Meccanismi molecolari ed epigenetici implicati nella patogenesi delle distrofie miotoniche

Myotonic dystrophies type 1 (DM1) and type 2 (DM2) are autosomal dominant diseases respectively caused by expansion of [CTG]n triplet repeats in 3’ untranslated region (UTR) of dystrophia myotonica protein kinase gene (DMPK), and to the expansion of [CCTG]n tetranucleotide repeats in intron 1 of the zinc finger protein-9 gene (CNBP). In DM pathogenesis, untranslated CUG/CCUG-RNAs interfere with the normal activity of RNAbinding proteins, as MBNL1 and CUGBP1. These proteins are antagonists in the regulation of splicing during skeletal muscle and heart development and their alteration leads to spliceopathy in DM cells and tissues, as the skeletal muscle and the brain. First aim of this work was to verify the expression of known splicing markers of DM in blood peripheric mononuclear cells (PBMCs) of patients, in order to identify possible noninvasive biomarkers sensitive to the disease progression and useful for ongoing clinical trials. To this purpose, RT-PCR was performed to determine the expressions of INSR, BIN1, SOS1, MBNL1 and MBNL2 alternative transcript isoforms in lymphocytes from DM1, DM2 patients and controls. Moreover, relative mRNA transcript levels of MBNL1 and MBNL2 isoforms, containing or lacking exon7, have been evaluated by qPCR. In lymphocytes from DM patients, RT-PCR did not reveal splicing defects of the INSR, BIN1, and SOS1 genes compared to control samples. However, we found an aberrant expression of MBNL1 and MBNL2 ex7-containing isoforms in DM2 patients, not detected in both DM1 and control samples. qPCR analysis has confirmed this result for both transcripts with a statistically significant difference on the levels of isoforms of MBNL1ex7 and MBNL2ex7 transcripts between DM2 and control samples. Despite the variability of expression between DM2 samples, our data suggest that in DM2 patients could be active different mechanisms of splicing regulation respect to DM1 patients. Given that DM splicing biomarkers SOS1, BIN1 and INSR were absent in PBMCs, we researched new molecular biomarkers of diseases specific for the tissue analysed. RNA from lymphocytes of DM1, DM2 and healthy controls has been isolated to perform a whole genome expression analysis of alternative splicing by SurePrint G3 Human Exon Microarrays. EASANA bioinformatics analysis has been performed to detect exons differentially expressed between DM and control samples and to select only those with a statistically significant splicing index (fold-changes ≥2.0 and p-values ≤0.05). 2 Preliminary results showed a global deregulation in the expression levels of 421 and 2407 genes and of 786 exons and 2658 exons in DM1 and DM2 samples, respectively. These genes are involved in different pathways linked to the DM pathogenesis and its clinical phenotype: signalling in immune system, circadian rhythm, apoptosis and mTOR signalling. RT-PCR is currently in progress to validate the mis-regulated splice events identified by the microarray analysis as non-invasive biomarkers for DM diseases. Epigenetic modifications have been reported in many repeat expansion disorders, including DM1, either as a mechanism to explain somatic repeat instability or transcriptional alterations in disease genes. Based on these evidences, another purpose of our work was to determine the effect of DM2 mutation on the methylation status of CpG islands localized in the 5′ promoter region and in the 3′ end of the [CCTG]n expansion of the CNBP gene. By bisulphite pyrosequencing, we characterized the methylation profile of two different CpG islands within these regions, either in whole blood and skeletal muscle tissues of DM2 patients and controls. Moreover, we compared the relative mRNA transcript levels of CNBP gene in lymphocytes and in skeletal muscle tissues from controls and DM2 patients. We found that CpG sites located in the promoter region showed hypomethylation, whereas CpG sites at 3′ end of the CCTG array are hypermethylated. Statistical analyses did not demonstrate any significant differences in the methylation profile between DM2 patients and controls in both tissues analysed. According to the methylation analysis, CNBP gene expression levels are not significantly altered in DM2 patients. These results show that [CCTG]n repeat expansion, differently from the DM1 mutation, does not influence the methylation status of the CNBP gene and suggest that other molecular mechanisms are involved in the pathogenesis of DM2. Finally, considering the higher oxidative stress in DM1 patients, this work has focused on the response to DNA damage and on the repair systems to assess if these systems are implied in somatic CTG instability. After having characterized DM1 primary fibroblasts, we treated them and healthy control fibroblasts (WT) with different genotoxic agent: hydrogen peroxide (200 μM) and X-rays (2Gy). After treatment, we have evaluated several cellular and molecular parameters. Furthermore, X-rays treatment (2 Gy) allowed us to evaluate NHEJ and HR working by the analysis of breaks, fragments and translocations. After 24 hrs from treatment, the analysis of the karyotype of DM1 and WT cells allowed us to exclude a possible misregulation of both 3 HR and NHEJ. In fact, the increase of chromosome aberration was similar in DM1 and in WT fibroblasts for all the targets evaluated. After treatment with H2O2 (200 μM, 1 hr), the analysis of Abnormal Nuclear Morphologies (ANMs) showed a greater increase of all the markers analysed (MNs; NPBs and NBUDs) in DM1 fibroblasts respect to WT at 48 hrs (especially in DM1 cells) persisting up to 72 hrs. Analysis of BER and MMR genes expression levels before treatment with H2O2 has revealed their down-regulation in DM1 lymphocytes. However, transcriptional activation of these genes in the DM1 samples, after induction of H2O2 damage, did not show differences compared to the control samples, revealing that the two DNA repair systems function correctly in DM1 fibroblasts. Overall, the results of this study show a higher intrinsic level of genomic instability in DM1 cells, but also a greater increase in DNA damage induced by H2O2. A study of the mechanisms that correlate the oxidative stress response with the formation of the observed nuclear anomalies could clarify the role of these mechanisms in the pathogenesis of DM1.