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ASSOCIATION OF TLR4 SNPs AND SARCOIDOSIS IN GREEK

PATIENTS

Journal: Genetic Testing

Manuscript ID: GTMB-2009-0117

Manuscript Type: Original Articles

Date Submitted by the Author:

23-Jul-2009

Complete List of Authors: Iliadi, Alexandra; Athens University, Chemistry

Makrythanasis, Periklis; Athens University, Medical Genetics Tzetis, Maria; Athens University, Medical genetics Tsipi, Maria; Athens University, Medical Genetics Traeger-Synodinos, Joanne; Athens University, Medical Genetics Ioannou, Penelope; Athens University, Chemistry Rapti, Aggeliki; Sotiria Chest Disease Hospital, 6th Departmrnt of pulmonary medicine Kanavakis, Emmanuel; Athens University, Medical Genetics Christopoulos, Theodore; University of Patras, Chemistry

Keyword: DNA Testing, Genetics, Human Genetics, Mutation Detection, Mutations

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ASSOCIATION OF TLR4 SNPs AND SARCOIDOSIS IN GREEK PATIENTS

Alexandra Iliadi1, Periklis Makrythanasis2, 3, Maria Tzetis2*, Maria Tsipi2,

Jan Traeger-Synodinos2, Penelope C. Ioannou1, Aggeliki Rapti4,

Emmanuel Kanavakis2, Theodore K. Christopoulos5,6

1Laboratory of Analytical Chemistry, Department of Chemistry, Athens

University, Athens, Greece

2 University of Athens, Medical School, “Aghia Sofia” Children’s Hospital,

Dept. of Medical Genetics, Athens, Greece

3 Department of Genetic Medicine and Development, Faculty of Medicine,

University of Geneva

4 6th Department of Respiratory Medicine, Sotiria Chest Disease Hospital

5 Department of Chemistry, University of Patras, Patras 26500, Greece

6 Foundation for Research and Technology Hellas, Institute of Chemical

Engineering and High Temperature Chemical Processes (FORTH/ICE-HT),

Patras 26504, Greece

Address for correspondence:

Maria Tzetis, PhD

University of Athens, Medical School,

“Aghia Sofia” Children’s Hospital,

Dept. of Medical Genetics, Athens, Greece

Tel: +030 210 7467460

Fax:+302107795553

E-mail: mtzetis@med.uoa.gr

Running Title: TLR4 gene polymorphisms and Sarcoidosis

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Abstract

The present study investigates the potential role of Toll-like receptor 4

(TLR4) Asp299Gly and Thr399Ile single nucleotide polymorphisms (SNPs) as

risk factors in the development of sarcoidosis using a novel high-throughput

microtiter well-based bioluminometric genotyping assay. 119 Greek patients

with sarcoidosis and 209 control subjects were genotyped for the two SNPs of

the TLR4 gene. The genotypes observed were in Hardy–Weinberg

equilibrium. The heterozygote frequency for both SNPs in sarcoidosis group

and control population was 13.4% (16/119) and 10.5% (22/209), respectively.

The minor genotype was found to be the same for both sarcoidosis and

control groups and similar to that found in other Caucasian populations. No

significant association of Asp299Gly and Thr399Ile polymorphisms with

increased susceptibility to sarcoidosis was found (P = 0.61 and OR =1.183).

In conclusion, genotype data for the TLR4 Asp299Gly and Thr399Ile

polymorphisms in the Greek population were found to be in linkage

disequilibrium and no contribution in the pathogenesis of sarcoidosis was

established. Furthermore, in course of the present study, we demonstrated a

very simple and sensitive high- throughput bioluminometric assay for

genotyping Asp299Gly and Thr399Ile polymorphisms in the TLR4 gene.

Key words

TLR4, sarcoidosis, bioluminometric assay, polymorphisms, SNPs

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Introduction

Sarcoidosis, from the Greek sarx, meaning "flesh", is a multi-factorial

systemic disease of unknown etiology, characterized by an increased

inflammatory activity, causing non-caseating granulomas and an exaggerated

cellular immune response. Most commonly, sarcoidosis affects young adults

of both sexes in the age-group from 20 to 29 years, with a slight

preponderance for women having been reported by most studies (Nunes et al.

2007).

Sarcoidosis is considered as a complex disease of unknown etiology. It

is most likely caused by the interaction of environmental factors and multiple

genes which contribute to the wide phenotypic variability concerning

progression and prognosis of the disease (Spagnolo et al. 2007). An

infectious etiology of sarcoidosis has long been suspected. Recently, scientific

evidence provided a strong link between infectious agents and sarcoidosis

Ezzie and Crouser 2007). On the other hand, the fact that sarcoidosis varies

across ethnic groups, with African Americans and Northern Europeans having

higher rates of disease than Southern Europeans and Japanese, strongly

suggests that a genetic predisposition exists (Rossman and Kreider 2007,

Kreider et al. 2005, Margolis and Loweder 2007). Despite the evidence for a

genetic predisposition to sarcoidosis, not a single gene has been identified as

the “sarcoidosis gene” until now. All current findings suggest that multiple

genes are involved in the pathogenesis of sarcoidosis. Understanding the

genetics of sarcoidosis will lead to the development of new diagnostic and

prognostic tests and new therapeutic approaches (Smith et al. 2008).

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Even though multiple organs can be affected, sarcoidosis primarily

affects lung and lymph nodes in the majority of the patients suggesting that an

inhaled “sarcoidosis” antigen to genetically susceptible individuals triggers the

inflammatory response and leads to the formation of the pathognomonic

granulomas (Newman et al. 1997, Schurmann et al. 2008). After exposure to

an antigen, antigen presenting cells (APCs) such as macrophages, secrete

numerous cytokines TNF-α, IL-12, IL-18 and INF-γ. Being responsible for the

attraction and activation of lymphocytes, these cytokines seem to play critical

role in the development and integrity of granuloma..

Toll-Like Receptors (TLRs) are pattern recognition receptors (PRRs)

and play a fundamental role in pathogen recognition. They are membrane

receptors mainly expressed on the surface of APC cells and recognize

pathogen-associated molecular patterns (PAMPs) expressed on microbial

components. Because of the influence of environmental factors, including

specific microorganisms, on sarcoidosis development, TLR could be of

importance for the pathogenesis of sarcoidosis (Janssens and Beyaert 2003,

Takeda and Akira 2004, Akira et al. 2001).

Two different single nucleotide substitutions of the TLR4 gene,

[rs4986790 Asp299Gly (A>G) and rs4986791 Thr399Ile (C>T)], are reported

to cause modification of the receptor’s extracellular region and therefore

influence the receptor’s ability for recognition and ligation (Schroder and

Schumann 2005). A significant association between the chronic course of

sarcoidosis and TLR4 mutations in German sarcoidosis patients and healthy

unrelated controls was reported by Pabst et al (Pabst et al. 2006). In another

study performed by Veltkamp et al in Dutch sarcoidosis patients and healthy

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controls no differences were found in allelic distributions (P = 0.79) or within

the different clinical entities of the sarcoidosis group (P = 0.44) (Veltkamp et

al. 2006). Importantly, there were no differences between the Dutch and

German sarcoidosis patients (P = 0.62). However, the allelic distribution of the

Asp299Gly differed significantly between both control groups (P = 0.04). In a

recent study of 100 sarcoidosis patients of Greek origin no correlation was

found between the disease and the Asp299Gly and Thr399Ile polymorphisms

in the TLR4 gene (Gazouli et al. 2006).

The aim of the present work was to clarify the controversial findings of

the previous studies by genotyping a group of Greek sarcoidosis patients. At

the same time, we demonstrated a novel high- throughput bioluminometric

assay for genotyping Asp299Gly and Thr399Ile polymorphisms in the TLR4

gene. Our results were compared to a group of ethnically matched controls.

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Materials and Methods

Human subjects

The patient group consisted of 119 Greek sarcoidosis patients (mean

age 53.3 ± 13.5; range 26-79 years), with mean age of disease onset

43.1±13.3 and a general population control group (n = 209) (mean age

30.7±5.70; range 22-45 years), while the male/female ratio was 27/92 and

70/139 among patients and controls, respectively.

Whole blood samples from the patients were obtained from the

Outpatient Clinic of the Respiratory Department of the University of Athens

and the 6th Department of Respiratory Medicine of the "Sotiria" Chest

Disease Hospital of Athens. In all patients, sarcoidosis was diagnosed when

typical clinico-radiological findings were supported by histological evidence of

noncaseating epithelioid cell granulomas. A standard radiographic staging for

sarcoidosis is used. The main characteristics of each stage are as follows:

stage I, characterized by bilateral hilar lymphadenopathy (BHL); stage II,

additional parenchymal infiltrates without BHL; stage III, only pulmonary

infiltrates can be seen without BHL; and stage IV, irreversible fibrosis with

loss of lung volume (Costabel and Hunninghake 1999). Detailed clinical data

of the patient population are shown in Table 1.

The control population consisted of healthy partners of In Vitro

Fertilization (IVF) couples or general population subjects that came to the

Department of Medical Genetics for Cystic Fibrosis Transmembrane

conductance Regulator (CFTR) gene screening. None of the controls had a

history of pulmonary or other inflammatory disease. All individuals included in

the study were of Greek origin. The study was approved by the ethics

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committee of the University of Athens and all subjects participating signed an

informed consent.

Data sources

TLR4 (MIM#603030) gene sequences were downloaded from

GenBank (www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=nucleotide).

Isolation of Genomic DNA

Genomic DNA was obtained from 3 ml of peripheral blood, using the

BioRobot® M48 System (Qiagen, Hilden, Germany) and the commercially

available kit MagAttract® DNA Blood Midi M48 Kit (Qiagen).

Genotyping of TLR-4 Asp299Gly and Thr399Ile polymorphisms

A high-throughput microtiter well-based bioluminometric genotyping

assay was used to detect the presence of TLR4 Asp299Gly and Thr399Ile

SNPs (Iliadi et al. 2008). Briefly, a 634 bp region spanning the two

polymorphic sites is amplified by PCR of genomic DNA samples. The product is

subjected directly (without purification) to two primer extension (PEXT) reactions (3

cycles each) using normal and mutant primers in the presence of biotin-dUTP. The

biotinylated nucleotide is incorporated in the extended primer carrying a (dA)24

segment at its 5’end. The products are captured in microtiter wells coated with

streptavidin and are detected using a (dT)30-aequorin conjugate.

Homozygotes and heterozygotes at each site were distinguished by the allelic

fraction (AF), which is calculated from the equation: AF = LN/(LN+LM), where

LN and LM are the luminescence signals obtained from PEXT reaction with the

normal (N) and mutant (M) primer, respectively. The theoretical value of allelic

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fraction for a normal (A/A), heterozygote (A/B) and a mutant homozygote

(B/B) sample is 1, 0.5 and 0, respectively.

Statistical Analysis

Genotyping data for the two TLR4 SNPs in sarcoidosis and control

groups were tested for Hardy-Weinberg Equilibrium. Fisher’s exact test was

applied to test for Hardy-Weinberg proportions. The genotype frequencies of

each SNP were compared between patients and the control group using the

Pearson’s χ2 test and Fisher’s exact test. The odds ratios (OR) with 95%

confidence intervals (95% CI) were also calculated. Statistical analysis was

performed using PLINK and R softwares.

Results

Sarcoidosis risk and TLR4 polymorphisms

Patients with sarcoidosis and matched controls were genotyped for the

two TLR4 SNPs using the bioluminometric PEXT assay [18].The minor allele

frequency (MAF) for both polymorphisms in the sarcoidosis patients was

0.067 and for the controls 0.057. The comparison was done using PLINK and

R and 4 different setups were tested, i) genotypes AA/AB/BB, ii) allele

frequency (A/B), iii) dominant effect of the minor allele by comparing the

frequency of the homozygotes for the major allele to the frequency of the

homozygotes for the minor allele and the frequency of the heterozygotes

(AA/AB+BB) and iv) recessive effect of the minor allele by doing the opposite

assumption (AA+AB/BB). Additionally, for the allelic test, odds ratios along

with confidence intervals were also calculated (Table 2). None of the P values

reached the significance level of 0.05. The majority of sarcoidosis patients

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and controls were found to be heterozygous for the major allele. Only one

healthy control was homozygous for the minor allele for both polymorphisms.

Both polymorphisms were found to be in Hardy-Weinberg equilibrium.

Discussion

Toll-Like Receptors, TLRs, are a class of single membrane-spanning

non-catalytic receptors that participate in the recognition process of

pathogenic organisms. These pathogen recognition receptors (PRR) are

expressed mostly on the surface of antigen presenting cells (APC) such as

dendritic, macrophages and neutrophils and appear to respond to different

stimuli (Janssens and Beyaert, 2003, Alberts et al. 2002, Armant and Fenton

2002). Their main role is the induction of inflammatory reaction and the

consolidation of adaptive immunity. TLR4 is the best characterized gene of

this family of receptors. It binds to LPS, unique to the membrane of the Gram-

negative bacteria, as well as to a number of host protein molecules, such as

heat shock proteins (HSP), which are released at sites of damage or infection.

After the ligand binding, a signal transduction starts the production of pro-

inflammatory cytokine, enhances surface expression of costimulatory

molecules, and activates caspase and IFN- inducible gene expression.

Furthermore, they play a crucial role in dendritic cell maturation and the

activation of the naive T cells, so they are involved in linking innate and

adaptive immunity (Tavener and Kubes 2005, O’Neill 2006). Recently, it has

been demonstrated that two common SNPs in exon 3 of TLR4 (A896G and

C1196T) are associated with airway hypo-responsiveness to inhaled

endotoxin (Arbour et al. 2000). The A896G (Asp299Gly) and C1196T

(Thr399Ile) are responsible for the modification of the receptor’s extracellular

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region. This modification influences the receptor’s ability for recognition and

ligation (Raby et al. 2002). Cross reactivity between endogenous ligands,

such as HSP60 and HSP70 and mycobacterial HSPs was recently suggested

as a disease mechanism of sarcoidosis (Ferwerda et al. 2007). The specific

role of TLR4 combined with the high frequency of these two SNPs (5-11% in

Caucasian populations) and the influence of environmental factors, including

specific microorganisms, on sarcoidosis development, lead to the theory that

these SNPs may be of importance for the development of the disease.

Pabst et al (Pabst et al. 2006) genotyped 141 Caucasian patients with

sarcoidosis and 141 healthy unrelated controls for the A896G and C1196T

SNPs using PCR followed by restriction fragment length polymorphism

(RFLP) analysis. They found that the two SNPs occurred more frequently in

sarcoidosis patients (7.8%) as compared to healthy controls (2.84%) but with

no statistical significance (P = 0.07). When 108 chronic sarcoidosis patients

were compared for the same two SNPs with the healthy control group, a

significant correlation between chronic course of the disease and the

prevalence of the two TLR4 SNPs was found (9.26% in chronic sarcoidosis

patients vs 2.84% in healthy control, P = 0.01). On the other hand, there was

no difference in the subgroup of patients with acute sarcoidosis and the

control group (P = 0.93). This finding, however, was not replicated in three

other association studies.

In another study, 156 Dutch sarcoidosis patients and 200 healthy

controls were genotyped using dual-labeled fluorescent oligonucleotides

(Veltkamp et al. 2006). No differences were found in allelic distributions

between patients and controls (P = 0.79) or within the different clinical entities

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of the sarcoidosis group (P = 0.44). The difference between the two studies,

that could have contributed to the conflicting results, lies in the low frequency

of the minor allele (G) for Asp299Gly in the control group of German patients

(Pabst et al. 2006).

Schürmann et al. (Schurmann et al. 2008) genotyped 1203 German

sarcoidosis patients from 997 families, 1084 relatives and 537 control subjects

for both SNPs using the Taqman technique. The minor allele frequency of the

control group (0.056) was close to the Dutch control group (0.063) (Veltkamp

et al. 2006) and differed from that of the other German study (0.028) (Pabst et

al. 2006). No linkage was detected for the TLR4 SNPs and sarcoidosis.

However, they proposed another as yet unidentified variant within or close to

TLR4 that might confer susceptibility to sarcoidosis. A more recent study in

healthy individuals of German and Dutch descent, gave frequencies of the

minor allele very close to the above studies (0.057 and 0.072, respectively)

(Ferwerda et al. 2007).These data support the assumption that the

underestimation of the minor allele frequency of the original German study

(Pabst et al. 2006) contributed to significant differences between the control

group and the chronic sarcoidosis group.

Another study (Gazouli et al. 2006) on 92 Greek sarcoidosis patients

and 143 healthy individuals for the same two SNPs reported MAF for

Asp299Gly of 0.045% and 0.0267% in patients and healthy controls,

respectively. MAF for Thr399Ile was found 0.025% and 0.0133% in patients

and controls, respectively. The MAF for both SNPs detected in this study

differs from our findings. However, no association with sarcoidosis risk was

indicated (P = 0.317, OR = 1.72), in concordance with previous studies.

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In conclusion, according to our findings, the TLR4 Asp299Gly and

Thr399Ile SNPs are not associated with sarcoidosis in Greek subjects. Our

results were in concordance with the results obtained from other studies

(Pabst et al. 2006, Veltkamp et al. 2006, Gazouli et al. 2006). Taking into

account the important role of TLR4 in the consolidation of immune response,

further investigations in other regions of the gene are required in order to fully

understand its role in disease development.

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‘‘Author Disclosure Statement.’’

All authors declare that they have no conflict of interest

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Alexandra Iliadi

Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens, Greece ailiadi@biol.uoa.gr

Periklis Makrythanasis

University of Athens, Medical School, “Aghia Sofia” Children’s Hospital, Dept. of Medical Genetics, Athens, Greece

Department of Genetic Medicine and Development, Faculty of Medicine, University of Geneva Periklis.makrythanasis@unige.ch

Maria Tzetis

University of Athens, Medical School, “Aghia Sofia” Children’s Hospital, Dept. of Medical Genetics, Athens, Greece mtzetis@med.uoa.gr

Maria Tsipi

University of Athens, Medical School, “Aghia Sofia” Children’s Hospital, Dept. of Medical Genetics, Athens, Greece mariatsipi@hotmail.com

Jan Traeger-Synodinos

University of Athens, Medical School, “Aghia Sofia” Children’s Hospital, Dept. of Medical Genetics, Athens, Greece jtraeger@med.uoa.gr

Penelope C. Ioannou

Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens, Greece ioannou@chem.uoa.gr

Aggeliki Rapti

6th Department of Respiratory Medicine, Sotiria Chest Disease Hospital aggelikirapti@hotmail.com

Emmanuel Kanavakis

University of Athens, Medical School, “Aghia Sofia” Children’s Hospital, Dept. of Medical Genetics, Athens, Greece ekanavak@med.uoa.gr

Theodore K. Christopoulos

Department of Chemistry, University of Patras, Patras 26500, Greece

Foundation for Research and Technology Hellas, Institute of Chemical Engineering and High Temperature Chemical Processes (FORTH/ICE-HT), Patras 26504, Greece

tkc@chemistry.upatras.gr

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Table 1: Clinical characteristics of patients with sarcoidosis

Sex Females: 92

Males: 27

Age (yrs, mean ± SD) 53 ±13

Age of onset (yrs ± SD) 43 ±13

Stage I: 48 (40.4%)

II: 60 (50%)

III: 10 (8.4%)

IV: 1 (0.8%)

Pulmonary Function tests (mean ± SD)

FEV1: 93 ± 20

FVC: 99 ± 18

Biopsy 50/119 (42%)

skin: 30

lung: 14

lymph: 6

Extrapulmonary localizations 36/119 (30.3%)

eye: 5

skin: 24

other: 7

Disease duration (months, mean ±SD)

50 ± 33

Follow up available (>1yr) 106/119 (88.8%)

Remission: 79/119 (66.7%)

Persistence: 13/119 (11.2%)

Patients with relapses 18/119 (15.1%)

(9 patients with >2 relapses)

Page 18 of 19

Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801

Genetic Testing

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For Peer Review

Iliadi et al

Table 2: Genotype frequencies in Sarcoidosis and control group for TLR4

GENE Controls Sarcoidosis P values

TLR4 Genotype

Counts

(frequency)

Counts

(frequency)

Genotypes Dominant Recessive Allelic OR*

G/G 1 (0.48%) 0 (0%)

A/G 22 (10.5%) 16 (13.4%)

Asp299Gly

(rs4986790) A/A

187

(89.5%) 103 (86.6%)

0.6655 0.595 1 0.6143‡

1.183

(0.3756-

3.2070)

T/T 1 (0.48%) 0 (0%)

C/T 22 (10.5%) 16 (13.4%) Thr399Ile

(rs4986791) C/C 186 (89%) 103 (86.6%)

0.6655 0.595 1 0.6143‡

1.183

(0.3756-

3.2070)

P values calculated by the PLINK software; ap values calculated by the R software (double-sided Fisher’s exact test);

*OR along with confidence intervals calculated for the

allelic test.

Page 19 of 19

Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801

Genetic Testing

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