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

Vascular Endothelial Vascular Endothelial Growth Factor and Growth Factor and

Tumor Necrosis Factor Tumor Necrosis Factor Gene Polymorphisms in Gene Polymorphisms in Turkish Patients With Turkish Patients With

SarcoidosisSarcoidosisEkrem Cengiz Seyhan, Sedat Altın, Ekrem Cengiz Seyhan, Sedat Altın,

Erdoğan Çetinkaya, Atayla Gençolu, Erdoğan Çetinkaya, Atayla Gençolu, Sinem TimurSinem Timur

Yedikule Göğüs Hastalıkları ve Göğüs Yedikule Göğüs Hastalıkları ve Göğüs Cerrahisi Eğitim ve Araştırma Hastanesi Cerrahisi Eğitim ve Araştırma Hastanesi

Introduction Introduction

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

environmentenvironment. .

IntroductionIntroduction

Existing familiar sarcoidosis cases, some relations Existing familiar sarcoidosis cases, some relations between HLA and disease, different clinical between HLA and disease, different clinical presentations in different races shows existance presentations in different races shows existance of some predisposition genes in sarcoidosis of some predisposition genes in sarcoidosis

Particular gene polymorphisms were reported Particular gene polymorphisms were reported recently to play role in increased or decreased recently to play role in increased or decreased susceptability to sarcoidosis and also in the susceptability to sarcoidosis and also in the severity and progress of the disease. severity and progress of the disease.

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)

ACEACE (Angiotensin converting enzyme)(Angiotensin converting enzyme)

CCR2 CCR2 (C-c chemokine receptor)(C-c chemokine receptor) CFTR CFTR (Cystic Fibrosis Transmembrane (Cystic Fibrosis Transmembrane

Regulator)Regulator)

HLA-BHLA-B HLA-CHLA-C HLA-DPB1HLA-DPB1 IL-1 IL-1 (Interleukin 1)(Interleukin 1)

NRAMP NRAMP (Natural resistance associated (Natural resistance associated macrophage protein)macrophage protein)

TAP 2 TAP 2 (Transporter associated with antigen (Transporter associated with antigen processing 2)processing 2)

TNF - TNF - αα TNF - TNF - ββ VDR VDR (Vitamin D receptor)(Vitamin D receptor)

IntroductionIntroduction

Main defining pathological feature of sarcoidosis Main defining pathological feature of sarcoidosis is chronic inflammation resulting in is chronic inflammation resulting in nonnecrotizing epitheloid granuloma formation nonnecrotizing epitheloid granuloma formation produced by center of epithelioid and produced by center of epithelioid and multinuclear inflamatory cells surrounded by multinuclear inflamatory cells surrounded by mononuclear inflamatory cells and fibroblasts. mononuclear inflamatory cells and fibroblasts.

Granulomatous inflammation in sarcoidosis are Granulomatous inflammation in sarcoidosis are regulated by complex relations between T cells, regulated by complex relations between T cells, mononuclear phagocytes, fibroblasts, B cells and mononuclear phagocytes, fibroblasts, B cells and dendritic cells.dendritic cells.

Relation between these Relation between these cells are regulated by cells are regulated by cytokines and direct cytokines and direct contact between cells.contact between cells.

There is growing There is growing evidence for the evidence for the contribution of genetic contribution of genetic polymorphisms to polymorphisms to interindividual interindividual differences in the differences in the regulatory mechanisms regulatory mechanisms of cytokine production. of cytokine production.

((PhysiologicalPhysiological SNP- SNP-VarVariationiation))

Phenotipical varietyPhenotipical variety

(F(Functionalunctional SNP) SNP)Susceptability to diseasesSusceptability to diseases

PharmacogeneticPharmacogenetic

(Mut(Mutationation))DiseaseDisease

Tumor necrosis factor alpha (TNF-Tumor necrosis factor alpha (TNF-αα) is secreted ) is secreted from activated macrophages and thought to be a from activated macrophages and thought to be a principal cytokine in the pathogenesis of principal cytokine in the pathogenesis of sarcoidosis due to its privotal role in the sarcoidosis due to its privotal role in the granuloma formation. granuloma formation.

There is evidence that polymorphisms in the There is evidence that polymorphisms in the promoter region of TNF- promoter region of TNF- αα gene affect the amount gene affect the amount of of TNF- TNF- αα production. production.

TNF-α gene

promoter

-857

-857 T increase in TNF-α gene expression

susceptible to Sarcoidosis

TNF-α and Sarcoidosis

Vascular endothelial growth factor (VEGF), which Vascular endothelial growth factor (VEGF), which is produced in activated macrophages, an is produced in activated macrophages, an endothelial cell-specific mitogen that promotes endothelial cell-specific mitogen that promotes angiogensis, is a potent mediator of vascular angiogensis, is a potent mediator of vascular permeability, and activates monocytes.permeability, and activates monocytes.

VEGF is one of the cytokines that promotes VEGF is one of the cytokines that promotes granuloma formation in sarcoidosis by activating granuloma formation in sarcoidosis by activating monocytes.monocytes.

Polymorphism at +183 and -627 of VEGF gene showed to be responsibıl for VEGF production factor in sarcoidosis..

VEGF gene binding sitefor Enhancing Binding Protein 4 (EBP4)

+813

+813 T reduce binding affinity of EBP4

decrease in VEGF expression

protective effect on Sarcoidosis

VEGF and Sarcoidosis

AimAim

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.

MethodMethod Patients who were histopatologicaly diagnosed as Patients who were histopatologicaly diagnosed as

sarcoidosis in between 2004-2006 in Yedikule sarcoidosis in between 2004-2006 in Yedikule Chest Disease and Chest Surgery Education and Chest Disease and Chest Surgery Education and Research Hospital were inculuded in to the studyResearch Hospital were inculuded in to the study

Authorization from our Hospital Ethics Committee Authorization from our Hospital Ethics Committee and confirmed consent from all patients were and confirmed consent from all patients were takentaken

55 patient, 36 women, 19 men55 patient, 36 women, 19 men Age interval 17-65, Age interval 17-65, Mean age 40.1Mean age 40.1±±9.39.3

MethodMethod

55 healthy volunter without any chronic disease 55 healthy volunter without any chronic disease history, and without any pathological finding on history, and without any pathological finding on their chest x-ray, physical examination and their chest x-ray, physical examination and routine laboratory blood testing were included routine laboratory blood testing were included into the study as contol group. into the study as contol group.

55 healthy volunter 33 women, 22 men55 healthy volunter 33 women, 22 men Age interval 19-76, Age interval 19-76, Mean age 38.3 Mean age 38.3 ±±9.79.7

Table 1. Dermographical characteristics of the Table 1. Dermographical characteristics of the

casescases

CharactersCharacters SarcoidosisSarcoidosis

(n=55)(n=55)VolunterVolunter

(n=55)(n=55)P valueP value

Men/womenMen/women 19/3619/36 22/3322/33 >0.05>0.05

AgeAge

meanmean±±SDSD

intervalinterval40.140.1±±9.39.3

17-6517-6538.3 38.3 ±±9.79.7

19-7619-76>0.05>0.05

Smoking (+)Smoking (+) 1414 2323 >0.05>0.05

Genotype AnalysisGenotype Analysis

VEGF 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

Genotype AnalysisGenotype Analysis

TNF-α genotype analysisgenotype 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

VEGFVEGF TNF-α

813

TC

813

TT

813

CC

PC

R

C T C T C T

857 CC 857 CT 857 TT

(PCR- RFLP) (ARMS)

MoleMolecucullaar Analr Analysis Tecniquesysis Tecniques (PCR, REA, ARMS, Heteroduplex, SSCP, (PCR, REA, ARMS, Heteroduplex, SSCP, DGGE, DGGE, sequences andsequences and chip) chip)

Statistical analysisStatistical analysis

Allel and genotip frequencies are evaluated by Allel and genotip frequencies are evaluated by using Pearson Chi-Square test.using Pearson Chi-Square test.

P value lower than 0,05 accepted to be P value lower than 0,05 accepted to be meaningfull.meaningfull.

RESULTS RESULTS

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

casescases

PolimorphisPolimorphismm

Sarcoidosis Sarcoidosis patients patients

(n=55)(n=55)

Healty Healty voluntersvolunters

(n=55)(n=55)

P valueP value Risk ratioRisk ratio(%95 safety (%95 safety interval)interval)

Genotype Genotype frequency frequency

CCCC

CTCT

TTTT

32 (%58.2)32 (%58.2)

22 (%40)22 (%40)

1 (%1.8)1 (%1.8)

31 (%56.3)31 (%56.3)

21 (%38.3)21 (%38.3)

3 (%5.4)3 (%5.4)

0.840.84

0.830.83

0,310,31

0.92 (0.4-1.9)0.92 (0.4-1.9)

0.92 (0,4-1.9)0.92 (0,4-1.9)

3.11 (0.3-3.1)3.11 (0.3-3.1)

Allele Allele frequencyfrequency

CC

TT

86 (%78)86 (%78)

24 (% 22)24 (% 22)

83 (%75)83 (%75)

27(%25)27(%25)0.860.86

0.880.881.05 (0.5-2.1)1.05 (0.5-2.1)

0.94 (0,4-1.1)0.94 (0,4-1.1)

Graph 1. TNF -857 gene polymorphism frequency in cases

0

10

20

30

40

50

60

70

CC CT TT

%

Sarcoidosis Healthy volunters

Table 3. VEGF+813 gene polimorphism allele and genotype Table 3. VEGF+813 gene polimorphism allele and genotype

frequency in sarcoidosis and healthy casesfrequency in sarcoidosis and healthy cases

PolimorphisPolimorphismm

Sarcoidosis Sarcoidosis patients patients

(n=55)(n=55)

Healty Healty volunters volunters (n=55) (n=55)

P valueP value Risk ratioRisk ratio(%95 safety (%95 safety interval)interval)

Genotype Genotype frequency frequency

CCCC

CTCT

TTTT

43 (%78)43 (%78)

9 (%16)9 (%16)

3 (%6)3 (%6)

33 (%60)33 (%60)

21 (%38)21 (%38)

1 (%2)1 (%2)

0.030.03

0.010.01

0,310,31

0.4 (0.01-0.9)0.4 (0.01-0.9)

3.15 (1.2-7.7)3.15 (1.2-7.7)

3.11 (0.03-3.11 (0.03-3.1)3.1)

Allele Allele frequencyfrequency

CC

TT

95 (%86)95 (%86)

15 (%14)15 (%14)

87 (%79)87 (%79)

23 (%21)23 (%21)0.010.01

0.0010.0010.2 (0.1-0.6)0.2 (0.1-0.6)

3.37 (1.5-7.1)3.37 (1.5-7.1)

Graph 2. VEGF + 813 gene polymorphism frequency in cases

CC CT TT

%

Sarcoidosis Healthy

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

Organ Organ involvemeinvolvementnt

CC (%)CC (%)

(n=32)(n=32)CT (%)CT (%)

(n=22)(n=22)TT(%)TT(%)

(n=1)(n=1)P valueP value

Eye (n=15)Eye (n=15) 9 (28)9 (28) 6 (27)6 (27) 00 >0.05>0.05

Skin Skin (n=12)(n=12)

7 (21)7 (21) 5 (22)5 (22) 00 >0.05>0.05

LiverLiver

(n=7)(n=7)4 (12.5)4 (12.5) 3 (13)3 (13) 00 >0.05>0.05

HearthHearth

(N=3)(N=3)2(6)2(6) 1(4.5)1(4.5) 00 >0.05>0.05

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

Organ Organ involvementinvolvement

CC (%)CC (%)

(n=43)(n=43)CT (%)CT (%)

(n=9)(n=9)TT(%)TT(%)

(n=3)(n=3)P valueP value

Eye (n=15)Eye (n=15) 13 (30)13 (30) 2 (22)2 (22) 00 >0.05>0.05

Skin (n=12)Skin (n=12) 9 (20)9 (20) 2 (22)2 (22) 1 (33)1 (33) >0.05>0.05

Liver (n=7)Liver (n=7) 6 (13)6 (13) 1 (11)1 (11) 00 >0.05>0.05

Hearth (N=3)Hearth (N=3) 2(4.6)2(4.6) 1(11)1(11) 00 >0.05>0.05

Graph 3. TNF gene polymorphism frequency in sarcoidosis cases with high or normal ACE levels

56

44 50

50

0

10

20

30

40

50

60

70

80

90

100

CC CT

%

Normal ACE

High ACE

Graph 4. VEGF gene polymorphism frequency in sarcoidosis patients with high or normal ACE levels

53 55 50

47 45 50

0

10

20

30

40

50

60

70

80

90

100

CC CT TT

%

Normal ACEHigh ACE

DISCUSSIONDISCUSSION

Literatures Literatures Case number(patient

/control)

Studied gene Susceptability to disease

Relation to clinic

Grutter -2002

96/222(İngiliz)

TNF-857,-307,-863,-237

Increase with -857Increase with -857 -307 / löfgrenSyndrome

Kauzaki-2003

103/146(Japon)

VEGF+813, -627

Decrease with +813Decrease with +813No clinical

relation

Swider-2005 78/50(Alman)

HLA-DRB1TNF-α -308 No relation with gene

polymorphism

-308 / löfgrenSyndrome

Yamag-2000 110/161(Japon)

TNF- α -308, -244,-238 No relation with gene

polymorphism

Relation with Relation with clinical clinical severityseverity

Seitzer-1997 101/206(Alman)

TNF-α -308 No relation with gene

polymorphism

Relation with Relation with clinical clinical severityseverity

Our study-2005

55/55(Turk)

TNF-857 / VEGF+817

No relation with TNF, decreased

susceptability with VEGF

No relatio

n

Grutters and colleagues showed that TNF -857 T Grutters and colleagues showed that TNF -857 T allele found increases frequency in Britsh and Dutch allele found increases frequency in Britsh and Dutch patients with sarcoidosis.patients with sarcoidosis.

They found a lower frequency of TNF -857 T in They found a lower frequency of TNF -857 T in Löfgren patients compared to non-Löfgren patients, Löfgren patients compared to non-Löfgren patients, but this difference did not reach stattistical but this difference did not reach stattistical significance. significance.

Am J Respir Crit Care Med 2002, Vol 165.1119-1124Am J Respir Crit Care Med 2002, Vol 165.1119-1124

Transcriptional promoter activity of the rarer TNF -Transcriptional promoter activity of the rarer TNF -857 T allele was shown to be higher than that of the 857 T allele was shown to be higher than that of the common allele in activated blood mononuclear cells common allele in activated blood mononuclear cells from Japanese donors by Higuchi and colleagues.from Japanese donors by Higuchi and colleagues.

Tissue Antigens 1998;51:605-Tissue Antigens 1998;51:605-612612

Morohashi and colleagues found that in VEGF gene Morohashi and colleagues found that in VEGF gene polymorphisms the T allele at +813 may decrease polymorphisms the T allele at +813 may decrease susceptability to sarcoidosis.susceptability to sarcoidosis.

They found no significant association between the They found no significant association between the genotypes of VEGF at -627 or at +813 and the genotypes of VEGF at -627 or at +813 and the organ involvement.organ involvement.

Chest 2003; 123:1520-1526Chest 2003; 123:1520-1526

ConclusionConclusion

There was no difference TNF-There was no difference TNF-αα gene gene polymorphism at the T allele at -857 location in polymorphism at the T allele at -857 location in between healthy and sarcoidosis patients.between healthy and sarcoidosis patients.

We suggest that in VEGF gene polymorphisms at We suggest that in VEGF gene polymorphisms at the T allele at + 813 may decrease susceptibility the T allele at + 813 may decrease susceptibility to sarcoidosis. to sarcoidosis.

There was no significant relation between TNF-There was no significant relation between TNF-αα, , VEGF gene polymorphisims and stage of the VEGF gene polymorphisims and stage of the disease, organ involvement, recurrens rates, ACE disease, organ involvement, recurrens rates, ACE levels and respiratory function tests. levels and respiratory function tests.