaicism was suspected based on a small chromatogram peak, Andrea M. Atherton, MS, which was less than the heterozygous peak of the mutated allele. Consistent with mosaicism, the CGC mosaic paternal samples had notable variability in the ratio of mutant vs wild-type allele in various Ann C. Modrcin, MD tissues (compared with the fully heterozygous daughter), with the highest paternal mutant levels in Majed Dasouki, MD saliva (32.7%) and lowest in dermal fibroblasts (13.9%). Targeted clinical re-examination of the father A. Reghan Foley, MD revealed a sensorimotor neuropathy that was previously clinically unrecognized. Carsten G. Bönnemann, Conclusions: These findings expand the phenotypic spectrum of SOX10-related neurocristop- MD athy. Mutations in SOX10 should be considered in patients presenting with a complicated form of hereditary spastic paraplegia that includes neuropathy and deafness. Diagnostic workup may be complicated, as SOX10 mutations can present in a mosaic state, with a mild clinical Correspondence to manifestation. Neurol Genet 2017;3:e151; doi: 10.1212/NXG.0000000000000151 Dr. Bönnemann: [email protected] GLOSSARY CV 5 conduction velocity; HSP 5 hereditary spastic paraplegia; NCC 5 neural crest cell; NMD 5 nonsense-mediated decay; RT 5 real time; SRY 5 sex determining region Y; WS4 5 Waardenburg syndrome type 4; WT 5 wild type.

SOX10 (SRY [sex determining region Y]-box 10) is a SRY-related transcription factor that plays a critical role in the early development of the pluripotent neural crest lineage and is necessary for cell fate determination and cell lineage development.1–3 These cell types include neurons and glia of the peripheral nervous system, Schwann cells, enteric neurons, facial skeleton and connective tissues, and melanocytes of the skin and of the inner ear.4–6 A defect in the neural crest cell (NCC) lineage can cause a clinical and genetic heterogeneous group of NCC disorders known as neurocristopathies.7–9 Historically, SOX10 mutations were known to cause a relatively restricted auditory- pigmentary phenotype known as Waardenburg syndrome type 4C (WS4), manifesting as Waardenburg syndrome with Hirschsprung disease (OMIM 613266).10,11 It was not until more recently that a distinct neurologic phenotype resulting from SOX10 mutations was recognized, which includes peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome and Hirschsprung disease (PCWH, OMIM Supplemental data at Neurology.org/ng From the Neuromuscular and Neurogenetic Disorders of Childhood Section (S.D., D.B.-G., P.Y., Y.H., P.M., D.E., A.R.F., C.G.B.), and National Eye Institute (W.M.Z.), National Institutes of Health, Bethesda, MD; Children’s National Medical Center (D.B.-G.), Washington, DC; Department of Neurology (A.H.), The Johns Hopkins University School of Medicine, Baltimore, MD; Children’s Mercy Hospital (A.M.A., A.C.M.), Kansas City, MO; and Department of Neurology (M.D.), University of Kansas Medical Center, Kansas City, KS. Funding information and disclosures are provided at the end of the article. Go to Neurology.org/ng for full disclosure forms. 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.

Neurology.org/ng Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology 1 609136).9 Here, we present 2 sisters with regular clinical diagnostic workup and were evaluated by standard a unique presentation of early-onset bilateral light and electron microscopy protocols. sensorineural hearing loss, progressive distal Mutation detection. Whole-exome sequencing on blood sam- lower extremity spasticity, hypomyelinating ples obtained from patient 1 (P1), patient 2 (P2), 1 unaffected sensorimotor neuropathy, mild pigmentary brother, and the parents was performed at the NIH Intramural Sequencing Center using the Illumina (San Diego, CA) TruSeq abnormalities, and normal cognition. A Exome Enrichment Kit, and Illumina HiSeq 2500 sequencing in- novel truncating mutation in SOX10 was struments. Variants were analyzed using VarSifter12 and searched identified in each sister through exome for in the National Heart, Lung, and Blood Institute Exome Sequencing Project database (evs.gs.washington.edu/EVS/) and sequencing and was found to be inherited Exome Aggregation Consortium database (exac.broadinstitute. fromamosaicfather.Somaticmosaicism org). PCR amplification of exon 4 of SOX10 in the complete was confirmed through quantitative analysis family was followed by Sanger sequencing on an ABI 3130 31 of the relative ratio of the mutant allele in capillary sequencer, in forward and reverse directions. Results were then confirmed in an outside Clinical Laboratory blood (23.3%), dermal fibroblasts (13.9%), Improvement Amendment–certified laboratory. and saliva (32.7%). Quantification of mutant vs normal allele. Genomic DNA METHODS Standard protocol approvals, registrations, was extracted from blood, dermal fibroblasts, and saliva using Custom TaqMan SNP Genotyping Assays (Life Technologies, and patient consents. The study was approved by the Institu- Grand Island, NY), consisting of mutation-specific primers, tional Review Board of the National Institute of Neurological and fluorescent-labeled allele discrimination probes were de- Disorders and Stroke, NIH (Protocol 12-N-0095). Written signed for the mutation using a custom design tool provided by informed consent and appropriate assent were obtained by Applied Biosystems (Grand Island, NY [table e-1 at Neurology. a qualified investigator. Medical history was obtained, and clinical org/ng]). Twenty-five nanograms of genomic DNA was used evaluations were performed as part of the standard neurologic and with total volume of 5 mL for each reaction. The master mix was ophthalmologic evaluations. Genomic DNA was obtained from ordered from the manufacturer (4371353; Life Technologies, blood, saliva, and dermal fibroblasts based on standard proce- Grand Island, NY). The fluorescent readings were recorded after dures. Muscle and nerve biopsies were obtained as part of the 40 amplification cycles. The amplification was performed using the QuantStudio 6 Flex real-time (RT) PCR system (Applied Figure 1 Clinical findings Biosystems), and signals were recorded by QuantStudio Real- time PCR software v1.1. Reactions were run in triplicates. COL6A1 was used as an endogenous quantity control for the samples. Quantitative analysis of the percentage of mosaicism was performed as previously reported.13 Mutant allele quantities were calculated by using the DD cycle threshold (DDCt) method: DDCt 5 DCt (mosaic) 2 DCt (het). (DCt was defined as the difference between the mean Ct values of mutant and wild type [WT].)

RESULTS Case report. Patient 1 (P1) is a 19-year-old woman with a history of lower extremity spasticity, neuropathy, and bilateral early-onset deafness who presented for diagnostic evaluation. First concerns arose prenatally with reduced fetal movements. The hearing loss was first recognized at 2 months of age, and brain MRI revealed the absence of the semicircular canals bilaterally. She had early gross motor delays and began walking with an unsteady gait at 3 years of age. By 7 years of age, she had developed a prominent waddling-type gait, bilateral foot drop, and had frequent falls. By 9 years of age, she could only ambulate short distances independently, and by 13 years of age, she was unable to walk independently without assistance. Progressive lower extremity spasticity has been noted since 16 years of age. P1 reportedly had evidence of some early fine motor difficulties. She has no history of seizures or learning difficulties. She has a history of urinary urgency and Cutaneous hypopigmentation is seen in P1 (A) and P2 (E). Photographs demonstrating scissoring-type gait with evidence of proximal lower extremity weakness in P1 (B–D) constipation (with no history of Hirschsprung and P2 (F–H). disease).

2 Neurology: Genetics Neurologic examination at 19 years of age revealed Reflexes were absent at the biceps and Achilles ten- mild dysmorphic facial features, including a high- dons bilaterally, 11 at the brachioradialis, and 31 at arched palate, a small-sized mouth, and thin, up- the patellae bilaterally. Babinski response was equiv- turned nostrils. There was evidence of a kyphotic ocal. There was prominent lower extremity spasticity posture. Skin examination was notable for areas of hy- with evidence of a spastic catch bilaterally. Ambula- popigmentation on the face and the flexor surfaces of tion was dependent on full truncal support. She had the elbows (figure 1A). There was a head tremor at a scissoring gait with circumduction of the legs and rest. Extraocular movements were full. Occasional bilateral foot drop (figure 1, B–D). Rapid alternating fine nystagmus was noted during funduscopic exam- movements were slow with reduced amplitude and ination only. Facial strength was normal. Upper evidence of mirror movements. There was dysmetria extremity strength was approximately 5/5 (the Med- on finger-to-nose testing which was increased with ical Research Council grade) except for the thumb eyes closed (fixed target). abduction which was 41/5, and lower extremity Nerve conduction studies (19 years of age) were strength was in the 52/5 range. Sensation was consistent with a demyelinating sensory and motor reduced to pinprick and vibration in a length- neuropathy, evidenced by diffuse, symmetric reduc- dependent fashion. Proprioception was normal. tion in nerve conduction velocities (CVs) (peroneal

Figure 2 Chronic hypomyelinating neuropathy

Plastic-embedded semithin sections (A and B) show a uniform reduction in large and small myelinated fiber density in P1. Several thinly myelinated fibers (arrowheads) and a few primordial onion bulb formations are present (arrows). Multiple empty Schwann cell nuclei are seen (S). Electron microscopy images (C and D) show similar findings. An onion bulb (C, lower right arrow) and an empty onion (C, upper right arrow) are highlighted. D is a higher magnification image of the area of the box in C. There are several empty Schwann cells and Remak bundles (S), suggestive of loss of unmyelinated fibers. Some larger axons appear to have no myelin (*), suggestive of defective myelination. (Scale bars: A: 50 mm, B: 20 mm, C: 10 mm, and D: 2 mm.)

Neurology: Genetics 3 fetal movements in utero as well as breech position Figure 3 Ophthalmologic findings at delivery. She was diagnosed with profound sensorineural hearing loss at 12 months of age, and brain MRI performed in infancy reportedly revealed the absence of 1 semicircular canal. P2 also has a history of delayed gross motor development (walking independently at 2½ years of age) and mild early fine motor difficulties. She was noted to have a wide- based gait at 2½ years of age, developed spasticity over time, and by 9 years of age was using a wheel- chair for longer distances. At 15 years of age, she can ambulate with minimal assistance (figure 1, F–H). She had been diagnosed with a progressive demyelinating sensorimotor neuropathy. On examination, upper extremity strength was approximately 5/5 except for finger extension and thumb abduction which were 41/5. Lower extremity strength was subgravity in hip flexion, hip abduction, hip adduc- tion, dorsiflexion, eversion, plantarflexion, and inversion. Sensation was reduced to pinprick and vibration in a length-dependent fashion. Proprio- ception was normal. Reflexes were absent at the biceps and Achilles tendons bilaterally, 11 at the brachioradialis, and 41 at the patellae bilaterally with cross-adductor spread. There was a spastic catch in the bilateral lower extremities. Rapid alter- (A) Color and red-free fundus photography of the right eye of P1 showing pigmentary nating movements were slow with reduced ampli- changes in a localized area temporal to the macula. Arrows point at the area of patchy hypopigmentation corresponding to the changes noted on optical coherence tomography. (B) tude. There was evidence of dysmetria with eyes Optical coherence tomography of P1 indicating an irregular bowing back of the retina closed only. Skin examination was notable for mul- (arrows) with a change in the reflectance of the choroid in the area corresponding to the pig- tiple areas of hypopigmentation (figure 1E). mentary changes noted on examination. There is no interruption of the retinal layers and no loss of retinal cells. Detailed clinical information for both sisters is summarized in table 1. P1 and P2’s mother and father did not report any motor: CV 11.7 m/s with amplitude 1.5 mV; tibial symptoms. On further questioning, however, the 39- motor: CV 19.4 m/s with amplitude 2.3 mV) and year-old father (P3) reported a history of frequent prolonged distal latencies. A sural nerve biopsy per- ankle sprains, apparent high-arched feet, and mild formed at 8 years of age was consistent with a chronic hammertoe deformities, suggestive of a chronic neu- predominantly hypomyelinating neuropathy (figure 2), ropathy of likely genetic etiology. On neurologic and muscle biopsy of the quadriceps performed at the examination, he was found to have a symmetric, same time was suggestive of neurogenic changes with length-dependent sensory greater than motor neurop- evidence of fiber-type grouping. X-ray of the spine athy with reduction of pain and vibration sensation in showed a 14-degree curvature of the thoracic and lum- the distal lower extremities. He had reduced reflexes bar spine from T11 to L3. throughout, including in the upper extremities Fundoscopic examination at age 19 years showed (table 1). Proprioception was normal. His ophthal- a well-developed foveal structure but with evidence mologic examination was unremarkable except for of bilateral patches of thinning and anomalous pig- mild myopia. Nerve conduction studies showed mentation (mostly depigmented) temporal to the absent sural sensory nerve action potential, suggest- central macula (figure 3A). Optical coherence tomog- ing the possibility of an existing neuropathy given raphy indicated bowing out of the retina/choroid the absent response at his age. He was found to have underlying the patchy areas of anomalous pigmenta- baseline sinus bradycardia and orthostasis (with an tion (figure 3B), suggestive of a developmental anom- increase in the heart rate by more than 20 beats aly of embryogenesis likely affecting the sclera in when transitioning from supine to standing), which those regions. may be suggestive of a mild autonomic neuropathy Patient 2 (P2) is a 15-year-old girl and the youn- as well. Other quantitative autonomic testing was gersisterofP1.Familyhistoryisshowninfigure4. unremarkable. Detailed clinical information can be She presented similarly to her sister with decreased found in appendix e-1.

4 Neurology: Genetics Figure 4 Pedigree and molecular characterization of the family

Family history showing the mosaic father (P3), his unaffected spouse (M), the 2 affected daughters (P1 and P2), and 3 unaffected sons (B1, B2, and B3). Genomic DNA sequence chromatograms show the somatic c.1140C.A; p.Y380X SOX10 mutation in the mosaic father and 2 affected daughters. The mosaic mutation sequence is seen as a lower-height peak in parent (P3) compared to the fully heterozygous mutation in the affected children P1 and P2.

Identification of a SOX10 mutation. Exome sequencing Paternal mosaicism was confirmed through quanti- identified a heterozygous mutation in SOX10: tative RT-PCR analysis of mutant allele vs WT allele (c.1140C.A [p.Y380X] [NM_006941.3]) in both in blood, dermal fibroblasts, and saliva in the affected sisters. This nonsense mutation was not present in patients compared with the mosaic and control par- the control database of the National Heart, Lung, ent. Although no symptoms were volunteered by the and Blood Institute Exome Sequencing Project and parent with the mosaicism, on careful clinical evalu- Exome Aggregation Consortium. The mutation was ation, he was found to have a mild sensory neurop- confirmed through Sanger sequencing, and familial athy and mild autonomic dysfunction. segregation studies were negative in the 3 asymptom- Mosaicism is a result of a postzygotic mitotic muta- atic brothers of P1 and P2 and their mother. Paternal tional event with the mutation confined to somatic mosaicism was suspected because a small peak of the cells (somatic mosaicism), germline cells (gonadal mutated allele was observed in the father’s chromato- mosaicism), or both, depending on the developmental gram (figure 4). stage and lineage at which the event occurred. The clinical manifestations of somatic mosaicism are highly Mutant vs normal allele quantification. To confirm and determine the degree of paternal mosaicism, the rela- variable and depend on the type of mutation, the tissue tive ratio of the mutant allele in the father was ana- distribution, and the relative mutation load. Recent ad- lyzed in different somatic tissues, with 3 pairs of vances in genetic technologies have shown that paren- RT PCR probes specific to either SOX10 mutant or tal mosaicism underlying the transmission of WT allele. Genomic DNA from saliva showed the mutations from parents without a clinical phenotype highest proportion of approximately 32.7% of the to children with a simplex genetic disorder is more 14 mutant allele, while the mutant ratio was 23.3% in common than previously appreciated. Mosaicism peripheral blood and 13.9% in dermal fibroblasts. can thus complicate the diagnostic workup and the goal of providing accurate genetic counseling. DISCUSSION Here, we present 2 sisters with The known neurologic manifestations of a unique phenotype of complicated hereditary spastic SOX10-related neurocristopathy include peripheral paraplegia (HSP) manifesting as progressive lower demyelinating neuropathy, central dysmyelinating extremity spasticity, demyelinating neuropathy, and leukodystrophy, Waardenburg syndrome, and early-onset sensorineural hearing loss with normal Hirschsprung disease (PCWH). Spasticity is rare, cognition, resulting from a novel mutation in SOX10. having been reported in 4 different patients and at

Neurology: Genetics 5 Table 1 Clinical findings

Patient/sex P1/F P2/F P3/M

SOX10 mutation c.1140C.A; p.Y380X c.1140C.A; p.Y380X c.1140C.A; p.Y380X

Mutation status Heterozygous Heterozygous Mosaic

Age at examination 19 y 15 y 39 y

Fetal movements/birth Reduced/uncomplicated Reduced/C-section (breech) NA

Gross motor development Sat 10 mo; walked 2½ y; unsteady Sat 10 mo; walked 2½ y; decreased NL gait 7 y; lost independent ambulation endurance 10 y 13 y

Speech delay 122

EMG/NCS (age study) Severe demyelinating sensorimotor Moderate demyelinating Mild distal sensory neuropathy (CV polyneuropathy (CV range 10–31 m/s); sensorimotor polyneuropathy (CV range 38–61 m/s); needle EMG NL needle EMG with mixed myopathic and range 18–27 m/s); needle EMG with (39 y) neurogenic MUPs (19 y) myopathic recruitment/MUPs (11 y)

Brain MRI (age of study) Absence of semicircular canals; Absence of 1 semicircular canal; NL (39 y) normal brain (2 y); images not normal brain (10 mo); images not available for rereview available for rereview

Urinary incontinences Daily urinary urgency and leakage 22

Constipation/Hirschsprung disease 1/21/22/2

Hearing loss/age at diagnosis/type Severe (bilateral)/2 mo/Mondini Severe (bilateral)/newborn screen/ 2/2/2 malformation and absence of the Mondini malformation and absence of semicircular canals 1 semicircular canal

Cochlear implants/ (age) 1 (26 mo) 1 (12 mo) 2/NA

Nystagmus Horizontal bilateral nystagmus noted Noted at 5 wk, resolved at 3 y 2 at 1 mo, resolved at 3 y

Bright blue irides/iris heterochromia 2/22/22/2

Hypopigmentation (eyes) Pigmentary change temporal to Pigmentary change temporal to 2 macula macula

Hypopigmentation (skin) Cubital fossae, cheeks Cheeks, neck, upper chest, cubital 2 fossae, back

Hyperpigmentation (skin) Right lower leg (4 cm light brown 22 patch)

White forelock 222

Abbreviations: 15present; 25absent; CV 5 conduction velocity; F 5 female; M 5 male; MUP 5 motor unit potential; NA 5 not available; NCS 5 nerve conduction study; NL 5 normal; UE 5 upper extremities.

times associated with additional PCWH manifesta- haploinsufficiency, which causes the restricted and tions including seizures and cognitive impairment, classic WS4 phenotype of Waardenburg syndrome features which were notably absent in our pa- and Hirschsprung disease, without peripheral or tients.9,15,16 Moreover, our patients did not have CNS involvement. By contrast, mutations in the last distinct symptoms of Waardenburg syndrome or exon escape NMD and generate a stable truncated Hirschsprung disease. Both sisters have a history SOX10 mutant protein with increased DNA- of constipation, and after deep phenotyping, cuta- binding affinity, which acts in a dominant-negative neous and macular hypopigmentation were identi- manner, and cause the severe PCWH neurocristop- fied in both, compatible with a “forme fruste” of athy with neurologic manifestations. More proximal Waardenburg syndrome. mutations within exon 5 (Q234X, Q250X, and The SOX10 gene consists of 5 exons and contains S251X) exhibit a stronger dominant-negative effect a high mobility group, a DNA-binding domain and and lead to more severe congenital-onset symptoms a C-terminal transactivation domain.17 Truncating and possible neonatal death.18–20 The novel mutation SOX10 mutations can lead to PCWH or WS4, and (Y380X) identified in our family is very close to the recent work has suggested that the 2 distinct pheno- C-terminal end of SOX10, with only 2 mutations types may be explained by 2 different molecular reported that are more distal9: the X467K mutation, pathogenic mechanisms.9 Truncating mutations in which was identified in a patient with severe motor any exons, except the last one, lead to mutant and cognitive delays, and the 1400del12 mutation, mRNA which is recognized and degraded through which was found in a patient with complete defi- nonsense-mediated decay (NMD). This results in ciency of brain myelination and seizures who never

6 Neurology: Genetics reached independent ambulation.16,21 It is of interest We have presented 2 sisters with a unique phe- that in our family, the Y380X mutation is not asso- notype of early-onset bilateral sensorineural hear- ciated with cognitive impairment or seizures, indicating loss, progressive distal lower extremity ing that there may be other protective factors at play. spasticity, demyelinating sensorimotor neuropa- SOX10 is involved in the early development of the thy, mild pigmentary abnormalities, and normal NCCs and is involved in Schwann cells and oligoden- cognition. Our report expands the phenotypic drocytes later in development as well as into adult- spectrum of SOX10-related neurocristopathy. Mu- hood.17 Sural nerve pathology in patient 1 showed tations in SOX10 should be considered in patients findings consistent with a chronic hypomyelinating presenting with a phenotype of complicated HSP neuropathy with reduction of myelinated fibers and with hypomyelinating neuropathy and deafness, defective myelination of axons. Because of the presence while SOX10 somatic mosaicism may manifest of cochlear implants, both sisters were unable to withonlyamildsensoryneuropathy. undergo brain MRI to evaluate for possible central demyelination. The progressive weakness and spastic- AUTHOR CONTRIBUTIONS ity seen in these 2 patients may potentially suggest S. Donkervoort: study concept and design, analysis, interpretation of genetic and clinical data, and drafting manuscript. D. Bharucha-Goebel aroleofSOX10 that is not only restricted to periods and P. Yun: acquisition and interpretation of clinical data and revision of neural development but is also involved in the pro- of manuscript. Y. Hu: analysis and interpretation of genetic data. P. Mo- duction and maintenance of myelin and axonal health. hassel, A. Hoke, W.M. Zein, AM. Atherton, AC Modrcin, and M Da- souki: acquisition and interpretation of clinical and biopsy data. AR An understanding of the underlying pathogenic Foley and C.G. Bönnemann: supervising the acquisition and interpreta- disease mechanism is essential for developing targeted tion of clinical and genetic data and critical revision of the manuscript for therapeutic interventions. Clinical and genetic data important intellectual content. All authors critically reviewed and evaluated in this family show that a reduction in approved the final manuscript. the relative dose of the mutant allele, as seen in the ACKNOWLEDGMENT mosaic parent, ameliorates the clinical severity, indi- The authors thank the family for their participation in the study. They cating that as a therapeutic approach in PCWH, thank the NIH Intramural Sequencing Center and Dr. Hakonarson and knockdown of the mutant allele expression could be Dr. Guo from The Center of Applied Genomics, Children’s Hospital of achieved through antisense-mediated or RNAi-based Philadelphia, for performing the exome sequencing. They thank Dr. Anne Rutkowski and CureCMD for their help with patient recruitment. therapy. Careful titration is essential, as haploinsufficiency leads to the classic WS4 phenotype. The WS4 STUDY FUNDING presentation may be more developmental in origin; Work in C.G. Bönnemann’s laboratory is supported by intramural funds therefore, the presence of haploinsufficiency postna- by the National Institute for Neurological Disorders and Stroke/NIH. Exome sequencing was funded through the Clinical Center Genomics tally (as induced via allele-specific knockdown) may Opportunity (CCGO), which is sponsored by the National Human have less of a clinical effect. Moreover, this approach Genome Research Institute (NHGRI), the NIH Deputy Director for may potentially only alleviate the progressive neuro- Intramural Research, and the NIH Clinical Center. muscular symptoms, as the classic WS4 symptoms DISCLOSURE are present at birth. Dr. Bharucha-Goebel, Mohassel, Hoke, Zein, Modrcin, Dasouki, Foley, Based on this family history of 2 affected sisters Bönnemann, Ms. Donkervoort, Yun, Hu, and Mr. Ezzo report no dis- born to apparently unaffected parents, autosomal closures. Andrea M. Atherton is now a full-time employee of Shire Phar- recessive inheritance was initially suspected, and the maceuticals. Go to Neurology.org/ng for full disclosure forms. family was counseled accordingly. A confirmed Received September 23, 2016. Accepted in final form March 22, 2017. genetic diagnosis allowed for accurate genetic counseling as the recurrence risk in future pregnancies of REFERENCES the patients is 50%, which is higher than that previ- 1. Kim J, Lo L, Dormand E, Anderson DJ. SOX10 main- ously anticipated. Genetic counseling for the mosaic tains multipotency and inhibits neuronal differentiation of parent (the father) was more complicated, as recur- neural crest stem cells. Neuron 2003;38:17–31. rence risk depends on the ratio of mutated germline 2. Southard-Smith EM, Kos L, Pavan WJ. Sox10 mutation disrupts neural crest development in Dom Hirschsprung progenitor cells, which cannot be predicted from mouse model. Nat Genet 1998;18:60–64. analysis of somatic tissues. In addition to his 2 3. Kapur RP. Early death of neural crest cells is responsible for affected daughters, the mosaic parent at present has total enteric aganglionosis in Sox10(Dom)/Sox10(Dom) 3 unaffected children. In reviewing families with mouse embryos. Pediatr Dev Pathol 1999;2:559–569. parental mosaicism for dominant mutations causing 4. Le Douarin NM, Kalcheim C. The Neural Crest, 2nd ed. osteogenesis imperfecta and retinoblastoma, the Cambridge, United Kingdom: Cambridge University Press; 1999. recurrence rate was estimated to be 27% and 10%, 5. Cordero DR, Brugmann S, Chu Y, Bajpai R, Jame M, 22,23 respectively. Caution is necessary when counsel- Helms JA. Cranial neural crest cells on the move: their ing for somatic mosaicism, as an individual’s recur- roles in craniofacial development. Am J Med Genet A rence risk may be as high as 50%. 2011;155A:270–279.

Neurology: Genetics 7 6. Bondurand N, Dastot-Le Moal F, Stanchina L, et al. Dele- 16. Inoue K, Tanabe Y, Lupski JR. Myelin deficiencies in both tions at the SOX10 gene locus cause Waardenburg syndrome the central and the peripheral nervous systems associated types 2 and 4. Am J Hum Genet 2007;81:1169–1185. with a SOX10 mutation. Ann Neurol 1999;46:313–318. 7. Bolande RP. The neurocristopathies: a unifying concept of 17. Kuhlbrodt K, Herbarth B, Sock E, Hermans-Borgmeyer I, disease arising in neural crest maldevelopment. Hum Pathol Wegner M. Sox10, a novel transcriptional modulator in 1974;5:409–429. glial cells. J Neurosci 1998;18:237–250. 8. Read AP, Newton VE. Waardenburg syndrome. J Med 18. Inoue K, Shilo K, Boerkoel CF, et al. Congenital hypo- Genet 1997;34:656–665. myelinating neuropathy, central dysmyelination, and 9. Inoue K, Khajavi M, Ohyama T, et al. Molecular mechanism Waardenburg-Hirschsprung disease: phenotypes linked for distinct neurological phenotypes conveyed by allelic trun- by SOX10 mutation. Ann Neurol 2002;52:836–842. cating mutations. Nat Genet 2004;36:361–369. 19. Pingault V, Girard M, Bondurand N, et al. SOX10 muta- 10. Omenn GS, McKusick VA. The association of Waardenburg tions in chronic intestinal pseudo-obstruction suggest syndrome and Hirschsprung megacolon. Am J Med Genet a complex physiopathological mechanism. Hum Genet 1979;3:217–223. 2002;111:198–206. 11. Pingault V, Bondurand N, Kuhlbrodt K, et al. SOX10 20. Touraine RL, Attie-Bitach T, Manceau E, et al. Neuro- mutations in patients with Waardenburg-Hirschsprung logical phenotype in Waardenburg syndrome type 4 cor- disease. Nat Genet 1998;18:171–173. relates with novel SOX10 truncating mutations and 12. Teer JK, Green ED, Mullikin JC, Biesecker LG. VarSifter: expression in developing brain. Am J Hum Genet 2000; Visualizing and analyzing exome-scale sequence variation data 66:1496–1503. on a desktop computer. Bioinformatics 2012;28:599–600. 21. Sham MH, Lui VC, Chen BL, Fu M, Tam PK. Novel 13. Hsu AP, Sowerwine KJ, Lawrence MG, et al. Intermediate mutations of SOX10 suggest a dominant negative role in phenotypes in patients with autosomal dominant hyper- Waardenburg-Shah syndrome. J Med Genet 2001;38: IgE syndrome caused by somatic mosaicism. J Allergy Clin E30. Immunol 2013;131:1586–1593. 22. Sippel KC, Fraioli RE, Smith GD, et al. Frequency of 14. Campbell IM, Yuan B, Robberecht C, et al. Parental somatic and germ-line mosaicism in retinoblastoma: im- somatic mosaicism is underrecognized and influences plications for genetic counseling. Am J Hum Genet 1998; recurrence risk of genomic disorders. Am J Hum Genet 62:610–619. 2014;95:173–182. 23. Pyott SM, Pepin MG, Schwarze U, Yang K, Smith G, 15. Pingault V, Pierre-Louis L, Chaoui A, et al. Phenotypic Byers PH. Recurrence of perinatal lethal osteogenesis im- similarities and differences in patients with a p.Met112Ile perfecta in sibships: parsing the risk between parental mutation in SOX10. Am J Med Genet Part A 2014;164A: mosaicism for dominant mutations and autosomal reces- 2344–2350. sive inheritance. Genet Med 2011;13:125–130.

8 Neurology: Genetics Genetic analysis of age at onset variation in spinocerebellar ataxia type 2

K.P. Figueroa, MS ABSTRACT Hilary Coon, PhD Objective: To examine heritability of the residual variability of spinocerebellar ataxia type 2 Nieves Santos, MS (SCA2) age at onset (AO) after controlling for CAG repeat length. Luis Velazquez, MD, Methods: From 1955 to 2001, dates of birth, CAG repeat lengths, AO, sex, familial inheritances, PhD, DrSc and clinical manifestations were collected for a large Cuban SCA2 cohort of 382 affected indi- Luis Almaguer Mederos, viduals, including 129 parent-child pairs and 69 sibships. Analyses were performed with log- PhD transformed AO in the GENMOD procedure to predict AO using repeat length, taking into account Stefan M. Pulst, MD, family structure. Because all relationships were first degree, the model was implemented with an Dr med exchangeable correlation matrix. Familial correlations were estimated using the Pedigree Analy- sis Package to control for similarity due to genetic relatedness.

Correspondence to Results: For the entire sample, the mutant CAG repeat allele explained 69% of AO variance. When Dr. Pulst: adjusted for pedigree structure, this decreased to 50%. Evidence for imprinting or sex-specific [email protected] effects of the CAG repeat on AO was not found. For the entire sample, we determined an upper bound for heritability of the residual variance of 33% (p 5 0.008). Heritability was higher in sib- sib pairs, especially in female sib-sib pairs, than in parent-child pairs. Conclusions: We established that a large proportion of AO variance in SCA2 was determined by genetic modifiers in addition to CAG repeat length. The genetic structure of heritability of the residual AO variance was surprisingly similar to Huntington disease, suggesting the presence of recessive modifying alleles and possibly X-chromosome–linked modifiers. Neurol Genet 2017;3:e155; doi: 10.1212/NXG.0000000000000155

GLOSSARY AO 5 age at onset; FC 5 father-child; HD 5 Huntington disease; MC 5 mother-child; PAP 5 Pedigree Analysis Package; PC 5 parent-child; polyQ 5 polyglutamine; RL 5 repeat length; SCA2 5 spinocerebellar ataxia type 2; SNP 5 single nucleotide polymorphism.

Ten human neurodegenerative diseases, including spinocerebellar ataxia type 2 (SCA2), are caused by expansions of a coding CAG repeat. These diseases are referred to as polyglutamine (polyQ) diseases. Common to all polyQ diseases is a decrease in age at onset (AO) with increasing repeat lengths (RLs). The square of the correlation coefficient (r2) ranges from 0.5 to 0.8.1–4 Despite this correlation, there is great variation of AO within each repeat class. This suggests the existence of other factors influencing disease onset and progression. A study of the Venezuelan Huntington disease (HD) population estimated that about 37% of the residual AO variance was attributable to additive genetic effects.5 Analysis of a second, multinational HD sample determined that 56% of the residual AO variance was heritable and revealed suggestive evidence for genetic modifiers on 4p16, 6p21–23, and 6q24–26.6,7 In an analysis of 148 Cuban SCA2 patients in 57 sibships, we determined that about half of the residual AO variance was heritable.3 SCA2 is relatively rare worldwide but has a prevalence of 40/100,000 in the Northeastern part of Cuba.8 This founder population9 provides a resource for genetic studies similar to the

From the Department of Neurology (K.P.F., S.M.P.), Department of Psychiatry (H.C.), University of Utah, Salt Lake City; Department of Surgery (N.S.), University of Miami, FL; and Center for the Research and Rehabilitation of Hereditary Ataxias (L.V., L.A.M.), Holguin, Cuba. Funding information and disclosures are provided at the end of the article. Go to Neurology.org/ng for full disclosure forms. 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.

Neurology.org/ng Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology 1 HD population in Venezuela.5 As in other to predict AO using RL, taking into account family structure. polyQ diseases, the mutant SCA2 allele ex- GENMOD uses generalized estimating equations to account for the nonindependence among related individuals. plains between 0.55 and 0.7 of the AO vari- Sensitivity analysis was not performed to determine bias due ance in SCA2.3,10 to aspects of sampling. However, certain aspects of sensitivity We report an analysis of factors influencing analysis were performed by reanalysis of data using different con- AO in the Cuban SCA2 cohort. We deter- stellations of relatives, parent-offspring and sib pairs. Because all relationships were first degree, the model was implemented with mined sex and imprinting effects on RL and an exchangeable correlation matrix. AO, as well as familial correlations for Significance of familial resemblance was estimated using the parent-child (PC) and sibling pairs. Pedigree Analysis Package (PAP).12 Familial correlations were estimated using the PAP to control for similarity due to genetic relatedness. METHODS Standard protocol approvals, registrations, An upper bound of heritability was computed in SOLAR.13 and patient consents. Informed consent was obtained, and SOLAR tests the likelihood of heritability due to multiple gene studies were approved by review boards in Holguin and Utah. effects (a polygenic model) of AO after taking into account RL. A total of 382 patients were recruited from the SCA2 popu- The estimate is an upper bound of genetic effects because only lation in Holguin province, Cuba from 1955 to 2001. Dates of first-degree relatives were in the study cohort, and familial birth, CAG RLs, AO, sex familial inheritance, and clinical man- similarity, which is used to estimate heritability, includes both ifestations were collected. genetic and shared family environmental factors. Heritability was computed on all available data, then using only siblings Determination of AO. AO was defined as the first sign of and using only PC pairs to determine the impact of these ataxia, usually manifested by unsteadiness of gait. AO was deter- relationships on the estimates. mined by 1 interviewer (N.S.) using self-report and, whenever possible, report of other family members. Usually, individuals and RESULTS family members recalled specific life stages at which function was For the analysis of residual variance and impaired, such as ability to perform military service, ability to ride heritability, information from 326 SCA2 patients a bicycle, to carry a child, or to handle tools. AO was virtually was used to identify 69 sibships and 129 PC pairs always related to balance problems. Although muscle cramps are (figure 1). Table 1 provides descriptive characteristics common in the Cuban SCA2 population, noncerebellar symp- for the total cohort and for several subcohorts toms were not used to determine AO in this sample. Faulty described below. Also shown are correlations between estimation of AO in individuals would result in a reduced esti- AO and CAG RL, both uncorrected and corrected for mate of familiality and an increased estimate of nonshared environmental components and stochastic factors. pedigree structure. The correlations were all highly DNA was extracted from venous blood and amplified using significant (p , 0.001). established primer pairs.11 All samples were analyzed at CIRAH Subcohort 1: RL. When we restricted the analysis to (Holguin) using conditions previously described and confirmed in our laboratory in the United States.11 205 adult onset samples with an RL from 32 to 40 Analyses were performed using SAS/STAT software (sas.com) repeats, we found that the variance in AO explained with log-transformed AO. We used the GENMOD procedure by RL (as measured using the R2 statistic from the

Figure 1 Age at onset and CAG repeat of 326 Cuban spinocerebellar ataxia type 2 patients

Box plot of age of disease onset and CAG repeat length of the expanded allele. Outliers for each CAG repeat were included in the analysis and are denoted in red asterisks. Bars denote interquartile range 3 1.5.

2 Neurology: Genetics Important for genetic counseling, 2 of the large ex- Table 1 Age at onset (AO) and CAG repeat correlations pansions occurred in parents with only 36 repeats.

Mean Mean repeat R2 due to repeat R2 due to repeat In our adjusted analyses, sibship membership ac- a a Cohort N AO (SD) length (SD) (uncorrected) (corrected) counted for a substantial proportion of variance in Total 326 33 (14) 40 (4) 0.69 0.50 AO (F 5 1.73; p 5 0.001; R2 5 0.23). The entries Males 173 33 (15) 40 (5) 0.69 0.56 in the last column of table 1 therefore reflect variance

Females 153 33 (13) 40 (4) 0.70 0.40 explained by AO above and beyond variance explained by sibship membership. Note that the effect CAG 32–40 205 40 (11) 38 (2) 0.38 0.22 of repeat remained considerable in each of the mod- CAG 41–77 121 21 (8) 44 (5) 0.57 0.51 els; although when RL was smaller, variance explained Parents 84 39 (13) 39 (2) 0.57 0.57 was reduced. Variance explained was also greater for Siblings 183 33 (14) 40 (3) 0.62 0.23 paternal vs maternal transmissions, although this Paternal 70 27 (14) 43 (6) 0.81 0.72 could be due to overall larger repeats observed in

Maternal 78 28 (12) 41 (4) 0.58 0.34 paternal transmissions. We examined the effect of RL on AO in different a Uncorrected/corrected for family structure. male-female PC combinations. In the models run separately by maternal vs paternal inheritance, there model) was lowered; the R2 uncorrected for family was a significant sex effect and sex 3 repeat interac- structure was 0.38 and 0.22 when corrected. For tion. The cause of this sizable interaction may be the these RLs, the mutation explained slightly less than reduction in the differential prediction of repeat on 25% of the variance when corrected for pedigree AO in the mother-daughter pairs. structure, indicating that genetic and environmental Effects of imprinting have not been studied in the modifiers played a greater role in patients with the dominant ataxias except for small samples that could shorter and more common RLs. only detect large effects. There was no difference in Subcohort 2: Effect of birth year. Our birth year analysis the mean repeat number or AO between paternal is based on 382 affected subjects with known AO and and maternal transmissions in our cohort of 326 sub- year of birth. The overall distribution of AO had jects in whom the sex of the transmitting parent was a mean of 31.22 years, SD of 16.38, and a range of known. The proportion of the variance in AO ex- 2–65 years. We observed possible sampling bias re- plained by the mutant allele was also similar in both sulting from a loss of subjects with longer repeats patient groups. Thus, we did not find evidence for born before 1960. These individuals had likely died imprinting effects on AO. before being examined, leading to an enrichment in Familial correlation is defined as the correlation this cohort of individuals with shorter pathologic re- of a trait among family members, used as a tool to peats. This observation also implies that shorter path- investigate genetic influences on a defined rela- ologic repeats were underrepresented in the cohort tionship. A value greater than zero implies genetic born after 1960 because these subjects had not shown influence on that trait. We estimated familial cor- evidence of disease at the time of examination. This relations in 129 parent-offspring pairs. These ascertainment bias may also produce unusual effects results show an interesting difference in parent- when looking at parental transmissions, as paternal offspring resemblance such that similarity is stron- transmissions tend to expand more than maternal. ger in mother-child (MC) relationships (table 2). There was no difference in the AO between males The test for significance of this difference was ob- (33.12 6 14.47) and females (33.06 6 13.34). Mean tained in the PAP by comparing 22timesthe CAG repeats for both sexes were virtually identical natural log of the likelihood (22lnL) of the model (males: 40.28 6 4.46; females 40.12 6 3.71). How- estimating a single PC correlation (PC model) with ever, the corrected R2 was higher for males than that 22lnL of the model allowing for different esti- for females, although this difference did not reach mates for father-child and mother-child (FC, MC statistical significance (table 1). model). For all traits, the model allowing for dif- We analyzed instability of the SCA2 repeat in ferent correlation estimates for MC vs FC pairs fits 129 PC pairs. The repeat was more unstable in better than the model with a single PC estimate. paternal vs maternal transmissions (figure 2, For both unadjusted AO and RL, MC correlations A and B). On average, the repeat increased by were higher (p 5 0.05 and p , 0.0001, respec- 4.8 6 5.4 units (range 21to36units),whenin- tively). A similar trend was observed for AO herited from the father, but only by 1.6 6 2.0 units adjusted for repeat, but the difference did not (range 21to11units),wheninheritedfromthe reach significance. Freeing all parent-offspring mother. Of the 6 expansions .10 units, 5 occurred relationships to test significance of sex of the child whentherepeatwaspassedthroughthefather. (mother-son, mother-daughter, father-son, and

Neurology: Genetics 3 Figure 2 Transmission of expanded CAG repeat

(A) Paternal. (B) Maternal. Box plot of age at onset and CAG repeat length of the expanded allele. Outliers for each CAG repeat denoted in red asterisks. Bars denote interquartile range 3 1.5. Note the higher instability of repeats seen in paternal vs maternal transmission.

father-daughter relationships) did not result in any above, heritability of the residual variance was higher further improvement in the fit of the model for any when estimated only from siblings (58%, p 5 0.001) of the traits. than that from parent-offspring pairs (10%, p 5 0.02). The higher value in sib pairs may reflect the Heritability of residual AO variance. To estimate fami- existence of recessive modifying alleles. Alternatively, liality or an upper bound for heritability of residual siblings may share additional family environment familial variance (adjusted AO), we used polygenic over and above parent-offspring pairs. variance component analysis in SOLAR applied to Because lower heritability for male-male sibs was AO data in the full cohort of 326 individuals. This observed in HD, we analyzed for differences between set contained a total of 129 parent-offspring pairs. male-male vs female-female sib pairs. Again, heritabil- Most were in families with a single child, although 44 ity estimates for both subgroups and female sibs pairs were in families with 2 siblings, and 25 pairs showed more than twice the heritability of the resid- were in families with 3 or more siblings. We deter- ual than male sibs. However, the difference itself did mined an upper bound for heritability of the residual not reach statistical significance. variance of 33% (p 5 0.008). As already suggested by Sibling correlation differences by sex were ana- our analyses of specific familial resemblance described lyzed in more detail using PAP, similar to our PC

4 Neurology: Genetics Table 2 Parent-offspring correlations (estimated using the Pedigree Analysis Package)

Correlation of log (AO) Correlation of Correlation of log (AO) HD5 correlation for Relationship N unadjusted (SE) repeat length (SE) adjusted for repeat (SE) HD,5 N residual AO (SE)

Father-son 35 0.32 (0.12) 0.02 (0.13) 0.07 (0.17) NR

Mother-daughter 34 0.56 (0.11) 0.74 (0.10) 0.03 (0.15) NR

Mother-son 35 0.46 (0.12) 0.79 (0.10) 0.23 (0.15) NR

Father-daughter 25 0.15 (0.13) 0.08 (0.13) 0.12 (0.18) NR

Father-child (FC) 60 0.08 (0.12) 0.02 (0.11) 0.01 (0.14) NR

Mother-child (MC) 69 0.47 (0.12) 0.77 (0.08) 0.14 (0.13) NR

Parent-child (PC) 129 0.20 (0.10) 0.13 (0.10) 0.03 (0.10) 202 0.10 (0.11)

x2 ([22lnL (PC)] 2 3.98 (1df); p 5 0.05 17.65 (1df); p , 0.0001 1.94 (1df); p 5 0.16 [22lnL (FC, MC)])a

Abbreviations: AO 5 age at onset; HD 5 Huntington disease; NR 5 not reported. a The difference between 22 times the natural log of the ratio of likelihoods of these nested models ([22lnL (PC)] 2 [22lnL (FC 5 MC)]) produces a x2 test (df 5 1) of whether the FC correlation is significantly different from the MC correlation.

analyses as described above. These correlations are may facilitate genetic analysis. On the other hand, comparable with the intraclass correlations reported findings in founder populations may be difficult to on this data set, although the PAP generates maxi- generalize to other populations. It is unknown if the mum likelihood estimates. PAP additionally allowed common founder haplotype is European, African, us to test for significant differences by sex (table 3). or Native American, and there is evidence of extensive Male-male pairs showed lower correlations for both population admixture in Cuba.17 unadjusted AO (p 5 0.05) and adjusted AO (p , The presence of founder alleles may explain why 0.0001). The adjusted AO correlations among sibling our prior evidence for variation in CACNA1A as pairs were remarkably similar to those reported in a modifier of AO3 did not generalize to a European a large Venezuelan pedigree.5 and US American population.18 Detailed comparisons can only be made with 2 studies of HD with DISCUSSION In SCA2 and other polyQ diseases, large sample sizes.5,6 the longer the length of the CAG repeat, the earlier Despitethelargesamplesize,ourstudywasnot the age of disease onset.2,14,15 Surprisingly, effects of population based. Therefore, it shares with previ- sex, imprinting, and modifier alleles have received ous studies inherent biases of referral for genetic little attention in SCA2 or in other SCAs. Alleles of testing by medical providers, self-referral in moti- the retinoic acid–induced gene 1 were identified as vated families, and ascertainment bias based on AO modifiers for SCA2.16 inclusion of gene carriers who become affected first We examined the relationship between CAG in a given family. Our cohort did not include repeat and AO in the Cuban SCA2 founder popula- asymptomatic gene carriers or individuals at risk tion. The study of founder populations has certain for the disease. inherent advantages, as they tend to be more homog- In the Cuban SCA2 population, 69% of the enous both genetically and environmentally, which observed variation on AO was explained by the

Table 3 Familial correlations of AO and CAG repeat

Correlation of Correlation of Correlation of log (AO) HD5 correlation for Relationship N log (AO) unadjusted repeat length adjusted for repeat HD,5 N residual AO (SE)

Male-male sib pairs (MM) 56 0.60 (0.05) 0.84 (0.04) 0.20 (0.12) 108 0.18 (0.20)

Female-female sib pairs (FF) 38 0.76 (0.04) 0.75 (0.06) 0.52 (0.13) 113 0.49 (0.13)

Male-female sib pairs (MF) 91 0.74 (0.05) 0.79 (0.04) 0.40 (0.10) 220 0.40 (0.11)

All sib pairs (sib-sib) 185 0.71 (0.05) 0.79 (0.03) 0.33 (0.09) 441 0.40 (0.09)

x2 ([22lnL (sib-sib)] 2 6.04 (2df); p 5 0.05 1.62 (2df); p 5 0.45 29.12 (2df); p , 0.0001 [22lnL (MM 5 MF 5 FF)])a

Abbreviations: AO 5 age at onset; HD 5 Huntington disease. a The difference between 22 times the natural log of the ratio of likelihoods of these nested models ([22lnL (sib-sib)] 2 [22lnL (MM 5 MF 5 FF)]) produces a x2 test (df 5 2) of whether the sib correlations differ by sex.5

Neurology: Genetics 5 RL. Although this is in good agreement with other than male-male sibs (table 3). This was noted in the studies,1,2,10,14,19,20 it is difficult to perform precise Venezuelan pedigrees as well,5 but the authors were comparisons, as most studies used a simple regres- cautious in commenting on them, as the large stan- sion without taking pedigree structure into dard errors associated with these estimates did not consideration. result in statistical significance. It is intriguing that Variability of AO in SCA1 individuals appeared to our data not only show similar qualitative differences cluster within families, suggesting the presence of but display virtually identical correlation coefficients shared environmental and genetic factors on AO.21 for sex effects in sibling pairs. Sex effects also extend In one study, the effect of the normal allele in SCA2 to PC pairs, as MC pairs had higher correlations than was examined and found to have little influence on FC pairs. AO.2 The normal allele in Cuban SCA2 is not highly What model may explain the greatly reduced variable; only 31 patients carried a 23 CAG ATXN2 brother-brother correlations of AO after correction allele preventing us from examining this effect. for CAG repeat size? Of note, imprinting does not The concept of familiality encompasses shared explain the observations, as the SCA2 repeat has the environmental and shared genetic factors and may same effect on AO in females and in males, as well be difficult to differentiate from heritability in human as after passage through the paternal or maternal populations. In the strict sense, heritability denotes germline. Thus, one needs to search for mechanisms the proportion of phenotypic variance that can be that largely act stochastically, affecting males only or attributed to additive genetic variance. Our estimate at least preferentially. These could be related to that 33% of the residual AO variance in SCA2 indi- endogenous male factors such as hormonal differen- viduals was familial was lower than those of our pre- ces or to male-specific lifestyle choices such as smok- vious analyses. However, this discrepancy was ing or alcohol use. Furthermore, males share explained, when we examined residual heritability X-chromosomes only 50% of the time, whereas fe- separately in sibling and PC pairs. An upper bound males all receive the same X from the father and share estimate of heritability as estimated using SOLAR the other one 50% of the time. Finally, the possibility was higher in siblings, and at 58%, it was close to remains that there is greater somatic instability in the previously reported value of 55% using the coef- males, which would show as a stochastic variance ficient of intraclass correlation in sibships to estimate component in our analyses. heritability. We did not collect epidemiologic data to analyze We can compare our analyses to 1 study of whether the environment experienced by males was SCA210 and 2 studies in HD.5,6 In SCA2 pedigrees, more variable than that for females. For example, if the CAG repeat determined 73% of AO variance and tobacco or alcohol use were important in SCA2 path- by incorporating familial dependency, the repeat ogenesis, and women were largely abstinent with influence actually increased to 80%.10 In one study highly variable use among men, this might explain of HD, the CAG repeat in the HD gene explained reduced heritability in male siblings. 67% of AO variance.6 When the size of the normal The nature of the recently identified modifiers for allele was taken into consideration, this value AO in HD is important with regard to this study.22 increased to 68%. Heritability of the residual variance This study found connection between AO and several was 56%. pathways subgrouped into 3 clusters by gene mem- An analysis of the large Venezuelan HD pedigree bership in the following: DNA repair, mitochondrial showed surprising similarities to our studies.5 fission, and oxidoreductase activity. Several of the Variance-components analysis was used to estimate single nucleotide variants (single nucleotide polymor- the between and within sibling pair variances allowing phisms [SNPs]) were replicated in separate cohorts of for the computation of the sibling intraclass correla- HD and SCA patients.23 Some of the SNPs were tion. The estimated sibling correlation for the residual validated for SCA2 in particular. Although the 2 stud- AO was 0.42, leading to an upper estimate for heri- ies defined common SNPs and identified potentially tability of 84%. This value is higher than that of our common pathways related to DNA repair, the major- SCA2 siblings, who only showed a correlation of 0.33. ity of the residual AO variance remained unex- Both studies found a much lower correlation for PC plained.23 This points to the potential presence of than sibling pairs, which was 0.10 in both studies. This rare variation with large effects or to modifiers acting may point to the presence of recessive modifying alleles only in a specific setting of gene-environment inter- but could also point to fewer shared environmental actions that is not shared across different cohorts.3 factors across generations than for siblings. AUTHOR CONTRIBUTIONS Analysis of the Venezuelan pedigree revealed K.P. Figueroa: study concept and design, analysis, interpretation of data, another striking similarity with Cuban SCA2. and critical revision of manuscript for intellectual content. Dr. Coon: Female-female sib pairs had much higher correlation analysis and interpretation of data. Nieves Santos, Dr. Velazquez, and

6 Neurology: Genetics Dr. Mederos: acquisition of data. Dr. Pulst: study concept and design, onset of Huntington disease. Am J Med Genet A 2003; analysis, interpretation of data, and critical revision of manuscript for 119A:279–282. intellectual content. 7. Li JL, Hayden MR, Almqvist EW, et al. A genome scan for modifiers of age at onset in Huntington disease: the ACKNOWLEDGMENT HD MAPS study. Am J Hum Genet 2003;73:682–687. The authors thank their Cuban patients for participating in these investi- 8. Velazquez Perez L, Cruz GS, Santos Falcon N, et al. gations. This publication is dedicated to the memory of Carmen Molecular epidemiology of spinocerebellar ataxias in Warschaw, a great supporter of neurogenetics and neurology research. Cuba: insights into SCA2 founder effect in Holguin. Neu- – STUDY FUNDING rosci Lett 2009;454:157 160. 9. Hernandez A, Magarino C, Gispert S, et al. Genetic map- This work was supported by grants from the NIH (R37 NS33123 to S.M.P., and R01 MH099134 and R01 MH094400 to H.C.), the ping of the spinocerebellar ataxia 2 (SCA2) locus on chro- – – National Ataxia Foundation (S.M.P.), and the Carmen and Louis War- mosome 12q23 q24.1. Genomics 1995;25:433 435. schaw Endowment for Neurology (S.M.P.). 10. van de Warrenburg BP, Hendriks H, Durr A, et al. Age at onset variance analysis in spinocerebellar ataxias: a study in DISCLOSURE a Dutch-French cohort. Ann Neurol 2005;57:505–512. K.P. Figueroa reports no disclosures. Dr. Coon has received research sup- 11. Pulst SM, Nechiporuk A, Nechiporuk T, et al. Moder- port from NIMH and the Simons Foundation. Nieves Santos, Dr. Velaz- ate expansion of a normally biallelic trinucleotide repeat quez, and Dr. Mederos report no disclosures. Dr. Pulst serves on the in spinocerebellar ataxia type 2. Nat Genet 1996;14: editorial boards of Journal of Cerebellum, NeuroMolecular Medicine, Con- 269–276. tinuum, Experimental Neurology, Neurogenetics, and Nature Clinical Practice 12. Hasstedt S. jPAP: document-driven software for genetic Neurology and as Editor-in-Chief of Current Genomics and of Neurology® analysis. Genet Epidemiol 2005;29:255. Genetics; and holds patents for the following: Nucleic acids encoding 13. Almasy L, Blangero J. Multipoint quantitative-trait linkage ataxin-2 binding proteins; Nucleic acid encoding Schwannomin-binding- proteins and products related thereto; Transgenic mouse expressing a poly- analysis in general pedigrees. Am J Hum Genet 1998;62: – nucleotide encoding a human ataxin-2 polypeptide; Methods of detecting 1198 1211. spinocerebellar ataxia-2 nucleic acids; Nucleic acid encoding spinocerebel- 14. Geschwind DH, Perlman S, Figueroa CP, Treiman LJ, lar ataxia-2 and products related thereto; Shwannomin-binding-proteins; Pulst SM. The prevalence and wide clinical spectrum of and Compositions and methods for spinocerebellar ataxia. In addition, he the spinocerebellar ataxia type 2 trinucleotide repeat in receives publishing royalties from Churchill Livingston, AAN Press, Aca- patients with autosomal dominant cerebellar ataxia. Am ’ demic Press, and Oxford University Press; has served on the speakers J Hum Genet 1997;60:842–850. bureau of Athena Diagnostics, Inc.; receives research support from NIH, 15. Schols L, Gispert S, Vorgerd M, et al. Spinocerebellar Target ALS, National Ataxia Foundation, and ISIS Pharmaceuticals; has ataxia type 2: genotype and phenotype in German kin- consulted for Ataxion Therapeutics; is a stockholder of Progenitor Life dreds. Arch Neurol 1997;54:1073–1080. Sciences; has received license fee payments from Cedars-Sinai Medical Center; has given expert testimony for Hall & Evans, LLC; and receives 16. Hayes S, Turecki G, Brisebois K, et al. CAG repeat length an honorarium from the AAN as the Editor of Neurology: Genetics.Goto in RAI1 is associated with age at onset variability in spi- Neurology.org/ng for full disclosure forms. nocerebellar ataxia type 2 (SCA2). Hum Mol Genet 2000; 9:1753–1758. Received January 3, 2017. Accepted in final form April 3, 2017. 17. Mendizabal I, Sandoval K, Berniell-Lee G, et al. Genetic origin, admixture, and asymmetry in maternal and pater- REFERENCES nal human lineages in Cuba. BMC Evol Biol 2008;8:213. 1. van de Warrenburg BP, Sinke RJ, Verschuuren-Bemelmans 18. Tezenas du Montcel S, Durr A, Bauer P, et al. Modulation CC, et al. Spinocerebellar ataxias in the Netherlands: preva- of the age at onset in spinocerebellar ataxia by CAG tracts lence and age at onset variance analysis. Neurology 2002;58: in various genes. Brain 2014;137:2444–2455. 702–708. 19. Riess O, Laccone FA, Gispert S, et al. SCA2 trinucleotide 2. Cancel G, Durr A, Didierjean O, et al. Molecular and expansion in German SCA patients. Neurogenetics 1997; clinical correlations in spinocerebellar ataxia 2: a study of 1:59–64. 32 families. Hum Mol Genet 1997;6:709–715. 20. Giuffrida S, Lanza S, Restivo DA, et al. Clinical and 3. Pulst SM, Santos N, Wang D, et al. Spinocerebellar ataxia molecular analysis of 11 Sicilian SCA2 families: influence type 2: polyQ repeat variation in the CACNA1A calcium of gender on age at onset. Eur J Neurol 1999;6:301–307. channel modifies age of onset. Brain 2005;128:2297– 21. Ranum LP, Chung MY, Banfi S, et al. Molecular and 2303. clinical correlations in spinocerebellar ataxia type I: evi- 4. van de Warrenburg BP, Sinke RJ, Kremer B. Recent ad- dence for familial effects on the age at onset. Am J Hum vances in hereditary spinocerebellar ataxias. J Neuropathol Genet 1994;55:244–252. Exp Neurol 2005;64:171–180. 22. Genetic Modifiers of Huntington’s Disease Consortium. 5. Wexler NS, Lorimer J, Porter J, et al. Venezuelan kindreds Identification of genetic factors that modify clinical onset reveal that genetic and environmental factors modulate of Huntington’s disease. Cell 2015;162:516–526. Huntington’s disease age of onset. Proc Natl Acad Sci 23. Bettencourt C, Hensman-Moss D, Flower M, et al. DNA USA 2004;101:3498–3503. repair pathways underlie a common genetic mechanism 6. Djousse L, Knowlton B, Hayden M, et al. Interaction of modulating onset in polyglutamine diseases. Ann Neurol normal and expanded CAG repeat sizes influences age at 2016;79:983–990.

Neurology: Genetics 7 Previously unrecognized behavioral phenotype in Gaucher disease type 3

Magy Abdelwahab, MD, ABSTRACT PhD Objective: To provide a comprehensive description of abnormal behaviors in patients with Michael Potegal, PhD Gaucher disease type 3 (GD3) and relate these behaviors to demographic, neurodevelopmental, Elsa G. Shapiro, PhD and neurologic characteristics. Igor Nestrasil, MD, PhD Methods: Thirty-four Egyptian patients with GD3 (mean age of 7.9 years) were enrolled in the study. They were selected based on parent report and/or physician observation of one or more abnormal behaviors documented in 2 settings and by 2 different individuals and/or by video Correspondence to Dr. Abdelwahab: recording. Behaviors were grouped into 4 categories: Crying/Withdrawal, Impatience/Overactiv- [email protected] ity, Anger/Aggression, and Repetitive Acts. Baseline and follow-up 6–12 monthly neurologic evaluations included IQ assessment and an EEG. All patients were receiving enzyme replacement therapy (30–60 IU/kg every 2 weeks) and were followed for periods of 3–10 years. Results: Supranuclear palsy of horizontal gaze, and of both horizontal and vertical gaze, bulbar symptoms, seizures, convergent strabismus, abnormal gait, and neck retroflexion were present in 97.1%, 50%, 55.9%, 29.4%, 29.4%, 20.6%, and 4.4% of patients, respectively. The most abnormal behavioral features were excessive anger (88.2%) and aggression (64.7%), and both were significantly higher in males. Anger/Aggression scores were highly correlated with IQ but not with either EEG/Seizure status or neurologic signs. Conclusions: We describe behavioral problems with a unique pattern of excessive anger and aggression in patients with GD3. Defining these components using quantitative behavioral scoring methods holds promise to provide a marker of neurologic disease progression and severity. Neurol Genet 2017;3:e158; doi: 10.1212/NXG.0000000000000158

GLOSSARY ERT 5 enzyme replacement therapy; GBA 5 glucocerebrosidase; GD3 5 Gaucher disease type 3; VSGP 5 vertical supranuclear gaze palsy.

Gaucher disease (GD) results from a glucocerebrosidase (GBA) mutation leading to glucocere- broside accumulation in multiple organs. Based on the genotype and/or the presence of neurologic involvement, 3 clinical subtypes are distinguished. Type 1 is nonneuronopathic; types 2 and 3 are neuronopathic. Type 3 (GD3) has a broad phenotypic spectrum1,2 with more than 50% of GD3 cases manifesting in infancy. The usual neurologic presentation includes developmental delay, supranuclear gaze palsy, bulbar symptoms, neurocognitive impairment, and seizures. Enzyme replacement therapy (ERT) has been reported to have no effect3,4 or lead to the stabilization5,6 or even improvement of neurologic function.7 Reports on GD3 describe the physical manifestations and neurologic spectrum, but none have referred to the behavioral aspects of the disease.8–11 However, more than half of parents of children in our GD3 cohort complain about a set of abnormal behaviors, including extreme anger and aggression, persistent crying, excessive motor activity, and bizarre repetitive acts. The Supplemental data at Neurology.org/ng From the Department of Pediatric Hematology (M.A.), Cairo University Pediatric Hospital, Egypt; and Program in Occupational Therapy (M.P.), and Division of Clinical Behavioral Neuroscience (E.G.S., I.N.), Department of Pediatrics, University of Minnesota, Minneapolis. Funding information and disclosures are provided at the end of the article. Go to Neurology.org/ng for full disclosure forms. 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.

Neurology.org/ng Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 who did not show any behavioral abnormalities were excluded. Table 1 Demographic and descriptive data of patients with GD3 The GD3 group showing behavioral problems represents 52% of the Cairo University Pediatric Hospital GD3 cohort contain- Demographic data No. of patients % of total ing 65 patients in total. Definitive diagnosis of GD was typically Sex prevalence made between 9 and 15 months of age from reduced leukocyte b , m Male 21 61.8 -GBA activity ( 1 mol/g of protein/h); type 3 diagnosis is determined by genotyping and/or the presence of neurologic Female 13 38.2 manifestation and persistence beyond infancy. Genetic testing Consanguinitya 30 88.2 of 27 children was performed by full sequencing of the gene for

Age, y, mean 6 SD common mutations associated with GD3. All were homozygous for L444P. 6 Current 8.7 5.1 Since diagnosis, the patients have been followed for peri- At diagnosis 1.0 6 0.4 ods of 3–10 years. They underwent systematic neurologic

Family history of previous induced abortionsb 6 17.6 re-evaluation at 6-month intervals. The data presented here were derived from the retrospective review of the records Similarly affected sibling/family member 19 55.8 (dead or alive) of a single evaluation of each child between 2006 and 2015. The patients’ demographic and descriptive data are shown in Patients receiving imiglucerase (ERT) 32 94.1 table 1. Patients receiving velaglucerase (ERT) 2 5.9 Procedures. Abnormal behaviors. Table 2 shows the list of Duration of ERT, y, mean 6 SD 6.9 6 4.9 abnormal behaviors, which was developed through observation

Improvement of neurologic and/or other 27 79.4 of patients in the clinic and discussions with their parents about systemsc on ERT frequency and variation among these behaviors that occurred

Worsening of neurologic and/or other 7 20.6 outside the clinic. Therefore, some descriptors and categories systemsc on ERT may be colloquial and culture specific. Behaviors of 18 patients were documented by video recording; 12 of these videos were Abbreviations: ERT 5 enzyme replacement therapy; GD3 5 Gaucher disease type 3. reviewed with coauthors (E.G.S. and I.N.) to identify, confirm, Four children with severe GD3 physical phenotype had abnormal facies characterized by and categorize behaviors. All behaviors included in this report low set ears, hypertelorism, and ptosis. Three children with GD3 underwent splenectomy. a Consanguinity: first-degree maternal or paternal cousins. were documented in at least 2 settings, including hospital b Induced abortion when the fetus was tested as homozygous for L444P by chorionic villous (observation by the examining physician during a clinic visit), sampling. school, and/or home. All these behaviors were witnessed by c Other systems: pulmonary (recurrent wheezy chest and chest infections), mesenteric and more than one person, including parents, siblings, peers, treat- mediastinal lymphadenopathy, bone complications (bone aches, fractures, and avascular ing physician, and sometimes strangers who reported it to necrosis), kyphoscoliosis, and dental problems. parents. Behaviors were grouped into 4 categories based on similarity treating physician (M.A.) has noted behavioral of form and/or function, co-occurrence, and/or sequential occurrence. Scales for each category consisted of 1 point for each of the changes and tendency for aggressive behav- behaviors assigned to that category: Anger/Aggression (0–14), 2,12 ior, but other than those preliminary re- Impatience/Overactivity (0–3), Crying/Withdrawal (0–4), and ports, the behavioral phenotype of GD3 has Repetitive Acts (0–3). The larger number of Anger/Aggression not been systematically studied and is cur- items represents both the diversity of behaviors in this category and their salience to observers. rently unknown. Cognitive, EEG, and clinical neurologic assessment. The The aims of this retrospective exploratory same clinician (M.A.) conducted baseline and follow-up neuro- study were to characterize these behavioral pat- logic evaluations every 6–12 months, individualized according to the patient’s condition. Other evaluations included IQ assess- terns and relate them to neurodevelopmental ment and an EEG recorded under standard International 10–20 and neurologic characteristics. Such founda- system. Wechsler scales for preschool, school age, and adult tional knowledge will permit the development subjects were used to assess IQ. For children younger than 4 of working hypotheses for a subsequent pro- years, the Vineland Adaptive Behaviour Scales II was used to estimate IQ.13 spective study. Based on a detailed single clini- EEGs were graded as follows: 0 5 normal, 1 5 nonepilepti- cal observation as well as neurologic and form abnormality, and 2 5 epileptiform abnormality.14 If clinical treatment-related response, we describe the seizures were present, an additional point was given. The conti- abnormal behaviors occurring in the cohort of nuity of this 4-point scale was indicated by the observation that the percentage of patients with EEG scores of 0, 1, and 2 who 34 Egyptian patients diagnosed with GD3. were known to have seizures was 8%, 20%, and 50%, respectively. The neurologic signs listed in table e-1 at Neurology.org/ng METHODS Standard protocol approvals, registrations, were scored as absent or present (0, 1). and patient consents. The protocol was approved by the local Statistical analysis. Descriptive statistics were used to Ethical Committee. All patients/parents/legal guardians of pa- describe results by percentages, mean values, and SDs. To tients provided their written informed consent. assess changes in behavior with age, the symptom profiles of Patients. Thirty-four Egyptian patients with GD3 whose pa- the children were ordered by age and examined for patterns rents reported and/or physician (M.A.) observed one or more of occurrence. Nonparametric Spearman rank-order correla- abnormal behaviors were enrolled in the study, while those tion was used to test for association between variables; the

2 Neurology: Genetics RESULTS All enrolled children presented initially Table 2 Abnormal behaviors grouped by category: Descriptors, number, and percentages of patients with Gaucher disease type 3 showing with organomegaly, motor, and rarely global delay. abnormal behavior Nineteen children had neurologic symptoms initially; the other 15 developed neurologic symptoms Descriptors Definitions/examples No. % subsequently. Prevalence of neurologic manifesta- Anger/Aggression tions noted in our type 3 GD cohort is shown in Excessive anger Inappropriately intense anger to 30 88.2 table e-1. Thirty-two patients were started on nonprovocative or trivially provocative – events ERT, imiglucerase (30 60 IU/kg every 2 weeks) and 2 patients on velaglucerase alfa (60 IU/kg every Physically aggressive Includes grabbing other children by the 22 64.7 hair, hitting, biting, and strangling 2 weeks). Patients received ERT regularly except

Oppositional Defies adults 20 58.8 duringthe2-yearworldwideenzymeshortagein – Verbally aggressive Insults others 17 50.0 2009 2011.

Age-inappropriate tantrums Tantrums occurring after age 4, lasting 15 44.1 IQ. The IQ for the group was 71.1 6 10.5 (mean 6 10–15 min in the clinic and, reportedly, for hours at home SD); for females and males, it was 74.3 6 8.9 and

Explosive temper Sudden outbursts of anger 13 38.2 68.9 6 11.0, respectively. These results suggest bor-

Unpredictable aggression Brief, sudden, and surprising physical 12 35.3 derline intellectual functioning overall for this group. attacks (e.g., biting) in the context of Compared with population norms, they were 2 SDs otherwise amicable social interaction below the mean. Only 1 patient had an IQ in the Proactive aggression Smiling or laughing while hitting or 12 35.3 . committing other aggressive acts average range ( 85).

Touchy Easily annoyed by others 9 26.5 EEG status/clinical seizures. Five patients (14.7%) had Blames Accuses others of making mistakes and/ 8 23.5 EEG ratings of 1; 16 (47%) had EEG ratings of 2. or of misbehaving Half of those with ratings of 2 had clinical seizures. Curses Uses obscenities 8 23.5 Initial EEG recording showed a clear epileptic focus Vindictive Takes revenge for real or imagined 8 23.5 in 10 children, 8 of whom had clinical seizures that provocations over hours or days ranged from focal or focal with secondary generaliza- Threatens Threats to injure, kill, or “slaughter” 8 23.5 tion to generalized or myoclonic types. Seizure fre- Self-injurious Slaps own face in frustration 6 17.6 quency varied from 1 to 2 per month to several per Impatience/Overactivity day. Patients were treated with valproic acid at doses Interrupts others Speaks out while others are talking 10 29.4 up to 60 mg/kg/d with variable seizure control. EEG

Impatient Cannot wait his/her turn 8 23.5 epileptic foci resolved in 4 children spontaneously in

Excess activity/hyposomnia Continual pacing, climbing up and down 5 14.7 later childhood over a 3-year period. chairs and other objects; never appearing to tire; sleeping for hours less Qualitative behavioral observations. A wide range of than typical for age and sometimes complete lack of sleep abnormal behaviors was observed. The frequency var-

Crying/Withdrawal ied from daily to monthly depending on the type and

Demanding Nags, makes excessive demands on 20 58.8 precipitating factor(s) such as a younger sibling smil- others ing at the patient or touching him, but in many chil- Social withdrawal Retreats from social interaction 17 50.0 dren, they have been going on for years. Table 3

Clings Age-inappropriate clinging to caretaker 8 23.5 displays the number (and percentage) of patients with GD3 showing each abnormal behavior, with exam- Cries excessively Continuous crying, with or without tears 7 20.6 ples, grouped by category. Repetitive Acts Anger/Aggression. These behaviors were directed to Recurring behaviors Stereotypies, punding, shredding paper 10 29.4 and plastic bags, tying-untying hair in peers, siblings, parents, and other adults/authority fig- a knot, pacing up and down in a room, ures including the treating physician. Angry and and repeating same housecleaning movements for hours aggressive acts were the most common, prominent,

Object obsession Obsessed with a specific object 6 17.6 and troublesome of the abnormal behaviors and were

Hyperphagia Obsessed with food, compulsive eating 4 8.8 reported to have had a major negative effect on patients’ and parents’ quality of life. For 23 (69.7%) Descriptors are ordered by category and by prevalence within category. patients, these behaviors were the parents’ main concern or sole complaint. Mann-Whitney U test was used to assess male-female differ- Based on the median age at which the various ences. Neurologic signs that were significantly associated with aggressive behaviors appeared, tantrums past toddler one or more behavior categories were identified by the Pratt measure15 in exploratory multiple regressions of behavior on age, oppositional behavior, physical aggression, and the full set of signs. Significance values were adjusted for angry self-injury appeared earliest to develop; these multiple tests using the false discovery rate protocol.16 behaviors tended to be the most prevalent across

Neurology: Genetics 3 threats were generally the last to appear and tended Table 3 Descriptive statistics for quantified behavioral observations to be least prevalent.

Female (n 5 13), Male (n 5 21), Total (N 5 34), Impatience/Overactivity. Parents reported that mean 6 SD mean 6 SD mean 6 SD a child’s overactivity in social gatherings was disrup- Age, y 9.7 6 6.2 8.1 6 4.3 8.7 6 5.1 tive, embarrassing, tiring, and sometimes impossible Anger/Aggression (0–14 scale) 3.3 6 2.0 7.0 6 3.3 5.5 6 3.4 to deal with.

Impatient/Overactive (0–3 scale) 0.2 6 0.4 1.0 6 1.1 0.7 6 1.0 Crying/Withdrawal. Children with these characteristics were wary of strangers, socially withdrawn, and Crying/Withdrawn (0–4 scale) 1.8 6 1.0 1.4 6 0.9 1.5 6 1.0 clung to and were demanding of their parents. Repetitive Acts (0–3 scale) 0.4 6 0.8 0.7 6 0.9 0.6 6 0.9 Repetitive Acts. These varied in type and complexity; they often could not be interrupted.

the group. Unpredictable and proactive aggression, Quantitative behavioral results. Anger/Aggression. All but using obscenities and vindictive behavior, tended to one of the 34 subjects had scores $3 on the Anger/ appear subsequently and were less prevalent; being Aggression measure. Mean values for this measure, touchy/easily annoyed, explosive temper, blaming calculated for successive 3-year periods, increased others, verbal aggression and insults, and homicidal for females and almost doubled for males from value at 1–3 years of age to a peak at 4–6-year-olds, then fell back to near values observed at 1–3-year-olds by Figure Age trends for all 4 abnormal behavior categories adolescence (figure). Males also showed higher Anger/ Aggression than females (U 5 162, p , 0.02, table 3). Anger/Aggression scores were highly negatively correlated with IQ but not with either the EEG/ Seizure status or neurologic signs (table 4). Anger/ Aggression scores were correlated with Impatient/ Overactive scores (r 5 0.45, p , 0.01). Impatience/Overactivity. Four of the seven 7–9-year- olds were reported to show one or more signs of Impatience/Overactivity, which was the age range for highest scores on this scale. Males were more Impatient/Overactive than females according to a Mann-Whitney test (U 5 136.5, p , 0.02). Across all subjects, there was a near-significance for Impatience/Overactivity scores to be correlated with vertical supranuclear gaze palsy (VSGP) (table 4). Crying/Withdrawal. One or more Crying/With- drawal behaviors were shown by 10 of the 12 youn- gest children (1–5 years old) in contrast to Impatient/Overactive and Repetitive Acts, which were each shown by only 2 of these 12 children. These scores appeared to decline after age 9 (figure). Crying/Withdrawn scores were nonsignificantly higher in females (figure). Over all subjects, these scores were associated with EEG/Seizure status (table 4). Crying/Withdrawal scores were not correlated with any of the other 3 behavior categories. Repetitive Acts. Repetitive Act scores were nonsignificantly higher for males. These scores were correlated with Impatient/Overactive scores (r 5 0.47, p , 0.005). Repetitive Act scores were also correlated with ptosis (r 5 0.42, p , 0.02); correlations with EEG status and with strabismus tended to approach significance as showed in table 4.

DISCUSSION Abnormal behavioral features in the patients with GD3 have not been described in (A) Males. (B) Females. detail. The largest GD type 3 study analyzing the

4 Neurology: Genetics Table 4 EEG/Seizure status and neurologic sign correlations with behavior categories

Anger/Aggression Repetitive Acts Cry/Withdrawal Impatient/Overactive

IQ 20.52*** 20.21 20.16 20.06

Strabismus 20.18 0.38* 0.11 0.18

Ptosis (with abnormal facies) 20.12 0.42** 0.31* 20.01

EEG/Seizure status 0.08 0.34* 0.34* 0.26

VSGP 0.29 0.07 20.20 0.31*

Abbreviation: VSGP 5 vertical supranuclear gaze palsy. *p , 0.1, **p , 0.05, ***p , 0.02.

Neurological Outcomes Subregistry of the Interna- physical aggression typically increase up through tional Collaborative Gaucher Group, which age 3, then decline.23 Although the greater aggres- included 40/131 Egyptian patients mostly homo- sion in males was to be expected,24 the overall anger zygous L444P, contained no reference to abnormal intensity and aggression severity in these patients behaviors.17 The prevalence of supranuclear gaze with GD3 appear to be considerably greater than palsy and convergent strabismus in our study normal and significantly affect quality of life for cohort resemble the registry data, but bulbar symp- them and their parents. We identified a strong cor- toms and seizures were more prevalent in our relation between IQ and the Anger/Aggression cohort, suggesting more severe CNS involvement. scale, which replicates the relationship found in Abnormal behaviors that occur in various genetic other neurodegenerative disease conditions.25,26 diseases and other clinical conditions include changes Whether other features of GD3, such as physical in attention and activity as well as simple and complex disability/discomfort, exacerbate anger and aggres- repetitive acts (e.g., motor stereotypies, object manip- sion remains to be determined. ulation, and compulsive acts).18 However, the profile Elevated rates of EEG abnormality and frank clin- of behavioral abnormalities varies in different clinical ical seizures are well known in children with GD3. In conditions. Some of these behaviors appear in many general, EEG abnormalities and clinical seizures are different conditions, presumably due to a confluence associated with a range of behavioral problems, most of genetic/developmental dysfunction in commonly often internalizing anxiety and depression, but also affected brain systems together with the shaping of extending to externalizing inattention and acting behavior by responses from caregivers and others.19 out.27 Some forms of juvenile epilepsy remit sponta- Our cohort showed a wide spectrum of behavioral neously during development.28–30 At least 4 patients abnormalities. Angry and aggressive behaviors were with GD3 seen in the Cairo Hospital have had appar- the most prevalent and the greatest cause of concern. ently spontaneous normalization of EEG and/or These included both reactive and proactive aggres- remission of seizures in later childhood. These obser- sion. Reactive aggression is a response to perceived vations raise the question of whether changes in EEG provocation or challenge to personal autonomy or might be associated with reductions in abnormal social norms and is often accompanied by anger. Pro- behavior. active aggression, such as deliberate harassment and Anger/Aggression and Repetitive Acts were both bullying, without provocation, is committed to dom- significantly associated with Impatient/Overactive inate others and/or for material gain, e.g., attention, behaviors. Such associations are common across control of toys etc.20 Children have been observed clinical conditions.24 On the other hand, these 3 to smile or laugh while committing proactive categories had different patterns of association with aggression.21,22 other independent variables: While Impatient/ The sequence in which tantrums, oppositional Overactive behaviors were just marginally associ- behavior, and physical aggression develop early; ated with VSGP, higher Anger/Aggression was proactive aggression, the use of obscenities and vin- strongly associated with lower IQ as noted, while dictive behavior appear later, and blaming others, Repetitive Acts correlated with the EEG/Seizure verbal aggression and insults, and homicidal threats score and with 2 neurologic signs. One possible are last to appear; reflects the children’s increasing interpretation of this pattern is that a primary dyscognitive and verbal capacity. However, the pro- function in corticobasal ganglia pathways regulat- gressive increases in angry and aggressive behaviors ing attention/activity reduces both inhibitory through age 4–6 differ from the normal trajectory. prefrontal control over several different neurologic Very large scale studies with hundreds to thousands subsystems such as temporal lobe/hypothalamic of children have determined that trajectories of circuits that control anger and aggression31 and

Neurology: Genetics 5 dopaminergic mesolimbic circuits that foster the answered by longitudinal follow-up. This work repetition of behaviors that are reinforcing.32,33 Be- should involve standardized scales of aggression and haviors in GBA1 genotype D409V/null mice seem other behaviors that are available in Arabic transla- similar in that according to their handlers. Mice tion, e.g., the Child Behavior Checklist37 to track were hyperactive, jumpy, and aggressive toward behavior and neurologic function together over time. other mice and humans, biting far more often than Standardized scales would also facilitate the applica- wild-typemiceormicewithotherGBA1 tion of The Diagnostic and Statistical Manual of mutations.34 Mental Disorders, 5th edition or the International Crying/Withdrawal behaviors appeared early in Classification of Diseases, 10th revision diagnostic cat- development and were not associated with any of egories, e.g., the Impatient/Overactive category may the other behavioral patterns. Crying and social with- well correspond to attention-deficit/hyperactivity dis- drawal are internalizing behaviors to which females order. Future studies should also include comparisons are more susceptible than males,35 and the mean with healthy siblings or neurotypicals as well as with Cry/Withdrawal score was the only one that was those with the nonneuronopathic GD type 1 who higher for females, albeit nonsignificantly. These be- have equivalent medical problems. haviors were associated with the EEG/Seizure score. Abnormal behaviors are a phenotypic feature of Sixty-two percent of patients had abnormal EEGs GD3 in the Egyptian cohort, the largest GD3 cohort and/or seizures consistent with the neurologic reported worldwide. The behavior patterns are clearly involvement of GD3. This is far higher than the rate complex. Our working hypothesis is that they may of EEG abnormalities in childhood populations.36 involve a Crying/Withdrawal component that mani- EEG abnormalities and clinical seizures are associated fests early, affects both males and females, and may with a range of behavioral problems, most often inter- relate to epileptiform activity in the brain. A second, nalizing anxiety and depression27 which may relate to apparently independent component of Impatience/ the Cry/Withdrawal pattern of GD3. Spontaneous Overactivity primarily involves males, can be associ- recovery from childhood seizures is well known.30 ated with bizarre repetitive acts and/or highly salient Such recovery may contribute to the reduction of intense anger and severe aggression. The repetitive the Crying/Withdrawal pattern after 7–9 years of age. acts may be associated with several neurologic signs. That these behaviors are intrinsic to the disease The Anger/Aggression component, which may start and not due to additional shared genes/inherited later and persist longer than Crying/Withdrawal, is traits is suggested by the observation that these pa- clearly associated with lower cognitive ability. tients, while derived from consanguineous matings, These components defined by quantitative behav- are not from a tightly inbred population with ioral scoring methods might serve as markers of neu- a founder. They are from small, independent (at least rologic disease progression and severity. EEG for many generations) groups. recording together with structural and functional For this initial study, we assessed behaviors with brain imaging methods can provide insight into the descriptors developed in collaboration with some pa- localization of the underlying pathophysiologic pro- rents and therefore most likely to be recognized and cesses in Gaucher type 3, which would guide future understood by other parents within the Egyptian cul- treatments targeting the CNS disease. ture. This approach has produced statistically significant results but may limit their replicability. AUTHOR CONTRIBUTIONS Behaviors were recorded as present/absent for sim- Magy Abdelwahab, study concept and design, recruited and studied the patients and gathered all data, interpreted the data, and edited the man- plicity, but frequency measures might provide data uscript. Michael Potegal, analysis and interpretation of data and writing for more sensitive analyses of behavioral profiles and and editing the manuscript. Elsa Shapiro, analysis and interpretation of correlations with neurologic function. The use of data and editing and critical revision of manuscript for intellectual con- cluster analyses to determine data-driven groupings tent. Igor Nestrasil, analysis and interpretation of data and writing and editing the manuscript. of behavior was precluded by systematic shifts in behaviors with age. The abnormal development of anger ACKNOWLEDGMENT and aggression in children with GD3 was highlighted The authors are grateful to the patients and their families who participated by comparison with published data on normal devel- in this study as well as the Center for Neurobehavioral Development opmental trajectories of these behaviors. As noted (CNBD) for providing the spaces for the video assessments with coauthors ’ (E.G.S. and I.N.). They thank Dr. Robin Rumsey for her help in distin- below, comparison of affected children s behaviors guishing behavioral abnormalities during initial video assessments; to that of their healthy siblings would provide a useful Dr. Greg Grabowski for the observations of the genetically intrinsic nature control for family-related variables in the future. of the behavioral abnormalities; Dr. Pramod Mistry, Yale University, This retrospective study has raised questions about for help with GBA1 sequencing; Dr. Khaled Eid for technical help; Dr. Mohamed Abd Elmonem, Cairo University, Egypt, for help with developmental changes with age, disease progression, GBA1 genotyping; and Genzyme A Sanofi Company, Shire Plc, and the and treatment effects in GD3 that can only be Hope project for providing ERT product.

6 Neurology: Genetics STUDY FUNDING the report form for the EEG findings. The International No targeted funding reported. Federation of Clinical Neurophysiology. Electroencepha- logr Clin Neurophysiol Suppl 1999;52:21–41. DISCLOSURE 15. Thomas DR, Hughes E, Zumbo BD. On variable Dr. Magy Abdelwahab has received travel funding from a nonprofit importance in linear regression. Soc Indic Res 1998; entity. Dr. Michael Potegal reports no disclosures. Dr. Elsa Shapiro 45:253–275. has served on scientific advisory boards for ReGenXBio, Armagen, Shire, 16. Benjamini Y, Drai D, Elmer G, Kafkafi N, Golani I. SOBI, BioMarin, and Eloxx; has received travel funding/speaker hono- Controlling the false discovery rate in behavior genetics raria from Shire and BioMarin; has been a consultant for ReGenXBio, research. Behav Brain Res 2001;125:279–284. Armagen, Shire, SOBI, BioMarin, Eloxx, Alexion, Chiesi, BluebirdBio, 17. Tylki-Szymanska A, Vellodi A, El-Beshlawy A, Cole JA, Sangamo, Aeglea, and Lysogene; and is a partner in Shapiro & Delaney, Kolodny E. Neuronopathic Gaucher disease: demo- LLC. Dr. Igor Nestrasil has been a consultant for Armagen, BioMarin, Lysogene, ReGenXBio, and ICON and has received research support graphic and clinical features of 131 patients enrolled from Shire, Genzyme, BioMarin, NIH, The Penn Medicine Orphan in the International Collaborative Gaucher Group Neu- Disease Center, and the University of Pennsylvania. Go to Neurology. rological Outcomes Subregistry. J Inherit Metab Dis org/ng for full disclosure forms. 2010;33:339–346. 18. Moss J, Oliver C, Arron K, Burbidge C, Berg K. The Received November 23, 2016. Accepted in final form April 7, 2017. prevalence and phenomenology of repetitive behavior in genetic syndromes. J Autism Dev Disord 2009;39: REFERENCES 572–588. 1. Benko W, Ries M, Wiggs EA, Brady RO, Schiffmann R, 19. Muehlmann AM, Lewis MH. Abnormal repetitive behav- Fitzgibbon EJ. The saccadic and neurological deficits in iours: shared phenomenology and pathophysiology. type 3 Gaucher disease. PLoS One 2011;6:e22410. J Intellect Disabil Res 2012;56:427–440. 2. Abdelwahab M, Blankenship D, Schiffmann R. Long-term 20. Hubbard JA, McAuliffe MD, Morrow MT, Romano LJ. follow-up and sudden unexpected death in Gaucher dis- Reactive and proactive aggression in childhood and ado- ease type 3 in Egypt. Neurol Genet 2016;2:e55. doi: 10. lescence: precursors, outcomes, processes, experiences, and 1212/NXG.0000000000000055. measurement. J Pers 2010;78:95–118. 3. Erikson A, Bembi B, Schiffmann R. Neuronopathic forms 21. Arsenio W, Cooperman S. Children’s conflict-related of Gaucher’s disease. Baillieres Clin Haematol 1997;10: emotions: implications for morality and autonomy. New 711–723. Dir Child Dev 1996:25–39. 4. Campbell PE, Harris CM, Vellodi A. Deterioration of the 22. Benenson JF, Carder HP, Geib-Cole SJ. The development auditory brainstem response in children with type 3 of boys’ preferential pleasure in physical aggression. Gaucher disease. Neurology 2004;63:385–387. Aggress Behav 2008;34:154–166. 5. Altarescu G, Hill S, Wiggs E, et al. The efficacy of enzyme 23. Fanti KA, Henrich CC. Trajectories of pure and co- replacement therapy in patients with chronic neurono- occurring internalizing and externalizing problems from pathic Gaucher’s disease. J Pediatr 2001;138:539–547. age 2 to age 12: findings from the National Institute of 6. Erikson A, Astrom M, Mansson JE. Enzyme infusion Child Health and Human Development Study of Early therapy of the Norrbottnian (type 3) Gaucher disease. Child Care. Dev Psychol 2010;46:1159–1175. Neuropediatrics 1995;26:203–207. 24. Potegal M, Archer J. Sex differences in childhood anger 7. Vellodi A, Bembi B, de Villemeur TB, et al. Management and aggression. Child Adolesc Psychiatr Clin N Am 2004; of neuronopathic Gaucher disease: a European consensus. 13:513–528, vi–vii. J Inherit Metab Dis 2001;24:319–327. 25. Oliver C, Petty J, Ruddick L, Bacarese-Hamilton M. The 8. Choy FY, Zhang W, Shi HP, et al. Gaucher disease among association between repetitive, self-injurious and aggressive Chinese patients: review on genotype/phenotype correla- behavior in children with severe intellectual disability. tion from 29 patients and identification of novel and rare J Autism Dev Disord 2012;42:910–919. alleles. Blood Cells Mol Dis 2007;38:287–293. 26. Schroeder SR, Marquis JG, Reese RM, et al. Risk fac- 9. Goker-Alpan O, Wiggs EA, Eblan MJ, et al. Cognitive tors for self-injury, aggression, and stereotyped behavior outcome in treated patients with chronic neuronopathic among young children at risk for intellectual and devel- Gaucher disease. J Pediatr 2008;153:89–94. opmental disabilities. Am J Intellect Dev Disabil 2014; 10. Davies EH, Erikson A, Collin-Histed T, Mengel E, 119:351–330. Tylki-Szymanska A, Vellodi A. Outcome of type III 27. Dunn DW, Austin JK, Perkins SM. Prevalence of psy- Gaucher disease on enzyme replacement therapy: review chopathology in childhood epilepsy: categorical and of 55 cases. J Inherit Metab Dis 2007;30:935–942. dimensional measures. Dev Med Child Neurol 2009; 11. Tajima A, Yokoi T, Ariga M, et al. Clinical and genetic 51:364–372. study of Japanese patients with type 3 Gaucher disease. 28. Cavazzuti GB, Cappella L, Nalin A. Longitudinal study of Mol Genet Metab 2009;97:272–277. epileptiform EEG patterns in normal children. Epilepsia 12. Abdelwahab M. Management of type 3 Gaucher disease. 1980;21:43–55. Int J Clin Rev 2012;10:93–99. 29. Loiseau P, Pestre M, Dartigues JF, Commenges D, 13. Delaney KA, Rudser KR, Yund BD, Whitley CB, Haslett Barberger-Gateau C, Cohadon S. Long-term prognosis PA, Shapiro EG. Methods of neurodevelopmental assess- in two forms of childhood epilepsy: typical absence ment in children with neurodegenerative disease: Sanfilip- seizures and epilepsy with rolandic (centrotemporal) po syndrome. JIMD Rep 2014;13:129–137. EEG foci. Ann Neurol 1983;13:642–648. 14. Noachtar S, Binnie C, Ebersole J, Mauguiere F, Sakamoto 30. Sillanpaa M, Jalava M, Kaleva O, Shinnar S. Long-term A, Westmoreland B. A glossary of terms most commonly prognosis of seizures with onset in childhood. N Engl J used by clinical electroencephalographers and proposal for Med 1998;338:1715–1722.

Neurology: Genetics 7 31. Potegal M. Temporal and frontal lobe initiation and reg- 35. Zahn-Waxler C, Crick NR, Shirtcliff EA, Woods KE. The ulation of the top-down escalation of anger and aggression. origins and development of psychopathology in females Behav Brain Res 2012;231:386–395. and males. In: Cicchetti D, Cohen DJ, editors. Develop- 32. Fasano A, Petrovic I. Insights into pathophysiology of mental Psychopathology: Volume 1 Theory and Method. punding reveal possible treatment strategies. Mol Psychia- Hoboken: Wiley; 2006. try 2010;15:560–573. 36. Shelley BP, Trimble MR. “All that spikes is not fits,” 33. McBride SD, Parker MO. The disrupted basal ganglia and mistaking the woods for the trees: the interictal spikes— behavioural control: an integrative cross-domain perspec- an “EEG chameleon” in the interface disorders of brain tive of spontaneous stereotypy. Behav Brain Res 2015;276: and mind: a critical review. Clin EEG Neurosci 2009;40: 45–58. 245–261. 34. Dai M, Liou B, Swope B, et al. Progression of behavioral 37. Achenbach TM, Ruffle TM. The child behavior check- and CNS deficits in a viable Murine model of chronic list and related forms for assessing behavioral/emotional neuronopathic Gaucher disease. PLoS One 2016;11: problems and competencies. Pediatr Rev 2000;21: e0162367. 265–271.

8 Neurology: Genetics Intramyocellular lipid excess in the mitochondrial disorder MELAS MRS determination at 7T

Sailaja Golla, MD ABSTRACT Jimin Ren, PhD Objective: There is a paucity of objective, quantifiable indicators of mitochondrial disease avail- Craig R. Malloy, MD able for clinical and scientific investigation. Juan M. Pascual, MD, Methods: To this end, we explore intramyocellular lipid (IMCL) accumulation noninvasively by 7T PhD magnetic resonance spectroscopy (MRS) as a reporter of metabolic dysfunction in MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). We reasoned that mitochondrial dysfunction may impair muscle fat metabolism, resulting in lipid deposition (as is Correspondence to Dr. Pascual: sometimes observed in biopsies), and that MRS is well suited to quantify these lipids. Juan.Pascual@UTSouthwestern. edu Results: In 10 MELAS participants and relatives, IMCL abundance correlates with percent mitochondrial DNA mutation abundance and with disease severity. Conclusions: These results indicate that IMCL accumulation is a novel potential disease hallmark in MELAS. Neurol Genet 2017;3:e160; doi: 10.1212/NXG.0000000000000160

GLOSSARY ATP 5 adenosine triphosphate; IMCL 5 intramyocellular lipid; MRS 5 magnetic resonance spectroscopy; mtDNA 5 mitochondrial DNA; ppm 5 parts per million; TE 5 echo time.

Proper diagnosis and effective monitoring of patients with mitochondrial diseases remains a challenge in part because plasma and urine metabolite concentrations are nonspecific, tissue biopsies have limited acceptability and are prone to sampling error, and longitudinal measurements with invasive procedures are difficult.1 MELAS syndrome (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes) is a maternally inherited and relatively prevalent mitochondrial disorder. Multiple studies have reported magnetic resonance spectroscopy (MRS) findings in the brain including elevated lactate, but studies of metabolism in skeletal muscle by MRS methods have been limited. For example, a combined examination with 31P and 1H NMR spectroscopy has characterized mitochondrial function and intramyocellular lipids (IMCLs) in one individual with MELAS.2 This study points to the advantages of MRS in examinations of patients with suspected mitochondrial disorders—subject acceptability, safety, and detailed metabolic information specific to an organ. The purpose of this study was to examine the feasibility of 1H MRS at 7 Tesla (7T) in MELAS participants and maternal relatives. We separately determined normal IMCL in healthy controls; results were compared with an earlier cohort of healthy controls. In MELAS, a robust correlation was found between IMCL abundance and percent A3243G mutant mitochondrial DNA (mtDNA) abundance in the blood. The study was well tolerated by all participants. The high chemical shift dispersion at 7T was advantageous because the IMCL and other metabolite

From the Rare Brain Disorders Program (S.G., J.M.P.), Department of Neurology and Neurotherapeutics, Department of Pediatrics (S.G., J.M.P.), Advanced Imaging Research Center (J.R., C.R.M.), Department of Radiology (J.R., C.R.M.), Department of Internal Medicine (C.R.M.), Department of Physiology (J.M.P.), and Eugene McDermott Center for Human Growth & Development/Center for Human Genetics (J.M.P.), The University of Texas Southwestern Medical Center, Dallas. Funding information and disclosures are provided at the end of the article. Go to Neurology.org/ng for full disclosure forms. 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.

Neurology.org/ng Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 signals were easily resolved. Thus, 1H MRS at a constant repetition time of 2 seconds. A typical number of scans 7T may provide an avenue for investigating were 192 (or 6.4 minutes for TE 140 milliseconds) and 256 (or 8.5 minutes for TE 280 milliseconds). No water suppression disease severity as well as the response of skel- pulses were applied during data acquisition to avoid signal etal muscle to therapies. reduction due to possible magnetization-transfer effects on metabolites as we previously described.3 The 1H chemical shifts of METHODS Standard protocol approvals, registrations, all metabolite resonances arising from muscle were referenced to and patient consents. This study was approved by the Insti- creatine methyl protons set to 3.02 parts per million (ppm) (Cr3). tutional Review Board of the University of Texas Southwestern The area of each metabolite 1H resonance signal was determined Medical Center. Informed written consent was obtained from by fitting the spectrum to a Voigt lineshape (variable proportions all participants or their legal guardians as appropriate. They of lorentzian plus gaussian) using ACD software (Advanced were studied over 4 months in one visit (each lasting less than Chemistry Development, Inc., Toronto, ON, Canada). The 3 hours). asymmetry of the extramyocellular lipid signal was fitted by 2 Voigt lineshapes as described.3 For quantitative comparison General methods. Mutant mtDNA abundance (expressed as between the metabolites, the area of creatine methyl 1H resonance percent of mutant DNA relative to total blood DNA) was rou- signal (Cr3) was used as the internal standard (30 mmol/kg wet tinely determined in the blood by quantitative PCR. Because tissue weight). All MRS data quantifications are expressed in – mutation abundance can vary with age by 0.6% 1.9% per year millimoles per kilogram wet tissue weight. Errors denote mean 6 in each person, participants were genotyped within 3 years SD where indicated. preceding enrollment. All participants’ symptoms and medical history were obtained using a standardized questionnaire. Com- RESULTS All the participants tested positive for the plete hematologic, endocrine, chemical, and lipid profiles . (including blood cell count, blood electrolytes, transaminases, m.3243A G mutation except those identified as lactate, urea nitrogen, creatinine, amino acids, creatine kinase, maternal relatives (table). As a group, the MELAS free fatty acids, thyroid-stimulating hormone, T4, lipid panel participants exhibited the common features of the [total, low-density lipoprotein and high-density lipoprotein disorder such as lactic acidosis, stroke-like episodes, cholesterol, and triglycerides], fasting blood glucose, and hearing loss, neuropathy and, in some, diabetes as hemoglobin A1c [HbA1c]) were measured by standard clinical indicated in the table. Elevated IMCLs were observed laboratory methods and compared with normal values established in each laboratory. in all the MELAS participants examined by MRS (figure 1). Of the 8 participants with MELAS (table), Skeletal muscle proton MRS. The details of the examination the average concentration of IMCLs was 26 6 18 have been published. Briefly, leg calf muscles of rested (.2 hours) mmol/kg muscle mass. The average concentration of participants were subject to MRS in a 7T system (Achieva; Philips Medical Systems, Cleveland, OH) without sedation. Scan IMCL among participants with the more severe sessions lasted less than 1 hour. Participants were positioned phenotype (participants 4, 7, 8, and 10) tended to be supine with their legs parallel to the magnetic field and the left higher, 33 mmol/kg muscle mass. The association calf sitting on the center of a partial volume quadrature transmit/ between IMCL abundance and percent mutant receive coil customized to fit the shape of a human calf. Axial, mtDNA abundance depended on the coexistence of coronal, and turbo spin-echo images were first acquired to guide diabetes (figure 2). voxel placement within the soleus muscle. Single-voxel 1H MRS spectra were collected from the soleus with a volume of 3–5 mL, The ratio of the trimethylamine signal from carni- using a STimulated Echo Acquisition Mode (STEAM) sequence, tine at 3.20 ppm relative to the creatine methyl signal at long echo time (TE) of both 140 and 280 milliseconds, and at 3.02 ppm (carnitine/Cr3) is typically ,1or;1;

Table Characteristics of the participants and IMCL values

m.A3243G IMCL, mmol/kg Participant Age, y Sex Diabetes MELAS findings abundance, % wet weight

1 27 F No None, maternal relative 0 7.2

2 52 F No Elevated blood lactate 19 7.0

3 25 F No Deafness 44 12.9

4 16 F No Elevated blood lactate and migraine 50 10.6

5 18 M No Elevated blood lactate, cerebral infarction, and dementia 71 18.4

6 39 F Yes None, maternal relative 0 19.6

7 47 F Yes Elevated blood lactate and distal neuropathy 0 23

8 43 F Yes Elevated blood lactate, migraine, and short stature 20 40.3

9 26 F Yes Elevated blood lactate and distal neuropathy 25 37.5

10 26 M Yes Elevated blood lactate, deafness, upgaze palsy, and cerebral infarction 25 58.8

Abbreviation: IMCL 5 intramyocellular lipid.

2 Neurology: Genetics Figure 1 Proton spectra of skeletal muscle

MRS of 2 soleus muscles (participants 1 [A] and 2 [B]). Proton spectra were acquired at 2 echo times as described in the text. Solid black lines are plots of the spectra. IMCL CH2 represents resonances attributed to intramyocellular lipids, whereas EMCL CH2 is considered to arise from extramyocellular lipids. Carnitine CH3 5 signal arising from carnitine; creatine CH3 5 signal emanating from carbon 3 of creatine; H2O 5 water spectrum; MRS 5 magnetic resonance spectroscopy; ppm 5 parts per million. the relative signals are sensitive to acquisition condi- detected by nerve conduction monitoring despite the tions. With a TE of 140 milliseconds, the signals are appearance of subjective benefits such as increased typically ;1:1 or nearly equally sized “twin peaks.” physical endurance. A key limitation is the difficulty MELAS participants displayed significantly higher of acquiring objective measurements of metabolic trimethylamine/Cr3 ratio (1.46 6 0.40). efficacy in the target tissue.4 The relation between IMCL and plasma lactate, Our study demonstrated an increase in the con- urea nitrogen, creatinine, free fatty acids, triglycerides, centration of IMCLs among participants with and total cholesterol was also examined. No significant MELAS syndrome compared with historical healthy correlation was found. controls. A simple explanation is that skeletal mitochondrial function in MELAS is substantially DISCUSSION There is a paucity of biomarkers and impaired, resulting in diminished oxidation of fatty of noninvasive methods available for the study of acids and accumulation of IMCL in the cytosol.5 mitochondrial disorders. These obstacles limit clinical Among the cytoplasmic lipids that accumulate in rela- trials. For example, our study of dichloroacetate (a tion to metabolic dysfunction are diacylglycerol and lactate-reducing drug) in a mitochondrial disorder ceramide. Extensive evidence links these lipids with was discontinued when toxic neuropathy was human mitochondrial dysfunction and with

Figure 2 IMCL, mtDNA, and diabetes correlations

IMCL abundance correlations with percent mutant mtDNA (m.A3243G) in the blood. (A) Correlations for nondiabetic (i.e., HbA1c ,6% and no history of diabetes) participants. (B) Correlations for diabetic participants (HbA1c .6% and a history of diabetes). Correlation coefficients with a straight line are given in each panel. IMCL 5 intramyocellular lipid; mtDNA, mitochondrial DNA.

Neurology: Genetics 3 mitochondrial-related insulin resistance. For example, board of Neuroscience Letters and has received research support from NIH/ in diabetes, slowed postexercise mitochondrial aden- National Institute of Neurological Disorders and Stroke and NIH/NIMH. Go to Neurology.org/ng for full disclosure forms. osine triphosphate (ATP) resynthesis is related to 6 increased insulin resistance, whereas insulin- Received March 6, 2017. Accepted in final form April 7, 2017. stimulated rates of mitochondrial ATP synthesis are reduced in otherwise healthy insulin-resistant partic- REFERENCES ipants.7 Accumulation of these lipids has also been 1. DiMauro S, Paradas C. Mitochondrial disorders due to observed by MRS in relation to insulin resistance in mutations in the mitochondrial genome. In: Rosenberg ’ otherwise healthy8,9 and diabetic participants. An ear- RN, Pascual JM, editors. Rosenberg s Molecular and Genetic Basis of Neurological and Psychiatric Disease, lier case report did not find a significant increase in 5th ed. San Diego: Academic Press; 2015. IMCL in a single participant using skeletal water as an 2. Szendroedi J, Schmid AI, Meyerspeer M, et al. Impaired 2 internal reference and a TE of 30 milliseconds. This mitochondrial function and insulin resistance of skeletal study used relatively long echo times to suppress muscle in mitochondrial diabetes. Diabetes Care 2009;32: water signal, so differences in acquisition and quanti- 677–679. tation methods may be important. 3. Ren J, Sherry AD, Malloy CR. 1H MRS of intramyocellular lipids in soleus muscle at 7 T: spectral simplification This study found an unexpected increase in the by using long echo times without water suppression. Magn carnitine/creatine ratio. Because this MRS method only Reson Med 2010;64:662–671. detects the ratio of NMR-visible metabolites and not 4. Kaufmann P, Engelstad K, Wei Y, et al. Dichloroacetate absolute concentration, this altered ratio could be due causes toxic neuropathy in MELAS: a randomized, con- to some combination of altered relaxation times, trolled clinical trial. Neurology 2006;66:324–330. increased carnitine, decrease creatine, or some combina- 5. Kiens B. Skeletal muscle lipid metabolism in exercise and – tion of these factors. Further studies will clarify any insulin resistance. Physiol Rev 2006;86:205 243. 6. Cree-Green M, Newcomer BR, Brown MS, et al. Delayed potential racial differences in IMCL abundance in rela- skeletal muscle mitochondrial ADP recovery in youth with 10 tion to insulin resistance in MELAS and the presence type 1 diabetes relates to muscle insulin resistance. Diabe- of IMCL in other mitochondrial diseases. tes 2015;64:383–392. 7. Petersen KF, Dufour S, Shulman GI. Decreased insulin- AUTHOR CONTRIBUTIONS stimulated ATP synthesis and phosphate transport in mus- Conception and design of the study: J.M.P. and C.R.M. Acquisition and cle of insulin-resistant offspring of type 2 diabetic parents. analysis of data: S.G., J.R., C.R.M., and J.M.P. Drafting of the manu- PLoS Med 2005;2:e233. script: S.G., J.R., C.R.M. and J.M.P. Drafting of the figures: S.G., J. 8. Sinha R, Dufour S, Petersen KF, et al. Assessment of R., C.R.M., and J.M.P. skeletal muscle triglyceride content by (1)H nuclear magnetic resonance spectroscopy in lean and obese adolescents: STUDY FUNDING relationships to insulin sensitivity, total body fat, and cen- This work was supported by a pilot grant to J.M.P. and C.R.M. under tral adiposity. Diabetes 2002;51:1022–1027. NIH grant UL1TR001105 and by a gift of the Hegi Foundation to 9. Goodpaster BH, Thaete FL, Simoneau JA, Kelley DE. Children’s Medical Center Dallas to support the work of S.G. and J. Subcutaneous abdominal fat and thigh muscle composi- M.P. J.M.P. and C.R.M. are also supported by NIH grants NS077015, tion predict insulin sensitivity independently of visceral NS078059, EB015908, and RR024982. fat. Diabetes 1997;46:1579–1585. DISCLOSURE 10. Misra A, Sinha S, Kumar M, Jagannathan NR, Pandey RM. Proton magnetic resonance spectroscopy study of Dr. Golla reports no disclosures. Dr. Ren has served on the editorial boards of the Journal of Analytical & Molecular Techniques, BioMed Research Inter- soleus muscle in non-obese healthy and type 2 diabetic national,andCurrent Metabolomics. Dr. Malloy has served on the editorial Asian Northern Indian males: high intramyocellular lipid board of Tomography and has received publishing royalties for writing content correlates with excess body fat and abdominal occasional book chapters (,$100). Dr. Pascual has served on the editorial obesity. Diabet Med 2003;20:361–367.

4 Neurology: Genetics Clinical/Scientific Notes

Jens Reimann, MD* CAMPTOCORMIA AND SHUFFLING GAIT DUE TO vastus lateralis muscle was normal. Repeated neu- Diana Lehmann, MD* A NOVEL MT-TV MUTATION: DIAGNOSTIC rography revealed a mild sensorimotor polyneuro- Steven A. Hardy, PhD PITFALLS pathy. Levodopa treatment and CSF drainage under Gavin Falkous, MPhil the suspicion of Parkinson disease or normal pressure Charlotte V.Y. Knowles, Camptocormia, the disabling flexion of the spine in hydrocephalus (NPH) due to enlarged lateral ventricles MBiolSci upright, but not supine position, has been reported in an otherwise unremarkable brain MRI and urge Rachel L. Jones, BSc in a range of central nervous and neuromuscular con- incontinence were unsuccessful. DAT scan, EEG, Wolfram S. Kunz, PhD ditions and is associated with aging, too. In many cases, Holter ECG, blood pressure monitoring, orthostatic Robert W. Taylor, PhD, e.g., Parkinson disease, further clinical symptoms will test, and CSF analysis, including biomarkers for neuro- FRCPath* clarify its association, if not pathophysiology. In others, degeneration (beta-amyloid, tau, and phospho-tau), Cornelia Kornblum, a tangle of signs and symptoms obscures the etiology. revealed no abnormalities. Mini-Mental State exami- MD* Here, we present a new solution to this challenging and nation and Montreal Cognitive Assessment indicated complex clinical problem. Neurol Genet mild cognitive impairment. Maximum serum creatine 2017;3:e147; doi: 10.1212/ A 71-year-old Caucasian woman presented with kinase was 236 U/L (,170 U/L). Genetic and anti- NXG.0000000000000147 a 3-year history of unstable, short-stepping slow, body analysis ruled out facioscapulohumeral muscular shuffling gait and complained of deteriorating hand- dystrophy types 1 and 2 as well as myasthenic syn- writing. Her medical history included basalioma, dromes, respectively. malignant melanoma, hypothyreosis, bilateral cata- Biceps brachii muscle biopsy showed angular ract and hypoacusis, gastroesophageal reflux, predia- atrophic fibers, a few degenerating fibers and betes, and thoracal and lumbal disc herniation. increased lipofuscin deposition. Modified Gomori While her mother and a brother suffer from type trichrome staining and oxidative enzyme reactions II diabetes and a sister has a thyroid disorder, no revealed ragged red fibers and ;6% cytochrome other recurrent, neuromuscular, or movement disor- c oxidase (COX)-deficient fibers (figure, B). Occa- ders are known in the family. A brother died in sional ragged red fibers appeared COX positive. In childhood of unclear causes, a further brother in skeletal muscle tissue homogenate, activities of adulthood of a liver condition. Her son, daughter, mitochondrial respiratory chain complexes I and and 2 grandsons are well. Clinical examination re- IV normalized against citrate synthase activity were vealed slow horizontal saccades. Deep tendon re- mildly decreased to 0.07 (controls: 0.11 6 0.03 flexes were brisk, but for diminished ankle reflexes. [n 5 11]) and 1.38 (2.7 6 0.5 [n 5 11]) U/g, respec- Babinski response was equivocal on the right. Mus- tively, while quadruple OXPHOS immunofluores- cle tone and bulk appeared normal, but there was cence1 confirmed the presence of fibers lacking both weakness of proximal lower limb muscles and foot extensors (MRC 4). Steps were short and their num- complex I (NDUFB8) and complex IV (COX-1) ber for 180° turn increased. Positive Romberg test expression, confirming a multiple respiratory chain and inability to tandem walking suggested sensory defect (figure, C). Mitochondrial DNA (mtDNA) ataxia, but sensory examination was otherwise nor- sequencing revealed a previously unreported hetero- . mal. At follow-up, a slightly stooped posture pro- plasmic m.1660G A MT-TV variant present at gressed into camptocormia (figure, A) without signs highest levels in the muscle (35% mutation load), with of dystonia or muscle rigidity. Mild weakness of lower levels in urinary epithelial sediments (13%) and proximal arm muscles was found. Spinal MRI blood (9%), consistent with the segregation pattern of showed cervical stenosis without signs of myelopa- a pathogenic mtDNA mutation. Single-fiber segrega- thy, but fatty replacement of paraspinal muscles. tion studies clearly confirmed pathogenicity, showing Initial neurophysiologic examinations showed pro- a statistically significant higher m.1660G.Amutation longed cortical latency after tibial nerve stimulation, load in COX-deficient fibers (94.30 6 0.76 [n 5 20]) but transcranial stimulation gave normal total and than in COX-positive fibers (22.17 6 6.49 [n 5 18], central motor conduction times, and EMG of the p , 0.0001, unpaired t test) (figure, D).

Neurology.org/ng Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Figure Clinical, histopathologic, and molecular genetic characterization of the m.1660G.A MT-TV mutation

(A) Camptocormia as the main clinical feature of the patient harboring the novel 1660G.A MT-TV mutation. (B) Serial hematoxylin and eosin (B.a), modified Gomori trichrome staining (B.b), succinate dehydrogenase (SDH) (B.c), and cytochrome c oxidase (COX)-SDH histochemistry (B.d) showing ragged red fibers and COX-deficient fibers (scale bar 5 50 mm). (C) Result of the quadruple OXPHOS immunofluorescence analysis, confirming the presence of fibers lacking both complex I (NDUFB8) and complex IV (COX-1) expressions. (D) Single muscle fiber mutation load segregation. The graph shows the mutation load measured in individual COX-positive (closed dots) and COX-deficient fibers (open dots) laser microdissected from the patient muscle biopsy. (E) Schematic representation of the cloverleaf structureofthemitochondrial(mt)-tRNAVal molecule and the corresponding location of the pathogenic mutation (marked in red) and previous reported mt-tRNA Val mutations (black). (F) Phylogenetic conservation of the appropriateregionsofthemt-tRNAValgenesequenceforthe m.1660G.A mutation.

2 Neurology: Genetics Camptocormia has been reported in association Disclosure: Dr. Reimann serves as an associate editor for BMC with myopathic and mitochondrial defects2–7 with Neurology. Dr. Lehmann receives funding from the European Academy of Neurology. Dr. Hardy, Mr. Falkous, Ms. Knowles, recent research suggesting limb muscle biopsy as a rec- and Ms. Jones report no disclosures. Prof. Kunz is supported by the ommended diagnostic procedure.7 Here, we demon- Deutsche Forschungsgemeinschaft (KU911/21-1). Prof. Taylor is strate a rare late-onset mitochondrial disorder due to supported by the Wellcome Trust Centre for Mitochondrial Research a novel pathogenic MT-TV mutation (figure, E and (096919Z/11/Z), the MRC Centre for Translational Research in Neuromuscular Disease Mitochondrial Disease Patient Cohort F) mimicking much more common clinical condi- (UK) (G0800674), the Lily Foundation, the UK NIHR Biomedical tions like NPH, subcortical artherosclerotic encepha- Research Centre for Aging and Age-related disease award to the lopathy, or extrapyramidal movement disorders. Newcastle upon Tyne Foundation Hospitals NHS Trust, and the UK NHS Highly Specialized “Rare Mitochondrial Disorders of Particularly, the coexistence of a shuffling gait, Adults and Children” Service. Prof. Kornblum has received travel peripheral neuropathy, axial weakness, and bent spine grants and honoraria for clinical advisory board activities from at an advanced age may masquerade a mitochondrial Stealth Biotherapeutics; has received travel grants and speaker hon- pathophysiology and lead to erroneous diagnosis and oraria from Sanofi Genzyme; has received travel grants from Mari- gold Foundation Canada and Deutsche Gesellschaft für treatment. Our finding adds to the spectrum of dif- Muskelkranke e.V.; and has received funding from the German ferential diagnostic considerations in gait and balance Ministry of Education and Research (BMBF: 01GM0862), the disorders in the elderly and underlines the importance Deutsche Gesellschaft für Muskelkranke e.V. (Me4/1), and the Mari- of skeletal muscle biopsy as a major diagnostic tool in gold Foundation, Canada. Go to Neurology.org/ng for full disclosure forms. The Article Processing Charge was funded by the Wellcome these patients. Mitochondrial disorders frequently Trust. lead to multisystemic disease and may manifest even This is an open access article distributed under the terms of the in late adulthood where symptomatic treatment op- Creative Commons Attribution License 4.0 (CC BY), which permits tions and tailored clinical advice are of utmost impor- unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. tance for affected patients. Received November 29, 2016. Accepted in final form February 24, 2017. *These authors contributed equally to the manuscript. Correspondence to Dr. Reimann: [email protected] From the Department of Neurology (J.R., C.K.), Department of Ep- ileptology (W.S.K.), Life and Brain Centre (W.S.K.), and Centre for 1. Rocha MC, Grady JP, Grunewald A, et al. A novel immuno- Rare Diseases Bonn (ZSEB) (C.K.), University Hospital of Bonn, fluorescent assay to investigate oxidative phosphorylation defi- Germany; Department of Neurology (D.L.), University of Halle/ ciency in mitochondrial myopathy: understanding mechanisms S., Germany; and Wellcome Trust Centre for Mitochondrial Research (D.L., S.A.H., G.F., C.V.Y.K., R.L.J., R.W.T.), Institute and improving diagnosis. Sci Rep 2015;5:15037. of Neuroscience, The Medical School, Newcastle University, 2. Sakiyama Y, Okamoto Y, Higuchi I, et al. A new phenotype Newcastle upon Tyne, UK. of mitochondrial disease characterized by familial late-onset Author contributions: J.R.: analysis and interpretation of the clinical predominant axial myopathy and encephalopathy. Acta – and histological data and drafting and revision of the manuscript. Neuropathol 2011;121:775 783. D.L.: analysis and interpretation of immunohistochemical and 3. Delcey V, Hachulla E, Michon-Pasturel U, et al. Campto- molecular genetic data, preparation of manuscript and figures, and cormia: a sign of axial myopathy: report of 7 cases [in statistical analysis. S.A.H.: analysis and interpretation of molecular French]. Rev Med Interne 2002;23:144–154. genetic data. G.F.: analysis and interpretation of histochemical and 4. Gomez-Puerta JA, Peris P, Grau JM, Martinez MA, histological data. C.V.Y.K. and R.L.J.: analysis and interpretation of Guanabens N. Camptocormia as a clinical manifestation molecular genetic data. W.S.K.: analysis and interpretation of bio- of mitochondrial myopathy. Clin Rheumatol 2007;26: chemical and genetic data. R.W.T.: drafting and revision of the 1017–1019. manuscript and figures for important intellectual content and study supervision and coordination. C.K.: analysis or interpretation of the 5. Schabitz WR, Glatz K, Schuhan C, et al. Severe forward ’ clinical data, drafting and revision of the manuscript, and study flexion of the trunk in Parkinson s disease: focal myopathy coordination. of the paraspinal muscles mimicking camptocormia. Mov – Acknowledgment: The authors thank Mrs. Karin Kappes-Horn, Disord 2003;18:408 414. Department of Neurology, University Hospital of Bonn, Germany, 6. Serratrice G, Pouget J, Pellissier JF. Bent spine syndrome. for her invaluable technical assistance with the diagnostic histopa- J Neurol Neurosurg Psychiatry 1996;60:51–54. thology and respiratory chain biochemistry. 7. Chanson JB, Lannes B, Echaniz-Laguna A. Is deltoid Study funding: This study was funded by a Wellcome Trust Strategic muscle biopsy useful in isolated camptocormia? A pro- Award (096919Z/11/Z). spective study. Eur J Neurol 2016;23:1086–1092.

Neurology: Genetics 3 Clinical/Scientific Notes

Ricardo H. Roda, MD, SCA8 SHOULD NOT BE TESTED IN ISOLATION evaluation at 53 years of age, he was found to have PhD FOR ATAXIA dysarthria, truncal ataxia, leg dysmetria, and an Alice B. Schindler, MS ataxic and spastic gait—a syndrome similar to that Craig Blackstone, MD, Spinocerebellar ataxia types 1 (SCA1, OMIM# of the proband. PhD 164400) and 8 (SCA8, OMIM# 608768) are auto- Testing of the proband for pathogenic mutations somal dominant, inherited ataxias. SCA1 is caused by in SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, Neurol Genet SCA10, SCA17, and DRPLA loci revealed potentially 2017;3:e150; doi: 10.1212/ abnormal expansion of a CAG triplet repeat in the NXG.0000000000000150 ATXN1 gene, while SCA8 results from CAG and pathogenic abnormalities at 2 loci (figure). There complementary CUG expansions from a bidirection- were 45 and 30 CAG repeats, respectively, in the 2 alleles at the SCA1 locus (normal #35, borderline ally transcribed locus comprising the ATXN8OS and 36–46, full mutation $47), while at the SCA8 locus, ATXN8 genes.1 In both cases, the expansions are there were 795 and 24 CTA/CTG repeats (normal thought to act as toxic gain-of-function mutations, #50, borderline 51–70, full mutation $71). He was although loss of normal protein function could also thus deemed to have potentially pathogenic muta- play a role. Although expansions in SCA1 have been tions in both SCA1 and SCA8 loci. His unaffected clearly determined as pathogenic, those in SCA8 have brother had a normal neurologic examination as well been more difficult to study. Some investigators have as normal numbers of triplet repeats in both SCA1 suggested that very large expansions of repeats in and SCA8 (figure, III.4). SCA8 may not be pathogenic at all, since these might Since the possibility remained that both expanded be unstable and in fact have been reported both in alleles could have been inherited from his affected clinically affected and unaffected persons.2–6 To fur- father, but DNA from the father was unfortunately ther complicate matters, pathogenic expansions in not available for testing, we pursued genetic testing SCA1 along with SCA8 have been reported to coex- of his mother, who did not have any history or com- ist, just as for SCA1 and SCA6.7 plaints suggestive of ataxia. Although she was unavail- At presentation, the proband was 56 years old, able to us for detailed examination, we were able to with a history of progressive difficulty walking and slurred speech over the prior 3 years (figure, obtain a blood specimen for DNA testing. She had III.5). He provided informed consent to participate 30 and 29 CAG repeats at SCA1, both within the in a clinical research protocol (00-N-0043) normal range, and 1,051 and 24 CTA/CTG repeats approved by the NIH Combined NeuroScience at SCA8. This indicated that the proband likely in- Institutional Review Board. On examination, cog- herited the massive SCA8 expansion from his clini- nition and language were normal. Speech was dys- cally unaffected mother and the borderline SCA1 arthric. Extraocular movements were normal. expansion from his affected father. Thus, the SCA1 Muscle bulk and power were normal throughout, repeat was, unexpectedly, most likely responsible for and there were no tremors or dystonia. Reflexes his symptoms, although the long SCA8 expansion were also normal, but there was dysmetria on could conceivably influence the expression of the finger-to-nose testing as well as truncal instability. SCA1 phenotype. The proband could ambulate independently, but This case illustrates the difficulty in determining – he had difficulty with tandem gait. Brain MRI re- pathogenic loci in cases where multiple ataxia vealed mild generalized cerebral volume loss and causing genes test positive. Reduced penetrance of prominent atrophy of the cerebellar hemispheres, SCA8 further complicates the issue. In this case, particularly the vermis. Nerve conductions studies determination of the gene most likely pathogenic were consistent with a demyelinating sensorimotor required testing of additional family members. This polyneuropathy. Detailed family history revealed type of comprehensive genetic testing is especially that his father suffered from a similar syndrome important for providing accurate genetic counseling anddiedat74yearsofage(figure,II.6).Wewere to the affected families. Furthermore, this case able to review his medical records, and on strongly suggests that SCA8 should not be evaluated

Neurology.org/ng Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Figure Four generations of the family afflicted with inherited ataxia

The number of triplet nucleotide repeats at the spinocerebellar ataxia type 1 (SCA1) and SCA8 loci, where available, is shown to the right. Filled symbols represent afflicted individuals. Patient III.5 is the proband, while III.4 is the unaffected brother. Patients III.4 and III.5 were examined, but only DNA was available for II.7.

in isolation as a candidate gene and that its patho- License 4.0 (CC BY-NC-ND), which permits downloading and genicity should be weighed based on the presence or sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from absence of variants at other loci. the journal. From Neuromuscular Medicine (R.H.R.), Department of Neurology, Received December 28, 2016. Accepted in final form March 13, 2017. Johns Hopkins University School of Medicine, Baltimore, MD; and Neurogenetics Branch (R.H.R., A.B.S., C.B.), National Institute of Correspondence to Dr. Roda: [email protected] Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD. 1. van de Warrenburg BPC, Sinke RJ, Kremer B. Recent advances in hereditary spinocerebellar ataxias. J Neuropathol Author contributions: Ricardo H. Roda was responsible for study – concept and design, acquisition of data, and analysis and interpre- Exp Neurol 2005;64:171 180. tation. Alice B. Schindler was responsible for acquisition of data and 2. Sobrido M-J, Cholfin JA, Perlman S, Pulst SM, Geschwind analysis and interpretation. Craig Blackstone was responsible for DH. SCA8 repeat expansions in ataxia: a controversial asso- analysis and interpretation and critical revision of the manuscript. ciation. Neurology 2001;57:1310–1312. Acknowledgment: The authors thank Elizabeth Hartnett for helping 3. Juvonen V, Hietala M, Paivarinta M, et al. Clinical and with patient scheduling. genetic findings in Finnish ataxia patients with the spino- Study funding: This work was supported by the Intramural Research cerebellar ataxia 8 repeat expansion. Ann Neurol 2000;48: Program of the National Institute of Neurological Disorders and 354–361.

Stroke, NIH. 4. Stevanin G, Herman A, Dürr A, et al. Are (CTG)n expan- Disclosure: Ricardo H. Roda has received research support from the sions at the SCA8 locus rare polymorphisms? Nat Genet Intramural Research Program of the National Institute of Neurolog- 2000;24:213. ical Disorders and Stroke (NIH). Alice B. Schindler has received 5. WorthPF,HouldenH,GiuntiP,DavisMB,Wood research support from the NIH (nongrant funded). Craig Blackstone NW. Large, expanded repeats in SCA8 are not confined serves on the editorial boards of the Journal of Clinical Investigation, to patients with cerebellar ataxia. Nat Genet 2000;24: the Journal of Neuromuscular Diseases, and Annals of Neurology; 214–215. has received research support from the National Institute of Neurolog- 6. Ranum LPW, Moseley ML, Leppet M, et al. Massive ical Disorders and Stroke Intramural Research Program (NIH); and receives license fee payments for a monoclonal antibody against the CTG expansions and deletions may reduce penetrance human atlastin-1 protein (licensed to EMD Millipore Corporation). of spinocerebellar ataxia type 8. Am J Hum Genet Go to Neurology.org/ng for full disclosure forms. The Article Processing 1999;65:A466. Charge was funded by the NIH Intramural Research Program. 7. Sulek A, Hoffman-Zacharska D, Zdzienicka E, Zaremba J. This is an open access article distributed under the terms of the SCA8 repeat expansion coexists with SCA1—not only with Creative Commons Attribution-NonCommercial-NoDerivatives SCA6. Am J Hum Genet 2003;73:972–974.

2 Neurology: Genetics Clinical/Scientific Notes

Elina Kari, MD* COMPOUND HETEROZYGOUS MUTATIONS IN gnomAD beta browser of 126,216 exome Isabelle Schrauwen, PhD* MASP1 IN A DEAF CHILD WITH ABSENT sequenced and 15,136 whole-genome sequenced Lorida Llaci, BS COCHLEAR NERVES individuals, with no homozygotes reported. Both Laurel M. Fisher, PhD variants were predicted damaging when assessed John L. Go, MD Abnormal cochleovestibular nerves (i.e., as absent, with 2 separate integrative pathogenicity prediction Marcus Naymik, MS aplastic, or deficient) are a rare congenital malforma- tools that implement diverse annotations into James A. Knowles, MD, tion that have a devastating impact on hearing and a single overall prediction, i.e., Combined Anno- PhD language development. To date, there have been no tation Dependent Depletion (CADD) score (33 Matthew J. Huentelman, genes identified associated with this abnormality. and 22.7, respectively) and a random forest analysis PhD with Integrating Molecular Heuristics and Other Rick A. Friedman, MD, Tools for Effect Prediction (IMHOTEP; based Case description. A healthy male child was born PhD on ENST00000296280 and ENST00000337774, with profound sensorineural hearing loss (SNHL) respectively). Both variants are also conserved and was referred for cochlear implantation (CI). Neurol Genet among species based on the genomic evolutionary 2017;3:e153; doi: 10.1212/ Auditory brainstem response thresholds were absent rate profiling method. Last, p.Thr644Met and p. NXG.0000000000000153 or profound across all frequencies. His facial nerve Ala17Ser are located within important protein do- function was normal on examination, and he did mains in MASP1: p.Thr644Met affects a very not have any motor delays. Vestibular testing was conserved trypsin-like serineproteasedomain, not performed. His evaluation included high- likely affecting catalytic-proteolytic enzyme activity resolution CT and MRI of the temporal bones. CT and p.Ala17Ser affects the CUB domain, which is revealed bony cochlear modioli, normal cochlear often involved in oligomerization and/or recogni- partitioning, narrow or absent cochlear apertures, tion of substrates and binding partners. All of the enlarged vestibules, dysplastic semicircular canals, and preceding suggest these to be function-altering, bifid internal auditory canals (IACs). MRI revealed deleterious, and disease-causal variants. only 1 nerve in the lateral IAC. On the left, the IAC Mutations of this gene have been associated previ- was too narrow in caliber to determine the contents, ously with 3MC syndrome (Carnevale, Mingarelli, but findings suggested a single nerve in the lateral Malpuech and Michels, or craniofacial-ulnar-renal IAC (figure). Findings were consistent with abnormal syndrome).1 Affected individuals present with a range cochleovestibular nerves bilaterally with likely absent of anomalies that lead to abnormal facial/limb/vesi- cochlear nerves. corenal development, cleft lip and/or palate, cognitive The child underwent a single cochlear implant dysfunction, and craniosynostosis.2 Patients exhibit and demonstrated no benefit. He ultimately under- variable hearing and vestibular dysfunction. How- went an auditory brainstem implant (ABI) at age 3. ever, our patient does not have any other clinical His postoperative hearing and language outcomes features consistent with 3MC syndrome other than are evolving, and the data are unavailable at this time. his SNHL and vestibular anomalies, expanding the Methods. Institutional review board approval was clinical spectrum of MASP1 mutations. obtained. Exome sequencing was performed in the proband and parents using the TruSeq Exome Discussion. Nearly 2–3 per 1,000 newborns suffer Library Prep Kit followed by 100 bp paired-end from hearing loss ranging from mild to profound in sequencing on a HiSeq 2500 instrument. We iden- the United States each year.3 Children with profound tified a compound heterozygote mutation in MASP1 SNHL are potentially considered for a CI or an ABI.4 in the propositus (c.1931C.T[p.Thr644Met] and However, current clinical imaging protocols are c.49G.T[p.Ala17Ser]), each inherited from one unable to consistently predict cochlear nerve status parent, which was confirmed by Sanger sequencing. to guide surgeons’ choice of auditory prosthesis.5 Both variants are rare in the ExAC Browser database Improving preoperative imaging characterization is of 60,706 unrelated individuals (3.049e-4 and a subject of widespread research but has not yet 2.481e-05 allele frequencies, respectively), and the reached clinical use.

Neurology.org/ng Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Figure Imaging, pedigree, and anger sequencing of a child with absent cochlear nerve

(A) Coronal high-resolution CT of a bifid and narrow internal auditory canal (IAC). (B) Axial high-resolution CT showing a pinpoint cochlear aperture (arrow) and an enlarged vestibule (arrowhead). Cochlear partitioning was normal, but the modiolus was bony and semicircular canals were dysplastic (not shown). (C) Axial high- resolution heavily T2-weighted (constructive interference in steady state [CISS]/fast imaging employing steady-state acquisition [FIESTA]) MRI of IACs bilaterally showing narrow IACs, one nerve in right IAC (short arrow) and nothing seen in left IAC (long arrow). Oblique cross-sectional imaging confirmed findings (not shown). (D) Family pedigree showing the mutations in MASP1. (E) Sanger sequencing traces showing the mutations inherited in the pedigree.

The variability of hearing outcomes in children collection of data and interpretation of data. James A. Knowles: with abnormal cochleovestibular nerves receiving interpretation of data. Matthew J. Huentelman: interpretation of data. Rick A. Friedman: interpretation of data and final manuscript CIs/ABIs coupled with the current inability to predict preparation. 4–7 their outcomes leads to children enduring multiple Acknowledgment: The authors thank the family for participating in assessments and interventions. The length of time to this study and Keri Ramsey for her help with sample collection. determine which treatment will provide benefit often Study funding: TGen, Translational Genomics Research Institute: ’ exceeds the sensitive periods for auditory develop- this work was supported by private donations to TGen s Center for Rare Childhood Disorders. Rick Friedman: NIH R01 DC010856. ment, delaying spoken language. Disclosure: Dr. Kari has received travel funds from Cochlear Corpo- MASP1 encodes mannan-binding lectin serine pro- ration and travel funds/speaker honoraria from Asan Medical Center. tease 1 that is involved in complement activation. Pre- She also reports the following disclosures regarding her husband: he is vious studies show that MASP1 is involved to direct the a consultant for Otonomy, Inc.; has received research support from NIH/NIDCD; has stock in Otonomy, Inc.; and receives royalties from migration of neural crest cells during embryonic devel- sales of Otiprio from Otonomy. Dr. Schrauwen has received research 2 opment, and mutations cause a spectrum of human support from Arizona Alzheimer’s Disease Core Center (ADCC). malformation syndromes as previously described, which Ms. Llaci, Dr. Fisher, Dr. Go, and Mr. Naymik report no disclosures. demonstrate the involvement of MASP1 in facial, Dr. Knowles has received research support from NIH/NIMH and the Della Martin Foundation. Dr. Huentelman has received research umbilical, and ear development during the embryonic support from NIH/National Institute of Neurological Disorders and period. Zebrafish morphants also develop pigmentary Stroke. Dr. Friedman is a co-founder for Otonomy Inc., which has no defects and severe craniofacial abnormalities.2 relationship to this publication. He also serves as a consultant for, has In this report, we expand the spectrum of pheno- stock in, and receives royalties (from sales of Otiprio) from Otonomy, Inc.; has received travel funding/speaker honoraria from Cochlear typic variability caused by MASP1 mutations and Corporation and Asan Medical University; and has received research suggest that MASP1 screening should be considered support from NIH. Go to Neurology.org/ng for full disclosure forms. in patients with nonsyndromic profound SNHL and The Article Processing Charge was funded by the authors. abnormal cochleovestibular nerves. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives Li- cense 4.0 (CC BY-NC-ND), which permits downloading and shar- * – These authors contributed equally to this work as co first authors. ing the work provided it is properly cited. The work cannot be From the Tina and Rick Caruso Department of Otolaryngology– changed in any way or used commercially without permission from Head and Neck Surgery (E.K., L.M.F., J.L.G., J.A.K., R.A.F.), the journal. Keck University of Southern California School of Medicine, Los Received December 17, 2016. Accepted in final form March 27, 2017. Angeles; and TGen (I.S., L.L., M.N., M.J.H.), Translational Ge- nomics Research Institute, Phoenix, AZ. Correspondence to Dr. Kari: [email protected] Author contributions: Elina Kari: collection of data, interpretation of data, writing of the manuscript, and the corresponding author. 1. Atik T, Koparir A, Bademci G, et al. Novel MASP1 muta- Isabelle Schrauwen: collection of data, DNA sequencing, analysis of tions are associated with an expanded phenotype in 3MC1 data, and writing of the manuscript. Lorida Llaci: DNA sequencing syndrome. Orphanet J Rare Dis 2015;10:128. and sample preparation. Laurel M. Fisher: collection of data and 2. Rooryck C, Diaz-Font A, Osborn DPS, et al. Mutations in interpretation of data. Marcus Naymik: bioinformatics. John L. Go: lectin complement pathway genes COLEC11 and MASP1

2 Neurology: Genetics cause 3MC syndrome. Nat Genet 2011;43:197–203. Sup- patients with “absent cochlear nerves” can derive benefit from plementary data. cochlear implantation. Accepted, Presentation at The Ameri- 3. CDC. Hearing loss in children. Available at: cdc.gov/ can Neurotological Society Spring Conference, April 28–29, ncbddd/hearingloss/data.html. Accessed January 1, 2016. 2017, San Diego, CA. 4. CollettiL,WilkinsonEP,CollettiV.Auditorybrain- 6. Birman CS, Powell HRF, Gibson WPR, Elliott EJ. stem implantation after unsuccessful cochlear implanta- Cochlear implant outcomes in cochlea nerve aplasia and tion of children with clinical diagnosis of cochlear nerve hypoplasia. Otol Neurotol 2016;37:438–445. deficiency. Ann Otol Rhinol Laryngol 2013;122:605– 7. Young NM, Kim FM, Ryan ME, Tournis E, Yaras S. 612. Pediatric cochlear implantation of children with eighth 5. Kari E, Go JL, Loggins J, Emmanuel N, Fisher LM. Abnor- nerve deficiency. Int J Pediatr Otorhinolaryngol 2012; mal cochleovestibular nerves and pediatric hearing outcomes: 76:1442–1448.

Neurology: Genetics 3 Clinical/Scientific Notes

Sara Chadwick Reichert, BIALLELIC TOR1A VARIANTS IN AN INFANT WITH half-sibling with microcephaly and developmental MS, MPH SEVERE ARTHROGRYPOSIS delays who has no contractures. The family has Pedro Gonzalez-Alegre, declined genetic evaluation for this sibling. MD, PhD Genetic workup included karyotype and chromo- DYT1 early-onset primary dystonia (DYT1) is Gunter H. Scharer, MD somal microarray, testing for congenital myasthenia a well-described dystonia caused by an in-frame gravis, distal arthrogryposis, spinal muscular atrophy, GAG nucleotide deletion in the TOR1A gene, Neurol Genet 1 and spinal muscular atrophy with respiratory disease 2017;3:e154; doi: 10.1212/ c.907_909delGAG. The only phenotype linked to —all unremarkable. WES, using the proband and NXG.0000000000000154 TOR1A is dystonia.2 Homozygous GAG deletions or his parents, was reported negative for characterized compound heterozygosity for mutations in TOR1A genetic etiologies relating to known genes and pheno- have never been reported in humans. types. However, 2 TOR1A variations—the known Arthrogryposis, defined as multiple congenital c.907_909delGAG (p.E303del) mutation (paternally contractures, affects 1 in 3,000–5,000 births.3 The inherited) and a c.961delA (p.T321Rfs*6) variant underlying etiology for arthrogryposis is broad, and (maternally inherited)—were listed as suspected candi- includes neurologic disease, maternal illness, myo- dates for a potential novel genetic etiology. These find- pathic processes, among many others.3 The number ings refer to a gene-disease relationship and/or of genetic factors causing arthrogryposis is vast, with mechanism not previously proposed or with limited over 150 genes currently identified.3 evidence. We report an infant with a severe congenital phenotype characterized by arthrogryposis, respiratory Discussion. In DYT1, one-third of carriers of the failure, and feeding difficulties found to have biallelic common mutation develop dystonia during child- mutations in TOR1A identified by whole-exome hood or adolescence with previously normal devel- sequencing (WES). opment.2 The factors that determine penetrance Case. The proband was born at 37 weeks to his 24- remain largely unknown. In affected individuals, year-old G2P1001 mother via cesarean section. He brain imaging studies are normal. weighed 2,150 g (2%), he was 44 cm long (2%), The protein coded by TOR1A, torsinA, contains his head circumference was 32 cm (12%), and he 332 amino acids and is widely expressed in the endo- had APGAR scores of 5 and 7 at 1 and 5 minutes, plasmic reticulum and is thought to function as an respectively. The pregnancy was complicated by adenosine triphosphate–regulated chaperone in the intrauterine growth restriction, reduced fetal move- secretory pathway and the nuclear envelope.1 Overall, ments, and arthrogryposis. Invasive prenatal genetic published research supports that DYT1 is caused by testing was declined, and no teratogenic exposures the loss of torsinA function, likely due to a dominant or maternal illnesses were reported. Following deliv- negative effect of the mutant torsinA over the wild- ery, examination was notable for contractures in all type protein.4 Animal models of DYT1 have been extremities and skeletal anomalies, including bilateral developed and demonstrate that torsinA is essential hip dislocations, bilateral clubfeet, 11 sets of ribs, and for postnatal life, as TOR1A2/2, homozygous scoliosis (figure). He developed restrictive lung dis- TOR1ADGAG/DGAG, and TOR1A2/DGAG mice die ease requiring tracheostomy with ventilator support, within 48 hours of birth.5 Of interest, the animal and a G-tube was placed for swallowing discoordina- models described above appear normal at birth except tion. Brain MRI at 3 weeks of age was unremarkable for a smaller size than their littermates, however, do (figure). He is currently 7 months old and has no not feed or generate sounds, suggesting oropharyn- spontaneous or involuntary movements, although geal dysfunction. Gross neuroanatomy is normal, but he responds to voices and makes eye contact. they have ultrastructural defects in neurons. Mice There is no extended family history of arthrog- with restricted conditional deletion of torsinA exhibit ryposis or dystonia; however, a paternal aunt and longer survival.6 uncle died during childhood from a nonspecific Our proband exhibits several features seen in ani- neurologic condition (figure). He has a maternal mal models, including oropharyngeal dysfunction

(A) Pedigree—the proband is the first child born to his parents and is designated by the arrow. The family history is unremarkable for symptoms of arthrogryposis or DYT1 dystonia; however, there is a paternal aunt and uncle who died of an unknown childhood onset neurologic disease. (B) Brain MRI was performed at 3 weeks of age. Sagittal view of the cerebellum and axial view of the caudate are unremarkable for the proband’s age. (C) Chest and abdomen X-rays taken at birth show multiple skeletal anomalies including 11 pairs of ribs, bilateral hip dislocations, and scoliosis.

and delayed growth with no gross neuroanatomical should be considered as a potential genetic cause defects appreciated by MRI. The c.907_909delGAG in the evaluation of arthrogryposis. mutation is causative of DYT1. The c.961delA variant has not been previously associated with disease, From the Department of Medical Genetics and Genomics (S.C.R., G.H.S.), Children’s Minnesota, Minneapolis; Department of but is expected to result in a translational frameshift Neurology (P.G.-A.), Perelman School of Medicine at the University at the 39 terminus, deleting the last 12 amino acids of Pennsylvania, Philadelphia; and Raymond G. Perelman Center ofthetypicalprotein.Thiswouldabolishahighly for Cellular & Molecular Therapeutics (P.G.-A.), The Children’s conserved essential domain, the sensor-II domain, Hospital of Philadelphia, PA. 7 Author contributions: Ms. Reichert contributed to report concept, likely resulting in a protein with reduced function. acquisition of data, analysis and interpretation, and critical revision To demonstrate pathogenicity, identification of of the manuscript for important intellectual content. Dr. Gonzelez- more patients or robust biological evidence is Alegre contributed to analysis and interpretation and critical revision required. Nevertheless, given our current knowl- of the manuscript for important intellectual content. Dr. Scharer contributed to acquisition of data, analysis and interpretation, and edge, it is reasonable to speculate that these com- critical revision of the manuscript for important intellectual content. pound heterozygous deleterious mutations result in Study funding: No targeted funding reported. the severe congenital phenotype observed. Disclosure: Ms. Reichert reports no disclosures. Dr. Gonzalez-Alegre This represents a rare case of TOR1A com- has received compensation from TEVA for participation in the Hun- ’ pound heterozygous mutations in a patient with tington s Disease Advisory Board. Dr. Scharer reports no disclosures. Go to Neurology.org/ng for full disclosure forms. The Article Process- congenital arthrogryposis. This information is ing Charge was funded by the authors. valuable for researchers investigating biological This is an open access article distributed under the terms of the bases of DYT1. Moreover, TOR1A mutations Creative Commons Attribution-NonCommercial-NoDerivatives

2 Neurology: Genetics License 4.0 (CC BY-NC-ND), which permits downloading and 4. Torres GE, Sweeney AL, Beaulieu JM, Shashidharan P, sharing the work provided it is properly cited. The work cannot be Caron MG. Effect of torsinA on membrane proteins reveals changed in any way or used commercially without permission from a loss of function and a dominant-negative phenotype of the the journal. dystonia-associated DeltaE-torsinA mutant. Proc Natl Acad Received February 13, 2017. Accepted in final form April 5, 2017. Sci USA 2004;101:15650–15655. 5. Goodchild RE, Kim CE, Dauer WT. Loss of the dystonia- Correspondence to Ms. Reichert: [email protected] associated protein torsinA selectively disrupts the neuronal nuclear envelope. Neuron 2005;48:923–932. 1. Ozelius LJ, Hewett JW, Page CE, et al. The early-onset 6. Liang CC, Tanabe LM, Jou S, Chi F, Dauer WT. TorsinA torsion dystonia gene (DYT1) encodes an ATP-binding hypofunction causes abnormal twisting movements and protein. Nat Genet 1997;17:40–48. sensorimotor circuit neurodegeneration. J Clin Invest 2. Bressman SB, Sabatti C, Raymond D, et al. The DYT1 2014;124:3080–3092. phenotype and guidelines for diagnostic testing. Neurology 7. ZhuL,WrablJO,HayashiAP,RoseLS,ThomasPJ. 2000;54:1746–1752. The torsin-family AAA1 protein OOC-5 contains a crit- 3. Hall JG. Arthrogryposis (multiple congenital contractures): ical disulfide adjacent to Sensor-II that couples redox diagnostic approach to etiology, classification, genetics, and state to nucleotide binding. Mol Biol Cel 2008;19: general principles. Eur J Med Genet 2014;57:464–472. 3599–3612.

Neurology: Genetics 3 Clinical/Scientific Notes

Rubina Dad, MPhil FEBRILE ATAXIA AND MYOKYMIA BROADEN NM_001478: c.C1358G; p.Pro453Arg. No other Susan Walker, PhD THE SPG26 HEREDITARY SPASTIC PARAPLEGIA known SPG, myokymia, or ataxia-related genes har- Stephen W. Scherer, PhD PHENOTYPE bored clinically relevant variants (table e-3). Sanger Muhammad Jawad sequencing in all family members confirmed that the Hassan, PhD Hereditary spastic paraplegias (SPGs) are among the B4GALNT1 change was homozygous in proband and Mohammad Domaia genetically most diverse neurologic disorders with sisters and heterozygous in healthy brother and Alghamdi, MD over 70 loci identified.1,2 The recessively inherited parents. It was not present in the 1000 Genomes Berge A. Minassian, MD SPG26 is caused by mutations in B4GALNT1, en- European, American, Asian, or African databases, Reem A. Alkhater, MD, coding the b-1-4-N-acetyl-galactosaminyl transferase dbSNP or ExAC. The variant was predicted damag- FRCP (C), MSc which functions in the biosynthesis of complex ing by PolyPhen2 and CADD and evolutionarily glycosphingolipids. To date, 12 families have been conserved (PhyloP). No further functional conse- Neurol Genet 2017;3:e156; doi: 10.1212/ reported in 3 publications, with a broad phenotypic quences could be predicted. NXG.0000000000000156 spectrum within and between families (table 1). We A summary of all SPG26 published cases (table 1) add a new family to the literature with 3 affected reveals spastic paraparesis as a commonly shared core members and a remarkable phenotype of purely symptom. The disease extends to other systems and fever-induced ataxia with myokymia. We also review includes dysarthria, ataxia, intellectual disability, and all published cases3–5 to encapsulate the current psychiatric manifestations, none of which is uniformly knowledge of the neurologic features and spectrum present across families and even within families. Onset of this disease. is in childhood, usually before age 10, in all families Our proband presented at age 5 years with severe except 1, with a missense mutation and onset between fever-induced ataxia and myokymia, the latter in the 28 and 39 years. In this family, despite late onset, flexor muscles of the hands and feet, which addition- progression was rapid, with severe muscle wasting.3 ally exhibited carpopedal spasm. Presumed postviral Our family, with another missense mutation, appears cerebellitis, he was treated with methylprednisolone to be the mildest to date among childhood-onset cases, and IV immunoglobulin and recovered fully in and the phenotype is particularly mild in the 2 girls a week. He had 4 additional identical episodes until (table 1). They had a later age at onset than the boy (8 age 10, all induced by a febrile illness (39–40°C), vs 5), and, despite being older (15 and 21 vs 11), have each self-resolving, untreated, following deferves- no muscle wasting or ataxia and only mild spasticity. cence. Examination at age 11 shows proximal muscle Finally, the male patient manifests a symptom set not weakness, mild lower limb spasticity, preserved deep previously reported, namely febrile ataxia and myoky- tendon reflexes and sensation, intact cognition, nor- mia with full recovery after each attack. mal brain MRI, and evidence of peripheral neuropa- Complex glycosphingolipids (e.g., GM1 or GD1a thy on nerve conduction study. His parents are gangliosides) are crucial components of plasma mem- first-degree cousins, and he has 2 older sisters and 1 branes but are by far most abundant in nervous tissue. older brother. The sisters are lean and tall with rela- Their 2 principal roles are to mediate cell-cell interactive microcephaly, and on examination have reduced tions and regulate membrane protein functions. muscle bulk, proximal muscle weakness, and mildly Additional functions include endocytosis, signal spastic gait. The elder sister had had Achilles tendon transduction, and synaptic plasticity. Complex release surgery at age 8 but continued to have lower glycosphingolipids represent a family of many mole- Supplemental data at limb spasticity. Her and affected brother’s Spastic cules, the shared “glyco” component consisting of 4 Neurology.org/ng Paraplegia Rating Scale scores were 4/52 and 13/52, glucans serially added to the sphingolipid during bio- respectively (tables e-1 and e-2 at Neurology.org/ng). synthesis. B4GALNT1 catalyzes the addition of the The older brother was and remains healthy. Whole- third (N-acetyl-b-D-galactosamine) to the second exome sequencing (e-Methods) for proband and 1 glucan, and the absence of the enzyme results in non- sister revealed a single significant shared homozygous progression to the more complex forms (e.g., from variant, namely B4GALNT1 chr12:58021427:G.C; GM3 to GM2 and GM1).6 B4galnt1 knockout mice

Table 1 Summary of clinical features of all published cases with B4GALNT1 mutations (NM_001478) erlg:Genetics Neurology: Present study Reference 5 Reference 4 Reference 3

Families Saudi Bedouin Bedouin Spanish Tunisian Brazilian Algerian Portuguese French German Kuwaiti Italian Amish

Affected 2 girls 1 boy 4 5 3 4 5 1 4 1 3 5 2 4

Age at 8 5 Early 6–77–83–19 9–14 11 2–3 Early Early 6–11 28–39 Second year onset, y childhood of life

Dysarthria No Mild Severe Severe NS 1 yes, 3 no No No Mild Mod No–sev No–sev NS NS

Spasticity Mild Mod Severe Severe Yes, NOS Mod–sev Mod–sev Mod Mild–mod Severe Mod–sev Mod Mod–sev Mod

Ataxia No Febrile Severe Severe Yes No Mild Moderate Mild–mod Severe No Severe Mod–sev Mod–sev

Reflexes Normal Normal YY Brisk Brisk Brisk Brisk Y NS Normal YYY

Muscle No No Severe Severe Moderate No–sev No No Mild–sev Severe Mod–sev Severe Severe Mild–mod wasting

Other Cataract Dysmorphism Seizure

Behavior Normal Normal Autism, Emotional Mild–mod ID Mild–mod ID Mild–mod ID Mild–mod ID Mild–mod ID Mild–mod ID Mild–mod ID Emotional Mild– Autism, and mod–sev lability, mild lability, severe ID mild–mod ID cognition ID ID severe ID

Brain or Normal Normal Normal Normal Cerebral Normal in 1, Normal Cerebral Subcortical Cerebral atrophy Normal (CT) Normal Congenital ND spinal MRI atrophy ND in 3 atrophy periventricular spinal WMH stenosis

NCS/EMG Normal Axonal ND ND Axonal Normal in 1, Normal Axonal Pes cavus and Axonal neuropathy Axonal Normal Decreased Pes cavus neuropathy neuropathy; ND in 3 neuropathy clinical neuropathy sensory and clinical pes cavus peripheral amplitudes peripheral neuropathy neuropathy

Mutations c.C1358G; c.1003- c.1458_1459 c.395delC; p. c.898C.T; c.682C.T; p. c.263 dupG; c.358C.T; c.917_ 922dup; p.Thr c.1298A.C; c.1458 c.852 c.1514 p.Pro453Arg 2A.G insA; p. Pro132Glnfs*7 p.Arg300Cys Arg228* p.Leu89fs*13 p.Gln120* 307_ Val308dup; p.Asp433Ala dup; p. G.C; p. G.A; p. Leu487*fs c.1315_1317delTTC; Leu487Thr Lys284Asn Arg505His p.Phe 439del fs*77

Abbreviations: ID 5 intellectual disability; Mod 5 moderate; NCS 5 nerve conduction study; ND 5 not determined; NOS 5 not otherwise specified; NS 5 not specified; Sev 5 severe; WMH 5 white matter hyperintensity. The downward arrow indicates decreased deep tendon reflex. are healthy, suggesting that shorter gangliosides (e.g., Journal, Genome Medicine, the Journal of Neurodevelopmental GM3) largely compensate for absent longer forms.7 Disorders, Autism Research, PathoGenetics, Comparative and Functional Genomics, BMC Medical Genomics, and Cytogenet- This appears to be the case also in humans, as evi- ics and Genome Research; and has received research support from denced by the patients discussed here, but only for Genome Canada/Ontario Genomics Institute, Canadian Institutes of the years until disease onset. After that, there is pro- Health Research, Canadian Institute for Advanced Research, gressive neurologic decompensation. Precisely, which McLaughlin Centre, Canada Foundation for Innovation, government of Ontario, NIH, Autism Speaks, and SickKids Foundation. complex ganglioside deficiency(ies) in which cell Berge A. Minassian holds patents for diagnostic testing of the following types, regions or pathways underlie the upper and genes: EPM2A, EPM2B, MECP2, and VMA21; receives license fee lower motor neuron disease and other aspects of payments/royalty payments for aforementioned patents; and has received research support from National Institute of Neurological Disorders and SPG26 await future studies. This knowledge will help Stroke of the NIH. Go to Neurology.org/ng for full disclosure forms. The elucidate the roles of complex glycosphingolipids in Article Processing Charge was funded by the authors. the development and function of the central and This is an open access article distributed under the terms of the peripheral nervous systems. Creative Commons Attribution-NonCommercial-NoDerivatives Li- cense 4.0 (CC BY-NC-ND), which permits downloading and shar- From the Atta-ur Rahman School of Applied Biosciences (R.D., M.J. ing the work provided it is properly cited. The work cannot be H.), National University of Sciences and Technology (NUST), Paki- changed in any way or used commercially without permission from stan; Program in Genetics and Genome Biology (R.D., B.A.M.), the journal. Division of Neurology (B.A.M.), Department of Paediatrics, and Received December 2, 2016. Accepted in final form April 13, 2017. The Centre for Applied Genomics, Genetics and Genome Biology (S.W., S.W.S.), The Hospital for Sick Children; Department of Correspondence to Dr. Alkhater: [email protected] Molecular Genetics (S.W.S.), and McLaughlin Centre (S.W.S.), 1. Noreau A, Dion PA, Rouleau GS. Molecular aspects of University of Toronto, Ontario, Canada; and Department of Pedi- hereditary spastic paraplegias. Exp Cell Res 2014;325: atrics (M.D.A., R.A.A.), Division of Neurology, Johns Hopkins Ara- 18–26. mco Healthcare, Dhahran, Saudi Arabia. 2. Chrestian N, Dupré N, Gan-Or Z, et al. Clinical and genetic Author contributions: Study concept and design by Berge A. Minassian study of hereditary spastic paraplegia in Canada. Neurol Genet and Reem A. Alkhater. Acquisition of clinical data by Reem A. Alkhater and Mohammad Domaia Alghamdi. Acquisition of data 2016;3:e122. doi: 10.1212/NXG.0000000000000122. by Susan Walker and Stephen W. Scherer. Analysis and interpreta- 3. Harlalka GV, Lehman A, Chioza B, et al. Mutations in tion of data by Rubina Dad and Susan Walker. Study supervision B4GALNT1 (GM2 synthase) underlie a new disorder of by Berge A. Minassian, Reem A. Alkhater, and Muhammad ganglioside biosynthesis. Brain 2013;136:3618–3624. Jawad Hassan. Critical revision of manuscript for intellectual 4. Boukhris A, Schule R, Loureiro JL, et al. Alteration of content, coinvestigators: Berge A. Minassian, Reem A. Alkhater, ganglioside biosynthesis responsible for complex hereditary and Mohammad Domaia Alghamdi. spastic paraplegia. Am J Hum Genet 2013;93:118–123. Acknowledgment: The authors thank the family members for partic- 5. Wakil S, Monies D, Ramzan K, et al. Novel B4GALNT1 ipation in the study. Rubina Dad acknowledges her funding by the mutations in a complicated form of hereditary spastic para- Higher Education Commission of Pakistan under the International plegia. Clin Genet 2014;86:500–501. Research Support Initiative Program (HEC-IRSIP). 6. Schnaar RL. Brain gangliosides in axon-myelin stability and Study funding: No targeted funding reported. axon regeneration. FEBS Lett 2010;584:1741–1747. Disclosure: Rubina Dad, Susan Walker, Muhammad Jawad Has- 7. Takamiya K, Yamamoto A, Furukawa K, et al. Mice san, Mohammad Domaia Alghamdi, and Reem A. Alkhater report with disrupted GM2/GD2 synthase gene lack complex no disclosures. Stephen Scherer has served on the scientific advisory board of Population Diagnostics; has served on the editorial boards of gangliosides but exhibit only subtle defects in their Genomic Medicine, Genes, Genomes, Genetics, Journal of Per- nervous system. Proc Natl Acad Sci USA 1996;93: sonalized Medicine, The Open Genomics Journal, The Hugo 10662–10667.

Neurology: Genetics 3

Table of Contents Neurology.Org/Ng  Online ISSN: 2376-7839 Volume 3, Number 3, June 2017

Table of Contents Neurology.Org/Ng Online ISSN: 2376-7839 Volume 3, Number 3, June 2017