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Unveiling the role of methylation prone (MP) and untranslated (UTRs) regions in the clinical variability of Spinocerebellar Ataxia Type 3(SCA3) /Machado-Joseph Disease (MJD): a genome-wide approach
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Abstract(s)
A Ataxia espinocerebelosa do tipo 3 (SCA3), também conhecida como doença de Machado-Joseph (MJD), é uma doença neurodegenerativa rara de manifestação tardia e pertencente ao grupo das doenças de poliglutamina (poliQ). O gene causal desta doença, ATXN3, apresenta como mutação causal a expansão do motivo repetitivo CAG. Este gene codifica a proteína ATXN3, uma proteína ubíqua que pode estar envolvida nos mecanismos de transcrição.
As regiões não traduzidas (UTRs), 5’ e 3’ UTRs, juntamente com elementos cis-acting (tais como os promotores dos genes), são essenciais para a regulação da expressão génica. Além disso, também a metilação do ADN tem um papel importante na referida regulação.
O principal objetivo desta tese de doutoramento foi analisar o impacto das regiões não traduzidas e das regiões propensas à metilação, na variabilidade clínica da SCA3. Para este efeito, estudámos a variação encontrada em 3'UTR do gene ATXN3 e analisámos o impacto destas variantes na variação da idade de início da doença nos doentes com SCA3.
Reportámos nove variantes presentes em 3'UTR do gene ATXN3 que podem ter um papel na sua própria regulação génica. Na coorte Açoriana, composta por doentes com SCA3, nenhuma das variantes que foram identificadas apresentou um efeito modulador significativo na variação da idade de início da doença, e não fomos capazes de replicar os resultados para a variante rs709930 (e rs910369) obtidos por outro grupo de investigadores. Neste estudo, conseguimos identificar haplótipos associados à doença e que podem fazer a discriminação dos alelos expandidos; este facto pode ser relevante para o desenvolvimento de novas estratégias de allele-specific silencing.
Estudámos também a variação encontrada nas UTRs de diferentes genes que foi obtida através da sequenciação alargada do exoma e efetuámos correlações genótipo- fenótipo para avaliar o impacto de tais variantes/genes na variação da idade de início dos doentes com SCA3. Neste estudo identificámos quatro genes candidatos a modificadores da variação da idade de início da doença: GLI4, TGFA, ERBB4 e MPHOSPH9. Os alelos alternativos das variantes no gene GLI4, apresentaram uma modificação prevista nos locais de ligação dos factores de transcrição e nos factores de transcrição, que se ligam a esses locais; este gene apresentou ainda uma variação na idade de início da SCA3 de três anos (a idade de início é mais tardia). Os restantes genes modificadores (TGFA, ERBBB4 e MPHOSPH9) identificados neste estudo, apresentam variantes em 3’UTRs, são conhecidos como interatores do gene ATXN3, e influenciam significativamente a variação na idade de início da doença. Também conseguimos obter oito pathways estatisticamente enriquecidas, duas delas contendo genes com efeito modificador (TGFA e ERBBB4): HIF1α Signaling e Neuregulin Signaling. Estas pathways podem ser importantes para outras aplicações terapêuticas nesta doença.
Por fim, efetuámos um estudo piloto de metilação genome-wide. Tanto quanto nos é possível saber, este é o primeiro estudo que investiga as alterações de metilação do ADN em doentes com SCA3 e em controlos emparelhados. Como o tamanho da amostra foi pequeno, não esperávamos a ocorrência de CpGs diferencialmente metiladas, entre doentes e controlos. A análise de enriquecimento da lista de 1279 genes que obtivemos, mostrou um enriquecimento para pathways envolvidas em doenças neurodegenerativas, tais como a Calcium signaling pathway. Também realizámos uma análise com epigenetic clocks (Hannum e Horvath) para doentes com SCA3 e controlos emparelhados. Não conseguimos encontrar diferenças entre doentes e controlos para a idade de metilação do ADN, para ambos epigenetic clocks . No caso da diferença da idade de metilação do ADN e a diferença da idade de metilação residual do ADN, também não fomos capazes de encontrar diferenças entre os dois grupos para ambos os epigenetic clocks. Estes resultados podem ser explicados pelo tamanho da amostra e pelo poder limitado deste estudo.
Esta tese de doutoramento tentou contribuir para a descoberta do papel das regiões não traduzidas e às regiões propensas à metilação na variabilidade clínica da SCA3. Para este efeito, obtivemos: a) variantes com impacto previsto em 3'UTR do gene ATXN3; b) genes candidatos a modificadores, com variantes em UTRs; c) nenhum resultado significativo para o primeiro estudo a nível do genoma sobre a metilação do ADN, comparando doentes e controlos; serão necessários mais estudos, num número maior de doentes e controlos para obter um perfil de metilação para a SCA3.
ABSTRACT: Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD) is a rare late-onset neurodegenerative disorder, which belongs to the polyglutamine (polyQ) group of diseases. The causal gene of this disease, ATXN3, presents CAG repeat expansion as the causative mutation. This gene encodes the protein ATXN3 an ubiquitous protein that can be involved in transcription mechanisms. Untranslated regions (UTRs), 5’ and 3’ UTRs, along with cis-acting elements (such as gene promoters), are essential for the regulation of gene expression. Also, DNA methylation has an important role in such regulation. The main goal of this thesis was to analyse the impact of untranslated and methylation prone regions in the clinical variability of SCA3. For this purpose, we studied the variation found in 3’UTR of ATXN3 and analysed the impact of these variants in the variance of the onset of SCA3 patients. We reported nine variants present in the 3’UTR of ATXN3 that can have a role in its own expression regulation. In the Azorean SCA3 cohort studied, none of the variants that were identified presented a significant modulatory effect on the disease onset and we were not able to replicate results for rs709930 (and rs910369) obtained by other group of researchers. In this study, we were able to identify disease-associated haplotypes that can discriminate expanded allele; this fact can be relevant for the design of new allele-specific silencing strategies. We also studied the variation found in the UTRs of different genes that was obtained through an extended exome sequencing and performed genotype-phenotype correlations to evaluate the impact of such variants/genes in the onset of SCA3 patients. In this study we identified four candidate modifier genes of the disease onset: GLI4, TGFA, ERBB4 and MPHOSPH9. The alternative alleles of the variants in GLI4, presented a predicted modification in transcription factors binding sites and in transcription factors, that bind to those sites; this gene showed a delay of three years in SCA3 onset. The remaining modifier genes (TGFA, ERBB4 and MPHOSPH9) identified in this study, are known ATXN3 interactors, and significantly influence the onset of the disease. These genes presented variants in the 3’UTRs. We were also able to obtain eight statistically enriched pathways, two of them containing genes with a modifier effect on the onset (TGFA and ERBB4): HIF1α Signaling and Neuregulin Signaling. These pathways can be important for further therapeutic applications in this disease. Finally, we performed an epigenome-wide pilot study. As far as we are aware, is the first study investigating genome-wide DNA methylation changes in SCA3 patients compared to matched controls. As the sample size was small, we were not expecting genome-wide significant differentially methylated CpG sites, between patients and controls. Enrichment analysis of the obtained list of 1279 genes showed an enrichment for pathways involved in neurodegenerative diseases such as Calcium signaling pathway. We also performed an analysis of epigenetic clocks (Hannum and Horvath) for SCA3 patients and matched healthy controls. We were not able to find differences between patients and controls for DNA methylation age for both epigenetic clocks. For DNA methylation age acceleration difference and DNA methylation age acceleration difference residuals we were not able to find differences between the two groups for both clocks. These results can be explained by sample size and the limited power of this study. This PhD thesis tried to contribute to the unveiling the role of untranslated and methylation prone regions in the clinical variability of SCA3. For this purpose, we obtained: a) variants with predicted impact in 3’UTR of ATXN3 gene; b) candidate modifier genes, with variants in UTRs; c) no significant results for the first genome-wide study on DNA methylation comparing patients and controls; further studies in a higher number of patients and controls are required to obtain a methylation profile for SCA3.
ABSTRACT: Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD) is a rare late-onset neurodegenerative disorder, which belongs to the polyglutamine (polyQ) group of diseases. The causal gene of this disease, ATXN3, presents CAG repeat expansion as the causative mutation. This gene encodes the protein ATXN3 an ubiquitous protein that can be involved in transcription mechanisms. Untranslated regions (UTRs), 5’ and 3’ UTRs, along with cis-acting elements (such as gene promoters), are essential for the regulation of gene expression. Also, DNA methylation has an important role in such regulation. The main goal of this thesis was to analyse the impact of untranslated and methylation prone regions in the clinical variability of SCA3. For this purpose, we studied the variation found in 3’UTR of ATXN3 and analysed the impact of these variants in the variance of the onset of SCA3 patients. We reported nine variants present in the 3’UTR of ATXN3 that can have a role in its own expression regulation. In the Azorean SCA3 cohort studied, none of the variants that were identified presented a significant modulatory effect on the disease onset and we were not able to replicate results for rs709930 (and rs910369) obtained by other group of researchers. In this study, we were able to identify disease-associated haplotypes that can discriminate expanded allele; this fact can be relevant for the design of new allele-specific silencing strategies. We also studied the variation found in the UTRs of different genes that was obtained through an extended exome sequencing and performed genotype-phenotype correlations to evaluate the impact of such variants/genes in the onset of SCA3 patients. In this study we identified four candidate modifier genes of the disease onset: GLI4, TGFA, ERBB4 and MPHOSPH9. The alternative alleles of the variants in GLI4, presented a predicted modification in transcription factors binding sites and in transcription factors, that bind to those sites; this gene showed a delay of three years in SCA3 onset. The remaining modifier genes (TGFA, ERBB4 and MPHOSPH9) identified in this study, are known ATXN3 interactors, and significantly influence the onset of the disease. These genes presented variants in the 3’UTRs. We were also able to obtain eight statistically enriched pathways, two of them containing genes with a modifier effect on the onset (TGFA and ERBB4): HIF1α Signaling and Neuregulin Signaling. These pathways can be important for further therapeutic applications in this disease. Finally, we performed an epigenome-wide pilot study. As far as we are aware, is the first study investigating genome-wide DNA methylation changes in SCA3 patients compared to matched controls. As the sample size was small, we were not expecting genome-wide significant differentially methylated CpG sites, between patients and controls. Enrichment analysis of the obtained list of 1279 genes showed an enrichment for pathways involved in neurodegenerative diseases such as Calcium signaling pathway. We also performed an analysis of epigenetic clocks (Hannum and Horvath) for SCA3 patients and matched healthy controls. We were not able to find differences between patients and controls for DNA methylation age for both epigenetic clocks. For DNA methylation age acceleration difference and DNA methylation age acceleration difference residuals we were not able to find differences between the two groups for both clocks. These results can be explained by sample size and the limited power of this study. This PhD thesis tried to contribute to the unveiling the role of untranslated and methylation prone regions in the clinical variability of SCA3. For this purpose, we obtained: a) variants with predicted impact in 3’UTR of ATXN3 gene; b) candidate modifier genes, with variants in UTRs; c) no significant results for the first genome-wide study on DNA methylation comparing patients and controls; further studies in a higher number of patients and controls are required to obtain a methylation profile for SCA3.
Description
Tese de Doutoramento, Biologia, 22 de junho de 2023, Universidade dos Açores.
Keywords
Machado-Joseph Disease Ataxia Espinocerebelosa
Citation
Melo, Ana Rosa Vieira. (2022). "Unveiling the role of methylation prone (MP) and untranslated (UTRs) regions in the clinical variability of Spinocerebellar Ataxia Type 3(SCA3) /Machado-Joseph Disease (MJD): a genome-wide approach". 153 p. (Tese de Doutoramento em Ciências Biológicas). Ponta Delgada: Universidade dos Açores, 2022. Disponível em http://hdl.handle.net/10400.3/7010