Genomic Variation in Educational Attainment Modifies Alzheimer

Volume 5, Number 2, April 2019 Neurology.org/NG

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ARTICLE Genomic variation in educational att ainment modifi es Alzheimer disease riske310

ARTICLE Homozygous TRPV4 mutation causes congenital distal spinal muscular atrophy and arthrogryposis e312

ARTICLE Lithium chloride corrects weakness and myopathology in a preclinical model of LGMD1D e318

ARTICLE Muscular dystrophy with arrhythmia caused by loss-of-function mutations in BVES e321 Academy Officers Neurology® is a registered trademark of the American Academy of Neurology (registration valid in the United States). Ralph L. Sacco, MD, MS, FAAN, President Neurology® Genetics (eISSN 2376-7839) is an open access journal published James C. Stevens, MD, FAAN, President Elect online for the American Academy of Neurology, 201 Chicago Avenue, Ann H. Tilton, MD, FAAN, Vice President Minneapolis, MN 55415, by Wolters Kluwer Health, Inc. at 14700 Citicorp Drive, Bldg. 3, Hagerstown, MD 21742. Business offices are located at Two Carlayne E. Jackson, MD, FAAN, Secretary Commerce Square, 2001 Market Street, Philadelphia, PA 19103. Production offices are located at 351 West Camden Street, Baltimore, MD 21201-2436. Janis M. Miyasaki, MD, MEd, FRCPC, FAAN, Treasurer © 2019 American Academy of Neurology. Terrence L. Cascino, MD, FAAN, Past President Neurology® Genetics is an official journal of the American Academy of Neurology. Journal website: Neurology.org/ng, AAN website: AAN.com Executive Office, American Academy of Neurology Copyright and Permission Information: Please go to the journal website (www.neurology.org/ng) and click the Permissions tab for the relevant Catherine M. Rydell, CAE article. Alternatively, send an email to [email protected]. Chief Executive Officer General information about permissions can be found here: https://shop.lww.com/ journal-permission. 20l Chicago Ave Disclaimer: Opinions expressed by the authors and advertisers are not Minneapolis, MN 55415 necessarily those of the American Academy of Neurology, its affiliates, or of the Publisher. The American Academy of Neurology, its affiliates, and the Tel: 612-928-6000 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 Editorial Office or non-use of any of the material contained in this publication. Patricia K. Baskin, MS, Executive Editor Advertising Sales Representatives: Wolters Kluwer, 333 Seventh Avenue, Kathleen M. Pieper, Senior Managing Editor, Neurology New York, NY 10001. Contacts: Eileen Henry, tel: 732-778-2261, fax: 973-215- 2485, [email protected] and in Europe: Craig Silver, tel: +44 Lee Ann Kleffman, Managing Editor, Neurology® Genetics 7855 062 550 or e-mail: [email protected]. Sharon L. Quimby, Managing Editor, Neurology® Clinical Practice Careers & Events: Monique McLaughlin, Wolters Kluwer, Two Commerce fl Square, 2001 Market Street, Philadelphia, PA 19103, tel: 215-521-8468, fax: 215- Morgan S. Sorenson, Managing Editor, Neurology® Neuroimmunology & Neuroin ammation 521-8801; [email protected]. Andrea Rahkola, Production Editor, Neurology Reprints: Meredith Edelman, Commercial Reprint Sales, Wolters Kluwer, Two Robert J. Witherow, Senior Editorial Associate Commerce Square, 2001 Market Street, Philadelphia, PA 19103, tel: 215-356-2721; Karen Skaja, Senior Editorial Associate [email protected]; [email protected]. Special projects: US & Canada: Alan Moore, Wolters Kluwer, Two Kaitlyn Aman Ramm, Editorial Assistant Commerce Square, 2001 Market Street, Philadelphia, PA 19103, tel: Kristen Swendsrud, Editorial Assistant 215-521-8638, [email protected]. International: Andrew Wible, Senior Manager, Rights, Licensing, and Partnerships, Wolters Kluwer; Justin Daugherty, Editorial Assistant [email protected]. Madeleine Sendek, MPH, Editorial Assistant

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e315 Clinical, genetic, and pathologic characterization of FKRP Mexican founder mutation c.1387A>G A.J. Lee, K.A. Jones, R.J. Butterfield, M.O. Cox, C.G. Konersman, C. Grosmann, J.E. Abdenur, M. Boyer, B. Beson, C. Wang, J.J. Dowling, M.A. Gibbons, A. Ballard, J.S. Janas, R.T. Leshner, S. Donkervoort, C.G. Bonnemann,¨ D.M. Malicki, R.B. Weiss, S.A. Moore, and K.D. Mathews Open Access

e316 Loss-of-function mutations in Lysyl-tRNA synthetase cause various leukoencephalopathy phenotypes C. Sun, J. Song, Y. Jiang, C. Zhao, J. Lu, Y. Li, Y. Wang, M. Gao, J. Xi, S. Luo, M. Li, K. Donaldson, S.N. Oprescu, T.P. Slavin, S. Lee, P.L. Magoulas, A. Lewis, L. Emrick, S.R. Lalani, Z. Niu, M.L. Landsverk, M. Walkiewicz, R.E. Person, H. Mei, J.A. Rosenfeld, Y. Yang, A. Antonellis, Y.-M. Hou, J. Lin, and V.W. Zhang Open Access

e317 Somatic expansion of the C9orf72 hexanucleotide repeat does not occur in ALS spinal cord tissues J.P. Ross, C.S. Leblond, H. Catoire, K. Volkening, M. Strong, L. Zinman, J. Robertson, P.A. Dion, and G.A. Rouleau Open Access

e318 Lithium chloride corrects weakness and myopathology in a preclinical model of LGMD1D A.R. Findlay, R. Bengoechea, S.K. Pittman, T.-F. Chou, H.L. True, and C.C. Weihl Open Access

e320 Novel PNKP mutations causing defective DNA strand break repair and PARP1 hyperactivity in MCSZ Editorial I. Kalasova, H. Hanzlikova, N. Gupta, Y. Li, J. Altmuller,¨ J.J. Reynolds, G.S. Stewart, B. Wollnik, G. Yigit, and K.W. Caldecott e313 Unraveling the genetic complexity of Alzheimer Open Access disease with Mendelian Randomization S. Bandres-Ciga and F. Faghri e321 Muscular dystrophy with arrhythmia caused by BVES Open Access Companion article, e310 loss-of-function mutations in W.DeRidder,I.Nelson,B.Asselbergh,B.DePaepe,M.Beuvin, Articles R. Ben Yaou, C. Masson, A. Boland, J.-F.Deleuze,T.Maisonobe, B. Eymard, S. Symoens, R. Schindler, T. Brand, K. Johnson, A. Topf,¨ V. Straub, P. De Jonghe, J.L. De Bleecker, G. Bonne, and e310 ﬁ Genomic variation in educational attainment modi es J. Baets Alzheimer disease risk Open Access N.S. Raghavan, B. Vardarajan, and R. Mayeux Open Access Editorial, e313 e322 Autosomal dominant optic atrophy and cataract “plus” phenotype including axonal TRPV4 e312 Homozygous mutation causes congenital neuropathy distal spinal muscular atrophy and arthrogryposis A. Horga, E. Bugiardini, A. Manole, F. Bremner, Z. Jaunmuktane, J. Velilla, M.M. Marchetti, A. Toth-Petroczy, C. Grosgogeat, L. Dankwa, A.P. Rebelo, C.E. Woodward, I.P. Hargreaves, A.H. Bennett, N. Carmichael, E. Estrella, B.T. Darras, N.Y. Frank, A. Cortese, A.M. Pittman, S. Brandner, J.M. Polke, J. Krier, R. Gaudet, and V.A. Gupta, on behalf of Brigham Genomics R.D.S. Pitceathly, S. Zuchner,¨ M.G. Hanna, S.S. Scherer, Medicine H. Houlden, and M.M. Reilly Open Access Open Access TABLE OF CONTENTS Volume 5, Number 2, April 2019 Neurology.org/NG

Clinical/Scientific Notes Views and Reviews e314 Mitochondrial cerebellar ataxia, renal failure, e323 Antisense oligonucleotides: A primer neuropathy, and encephalopathy (MCARNE) D.R. Scoles, E.V. Minikel, and S.M. Pulst P.S. Ng, M.V. Pinto, J.L. Neff, L. Hasadsri, E.W. Highsmith, M.E. Fidler, Open Access R.H. Gavrilova, and C.J. Klein Open Access e319 Leaky splicing variant in sepiapterin reductase deﬁciency: Are milder cases escaping diagnosis? Y. Nakagama, K. Hamanaka, M. Mimaki, H. Shintaku, S. Miyatake, N. Matsumoto, K. Hirohata, R. Inuzuka, and A. Oka Open Access

Cover image Red-free photographs of the optic discs of a patient, showing mild temporal pallor of both optic discs. Stylized by Kaitlyn Aman Ramm, Neurology Editorial Assistant. See e322 EDITORIAL OPEN ACCESS Unraveling the genetic complexity of Alzheimer disease with Mendelian Randomization

Sara Bandres-Ciga, PhD, and Faraz Faghri, MS Correspondence Dr. Bandres-Ciga Neurol Genet 2019;5:e313. doi:10.1212/NXG.0000000000000313 [email protected]

Genome-wide association studies (GWASs) have changed the way we conceive human RELATED ARTICLE genetics and have led to the discovery of thousands of risk variants involved in disease etiology. However, despite tremendous advances made in understanding the genetic ar- Genomic variation in chitecture underlying disease, there remains an underinvestigated component of risk, educational attainment namely phenotypic traits that can predispose or protect individuals to disease. The avail- modifies Alzheimer disease ability of large amounts of GWAS data affords the opportunity to investigate the relationship risk 1 between myriad traits. Page e310 InthecurrentissueofNeurology® Genetics,Raghavanetal.2 aim at determining the putative causal relationship between educational attainment and Alzheimer disease (AD). The authors use Mendelian Randomization, the gold standard for causality in genetic studies, as a statistical approach that uses genetic data in the form of SNPs to study whether an exposure exerts a causal effect in an outcome. This promising methodology sits at the interface between observational epidemiology and interventional trials and aims at addressing the question of whether an observational association between a risk or protective factor and a disease of interest is consistent with a causal effect by focusing usually only on genome-wide significant SNPs. One of the key strengths of this method is that it relies on genetic variants that are fixed at conception and remain constant over the lifespan of an individual and that are randomized during gametogenesis, which means that genetic variants are not associated with all the confounder factors that affect an observational study.3

In a simple way, SNPs genome-wide associated with a certain exposure modify the risk of that exposure, which in turn aﬀects the disease of interest. Raghavan et al. not only consider SNP genome-wide related to educational attainment as instrumental variables but also use genetic regions surrounding individually associated SNPs to nominate genes that might contribute to the disease.

The authors identify a causal inverse relationship between educational attainment and AD and demonstrate that an increase of 4.2 years of educational attainment is associated with 37% reduction in AD, exerting a notable protective eﬀect. When focusing on individual loci, the authors identify 6 regions that signiﬁcantly replicate the causal association and nominate the following genes: the leucine-rich repeat-containing 7 (LRCC7), the prostaglandin E receptor 3 (PTGER3), and the neuronal growth regulator precursor (NEGR1) genes as the main drivers of this relationship.

Mendelian Randomization has the potential to significantly contribute to our understanding of environmental and protective factors in Alzheimer disease; however, this method depends on assumptions, and the plausibility of these assumptions must be assessed. To verify the consistency of their findings, the authors perform a set of sensitivity analyses to account for confounding effects that

From the Molecular Genetics Section (S.B.-C., F.F.), Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD; Instituto de Investigacion´ Biosanitaria de Granada (S.B.-C.), Granada, Spain; and Department of Computer Science (F.F.), University of Illinois at Urbana-Champaign, Urbana.

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

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 might be violating any core assumption. No evidence of reverse Study funding causation, horizontal pleiotropy, or heterogeneity is identiﬁed. No targeted funding reported.

The reported findings should be interpreted in the context of existing evidence from other research studies using different Disclosure designs, and clinical guidelines should not be elaborated S. Bandres-Ciga and F. Faghri report no disclosures. uniquely based on Mendelian Randomization results. To Disclosures available: at Neurology.org/NG. make a definite conclusion that might be helpful from the clinical perspective to guide disease prevention, replicating References fi 1. Buniello A, MacArthur JAL, Cerezo M, et al. The NHGRI-EBI GWAS Catalog of these ndings in non-European populations with variable published genome-wide association studies, targeted arrays and summary statistics educational background and experiences remains key. 2019. Nucleic Acids Res 2019;47:D1005–D1012. 2. Raghavan N, Vardarajan B, Mayeux R. Genomic variation in educational attainment modifies Alzheimer disease risk. Neurol Genet 2019;5:e310. doi: 10.1212/ Author contributions NXG.0000000000000310. 3. Davey Smith G, Hemani G. Mendelian randomization: genetic anchors for Both authors contributed equally to the initial manuscript causal inference in epidemiological studies. Hum Mol Genet 2014;23: preparation, manuscript editing, and commentary. R89–R98.

2 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG ARTICLE OPEN ACCESS Genomic variation in educational attainment modiﬁes Alzheimer disease risk

Neha S. Raghavan, PhD, Badri Vardarajan, PhD, and Richard Mayeux, MD Correspondence Dr. Mayeux Neurol Genet 2019;5:e310. doi:10.1212/NXG.0000000000000310 [email protected] Abstract Objective To determine the putative protective relationship of educational attainment on Alzheimer disease (AD) risk using Mendelian randomization and to test the hypothesis that by using genetic regions surrounding individually associated single nucleotide polymorphisms (SNPs) as the instrumental variable, we can identify genes that contribute to the relationship.

Methods We performed Mendelian randomization using genome-wide association study summary statistics from studies of educational attainment and AD in two stages. Our instrumental variable comprised (1) 1,271 SNPs signiﬁcantly associated with educational attainment and (2) individual 2-Mb regions surrounding the genome-wide signiﬁcant SNPs.

Results A causal inverse relationship between educational attainment and AD was identified by the − 1,271 SNPs (odds ratio = 0.63; 95% confidence interval, 0.54–0.74; p = 4.08 x 10 8). Analysis of individual loci identified 2 regions that significantly replicated the causal relationship. Genes within these regions included LRRC2, SSBP2, and NEGR1; the latter a regulator of neuronal growth.

Conclusions Educational attainment is an important protective factor for AD. Genomic regions that sig- niﬁcantly paralleled the overall causal relationship contain genes expressed in neurons or involved in the regulation of neuronal development.

From the The Gertrude H. Sergievsky Center (N.S.R., B.V.), Columbia University; The Institute for Genomic Medicine (N.S.R.), Columbia University; The Taub Institute for Research in Alzheimer’s Disease and the Aging Brain (B.V.), Columbia University; The Department of Neurology (R.M.), Columbia University and The New York Presbyterian Hospital; The Department of Epidemiology (R.M.), Joseph P. Mailman School of Public Health, Columbia University, New York, NY.

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

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary AD = Alzheimer disease; CI = conﬁdence interval; GWAS = genome-wide association study; IGAP = International Genomics of Alzheimer’s Project; IVW = inverse variance weighted; LD = linkage disequilibrium; OR = odds ratio; SNP = single nucleotide polymorphism.

Education has consistently been identified as an important no appreciable bias was expected. The study population in both antecedent factor in Alzheimer disease (AD), whereby ad- GWASs were of European descent. vanced educational attainment is thought to reduce AD risk.1,2 In addition, educational attainment and AD may be SNP selection genetically related based on the genome-wide correlation of TwoSampleMR, an R package that performs Mendelian ran- − 9 −0.31 (p =4×104).3 However, confounding factors that domization using data from MR-Base, was used in R (R v.3.3.1) affect educational attainment such as socioeconomic status, to perform all analyses. Mendelian randomization was per- ff nutrition, and ethnicity blur the relationship. formed using two di erent schemes for selecting SNPs for the instrumental variable: Mendelian randomization limits confounding by using instrumental variables associated with a risk factor to establish 1. 1,271 SNP analysis: the instrumental variables consisted effects on the outcome. The largest (n = 1,131,881) genome- of the 1,271 approximately independent SNPs (all with −8 fi wide association study (GWAS) of educational attainment4 p <5×10 )signicantly associated with educational identified 1,271 significantly associated single nucleotide attainment and were used to test for causality to AD. polymorphisms (SNPs), and a separate 2013 GWAS from the Independence for SNPs in the analysis was established 2 ≥ International Genomics of Alzheimer’s Project (IGAP) pro- using linkage disequilibrium (LD) clumping (r 0.001 vided AD SNP association data. The data created an oppor- within a 10,000-kb window). tunity to examine the role of educational attainment in AD 2. Individual locus analyses: Mendelian randomization was through Mendelian randomization. performed independently on each of the 1,271 loci using SNPs within 1-Mb upstream and downstream of individual In GWAS analyses, the significantly associated tag SNPs may SNPs. We used more liberal inclusion criteria including not be the causal SNP.5 When pleiotropic effects and het- SNPs associated with educational attainment at p <0.01 2 ≥ erogeneity remain minimal, the instrumental variable com- and clumped at r 0.1 within a 250-kb window. posed of the index and all associated SNPs in a region improves the validity of the relationship,6 explains a greater Of the 1,271 independent loci, SNPs found within a 2-Mb percentage of variation in the phenotype, and reduces het- region of another were merged together, resulting in 441 erogeneous effects of using fewer SNPs.7 independent loci. Mendelian randomization analyses In addition to testing the overall hypothesis that educational The strictest LD threshold was applied for the first analysis, attainment has a protective effect on AD risk, we tested the using default settings in MR-Base; for analysis 2, we allowed hypothesis that a subset of genetic loci related to educational for more SNPs with still minimal LD to be included in the attainment might individually significantly reflect the overall analysis, as we hypothesized that this would strengthen the protective relationship with AD. To maximize detection, we per-loci analysis. Those SNPs remaining after LD clumping included independent SNPs in a 2-Mb region surrounding were queried within the IGAP summary statistics, if they were each of the 1,271 SNPs significantly associated with educa- 3 not present in that data set, genetic proxies were found, and tional attainment. finally SNPs for which no proxies could be found were excluded. Next, SNPs were harmonized for the effect allele Methods between the 2 GWAS data sets or removed if harmonization predictions were inconclusive. For the joint 1,271 SNP anal- We used available summary statistics from the analysis of all ysis, 387 remained after LD clumping, and 13 SNPs were discovery data excluding the 23andMe cohort in the largest neither found in the IGAP nor had appropriate proxies and GWAS meta-analysis of educational attainment3 (n = 766,345). thus removed. Finally, 57 SNPs were removed because of low Summary statistics from the IGAP GWAS of AD,8 consisting of confidence that the effect allele of the exposure corresponded 54,162 individuals and 7,055,881 analyzed SNPs, were used for to the same allele in the outcome. Therefore, a total of 317 the outcome. There is a small overlap of samples used from SNPs were included in the initial joint SNP analysis. For the the educational attainment and AD GWAS. Sample overlap regional analyses, SNPs were similarly removed if not found in a 2-sample Mendelian randomization can cause bias in in the IGAP or if allele harmonization failed. The inverse results; however, because of the large sample size of the edu- variance weighted (IVW) method was used to calculate odds cational attainment GWAS and minimal overlap of the sample, ratios (ORs). Results are reported as the OR (±95% confidence

Table Results from inverse variance weighted method and sensitivity analyses for Mendelian randomization analyses

Inverse variance weighted method Sensitivity analysis

− Top educational attainment SNPs (317 SNPs included) Odds ratio = 0.63; 95% CI, 0.54–0.74; p =4.08×10 8 Egger regression: intercept = 0.003; p value = 0.43

Inverse variance weighted Sensitivity method analysis

Egger regression No. of Odds ratio (intercept ± Locusa Chromosome Gene(s)b Proposed role of gene(s) SNPsc (95% CI) p Value SE, p value)

− rs7552964, rs10789285, rs1024268, rs663251, rs481940, rs72677177, rs34122915, 1 LRRC7/PTGER3/ Necessary for synaptic spine 89 0.31(0.19–0.50) 1.92 × 10 6 0.013 ± 0.006, p rs34305371, rs2568955, rs1445591, rs12028229, rs11210228, rs74091672, NEGR1/FPGT- architecture and function/1 value = 0.05 rs11210400, rs1569092, rs28482086 TNNI3K/ receptor for prostaglandin TNNI3K/TYW3 E2/cell adhesion and/or regenerative axon sprouting/ read-through transcription/ MAPKKK family involved in cardiac physiology/stabilizes codon-anticodon interactions

− rs1910005 5 SSBP2 DNA damage response; 17 0.07 5.8 × 10 5 −0.008 ± 0.017, maintenance of genome (0.02–0.25) p value = 0.66 stability; telomere repair

Abbreviation: SNP = single nucleotide polymorphism. Top: 1,271 SNP Analysis. Bottom: Individual locus analyses. a SNPs significantly associated with educational attainment in Lee et al.,4 in this locus. b The gene or closest gene to the SNP. c Number of SNPs that were included in the regional analysis. 3 interval [CI]) of AD risk per SD increase in educational at- involved in regulation of neuronal development. For example, tainment in each test. one of the SNPs is within the LRCC7 gene, which is crucial to dendritic spine architecture and function and may be involved Sensitivity analyses in bipolar disorder.14 Another SNP within the intronic region Mendelian randomization requires that that the instrumental of the NEGR1 gene is also highly expressed in neurons and variables meet 3 requirements: it must be associated with the has been found to be associated with major depressive dis- risk factor, not associated with any confounder of the risk factor order.15 An SNP was found in the PTGER3 gene, which is or outcome, and is only associated with the outcome through thought to be involved in the modulation of neurotransmitter the risk factor. Sensitivity analyses following the initial analysis release in neurons. provide confidence that assumptions of the instrumental variables were not broken. In addition to the IVW, the weighted One limitation of using the Mendelian randomization method median method was used to measure causality and provided for outcomes with other strong nongenetic factors risk factors consistent results with IVW when at least 50% of the in- is that the CIs tend to be large. However, this outcome is strumental variables were valid. Next, the intercept of the preferable to the bias that is inherent in epidemiologic studies, Mendelian randomization–Egger test was used to determine which unavoidably include confounders.16 Taken together, potential horizontal pleiotropy or an effect of the instrumental the results presented here, along with earlier reports, establish variables on a phenotype other than the outcome. As the in- a putative causal relationship between educational attainment tercept neared zero in the mendelian randomization–Egger and AD. test, horizontal pleiotropy was reduced. The Steiger test of directionality was used to confirm directionality of the effect, The key next steps are to replicate these findings in diverse i.e., that the SNPs first affected educational attainment and ethnic groups with more variable educational experiences and subsequently that AD risk was affected through educational to identify and validate specific variants within these loci that attainment. Finally, the radial regression analyses were per- account for the association. formed for the inverse variance methods, which identify any 10 significant SNP outliers using the Cochran’sQ-statistic. Author contributions All authors of the study contributed to the study design, study analysis, and writing and editing. Results Using the 1,271 SNPs associated with educational attainment, Acknowledgments ’ we detected statistically significant evidence for causation The authors thank the International Genomics of Alzheimer s between educational attainment and AD such that an increase Project and Lee et al. for providing summary statistics for this of 4.2 years of educational attainment was associated with project. a 37% reduction in AD risk (OR—scaled per SD, 4.2 years = − 0.63; 95% CI, 0.54–0.74; p = 4.08 × 10 8). Study funding This work was supported by grants from the National In- In the per-loci analyses of the 1,271 regions, 2 independent stitute on Aging from the NIH (U01AG032984 and SNP regions demonstrated a statistically significant inverse RO1AG041797) and the National Center for Advancing relationship between educational attainment and AD risk Translational Sciences (TL1TR001875). − (Bonferroni-corrected threshold p <1.2×10 4). These regions include the neuronal growth regulator precursor (NEGR1) Disclosure gene, leucine-rich repeat containing 7 (LRCC7)gene,and N.S. Raghavan reports no disclosures. B. Vardarajan has prostaglandin E receptor 3 (PTGER3)gene(table1). served as a consultant for BISC Global. R. Mayeux has served as a consultant for the Alzheimer’s Disease Center at Rush Sensitivity analyses were performed for all Mendelian ran- University Medical School and has received research support domization analyses. There was no indication of pleiotropy, from the National Institute on Aging. Full disclosure form reverse causality, or heterogeneity, and the weighted median information provided by the authors is available with the full method showed consistent results with IVW overall in the text of this article at Neurology.org/NG. significant analyses. Publication history Received by Neurology: Genetics July 15, 2018. Accepted in final form Discussion December 18, 2018. 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Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 5 ARTICLE OPEN ACCESS Homozygous TRPV4 mutation causes congenital distal spinal muscular atrophy and arthrogryposis

Jose Velilla, BS,* Michael Mario Marchetti, BS,* Agnes Toth-Petroczy, PhD, Claire Grosgogeat, BS, Correspondence Alexis H. Bennett, BS, Nikkola Carmichael, MS, CGC, Elicia Estrella, MS, CGC, Basil T. Darras, MD, Dr. Gupta [email protected] Natasha Y. Frank, MD, Joel Krier, MD, Rachelle Gaudet, PhD, and Vandana A. Gupta, PhD, on behalf of Brigham or Dr. Gaudet Genomics Medicine [email protected] Neurol Genet 2019;5:e312. doi:10.1212/NXG.0000000000000312 Abstract Objective To identify the genetic cause of disease in a form of congenital spinal muscular atrophy and arthrogryposis (CSMAA).

Methods A 2-year-old boy was diagnosed with arthrogryposis multiplex congenita, severe skeletal abnormalities, torticollis, vocal cord paralysis, and diminished lower limb movement. Whole- exome sequencing (WES) was performed on the proband and family members. In silico modeling of protein structure and heterologous protein expression and cytotoxicity assays were performed to validate pathogenicity of the identiﬁed variant.

Results WES revealed a homozygous mutation in the TRPV4 gene (c.281C>T; p.S94L). The identification of a recessive mutation in TRPV4 extends the spectrum of mutations in recessive forms of the TRPV4-associated disease. p.S94L and other previously identified TRPV4 variants in different protein domains were compared in structural modeling and functional studies. In silico structural modeling suggests that the p.S94L mutation is in the disordered N-terminal region proximal to important regulatory binding sites for phosphoinositides and for PACSIN3, which could lead to alterations in trafficking and/or channel sensitivity. Functional studies by Western blot and immunohistochemical analysis show that p.S94L increased TRPV4 activity- based cytotoxicity and resultant decreased TRPV4 expression levels, therefore involves a gain- of-function mechanism.

Conclusions This study identiﬁes a novel homozygous mutation in TRPV4 as a cause of the recessive form of CSMAA.

*Co-first authors.

From the Department of Molecular and Cellular Biology (J.V., R.G.), Harvard University, Cambridge; Division of Genetics (M.M.M., A.T.-P., C.G., A.H.B., N.C., B.T.D., N.Y.F., J.K., V.A.G.), Brigham Genomic Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston; Division of Genetics (E.E.), Boston Children’s Hospital; and Division of Neurology (B.T.D.), Boston Children’s Hospital, Harvard Medical School, MA.

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary CMT = Charcot-Marie-Tooth; cryoEM = cryoelectron microscopy; CSMAA = congenital spinal muscular atrophy and arthrogryposis; dHMN = distal hereditary motor neuropathy; PIP2 = phosphatidylinositol 4,5-bisphosphate; PRR = proline- rich region; SPSMA = scapuloperoneal spinal muscular atrophy; TRPV = transient receptor potential vanilloid; WES = whole- exome sequencing; WT = wild-type.

Hereditary neuropathies are a clinically and genetically het- homozygous mutation in a patient presenting with CSMAA. erogeneous group of diseases with an estimated prevalence of Follow-up functional studies reveal distinct disease mecha- 1:2,500. Clinical manifestations of hereditary neuropathies nisms for different TRPV4 variants and provide novel insights include slow progressive distal weakness and muscle wasting that may inform future therapeutic strategies for these with or without sensory loss. Hereditary neuropathies are patients. classified into 3 broad categories on the basis of the clinical phenotype. Charcot-Marie-Tooth (CMT) disease or hereditary motor and sensory neuropathy typically exhibits in- Methods volvement of both motor and sensory systems, hereditary Standard protocol approvals, registrations, sensory and autonomic neuropathy involves sensory deficits and patient consents and/or autonomic dysfunction, and distal hereditary motor The proband, both parents, and the unaffected sibling were neuropathy (dHMN) predominantly involves motor deficits. enrolled, and informed consent was obtained from partic- These groups can be further classified into many subtypes ipants in accord to an institutional review board–approved depending on electrophysiologic criteria, pathologic defects, study at Boston Children’s Hospital and Brigham and mode of inheritance, and molecular genetic defects. These Women’s Hospital. diseases are genetically highly heterogeneous with mutations ff 1,2 in at least 80 di erent genes associated with these subtypes. Whole-exome sequencing – ff Despite this progress, 30% 70% of people a ected with DNA extraction from blood samples was performed by the neuropathies do not have a genetic diagnosis because of Research Connection Biobank Core (Boston Children’s clinical and genetic heterogeneity. Some of the disease Hospital) using the QIAamp DNA Mini kit (Qiagen). WES symptoms can be controlled with generic drugs. However, the was performed by the Yale Genome Center. DNA samples fi identi cation of the underlying genetic lesion is necessary for from the proband and parents were sent for WES. Samples accurate disease prognosis, management, and family planning were prepared as an Illumina sequencing library and enriched and may ultimately lead to personalized treatments. for exomic sequences using the Agilent V5 Sureselect kit. The captured libraries were sequenced using Illumina HiSeq 2000 Mutations in the transient receptor potential vanilloid 4 Sequencers at Lab Corp. FASTQs generated from exome (TRPV4) cation channel gene are a rare cause of dominant sequencing were filtered and aligned, and variants were fil- 3,4 inherited axonal neuropathies and skeletal dysplasias. tered and annotated, as previously described.6 In short, first, TRPV4 mutations underlie a wide spectrum of clinical pre- we apply agnostic filtering, and we use pedigree-based in- sentation and are associated with dHMN, scapuloperoneal heritance mode filtering for de novo and recessive variants, as spinal muscular atrophy (SPSMA), congenital spinal muscu- well as filter for rare variants only, based on large population lar atrophy and arthrogryposis (CSMAA), autosomal domi- databases (gnomAD, gnomad.broadinstitute.org). In the nant axonal CMT type 2C, and congenital distal spinal second step, we apply several knowledge-based filters on the 5 muscular atrophy (dSMA). TRPV4-related neuropathies are genes (e.g., known functional and disease associations, ex- frequently associated with vocal cord paralysis and occasion- pression level data, and model organism data) and on the ally with sensorineural hearing loss. TRPV4 mutations can variants (e.g., evolutionary conservation and structural con- also result in autosomal dominant skeletal dysplasias. Typi- straints). Finally, the variants were prioritized during ff cally, di erent mutations in the TRPV4 gene are associated a crowdsourcing case conference of interdisciplinary audi- with either neuropathies or skeletal dysplasia; however, some ence.6 patients exhibit both clinical phenotypes. So far, >20 different fi mutations in TRPV4 have been identi ed in patients with To validate the TRPV4 variant, PCR products for the pro- neuropathies. However, functional validation of pathogenicity band, unaffected sibling, and parents were analyzed by stan- for many of these variants is still lacking, thus preventing dard Sanger sequencing (Dana-Farber/Harvard Cancer a conclusive genetic diagnosis in these patients. To improve Center DNA Sequencing Facility). the genetic diagnosis of patients affected with neuropathies, we use whole-exome sequencing (WES) combined with In silico modeling of TRPV4 mutations functional validation studies to establish the pathogenicity of TRPV4 mutations were mapped onto the cryoelectron mi- variants identified by WES. This work has identified a TRPV4 croscopy (cryoEM) structure of Xenopus tropicalis TRPV4

2 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG (PDB ID: 6bdj)7 after aligning the XtTRPV4 and human media) × 100. Statistical significance was determined using TRPV4 sequences using Clustal Omega. Figure 2 was gen- unpaired the Student t test. erated using PyMOL (Schrodinger). Western Blot Cloning of mutant constructs HEK293 cells were transfected with the respective vector To generate TRPV4 mutant plasmids, p.S94L, p.R315W, and using polyethylenimine (PEI to DNA ratio of 3:1). Cells were p.T701I variants were incorporated into TRPV4 cDNA in incubated for 12 hours at 37°C, then shifted to 33°C, and plasmid pcDNA3.1-TRPV4-FLAG using a Q5 site-directed harvested 48 hours after transfection. Western blots were mutagenesis kit (New England Biolabs). The mutagenesis performed according to standard protocols, using 1:2,000 M2 primer sequences were S94L forward: 59-TAT GAG TCC anti-FLAG (Sigma, F-3165) to detect TRPV4, 1:10,000 anti- TTG GTG GTG CCT-39, S94L reverse: 59-TAG GGT GGA GAPDH (Abcam, 8245), and 1:1000 alkaline phosphatase CTC CAG CAG-39, R315W forward: 59-GGC GGA CAT conjugated anti-mouse secondary antibody (Sigma, A3562). GTG GCG CCA GGA-39, R315W reverse: 59-TTC TTG Membranes were developed using the 1-Step NBT/BCIP TGG GGG TTC TCC GTC AGGT-39, T701I forward: 59- reagent (Thermo Fisher Scientific, 34042). Densitometric CTG CTG GTG ATC TAC ATC ATC-39, and T701I re- analysis was performed with ImageJ (imagej.nih.gov/ij/). verse: 59-GAT GAT GAA GAC CAC GGG-39. The full Data are presented as the mean ± SD, n = 3, 2-tailed t test, *p coding sequences were confirmed using Sanger sequencing. < 0.05.

Cytotoxicity assay Immunofluorescence analysis ’ fi Human HEK293 cells were cultured in Dulbecco sModi ed For immunofluorescence, HEK293 cells were plated in Eagle Media supplemented with 10% fetal bovine serum. Cells 8-chambered slides, and transfections were performed with were transfected with the respective TRPV4 or empty vector lipofectamine 3000 (Thermo Fisher Scientific). Twenty-four fi using Lipofectamine 3,000 (Thermo Fisher Scienti c) and hours after transfection, cells were fixed in 4% para- μ cultured in the presence or absence of HC-067047 (5 M). Cell formaldehyde, and immunofluorescence was performed as death analysis was performed 24 hours after transfection using described previously9 using M2 anti-FLAG antibody (Sigma, the Cytotoxicity Detection Kit (Roche Diagnostics, Indian- 1:250) and sodium potassium ATPase antibody (Abcam apolis, IN) at 25°C according to the manufacturer’s instructions 8 76020,1:100). Nuclear staining was performed using DAPI as described previously (n =3). Cytotoxicity was calculated by (BioLegend, 422801). subtracting the absorbance of the background control (normal media) from the absorbance of the experimental samples (supernatant from cells transfected with wild-type [WT] or mutant plasmid). Results are expressed as a percentage relative to Results the absorbance of the high control (supernatant from cells Clinical presentation treated with lysis buffer) as Cytotoxicity (%) = Experimental The male proband was born to unrelated healthy parents of value-control (normal media)/high control-control (normal Puerto Rican descent after 37 weeks of gestation (figure 1A).

Figure 1 Identification of TRPV4 homozygous mutation

(A) Pedigree of the family of the affected patient; the proband is indicated by the arrow. (B) Sanger sequencing pherograms show homozygosity for c.281C>T in TRPV4 in the proband and heterozygosity in both parents. The position of the variant is marked as *. (C) Protein sequence alignment of TRPV4 orthologs showing conservation in the region including p.S94L in higher vertebrates. (D) Schematic diagram of the TRPV4 protein demonstrating localization of p.S94L on the N-terminal intracellular region. Numbers 1–6 correspond to the 6 ankyrin repeats.

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 3 He has a healthy sister. The proband was delivered by ce- (p.S94L). Sanger sequencing confirmed the homozygosity of sarean section because of breech position and arthrogryposis this variant in the proband (BGM0049-1) and heterozygous multiplex congenita on ultrasound. At birth, the proband inheritance from both parents (BGM0049-2 and BGM0049- exhibited a right clubfoot, a left congenital vertical talus, and 3) (figure 1B). The unaffected sister was found to be homo- bilateral flexion deformities of the knees and torticollis. X-rays zygous for the normal variant (not shown). The other 14 showed dislocated hips with wide proximal femurs. Ultra- variants/genes could be ruled out by having low population sound revealed that the patient exhibited dysplastic acetabuli constraints, known unrelated disease associations, unrelated on both sides. An ultrasound of the spine was performed, protein functions, and lack of expression in relevant tissues which showed that the spinal cord was at the L3 level that is (table e-1), corroborating the potential causal effect of the slightly lower than the expected level. Bilateral flexible lar- TRPV4 variant. The missense variant is at a highly conserved yngoscopy was performed through the left naris that showed position in most of the vertebrate species and is localized to nonobstructive adenoid hypertrophy, normal tongue base, the N-terminus of the TRPV4 protein (figure 1, C and D). and structurally normal larynx with no evidence of lar- yngomalacia. Vocal cords were clearly visible and appeared to Structural model be immobile bilaterally in the paramedian position. The A recent cryoEM structure of Xenopus tropicalis TRPV4 was proband also exhibited inspiratory stridor. Examination of the used to model the position of p.S94L and other previously right and left tympanic membranes showed an evidence of described neuropathy-causing TRPV4 mutations (figure 2).7 middle ear effusions bilaterally and normal external ear and Consistent with previous proteolysis protection10 and external auditory canals. Visual reinforced audiometry showed Nuclear Magnetic Resonance studies,11 the N-terminal a moderate conductive hearing loss for at least 1 ear at 500 and region—up to residue 147 (human numbering), thus in- 2000 Hz. Tympanograms were flat bilaterally consistent with cluding p.S94—was disordered and not modeled in the cry- clinical findings. oEM structure. However, p.S94L is located N-terminal to 2 important regulatory regions: a phosphoinositide-binding At age 2 years, the proband could sit independently and walk domain; residues 121–125 in humans) that interacts with 10 on his knees. However, he was unable to stand or walk in- phosphatidylinositol 4,5-bisphosphate (PIP2) and a proline- dependently. Except for lower limb movement, the proband rich region (PRR; residues 135–144 in humans) that interacts has normal speech, fine motor and gross motor skills in his with the SH3 domain of PACSIN3.12 PACSIN3 enhances ffi upper limbs, normal hearing, and visual development and TRPV4 tra cking to the cellular membrane. PIP2 binding exhibits a static condition. EMG examination revealed normal enhances TRPV4 responses to several stimuli, whereas right sural sensory response and normal left ulnar-DV (Digit PACSIN3 decreases responses to these same stimuli. This 5) sensory response. Normal left tibial abductor hallucis and antagonistic interaction between 2 key regulators, PIP2 and median abductor pollicis brevis motor response for the age PACSIN3, is likely mediated through PACSIN3-mediated was also noted. Concentric needle examination of selected structural rearrangements of the PRR.10,13 Therefore, our muscles showed fibrillation potentials in the right vastus lat- structural modeling suggests that the p.S94L mutation could eralis muscle with late and fast firing motor unit potentials in potentially affect the regulation and/or sensitivity of TRPV4. left deltoid, right tibialis anterior, and right iliopsoas muscles. The electrophysiologic findings are suggestive of generalized Functional modeling of TRPV4 variants motor axonopathy with coexisting denervation and reinner- Functional studies have shown a gain-of-function mechanism 14 vation changes. for previously known dominant mutations in TRPV4. To validate the pathogenicity of the p.S94L variant, functional Whole-exome sequencing studies were performed by analyzing the expression levels and WES was performed on the proband and parents. The pres- localization of the mutant protein. To compare the magnitude ence of a single affected male individual in this family is of the functional deficits, known neuropathy-causing consistent with autosomal recessive inheritance, X-linked in- p.R269C and p.R315W TRPV4 mutants were analyzed in heritance, or with a dominant de novo mutation. Therefore, parallel. In addition, a mutation p.T701I has been reported as pedigree and population-based filtering was performed to a cause of neuropathy. p.T701I is localized to the trans- identify potentially damaging homozygous, X-linked, com- membrane domain S6 helix (figure 2), near the vanilloid- pound heterozygous, and de novo variants. We only consid- binding site in the homologous ion channel TRPV1.15 ered rare coding and splice variants that have <0.1–1% allele Currently, there are no functional studies establishing the in the general healthy population in the gnomAD database pathogenicity of this variant in human diseases or TRPV4 (gnomad.broadinstitute.org) depending on the dominant or protein functions. Therefore, we investigated the effect of recessive inheritance mode that is consistent with the preva- p.S94L, p.R269C, p.R314W and p.T701I on the expression lence of the disease. In total, 15 potential genes carried level and subcellular localization of TRPV4. To investigate the a variant (table e-1, links.lww.com/NXG/A140) that were effect of different variants on protein expression, TRPV4-WT further prioritized using our knowledge-based and crowd- and mutant constructs were transiently expressed in HEK293 sourcing pipeline as recently reviewed.6 The top candidate cells, and expression was analyzed by Western blot (figure 3). was a homozygous missense variant in TRPV4 c.281C>T; Protein analysis by Western blot showed reduced level of

4 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Figure 2 Structure modeling of TRPV4 mutations

Structural model based on the cryoEM structure of Xenopus tropicalis TRPV4 (PDB ID: 6bbj). The structure, corresponding to residues 148–788 (human numbering), does not include disordered N- and C-terminal regions. The N-terminal region is schematized as a dotted line for each subunit, with the phosphoinositide-binding domain (PBD; residues 121–125 in human) and proline-rich region (PRR, residues 135–144) indicated. Residue positions for neuropathy-causing mutations (D62N, P97R, R186Q, R232C/S, R237G/L, R269C/H, R315W, R316C/H, and T701I) and disease mutations with mixed phenotypes (G78W, A217S, E278K, S542Y, V620Y, and T740I) are green and yellow, respectively. Compound heterozygous neuromuscular disease mutations N833S and E840K are not illustrated because they are in the disordered cytoplasmic C-terminal region (residues 789–871). The position of p.S94L is indicated in purple. p.S94L protein and reduced levels of p.R269C and p.R315W. Discussion In contrast, p.T701I exhibited levels of protein expression similar to those of WT TRPV4 (figure 3, A and B). As pre- The distal hereditary motor neuropathies (dHMNs) are vious studies have reported cytotoxic effects of mutant a genetically heterogeneous group of diseases characterized by TRPV4 proteins, cytotoxicity analysis was performed in distal lower motor neuron weakness. This is in contrast to HEK293 cells transfected with control and mutant plasmids. CMT disease and the hereditary sensory neuropathies where Quantification showed significantly higher levels of cell death sensory involvement forms a major component of the disease. in the p.S94L transfected cells in comparison to WT TRPV4- Many forms of dHMN can exhibit a minor sensory compo- transfected cells. Similarly, p.R269C and p.R315W also nent, and there is an overlap between axonal forms of CMT resulted in the increased cytotoxicity. Of interest, no changes (CMT2) and dHMN. Autosomal dominant mutations in in cytotoxicity were observed in p.T701I transfected cells in TRPV4 are associated with diverse clinical presentations in- comparison to WT TRPV4-transfected cells. HEK293 cells cluding dHMN, SPSMA, CSMAA, and autosomal dominant transfected with control and mutant plasmids were also cul- axonal CMT type 2C (CMT2C) disease. TRPV4 mutations tured in the presence of a TRPV4 channel antagonist (HC- also result in several forms of autosomal dominant skeletal 067047) that reversed the cytotoxicity effects observed in p. dysplasias. The proband exhibited arthrogryposis associated S94L, p.R269C, and p.315W transfected cells (figure 3C). with distal muscle weakness with normal sensory response The presence of HC-067047 also resulted in an increased and therefore clinically diagnosed as CSMAA, a subtype of expression of p.S94L, p.R269C, and p.R315W; however, no dHMN. Congenital arthrogryposis could result from a defect expression changes were detected for p.701I protein (figure in skeletal or neuromuscular system. As both skeletal and 3B). To examine the subcellular localization of p.S94L and neurologic deficiencies were present in the proband during other mutants, immunofluorescence analyses were performed early childhood, it is hard to predict which of these contrib- (figure 4). Wild-type TRPV4 primarily localized to the plasma uted to primary disease pathology. TRPV4 mutations are membrane as has been reported for endogenous TRPV4 pro- typically associated with either neuropathies or skeletal dys- tein. p.S94L and previously reported p.R269C and p.R315W plasia; however, the presence of both skeletal and neurologic exhibited localization to the plasma membrane. Interestingly, phenotypes has been seen in several previously reported the p.T701I mutant protein showed a perinuclear localization cases.16 This suggests that genotype-phenotype association in pattern, and in contrast to WT protein, no protein was detected TRPV4-related diseases is not stringent and may lead to both on the plasma membrane (figure 4). skeletal and neurologic abnormalities.

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 5 Figure 3 Influence of TRPV4 mutations on protein expression level and cytotoxicity

(A–B) Western blot (A) and quantification based on 3 experimental repeats (B) from HEK293 cells transiently expressing TRPV4-FLAG, p.S94L-FLAG, p.R269C-FLAG, p.R315W-FLAG, and p.T701I-FLAG or empty vector collected 48 hours after transfection and incubated in the absence or presence of HC-067047 (5 μM). Cell lysates were probed with antibodies against FLAG tag to detect transfected wild-type (WT) and mutant TRPV4 proteins and GAPDH as the loading control. (C) Cytotoxicity analysis in cells transfected with control or mutant TRPV4 plasmids. Differences in the protein levels or cytotoxicity were considered significant with *p < 0.05.

Several functional studies have demonstrated a gain-of- of protein expression levels by the antagonist suggests that it is function mechanism for mutant TRPV4-mediated neuropa- an increase in the channel activity of the p.S94L variant that thies.14 Mutant TRPV4 proteins frequently exhibit normal causes its reduced expression levels. Previous work has also localization; however, they demonstrate increased calcium identified reduced stability of many dominant variants in the channel activity at both basal and activated levels. This results intracellular ankyrin repeat domain of TRPV4.17 A recent in increased intracellular calcium concentration leading to study also identified biallelic heterozygous mutations located cytotoxicity. in the C-terminus of the TRPV4 channel in 2 siblings with neuropathy associated with severe intellectual disability.18 Following a combination of WES and functional analysis, we These mutant proteins demonstrated normal membrane lo- identified a homozygous missense variant, p.S94L, in TRPV4 calization; however, they showed reduced channel function. as a cause of a severe form of dHMN. This serine residue is The severe phenotype in the proband is consistent with the highly conserved in mammals, and only 6 heterozygous and observed cytotoxicity of the p.S94L variant in the HEK293 no homozygous individuals are reported in a control pop- cells. ulation of 246210 people (Genome Aggregation Database, gnomAD), implicating that this as a crucial amino acid. Here, The p.S94L mutation is located in the N-terminal in- we demonstrate that the TRPV4 p.S94L variant resulted in tracellular region upstream of the ankyrin repeat domain. increased cytotoxicity, as previously reported for other Previous studies have shown that dominant mutations in neuropathy-causing pathogenic TRPV4 variants. A limitation this region result in neuropathy in affected individuals.19 In of this work is that these studies were performed in HEK293 comparison to previously known TRPV4 variants such as cells rather than neuronal-like cells. However, HEK293 cells p.R269C and p.R315W, higher levels of p.S94L TRPV4 have commonly been used to determine aberrant channel protein were detected in transfected HEK293 cells (figure function for dominant TRPV4 mutations, allowing compar- 3, A and B). Similarly, we observed a lower level of cyto- isons of our results with previous work.3 Furthermore, p.S94L toxicity in p.S94L transfected cells in comparison with mutation resulted in reduced levels of the mutant protein that p.R269C and p.R315W transfected cells (figure 3C). were rescued by the TRPV4 channel antagonist as previously Furthermore, the facts that the proband’sparentswhoare observed for other mutations in neuronal cells.14 The rescue heterozygous for the TRPV4 p.S94L variant did not exhibit

6 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG variants but also for the design of speciﬁc therapies as both Figure 4 Subcellular localization of mutant TRPV4 proteins agonists and antagonists of TRPV4 channels are available.

Data availability The research material supporting this publication can be accessed by contacting the corresponding author at “vgupta@ research.bwh.harvard.edu.”

Acknowledgments The authors thank the Brigham Genomics Medicine for clinical and genomic data analyses. Brigham Genomics Medicine acknowledgments and full collaborator list are provided in Appendix 2.

Study funding This work was supported by K01 AR062601 (VAG) and the Eleanor, Miles Shore Fellowship for Scholars in Medicine and Brigham and Women’s Hospital Career Development Award (VAG), a Brigham Biomedical Re- search Institute Director’s Transformative Award (Brig- ham Genomic Medicine), and the American Heart Association 16GRNT27250119 (RG).

Disclosure J. Velilla, M.M. Marchetti, A. Toth-Petroczy, C. Grosgogeat, A.H. Bennett, N. Carmichael, and E. Estrella report no disclosures. B.T. Darras has served on the scientific advisory boards of Hoffman-La Roche, Cytokinetics, Inc, BMS, Inc, Sarepta Therapeutics, Biogen, AveXis, and PTC Therapeu- tics; has received travel funding and/or speaker honoraria from Biogen; has received publishing royalties from UpTo- Date; and has received research support from PTC Therapeutics, Valerion Therapeutics (MTM), PTC Phar- Representative images of HEK293 cells transiently expressing TRPV4-Flag, p.S94L-Flag, p.R269C-Flag, p.R315W-Flag, and p.T701I proteins. Immuno- maceuticals, Sarepta Therapeutics, Biogen, Summit, AveXis, fluorescence was performed with anti-Flag tag antibody to detect TRPV4- Flag proteins and Na-K-ATPase to label the plasma membrane. Nuclei were Roche, Fibrogen, Santhera, Cytokinetics, NIH/NINDS, SMA stained with DAPI. Scale bar = 20 μm. Foundation, Muscular Dystrophy Association, and the Slaney Family Fund for SMA. N.Y. Frank holds patents assigned to Brigham and Women’s Hospital, Boston, MA, and licensed to any phenotype and that 6 heterozygous individuals are Ticeba GmbH (Heidelberg, Germany) and Rheacell GmbH reported in the gnomAD database suggest reduced pene- & Co. KG (Heidelberg, Germany); serves as an advisor to trance of this variant in the heterozygous state. Nonethe- Veritas Genetics, Inc. (Danvers, MA); and her spouse less, many heterozygous TRPV4 variants exhibit reduced Dr. Markus Frank serves as a scientific advisor to Ticeba penetrance in adult carriers, and for this reason, the GmbH and Rheacell GmbH & Co. KG. J. Krier has received parents of the proband were counseled to routine follow- research support from the NIH. Rachelle Gaudet’sspouseis up in a neurology clinic. Of interest, most human disease- employed by and holds stock/stock options in Bristol- causing variants studied in this work exhibited a reduced Myers Squibb, and she has received research support from protein expression level, whereas the p.T701I variant al- the NIH. V.A. Gupta was supported by K01 AR062601 tered the cellular localization of TRPV4 protein from the (VAG) and the Eleanor and Miles Shore Fellowship for plasma membrane to the perinuclear area. This mis- Scholars in Medicine and Brigham and Women’sHospital localization may result in altered calcium channel function Career Development Award (VAG). Disclosures available: in affected patients. Together, these results demonstrate Neurology.org/NG. that different mutations in TRPV4 affect the channel function by different mechanisms. Understanding the Publication history functional alterations in mutant TRPV4 channels is es- Received by Neurology: Genetics June 13, 2018. Accepted in final form sential not only for determining the pathogenicity of novel January 22, 2019.

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 7 Appendix 1 Author contributions Appendix 2 (continued)

Name Location Role Contribution Name Location Role Contribution

Jose Velilla, Harvard Author Major role in the acquisition of Robert Green, Brigham and Site Review of clinical, BS University data MD, MPH Women’s Investigator genetic and Hospital and bioinformatic Michael M Brigham and Author Major role in the acquisition of Harvard results Marchetti, Women’s data Medical School BS Hospital Wolfram Brigham and Site Review of clinical, Agnes Toth- Brigham and Author Interpreted the data and Goessling, MD, Women’s Investigator genetic and Petroczy, Women’s revised the manuscript for PhD Hospital and bioinformatic PhD Hospital intellectual content Harvard results Medical School Claire Brigham and Author Major role in the acquisition of Grosgogeat Women’s data Alireza Brigham and Site Review of clinical, Hospital Haghighi, MD, Women’s Investigator genetic and PhD Hospital and bioinformatic Alexis H. Brigham and Author Major role in the acquisition of Harvard results Bennett, MS Women’s data Medical School Hospital Elizabeth Fieg, Brigham and Coordinator Coordinated Nikkola Brigham and Author Interpreted the data and MS, CGC Women’s and Genetic communication Carmichael, Women’s revised the manuscript for Hospital Counselor among team and MS Hospital intellectual content review of clinical and genetic data Elicia Boston Author Designed and conceptualized Estrella, MS Children’s the study, analyzed the data, Calum Brigham and Site Review of clinical, Hospital and drafted the manuscript MacRae, MD, Women’s Investigator genetic and for intellectual content PhD Hospital and bioinformatic Harvard results Basil T. Boston Author Designed and conceptualized Medical School Darras, MD Children’s the study, analyzed the data, Hospital and drafted the manuscript Soumya Brigham and Site Review of clinical, for intellectual content Raychaudhuri, Women’s Investigator genetic and MD, PhD Hospital and bioinformatic Natasha Y. Brigham and Author Interpreted the data and Harvard results Frank, MD Women’s revised the manuscript for Medical School Hospital intellectual content Christine Brigham and Site Review of clinical, Joel Krier, Brigham and Author Interpreted the data and Seidman, MD Women’s Investigator genetic and MD Women’s revised the manuscript for Hospital and bioinformatic Hospital intellectual content Harvard results Medical School Rachelle Harvard Author Designed and conceptualized Gaudet, PhD University the study, interpreted the data, Nikolaos Brigham and Site Review of clinical, and revised the manuscript for Patsopolous, Women’s Investigator genetic and intellectual content MD, PhD Hospital and bioinformatic Harvard results Vandana A. Brigham and Author Designed and conceptualized Medical School Gupta, PhD Women’s the study, analyzed the data, Hospital and drafted the manuscript Onuralp Brigham and Site Review of clinical, for intellectual content Soylemez, PhD Women’s Investigator genetic and Hospital and bioinformatic Harvard results Medical School

Appendix 2 Brigham Genomics Medicine co-investigators References Name Location Role Contribution 1. Rossor AM, Polke JM, Houlden H, Reilly MM. Clinical implications of genetic advances in Charcot-Marie-Tooth disease. Nat Rev Neurol 2013;9:562–571. Richard L. Brigham and Chief Team lead and 2. Pareyson D, Saveri P, Pisciotta C. New developments in Charcot-Marie-Tooth Maas, MD, PhD Women’s -Investigator review of clinical, neuropathy and related diseases. Curr Opin Neurol 2017;30:471–480. Hospital and Brigham genetic and 3. Landour´e G, Zdebik AA, Martinez TL, et al. Mutations in TRPV4 cause Charcot- Harvard Genomic bioinformatic Marie-Tooth disease type 2C. Nat Genet 2010;42:170–174. Medical School Medicine results 4. Rock MJ, Prenen J, Funari VA, et al. Gain-of-function mutations in TRPV4 cause autosomal dominant brachyolmia. Nat Genet 2008;40:999–1003. Shamil Brigham and Co-Director Team lead and 5. Fawcett KA, Murphy SM, Polke JM, et al. Comprehensive analysis of the TRPV4 gene Sunyaev, PhD Women’s Brigham review of clinical, in a large series of inherited neuropathies and controls. J Neurol Neurosurg Psychiatry Hospital and Genomic genetic and 2012;83:1204–1209. Harvard Medicine bioinformatic 6. Haghighi A, Cassa CA, Krier JB, et al. An integrated clinical program and crowd- Medical School results sourcing strategy for genomic sequencing and mendelian disease gene discovery. NPJ Genom Med 2018;3:21. Christopher Brigham and Site Review of clinical, 7. Deng Z, Paknejad N, Maksaev G, et al. Cryo-EM and X-ray structures of TRPV4 Cassa, PhD Women’s Investigator genetic and reveal insight into ion permeation and gating mechanisms. Nat Struct Mol Biol 2018; Hospital and bioinformatic results 25:252–260. Harvard 8. Sullivan JM, Zimanyi CM, Aisenberg W, et al. Novel mutations highlight the key role Medical School of the ankyrin repeat domain in TRPV4-mediated neuropathy. Neurol Genet 2015;1: e29. doi: 10.1212/NXG.0000000000000029.

8 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG 9. Bennett AH, O’Donohue MF, Gundry SR, et al. RNA helicase, DDX27 regulates 14. Fecto F, Shi Y, Huda R, Martina M, Siddique T, Deng HX. Mutant TRPV4-mediated skeletal muscle growth and regeneration by modulation of translational processes. toxicity is linked to increased constitutive function in axonal neuropathies. J Biol PLoS Genet 2018;14:e1007226. Chem 2011;286:17281–17291. 10. Garcia-Elias A, Mrkonjic S, Pardo-Pastor C, et al. Phosphatidylinositol-4,5- 15. Gao Y, Cao E, Julius D, Cheng Y. TRPV1 structures in nanodiscs reveal mechanisms biphosphate-dependent rearrangement of TRPV4 cytosolic tails enables channel of ligand and lipid action. Nature 2016;534:347–351. activation by physiological stimuli. Proc Natl Acad Sci U S A 2013;110: 16. Cho TJ, Matsumoto K, Fano V, et al. TRPV4-pathy manifesting both skeletal dysplasia and 9553–9558. peripheral neuropathy: a report of three patients. Am J Med Genet A 2012;158A:795–802. 11. Goretzki B, Glogowski NA, Diehl E, et al. Structural Basis of TRPV4 N Ter- 17. Inada H, Procko E, Sotomayor M, Gaudet R. Structural and biochemical con- minus Interaction with Syndapin/PACSIN1-3 and PIP2. Structure 2018;26: sequences of disease-causing mutations in the ankyrin repeat domain of the human 1583–1593.e5. TRPV4 channel. Biochemistry 2012;51:6195–6206. 12. Cuajungco MP, Grimm C, Oshima K, et al. PACSINs bind to the TRPV4 cation 18. Thibodeau ML, Peters CH, Townsend KN, et al. Compound heterozygous TRPV4 channel. PACSIN 3 modulates the subcellular localization of TRPV4. J Biol Chem mutations in two siblings with a complex phenotype including severe intellectual 2006;281:18753–18762. disability and neuropathy. Am J Med Genet A 2017;173:3087–3092. 13. D’hoedt D, Owsianik G, Prenen J, et al. Stimulus-speciﬁc modulation of the cation 19. Fiorillo C, Moro F, Brisca G, et al. TRPV4 mutations in children with congenital distal channel TRPV4 by PACSIN 3. J Biol Chem 2008;283:6272–6280. spinal muscular atrophy. Neurogenetics 2012;13:195–203.

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 9 ARTICLE OPEN ACCESS Clinical, genetic, and pathologic characterization of FKRP Mexican founder mutation c.1387A>G

Angela J. Lee, BA,* Karra A. Jones, MD, PhD,* Russell J. Butterfield, MD, PhD, Mary O. Cox, BS, Correspondence Chamindra G. Konersman, MD, Carla Grosmann, MD, Jose E. Abdenur, MD, Monica Boyer, NP, Dr. Jones [email protected] Brent Beson, MD, Ching Wang, MD, James J. Dowling, MD, PhD, Melissa A. Gibbons, MS, Alison Ballard, NP, Joanne S. Janas, MD, Robert T. Leshner, MD, Sandra Donkervoort, MS, CGC, Carsten G. Bonnemann,¨ MD, Denise M. Malicki, MD, PhD, Robert B. Weiss, PhD, Steven A. Moore, MD, PhD, and Katherine D. Mathews, MD

Neurol Genet 2019;5:e315. doi:10.1212/NXG.0000000000000315 Abstract Objective To characterize the clinical phenotype, genetic origin, and muscle pathology of patients with the FKRP c.1387A>G mutation.

Methods Standardized clinical data were collected for all patients known to the authors with c.1387A>G mutations in FKRP. Muscle biopsies were reviewed and used for histopathology, immunostaining, Western blotting, and DNA extraction. Genetic analysis was performed on extracted DNA.

Results We report the clinical phenotypes of 6 patients homozygous for the c.1387A>G mutation in FKRP. Onset of symptoms was <2 years, and 5 of the 6 patients never learned to walk. Brain MRIs were normal. Cognition was normal to mildly impaired. Microarray analysis of 5 homozygous FKRP c.1387A>G patients revealed a 500-kb region of shared homozygosity at 19q13.32, including FKRP. All 4 muscle biopsies available for review showed end-stage dystrophic pathology, near absence of glycosylated α-dystroglycan (α-DG) by immunoﬂuores- cence, and reduced molecular weight of α-DG compared with controls and patients with homozygous FKRP c.826C>A limb-girdle muscular dystrophy.

Conclusions The clinical features and muscle pathology in these newly reported patients homozygous for FKRP c.1387A>G conﬁrm that this mutation causes congenital muscular dystrophy. The clinical severity might be explained by the greater reduction in α-DG glycosylation compared with that seen with the c.826C>A mutation. The shared region of homozygosity at 19q13.32 indicates that FKRP c.1387A>G is a founder mutation with an estimated age of 60 generations (;1,200–1,500 years).

*These authors contributed equally to the manuscript.

From the University of Iowa (A.J.L.), Carver College of Medicine; Department of Pathology (K.A.J., M.O.C., S.A.M.), University of Iowa; Departments of Pediatrics and Neurology (R.J.B.), University of Utah; Department of Neurology (C.G.K.), University of California San Diego; Department of Neurology (C.G.), Gillette Children’s Specialty Healthcare; Division of Metabolic Disorders (J.E.A., M.B.), CHOC Children’s; Department of Neurology (B.B.), Integris Southwest Medical Center; Departments of Pediatrics and Neurology (C.W.), Driscoll Children’s Hospital; Departments of Paediatrics and Molecular Genetics (J.J.D.), Hospital for Sick Children, University of Toronto; Departments of Pediatrics and Neurology (M.A.G., J.S.J.), University of Colorado; Department of Physical Medicine and Rehabilitation (A.B.), University of Colorado; Department of Neurosciences (R.T.L.), University of California San Diego; National Institutes of Health (S.D., C.G.B.), Institute of Neurological Disorders and Stroke; Department of Pathology (D.M.M.), University of California San Diego; Department of Human Genetics (R.B.W.), University of Utah; and Departments of Pediatrics and Neurology (K.D.M.), University of Iowa.

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

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary CMD = congenital muscular dystrophy; DSHB = Developmental Studies Hybridoma Bank; EF = ejection fraction; H&E = hematoxylin and eosin; HRC = Haplotype Reference Consortium; IF = immunoﬂuorescence; IRB = institutional review board; LGMD2I = limb-girdle muscular dystrophy type 2I; SNP = single nucleotide polymorphism; WGA = wheat germ agglutinin; α-DG = α-dystroglycan; β-DG = β-dystroglycan.

Dystroglycanopathies are muscular dystrophies resulting using a standardized data collection form, and the deidentified from hypoglycosylation of α-dystroglycan (α-DG), a protein information was collated centrally. – in the dystrophin-glycoprotein complex.1 3 More than 17 genes are required for proper α-DG functional glycosylation; Genotype and haplotype analysis FKRP is one of the most commonly mutated genes.4 It was The FKRP mutations were identified or confirmed through recently shown that FKRP functions as a ribitol 5-phosphate clinical testing in Clinical Laboratory Improvement transferase.5 Amendments-certified laboratories. Identification of the FKRP mutation for patient 4 was initially done through whole-exome FKRP mutations result in highly variable phenotypes, ranging sequencing using Broad dual-barcoded library construction from severe congenital muscular dystrophy (CMD) to mild followed by the Illumina Rapid Capture Exome enrichment kit limb-girdle muscular dystrophy type 2I (LGMD2I).6,7 The with 38 Mb target territory (29 Mb baited). most common founder mutation (c.826C>A, p.Leu276Ile) is associated with an LGMD2I phenotype.8,9 Muscle biopsies Genome-wide single nucleotide polymorphism (SNP) gen- from patients with LGMD2I show mild to moderate dystro- otyping was performed on genomic DNA using Illumina phic changes and highly variable partial reduction in immu- Human Infinium Omni2.5Exome-8 v1.3 BeadChips. Samples nostaining for glycosylated α-DG.10 were processed on an Illumina iScan system using standard Illumina protocols, and genotypes were called with Illumina In 2007, a novel homozygous FKRP mutation (c.1387A>G, GenomeStudio software. Genotypes were cleaned using 13 p.Asn463Asp) was identified in 2 Mexican American girls; the PLINK 1.9 software, yielding an average genotyping rate of authors suggested a possible founder mutation.11 The 2 99.8% and between 2,594,691 to 2,602,822 genotypes per patients had hypotonia at birth and never achieved the ability sample. Phased haplotypes for chromosome 19 were com- to stand or walk. Both girls had a marked reduction in gly- puted from unphased genotypes using the Eagle2 software cosylated α-DG and decreased laminin α2 (merosin) immu- and the Haplotype Reference Consortium (HRC r.1.1) ref- nostaining.11 A third Mexican patient homozygous for FKRP erence panel of human haplotypes, executed on the Michigan 14 c.1387A>G had a slightly milder clinical course with in- Imputation Server. The age of the founder mutation was dependent ambulation between 14 and 24 months of age12; estimated using the Gamma method assuming a correlated no muscle biopsy immunostaining was reported. “tree-like” genealogy applied to the genetic length of the ancestral segment lengths surrounding the FKRP mutation.15 We have identified 6 additional patients homozygous for the Ancestry inference combined patient genotypes with 1000 FKRP c.1387A>G variant and 3 compound heterozygous Genomes Project Illumina Omni2.5 genotypes merged from patients with the FRKP c.1387A>G and the FKRP c.826C>A ALL.chip.omni_broad_sanger_combined.20140818.snps.ge- mutations. Here, we describe the clinical and pathologic fea- notypes.vcf.gz (ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/ tures of these cases and provide genetic evidence that 20130502/supporting/hd_genotype_chip). The merged ge- c.1387A>G is a founder mutation originating in pre- notype set was pruned for linked SNPs using PLINK indep- Columbian central Mexico. pairwise functionality with the arguments 1000 50 0.2, keeping 135,080 unlinked SNPs for subsequent ancestry analysis using the program ADMIXTURE with K = 3 ancestral populations.16 Methods Muscle biopsy evaluation Patient data collection All available muscle biopsies from these patients were Standardized clinical data were collected for all patients reviewed and re-evaluated (K.A.J. and S.A.M.). Frozen sec- known to the authors with a c.1387A>G mutation in FKRP. tions of skeletal muscle were evaluated at The University of Patients were identified through diagnostic testing in the Iowa using standard hematoxylin and eosin (H&E) staining Department of Pathology at The University of Iowa, personal and immunofluorescence (IF). Immunostaining was per- communications, or through patient participation in the Iowa formed using the following antibodies: dystrophin, carboxy Wellstone Center dystroglycanopathy natural history study terminus (rabbit polyclonal ab15277; Abcam, Cambridge, (clinical trials identifier NCT00313677). The clinical teams UK); α-DG (clone IIH6; Developmental Studies Hybridoma involved in the patients’ care abstracted the clinical data, in- Bank (DSHB), The University of Iowa), β-DG (clone 7D11; cluding results from genetic testing, from the medical records DSHB), and merosin (laminin α2) (clone 5H2; Millipore

2 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Sigma, Massachusetts, US). Secondary antibodies used milestones) before age 1 year. All patients learned to sit, but included goat anti-rabbit immunoglobulin G (IgG), goat anti- most (5 of the 6) patients never walked independently. Many mouse IgM, or goat anti-mouse IgG all labeled with Alexa- started using a wheelchair by age 1–2 years. One patient Fluor488 (Life Technologies, Carlsbad, CA). Immunostains walked at 2.5 years but required a wheelchair fulltime at age 8 were analyzed in a blinded manner by standard fluorescence years. Cognition is normal to mildly impaired; brain imaging microscopy. The intensity of staining with each antibody was (MRI or CT) and vision are normal. Most patients are cur- graded from zero (absent) to 3+ (normal expression). Con- rently speaking in sentences. Creatine kinase levels were >10× trol human skeletal muscle was included with research patient normal (average 11,695 IU/L). All 3 of the patients who material on each glass slide immunostained in the study. underwent echocardiogram had normal ejection fractions (EFs) at ages 3, 9, and 19 years. Western blotting Pooled cryosections cut from selected muscle biopsies were The average current age of the 3 patients with compound used for Western blotting at The University of Iowa. Wheat heterozygous mutations in FKRP (c.1387A>G and germ agglutinin (WGA) glycoprotein preparations were c.826C>A) is 19.3 years (range 7–29 years). They met initial performed and samples run on 3%–15% gradient gels as developmental milestones on time (sitting, walking, and 17,18 previously described. Antibodies used for blotting in- talking) when details were known. They presented with hy- cluded IIH6 (gift from Kevin P. Campbell, The University of potonia, difficulty with stairs, and muscle hypertrophy in Iowa) and AF6868 (R&D Systems, Minneapolis, MN). Blots childhood from age <2 years to <10 years. The youngest were imaged on an Odyssey infrared fluorescence imaging patient (7 years) is still ambulatory. The other 2 patients system (Li-Cor Biosciences, Lincoln, NE). became wheelchair dependent at ages 12 and 16 years. Cog- nition is normal. Ejection fraction on echocardiogram was Standard protocol approvals and normal for the youngest patient (EF 59% at age 7 years) but patient consents was decreased for the 2 other patients (EFs 44% at age 22 The University of Iowa institutional review board approved years and 35–40% at age 21 years). this study (IRB# 201703860). Initial sequencing for patient 4 was approved by the NIH/NINDS Institutional Review Genetic analysis Board (IRB# 12-N-0095). Informed consent was obtained All homozygous FKRP c.1387A>G patients report Hispanic from all participants who had muscle biopsy tissue stored in ethnicity. Two compound heterozygous FKRP c.1387A>G/ the Iowa Wellstone Center Tissue Repository. Letters of c.826C>A patients (siblings) reported Hispanic ethnicity, agreement were obtained from all collaborating clinicians. with the mother carrying FKRP c.1387A>G. The other patient with the FKRP c.1387A>G/c.826C>A genotype repor- Data availability ted a father with Hispanic ethnicity. All 3 homozygous FKRP Study data for the primary analyses presented in this manu- c.1387A>G cases in the literature also reported Hispanic script are available upon reasonable request from the corre- ethnicity. Reported family origins of current and published sponding and senior author. cases localize to central Mexico (figure 1A). Genome-wide SNPs from 5 unrelated homozygous c.1387A>G patients Results (patients 0, 3, 4, 5, and 6) and 1 compound heterozygous c.1387A>G/c.826C>A patient (patient 9) were compared Clinical with 1000 Genomes Project populations with varying degrees Clinical data were collected on 6 patients from 5 families of continental Native American, European, and African ad- (patients 1–6) who are homozygous for the c.1387A>G mixture. The genomic ancestry of FKRP c.1387A>G patients FKRP mutation and 3 patients from 2 families with compound showed largely Native American fractions (37%–74%) fol- heterozygous FKRP mutations, c.1387A>G and c.826C>A lowed by European (22%–53%), consistent with cosmopoli- (patients 7–9). Genotypes and clinical data are summarized in tan Mexican ancestry (figure 1B). Patient 9, with c.1387A>G/ table 1. Homozygous c.1387A>G mutations were found in c.826C>A genotype, had a European ancestry fraction of 72%, a seventh patient (patient 0) through clinical testing in The consistent with 1 Hispanic parent. Three additional homo- University of Iowa’s Molecular Pathology Laboratory, but we zygous FKRP c.826C>A patients showed predominantly were unable to obtain clinical information. This seventh ho- European ancestry (patients A, B, and C, figure 1B). Fine- mozygous c.1387A>G patient was only included in the ge- scale heterozygosity analysis surrounding the FKRP locus on netic analysis. chromosome 19 revealed a ;500-kb region of shared homozygosity between the FKRP c.1387A>G patients, and the The average current age of the homozygous patients is 9.3 decay of haplotype sharing (figure 1C) indicated that years (range 4–19 years). All are of Hispanic ethnicity; some c.1387A>G is a founder mutation. In the 3 homozygous individuals reported a history of family members emigrating FKRP c.826C>A patients of European ancestry, a smaller from central Mexico (figure 1A). Three of the 6 patients are ;150-kb region of shared homozygosity confirmed that male. All patients for whom details of early course are available c.826C>A is also a founder mutation. Phased haplotypes from had onset of symptoms (hypotonia and delayed motor the compound heterozygous FKRP patient (c.1387A>G/

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 3 Table 1 Summary of clinical data

Patient 1 2a 3a 456 7b 8b 9

Allele 2c c.1387A>G c.1387A>G c.1387A>G c.1387A>G c.1387A>G c.1387A>G c.826C>A c.826C>A c.826C>A

Age (y)/sex 4/M 4/F 6/M 9/M 14/F 19/F 7/F 22/M 29/M

Ethnicity HH H HHHHHH/C

Consanguinity YN N YUNNNN

Age at onset <2 y 9 mo 4 mo Birth 8 mo <2 y <2 y <2 y 2–10 y

First sitting 5 mo <1 y Normal age 6 y 1 y 8 mo Normal Normal <1 y age age

First walking NA NA NA NA NA 2.5 y 1 y 1 y 9 mo

First words 18–21 mo 1 y Normal age 1 y Normal 18 mo Normal 1y 1y age age

Cognitive Mild Normal Mild Normal Normal Mild Normal Normal Normal function impairment impairment impairment

Age FT NA 1 y 2–3 y 1 y NA 8 y NA 12 y 16 y wheelchair

Respiratory None None Nocturnal NIV Cough None Trach/vent at None None NIV at 24 support at 5 y assist 14 y y

CK 2,800e 26,810 (RR: 22,170 (RR: 2,657 (RR: U 4,038 (RR: 14,451e UU 20–200) 20–200) 4–87) 28–170)

Brain imaging Normal CT Normal MRI Normal MRI Normal U Normal MRI U Normal U MRI MRI

EFd (age) 64% (3 y) U U 64% (9 y) U 61% (19 y) 59% (7 y) 44% (22 35–40% y) (21 y)

Muscle bx age NA NA 2 y 2 y 10 mo 4 y NA NA 9 y

Abbreviations: Bx = biopsy; C = Caucasian; CK = creatine kinase; EF = ejection fraction; FT wheelchair = full-time wheelchair use; H = Hispanic; NA = not applicable; NIV = noninvasive ventilation; normal age = specific age is not known but considered within a normal range; U = unknown. a Patients 2 and 3 are siblings. b Patients 7 and 8 are siblings. c Allele 1 for all patients is c.1387A>G. d EF measured by echocardiogram. e CK reference range unknown. RR: CK reference range in U/L.

c.826C>A) revealed a compound diplotype of the 2 founder Muscle biopsies from the patient homozygous for c.826C>A mutations (figure 1D) and confirmed that these 2 founder and the patient compound heterozygous for c.1387A>G and mutations occurred on different ancestral chromosomes. The c.826C>A (patient 9) showed similar mild to moderate dys- range of physical lengths of the c.1387A>G ancestral seg- trophic changes on H&E (figure 2, A and B, respectively). ments were 0.98 Mb in patient 3, 1.38 Mb in patient 5, 1.52 These included increased fiber size variation with scattered Mb in patient 6, 2.32 Mb in patient 0, and 4.48 Mb in patient atrophic and hypertrophic fibers, necrotic fibers undergoing 4. The estimated age of the c.1387A>G founder mutation was myophagocytosis, and grouped regeneration. In contrast, 59.9 generations (95% confidence interval 10.8–123.5), muscle biopsies from 4 patients homozygous for c.1387A>G which is ;1,200–1,500 years old, assuming 20- to 25-year (patients 3–6) showed severe dystrophic pathology on H&E average generation spans. staining including marked endomysial fibrosis and fatty replacement, large variation in fiber size with atrophic and very Muscle biopsy histopathology large hypertrophic fibers, conspicuous myonecrosis/ Muscle biopsies from patients with FKRP c.1387A>G muta- myophagocytosis, and grouped regeneration (figure 2, C tions (4 homozygous and 1 compound heterozygous and D). Some biopsies could be classified as “end stage” be- c.1387A>G/c.826C>A) were reviewed and compared with the cause of the extensive loss of muscle fibers. muscle biopsy from a patient homozygous for the FKRP c.826C>A common founder mutation (patient D; biopsy at age Immunostaining 25 years). The average age at muscle biopsy for homozygous IF staining was evaluated centrally in a blinded manner (K.A.J. FKRP c.1387A>G patients was 2.2 years. The compound and S.A.M.). Table 2 outlines IF staining quantification heterozygous patient had a muscle biopsy at age 9 years. results. The 4 patients homozygous for c.1387A>G (patients

4 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Figure 1 Comparative ancestry and FKRP haplotype sharing

(A) Map of reported family origins of patients homozygous for FKRP c.1387A>G. Blue markers represent patients 2, 3 (siblings), and 6, pink markers represent patient 9’s distant grandparents, and purple markers represent 3 previously reported homozygous FKRP c.1387A>G cases. (B) Global ancestry proportions estimated with ADMIXTURE (K = 3) for FKRP patients 0, 3, 4, 5, 6, 9, A, B, and C, compared with 1000 Genomes Project samples from unrelated Native Americans (MXL, 34 samples; PEL 20 samples; CLM 20 samples), Europeans (IBS, 20 samples; CEU 20 samples), and African Americans (ASW, 20 samples). Continental ancestry fraction is shown as Native American (red), European (orange), and African (blue). (C) Heterozygosity for 701 SNPs from chr19:46,664,561-47,933,257 (hg19), with shared homozygous regions for c.1387A>G highlighted in blue and c.826C>A in green. (D) Phased haplotypes from patient 9 (heterozygous c.1387A>G/c.826C>A), patient 5 (c.1387A>G), and patient B (c.826C>A) with red/gray indicating the allele at each SNP position and the minimally shared homozygous regions highlighted in blue/green. SNP = single nucleotide polymorphism.

3–6) all showed largely decreased to absent glycosylated binding. The smaller molecular weight of α-DG in the 2 α-DG positivity (0–1+) with the IIH6 antibody, and mild patients homozygous for FKRP c.1387A>G suggests that this variable decreases in β-DG, dystrophin, and merosin. The mutation results in a greater degree of α-DG hypoglycosylation. patient heterozygous for c.1387A>G/c.826C>A (patient 9) and a patient homozygous for c.826C>A (patient D) both showed a much more variable pattern of α-DG glycosylation Discussion loss with some fibers retaining a normal staining intensity We present the clinical features, genetic analysis, and muscle (0–3+). These biopsies also showed mild variable decreases in pathology of 6 individuals from 5 unrelated families who are β-DG, dystrophin, and merosin. Representative images of IF homozygous for FKRP c.1387A>G and 3 compound het- staining are shown in figure 3 with normal control (figure 3, erozygous patients from 2 unrelated families for FKRP A–D), homozygous c.826C>A (patient D; figure 3, E–H), c.1387A>G and c.826C>A. Our results expand on the phe- and 2 of the homozygous 1387A>G patients (patients 3 and notype of the 3 previously reported cases with this founder 4; figure 3, I–L and M–P, respectively). mutation. Five of our cases presented with a typical CMD phenotype and never walked independently, consistent with Western blotting previous reports.6,11,12 The remaining patient presented be- Gradient gel separation of WGA preparations derived from fore age 2 years with delayed acquisition of motor skills, and frozen muscle biopsies showed that each patient with FKRP although the patient acquired independent walking, this was mutations has reduced molecular weight α-DG (figure 4). lost by age 8 years. None of our patients had overt abnor- These same patients have lost α-DG functional glycosylation malities in eye or brain development, reported in some cases – as demonstrated by the absence or near absence of IIH6 of FKRP-related CMD with different mutations.19 22 Based

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 5 Figure 2 Histopathology

(A) Representative image of muscle biopsy from a patient homozygous for the European common mutation in FKRP c.826C>A (patient D) showing mild to moderate dystrophic changes. (B) Repre- sentative image of muscle biopsy from patient 9 (heterozygous for c.1387A>G and c.826C>A) showing similar changes to the biopsy in part A. (C and D) Representative images from patients 3 and 4 (both homozygous for c.1387A>G) showing a very severe dystrophic, nearly end-stage histopathology. Scale bar = 100 μm, equivalent for all photomicrographs.

on all known cases to date, FKRP c.1387A>G mutations do the 3 compound heterozygous patients had decreased EF on not appear to be associated with the more severe muscle-eye- echocardiogram, ages 21 and 22 years at the time of evalua- brain phenotype. tion. The probability of cardiomyopathy in patients with FKRP mutations increases with age, most commonly occur- – In contrast, the patients with compound heterozygous ring in adulthood,23 25 and therefore, patients should be ap- mutations (c.1387A>G/c.826C>A) had a milder and more propriately screened. slowly progressive course; all achieved walking at the expected age of 1 year. This is consistent with what is reported in other Three of the 6 patients homozygous for the FKRP c.1387A>G patients who have compound heterozygous mutations with 1 mutation use some sort of respiratory support in our series, c.826C>A allele.8 despite the young average age of the cohort, and a single patient with compound heterozygous mutations started using Cardiac function may be impaired in both CMD and noninvasive ventilation at age 24 years. These observations – LGMD2I because of FKRP mutations.23 25 None of the emphasize the importance of monitoring respiratory status in patients homozygous for FKRP c.1387A>G who underwent these patients. testing in our series have cardiomyopathy, but they were young at the last echocardiogram (ages 3, 9, and 19 years) and Microarray analysis conﬁrms that the FKRP c.1387A>G allele may develop abnormal cardiac function later in life. Two of is part of a ;500-kb shared homozygous segment on

Table 2 Immunofluorescence staining quantification

Patient number 3 4569 Da

Genotype c.1387A>G c.1387A>G c.1387A>G c.1387A>G c.1387A>G/c.826C>A c.826C>A

α-DG (IIH6) 0–1+ 0–1+ 0–1+ 0–1+ 0–3+ 0–3+

β-DG 1–3+ 2–3+ 3+ 2 2–3+ 3+

Dystrophin 2–3+ 3+ 2–3+ 3+ 2–3+ 2–3+

Merosin 1–3+ 2–3+ 3+ 3+ 2–3+ 3+

Abbreviation: α-DG = α-dystroglycan. In this scoring system, zero = absent and 3+ = normal. a Patient D is representative of a homozygous c.826C>A, LGMD2I patient. The biopsy is included for comparative purposes.

6 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Figure 3 Immunofluorescence staining

(A–D) Normal control muscle staining patterns for each of the antibodies. (E–H) Representative images from patient D homozygous for the European common mutation (c.826C>A). (I–L) Representative images from patient 3 homozygous for c.1387A>G. (M–P) Representative images from patient 4 homozygous for c.1387A>G. Scale bar = 50 μm, equivalent for all photomicrographs. chromosome 19. The size of the homozygous interval and also showed variable, slightly reduced expression of merosin, haplotype analysis indicate that the allele originated from which is comparable to findings in 2 previously reported a common ancestor approximately 60 generations ago. homozygous FKRP c.1387A>G cases.11 By Western blotting, Compared with the smaller ;150-kb homozygous segment α-DG glycosylation was reduced to a greater degree in associated with the c.826C>A allele of European origin, the patients homozygous for FKRP c.1387A>G compared with c.1387A>G allele has a more recent origin, but still likely patients with either compound heterozygous mutations predates European settlement in the Americas. Family history c.1387A>G/c.826C>A or homozygous FKRP c.826C>A information for the cases reported here (and previously11,12) mutations. It has been previously suggested that there is suggests an origin in central Mexico (figure 1A). a relationship between the level of α-DG glycosylation and clinical phenotype in patients with FKRP mutations32; how- The common European founder mutation (c.826C>A, ever, this relationship could not be confirmed in other – p.Leu276Ile) is found in many European populations, and the studies.33 35 Our finding of further decreased α-DG glyco- mutation is thought to have occurred once in a common sylation in those homozygous for c.1387A>G compared with ancestor.6,7,26,27 FKRP c.826C>A has an increased prevalence those with milder phenotypes supports the interpretation that in Scandinavian countries,28 leading to the speculation that the degree of α-DG hypoglycosylation is relevant for the se- the founder mutation occurred in the Scandinavian pop- verity of the phenotype. ulation. Other founder mutations have also been reported as – summarized in table 3.29 31 The characterization of the phenotype associated with homozygous FKRP c.1387A>G mutations provided in this re- Muscle biopsies of patients homozygous for FKRP port adds to those previously described and will aid in the c.1387A>G showed severe dystrophic histopathology, and diagnosis and counseling regarding prognosis of patients with immunostaining showed greatly decreased to absent fully this rare mutation. Genetic analysis indicates that this muta- glycosylated α-DG, consistent with other dystroglycano- tion is of Mexican origin, and further genetic analysis of the pathies manifesting a CMD phenotype.6 The muscle biopsies specific population of origin may be of interest. In addition,

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 7 Figure 4 Western blotting

The antipeptide antibody (AF6868) shows greatly reduced molecular weight for α-DG in each of the patients with FKRP mutations. Fully glycosylated control (C) α-DG is >150 kd, whereas the α-DG from homozygous c.1387A>G patients (3 and 5) ranges from ;65–90 kd, and the α-DG from homozygous c.826C>A (D) or compound heterozygous c.1387A>G/c.826C>A (9) patients ranges from ;75–90 kd. The smaller molecular weight α-DG observed in homozygous c.1387A>G patients suggests a greater degree of hypoglycosylation than that of c.826C>A patients. Each patient with FKRP mutations has lost functional glycosylation and no longer binds the anti-glycoepitope antibody (IIH6). The AF6868 antibody binds to epitopes on both α-DG and β-DG. The β-DG bands show the relative amounts of protein loaded in each lane. Lanes were loaded equivalently in both gels. These images are representative of blots performed 3 or 4 times for each patient sample.

our findings are consistent with the idea that the more severe Boyer, B. Beson, C. Wang, J.J. Dowling, M.A. Gibbons, and A. clinical phenotype associated the c.1387A>G mutation is Ballard: acquisition of data and revision of the manuscript. J.S. explained, in part, by the greater reduction of α-DG glyco- Janas, R.T. Leshner, S. Donkervoort, C.G. Bönnemann, and sylation relative to other genotypes examined; however, D.M. Malicki: acquisition of data and revision of the manu- clarity on this issue requires additional study. script. R.B. Weiss: study design, analysis/interpretation of data, and drafting/revision of the manuscript. S.A. Moore: study Author contributions concept/design, analysis/interpretation of data, and revision of A.J. Lee and K.A. Jones: study concept/design, analysis/ the manuscript. K.D. Mathews: study concept/design, interpretation of data, and drafting/revision of the manuscript. analysis/interpretation of data, and revision of the manuscript. R.J. Butterfield: study design, acquisition/analysis/ interpretation of data, and revision of the manuscript. M.O. Acknowledgment Cox: analysis/interpretation of data and revision of the man- The authors acknowledge The University of Iowa uscript. C.G. Konersman, C. Grosmann, J.E. Abdenur, M. histotechnologist Terese Nelson for performing the

Table 3 Founder mutations in FKRP

Mutation Protein change Population Phenotype Author (year)

c.826C>A p.Leu276Ile Europeana LGMD2I Frosk et al. (2005)26

c.1364C>A p.Ala455Asp Tunisian CMD with brain involvement Louhichi et al. (2004)30

c.545A>G p.Tyr182Cys Chinese Asymptomatic Fu et al. (2016)31

c.1100C>T p.Ile367Thr South African Afrikaner LGMD2I Mudau et al. (2016)29

c.1387A>G p.Asn463Asp Mexican CMD without brain involvement

Abbreviations: CMD = congenital muscular dystrophy; LGMD2I = limb-girdle muscular dystrophy type 2I. Reported founder mutations in FKRP. Phenotypes described are for patients homozygous for these mutations. a Genetic analysis was performed in the Hutterites, and a shared region was also identified in samples of other European populations. Further research showed the highest prevalence of FKRP c.826C>A in Scandinavian populations.28

8 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG histology and immunofluorescence staining. Initial se- References quencing and analysis for patient 4 was provided by the 1. Ibraghimov-Beskrovnaya O, Ervasti JM, Leveille CJ, Slaughter CA, Sernett SW, Campbell KP. Primary structure of dystrophin-associated glycoproteins linking dys- Broad Institute of MIT and Harvard Center for Mendelian trophin to the extracellular matrix. Nature 1992;355:696–702. Genomics (Broad CMG) and was funded by the National 2. Ervasti JM, Campbell KP. A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J Cell Biol 1993;122:809–823. Human Genome Research Institute, the National Eye 3. Henry MD, Campbell KP. Dystroglycan inside and out. Curr Opin Cell Biol 1999;11: Institute, and the National Heart, Lung and Blood 602–607. 4. Cohn RD. Dystroglycan: important player in skeletal muscle and beyond. Neuro- Institute grant UM1 HG008900 to Daniel MacArthur muscul Disord 2005;15:207–217. and Heidi Rehm. 5. Kanagawa M, Kobayashi K, Tajiri M, et al. Identification of a post-translational modification with ribitol-phosphate and its defect in muscular dystrophy. Cell Rep 2016;14:2209–2223. Study funding 6. Brockington M, Blake DJ, Prandini P, et al. Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin This research was funded by a Paul D. Wellstone Muscular alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan. Am J Hum Dystrophy Cooperative Research Center grant (NIH U54 Genet 2001;69:1198–1209. 7. Brockington M, Yuva Y, Prandini P, et al. Mutations in the fukutin-related NS053672). M.B. is partially supported by a grant from the protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder Fry Family Foundation (16084001). allelic variant of congenital muscular dystrophy MDC1C. Hum Mol Genet 2001; 10:2851–2859. 8. Stensland E, Lindal S, Jonsrud C, et al. Prevalence, mutation spectrum and phenotypic Disclosure variability in Norwegian patients with Limb Girdle Muscular Dystrophy 2I. Neuro- A.J. Lee received funding from the Iowa Wellstone Muscular muscul Disord 2011;21:41–46. 9. Kang PB, Feener CA, Estrella E, et al. LGMD2I in a North American population. Dystrophy Cooperative Research Center, U54, NS053672. BMC Musculoskelet Disord 2007;8:115. K.A. Jones reports no disclosures. R.J. Butterfield is supported 10. Yamamoto LU, Velloso FJ, Lima BL, et al. Muscle protein alterations in LGMD2I patients with different mutations in the Fukutin-related protein gene. J Histochem by NIH grant 1K08NS097631-01. He is receiving funding via Cytochem 2008;56:995–1001. contracts for clinical trials from PTC Therapeutics, Sarepta 11. MacLeod H, Pytel P, Wollmann R, et al. A novel FKRP mutation in congenital fi muscular dystrophy disrupts the dystrophin glycoprotein complex. Neuromuscul Therapeutics, P zer, Marathon, Biogen, Summit Therapeu- Disord 2007;17:285–289. tics, Santhera Pharmaceuticals, and aTyr Pharmaceuticals. He 12. Navarro-Cobos MJ, Gonzalez-Del Angel A, Estandia-Ortega B, et al. Molecular fi analysis confirms that FKRP-related Disorders are underdiagnosed in Mexican serves on the scienti c advisory boards of Sarepta Thera- patients with neuromuscular diseases. Neuropediatrics 2017;48:442–450. peutics, Biogen, PTC Therapeutics, and Wave Life Sciences. 13. Chang CC, Chow CC, Tellier LC, Vattikuti S, Purcell SM, Lee JJ. Second-generation PLINK: rising to the challenge of larger and richer datasets. Gigascience 2015;4:7. M.O. Cox received funding from the Iowa Wellstone Mus- 14. Das S, Forer L, Schönherr S, et al. Next-generation genotype imputation service and cular Dystrophy Cooperative Research Center, U54, methods. Nat Genet 2016;48:1284–1287. 15. Gandolfo LC, Bahlo M, Speed TP. Dating rare mutations from small samples with NS053672. C.G. Konersman previously served as a consultant dense marker data. Genetics 2014;197:1315–1327. of Sarepta Therapeutics for their medication, ExonDys51. C. 16. Alexander DH, Novembre J, Lange K. Fast model-based estimation of ancestry in unrelated individuals. Genome Res 2009;19:1655–1664. Grosmann sits on an advisory board of Sarepta Therapeutics 17. Michele DE, Barresi R, Kanagawa M, et al. Post-translational disruption of and receives funding from Sarepta and Italfarmaco through dystroglycan-ligand interactions in congenital muscular dystrophies. Nature 2002; clinical trial contracts. J.E. Abdenur and M. Boyer report no 418:417–422. 18. Willer T, Lee H, Lommel M, et al. ISPD loss-of-function mutations disrupt dystro- disclosures. B. Beson receives MDA grant money for his glycan O-mannosylation and cause Walker-Warburg syndrome. Nat Genet 2012;44: neuromuscular clinic. C. Wang reports no disclosures. J.J. 575–580. 19. Quijano-Roy S, Marti-Carrera I, Makri S, et al. Brain MRI abnormalities in muscular Dowling is a scientific advisory board member of the RYR1 dystrophy due to FKRP mutations. Brain Dev 2006;28:232–242. Foundation, the Muscular Dystrophy Association, and 20. Beltran-Valero de Bernabe D, Voit T, Longman C, et al. Mutations in the FKRP gene can cause muscle-eye-brain disease and Walker-Warburg syndrome. J Med Genet Dynacure. He does occasional biomedical consulting for GLG 2004;41:e61. and Guidepoint consulting. M.A. Gibbons, A. Ballard, J.S. 21. Mercuri E, Topaloglu H, Brockington M, et al. Spectrum of brain changes in patients with congenital muscular dystrophy and FKRP gene mutations. Arch Neurol 2006;63: Janas, R.T. Leshner, S. Donkervoort, C.G. Bönnemann, and 251–257. D.M. Malicki report no disclosures. R.B. Weiss is supported 22. Van Reeuwijk J, Olderode-Berends MJ, Van den Elzen C, et al. A homozygous FKRP start codon mutation is associated with Walker-Warburg syndrome, the severe end of by NIH grant NS085238. S.A Moore has fee for service the clinical spectrum. Clin Genet 2010;78:275–281. contracts with Sarepta Therapeutics, Inc. and Flagship Bio- 23. Kefi M, Amouri R, Chabrak S, Mechmeche R, Hentati F. Variable cardiac involvement in Tunisian siblings harboring FKRP gene mutations. Neuropediatrics 2008;39: sciences. He received funding from the Iowa Wellstone 113–115. Muscular Dystrophy Cooperative Research Center, U54, 24. Margeta M, Connolly AM, Winder TL, Pestronk A, Moore SA. Cardiac pathology exceeds skeletal muscle pathology in two cases of limb-girdle muscular dystrophy type NS053672. K.D. Mathews received funding from the Iowa 2I. Muscle Nerve 2009;40:883–889. Wellstone Muscular Dystrophy Cooperative Research Cen- 25. Poppe M, Bourke J, Eagle M, et al. Cardiac and respiratory failure in limb-girdle muscular dystrophy 2I. Ann Neurol 2004;56:738–741. ter, U54, NS053672. She also receives research funding from 26. Frosk P, Greenberg CR, Tennese AA, et al. The most common mutation in FKRP the CDC (U01 DD000189) and clinical trial support related causing limb girdle muscular dystrophy type 2I (LGMD2I) may have occurred only to DMD from Sarepta, Pfizer, Santhera, Roche, FibroGen, once and is present in Hutterites and other populations. Hum Mutat 2005;25:38–44. 27. Walter MC, Petersen JA, Stucka R, et al. FKRP (826C>A) frequently causes limb- and Italfarmaco. She has served on the advisory boards of girdle muscular dystrophy in German patients. J Med Genet 2004;41:e50. Sarepta and Santhera. Full disclosure form information pro- 28. Sveen ML, Schwartz M, Vissing J. High prevalence and phenotype-genotype correlations of limb girdle muscular dystrophy type 2I in Denmark. Ann Neurol 2006;59: vided by the authors is available with the full text of this article 808–815. at Neurology.org/NG. 29. Mudau MM, Essop F, Krause A. A novel FKRP-related muscular dystrophy founder mutation in South African Afrikaner patients with a phenotype suggestive of a dys- trophinopathy. S Afr Med J 2016;107:80–82. Publication history 30. Louhichi N, Triki C, Quijano-Roy S, et al. New FKRP mutations causing congenital fi muscular dystrophy associated with mental retardation and central nervous system Received by Neurology: Genetics October 17, 2018. Accepted in nal abnormalities. Identification of a founder mutation in Tunisian families. Neuro- form January 2, 2019. genetics 2004;5:27–34.

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 9 31. Fu X, Yang H, Wei C, et al. FKRP mutations, including a founder mutation, cause homozygous for common FKRP mutation. Neuromuscul Disord 2017;27: phenotype variability in Chinese patients with dystroglycanopathies. J Hum Genet 619–626. 2016;61:1013–1020. 34. Boito CA, Fanin M, Gavassini BF, Cenacchi G, Angelini C, Pegoraro E. Biochemical 32. Brown SC, Torelli S, Brockington M, et al. Abnormalities in alpha-dystroglycan and ultrastructural evidence of endoplasmic reticulum stress in LGMD2I. Virchows expression in MDC1C and LGMD2I muscular dystrophies. Am J Pathol 2004;164: Arch 2007;451:1047–1055. 727–737. 35. Jimenez-Mallebrera C, Torelli S, Feng L, et al. A comparative study of alpha- 33. Alhamidi M, Brox V, Stensland E, Liset M, Lindal S, Nilssen O. Limb girdle dystroglycan glycosylation in dystroglycanopathies suggests that the hypo- muscular dystrophy type 2I: No correlation between clinical severity, glycosylation of alpha-dystroglycan does not consistently correlate with clinical histopathology and glycosylated alpha-dystroglycan levels in patients severity. Brain Pathol 2009;19:596–611.

10 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG ARTICLE OPEN ACCESS Loss-of-function mutations in Lysyl-tRNA synthetase cause various leukoencephalopathy phenotypes

Chong Sun, MD,* Jie Song, MD,* Yanjun Jiang, PhD,* Chongbo Zhao, MD, Jiahong Lu, MD, Yuxin Li, MD, Correspondence Yin Wang, MD, Mingshi Gao, MD, Jianying Xi, MD, Sushan Luo, MD, Meixia Li, MS, Kevin Donaldson, MS, Dr. Zhang [email protected] Stephanie N. Oprescu, BS, Thomas P. Slavin, MD, Sansan Lee, MS, Pilar L. Magoulas, MS, Andrea M. Lewis, MS, or Dr. Lin Lisa Emrick, MD, Seema R. Lalani, MD, Zhiyv Niu, PhD, Megan L. Landsverk, PhD, Magdalena Walkiewicz, PhD, [email protected] Richard E. Person, PhD, Hui Mei, PhD, Jill A. Rosenfeld, MS, Yaping Yang, PhD, Anthony Antonellis, PhD, Ya-Ming Hou, PhD, Jie Lin, MD,† and Victor W. Zhang, MD, PhD†

Neurol Genet 2019;5:e316. doi:10.1212/NXG.0000000000000316 Abstract Objective To expand the clinical spectrum of lysyl-tRNA synthetase (KARS) gene–related diseases, which so far includes Charcot-Marie-Tooth disease, congenital visual impairment and microcephaly, and nonsyndromic hearing impairment.

Methods Whole-exome sequencing was performed on index patients from 4 unrelated families with leukoencephalopathy. Candidate pathogenic variants and their cosegregation were conﬁrmed by Sanger sequencing. Eﬀects of mutations on KARS protein function were examined by aminoacylation assays and yeast complementation assays.

Results Common clinical features of the patients in this study included impaired cognitive ability, seizure, hypotonia, ataxia, and abnormal brain imaging, suggesting that the CNS involvement is the main clinical presentation. Six previously unreported and 1 known KARS mutations were identiﬁed and cosegregated in these families. Two patients are compound heterozygous for missense mutations, 1 patient is homozygous for a missense mutation, and 1 patient harbored an insertion mutation and a missense mutation. Functional and structural analyses revealed that these mutations impair aminoacylation activity of lysyl-tRNA synthetase, indicating that defective KARS function is responsible for the phenotypes in these individuals.

Conclusions Our results demonstrate that patients with loss-of-function KARS mutations can manifest CNS disorders, thus broadening the phenotypic spectrum associated with KARS-related disease.

*These authors contributed equally to the manuscript.

†These authors share senior authorship.

From the Department of Neurology (C.S., J.S., C.Z., J. Lu, J.X., S. Luo, J. Lin), Huashan Hospital, Fudan University, Shanghai, China; Baylor Genetic Laboratories (Y.J., Z.N., M.L.L., M.W., R.E.P., H.M., Y.Y.), Houston, TX; Department of Radiology (Y.L.), Huashan Hospital, Fudan University; Department of Pathology (Y.W., M.G.), Huashan Hospital, Fudan University, Shanghai, China; Department of Biochemistry and Molecular Pharmacology (M.L., K.D., Y.-M.H.), Thomas Jefferson University, Philadelphia, PA; Department of Human Genetics (S.N.O., A.A.), University of Michigan Medical School, Ann Arbor, MI; Department of Pediatrics and Department of Obstetrics and Gynecology (S.L.), University of Hawaii School of Medicine, Honolulu, HI; Department of Medical Oncology and Therapeutics Research (T.P.S.), Division of Clinical Cancer Genetics, City of Hope National Medical Center, Duarte, CA; Department of Molecular and Human Genetics (P.L.M., A.L.M., L.E., S.R.L., Z.N., M.L.L., J.A.R., M.W., R.E.P., H.M., J.A.R., Y.Y., V.W.Z.), Baylor College of Medicine, Houston, TX; and AmCare Genomics Lab (V.W.Z.), Guangzhou, China.

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary ARS = aminoacyl-tRNA synthetase; 5-FOA =5-ﬂuoroorotic acid; OXPHOS = oxidative phosphorylation system.

Aminoacyl-tRNA synthetases (ARSs) play key roles in members were also tested to evaluate the mode of inheritance charging specific tRNAs with cognate amino acids and are and disease segregation. critical for enabling protein translational fidelity and cellular integrity. Pathogenic mutations in different ARSs have been Functional studies reported in patients with a variety of clinical presentations Aminoacylation assays in vitro were performed as previously 28,34,35 including cardiomyopathy, cancer, autoimmune disorders, described. The initial rate of aminoacylation as a func- – and diabetes.1 3 Of interest, a number of mutations in genes tion of tRNA concentration was fit to a hyperbola equation, encoding ARSs have been linked to neurologic diseases, in- from which the Michaelis constant (Km) for tRNA and the cluding inherited peripheral neuropathy, sensorineural deafness, catalytic turnover (kcat) were derived. Analysis of the catalytic ffi leukodystrophies, or leukoencephalopathies, summarized in e ciency (kcat/Km) of aminoacylation was presented for each – table e-1, links.lww.com/NXG/A143.4 27 mutant enzyme.

KARS is one of the 3 bifunctional ARSs and catalyzes the Yeast complementation assays were performed using a hap- specific attachment of L-lysine to cognate tRNA molecules. loid Saccharomyces cerevisiae strain with the endogenous KRS1 28 Compound heterozygous disease–associated KARS mutations gene deleted (ΔKRS1) in both solid and liquid -Leu- were first identified in a single patient with Charcot-Marie-Tooth Ura media (Teknova, Hollister, CA) containing 0.1% disease.28 Later, other KARS mutations were also reported in 3 5-fluoroorotic acid (5-FOA) (Boeke, Trueheart et al. 1987). unrelated families with autosomal recessive nonsyndromic Each human KARS variant (GenBank accession number hearing impairment,19 in 1 patient with a suspected mitochon- AAG30114.1, NM_005548) was modeled in yeast KRS1 drial disorder,29 in 2 siblings with severe infantile visual loss and (GenBank accession number AAA66916.1) using Gateway progressive microcephaly,15 andinaboywithcombinedre- technology (Invitrogen). The human KARS residues p.L233, spiratory chain complex deficiencies (I and IV).30 However, p.E427, p.R505, p.P533, p.T587, and p.L596 correspond to dysfunctions of CNS involved in leukoencephalopathy have not the following yeast residues, respectively: p.L208, p.E403, been reported or observed as a major clinical presentation in p.R480, p.P508, p.T562, and p.L571. these affected individuals. In this study, 4 unrelated nonconsanguineous families with Results leukoencephalopathy were identified by whole-exome sequencing analysis. Functional and structural analyses revealed Patient history and clinical presentations that each variant was a loss-of-function KARS mutation. Our Patient 1 is a 26-year-old woman who developed progressive findings highlight the association of KARS mutations in neurocognitive decline at age 25 years. Her key clinical fea- patients with CNS involvement and broaden the phenotypic tures included hypotonia, mild intellectual disability, slurred spectrum associated with KARS-related disease. speech, ataxia, and abnormal movement, as well as congenital hearing loss. Two KARS variants c.1514G>A (p.R505H) and c.1597C>T (p.P533S) were identified in the compound heterozygous state. The EMG and histochemical analysis of Methods muscle biopsy did not reveal any myogenic or neurogenic Standard protocol approval, registrations, and damage. Brain MRI showed symmetric hyperintensity in bi- patient consents lateral frontal white matter, extending along the anterior limb Patient blood specimens were submitted to Baylor Genetics, of inner capsule on FLAIR and DWI (figure 1, A and B). previously Medical Genetics Laboratories at Baylor College of Magnetic resonance spectroscopy was performed with Stim- Medicine, Houston, TX, for WES-based analyses. Additional ulated Echo Acquisition Mode sequence, and data were an- patient specimens were from Fudan University Huashan alyzed with the LC Model (version 6.3). The spectroscopy Hospital, Shanghai, China. The ethical review boards of the showed distinctly reduced N-acetylaspartate and slightly ele- participating institutions approved this study. vated lactate peak in the right frontal lesion (figure 1E) compared with that in the ipsilateral normal white matter Exome sequencing in probands and (figure 1F). The brother of patient 1, who also harbored the family studies same 2 variants, had hearing loss, and his brain MRI per- Library preparation, exome capture, HiSeq next-generation formed at age 16 years showed bilateral abnormality in the sequencing, and data analyses were conducted as periventricular white matter (figure 1C). The parents were – described.31 33 Variants identified in KARS were further val- clinically unaffected and had normal MRI scans, and each was idated by Sanger sequencing in these patients. Family heterozygous for one of these 2 variants. Therefore, variants

2 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Figure 1 Representative cases for novel KARS mutation with leukoencephalopathy

Characteristics of patients for novel KARS mutations with leukoencephalopathy. (A,B) Brain imaging of Patient 1 showed bilateral FLAIR and DWI signal hyperintensity in the white matter of the frontal lobe. (C) The affacted brother of Patient 1 has also abnormality in the white matter. (D) Brain MRI of Patient 4 showed bilateral T2 signal hyperintensity in the white matter of periventricular area. (E) The MRS showed reduced NAA and elevated lactate peak in the right frontal lesion of Patient 1. (F) The MRS showed the presence of NAA and lactate in the normal white matter of Patient 1.

c.1514G>A (p.R505H) (from the mother) and c.1597C>T compound heterozygous for a novel insertion variant c.1281_ (p.P533S) (from the father) segregate with leukoencephalop- 1282insAGA (p.E427_L428insR) and a novel missense variant athy in an autosomal recessive manner in the family of patient 1. c.1786C>T (p.L596F). She was deceased after 2 years of neurologic features appeared. Patient 4 is an 11-year-old girl whose clinical presentations Patient 2 is a 35-year-old man who developed progressive include global developmental delay, hypotonia, mild in- neurocognitive decline, hypertonia, seizures, ataxia, and abnor- tellectual disability, congenital bilateral profound sensori- mal movement over 3 years. Other clinical features included neural hearing loss, history of seizure disorder, mildly elevated congenital hearing loss, likely secondary hypothyroidism and lactate, elevated CSF total protein, and developmental re- possible left eye blindness. Brain MRI revealed abnormality in gression. Family history was remarkable for a sister with bi- the white matter, and EEG was normal. Family history indicated lateral sensorineural hearing loss. Her brain MRI and CT a sister with congenital deafness and hydrocephaly and 2 second showed normal at age 1 year. However, the second MRI at age cousins with congenital deafness. He was compound heterozy- 11 years showed extensive abnormality of the deep white gous for a novel missense variant c.881T>C (p.I294T) and matter of both cerebral hemispheres with increased T2 a reported variant c.1760C>T (p.T587M). hyperintensity and involvement of the corticospinal tracts with sparing of the subcortical U fibers (figure 1D). Mild Patient 3 is a 3-year-old boy present with developmental delay cerebellar volume loss with prominence of the fourth ventricle and regression, failure to thrive, microcephaly, and progressive was also observed. The patient died at age 12 years after hypotonia. Other clinical presentations include hearing and a rapid deterioration in her neurologic status over a period of 2 vision loss, nystagmus, hyperreflexia, progressive joint con- years. Her autopsy showed severe bilateral spongiform leu- tractures, febrile seizures, dysphagia, renal tubular acidosis type kodystrophy involving the internal capsule, frontal, parietal, I, mild hydronephrosis of the left kidney, and abnormal liver and occipital white matter. There was symmetric white matter ultrasound. Head CT showed increasing periventricular and loss in the descending corticospinal tracts, brainstem, and cerebellar nuclei calcifications and cerebral atrophy. He was spinal cord. Dystrophic calcifications were noted in basal

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 3 ganglia, frontal, and parietal lobes. She harbored a homozy- by 20- and 200-fold relative to the wild-type enzyme, re- gous missense variant c.697C>G (p.L233V). Her mother was spectively (figure 3). Of interest, although p.P533S mutation found to be heterozygous for this variant. But the father’s caused a 10-fold reduction relative to p.R505H, p.R505H sample was not enough for further testing. mutation showed a more severe reduction in cellular growth in yeast complementation assays. Thus, these 2 mutations Computational evaluation of KARS variants identified in this patient affect critical amino acids and reduce Six novel and 1 known KARS variants were identified by WES tRNA charging capacity, suggesting a pathogenic role in in the index patients described here (table). All variants are causing the patient phenotype. rare, and none is found in the homozygote state in the ExAC database (table e-2, links.lww.com/NXG/A144). Sequence Patient 2 has a novel variant c.881T>C (p.I294T) and alignments of KARS proteins from bacteria to human showed a known mutation c.1760C>T (p.T587M). The Ile294 resi- that all the affected amino acids are highly conserved (figure 2F). due forms a hydrophobic interaction with Leu597 located A variety of in silico prediction programs were used to predict within the same monomer to form the dimeric interface. the possible effect of each amino acid substitution (table e-2). Replacement of Ile294 with Thr introduces an extra hydroxyl All 8 novel variants were considered as likely pathogenic, group, which may alter the nature of hydrophobic core. The based on predictions by PolyPhen-2, Sorting Intolerant p.I294T mutant showed a reduction in tRNA charging by 13- from Tolerant, MutationTaster, MutationAssessor, etc. fold relative to the wild-type enzyme (figure 3). The p.T587M (table e-2). variant, a previously reported mutation, almost completely eliminated the enzyme activity. Functional studies fi As shown in gure 3, all KARS mutations studied (p.L233V, Patient 3 has a novel c.1281_1282insAGA (p.E427_ p.I294T, p.R505H, p.P533S, p.T587M, and p.L596F) re- L428insR) insertion mutation and a novel c.1786C>T duced enzyme kinetics by at least 13-fold compared with that (p.L596F) missense mutation. Structural analysis indicated of wild-type KARS, indicating that these mutations impaired that Glu427 and Leu428 are located in a helix tightly packed KARS aminoacylation activity. against another helix consisting of residues 468–484. The insertion of a bulky amino acid Arg into the middle of the first ff To further evaluate the deleterious e ects of KARS mutations, helix will disrupt its secondary structure, affecting the enzyme yeast complementation assays were performed by modeling stability. In addition, Leu596 is embedded in the protein in- each KARS variant in the S. cerevisiae ortholog KRS1. As terior and packed tightly with adjacent hydrophobic residues. fi shown in gure 4A, yeast expressing p.E427_Lins428R and The replacement with Phe introduced a large bulky side chain p.R505H KRS1 demonstrated dramatically reduced, but not creating stereochemical clashes and causing protein instability. ablated, growth. In addition, yeast expressing p.L233V, Yeast expressing p.E427L_ins428R KRS1 showed a severe re- p.P533S, p.T587M, and p.L596F KRS1 showed a slight but duction of yeast viability compared with wild-type strain, in- fi signi cant reduction of yeast viability compared with wild- dicating that it is a loss-of-function allele, whereas the p.L596F type KRS1. These results suggested that p.L233V, p.P533S, mutation caused a slight but significant reduction in yeast via- p.T587M, p.L596F, p.R505H, and p.E427_Lins428R are bility. On the other hand, the tRNA charging activity of p.L596F hypomorphic alleles. Similar results were obtained from mutant is reduced by 571-fold, with a 2.2-fold increase in Km,but growth curve analyses in liquid media containing 5-FOA fi nearly a 240-fold reduction in kcat ( gure 3). (figure 4B). Patient 4 has a single nucleotide variation, c.697C>G Mechanisms of pathogenicity for impaired (p.L233V), and a copy number loss of KARS at this locus, function of mutated residues which were inherited from each parent respectively. The Patient 1 harbors 2 novel KARS missense variants Leu233 is located in the anticodon binding domain. This (c.1514G>A [p.R505H] and c.1597C>T [p.P533S]) in the variant reduced enzyme activity by 13-fold relative to the wild- compound heterozygous state. Close examination of the type protein and slightly decreased yeast viability in yeast crystal structure revealed that Arg505 forms a hydrogen-bond complementation studies (figures 3 and 4). Thus, the ab- network with Asp374 and Glu512 of KARS and an adjacent normal kinetic aspect of this mutation affected the catalytic water molecule.36 Replacement with the side chain of His behavior of the enzyme and may likely contribute to disease would eliminate the positively charged side chain of Arg and pathogenesis. thus impair their hydrogen-bond network (figure 2, B and C). Pro533 is located at the transition point of a helix to a β-strand of KARS protein consisting of amino acids 520–545 to form Discussion dimeric interface. The replacement with Ser would not permit the required peptide bond turn of Pro and thus would be KARS mutations have been linked to neurologic disorders expected to have a detrimental effect on the protein secondary with different clinical manifestations. Compound heterozy- structure. Consistent with the predictions from structural gous mutations p.L133H and p.Y173Sfs*7 were first identified analysis, both mutations decreased in tRNA charging activity in a patient with Charcot-Marie-Tooth disease.28 Two

Table Summary of key clinical manifestations of affected individuals

Summary of clinical presentation of affected individuals Molecular study Age Index at Hearing number onset Sex CNS Brain imaging loss EEG NCV + EMG Other systems Allele 1 Allele 2 Status

1 26 y F Hypotonia/spasticity, mild intellectual Bilateral frontal Y Normal Normal N c.1514G>A c.1597C>T Compound disability, slurred speech, ataxia, and white matter heterozygous abnormal movement

p.R505H p.P533S

2 35 y M Neurocognitive decline, spasticity, Bilateral Y Diffuse slow UN Primary hypothyroidism c.881T>C c.1760C>T Compound seizures, ataxia, and abnormal periventricular activity heterozygous movements white matter

p.I294T p.T587M

3 3 y M Failure to thrive, developmental delay, Cerebellar nuclei Y UN UN Vision loss, abnormal renal c.1281_ c.1786C>T Compound developmental regression, calcifications function, abnormal liver 1282insAGA heterozygous microcephaly, nystagmus, hypotonia, ultrasound, and progressive hypertonia/spasticity, hyperreflexia, joint contractures febrile seizures, and dysphagia

p.E427_ p.L596F L428insR

4 11 y F Developmental delay, hypotonia/ Bilateral deep Y Diffuse slow Normal N c.697C>G c.697C>G Homozygous spasticity, mild intellectual disability, white matter activity with seizures, bilateral hand tremor and spike activity ataxia, mildly elevated lactate, elevated CSF total protein, and slurred speech

p.L233V p.L233V

BAB56428 UN UN Developmental delay and dysmorphic UN UN UN Neurogenic Charcot-Marie-Tooth, self- c.398T>A c.524_ Compound features damages abusive behavior, and 525insTT heterozygous vestibular schwannoma

p.L133H p.Y173Sfs*7

109829 6 m M Hypotonia, global developmental delay, N Y N UN Increased mtDNA levels in c.683C>T c.1760C>T Compound strabismus, ophthalmoplegia, dystonia, muscle and abnormal heterozygous elevated plasma alanine, and CSF brainstem auditory-evoked lactate potentiala

p.P228L p.T587M

4338 V119 UN M N UN Y UN UN Autosomal recessive c.1129G>A c.1129G>A Homozygous nonsyndromic hearing impairment (ARNSHI)

p.D377N p.D377N

Continued 5 mutations p.D377N and p.Y173H were found in the homozygous state in patients with autosomal recessive nonsyndromic hearing impairment.19 Compound heterozygous

heterozygous heterozygous mutations p.P228L and p.T587M were identiﬁed in a patient presenting with development delay, hypotonia, and ophthalmoplegia.29 Meanwhile, patients with congenital visual impairment and progressive microcephaly were reported to

” be compound heterozygous for mutations p.R438W and p.E525K.15 Recently, compound heterozygous mutations (p.V448D and p.I318T) were identified in a boy with combined mitochondrial complex deficiencies.30 In this study, we identified Molecular study p.R438W p.E525K p.R438W p.E525K 6 novel KARS mutations in patients with leukoencephalopathy. hways bilaterally. We also found that the previously reported mutation p.T587M is associated with leukoencephalopathy. Each mutation exerts a loss-of-function effect in at least 1 of the following assays: aminoacylation assay and yeast complementation assay, suggesting that defective KARS charging function is an important component of leukoencephalopathy pathogenesis in our patients. Our results are consistent with the notion that impaired enzyme function is a common characteristic of disease- associated ARSs mutations.

In the patients examined here, CNS involvement is the main clinical presentation. Common clinical features include im- UN NUN N c.1312C>T c.1573G>A Compound paired c.1312C>T cognitive c.1573G>A Compound ability, seizure, hypotonia, and ataxia. Brain MRI or CT revealed abnormalities in the white matter in all patients. Our results suggest that leukoencephalopathy in these patients is caused by KARS defects and that there are

epileptiform discharges epileptiform discharges novel clinical features from the previously described KARS- associated neurologic diseases. Hearing loss EEG NCV + EMG Other systems Allele 1 Allele 2 Status N Generalized N Generalized Like other bifunctional ARSs, KARS has cytoplasmic and mitochondrial isoforms, which result from alternative splicing ﬁ 36,37

(continued) of the rst 3 exons. Previously, KARS mutations have been implicated in peripheral neuropathies and sensorineural diseases. In this study, we showed that KARS is also involved in CNS diseases. The mechanisms underlying the tissue

Thinning of the central cerebral white matter and corpus callosum Thinning of the central cerebral white matter and corpus callosum speciﬁcity of KARS-associated neurologic disorders are unclear. To date, 4 cytoplasmic ARSs (YARS, AARS, HARS, and MARS), but not their mitochondrial counterparts (YARS2, AARS2, HARS2, and MARS2), are associated with peripheral neuropathy, indicating that this disease might be caused by dysfunctions of cytoplasmic protein translation. Mutations in KARS and another bifunctional enzyme (GARS) had also been implicated in peripheral neuropathy. We propose that the dysfunction of cytoplasmic KARS or GARS, but not of mitochondrial KARS or GARS, is involved in peripheral neuropathy. Conversely, most of the mitochondrial ARSs nystagmus, microcephaly, seizures, and global developmental delay nystagmus, microcephaly, seizures, and global developmental delay Summary of clinical presentation of affected individuals CNS Brain imaging defects have been found to aﬀect the CNS. A functional CNS has an extremely high demand for energy. The oxidative

brainstem auditory-evoked potential study at 10 months was markedly abnormal, suggesting severe peripheral conduction defects in the auditory pat phosphorylation system (OXPHOS) is a key functional unit “ in mitochondria, and it is the major source of cellular adenosine triphosphate. Defects in mitochondrial ARSs aﬀect 9 m M Visual impairment/pendular 4 m F Visual impairment/pendular Age at onset Sex mitochondrial protein synthesis and lead to mitochondrial 38 Summary of key clinical manifestations of affected individuals 15 OXPHOS dysfunction (tables e-1 and e-2, links.lww.com/ 15 NXG/A143 and links.lww.com/NXG/A144). As expected, In the original text is Table a Brother Index number Sister Abbreviations: M = male; F = female; Y = yes; N = no; UN = unknown. CNS involvement as the main or sole clinical presentation had

6 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Figure 2 Summary of KARS mutations

(A) Schematic representation of the KARS gene and the distribution of published mutations (black, above) and mutations found in our patient cohort (red, below). (B) Ribbon diagram of the complex structure model of human lysyl-tRNA synthetase (PDB ID: 3BJU) and mapping the missense mutations onto the structure model. One monomer colored in cyan and the other one in green. The mutation is drawn as a ball-and-stick model and colored in red. (C) Close-up view of the in silico analysis for mutation p.Arg505His. (D) View for mutation p.Pro533Ser. (E) View for mutation p.Thr587Met. (F) Cross species sequence alignment of amino acids. The corresponding positions are indicated in red text.

been reported in most patients with pathogenic mutations in dysfunction of mitochondrial KARS, but not cytoplasmic KARS, mitochondrial ARSs, including DARS2, EARS2, MARS2, FARS2, more likely to be contributing to the pathogenesis of leu- RARS2, VARS2, TARS2, NARS2,andCARS2.Meanwhile,the koencephalopathy reported in this study. This work provides elevated level of lactate observed in some of our patients is also a framework to link the dysfunction of mitochondrial KARS with consistent with mitochondrial dysfunction. Therefore, leukoencephalopathy associated with disorders of the CNS.

Figure 3 Summary of the mutations identified in KARS and their effects on tRNA charging

The column “ratio to WT” indicates the decrease in tRNA charging relative to the WT enzyme for each mutant. All these mutations have a deleterious effect, ranging from 13- to nearly 107-fold.

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 7 Figure 4 Yeast complementation analysis of mutant KRS1 alleles

(A) A haploid yeast strain deleted for endogenous KRS1 was transformed with a LEU2- bearing pRS315 vector containing wild-type KRS1, the indicated mutant form of KRS1,orno insert (“Empty”). Cultures for each strain (labeled along the top) were grown for 2 days in liquid medium and spotted on solid medium containing 5-FOA to determine whether the KRS1 alleles complement loss of KRS1 at 30°C. (B) Cultures for each indicated strain (labeled at right) were grown for 2 days in liquid medium and then diluted in liquid medium containing 5-FOA to determine whether the KRS1 alleles complement loss of KRS1 at 30°C. The optical density (OD600, y-axis) was evaluated for each culture at the indicated time points (x- axis), and error bars indicate SD. 5-FOA = 5- fluoroorotic acid.

Author contributions of Molecular and Human Genetics at Baylor College of C. Sun, J. Song, Y. Jiang, C. Zhao, J. Lin, Y. Li, Y. Wang, M Medicine derives revenue from the chromosomal microarray Gao, J. Xi, S. Luo, M. Li, K. Donaldson, S.N. Oprescu, T.P. analysis (CMA) and clinical exome sequencing oﬀered in the Slavin, S. Lee, P.L. Magoulas, A.M. Lewis, L. Emrick, S.R. Baylor Genetics Laboratory (BMGL; bmgl.com/BMGL/ Lalani, Z. Niu, M.L. Landsverk, M. Walkiewicz, R.E. Person, Default.aspx). V.W. Zhang is employed by and receives a sal- H. Mei, J.A. Rosenfeld, Y. Yang, A. Antonellis, Y-M. Hou, ary from AmCare Genomics Lab. Exome and other panel J. Lin, and V.W. Zhang designed the study, performed sequencing are among the commercially available tests avail- experiments, and collected and analyzed data. C. Sun, J. Song, able at AmCare Genomics Lab. C. Sun and J. Song report no Y. Jiang, J. Lin, and V.W. Zhang wrote the manuscript and disclosures. Y. Jiang has been employed by Baylor Genetics critically revised the manuscript for important intellectual Laboratories. C. Zhao has received foundation/society re- content. J. Lin and V.W. Zhang supervised the study. search support from the Shanghai Committee of Science and Technology. J. Lu, Y. Li, Y. Wang, M. Gao, J. Xi, S. Luo, M. Li, Acknowledgment K. Donaldson, and S.N. Oprescu report no disclosures. T.P. The authors thank the families and patients for their Slavin has received research support from the NIH/NCI, participation to this study. (1K08CA234394), Oxnard Foundation, Stop Cancer Foun- dation, and the Israeli Cancer Research Foundation. S. Lee Study funding and P.L. Magoulas report no disclosures. A.M. Lewis has re- This work was supported by the National Natural Science ceived travel funding or speaker honoraria from Texas Parent Foundation of China (Nos. 81301203 and 81401035), to Parent Conference. L. Emrick has served on the advisory Shanghai Health and Family Planning Commission key board of the American Board of Psychiatry and Neurology projects (Nos. 201440019 and 15DZ1208002), NIH and has received government research support from the NIH. GM108972 and NIH GM114343 (to YMH), NIH S.R. Lalani has received publishing royalties from UpToDate. GM118647 (to AA), and a China Scholarship (to ML). Z. Niu reports no disclosures. M.L. Landsverk has been employed by Ambry Genetics. M. Walkiewicz has provided Disclosure services for Baylor Miraca Genetics Laboratories. R.E. Person Baylor College of Medicine (BCM) and Miraca Holdings Inc. has been employed by GeneDX. H. Mei has been employed have formed a joint venture with shared ownership and by GeneDX and Baylor Genetics. J.A. Rosenfeld has served on governance of the Baylor Genetics Laboratories (BMGL), the editorial boards of Prenatal Diagnosis and Molecular Syn- which performs clinical exome sequencing. The Department dromology and has received government research support

8 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG from the NIH. Y. Yang and A. Antonellis report no dis- 17. Pierce SB, Chisholm KM, Lynch ED, et al. Mutations in mitochondrial histidyl tRNA synthetase HARS2 cause ovarian dysgenesis and sensorineural hearing loss of Perrault closures. Y.-M. Hou has received government research sup- syndrome. Proc Natl Acad Sci USA 2011;108:6543–6548. port from the NIH and has received foundation/society 18. Pierce SB, Gersak K, Michaelson-Cohen R, et al. Mutations in LARS2, encoding mitochondrial leucyl-tRNA synthetase, lead to premature ovarian failure and hearing support from the Packard Foundation. J. Lin has received loss in Perrault syndrome. Am J Hum Genet 2013;92:614–620. government research support from the National Natural 19. Santos-Cortez RL, Lee K, Azeem Z, et al. Mutations in KARS, encoding lysyl-tRNA synthetase, cause autosomal-recessive nonsyndromic hearing impairment DFNB89. Science Foundation of China and the Shanghai Health and Am J Hum Genet 2013;93:132–140. Family Planning Commission. V.W. Zhang has been 20. Puffenberger EG, Jinks RN, Sougnez C, et al. Genetic mapping and exome sequencing employed by AmCare Genomic Lab. Disclosures available: identify variants associated with five novel diseases. PLoS One 2012;7:e28936. 21. Dallabona C, Diodato D, Kevelam SH, et al. Novel (ovario) leukodystrophy related to Neurology.org/NG. AARS2 mutations. Neurology 2014;82:2063–2071. 22. Sahin S, Cansu A, Kalay E, et al. Leukoencephalopathy with thalamus and brainstem involvement and high lactate caused by novel mutations in the EARS2 gene in two Publication history siblings. J Neurol Sci 2016;365:54–58. Received by Neurology: Genetics June 26, 2017. Accepted in final form 23. Simons C, Griffin LB, Helman G, et al. Loss-of-function alanyl-tRNA synthetase February 14, 2019. mutations cause an autosomal-recessive early-onset epileptic encephalopathy with persistent myelination defect. Am J Hum Genet 2015;96:675–681. 24. Steenweg ME, Ghezzi D, Haack T, et al. Leukoencephalopathy with thalamus and References brainstem involvement and high lactate “LTBL” caused by EARS2 mutations. Brain 1. Park SG, Schimmel P, Kim S. Aminoacyl tRNA synthetases and their connections to 2012;135:1387–1394. disease. Proc Natl Acad Sci USA 2008;105:11043–11049. 25. van Berge L, Hamilton EM, Linnankivi T, et al. Leukoencephalopathy with brainstem 2. Antonellis A, Green ED. The role of aminoacyl-tRNA synthetases in genetic diseases. and spinal cord involvement and lactate elevation: clinical and genetic characterization Annu Rev Genomics Hum Genet 2008;9:87–107. and target for therapy. Brain 2014;137:1019–1029. 3. Abbott JA, Francklyn CS, Robey-Bond SM. Transfer RNA and human disease. Front 26. 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Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 9 ARTICLE OPEN ACCESS Somatic expansion of the C9orf72 hexanucleotide repeat does not occur in ALS spinal cord tissues

Jay P. Ross, BSc, Claire S. Leblond, PhD, H´el`ene Catoire, PhD, Kathryn Volkening, PhD, Correspondence Michael Strong, MD, FRCPC, FANN, FCAHS, Lorne Zinman, MD, MSc, FRCPC, Janice Robertson, PhD, Dr. Rouleau [email protected] Patrick A. Dion, PhD, and Guy A. Rouleau, MD PhD, FRCPC

Neurol Genet 2019;5:e317. doi:10.1212/NXG.0000000000000317 Abstract Objective To test for somatic C9orf72 hexanucleotide repeat expansion (HRE) and hexanucleotide repeat length instability in the spinal cord of amyotrophic lateral sclerosis (ALS) cases.

Methods Whole and partial spinal cords of 19 ALS cases were dissected into transversal sections (5 mm thick). The presence of C9orf72 HRE was tested in each independent section using Repeat- Primed PCR and amplicon-size genotyping. Index measures for the testing of mosaicism were obtained through serial dilutions of genomic DNA from an individual carrying a germline C9orf72 HRE in the genomic DNA of an individual without a C9orf72 HRE.

Results None of the sections examined supported the presence of a subpopulation of cells with a C9orf72 HRE. Moreover, the C9orf72 hexanucleotide repeat lengths measured were identical across all the spinal cord sections of each individual patient.

Conclusions We did not observe somatic instability of the C9orf72 HRE in disease relevant tissues of ALS cases.

From the Department of Human Genetics (J.P.R., G.A.R.), McGill University, Montréal, Quebec, Canada; Montreal Neurological Institute and Hospital (J.P.R., H.C., P.A.D., G.A.R.), McGill University, Montréal, Quebec, Canada; Pasteur Institute (C.S.L.), University Paris Diderot, Sorbonne Paris Cité, Paris, France; Department of Neurology and Neurosurgery (H.C., P.A.D., G.A.R.), McGill University, Montréal, Quebec, Canada; Department of Clinical Neurological Sciences and Robarts Research Institute (K.V., M.S.A.N.N.), Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada; Sunnybrook Health Sciences Centre (L.Z.); and Tanz Centre for Research in Neurodegenerative Diseases (J.R.), University of Toronto, Toronto, Ontario, Canada.

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary ALS = amyotrophic lateral sclerosis; HRE = hexanucleotide repeat expansion; RPPCR = repeat-primed PCR.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative sequences are particularly of interest for somatic mutation disease characterized by rapid and progressive loss of motor analysis because their emergent secondary structures can neurons.1 Although germline mutations in several genes lead to expansion or contraction of repeat lengths.4 It is have been identified, the C9orf72 hexanucleotide repeat also notable that the C9orf72 HRE can lead to cell-to-cell expansion (HRE) is currently one of the most prevalent transmission of dipeptide repeat proteins,5 and as such, it and penetrant cause of ALS.1 In the general population, is conceivable that a small population of C9orf72 HRE cells C9orf72 contains less than 30 GGGGCC repeats in the first nested in the nervous system could potentiate ALS. intron,whereasinALScasesthenumberofrepeatsranges between hundreds to thousands.1 Because it is difficult to Recently, somatic recombination of APP has been demon- precisely size the repeat length above 30,2 many aspects of stratedtooccurinAlzheimer’s disease neurons.6 Because C9orf72-related ALS have not been thoroughly somatic expansion of C9orf72 hexanucleotide repeats is investigated. a potential mechanism for ALS pathogenesis and because routine blood DNA testing would not identify such somatic Somatic mutations have been hypothesized as a possible events,2 we tested DNA extracted from finely sectioned cause of ALS in cases who do not have germline mutations spinal cords of 19 patients with ALS for low levels of the in genes known to be associated with the disease.3 Repeat C9orf72 HRE.

Table Description of the ALS patient cohort

Individual No. sections Age Sex Site of onset Germline mutations

ALS01 108 50 M Left hand None

ALS02 75 69 F Right hand None

ALS03 96 62 F Bulbar None

ALS04 70 79 M Right leg None

ALS05 118 78 M Bulbar NEK1 p.P318L

ALS06 78 58 F Right hand None

ALS07 78 Left foot None

ALS08 88 Left hand None

ALS09 82 Right foot None

ALS10 22 66 M None

ALS11 28 62 M None

ALS12 30 61 M TBK1 p.L306I, CCNF p.E396D, SPG11 p.R1992Q

ALS13 17 57 M None

ALS14 38 66 F None

ALS15 11 78 F Bulbar None

ALS16 21 62 M None

ALS17 31 71 M None

ALS18 22 69 F None

ALS19 31 67 F SPAST p.R221C

Abbreviation: ALS = amyotrophic lateral sclerosis. Site of onset refers to the initial location of ALS symptoms, Sections refer to the number of ;5 mm spinal cord samples generated from each spinal cord. A total of 1,053 unique sections were tested.

2 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Figure 1 C9orf72 HRE mosaicism RPPCR profiles

Genomic DNA from an individual with germline C9orf72 HRE was diluted in genomic DNA from an individual without germline C9orf72 HRE at various percentages. HRE = hexanucleotide repeat expansion; RPPCR = repeat-primed PCR.

Methods blood was not available) to accurately size germline hexanucleotide repeat alleles. Repeat-primed PCR (RPPCR)9 was Samples performed on all sampled sections of each patient to assess for The spinal cords from 19 ALS cases were included in this study. the C9orf72 HRE and to estimate the lengths of C9orf72 alleles DNA obtained from prior blood samplings of these cases in each section. GeneMapper v4.0 (Applied Biosystems) was established them all to be negative for the C9orf72 HRE. used to visualize and estimate reaction fragment sizes. Lengths Samples were collected from 3 institutions: the Montreal of C9orf72 hexanucleotide repeat amplicons were measured Neurological Institute and Hospital in Montréal, Québec; the using GeneMapper compared to the GeneScan-500 LIZ Size Sunnybrook Health Sciences Centre in Toronto, Ontario; and Standard (Applied Biosystems). Peaks from the RPPCR pro- the ALS Clinic at the London Health Science Centre in London, files were chosen based on the genotyping method results to Ontario. Average patient age at donation was 65.9 years, with represent C9orf72 alleles, which were plotted to assess variation 7 a male-to-female ratio of 1.29. A targeted sequencing approach within normal-length C9orf72 hexanucleotide repeat lengths. was used to test for rare (minor allele frequency < 0.001) protein- altering germline mutations in genes known to be ALS risk fac- HRE mosaicism index measures tors. Information regarding the ALS cases is listed in table. Genomic DNA from a patient previously established as a C9orf72 HRE carrier was diluted in genomic DNA from Standard protocol approvals, registrations, an ALS patient without the HRE to generate a percentage and patient consents of HRE within a sample (0%, 5%, 10%, 20%, 30%, 40%, All participants signed an informed consent form that was 50%, and 100%). These dilutions were index measures for approved by the ethical review boards of institutions that the testing of C9orf72 HRE mosaicism within a section; contributed the material. their RPPCR profiles enabled us to assess the sensitivity of the method for each HRE dilution. RPPCR fragment length Tissue sectioning and DNA extraction profiles were visually compared between every spinal cord Spinal cords were manually portioned into transverse sections section and the mosaicism index measures. of approximately 5 mm thickness. Sections were then separated along the coronal plane into dorsal and ventral halves, with only Data availability statement fi fi the ventral areas being used in the present study. Each ventral The authors con rm that the data necessary for con rming the portion was separated into left and right ventral horns. Geno- conclusions of this study are available within the article and its mic DNA was extracted using standard salting-out methods supplementary material. Raw data is available upon request. from approximately half of both the left and right ventral portions of every section available from each spinal cord. Results C9orf72 HRE reactions Mosaicism detection C9orf72 HRE genotyping8 was performed on blood DNA Varying proportions of the C9orf72 HRE diluted in wild-type samples (or sampling of the cervical area of the cerebellum if DNA displayed unique profiles on RPPCR fragment sizing

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 3 (figure 1). We were able to detect as low as 5% mosaicism Our study did not find evidence for C9orf72 HRE somatic based on the profiles generated by our assay. expansion in the spinal cords of patients with ALS. This does not preclude the possibility that very low levels of expansion Spinal sample testing may exist in patients with ALS. However, as we were able to A total of 1,053 individual sections were tested by RPPCR detect the levels of mosaicism at or above 5%, lower- in the spinal cords of patients with ALS. No section showed frequency somatic mutations would have had to occur late in evidence of C9orf72 HRE at or above a 5% mosaicism level neural tissue development. in any of the spinal cords tested. All sections from the same spinal cord showed the same profile of RPPCR fragments, The lengths of C9orf72 hexanucleotide repeats across all and RPPCR peaks (chosen by the amplicon genotyping sections of the same spinal cord were identical. This result method sizing) showed that repeat sizing did not signifi- confirms that C9orf72 hexanucleotide repeats are stable cantly change across a spinal cord (figure 2). when in the normal range10 and that if instability does occur, it is restricted to expanded alleles.2 In C9orf72 expression Discussion vectors, the number of hexanucleotide repeats has been reported to contract or expand above a critical number of Because of the high penetrance of the C9orf72 HRE and the repeats.4 Changes in C9orf72 hexanucleotide repeat length accumulation of repeat RNA fragments and dipeptide might occur more readily in artificial systems, and in human proteins,1,9 its pathologic mechanism must have a strong (albeit neural cells there may be a mechanism to prevent frequent time-dependent) effect. Therefore, there must be a threshold or alterations. Very large C9orf72 HRE can exhibit a range of concentration at which the products and effects of C9orf72 HRE repeat lengths across tissues of an individual10; however, are toxic to cells and tissues. It is possible that low levels of these pathogenic expansions likely occur in most or all cells C9orf72 HRE not detectable by germline testing could be suf- of an individual and the exact number of repeats triggering ficient to cause disease through accumulation of products. the disease remains to be established.

Figure 2 RPPCR fragment sizes in the spinal cord sections of patients with ALS

RPPCR peaks representing the measured C9orf72 HRE alleles were chosen based on the results of the amplicon genotyping method. ALS = amyotrophic lateral sclerosis; HRE = hexanucleotide repeat expansion; RPPCR = repeat-primed PCR.

4 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Our study is limited by sample size, as it is difficult to acquire large numbers of spinal cords from patients with ALS. Based Appendix (continued) on our results, if somatic expansion occurs at the level de- Name Location Role Contribution tectable by our assays, it is likely that it does not account for a large proportion of ALS cases, not occurring in large clusters Claire S. Pasteur Institute, Author Design and concept Leblond, PhD University Paris of study; of neuronal cells. However, as we sampled exclusively from Diderot, Sorbonne experimental the ventral spinal cord, our assay did not test for somatic Paris Cité, Paris, procedures; France analysis and events in dorsal neurons or glial cells, which could be sources interpretation of of pathogenic protein seeding. the data; and drafting the manuscript for Study of the C9orf72 HRE remains difficult because of the intellectual content technological limitations of sequencing GC-rich and re- Hélène McGill University, Author Experimental petitive regions of the genome. Techniques such as single Catoire, PhD Montréal, QC, procedures; and Canada drafting the cell and long-read sequencing may allow detection of very manuscript for low-level somatic events and precise measurement of the intellectual C9orf72 HRE length. content Kathryn Schulich School of Author Experimental Acknowledgment Volkening, PhD Medicine and procedures; and The authors thank the participants for their contribution to the Dentistry, Western drafting the University, London, manuscript for study. The authors would like to thank Cynthia Bourassa, Fulya Ontario, Canada. intellectual Akçimen, Boris Chaumette, Calwing Liao, and Qin He for their content fi assistance in reviewing the manuscript and scienti ccontent. Michael Schulich School of Author Clinical Thanks are also due to Vessela Zaharieva and Cathy Mirarchi for Strong, MD, Medicine and assessment of FRCPC, FANN, Dentistry, Western patients; their assistance in clinical coordination. The authors thank the FCAHS University, London, experimental Douglas Bell Canada Brain Bank (McGill University), Sunny- Ontario, Canada. procedures; and drafting the brook Health Sciences Centre (University of Toronto), and the manuscript for Schulich School of Medicine and Dentistry of Western University intellectual for providing the material and relevant clinical information. content Lorne Zinman, Sunnybrook Health Author Clinical assessment Study funding MD, MSc, Sciences Centre, of patients; and FRCPC Toronto, ON, drafting the Amyotrophic Lateral Sclerosis Society of Canada. Canada manuscript for intellectual content Disclosure Janice University of Author Drafting the J.P. Ross has received a doctoral student fellowship from the Robertson,PhD Toronto, Toronto, manuscript for ALS Society of Canada and a Canadian Institutes of Health ON, Canada intellectual content Research Frederick Banting & Charles Best Canada Grad- Patrick A. McGill University, Author Design and concept uate Scholarship (FRN 159279). C.S. Leblond, H. Catoire, Dion, PhD Montréal, QC, of study; Canada interpretation of the K. Volkening, M. Strong, L. Zinman, J. Robertson, and P.A. data; and drafting or Dion reports no disclosures. G. A Rouleau has received revising the manuscript for support from the Canadian Institute of Health Research intellectual content (CIHR). Full disclosure form information provided by Guy A. McGill University, Author Design and concept the authors is available with the full text of this article at Rouleau, MD Montréal, QC, of study; Neurology.org/NG. PhD, FRCPC Canada interpretation of the data; and drafting or revising the Publication history manuscript for Received by Neurology: Genetics October 26, 2018. Accepted in final intellectual content form February 19, 2019.

Appendix Author contributions References Name Location Role Contribution 1. Taylor JP, Brown RH Jr, Cleveland DW. Decoding ALS: from genes to mechanism. Nature 2016;539:197–206. Jay P. Ross, BSc McGill University, Author Design and concept 2. Pamphlett R, Cheong PL, Trent RJ, Yu B. Can ALS-associated C9orf72 repeat Montr´eal, QC, of study; expansions be diagnosed on a blood DNA test alone? PLoS One 2013;8: Canada experimental e70007. procedures; analysis 3. Leija-Salazar M, Piette C, Proukakis C. Review: somatic mutations in neuro- and interpretation of degeneration. Neuropathol Appl Neurobiol 2018;44:267–285. the data; and drafting 4. Thys RG, Wang YH. DNA replication dynamics of the GGGGCC repeat of the the manuscript for C9orf72 gene. J Biol Chem 2015;290:28953–28962. intellectual content 5. Westergard T, Jensen BK, Wen X, et al. Cell-to-Cell transmission of dipeptide repeat proteins linked to C9orf72-ALS/FTD. Cell Rep 2016;17:645–652.

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 5 6. Lee MH, Siddoway B, Kaeser GE, et al. Somatic APP gene recombination in Alz- 9. DeJesus-Hernandez M, Mackenzie IR, Boeve BF, et al. Expanded GGGGCC hex- heimer’s disease and normal neurons. Nature 2018;563:639–645. anucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked 7. O’Roak BJ, Vives L, Fu W, et al. Multiplex targeted sequencing identiﬁes recurrently FTD and ALS. Neuron 2011;72:245–256. mutated genes in autism spectrum disorders. Science 2012;338:1619–1622. 10. Nordin A, Akimoto C, Wuolikainen A, et al. Extensive size variability of the 8. Renton AE, Majounie E, Waite A, et al. A hexanucleotide repeat expansion in C9ORF72 GGGGCC expansion in C9orf72 in both neuronal and non-neuronal tissues in 18 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 2011;72:257–268. patients with ALS or FTD. Hum Mol Genet 2015;24:3133–3142.

6 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG ARTICLE OPEN ACCESS Lithium chloride corrects weakness and myopathology in a preclinical model of LGMD1D

Andrew R. Findlay, MD,* Rocio Bengoechea, PhD,* Sara K. Pittman, BS, Tsui-Fen Chou, PhD, Correspondence Heather L. True, PhD, and Conrad C. Weihl, MD, PhD Dr. Findlay [email protected] Neurol Genet 2019;5:e318. doi:10.1212/NXG.0000000000000318 Abstract Objective To understand DNAJB6’s function in skeletal muscle and identify therapeutic targets for limb- girdle muscular dystrophy 1D (LGMD1D).

Methods DNAJB6 knockout (KO) myoblasts were generated with Crispr/cas9 technology, and diﬀer- entially accumulated proteins were identiﬁed using stable isotope labeling, followed by quantitative mass spectrometry. Cultured KO myotubes and mouse muscle from DNAJB6b-WT or DNAJB6b-F93L mice were analyzed using histochemistry, immunohistochemistry, and im- munoblot. Mouse functional strength measures included forelimb grip strength and inverted wire hang.

Results DNAJB6 inactivation leads to the accumulation of sarcomeric proteins and hypertrophic myotubes with an enhanced fusion index. The increased fusion in DNAJB6 KO myotubes correlates with diminished glycogen synthase kinase-β (GSK3β) activity. In contrast, LGMD1D mutations in DNAJB6 enhance GSK3β activation and suppress β-catenin and NFAT3c signaling. GSK3β inhibition with lithium chloride improves muscle size and strength in an LGMD1D preclinical mouse model.

Conclusions Our results suggest that DNAJB6 facilitates protein quality control and negatively regulates myogenic signaling. In addition, LGMD1D-associated DNAJB6 mutations inhibit myogenic signaling through augmented GSK3β activity. GSK3β inhibition with lithium chloride may be a therapeutic option in LGMD1D.

*These authors contributed equally to the manuscript.

From the Washington University School of Medicine (A.R.F., R.B., S.K.P., H.L.T., C.C.W); Department of Neurology (A.R.F., R.B., S.K.P., C.C.W), Hope Center for Neurological Diseases, St. Louis, MO; Harbor-UCLA Medical Center (T.-F.C.), Department of Pediatrics, Division of Medical Genetics, Torrance, CA; Department of Cell Biology and Physiology (H.L.T.), Saint Louis, MO.

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary CSA = cross-sectional area; GFP = green ﬂuorescent protein; GO = Gene Ontology; GSK3β = glycogen synthase kinase-β; HSP = heat shock protein; KO = knockout; LGMD1D = limb-girdle muscular dystrophy 1D; LC-MS/MS = Liquid chromatography with tandem mass spectrometry; NFATc3 = nuclear factor of activated T cells cytoplasmic 3; PBS = phosphate-buﬀered saline; SILAC = stable isotope labeling with amino acids in cell culture; WT = wild type.

Protein chaperones, or heat shock proteins (HSPs), are in- rabbit DNAJB6 (Abcam, ab75196), anti-rabbit αβ-crystallin creasingly recognized as critical for skeletal muscle health.1 (Enzo, ADI-SPA-223), anti-mouse hnRNPA2/B1 (Sigma, Recently, mutations in DNAJB6, an HSP40 co-chaperone, R4653), anti-rabbit alpha-actinin (Abcam ab68167), anti-mouse were identified to cause limb-girdle muscular dystrophy 1D keratin 18 (Abcam, ab668), anti-rabbit GSK3β-P(ser-9) (Cell (LGMD1D), also known as LGMD D1 DNAJB6-related, Signaling, 9336), anti-rabbit GSK3β (Cell Signaling, 9315), anti- a childhood- or adult-onset, dominantly inherited, progressive goat FHL-1 (Abcam ab23937), and anti-mouse myosin (Sigma- – myopathy with vacuolar and aggregate myopathology.2 5 Aldrich, M1570). Secondary antibodies include anti-mouse HRP DNAJB6 is ubiquitously transcribed and mediates proper (Pierce), anti-rabbit HRP (Cell Signaling), anti-goat HRP (Santa folding and disaggregation of proteins by HSP70.6,7 It has 2 Cruz), and anti-mouse AlexaFluor (488). isoforms: DNAJB6a, which localizes to the nucleus, and DNAJB6b, which localizes diffusely.4,8 In skeletal muscle, Western blot DNAJB6b localizes to the Z disc and is thought to be the Muscle tissues and cultured cells were homogenized using ff isoform responsible for disease pathogenesis of LGMD1D.4,8 RIPA lysis bu er (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, LGMD1D mutations in DNAJB6 reside within the G/F do- 1% NP-40, 0.25% Na-deoxycholate and 1 mM EDTA) sup- main, a region important for client protein handling.4,8 plemented with protease inhibitor cocktail (Sigma-Aldrich), DNAJB6’s role in normal muscle and the pathomechanism of and lysates were centrifuged at 21380g for 10 minutes. Protein disease mutations is unknown. concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific). Aliquots of lysates were solubilized ff In addition to its role in protein homeostasis, DNAJB6 also in Laemmli bu er, and equal amounts of proteins were separated acts as a tumor suppressor through its interaction with glycogen on 12% sodium dodecyl sulfate polyacrylamide gels. Proteins synthase kinase-β (GSK3β).9,10 GSK3β activity is dependent on were transferred to nitrocellulose membrane, and the membrane ff DNAJB6 chaperoning a multiprotein complex to maintain its was blocked with 5% nonfat dry milk in phosphate-bu ered dephosphorylated (active) state.10 Active GSK3β negatively saline (PBS) with 0.1% Tween-20 for 1 hour. The membrane regulates several myogenic signaling pathways, including was then incubated with primary antibody, in 5% nonfat dry milk β-catenin and nuclear factor of activated T cells cytoplasmic 3 overnight at 4°C, and then secondary antibody conjugated with – (NFATc3) signaling.10 15 DNAJB6 has also been shown to di- horseradish peroxidase. Enhanced chemiluminescence (GH rectly interact with and inhibit NFATc3 transcriptional activity Healthcare, UK) was used for protein detection. Immunoblots by recruiting class II histone deacetylases.16 In this study, we were obtained using the G:BoxChemi XT4, Genesys Version explored DNAJB6’sroleinskeletalmuscleanditsimpacton 1.1.2.0 (Syngene). Densitometry was measured with ImageJ myogenesis and related signaling pathways. We also investigated software (NIH). ’ DNAJB6 disease mutation s impact on these pathways and their Cell culture contribution to LGMD1D pathogenesis. HeLa cells were maintained in Dulbecco’s Modified Eagle medium (DMEM, Gibco #11965-084), 10% fetal bovine serum (FBS, Atlanta Biologicals #S10350H), and 50 μg/mL Methods penicillin and streptomycin (P/S, Sigma #P4333) at 37°C with 5% CO2. C2C12 cells were maintained in proliferation Generation of DNAJB6 knockout (KO) media (DMEM with 20% FBS, 50 μg/mL P/S) and switched to C2C12 myoblasts differentiation media (DMEM with 2% horse serum, 50 μg/mL DNAJB6 KO C2C12 cells were generated using 2 guide P/S) to form myotubes. Transfection of cells was performed RNAs targeting 2 introns of DNAJB6 to generate a 5.5-kb out- with Lipofectamine 2000 (Life Technologies #11668019) of-frame deletion (figure e-1A, links.lww.com/NXG/A147). according to the manufacturer’s instruction. We used this strategy to avoid modifying other DNAJ genes. Clones were screened for homozygosity of the 5.5-kb deletion Plasmid construction via sequencing (figure e-1B). Absence of DNAJB6 protein Mammalian constructs of DNAJB6b were cloned using site- expression was confirmed via western blot (figure 1A). directed mutagenesis, digested with HindIII/XhoI, and ligated into vector pcDNA3.1 containing a green fluorescent protein Antibodies (GFP) tag. DNAJB6b F89I and P96R mutations were generated Antibodies used were the following: anti-rabbit GAPDH (Cell with the Quik Change Mutagenesis Kit (Agilent Technologies Signaling, 2118), anti-rabbit desmin (Abcam, ab8592), anti- #200517).

2 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Immunofluorescence decreased in DNAJB6 KO C2C12 cells if there was at least C2C12 cells were grown, stained, and imaged directly on cell a 0.6-fold change in forward KO/WT experiments and a 1.5- culture plastic. Cells were washed 3 times with PBS, fixed in 4% fold change in reverse WT/KO experiments. PFA for 10 minutes, permeabilized with 0.1% Triton X-100 in PBS for 10 minutes, and then blocked with 3% BSA in PBS for Ontological analysis ff fi 30 minutes to 1 hour at room temperature. Cells were stained All di erentially expressed proteins were classi ed broadly with primary antibody (anti-mouse myosin) at 4°C overnight, into several catalogs according to the Gene Ontology (GO) followed by washing 3 times with PBS. Cells were incubated annotation (geneontology.org). Overrepresentation analyses with Alexa 488 Fluor-conjugated secondary antibody at RT of GO terms, including biological process, molecular func- for 1 hour and mounted with Mowiol media containing 49,6- tion, and cellular component, were performed using the diamidino-2-phenylindole. C2C12 differentiation experiments ConsensusPathDB-human database system (cpdb.molgen. were performed in triplicate. Fusion index was determined as mpg.de/CPDB), which is a molecular functional interaction a ratio of nuclei number within multinucleated myosin-positive database. All proteins detected in SILAC experiments were myotubes to the total number of nuclei. Nuclei were counted used as background for comparison. The GO level 2 and 3 ff from 10 random fields taken with 10× objective equipped in categories and a p value cuto of 0.01 were selected. a NIKON Eclipse 80i fluorescence microscope. Myosin stained myotubes with irregular staining pattern were counted and di- Luciferase assays vided by the total number of myosin-positive myotubes to C2C12 myoblasts were transfected with 20 ng Renilla control quantify the percent of myotubes with irregular myosin staining. reporter (pRL-TK) and either 1 μg of the β-catenin luciferase reporter (TOPflash, Addgene plasmid #12456) or 1 μgofan Electron microscopy NFAT-sensitive luciferase reporter (pGL3-NFAT luciferase, C2C12 cells were differentiated for 6 days, rinsed brieflyin Addgene plasmid #17870). PBS, and fixed immediately with Karnovsky fixative at 4°C for 24 hours. Fixed myotubes were embedded in plastic and HeLa cells were transfected with 0.5 μg of GFP control or GFP- sectioned for imaging with a JEOL JEM-1400 Plus 120 kV tagged DNAJB6b construct, 20 ng Renilla control reporter, and Transmission Electron Microscope equipped with an AMT either 0.5 μgoftheβ-catenin luciferase reporter or 0.5 μgofan XR111 high-speed 4 k × 2 k pixel phosphor-scintillated 12-bit NFAT-sensitive luciferase reporter. For β-catenin luciferase charge coupled device camera. experiments, HeLa cells were treated with 20 mM lithium chloride (LiCl) (Sigma-Aldrich 203637) for 12 hours before Stable isotope labeling by amino acids and measuring luminescence to stimulate β-catenin transcriptional mass spectrometry activity. For NFATc3 luciferase experiments, HeLa cells were For “forward” stable isotope labeling with amino acids in cell transfectedwith0.5μg of plasmid encoding NFATc3 to stim- culture (SILAC) experiments, KO C2C12 cells were cultured ulate transcriptional activity (pBS mNFATc3-EE, Addgene “ ” 13 in heavy media in which Arg and Lys were replaced by C6- plasmid #17868). Cells were lysed, and luciferase activities were 13 15 Arg and C6, N2-Lys, whereas C2C12 DNAJB6 wild-type measured using the Dual-Glo luciferase kit (Promega E2920) “ ” 12 (WT) cells were grown in light media containing C6-Arg and a microplate luminometer (BioTek Instruments). Lysates 12 14 “ ” μ and C6, N2-Lys (Thermo Fisher). For reverse experi- (75 L) were pipetted into 96-well plates in triplicate. Data were ments, DNAJB6 KO C2C12 cells were grown in “light” media calculated as luciferase/Renilla signal. Fold change was calculated and WT C2C12 cells were grown in “heavy” media. Two based on change from unstimulated baseline (e.g., LiCl treated vs biological replicates were used for each condition. Myoblasts untreated or NFATc3 transfected vs control transfected). were passaged 6 times to allow for sufficient incorporation of label. For mass spectrometry analyses, we used the same methodology as the one used in previous studies with minor Animal studies modification.17,18 Briefly, total cell lysate was generated using 9 M Urea in 20 mM HEPES, pH 8.0 containing protease Animal and experimental protocols inhibitor and phosphatase inhibitor tablets. Equal amount Transgenic human-V5-DNAJB6b mice with MCK promoter 8 (0.3 mg) for each cell type was mixed and digested using were previously generated. DNAJB6b-F93L mice develop 8 Lys-C and trypsin digestion protocol as described.17,18 The a prominent myopathy and are weak by age 3 months. Mice peptides were loaded onto an Easy Nano-LC Q-Exactive were housed in a temperature-controlled environment with Orbitrap, and the peak lists were generated using Proteome 12-hour light-dark cycles and received food and water ad Discoverer software (Thermo Fisher Scientific).18 Only pro- libitum. Mice were killed, and skeletal muscle was dissected. teins that were present in all biological replicates in both For western blot analysis, muscle was flash frozen in liquid forward and reverse SILAC experiments were considered for nitrogen and stored at −80°C. further data analysis. Proteins were considered increased in DNAJB6 KO C2C12 cells if there was at least a 1.5-fold Wire screen holding and grip test change in forward KO/WT experiments and a 0.6-fold change Grip strength testing consisted of 5 separate measurements using in reverse WT/KO experiments. Proteins were considered a trapeze bar attached to a force transducer that recorded peak-

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 3 Figure 1 DNAJB6 knockout results in accumulation of sarcomeric proteins and altered myofibrillogenesis

(A) Western blot demonstrating the absence of both DNAJB6 isoforms in Crispr/Cas9-generated DNAJB6 KO C2C12 myoblasts and during differentiation into myotubes. (B) Bright-field image demonstrating normal morphology of DNAJB6 KO myoblasts. (C) Flowchart of forward and reverse SILAC labeling combined with LC-MS/MS for comparative analysis of protein expression in WT and DNAJB6 KO myoblasts. (D) Quantitation overlap of the detected proteins in the 2 forward and 2 reverse SILAC labeling experiments and those that differentially accumulated in WT vs DNAJB6 KO C2C12 cells. (E) Ontological analysis of proteins increased in DNAJB6 KO myoblasts. (F) Ontological analysis of proteins decreased in DNAJB6 KO myoblasts. (G) Western blot of WT and KO myoblasts confirming increased levels of several proteins identified in SILAC analysis. (H) Differentiation of KO myoblasts into myotubes reveals altered myofibrillar organization on myosin staining and electron microscopy. White arrows demonstrate Z discs in WT myotubes with well-organized sarcomeres. Dark arrows demonstrate Z discs in KO myotubes with poor myofibrillar organization. (I) Quantitation of myotubes with irregular myosin staining. Error bars represent the standard error of 3 independent experiments. KO = knockout; LC-MS/MS = liquid chromatography with tandem mass spectrometry; SILAC = stable isotope labeling with amino acids in cell culture; WT = wild type.

4 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG above a cage. Latency for the mouse to release the mesh Table Increased proteins in DNAJB6 KO myoblasts is recorded, and the average hanging time of 3 trials was

KO/WT used. Gene Protein average Histochemistry *PDLIM3 PDZ and LIM domain protein 3 6.89 Isolated muscle was mounted using tragacanth gum PTGIS Prostacyclin synthase 4.30 (Sigma, G1128) and quick frozen in liquid nitrogen– − *ACTN3 Alpha-actinin-3 4.22 cooled 2-methylbutane. Samples were stored at 80°C until sectioning into 10-μm sections. Hematoxylin and eosin staining SERPINB9B Serpin B9 3.99 was performed as previously described.19 Images were taken TPPP3 Tubulin polymerization-promoting 3.30 with a 5-megapixel color charge coupled device (Nikon, Tokyo, protein family member 3 Japan), and the muscle ﬁber cross-sectional area (CSA) was FTL1 Ferritin light chain 1 3.24 measured using ImageJ software. Two separate individuals, blinded to the treatment status of mice, took photographs and NAPA Alpha-soluble NSF attachment 3.16 protein measured the CSA. Four representative images from each animal’s tibialis anterior were used to determine the average cross- *CRYAB Alpha-crystallin B chain 3.00 sectional area. *AKAP12 Isoform 2 of A-kinase anchor 2.92 protein 12 Lithium treatment RPL14 60S ribosomal protein L14 2.69 After oxygen exposure, mice were anesthetized with iso- ﬂuorane and injected intraperitoneally with LiCl (250 mg/kg EXOC1 Exocyst complex component 1 2.42 body weight; Sigma-Aldrich) or dimethyl sulfoxide daily for 1 *FHL1 Four and a half LIM domains protein 1 2.34 month.

SGPL1 Sphingosine-1-phosphate lyase 1 2.26 Statistical analysis *JUP Junction plakoglobin 2.18 Results are presented as mean and standard error of the mean.

PAK2 Serine/threonine protein kinase PAK 2 2.13 Statistical analyses were performed using paired t tests.

DNM1L Isoform 4 of dynamin-1-like protein 2.09 Standard protocol approvals, registrations, *DES Desmin 2.05 and patient consents All animal experimental protocols were approved by the Ani- TUBB3 Tubulin beta-3 chain 1.97 mal Studies Committee of Washington University School of GAA Lysosomal alpha-glucosidase 1.96 Medicine.

UGP2 Isoform 2 of UTP—glucose-1- 1.95 phosphate uridylyltransferase Data availability policy

CTSL Cathepsin L1 1.86 The supplementary data can be accessed via links.lww.com/ NXG/A145 and links.lww.com/NXG/A147. The authors will *DNAJB4 DnaJ homolog subfamily B member 4 1.83 share the data of this study by request from any qualiﬁed *HSPB1 Isoform B of heat shock protein beta-1 1.82 investigator. Any data not published within the article are available in a public repository and will be shared by request MVP Major vault protein 1.81 from any qualiﬁed investigator. ABRACL Costars family protein ABRACL 1.80

*PDLIM1 PDZ and LIM domain protein 1 1.78 Results KLC2 Kinesin light chain 2 1.75 DNAJB6 KO myoblasts accumulate sarcomeric Abbreviations: KO = knockout; SILAC = stable isotope labeling with amino acids in cell culture; WT = wild type. proteins and form altered myofibrillar Note the many sarcomeric and chaperone proteins increased in DNAJB6 KO structures myoblasts (* and bold). Proteins meeting SILAC threshold of KO/WT ratio of ’ >1.5 and WT/KO ratio of <0.6. To understand DNAJB6 s role in skeletal muscle, we generated C2C12 myoblasts that lack both isoforms of DNAJB6 (DNAJB6a and DNAJB6b) using CRISPR/cas9 technology generated force while mice were pulled backward by their tail (figure 1A). These undifferentiated cells were viable and (Stoelting, Wood Dale, IL). The resulting measurement was morphologically similar to control C2C12 cells (figure 1B). recorded, and the average of the highest 3 measurements was To identify differentially accumulated proteins, we performed determined to give the strength score. Another quantita- SILAC. SILAC experiments were conducted in duplicate, tive strength measurement was performed by wire screen including 2 forward (KO/WT) and 2 reverse labeling (WT/ holding test. Mice were placed on a grid where it stood KO) of control and DNAJB6 KO C2C12 myoblasts (figure using all 4 limbs. Subsequently, the grid was inverted 15 cm 1C depicts the setup for SILAC-based proteomics). Using

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 5 Figure 2 Absence of DNAJB6 impairs GSK3β dephosphorylation and enhances myotube formation

(A) C2C12 WT and DNAJB6 KO myoblasts differentiated into myotubes for 6 days and stained for myosin. DNAJB6 KO myoblasts fused to form large myotubes with enhanced fusion index (B). Error bars represent the standard error of 3 independent experiments. (C) Western blot confirmed increased inactive GSK3β- P(ser-9) in KO myoblasts. GSK3β-P(ser-9)/GSK3β-total ratio is given below western blot. (D) Increased β-catenin transcriptional activity in DNAJB6 KO myoblasts compared with WT myoblasts measured by TOPFLASH luciferase assay. (E) Increased NFATc3 transcriptional activity in DNAJB6 KO myoblasts compared with WT myoblasts. Error bars represent the standard error of at least 2 independent experiments. GSK3β = glycogen synthase kinase-β;KO= knockout; NFATc3 = nuclear factor of activated T cells cytoplasmic 3; WT = wild type.

liquid chromatography with tandem mass spectrometry 1). Ontological analysis of the 27 proteins increased in KO analysis, we quantified 2035 proteins in the 2 forward cells identified categories such as sarcomere, Z disc, and experiments, 2215 proteins in the 2 reverse experiments, and muscle contraction (figure 1E). Notably, these proteins in- a total of 2445 distinct proteins overall (figure 1D). Nearly cluded several Z-disc proteins such as desmin, α-actinin, 75% of the proteins quantified were present in both forward FHL-1, and PDZ-Lim domain proteins 1 and 3. It also and reverse experiments (figure 1D). For subsequent data identified the co-chaperone DNAJB4 and small HSPs analysis, we used only proteins present in both duplicates of HSPB1 and HSPB5 (αβ-crystallin). Reversal of thresholds to the forward and reverse experiments (figure 1D). To identify identify proteins decreased in DNAJB6 KO cells identified proteins that accumulate in DNAJB6 KO cells, we used 2 46 proteins belonging to several ontological categories in- thresholds: a 1.5-fold change in forward KO/WT experiments cluding laminin complex and collagen catabolic process and a 0.6-fold change in reverse WT/KO experiments. Based (figure 1, D and F, table e-1, links.lww.com/NXG/A145). on these criteria, 27 proteins were identified (figure 1D, table Immunoblotting of lysates from control and DNAJB6 KO

6 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Figure 3 DNAJB6 disease mutation impact on GSK3β dephosphorylation and downstream signaling pathways

(A) Western blot of skeletal muscle lysates from 3-month old LGMD1D mutant mice (F93Lb, n = 2) vs control (WTb, n = 2) demonstrating significantly reduced inactive GSK3β- P(ser-9) in mutant mice. Error bars represent the standard error. (B) Quantitation of western blot. Dual- luciferase assay demonstrating reduced β-catenin (C) and NFATc3 (D) transcriptional activity in HeLa cells transfected with mutant DNAJB6b constructs compared with unstimulated controls. β-catenin transcriptional activity was stimulated via treatment with 20 mM LiCl for 12 hours. NFATc3 transcriptional activity was stimulated by overexpression of NFATc3 via transient transection. Error bars in C and D represent the standard error from 3 separate experiments. (E) Proposed model for DNAJB6’s spectrum of impact on GSK3β activation and myogenesis. GSK3β = glycogen synthase kinase-β; LGMD1D = limb- girdle muscular dystrophy 1D; NFATc3 = nuclear factor of activated T cells cytoplasmic 3; WT = wild type.

myoblasts for several of these proteins confirmed their ac- and disorganized sarcomere organization on electron micros- cumulation (figure 1G). Skeletal muscle from LGMD1D copy (figure1,HandI).Overall,thissuggeststhattheabsence mice similarly demonstrates an accumulation of several of of DNAJB6 leads to impaired organization and accumulation of these Z-disc proteins.8 When differentiated into myotubes, sarcomeric proteins. These findings are similar to the myofi- DNAJB6 KO cells contained abnormal myofibrillar struc- brillar abnormalities noted in mouse and human LGMD1D tures characterized by irregular myosin immunofluorescence skeletal muscle.3,8

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 7 Figure 4 GSK3β inhibition improves muscle strength in LGMD1D mice

(A) Diagram demonstrating DNAJB6’s role in chaperoning the dephosphorylation of GSK3β-P(ser-9) and treatment strategy with GSK3β inhibitor, LiCl. (B) Functional testing (wire hang and grip strength) of 3-month-old DNAJ6b-WT and DNAJB6b-F93L mice treated daily with LiCl vs no treatment (NT). Two independent experiments were performed for a total of 8 mice per condition. Strength of DNAJB6b-F93L mice treated with LiCl significantly improves over the course of the month and reaches levels similar to DNAJB6b-WT mice. Error bars represent the standard error. *p < 0.05, **p< 0.01, ***p < 0.005. GSK3β = glycogen synthase kinase-β; WT = wild type.

KO of DNAJB6 in myoblasts impairs GSK3β state in muscle. We used skeletal muscle lysate from activation and enhances myogenesis 3-month-old LGMD1D model mice that express a V5- When differentiated for 6 days, C2C12 myoblasts form tagged DNAJB6b-F93L transgene and control mice that myotubes with a fusion index (nuclei contained in express a DNAJB6b-WT transgene.8 Of interest, we found myotubes/total nuclei) of approximately 20% (figure 2, A a near complete absence of GSK3β-P(ser-9) in the and B). Surprisingly, we found that DNAJB6 KO myotubes LGMD1D mice (figure 3, A and B). This suggested that have an enhanced fusion index (77%) and form enlarged DNAJB6 disease mutations increase GSK3β de- myotubes (figure 2, A and B). This suggested that DNAJB6 phosphorylation to its active state. We next evaluated the may play a role in myogenic signaling pathways. DNAJB6 is impact of DNAJB6 mutations on myogenic signaling path- known to chaperone the dephosphorylation (activation) of ways downstream of GSK3β. We used a dual luciferase assay GSK3β, an important signaling kinase that suppresses sev- to measure β-catenin and NFATc3 transcriptional activity in eral myogenic signaling pathways.10,14,15,20 Compared with HeLa cells transfected with GFP-tagged DNAJB6 con- WT, DNAJB6 KO myoblasts and myotubes contained in- structs. We treated cells with 20 mM LiCl, a GSK3β increased levels of phosphorylated (inactive) GSK3β-P(ser-9) hibitor, to stimulate β-catenin transcriptional activity (figure (figure 2C). This suggests that the absence of DNAJB6 3C). We overexpressed NFATc3 via transient transfection of is associated with impaired GSK3β-P(ser-9) de- cells to stimulate NFATc3 transcriptional activity. We found phosphorylation and supports DNAJB6’s proposed role in that both β-catenin and NFATc3 transcriptional activity GSK3β activation.10 GSK3β9s impact on myogenesis is were suppressed by the presence of various DNAJB6 thought to be mediated through suppression of β-catenin mutants (figure 3, C and D). – and NFATc3 signaling.13 15 We therefore evaluated β-catenin and NFATc3 transcriptional activity using dual lucif- These findings illustrate DNAJB6’s spectrum of impact on erase assays. We found that both NFATc3 and β-catenin GSK3β9s activity: absence of DNAJB6 is associated with transcriptional activity were increased in DNAJB6 KO impaired dephosphorylation of GSK3β-P(ser-9), whereas myoblasts (figure 2, D and E). These findings suggest that dominant disease-causing mutations in DNAJB6 enhance the enlarged myotubes and enhanced fusion index resulting dephosphorylation GSK3β-P(ser-9) to its active form from loss of DNAJB6 may be related to impaired GSK3β- (figure 3E). P(ser-9) dephosphorylation and increased β-catenin and NFATc3 transcriptional activity. GSK3β inhibitor LiCl improves strength, muscle mass, and histopathology in LGMD1D mice LGMD1D mutations enhance GSK3β To test the therapeutic potential of GSK3β inhibition (figure dephosphorylation in skeletal muscle 4A), we treated 3-month-old control mice overexpressing We next investigated the impact of dominant DNAJB6 DNAJB6b-WT and mutant mice overexpressing DNAJB6b- disease-causing mutations on GSK3β9s phosphorylation F93L with either vehicle (dimethyl sulfoxide) or LiCl via

8 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Figure 5 GSK3β inhibition improves muscle size in LGMD1D mice

(A) Mass of selected muscles after 1 month of treatment. Note visible difference in the size of F93L muscles treated with LiCl. (B) H&E staining of TA from LiCl treated or untreated F93L mice illustrating increased muscle fiber size and reduced fiber size variability. Two representative examples are provided from the tibialis anterior. (C) Quantitation of fiber CSA profile demonstrates an overall increase in the muscle fiber size after LiCl treatment in F93L mice and no change in LiCl-treated control mice. (D) Average muscle fiber cross-sectional area of LiCl-treated F93L mice significantly improves but does not reach control levels. LiCl does not alter the muscle fiber size of control mice. Error bars from A and D represent the standard error. (E) Western blot of skeletal muscle lysates in LiCl-treated and untreated mice illustrates no improvement in accumulated sarcomeric proteins or RNA-binding proteins in F93L mice. WT = wild type. daily intraperitoneal injection for 1 month. Results were Discussion obtained from 4 mice per group. Two independent experiments were performed for a total of 8 mice per condition. This study demonstrates that DNAJB6’sroleinskeletal We used a dose of 250 mg/kg body weight to mimic ther- muscle involves not only sarcomeric protein quality con- apeutic serum levels in humans.21,22 Skeletal muscle function trol but also suppression of myogenic signaling pathways of LiCl-treated DNAJB6b-F93L mice, as measured by grip important for myoblast differentiation, myotube fusion, strength and inverted wire hang test,normalizedtothatof skeletal muscle hypertrophy, and regeneration. Many control mice over the course of the month (figure 4B). The other skeletal muscle chaperones also have dual roles in mass and visible size of selected muscles and muscle fiber protein quality control and modulation of signaling path- CSA were significantly increased in DNAJB6b-F93L mice ways. The co-chaperone BAG3 facilitates autophagic sorting treated with LiCl (figure 5, A-D). Despite improvements in of damaged filamin to lysosomes while also engaging in strength and muscle fiber size, western blot analyses of YAP/TAZ mechanotransduction signaling.23 αβ-crystallin not skeletal muscle lysates demonstrated no change in accu- only functions as a desmin chaperone at the Z disc but also mulated sarcomeric proteins or RNA-binding proteins seen modulates NF-κβ and TGF-β2 signaling.24,25 HSP70 and with DNAJB6 mutations (figure 5E). HSP90 not only play key roles in myofibrillogenesis but also act

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 9 as circulating cachexins by activating the p38β-MAPK-C/EBPβ GSK3β and may also act indirectly through enhanced phos- catabolic signaling pathway.26 Although these dual functions phorylation of GSK3β.33 Although GSK3β activity suppresses may initially seem unrelated, regulation of protein synthesis is myogenesis and blunts skeletal muscle regeneration, it has central to both these stress signaling pathways and maintaining also been linked to formation of aggregates in several dis- balanced protein homeostasis. It is therefore not surprising to orders, making GSK3β inhibition a good strategy for find chaperones that coordinate protein quality control with LGMD1D treatment.34,35 LiCl also stimulates autophagy in- signaling pathways regulating protein synthesis. dependent of GSK3β, which may benefit LGMD1D further.36 Given LiCl’s dual impact on GSK3β and autophagy, many Our comparisons between DNAJB6 KO and dominant have used it to treat in vivo models of neurologic disorders LGMD1D models facilitated many interesting observations of with protein aggregate pathology and GSK3β dysfunction, DNAJB6’s dual roles in skeletal muscle. For instance, KO of such as Huntington disease, Alzheimer disease, SOD1 DNAJB6 in myoblasts and myotubes recapitulated the myo- amyotrophic lateral sclerosis, Parkinson disease, and inclusion – – fibrillar disorganization and accumulation of sarcomeric pro- body myositis.33 35,37 40 In addition to showing therapeutic teins seen in LGMD1D patients and animal models.3,8 In efficacy in these animal models, LiCl improved biochemical general, protein aggregation in myopathies may occur from markers of disease and histopathologic evidence of – mutations causing a protein itself to misfold and aggregate aggregates.34,37 40 These previous studies raise the possibility (e.g., desmin) or from mutations in protein quality control that the significant improvement of LGMD1D mice treated machinery (e.g., αβ-crystallin) causing other proteins to with LiCl was due to enhanced autophagy and clearance of aggregate (desmin). With DNAJB6, the absence of sarco- accumulated sarcomeric proteins. Of interest, the accumu- meric chaperone activity could explain the altered myofi- lations of sarcomeric and RNA binding proteins in mutant brillar assembly and accumulation of sarcomeric proteins in mousemusclewerenotalteredbyLiCltreatment(figure KO cells. Other DNAJB6 KO models have similarly 5E). This lack of improvement may simply be due to resulted in aggregation of client proteins.27,28 Because pathologic changes lagging behind functional improve- LGMD1D is not due to a KO or loss of DNAJB6, one could ments. However, it may actually suggest that LGMD1D speculate that the vacuolar and aggregate myopathology pathogenesis is due not only to DNAJB6 dysfunction seen with disease mutations results from a dominant neg- in protein quality control but also to GSK3β-related sig- ative effect on DNAJB6’s sarcomeric chaperone activities. naling pathways. Further studies are required to clarify, Lending support to this theory, mutations in VCP, BAG3, however, that LiCl may be an ideal therapeutic option for and CRYAB are also thought to cause myopathies with LGMD1D. vacuoles and aggregates from a dominant negative – mechanism.29 31 Study funding This work was supported by NIH AG031867 (C.C.W.), However, several findings in this study do not fit this theory. AG042095 (C.C.W.), and AR068797 (C.C.W. and H.L.T.) Specifically, dominant mutations and absence of DNAJB6 had and the Muscular Dystrophy Association (C.C.W.). opposite effects on GSK3β phosphorylation status and downstream myogenic signaling pathways. We found an increased size Disclosure of DNAJB6 KO myotubes owing to an enhanced fusion index. A.R. Findlay has received foundation/society research This may be related to impaired dephosphorylation of GSK3β- support from the American Academy of Neurology. R. P(ser-9) and derepression of downstream myogenic signaling Bengoechea has served on the editorial board of BioMed – pathways such as β-catenin and NFATc3.9 12,32 Contrary to KO Central Neuroscience. S.K. Pittman reports no disclosures. cells, dominant DNAJB6 mutations were associated with an T.-F. Chou has served on the editorial board of the Journal enhanced dephosphorylation of GSK3β-P(ser-9) and sup- of Cancer Sciences; holds patents in Methods and Compo- pressed β-catenin and NFATc3 transcriptional activity. Although sitions for Inhibition of the Transitional Endoplasmic Re- DNAJB6 is known to chaperone the dephosphorylation of ticulum ATPase; and has received government funding GSK3β-P(ser-9)tomaintainitinanactivestate,theexact from the NIH. H.L. True has served on the editorial board mechanism linking DNAJB6 disease mutations with enhanced of PLOS Pathogens and has received government funding GSK3β-P(ser-9) dephosphorylation is not clear. One could from the NIH. C.C. Weihl has served on advisory boards of speculate that it may be related to a gain of function from in- Novartis, Acceleron, and Sarepta; has served on the edi- creased stability of the mutant DNAJB6 protein; however, fur- torial board of Neuromuscular Disorders;hasreceivedgov- ther studies are needed.4,8 ernment funding from the NIH; and has received foundation/society research support from the Muscular As DNAJB6 appears to have multiple functions within skeletal Dystrophy Association and the Myositis Association. Dis- muscle, it is not surprising that mutations may cause disease closures available: Neurology.org/NG. through multiple mechanisms. LiCl is well suited to address alterations in both GSK3β signaling and protein aggregation. Publication history It acts as a competitive inhibitor of the adenosine tri- Received by Neurology: Genetics October 19, 2018. Accepted in final phosphate–magnesium-dependent catalytic activity of form February 4, 2019.

10 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG 14. van der Velden JL, Schols AM, Willems J, Kelders MC, Langen RC. Glycogen syn- ff Appendix Author contributions thase kinase 3 suppresses myogenic di erentiation through negative regulation of NFATc3. J Biol Chem 2008;283:358–366. 15. Vyas DR, Spangenburg EE, Abraha TW, Childs TE, Booth FW. GSK-3beta negatively Name Location Role Contribution regulates skeletal myotube hypertrophy. Am J Physiol Cell Physiol 2002;283: – Andrew R. Washington Author Design and conceptualization C545 C551. Findlay, MD University St. of the study, acquisition of 16. Dai YS, Xu J, Molkentin JD. The DnaJ-related factor Mrj interacts with nuclear factor Louis data, interpretation of data, of activated T cells c3 and mediates transcriptional repression through class II histone – writing of the manuscript, and deacetylase recruitment. Mol Cell Biol 2005;25:9936 9948. revision of the manuscript for 17. Kato M, Chou TF, Yu CZ, DeModena J, Sternberg PW. LINKIN, a new trans- intellectual content. membrane protein necessary for cell adhesion. Elife 2014;3:e04449. 18. Sapir A, Tsur A, Koorman T, et al. Controlled sumoylation of the mevalonate pathway Rocio Washington Author Acquisition of data and enzyme HMGS-1 regulates metabolism during aging. Proc Natl Acad Sci USA 2014; Bengoecha, University St. revision of the manuscript for 111:E3880–E3889. PhD Louis intellectual content. 19. Kim HJ, Kim NC, Wang YD, et al. Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS. Nature 2013;495: Sara K. Washington Author Acquisition of data. 467–473. Pittman, BS University St. 20. Pansters NA, Schols AM, Verhees KJ, et al. Muscle-specific GSK-3β ablation accel- Louis erates regeneration of disuse-atrophied skeletal muscle. Biochim Biophys Acta 2015; 1852:490–506. Tsui-Fen Harbor- Author Acquisition of data, 21. Patel NC, DelBello MP, Bryan HS, et al. Open-label lithium for the treatment of Chou, PhD UCLA interpretation of data, and adolescents with bipolar depression. J Am Acad Child Adolesc Psychiatry 2006;45: Medical revision of the manuscript for 289–297. Center intellectual content. 22. Nair AB, Jacob S. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm 2016;7:27–31. Heather L. Washington Author Revision of the manuscript for 23. Ulbricht A, Eppler FJ, Tapia VE, et al. Cellular mechanotransduction relies on tension- True, PhD University St. intellectual content. induced and chaperone-assisted autophagy. Curr Biol 2013;23:430–435. Louis 24. Adhikari AS, Singh BN, Rao KS, Rao CM. αB-crystallin, a small heat shock protein, modulates NF-κB activity in a phosphorylation-dependent manner and protects Conrad C. Washington Author Design and conceptualization muscle myoblasts from TNF-α induced cytotoxicity. Biochim Biophys 2011;1813: Weihl, MD, University St. of the study, interpretation of 1532–1542. PhD Louis data, and revision of the 25. Nahomi RB, Pantcheva M, Nagaraj RH. αB-Crystallin is Essential for the TGF-β2- manuscript for intellectual mediated Epithelial to Mesenchymal Transition of Lens Epithelial Cells. Biochem J content. 2016;473:1455–1469. 26. Zhang G, Liu Z, Ding H, et al. Tumor induces muscle wasting in mice through releasing extracellular Hsp70 and Hsp90. Nat Commun 2017;8:589. 27. Watson ED, Geary-Joo C, Hughes M, Cross JC. The Mrj co-chaperone mediates keratin turnover and prevents the formation of toxic inclusion bodies in trophoblast References cells of the placenta. Development 2007;134:1809–1817. 1. Smith DA, Carland CR, Guo Y, Bernstein SI. Getting folded: chaperone proteins in muscle 28. Aprile FA, Källstig E, Limorenko G, Vendruscolo M, Ron D, Hansen C. The mo- development, maintenance and disease. Anat Rec (Hoboken) 2014;297:1637–1649. lecular chaperones DNAJB6 and Hsp70 cooperate to suppress α-synuclein aggrega- 2. Harms MB, Sommerville RB, Allred P, et al. Exome sequencing reveals tion. Sci Rep 2017;7:9039. DNAJB6 mutations in dominantly-inherited myopathy. Ann Neurol 2012;71: 29. Kitami MI, Kitami T, Nagahama M, et al. Dominant-negative effect of mutant valosin- 407–416. containing protein in aggresome formation. FEBS Lett 2006;580:474–478. 3. Sandell S, Huovinen S, Palmio J, et al. Diagnostically important muscle pathology in 30. Myers VD, McClung JM, Wang J, et al. The multifunctional protein BAG3: A novel DNAJB6 mutated LGMD1D. Acta Neuropathol Commun 2016;4:9. therapeutic target in cardiovascular disease. JACC Basic Transl Sci 2018;3: ff 4. Sarparanta J, Jonson PH, Golzio C, et al. Mutations a ecting the cytoplasmic func- 122–131. tions of the co-chaperone DNAJB6 cause limb-girdle muscular dystrophy. Nat Genet 31. Selcen D, Engel AG. 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Seki N, Hattori A, Hayashi A, Kozuma S, Miyajima N, Saito T. Cloning, tissue clusion body myositis model via glycogen synthase kinase-3β. Ann Neurol 2008;64: expression, and chromosomal assignment of human MRJ gene for a member of the 15–24. DNAJ protein family. J Hum Genet 1999;44:185–189. fi 35. Beurel E, Grieco SF, Jope RS. Glycogen synthase kinase-3 (GSK3): regulation, 8. Bengoechea R, Pittman SK, Tuck EP, True HL, Weihl CC. Myo brillar disruption – and RNA-binding protein aggregation in a mouse model of limb-girdle muscular actions, and diseases. Pharmacol Ther 2015;148:114 131. dystrophy 1D. Hum Mol Genet 2015;24:6588–6602. 36. Motoi Y, Shimada K, Ishiguro K, Hattori N. Lithium and autophagy. ACS Chem – 9. Meng E, Shevde LA, Samant RS. Emerging roles and underlying molecular mecha- Neurosci 2014;5:434 442. nisms of DNAJB6 in cancer. Oncotarget 2016;7:53984–53996. 37. Pouladi MA, Brillaud E, Xie Y, et al. NP03, a novel low-dose lithium formulation, is 10. Mitra A, Menezes ME, Pannell LK, et al. DNAJB6 chaperones PP2A mediated de- neuroprotective in the YAC128 mouse model of Huntington disease. Neurobiol Dis – phosphorylation of GSK3β to downregulate β-catenin transcription target, osteo- 2012;48:282 289. pontin. Oncogene 2012;31:4472–4483. 38. Zhang X, Heng X, Li T, et al. Long-term treatment with lithium alleviates memory fi β ’ 11. Mitra A, Menezes ME, Shevde LA, Samant RS. DNAJB6 induces degradation of beta- de cits and reduces amyloid- production in an aged Alzheimer s disease transgenic catenin and causes partial reversal of mesenchymal phenotype. J Biol Chem 2010;285: mouse model. J Alzheimers Dis 2011;24:739–749. 24686–24694. 39. Fornai F, Longone P, Cafaro L, et al. Lithium delays progression of amyotrophic 12. Menezes ME, Mitra A, Shevde LA, Samant RS. DNAJB6 governs a novel regulatory lateral sclerosis. Proc Natl Acad Sci USA 2008;105:2052–2057. loop determining Wnt/β-catenin signalling activity. 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Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 11 ARTICLE OPEN ACCESS Novel PNKP mutations causing defective DNA strand break repair and PARP1 hyperactivity in MCSZ

Ilona Kalasova, PhD,* Hana Hanzlikova, PhD,* Neerja Gupta, MD, DM, Yun Li, MD, Janine Altmuller,¨ MD, Correspondence John J. Reynolds, PhD, Grant S. Stewart, PhD, Bernd Wollnik, MD, Gokhan¨ Yigit, PhD, and Dr. Hanzlikova hana.hanzlikova@ Keith W. Caldecott, PhD img.cas.cz, or Dr. Gupta [email protected], or Neurol Genet 2019;5:e320. doi:10.1212/NXG.0000000000000320 Dr. Caldecott [email protected] Abstract Objective To address the relationship between novel mutations in polynucleotide 5’-kinase 3’-phosphatase (PNKP), DNA strand break repair, and neurologic disease.

Methods We have employed whole-exome sequencing, Sanger sequencing, and molecular/cellular biology.

Results We describe here a patient with microcephaly with early onset seizures (MCSZ) from the Indian sub-continent harboring 2 novel mutations in PNKP, including a pathogenic mutation in the fork-head associated domain. In addition, we conﬁrm that MCSZ is associated with hyperactivation of the single-strand break sensor protein protein poly (ADP-ribose) polymerase 1 (PARP1) following the induction of abortive topoisomerase I activity, a source of DNA strand breakage associated previously with neurologic disease.

Conclusions These data expand the spectrum of PNKP mutations associated with MCSZ and show that PARP1 hyperactivation at unrepaired topoisomerase-induced DNA breaks is a molecular feature of this disease.

*These authors contributed equally. From the Department of Genome Dynamics (I.K., H.H., K.W.C.), Institute of Molecular Genetics of the Czech Academy of Sciences, Czech Republic; Genome Damage and Stability Centre (H.H., K.W.C.), School of Life Sciences, University of Sussex, Falmer, Brighton, UK; Institute of Human Genetics (Y.L., B.W., G.Y.), University Medical Center Gottingen,¨ Germany; Cologne Center for Genomics (J.A.), University of Cologne, Germany; Institute of Cancer and Genomic Sciences (J.J.R., G.S.S.), College of Medical and Dental Sciences, University of Birmingham, UK; and Division of Genetics (N.G.), Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India.

The Article Processing Charge was funded by the ERC. 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.

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary CCG = Cologne Center for Genomics; CPT = camptothecin; FHA = fork-head associated; HRP = horseradish peroxidase; MCSZ = microcephaly with early onset seizures; PARP1 = protein poly (ADP-ribose) polymerase 1; PNKP = polynucleotide 59- kinase 39-phosphatase; SCAN-1 = spinocerebellar ataxia with axonal neuropathy-1; SSBR = single-strand break repair; WES = whole-exome sequencing.

Mutations in DNA single-strand break repair (SSBR) associated with developmental delay, microcephaly, and proteins are associated with hereditary neurologic seizures whereas the latter is a degenerative disease asso- disease.1,2 Recently, using a mouse model in which the ciated with cerebellar atrophy, ataxia, and oculomotor SSBR protein Xrcc1 is mutated, we demonstrated that apraxia. Intriguingly, some individuals with PNKP muta- hyperactivation of the SSB sensor protein poly(ADP- tions exhibit phenotypic aspects of both disorders, and in ribose) polymerase 1 (PARP1) is a likely source of the some cases can overlap with Charcot-Marie-Tooth neuropathology induced by SSBs.3 During SSBR, the en- disease.9,10 To explain the molecular basis and phenotypic zyme polynucleotide 59-kinase 39-phosphatase (PNKP) diversity of PNKP-associated disease will require an un- can employ either DNA 39-phosphatase and/or DNA 59- derstanding of the spectrum of PNKP mutations associated kinase activities to restore ligatable termini at DNA strand with disease, and of the inﬂuence of these mutations on breaks, and is recruited to SSBs by interacting with XRCC1 DNA repair. Here, we identify 2 novel disease mutations in via an amino-terminal fork-head associated (FHA) PNKP in a patient with MCSZ from the Indian sub- – domain.4 6 Strikingly, PNKP mutations are associated with continent. We identify a defect in DNA strand break repair 2 apparently distinct neurologic diseases: microcephaly with in patient-derived primary ﬁbroblasts and that these MCSZ early onset seizures (MCSZ) and ataxia with oculomotor cells are associated with hyperactivation of the SSB sensor apraxia 4.7,8 The former is a neurodevelopmental disease protein, PARP1.

Figure 1 Novel PNKP mutations in an individual from India with MCSZ

(A), patient MRI. T2 axial and FLAIR axial images showing microcephaly with diffuse white matter signal changes. There is mild ventriculomegaly with generalized atrophy and cystic changes in bilateral temporal lobes. There is mild hypoplasia of inferior vermis as seen on sagittal T2 weighted image. (B), Pedi- gree of the proband (black square). The tri- angles indicate pregnancies that did not go to term. Note that one of these (black triangle) was associated with antenatal detection of microcephaly. (C), Electropherograms showing the compound heterozygous PNKP mutations in the proband and heterozygosity in the parents. (D), cartoon of PNKP and its functional domains. The proband mutations, confirmed by Sanger sequencing, are shown in red. Epit- opes of the N- and C-terminal polyclonal antibodies used in this study are indicated with horizontal blue lines. The inset shows the impact of the deletion caused by c.1295_ 1298+6del on the splice site between exon 14 and intron 14. FLAIR = fluid-attenuated inversion recovery; MCSZ = microcephaly with early onset seizures; PNKP = polynucleotide 59- kinase 39-phosphatase.

2 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Figure 2 PNKP protein levels and PNKP activity in MCSZ patient-derived fibroblasts

(A), western blot showing PNKP protein levels in 1BR and PNKP patient fibroblasts after 6 hours incubation with DMSO vehicle or 10 μM proteasome inhibitor MG132. Two different antibodies were employed, directed at the N- or C-terminus as indicated. p53 and GAPDH detection was employed as a positive control for proteasome inhibition by MG132 and as a loading control, respectively. Asterisks denote nonspecific bands. (B), PNKP indirect immunofluorescence with the antibodies described above in 1BR and PNKP patient fibroblasts. Scale bar 50 μm (C), PNKP DNA 39-phosphatase and DNA 59-kinase activity in wild type 1BR and PNKP patient cell extracts. Top, A cartoon of the PNKP oligonucleotide duplex substrate, harboring a SSB with 39-phosphate and 59-hydroxyl termini. Bottom; reaction products of PNKP activity. The positions of the substrate (39-P) and product (39-OH) of PNKP 39-phosphatase activity (left panel), and of the substrate (59-OH) and product (59-P) of the 59-kinase activity (right panel) are shown. The 40-mer ligation product of the repair reaction, which is labeled with both TAMRA and FAM, is indicated by an arrow in the top panel and is shown in the bottom panels with high contrast (D), PNKP DNA 39-phosphatase activity in cell extracts from 1BR and PNKP patient fibroblasts additionally transfected with PNKP siRNA. The 40-mer ligation products are highlighted in the middle (high contrast) panel. The PNKP protein levels in the siRNA transfected 1BR and patient fibroblasts are shown at the bottom. MCSZ = microcephaly with early onset seizures; PNKP = polynucleotide 59-kinase 39-phosphatase; TAMRA = tetramethylrhodamine.

Methods receding forehead, high nasal bridge, bilateral up slant, receding chin, dental crowding and teeth caries, spasticity, Patient case report brisk reflexes, and striatal toe. Ataxia could not be ascer- The patient is currently a 8.5-year-old boy who was born at tained. He has a café-au-lait spot on his right upper thigh and term with microcephaly (occipito-frontal circumference 29 dark hyperpigmented skin. He also has a large amplitude cm, –3 SD) and below-normal weight (−2.8 SD). Seizures stereotype lower limb movements and midline hand ster- began at day 15 of his life and he had severe developmental eotypes. Hearing evaluation revealed an absence of waves at delay. He established head control at 6 months and started 50dBintheleftear.Atyear4.5,T2axialandfluid-attenuated sitting with support at month 7, but never attained walking. inversion recovery axial MRI images revealed microcephaly He developed monosyllable speech at month 8 but since then with diffuse white matter signal changes (figure 1A). There has exhibited a complete developmental arrest. Examination was mild ventriculomegaly with generalized atrophy and at ;year 8.5 revealed severe microcephaly (−14.9 SD), short cystic changes in bilateral temporal lobes. Mild hypoplasia of stature (−6.86 SD), and failure to thrive (weight = −5.83SD). the inferior vermis was evident on the sagittal T2 weighted He is a thin child with a slender build, convergent squint, image. His CT scan brain did not show any calcifications.

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 3 Figure 3 Elevated levels of TOP1-induced DNA breaks and poly(ADP-ribose) in MCSZ fibroblasts

(A) The accumulation of CPT-induced DNA strand breaks in 1BR and patient fibroblasts, quantified by alkaline comet assays. Cells were treated with DMSO vehicle or 10 μM CPT for 45 minutes. Data are the average comet tail moment of 100 cells per sample and are the mean (±SEM) of 4 independent experiments. (B) Representative ScanR images (left) and quantitation (right) of poly (ADP-ribose) levels in control 1BR and PNKP patient fibroblasts incubated in the presence or absence of CPT as above. Data are the mean (±SEM) of 3 independent experiments. CPT = camptothecin; DMSO = dimethyl sulphoxide; MCSZ = microcephaly with early onset seizures; PNKP = polynucleotide 59’-kinase 39’-phosphatase; SEM = standard error of the mean.

EEG at 5 years revealed resolving hypsarryhthmia and reads, a minimum quality score of 10), rare (minor allele evolving multifocal epilepsy. His karyotype, liver function, frequency < 0.5% in the databases of the exome aggregation and renal function were all normal. His serum uric acid was consortium and single nucleotide polymorphisms) auto- 1.2mg/dL.Hisserumα fetoprotein levels are 22.9 ng/mL somal recessive variants. (normal range 0.89–8.78 ng/mL). His lipid profile showed mildly high triglycerides 161 (normal range 50–150) with Mutation screening low density lipoprotein/high density lipoprotein ratio 3.44. Variants identified by WES were amplified from DNA of the He is at present non-ambulatory, can sit with support, and index patients, and its parents and PCR products were se- does not have seizures. quenced by BigDye Terminator method on an ABI 3500 × L Genetic Analyzer (Life Technologies, Germany). Iden- Whole exome sequencing tified mutations were re-sequenced in independent Whole-exome sequencing (WES) was performed on DNA experiments. extracted from blood lymphocytes of the index patient using the SureSelectXT Human All Exon V6 enrichment kit Primary human fibroblasts (Agilent technologies) on an Illumina HiSeq4000 se- Control human fibroblast 1BR3 (denoted 1BR in the text) quencer. In total, we obtained a mean coverage of 84 reads, and PNKP patient-derived skin fibroblasts, obtained with and 96.3% of target were covered more than 10×. WES data appropriate patient consent, were grown in minimum essen- analysis and variant filtering was conducted using the tial media (Gibco) supplemented with 15% fetal bovine se- exome analysis pipeline “varbank” of the Cologne Center rum, 2 mM glutamine, the antibiotics penicillin (100 units/ for Genomics (CCG, varbank.ccg.uni-koeln.de/) and data mL), and streptomycin (100 μg/mL) in a humidified atmo- fi were ltered for high-quality (coverage of more than 6 sphere of 5% CO2 at low oxygen (5%) at 37°C.

4 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Antibodies, western blotting, and indirect mock-treatment or treatment with 10 μM CPT for 60 minutes immunofluorescence microscopy at 37°C. The average comet tail moment in 100 cells per Primary antibodies were anti-pan-ADP-ribose binding re- sample was evaluated by Comet Assay IV software (Percep- agent (MABE1016, Millipore), anti-PNKP FHA (SK3195), tive Instruments). anti-PNKP C-terminal (ab18107, Abcam), anti-p53 (OP29, Millipore), and anti-GAPDH (sc-47724, Santa Cruz). Sec- Standard protocol approvals, registrations, ondary antibodies were horseradish peroxidase (HRP) and patient consents conjugated goat anti-rabbit (170-6515, Bio-Rad) and goat Ethical approval and informed parental consent for the use anti-mouse (170-6516, Bio-Rad) and for indirect immuno- and publication of patient information and patient derived cell fluorescence donkey anti-rabbit Alexa 488 (A21206, Invi- lines was obtained from the appropriate Institutional and trogen). For western blotting, cells were lysed in sodium regional committees. No vertebrate animal models were dodecyl sulphate (SDS) sample buffer and subjected to SDS- employed in this work. PAGE, transferred onto nitrocellulose membrane and fi detected by speci c antibodies combined with HRP conju- Data availability fl gated secondary antibodies. For indirect immuno uores- Data will be provided on request at https://sussex.figshare. cence microscopy, cells were cultured on glass coverslips and com/10.25377/sussex.7836653. treated where indicated with 10 μM camptothecin (CPT) (Sigma) for 45 minutes. Cells were fixed with 4% para- formaldehyde and immunostained as described previously.3 Results Images were taken using a DMi6000 microscope (Leica) with 40x dry objective. Automated wide-field microscopy Individuals with neurologic disease resulting from mutations was performed on ScanR system (Olympus) with ScanR in PNKP protein have been identified in the Americas, Image Acquisition and Analysis Software, 40x/0.95NA Europe, Middle East, and Japan, but surprisingly not yet on (UPLSAPO 2 40X) dry objective. the Indian subcontinent. Here, we describe the first Indian patient with biallelic mutations in PNKP. The proband is an PNKP biochemical activity assays 8.5 year old boy with microcephaly, early-onset seizures, and PNKP substrate was prepared by annealing equimolar amounts developmental delay (MCSZ). Seizures were detected from of the fluorophore-labeled deoxyriboligonucleotides (Midland day 15 of life and MRI revealed microcephaly with diffuse Certified Reagent Company) “S1” [5’-(TAMRA)-TAG- white matter signal changes and generalized atrophy in the CATCGATCAGTCCTC-39-P], “S2” [59-OH-GAGGTC- bilateral temporal lobes (figure 1A). A detailed case report is TAGCATCGTTAGTCA-(6-FAM)-3’] and a complementary presented in the Methods. strand oligonucleotide [59-TGACTAACGATGCTA- GACCTCTGAGGACTGATCGATGCTA-3’] in annealing WES on DNA extracted from blood lymphocytes from the buffer (10 mM Tris pH 7.5, 200 mM NaCl, 1 mM EDTA). proband identified 2 heterozygous variants in PNKP (NM_ Cell-free protein extracts were prepared in lysis buffer [25 mM 007254.3); c.63dupC and c.1295_1298+6delCCAGGTAGCG Tris, pH 7.5, 10 mM EDTA, 10 mM EGTA, 100 mM NaCl, 1% (figure 1B and C). Sanger sequencing confirmed the presence of Triton X-100, cOmplete protease inhibitors (Roche)] and in- both mutations in the proband and their heterozygosity in the cubated (25 μg total protein) with 50 nM substrate and 1 μM parents. c.63dupC is a 1 base pair duplication in exon 2 and was single-stranded nuclease competitor oligonucleotide [59- inherited from the mother, and c.1295_1298+- AAAGATCACAAGCATAAAGAGACAGG-3’] in reaction 6delCCAGGTAGCG is a 10 base pair deletion that was buffer (25 mM Tris, pH 7.5, 130 mM KCl, 10 mM MgCl2, inherited from the father (figure 1B and C). The 1 base pair 1mMDTT,1mMATP)at37°Cfor10minutesin50μL duplication is predicted to cause a frameshift resulting in reactions and were terminated by addition of 50 μL quenching nonsense-mediated mRNA decay and/or truncated PNKP buffer (90% formamide, 50 mM EDTA, 0.006% Orange G). 10 protein (p.Ile21Hisfs*37) and is the first unambiguously dele- μL was separated on 20% denaturing polyacrylamide gels and terious mutation identified in the FHA domain (figure 1D). The analyzed on a PharosFX Molecular Imager System (Bio-Rad). 10 base pair deletion spans the last 4 nucleotides of exon 14 and Where indicated, cells were transfected with mix of either the first 6 nucleotides of intron 14 (figure 1C) and deletes PNKP siRNA #1: 59-CCGGAUAUGUCCACGUGAA-39 and a donor splice-site, leading most likely to nonsense-mediated PNKP siRNA #2: 59-GGAAACGGGUCGCCAUCGA-39 or mRNA decay and/or a synthesis of a truncated protein. non-target siRNA #1: 59-UGGUUUACAUGUCGACUAA-39 and non-target siRNA #2: 59-UGGUUUACAUGUUGU- To define the consequence of the 2 novel PNKP mutations at GUGA-39) using Lipofectamine RNAiMAX (Life Technolo- the molecular and cellular level, we employed primary fibro- gies) 72 hours before lysis. blasts established from a skin biopsy from the proband. Western blotting using antibodies targeting either the Cellular DNA strand break assays N-terminal or C-terminal regions of PNKP failed to detect The steady-state level of DNA strand breaks was evaluated by PNKP protein in the patient-derived fibroblasts, even if we alkaline comet assays essentially as described11 following incubated the cells with the proteasome inhibitor MG132 to

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 5 prevent the degradation of the mutant PNKP (figure 2A). wondered whether PARP1 hyperactivation might also be Incubation with MG132 increased the level of p53 protein, a feature of these MCSZ cells. Indeed, strikingly, PNKP however, confirming that the proteasome was inhibited in patient fibroblasts accumulated much higher levels of the these experiments. The impact of the 2 novel mutations on poly (ADP-ribose) product of PARP1 activity than did PNKP protein levels was also observed by indirect immuno- 1BR control fibroblasts during treatment with CPT fluorescence, with both N-terminal and C-terminal PNKP (figure 3B). antibodies again detecting little or no PNKP in the patient- derived fibroblasts (figure 2B). Our data identify 2 novel PNKP mutations in a patient with MCSZ from India. Of interest this patient exhibits one of the Next, we measured the level of PNKP activity using a sensitive most severe forms of MCSZ documented, with a level of biochemical assay.12 This assay uses an oligonucleotide du- microcephaly and growth retardation comparable to patients plex substrate possessing an internal single-nucleotide gap with ataxia telangiectasia and Rad3 related-Seckel Syn- with 39-phosphate and 59-hydroxyl termini, which is labeled at drome.16 Despite this, in keeping with the functional analysis the ends of the duplex with the fluorescent dyes tetrame- of other identified PNKP mutations,15 cells derived from this thylrhodamine and 6-carboxyfluorescein (figure 2C). The 39- patient retain some residual PNKP activity. This is consistent phosphate and 59-hydroxyl termini in this substrate were with the observation that complete deletion of PNKP in completely repaired by incubation with cell extract from wild mouse is embryonic lethal.17 Finally, we have confirmed that type fibroblasts within 10 minutes. Although greatly reduced, PARP1 hyperactivation is a cellular hallmark of MCSZ cells, DNA 39-phosphatase and 59-kinase activities were also highlighting this phenomenon in the development of a range detected in the patient cell extract as indicated by the of clinically distinct neuro-pathologies. formation of a small amount of fully repaired reaction product (“ligation product”) and also in the case of 39-phosphatase Author contributions activity by the appearance of a small amount of I. Kalasova: designed and conducted the experiments. H. dephosphorylated oligonucleotide (figure 2C left panel,“39- Hanzlikova: designed, conducted, and coordinated the OH”). This residual activity in the patient-derived fibroblasts experiments. N. Gupta: identified and characterized the pa- was greatly reduced or ablated if the patient cells were first tient and initiated the study. J. Altmüller: conducted WES. Y. transfected with PNKP siRNA, indicating that it reflected Li: analyzed the WES Data and identified the mutations. J. residual levels of PNKP (figure 2D). It is currently unclear Reynolds: initiated and consulted on the study. G. Stewart: how the residual PNKP activity is generated in the patient initiated and consulted on the study. B. Wollnik: supervised cells, especially with respect to 59-kinase activity, because both the WES. G. Yigit: supervised the WES. K.W Caldecott: mutations are predicted to result in translational frameshifts managed and coordinated the study. and to truncate either almost all of the protein (in the case of the maternal allele, c.63dupC) or a large region of the kinase Acknowledgment domain (in the case of the paternal allele, c.1295_1298+6del) This work was funded by an ERC advanced investigator award (figure 1D). Possible explanations include a low level of al- to KWC (SIDSCA; 694996). Access to the Olympus ScanR ternative splicing, an alternative translation start site down- and Leica microscope at the Light Microscopy Core Facility, stream of the premature stop codon in the maternal PNKP IMG CAS, Prague was supported by MEYS (LM2015062), allele, a small amount of properly spliced mRNA arising from OPPK (CZ.2.16/3.1.00/21547), and NPU the authors the paternal allele, or the presence of a small amount of an (LO1419). This work used instruments provided by C4Sys alternative 59-kinase. infrastructure. GSS is funded by a CR-UK programme grant (C17183/A23303) and JJR is funded by the University of We next examined whether the greatly reduced level of PNKP Birmingham. protein and activity in the patient cells affected the rate of DNA strand break repair. DNA strand breaks induced by Study funding abortive topoisomerase 1 (TOP1) activity possess termini The study was funded by an MRC Programme grant to KWC that are substrates for both the DNA kinase and DNA (MR/P010121/1) and an ERC Advanced Investigator Award phosphatase activities of PNKP. Indeed, the PNKP patient to KWC (SIDSCA; 694996). fibroblasts accumulated higher levels of DNA strand breaks than 1BR control cells during incubation with TOP1 ‘poison’ Disclosure CPT (figure 3A). This observation is significant, because I. Kalasova, H. Hanzlikova, and N. Gupta report no dis- defects in the repair of TOP1-induced DNA breaks are also closures. J. Altmüller has nothing to disclose. Y. Li reports no observed in cells from other MCSZ patients and from patients disclosures. J. Reynolds has received academic research sup- with spinocerebellar ataxia with axonal neuropathy-1 and ataxia port from the University of Birmingham and has received telangiectasia, illustrating their relevance to neurodegenerative foundation/society support from Cancer Research UK. Grant – disease13 15. Importantly, hyperactivation of the SSB sensor Stewart has served on the Editorial Board of Oncogene and protein PARP1 is observed in and is causally linked with Molecular and Cellular Biology and has received foundation/ SSBR-defective cerebellar ataxia.3 Consequently, we society support from Cancer Research UK. B. Wollnik and G.

6 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Yigit report no disclosures. Keith Caldecott has served on the 7. Shen J, Gilmore EC, Marshall CA, et al. Mutations in PNKP cause microcephaly, seizures and defects in DNA repair. Nat Genet 2010;42:245–249. Editorial Board for DNA Repair and has received government 8. Bras J, Alonso I, Barbot C, et al. Mutations in PNKP cause recessive ataxia with research support from the Medical Research Council, Cancer oculomotor apraxia type 4. Am J Hum Genet 2015;96:474–479. 9. Leal A, Bogantes-Ledezma S, Ekici AB, et al. The polynucleotide kinase 3’-phos- Research UK, and the European Research Council. Full dis- phatase gene (PNKP) is involved in Charcot-Marie-Tooth disease (CMT2B2) pre- closure form information provided by the authors is available viously related to MED25. Neurogenetics 2018;19:215–225. with the full text of this article at Neurology.org/NG. 10. Pedroso JL, Rocha CRR, Macedo-Souza LI, et al. Mutation in PNKP presenting initially as axonal Charcot-Marie-Tooth disease. Neurol Genet 2015;1:e30. 11. Breslin C, Clements PM, El-Khamisy SF, Petermann E, Iles N, Caldecott KW. Publication history Measurement of chromosomal DNA single-strand breaks and replication fork pro- Received by Neurology: Genetics November 6, 2018. Accepted in final gression rates. Meth Enzymol 2006;409:410–425. form February 7, 2019. 12. Dobson CJ, Allinson SL. The phosphatase activity of mammalian polynucleotide kinase takes precedence over its kinase activity in repair of single strand breaks. Nucleic Acids Res 2006;34:2230–2237. References 13. El-Khamisy SF, Saifi GM, Weinfeld M, et al. Defective DNA single-strand break repair 1. Jiang B, Glover JNM, Weinfeld M. Neurological disorders associated with DNA in spinocerebellar ataxia with axonal neuropathy-1. Nature 2005;434:108–113. strand-break processing enzymes. Mech Ageing Dev 2017;161(pt A):130–140. 14. Katyal S, Lee Y, Nitiss KC, et al. Aberrant topoisomerase-1 DNA lesions are patho- 2. Yoon G, Caldecott KW. Nonsyndromic cerebellar ataxias associated with disorders of genic in neurodegenerative genome instability syndromes. Nat Neurosci 2014;17: DNA single-strand break repair. Handb Clin Neurol 2018;155:105–115. 813–821. 3. Hoch NC, Hanzlikova H, Rulten SL, et al. XRCC1 mutation is associated with PARP1 15. Reynolds JJ, Walker AK, Gilmore EC, Walsh CA, Caldecott KW. Impact of PNKP hyperactivation and cerebellar ataxia. Nature 2016;541:87–91. mutations associated with microcephaly, seizures and developmental delay on 4. Jilani A, Ramotar D, Slack C, et al. Molecular cloning of the human gene, PNKP, enzyme activity and DNA strand break repair. Nucleic Acids Res 2012;40: encoding a polynucleotide kinase 3’-phosphatase and evidence for its role in repair of 6608–6619. DNA strand breaks caused by oxidative damage. J Biol Chem 1999;274: 16. O’Driscoll M, Ruiz-Perez VL, Woods CG, Jeggo PA, Goodship JA. A splicing mu- 24176–24186. tation affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) 5. Karimi-Busheri F, Daly G, Robins P, et al. Molecular characterization of a human results in Seckel syndrome. Nat Genet 2003;33:497–501. DNA kinase. J Biol Chem 1999;274:24187–24194. 17. Shimada M, Dumitrache LC, Russell HR, McKinnon PJ. Polynucleotide kinase- 6. Loizou JI, El-Khamisy SF, Zlatanou A, et al. The protein kinase CK2 facilitates repair phosphatase enables neurogenesis via multiple DNA repair pathways to maintain of chromosomal DNA single-strand breaks. Cell 2004;117:17–28. genome stability. EMBO J 2015;34:2465–2480.

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 7 ARTICLE OPEN ACCESS Muscular dystrophy with arrhythmia caused by loss-of-function mutations in BVES

Willem De Ridder, MD,* Isabelle Nelson, PhD,* Bob Asselbergh, PhD, Boel De Paepe, PhD, Maud Beuvin, MSc, Correspondence Rabah Ben Yaou, MD, C´ecile Masson, MSc, Anne Boland, PhD, Jean-François Deleuze, PhD, Dr. Baets [email protected] Thierry Maisonobe, MD, Bruno Eymard, MD, PhD, Sofie Symoens, PhD, Roland Schindler, PhD, Thomas Brand, PhD, Katherine Johnson, PhD, Ana Topf,¨ PhD, Volker Straub, MD, PhD, Peter De Jonghe, MD, PhD, Jan L. De Bleecker, MD, PhD, Gis`ele Bonne, PhD,† and Jonathan Baets, MD, PhD†

Neurol Genet 2019;5:e321. doi:10.1212/NXG.0000000000000321 Abstract Objective To study the genetic and phenotypic spectrum of patients harboring recessive mutations in BVES.

Methods We performed whole-exome sequencing in a multicenter cohort of 1929 patients with a suspected hereditary myopathy, showing unexplained limb-girdle muscular weakness and/or elevated creatine kinase levels. Immunohistochemistry and mRNA experiments on patients’ skeletal muscle tissue were performed to study the pathogenicity of identiﬁed loss-of-function (LOF) variants in BVES.

Results We identiﬁed 4 individuals from 3 families harboring homozygous LOF variants in BVES, the gene that encodes for Popeye domain containing protein 1 (POPDC1). Patients showed skeletal muscle involvement and cardiac conduction abnormalities of varying nature and severity, but all exhibited at least subclinical signs of both skeletal muscle and cardiac disease. All identiﬁed mutations lead to a partial or complete loss of function of BVES through nonsense- mediated decay or through functional changes to the POPDC1 protein.

Conclusions We report the identiﬁcation of homozygous LOF mutations in BVES, causal in a young adult- onset myopathy with concomitant cardiac conduction disorders in the absence of structural heart disease. These ﬁndings underline the role of POPDC1, and by extension, other members of this protein family, in striated muscle physiology and disease. This disorder appears to have a low prevalence, although it is probably underdiagnosed because of its striking phenotypic variability and often subtle yet clinically relevant manifestations, particularly concerning the cardiac conduction abnormalities.

*These authors contributed equally to the manuscript as first authors.

†These authors contributed equally to the manuscript as last authors.

From the Neurogenetics Group (W.D.R., P.D.J., J.B.), University of Antwerp; the Laboratory of Neuromuscular Pathology (W.D.R., P.D.J., J.B.), Institute Born- Bunge, University of Antwerp; the Neuromuscular Reference Centre (W.D.R., P.D.J., J.B.), Department of Neurology, Antwerp University Hospital, Belgium; Sorbonne Université (I.N., M.B., R.B.Y., G.B.), INSERM U974, Center of Research in Myology, Institute of Myology, G.H. Pitié-Salpêtrière Paris, France; Histology and Cellular Imaging (B.A.), Neuromics Support Facility, VIB-UAntwerp Center for Molecular Neurology, University of Antwerp; Laboratory for Neuropathology (B.D.P., J.D.B.), Division of Neurology, Ghent University Hospital, Belgium; AP-HP, Centre de Référence de Pathologie Neuromusculaire Nord/Est/Ile-deFrance (R.B.Y., B.E.), G.H. Pitié-Salpêtrière, Bioinformatics Unit (C.M.), Necker Hospital, AP-HP, and University Paris Des- cartes, ; Centre National de Recherche en Génomique Humaine (CNRGH) (A.B., J.F.D.), Institut de Biologie François Jacob, CEA, Université Paris-Saclay, Evry; Laboratoire de Neuro- pathologie (T.M.), G.H. Pitié-Salpêtrière, Paris, France; Center for Medical Genetics (S.S.), Ghent University Hospital, Belgium; Developmental Dynamics, Imperial Centre for Experimental and Translational Medicine (R.S., T.B.), Imperial College London; John Walton Muscular Dystrophy Research Centre (K.J., A.T., V.S.), MRC Centre for Neuromuscular Diseases, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom.

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary AV = atrioventricular; CADD = Combined Annotation-Dependent Depletion; CK = creatine kinase; EPS = electrophysiology study; EPS = electrophysiology study; ExAC = Exome Aggregation Consortium; IHC = immunohistochemical; KI = knock-in; LGMD = limb-girdle muscular dystrophy; LOF = loss of function; NMD = nonsense-mediated mRNA decay; PCCD = progressive cardiac conduction disorder; SND = sinus node dysfunction; WES = whole-exome sequencing.

Limb-girdle muscular dystrophies (LGMDs) comprise Here, we present 4 individuals from 3 families harboring a phenotypically and genetically heterogeneous group of homozygous loss-of-function (LOF) mutations in BVES.We autosomally inherited myopathies characterized by pro- show that the skeletal muscle and cardiac involvement gressive proximal muscle weakness.1 Cardiac involvement is resulting from these BVES mutations is highly variable and common in LGMDs,2 and practice guidelines recommend emphasizes the relevance of BVES mutations with regard to referring patients for cardiac assessment.3 Hereditary car- hereditary cardiac conduction disorders. diac conduction disorders without structural cardiac disease are rare, but an increasing number of culprit genes are being identified.4 Methods Standard protocol approvals, registrations, Previously, a homozygous missense mutation (p.Ser201- and patient consents Phe) in the blood vessel epicardial substance (BVES)gene Ethical approval was granted by the relevant local ethical has been identified in 3 individuals from a single family, the committees of the participating centers. All patients provided eldest presenting with an overt LGMD phenotype and all 3 their written consent for participation in the study. showing elevated creatine kinase (CK) levels and a second- degree atrioventricular (AV) block.5 The disease was orig- Patients and clinical evaluation inally classified as LGMD2X (OMIM #616812). BVES We studied 4 patients harboring rare variants in BVES, encodes for a 360 amino acid protein also known as identified by whole-exome sequencing (WES) of a cohort of POPDC1, part of the Popeye domain containing (POPDC) 1929 unsolved cases with limb-girdle muscular weakness and/ family of proteins, which are cyclic 39,59-adenosine mono- or an elevated CK level, established through an international phosphate (cAMP)-binding transmembrane proteins that collaboration between different clinical and genetic centers: are abundantly expressed in striated muscle.6 In patients’ the MYO-SEQ project, the Myocapture project, and the muscle, a marked reduction was observed in the membrane Center for Medical Genetics of the Ghent University Hos- localization of POPDC1 and POPDC2.5 In zebrafish popdc1 pital. Patients 1 and 2 (family A) are siblings of consanguin- morphants and popdc1p.Ser191Phe knock-in (KI) mutants, eous parents (figure 1). Patient 3 (family B) and patient 4 skeletal muscle abnormalities and AV conduction blocks (family C) are isolated cases, with the maternal grandfather have been noted.5 In addition, previously reported homo- and paternal grandmother of patient 4 being first cousins. zygous Popdc1 null mutant mice showed delayed skeletal muscle regeneration and an age-dependent stress-induced Medical history taking and physical examination were focused sinus node dysfunction (SND).7,8 on neuromuscular and cardiac symptoms and signs. Muscle

Figure 1 Segregation analysis of the 3 identified BVES mutations

Pedigrees of families A, B, and C are shown. Unaffected family members for whom DNA was available for segregation analysis of the BVES variants in the respective families, I-1, II-1, II-2, III-1, III-2, III-3, and III-4, were heterozygous for the respective variants.

2 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG strength was evaluated according to the Medical Research Leica), for membrane counterstaining. The following sec- Council scale. Cardiac function was assessed by ECG, Holter ondary antibodies were used for detection of the signal: Alexa monitoring, and echocardiography in all patients. In addition, Fluor 488–conjugated goat anti-rabbit (A11034, Life Tech- arm ergometer stress testing was performed in patient 3 and nologies) and CY3labeled goat antimouse (115-166-071, bicycle ergometer stress testing and a cardiac electrophysiol- Jackson ImmunoResearch). ogy study (EPS) in patient 2. Microscopy and image analysis Muscle MRI Images were acquired with a Zeiss LSM700 laser scanning Muscle MRI studies were performed for patients 1, 2, and 3 confocal microscope using a 20×/0.8 Plan Apochromat ob- on 1.5-T MRI platforms at the respective centers. Cross jective. To avoid crosstalk between fluorescence channels, sections at the pelvic, thigh, and calf levels were assessed on line-by-line sequential scanning was used. All images (16-bit, axial T1-weighted images to evaluate patterns of muscle in- 512 × 512 pixels, 417 nm × 417 nm per pixel) were taken with volvement. Fatty replacement of muscle was graded according identical excitation and detection settings to allow compari- to the Mercuri scale.9 son of fluorescence intensities. For each sample, 5 images were acquired at random positions. Intensities at the sarco- Analysis of WES data lemma were quantified using the Fiji distribution of For family A, WES was performed by the CNRGH on DNA ImageJ.15,16 Raw image files (LSM5) were loaded in Fiji, and samples from patients 1 and 2 and their mother. Variants were background intensity levels were corrected by subtracting the annotated and filtered using an in-house-developed software mean intensity of the Gaussian blurred image (sigma = 20 system (PolyWeb).10 WES data of patient 3 were processed by pixels) from the original image. On each image, 6 random the Genomics Platform at the Broad Institute MIT and Harvard membrane segments were delineated (average line length (Boston, MA) and analyzed by the team at the John Walton per segment >50 μm) using the segmented line tool on the Muscular Dystrophy Research Centre, Newcastle University, as sarcolemma channel and were stored as ImageJ ROI files. described previously.11 For patient 4, WES was performed using Intensities in both channels (POPDC1/POPDC2 and the SureSelect XT Human All Exon V6 enrichment kit (Agilent SGCA) were quantified as mean intensities of these lines, Technologies), followed by paired-end sequencing (2 × 150 bp) set at a line width of 5 pixels (2 μm). The analysis pro- on the HiSeq3000 sequencer (Illumina). Reads were mapped, cedure was used as an ImageJ macro, measuring all images and variants were called and annotated with the BCBio pipeline. in batch.

BVES (reference sequence: NM_001199563) was analyzed for mRNA studies biologically relevant variants. Population frequencies were es- Total RNA was extracted from muscle biopsies (controls and timated using Exome Aggregation Consortium (ExAC),12 last patients) using standard methods (TRIzol). First-strand accessed in August 2018. MutationTaster213 and the Combined cDNA was synthesized using the SuperScript III First- Annotation Dependent Depletion (CADD)14 tool (version Strand Synthesis System (Invitrogen–Thermo Fisher Scien- v1.3) were used as in silico prediction algorithms to predict tific) with Oligo-dTs. The obtained cDNA was used for the pathogenicity of the identified variants. Variants with Sanger sequencing and quantitative PCR (qPCR) experi- CADD scores >20 represent the 1% highest ranked variants ments with SYBR green I dye incorporation (LightCycler 480 genome wide with regard to potential deleteriousness. In System; Roche, Basel, Switzerland). The average Ct value silico splice site predictions were obtained using Alamut obtained with multiple BVES primers (figure 2A) was nor- Batch Software v.2.8 (Interactive Biosoftware). Variants malized against the housekeeping gene RPLP0; then, fold were validated by Sanger sequencing, and segregation changes in mRNA levels were calculated relative to the con- studies were performed with available DNA samples. trol sample.

Muscle biopsies Experiments were performed in triplicate. All primer Muscle biopsies of quadriceps or deltoid muscle were sequences are available upon request. obtained for patients 2, 3, and 4 and analyzed following standard histologic and immunohistochemical (IHC) proce- Data analysis was performed with qBase + (Version 3.1; dures. Standard IHC stainings, including those for dystrophin, Biogazelle, Zwijnaarde, Belgium). Data are presented using α-, β-, and γ-sarcoglycans, α- and β-dystroglycan, caveolin 3, GraphPad Prism (GraphPad Software, La Jolla, CA) as mean and telethonin, were evaluated. In addition, IHC stainings values with standard error of the mean. Data were analyzed were performed for POPDC1 and POPDC2. Frozen 10-μm using the unpaired t test. p < 0.05 was considered statistically sections of skeletal muscle biopsies of patients and 2 controls significant. were mounted on Superfrost Plus glass slides (Thermo Fisher Scientific) and used for IHC using the following primary Data availability statement antibodies as described5: anti-POPDC1 (HPA014788, Sigma- Anonymized data will be shared by request from any qualified Aldrich) and anti-POPDC2 (HPA024255, Sigma-Aldrich), investigator, only for purposes of replicating procedures and combined with anti-α-sarcoglycan (SGCA) (NCL-a-sarc, results.

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 3 Figure 2 BVES gene structure and mRNA analysis

(A) BVES mRNA transcript variant C (NM_ 001199563) contains 8 exons, of which 7 coding. Transcript length is 5,567 base pairs. Untranslated regions are filled in grey, translated in black. The location of the 3 identified variants is marked with an arrow, position of the amplified fragments for quantitative and quantitative PCR experiments with dashed lines. (B) Qualitative PCR: agarose gel of PCR products (amplicons A, B, and C) amplified from cDNA from control (C1) and patient 2 (P2). For every amplicon, the 2 lanes on the right contain mRNA without reverse transcriptase and H2O, respectively. (C and D) BVES quantitative mRNA analysis for amplicons A and C, respectively. BVES mRNA levels were first normalized internally to RPLP0 mRNA levels; subsequently, BVES mRNA levels of the patients were normalized (as a percentage) to the mean of the 2 controls. For both amplicons A and C, there is a significant difference in mRNA levels between both controls and patients 2 and 3, respectively, which is not the case for patient 4. Error bars: standard error of the mean (SEM). *p < 0.05, **p < 0.01, using the unpaired t test. bp = base pair; Ex = exon.

Results on ECG and Holter monitoring, with an echocardiography showing no structural abnormalities. There were no symptoms Clinical findings and case descriptions suggestive of neuromuscular disease, and clinical examination at A summarized overview of the highly variable clinical symp- age 20 years was normal. However, high CK levels (1,074–3,600 toms is provided in table 1. IU/L) were measured repeatedly, EMG disclosed myopathic abnormalities, and muscle biopsy showed mild dystrophic Patients 1 and 2 are affected siblings, among 5, of consan- changes with normal routine IHC and Western blot studies. guineous parents (first cousins). Patient 1, a 41-year-old Intercurrently, the patient manifested a symptomatic S1 radi- woman, manifested complaints of exercise-induced myalgia culopathy on the right side at age 22 years, with residual and fatigue of the lower limb muscles and breathlessness from weakness and atrophy of the right gastrocnemius and soleus age 27 years. No further skeletal muscle signs or symptoms muscles. An EPS at age 27 years confirmed an AV nodal block, were noted, and clinical neurologic examination was normal. with a normal His-ventricular interval and without SND or CK levels, repeatedly measured at that time, were elevated in ventricular hyperexcitability. During follow-up, this patient the range of 13003661 IU/L. EMG was normal, and cardiac manifested multiple presyncopal episodes, and the cardiac assessment revealed a first-degree AV block in the absence of conduction abnormalities progressed toward a second-degree structural cardiac disease on echocardiography. Muscle symp- Mobitz type 2 AV block combined with an incomplete right toms and cardiac function remained stable during follow-up. bundle branch block. A new EPS at age 29 years revealed a combined AV nodal and infrahissian block (His-ventricular Patient 2, a currently 37-year-old man, was first investigated at interval ranging 55–80 ms), again without SND or hyperexcit- age 19 years, presenting with presyncopal episodes and palpi- ability. Pacemaker implantation was advised but refused by the tations. During cardiac workup, a first-degree AV block and patient. Bicycle ergometer testing performed at age 35 years a transient second-degree Mobitz type 1 AV block were noted showed a second-degree Mobitz type 2 AV block during

4 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Table 1 Phenotypic information for the patients harboring homozygous mutations in BVES

Patient 1 Patient 2 Patient 3 Patient 4

Sex Female Male Female Male

Ethnicity North African North African Caucasian (Belgium) Caucasian (Belgium)

BVES variant, cDNA position c.816+2T>C c.816+2T>C c.262C>T c.1A>G

Age at onset, y 27 19 35 39

Presenting symptoms Exercise intolerance Palpitations, faintness, Proximal weakness LL Myalgia and high CK and elevated CK

Age at the last examination, y 41 37 65 44

Weakness

Proximal UL No No Yes No

Proximal LL No No Yes No

Distal UL No No Yes No

Distal LL No No Yes No

Other No Right calf muscles Periscapular No

Ambulation status Ambulatory Ambulatory Wheelchair use Ambulatory

Serum CK (IU/L) 1,300–3,661 1,074–5,500 1918 3,500–4,000

EMG (age, y) Normal (28) Myopathic (30) Myopathic (45) Myopathic (43)

Resting ECG First-degree AV block First-degree AV block Normal Normal

Echocardiography Normal Normal Aortic valve stenosis Normal

Holter monitoring First-degree AV block Second-degree AV block Nocturnal first-degree AV Nocturnal second-degree (Mobitz type 2), iRBBB block AV block (Mobitz type 2)

Bicycle/arm ergometer stress testing NA Bicycle ergometer: Mobitz Arm ergometer: no NA type 2 second-degree AV increase in the heart rate block during recovery during the test; borderline first-degree AV block

Biopsy (age, y) NA Myopathic (21) Myopathic (45) Myopathic (43)

Biopsied muscle NA Left deltoid Quadriceps Quadriceps

Abbreviations: CK = creatine kinase; UL = upper limb; LL = lower limb; AV = atrioventricular; iRBBB = incomplete right bundle branch block; NA = not assessed. recovery. Apart from right S1 radiculopathy sequelae, no addi- increase in the heart rate (85/min during the whole test) was tional muscle weakness or wasting was noted during follow-up, observed during arm ergometer testing, halted because of ex- but CK levels stayed consistently elevated (maximal 5,500 IU/ haustion at 50 W. Both parents were deceased, and no neuro- L). Patient 3, a 65-year-old woman, developed slowly muscular or cardiac problems were reported. Of 5 siblings, 1 was progressive proximal weakness in the lower limbs in her mid- deceased, and none reported muscle or cardiac symptoms. One thirties. Being very athletic up to that point, she noticed diffi- sister agreed to be formally examined, and clinical examination culties in walking uphill and climbing stairs. A few years later, she was unremarkable. manifested slowly progressive proximal weakness in the upper limbs too. When she was referred for neuromuscular workup for Patient 4, a currently 44-year-old man, reported mild myalgia the first time at age 43 years, she climbed stairs on her hands and of the calves at age 39 years, 6 months after starting fibrate feet. Clinical neurologic examination at that time confirmed therapy for hypertriglyceridemia. A CK value of 8,000 IU/L proximal weakness in the lower limbs, as well as weakness of the was measured, and he was referred to a cardiologist. The fibrate anterior tibial muscles. Scapular winging was found. The CK therapy was interrupted, muscle complaints diminished, and level was 1,161 IU/L, and EMG revealed myopathic changes. control CK values averaged around 3,500–4,000 U/L. Initial Over the following years, the muscle weakness was slowly cardiac workup, including ECG, echocardiography, and Holter progressive, leading to loss of ambulation. Thorough cardiac monitoring, yielded normal results. The cardiologist referred the workup revealed a first-degree AV block at night and a border- patient for neuromuscular workup. By that time, muscle com- line first-degree AV block during exercise. Furthermore, no plaints had resolved. A myopathic recruitment pattern was noted

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 5 on EMG though, as well as nonspecific myopathic features on database, and in silico prediction algorithms were in favor of muscle biopsy. Clinical examination of 2 brothers, a 7-year-old pathogenicity. Segregation analyses were performed with son, and a 5-year-old daughter was unremarkable. For the available DNA samples (figure 1). Only affected individuals parents and the 2 brothers, a normal CK value was measured. harbored the variant in the homozygous state. The c.816+2- Clinically, the patient remained stable during 5 years of follow- T>C variant in intron 6 of BVES affects the highly conserved up. Cardiac workup was, however, repeated and revealed a first- canonical T of the splice donor site and is predicted to cause degree AV block and a nocturnal second-degree Mobitz type 2 skipping of exon 6 and the loss of 56 amino acids (p.Val217_ AV block. None of the patients presented with contractures, Lys272del) within the Popeye domain of BVES.Twopotential rigid spine, clinical myotonia, or myotonic discharges on EMG. cryptic splice sites could be activated alternatively: in intron 6 (c.816+46, p.Lys272fs4*) or within exon 6 (c.749*, Muscle imaging p.Arg250Argfs20*). The c.262C>T variant (rs796206315) Muscle MRI studies of patients 1, 2, and 3 are shown in figure 3. introduces a premature stop codon at amino acid position 88, For patient 1, muscle MRI performed at age 28 years and located in the second transmembrane domain of this short repeated at age 39 years did not reveal any selective muscle protein. According to the MutationTaster2 algorithm,13 the involvement. For patient 2, initial CT imaging of muscle at age c.1A>G variant is predicted to result in a loss of the initiating 20 years was normal. At age 30 years, muscle MRI showed methionine (cDNA position 218) and potential activation of moderate fatty replacement and atrophy of the right gastroc- a downstream translation initiation site at cDNA position 354, nemius, soleus, and biceps femoris muscles, visualizing the resulting in a new reading frame with insertion of a premature clinically evident residual atrophy and weakness due to the S1 stop codon at amino acid position 2. As this potential alter- radiculopathy. Additional muscle MRI studies at age 35 years native initiation site is not embedded in a strong Kozak se- confirmed these findings. quence, other initiation sites might be activated at cDNA position 423 or 431. In both cases, this would lead to an in- Muscle MRI of patient 3, showing the most severe skeletal frame deletion of 66 or 72 amino acids, respectively. Activation muscle phenotype, revealed a pattern of selective muscle in- of different alternative start codons located more downstream volvement with preferential affection of the posterior thigh could, however, also result in a shift of the reading frame with compartment. In addition, an asymmetric moderate in- insertion of a premature stop codon. volvement of the left lateral vastus and relative sparing of distal leg muscles were observed. Molecular consequences at the mRNA level To provide direct evidence for the predicted alterations at the Genetic findings transcript level, Sanger sequencing and qPCR experiments We identified 3 different homozygous variants in BVES in 3 were performed with mRNA extracted from muscle biopsies families (table 2). Variants were absent from the ExAC control of patients 2, 3, and 4. PCR products of BVES cDNA

Figure 3 Muscle MRI findings in 3 patients harboring homozygous mutations in BVES

Axial T1-weighted images are shown for patients 1, 2, and 3, performed at ages 39, 35, and 56 years, respectively.

6 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Table 2 BVES mutations identified in the present study

Variant location (hg19) Predicted deleteriousness

Genomic (chr. Protein CADD ExAC allele Patient 6) Codingchange Genotype Variant MutationTaster2 score frequency

1 105564574A>G c.816+2T>C p.? Hom. Splice No data No data 0 donor

2 105564574A>G c.816+2T>C p.? Hom. Splice No data No data 0 donor

3 105577343G>A c.262C>T p.Arg88Ter Hom. Stop Disease causing 39 0 gained

4 105581452T>C c.1A>G p.? Hom. Start lost Disease causing 24.1 0

Abbreviations: Hom. = homozygous; CADD = Combined Annotation-Dependent Depletion; start lost = variant in the start codon; ExAC = Exome Aggregation Consortium. amplicons A, B, and C (figure 2A) could all be sequenced samples. These results confirm that the predicted LOF (data not shown), and all variants were confirmed at the mutations in BVES indeed lead to a loss of membrane local- cDNA level. Gel electrophoresis of the amplicon B encom- ization and consequently the main function of POPDC1 and passing exon 4 up to and including 8 revealed a shorter product POPDC2 in patient muscle. for patient 2, sized approximately 350 base pairs (bp) instead of the expected size of 507 bp (figure 2B). The sequencing of this Discussion fragment confirmed that exon 6 (168 bp long) was indeed spliced out. Nevertheless, fragment C could be amplified for In the present study, we identified 3 different new homozy- patient 2, although the forward primer is located in exon 6. gous LOF mutations in BVES in 4 individuals from 3 families, qPCR data, however, showed that these mRNA levels for showing early adult-onset skeletal muscle and cardiac con- amplicon C are close to 0 compared with the controls, induction abnormalities of varying nature and severity. dicating that the absolute level of mutant mRNA in which exon 6 is not skipped is extremely low. Furthermore, mRNA levels The presenting symptoms vary between individuals, with for amplicon A are markedly decreased as well, indicating patient 2 presenting with cardiac symptoms due to an AV nonsense-mediated mRNA decay (NMD) of the mutant conduction defect within the second decade and patients 1, 3 transcript in any case. For patient 3, qPCR for amplicons A and and 4 with skeletal muscle symptoms or signs. Only patient 3 C revealed a marked decrease in BVES mRNA relative to the showed progressive proximal muscle weakness, whereas patient controls (figure 2, C and D). For patient 4, BVES mRNA levels 1 had symptoms of exercise intolerance, and patient 4 had are similar to these in the controls. permanently high CK levels (>3,500 IU/L after interruption of fibrate therapy) with transient complaints of myalgia. During Reduction in membrane localization of follow-up, all patients appeared to have at least subclinical signs POPDC1 and POPDC2 of both skeletal muscle and cardiac involvement. In patient 2 Nonspecific myopathic features such as increased fiber size with a predominant cardiac phenotype, a chronically elevated variation were noted on muscle biopsies of patients 2, 3, and 4. CK level (>1,000 U/L) was noted repeatedly, as well as In addition, for patient 2, a few necrotic fibers were observed. myopathic features on EMG and muscle biopsy. In patients 1, Standard IHC stainings were normal. 3, and 4, initially showing a predominant skeletal muscle phenotype of variable severity, a progressive cardiac conduction For the previously described patients harboring the disorder (PCCD) became apparent during follow-up. p.Ser201Phe in BVES in homozygosity, defective POPDC1 membrane trafficking was reported to result in strongly re- Intrafamilial variability of the phenotype linked to mutations duced membrane localization of POPDC1 and POPDC2.5 in BVES has already been shown in the originally reported To examine POPDC1 and POPDC2 at the plasma mem- consanguineous family in which a pseudodominant in- brane, we performed IHC stainings in patient and control heritance pattern of the p.Ser201Phe variant was described5: muscle samples. Both POPDC1 and POPDC2 were abun- the grandfather presented with an LGMD phenotype at age dantly present at the plasma membrane of control skeletal 40 years, complicated by a symptomatic second-degree AV muscle. In all patient samples, however, both POPDC1 and block at age 59 years requiring pacemaker implantation, and POPDC2 were drastically diminished at the sarcolemma the grandsons with symptomatic second-degree AV block, (figure 4, A-D). Levels of SGCA, used as a control marker for respectively, at age 17 years and 12 years. Cardiac conduction sarcolemmal proteins, remained normal in the patient sam- disorders seemed to be confined to the AV node.5 However, ples with similar staining patterns and intensities as for control for patient 2, definite His-bundle involvement was shown on

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 7 Figure 4 Reduction of POPDC1 and PODPC2 at the sarcolemma in muscle of patients harboring homozygous mutations in BVES

(A and B) Representative muscle sections of patients and controls immunostained for POPDC1 and POPDC2, respectively, and α-sarcoglycan (SGCA) as sarcolemmal marker. All images were acquired with identical settings and are displayed in the figure with identical intensity scaling for each channel. Scale bar = 50 μm. (C and D) Fluorescence intensities measured at the sarcolemma (n = 30 sarcolemmal segments at random positions on the section). AU = arbitrary unit.

an EPS at age 29 years. Although not fulﬁlling the exact cri- Similarly, heterogeneity was observed on muscle imaging teria of chronotropic incompetence,17 the results of the arm studies. For patient 3, a pattern of selective muscle in- ergometer testing in patient 3 are strongly indicative of SND. volvement was shown, with muscles of the posterior com- Because of lower limb weakness, only less standardized arm partment of the thighs being most severely involved. Muscle ergometer testing could be performed for this wheelchair- MRI studies of patient 1 yielded normal results. The selective using patient. During an incremental dynamic exercise test, unilateral atrophy and fatty replacement of soleus, gastroc- work load was limited to 50 W, mainly because of weakness of nemius, and biceps femoris muscles in patient 2 may be due to the biceps muscles. S1 radiculopathy.

8 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG As we identified additional unrelated families with a BVES- Of interest, the Popdc1 null mutant mice revealed an age- related myopathy, we propose that this disorder should be dependent stress-induced SND with chronotropic in- classified as “LGMD R25 BVES-related” according to the competence,8 a condition we also strongly suspect in patient recently published novel European Neuromuscular Centre 3. Further studies are needed to unveil the exact functional classification of LGMDs. “LGMD2X” was excluded from the consequences of the complete loss of function of the LGMD nomenclature, based on the criterion that the con- POPDC1 protein in skeletal muscle and the heart. Crucially, dition must be described in at least 2 unrelated families with POPDC2 appears to be a direct interactor of POPDC1, and affected individuals.18 All other criteria of the novel definition aberrant trafficking of POPDC1 also impairs membrane of an “LGMD” are fulfilled too. Each patient achieved in- transport of POPDC2. Heteromeric complexes might be dependent walking and has an elevated CK activity. De- formed, potentially explaining the secondary loss of mem- generative changes on muscle imaging are clearly shown on brane localization of POPDC2, similarly shown for patients muscle MRI of patient 3 of the current study and dystrophic harboring the p.Ser201Phe missense mutation.5 This appears changes on histology, noted for the same patient, had already to be pathomechanistically relevant, as popdc2 zebrafish been described for the eldest patient of the originally reported morphants show a severe muscular dystrophy phenotype and family too.5,18 IHC experiments revealed a pattern of con- cardiac abnormalities too.19 sistent reduction of POPDC1 and POPDC2 at the sarcolemma, similar to the pattern described in muscle of patients Our findings stress the importance of a thorough cardiac harboring the homozygous p.Ser201Phe missense mutation workup in (unsolved) LGMD patients. Cardiac workup is and the popdc1p.Ser191Phe KI zebrafish.5 Of note, for patient 3, often focused on structural cardiac evaluation,3 but as in harboring the homozygous p.Arg88Ter variant, no antibody myotonic dystrophy,2 extensive and repeated screening for targeting an epitope at the N-terminal side of the premature arrhythmias in the absence of structural heart disease is defi- stop codon was available. In addition, mRNA studies are nitely relevant in case of a BVES-related myopathy. supportive of a LOF mechanism at the mRNA level for the BVES variants in patients 2 and 3, most likely due to NMD. Furthermore, this study highlights the diagnostic difficulties For patient 2, qualitative and quantitative PCR data illustrate that can be faced in case of pauci- or asymptomatic hyper- that different splice variants are transcribed, though ultimately CKemia.20 We note chronically elevated CK values (>3 times leading to significantly decreased BVES mRNA levels. The the upper limit of normal) in all patients. In the absence of low level of remaining mRNA probably mainly consists of marked dystrophic features on muscle biopsy, this may be an mRNA with in-frame skipping of exon 6, encoding for 56 indication of membrane instability linked to the interaction of amino acids (p.Val217_Lys272del), which are part of the POPDC1 with dystrophin, dysferlin, and caveolin 3.6,21 In Popeye domain, the crucial functional domain of the protein. addition, ultrastructural analysis of muscle of the grandfather For patient 4, BVES mRNA levels were similar to these in the of the p.Ser201Phe family revealed membrane dis- controls. Apparently, the mutant mRNA is not targeted by continuities.5 When further neuromuscular workup is advised NMD, yet a nonfunctional protein is translated, which is not in this clinical setting according to guidelines,20,22 cardiac recognized by the anti-POPDC1 antibody, raised against the screening could be relevant too, regardless of the results of the C-terminal part of the POPDC1 protein. In case of an in- neuromuscular workup. The other way around, our findings frame deletion of 66 or 72 amino acids at the N-terminal end suggest that BVES should be included in a candidate gene list of the protein, the antibody should still recognize this trun- for PCCDs presenting as primary electrical disease. The cated POPDC1 protein. This observation most likely search for culprit genes has long been complicated by the fact implies that there is a shift of the reading frame, with that most cardiac conduction disorders are sporadic, as they a premature stop codon less than 55 nucleotides upstream are highly prevalent and usually associated with diverse (ac- of the last exon-exon border. In that case, NMD will be quired) structural heart disease. Channelopathies are evi- skipped and a protein with a different amino acid sequence dently the predominant hereditary entities associated with is formed, not recognized by the anti-POPDC1 antibody. PCCD.4,23 Furthermore, a short clinical neuromuscular All mutations identified lead to a partial or complete loss evaluation, including CK measurement, could be of value of function of BVES through NMD or through functional when a hereditary PCCD is suspected. changes to the POPDC1 protein. Identification of LOF mutations in BVES underlines by ex- By identifying and elaborating on LOF mutations in BVES,we tension the role of the POPDC protein family in striated expand the genetic spectrum of the disorder and provide muscle physiology and disease.6 We present 4 individuals pathomechanistic insights in the disorder. Only the from 3 families harboring homozygous LOF variants in BVES, p.Ser201Phe missense mutation had been previously identi- showing early adult-onset skeletal muscle and cardiac con- fied in a single family. Identification of patients harboring duction abnormalities of varying nature and severity. Overall, LOF mutations in BVES could, however, be anticipated based this recessive disorder linked to mutations in BVES appears to on functional studies in homozygous zebrafish popdc1 mor- have a low prevalence, but is probably underdiagnosed be- phants and Popdc1 null mutant mice, showing a similar phe- cause of its striking phenotypic variability and often subtle yet notype compared with the popdc1p.Ser191Phe KI zebrafish.5,7,8 clinically relevant manifestations.

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 9 Acknowledgment J.L. De Bleecker has served on the advisory boards of Sanofi The authors thank the patients and families for their Genzyme, Pfizer, and CSL Behring and has received travel cooperation and contributions; Sophie D’hose, technician, funding or speaker honoraria from Sanofi Genzyme and Laboratory for Neuropathology, Division of Neurology, CSL Behring. G. Bonne has served on the editorial boards Ghent University Hospital, for laboratory assistance; and of the Journal of Neuromuscular Disease and Neuromuscular Ursula Herbort-Brand, technician, Imperial College London, Disorders; has received academic research support from for laboratory assistance. ANR-BMBF; and has received foundation/society research support from Association Institut de Myologie, GIS Mal- Study funding adies Rare “Plateforme Mutations,” and CURE-CMD The study received financial support from Sanofi Genzyme, Translational Award. J. Baets reports no disclosures. Full Ultragenyx, LGMD2I Research Fund, Samantha J. Brazzo disclosure form information provided by the authors is Foundation, LGMD2D Foundation, Kurt+Peter Foundation, available with the full text of this article at Neurology. Muscular Dystrophy UK and Coalition to Cure Calpain 3, the org/NG. Association Belge contre les Maladies Neuromusculaire (ABMM)—Aide à la Recherche ASBL, the Institut National Publication history de la Santé et de la Recherche Médicale, the Sorbonne Uni- Received by Neurology: Genetics November 21, 2018. Accepted in final versité- FacultédeMédecine, the Association Française con- form February 12, 2019. tre les Myopathies and France Génomique (Myocapture Project), National Research Agency, and Investment for the Future (Grant No. ANR-10-INBS-09). J.B. is supported by a Senior Clinical Researcher mandate of the Research Appendix Author contributions — Fund Flanders (FWO). T.B. is supported by grants from Name Location Role Contribution the British Heart Foundation (PG/14/46/30911 and PG/ Willem De University of Author Acquisition and 14/83/31128) and the Magdi Yacoub Institute. Ridder, MD Antwerp, interpretation of patient, Antwerp, imaging, and Belgium histopathology data, Disclosure analysis of genetic data, W. De Ridder, I. Nelson, and B. Asselbergh report no dis- and manuscript writing closures. B. De Paepe has served on the editorial boards of Isabelle Institute of Author Acquisition and Clinical Medicine and Diagnostics and ISRN Pathology and has Nelson, PhD Myology, G.H. interpretation of genetic received foundation/society research support from the As- Pitié-Salpêtrière data and manuscript Paris, France writing sociation Belge contre les Maladies neuromusculaires. M. Beuvin, R. Ben Yaou, C. Masson, and A. Boland report no Bob University of Author Interpretation of Asselbergh, Antwerp, histopathology data disclosures. J.-F. Deleuze has served on the editorial board of PhD Antwerp, Human Genetics; holds patents in genetic diagnostics; and has Belgium

received government research support from the French Na- Boel De Ghent University Author Interpretation of tional Research Agency. T. Maisonobe, B. Eymard, and S. Paepe, PhD Hospital, Ghent, histopathology data Symoens report no disclosures. R. Schindler has been Belgium employed by Domainex Ltd. T. Brand has served on the Maud Beuvin, Institute of Author Acquisition and MSc Myology, G.H. interpretation of protein editorial boards of the Journal of Cardiovascular Development Pitié-Salpêtrière expression data and Disease, Cellular Signaling, and PLoS One and has received Paris F-75013, (immunohistopathology) foundation/society support from the British Heart Founda- France tion and AFM Telethon. K. Johnson and A. Töpf report no Rabah Ben Institute of Author Acquisition and disclosures. V. Straub has served on the advisory boards of Yaou, MD Myology, G.H. interpretation of patient, Pitié-Salpêtrière imaging, and Audentes Therapeutics, Summit Therapeutics, Biogen, Paris F-75013, histopathology data and Exonics Therapeutics, Sanofi Genzyme, Sarepta Therapeu- France manuscript writing tics, Wave Therapeutics, and Roche; has received travel Cécile University Paris Author Interpretation of genetic funding or speaker honoraria from Sanofi Genzyme; has Masson, MSc Descartes, F- data 75015, Paris, served on the editorial boards of Neuromuscular Disorders and France Journal of Neuromuscular Diseases; has consulted for Bayer, Anne Boland, Université Paris- Author Acquisition of genetic Wave Therapeutics, and UCB; has received commercial re- PhD Saclay, F-91057, data search support from Sanofi Genzyme; has received govern- Evry, France

ment funding support from the European Commission and JeanFrançois, Universit´e Paris- Author Acquisition of genetic the UK Medical Research Council; and has received Deleuze, PhD Saclay, F-91057, data foundation/society research support from the Parent Project Evry, France Muscular Dystrophy, Association Francaise contre les Myo- Thierry Universit´e Paris- Author Acquisition and pathies, the LGMD2I Research Fund, Muscular Dystrophy Maisonobe, Saclay, F-91057, interpretation of MD Evry, France histopathology data UK, and Duchenne-UK. P. De Jonghe reports no disclosures.

10 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG References Appendix (continued) 1. Vissing J. Limb girdle muscular dystrophies: classification, clinical spectrum and emerging therapies. Curr Opin Neurol 2016;29:635–641. Name Location Role Contribution 2. Silvestri NJ, Ismail H, Zimetbaum P, Raynor EM. Cardiac involvement in the muscular dystrophies. Muscle Nerve 2018;57:707–715. 3. Narayanaswami P, Weiss M, Selcen D, et al. Evidence-based guideline summary: Bruno G.H. Pitié- Author Acquisition and diagnosis and treatment of limb-girdle and distal dystrophies: report of the guideline Eymard, MD, Salpêtrière, F- interpretation of patient development subcommittee of the American Academy of Neurology and the practice PhD 75013, Paris, data issues review panel of the American Association of Neuromuscular & Electro- France diagnostic Medicine. Neurology 2014;83:1453–1463. 4. Rezazadeh S, Duff HJ. Genetic determinants of hereditary bradyarrhythmias: Sofie Ghent University Author Analysis of genetic data a contemporary review of a diverse group of disorders. Can J Cardiol 2017;33: Symoens, Hospital, Ghent, 758–767. PhD Belgium 5. Schindler RF, Scotton C, Zhang J, et al. POPDC1(S201F) causes muscular dystrophy and arrhythmia by affecting protein trafficking. J Clin Invest 2016;126: Roland Imperial College Author Critical revision of the 239–253. Schindler, London, London, manuscript for 6. Brand T, Schindler R. New kids on the block: the Popeye domain containing PhD W12 0NN, important intellectual (POPDC) protein family acting as a novel class of cAMP effector proteins in striated United Kingdom content muscle. Cell Signal 2017;40:156–165. 7. Andree B, Fleige A, Arnold HH, Brand T. Mouse Pop1 is required for muscle re- Thomas Imperial College Author Critical revision of the generation in adult skeletal muscle. Mol Cell Biol 2002;22:1504–1512. Brand, PhD London, London, manuscript for 8. Froese A, Breher SS, Waldeyer C, et al. Popeye domain containing proteins are W12 0NN, important intellectual essential for stress-mediated modulation of cardiac pacemaking in mice. J Clin Invest United Kingdom content 2012;122:1119–1130. 9. Mercuri E, Pichiecchio A, Allsop J, Messina S, Pane M, Muntoni F. Muscle MRI in Katherine Newcastle Author Analysis of genetic data inherited neuromuscular disorders: past, present, and future. J Magn Reson Imaging Johnson, PhD University, 2007;25:433–440. Newcastle upon 10. Gordon CT, Petit F, Kroisel PM, et al Mutations in endothelin 1 cause recessive Tyne, United auriculocondylar syndrome and dominant isolated question-mark ears. Am J Hum Kingdom Genet 2013;93:1118–1125. 11. Peric S, Glumac JN, Topf A, et al. A novel recessive TTN founder variant is a common ¨ Ana Topf, PhD Newcastle Author Analysis of genetic data cause of distal myopathy in the Serbian population. Eur J Hum Genet : EJHG 2017; University, 25:572–581. Newcastle upon 12. Lek M, Karczewski KJ, Minikel EV, et al. Analysis of protein-coding genetic variation Tyne, United in 60,706 humans. Nature 2016;536:285–291. Kingdom 13. Schwarz JM, Cooper DN, Schuelke M, Seelow D. MutationTaster2: mutation prediction for the deep-sequencing age. Nat Methods 2014;11:361–362. Volker Newcastle Author Critical revision of the 14. Kircher M, Witten DM, Jain P, O’Roak BJ, Cooper GM, Shendure J. A general Straub, MD, University, manuscript for framework for estimating the relative pathogenicity of human genetic variants. Nat PhD Newcastle upon important intellectual Genet 2014;46:310–315. Tyne, United content 15. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for Kingdom biological-image analysis. Nat Methods 2012;9:676–682. 16. Rueden CT, Schindelin J, Hiner MC, et al. ImageJ2: ImageJ for the next generation of Peter De University of Author Acquisition of patient scientific image data. BMC Bioinformatics 2017;18:529. Jonghe, MD, Antwerp, and imaging data and 17. Brubaker PH, Kitzman DW. Chronotropic incompetence: causes, consequences, and PhD Antwerp, critical revision of the management. Circulation 2011;123:1010–1020. Belgium manuscript for 18. Straub V, Murphy A, Udd B. 229th ENMC international workshop: limb important intellectual girdle muscular dystrophies - nomenclature and reformed classification content Naarden, The Netherlands, 17-19 March 2017. Neuromuscul Disord 2018;28: 702–710. Jan L. De Ghent University Author Acquisition and 19. Kirchmaier BC, Poon KL, Schwerte T, et al. The Popeye domain containing 2 Bleecker, MD, Hospital, Ghent, interpretation of patient, (popdc2) gene in zebrafish is required for heart and skeletal muscle development. Dev PhD Belgium imaging, and Biol 2012;363:438–450. histopathology data 20. Kyriakides T, Angelini C, Schaefer J, et al. EFNS guidelines on the diagnostic approach to pauci- or asymptomatic hyperCKemia. Eur J Neurol 2010;17: Gisèle Bonne, Institute of Author Study supervision, study 767–773. PhD Myology, G.H. concept and design, and 21. Alcalay Y, Hochhauser E, Kliminski V, et al. Popeye domain containing 1 (Popdc1/ ˆ Pitié-, Salpetrière critical revision of the Bves) is a caveolae-associated protein involved in ischemia tolerance. PLoS One 2013; Paris F-75013, manuscript for 8:e71100. France important intellectual 22. Silvestri NJ, Wolfe GI. Asymptomatic/pauci-symptomatic creatine kinase elevations content (hyperckemia). Muscle & nerve 2013;47:805–815. 23. Baruteau AE, Probst V, Abriel H. Inherited progressive cardiac conduction disorders. Jonathan University of Author Acquisition and Curr Opin Cardiol 2015;30:33–39. 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Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 11 ARTICLE OPEN ACCESS Autosomal dominant optic atrophy and cataract “plus” phenotype including axonal neuropathy

Alejandro Horga, MD, Enrico Bugiardini, MD,* Andreea Manole, PhD,* Fion Bremner, FRCOphth, PhD, Correspondence Zane Jaunmuktane, MD, FRCPath, Lois Dankwa, MS, Adriana P. Rebelo, PhD, Catherine E. Woodward, BSc, Dr. Reilly [email protected] Iain P. Hargreaves, PhD, Andrea Cortese, MD, Alan M. Pittman, PhD, Sebastian Brandner, MD, FRCPath, James M. Polke, PhD, Robert D.S. Pitceathly, MRCP, PhD, Stephan Zuchner,¨ MD, PhD, Michael G. Hanna, MD, FRCP, Steven S. Scherer, MD, PhD, Henry Houlden, MD, PhD, and Mary M. Reilly, MD, FRCP

Neurol Genet 2019;5:e322. doi:10.1212/NXG.0000000000000322 Abstract Objective To characterize the phenotype in individuals with OPA3-related autosomal dominant optic atrophy and cataract (ADOAC) and peripheral neuropathy (PN).

Methods Two probands with multiple aﬀected relatives and one sporadic case were referred for evaluation of a PN. Their phenotype was determined by clinical ± neurophysiological assessment. Neuropath- ologic examination of sural nerve and skeletal muscle, and ultrastructural analysis of mitochondria in ﬁbroblasts were performed in one case. Exome sequencing was performed in the probands.

Results The main clinical features in one family (n = 7 affected individuals) and one sporadic case were early-onset cataracts (n = 7), symptoms of gastrointestinal dysmotility (n = 8), and possible/ confirmed PN (n = 7). Impaired vision was an early-onset feature in another family (n = 4 affected individuals), in which 3 members had symptoms of gastrointestinal dysmotility and 2 developed PN and cataracts. The less common features among all individuals included symptoms/signs of autonomic dysfunction (n = 3), hearing loss (n = 3), and recurrent pancreatitis (n = 1). In 5 individuals, the neuropathy was axonal and clinically asymptomatic (n = 1), sensory-predominant (n = 2), or motor and sensory (n = 2). In one patient, nerve biopsy revealed a loss of large and small myelinated fibers. In fibroblasts, mitochondria were frequently enlarged with slightly fragmented cristae. The exome sequencing identified OPA3 variants in all probands: a novel variant (c.23T>C) and the known mutation (c.313C>G) in OPA3.

Conclusions A syndromic form of ADOAC (ADOAC+), in which axonal neuropathy may be a major feature, is described. OPA3 mutations should be included in the diﬀerential diagnosis of complex inherited PN, even in the absence of clinically apparent optic atrophy.

*These authors contributed equally to the manuscript.

From the Department of Neuromuscular Diseases (A.H., A.C., M.G.H., M.M.R.), UCL Queen Square Institute of Neurology and the National Hospital for Neurology and Neurosurgery, University College London Hospitals; Department of Molecular Neuroscience (A.M.P., H.H.), UCL Queen Square Institute of Neurology; Department of Neuro-ophthalmology (F.B.F.R.C.O.), the National Hospital for Neurology and Neurosurgery, University College London Hospitals; Division of Neuropathology (Z.J., S.B.), the National Hospital for Neurology and Neurosurgery, University College London Hospitals; Department of Clinical and Movement Neurosciences (Z.J.), UCL Queen Square Institute of Neurology, London, United Kingdom; Department of Neurology (L.D., S.S.S.), Perelman School of Medicine, University of Pennsylvania, Philadelphia; Department of Human Genetics and Hussman Institute for Human Genomics (A.P.R., S.Z.), University of Miami, FL; Department of Neurogenetics (C.E.W., J.M.P.), the National Hospital for Neurology and Neurosurgery, University College London Hospitals; Neurometabolic Unit (I.P.H.), the National Hospital for Neurology and Neurosurgery, University College London Hospitals; and Department of Neuro- degenerative Disease (S.B.), UCL Queen Square Institute of Neurology, London, United Kingdom.

The Article Processing Charge was funded by University College London. 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.

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary AA = amino acid; ADOAC = autosomal dominant optic atrophy and cataracts; DOA = dominant optic atrophy; EM = electron microscope; ETC = electron transport chain; MGA3 = 3-methylglutaconic aciduria type III (Costeﬀ syndrome); MTS = mitochondrial targeting sequence; NCS = nerve conduction study; OA = optic atrophy; OPA3 = Optic atrophy 3 protein; PN = peripheral neuropathy.

– Mutations in OPA3 (figure 1), encoding a mitochondrial ADOAC [MIM 165300]).14 18 Additional features such as protein likely involved in the regulation of mitochondrial hearing loss, cerebellar and extrapyramidal signs, and dysau- – fission,1 5 are associated with 2 distinct disorders that share tonomic or gastrointestinal symptoms have been reported less the common feature of bilateral optic atrophy (OA; table e-1, consistently in patients with ADOAC (table e-2). Symptoms links.lww.com/NXG/A146). Recessive, loss-of-function or signs suggestive of peripheral neuropathy (PN) have been mutations cause 3-methylglutaconic aciduria type III described to date in 4 individuals with dominant OPA3 (MGA3 [MIM 258501]), also known as Costeff syndrome, mutations,14,15,18 although it was confirmed by nerve con- a rare disorder that is clinically characterized by OA and ex- duction studies (NCSs) in only one of them.18 trapyramidal dysfunction with onset in the first decade of life, subsequent development of spastic paraparesis and cerebellar Here, we describe 2 families and one sporadic case with signs, and increased urinary excretion of 3-methylglutaconic a syndromic form of ADOAC in which PN was a major – and 3-methylglutaric acids.6 13 Dominant mutations, in con- clinical feature. This report broadens the clinical and genetic trast, lead to OA that typically presents within the first 2 spectrum of ADOAC and indicates that OPA3 should be decades of life and is often associated with cataracts with included in the differential diagnosis of the complex inherited variable age at onset (autosomal dominant OA and cataract; PN19 even in the absence of clinically apparent OA.

Figure 1 Schematic of the OPA3 gene and OPA3 protein isoform b

The OPA3 gene (NCBI RefSeq NG_013332.1; top figure), located in chromosome 19q13.32, contains 3 exons (1, 2, and 3; boxes containing exon numbers) and spans 57.4 kb. The coding regions of exons 2 and 3 and their exon-intron boundaries are highly similar and may have originated by segmental duplication.3 OPA3 exons are alternatively spliced to generate 2 mRNA transcripts: transcript variant 2 (exon 1 plus exon 2 [NM_025136.3]) and transcript variant 1 (exon1 plus exon 3 [NM_001017989.2]). Transcript variant 2 seems to be the predominant transcript in most tissues and encodes a 179 amino acid (AA) protein (OPA3 isoform b [NP_079412.1]; bottom figure). Transcript variant 1 encodes a 180 AA protein (OPA3 isoform a [NP_001017989]; not shown). OPA3 isoform b amino acids 1–48 are encoded by exon 1 (dark green) and amino acids 48–179 are encoded by exon 2 (light green). Its N-terminal region contains a putative mitochondrial targeting sequence in AAs 1–18 (as predicted by in silico analysis with MitoProt and TargetP) or 1–30 (as indicated by functional studies with deletion mutants). In silico analysis with MitoFates predicts a mitochondrial processing peptidase cleavage site (red arrow) and 2 TOM20 recognition motifs (AAs 8–12 and 48–52; red boxes). An additional mitochondrial sorting/cleavage signal at position 25–29 (purple box) has been proposed by some authors.3,8 The localizations of reported dominant and recessive mutations are shown in the figure (one-letter AA abbreviations are used for simplicity; blue boxes indicate mutations reported in the present study). p.Leu11Gln (p.L11Q) was homozygous in 2 siblings with optic atrophy, extrapyramidal signs including dystonia, and pyramidal signs or ataxia, and it was heterozygous in their mother with later-onset dystonia.30 No pathogenic mutations have been described in AAs 48-180 of OPA3 isoform a encoded by exon 3 (not shown).

2 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Methods using standard methods. Cells were grown in high glucose Dulbecco’s modified Eagle’s medium, supplemented with Patients 10% fetal bovine serum, penicillin/streptomycin, and 0.05 The 3 probands were identified at the outpatient clinics of the mg/mL uridine. For electron microscopy (EM), fibroblasts National Hospital for Neurology and Neurosurgery, London, were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate ff UK, and the Charcot-Marie-Tooth disease (CMT) Center of bu er and 5 mM CaCl2 overnight, then treated with 1% os- Excellence at the University of Pennsylvania, Philadelphia, mium tetroxide for 3 hours at 4°C and embedded in Araldite PA. These probands were recruited into local research pro- CY-212 resin. Ultrathin sections (70 μm) were stained with tocols to determine the genetic etiology in patients with lead citrate and uranyl acetate. Images were taken on a Philips inherited neuropathies using next-generation sequencing. CM10 transmission electron microscope fitted with a Mega- Diagnostic laboratory tests, neurophysiological studies, MRI View G3 camera and RADIUS Software (Olympus). Mito- scans, and tissue biopsies were performed and analyzed using chondria morphology in fibroblasts was measured blind to standard protocols. disease status.

Sequencing Standard protocol approvals, registrations, DNA samples were extracted from peripheral blood leuko- and patient consents cytes and skeletal muscle biopsy specimens using commercial Research protocols were approved by local institutional re- kits. Exome sequencing was performed after target capture view board and/or ethics committee. Patients gave written using the Illumina TruSeq Exome, Agilent SureSelect Focused informed consent to participate. Exome, or Agilent SureSelect Human All Exon kit. The Illumina HiSeq2000 or HiSeq2500 instruments were used to produce 100 Data availability bp paired-end sequence reads. The following software tools were Anonymized data not published within this article will be used to align sequence reads to the human genome assembly made available by request from any qualified investigator. 19 (GRCh37) and to call and annotate variants: Novocraft NovoAlign, Burrows-Wheeler Aligner, Picard, Genome Analysis Results Toolkit, SAMtools, and ANNOVAR.20 After filtering, candidate variants were evaluated in silico to predict their effects, and vali- Families and overall phenotype dation and cosegregation analysis of OPA3 variants in families A, Three unrelated probands were referred to our PN clinics for B, and C were performed by Sanger sequencing (see supple- evaluation. Probands AII-2 and BIII-2 had affected relatives of mentary material for details, links.lww.com/NXG/A146). 3 generations based on history (AI-1, AII-1, AIII-2, AIII-3, AIII-4, BI-1, and BII-3) or examination (AIII-1 and BII-1), Mitochondrial DNA (mtDNA) was assessed for large-scale while proband CII-1 was a sporadic case (figure 2). The rearrangements using long-range PCR and Southern blot of clinical information of all affected individuals is summarized in total genomic DNA extracted from skeletal muscle. The entire table 1, and their clinical description is provided in the sup- mtDNA sequence was analyzed with Affymetrix GeneChip plementary material (links.lww.com/NXG/A146). Human Mitochondrial Resequencing Array 2.0 as described elsewhere.21 Family A The phenotype in the affected members of family A (n = 7) Cell imaging was mostly characterized by early-onset cataracts, gastroin- Fibroblast cell lines were established from skin biopsies of testinal dysmotility symptoms, and PN (based on symptoms/ proband AII-2 and a healthy age- and sex-matched control signs or neurophysiological studies), although the data on

Figure 2 Pedigrees of families and segregation analysis of variants c.23T>C (p.Met8Thr) and c.313C>G (p.Gln105Glu) in OPA3 (NM_025136.3)

OPA3 variants found in each family and the genotype of tested individuals are indicated in red. Arrow = probands.

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 3 Table 1 Summary of clinical features in affected individuals from families A, B, and C

Bilateral Gastrointestinal Peripheral PN on Bilateral optic dysmotility neuropathy NCS/ Hearing Individual cataracts atrophy symptoms symptoms/signs EMG loss Other features

AI-1 + n/a + ± n/a + <5 y 50s

AII-1 + n/a + ± n/a n/a <5 y 40s

AII-2 + + + + + + Autonomic <5 y on examination 49 y 30s 40s axonal S>M 50s features, mild ptosis

AIII-1 + ±a + + + + Parenteral <3 y 10s 10s axonal S&M nutrition from his 20s

AIII-2 n/a n/a ± n/a n/ab n/a

AIII-3 + n/a ± n/a + n/a early-onset

AIII-4 + n/a ± + n/a n/a surgery ≥15 y

BI-1 n/a ±a n/a n/a n/a — 10 y

BII-1 + + ±+ + — on examination 65 y 10 y on examination 65 y axonal S>M

BII-3 n/a + ± n/a n/a — 7y

BIII-2 + + + + + + Orthostatic surgery 44 y 5y 5y 20s axonal S>M 50s hypotension

CII-1 + + + + + — Recurrent surgery 14 y on examination 32 y 10s ≤16 y axonal S&M pancreatitis, hypertension, PRES

Abbreviations: n/a = not available or unknown; M = motor; NCS/EMG = nerve conduction studies/electromyography; PN = peripheral neuropathy; PRES = posterior reversible encephalopathy syndrome; S = sensory; ± mild or possible symptoms reported by relatives. The age at onset of symptoms is indicated below the clinical features when available. a Bilateral visual loss but no confirmed diagnosis of optic atrophy. b Abnormal neurophysiological studies.

these features were incomplete for 3 individuals. Reduced Family C visual acuity was first detected in proband AII-2 during the Proband CII-1 was the only affected individual in this family. evaluation of her PN at age 49, and OA was subsequently Her phenotype was similar to that of family A: early-onset confirmed (figure 3, A and B). A reduced visual acuity was cataracts, gastrointestinal dysmotility symptoms, and PN. In also observed in AIII-1, but we had no confirmation of an addition, she had recurrent episodes of pancreatitis from age underlying OA. Impaired vision was not reported for other 11. Bilateral OA was detected by reverse phenotyping: after relatives. Intrafamilial phenotypic variability was evident, a genetic diagnosis of her PN was achieved, by optical co- with patient AIII-1 having more severe symptoms than his herence tomography. siblings, and an earlier and more severe presentation than AII-2. Peripheral neuropathy Nine of the 12 affected individuals from the 3 families had Family B a possible or confirmed PN. It was considered possible in 4 Early-onset, bilateral visual impairment leading to severely individuals (AI-4, AIII-2, AIII-3, and AIII-4) based on the reduced visual acuities by the age of 30–40 was the initial history provided by relatives. In 5 patients (AII-2, AIII-1, BII- clinical feature in all affected members of family B (n = 4; 1, BIII-2, and CII-1), we confirmed the diagnosis based on originally reported in 199622). Three of them had a diagnosis clinical and neurophysiological assessment (see supplemen- of OA and reported symptoms of gastrointestinal dysmotility. tary material for an extended description, links.lww.com/ PN and cataracts were detected in proband BIII-2 and her NXG/A146). Detailed results of NCS/EMG were available in father BII-1 on follow-up examinations in their adulthood. 4 cases (table e-3).

4 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Figure 3 Bilateral optic atrophy and sural nerve biopsy of patient AII-2

(A) Red-free photographs of the optic discs of patient AII-2 show mild temporal pallor of both optic discs. (B) Optical coherence tomography measurements of the retinal nerve fiber layer thickness around both optic discs of patient AII-2 confirm significant thinning in the temporal quadrants consistent with an optic neuropathy. (D) Semi-thin resin section of the sural nerve, stained with methylene blue azure-basic fuchsin, shows a fascicle with severe loss of large (red arrowhead) and small myelinated fibers with no apparent active axonal degeneration and minimal regeneration (scale bar = 25 μm). (E) Electron microscopy shows frequent denervated Schwann cell profiles and bands of Bungner¨ (blue arrowheads) in keeping with widespread fiber loss. The mitochondria (yellow arrowheads) show no apparent pathology (scale bar = 1 μm).

Asymptomatic neuropathy AIII-1 had pes cavus and limb weakness since his teens and In patient BII-1, the PN was asymptomatic and detected on had been diagnosed with inherited neuropathy in his early examination at age 65. NCS were consistent with an axonal 20s; he was examined by us at age 31 as part of the family motor and sensory neuropathy. evaluation. Both patients exhibited motor and sensory deficits in upper and lower limbs on examination. Proband CII-1 had Sensory-predominant neuropathy 4 neurophysiological studies performed between ages 16 and Probands AII-2 and BIII-2 had a slowly progressive, pre- 32, which revealed a severe motor and sensory axonal neu- dominantly sensory neuropathy. AII-2 developed sensory ropathy. In patient AIII-3, a previous neurophysiological symptoms in her feet in her late 40s and was referred to us for study at age 29 confirmed the same diagnosis. the evaluation of suspected PN at age 49. BIII-2 developed sensory symptoms in her feet in his 20s; her PN was con- Other clinical features firmed in her early 30s, but she was referred again to us at age Symptoms of gastrointestinal dysmotility of variable severity 52 because of symptom worsening. Both patients described were reported for most individuals. In the best documented reduced sensation restricted to the lower limbs. On serial cases, symptoms included intermittent constipation (AII-2); examinations, we observed distal sensory loss in the upper and episodes of nausea, vomiting, and abdominal pain (CII-1); lower limbs and signs of mild motor involvement. In both gastroparesis and episodes of intestinal pseudo-obstruction cases, NCS revealed a length-dependent axonal sensory (BIII-2); and intestinal pseudo-obstruction requiring paren- neuropathy. NCS/EMG signs of distal motor involvement teral nutrition (AIII-1). Patient BIII-2 required emergency were detected only in proband BIII-2. surgery for intestinal intussusception in her 50s, and patient AII-1 was also reported to require surgery for her intestinal Motor and sensory neuropathy motility problems. Proband CII-1 and patient AIII-1 had a moderate-to-severe progressive motor and sensory neuropathy with onset in their All probands had features suggestive of autonomic nervous childhood or teens. Proband CII-1 had frequent ankle sprains system dysfunction, including orthostatic tachycardia (AII-2), as a child and was referred to us at age 16 when a PN was postural hypotension (BIII-2), and episodes of hypertension suspected during physical therapy for a foot fracture. Patient in the context of gastrointestinal symptoms that, in one

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 5 occasion, lead to a posterior reversible encephalopathy syn- In proband AII-2, we identified the heterozygous missense drome (CII-1). variant c.23T>C in exon 1 of OPA3 (table e-5, links.lww.com/ NXG/A146). Sanger sequencing detected the mutation in the Patient AIII-1 developed hearing loss concurrently to other proband and her affected daughter (AIII-4) but not in the symptoms. Proband AII-2 complained of mild hearing loss in unaffected son (AIII-5), who was homozygous for the wild- her 50s, and a bilateral auditory neuropathy was confirmed in type allele (figure 2). c.23T>C is a novel variant absent from her 60s. Patient AI-1 and proband BIII-2 developed hearing public databases and from an in-house control exome database. loss in their 50s. The less common clinical features are shown It affects an evolutionarily conserved nucleotide and is pre- in table 1. dicted to be deleterious by several bioinformatics tools (table e-6). c.23T>C leads to the substitution of nonpolar, hydro- Neuropathology phobic methionine for polar threonine at amino acid (AA) Patient AII-2 underwent sural nerve and quadriceps muscle position 8 of OPA3 (p.Met8Thr). This position lies within the biopsy at age 57. Nerve biopsy showed a severe loss of my- N-terminal mitochondrial targeting sequence (MTS) predicted elinated fibers that was not selective for large or small fibers, by MitoProt and TargetP software tools (AA 1-18) or by with no apparent active axonal degeneration and minimal functional studies with deletion mutants (AA 1-30),2 in which regeneration, and no evidence of demyelination (figure 3, C another dominant mutation has been identified (figure 1).16 In and D). Muscle biopsy revealed occasional angular atrophic silico analysis with MitoFates suggests that methionine 8 would fibers, 2 cytochrome oxidase-negative fibers, and one ragged- be the first of 5 AA of a putative consensus motif recognized by red fiber. The activity of mitochondrial electron transport the mitochondrial import receptor subunit TOM20 (figure 1) chain (ETC) complexes I, II + III, and IV was within control and that p.Met8Thr would eliminate this recognition motif. ranges, and screening for single/multiple deletions and point The functional significance of hydrophobic AA at position 8 of mutations of mtDNA in muscle were negative. OPA3 is supported by multiple sequence alignment of OPA3 homologs, which shows conservation of methionine and iso- EM of mitochondria leucine at that position from human to zebra fish (figure e-1). Ultrastructural examination of mitochondria with EM was Based on these data, and the previous association of dominant performed in the cultured fibroblasts from proband AII-2 and OPA3 mutations with OA, cataracts, and PN, we considered a healthy control. On EM images, we frequently observed c.23T>C as the most plausible genetic etiology in family A. abnormally enlarged mitochondria coupled with slightly fragmented mitochondrial cristae and a concomitant reduction in In probands BIII-2 and CII-1, we identified the heterozygous the area of mitochondrial cristae (figure 4). missense mutation c.313C>G (p.Gln105Glu) in exon 2 of OPA3. The mutation was validated by Sanger sequencing in Genetic studies both cases. In family B, DNA samples from relatives were not In probands AII-2, BIII-2, and CII-1, analysis of common available for cosegregation analysis. In family C, the mutation genes associated with inherited neuropathy and/or OA yielded was not found in either parent, confirming that it had occurred negative results. Exome sequencing was then performed on these de novo in the proband (figure 2). c.313C>G is not reported patients as 3 independent studies. The analysis focused on in public databases but is the most frequent mutation reported nonsynonymous, splice-site, and coding indel variants with mi- in families with dominant OPA3-related OA, sometimes in nor allele frequency <0.1% in the Exome Aggregation Consor- association with additional features (tables e-1 and e-2, links. tium data set, located in genes known to cause inherited lww.com/NXG/A146).14,16,17 This variant was therefore neuropathy and/or OA (table e-4, links.lww.com/NXG/A146). classified as likely pathogenic.

Figure 4 Ultrastructural examination of mitochondria in cultured skin fibroblasts

(A) Representative electron micrographs showing the ultrastructure of mitochondria in control and patient AII-2 fibroblasts (scale bar = 1 μm). (B) Patient AII-2 mitochondria (n = 94) display a significant increase in area as compared to control mitochondria (n = 123). (C) Data are presented as box plots illustrating 80% of the data distribution; 10th, 25th, median, 75th, and 90th percentiles are shown for these box plots. *p < 0.0005 (Mann-Whitney U test).

6 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Discussion disorders, for which both neuropathic and myopathic mechanisms have been proposed.22 Here, we describe the members from 3 families in which a novel missense variant (c.23T>C, p.Met8Thr) and a known The precise function of the OPA3 protein and the molecular mutation (c.313C>G, p.Gln105Glu) in OPA3 were the most mechanisms by which heterozygous missense mutations in likely underlying cause of a complex phenotype consisting of OPA3 cause ADOAC/+ are unknown. OPA3 predominantly – OA, cataracts, gastrointestinal dysmotility, axonal neuropathy, localizes to the mitochondria2 4 but its intramitochondrial and possibly autonomic dysfunction and hearing loss. This topology is still discussed: the mouse homolog of OPA3 observation, together with 2 former reports of similar phe- copurifies with the inner mitochondrial membrane,1 but the notypes associated with the missense substitutions studies on subcellular fractions of HeLa cells suggest that 15,18 p.Leu79Val and p.Gln105Glu, demonstrates that the OPA3 may be anchored to the mitochondrial outer mem- dominant OPA3 mutations can cause syndromic forms of brane with the C-terminus exposed to the cytoplasm.2 ADOAC (ADOAC “plus”). Overexpression of OPA3 induces mitochondrial fragmentation, whereas downregulation leads to more elongated and We also confirm that an axonal neuropathy may be a major tubular mitochondria.2,5 These data support that OPA3 is clinical feature of ADOAC/+. In 4 patients from our series, a mitochondrial membrane protein implicated in the regula- the PN was indeed a major cause of disability or was severe tion of mitochondrial fission and morphology. enough to motivate the referral to our centers. In 5 patients with a well-documented axonal neuropathy, we observed 3 Heterozygous carriers of the recessive OPA3 mutation c.143- clinical presentations: asymptomatic, sensory-predominant, 1G>C, which abolishes mRNA expression, are and motor and sensory. However, given the differences in the asymptomatic.8,23 This suggests that dominant missense age of symptoms onset and overall disease severity between mutations have a dominant-negative effect or result in a gain-of- patients, and the later development of motor signs in patients function rather than haploinsufficiency. Fibroblasts from with sensory-predominant forms, it is possible that the dif- ADOAC patients with the p.Val3_Gly4insAlaPro mutation, ferent PN presentations may simply reflect a variable ex- located in the N-terminal MTS of OPA3, showed increased pressivity of the same pathologic process rather than distinct fragmentation of the mitochondrial network,16 which mimics PN subtypes. A sensory ganglionopathy seemed unlikely in OPA3 overexpression and gives support to the gain-of-function patients with sensory-predominant forms, in whom the PN hypothesis. Similar findings were observed in HeLa cells was length-dependent and symmetrical, with almost normal transfectedwithanOPA3mutantcarryingthep.Gly93Ser joint position sense and no significant limb ataxia. mutation, located in the hydrophobic region of OPA3 (AA 83- 120) that is required for mitochondrial fragmentation.2 How- Despite the limited sample size, our report also suggests ever, no mitochondrial network abnormalities were detected in a higher prevalence of PN among patients with ADOAC/+ fibroblasts from one patient with the same mutation.14 There- than previously recognized. Possible reasons for this are that fore, it is possible that dominant mutations affecting different the PN may be asymptomatic and/or develop several decades regions of OPA3 may exert different deleterious effects. after the onset of the ophthalmologic problems. Therefore, long-term follow-up with neurologic evaluations may be Studies in a zebra fish model of MGA3 indicate that OPA3 necessary to confirm or exclude a PN in these patients. On the does not have a direct role in mitochondrial ETC function,24 other hand, data from this and previous studies indicate that and consistent with this, we found the normal activities of the dominant OPA3 mutations may exhibit considerable ETC complexes in skeletal muscle from patient AII-2. We did phenotypic variability, so it is also possible that the families or not detect large-scale mtDNA rearrangements in muscle, individuals with the same mutation may or may not develop which argues against a role of OPA3 in mtDNA maintenance. additional features such as PN. This point should be clarified However, EM analysis of mitochondria in fibroblasts from in future studies encompassing larger sample sizes. patient AII-2 revealed frequently enlarged mitochondria with abnormal cristae. Subtle alterations to the morphology of Symptoms of gastrointestinal dysmotility were reported mitochondrial cristae have also been identified in the retinal for most affected individuals from the present series, which so tissues of a mouse model of MGA3 caused by the homozy- far has only been described in 3 patients with ADOAC/ gous missense mutation p.Leu122Pro.4 Therefore, OPA3 15,16,18 +. Results from gastrointestinal investigations were not function might be important for the structural integrity of available for review, and thus, we could not confirm the mitochondrial cristae, and altered mitochondrial cristae ar- underlying pathophysiology. Its coexistence with cardio- chitecture could be an additional mechanism involved in the vascular dysautonomic features and/or PN in cases from pathogenesis OPA3-related disease. the present and previous studies raises the possibility of a myenteric plexus or extra-intestinal autonomic neuropa- Based on the findings from the present study, some parallels thy, although we cannot exclude other causes such as an can be drawn between OPA3- and OPA1-related diseases. enteric myopathy. Of note, gastrointestinal dysmotility is Mutations affecting OPA1, a dynamin-related guanosine tri- a well-recognized manifestation of certain mitochondrial phosphatase involved in mitochondrial dynamics, represent

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 7 a major cause of dominant OA (DOA), and ;20% of patients received stock in Disarm Therapeutics, and has worked as may present a DOA “plus” phenotype, including axonal an expert witness for Kaye Scholer. H. Houlden has re- neuropathy.25 Biallelic mutations in OPA1 can also cause Behr ceived research support from The Medical Research – syndrome,26 28 a severe, early-onset neuro-ophthalmologic Council (MRC) UK, The BRT, The MDA USA, Muscular syndrome that is clinically similar to MGA3. Of interest, the Dystrophy UK, Ataxia UK, Muscular Dystrophy UK, loss of OPA1 causes disruption of mitochondrial cristae RosetreesTrust,TheWellcomeTrust,andtheNational structures.29 Overall, these data may indicate shared patho- Institute for Health UCL/UCLH BRC. M.M. Reilly has genic mechanisms between OPA3 and OPA1 defects that served on the Editorial Boards for Brain, Neuromuscular warrant further research. Disorders, and JNNP; has consulted for Servier, Acceleron, Alynam, IONIS, Myotherix, and Inﬂectis; and has received Acknowledgment research support from the NIH, MDA, MRC, Wellcome SSS and LD thank Tanya Bardakjian and Diana Lee for their Trust, UCL CBRC, NIHR, Muscular Dystrophy Campaign, help. and the CRDC. Disclosures available: Neurology.org/NG.

Study funding Publication history M.M. Reilly, S.S. Scherer, A. Cortese, and L. Dankwa are Received by Neurology: Genetics November 16, 2018. Accepted in ﬁnal supported by the Inherited Neuropathy Consortium form February 1, 2019. (INC), which is a part of the NIH Rare Diseases Clinical Research Network (RDCRN) (U54NS065712). RDCRN is an initiative of the Oﬃce of Rare Diseases Research Appendix Author contributions (ORDR), NCATS, funded through a collaboration between NCATS and the NINDS. S.S. Scherer and L. Dankwa Name Location Role Contribution are also supported by the Judy Seltzer Levenson Memorial Alejandro University College Author Drafting/revising the Horga, MD London, London, manuscript for Fund for CMT Research. M.M. Reilly and S. Brandner are UK content; major role in also supported by the National Institute for Health Re- acquisition of data; study concept or search University College London Hospitals (UCLH) design; Analysis/ Biomedical Research Centre. A. Cortese is also funded by interpretation of data the Wellcome Trust (204841/Z/16/Z). H. Houlden is Enrico University College Author Drafting/revising the supported by the Medical Research Council and Wellcome Bugiardini, MD London, London, manuscript for Trust. UK content; Major role in acquisition of data; Study concept or Disclosure design; Analysis/ interpretation of data The present study is not industry-sponsored. A. Horga, E. Bugiardini, and A. Manole report no disclosures. F. Bremner Andreea University College Author Drafting/revising the Manole, PhD London, London, manuscript for has received funding for travel or speaker honoraria from UK content; Major role in Allergan and has served on the Editorial Board for BMC acquisition of data; Study concept or Opthalmology, Frontiers in Neuro-opthalmology, and Tropical design; Analysis/ Medicine. Z. Jaunmuktane has served on the Editorial Board of interpretation of data

Acta Neuropathologica. L. Dankwa, A.P. Rebelo, C.E. Wood- Fion Bremner, National Hospital Author Drafting/revising the ward, and I. Hargreaves report no disclosures. A. Cortese has FRCOphth, for Neurology and manuscript for PhD Neurosurgery, content; Major role in served on the Editorial Board for Journal of the Peripheral London, UK acquisition of data; Nervous System and has received foundation/society research Analysis/ support from the NIH and Wellcome Trust. A. Pittman interpretation of data reports no disclosures. Sebastian Brandner has served on Zane National Hospital Author Drafting/revising the advisory boards for the French Institute for Cancer, has served Jaunmuktane, for Neurology and manuscript for MD, FRCPath Neurosurgery, content; Major role in on the Editorial Board for Acta Neuropathologica, has received London, UK acquisition of data; publishing royalties from Elsevier, and has received govern- Analysis/ interpretation of data ment research support from the Department of Health. J.M. Polke, R.D.S. Pitceathly, and S. Z¨uchner report no Lois Dankwa, Perelman School Author Revising the MS of Medicine, manuscript; Major disclosures. M.G. Hanna has consulted for Novartis and Philadelphia, USA role in acquisition of received foundation/society research support from the data; Analysis/ interpretation of Medical Research Council, the Myositis Support Group, data and NIHR BRC. Steven S Scherer has received travel Adriana P. University of Author Revising the funding or speaker honoraria from Easai, has consulted for Rebelo, PhD Miami, Miami, USA manuscript; Major Disarm Therapeutics and Biogen, has received government role in acquisition of research support from the NIH, has received foundation/ data; Analysis/ interpretation of data society research support from the CMT association, has

8 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG References Appendix (continued) 1. Da Cruz S, Xenarios I, Langridge J, Vilbois F,ParonePA,MartinouJC.Proteomicanalysis of the mouse liver mitochondrial inner membrane. J Biol Chem 2003;278:41566–41571. Name Location Role Contribution 2. Ryu SW, Jeong HJ, Choi M, Karbowski M, Choi C. Optic atrophy 3 as a protein of the mitochondrial outer membrane induces mitochondrial fragmentation. Cell Mol Life Sci 2010;67:2839–2850. Catherine E National Hospital Author Revising the 3. Huizing M, Dorward H, Ly L, et al. OPA3, mutated in 3-methylglutaconic aciduria Woodward, for Neurology and manuscript; Analysis/ type III, encodes two transcripts targeted primarily to mitochondria. Mol Genet BSc Neurosurgery, interpretation of data; Metab 2010;100:149–154. London, UK Acquisition of data 4. Powell KA, Davies JR, Taylor E, Wride MA, Votruba M. Mitochondrial localization and ocular expression of mutant Opa3 in a mouse model of 3-methylglutaconicaciduria type III. Iain P. National Hospital Author Revising the Invest Ophthalmol Vis Sci 2011;52:4369–4380. Hargreaves, for Neurology and manuscript; Analysis/ 5. Ryu SW, Yoon J, Yim N, Choi K, Choi C. Downregulation of OPA3 is responsible for PhD Neurosurgery, interpretation of data; London, UK Acquisition of data transforming growth factor-beta-induced mitochondrial elongation and F-actin rearrangement in retinal pigment epithelial ARPE-19 cells. PLoS One 2013;8:e63495. ff Andrea University College Author Revising the 6. Coste H, Gadoth N, Apter N, Prialnic M, Savir H. A familial syndrome of infantile optic atrophy, movement disorder, and spastic paraplegia. Neurology 1989;39:595–597. Cortese, MD London, London, manuscript; Analysis/ ff UK interpretation of data 7. Elpeleg ON, Coste H, Joseph A, Shental Y, Weitz R, Gibson KM. 3-Methylglutaconic aciduria in the Iraqi-Jewish ’optic atrophy plus’ (Costeff) syndrome. Dev Med Child Neurol – Alan M. University College Author Revising the 1994;36:167 172. 8. Anikster Y, Kleta R, Shaag A, Gahl WA, Elpeleg O. Type III 3-methylglutaconic Pittman, PhD London, London, manuscript; ff UK Acquisition of data aciduria (optic atrophy plus syndrome, or Coste optic atrophy syndrome): identification of the OPA3 gene and its founder mutation in Iraqi Jews. Am J Hum Genet – Sebastian National Hospital Author Revising the 2001;69:1218 1224. 9. Kleta R, Skovby F, Christensen E, Rosenberg T, Gahl WA, Anikster Y. 3-Methylglutaconic Brandner, MD, for Neurology and manuscript; Analysis/ fi FRCPath Neurosurgery, interpretation of data; aciduria type III in a non-Iraqi-Jewish kindred: clinical and molecular ndings. Mol Genet – London, UK Acquisition of data Metab 2002;76:201 206. 10. Neas K, Bennetts B, Carpenter K, et al. OPA3 mutation screening in patients with unexplained 3-methylglutaconic aciduria. J Inherit Metab Dis 2005;28:525–532. James M. National Hospital Author Revising the ff Polke, PhD for Neurology and manuscript; Analysis/ 11. Ho G, Walter JH, Christodoulou J. Coste optic atrophy syndrome: new clinical case fi – Neurosurgery, interpretation of data; and novel molecular ndings. J Inherit Metab Dis 2008;31(suppl 2):419 423. London, UK Acquisition of data 12. Lam C, Gallo LK, Dineen R, et al. Two novel compound heterozygous mutations in OPA3 in two siblings with OPA3-related 3-methylglutaconic aciduria. Mol Genet – Robert D.S. University College Author Revising the Metab Rep 2014;1:114 123. ff Pitceathly, London, London, manuscript for 13. Yahalom G, Anikster Y, Huna-Baron R, et al. Coste syndrome: clinical features and – MRCP, PhD UK content; Study natural history. J Neurol 2014;261:2275 2282. concept or design; 14. Reynier P, Amati-Bonneau P, Verny C, et al. OPA3 gene mutations responsible for Acquisition of data autosomal dominant optic atrophy and cataract. J Med Genet 2004;41:e110. 15. Ayrignac X, Liauzun C, Lenaers G, et al. OPA3—related autosomal dominant optic fl – Stephan University of Author Drafting/revising the atrophy and cataract with ataxia and are exia. Eur Neurol 2012;68:108 110. Zuchner,¨ MD, Miami, Miami, USA manuscript for 16. Grau T, Burbulla LF, Engl G, et al. A novel heterozygous OPA3 mutation located in PhD content; Major role in the mitochondrial target sequence results in altered steady-state levels and fragmented – acquisition of data; mitochondrial network. J Med Genet 2013;50:848 858. fi Study supervision 17. Sergouniotis PI, Perveen R, Thiselton DL, et al. Clinical and molecular genetic ndings in autosomal dominant OPA3-related optic neuropathy. Neurogenetics 2015;16:69–75. Michael G. University College Author Drafting/revising the 18. Bourne SC, Townsend KN, Shyr C, et al. Optic atrophy, cataracts, lipodystrophy/ Hanna, MD, London, London, manuscript for lipoatrophy, and peripheral neuropathy caused by a de novo OPA3 mutation. Cold FRCP UK content; Study Spring Harb Mol Case Stud 2017;3:a001156. concept or design; 19. Rossor AM, Carr AS, Devine H, et al. Peripheral neuropathy in complex inherited – Study supervision; diseases: an approach to diagnosis. J Neurol Neurosurg Psychiatry 2017;88:846 863. Obtaining funding 20. Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 2010;38:e164. Steven S. Perelman School Author Drafting/revising the 21. Rahman S, Ecob R, Costello H, et al. Hearing in 44-45 year olds with m.1555A>G, Scherer, MD, of Medicine, manuscript for a genetic mutation predisposing to aminoglycoside-induced deafness: a population PhD Philadelphia, USA content; Major role in based cohort study. BMJ Open 2012;2:e000411. acquisition of data; 22. Hom XB, Lavine JE. Gastrointestinal complications of mitochondrial disease. Mito- – Study supervision chondrion 2004;4:601 607. 23. Gunay-Aygun M, Huizing M, Anikster Y. OPA3-related 3-methylglutaconic aciduria. Henry University College Author Drafting/revising the In: Adam MP, Ardinger HH, Pagon RA, et al, editors. GeneReviews® [Internet]. Houlden, MD, London, London, manuscript for Seattle: University of Washington, Seattle; 2013. ff PhD UK content; Major role in 24. Pei W, Kratz LE, Bernardini I, et al. A model of Coste Syndrome reveals metabolic and – acquisition of data; protective functions of mitochondrial OPA3. Development 2010;137:2587 2596. Study concept and 25. Chao de la Barca JM, Prunier-Mirebeau D, Amati-Bonneau P, et al. OPA1-related design; Study disorders: diversity of clinical expression, modes of inheritance and pathophysiology. – supervision; Neurobiol Dis 2016;90:20 26. Obtaining funding 26. Bonneau D, Colin E, Oca F, et al. Early-onset Behr syndrome due to compound heterozygous mutations in OPA1. Brain 2014;137:e301. “ ” Mary M. Reilly, University College Author Drafting/revising the 27. Carelli V, Sabatelli M, Carrozzo R, et al. Behr syndrome with OPA1 compound MD, FRCP London, London, manuscript for heterozygote mutations. Brain 2015;138:e321. UK content; Major role in 28. Marelli C, Amati-Bonneau P, Reynier P, et al. Heterozygous OPA1 mutations in Behr – acquisition of data; syndrome. Brain 2011;134:e169 e170. Study concept and 29. Olichon A, Baricault L, Gas N, et al. Loss of OPA1 perturbates the mitochondrial design; Study inner membrane structure and integrity, leading to cytochrome c release and apo- supervision; ptosis. J Biol Chem 2003;278:7743–7746. Obtaining funding 30. Arif B, Kumar KR, Seibler P, et al. A novel OPA3 mutation revealed by exome sequencing: an example of reverse phenotyping. JAMA Neurol 2013;70:783–787.

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 9 CLINICAL/SCIENTIFIC NOTES OPEN ACCESS Mitochondrial cerebellar ataxia, renal failure, neuropathy, and encephalopathy (MCARNE)

Peng Soon Ng, MBBS,* Marcus V. Pinto, MD,* Jadee L. Neff, MD, PhD, Linda Hasadsri, MD, PhD, Correspondence Edward W. Highsmith, PhD,† Mary E. Fidler, MD, Ralitza H. Gavrilova, MD, and Christopher J. Klein, MD Dr. Klein [email protected] Neurol Genet 2019;5:e314. doi:10.1212/NXG.0000000000000314

Mitochondrial NADH dehydrogenase 5 (MT-ND5) Asp393Asn missense mutation is established to cause mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS).1,2 We describe a case and family with this mutation and a divergent phenotype that eluded diagnosis. We suggest an expanded nomenclature, mitochondrial cerebellar ataxia, renal failure, neuropathy, and encephalopathy (MCARNE).

Case report A 60-year-old naturalist had a slowly progressive loss of feeling in his feet with gait ataxia over 10 years with associated memory declines. Renal failure was diagnosed at age 57 years with a creatinine level of 3.5 mg/dL compared with 5 years earlier at 1.4 mg/dL (0.8–1.3 mg/dL). He did not have hypertension, was not a drinker, and had no stroke history. At age 59 years, he underwent living nonrelative donor kidney transplant. He had hearing loss and required hearing aids by age 45 years.

On cognitive evaluation, he scored 32/38 on Kokmen Short Test of Mental Status with deficits in recall and construction with inability to draw a cube or remember 4 objects at 5 minutes. He had a wide-based gait with inability to perform tandem walking or heel to shin testing and required 2 ski poles to walk. He had a mild ataxic dysarthria with past-pointing on finger-to-nose testing. There were reduced ankle reflexes without the Babinski sign. Pin prick, vibration, and proprioception were reduced at the toes. Upper and lower extremity strength was normal.

Brain MRI showed moderate generalized cerebral and severe cerebellar atrophy without strokes (figure). Nerve conduction studies at age 52 years showed absent medial plantar and reduced ulnar and sural sensory amplitudes with large motor units on needle EMG limited to extremity muscles consistent with a length-dependent axonal polyneuropathy. Myopathic units were not seen. Renal ultrasound revealed small highly echogenic renal parenchyma consistent with chronic kidney disease. Renal biopsy (figure) at age 57 years demonstrated focal segmental and global glomerulosclerosis with moderate chronic tubulointerstitial nephropathy. No mitochondrial ultrastructural abnormalities were found on electron microscopy. Serum vitamin E, copper, ceruloplasmin, fasting glucose, vitamin B12, methylmalonic acid, folate, thyroid- stimulating hormone, serum and urine immunofixation, very-long-chain fatty acid levels, antinuclear antibodies, antibodies to extractable nuclear antigens, tissue transglutaminase antibodies, syphilis IgG, RPR, HIV serology, and peripheral acanthocytosis were normal or negative.

*These authors contributed equally.

†This author is deceased.

From the Department of Neurology (P.S.N., M.V.P., C.J.K.), Department of Laboratory Genetics and Genomics (J.L.N., L.H., E.W.H.), Department of Anatomic Pathology (J.L.N., M.E.F.), and Department of Clinical Genomics (R.H.G.), Mayo Clinic and Mayo Foundation, Rochester, MN.

(A) Brain MRI sagittal T1 FLAIR showing moderate generalized cerebral and severe cerebellar atrophy without evidence of previous strokes. (B) Kidney biopsy revealed a subset of glomeruli with segmental glomerulosclerosis (marked star) as is seen here at ×400 magnification with Masson trichrome stain. (C) Morphologically, the mitochondria were well within the range of normal variation as is seen in electron micrograph at ×13,000 magnification. (D) Pedigree chart; index case (arrow). (E) Methylene blue epoxy section of the patient’s brother’s sural nerve biopsy at ×500 magnification showing clusters of regenerating myelinated axons surrounded by concentric Schwann cell processes (arrows) with onion bulb–like formations.

His mother had ataxia and neuropathy. His only brother was Mitochondrial genome testing was ambiguous from lympho- diagnosed with “idiopathic neuropathy” at age 50 years with cytes with ;2% of the DNA sequenced, querying MT-ND5 negative evaluations for cause without ataxia or hearing loss. m.13513G>A Asp393Asn. A sequencing error vs mitochon- He was considered a possible living kidney donor and ex- drial heteroplasmy was raised as the possibilities to explain cluded when asymptomatic idiopathic nephropathy was this result by the performing laboratory. Given the very small found. Creatinine clearance was at 50 mL/min/m2 (normal > percentage of lymphocytes with the variant and because ear- 60 mL/min/m2). That brother had undergone a whole sural lier patients with MT-ND5 Asp393Asn had not been identi- – nerve biopsy, which showed decreased density of myelin- fied with this phenotype,1 4 uncertainty remained to the ated nerve fibers and frequent clusters of regenerating diagnosis. However, when mitochondrial genomic analysis of fibers surrounded by concentric Schwann cell processes the kidney was performed, 62% of ;7,000 DNA reads (depth (figure), similar to other confirmed mitochondrial muta- of coverage) had MT-ND5 Asp393Asn mutation. The mu- tion cases.3 His sister does not have neuropathy or ne- tation provided a unifying diagnosis for cerebellar ataxia, renal phropathy at age 72 years. His son, daughter, and brother’s failure, neuropathy, and encephalopathy. Low-dose coenzyme son are healthy at age 23, 19, and 27 years, respectively 10 and carnitine were started, and yearly neurologic and (figure). His sister has no children. Cerebellar ataxia ge- cardiac evaluations were recommended. netic testing for Fragile-X, APTX, SCA1, SCA2, SCA3, SCA5, SCA6, SCA7, SCA8, SCA10, SCA13, SCA14, SCA17, DRPLA, FRDA, AOA1, POLG1, TTPA, SIL1, Discussion SETX, and KCNC3 was all unremarkable. Evaluations for acidemia showed a borderline lactate 2.2 mmol/L (normal MT-ND5 mutation can present with the phenotype of 0.7–2.1 mmol/L) and pyruvate 0.152 mmol/L (normal MCARNE. This case expands the reported manifestations of 0.03–0.107 mmol/L) level. MT-ND5 mutations beyond Leigh syndrome,3,4 Leber

2 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG hereditary optic neuropathy, and MELAS.5 We suspect that Disclosure other mitochondrial mutations could present similarly, and P.S.NgandM.V.Pintoreportnodisclosures.J.L.Neff has the MCARNE phenotype will be important to recognize. received research support from Duke University. Mitochondrial ultrastructural kidney tissue abnormalities are L. Hasadsri reports no disclosures. Dr Highsmith is de- absent, and tubulointerstitial nephropathy can be reported as ceased; disclosures are not included for this author. occurred in our case, and glomerular and cystic kidney M.E. Fidler reports no disclosures. R.H. Gavrilova has changes were not seen in our patient.6 Also highlighted is the served on the scientific advisory board of Mitochondrial importance of having DNA sequencing from the kidney with Medicine. C.J. Klein has served on the scientificadvisory a high depth of coverage (×7,000 in our case) so as not to miss boards of CMTA Research and Therapeutics; has received mitochondrial mutation heteroplasmy.7 funding for travel and/or speaker honoraria from Akcea; and serves on the editorial board of Neurology®.Goto Author contributions Neurology.org/NG for full disclosures. P.S. Ng: concept and design, acquisition of data, MRI images, figure legends, analysis and interpretations, and drafting and revisions of the manuscript. M.V. Pinto: acquisition of data, Publication history fi analysis and interpretations, and drafting and critical revisions Received by Neurology: Genetics August 23, 2018. Accepted in nal form of the manuscript. J.L. Neff: pathology images, figure legends, January 7, 2019. and manuscript revisions. L. Hasadsri: data analysis of DNA References sequencing and kindred evaluation, mitochondrial test crea- 1. Santorelli FM, Tanji K, Kulikova R, et al. Identification of a novel mutation in the tion, and critical edits in response to reviewers. E.W. High- mtDNA ND5 gene associated with MELAS. Biochem Biophys Res Commun 1997; 238:326–328. smith: acquisition of data, analysis and interpretations, critical 2. Sara S, Jorida C, Jiesheng L, et al. The G13513A mutation in the ND5 gene of revisions of the manuscript, and study supervision. M.E. mitochondrial DNA as a common cause of MELAS or Leigh syndrome: evidence from 12 cases. Arch Neurol 2008;65:368–372. Fidler: review of pathology images and critical revisions of the 3. Vital A, Vital C. Mitochondria and peripheral neuropathies. J Neuropathol Exp Neurol manuscript. R.H. Gavrilova: acquisition of data, analysis and 2012;71:1036–1046. 4. Chol M, Lebon S, Bénit P, et al. The mitochondrial DNA G13513A MELAS interpretations, and critical revisions of the manuscript. C.J. mutation in the NADH dehydrogenase 5 gene is a frequent cause of Leigh- Klein: concept and design, acquisition of data, analysis and like syndrome with isolated complex I deficiency. J Med Genet 2003;40: 188–191. interpretations, drafting and critical revisions of the manu- 5. Pulkes T, Eunson L, Patterson V, et al. The mitochondrial DNA G13513A transition script, and study supervision. in ND5 is associated with a LHON/MELAS overlap syndrome and may be a frequent cause of MELAS. Ann Neurol 1999;46:916–919. 6. Seidowsky A, Hoffmann M, Glowacki F, et al. Renal involvement in MELAS Study funding syndrome—a series of 5 cases and review of the literature. Clin Nephrol 2013;80: 456–463. This work was supported by the Mayo Clinic Center for 7. Ye F, Samuels DC, Clark T, Guo Y. High-throughput sequencing in mitochondrial Individualized Medicine. DNA research. Mitochondrion 2014;17:157–163.

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 3 CLINICAL/SCIENTIFIC NOTES OPEN ACCESS Leaky splicing variant in sepiapterin reductase deﬁciency Are milder cases escaping diagnosis?

Yu Nakagama, MD,* Kohei Hamanaka, MD, PhD,* Masakazu Mimaki, MD, PhD, Haruo Shintaku, MD, PhD, Correspondence Satoko Miyatake, MD, PhD, Naomichi Matsumoto, MD, PhD, Koji Hirohata, MD, Ryo Inuzuka, MD, PhD, and Dr. Nakagama [email protected] Akira Oka, MD, PhD

Neurol Genet 2019;5:e319. doi:10.1212/NXG.0000000000000319

Sepiapterin reductase deficiency (SRD), an extremely rare but treatable neurotransmitter fi 1 disease, is an enzyme defect in the nal step of tetrahydrobiopterin (BH4) synthesis. Unlike fi other forms of BH4-de cient dopa-responsive dystonia, SRD uniquely does not manifest hyperphenylalaninemia and thus slips through detection by newborn screening. Owing to its variable presenting features and need for a sensitive method of CSF analysis, diagnosis of SRD may be compromised in mild phenotypes.2

We describe a novel splice site variant leading to leaky splicing control of the SPR gene. Our observation adds evidence to the notion that leaky splicing may take part in SRD heterogeneity and evokes the image of an iceberg beneath the water: patients at the milder end of the spectrum escaping recognition.

Case report An 8-month-old girl presented with postural limb dystonia that worsened in the evening. Brain imaging, EEG, routine blood, urine, and CSF testing were nondiagnostic. Recognition of her episodic oculogyric crises and convergence spasms prompted us to analyze her CSF for pterins and biogenic amines. CSF homovanillic acid (132 nmol/L) and 5-hydroxyindoleacetic acid (11.5 nmol/L) were decreased (normal range: 295–932 nmol/L and 114–336 nmol/L, re- 3 spectively). The CSF BH4 level, analyzed by the method described by Fukushima and Nixon, was below the detection limit, whereas total biopterin (27.06 nmol/L) and neopterin (22.06 nmol/L) levels were within the normal range, suggesting that most of the patient’s total biopterin was a sum of biopterin and dihydrobiopterin. Findings were suggestive of monoamine ﬁ neurotransmitter disease due to BH4 de ciency. L-dopa/carbidopa therapy completely suppressed her dystonia and resulted in near-normal psychomotor development.

Genetic analysis established the diagnosis of SRD by identifying compound heterozygous variants in the SPR gene (NM_003124.4): c.512G>A and c.304+1_+12del. The former is a novel missense variant, absent in the Exome Aggregation Consortium (ExAC) and gnomAD databases, estimated to substitute a well-conserved cysteine for tyrosine, and predicted as damaging according to in silico analyses. The latter, also absent in the ExAC and gnomAD databases, destroys the 59 splice donor site in intron 1, rendering the gene prone to aberrant splicing (ﬁgure, A).

*These authors contributed equally to this work.

From the Department of Pediatrics (Y.N., K. Hirohata, R.I., A.O.), Graduate School of Medicine, The University of Tokyo; Department of Human Genetics (K. Hamanaka, S.M., N.M.), Graduate School of Medicine, Yokohama City University; Department of Pediatrics (M.M.), School of Medicine, Teikyo University, Tokyo; and Department of Pediatrics (H.S.), Graduate School of Medicine, Osaka City University, Osaka, Japan.

The Article Processing Charge was funded by the authors.

Patient consent: Written consent for publication was obtained from the patient’s family. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Study funding Figure Mutational and splicing analyses Supported by grants from Morinaga Hoshikai and AMED (JP18ek0109280 and JP18ek0109301).

Disclosure Y. Nakagama has received government research support from JSPS Kakenhi and has received foundation/society research support from Morinaga Hoshikai. K. Hamanaka and M. Mimaki report no disclosures. H. Shintaku has received government research support from the AMED. S. Miyatake has received foundation/society research support from the Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics, JSPS KAKENHI, and The Ichiro Kanehara Foundation for the Promotion of Medical Science & Medical Care. N. Matsumoto has served on the editorial boards of Clinical Genetics, Journal of Human Genetics, and American Journal of Medical Genetics and has received foundation/society research support from the AMED, JSPS KAKENHI, and Takeda Science Foundation. K. Hirohata reports no disclosures. R. Inuzuka has served on the editorial board of Journal of Pediatric Cardiology and (A) The patient was compound heterozygous for an exonic c.512G>A and an intronic c.304+1_+12del (SPR, NM_003124.4) (B and C) The destroyed splice Cardiac Surgery and has received government research site and retention of intron 1 resulted in a larger size 899-bp band (B, arrow) specific to the patient. Because allelic origin was identifiable based on the support from JSPS KAKENHI. A. Oka has received funding c.512G>A variant, sequencing the normally spliced 319-bp product (B, ar- for travel or speaker honoraria from Otsuka Pharmaceuti- rowhead) showed significant wild-type splicing from the allele carrying c.304+1_+12del (C). Primers were designed as depicted, and sequences are cal, UCB Japan, Nobelpharma, Janssen Pharmaceutical, available upon request. Eisai, BioMarin Pharmaceutical, Novartis Pharma K.K., GE Healthcare Japan, Teijin Pharma Limited, Shionogi & Co., Shire Japan, Bayer Yakuhin, and SRL Inc.; has served on the editorial board of Pediatrics International;has Next, splicing analysis was performed, using blood cell tran- commercial research support from Maruho Co, Pfizer scripts extracted from the patient and a healthy control. Pri- Japan, Astellas Pharma, Novartis Pharma K.K., Chugai mers were designed to flank intron 1 and exon 2 of the SPR Pharmaceutical Co., and Eli Lilly Japan K.K.; and has gene and to specifically amplify the RNA sequences (figure received government funding from The Ministry of B). Reverse transcription-PCR–based splicing analysis not Health Labour and Welfare Japan. Disclosures available: only confirmed aberrant splicing causing intron retention Neurology.org/NG. (figure B, arrow) but also discovered evidence for leaky splicing control related to c.304+1_+12del. Because the allelic Publication history origin was identifiable based on the presence or absence of Received by Neurology: Genetics October 26, 2018. Accepted in final c.512G>A, directly sequencing the shorter 319-bp amplicon form February 8, 2019. (figure B, arrowhead) showed significant wild-type splicing from the allele carrying c.304+1_+12del (figure C).

Discussion Appendix Author contributions Name Location Role Contribution Leaky splicing control contributes to phenotypic variation by aﬀecting disease onset and/or severity. The extent of Yu The University Author Interpreted clinical data, Nakagama, of Tokyo, performed splicing analysis, leaky wild-type transcription determines, for example, re- MD Tokyo and drafted the manuscript. sidual acid alpha-glucosidase activity in Pompe disease and ﬁ 4 Masakazu Teikyo Author Interpreted clinical data and relates to a speci c-form of adult-onset disease. As for SRD, Mimaki, MD, University, revised the manuscript. others have reported the possibility of leaky splicing causing PhD Tokyo 5 intrafamilial heterogeneity. In the report, however, splicing Haruo Osaka City Author Performed CSF analysis for was assessed indirectly using the minigene system. Our re- Shintaku, University, pterins and amines. port proves by directly analyzing patient RNA that leaky MD, PhD Osaka splice site variants indeed underlie SRD. Phenotypic vari- Kohei Yokohama City Author Performed genetic analysis ability owing to such leaky splicing control may further Hamanaka, University, and interpreted results. MD, PhD Yokohama expand the SRD spectrum.

2 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG References Appendix (continued) 1. BonaféL,Thöny B, Penzien JM, Czarnecki B, Blau N. Mutations in the sepiapterin reductase gene cause a novel tetrahydrobiopterin-dependent monoamine- fi Name Location Role Contribution neurotransmitter de ciency without hyperphenylalaninemia. Am J Hum Genet 2001;69:269–277. 2. Ng J, Papandreou A, Heales SJ, Kurian MA. Monoamine neurotransmitter disorders– Satoko Yokohama City Author Performed genetic analysis clinical advances and future perspectives. Nat Rev Neurol 2015;11:567–584. Miyatake, University, and interpreted results. 3. Fukushima T, Nixon JC. Analysis of reduced forms of biopterin in biological tissues MD, PhD Yokohama and fluids. Anal Biochem 1980;102:176–188. 4. Boerkoel CF, Exelbert R, Nicastri C, et al. Leaky splicing mutation in the acid maltase Naomichi Yokohama City Author Performed genetic analysis gene is associated with delayed onset of glycogenosis type II. Am J Hum Genet 1995; Matsumoto, University, and interpreted results. 56:887–897. MD, PhD Yokohama 5. Arrabal L, Teresa L, Sánchez-Alcudia R, et al. Genotype-phenotype correlations in sepiapterin reductase deficiency. A splicing defect accounts for a new phenotypic Koji The University Author Interpreted clinical data and variant. Neurogenetics 2011;12:183–191. Hirohata, of Tokyo, revised the manuscript. MD Tokyo

Ryo Inuzuka, The University Author Critically revised the MD, PhD of Tokyo, manuscript. Tokyo

Akira Oka, The University Author Critically revised the MD, PhD of Tokyo, manuscript. Tokyo

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 3 VIEWS AND REVIEWS OPEN ACCESS Antisense oligonucleotides A primer

Daniel R. Scoles, PhD, Eric V. Minikel, MS, and Stefan M. Pulst, MD, Dr med Correspondence Dr. Scoles Neurol Genet 2019;5:e323. doi:10.1212/NXG.0000000000000323 [email protected] or Dr. Pulst [email protected] Abstract There are few disease-modifying therapeutics for neurodegenerative diseases, but successes on the development of antisense oligonucleotide (ASO) therapeutics for spinal muscular atrophy and Duchenne muscular dystrophy predict a robust future for ASOs in medicine. Indeed, existing pipelines for the development of ASO therapies for spinocerebellar ataxias, Huntington disease, Alzheimer disease, amyotrophic lateral sclerosis, Parkinson disease, and others, and increased focus by the pharmaceutical industry on ASO development, strengthen the outlook for using ASOs for neurodegenerative diseases. Perhaps the most signiﬁcant advantage to ASO therapeutics over other small molecule approaches is that acquisition of the target sequence provides immediate knowledge of putative complementary oligonucleotide therapeutics. In this review, we describe the various types of ASOs, how they are used therapeutically, and the present eﬀorts to develop new ASO therapies that will contribute to a forthcoming toolkit for treating multiple neurodegenerative diseases.

From the Department of Neurology (D.R.S., S.M.P.), University of Utah, Salt Lake City, UT; and Center for the Science of Therapeutics (E.V.M.), Broad Institute of MIT and Harvard, Cambridge, MA.

The Article Processing Charge 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.

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary AD = Alzheimer disease; ALS = amyotrophic lateral sclerosis; ASO = antisense oligonucleotide; cET = constrained ethyl; DMD = Duchenne muscular dystrophy; ESE = exonic splicing enhancer; FAP = familial amyloid polyneuropathy; FDA = Food and Drug Administration; FTD = frontotemporal dementia; HD = Huntington disease; LNA = locked nucleic acid; MOE = methoxyethyl; OMe = O methyl; PD = Parkinson disease; PMO = phosphorodiamidate morpholino; PNA = peptide nucleic acid; PO = phosphodiester; PS = phosphorothioate; SCA = spinocerebellar ataxia; SMA = spinal muscular atrophy; SMN = survival motor neuron; SSO = splice-switching oligonucleotide; STAU1 = Staufen1.

The genetic revolution led to the identification of many neu- linkages can improve various properties increasing ASO rologic disease genes. The initial hope that finding the mutated suitability as drugs. Most of these modifications alter phar- protein and placing it into a known cellular pathway would lead macokinetics (improved nuclease resistance resulting in to rapid development of therapies has remained largely un- a longer half-life), pharmacodynamics (superior affinity for fulfilled. The ability to target the disease gene or its encoded the target RNA), or endocytic uptake, which is controlled by messenger RNAs (mRNAs) has opened new opportunities for specific sets of cell surface proteins.10,11 But, with the excep- therapy development. Of the many ways to target the expres- tion of the phosphorothioate (PS) modification to the ASO sion of RNA, this review will focus on the use of antisense backbone, most also preclude cleavage by RNase H, which is oligonucleotides (ASOs) for therapy of neurologic diseases. the desired mechanism of action for many ASOs. Thus, many RNase H ASOs are designed as chimeras, where different Therapeutic ASOs range from 18 to 30 base pairs (bp) in bases are a mix of different chemistries, or as gapmers, where length. They modify expression of a target mRNA, by either some modifications are placed on the “wings” and not the altering splicing or by recruiting RNase H leading to target central bases. Yet, for ASOs intended to alter mRNA splicing degradation. RNase H is a ubiquitous cellular enzyme that or translation, chemical compositions that do not support recognizes DNA:RNA hybrids and cleaves the RNA in the RNase H can be optimal. Thus, ASOs are highly versatile, hybrid. The mode of ASO action depends on the target se- customizable therapeutic tools. Some of the different ASO quence and the precise ASO chemistry used in the design. An compositions and their resultant effects are discussed here, illustration of ASO action depending on target type and ASO and the relevant structures are presented in figure 2. chemistry is provided in figure 1. Oligonucleotide phosphate Leading the way currently in medical use is the ASO drug linkage modifications nusinersen that is approved for treating multiple forms of Modifications to the oligonucleotide phosphate linkages spinal muscular atrophy (SMA).1 Other ASO therapeutics predominantly assist in nuclease avoidance. In the phos- that are Food and Drug Administration (FDA) approved phorodiamidate morpholino (PMO), the phosphodiester include eteplirsen for Duchene muscular dystrophy (DMD)2 (PO) linkages in the oligonucleotide backbone are replaced and inotersen for familial amyloid polyneuropathy (FAP).3 with nonionic phosphorodiamidate linkages leading to resistance to PO.12 Other ASO types have PS modifications that ASOs targeting HTT for Huntington disease (HD),4 SOD1 and result in resistance to a broad spectrum of nucleases, support C9ORF72 for amyotrophic lateral sclerosis (ALS),5,6 and MAPT RNase H activity, and increase protein binding, which also 10,11 (TAU) for Alzheimer disease (AD)7 are in early-phase clinical improves tissue uptake. trials. Most current ASO therapeutics do not cross the blood- brain barrier and therefore for targets in the CNS have to be Morpholinos delivered by intraventricular injection in mice or lumbar puncture Morpholinos are oligonucleotides with unique modifications in humans. Eteplirsen and inotersen have targets that are not in to the ribose sugar that lead to greater target affinity and 12 the CNS and are delivered by intravenous or subcutaneous in- facilitate nuclease avoidance. This modification reduces 12 jection, respectively. On the other hand, systemic side effects are oligonucleotide-protein interactions. Eteplirsen, a 30-bp limited. Uptake into the CNS is an active process and is not morpholino-based ASO drug for the treatment of Duchenne uniform for all cell types or neurons. Chemical modifications can muscular dystrophy (DMD), represents the only FDA- dramatically increase the half-life of ASOs and minimize toxicity. approved morpholino therapy for a neurodegenerative (or neuromuscular) disease. Oligonucleotide chemistry Methoxyethyl oligonucleotides A chemical modification included in most so-called second- Natural, or unmodified, nucleic acids are susceptible to nu- generation ASOs is 29 O-methoxyethyl (MOE). MOE ASOs clease degradation and have poor protein binding and thus have an MOE modification at the 29-position of the ribose inefficient tissue uptake precluding their use as drugs.8,9 sugar. This change enables enhanced binding affinity to the Multiple types of modifications made to nucleotides, and their target mRNA and is considerably less toxic than the 29-O

2 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Figure 1 ASO functions

Target mRNA fates depending on ASO mechanism of action that is determined by where the ASO is targeted and by ASO chemistry. ASO = antisense oligonucleotide; DMD = Du- chenne muscular dystrophy; ESE = exonic splicing enhancer; ISS = intronic splicing silencer; LncRNA = long noncoding RNA; SMN = survival motor neuron.

methyl (OMe) modification. In addition, MOE is sufficiently ribose sugar molecule (figure 2). This bond effectively locks nuclease resistant that some MOE nucleotides can be syn- the base into a conformation predominantly characterizing thesized with normal PO linkages so that a mix of PO and PS the RNA ribose sugar and prevents the conformation char- linkages can be used to fine tune the pharmacokinetics of the acteristic of the deoxyribose sugar.14 The benefit of locked ASO. This can facilitate more rapid distribution into tissue nucleic acids (LNAs) is that they can produce both increased while keeping the terminal elimination rate slow.13 MOE target specificity and reduced recognition by nucleases. LNAs modifications also reduce plasma protein binding, which seem can hybridize to both DNAs and RNAs forming highly stable to shift ASOs away from hepatic metabolism and toward the double-helix duplexes.14 LNAs can be incorporated into kidney for excretion in urine.13 29-MOE PS ASOs delivered to siRNAs15 and gapmer ASOs supporting RNAse H activity.16 the CSF can have biological half-lives exceeding 6 LNA ASOs can be more potent in vivo than their 29-MOE months.50,58 analogs.17

Constrained nucleic acids LNAs have been associated with increased liver toxicity18 but Nucleotides that are covalently modified to limit conforma- can be used in combination with unmodified bases to reduce tion are referred to as constrained or locked. Nucleic acids are toxicity while improving ASO efficacy.19,20 To get around considered “locked” when they have a methylene bridge hepatotoxicities associated with LNA chemistry, chemists connection made between 29-oxygen and the 49-carbon of the have created a sort of LNA/MOE hybrid by adding a methyl-

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 3 Figure 2 Structural elements commonly used in ASOs

cET = contrained ethyl; MOE = 29 O-methoxyethyl; LNA = locked nucleic acid. Please see the text for descriptions of the structures.

bridge characteristic of LNAs to MOE oligonucleotides. The RNase H cleavage of target mRNAs.26 However, a PNA ASO result is ASOs with reduced liver toxicity and increased po- targeting exon skipping in the DMD gene was poorly effective tency.21 There are multiple types of constrained ethyl (cET) compared with an ASO with the 29-OMe modification.27 and MOE oligonucleotides (S-cEt, R-cEt, S-cMOE, and R-cMOE), where S and R refer to the left and right chiral 59-methylcytosine modification structures, respectively. ASOs with these structures hybridize Methylation of cytosines at the 59 position is one way to to target RNAs with affinities like their corresponding LNAs increase ASO specificity. For example, all cytosines in an ASO 5 and have improved liver toxicity and increased resistance to against SOD1 included the 59-methylcytosine modification. nuclease degradation compared with the LNA chemistry. This inclusion can enhance base pairing by modifying the hydrophobic nature of the ASO. On the other hand, 59- Stereopure PS ASOs methylcytosine-thymidine repeats can increase cytotoxicity,28 PS ASOs are usually stereorandom with regard to chiral PS and CpG motifs can stimulate immunoreactivity that can be at centers, each of which has 2 distinct stereochemical config- least partially alleviated by including 59-methylcytosine.29,30 urations, making 219 stereoisoforms possible for a 20mer ASO with 19 linkages. Although it is recognized that the 2 stereo- Gapmers, mixed chemistry, and target fate isomers (Rp and Sp) differ in their binding affinity and sus- Gapmer ASOs have “wings” on either side of LNA or MOE – ceptibility to degradation,22 24 there are tradeoffs between the modified bases flanking a tract of unmodified bases with PS two, and it is still debated whether stereopure isomers can be linkages usually throughout the ASO backbone. Mixed more potent than stereorandom ASOs. Studies in cell culture chemistry is intended to maximize resistance to nucleases and 31 have failed to show any potency differences between stereopure minimize toxicity while supporting RNAse H activity. The and stereorandom ASOs. On the other hand, 1 study reported target mRNAs cleaved by RNAse H will be degraded in either that ASOs with repeated left-left-right (or SSR) chiral PS the nucleus or the cytoplasm by the exosome complex and 32 centers optimized ASO recognition by RNAse H and resulted XRN exonucleases (figure 1). Lead optimization may in- in greater potency in vivo.22 Therapeutic development of clude a screen for optimal structure activity relationship. For stereopure ASOs is the strategy used at WAVE Life Sciences. PS ASOs, this may combine cET, LNA, MOE, cMOE, and 59- methylcytosine chemistries. Tracts of 4 guanosines should be Peptide nucleic acid avoided because they can result in complex ASO structures.30 The peptide nucleic acid (PNA) has a peptide in the position of For gapmer ASOs, this may include unmodified bases. Splice- the ribose sugar. PNAs have typically been used for modulating switching oligonucleotides (SSOs) will exclude a gap feature transcription and other applications not supporting RNase H as RNase H activity that could lead to target degradation is activity.25 But, PNA gapmers have been shown to support unwanted.

4 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG Development of ASO therapeutics for into symptomatic mice, ASO7 improved motor function and restored proteomic and physiologic Purkinje cell neurodegenerative diseases abnormalities.36,38 The study provided proof of concept of At the time of writing this review, only 3 ASO therapeutics lowering of ATXN2 for treatment of ATXN2-related diseases ffi have been approved by the FDA for neurodegenerative or and the impetus for identifying more e cacious SCA2 ASO muscular dystrophy diseases. These are nusinersen for SMA, for use in humans. eteplirsen for Duchenne muscular dystrophy (DMD), and inotersen for FAP. A handful of others are in clinical trials or in SCA3 (Machado-Joseph disease) is caused by a CAG repeat ff preclinical development (table). expansion in exon 10 of the ATXN3 gene. Di erent strategies have been used to develop SCA3 ASOs. This has included Spinocerebellar ataxias allele-specific ASOs antisense to the expanded CAG repeat Three Spinocerebellar ataxias (SCAs), all caused by DNA that sterically block translation,39 splice-switching MOE CAG repeat expansion encoding a polyglutamine (polyQ) ASOs to exclude exon 10 encoding the expanded repeat,40 repeat, have been investigated as diseases targetable by ASO and MOE gapmer ASOs targeting wild-type and mutant treatment in mouse models. SCA2 is caused by CAG repeat ATXN3 alleles41,42 (figure 1). expansion in exon 1 of the ATXN2 gene.33 The phenotypes in 2 SCA2 mouse models have been well characterized at the SCA7 is characterized by cerebellar ataxia and progressive – morphologic, physiologic, and transcriptome levels.34 36 cone-rod dystrophy and caused by CAG repeat expansion in ATXN2 mutation is also associated with a substantial rise in the ATXN7 gene. ASO therapies are ideally suited for treating the expression of the stress granule protein Staufen1 the eye, as they can easily be injected intravitreally. Although (STAU1) and production of ATXN2/STAU1/TIA1– no longer in use, 2 decades ago, fomivirsen became the first positive stress granules.37 After screening for ASOs targeting ever FDA-approved ASO drug, injected into the vitreous of ATXN2 in a human cell line, we took the best ASOs reducing the eye for cytomegalovirus retinitis.43 Niu et al.44 developed expression to in vivo testing by injection into the lateral proof-of-concept data in mice for the use of an ASO therapy ventricle of the mouse. These ASOs had a gapmer design. for blindness in SCA7. Of note, the ASO therapy was effective Several ASOs induced a glial or microglial response, a com- in mice with symptomatic eye disease. mon off-target effect of some ASOs, and were not further evaluated. Huntington disease HD is caused by a CAG repeat expansion in an encoded The top lead ASO was designated ASO7 and lowered ATXN2 region of the HTT gene. PolyQ expansion in huntingtin expression in SCA2 mouse cerebella by >60% for up to 13 causes a gain of toxic function. Development of ASOs tar- weeks, without activating markers of gliosis. When injected geting HTT has followed different approaches. One targets

Table ASO therapeutics for neurodegenerative disease

Drug Indication Target ASO chemistry Status

Nusinersen SMA SMN2, exon-7 inclusion ASO, full 29-MOE FDA approved

Eteplirsen DMD DMD, exon-51 skipping Morpholino FDA approved

Inotersen FAP TTR expression ASO MOE gapmer FDA approved

WVE-210201 DMD DMD, exon-51 skipping Stereopure ASO Phase 1 clinical trial

RG6042 HD HTT expression ASO MOE gapmer Phase 3 clinical trial

WVE-120101 HD HTT expression Stereopure ASO Phase 1/2 clinical trial

WVE-120102 HD HTT expression Stereopure ASO Phase 1/2 clinical trial

IONIS-MAPTRx AD Tau expression ASO MOE gapmer Phase 1/2 clinical trial

BIIB078 ALS C9ORF72 expression ASO MOE Phase 1 clinical trial

IONIS-SOD1Rx ALS SOD1 expression ASO MOE gapmer Phase 1 clinical trial

ATXN2 ASO SCA2 ATXN2 expression ASO MOE gapmer Preclinical development38

ATXN3 ASO SCA3 ATXN3 expression ASO MOE gapmer Preclinical development42

Abbreviations: AD = Alzheimer disease; ALS = amyotrophic lateral sclerosis; ASO = antisense oligonucleotide; DMD = Duchenne muscular dystrophy; FAP= familial amyloid polyneuropathy; FDA = Food and Drug Administration; HD = Huntington disease; MOE = methoxyethyl; SCA = spinocerebellar ataxia; SMA = spinal muscular atrophy.

Neurology.org/NG Neurology: Genetics | Volume 5, Number 2 | April 2019 5 both mutant and nonmutant alleles, whereas another ap- in a mouse seizure model.7 Transgenic mice expressing a hu- proach is allele specific whereby ASOs were screened that man P301S mutant Tau that were treated with Tau ASO had target single nucleotide polymorphisms in HTT in linkage reduced neuronal Tau aggregates and prolonged lifespan from disequilibrium with expanded CAG repeats.45,46 This strategy 312 to 348 days.57 Tau was also reduced in the CNS of may potentially target >75% of HD mutation carriers.47 cynomolgus monkeys following 6 weeks of Tau ASO treat- Currently, 3 ASOs targeting HTT are in clinical trials (table). ment delivered intrathecally.57 ASOs targeting MAPT splicing to exclude a mutant exon also reduced Tau abundance in Amyotrophic lateral sclerosis neuroblastoma cells and in an MAPT AD mouse model.58 ATXN2 as a target for ALS: Although mutations in several There is another splice-switching ASO strategy for AD that genes cause ALS, a number of studies point to ATXN2 as targets the amyloid precursor protein (APP)genetoprevent a therapeutic target for ALS. Over the past decade, it has inclusion of its exon 17 to block APP processing and Aβ pro- become well established that intermediate CAG repeat duction.59 And there is yet another, where an ASO is used to 48,49 expansions in the ATXN2 gene increase the risk of ALS. correct splicing of the APOE gene encoding apolipoprotein E.60 Intertwined with this discovery was the finding that reducing ATXN2 expression improved transactive response DNA- Parkinson disease binding protein 43 (TDP-43) toxicity in both yeast and flies.49 Reduction of LRRK2 abundance is predicted to be thera- When endogenous Atxn2 was reduced in TDP-43 transgenic peutic for Parkinson disease (PD). This is supported by mice by crossing with Atxn2 knockout mice, survival was observations that disease-causing mutations in LRRK2 are significantly improved, TDP-43–positive stress granules were associated with elevated α-synuclein expression.61 Targeting eliminated in motor neurons, and gait scores improved.50 Lrrk2 in wild-type mice was well tolerated supporting that PD Similarly, the survival of TDP-43 transgenic mice was im- is not associated with LRRK2 loss of function.62 MOE- proved by treating with an ASO targeting the Atxn2 gene.50 gamper ASOs reducing LRRK2 in mice treated with α-synu- The effect of targeting ATXN2 on TDP-43 aggregations clein preformed fibirls were also associated with reduced might be explained by related effects on STAU1.37 aggregations of phospho(S129)-α-synuclein.63 Some effort has also been made to develop an ASO therapeutic targeting Targeting SOD1 for ALS α-synuclein for PD.64 Approximately 10% of familial ALS cases are caused by 51 mutations in SOD1 altering its function. Phase I clinical Spinal muscular atrophy and nusinersen testing of the first-in-human SOD1 ASO, intended to lower SMA is caused by loss-of-function mutations in the SMN1 SOD1 expression, demonstrated that the drug was well tol- gene resulting in the loss of the survival motor neuron (SMN) erated when infused into the CSF, but abundance of the protein. SMA severity is inversely correlated with copy 52 mutant SOD1 protein in CSF was reduced by only ;12%. A number of the homologous SMN2 gene. The cDNA encoded reformulated version of the drug designated IONIS-SOD1Rx by SMN2 is identical to that encoded by SMN1 except for lack (BIIB067) is presently undergoing phase 1 clinical trials by of exon 7. The ASO drug nusinersen is an SSO that functions 53 Ionis Pharmaceuticals and Biogen. by blocking an intronic splicing silencer element in the SMN2 intron 7 preventing the spliceosome from excluding exon 7 Targeting C9ORF72 for ALS (figure 1). The result of nusinersen action is expression of the GGGGCC repeat expansions in the C9ORF72 gene are functional, full-length SMN protein from the SMN2 gene. In 54,55 − − causative of ALS and frontotemporal dementia (FTD). Smn1 / ; SMN2+/+ mice, SSOs that restore SMA2 exon 7 C9ORF72 repeat expansions result in loss of expression of the splicing also restored tail and ear necrosis phenotypes.65 normal C9ORF72 gene and gain of C9ORF72 mRNA aggre- Nusinersin was well tolerated in patients with SMA1,66,67 and gates and repeat associated non-AUG (RAN) translation was approved by the FDA for use in humans for the treatment products. Mice with expanded C9ORF72 treated by intra- of SMA in December of 2016.27 cerebroventricular injection with ASOs that interfere with translation of GGGGCC expanded C9ORF72 had reduced Duchenne muscular dystrophy and eteplirsen mRNA foci and RAN translation products, associated with 56 DMDiscausedbymutationintheDMD gene encoding dys- improved anxiety and cognitive function phenotypes. With trophin, which is one of the largest gene in the human genome. proof of concept established, Ionis Pharmaceuticals and Most of the DMD mutations causing DMD result in premature Biogen have undertaken a phase 1 clinical trial of an ASO dystrophin truncations. The predominant ASO strategy for therapeutic targeting C9ORF72 for ALS. treating DMD is employment of ASOs to exclude exons resulting Alzheimer disease and tauopathies in DMD proteins with partially restored functions. Eteplirsen, an ASO that has a PMO oligomer structure, interacts with the DMD Targeting Tau for AD and tauopathies pre-mRNA at exon 51 resulting in exon 51 exclusion. As illus- Lowering Tau abundance by targeting expression of the trated in figure 1, it is used in DMD patients who have a deletion MAPT gene may be therapeutic for AD and FTD. Targeting including exons 49 and 50 resulting in truncated dystrophin; Mapt in mice with a MOE gapmer ASO reduced Tau ex- exclusion of exon 51 restores the reading frame from exon 48 to pression throughout the CNS and protected against seizures exon 52, partially restoring DMD function.68 Approved by the

6 Neurology: Genetics | Volume 5, Number 2 | April 2019 Neurology.org/NG FDA in 2016 for the treatment of DMD, eteplirsen can be used to Author contributions treat approximately 14% of DMD cases,69 and efforts are ongoing D.R. Scoles wrote the manuscript and produced the figures. to develop additional ASOs targeting other exons to treat DMD E.V. Minikel further contributed to ASO chemistry. S.M. Pulst cases caused by other mutations in the dystrophin gene.68 Wave further contributed to components on movement disorders Life Sciences has also initiated a phase 1 clinical trial for testing and neuromuscular diseases. a stereopure ASO for DMD exon 51 exon skipping. Acknowledgment Familial amyloid polyneuropathy A portion of this work was supported by grants R01NS097903, and inotersen R21NS081182, R37NS033123, and U01NS103883 from the Missense mutation in the TTR gene encoding transthyretin is National Institutes of Neurological Disorders and Stroke the cause of hereditary transthyretin amyloidosis (ATTR). (NINDS). EVM is supported by F31 AI22592 (National TTR mutations result in transthyretin misfolding and pro- Institute of Allergy and Infectious Diseases). gressive accumulation of amyloid deposition in many tissues resulting in polyneuropathy, multiorgan dysfunction, and Study funding cardiomyopathy. Multiorgan failure and cardiac arrest pose No targeted funding reported. a greatest risk of death for ATTR patients. Lowering the total expression of TTR at its source in the liver is an effective Disclosure strategy for ATTR, which is achieved by systemic treatment D.R. Scoles has received research support from the NINDS using the 29-MOE ASO drug inotersen,3 now approved by the (U01NS103883 and R01NS097903) and Harrington Dis- FDA for FAP. Inotersen is treated systemically with delivery covery Institute. E.V. Minikel has received funding for travel made by weekly subcutaneous injections. In the brain, trans- and/or speaker honoraria from Illumina and has received thyretin is produced by the choroid plexus, and TTR muta- research support from Charles River Laboratories, NIH tions cause leptomeningeal amyloidosis that potentially could (F31 AI22952), and Prion Alliance. S.M. Pulst serves on the also be treated by ASO therapy targeting TTR expression.70 editorial boards of Journal of Cerebellum, NeuroMolecular Medicine, Experimental Neurology, Neurogenetics, Nature Clinical Practice, and Neurology: Genetics; holds patents for Controlling off-target effects Nucleic acids encoding ataxin-2 binding proteins; Nucleic acid encoding Schwannomin-binding proteins and products Ensuring target specificity is a critical step in ASO therapeutic related thereto; Transgenic mouse expressing a polynucleotide development. One useful approach is to perform quantitative encoding a human ataxin-2 polypeptide; Methods of detecting PCR to assess expression of mRNAs with target sequences SCA-2 nucleic acids; Nucleic acid encoding SCA-2 and prod- including mismatches to the ASO candidate to ensure un- ucts related thereto; Schwannomin-binding-proteins; and changed expression. Another powerful method is to perform Compositions and methods for SCA; has received publish- transcriptome analysis (RNA-seq) using tissues from mice ing royalties for The Ataxias (Churchill Livingston, 2007), that are null for the mRNA target, following a treatment trial Genetics in Neurology (ANN Press, 2005), Genetics of with the ASO candidate, where differentially expressed genes Movement Disorders (Academic Press, 2003), Neurogenetics would indicate off-targets or perhaps incidental cytotoxicity. (Oxford University Press, 2000), and Molecular Genetic Testing in Neurology, 2nd to 5th (AAN Press, 1996); serves A number of chemical modifications have been developed to or has served as a consultant for Ataxion Therapeutics; has improve the pharmacokinetic and pharmacodynamic prop- received research support from the NIH (RC1NS068897, erties of ASOs. PS or PMO backbones are often used. Mod- RC4NS073009, R21NS081182, R21NS079852, and ifications at the 29 position of the sugar can improve affinity, R01NS33123) and the National Ataxia foundation; and has protein binding, and nuclease resistance. Modifications received license fee payments for technology or inventions spanning the 29 and 49 positions to conformationally con- from the Cedars-Sinai Medical Center. Disclosures available: strain the sugar can give dramatic improvements in affinity. All Neurology.org/NG. modifications except PS prohibit RNase H cleavage, but this can be rescued by a “gapmer” design where the modification is Publication history included only in the “wings” of the oligo, and the central bases Received by Neurology: Genetics December 17, 2018. Accepted in final are just PS. Although there are a number of new ASO ther- form February 14, 2019. apies for neurodegenerative diseases in preclinical development and in clinical trials, there are only 2 therapies for References 1. 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