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1 2006, Vol. 50, No. 2 Printed in the Republic of Korea RFLP 와 DHPLC 를이용한베체트병환자에서 Interleukin-6 유전자다형성의분석 Áû xá Á Á * w w yw» w ( ) Analysis of Interleukin-6 Gene Polymorphisms in Behcet s Disease Using RFLP and DHPLC Sang-Bum Park, Youn-Hyoung Nam, Jae-Sik Lee, Won Kang, and Won-Cheoul Jang* Department of Chemistry, School of Advanced Science and Basic Science Research Institute, Dankook University, Cheonan, , Korea (Deceived December 26, 2005). p x p y,,, v,,» e w y. p ù» w y x ù y g x. w (major histocompatibility complex, MHC) non-mhc gene w p w. IL-6 x w p w y w. x VNTR (variable number of tandem repeat), RFLP (restriction fragment length polymorphism), DHPLC (denaturing high performance liquid chromatography) w y w. x, p y IL-6prom x IL-6vntr x x ƒ ùkû. IL-6vntr * C ƒ w p ùkû. y w» w ƒ v w. : BD(behcet s disease), IL-6, DHPLC, RFLP, VNTR ABSTRACT. Behcet's disease (BD) is a systemic vasculitis characterized by recurrent oral and genital ulcers, and ocular inflammation, and which may involve the joints, skin, central nervous system and gastrointestinal tract. Although the exact pathogenesis for BD is not completely understood, it has been suggested that the disease is triggered in genetically susceptible individuals by environmental factors, such as microbial agents. It is noted that multiple genes, including MHC (major histocompatibility complex) and non-mhc genes, are implicated in the pathogenesis of BD. This study tries to determine whether IL-6 gene polymorphisms are associated with susceptibility to Behcet's disease in Koreans. Gene polymorphisms were typed by VNTR (variable number of tandem repeat), RFLP (restriction fragment length polymorphism), DHPLC (denaturing high performance liquid chromatography). There were no evidences for genetic association conferred by the IL-6prom polymorphism. However, significant differences in the IL-6vntr genotype and allele frequencies were found between patients with BD and controls. The IL-6vntr * C allele appeared to be an additional susceptibility gene to Korean BD. Further studies in other populations and gene are required to confirm these results. Keywords: BD(behcet s disease), IL-6, DHPLC, RFLP, VNTR 123

2 124 Áû xá Á Á p,, v, x,,, s» e w y wš w y. 1-4 yw ù» x ù l y y ƒ w y y jš š š. p ƒ w ƒ ¼ w HLA-B51 ù sww Silk Road w ù w y w. Ohno p j (Silk Road disease) š w. ù y 50% ƒ HLA-B51. ù l j Glu HLA-B51 sww HLA-B locusƒ p» w ƒ 20% w š šw š HLA-B51 MHC ù non-mhc ƒ w w endothelial nitric oxide synthases gene polymorphism (enos), intercellular adhesion molecule-1 gene polymorphisms (ICAM) IL-1 gene cluster polymorphisms MHC ƒ ew 6 w p» w š š ù šƒ p ü ü w y y ƒ š w. ù p y w y p ƒ. 30 wš ƒ û ƒ. w r 30-40% ƒ, ƒ 15-20% w ù w x e w y. 7p21 ew 13 interleukin (IL) 6 w š, w IL-6 x y ƒ y š, y» p y x IL-6ƒ ƒ š IL-6 mrna x ƒ š š š. w p IL-6vntr (variable number of tandem repeat polymorphisms in the 3' flanking region of the IL6 gene) IL-6prom (single nucleotide polymorphism at -174G/C in the IL6 gene promoter) w. w HLA-B51 y y IL- 6 x w ³ w, p w ƒ» w. x w p ü international study group» w 89 p y ù û ƒ ew 123 w.»» x genomic DNA w AccuPrep Genomic Extraction Kit, Taq. polymerase, PCR w AccuPrep PCR Purification Kit, RFLP w w w z SfaN1 Bioneer t w.» w agarose QA-Agarose TM (Q-bio gene) w š, DHPLC w triethylammonium acetate (TEAA) Transgenomic, acetonitrile (ACN) Merck w w.»» DNA s GeneAmp PCR System 2700 (Perkin-Elmer, USA) w š, PCR y Agaro Power TM (Bioneer)» e y w. DHPLC WAVE SYSTEM(Transgenomic, USA) w. x p y x genomic DNA w, IL-6 PCR sw z

3 Interleukin-6 Gene Polymorphisms in Behcet s Disease 125 IL-6prom RFLP DHPLC w w š IL-6vntr NuSieve agarose gel y w. x genomic DNA AccuPrep TM Genomic Extrction kit (Bioneer, Korea) w w DNA 25 ng/µl w PCR wš» w» w -20 C w o. Analysis of IL-6prom and IL-6vntr wz (PCR). Forward primer 5'-GAGCCA- GAACACAGAAGAAC-3' reverse primer 5'- CAGAATGAGCCTCAGACATC-3' w IL- 6prom PCR š forward primer 5'- GCAACTTTGAGTGTGTCACG-3' reverse primer 5'- TGACGTGATGGATGCAACAC-3' w IL-6vntr PCR ƒƒ 25 µl PCR 0.2 ml PCR p z, DNA thermal cycler (Applied Biosystems, USA, GeneAmp PCR System 2700, USA) w DNA s g. AccuPrep TM PCR Purification Kit DNA PrepMate TM Kit w PCR product w. RFLP. IL-6prom PCR w z SfaN1(Bioneer, Korea) w w š w 5' g^gacc 3' GeneAmp PCR System 2700(Applied Biosystems, USA) w. 0.2 ml p RFLP 10 µl š w z 37 o C 3, w z ƒ y 65 o C 20 g PCR w. 2%(w/v) ƒ w Mupid-α(Advance, Japan)» e RFLP w. RFLP-size based DHPLC w. IL- 6prom PCR e š š RFLP w z j»» w DHPLC» x w. f x k y w» w size standard (Trangenomic, USA) 5 µl 3 w w. z RFLP f 50 o C 0.75 ml/min w 5 µl w WAVEMAKER TM software w w gradient w š UV» 260 nm w. Double strand based DHPLC w. IL-6prom PCR 95 C 10 o k z 45 x x w x k z, f 58 o C š 5uL w 260 nm x q w. NuSieve agarose gel electrophoresis. IL-6vntr PCR 3%(w/v) NuSieve agarose gel (Cambrex Bio Science Rockland, Inc., Rockland, Me, USA) w Mupid-a (Advance, Japan)» e PCR w. NuSieve agarose w j» r» ƒ w. m. SPSS version 10.0 for Windows (SPSS Inc., Chicago, IL, USA) w chi-square t-test wš, ƒ allele p ù» w odds ratio 95% confidence interval w. w p wš v w p ww allele w corrected p(p corr) w, 0.05 w m w. š Analysis of IL-6prom RFLP IL-6prom PCR w z, SfaN1 w z w RFLP w z ƒ» y w. Fig. 1 lane 1 DNA molecular weight marker š, lane 2 w z w 568 bp PCR. Lane 3 G/C genotype 568 bp, 379 bp, 189 bp 3 š, lane 4 G/G genotype 379 bp, 189 bp 2 PCR ƒ. Size based DHPLC RFLP w PCR e š j k DHPLC w. f 50 o C w PCR ù xk j» w., Fig. 2 G/C genotype RFLP 3 vj ùkûš, G/G genotype 2 vj. 2006, Vol. 50, No. 2

126 Áû xá Á Á Double strand based DHPLC RFLP w PCR e š 95 o C 10 k z 45 x w x k z DHPLC w. f 58 o C w DNA ù t xk w x w. Fig. 3 G/C genotype 3 vj ùkûš, G/G genotype v j xk. Fig. 1. Analysis of a single nucleotide polymorphism at -174 (G/C) in the IL-6 gene promoter.

4 126 Áû xá Á Á Double strand based DHPLC RFLP w PCR e š 95 o C 10 k z 45 x w x k z DHPLC w. f 58 o C w DNA ù t xk w x w. Fig. 3 G/C genotype 3 vj ùkûš, G/G genotype v j xk. Fig. 1. Analysis of a single nucleotide polymorphism at -174 (G/C) in the IL-6 gene promoter. Three DNA fragments are shown after digestion with SfaNI: lane 1, size marker; lane 2, undigested 568 bp PCR product; lane 3, GC genotype; lane 4, GG genotype. Analysis of IL-6vntr IL-6vntr 760 bp (allele A), 680 bp (allele B), 640 bp (allele C), 610 bp (allele D) 4 x w. NuSieve agarose» w x y w (Fig. 4). v AB, 2 (1.6%); BB, 117 (95.1%); BC, 3 (2.4%); CC, 1 (0.8%) y š, p y Fig. 2. Elution profiles of RFLP analysis of the G to C transition located at the -174G/C of IL-6prom gene; a) undigested, b) G/C genotype, c) G/G genotype.

Interleukin-6 Gene Polymorphisms in Behcet s Disease 127 Fig. 3. Chromatograms produced DHPLC analysis of wild type and mutant type in IL-6prom. v BB, 77 (86.5%); BC, 12 (13.5%) ù kû.

5 Interleukin-6 Gene Polymorphisms in Behcet s Disease 127 Fig. 3. Chromatograms produced DHPLC analysis of wild type and mutant type in IL-6prom. v BB, 77 (86.5%); BC, 12 (13.5%) ù kû. AB type CC type ƒ û š v. w BB type BC type 98.6% w. p y BC genotype C alleleƒ v ùkû (Table 2). p y v 89 w v 123 DNA w PCR w. IL-6prom IL-6vntr, HLA-B51 gene x RFLP DHPLC w. IL-6prom p, IL-6vntr Fig. 4. PCR genotyping of a variable number of tandem repeat polymorphism in the 3' flanking region of the IL-6 gene. Lane 1 is for size marker, and lane 2 to 10 represents the genotype BB, BB, BC, BB, BB, BB, AB, BB, and CC, respectively. Table 2. Genotype and allele frequencies of the IL-6vntr polymorphisms in the Behcet s group and controls Controls No (%) Behcet s group No (%) Genotype BB 117 (97.5) 77 (86.5) BC 3 (2.5) 12 (13.5) Allele B 239 (98.0) 166 (93.3) C 5 (2.0) 12 (6.7) P=0.005 (P corr=0.01) and P=0.022 (P corr=0.044) for comparisons of the IL-6vntr between Behcet s group and controls. p y û ƒ š š (BB: û, 45.5% vs., 54.5%; BC: û, 8.3% vs., 91.7%; P=0.024, Pcorr=0.048) HLA- Table 1. Association between Behcet's disease (BD) and the IL-6vntr polymorphism after stratification of the subjects according to the results of HLA-B51 testing HLA-B51 negativity HLA-B51 positivity Control No (%) BD No (%) Control No (%) BD No (%) Genotype BB 103 (97.2) 33 (82.5) 14 (100) 44 (89.8) BC 3 (2.8) 7 (17.5)* 0 5 (10.2) Allele B 211 (97.7) 73 (91.3) 28 (100) 93 (94.9) C 5 (2.3) 7 (8.8)** 0 5 (5.1) * P=0.005 (P corr=0.01) and ** P=0.02 (P corr=0.04) for comparison between Behcet s group and controls in the HLA-B51-negative subjects P=0.578 and P=0.586 for comparison between Behcet s group and controls in the HLA-B51-positive subjects. 2006, Vol. 50, No. 2

6 128 Áû xá Á Á Table 3. Haplotype analysis between the IL-6prom and the IL-6vntr polymorphisms using the EH program Haplotype frequency in control subjects Haplotype frequency in Behcet s group P* OR IL-6prom*G/IL-6vntr*B IL-6prom*G/IL-6vntr*C IL-6prom*C/IL-6vntr*B IL-6prom*C/IL-6vntr*C * P values were estimated between subjects with and without each haplotype. B51 ùkþ (Table 1). w p y IL-6vntr * C (OR 3.5) x IL-6prom * G/IL-6vntr * C haplotype (OR 7.3) ùkû (Table 3). p ƒ w, MHC non- MHC p w ƒ w š š, ü x üv endothelial nitric oxide synthase (enos) x, IL-18 x, SLC11A1 x intercellular adhesion molecule-1 (ICAM-1) x non- MHC p w š. š TNF (promoter) ew TNF -1031C ƒ HLA-B51 w p x šw š, ü Lee TNFa -308 G/A, TNFß +252 G/A TNFR2 196 R/ M w p šw. p yw» w IL6vntr w ù e MHC non-mhc ƒ v w ƒ. 2004w w w. 2. Bowcock, A. M.; Kidd, J. R.; Lathrop, G. M., Daneshvar L, May LT, Ray A, et al. Genomics, 1988, 3, Xing, Z.; Gauldie, J.; Cox, G.; Baumann, H.; Jordana, M.; Lei, X. F., et al. J Clin Invest, 1998, 101, Fishman, D.; Faulds. G.; Jeffery, R.; Mohamed-Ali, V.; Yudkin, J. S.; Humphries, S., et al. J Clin Invest, 1998, 102, Ogilvie, E. M.; Fife, M. S.; Thompson, S. D.; Twine, N.; Tsoras, M.; Moroldo, M., et al. Arthritis Rheum, 2003, 48, Murray, R. E.; McGuigan, F.; Grant, S. F.; Reid, D. M.; Ralston, S. H., Bone, 1997, 21, Linker-Israeli, M.; Wallace, D. J.; Prehn, J.; Michael, D.; Honda, M.; Taylor, K. D., et al. Genes Immun, 1999, 1, Bagli, M.; Papassotiropoulos, A.; Knapp, M.; Jessen, F.; Luise, R. M.; Maier, W., et al. Neurosci Lett, 2000, 283, Georges, J. L.; Loukaci, V.; Poirier, O.; Evans, A.; Luc, G.; Arveiler, D., et al. J Mol Med, 2001, 79, Rauramaa, R.; Vaisanen, S. B.; Luong, L. A.; Schmidt- Trucksass, A.; Penttila, I. M.; Bouchard, C., et al. Arterioscler Thromb Vasc Biol, 2000, 20, Yamakawa, Y.; Sugita, Y.; Nagatani, T.; Takahashi, S.; Yamakawa, T.; Tanaka, S., et al. J Dermatol Sci, 1996, 11, Hirohata, S.; Oka, H.; Mizushima, Y., Cell Immunol, 1992, 140, Chang, H. K.; Cheon, K. S., J Korean Med Sci, 2002, 17, x 1. Verity, D. H.; Marr, J. E.; Ohno, S.; Wallace, G. R., Stanford MR. Tissue Antigens, 1999, 5421, 3-20.

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