Ekrem Cengiz Seyhan, Sedat Altın, Erdoğan Çetinkaya, Atayla Gençolu , Sinem Timur

1. VascularEndothelialGrowthFactor and TumorNecrosisFactor Gene Polymorphisms in Turkish PatientsWithSarcoidosis Ekrem Cengiz Seyhan, Sedat Altın, Erdoğan Çetinkaya, Atayla Gençolu, Sinem Timur Yedikule Göğüs Hastalıkları ve Göğüs Cerrahisi Eğitim ve Araştırma Hastanesi

2. Introduction • Sarcoidosis is a complex systemic granulomatous disease with multiorgan involvement which is thought to be product of genetic susceptability and an unknown antigenic stimulus from the environment.

3. Introduction • Existing familiar sarcoidosis cases, some relations between HLA and disease, different clinical presentations in different races shows existance of some predisposition genes in sarcoidosis • Particular gene polymorphisms were reported recently to play role in increased or decreased susceptability to sarcoidosis and also in the severity and progress of the disease.

5. 2020 Targets / Mapping Susceptability Genes for Diseases (Case/Control SNP Maps , making HAP-MAP) Cancer(Lung Ca - 17 gene-35 SNP) Hipertension/Atherosclerosis ( Stroke-14 gene-28 SNP) Diabetes(46 gene-117 SNP) Astma( 13 gene-44 SNP) Alzheimer/Schizophrenia(172 gene-380 SNP)

6. ACE(Angiotensin converting enzyme) CCR2 (C-c chemokine receptor) CFTR (Cystic Fibrosis Transmembrane Regulator) HLA-B HLA-C HLA-DPB1 IL-1 (Interleukin 1) NRAMP (Natural resistance associated macrophage protein) TAP 2 (Transporter associated with antigen processing 2) TNF - α TNF - β VDR (Vitamin D receptor)

7. Introduction • Main defining pathological feature of sarcoidosis is chronic inflammation resulting in nonnecrotizing epitheloid granuloma formation produced by center of epithelioid and multinuclear inflamatory cells surrounded by mononuclear inflamatory cells and fibroblasts. • Granulomatous inflammation in sarcoidosis are regulated by complex relations between T cells, mononuclear phagocytes, fibroblasts, B cells and dendritic cells.

8. Relation between these cells are regulated by cytokines and direct contact between cells. There is growing evidence for the contribution of genetic polymorphisms to interindividual differences in the regulatory mechanisms of cytokine production.

11. (Physiological SNP-Variation)Phenotipical variety (Functional SNP)Susceptability to diseasesPharmacogenetic (Mutation)Disease

12. Tumor necrosis factor alpha (TNF-α) is secreted from activated macrophages and thought to be a principal cytokine in the pathogenesis of sarcoidosis due to its privotal role in the granuloma formation. • There is evidence that polymorphisms in the promoter region of TNF- α gene affect the amount of TNF- α production.

13. TNF-α and Sarcoidosis -857 TNF-α gene promoter -857 T increase in TNF-α gene expression susceptible to Sarcoidosis

14. Vascular endothelial growth factor (VEGF), which is produced in activated macrophages, an endothelial cell-specific mitogen that promotes angiogensis, is a potent mediator of vascular permeability, and activates monocytes. • VEGF is one of the cytokines that promotes granuloma formation in sarcoidosis by activating monocytes. • Polymorphism at +183 and -627 of VEGF gene showed to be responsibıl for VEGF production factor in sarcoidosis.

15. VEGF and Sarcoidosis +813 VEGF gene binding site for Enhancing Binding Protein 4 (EBP4) +813 T reduce binding affinity of EBP4 decrease in VEGF expression protective effect on Sarcoidosis

16. Aim • Polymorphism at -857 of TNF gene thought to be a predispositive factor in sarcoidosis and blamed to be responsibıl in patogenesis of the disease where as polymorphism at + 813 VEGF gene thought to decrease vulnerability to sarcoidosis. In our study we examined and compared these polymorphisms between healthy Turkish population and Turkish patients with sarcoidosis.

17. Method • Patients who were histopatologicaly diagnosed as sarcoidosis in between 2004-2006 in Yedikule Chest Disease and Chest Surgery Education and Research Hospital were inculuded in to the study • Authorization from our Hospital Ethics Committee and confirmed consent from all patients were taken • 55 patient, 36 women, 19 men • Age interval 17-65, • Mean age 40.1±9.3

18. Method • 55 healthy volunter without any chronic disease history, and without any pathological finding on their chest x-ray, physical examination and routine laboratory blood testing were included into the study as contol group. • 55 healthy volunter 33 women, 22 men • Age interval 19-76, • Mean age 38.3 ±9.7

19. Table 1. Dermographical characteristics of the cases

20. Genotype Analysis • VEGF genotype analysis (BURÇ genetic lab): • Peripheric blood • DNA isolation • PCR (208 bp) • F: 5’-aag gaa gag gag act ctg cgc aga gc-3’ • R: 5’-taa atg tat gta tgt ggg tgg gtg tgt cta cag g-3 • RFLP (Nla lll) • 813 CC : 208 bp • 813 TT: 86 bp + 122 bp • Agarose Gel Electrophoresis

21. Genotype Analysis • TNF-αgenotype analysis (BURÇ genetik lab): • Peripheric blood • DNA isolation • ARMS PCR (270 bp) • C: 5’- aag gat aag ggc tca gag ag-3’ • N: 5’-cta cat ggc cct gtc ttc g-3’ • M: 5’-t cta cat ggc cct gtc ttc a-3’ • Agarose Gel Electrophoresis

22. 813 TC 813 TT 813 CC PCR 857 CC 857 CT 857 TT C T C T C T VEGF VEGF TNF-α (PCR- RFLP) (ARMS)

23. Molecular Analysis Tecniques (PCR, REA, ARMS, Heteroduplex, SSCP, DGGE, sequences and chip)

24. Statistical analysis • Allel and genotip frequencies are evaluated by using Pearson Chi-Square test. • P value lower than 0,05 accepted to be meaningfull.

25. RESULTS

26. Table 2. TNF -857 gene polimorphism allele and genotype frequency in sarcoidosis and healthy cases

28. Table 3. VEGF+813 gene polimorphism allele and genotype frequency in sarcoidosis and healthy cases

30. Table 4. Organ involvement ratio of TNF – 857 polymorphisms in patients with sarcoidosis

31. Table 5. Organ involvement ratio of VEGF +813 polymorphisms in patients with sarcoidosis

34. DISCUSSION

36. Grutters and colleagues showed that TNF -857 T allele found increases frequency in Britsh and Dutch patients with sarcoidosis. • They found a lower frequency of TNF -857 T in Löfgren patients compared to non-Löfgren patients, but this difference did not reach stattistical significance. Am J Respir Crit Care Med 2002, Vol 165.1119-1124

37. Transcriptional promoter activity of the rarer TNF -857 T allele was shown to be higher than that of the common allele in activated blood mononuclear cells from Japanese donors by Higuchi and colleagues. Tissue Antigens 1998;51:605-612

38. Morohashi and colleagues found that in VEGF gene polymorphisms the T allele at +813 may decrease susceptability to sarcoidosis. • They found no significant association between the genotypes of VEGF at -627 or at +813 and the organ involvement. Chest 2003; 123:1520-1526

39. Conclusion • There was no difference TNF-α gene polymorphism at the T allele at -857 location in between healthy and sarcoidosis patients. • We suggest that in VEGF gene polymorphisms at the T allele at + 813 may decrease susceptibility to sarcoidosis. • There was no significant relation between TNF-α, VEGF gene polymorphisims and stage of the disease, organ involvement, recurrens rates, ACE levels and respiratory function tests.