VRK1 (Y213H) homozygous mutant impairs Cajal bodies in a hereditary case of distal motor neuropathy

Abstract

Background Distal motor neuropathies with a genetic origin have a heterogeneous clinical presentation with overlapping features affecting distal nerves and including spinal muscular atrophies and amyotrophic lateral sclerosis. This indicates that their genetic background is heterogeneous. Patient and methods 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 this study, we performed whole‐exome sequencing, molecular modelling, cloning and expression of mutant gene, and biochemical and cell biology analysis of the mutant protein. Results A novel homozygous recessive mutation in the human VRK1 gene, coding for a chromatin kinase, causing a substitution (c.637T > C; p.Tyr213His) in exon 8, was detected in a patient presenting since childhood a progressive distal sensorimotor neuropathy and spinal muscular atrophy syndrome, with normal intellectual development. Molecular modelling predicted this mutant VRK1 has altered the kinase activation loop by disrupting its interaction with the C‐terminal regulatory region. The p.Y213H mutant protein has a reduced kinase activity with different substrates, including histones H3 and H2AX, proteins involved in DNA damage responses, such as p53 and 53BP1, and coilin, the scaffold for Cajal bodies. The mutant VRK1(Y213H) protein is unable to rescue the formation of Cajal bodies assembled on coilin, in the absence of wild‐type VRK1. Conclusion The VRK1(Y213H) mutant protein alters the activation loop, impairs the kinase activity of VRK1 causing a functional insufficiency that impairs the formation of Cajal bodies assembled on coilin, a protein that regulates SMN1 and Cajal body formation.

Dysfunctional Homozygous VRK1-D263G Variant Impairs the Assembly of Cajal Bodies and DNA Damage Response in Hereditary Spastic Paraplegia

“…Control plasmids expressing murine kinase-active (pLenti-C-HA-IRES-BSD-mVRK1) and kinase-dead (K179E) (pLenti-C-HA-IRES-BSD-mVRK-K179E) have already been reported. 8 , 22 Full methods and plasmids coding used for some substrates are described in eMethods, links.lww.com/NXG/A468 .…”

“…They include several of the clinical phenotypes associated with motor neurons, including SMA, 4 - 6 ALS, 7 and Charcot-Marie Tooth. 8 , 9 Functionally, the VRK1 gene is implicated in the regulation of cell proliferation, 10 , 11 transcription, 12 - 14 autophagy, 15 chromatin compaction, 16 , 17 DNA damage responses, 18 , 19 Cajal body (CB) stability and assembly, 20 - 22 and neuronal migration. 23 Most of the known VRK1 mutants are very uncommon variants in the population, which are recessive, and their neurologic phenotype is only detected in either homozygous or compound heterozygous individuals.…”

VRK1 Depletion Facilitates the Synthetic Lethality of Temozolomide and Olaparib in Glioblastoma Cells

“…Cells were incubated with antibiotic-free medium. The VRK1 siRNA used in this work are highly specific, and their effects are rescued by kinase-active VRK1 (human or murine), but they are not rescued by kinase-dead VRK1 (Sanz-Garcia et al, 2012;Cantarero et al, 2015;Salzano et al, 2015;Monsalve et al, 2016;Martin-Doncel et al, 2019;Marcos et al, 2020). Furthermore, kinase-dead VRK1 (K179E) also does not rescue the effects in response to DDR (Sanz-Garcia et al, 2012;Cantarero et al, 2015;Salzano et al, 2015;Monsalve et al, 2016;Martin-Doncel et al, 2019;Marcos et al, 2020).…”

“…The in vitro kinase assay was performed in reaction buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl 2 , 0.5 mM DTT, and 150 mM KCl, 5 mM ATP) and 250 ng of recombinant H3 as previously reported in a reaction volume of 40 µl during 45 min at 30 • C (Martin-Doncel et al, 2019). H3T3ph was detected with a rabbit polyclonal antibody (Upstate-Millipore) (Salzano et al, 2015;Moura et al, 2018;Marcos et al, 2020).…”

VRK1 Phosphorylates Tip60/KAT5 and Is Required for H4K16 Acetylation in Response to DNA Damage

“…A549 cells lines were infected with lentiviral particles containing the vector pLentiC-HA-IRES-BSD (OriGene Technologies, Rockville, MD, USA) expressing murine VRK1 or kinase-dead murine VRK1 (K179E) were cloned and selected with blasticidine [ 76 ].…”