ATXN1 repeat expansions confer risk for amyotrophic lateral sclerosis and contribute to TDP-43 mislocalization

Abstract

Increasingly, repeat expansions are being identified as part of the complex genetic architecture of amyotrophic lateral sclerosis. To date, several repeat expansions have been genetically associated with the disease: intronic repeat expansions in C9orf72, polyglutamine expansions in ATXN2 and polyalanine expansions in NIPA1. Together with previously published data, the identification of an amyotrophic lateral sclerosis patient with a family history of spinocerebellar ataxia type 1, caused by polyglutamine expansions in ATXN1, suggested a similar disease association for the repeat expansion in ATXN1. We, therefore, performed a large-scale international study in 11 700 individuals, in which we showed a significant association between intermediate ATXN1 repeat expansions and amyotrophic lateral sclerosis (P = 3.33 × 10^-7). Subsequent functional experiments have shown that ATXN1 reduces the nucleocytoplasmic ratio of TDP-43 and enhances amyotrophic lateral sclerosis phenotypes in Drosophila, further emphasizing the role of polyglutamine repeat expansions in the pathophysiology of amyotrophic lateral sclerosis.

Keywords: DNA repeat expansion; amyotrophic lateral sclerosis; genetic association study; trinucleotide repeat expansions.

Graphical Abstract

Figure 1

Pedigree with co-occurrence of SCA1 and ALS. The index patient (arrow) was diagnosed with ALS and reported a positive family history for spinocerebellar ataxia type 1 (SCA1) in four other family members. No DNA samples from family members diagnosed with SCA1 were available for analysis.

Figure 2

Distribution of ATXN1 CAG/CAT repeat length. Proportion of total alleles grouped per ATXN1 repeat length determined via PCR analysis in a cohort of 2672 individuals affected with ALS (gray) and 2416 geographically matched controls (black) from four different cohorts (Belgium, France, Ireland and The Netherlands).

Figure 3

ATXN1 polyglutamine repeat expansion meta-analysis. Forest plot for the fixed-effect Mantel–Haenszel meta-analysis of the effect of expanded (≥33) ATXN1 CAG/CAT repeats on ALS risk in three different datasets grouped per country of origin: previous reports, PCR-genotyped cohort and WGS-genotyped cohort. In addition, individual-level data of all three datasets were combined in a single logistic regression analysis (Joint analysis), which was corrected for the country of origin and method of genotyping. Weights depending on number of participants. CI, confidence interval. *Conforti et al. used a different cut-off for expanded/non-expanded status (≥32 CAG/CAT repeats). However, since the most frequent alleles in their data [28/29] seem to also have shifted one repeat unit compared to the Italian population in Lattante et al. and our data [29/30], we did not alter the expansion status.

Figure 4

Presence and number of CAT interruptions in ATXN1 CAG repeat expansion. (A and B) Plots show the results after genotyping 1418 repeat alleles (849 ALS; 569 control) from 150 bp WGS reads that span the full repeat. (A) Number of CAT interruptions per repeat allele. (B) Correlation between the total repeat size, including both CAG and CAT, and the longest stretch of uninterrupted CAG per allele for both ALS affected (blue) and unaffected (orange). CAT interruptions usually and exclusively appear after the first 12–17 CAG repeats, resulting in a significant correlation between the total and uninterrupted CAG repeat size (Kendall’s tau cor., P < 2.2e−16 for both ALS and controls) and therefore a similar distribution (margin panels; prop.tot = proportion of total alleles). There were two exceptions (red border): one ALS-affected allele had no interruptions, probably because of its short length (13), and one unaffected sample seemed to carry an uninterrupted stretch of 30 CAG.

Figure 5

Effect of ATXN1 repeat expansion on survival and age at onset in ALS. (A and B) Plots of time-dependent probabilities in 1890 ALS patients with either ATXN1 normal (<33, orange) or expanded (≥33, blue) CAG/CAT repeat expansion. (A) Survival after the onset of disease in months, corrected for: sex, age at onset, bulbar site of onset and presence of C9ORF72 expansion. (B) Age at onset of the disease in years corrected for: sex, site of onset and the presence of a C9orf72 repeat expansion. No significant effects were found.

Figure 6

HeLA cells were transfected with mCherry-tagged ataxin-1 containing 27 polyglutamine repeats (mCherry-Atx227Q) or control vector (mCherry). (A) TDP-43 protein levels are not altered in ataxin-1 expressing cells (uncropped blot image in Supplementary Fig. 1). (B) Quantification of TDP-43 levels normalized to loading control GAPDH (glyceraldehyde 3-phosphate dehydrogenase). Unpaired t-test, two-sided, P-value: 0.1312. (C) The presence of cytoplasmic inclusion bodies (IB) correlates with TDP-43 mislocalization in ataxin-1-expressing cells. TDP-43 does not accumulate in nuclear or cytoplasmic ataxin-1 IB but does mislocalize to the cytoplasm in cells with IB. (D) Quantification of TDP-43 mislocalization in controls cells (mCherry) and cells without (−IB_cyto) or with cytoplasmic ataxin-1 IB (+IB_cyto). (E) Cytoplasmic IB also correlate with GFP-tagged KNPA2 mislocalization to the cytoplasm. (F) Quantification of KPNA2 mislocalization. (D and F) One-way ANOVA, ****P < 0.0001.

Figure 7

Ataxin-1 polyQ modifies eye phenotypes in Drosophila. (A) Scheme indicating assessment of genetic modifiers. (B) Effect of eye phenotype after co-expression of eGFP, 2Q ataxin-1 and 82Q ataxin-1 in wild type (top) and TDP-43- (middle) and GR36 (bottom)-expressing flies. (C) Fraction of flies per necrotic eye score rank (darker shading equals higher score). Right panel: flies overexpressing ATXN1.82Q only show a clear degenerative phenotype characterized by a moderate rough eye phenotype, but only very small necrotic spots. Middle panel: flies co-expressing TDP-43 and ATXN1 polyQ with a repeat length of 82 with a severe eye phenotype are significantly enriched compared to flies expressing TDP-43 and ATXN1 with a polyQ repeat length of 2 (P = 2.65 × 10⁻⁴); there was no significant difference with eGFP and 2 polyQ. Left panel: flies co-expressing GR(36) and ATXN1 polyQ with a repeat length of 82 with a severe eye phenotype are significantly enriched compared to flies expressing GR(36) and ATXN1 with a polyQ repeat length of 2 (P < 2.0 × 10⁻¹⁶); there was no significant difference with eGFP and 2 polyQ. Statistical analysis using linear by linear association test, n > 50 per genotype.