Polymorphisms Of DBP And Proteome Analysis In Multiple Sclerosis

Multiple Sclerosis(MS) is a chronic inflammatory disease that affects the central nervous system consisting of the brain and spinal cord. In this disease the nervous system is affected by body's immune response that results in demyelination, where the sheath of neurons of central nervous system is damaged, causing further damage to the underlying nerve fiber. These are the very nerve fibers that help communicate in between the brain and all other parts of the body. Depending on the amount of nerve damage, communication between the brain and other parts of the body is either diminished or totally missing.A variety of reasons are related to the occurrence of MS, such as genetic factor, geographical factor and viral infection. It was shown that the concentration decrease of Vitamin D in body are intimate related to MS. Vitamin D binding protein (DBP) is a multifunctional and highly polymorphic plasma protein with many important functions. These include transport of vitamin D metabolites, control of bone development, binding offatty acids, sequestration of actin and a roles in modulating immune and inflammatory responses. Since MS is postulated to be a T cell-mediated autoimmune disease, the concentration of DBP may associate to the occurrence of MS.It has been discovered that DBP polymorphisms in exon 11 [Asp (GAT)/Glu (GAG) at codon 416, Thr (ACG)/Lys (AAG) at codon 420] could be determined by PCR-restriction fragment length polymorphism. However, while the association between MS and DBP polymorphisms was explored, the results of the study were still controversial in differ race people.Proteomics is the study of separating and identifying the entire protein components (proteome) of a cell, tissue or fluid at different time points, to compare the protein alterations of the disease. Two-dimensional electrophoresis is the best technology to separate complex protein compound, which is combined by isoelectrofocusing electrophoresis and sodium dodecyl sulfate polyacrylamide gel electropheresis. Mass spectrum is an important technique to identify proteins by means of determining molecular weight measuring charge-to-mass ratio of charged atoms, molecules or molecular fragments.In this study, we investigated the association of two polymorphisms at the restriction site of Sty I and Haeâ…¢in the DBP gene between MS patients and the health in Han pepple of Shandong province; and the plasma protein alterations of MS patients by means of the proteomic analysis with two-dimensional electrophoresis (2-DE), followed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), to explore the pathogenesy, diagnosis, prognosis and treatment on the molecular level of MS.Objective:To investigate the association of two polymorphisms (codon 416 and codon 420) in the DBP gene and multiple sclerosis(MS) in Han people of Shandong province, and the relations between the differential expression proteins in the plasma of the patients and the disease. These results may be give information for studying the pathophysiological process, the clinical diagnosis and treatment of the diseases.Methods:The case-control study involving 46 patients with clinical determined multiple sclerosis (MS) and 43 healthy controls. DBP polymorphisms in exon 11 at codon 416 and 420 were determined by PCR-restriction fragment length polymorphism. DNA was extracted from blood.A region of exon 11 was specifically amplified by PCR and the PCR product was digested by Haeâ…¢and Styâ… . Digested DNA fragments were electrophoresed by agarose gel and visualized by ultraviolet light.Venous blood were extracted from patients with MS and the controls respectively, and the proteins were separated by the two-dimensional electrophoresis (2-DE) and the differential expression protein spots were selected with the ImageMaster 2D-gel software. The peptides mass fingerprint (PMF) was got using the matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) to identify these differential proteins by SWISSPROT database. The differential expression protein were selected to be analized the relationship between MS.Results:The frequencies of DBP genotypes at codons 416 and 420 between the MS patients and the control group were not significantly different (p>0.05; p>0.05); phenotypes frequencies of Gc were no significant difference (P> 0.05).2-DE maps of plasma with MS and the controls group were got. There were seven protein spots expressed differentially on the 2-DE maps, in which five up-regulated such as DBP, ApoE,HPT,C4b,ACTB etc. and two down-regulated such as ApoA and RRBP were identified in MS.Conclusions:None of the polymorphisms at 416 site and 420 site of DBP gene showed any association with the occurrence of MS in Han people of Shandong province. Expressions of DBP in MS group increased relativing to that in the controls.The differential expressions researches of the proteins were assistant to explore the pathogenesis, differential diagnosis and drug targets of MS on molecular level.