A conserved eEF2 coding variant in SCA26 leads to loss of translational fidelity and increased susceptibility to proteostatic insult

Katherine E. Hekman 2 Guo-Yun Yu 0 Christopher D. Brown 1 Haipeng Zhu 2 Xiaofei Du 2 Kristina Gervin 6 Dag Erik Undlien 6 April Peterson 1 Giovanni Stevanin 5 H. Brent Clark 4 Stefan M. Pulst 3 Thomas D. Bird 7 Kevin P. White 1 Christopher M. Gomez 2 0 SAIC-Frederick Inc, NCI-Frederick, Frederick, MD , Division of Cancer Epidemiology and Genetics , NCI/NIH, Bethesda, MD 20892, USA 1 Department of Human Genetics, Institute for Genomics and Systems Biology, University of Chicago , Chicago, IL 60637, USA 2 Department of Neurology 3 Department of Neurology, University of Utah , Salt Lake City, UT 84132, USA 4 Department of Laboratory Medicine and Pathology, Neurology, and Neurosurgery, University of Minnesota , Minneapolis, MN 55455, USA 5 Centre de Recherche de l'Institut du Cerveau et de la Moelle e pinie` re , INSERM, UPMC Univ. Paris 6 , UMR_S 975, CNRS 7225, EPHE, Ho pital Pitie -Salpe trie` re, Paris, France 6 Department of Medical Genetics, Oslo University Hospital and University of Oslo , Oslo, Norway 7 Department of Neurology, Geriatric Research Center, VA Medical Center, University of Washington , Seattle, WA 98195, USA The autosomal dominant spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of disorders exhibiting cerebellar atrophy and Purkinje cell degeneration whose subtypes arise from 31 distinct genetic loci. Our group previously published the locus for SCA26 on chromosome 19p13.3. In this study, we performed targeted deep sequencing of the critical interval in order to identify candidate causative variants in individuals from the SCA26 family. We identified a single variant that co-segregates with the disease phenotype that produces a single amino acid substitution in eukaryotic elongation factor 2. This substitution, P596H, sits in a domain critical for maintaining reading frame during translation. The yeast equivalent, P580H EF2, demonstrated impaired translocation, detected as an increased rate of 21 programmed ribosomal frameshift read-through in a dual-luciferase assay for observing translational recoding. This substitution also results in a greater susceptibility to proteostatic disruption, as evidenced by a more robust activation of a reporter gene driven by unfolded protein response activation upon challenge with dithiothreitol or heat shock in our yeast model system. Our results present a compelling candidate mutation and mechanism for the pathogenesis of SCA26 and further support the role of proteostatic disruption in neurodegenerative diseases. - The autosomal dominant spinocerebellar ataxias (SCAs) are a genetically and clinically heterogeneous group of neurodegenerative disorders. To date, 31 unique subtypes attributed to distinct genetic loci have been identified, with a collective incidence of 50/100 000 (1). All subtypes share the common endpoint of Purkinje cell death and atrophy of the cerebellum (2). For 20 of the 31 subtypes, genetic bases have been identified (2 38). Six have been attributed to polyglutamine expansions in unrelated proteins (SCAs 1, 2, 3, 6, 7 and 17) (3 6,9,10,33), five to untranslated repeat expansions (SCAs 8, 10, 12, 31 and 36) (11 13,15,27,29), one to various deletions in a single gene (SCA15/16) (34), and To whom correspondence should be addressed. Tel: +7 737026390; Fax: +7 737025670; Email: These authors contributed equally to this manuscript. The locus for SCA26 was mapped to a 15.55 cM region with maximum logarithm of odds scores (6.06 14.61) between markers D19S886 and D19S894 (39), determined by obligate recombinants. Large-scale genomic rearrangements were not examined, but CAG repeat expansions were excluded (39) by repeat expansion detection (40). This region contained 3.5 Mb and 104 known genes. We designed an Agilent 1 M feature custom capture array to tile across all coding and noncoding non-repetitive bases within the critical interval and sequenced four individuals: two sibship pairs composed of affected/unaffected (without disease haplotype) individuals separated by six meioses (Supplementary Material, Fig. S1). Given the dominant mode of inheritance and the presumed low frequency of the causal allele, genotypes were filtered to identify heterozygous variants shared by both affecteds, but not present in either unaffected or dbSNP (build 131) or 1000 genomes project data (41). This process identified 38 variants that co-segregated with the disease phenotype Characterization of the substituted protein The mRNA produces a 95 kDa protein of 858 amino acids with six domains (NCBI protein ID AAI26260.1). The affected amino acid residue, P596, and its surrounding residues are highly conserved, from Arabidopsis thaliana to Homo sapiens (Fig. 3), indicating that the position has evolved under strong purifying selection. Given the predicted deleterious effect of the P596H variant, we tested whether DAMAGING Four family members were used for critical interval sequencing via custom capture array. The P596H amino acid substitution in eEF2 is the only highly evolutionarily conserved variant predicted to be damaging. REF, reference nucleotide; ALT, alternate found upon sequencing; GERP, genomic evolutionary rate profiling (43); SIFT, Sorting Tolerant From Intolerant algorithm (42); SYN, synonymous variant. eEF2 protein containing the P596H substitution was able to fold properly. cDNAs encoding reference and the P596H eEF2 were cloned, in-frame into the pCDNA3.1 vector with a C-terminal 3XFLAGTM epitope tag and transiently transfected into HEK293 cells. P596H and reference eEF2 proteins were expressed at comparable levels, with no evidence of unique degradation products, suggesting that the P596H protein is properly expressed and post-translationally processed (Supplementary Material, Fig. S2). The ubiquitously expressed eEF2 protein has been highly characterized. Its primary function is to facilitate the translocation of the peptidyl-tRNA from the A-site to the P-site in the ribosome (44,45). eEF2 is predominantly found near the rough endoplasmic reticulum (rER), consistent with its essential role in all protein translation. Several neurodegenerative diseases known to be caused by point mutations in specific proteins [familial amyotrophic lateral sclerosis (fALS), SCA14] (22,46) exhibit altered subcellular localization, misfolding and/or aggregates of the mutant protein in affected brain tissues, although the role of these aggregates in disease pathogenesis is still undefined. To investigate whether P596H eEF2 has altered subcellular localization or forms prominent intracellular aggregates, we first performed immunocytochemical staining for the eEF2 protein and the rER marker, calnexin, in lymphoblastoid cell lines derived from an SCA26 patient and a phenotypically normal genetically unrelated individual. In both control and patient cells, eEF2 and calnexin were highly co-localized, as shown by the cytofluorogram (Pearsons coefficients of 0.935 for wild-type (WT) and 0.946 for P (...truncated)