Projecto nº:016592

Referência do Projecto:PTDC/DTP-PIC/2638/2014 (POCI-01-0125-FEDER-016592)

Título:Whole-exome sequencing of discordant and concordant affected sib pairs in spinocerebellar ataxia type 3 (SCA3): a tool to identify novel modifier genes and highlight disrupted molecular pathways.

Montante envolvidos:

Investimento total: 129.366,00 €

IBMC-Instituto de Biologia Molecular e Celular - 66.738,00€

Apoio FEDER: 56.727,30€

Apoio OE: 10.010,70€

Fundação Gaspar Frutuoso, FP – 62.628,00€

Apoio FEDER: 0,00€

Apoio OE: 62.628,00€

Localização do projecto: Porto e Açores, Portugal

Sintese do projecto:

Spinocerebellar ataxia type 3 (SCA3/DMJ) (ORPHA98757) is a neurodegenerative polyglutamine (polyQ) disorder, which exhibits marked clinical heterogeneity, manifested, amongst other aspects, in the wide distribution of the age at onset. SCA3 is the worldwide most frequent spinocerebellar ataxia, being particularly prevalent in the Portuguese population, namely in the Azores Islands; it is considered a fatal disease and remains without pharmacological intervention, culminating in death, which occurs approximately 20 years after onset. At the ATXN3 gene, the size of the CAG tract only explains about 50% of the variation in age at onset, reinforcing the importance of studying genetic factors underlying the remaining variance. The identification of genetic modifiers would allow the increase of onset prediction, promoting further understanding of the biological pathways disrupted in SCA3 and potentially suggesting new therapeutic targets. Given the common features underlying polyQ disorders, this knowledge would,moreover, be potentially transferable to other neurodegenerative diseases of the same group. Previous attempts to identify modifier loci in SCA3 have used a candidate gene approach, which has proven to be extremely limited. In this project we will use Whole-Exome Sequencing (WES) to analyze 6 discordant and 6 concordant pairs of affected SCA3 sibs belonging to Azorean families; discordant pairs will be matched to concordant, by SCA3 family. Discordant pairs

includ-016592e sibs presenting the same size of the expanded allele, but displaying a difference in age at onset of a minimum of 5 years; concordant pairs include sibs presenting the same size of the expanded allele as well as the same age at onset (maximum difference of 1 year). Exonic sequences will be enriched using Nextera Rapid Capture Exome (Illumina) and sequenced in a HiSeq 2500 (Illumina), at an average depth of 100x. After the bioinformatic pipeline, WES data will be filtered to retrieve variants which alter or potentially impair protein function, using several functional predicting tools. The filtered variants will then be screened in sib pairs; variants that distinguish discordant but are not present in concordant sibs from the same family will be given priority. Screening in sib pairs will allow narrowing down the number of variants whose respective genes could behave as genetic modifiers of SCA3. A meticulous analysis of the pathways in which these different genes are involved will then be conducted to allow prioritization of variants to be used in the subsequent stage of the project. Furthermore, we will access data on the expression levels of such genes in peripheral blood (MESCA3, PTDC/DTP-PIC/0370/2012, PI-MLima) and cerebellum (http://www.braineac.org/) of SCA3 patients; genes found to be differentially expressed in patients will be given high priority. Validation of the strongest resulting candidate modifiers will be performed using four distinct series (Azores, Mainland Portugal, UK and Brazil), comprising 350 patients. In this group of patients, after sequencing the variants of interest,

we will investigate the presence of an association with disease onset. To understand the functional role of validated modifiers we will modulate the expression of the corresponding orthologues in the C. elegans model of SCA3, using an interference RNA (RNAi) based strategy. We believe that the chances of success of this project are based on the following strengths: a) the availability of discordant and concordant sib pairs from the same homogeneous Azorean SCA3 families, as well as a defined strategy to explore them as a suitable model to test the molecular basis of disease variability, using a WES; b) the availability of SCA3 subjects from four independente series, in which potential modifiers can be tested; c) the availability of a SCA3 C. elegans model, in which further functional verification of the validated modifiers can be performed; d) the extended experience of the research team concerning the investigation of the clinical variability of SCA3, using both human subjects and animal models.

Galeria de fotos do projeto