RESEARCH ARTICLE VRK1 (Y213H) homozygous mutant impairs Cajal bodies in a hereditary case of distal motor neuropathy Ana T. Marcos1,* , Elena Martın-Doncel2,3,* , Patricia Morejo  n-Garcıa2,3 , In ~ igo Marcos- Alcalde 4,5  mez-Puertas , Paulino Go 4 , Marıa Segura-Puimedon6 , Lluis Armengol6 , Jose  M. Navarro-Pando1,7,8 & Pedro A. Lazo2,3 1 Unidad de Gen n Humana (INEBIR), Sevilla, Spain etica, Instituto para el Estudio de la Biologıa de la Reproduccio 2 Molecular Mechanisms of Cancer Program, Instituto de Biologıa Molecular y Celular del C ancer, Consejo Superior de Investigaciones Cientıficas (CSIC), Universidad de Salamanca, Salamanca, Spain 3 n Biomedica de Salamanca (IBSAL), Hospital Universitario de Salamanca, Salamanca, Spain Instituto de Investigacio 4 Molecular Modelling Group, Centro de Biologıa Molecular “Severo Ochoa”, CSIC-Universidad Auto noma de Madrid, Cantoblanco, Madrid, Spain 5 School of Experimental Sciences, Biosciences Research Institute, Universidad Francisco de Vitoria, Pozuelo de Alarco n, Madrid, Spain 6 Quantitative Genomic Medicine Laboratories, qGenomics, Espluges de Llobregat, Barcelona, Spain 7 Catedra de Reproduccio n y Genetica Humana, Facultad de Ciencias de la Salud, Universidad Europea del Atl antico, Santander, Spain 8 Fundacio n Universitaria Iberoamericana (FUNIBER), Barcelona, Spain Correspondence Abstract Pedro A. Lazo, Instituto de Biologıa Molecular y Celular del Cancer, CSIC- Background: Distal motor neuropathies with a genetic origin have a heteroge- Universidad de Salamanca, Campus Miguel neous clinical presentation with overlapping features affecting distal nerves and de Unamuno s/n, 37007 Salamanca, Spain. including spinal muscular atrophies and amyotrophic lateral sclerosis. This Tel: +34 923 294 804; Fax: +34 923 194 indicates that their genetic background is heterogeneous. Patient and methods: 795; E-mail: pedro.lazo@csic.es In this work, we have identified and characterized the genetic and molecular base of a patient with a distal sensorimotor neuropathy of unknown origin. For Funding Information this study, we performed whole-exome sequencing, molecular modelling, clon- E. M-D., and P. M-G. were supported by Junta ing and expression of mutant gene, and biochemical and cell biology analysis of n-Fondo Social Europeo (ESF), de Castilla y Leo the mutant protein. Results: A novel homozygous recessive mutation in the and Ministerio de Educacio n-FPU predoctoral human VRK1 gene, coding for a chromatin kinase, causing a substitution (FPU16/01883) fellowships, respectively. This (c.637T > C; p.Tyr213His) in exon 8, was detected in a patient presenting since work was funded by grants from Ministerio de childhood a progressive distal sensorimotor neuropathy and spinal muscular Ciencia, Innovacion y Universidades-Agencia n (SAF2016-75744-R, atrophy syndrome, with normal intellectual development. Molecular modelling Estatal de Investigacio RED2018-102801-T) and Consejerıa de predicted this mutant VRK1 has altered the kinase activation loop by disrupting Educacio n-Junta de Castilla y Leo n-ERDF (UIC- its interaction with the C-terminal regulatory region. The p.Y213H mutant pro- 258) to P.A.L. Ministerio de Ciencia, Innovacion tein has a reduced kinase activity with different substrates, including histones y Universidades-Agencia Estatal de H3 and H2AX, proteins involved in DNA damage responses, such as p53 and Investigacion (RTC-2017-6494-1 and RTI2018- 53BP1, and coilin, the scaffold for Cajal bodies. The mutant VRK1(Y213H) 094434-B-I00) and European Commission protein is unable to rescue the formation of Cajal bodies assembled on coilin, project "CONNECT-JPIAMR” Virtual Research in the absence of wild-type VRK1. Conclusion: The VRK1(Y213H) mutant pro- Institute to P.G-P. The Instituto de Biologıa Molecular y Celular del Cancer is cofunded by tein alters the activation loop, impairs the kinase activity of VRK1 causing a n-European Regional Junta de Castilla y Leo functional insufficiency that impairs the formation of Cajal bodies assembled Development Fund (CLC–2017–01). on coilin, a protein that regulates SMN1 and Cajal body formation. Received: 8 February 2020; Revised: 27 March 2020; Accepted: 31 March 2020 Annals of Clinical and Translational Neurology 2020; 7(5): 808–818 doi: 10.1002/acn3.51050 808 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. A. T. Marcos et al. Homozygous VRK1 (Y213H) Mutant in Distal Motor Neuropathy *Equal contributing first authors. In this work, we have identified a novel homozygous Introduction recessive mutation in the human VRK1 gene, and the Hereditary neuropathies are characterized by involvement mutant protein has altered the folding of its activation of motor, sensory, and/or autonomic nerve fibers,1 and loop that prevents the activation of the kinase activity are divided into three main categories: hereditary motor leading to a deficiency in the assembly of Cajal bodies. and sensory neuropathies (HMSN), also known as Char- cot–Marie-Tooth (CMT) disease, hereditary motor neu- Patient, Materials, and Methods ropathy, and hereditary sensory and autonomic neuropathy (HSAN).2 Distal neuropathies and spinal Clinical characteristics of the patient muscular atrophy (SMA) are progressive diseases affecting the lower motor neurons and characterized for a progres- The patient, son of consanguineous parents and currently sive muscle loss and weakness, and have overlapping 35 years, presented initial symptoms at 4 years with a symptoms.3 The most common forms of these diseases progressive distal muscle weakness in legs and arms that are associated with deletions or mutations in CMT genes, became much more severe with time. The child has a foot or in the exon of the SMN1 gene that is not compensated deformity with pes cavus and bilateral foot drop, leading by SMN23,4 and is involved in RNA processing mediated to unstable walk with distal amyotrophy of lower and by snRNP.5 However, there is heterogeneity in disease upper members. Electromyogram, performed at 9 years, presentation, evolution and manifestations, which has led detected a significant slowdown of motor and sensory to the identification of novel genes implicated in the nerve conductance velocity. At 16 years required foot sur- pathogenesis of these diseases.3 These include alterations gical correction to allow for adequate standing. The dis- in mechanisms that regulate RNA splicing and processing, ease progressed with time needing walking stick, and or in the subcellular structures where these functions take currently is wheel chair bound. At 24 years there was a place, such as Cajal bodies. significant loss of muscle strength, unable to raise from Cajal bodies (CBs) are assembled on coilin.6,7 CBs sedestation without help, and at 34 years the patient can- organization and assembly are lost by either SMN1 deple- not use hands for feeding or writing. Normal intellectual tion,8 or VRK1 depletion,7 and both form part of a com- development and normal speech. mon complex.6,7 Coilin also forms complexes with The parents are consanguineous first cousins, and the splicing snRNP.9–11 Moreover, both SMN112 and VRK113 father has epilepsy. Both parents have pes cavus. bind to chromatin. Coilin levels and its posttranslational modifications also affect RNA processing and splicing.14 Whole-exome sequencing In this context, VRK1 phosphorylates coilin in Ser184 and regulates its stability and assembly by protecting it For whole-exome sequencing (WES), DNA was extracted from ubiquitin-mediated proteasomal degradation in the from peripheral blood of the patient using the Maxwell cytosol, facilitating its nuclear accumulation,7 and also 16 system. DNA quality was determined measuring opti- interacts with Heterogeneous nuclear ribonucleoprotein cal density with a DeNovix DS-11. Human exome was A1 (hnRNP A1) and regulates telomerase activity and enriched using the MedExome SeqCap EZ assay (Roche- telomere maintenance.15 CBs alterations can be alternative Nimblegen, CA), and sequenced using a NextSeq 500 pathogenic routes leading to distal neuromotor syn- (Illumina, CA) equipment. An average 71x coverage dromes, and in which multiple genes have been impli- depth was achieved along the target regions (exons cated.16 and  75 intronic nucleotides lanking the exon–intron Several very rare recessive mutations in the human boundaries). Variants were identified by alignment VRK1 gene, either homozygous or compound heterozy- of reads against the Human reference genome sequence gous, have been detected in diseases affecting the motor (hg19), using BWA-0.7–12, GATK-3.5 procedures for the neuron, which have a phenotypic heterogeneity in their detection of single nucleotide variants and indels and clinical presentation.17–24 These VRK1 mutations are exome depth for deletions and duplications. Annotation recessive, and all of them are very rare, some hereditary, of the variants was performed using Annovar and others de novo. Among the distal motor neuropathy (2016Feb01) and filtering and prioritization was per- phenotypes associated with human VRK1 mutations are formed using public (1000 genomes, Genome Aggregation SMA,17,20,23–25 ALS,19,20 and pontocerebellar hypoplasia.17 Database and Exome Variant Server) databases in order to identify candidate variants related to the clinical ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 809 Homozygous VRK1 (Y213H) Mutant in Distal Motor Neuropathy A. T. Marcos et al. phenotype. Variants were classified following the Ameri- H2AX,13 p53,37,38 53BP1,39 and coilin.7 Full methods are can College of Medical Genetics and Genomics (ACMG) in Data S1. guidelines,26,27 Polyphen-228 and VarElect29 algorithms were used to predict the effect of the variants. Sanger Cell lines, transfections, and protein sequencing in the index case, parents, and siblings was analysis performed to confirm the variant and to determine its inheritance pattern. All molecular and cellular methods have been reported before and are detailed in cellular and in Data S1.34 Anti- bodies are in Table S1. Ethical compliance The genomic study was performed for diagnosis in a case Results of unknown origin and was approved by the Institutional review board. Informed consent was obtained from each Genetic findings subject. Whole-exome sequencing detected in the patient a homozygous mutation (NM_003384:exon8:c.T637C: Molecular modeling of structure p.Y213H) in exon 8 of the VRK1 gene. This mutation The 3D structure of the human Vaccinia-Related Kinase 1 was detected in heterozygosis in both consanguineous (VRK1) wild-type protein was obtained from the Protein parents, and the two siblings of the patient (Fig. 1). Data Bank (PDB id: 2LAV).30 The dynamic molecular Heterozygous carriers have no neurological pathology. modeling methods are described in Data S1. Molecular modeling of VRK1(Y213H) variant Cloning of altered VRK1 gene, plasmids, and protein and functional prediction mutagenesis Polyphen-2 analysis of this aminoacid substitution predicts Human VRK1 was expressed from mammalian expression the highest damaging score. VarElect analysis predicts a vector, pCEFL-HA-VRK1,31 and bacterial expression very high likelihood of causing a neuropathy and muscular pGEX-4T-VRK1.31–34 The Y213H mutation was intro- atrophy.29 To study the effect of the VRK1(Y213H) mutant duced in these plasmids with the GeneArt Site-Directed protein, the 3D structure of human VRK1 protein (2LAV) Mutagenesis System (Invitrogen-ThermoFisher). The pri- was obtained from the Protein Data Bank.30 This structure mers for human VRK1-p.Y213H were: forward (50 - includes the position of the protein C-terminal tail, as well AGGAGTTCATAAAGAACACAAAGAAGACCCCAAAA- as the structure of the VRK1 activation loop, where the 30 ), and reverse (50 -TTTTGGGGTCTTCTTTGTGT Tyr213 residue is located (Fig. S1). Tyr213 forms a T- TCTTTATGAACTCCT-30 ). VRK1 wild-type and the shaped stacking complex with Tyr311, located in an alpha p.Y213H variant proteins were expressed in E.coli from helix close to the activation loop (Fig. S1). This type of constructs made in plasmid pGEX4T-GST-VRK1 for bac- Tyr-Tyr interactions has been shown as contributing to terial expression,32 or in pCEFL-HA-VRK1 for expression structural protein stability,40 as is the case of the VRK1 in mammalian cells.34 The human Y213H mutation was activation loop. The mutant residue VRK1(Y213H) is introduced in murine VRK1 (mVRK1) and cloned in located in the activation loop of the kinase.30 To detect the pCEFL-Myc-mVRK1 to generate pCEFL-Myc-mVRK1 structural consequences of the Y213H mutant, models for (Y213H) plasmid. The primers used for murine VRK1- wild-type (VRK1-WT), and Y213H mutant (VRK1-Y213H) Y213H forward (50 - TGGAGTTCATAAAGAGCACAAG- proteins were subjected to 200 nsec of free molecular GAAGATCCCAAA-30 ) and reverse (50 - TTTGGGA dynamic (MD) simulation. The activation loop of the TCTTCCTTGTACTCTTTATGAACTCCA-3. Full methods VRK1-WT structure did not experience noticeable varia- are in Data S1. tions during the molecular dynamics trajectory, remaining virtually unchanged after 200 nsec of MD (Fig. 2A). The only variation suffered by the activation loop was the stabi- Kinase assays lization of the C-terminal end of the protein, through the Kinase assays were performed as previously formation of saline bridges between the negative amino described.31,34,35 Briefly, in vitro kinase assays with [32- acid Glu361 and the positive residues Lys211 and Arg219 P]-cATP were performed with GST-VRK1 wild-type, or of the loop (Fig. 2A). This interaction remained stable for the p.Y213H variant.7,31,36 Kinase assays with the follow- more than 40% of the total simulation time. After 200 ns ing substrates were previously published: histone H3,13,33 of molecular dynamics (MD), the 3D structure of the 810 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association A. T. Marcos et al. Homozygous VRK1 (Y213H) Mutant in Distal Motor Neuropathy Father T C A T A A A G A A T/C A C A A A G A A G A Proband son T C A T A A A G A A C A C A A A G A A G A Mother T C A T A A A G A A T/C A C A A A G A A G A Brother T C A T A A A G A A T/C A C A A A G A A G A Sister T C A T A A A G A A T/C A C A A A G A A G A Figure 1. Identification of a novel mutation in the human VRK1 gene coding for a nucleosomal kinase. Detection and confirmation of the VRK1 mutation in the five members of the family. VRK1(Y213H) mutant activation loop displayed a different protein, which are required for kinase activation,30,41 and arrangement (Fig. 2B). His213 continued to interact with this structural change predicts a loss of kinase activity. Tyr311 but in the form of a parallel-displaced stacking, which confers a completely different shape to the activation The VRK1(Y213H) variant kinase is loop. Because of this, the position of Lys211 suffered a functionally deficient large displacement. The behavior of the VRK1(Y213H) mutant differs of the VRK1-WT structure during the MD To determine the functional effect of the VRK1(Y213H) trajectory. The mutant does not have a close interaction mutant, we performed in vitro kinase assays using several between the specific residues of the activation loop and the of the known VRK1 substrates. These included histones residues located in the regulatory C-terminal tail of the H3 and H2AX associated with chromatin remodeling,13 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 811 Homozygous VRK1 (Y213H) Mutant in Distal Motor Neuropathy A. T. Marcos et al. A B Lys211 Lys211 Tyr311 Glu361 Tyr311 His213 Tyr213 Arg219 Arg219 Figure 2. Structural modeling of the human VRK1-Y213H variant protein. (A) Structure of the wild-type VRK1 activation loop after 200 nsec of free molecular dynamics. Position of C-terminal tail interacting with the activation loop is indicated (magenta). The activation loop is colored in green. Position of residues Tyr213, Tyr311 (blue, not in the activation loop), Lys211, Arg219 and Glu361 (grey, located in the C-terminal tail) is indicated. (B) Structure of the activation loop of VRK1-Y213H variant after 200 nsec of free molecular dynamics. Note the displacement of the loop compared to the wild-type structure and the different position of the mutated residue (His213). No interaction was detected between the mutated activation loop and the C-terminal tail of VRK1. coilin required for Cajal body formation,7 p53,42 and Effect of the VRK1(Y213H) mutant on the 53BP1 39 involved in different aspects of DNA damage formation of Cajal bodies responses.43 The mutant VRK1(Y213H) was compared with the wild-type VRK1. In the kinase assays, the effect Phosphorylation of coilin regulates the assembly and sta- of the p.Y213H mutant was common to all substrates. bility of Cajal bodies.7,44 Therefore, we studied the possi- There was a significant loss regarding the phosphorylation ble effect of the Y213H mutant on the formation of Cajal of histone H3, (Fig. 3A), histone H2AX (Fig. 3B), coilin bodies. For this purpose, we used Hela cells in which the (Fig. 3C), p53 (Fig. 3D), and 53BP1(Fig. 3E). endogenous human VRK1 was depleted by siRNA. These human VRK1 depleted cells were transfected with the murine VRK1, either wild-type (mVRK1) or containing Protein stability of the VRK1(Y213H) mutant the Y213H mutant (mVRK1-Y213H). In transfected cells Several of the known VRK1 mutants associated with neu- depleted of human endogenous VRK1 there was a loss of rological phenotypes also have a reduced protein stabil- Cajal bodies, and this effect was rescued in cells express- ity.34 Therefore, we tested the stability of p.Y213H ing the murine wild-type VRK1, but not by the murine compared to the normal VRK1 protein. Plasmids express- VRK1-Y213H mutant protein (Fig. 4). ing both proteins were transfected in 293T cells, which were treated with cycloheximide, and the level of protein Discussion was determined at different time points. The p.Y213H mutant protein was slightly less stable that normal VRK1 The heterogeneity of the neuromotor syndromes associ- (Fig. S3). ated with VRK1 pathogenic variants suggest that their Figure 3. Kinase activity of the wild-type VRK1 and mutant VRK1-Y213H using different phosphorylation substrates. (A) Phosphorylation of histone H3 by wild-type VRK1 (WT), VRK1-T213H and kinase-dead VRK1-K179E in Thr3 detected with a phospho-specific antibody. (B) Phosphorylation of histone H2AX in a radioactive kinase assay. (C) Phosphorylation of coilin in a radioactive kinase assay. (D) Phosphorylation of p53 in a radioactive kinase assay. (E) Phosphorylation of 53BP1 in a radioactive kinase assay. One representative gel is shown, and the three independent experiments are shown is Figure S2. 812 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association A. T. Marcos et al. Homozygous VRK1 (Y213H) Mutant in Distal Motor Neuropathy A VRK1 B VRK1 C VRK1 Y213H Y213H WT Y213H K179E WT VRK1 - + + WT kD VRK1: - + + H2AX + + + Coilin + kD VRK1 - + + + 75 + + kD VRK1 (160-214): H3 + + + + 75 75 20 VRK1 VRK1 H2AX-ph Coilin-ph 37 H3-T3ph 20 32P kD (160-214) 32P 20 kD H3 75 1.5 VRK1 75 20 VRK1 *** H2AX 37 Coilin H3-T3ph / H3 relative value WB (α-GST) (160-214) 1.0 ** WB (α-GST) / H2AX 1.0 relative values * 32P-Coilin/ Coilin 1.0 0.5 relative value 32P-H2AX 0.5 0.5 0.0 0 0 D VRK1 E VRK1 Y213H Y213H WT WT VRK1 - + + VRK1 - + + p53 (1-84) + kD kD + + 53BP1 (1-346) + + + VRK1 75 32P-53BP1 100 32P-p53 50 VRK1 75 32P 32P kD kD VRK1 75 53BP1 (1-346) 100 p53 (1-84) VRK1 75 50 WB (α-GST) WB (α-GST) *** ** 1.0 53BP1 1.0 Relative values 32P-p53 / p53 Relative values 32P-53BP1 / 0.5 0.5 0 0 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 813 Homozygous VRK1 (Y213H) Mutant in Distal Motor Neuropathy A. T. Marcos et al. A Coilin mVRK1 Coilin mVRK1 DAPI B *** P<0.0005 ns. 100 ns. ns. *** *** Si-Ct + Vector 15μ 80 m %Cells with CBs mVRK1 wt Si-Ct + 60 15μm 40 mVRK1- [Y213H] 20 Si-Ct + 15μm 0 siControl siVRK1-02 cells (n) 150 140 140 120 120 200 + Vector siVRK1 vector- myc mVRK1-myc mY213H-myc 15μm C mVRK1-Y213H mVRK1-Y213H mVRK1 wt mVRK1-MYC siVRK1 + mVRK1 15μm Ct Ct VRK1 50 kDa siVRK1 + -[Y213H] mVRK1 mVRK1-myc 50 kDa β-actin 37 kDa 15μm siControl siVRK1-02 Figure 4. Rescue of Cajal body formation by wild-type VRK1 and mutant VRK1-Y213H. Endogenous human VRK1 was depleted with si-VRK1-02 and cells were transfected with either murine VRK1 wild-type (muVRK1) or with the murine VRK1 Y213H variant (muVRK1-Y213H). (A) Images of confocal microscopy showing the presence or absence of Cajal bodies assembled on coilin (red). (B) Quantification of the presence or absence of Cajal bodies in cells that after depletion of endogenous human VRK1 were transfected with either wild-type of the Y213H mutant murine VRK1. (C) Immunoblot to show the depletion of endogenous human VRK1 and its replacement by either murine VRK1 or the murine Y213H VRK1 mutant. contribution is likely to be mediated by downstream substrates including histone H3, H2AX, p53, 53BP1, and direct targets. All pathogenic variants have in common coilin, all of them are pathogenically associated with heredi- two possible effects. Some have a reduction of the mutant tary neurological/neuromotor syndromes. Functionally, this protein stability, which leads to reduced protein levels. impaired VRK1 kinase activity implicates a loss of func- Others have a reduction of their kinase activity with tions, which have to be linked to distal motor neuropathies respect to several of its known specific target proteins,34 and SMA phenotype. The most likely intermediate step in some of which are already associated with neurological the pathogenesis VRK1(Y213H) is a consequence of the development and diseases, such as proteins regulating inability of the Y213H mutant protein to phosphorylate chromatin epigenetic changes, neural development or and regulate coilin, which can alter its associated proteins response to cellular stress and DNA damage. and functions. Coilin is the scaffold protein of Cajal In this report, we have shown that the VRK1(Y213H) Bodies45 discovered more than one hundred years ago,46 mutant protein is unable to phosphorylate several of its which forms a complex with SMN1 in SMA.6 However, the 814 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association A. T. Marcos et al. Homozygous VRK1 (Y213H) Mutant in Distal Motor Neuropathy function of Cajal bodies is not well understood, but it is a as distal motor neuropathies, spinal muscular atrophy ribonucleoprotein complex that participates in RNA pro- (SMA) or amyotrophic lateral sclerosis (ELA). However, cessing.47,48 In a murine coilin knockout models there is a they differed in their severity and age of presentation, failure to recruit SMN1 to defective or absent CBs.49,50 All although initial symptoms started during infancy in most human VRK1 mutant proteins, directly or indirectly fail to cases and are progressive. There are also important dif- form CBs7,34,51 and mimic the coilin knockout. Further- ferences in intellectual ability ranging from normal to more, SMN1 protects against mutant SOD1 toxicity.52 very severe deficiency. Some additional neurological Mutations in SOD1 disrupt the recruitment of SMN1 to symptoms are associated. This heterogeneity is unlikely Cajal bodies,53 and mutations in VCP (valosin) contribute to be only the result of the specific VRK1 mutant, since to development of ALS in KO mice,54 all these proteins molecularly they have a similar defective function,34 but form complexes with VRK1 (unpublished). rather by the contribution of additional variants in other Most of the known human VRK1 pathogenic variants genes, which differ among patients. However, when more are unable to form CBs,34 as it also occurs with the than one case occurs within a family sharing the same Y213H mutant. Moreover, in cells obtained from mutant, the phenotype is similar among the affected patients with compound R219L/W254L mutations, there family members.17 are no CBs in motor neurons derived from human An alternative pathogenic mechanism implicating the induced pluripotent stem cells.51 In a murine model, the contribution of VRK1 may be a consequence of its inter- human VRK1(R358X) mutant impairs cell cycle progres- action with GARS,41 a gene whose mutations are also sion and migration of neuronal progenitors in early associated with distal neuropathies.63–65 However, the role embryogenesis.55 Moreover, VRK1 partial knockdown in of aminoacyl-tRNA-synthetases in these diseases is mice causes a motor dysfunction.56 Nuclear bodies, such unknown, but may be a consequence of altering neurite as CBs, are regulated during embryogenesis,57 and their formation66 and peripheral axons.67,68 alteration is very likely to cause neurological disorders. We conclude that VRK1 mutations, associated with dis- In this context, VRK1 regulates the assembly and disas- tal neuromotor syndromes of unknown origin, should be sembly of CB mediated by a specific phosphorylation of considered as a rare underlying pathogenic mechanism. coilin that controls its stability and the formation of Since defective CBs assembly and associated functions are complexes with other proteins.7 Thus, functionally defi- common to a group of heterogeneous distal neuromotor cient VRK1 mutations alter the physiological dynamics syndromes, whose final neurological phenotype will be of CBs, and consequently of its associated proteins and determined by the genetic background of the affected functions. This has been shown to be the case for individual. Distal motor neuropathies are a consequence Y213H (this report) and for other mutants.34 The of a mutation in any of the different components of heterogeneity of the VRK1 mutations suggests that there nuclear suborganelles, such as CBs, and which lead to this might be differences in their specific protein interactions group of related neurological phenotypes. that lead to some heterogeneity among the associated neuromotor syndromes by altering the functionality of Acknowledgments Cajal bodies. Another target of VRK1, p53, regulates WRAP53 that The computational support of the “Centro de Com- is required for CB formation and maintenance of genome putaci on Cientıfica CCC-UAM” is gratefully recognized. integrity,58,59 but the activation of p53 by phosphoryla- tion is impaired in the case of the VRK1(Y213H) mutant Conflict of Interest protein. CBs are associated with ribonucleoproteins com- plexes involved in RNA processing, but the consequences The authors declare they have no competing conflict of of their alteration is unknown.47,60 In the CBs assembled interest. on coilin there are other proteins such as SMN1, associ- ated with SMA,4 VCP associated with amyotrophic lateral Author Contributions sclerosis (ALS),61 and ataxin 1 (ATXN1) associated with ataxia.62 Alterations in any of them cause a distal neuro- ATM, MS-P, and LA performed the genetic and sequenc- motor phenotype. ing study. EM-D and PM-G performed biochemical and All the VRK1 mutants are either homozygous or com- cell biology studies, IM-A and PG-P performed molecular pound heterozygous. The heterogeneity of the neurologi- structural analysis, JMN-P, and PAL planned the work cal phenotypes associated with VRK1 pathogenic variants and coordinated clinical and molecular studies. PAL has some common features. All patients presented a wrote the final version of the manuscript that was revised functional alteration of motor neuron function, mainly and approved by all authors. ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 815 Homozygous VRK1 (Y213H) Mutant in Distal Motor Neuropathy A. T. Marcos et al. 9. Enwerem II, Wu G, Yu YT, Hebert MD. Cajal body Informed Consent proteins differentially affect the processing of box C/D The parents and the affected patient provided written scaRNPs. PLoS One 2015;10:e0122348. consent to perform the genetic study. 10. Jady BE, Darzacq X, Tucker KE, et al. Modification of Sm small nuclear RNAs occurs in the nucleoplasmic Cajal body following import from the cytoplasm. EMBO J Consent to Publish 2003;22:1878–1888. Written informed consent for publication was obtained 11. Lemm I, Girard C, Kuhn AN, et al. 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WRAP53 Additional supporting information may be found online is essential for Cajal body formation and for targeting the in the Supporting Information section at the end of the survival of motor neuron complex to Cajal bodies. PLoS article. Biol 2010;8:e1000521. 59. Henriksson S, Farnebo M. On the road with WRAP53beta: Figure S1. Structural modeling of VRK1 Y213H variant. guardian of Cajal bodies and genome integrity. Front (A) 3D structure of wild-type VRK1 protein (PDB id: Genet 2015;6:91. 2LAV). The activation loop is colored in green. Position 60. Poole AR, Hebert MD. SMN and coilin negatively regulate of residues Tyr213, Tyr311 (blue, not in the activation dyskerin association with telomerase RNA. Biol Open loop), Lys211, Arg219 and Glu361 (grey, located in the 2016;5:726–735. C-terminal tail) are indicated. 61. Abramzon Y, Johnson JO, Scholz SW, et al. Valosin- Figure S2. Phosphorylation of several substrates by VRK1 containing protein (VCP) mutations in sporadic and VRK1-Y213H in triplicate. (A) Histone H3. (B) His- amyotrophic lateral sclerosis. Neurobiol Aging tone H2AX. (C) Coilin. (D) TP53. (E) 53BP1. 2012;33:2231.e1–2231.e6. Figure S3. Protein stability of the wild-type VRK1 and 62. Hong S, Ka S, Kim S, et al. p80 coilin, a coiled body- mutant VRK1-Y213H proteins. specific protein, interacts with ataxin-1, the SCA1 gene Table S1. List of primary and secondary antibodies used product. Biochim Biophys Acta 2003;1638:35–42. in this work. 63. Dubourg O, Azzedine H, Yaou RB, et al. The G526R Data S1. This refers to databases correctly placed before glycyl-tRNA synthetase gene mutation in distal hereditary in the text. motor neuropathy type V. Neurology 2006;66:1721–1726. 818 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association