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Genetic variability of vitamin D receptors in autoimmune diseases
Serbia (IFMSA-Serbia) - University of Nis, Nis
University of Nis, Medical faculty Research Center for Biomedicine Laboratory for functional genomics and proteomics Bul. dr Zorana Djindjica 81 Niš, Serbia
Prof. dr Dobrila Stankovic Đorđevic
Prof. dr Tatjana Jevtovic Stoimenov
Type of Research Project
- Clinical Project with Laboratory work
What is the background of the project?
Biological effects of the vitamin D active form (1,25- dihydroxycholecalciferol;1,25(OH)2D3) are exerted by its binding to the vitamin D receptor (VDR). VDR is expressed in tissues and cells that are not involved in calcium and phosphorus metabolsm, including the airways, as well as the immune cells (mast cells, macrophages, dendritic, natural killer, T, and B cells). In humans, VDR is coded by the VDR gene located at the position 12q13-14. Numbers of single nucleotide polymorphisms (SNPs) are identified in the VDR gen but among the most studied are SNPs located in exon 2 (rs2228570) intron 8 (rs1544410 and rs7975232), and exon 9 (rs731236), named by the restriction enzymes used for their detectio. Even the VDR SNPs are previously studied in autoimmune diseases patients of different populations, obtained results still remain contradictory and show differences between the studied populations. Moreover, to the best of our knowledge VDR SNPs where not studied in Serbian patients with multiple sclerosis, systemic lupus, rheumatoid arthritis, and inflammatory bowel diseases.
What is the aim of the project?
Main goals of the project are: I. understanding the pathogenesis of autoimmune diseases and mechanisms of vitamin D receptors variability under different epigenetic factors, nutrient-gene interactions and molecular classification, II. studying the correlations of genotype and phenotype vitamin D receptors gene in patients with autoimmune diseases (SLE, Diabetes Mellitus 1, multiple sclerosis andrheumatoid arthritis) III. identification of the risk groups among the patients with autoimmune diseases and malignancies with genetic variability of VDR.
What techniques and methods are used?
VDR genotyping by Polymerase Chain Reaction - Restriction Fragment Lenth Polymorphism (CR- RFLP)technique Blood samples and DNA preparation Blood samples is going to be collected in the vacutainers with ethylenediaminetetraaceticacid (EDTA) as an anticoagulant during theroutine laboratory testing by the cubital vein punction. DNA will be isolated from 200 ml of blood according to the protocol of QIAampDNA Blood Mini Kit (Quiagen GmbH, Hilden, Germany). PCR - RFLP is going to beused for the determination of VDR genevariability. Using KAPA2GFast HotStart ReadyMix (Kapa Biosystems Inc., Wilmington, MA. After the verification of PCR products restriction digestion will be carried out using FastDigest restriction enzymes (FermentasFmbH, St. Leon-Rot, Germany).Restriction fragmentsare going to be analyzed by 8% and 6% polyacrylamide gel Research plan: Phase 1: Initial first draft Activity 1: to get notification or authorization of this project from the ethic committee Activity 2: to collect blood samples from patients with rheumatoid arthritis, systemic lupus erhytematodes, ulcerative colitis, Crohn`s Activity 3: Workshop I:Distribution of investigated VDR gene polymorphism in patients with autoimmune disease. Phase 2: Standardization of PCR-RFLP Activity 1: genotyping VDR gene locus from obtained material by PCR-RFLP method in all groups of patients. Activity 2: Analyzing the obtained results Phase 3: Finalizations Activity 1: Statistic analyses of the results Activity 2: Publication of the obtained results Activity 3: Dissemination and reporting the results of proposed project: Workshop II
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks of the student will be performed on his/her own
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
understanding the pathogenesis of autoimmune diseases -molecular variability of VDR gene -primer designing -skills: isolation of DNA from blood; determination the concentration of DNA; amplification of target DNA; detection of gene polymorphism; -statistical analysis of obtained results; working in SPSS package program 20; Hi square test, Odds ratio, confidence interval
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Firs week will be organized to prepare the student for practical laboratory work by literature readings and by observation.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- No specific outcome is expected
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
The skills required from the students are: knowledge from biochemistry, good laboratory practise
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Students in biomedical fields
- Despotovic Milena R;Jevtovic-Stoimenov Tatjana M;Stankovic Ivana J;Basic Jelena T;Pavlovic Dusica D (2017) Vitamin D Receptor Gene Polymorphisms in Serbian Patients With Bronchial Asthma: A Case- Control Study; JOURNAL OF CELLULAR BIOCHEMISTRY; vol. 118; br. 11; str. 3986-3992 (Article)
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