Multipl skleroz klinik alt tiplerinde moleküler yolakların ve biyobelirteçlerin araştırılması

Multiple sclerosis (MS) is a neuro-inflammatory and neuro-degenerative disease of the central nervous system (CNS), in which myelin sheaths and axons are damaged. Inflammatory demyelination and other pathological changes disrupt the ability of the nervous system to communicate resulting the wide variety of signs and symptoms. Heterogeneous clinical presentation and course ıf the disease, without an effective diagnostic laboratory test, make it difficult to draw firm conclusions about the disease's states, drug response and in general prognosis which may lead to more severe forms in some cases. The purpose of this study is to find out molecular biomarkers and pathways associated with the etiopathogenesis of the disease and its major subclinical forms. To this aim, proteomic, bioinformatics and genomic approaches were used to reveal differentially expressed proteins and their related pathways in cerebrospinal fluids of patients with clinically isolated syndrome (CIS), relapsing remitting MS (RRMS) and progressive MS (PPMS). Cerebrospinal fluid (CSF) samples of 65 CIS, 72 RRMS, 42 PPMS and 42 controls including healthy subjects and other neurological disease samples were analyzed. For all samples, 2D-PAGE analyses were performed. At least 2 times differently expressed protein spots were identified by PDQuest® software and removed from the gel for MALDI-TOF-MS protein identification analysis. The results of proteomic studies were further confirmed by both ELISA and western blot methods for selected candidate proteins. KEGG pathways analysis was performed with genes corresponding the proteins to identify disease relevant specific molecular pathways. Further linkage and genome-wide association study (GWAS) were performed by using single nucleotide polymorphism (SNP) genotyping on the Illumina CytoSNP 300K array in 36 individuals from 10 informative MS families having more than one affected member. Nonparametric linkage (NPL) scores were calculated for each of 3118 SNP markers spaced at an average of 1 cM intervals using SimWalk multipoint NPL analysis. Subsequent fine mapping of regions showing higher NPL scores was performed. GWAS analysis was performed by using 18 MS and 60 healthy subjects data by comparing the 298114 different genome wide SNPs. Comparison of disease groups with controls identified a total of 151 proteins that are differentially expressed in clinically different MS subtypes. KEGG analysis using PANOGA tool revealed disease related pathways including aldosterone-regulated sodium reabsorption (p=8.02x10-5) which is important in the immune cell migration, renin-angiotensin (p=6.88x10-5) system that induces Th17 dependent immunity, notch signaling (p=1.83x10-10) pathway indicating the activated remyelination and vitamin digestion and absorption pathways (p=1.73x10-5). Linkage study including 10 MS families showed suggestive evidence of linkage (NPL scores above 1.7) to chromosomal regions of 13q12.2-14.11 and 21q22.12-22.3. Fine mapping of these regions revealed that the most promising loci for linkage were mapped to 13q13.3 and 21q22.2, with NPL scores of 1.82 and 1.85, respectively. Detailed search of these regions revealed inflammation and neurodegeneration associated genes some of which previously implicated in MS and also promising novel genes yet to be identified like N4BP2L2 and TRPC4 were found within the suggestive regions. On the other hand, GWAS analysis revealed disease associated SNPs with significant associations on disease pathogenesis associated genes like INS-IGF2 (p=4.39x10-7 MAF = Case:Control = 0.36/0.03), MGMT (p=9.66x10-6 MAF = Case:Control = 0.32/0.04). Individual comparison of genomic and proteomic data revealed 9 different genes correlated with 14 different proteins, in 4 different molecular pathways that are associated with proteomic data. Those genes and proteins are the most prominent molecular parameters to understand disease mechanisms and needed to be further verified with various studies. To conclude, proteomic investigation of CSF samples belonging to different MS phenotypes indicated that all MS clinical forms share common biological pathways such as renin-angiotensin system (RAS) and complement and coagulation cascade (CCC) pathways. There are also clinical subtypes specific and pathophysiology related pathways, which may have further therapeutic implications. Furthermore, our study implicated importance of salt metabolism in MS pathogenesis for the first time. On the other hand, genomic results also showed disease pathogenesis associated genetic variations and chromosomal regions. Our suggestive results provide a framework for deep sequencing to identify new susceptibility genes and novel variants associated with risk of MS.