Mutation of senataxin alters disease-specific transcriptional networks in patients with ataxia with oculomotor apraxia type 2

Abstract

Senataxin, encoded by the SETX gene, contributes to multiple aspects of gene expression, including transcription and RNA processing. Mutations in SETX cause the recessive disorder ataxia with oculomotor apraxia type 2 (AOA2) and a dominant juvenile form of amyotrophic lateral sclerosis (ALS4). To assess the functional role of senataxin in disease, we examined differential gene expression in AOA2 patient fibroblasts, identifying a core set of genes showing altered expression by microarray and RNA-sequencing. To determine whether AOA2 and ALS4 mutations differentially affect gene expression, we overexpressed disease-specific SETX mutations in senataxin-haploinsufficient fibroblasts and observed changes in distinct sets of genes. This implicates mutation-specific alterations of senataxin function in disease pathogenesis and provides a novel example of allelic neurogenetic disorders with differing gene expression profiles. Weighted gene co-expression network analysis (WGCNA) demonstrated these senataxin-associated genes to be involved in both mutation-specific and shared functional gene networks. To assess this in vivo, we performed gene expression analysis on peripheral blood from members of 12 different AOA2 families and identified an AOA2-specific transcriptional signature. WGCNA identified two gene modules highly enriched for this transcriptional signature in the peripheral blood of all AOA2 patients studied. These modules were disease-specific and preserved in patient fibroblasts and in the cerebellum of Setx knockout mice demonstrating conservation across species and cell types, including neurons. These results identify novel genes and cellular pathways related to senataxin function in normal and disease states, and implicate alterations in gene expression as underlying the phenotypic differences between AOA2 and ALS4.

Figure 1.

Expression of senataxin protein in patient and carrier fibroblasts and peripheral blood lymphoblasts. Western blotting was performed using nuclear protein from cultured lymphoblasts (LB) or fibroblasts (FB) from AOA2 patients or carriers (Family US2, Table 1) as well as a normal control (WT) using an antibody directed against senataxin (SETX). SMC1 is shown as a loading control.

Figure 2.

Weighted gene co-expression network analysis (WGCNA) in senataxin-haploinsufficient fibroblasts overexpressing mutant forms of senataxin identifies molecular pathways related to biological function. WGCNA was performed to identify groups of genes (modules) whose collective changes in gene expression most correlate with senataxin mutation, reflecting biologically relevant relationships. For clarity, only the most highly connected module members are shown. Genes with the highest connectivity (i.e. hubs) are indicated in red. (A) Skyblue module. (B). Green module.

Figure 3.

WGCNA in peripheral blood identifies a transcriptional signature for senataxin dysfunction in AOA2 patients. WGCNA was performed to identify gene modules whose collective changes in gene expression most correlate with AOA2 disease status. For clarity, only the most highly connected module members are shown. Genes with the highest connectivity (i.e. hubs) are indicated in red. (A) Blue module. (B). Turquoise module. (C) Modules from the peripheral blood weighted gene co-expression network (Supplementary Material, File 9) are shown in comparison with the network derived from human transfected fibroblast cell lines (Supplementary Material, File 6) or Setx knockout mouse cerebellum (Supplementary Material, File 12). Results are graphed by number of module members versus preservation score (Zsummary) calculated using the module preservation function of the WGCNA software package. Zsummary scores of 10 or greater (dashed line) indicate strong preservation, scores between 10 and 2 show moderate preservation, and scores less than 2 (dotted line) show weak or no preservation.

Figure 3.

WGCNA in peripheral blood identifies a transcriptional signature for senataxin dysfunction in AOA2 patients. WGCNA was performed to identify gene modules whose collective changes in gene expression most correlate with AOA2 disease status. For clarity, only the most highly connected module members are shown. Genes with the highest connectivity (i.e. hubs) are indicated in red. (A) Blue module. (B). Turquoise module. (C) Modules from the peripheral blood weighted gene co-expression network (Supplementary Material, File 9) are shown in comparison with the network derived from human transfected fibroblast cell lines (Supplementary Material, File 6) or Setx knockout mouse cerebellum (Supplementary Material, File 12). Results are graphed by number of module members versus preservation score (Zsummary) calculated using the module preservation function of the WGCNA software package. Zsummary scores of 10 or greater (dashed line) indicate strong preservation, scores between 10 and 2 show moderate preservation, and scores less than 2 (dotted line) show weak or no preservation.

Figure 4.

Model of senataxin (SETX) function in normal and disease states.