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Transcription

1 1394/1/9: 1394/3/15: 1394!" #$/335/!! TIM C>T "#$% 3 2 1!"#$ /0" 12 3, $- #$)*"#&'( &'( ) $&.'"./ 0"1$ 23 ) $ 2/5 '( )* '+$ %&!"#$ : )*'+$ "89 $& )5 (T cell immunoglobulin and mucin) TIM29.'8:;8 < "89 =>$?"@$ L16 )$ 1G HI#=$ J"8 K )I#=$ <.D )"EF G )56 $ / 23 :;8 /C!"#$ 'P6$ D63 )Q$ N.8$ ) )56 'P6$ D63 TIM C>T N.E$M N# E ).;$!"#$ ) 0"1$ 2 TIM C>T N.E$M L16 8E R'/.'+ TU8 247S DI5 '/+ F 2& ) N# E 138 $ F 2& ) 247S 2"!"#$ ) 0"1$ 140 : PCR-RFLP 6 7" '+ Y@" DNA TIM C>T H7"$ /X689.'+ TU8 F)88 7" J"8 *.DEF G $ (Polymerase chain reaction-restriction fragment length polymorphism).'+ TU8 SPSSZET8 TIM C>T < I$ L16 N# E "Q!"#$ ) 0"1$ E CT X689 8E :.(P= 0/009OR= 4/08) D S DI5!"#$ '8M >6 9#3 )5 /\M N]6 4$ :;8 D )"EF G 2.8 5q33/2 T$* TIM 29 : 247S DI5!"#$ TIM C>T N.E$M )* 2Q8 )I#=$ <. /.'+$ L16 N.E$M!"#$ T cell immunoglobulin, and mucin 29:.'( 847S :* I.$ E"& )"+E C_E `(SC3 C >#S ^Z$ *( 8 IUF 1E 8'1 &Z$ :! +ZM 'Q8 )U$./01# 234) $ 5& TIM C>T '" %& " "#$ :(335) 33 a s mghakemi@med.mui.ac.ir $ $%&' $%&' * +!!"# -1 $ $%&' $%&' * +..-.) /.!!"# - -2 $ $%&' $%&' * + 1/ $ $%&' $%&' * + -4 $ $%&' $%&' * * 74-5 $ $%&' $%&' + * 9:; :

2 Th1 '8 )f% (6) /. O 0e )f% ( UVB 3-_. Th2 3(T helper) TIM-4 TIM-3 3TIM-1.(7) /B 'gn a %A MG 3&.D MA '8 TIM-1 31*B.(8) /B 'gn )f% + TCD4 'DA + 0 0R -% (Thermal conductivity detector4 ) ie. 8a 1" 0 / 3Th2 '&. /% '8 & 1/# 8PR TIM-1 3&j'./. " b+% 3(Invariant natural killer T) inkt 3'D k% 8%N '8 B '8 '_.(8) #. Tc1 3Th1 'DA TIM-3 8 Th17 'DA ). 1-0 (T cell1) A /. )f% 1" 0 / " '8 & b% /# O gn '8 TIM-3 3&j'.(8) /B l" 0. / 'DC 0= %4 0S" -B NN2 '8 _R.(5) _ '1KB2 =0 = 0. D S db & 3Galectin-9 'D 3T 'DA 3B 'DA # 7'8 # 0 TIM-3 b% # '/Ba l" /B% TIM-3 0 8,% 0. 3(8) /2.(9-10) _ '8 n /. )f% TIM-3-galectin-9 * E% O Th1 0J gn )( (Multiple sclerosis MS)!" +," )* ( ' &%$# %4 )* /# ," '8 & *. /9 3= 2 <.(1) /. CB & 1(= AB 1 2/5 HI " GF% DE% MS.(1) /B # O BKN ML% BK 3JE 3'DBA" 0 1% 3JE " 0=.(2) B <% 0 TL% DPS 3RL= DPQ -B BK ".B # /#.(3) /B ( UVB MS (Genome-wide association studies) A GWAS 6 / % GWAS 0 1K &/[ 3'12 0. D /# ZOB MS 0 6 J /' U-R BK " 1" 0 1% 0=./B/# # MS CD58 3IL-2R 3IL-7R '1K )*RN.(3) B # STAT3 (TIM) T cell immunoglobulin and mucin 1K 3 '8 0. D BK " ' /B HI ' 12 UVB Q 0=% ' K2!" 0 # 1*B TIM.(4) D 0R_ 5q33/2 Z. TIM-4 TIM-3 3TIM-1 '1K a= b+% 3a= & 0. D.(5) /# ' )2 3K2 db EJ &c%n_ 1K & 740

3 RRMS 09 3 / (Relapsing-remitting multiple sclerosis) 0= 3. +*B +( " Q/9 Expanded disability status scale) B%B (1 8/=) _ 1" (EDSS 0V 1/ ) R !" /BN 0V 1/ ( & B 0P= 1 8VB /' './B/# rib /'# _ 1" 0 3/B &./B/# 1*' = & fb _ #-N s6 0. %N b% 30PJ 0' /# /F% 1(Ap #-N Z" aeb./_ T +. 0BDS /. D.# R 9 '0 1 2 cc /V 0 R ' (Ethylenediaminetetraacetic acid) EDTA 12 DNA /# 0R_ (Cinagen Co, Iran) Genomic DNA extraction D. A tj (Qiagen Inc, Germany) Qiagen./_ ui /B D.# P O 'gn )f% 8. Z/"./# 0 O 3Th1 'DA b% /# F% D*9 'U. /B S" ' 3&j' (4 db).(8) # 1- L% UVB 0J AI MPJ TIM-3 1K /%c.b H 1 a 0. D /# ZOB HI ' 0. = 12./B0#,Q CB B% /B% 1K & HI ')*RN 1 BZN '* &j' /Ba 0 8,% & 3Ta GF% 'D " )*RN o+% 5/' 3S9 0PJ./# ZOB MS TIM-3 1K -1541C>T /'# - 0PJ & (<) ' P= /Be KB q,i /F% 0 ' (Ap.1 P 0/360 0/200-31/3 ± 6/6 60 (43/66) 78 (56/33) (100) EDSS: expanded disability status score 32/1 ± 8/4 52 (37/14) 88 (62/67) 92 (65/70) 24 (17/10) 15 (10/70) 9 (6/40) 140 (100) ( -./ ± ) * ) ) 6 1 1/ EDSS 9:

4 MS TIM C>T _2 8K RFLP8,E R v%bk abeb 138 bp 61 bp MPJQ 3/p 2 v%bk 199 bp 0PJQ 3CC <a 138 bp 361 bp MPJQ TT Ma ' ' /# CT Ma ' v%bk abeb 3199 bp +,-)* PCR-RFLP # $# %& '(.1 TIM C>T.(1 #)!" 9(: : bp DNA ladder :M 23 CC ;&<= 9(: :5 4 6CT ;&<' PCR-RFLP: Polymerase chain reactionrestriction fragment length polymorphism 0BB &/[ 3CB DEp 1x f 0 rib R,% Mp 0 HI 'v%bk 1 D* N% D.# 0 % &P% D(= &P% CB./# 8 (Bioneer; Korea /B) ae 2 /2 D 0 CB %.D# DVJ 16 0I*B SPSS -RZB 0PJ & f 0 (version 16, SPSS Inc., Chicago, IL) 2 χ 1 2.DR_ Q A CB. = & BR $ % 0 *V D(= 'v%bk 2 BR $ % 0*V 3&j' w 0 0 TIM C>T v%bk Polymerase chain reaction- ) PCR-RFLP (restriction fragment length polymorphism U. A /.DR_ Q 0PJ /# 9x 'N (PCR) N OB 5 -TCCAGCCTGAGGCTCTTGTTT-3 # 35 -ATGCTCATTGTTGTTGGAACAG-3 fb )*RN /B_ 199 bp 0PJQ 310X R 3 µl9 30 µl)o9 (Deoxynucleotide triphosphate)./# y% U. 200 µm 1/5 mm 315 pmol N ' 0/5 µm 3dNTP TaqDNA- Polymerase.(Cinnagen Co, Iran) /# ZOB DNA 0/3 µl3mgcl ng (Techne Co, UK) a b% PCR 0B 1%B UN 09 Mp 0 # 0[ 30 0VQ 5 M/ 0 94ºC 0BG 30 M/ 0 58ºC 30BG 30 M/ 0 94ºC 09 (B 0BG 30 M/ 0 72ºC = 0VQ 5 M/ 0 72ºC (B 5 µl3pcr ZOB DEp /p 1/5 B x 1x f 0./# _2 8K PCR Mk,E % 199 bp '/B =./# R.. ZOB 1eB (Ultraviolet) UV (Afratoos, Iran) Green viewer DR zb.dr_ Q A '/B /'e D(= 10 µl3pcr U. DEp )-B2 1 µl 1x N v%bk &P% f 0 PCR 8,E 0 (Fermentas Co., Germany) BsaJI Gk/E./_ 0B 56ºC D" 2 M/ 742

5 )*RN 0. /' 1eB CB &.(2 8/=) U-R l" /B% 3TIM C > T 3&j'._ MS 0 6 /P ZOB _ '2 BR 0*V 3 8/= _ T 2 BR /' 1eB 0. D 0R_ 6 B# 3&j' } /'# _ e R 0*V T 2 9 R 0. e 4 3 & /QR ( ' &%$#!" # r*e -. +," )* & *. %4 )* U'. ~I% /. 09 +," '8 'ZN D/' 86 'D/Ba.(11) # qie 3+," o+% D(=./# A _ HI TIM C > T )*RN Logistic regression 1 2 P {J 1" 0 P < 0/050./_ A./# 0R_ fb U' N & '_ 1" 0 ) R 138 ' '_./R_ Q 0PJ 3/'# & fb 3_ '.D /2 1 8/= _ %BK $ %./B/# 1*' = Hardy-Weinberg 0P /'# _ tx _ %BK BR.D# _ 2./_ 0*Vχ o+% MS CT v%bk BR 1 2 (P = 0/009 3OR = 4/08) 1eB P " - TIM C>T?)'@ 9(: (-,& >.2 OR (CI %95) 4/08 (1/32-12/64) P 0/009 OR: odds ratio; CI: Confidence interval 134 (97/10) 4 (2/90) (100) 125 (89/10) 15 (10/90) (100)! CC CT TT 9: 85 " - TIM C>T?)'@ OR (CI %95) 4/08 (1/32-12/64) P 0/009 OR: odds ratio; CI: Confidence interval 272 (98/60) 4 (2/90) 276 (100) A (-,& > (94/60) 15 (10/90) 276 (100) &'% C T 9:

6 0. D 1eB &[ Hui DP= / % D%2 CT v%bk /2 D 0 0OB 0OB & 0. /# o+% D 9 &.D *' S9 U' N 0B_ ' 3&[ Han DP= 0PJ 0. v%bk & & P o+% CB /B 1eB / % D%2.(17) /# S9 U' N 'MA% 0OB /B% ƒq '0R 12./# MA JE " &j' B 3Th1 '8 TIM = /B# 1 /B '8 KR. 3Th17 ' *BK%N '8 & 1K & U(= GF% N 3/B UVB MS 0=.(18) D*B fb MS )*B 0. & 0 0=% 3/# / % D%2 0e UVB 0 % )/# 12 0PJ & &.) MS )*RN & ab U' N & '0R 3dO MS TIM C > T & o+% 0 %n- 'DP= e MPJ }/# 1K & UVB %tq f *+./# S MS /# #. 0B1N p9 0V & 0B1N # 0 B/+ "- +R D 1(Ap #-N Z" aeb = $ 1(Ap #-N Z" aeb 'gn Th1/Th2 '8 & 8P% 8P% UVB 3/# ZOB MPJ./# )( 'gn )f% E% Th1/Th2 & /B% AI ".(12) /B. M+G 0. TIM 1K 0= 3/' Q GF% DE% 8P% '8 8P% )f% 12 UVB 3 MPJ 'gn )f% 30OB Th1/Th2 /B ' _# /B% & D / M+G 0 -B ' _ & 1 R/' B" 0.(13-14) _ J TIM 1K B!" TIM-3 UVB 'gn )f% 0. /# " # D & 312 1K U(= = ' 0 /' Q GF% DE% 8.(15-16) /B. )*RN 'v%bk BR 30PJ & MS TIM C>T 12 o+% &j' ) R 0*V.DR_ Q 1(Ap DP= CT v%bk BR 0. 1eB 0PJ & CB /# ) R e MS & CT v%bk & P o+%.(or = 4/08 3P = 0/009) = 3+P " 'an 'O*= < 1K )*RN 1 a*+' + #-_ ' = TIM-3 & o+%./b MPJ }D#/B = / % D%2 3)*RN /# ZOB 0PJ./# V '12 CB

7 3/B0#!9 0PJ & 0. R._ -a Z(# Q2 r= 0 &/.D. &F% % 1kN 6( )B. 1B References 1. Alatab S, Hossein-nezhad A, Mirzaei K, Mokhtari F, Shariati G, Najmafshar A. Inflammatory profile, age of onset, and the MTHFR polymorphism in patients with multiple sclerosis. J Mol Neurosci 2011; 44(1): Lorentzen AR, Melum E, Ellinghaus E, Smestad C, Mero IL, Aarseth JH, et al. Association to the Glypican-5 gene in multiple sclerosis. J Neuroimmunol 2010; 226(1-2): Sadovnick AD. Genetic background of multiple sclerosis. Autoimmun Rev 2012; 11(3): Freeman GJ, Casasnovas JM, Umetsu DT, DeKruyff RH. TIM genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity. Immunol Rev 2010; 235(1): Rodriguez-Manzanet R, DeKruyff R, Kuchroo VK, Umetsu DT. The costimulatory role of TIM molecules. Immunol Rev 2009; 229(1): Lee J, Phong B, Egloff AM, Kane LP. TIM polymorphisms--genetics and function. Genes Immun 2011; 12(8): Zhao W, Li Y, Wang Z, Li K, Yang S. Molecular characterization of the porcine TIM-3 gene. Scand J Immunol 2011; 73(1): Yeung MY, McGrath M, Najafian N. The emerging role of the TIM molecules in transplantation. Am J Transplant 2011; 11(10): Zhu C, Anderson AC, Schubart A, Xiong H, Imitola J, Khoury SJ, et al. The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity. Nat Immunol 2005; 6(12): Hastings WD, Anderson DE, Kassam N, Koguchi K, Greenfield EA, Kent SC, et al. TIM-3 is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. Eur J Immunol 2009; 39(9): Xu G, Cheng L, Lu L, Zhu Y, Xu R, Yao X, et al. Expression of T-cell immunoglobulin- and mucin-domain-containing molecule-1 (TIM-1) is increased in a mouse model of asthma and relationship to GATA-3. Life Sci 2008; 82(11-12): Weinshenker BG. Epidemiology of multiple sclerosis. Neurol Clin 1996; 14(2): Kidd P. Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern Med Rev 2003; 8(3): Meyers JH, Sabatos CA, Chakravarti S, Kuchroo VK. The TIM gene family regulates autoimmune and allergic diseases. Trends Mol Med 2005; 11(8): Khademi M, Illes Z, Gielen AW, Marta M, Takazawa N, Baecher-Allan C, et al. T Cell Igand mucin-domain-containing molecule-3 (TIM-3) and TIM-1 molecules are differentially expressed on human Th1 and Th2 cells and in cerebrospinal fluid-derived mononuclear cells in multiple sclerosis. J Immunol 2004; 172(11): Nuchnoi P, Ohashi J, Kimura R, Hananantachai H, Naka I, Krudsood S, et al. Significant association between TIM1 promoter polymorphisms and protection against cerebral malaria in Thailand. Ann Hum Genet 2008; 72(Pt 3): Xu J, Yang Y, Liu X, Wang Y. The C>T and G>T of TIM-3 polymorphisms are associated with rheumatoid arthritis susceptibility in a Chinese Hui population. Int J Immunogenet 2011; 38(6): Monney L, Sabatos CA, Gaglia JL, Ryu A, Waldner H, Chernova T, et al. Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease. Nature 2002; 415(6871):

8 Journal of Isfahan Medical School Received: Vol. 33, No. 335, 3 rd Week, July 2015 Accepted: The Frequency of TIM C>T Polymorphisms and its Association with Multiple Sclerosis Fariba Mazrouei 1, Mazdak Ganjalikhani-Hakemi PhD 2, Rasoul Salehi PhD 3, Fereshteh Ale-Sahebfosoul PhD 4, Masoud Etemadifar MD 5, Hamid Zarkesh-Esfahani PhD 6 Abstract Original Article Background: Multiple sclerosis (MS) is an organ-specific autoimmune disease in central nervous system, which about 2.5 million people around the world are suffering from it. Various factors including environmental and genetic factors are involved in the incidence of the disease. T cell immunoglobulin and mucin (TIM) gene is one of the genetic factors which recently demonstrated that has critical role in autoimmune diseases. In this study, the frequency and the association of TIM C>T polymorphisms with multiple sclerosis disease in Isfahan, Iran, population were investigated. Methods: Blood samples were collected from 140 patients with multiple sclerosis and 138 healthy controls. After DNA extraction, the TIM C>T polymorphism was detected via polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques. The results were analyzed with the SPSS 16.0 software package. Findings: The frequency of CT genotype in patients with multiple sclerosis was higher than the control group and there was a significant association between multiple sclerosis and TIM C>T polymorphism in Isfahan population (P = 0.009, OR = 4.08). Conclusion: T cell immunoglobulin and mucin gene family (TIM) is located on human chromosome 5q33.2 and play a critical role in regulating immune responses including allergy, transplant tolerance and autoimmunity. This study was done based on the results of previous studies about the association of TIM C>T with rheumatoid arthritis, since the mechanisms of pathogenesis of this disease and multiple sclerosis are similar. Our results showed that TIM C>T polymorphism is associated with multiple sclerosis in Isfahan population. Keywords: Multiple sclerosis, T cell immunoglobulin and mucin (TIM) gen, Polymorphism Citation: Mazrouei F, Ganjalikhani-Hakemi M, Salehi R, Ale-Sahebfosoul F, Etemadifar M, Zarkesh-Esfahani H. The Frequency of TIM C>T Polymorphisms and its Association with Multiple Sclerosis. J Isfahan Med Sch 2015; 33(335): MSc Student, Department of Immunology, School of Medicine AND Student Research Committee, Isfahan University of Medical Sciences, Isfahan, Iran 2- Assistant Professor, Cellular and Molecular Immunology Research Center, Isfahan University of Medical Sciences, Isfahan, Iran 3- Associate Professor, Department of Cellular and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran 4- Assistant Professor, Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran 5- Professor, Department of Neurosciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran 6- Associate Professor, Department of Biology, School of Science, University of Isfahan, Isfahan, Iran Corresponding Author: Mazdak Ganjalikhani-Hakemi PhD, mghakemi@med.mui.ac.ir 746