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The use of role-assigned ionic additives with different adsorption energies and distinct electron-accepting abilities enables the construction of a multilayer solid electrolyte interphase (SEI) wit...

Introduction: Despite increasing numbers of homeless people, less attention has been paid to their mental health, particularly associated with crime victimization. This study sought to examine the ...

Background Hereditary cerebellar ataxias exhibit heterogeneous phenotypes and genotypes. To date, advancement of next-generation sequencing technologies have identified many causative genes for ataxia in various population. In this study, whole-exome sequencing (WES) was utilized to explore the genetic cause of ataxia among Korean patients who remained undiagnosed following routine investigation. Methods Patients with ataxia were enrolled in this study. We excluded patients with acquired, degenerative, and trinucleotide repeat ataxias, such as spinocerebellar ataxia 1 (SCA1), SCA2, SCA3, SCA6, SCA7, SCA8, SCA17, Dentatorubral-pallidoluysian atrophy, and Friedreich ataxia. WES was performed. After basic filtering based on population databases, we then performed primary filtering to screen for known ataxia-associated genes, followed by expanded filtering customized for individual patients. Results We enrolled 77 ataxia patients from 68 families. Eighteen families had pathogenic or likely pathogenic variants in 14 different genes, including NEU1, APTX, SPG7, HTRA1, POLG2, SYNE1, CACNA1G, CACNA1A, ITPR1, AHI1, SPG11, ANO10, ATM, and C5orf42, resulting in a diagnostic yield of 26.5%. Hereditary spastic paraplegia was the most common diagnosis. Adult-onset ataxias and those without family history were frequently encountered. Variants of unknown significance were found in 14 (20.6%) families, some of which were highly probable from the clinical perspective. Conclusion Using WES, we explored the molecular etiology of ataxia in patients whom were not diagnosed through routine clinical investigation. This study revealed unexpected rare disorders as well as the known ataxia-associated genes in a Korean population.

Additional file 1: Figure S1. (A) Schematic image of the E3 ubiquitin ligase candidates of SPAST. (B) After transfection with the Flag-SPAST-M1 construct, HEK293 cells were immunoprecipitated, and analyzed using western blotting. Figure S2. The RT-PCR band density in Fig. 1D is generated using ImageJ software. Figure S3. (A) Schematic representation of SPAST N-terminal deletion mutants. Interaction capacity between SPAST deletion mutants and FBXL17 is indicated by an asterisk. (B) BTB domain sequences of the identified BTB proteins and SPAST-M1 were represented by multiple sequence alignments (C) Schematic representation of FBXL17 N-terminal deletion mutants. Interaction capacity between FBXL17 mutants and SPAST is indicated with the symbol. Figure S4. (A) mRNA expression was analyzed under the conditions shown in Fig. 3A and quantified using ImageJ software. (B) mRNA expression was analyzed under the conditions shown in Fig. 3C. Data are mean ± standard deviation. (ns, not significant). Figure S5. (A) Multiple sequence alignment of SPAST proteins in three species. The nine lysine residues or the predicted phosphorylation sites of SPAST confirmed its interspecies functional conservation using multiple alignment software (lysine residues, marked in red boxes; predicted S/T residues, marked in blue boxes). (B) For in vivo ubiquitination assay, HEK293 cells were transfected with indicated plasmids followed by MG132 treatment, and total cell lysates immunoprecipitated with anti-Flag-agarose gel followed by western blotting with indicated antibodies. Figure S6. (A) SDS-PAGE analysis of purified His-E1, His-UbcH10b, His-FBXL17-ΔNT1 (317-717a.a.), GST-SPAST-M1, GST-KLHL, and GST. All human recombinant proteins were expressed in E. coli and purified as described in the Materials and Methods section. Purified proteins were separated using SDS-PAGE gel and visualized using Coomassie Blue staining. (B) The E1/E2 thioester assay with His-E1, His-UbcH10b, and Flag-ubiquitin. (C) HeLa cells were transduced with scrambled shRNA control lentivirus or shRNA lentivirus targeted against FBXL17. After incubation for 72 h, S-100 cytosolic extract was prepared from the cells and analyzed via western blotting using antibodies against SCFFBXL17 components. (D) SDS-PAGE analysis of purified WT or K554R of GST-SPAST-M1 and GST proteins from the E.coli recombinant protein expression system. Figure S7. (A) In vitro CK2 kinase assay against SPAST. Proteins were separated using SDS-PAGE after the in vitro kinase assay in the presence or absence of CK2 and stained with Coomassie Blue staining demonstrating equal loading of the substrate. (B) mRNA level of CK2α and CK2β was analyzed by real-time qPCR in HEK293 WT or CK2β-Cas9 stable cell line (left). Data are represented as mean + /– standard deviation calculated from three replicates. The PCR products were visualized by 2% agarose gel electrophoresis (right). (**p < 0.01; ns, not significant). Figure S8. (A) mRNA expression was analyzed and quantified using RT-PCR under the conditions shown in Fig. 5B. (B) mRNA expression was analyzed using RT-PCR under the conditions shown in Fig. 5C and quantified. Data are mean ± standard deviation. (*p < 0.05; **p < 0.01; ns, not significant). Figure S9. (A) Rencell CX cells were treated with 0.2, 0.5 μM MLN4924 for 2 d, and analyzed using western blotting with the indicated antibodies. (B) After transfection with pEGFP plasmid, the cells were differentiated for 2 d in the presence or absence of 0.5 μM MLN4924 and stained with GFP and acetylated α-tubulin antibodies. Fluorescence intensities of acetylated α-tubulin quantified with ImageJ software. Data are mean ± standard deviation calculated from three replicates. Scale bar: 20 μm (*p < 0.05). Figure S10. (A) Immunostaining of GFP and Acetylated α-tubulin in HeLa cells transfected with GFP empty or GFP-SPAST-M1 plasmids, and (B) fluorescence intensities of acetylated α-tubulin quantified using ImageJ software. Data are mean ±

Additional file 2: Table S1. Antibody list used in this study. Table S2. Reagents list used in this study. Table S3. Plasmid constructs used in the study. Table S4. The primer list for conventional RT-PCR. Table S5. The primer list for RT-qPCR. Table S6. The predicted ubiquitination sites of SPAST. Table S7. The predicted CK2 phosphorylation sites of SPAST. Table S8. SPAST exon 1 mutation and clinical summary found in HSP patients.