ELOVL5 mutations cause spinocerebellar ataxia 38

Abstract

Spinocerebellar ataxias (SCAs) are a heterogeneous group of autosomal-dominant neurodegenerative disorders involving the cerebellum and 23 different genes. We mapped SCA38 to a 56 Mb region on chromosome 6p in a SCA-affected Italian family by whole-genome linkage analysis. Targeted resequencing identified a single missense mutation (c.689G>T [p.Gly230Val]) in ELOVL5. Mutation screening of 456 independent SCA-affected individuals identified the same mutation in two further unrelated Italian families. Haplotyping showed that at least two of the three families shared a common ancestor. One further missense variant (c.214C>G [p.Leu72Val]) was found in a French family. Both missense changes affect conserved amino acids, are predicted to be damaging by multiple bioinformatics tools, and were not identified in ethnically matched controls or within variant databases. ELOVL5 encodes an elongase involved in the synthesis of polyunsaturated fatty acids of the ω3 and ω6 series. Arachidonic acid and docosahexaenoic acid, two final products of the enzyme, were reduced in the serum of affected individuals. Immunohistochemistry on control mice and human brain demonstrated high levels in Purkinje cells. In transfection experiments, subcellular localization of altered ELOVL5 showed a perinuclear distribution with a signal increase in the Golgi compartment, whereas the wild-type showed a widespread signal in the endoplasmic reticulum. SCA38 and SCA34 are examples of SCAs due to mutations in elongase-encoding genes, emphasizing the importance of fatty-acid metabolism in neurological diseases.

Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Family Trees, Haplotype Analysis, and ELOVL5 Mutation Analysis (A) Pedigrees of SCA38-affected families. Open symbols indicate unaffected family members, and solid black symbols indicate affected members. Lines above the symbols indicate individuals for whom DNA was available. The genotype of the ELOVL5 mutations is indicated below each tested subject. (B) Microsatellite genotypes with haplotype reconstruction of family SCA38-01-BS. Arrows indicate recombination events in healthy subjects IV-3 and IV-4; these events define the minimal region (boxed). On the right, distances of the markers from the top of chromosome 6p are indicated in Mb. (C) Electropherograms of missense mutations c.214C>G (p.Leu72Val) and c.689G>T (p.Gly230Val). (D) The amino acid sequence alignment of a portion of the human ELOVL5 in various orthologs. The two changed amino acids are conserved through vertebrates.

Figure 2

MRI Scans, ELOVL5 Expression, ELOVL5 Localization, and PUFA Serum Levels (A) MRI scans of an affected individual (III-10 in family SCA38-01-BS). Coronal, midsagittal, and horizontal sections are reported. Cerebellar atrophy with vermis involvement is shown; the cortex and brainstem are preserved. (B) The normal expression pattern of ELOVL5 in five human tissues, lymphoblastoid cells, and four brain areas (mean ± SD). ELOVL5 showed prominent expression in brain and lung. Gene expression was measured by quantitative PCR with FAM-labeled Universal ProbeLibrary LNA probes (Roche Diagnostics) (Table S6). (C–F) Micrographs of sections of healthy human (C and E) and murine (D and F) cerebellar cortex immunostained with ELOVL5 antibody. Human cerebellar sections were obtained from paraffin-embedded tissues of postmortem brain derived from a healthy subject. Sections were incubated with ELOVL5 polyclonal antibody (C15621, diluted 1:400, AssaybIoTech) and counterstained with hematoxylin. Higher magnifications of sections of human and mouse cerebellar cortex are shown in (E) and (F), respectively. Note the similar labeling pattern of human and mouse Purkinje cells and the presence of numerous labeled cells in the white matter. Abbreviations are as follows: ml, molecular layer; PCl, Purkinje cell layer; gl, granular layer; and wm, white matter. Scale bars represent 200 μm (C), 80 μm (D), 50 μm (E), and 20 μm (F). (G) Fatty-acid analysis in serum isolated from whole blood of four affected individuals (III-10 in family SCA38-01-BS, IV-8 and IV-9 in family SCA38-02-CA, and III-3 in family SCA38-03-TO). Fatty acids were analyzed in the serum from affected and control individuals by liquid chromatography-tandem mass spectrometry. Reduced AA and DHA were measured in affected individuals (mean ± SEM; AA, ∗p < 0.05; DHA,∗p < 0.05). Statistical analysis was performed with a one-tailed unpaired t test. (H) Quantitative real-time PCR on the affected individuals’ cDNA showed an increase in ELOVL5 expression in comparison to the TBP reference (mean ± SEM; ∗∗∗p < 0.001, Mann Whitney test; ∗∗p < 0.01, two-tailed unpaired t test, Welch’s correction). (I) ELOVL5 showed higher amounts in comparison to β-actin (ACTB) (mean ± SEM; ∗∗∗p < 0.001, Mann Whitney test; ∗∗p < 0.01, two-tailed unpaired t test, Welch’s correction). Total proteins were extracted from lymphoblasts of five affected individuals (III-6 and III-10 in family SCA38-01-BS and IV-8, IV-9, and IV-12 in family SCA38-02-CA) and five age- and origin-matched healthy controls. ELOVL5 amounts were normalized to ACTB. Different symbols indicate different affected individuals. The asterisk indicates an uncharacterized immunoblot signal as demonstrated in Elovl5-knockout mice (see Figure S3A). Lanes 1 and 2 are affected individuals IV-8 and IV-9, respectively, of family SCA38-02-CA; lanes 3 and 4 are two healthy controls.

Figure 3

Subcellular Localization of Altered ELOVL5 in COS7 Cells COS-7 cells transfected with wild-type or mutated (c.214C>G [p.Leu72Val] and c.689G>T [p.Gly230Val]) ELOVL5 cDNAs. The day before transfection, COS-7 (gray monkey kidney) cells were seeded at 90% confluence in 500 μl DMEM and 10% fetal bovine serum without antibiotic. In vitro transfections were performed with 500 ng of ScaI-digested linearized plasmid and 1 μl Lipofectamine 2000 according to the manufacturer’s (Life Technologies) instructions. After 24 hr, transfected cells were selected for 7 days with the use of G418 (Invivo Gen). Transfected COS-7 cells (5 × 104) were seeded on fibronectin-coated coverslips (24-well plates) and maintained with complete medium (DMEM, GIBCO) at 37°C. After 24 hr, cells were incubated with HaloTag TMR Direct Ligand at a final concentration of 1 μM (Promega) and the indicated primary antibodies. Samples were analyzed with an Olympus FV300 laser-scanning confocal microscope equipped with a blue argon (488 nm) laser, a green helium-neon (543 nm) laser, and FluoView 300 software (Olympus Biosystems). Cells were imaged with a 60× oil-immersion objective (1.4 NA). Images of optical sections (512 × 512 pixels) were digitally recorded and processed with BioView software (Center for Bio-Image Informatics). (A) The ER is shown in green (rabbit anti-calreticulin; C4606, 1:400, Sigma-Aldrich), and ELOVL5 is shown in red (HaloTag TMR Direct Ligand). The two proteins completely colocalized in cells expressing wild-type ELOVL5, as shown by the merge row. This pattern was observed in 96% of cells (n = 49) expressing wild-type ELOVL5, 23% of cells (n = 42) with p.Leu72Val, and 17.5% of cells (n = 40) with p.Gly230Val (Fisher’s exact test performed between the wild-type and altered proteins, two-sided, p