Mammary areas were stained with hematoxylin and eosin and martius yellowsoluble bluebrilliant crystal

The quantification of CpG sites’ methylation levels for all amplicons was performed using Epigenetic Sequencing Methylation analysis software. The software was repeatedly used to determine the methylation profile of several genes,,. The software algorithm allows analyzing the methylation percentage of each CpG site in amplicon without a cloning stage. However during the analysis CpG sites with bad quality of sequences, especially at the 30 and 50 ends of amplicons, were excluded. The exact number of SMA patients DNA samples analyzed for each CpG site of certain gene is presented below. We included the methylation level of controls determined with the Infinium HumanMethylation450 BeadChip from our previous study. However, the obtained data were not expected to be precisely matching our previous data, as methylation data generated by two different techniques might have discrepancy in values,. Comparison between type III-IV and type I SMA male patients demonstrated a 16% decrease in methylation levels of the CpG4 target site belonging to the 5’UTR of NCOR2. This is consistent with the data of whole genome methylation analysis that identified lower methylation levels of the CpG4 target site in healthy individuals. It might be concluded that the methylation level of these sites in type III-IV SMA patients is rather similar to the methylation level of healthy individuals. Our results are also in good concordance with ENCODE project data showing hypermethylation of the CpG target site in all cell types. Interestingly, these CpG sites overlap with signals for DNAse I hypersensitivity and histone modifications, thus indicating an active regulatory region. We did not reveal any significant difference in the expression level of NCOR2 between SMA patients of various types. This could be explained by small cohort size and unequal amounts of samples from type I and type III-IV SMA patients. Another explanation could be that NCOR2 expression levels might be regulated independently of regulatory regions DNA methylation, involving other epigenetic mechanisms. Such microRNAs can influence NCOR2 expression regulation. NCOR2 and CDK2AP1 genes play an important role in transcription regulation. NCOR2 encodes a SMRT protein. SMRT, together with the NCoR1 protein, forms a core of multisubunit complexes that contain one of three different classes of histone deacetylases and consequently repress transcription of different genes. It is interesting to note that the SMRT-NCoR complex is connected with HDAC1 and HDAC2 through mSin3A/B co-repressors. It was demonstrated that mSin3A is associated with the SMN protein. The CDK2AP1 gene product, CDK2AP1 protein, is a subunit of the NuRD complex, contained the histone deacetylases HDAC1, HDAC2 and the methyl-CpG-binding domain proteins MBD2 or MBD3. In the light of SMA interactions between SMRT and CDK2AP1 with histone deacetylases seems to be interesting, taking in the account that the main agents tested for SMA company website therapy are histone deacetylase inhibitors,. The region upstream of the RPL9 TSS was strongly hypomethylated in all SMA patient groups, although no difference was found in methylation level among different SMA types. This correlates with whole genome methylation analysis data showing a highly decreased methylation level of target CpG site in SMA patients comparing to healthy individuals. RPL9 DNA methylation alteration may be related to changes in different directions in the level of other ribosomal proteins which were described in SMA mice. Alzheimer’s disease was also characterized by alterations in ribosomal proteins, rRNAs, and ribosomes, along with decreases in some protein factors stabilizing methylation. Our finding of highly decreased methylation levels in RPL9 could be additional evidence of significant methylation disturbances attending neurodegenerative disorders. We did not observe significant differences in the methylation levels of CpG sites close to the TSS of the CHML gene among SMA patients. However a trend towards to an increase in methylation levels connected with mild SMA severity was observed. This is in accordance with our previous data on the whole genome methylation analysis showed significantly hypermethylated level for controls compared to SMA patients. Whole genome methylation analysis showed that, CpG site located within the 3’UTR region of ARHGAP22 was hypomethylated in healthy controls compared to intermediate methylation for SMA patients. Highly variable methylation levels of CpG sites for all analyzed groups in this study suggest different expression levels in SMA patients, but this is not obviously correlated with SMA severity.

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