Association of IL-17A and IL-17F single nucleotide polymorphisms with susceptibility to osteoarthritis in a Korean population

    Association of IL-17A and IL-17F single nucleotide polymorphisms with susceptibility to osteoarthritis in a Korean population Lin Han, Hwa Sung Lee, Jung Hwan Yoon, Won Suk Choi, Yong Gyu Park, Suk Woo Nam, Jung Young Lee, Won Sang Park PII: DOI: Reference:

S0378-1119(13)01347-4 doi: 10.1016/j.gene.2013.09.113 GENE 39113

To appear in:

Gene

Accepted date:

27 September 2013

Please cite this article as: Han, Lin, Lee, Hwa Sung, Yoon, Jung Hwan, Choi, Won Suk, Park, Yong Gyu, Nam, Suk Woo, Lee, Jung Young, Park, Won Sang, Association of IL-17A and IL-17F single nucleotide polymorphisms with susceptibility to osteoarthritis in a Korean population, Gene (2013), doi: 10.1016/j.gene.2013.09.113

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Association of IL-17A and IL-17F Single Nucleotide Polymorphisms

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with Susceptibility to Osteoarthritis in a Korean Population

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Lin Hana,c, Hwa Sung Leeb, Jung Hwan Yoona, Won Suk Choia, Yong Gyu Parkc, Suk

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Woo Nama, Jung Young Leea, Won Sang Parka

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Departments of aPathology, bOrthopedics and cBiostatistics, College of Medicine, The

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Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea

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Department of cPathology, Liaocheng People’s Hospital, No.67 Dongchang West

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Road, Liaocheng City, China

Corresponding Author: WonSangPark, M.D., Ph.D., Department of Pathology,

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College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea. Phone: 82-2-590-1192, Fax: 82-2-537-6586, E-mail: [email protected]

Running Head: IL-17 Polymorphisms and Knee OA

Lin Han and Hwa Sung Lee equally contributed to this paper.

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Abstract

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The damage incurred in osteoarthritis (OA) is mediated by a variety of cytokines,

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growth factors and inflammatory mediators. The importance of the interleukin-17 (IL-17) family in inflammatory and autoimmune disease is becoming increasingly

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apparent. Microsatellite association mapping reveals a primary osteoarthritis susceptibility locus on chromosome 6p12.3-q13. IL-17A and IL-17F genes resided on

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chromosome 6p12.3-q13 are believed to play an important role in the primary OA

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susceptibility. We investigated the allele and genotype of IL-17A G-197A and IL-17F

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T7488C in 302 OA patients and 300 healthy subjects as controls. We employed a

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PCR-SSCP assay to identify the genotypes IL-17A G-197A and IL-17F T7488C. For IL-17A G-197A, there were significant differences in frequencies of genotype and allele of IL-17A G-197A between OA patients and controls (both P<0.0001). For

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IL-17F T7488C, there were no significant differences in the alleles frequency and genotypes distribution for IL-17F T7488C between OA patients and controls (p=0.938 and p=0.1735, respectively). In conclusion, current study showed that polymorphism of IL-17A G-197A may be closely associated with susceptibility to the development of OA in the Korean population. However, there was no relationship between IL-17F T7488C polymorphism and OA susceptibility.

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Abbreviations: OA, osteoarthritis; IL, interleukin; GDF 5, growth differentiation factor

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5; RA, rheumatoid arthritis; OR, odds ratio; CI, confidence interval.

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Keywords: Genetic Polymorphism, Genetic Susceptibility, Osteoarthritis, IL-17A,

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IL-17F.

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1. Introduction

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Osteoarthritis (OA) is characterized as the destruction of the articular cartilage,

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subchondral bone alterations, and synovitis. Clinical manifestations of OA may include joint pain, swelling, stiffness, and even the loss of some bodily function. In

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Korea, radiographic and symptomatic knee OA affects 37.3% and 24.2% of older individuals, respectively.(Kim et al., 2010) Genetic factors combined with mechanical

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forces play an important role in both the incidence and the progression of OA.

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Research interest is increasing in the relationship between genetic polymorphisms

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and OA, for example, interleukin-6 (IL-6), IL-18 and growth differentiation factor 5

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(GDF 5).(Honsawek et al., 2011; Tawonsawatruk et al., 2011; Hulin-Curtis et al., 2012) Microsatellite association mapping reveals a primary osteoarthritis susceptibility locus on chromosome 6p12.3-q13.(Southam et al., 2004) Interestingly, both IL-17A and

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IL-17F genes reside on chromosome 6p12.3-q13. An investigation of the relationship between the risk of OA and polymorphisms of these two genes could be rewarding. The damage incurred by joint tissues is mediated by a variety of cytokines, growth factors, proteases, and inflammatory mediators. We chose the IL-17 gene as a candidate because of its following role in inflammatory disease. It is well known that IL-17 family members play an active role in inflammatory diseases, autoimmune diseases, and cancer.(Kolls and Linden, 2004) The IL-17 cytokine family is composed of IL-17A, B, C, D, E and F six members. IL-17A is a pro-inflammatory cytokine

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associated with many inflammatory diseases, such as rheumatoid arthritis (RA),

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ankylosing spondylitis, and systemic lupus erythematosus.(Paradowska et al., 2007;

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Chen et al., 2012; Rana et al., 2012) Recently, IL-17F garnered increasing attention

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due to its great similarity to IL-17A.(Chang and Dong, 2009) Both genes induce the expression of various cytokines, chemokines and adhesion molecules, suggesting

addition,

IL-17A

G-197A

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potential overlapping functions for IL-17A and IL-17F. (Chang and Dong, 2009) In

(rs2275913)

and

IL-17F

T7488C

(rs763780)

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polymorphisms have been shown to be positively associated with ulcerative colitis

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and RA.(Arisawa et al., 2008; Nordang et al., 2009). Thus, we hypothesized that

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IL-17A and IL-17F gene polymorphisms are associated with individual susceptibility

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to OA in a Korean population.

Here, we examined the frequencies of genotypes and alleles for the IL-17A –G-197A (rs2275913)and IL-17F T7488C (rs763780) polymorphisms in 302 patients

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with OA and 300 healthy individuals by the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and found that polymorphism of IL-17A –G-197A (rs2275913) may be closely associated with susceptibility to the development of OA in the Korean population.

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2. MATERIALS AND METHODS

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2.1 Tissue samples

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Specimens were obtained degenerative articular cartilage, meniscus, and

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ligament tissue from 302 OA patients who received total knee arthroplasty at Seoul St. Mary's Hospital of the Catholic University of Korea between 2004 and 2005. All

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patients were confirmed by radiology and pathology to have OA. Among the 302 patients, 57 (18.9%) were male, and 245 (81.1%) were female. The mean age was 60

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at the initial diagnosis. Because these patients received total knee arthroplasty, we

narrowing

grade

or

higher.

autoimmune

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diseases,

excluded

patients

post-traumatic

with OA,

RA, and

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polyarthritis-associated

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space

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obtained the specimens from those patients of Kellgren-Lawrence grade 4 or joint

infection-induced OA. We also excluded those with clinical and radiographic findings suggesting skeletal dysplasia and those with other malignant diseases such as bone

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tumors, secondary metastasis, alcohol/drug dependence, hepatic failure, and renal failure. The control group consisted of a total of 300 healthy individuals (136 females and 164 males). The mean age was 51. We excluded those individuals with joint pain, a limp, or limited joint movement and those with radiographic signs of joint space narrowing or osteophyte formation. The healthy individuals and patients belonged to the same ethnicity and geographical area. This study was approved by the Institutional Review Board (IRB) of the Catholic University of Korea, College of Medicine (MC12SNS10013).

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2.2 DNA extraction

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We extracted DNA templates from the paraffin-embedded knee joint tissue of OA

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patients. We cut these tissue samples into 4-5 μm slices and dissolved the paraffin

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with xylene, then washed the xylene with 100% ethanol. We suspended the tissues in an ice-cold Nonidet P-40 lysis buffer solution and treated them with proteinase K. We

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extracted the DNA by the phenol/chloroform/isoamyl alcohol extraction method and ethanol precipitation, following previous research.(Ikegawa, 2007) For healthy

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individuals, we obtained a leukocyte cell pellet from each blood sample through the

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Buffy coat by the centrifugation of 2 ml of whole blood. We used the cell pellet for the

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DNA extraction. We employed the Qiagen DNA Blood Mini Kit (Qiagen, Valencia, CA,

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USA) and followed the manufacturer's instructions to obtain the genomic DNA. We determined the purity and concentration of the extracted DNA by using the

USA).

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Nanodrop® ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE,

2.3 PCR-SSCP for IL-17A and IL-17F A PCR-SSCP assay was used to identify the IL-17A and IL-17F genotypes. For IL-17A (rs2275913), the primer sequences of sense and antisense were as follows: 5’-AAC AAG TAA GAA TGA AAA GAG GAC ATG GT-3’ and 5’-CCC CCA ATG AGG TCA TAG AAG AAT C-3’, respectively. For IL-17F (rs763780), sense and antisense primers were designed as 5’-GTG TAG GAA CTT GGG CTG CAT CAA T-3’ and

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5’-AGT GGA TAT GCA CCT CTT ACT GCA CA-3’, respectively. We performed each

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PCR procedure under standard conditions with a 10 µl PCR mixture containing 1 µL

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of template DNA, 0.5 μM of each primer, 0.2 μM of each deoxynucleotide

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triphosphate, 1.5 mM of MgCl2, 0.4 unit of the AmpliTaq gold polymerase (Perkin-Elmer, Foster City, CA, USA), 0.5 μCi of [32P]dCTP (Amersham,

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Buckinghamshire, UK), and 1 μl of 10X buffer. We denatured the reaction mixture for 12 min at 95 °C and incubated it for 40 cycles (denaturation for 30 s at 95 °C,

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annealing for 30 s at 59 °C/60°C, and elongation for 30 s at 72 °C). We continued the

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final extension for 5 min at 72 °C. After the amplification, we denatured the PCR

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products for 5 min at 95 °C in a 1:1 dilution of a sample buffer solution containing 98%

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formamide and 5 mmol/L NaOH. We loaded these products onto an SSCP gel (FMC Mutation Detection Enhancement system; Intermountain Scientific, Kaysville, UT) containing 10% glycerol. After the electrophoresis, we transferred the gels to 3 MM

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Whatman paper and dried them. We then performed the autoradiography by using Kodak X-OMAT film (Eastman Kodak, Rochester, NY, USA). We cut the DNA showing mobility shifts from the dried gels and amplified it for 40 cycles by using the same primer set. We confirmed the results of genotypes by sequencing the PCR products by using the ABI 3730XL Analyzer (Applied Biosystems, Foster City, CA, USA) (data not shown). Blinded to the status of the study cohort, one of the authors evaluated the results. We randomly selected more than 10% of the sample for repeat assays and found that the results were in complete agreement.

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2.4 Statistical analysis

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We conducted a two-tailed Fisher’s exact test to determine the differences in the

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percentages of genotypes and alleles between OA patients and controls. The

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strength of association between allele frequencies and the OA was estimated by calculating the odds ratios (OR) and 95% confidence intervals (95% CI) by logistic

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regression analysis using genotype or the number of allele as a regressor. An adjusted analysis was also performed by logistic regression analysis after adjustment

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for gender and age. A post-hoc power analysis was performed to determine the OR

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for which our sample provided 80% power with an alpha of 0.05 to detect a

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statistically significant association between groups (patients/controls) and genotypes

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(AA/GG for IL-17A G-197A and TT/CC for IL-17F T7488C), given the proportions of reference genotypes and the sums of genotype frequencies. It was estimated that we had 80% power to detect a 0.56-fold OR of AA/GG for IL-17A G-197A and a 3.13-fold

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OR of TT/CC for IL-17F T7488C. A p values <0.05 were considered to be statistically significant.

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3.1 IL-17A –G-197A polymorphism and the risk of OA

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3. Results

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A total of 302 OA patients and 300 healthy controls were enrolled in the study. Table 1 shows the distributions of genotypes and alleles for the IL-17A G-197A

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polymorphism (rs2275913) in OA patients and controls. Interestingly, there were significant differences in genotype and allele frequencies for IL-17A G-197A between

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OA patients and controls (both p<0.0001). The risk of OA in carriers with an A allele

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(G/A or A/A genotypes) was statistically higher compared to that of carriers with the

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G/G genotype. Because the typical age for OA onset among Koreans is approximately 60, we classified the patients into two age groups: ≤60 as “young”

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patients and >60 as “old” patients.(Kim et al., 2010) When we analyzed the relationships between IL-17A G-197A polymorphism and OA stratified according to

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age and sex, there were significant differences in genotype and allele frequencies between young patients and young controls (Table 2). However, there were no significant differences in genotype distribution and allele frequency for IL-17A G-197A between male patients and female patients and between old patients and old controls.

3.2 IL-17F T7488C polymorphism and the risk of OA Table 3 shows the distributions of genotypes and alleles for the IL-17F T7488C

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polymorphism (rs763780) in OA patients and controls. There were no significant

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differences in the alleles frequency and genotypes distribution for IL-17F

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T7488Cbetween OA patients and controls (p=0.938 and p=0.1735, respectively).

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When we analyzed the relationships between the IL-17F T7488C polymorphism genotype and OA stratified according to age and sex, there were also no significant

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differences in genotype distribution and allele frequency between male patients and female patients (data not shown). We could not estimate the relationships between

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zero frequency in control group.

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IL-17F T7488C polymorphism genotypes and OA stratified according to age due to

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4. Discussion

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OA is a degenerative disease of articular cartilage and subchondral bone

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causing gradual permanent compromise of joint function. While the biological onset of OA is not clearly understood, evidence suggests that OA is a complex inflammatory

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disease marked by increased joint inflammation and synovitis.(Koller et al., 1999) Inflammatory cytokines contribute to tissue destruction by disrupting the balance of

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the catabolic and anabolic activities of chondrocytes, the major cell type of cartilage

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tissue. Of these cytokines, Interleukin-17A (IL-17) family members play an active role

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in inflammatory diseases.(Kolls and Linden, 2004) In addition, microsatellite

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association mapping reveals that the investigation of the relationship between the risk of OA and polymorphisms of both IL-17A and IL-17F genes could be rewarding.(Southam et al., 2004)

cells

to

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IL-17A may have a role in T cell–triggered inflammation by stimulating stromal secrete

various

cytokines

and

growth

factors

associated

with

inflammation.(Ouyang et al., 2008) The increased levels of IL-17A induce a multitude of factors contributing to the degradation of the articular cartilage and erosion of the underlying bone in different ways. IL-17A has been shown to up-regulate NO production and also to increase the mRNA levels of inducible nitric oxide synthase (iNOS) in osteoarthritic cartilage. (Attur et al., 1997; Martel-Pelletier et al., 1999) What’s more, IL-17A can induce pro-inflammatory cytokines such as TNF-a, IL-1β

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and IL-6 from cartilage, synoviocytes, macrophages and bone cells.(Rifas and Avioli,

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1999; Katz et al., 2001) These pro-inflammatory cytokines collectively play a central

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role in the pathophysiology of cartilage degradation and inhibit cartilage repair, and

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therefore lead the cartilage into an autodestructive pathway in osteoarthritis.(Koshy et al., 2002) It has also been reported that IL-17A is approximately 10-fold more potent

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than IL-17F in chemokine responses.(Dubin and Kolls, 2009) Recently, in vitro stimulated T cells from healthy individuals possessing the 197A allele produced

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significantly more IL-17 than those without the 197A allele and induced higher

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luciferase activity than the 197G allele.(Espinoza et al., 2011) Considering these

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important functions of IL-17A in OA development, we examined the distributions of

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genotypes and alleles for the IL-17A G-197A polymorphism in OA patients and controls. Finally, we found that there were significant differences in genotype and allele frequencies for IL-17A G-197A between OA patients and controls (both

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p<0.0001). Our findings suggest that the A allele of the IL-17A G-197A gene may be closely associated with susceptibility to OA in Korean patients. The allele frequencies of IL-17A G-197A in healthy control were consistent with Japanese and Chinese populations.( Arisawa et al, 2008; Peng et al, 2013) Whereas, the frequency of GA genotype was found in 53.4% and 54.8% of Japanese and Chinese people,( Arisawa et al, 2008; Peng et al, 2013) we found that only 35.3% of Korean people exhibited the GA variants. These results indicate that there may be ethnic variation in the IL-17 polymorphism. Such national differences, even among people of same ethnicity, need

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to be considered when associating IL-17 polymorphism with disease risk

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determination in different populations.

collagenases

and

decreasing

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IL-17F stimulates the degradation of cartilage by increasing expression of expression

of

type

II

collagen

in

chondrocytes.(Tanigawa et al., 2011) The single-nucleotide polymorphism T7488C is

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in the third exon which causes a His-to-Arg substitution at amino acid

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161.(Kawaguchi et al., 2006) In IL-17F T7488C, T allele is significantly associated with the development of chronic immune thrombocytopenia and inflammatory bowel

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disease.(Chen et al., 2009; Saitoh et al., 2011) In this study, the genotypic and allelic

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frequencies of IL-17F T7488C in the healthy controls were consistent with those of

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Chinese and Japanese populations. (Peng et al, 2013; Hayashi R et al, 2012) When the genotypic and allelic frequencies of the healthy controls and patients were

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compared, there were no significant differences in the alleles frequency and genotypes distribution for IL-17F T7488C between OA patients and controls (p=0.0954 and P=0.0738, respectively). These results further support the report of Southam et al (2006) describing no association of IL-17F as coding for the OA susceptibility. However, both polymorphisms analyzed in this study were not in concordances with the Hardy-Weinberg Equilibrium (HWE). Deviations from HWE can point to either a sampling bias, mistyping of genotypes, or spurious gene associations because of population stratification. Although deviation from the HWE often indicates a poor quality of genotyping, it can also be caused to the small size of

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the sample group and variations of allelic frequencies according to ethnic

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background.(Esser and Tomluk, 2005; Levecque et al, 2003). In this study, all of the

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participants were Korean, and analyses restricted to this subgroup yielded results

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similar to our previous findings, indicating that population stratification is unlikely to have biased our findings. However, it is necessary to extend the study in larger

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populations in order to have more precise conclusions about the association of IL-17

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polymorphism with OA.

Although our study had limited statistical power probably due to their small

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sample size or the low frequency of some variant genotype, while limited, we showed

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that polymorphism of IL-17A G-197A may be closely associated with susceptibility to

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the development of OA in the Korean population. Further studies are strongly necessary to clarify the biochemical mechanisms of these differences in potency and

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the exact effects of these polymorphisms in regulating IL-17 expression.

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ACKNOWLEDGEMENTS

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by the Korea government (MEST) No. 2012M3A9D1054476

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This research was supported by the National Research Foundation grant funded

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Percent

Number

AA

141

46.7

97

AG

109

36.1

106

GG

52

17.2

Adjusted OR(95% CI)

32.3

1.00

1.00

35.3

0.707 (0.488-1.026)

0.940 (0.551-1.606)

32.3

0.369 (0.241-0.564)

0.477 (0.259-0.878)

0.616 (0.500-0.759)

0.710 (0.526-0.958)

Percent

300:300

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c

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391:213

97

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b

A:G allele frequency

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IL-17A –G-197A

Trend test

a

Crude OR (95% CI)

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Number

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Controls (N=300)

CR

Patients (N=302)

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Table 1. Genotype and allele distributions of the IL-17A G-197A gene polymorphism in OA patients and controls

a Adjusted for age (in year) and sex b Two-sided Fisher's exact test: for allele frequencies, p<0.0001; for genotype distribution, p<0.0001 c Calculated in the logistic regression model using the number of A alleles in the genotypes as a continuous variable

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IL-17A G-197A genotype percentages for OA patients and controls Adja OR(95% CI)

GG

AA vs. AG

AA vs. GG

101

94

0.900 (0.539-1.502)

0.323 (0.168-0.624)

5

3

0.775 (0.192-3.127)

1.464 (0.310-6.927)

57

56

51

0.983 (0.462-2.091)

0.573 (0.228-1.441)

40

50

46

0.881 (0.411-1.890)

0.439 (0.166-1.162)

Controls

IL-17A

AG

GG

AA

≤60

45

47

16

90

>60

96

62

36

7

male

24

20

13

female

117

89

39

AG

US

AA

IP

Adja OR (95% CI)

Patients

CR

Table 2. Subgroup analysis of

AC

TE D

CE P

sex

MA N

age

a adjusted for the other covariates [age (in years) as a continuous variable] presented in this table in a logistic regression model for each stratum

ACCEPTED MANUSCRIPT

Percent

Number

TT

226

74.8

236

TC

59

19.5

56

CC

17

5.6

T:C allele frequencyb

511:93

Crude OR (95% CI)

Adjusteda OR(95% CI)

78.7

1.00

1.00

18.7

1.100 (0.731-1.656)

1.106 (0.604-2.027)

2.7

2.219 (0.939-5.242)

1.774 (0.464-6.787)

1.275 (0.940-1.728)

1.202 (0.753-1.918)

Percent

US

Number

IP

Controls (N=300)

CR

Patients (N=302)

T

Table 3. Genotype and allele distributions of the IL-17F T7488C gene polymorphism in OA patients and controls

TE D 8

CE P

528:72

AC

Trend testc

MA N

IL-17F T7488C

a.

Adjusted for age (in year) and sex

b.

Two-sided Fisher's exact test: for allele frequencies, p=0.938; for genotype distribution, p=0.1735

c.

Calculated in the logistic regression model using the number of A alleles in the genotypes as a continuous variable

ACCEPTED MANUSCRIPT

IL-17A G-197A polymorphism is associated with susceptibility to OA in th

IP



T

Highlights



SC R

e Korean population.

The A allele of the IL-17A G-197A gene may play an important role in

There is no relationship between IL-17F T7488C polymorphism and OA

CE P

TE

D

MA

susceptibility.

AC



NU

OA.