Exome sequencing and transcriptomic analysis in jugular vein wall for detection of CCSVI biomarkers in Multiple Sclerosis

Chronic cerebrospinal venous insufficiency (CCSVI) is a vascular condition with impaired venous drainage from central nervous system, characterized by abnormal anatomy and flow in the neck veins. The described comorbidity with Multiple Sclerosis (MS) offers a unique model for the investigation of genes involved in vascular changes participating in the pathophysiology of MS, which could in turn reveal biomarkers of the disease. We used two experimental approaches, aimed at revealing genomic and transcriptomic components of the diseases. EXOME SEQUENCING: We selected candidate genes by systematically reviewing the literature of Genome Wide Association Studies (GWAS) performed on MS, which identified 96 loci including frequent intragenic SNPs (n=125) with p value ≤ 5X10⁻⁶, an arbitrary threshold potentially highlighting genes with remarkable disease association. Since we hypothesize that rare and functional mutations in candidate genes could contribute to MS/CCSVI, we applied Whole Exome Sequencing (WES) in families including 2-3 relatives affected by MS clinical phenotype with at least one member carrying also CCSVI. A WES-based pilot study in the 96 candidate MS susceptibility genes for SNPs with MAF≤0.04 was conducted in 11 subjects belonging to three families. These selection criteria revealed 13 exonic mutations (6 missense, 5 synonymous and 2 in UTR) and 26 intronic variants, part of which potentially affecting pre-mRNA splicing. Eight exonic variants were present only in affected members of the families. We are currently evaluating the detected SNPs by further Sanger sequencing in a cohort of MS patients and healthy controls to validate the spectrum of rare variants present in candidate genes for MS. These information could improve our knowledge of genetic components of the disease and provide, through expression studies, new hints on pathogenic mechanisms. TRANSCRIPTOMIC ANALYSIS: We aimed at characterizing the transcriptomic profile of vascular tissue from MS/CCSVI patients, an approach potentially revealing dysregulation of mRNA transcription/maturation/half-life, and thus able to suggest candidate genes for MS/CCSVI by independent experimental investigation. The RNA study was conducted using the Agilent platform 60K microarray on specimens of internal jugular vein wall from 4 patients with CCSVI/MS, who underwent surgical jugular angioplasty with autologous saphenous vein patch, and from 5 non-MS patients, who underwent endarterectomy for severe carotid stenosis. Overall, mRNA from 929 genes were found to be differentially expressed (411 up-regulated and 518 down-regulated). Among the 96 genes indicated by GWAS, only two displayed differential expression (p<0.05, two-fold change). At present we are conducting immunohistochemistry/proteomic approaches for validation of selected transcriptional changes in tissues/plasma from patients and controls. Genes with differential expression confirmed at the RNA and protein levels, are of potential interest for vascular aspects in MS, both as biomarkers and targets of mechanistic studies.