Blood DDIT4 and TRIM13 Transcript Levels Mark the Early Stages of Machado-Joseph Disease

Abstract

Objective: An abundance of select transcripts and proteins has been found to be dysregulated in blood samples of Machado-Joseph disease (MJD) carriers. Here, we aimed to: (1) identify blood transcriptional changes as potential biomarkers of MJD; (2) correlate levels of differentially expressed blood transcripts with MJD carriers features; and (3) evaluate whether the identified differential abundance of blood transcripts in MJD patients is preserved in MJD brains.

Methods: We used unbiased RNA microarray and quantitative polymerase chain reaction to assess transcript levels in blood and brain samples, and western blot analysis to evaluate the abundance of specific proteins in brain samples.

Results: We observed consistent dysregulation of DDIT4, TRIM13, and P2RY13 transcriptional levels in the blood of MJD patients from preclinical to symptomatic stages in Azorean and Brazilian cohorts. Combined blood DDIT4 and TRIM13 transcriptional levels show a very high accuracy to discriminate MJD carriers from matched controls (AUC ≥0.90). Levels of P2RY13 transcripts correlate with age at onset, and an abundance of DDIT4 and TRIM13 transcripts correlate with the expanded CAG repeat size in combined Azorean and Brazilian patients; and levels of TRIM13 transcripts correlate with age at onset of early-stage Azorean patients. Moreover, the abundance of TRIM13 protein is increased in the cerebral cortex of MJD patients.

Interpretation: Overall, blood DDIT4 and TRIM13 transcript levels are potential biomarkers of MJD. Cellular processes involving DDIT4, TRIM13, and P2RY13 appear to be commonly dysregulated in the blood and brain of MJD patients, indicating the involvement of these genes in MJD pathogenesis. ANN NEUROL 2025;98:107-119.

FIGURE 1

Overview of the experimental workflow used in this study. Peripheral blood samples from Machado–Joseph disease patients and controls from the Azorean cohort were used in (A) a genome‐wide expression microarray analysis and (B) technical validation by qPCR. (C) The expression levels were evaluated in 2 independent cohorts: (C.1) The expression levels of a set of dysregulated genes were assessed in a set of Azorean samples; and (C.2) genes considered as the most promising disease markers were evaluated in a set of patients and controls of the Brazilian cohort. (D) The expression levels of the validated genes were assessed in a set of Azorean PC individuals and early patients. (E) The expression of the validated genes was evaluated at the transcript and protein levels in postmortem brain samples from Machado–Joseph disease patients and controls. Human brain figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license. C = controls; CEP = controls of early patients; CPC = controls of preclinical participants; EP = early patients; P = patients; PC = preclinical participants.

FIGURE 2

Blood transcript levels of DDIT4, TRIM13, and P2RY13 are consistently dysregulated in 2 independent Machado–Joseph disease cohorts. Gene expression levels of DDIT4, TRIM13, P2RY13 S100P, SVBP, and USP49 in peripheral blood samples from Machado–Joseph disease carriers (preclinical participant [PC], and patient [P]) and controls (C) from the Azorean cohort (group 2) and MJD patients (P) and controls (C) from the Brazilian cohort (group 3). Dots represent transcript levels for each individual. Graphs show the mean (Azores) or median (Brazil) and the 95% confidence interval of the 2^−ΔCt values.

FIGURE 3

Blood transcript levels of DDIT4, TRIM13, and P2RY13 are dysregulated in initial stages of the disease. Genes expression levels in peripheral blood samples from preclinical (PC) participants and early patients (EP), and age‐ and sex‐matched paired controls (controls of preclinical participants [CPC]; controls of EP [CEP]) from group 4. Dots represent transcript levels for each individual. Graphs show the median and the 95% confidence interval of the 2^−ΔCt values.

FIGURE 4

Blood transcript levels of DDIT4 and TRIM13 differentiate with very high accuracy Machado–Joseph disease subjects from controls. Receiver operating characteristics curves show the ability of blood transcript levels of DDIT4, TRIM13, P2RY13 and the combination of DDIT4 and TRIM13 levels to differentiate Machado–Joseph disease subjects in different stages of the disease (PC, light gray line; EP, dark gray line; patients with >5 years of disease duration, black line) from matched controls.

FIGURE 5

DDIT4, TRIM13, and P2RY13 transcript levels are similar in postmortem brains of Machado–Joseph disease patients and controls. Transcript levels in pons, dentate cerebellar nucleus (DCN), and cerebral cortex (cortex) from Machado–Joseph disease patients (P) and controls (C). Dots represent transcript levels for each individual. Graphs show the mean and the standard error mean of the 2^−ΔCt values.

FIGURE 6

TRIM13 protein levels are altered in postmortem brains of Machado‐Joseph disease patients. Phosphate‐buffered saline (PBS)‐soluble (soluble) and sarkosyl‐soluble (insoluble) protein fractions were analyzed in dentate cerebellar nucleus (DCN) and cerebral cortex (cortex) from Machado‐Joseph disease patients (P) and controls (C). Left panels show immunoblots of indicated proteins in soluble and insoluble protein fractions. Right panels display quantification of band intensity, with values normalized to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Dots represent protein levels for each individual. Graphs show the mean and the standard error mean of protein relative to controls.