research article association of fas and fas ligand...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 176741 6 pageshttpdxdoiorg1011552013176741

Research ArticleAssociation of FAS and FAS Ligand Genes Polymorphism andRisk of Systemic Lupus Erythematosus

Bita Moudi1 Saeedeh Salimi12 Farzaneh Farajian Mashhadi13

Mahnaz Sandoughi4 and Zahra Zakeri4

1 Cellular and Molecular Research Center Zahedan University of Medical Sciences Zahedan 9816743175 Iran2Department of Clinical Biochemistry School of Medicine Zahedan University of Medical Sciences Zahedan 9816743175 Iran3Department of Pharmacology School of Medicine Zahedan University of Medical Sciences Zahedan 9816743175 Iran4Department of Internal Medicine School of Medicine Zahedan University of Medical Sciences Zahedan 9816743175 Iran

Correspondence should be addressed to Saeedeh Salimi sasalimiyahoocom

Received 25 August 2013 Accepted 17 September 2013

Academic Editors A La Cava and M Pinti

Copyright copy 2013 Bita Moudi et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

FASFASL pathway plays a critical role in maintaining peripheral immune tolerance therefore the apoptosis genes Fas andFas ligand (FasL) could be suitable candidate genes in human SLE susceptibility Materials and Methods In this case-controlstudy 106 SLE patients and 149 sex age and ethnicity matched healthy controls were genotyped for the Fas A-670G and FasLC-844T polymorphisms by polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) Results Thefrequency of -670AA genotype was significantly higher in SLE patients than control group and the risk of SLE was 21-fold greaterin subjects with AA genotype (119875 = 003) The frequency of -670A allele was significantly higher in SLE patients than in controlstoo (58 versus 49 119875 = 003) The -844CC genotype frequency was significantly higher in SLE patients than in healthy controlsand the risk of SLE was 28-fold greater in these subjects (119875 = 001) The C allele frequency was significantly higher in patientsthan in controls (69 versus 49 119875 = 0001) Increased SLE risk was observed in individuals with combined effect of Fas-670AAand FasL-844CC genotypes (119875 = 0001) Conclusion Fas-670AA and FasL-844CC genotypes were associated with SLE risk andcombined effect of -670AA and -844CC genotypes might increase SLE susceptibility

1 Introduction

Systemic lupus erythematosus (SLE) is a prototype autoim-mune disease that is easily confused with many disordersand can affect different organs including skin heart lungsblood vessels liver kidneys joints and nervous system Thiscomplication is characterized by malregulation of T and Bcells causing production of excessive amounts of differentautoantibodies and formation of immune complexes againstdifferent nuclear antigens [1 2]

The prevalence of SLE ranges from approximately 40cases per 100000 individuals among Northern Europeans tomore than 200 per 100000 persons among blacks [3] Thisdisease occurs in women ten times more frequently than inmen especially in women during their reproductive years[4 5] Similar to other autoimmune diseases SLE showed an

inheritance pattern and multiple candidate genes have beenstudied with SLE susceptibility [4]

Several evidence indicates that abnormalities in the apop-tosis (programmed cell death) process could be related todevelopment of SLE and the elimination of autoreactive Tor B cells is impaired in this disease [6 7] Delayed clear-ance of apoptotic cells by phagocytes was observed in SLEpatients [8] Also accelerated apoptosis of circulating cellswas observed in SLE patients which causes the specific lupusautoantigens for instance double stranded DNA (dsDNA)to be exposed on surface blebs [6 9]

The importance of apoptosis in immune tolerance wasdistinguished on a study of the genetic defects in FAS and itsligand (FASL) in mouse models of human SLE [10]

FASFAS ligand system is the main extrinsic pathway forthe initiation of apoptosis in numerous cells and tissues [11]

2 The Scientific World Journal

Table 1 The primer sequences annealing temperature restriction enzymes and fragments size of FAS and FASL polymorphisms

Target sequence Primer sequence Annealingtemperature Restriction enzyme PCR product RFLP

fragmentsFAS A-670Grs1800682

Forward 51015840-CTACCTAAGAGCTATCTACCGTTC-31015840Reverse 51015840-GGCTGTCCATGTTGTGGCTGC-31015840 [29] 61∘C MvaI (BstNI) 223 A 223

G 44 189FASL C-844Trs763110

Forward 51015840-CAGCTACTCGGAGGCCAAG-31015840Reverse 51015840-GCTCTGAGGGGAGAGACCAT-31015840 [19] 63∘C BseMI (BsrDI) 401 T 401

C 223 168

FAS is a membrane protein of the transmembrane tumornecrosis factor superfamily of death receptors and plays animportant role in apoptotic signaling in different cells Thisreceptor induces apoptosis by binding to its natural ligandFASL is a member of tumor necrosis factor superfamily andinitiates the death signal cascade which ultimately results inapoptotic cell death Although FAS is existing constitutivelyon the surface of resting cells in low levels it is expressedprominently on the surface of activated T cells [12]

FAS and FASL genes are located on chromosomes 10q241and 1q23 respectively [13 14] Several polymorphisms havebeen recognized in FAS gene and G to A replacement atnucleotide position -1377 (FAS G-1377A rs2234767) in thesilencer region as well as A to G replacement at nucleotideposition -670 (FAS A-670G rs1800682) in the enhancerregion are studied more than others [15] The FASL geneC-844T polymorphism is located in the binding site of tran-scription factor CAATenhancer-binding protein 120573 Amarkedly elevated basal expression of FASL is observedin subjects with the C allele compared with the T allele atnucleotide position -844 (FASL C-844T rs763110) in thepromoter region [16]

Several studies investigated the role of FAS and FASLgene polymorphisms in the etiology of SLE however therole of FAS and FASL gene polymorphisms in lupus has notbeen conclusively established [16ndash18] Therefore the presentstudywas designed to investigate the association of apoptosis-related genes FAS and FASL polymorphisms with SLEsusceptibility and clinical characteristics in Iran

2 Materials and Methods

21 Characteristics of Study Populations Approval of thestudy was obtained from the Ethics Committee of ZahedanUniversity of Medical Sciences This case-control studyperformed on 106 SLE patients (9 men and 97 women) witha mean age of 318 plusmn 78 years who fulfilled the 1998 Amer-ican College of Rheumatology (ACR) criteria was referredto rheumatology clinics of Ali-Ebne-Abitaleb hospital inZahedan from 2011 to 2013 The control group consists of 149age sex and ethnically matched volunteers (13 men and 139women) with a mean age of 3290 plusmn 133 years The healthycontrols were selected following a questionnaire survey toexclude any systemic inflammatory and autoimmune dis-eases with negative ANA test and no family relation withlupus patients A written informed consent was obtainedfrom all participants

22 Genotyping Genomic DNAwas extracted from ethylenediamine tetra-acetic acid (EDTA) anticoagulated peripheral

Table 2 Demographic characteristics of SLE patients and controls

Parameter SLE119873 = 106

Controls119873 = 103

P value 1199092

Age (year) 318 plusmn 78 3290 plusmn 133 053 004Sex (malefemale) 997 895 05 004Race N ()

Persian 49 (46) 53 (51) 027 027Balouch 57 (54) 50 (49)

blood byDNA isolation kit (Roche Germany) FAS and FASLpolymorphisms were identified by the PCR-RFLP method

The primer sequences annealing temperature restrictionenzymes and fragments size are shown in Table 1

Polymerase chain reaction was performed in thermalcycler (Eppendorf-Mastercycler Gradient-Germany) in a25 120583L final volume which contained 20 pmol of each primer01mmol of dNTP (Fermentas Lithuania) 03 120583g of genomicDNA 15mmolL of MgCl

2 25 120583L of 10X PCR buffer and 15

unit of TaqDNApolymerase (Fermentas Lithuania) accord-ing to the following protocol initial denaturation at 95∘C for5min 30 cycles of denaturation at 95∘C for 1min annealingfor 1min at 61∘C for FAS A-670G and 63∘C for FASL C-844T polymorphisms extension at 72∘C for 2min and finalextension at 72∘C for 5 minutes PCR and digested productswere separated by electrophoresis on a 2 agarose gel andvisualized by ethidium bromide staining

23 Statistical Analyses The statistical analysis of the datawas performed using the SPSS 150 software Quantitativedatawere presented asmeanplusmn standard deviationGenotypesand alleles were compared between groups by use of 1205942 testComparison of quantitative variants between two groups wasassessed by Studentrsquos 119905-test The association between quali-tative variables was analyzed using the chi-square or Fisherexact tests Variations with 119875 values less than 005 were con-sidered significant Odds ratio and 95 confidence intervalwere calculated to evaluate the strength of the associations

3 Results

Clinical and demographic characteristics of SLE patients andcontrol samples were shown in Table 2 The SLE patients andcontrols did not differ significantly with respect to sex ageand ethnic parameters Skin manifestations arthritis lupusnephritis and neuropsychiatric manifestations developed in83 87 22 and 14 of patients respectively during thecourse of their disease

The Scientific World Journal 3

Table 3 Genotype and allele frequencies of FAS A-670G and FASLC-844T polymorphisms in SLE patients and controls

SLE119873 = 106

Control119873 = 149

P value Odds ratio(95 CI)

FAS A-670GAA n () 34 (32) 37 (25) 003 21 (1ndash41)

AG n () 55 (52) 73 (49) 03 12(07ndash22)

GG n () 17 (16) 39 (26) Ref = 1A n () 123 (58) 147 (49) 003 14 (1-2)G n () 89 (42) 151 (51) Ref = 1

FAS ligand C-844T

CC n () 50 (472) 51 (342) 001 28(12ndash62)

CT n () 46 (434) 80 (47) 01 18(08ndash42)

TT n () 10 (94) 28 (188) Ref = 1

C n () 146 (69) 162 (54) 0001 19(13ndash27)

T n () 66 (31) 136 (46) Ref = 1

Allele and genotype frequencies of both polymorphismswere in Hardy-Weinberg equilibrium in case and controlgroups The frequency of the AA genotype of FAS A-670Gpolymorphism was significantly higher in SLE patients thanin controls and the risk of SLEwas 21-fold greater in individ-uals with AA genotype in comparison to GG genotype (OR21 (95 CI 1 to 41) 119875 = 003) The frequency of A allele wasstatistically higher in SLE group (58) than in controls (49)too (OR 14 (95 CI 1 to 2) 119875 = 003)

Furthermore the frequency of CC genotype of C-844Tpolymorphism of FASL gene was significantly higher in SLEpatients than in healthy controls and the risk of SLE was 28-fold higher in individuals with CC genotype in comparisonto TT genotype (OR 28 (95 CI 12 to 62) 119875 = 001) Thefrequency of C allele was 69 and 54 percent in SLE and controlgroups respectively and was significantly different (OR 19(95 CI 13 to 27) 119875 = 0001) (Table 3)

Analysis of the synergic effects of various genotypes onrisk of SLE showed that the individuals with the AATTgenotype for FASA-670G and FASLC-844T polymorphismsshowed 37-fold increase in SLE risk compared to GGTTgenotype (OR 37 (95 CI 11 to 125) 119875 = 003) Also 56-fold increased risk of SLE was found by comparing AATTgenotype with GGTT GGCT and AGTT genotypesbetween the case and control groups (OR 56 (95 CI 19to 156) 119875 = 0001) (Table 4)

Moreover no minor alleles frequency differences wereobserved between two ethnic groups and different manifes-tations

4 Discussion

Established evidence shows that failure of apoptosis orprogrammed cell death is considered to contribute to thedevelopment of autoimmune disorders characterized by the

impaired elimination of autoreactive T cells or B cells [6 7]Moreover it appears that increase of lymphocyte apoptosisand deficient phagocyte-mediated clearance of apoptoticcells could contribute to B-cell hyperactivity and subsequentautoantibodies overproduction [8] Apoptotic cells are themajor source of autoantigens production in SLE patho-genesis The autoantigens trigger autoantibody productionand subsequently induce systemic autoimmunity leading toformation of many apoptotic blebs [8 19 20]The acceleratedapoptosis may be a direct consequence of alterations inproteinsgenes related to programmed cell death such as FASand FASL [21]

Although the role of FAS-mediated apoptosis in immu-nity and elimination of autoreactive lymphocytes are clearbut the function of FAS or FASL as the markers of apoptosisin autoimmune disorders especially SLE needsmore investi-gations [22] Nevertheless several studies indicated that FASmay be involved in the defective apoptosis of T cells in SLEand resistance to the FAS-mediated apoptosis has beenobserved in T cells from SLE patients [23 24]

The FASFASL system stimulates immune tolerance bythe apoptosis induction and elimination of activated T Blymphocytes and macrophages [24]

Mouse model investigations indicated that FAS or FASLmutations could initiate autoimmune diseases The essentialrole could be played by the FASFASL system in the mainte-nance of immune tolerance and inhibition of autoimmunedisease has been clearly established [25]

In addition abnormal expression of FAS and FASL on Tand B lymphocytes in SLE patients has been demonstrated inseveral reports [26 27]

The FAS gene has a A to G substitution in the promoterregion at position -670 which decreases FAS production [15]Also higher basal expression of FASL gene is observed inassociation with the C allele compared with the T allele ofC-844T polymorphism of FASL [16]

Since these two polymorphisms could alter basal expres-sion of FAS and FASL genes recent studies have suggestedthat FAS and FASL gene polymorphisms may play importantroles in pathogenesis of SLE

In the present study a significant difference in AAgenotype frequency was observed between SLE patients andcontrols in comparison to GG genotype and the risk of SLEwas 21-fold higher in subjects with AA genotype Also thefrequency of A allele was significantly higher than G allele inSLE patients than in healthy controls

For the first timeWu et al in 1996 reported an associationbetween a 84 bp deletion within exon 4 (28 amino aciddeletion) of the FASL gene and SLE in USA This deletion inextracellular domain of the FASL gene was shown to causedefect in cell death and FASL-mediated apoptosis [28] Fur-thermore in 1996 Huang et al screened the whole 51015840 flankingregion of the FAS gene and identified the A-670G andG-1377A polymorphisms within the silencer and enhancerregions of this gene respectively [29] Then in 1999 theassociation between FAS A-670G polymorphism and SLEwas investigated in Australians and higher frequency of GGgenotype in SLE patients with photosensitivity or oral ulcerswas found [18]


Table 4 Synergic effect of FAS A-670G and FAS ligand C-844T polymorphisms in SLE patients and controls

FAS A-670GFASL C-844T SLE119873 = 106

Control119873 = 149

P value OR (95 CI)

GGTT 7 (66) 13 (99) Ref = 1AGCT 29 (274) 37 (259) 03 14 (05ndash42)AGCC 25 (236) 30 (21) 03 14 (05ndash46)AATT 2 (19) 8 (56) 03 045 (008ndash29)AACT 14 (132) 18 (126) 04 15 (05ndash5)AACC 18 (17) 9 (63) 003 37 (11ndash125)GGTT GGCT AGTT 11 (104) 30 (20) Ref = 1AGCT 29 (274) 37 (259) 006 21 (09ndash5)AGCC 25 (236) 30 (21) 005 23 (09ndash54)AATT 2 (19) 8 (56) 05 067 (013ndash33)AACT 14 (132) 18 (126) 05 21 (08ndash5)AACC 18 (17) 9 (63) 0001 56 (19ndash156)

An association between FAS A-670G polymorphism anddevelopment of anti-RNP antibodies was observed by Lee etal in SLE patients [30]

In consistent with present study Kanemitsu reported thatthe A allele of FAS A-670G but not G-1377A was significantlyassociated with SLE [31]

In contrast to present study Araste et al reported no dif-ferent allelic distributions at position -670 between patientsand controls in an Iranian population But they observedhigher GG genotype and G allele distribution at position-1377 in SLE patients Also they found higher soluble FASand FAS ligand levels in the patient group and lower amountsof serum anti-SSBLa in patients with the -670GG genotype[32]

Our data supported two separate meta-analysis resultsthat performed recently by Lee et al in 2012 and Xiang et alin 2013 [17 33] Lee et al carried out a meta-analysis tosearch the effect of FAS A-670G polymorphism on suscep-tibility to autoimmune rheumatic diseases They revealedan association between rheumatic diseases and the FAS A-670G polymorphism in the dominant model Moreover theyindicated an association between the FAS-670 G allele carrierand rheumatic diseases in theAsianTheyobserved a negativeassociation between the FAS-670 G allele and SLE suscep-tibility (OR = 0578 95 CI = 0358ndash0934 119875 = 0025) Inanother meta-analysis that conducted by Xiang et al similarresults were achieved [17 33]

Moreover we revealed that CC genotype of FASL C-844T polymorphism was significantly higher in SLE patientsthan in healthy controls and the frequency of C allele wasstatistically higher in SLE patients

Although several studies are performed to investigate theeffect of FASL gene mutations on SLE susceptibility to datethere are only a few published reports about the associationbetween C-844T polymorphism and SLE [16 33]

In 2003 Wu et al sequenced the coding region and prox-imal 1 kb of promoter region of FASL gene in 14 SLE patients30 rheumatoid arthritis patients and 7 normal subjectsThey found an SNP at -844 within the FASL promoter region

(T allele in addition to the C) in normal populations withan allele frequency of 082 for African Americans Also theyreported increased expression of FASL in individuals with-844CC genotype that could enhance autoimmunity risk Inthis study a significant association between -844CC genotypeand SLE was reported in the African Americans in compar-ison to ethnically matched healthy controls Although theyshowed higher frequency of -844CC genotype in CaucasianSLE patients the difference was not statistically significant[16]

Similarly in 2005 Chen et al revealed that -844CC geno-type and C allele were associated with lupus susceptibilityhowever they found that the -1094AC polymorphism is notassociated with SLE [33]

As mentioned above our findings are in line with thosereported by Wu et al and Chen et al results and supportedthe association of FAS ligandC-844T polymorphism and SLEhypothesis

Since the effect of a single SNP is generally minor it isbelieved that the combination effects of functionally relevantSNPs may contribute to increased SLE risk So we studiedthe combined polymorphism of FAS and FASL genes and SLEsusceptibility

Our results revealed that the combination of AACCgenotypes of FAS A-670GFASL C-844T polymorphismsmight increase SLE risk approximately 37-fold comparedto GGCC genotype Also 56-fold increased SLE risk wasobserved in individuals withAACC genotype in comparisonwith GGTT GGCT and AGTT genotypes To the best ofour knowledge the present study is the first investigationabout the synergic effect of FAS A-670G and FAS ligandC-844T polymorphisms on SLE susceptibility These resultsneed more study to explore gene-gene interactions and itsrelation to SLE susceptibility to identify individuals atincreased risk of SLE and to develop preventive strategies

There were not any differences inminor allele frequenciesbetween two ethnic groups and various SLE manifestations

In conclusion we found an association between AAgenotype of FAS A-670G polymorphism and CC genotype


of FASL C-844T polymorphism with SLE risk Also thecombination of AACC genotypes of FAS A-670G and FASLC-844T polymorphisms indicated higher SLE risk in thisIranian population

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgment

The authors would like to appreciate the research deputy ofZahedan University of Medical Sciences for financial supportof this research

References

[1] A Rahman andDA Iseberg ldquoMechanisms of disease Systemiclupus erythematosusrdquo The New England Journal of Medicinevol 358 pp 929ndash939 2008

[2] B L Kotzin ldquoSystemic lupus erythematosusrdquo Cell vol 85 no3 pp 303ndash306 1996

[3] A E Johnson C Gordon R G Palmer and P A Bacon ldquoTheprevalence and incidence of systemic lupus erythematosus inBirmingham England relationship to ethnicity and country ofbirthrdquo Arthritis and Rheumatism vol 38 no 4 pp 551ndash5581995

[4] S K Nath J Kilpatrick and J B Harley ldquoGenetics of humansystemic lupus erythematosus the emerging picturerdquo CurrentOpinion in Immunology vol 16 no 6 pp 794ndash800 2004

[5] V H A Rus andM C Hochberg ldquoSystemic lupus erythemato-susrdquo in Epidemiology of the Rheumatic Diseases A J H MSilman Ed pp 123ndash140 Oxford University Press 2nd edition2001

[6] F Andrade L Casciola-Rosen and A Rosen ldquoApoptosis insystemic lupus erythematosus clinical implicationsrdquoRheumaticDisease Clinics of North America vol 26 no 2 pp 215ndash2272000

[7] L E Munoz C Van Bavel S Franz J Berden M Herrmannand J Van Der Vlag ldquoApoptosis in the pathogenesis of systemiclupus erythematosusrdquo Lupus vol 17 no 5 pp 371ndash375 2008

[8] Y Ren J Tang M Y Mok A W K Chan A Wu and C SLau ldquoIncreased apoptotic neutrophils and macrophages andimpaired macrophage phagocytic clearance of apoptotic neu-trophils in systemic lupus erythematosusrdquo Arthritis andRheumatism vol 48 no 10 pp 2888ndash2897 2003

[9] D R Green ldquoOverview apoptotic signaling pathways in theimmune systemrdquo Immunological Reviews vol 193 pp 5ndash9 2003

[10] S Nagata and T Suda ldquoFas and Fas ligand lpr and gldmutationsrdquo Immunology Today vol 16 no 1 pp 39ndash43 1995

[11] S Nagata ldquoFas and Fas ligand a death factor and its receptorrdquoAdvances in Immunology vol 57 pp 129ndash144 1994

[12] T Kamradt and N A Mitchison ldquoTolerance and autoimmu-nityrdquo The New England Journal of Medicine vol 344 no 9 pp655ndash664 2001

[13] I Behrmann HWalczak and P H Krammer ldquoStructure of thehuman APO-1 generdquo European Journal of Immunology vol 24no 12 pp 3057ndash3062 1994

[14] T Takahashi M Tanaka J Inazawa T Abe T Suda andS Nagata ldquoHuman Fas ligand gene structure chromosomal

location and species specificityrdquo International Immunology vol6 no 10 pp 1567ndash1574 1994

[15] M Pinti L Troiano M Nasi et al ldquoGenetic polymorphisms ofFas (CD95) and FasL (CD178) in human longevity studies oncentenariansrdquo Cell Death and Differentiation vol 9 no 4 pp431ndash438 2002

[16] J Wu C Metz X Xu et al ldquoA novel polymorphic CAATenhancer-binding protein 120573 element in the FasL gene promoteralters Fas ligand expression a candidate background genein African American systemic lupus erythematosus patientsrdquoJournal of Immunology vol 170 no 1 pp 132ndash138 2003

[17] N Xiang X M Li G S Wang J H Tao and X P Li ldquoAssoci-ation of FAS gene polymorphisms with systemic lupus erythe-matosus a meta-analysisrdquo Molecular Biology Reports vol 40no 1 pp 407ndash415 2013

[18] Q R Huang V Danis M Lassere J Edmonds and N Mano-lios ldquoEvaluation of a new Apo-1Fas promoter polymorphismin rheumatoid arthritis and systemic lupus erythematosuspatientsrdquo Rheumatology vol 38 no 7 pp 645ndash651 1999

[19] T Sun X Miao X Zhang W Tan P Xiong and D LinldquoPolymorphisms of death pathway genes FAS and FASL inesophageal squamous-cell carcinomardquo Journal of the NationalCancer Institute vol 96 no 13 pp 1030ndash1036 2004

[20] P A Courtney A D Crockard KWilliamson A E Irvine R JKennedy and A L Bell ldquoIncreased apoptotic peripheral bloodneutrophils in systemic lupus erythematosus relations withdisease activity antibodies to double stranded DNA andneutropeniardquo Annals of the Rheumatic Diseases vol 58 no 5pp 309ndash314 1999

[21] B L Liphaus and M H B Kiss ldquoThe role of apoptosis proteinsand complement components in the etiopathogenesis of sys-temic lupus erythematosusrdquo Clinics vol 65 no 3 pp 327ndash3332010

[22] R De Maria and R Testi ldquoFas-FasL interactions a com-monpathogeneticmechanism in organ-specific autoimmunityrdquoImmunology Today vol 19 no 3 pp 121ndash125 1998

[23] V M Budagyan E G Bulanova N I Sharova M F NikonovaM L Stanislav andA A Yarylin ldquoThe resistance of activated T-cells from SLE patients to apoptosis induced by human thymicstromal cellsrdquo Immunology Letters vol 60 no 1 pp 1ndash5 1998

[24] H Fukuyama M Adachi S Suematsu et al ldquoRequirement ofFAS expression in B cells for tolerance inductionrdquo EuropeanJournal of Immunology vol 32 pp 223ndash230 2002

[25] T Takahashi M Tanaka C I Brannan et al ldquoGeneralized lym-phoproliferative disease in mice caused by a point mutation inthe Fas ligandrdquo Cell vol 76 no 6 pp 969ndash976 1994

[26] B Kovacs S-N C Liossis G J Dennis and G C TsokosldquoIncreased expression of functional Fas-ligand in activatedT cells from patients with systemic lupus erythematosusrdquoAutoimmunity vol 25 no 4 pp 213ndash221 1997

[27] N Suzuki and T Sakane ldquoAbnormal Fas and Fas ligand expres-sion of lymphocytes in patients with SLErdquo Nippon RinshoJapanese Journal of Clinical Medicine vol 54 no 7 pp 1955ndash1959 1996

[28] J Wu J Wilson J He L Xiang P H Schur and J D MountzldquoFas ligand mutation in a patient with systemic lupus erythe-matosus and lymphoproliferative diseaserdquo Journal of ClinicalInvestigation vol 98 no 5 pp 1107ndash1113 1996

[29] Q R Huang D Morris and N Manolios ldquoIdentification andcharacterisation of polymorphisms in the promoter region ofthe human Apo-1Fas (CD95) generdquo Molecular Immunologyvol 34 no 8-9 pp 577ndash582 1997


[30] Y H Lee Y R Kim J D Ji J Sohn and G G Song ldquoFas pro-moter -670 polymorphism is associated with development ofanti-RNP antibodies in systemic lupus erythematosusrdquo Journalof Rheumatology vol 28 no 9 pp 2008ndash2011 2001

[31] S Kanemitsu K Ihara A Saifddin et al ldquoA functional poly-morphism in Fas (CD95APO-1) gene promoter associatedwithsystemic lupus erythematosusrdquo Journal of Rheumatology vol29 no 6 pp 1183ndash1188 2002

[32] J M Araste E K Sarvestani E Aflaki and Z AmirghofranldquoFas gene polymorphisms in systemic lupus erythematosus andserum levels of some apoptosis-related moleculesrdquo Immunolog-ical Investigations vol 39 no 1 pp 27ndash38 2010

[33] J-Y Chen C-M Wang C-C Ma Y-H Chow and S-F LuoldquoThe minus844CT polymorphism in the Fas ligand promoter asso-ciates with Taiwanese SLErdquo Genes and Immunity vol 6 no 2pp 123ndash128 2005

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Table 1 The primer sequences annealing temperature restriction enzymes and fragments size of FAS and FASL polymorphisms

Target sequence Primer sequence Annealingtemperature Restriction enzyme PCR product RFLP

fragmentsFAS A-670Grs1800682

Forward 51015840-CTACCTAAGAGCTATCTACCGTTC-31015840Reverse 51015840-GGCTGTCCATGTTGTGGCTGC-31015840 [29] 61∘C MvaI (BstNI) 223 A 223

G 44 189FASL C-844Trs763110

Forward 51015840-CAGCTACTCGGAGGCCAAG-31015840Reverse 51015840-GCTCTGAGGGGAGAGACCAT-31015840 [19] 63∘C BseMI (BsrDI) 401 T 401

C 223 168

FAS is a membrane protein of the transmembrane tumornecrosis factor superfamily of death receptors and plays animportant role in apoptotic signaling in different cells Thisreceptor induces apoptosis by binding to its natural ligandFASL is a member of tumor necrosis factor superfamily andinitiates the death signal cascade which ultimately results inapoptotic cell death Although FAS is existing constitutivelyon the surface of resting cells in low levels it is expressedprominently on the surface of activated T cells [12]

FAS and FASL genes are located on chromosomes 10q241and 1q23 respectively [13 14] Several polymorphisms havebeen recognized in FAS gene and G to A replacement atnucleotide position -1377 (FAS G-1377A rs2234767) in thesilencer region as well as A to G replacement at nucleotideposition -670 (FAS A-670G rs1800682) in the enhancerregion are studied more than others [15] The FASL geneC-844T polymorphism is located in the binding site of tran-scription factor CAATenhancer-binding protein 120573 Amarkedly elevated basal expression of FASL is observedin subjects with the C allele compared with the T allele atnucleotide position -844 (FASL C-844T rs763110) in thepromoter region [16]

Several studies investigated the role of FAS and FASLgene polymorphisms in the etiology of SLE however therole of FAS and FASL gene polymorphisms in lupus has notbeen conclusively established [16ndash18] Therefore the presentstudywas designed to investigate the association of apoptosis-related genes FAS and FASL polymorphisms with SLEsusceptibility and clinical characteristics in Iran

2 Materials and Methods

21 Characteristics of Study Populations Approval of thestudy was obtained from the Ethics Committee of ZahedanUniversity of Medical Sciences This case-control studyperformed on 106 SLE patients (9 men and 97 women) witha mean age of 318 plusmn 78 years who fulfilled the 1998 Amer-ican College of Rheumatology (ACR) criteria was referredto rheumatology clinics of Ali-Ebne-Abitaleb hospital inZahedan from 2011 to 2013 The control group consists of 149age sex and ethnically matched volunteers (13 men and 139women) with a mean age of 3290 plusmn 133 years The healthycontrols were selected following a questionnaire survey toexclude any systemic inflammatory and autoimmune dis-eases with negative ANA test and no family relation withlupus patients A written informed consent was obtainedfrom all participants

22 Genotyping Genomic DNAwas extracted from ethylenediamine tetra-acetic acid (EDTA) anticoagulated peripheral

Table 2 Demographic characteristics of SLE patients and controls

Parameter SLE119873 = 106

Controls119873 = 103

P value 1199092

Age (year) 318 plusmn 78 3290 plusmn 133 053 004Sex (malefemale) 997 895 05 004Race N ()

Persian 49 (46) 53 (51) 027 027Balouch 57 (54) 50 (49)

blood byDNA isolation kit (Roche Germany) FAS and FASLpolymorphisms were identified by the PCR-RFLP method

The primer sequences annealing temperature restrictionenzymes and fragments size are shown in Table 1

Polymerase chain reaction was performed in thermalcycler (Eppendorf-Mastercycler Gradient-Germany) in a25 120583L final volume which contained 20 pmol of each primer01mmol of dNTP (Fermentas Lithuania) 03 120583g of genomicDNA 15mmolL of MgCl

2 25 120583L of 10X PCR buffer and 15

unit of TaqDNApolymerase (Fermentas Lithuania) accord-ing to the following protocol initial denaturation at 95∘C for5min 30 cycles of denaturation at 95∘C for 1min annealingfor 1min at 61∘C for FAS A-670G and 63∘C for FASL C-844T polymorphisms extension at 72∘C for 2min and finalextension at 72∘C for 5 minutes PCR and digested productswere separated by electrophoresis on a 2 agarose gel andvisualized by ethidium bromide staining

23 Statistical Analyses The statistical analysis of the datawas performed using the SPSS 150 software Quantitativedatawere presented asmeanplusmn standard deviationGenotypesand alleles were compared between groups by use of 1205942 testComparison of quantitative variants between two groups wasassessed by Studentrsquos 119905-test The association between quali-tative variables was analyzed using the chi-square or Fisherexact tests Variations with 119875 values less than 005 were con-sidered significant Odds ratio and 95 confidence intervalwere calculated to evaluate the strength of the associations

3 Results

Clinical and demographic characteristics of SLE patients andcontrol samples were shown in Table 2 The SLE patients andcontrols did not differ significantly with respect to sex ageand ethnic parameters Skin manifestations arthritis lupusnephritis and neuropsychiatric manifestations developed in83 87 22 and 14 of patients respectively during thecourse of their disease



SLE119873 = 106

Control119873 = 149


FAS A-670GAA n () 34 (32) 37 (25) 003 21 (1ndash41)

AG n () 55 (52) 73 (49) 03 12(07ndash22)

GG n () 17 (16) 39 (26) Ref = 1A n () 123 (58) 147 (49) 003 14 (1-2)G n () 89 (42) 151 (51) Ref = 1

FAS ligand C-844T

CC n () 50 (472) 51 (342) 001 28(12ndash62)

CT n () 46 (434) 80 (47) 01 18(08ndash42)

TT n () 10 (94) 28 (188) Ref = 1

C n () 146 (69) 162 (54) 0001 19(13ndash27)

T n () 66 (31) 136 (46) Ref = 1





4 Discussion













FAS A-670GFASL C-844T SLE119873 = 106

Control119873 = 149

P value OR (95 CI)




















Acknowledgment


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



SLE119873 = 106

Control119873 = 149


FAS A-670GAA n () 34 (32) 37 (25) 003 21 (1ndash41)

AG n () 55 (52) 73 (49) 03 12(07ndash22)

GG n () 17 (16) 39 (26) Ref = 1A n () 123 (58) 147 (49) 003 14 (1-2)G n () 89 (42) 151 (51) Ref = 1

FAS ligand C-844T

CC n () 50 (472) 51 (342) 001 28(12ndash62)

CT n () 46 (434) 80 (47) 01 18(08ndash42)

TT n () 10 (94) 28 (188) Ref = 1

C n () 146 (69) 162 (54) 0001 19(13ndash27)

T n () 66 (31) 136 (46) Ref = 1





4 Discussion













FAS A-670GFASL C-844T SLE119873 = 106

Control119873 = 149

P value OR (95 CI)




















Acknowledgment


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



FAS A-670GFASL C-844T SLE119873 = 106

Control119873 = 149

P value OR (95 CI)




















Acknowledgment


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgment


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article association of fas and fas ligand...

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