DNA methylation of TOMM40-APOE locus and Alzheimer’s disease pathology in the prefrontal cortex

Bezuch, Natalia Ewa (2018) DNA methylation of TOMM40-APOE locus and Alzheimer’s disease pathology in the prefrontal cortex. Masters by Research thesis (MSc), Manchester Metropolitan University.

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Abstract

Introduction: Apolipoprotein E (APOE) encoded by APOE gene is an important lipid transport molecule in the central nervous system. Carrying at least one APOE4 allele is the strongest known genetic risk factor for the sporadic Alzheimer’s Disease (AD). APOE isoforms can differentially influence the AD pathogenesis by affecting the synthesis and clearance of the Aβ as well as affecting the neurons independently of the Aβ, for instance by disrupting the mitochondria or cytoskeleton and leading to neurodegeneration and defective repair. TOMM40 is another gene that have been implicated in AD. Due to its close proximity to the APOE gene, it is thought to be in linkage disequilibrium with the APOE gene and therefore influence its transcription. APOE genotype could also have an effect on the methylation levels, as the 2 SNPs that account for the 3 APOE isoforms, either disrupt or create a CpG site- Rs7412 (C->T) removes a CpG site, whereas rs429358 (T->C) creates an extra CpG site. Previous studies suggested that methylation of the TOMM40-APOE locus may influence the APOE protein levels and thus associate with AD pathology. However, it is not fully understood how these genes are transcriptionally regulated. Aims: The main aim of this study was to investigate APOE methylation levels and protein expression in a number of aged (n=32) and AD (n=30) human brains and to test if the gene methylation and protein levels associate with the AD pathology. Methods: APOE protein levels were estimated using an ELISA. Genotyping was performed using Sequenom MassARRAY iPLEX platform. DNA methylation levels were measured using bisulphite pyrosequencing. Prefrontal cortex samples were stained with rabbit APOE polyclonal antibody and co-stained with mouse Glial Fibrillary Acidic Protein (GFAP) antibody (astrocyte marker) and visualised under fluorescent microscope. Results: As expected, the number of APOE4 carriers was significantly higher in the AD group. APOE genotype did not have an effect on the TOMM40-APOE locus methylation, nor on the APOE protein expression. APOE protein levels were not significantly associated with AD pathology. TOMM40 CpG2 negatively correlated with APOE protein levels (r=-0.270, P=0.042), however the association was no longer significant after adjustment for confounding factors. Methylation of the remaining CpG sites did not correlate with APOE protein expression. TOMM40-APOE locus methylation levels did not significantly associate with AD pathology. APOE protein levels positively correlated with longitudinal fluid intelligence, which in turn were negatively associated with Thal score. Conclusion: TOMM40-APOE locus methylation may not be affected by the genotype and the methylation levels do not have a direct effect on the AD pathology. APOE protein levels may have a domain-specific effect in the prefrontal cortex affecting longitudinal fluid intelligence. Further research, preferably in a larger sample group, is necessary to investigate the role of the TOMM40-APOE locus methylation in the AD pathology.