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The Human Genome Is Now Terminated and Available for All The Gene Expression Analysis and Genetic Studies in Multiple Sclerosis Author Tajouri, Lotfi Published 2005 Thesis Type Thesis (PhD Doctorate) School School of Health Sciences DOI https://doi.org/10.25904/1912/3873 Copyright Statement The author owns the copyright in this thesis, unless stated otherwise. Downloaded from http://hdl.handle.net/10072/366467 Griffith Research Online https://research-repository.griffith.edu.au GENE EXPRESSION ANALYSIS AND GENETIC STUDIES IN MULTIPLE SCLEROSIS By Lotfi Tajouri Bsc, Master of Cellular Physiology A Thesis submitted as a fulfilment for the degree of Doctor of Philosophy in the School of Health Science, Griffith University, Gold Coast, Queensland. AUSTRALIA. August 2004 Tajouri Page 1 1/11/2006 ABSTRACT Multiple Sclerosis (MS) is a neurodegenerative disease of the central nervous system (CNS). As part of this disorder the myelin sheath undergoes degeneration, leading to alterations in the conductivity of axons, and impaired function. The onset of the disease occurs in young adults and clinical pathology is characterised by varying severity. These include i) Relapsing Remitting MS (RR-MS), ii) Secondary Progressive MS (SP-MS) and iii) Primary Progressive MS (PP-MS). MS is more prevalent in women and accounts for more than two thirds of all MS sufferers. MS is considered to be a multifactorial disorder with both genetic and environmental components. The prevalence of MS is dependent on geographical localisation, with lower sunlight exposure linked to higher prevalence. Also, studies show an increased risk in close relatives, or in identical twins, indicating a significant genetic component to the disorder. There are a number of genes that may plausibly be involved in MS pathophysiology. These include myelin-related genes, such as the myelin basic protein (MBP), immune-related genes, such FCγ receptor and osteopontin, and heat shock proteins such as αβ crystallin. These candidate genes have been implicated in a variety of ways but usually through immunological and/or genetic studies. One of the most consistent findings in recent years has been the association of disease with alterations in the specific major histocompatibility complex (MHC) localised to chromosome 6p21.3, and includes MHC I, II, III. Tajouri Page 2 1/11/2006 Genome wide screens have permitted the identification of loci in the genome, which are associated with MS susceptibility. The number of genes involved in MS is unknown and several case-control association studies have been undertaken to reveal the involvement of potential candidate genes. In general terms, current research is aimed at determining allelic variation of candidate genes. Such genes have been implicated in MS because they reside within susceptible regions of the chromosome associated with MS or they have a plausible potential pathophysiological role in MS. Candidate loci investigated in this study, for association with MS susceptibility, include members of the nitric oxide synthase family of metabolic proteins (inducible NOS, iNOS/NOS2A and neuronal NOS, nNOS), methylenetetrahydrofolate reductase (MTHFR), catechol-O-methyl transferase (COMT), and vitamin D receptor (VDR). The MS population used in all studies consisted of over 100 MS cases and gender, age and ethnicity matched controls. In our study of inducible and neuronal NOS genes, PCR based assays were developed to amplify a region of both promoters that contained known microsatellite variation. Supporting phyisological data suggests that the neuroinflammatory aspects of MS are associated with aberrant NO production, which may be due to aberrant regulation of NOS activity. Specific amplified products were identified by fluorescent capillary electrophoresis and allele frequencies were statistically compared using chi-squared analysis. In the nNOS and iNOS study, no association was identified with allele frequency variation and MS susceptibility (nNOS: χ2=5.63, P=0.962; iNOS: χ2=3.4; P=0.082). Similarly, no differences in allele frequencies were observed for gender or clinical course for both markers (Pvalue>0.05). In short, results from this study indicate that the NOS Tajouri Page 3 1/11/2006 promoter variations studied do not play a significant role in determining susceptibility to MS in the tested population. The COMT and MTHFR genes are localised at 22q12-13 and 1p36.3 respectively, regions of the genome that have been found to be positively associated with MS susceptibility. In th our research, we set out to examine the G158A change in the 4 exon of the COMT gene. This functional mutation leads to an amino acid change (valine to methionine) that is directly associated with changes in the activity of COMT. The MTHFR enzyme plays a role in folate metabolism, and can be implicated in the turnover of homocysteine. Previous investigations have shown that high levels of homocysteine are encountered in MS patients, where it is also linked to demyelination in the CNS. In our study the aim was to examine the C677T variation (alanine to valine amino acid change) in the exon 4 coding region of the MTHFR gene and the G158A variation in the COMT gene. Restriction fragment length polymorphism (RFLP) analysis and gel electrophoresis was used to identify specific alleles for both COMT and MTHFR. However, as with the NOS study, no specific association was identified between MS susceptibility and variation for either of the tested COMT or MTHFR (Pvalue>0.05) variants. In a final genomic investigation of the MS population, three variations in the VDR gene were analysed for association with MS susceptibility and pathology. Using RFLP analysis, three VDR variants were investigated with genotypes detected using the Taq I, Apa I and Fok I restriction enzymes. In contrast to previous genotypic analyses, this study did show a positive association, specifically between the functional variation in exon 9 of the VDR gene and MS (Taq I, χ2= 7.22, P= 0.0072). Interestingly, the Apa I variant of VDR was Tajouri Page 4 1/11/2006 also found to be associated with MS (χ2=4.2, P=0.04). The Taq I and Apa I variants were also found to be in very strong and significant linkage disequilibrium (D’=0.96, Pvalue < 0.0001) and their associations were more prominent with the progressive forms of MS (SP– MS and PP–MS). In addition to genotypic analysis of a clinical population, additional research was undertaken to identify novel targets for MS susceptibility studies. Global gene expression analysis was undertaken using comparative subtractive fluorescent microarray technology to examine differences in gene activity (expression) in age and sex matched MS plaque tissue and anatomically matched normal white matter (NWM). MS plaques were obtained post mortem from MS sufferers with no drug history in the last two months before death and matched anatomically to healthy white matter from donors with no previous neurological disorders. Target arrays consisted of 5000 cDNAs and analysis was conducted using the Affymetrix 428 scanner. In this way, 139 genes were shown to be differentially regulated in MS plaque tissue compared to NWM. Of these, 69 genes showed a common pattern of expression in the chronic active and acute plaque tissues investigated (Pvalue<0.0001, ρ=0.73); while 70 transcripts were uniquely differentially expressed (≥1.5- fold) in either acute or chronic active lesions. To validate the gene expression profile results, quantitative real time reverse transcriptase (RT) PCR (Q-PCR) analysis was performed. 12 genes were selected because they were shown to be differentially expressed by array analysis in this study, or because of their involvement in MS pathology. These included transferrin (TF), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), glutathione S-transferase pi (GSTP1), crystallin, Tajouri Page 5 1/11/2006 alpha-B (CRYAB), phosphomannomutase 1 (PMM1), tubulin beta-5 (TBB5), inositol 1,4,5- trisphosphate 3-kinase B (ITPKB), calpain 1 (CAPNS1), osteopontin (SPP1 or OPN), as well as the signal transducer and activator of transcription 1 (STAT1) and protein inhibitor of activated STAT1 (PIAS1). Both absolute (copy number) and comparative differences in the relative levels of expression in MS lesions and NWM were determined for each gene. The results from this study revealed a significant correlation of real time PCR results with the microarray data, while a significant correlation was also found between comparative and absolute determinations of fold. As with the results of array analysis, a significant difference in gene expression patterning was identified between chronic active and acute plaque pathologies. For example, a >50-fold increase in SPP1 and ITPKB levels in acute plaques contrasted with the 5-fold or less increase in chronic active plaques (P<0.0.1, unpaired t-Test). Of particular note, gamma-amino butyric acid receptor γ2 (GABG2), integrin β5 (ITGB5), complement component 4B (C4B), parathyroid hormone receptor 1 (PTHR1) were found up-regulated in MS and glial derived neurotropic factor α2 (GDNFA2), insulin receptor (INSR), thyroid hormone receptor ZAKI4 (ZAKI4) were found down-regulated in MS. Data also revealed a decreased expression of the immune related genes STAT1 and PIAS1 in acute plaques. In conclusion, this research used both genomic analysis and technologies in gene expression to investigate both known and novel markers of MS pathology and susceptibility. The study developed tools that may be used for further investigation of clinical pathology in MS and have provided interesting initial expression
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