Predicting and tracking Machado-Joseph disease: biomarkers of diagnosis and prognosis.

  • Estado
  • Nome
    Mafalda Sofia Bastos Raposo
  • Entidade de acolhimento
    CIRN – Centro de Investigação de Recursos Naturais


Machado-Joseph disease/spinocerebellar ataxia type 3 (MJD/SCA3) is a rare late-onset neurodegenerative disorder, which is currently untreatable. In this polyglutamine (polyQ) disease, a CAG repeat expansion located at the ATXN3 gene, is the causative mutation. As a progressive disorder, MJD is characterized by different stages throughout its natural course, namely by a long preclinical period, during which molecular and even clinical alterations could be starting, many years before the ataxia onset. MJD onset is partially explained by the size of the CAG tract constituting its main trait biomarker. The incompleteness of this explanation, however, implies the existence of genetic modifiers, which should provide additional trait biomarkers. The identification of molecular trait biomarkers of MJD will allow to (1) improve the prediction of age at onset and (2) empower interventional trials, by allowing genetic stratification of patients. The characterization of MJD disease stages has so far been performed using mainly clinical scales or imaging data, while few studies investigated molecular biomarkers. The identification of such molecular state biomarkers would allow (1) the prediction of age at pathological onset (age at which cell damage is already present although a clinical phenotype is not yet evident), (2) monitoring different stages of disease progression, and (3) detecting subtle therapeutic benefits, in emergent clinical trials. The main goal of the present PhD project was to contribute to the development of molecular biomarkers for MJD. RNA and DNA, obtained from blood samples of preataxic subjects and MJD patients of Azorean background, including samples collected in two different moments, constituted the main resource used throughout the present PhD project. Samples from population controls were also used. The determination of the number of CAG repeats at the ATXN3 was performed for all MJD subjects studied. The homogeneous MJD cluster comprising a total of 88 patients was characterized. The average of the neurological examination score for spinocerebellar ataxia (NESSCA) available for 69 patients was 12±5 (mean ± standard deviation); 40% of the variance explanation is provided by the number of the CAG repeats in the expanded allele and disease duration. Ocular alterations, namely nystagmus, are clinical features commonly described in MJD. The evaluation of nystagmus in the early stages of the disease was undertaken using asymptomatic carriers and non-carriers of ATXN3 mutation. The frequency of nystagmus in asymptomatic carriers (17%) and its absence in non-carriers of the mutation suggests that nystagmus may appear before gait disturbance and can thus be considered an early sign of MJD. A candidate gene approach was used in this work to pursuit modifiers of the MJD onset. The CAG repeat number in loci associated with polyQ disorders, namely spinocerebellar ataxias (SCA) types 1, 2, 6, 7, 17, Huntington disease (HD), and dentato-rubro-pallydoluysian atrophy (DRPLA) was investigated as modulators of the age at onset in the Azorean MJD cohort; a negative correlation was found between the longer allele at ATXN1 and disease onset, significantly increasing by 1.5% the explanation of onset variance. Furthermore, and because a role of inflammation in MJD has been postulated, promoter variants leading to alterations in cytokines expression were studied, and their impact in the disease onset was analysed. Patients carrying the IL6*C allele had a significant earlier onset. A complex net of mechanisms, which includes transcriptional regulation, ubiquitin-proteasome system, autophagy, apoptosis as well as mitochondrial function, has been implicated in MJD pathogenesis. Results from the transcriptome-wide gene expression analyses, followed by several steps of validation, showed that even in a peripheral tissue, transcription dysregulation was detectable and that FCGR3B, P2RY13 and SELPLG were dysregulated in patients. To further investigate dysregulated mRNA levels as a way to identify candidate biomarkers, the expression patterns of nine candidate genes – HSPB1, DNAJB1, DNAJB12, DNAJB14, BAX, BCL2, SOD2, IL1B and IL6 genes were quantified. Decreased levels of IL6 and BCL2 mRNA were observed in preataxic subjects. Lower HSPB1 and BCL2 mRNA levels after the ataxia onset were, furthermore, evidenced. A significant decrease of BCL2 mRNA levels during disease progression was observed in all patients used in the exploratory longitudinal study. Mitochondrial DNA (mtDNA) damage and depletion has been inconsistently reported in MJD. In this work, the mtDNA common deletion was significantly more frequent in patients and preataxic carriers than in controls. In conclusion, although the clinical and physiological phenotype of MJD reflects mainly neuronal damage, novel peripheral molecular alterations were described in the present dissertation. The confirmation of the link between peripheral abnormalities and neurodegeneration offers a new window of opportunity for biomarker discovery. More steps of validation will be crucial to develop a battery of molecular biomarkers able to describe disease progression, as well as to be used as outcome measures in future preventive and/or disease-modifying clinical trials. Data from molecular biomarkers should also be further correlated with imaging and clinical measures. Efforts to test the promising molecular biomarkers in independent cohorts, as well as to investigate their potential in longitudinal studies are warranted.