Pharmacogenomics of responsiveness to interferon IFN-β treatment in multiple sclerosis: A genetic...

harmacogenomics of responsiveness tonterferon IFN-� treatment in multipleclerosis: A genetic screen of 100 type Interferon-inducible genes

Objectives: Interferon IFN-� is indicated for the treatment of multiple sclerosis. A significant proportion ofpatients show a poor clinical response to therapy. Type I interferon exerts its effect at least partially throughinteraction of specific transcription factors with interferon-stimulated response elements (ISREs), mostlylocated in promoter regions of its target genes. We hypothesized that polymorphisms may occur within orclose to ISRE elements, altering type I interferon inducibility and ultimately leading to a modified clinicalresponse in carriers.Methods: We selected 100 ISRE-containing genes and sequenced their promoter regions in small genomicdeoxyribonucleic acid pools of responding and nonresponding patients, as well as healthy control subjects. Aselection of polymorphisms discovered by this approach was scrutinized subsequently in a collection ofindividual deoxyribonucleic acid samples.Results: We identified 4 genes containing polymorphisms associated with response to recombinant IFN-�:IFNAR1 (P � .036), LMP7 (P � .002; odds ratio [OR], 6.37 [95% confidence interval (CI), 1.84-24.1]),CTSS (P � .02; OR, 0.38 [95% CI, 0.18-0.84]), and MxA (P � .015; OR, 3.37 [95% CI, 1.11-11.4]).Logistic regression analysis with treatment outcome used as the dependent variable and genotype as theindependent variable revealed 2 genes, LMP7 (OR, 0.55 [95% CI, 0.34-0.89]) and MxA (OR, 0.41 [95% CI,0.19-0.88]), that were independently associated with treatment response.Conclusions: Our work confirms and extends previous indications for a polygenic mechanism involved inbringing about responsiveness to recombinant IFN-�. The identification of 2 genes active in the antigenprocessing and presentation cascade; that is, LMP7, coding for the proteasome subunit �, and CTSS, codingfor cathepsin S; as potential response modifiers may identify this pathway as being of particular relevance tophenotypic expression of response heterogeneity. (Clin Pharmacol Ther 2005;78:635-46.)

Stephen Cunningham, PhD, Colin Graham, PhD, Michael Hutchinson, MD,Aidan Droogan, MD, Killian O’Rourke, MD, Chris Patterson, PhD,Gavin McDonnell, MD, Stanley Hawkins, FRCP, and Koen Vandenbroeck, PhD Belfast

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Interferon IFN-� was the first drug established as anffective disease-modifying treatment for patients with

rom the Applied Genomics Research Group, McClay Center for Phar-maceutical Sciences, School of Pharmacy, Queen’s University ofBelfast, Belfast; Centre of Medical Genetics, Belfast City Hospital,Belfast; Department of Neurology, St Vincent’s University Hospital,and University College, Dublin; Neurology Department, Royal Vic-toria Hospital, Belfast; Department of Epidemiology and PublicHealth, School of Medicine, Queen’s University of Belfast, Belfast;and School of Medicine, Queen’s University of Belfast, Belfast.

his work was supported by grant RRG11.5 from the NorthernIreland Health and Personal Social Services Research and Devel-opment (HPSS R&D) Office.

eceived for publication May 18, 2005; accepted Aug 23, 2005. d

elapsing-remitting and secondary progressive multipleclerosis (MS).1 The use of recombinant IFN-�rIFN-�) was originally based on the belief that theondition resulted from a viral or bacterial source,hich in genetically susceptible individuals could lead

o the development of demyelinating lesions. The in-

eprint requests: Koen Vandenbroeck, PhD, Chair in AppliedGenomics, School of Pharmacy, Queen’s University of Belfast, 97Lisburn Rd, Belfast, BT9 7BL UK.

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CLINICAL PHARMACOLOGY & THERAPEUTICS636 Cunningham et al DECEMBER 2005

ammatory nature of these lesions, as well as the geo-raphic distribution of the condition, initiated the con-ept of a slow or latent viral infection participating inisease pathogenesis.2 Thus it was straightforward toonsider the then just discovered type I IFNs with theirronounced antiviral activity as potentially useful ther-peutics for MS, and this was reinforced by studies thatemonstrated defective or weakened IFN synthesis inatients with MS compared with healthy persons.3

Treatment with rIFN-� decreases the relapse rateoth during and after treatment, affects disease progres-ion, and causes a substantial reduction in the formationf new (70%) and active (20%) magnetic resonancemaging lesions in relapsing-remitting patients with MSs compared with subjects receiving placebo.4-6 Al-hough rIFN-� treatment has been demonstrated to beffective, individual responses of MS patients to thisreatment are known to be heterogeneous, with up to0% of patients demonstrating treatment failure, poten-ially linked to genetic or environmental factors (oroth).5,7 Formation of neutralizing antibodies (NAbs)o rIFN-� may potentially affect treatment outcome,ith approximately 30% of all patients being NAb

iter-positive.8,9 However, to date, the significance ofAb in the classification of response status to IFN

reatment has not been fully clarified.Type I IFN exerts its effect at least partially through

he transcriptional regulation of IFN-stimulated genesISGs). The IFN-stimulated response element (ISRE)onsensus sequence GGAAAN(N)GAAAC is recog-ized as a functional cis-acting element necessary toonfer IFN inducibility.10,11 Comparative sequencenalysis together with transfection experiments hasemonstrated ISRE elements in the promoter of mostFN-inducible genes.10-12 Transcriptional factors thatnteract with ISRE elements have been described in theiterature.10-13

By applying the presence of an ISRE element withinhe promoter region of a gene as the selection criterion,00 genes containing regions with 70% homology tohe ISRE consensus sequence or greater were retrievedrom public databases and literature screens (Table Ind Appendix 1 [available online at http://journals.lsevierhealth.com/periodicals/ymcp]). ISREs mediateFN inducibility in the context of interaction with otherranscription factors, such as TFIIB and ribonucleiccid (RNA) polymerase II, that recognize distinct cis-cting response elements (eg, Inr and TATA box).14 Ineneral, such additional elements are located within aange not exceeding a few hundreds of base pairs onither side of the ISRE element. By sequencing

o ensure that the majority of polymorphisms in ISREs,s well as in any other transcription regulatory elementshat indirectly might affect IFN inducibility, would beetected. When ISREs were located further than 1ilobase away from the transcription start site or TATAox, larger fragments were examined. The initial screenas performed by use of a pooled deoxyribonucleic

cid (DNA) approach with polymorphisms showingistortions between the pools further scrutinized inndividual DNA samples.

ETHODSPatients and samples. All MS patients were diag-

osed as having clinically definite MS according to theriteria of Poser et al,15 had the relapsing-remittingorm of the disease, and had clinical data available foryears before treatment with rIFN-� up to 2 years after

nitiation of treatment. This time period provided anndication of initial response to treatment before declinento the secondary stage of the disease. As previouslyeported, response status of patients receiving rIFN-�hould not be judged in the first 6 to 9 months ofreatment because the treatment may not be effectiventil after this period.16 With this in mind, we haveermed a nonresponder to treatment as a patient whoseelapse rate remains the same or increases after thenitial 6- to 9-month period of treatment. A respondero treatment is a patient whose relapse rate, after thenitial 6- to 9-month period, is reduced by one third andho had no sustained progression (verified at 3 months)n the Expanded Disability Status Scale (EDSS) (�1oint if baseline EDSS �5.5 or �0.5 points if baselineDSS �5.5).16,17 The total number of MS patientstudied was 230 and included 94 classified respondersnd 68 nonresponders. The remainder of MS patients (n

68) did not fulfill the requirement to have theiresponse status unambiguously classified. A group of1 healthy control subjects was also studied. All MSatients within this study were, on an ongoing basis,xamined for the presence of a high NAb titer by use ofcytopathic effect inhibition assay.18 Those patientsith a sustained (after �2 determinations) high NAb

iter as determined by the cytopathic effect inhibitionssay were classified as patients with a high titer. Theurrent status of the NAb titer in relation to IFN treat-ent clinical response is not clearly understood. TheAb titer was, therefore, not applied as a criterion for

esponse status in this study but was considered sepa-ately. Written informed consent was attained from allarticipants before enrollment, and the right of with-rawal from this study was given to all patients. The

CLINICAL PHARMACOLOGY & THERAPEUTICS2005;78(6):635-46 Pharmacogenomics of IFN-� response in MS 637

able I. List of 100 genes included in pooled screen of Northern Irish samples

Gene name (symbol) Chromosomal locationReference

sequence ID

Guanylate-binding protein 1 (GBP1) 1p22.2 NM_002053IFN-induced gene 6-16 (G1P3, H6-16) 1p36 NM_002038TNF receptor p75/80 (TNFRSF1B, TNFp75/80, CD120b) 1p36.2-36.3 NM_001066IFN-stimulated gene 15 (G1P2, ISG15, IFI-15K) 1p36.33 NM_005101RNA-specific adenosine deaminase (ADAR) 1q21 NM_056655SPRR2A gene encoding small proline-rich protein (SPRR2A) 1q21 NM_005988Cathepsin S (CTSS) 1q21 NM_004079IFN-�–inducible protein (IFI-16) 1q22 NM_005311Myeloid cell nuclear differentiation antigen (MNDA) 1q22-23 NM_002432Fas ligand (FasLG) 1q24.3 NM_000639Cyclooxygenase 2 (PTGS2) 1q25.2 NM_000963Polymeric immunoglobulin receptor (PIGR) 1q31-41 NM_002644Vascular cell adhesion molecule 1 (VCAM1, CD106) 1q32 NM_001078Interleukin 10 (IL10) 1q32.1 NM_000572Mitogen-inducible gene 6 protein (Mig-6, GENE-33) 1q36 NM_018948Double-stranded RNA-dependent protein kinase (PRKR) 2p22 NM_002759Interleukin 1� (IL1B) 2q13 NM_000576Signal transducer and activator of transcription 1 (STAT1) 2q32.2 NM_007315Sp100 gene (Sp100) 2q37.1 NM_003113CC chemokine receptor 3 (CCR3) 3p21.31 NM_001837Synapsin II gene (SYN2) 3p25 NM_033625TNF-related apoptosis-inducing ligand (TNFSF10, TRAIL) 3q26 NM_003810Interleukin 8 (IL8) 4q13.3 NM_000584IFN-�–induced precursor/IP-10 chemokine (CXCL10, IP10) 4q21 NM_001565IFN-�–inducible T-cell � chemoattractant (BR1, ITAC,

CXCL9)4q21.1 NM_002416

CXCL 11 gene/chemokine IP-9 (CXCL11, IP9) 4q21.2 NM_005409Complement factor 1 precursor (IF) 4q25 NM_000204IFN regulatory factor 2 (IRF2) 4q34 NM_002199Interleukin 7 receptor (IL7R) 5p13.2 NM_002185Interleukin 3 (IL3) 5q23.3 NM_000558Interleukin 4 (IL4) 5q23.3 NM_000589Interleukin 5 (IL5) 5q23.3 NM_000879Interleukin 13 (IL13) 5q23.3 NM_002188IFN-stimulated gene 20 (ISG20) 5q26 NM_002201IFN regulatory factor 1 (IRF1) 5q31.1 NM_002198Cyclin-dependent kinase inhibitor 1A (p21, Cip1) 6p21.2 NM_000389MHC class II, DM � (DMA) 6p21.3 NM_006120MHC class 1 (HLAG) 6p21.3 NM_002127MHC class 1 (HLAC) 6p21.3 NM_002117Complement component C2 (C2) 6p21.3 NM_000063Transporter 1 ATP binding cassette subfamily (TAP1) 6p21.3 NM_000593Transporter 2 ATP binding cassette subfamily (TAP2,

RINGII)6p21.3 NM_000549

Complement factor B (Bf, GBG, CFAB, PBF2) 6p21.3 NM_001710MHC class 1 (HLAA) 6p21.3 NM_002116MHC class 1 (HLAB7) 6p21.3 NM_002397HLA-E 6p21.3 NM_005516HLA-F 6p21.3 NM_018950Low–molecular mass polypeptide 7 (PSMB8, LMP7) 6p21.3 NM_004159Tumor necrosis factor B (TNFB, LTA) 6p21.3 NM_000595Tumor necrosis factor A (TNFA) 6p21.3 NM_000594

able I—Cont’d

Gene name (symbol) Chromosomal locationReference

sequence ID

Interleukin 7 (IL7) 8q21.12 NM_000880c-myc Promoter binding protein (C-myc, IRLB) 8q24 NM_005848IFN-� (IFNB) 9p21.3 NM_002176Human mimecan gene (OGN) 9q22 NM_014057IFN-�1 (IFNA) 9p22 NM_024013Toll-like receptor (TLR-4) 9q32-33 NM_003266Mannose-binding lectin 2 (MBP2, MBP1) 10q20 NM_000242IFN-inducible gene IFI-60 (IFIT3, CIG-49, RIG-G) 10q24 NM_001549IFN-stimulated gene 54 (ISG54, IFI-54K, IFIT2, IFI54) 10q25 NM_001547IFN-inducible gene IFIT1 (ISG56, IFI56) 10q25 NM_001548Human TNF receptor–associated factor 6 (TRAF6) 11p12 NM_004620IFN regulatory factor 7H (IRF-7H) 11p15 NM_004029IFN-induced 14-kd transmembrane protein (IFITM3, 1-8U) 11p15 NM_021034IFN-induced gene 9-27, Leu13 antigen (UFUTM1, H9-27) 11p15.1 NM_003641Interleukin 1�–converting enzyme/caspase 1 (CASP1, ICE) 11q23 NM_001223Interleukin 22 (IL22) 12q15 NM_020525Signal transducer and activator of transcription 2 (STAT2) 12q13.3 NM_005419IFN-� (IFNG) 12q15 NM_0006192�-5� Oligoadenylate synthase (OAS1) 12q24 NM_002534Inducible nitric oxide synthase I (NOS1, NOS) 12q24.22 NM_000620TGF-�–stimulated clone (TSC22) 13q14 NM_006022Eukaryotic translation initiation factor (eIF) 2A (eIF-2�) 14q.23 NM_032025Tryptophanyl-tRNA synthase (WARS, IFP53) 14q32 NM_004184IFN-stimulated gene 12 (IFI27, ISG12) 14q32 NM_005532IgE switch region (IGHE) 14q32.3 NM_001019Promyelocytic leukemia (PML) 15q22 NM_002675Suppressor of cytokine signaling protein 1 (SOCS1) 16p13.13 NM_003745Protamine 3 (PRM3) 16p13.3 NM_021247Nitric oxide synthase 2A, inducible (NOS2A) 17q11 NM_000625Regulated upon activation, normal T cell–expressed

chemokine (CCL5, RANTES)17q11.2 NM_002985

Prepro-orexin gene, preprohypocretin (PPOX) 17q21-22 NM_001524Platelet endothelial cell adhesion molecule 1 (PECAM-1) 17q24 NM_000442Guanine nucleotide–binding protein � 13 (GNA13) 17q24.3 NM_006572PMA-inducible gene (PMAIP1) 18q21.31 NM_021127IP-30 chemokine (IFI-30, IP30, GILT) 19p13.11 NM_006332Intercellular adhesion molecule 1 (ICAM1) 19p13.2 NM_000201IFN regulatory factor 3 (IRF3) 19q13 NM_001571Biliary glycoprotein (CEACAM-1, BGP-1, CD66) 19q13.1-13.2 NM_001712FMS-related tyrosine kinase ligand 3 (FLT3LG) 19q13.3 NM_001459DNA-directed RNA polymerases III 39-kd polypeptide

(POLR3F, RPC39)20p11.23 NM_006466

Secretory leukocyte protease inhibitor (SLPI) 20q12 NM_003064IFN-�/-� receptor (IFNAR) 21q22.11 NM_000629IFN-induced protein p78 (Mx1, Mx78, MxA) 21q22.3 NM_002462A kinase (PRKA) anchor protein 4 (AKAP4) Xp11.2 NM_003886Synapsin I gene (SYN1) Xp11.23 NM_006950Cytochrome b558 heavy-chain gene (CYBB, GP91-PHOX) Xp21.1 NM_000397Thymosin �4 (TMSB4X) Xp22 NM_021109Toll-like receptor 7 (TLR7) Xp22.3 NM_016562CD40 ligand gene (CD40LG) Xq26.3-27.1 NM_000074

Indicated are the gene names, symbols that have been used in the literature to refer to these genes, and database accession numbers used to access their genomicequence and chromosomal location.

IFN, Interferon; TNF, tumor necrosis factor; RNA, ribonucleic acid; IP, inflammatory protein; MHC, major histocompatibility complex; TGF, transforming growth

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ommittee of Queen’s University Belfast (approvalo. 13/01), Belfast, Ireland.Polymorphism discovery by sequencing of DNA

ools. Genomic DNA was extracted from whole-bloodamples, and DNA concentrations were determined byptical density. Initial screening for polymorphic sitesithin the selected genes was performed by use of aooled DNA approach.19 Pools were made consistingf 10 individuals each, and in all, 4 pools were con-tructed, representing (1) IFN treatment responders, (2)onresponders, (3) patients with a high Nab titer toreatment, and (4) a healthy control group.

Gene selection and polymorphism screening. Theenes studied included a selection of (1) those cur-ently reported in the literature as having known orutative ISRE elements, (2) those contained withinublic transcription factor databases, and (3) thosehat were identified through the screening of pro-

oter regions by use of the consensus ISRE se-uence as a recognition motif and with allowance foregions of greater than 70% homology. Genes takenrom databases (National Center for Biotechnologynformation [NCBI], MutDB,20,21 SNPper,22 Ensem-le,23 and Genecard24,25) were subjected to compu-ational motif searches using online bioinformaticools available through the World Wide Web (Trans-ac,26,27 TRRD,28 MATCH,29 MatInspector,30

odel-Inspector, TESS,31 and Signal Scan32). Theegions containing ISRE elements were inspected forolymorphic sites by direct sequencing. Comprisedithin the selection of 100 genes are a few genes

hat, though lacking clear-cut ISRE motifs, producexpression products directly involved in IFN-� sig-al transduction (eg, type I IFN receptor). Polymer-se chain reaction (PCR) amplification was per-ormed by use of standard conditions, with a typicalCR reaction comprising 1� buffer, 1.5 to 3.0-mol/L magnesium chloride (Promega, Madison,is), 300-mol/L deoxyribonucleoside triphosphate

Promega), 300-nmol/L both forward and reverserimers (Operon Biotechnologies Inc, Cologne, Ger-any), Taq polymerase (Promega), and templateNA. Bidirectional sequencing of all fragments waserformed by use of both the PCR amplificationrimers and an overlapping pair of fragment-specificnternal primers. Sequencing was performedhroughout this study with the ABI Prism BigDyeerminator Kit v3.1 (Applied Biosystems, Fosterity, Calif) and was performed on an ABI 3100enetic analyzer (Applied Biossytems). Sequencelignment and comparison of the pooled data were

odes, Ann Arbor, Mich), and Sequencing Analysis3.7 software (Applied Biosystems). (All primersnd PCR conditions are included in Appendix 2available online at http://journals.elsevierhealth.com/eriodicals/ymcp].)

Allele estimation and pooled comparisons. Averageeak heights were taken across the sequenced fragmentf both alleles contained within a single-nucleotideolymorphism (SNP), with the sequence region sur-ounding the SNP and selecting peaks to measure thatre located in similar surroundings. All peak measure-ents were computationally performed with Sequenc-

ng Analysis v3.7 software (Applied Biosystems); Se-uencher, version 4.0.5 (Gene Codes); and Phred andhrap software.33,34 Assessment of this pooling strat-gy method and the sensitivity of detection was evalu-ted by use of 3 known SNPs, CTLA 49 A/G, inter-eukin (IL) 6 �174 G/C, and IL2 114 G/A, throughnalysis in individual and pooled DNA samples. By usef these SNPs, the allele frequencies were determinedn the individual samples and then in pooled DNAonsisting of the same individuals who had been typed.irect comparisons could then be made from the allele

requencies from both methods. Controlled pooledpiking was also conducted to test the sensitivity andetection frequency limits.35

Individual polymorphic genotyping. IndividualNA samples were used to analyze polymorphisms inenes as discovered in the pooled approach. Tech-iques used included fluorescent polarization-single-ase extension, restriction fragment length polymor-hism enzyme digestion, fragment analysis, andyrosequencing. (Genotyping assays and conditions arehown in Appendix 3 [available online at http://ournals.elsevierhealth.com/periodicals/ymcp].)

Statistical analysis. Differences in genotype fre-uencies and carriage rates between all MS patients andealthy control subjects and between stratified rIFN-�esponders and rIFN-� nonresponders were assessed byhi-square analysis,36 and the CLUMP program wassed for analysis of multiallelic polymorphic markersased on Monte Carlo simulation.37 Between respond-rs and nonresponders, gender stratification was alsonalyzed. Tests for Hardy-Weinberg equilibrium werepplied by use of a chi-square approximation test atach locus.38 Deviations from Hardy-Weinberg equi-ibrium were observed for 2 markers within the com-lete population and a number of times within MSases (stratified by response or unstratified). By use ofPSS, version 11.5 (SPSS, Chicago, Ill), logistic re-ression was performed to evaluate whether there was

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yzed, with the clinically defined outcome of treatments the dependent variable (Table V). P � .05 wasonsidered significant. Because this is an exploratorytudy, no corrections for multiple comparisons werepplied (see Discussion section).

ESULTSSequencing of DNA pools was carried out to uncover

olymorphisms in the promoters of 100 ISRE-ontaining genes (Table I). Genes selected included,mong others, those coding for cytokines (eg, IL4 andL5), receptors (eg, tumor necrosis factor [TNF] recep-or), proteases (eg, cathepsin S), and signal transductionactors (eg, IFN regulatory factors [IRFs]). Detectionimits of the pools used throughout this study weressessed by comparing allele information gathered byhe individual genotyping of samples with the allelerequency estimates or allele ratio estimates determinedy the direct sequencing of the corresponding pools.hese findings indicated that the frequency of the minorllele of the polymorphism must be greater than 20% toesolve from background peaks (data not shown).

Numerous polymorphic sites were identified, ofhich a small number showed peak distortions between

he pools. Confirmation of known SNPs, such as those

able II. Individual association analysis of 15arkers identified in pools*

Gene Marker

P value

Case/control Response

DMA Novel SNP NS NSPKR CCGn repeat NS NSIFNAR 408 C/T SNP .046† NSIFNAR GTn repeat NS .036‡IFNAR rs1012334 .042† NSIP9 Novel SNP NS NSISG12 rs381482 NS NSISG56 Novel repeat .047† NSLMP7 rs2071543 NS .002†CTSS rs1136774 NS .02†CTSS rs3754212 .003† NSIFP53 rs2234518 NS NSIFP53 rs7143004 NS NSMxA rs2071430 NS .015†MxA �123nt NS .018†

SNP, Single-nucleotide polymorphism; NS, nonsignificant.*Both significant and nonsignificant (P� .05) associations are shown. P

alues were calculated by use of standard chi-square analysis for comparison ofenotypes and are uncorrected.†Calculated by use of standard chi-square 3 � 2 tables with 2 df.‡Calculated by use of standard chi-square 9 � 2 tables with 8 df.

n the TNF� and TNF� genes (data not shown), under- h

cores the suitability of the pooling approach as aolymorphism discovery tool.19,39 Of the 100 genescreened in the pools, polymorphic markers were ob-erved in 32 genes (51 SNPs, 2 repeat elements, and 1ovel 3-nucleotide duplication) (Appendix 4 [availablenline at http://journals.elsevierhealth.com/periodicals/mcp]), and allele peak distortions larger than 10%etween the pools were observed for 21 of these genes.f these genes, 6 showed peak distortions betweenooled cases and controls, and 15 showed peak distor-ions between responding and nonresponding patients.hese 15 genes included IP9, ISG12, ISG20, ISG56,KR, Sp100, IFP53, LMP7, HLA-B, IF, HLA-F, CTSS,

FNAR, TNF�, and MxA genes. The polymorphismsemonstrating the most pronounced peak differences inhe pool comparison were further screened in individualNA samples. In total, 15 polymorphic sites within 10enes were genotyped. (Assays and conditions are in-luded in Appendix 3 [available online at http://ournals.elsevierhealth.com/periodicals/ymcp].) Theignificance of the association in cases versus controlsr between classified patient responder strata is given inable II, whereas Table III presents genotyping data for

he 7 SNPs showing significant associations and TableV shows genotyping data for 3 polymorphic repeatlements. Polymorphisms were not further scrutinizedn this stage for association with high NAb response toFN-�.

Analysis of polymorphic markers in the IP9, ISG12,nd IFP53 genes failed to reach a significant associa-ion when analyzed in the individual samples. TheFP53 gene, although having an SNP located within anSRE element, did not demonstrate an association withesponse on individual typing. A novel 3-base pair (bp)uplication was identified in ISG56 in the nonresponderool that was absent or below the detection thresholdrom the other pools. Individual genotyping showed theresence of all 3 possible genotypes. A number ofamples were sequenced to verify the presence andbsence of this repeat element. Although this elementas previously shown to be a marker for response to

FN,40 no associations or trends were observed in ourtudy regarding IFN response within MS for this ele-ent. A borderline significant association was observed

or susceptibility (P � .047).The IFN-inducible double-stranded RNA-activated

rotein kinase (PKR) plays an important role in mes-enger RNA translation by phosphorylating the � sub-nit of eukaryotic initiation factor 2. The location andunctionality of a regulatory ISRE within the promoterave been documented, and IFN inducibility of PKR

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epeat element41 was found to be distorted between thenitial pools representing rIFN-� responders and non-esponders. Individual DNA analysis of responders ver-us nonresponders failed to reach significance.

Analysis of the IFNAR1 gene in the pooled DNAsighlighted 2 potentially interesting polymorphic sites.he first, a C/T SNP, is located �408 bp from the ATGtart codon, whereas the second marker comprises aTn repeat element approximately ranging from �80

o �55 bp from the ATG start codon.42,43 Both poly-orphisms, in addition to a third polymorphic marker,

s1012334 (which is located in intron 3, a region ini-ially not screened in our DNA pools), were included inhe extended individual analysis of this gene. NeitherNP showed an IFN-� response association, althoughe found evidence for a weak association of both SNPs

n the comparison of cases versus controls (Table II). Inrecent study, rs1012334 was found to be associatedith response to rIFN-� in MS (P � .03),42 which wasot confirmed in our study. The GTn microsatelliteepeat showed a weak association with response toIFN-� when analyzed by use of normal chi-squareistribution (�2, 16.44; 8 df; P � .037). However, whennalyzed after pooling of minor alleles by use of the

able III. Raw genotyping data from individual typinn Table II)

Gene (marker) Genotype Control

IFNAR (408 C/T SNP) CC 57 (0.626)CT 24 (0.264)TT 10 (0.110)

IFNAR (rs1012334) TT 18 (0.198)TA 58 (0.637)AA 15 (0.165)

LMP7 (rs2071543) CC 66 (0.725)CA 20 (0.220)AA 5 (0.055)

CTSS (rs1136774) TT 22 (0.242)TC 46 (0.505)CC 23 (0.253)

CTSS (rs3754212) AA 36 (0.396)AG 51 (0.560)GG 4 (0.044)

MxA (rs2071430) GG 75 (0.824)GT 16 (0.176)TT 0 (0.000)

MxA (�123nt) GG 74 (0.813)GT 17 (0.187)

LUMP program37 (T2), this association was no longer c

ignificant (�2, 11.07; 6 df; P � .086). Thus, notwith-tanding differences, both our study and that of Sriramt al42 point toward the IFNAR gene as a potentialandidate in bringing about clinical response or nonre-ponse to IFN-� therapy. In a recent study Leyva et al44

id not find IFN-� response associations in MS with 2NPs in IFNAR1, one of which was the �408 C/TNP.LMP7 codes for the proteasome � subunit, is located

ithin the major histocompatibility complex (MHC)lass II at 6p21.3, and is involved in the degradation ofytosolic proteins and antigens before their presentationhrough the MHC class I pathway.45 LMP7 has beenighlighted previously as a putative type I IFN re-ponder gene. Specifically, on examination of IFN re-ponse in patients with chronic hepatitis, Sugimoto etl45 reported an association of an LMP7 SNP withustained response to treatment at a P value of .006.his SNP, rs2071543, is located 561 bp from theTG codon and was identified in our MS pools, where

t showed allelic differences with relation to response toFN treatment. Typing of this polymorphism in indi-idual samples was performed as previously de-cribed.45 The polymorphism showed a highly signifi-

P markers reaching significant association (P � .05

Count (%)

Case(unstratified) Responder Nonresponder

119 (0.529) 46 (0.495) 34 (0.500)91 (0.404) 41 (0.441) 29 (0.426)15 (0.067) 6 (0.064) 5 (0.074)46 (0.206) 17 (0.181) 13 (0.194)

161 (0.722) 70 (0.745) 53 (0.791)16 (0.072) 7 (0.074) 1 (0.015)

152 (0.676) 65 (0.691) 39 (0.574)45 (0.200) 25 (0.266) 14 (0.206)28 (0.124) 4 (0.043) 15 (0.220)65 (0.289) 17 (0.183) 25 (0.368)

116 (0.515) 59 (0.634) 30 (0.441)44 (0.196) 17 (0.183) 13 (0.191)

115 (0.509) 49 (0.521) 37 (0.544)83 (0.367) 34 (0.362) 22 (0.324)28 (0.124) 11 (0.117) 9 (0.132)

195 (0.859) 87 (0.935) 56 (0.812)31 (0.137) 6 (0.065) 12 (0.174)1 (0.004) 0 (0.000) 1 (0.014)

170 (0.752) 77 (0.828) 46 (0.667)56 (0.248) 16 (0.172) 23 (0.333)

g of SN

ant association (�2, 12.11; 2 df; P � .002) with

r6paiI

efaaoesswia.a.c

trAdpeooapat11Tldppt

by use ofr

esponse compared with nonresponse (odds ratio [OR],.37 [95% confidence interval (CI), 1.84-24.1] for com-arison of C carriers versus non-C carriers) (Tables IInd III). Thus our data and those of Sugimoto et alndicate that this SNP may constitute a generic type IFN response marker irrespective of disease treated.

The cathepsin S (CTSS) gene showed allelic differ-nces between case-control pools for 2 SNP markersound within 4 bases of each other. Both of these SNPsre contained within databases, identified as rs3754212nd rs1136774. Because of the close proximity to eachther, a suitable fluorescent polarization-single-basextension probe design was compromised. Individualample genotyping was, therefore, performed via pyro-equencing, which allowed for the typing of both SNPsithin a single reaction. Of the 2 polymorphisms stud-

ed within this study, rs3754212 showed significantssociation with susceptibility (�2, 11.58; 2 df; P �003), whereas the second SNP (rs1136774) showedssociation with rIFN-� response (�2, 7.81; 2 df; P �02), with an OR of 0.38 (95% CI, 0.18-0.84) for

able IV. Raw genotyping data from individual typin.05 in Table II)

Gene (marker) Allele* Control

PKR (CCGn repeat) 282 4 (0.02)291 2 (0.01)297 5 (0.03)300 12 (0.07)303 1 (0.01)306 0 (0.00)309 144 (0.84)312 3 (0.02)315 1 (0.01)318 0 (0.00)

IFNAR (GTn repeat) 254 91 (0.50)256 8 (0.04)260 22 (0.12)262 16 (0.09)264 3 (0.02)270 10 (0.05)272 19 (0.10)274 12 (0.07)276 1 (0.01)

ISG56 (duplication) 0 62 (0.681)1 29 (0.319)2 0 (0.000)

*Allele refers to the size assigned to each fragment during fragment analysisepeat heterozygote, and 2 indicates repeat homozygote.

omparison of C carriers versus non-C carriers. I

The MxA gene has been applied as a biomarker forhe activity of type I IFN and has consistently beeneported in studies examining IFN response.46,47

nalysis of this gene showed 2 SNPs with peakistortions in responder compared with nonresponderools in the initial pooling stage. These 2 SNP mark-rs were located 35 bp apart, with one on either sidef a single ISRE element within the promoter regionf the gene, as reported by Hijikata et al48 withnalysis of SNPs for responsiveness of hepatitis Catients to IFN treatment. In our study we found anssociation between each of these SNPs and responseo rIFN-� (P � .015 and OR of 3.37 [95% CI,.11-11.4] and P � .018 and OR of 2.41 [95% CI,.08-5.39] for rs2071430 and �123nt, respectively).hese polymorphisms are in strong linkage disequi-

ibrium, yielding D� (pairwise measure of linkageisequilibrium) values of 0.85 within the completeopulation and 0.88 within cases only. Previous re-orts have highlighted a potential relevance of one ofhese polymorphic sites, rs2071430, in relation to

ltiallele markers reaching significant association (P

Count (%)

Case(unstratified) Responder Nonresponder

2 (0.00) 1 (0.01) 1 (0.01)6 (0.01) 4 (0.02) 2 (0.02)

15 (0.04) 7 (0.04) 3 (0.03)32 (0.08) 15 (0.08) 7 (0.06)0 (0.00) 0 (0.00) 0 (0.00)1 (0.00) 0 (0.00) 1 (0.01)

357 (0.85) 153 (0.83) 104 (0.87)6 (0.01) 4 (0.02) 1 (0.01)0 (0.00) 0 (0.00) 0 (0.00)1 (0.00) 0 (0.00) 1 (0.01)

200 (0.45) 97 (0.52) 53 (0.40)36 (0.08) 7 (0.04) 12 (0.09)40 (0.09) 17 (0.09) 12 (0.09)55 (0.13) 27 (0.15) 16 (0.12)5 (0.01) 0 (0.00) 3 (0.02)

16 (0.04) 9 (0.05) 4 (0.03)51 (0.12) 16 (0.08) 22 (0.17)33 (0.08) 14 (0.07) 8 (0.06)4 (0.01) 1 (0.01) 2 (0.02)

162 (0.723) 71 (0.763) 47 (0.701)52 (0.232) 18 (0.194) 17 (0.254)10 (0.045) 4 (0.043) 3 (0.045)

a known size standard. In the case of ISG56, 0 indicates no repeat, 1 indicates

g of mu

FN-� therapy (in hepatitis).48,49

umpotf[rarae4c.p(swmc�a

petDrrro(sga

ItcdapmictCpkid

sLbFiatdcM(pPrchiir

Logistic regression analysis was performed with these of treatment outcome as the dependent variable andarker genotypes as the independent variable, by ap-

lying a forward stepwise approach (significance levelf 5%) for marker selection. This resulted in the iden-ification of 2 significant markers (Table V). The ORsor the LMP7 rs2071543 and MxA �123nt SNPs (0.5595% CI, 0.34-0.89] and 0.41 [95% CI, 0.19-0.88],espectively) are indicative of both the LMP7 A allelesnd the MxA 123nt T alleles being associated with aeduced probability of response to treatment. Furthernalysis indicated that there were significant differ-nces in response rates by gender (63% in men versus0% in women, P � .008) and between collectionenters (46% in center 1 versus 64% in center 2, P �03), and these variables were, therefore, considered asotential confounders. By contrast, age at disease onsetP � .51), duration of treatment (P � .46), and NAbtatus (P � .59) were not associated with response andere, therefore, not considered as confounders. Adjust-ent for gender and center resulted in slightly in-

reased ORs for both the LMP7 rs2071543 and MxA123nt markers (0.61 [95% CI, 0.37-1.02] [P � .06]

nd 0.46 [95% CI, 0.21-1.02] [P � .05], respectively).

ISCUSSIONThe screening of ISRE-containing genes by use of a

ooled DNA sequencing approach was found to beffective in the identification of polymorphisms withinhe fragments examined. After screening of pooledNA, individual genotyping of a cohort of 162

esponse-classified patients and 91 control subjects cor-oborated 4 genes associated with the response toIFN-�; that is, IFNAR1, LMP7, CTSS, and MxA. Twof these have been previously reported in the literatureLMP7 and MxA).45,48 Novel associations were ob-erved for IFNAR and CTSS. The confirmation of theseenes as response markers to rIFN-� requires furthernalysis and validation of results.

The polymorphisms in the IFNAR1 gene have noteen reported previously in relation to response asso-iation.42 Various reasons may account for this discrep-

able V. Logistic regression analysis

MarkerOdds ratio

(95% confidence inte

LMP7 (rs2071543)* 0.55 (0.34-0.89)MxA (�123nt)† 0.41 (0.19-0.88)

*Coded as number of A alleles (CC � 0, CA � 1, and AA � 2).†Coded as number of T alleles (GG � 0 and GT � 1).

ncy, among which differences in classification criteria r

f response status, clinical ascertainment of patients, oropulation effects on the frequency and linkage dis-quilibrium patterns of associated marker alleles maye of special importance.The CTSS gene was found to be associated with both

FN treatment response and disease susceptibility. Ca-hepsin S plays a significant role in the activation ofertain immune responses.50 A member of the pepti-ase C1 family, CTSS is a lysosomal cysteine protein-se that may participate in the degradation of antigenicroteins to peptides for presentation on MHC class IIolecules.50,51 There is accumulating evidence that

ntracellular and extracellular proteases of microgliaontribute to various events in the central nervous sys-em through both nonspecific and limited proteolysis.50

TSS was reported to be functional in the proteolyticrocessing of myelin basic protein in vitro51 and isnown to be a major determinant in the regulation ofntracellular trafficking of MHC class II molecules inendritic cells and microglia.50-52

It is of relevance to note that the gene showing thetrongest association with IFN-� response in MS,MP7, shares with cathepsin S the characteristic ofeing involved in antigen processing and presentation.urther investigation of these and other genes involved

n this specific pathway should elucidate whether, atny rate, this is the main determinant of response toype I IFN treatment. In any case it is remarkable that,espite numerous reports highlighting differences inytokine expression profiles after IFN-� treatment inS patients,53,54 in our study not a single cytokine

receptor) gene, out of about 10 scrutinized, containedolymorphisms showing association with response.ending further investigation, this suggests that alteredegulation of cytokine expression on IFN treatmentould be merely phenotypic. Nevertheless, exceptionas to be made to the fact that putative functionalntronic or IFN-responsive 3�-untranslated region SNPsnvolved in RNA transcript stability or transcriptionegulation of cytokines were not covered in our screen.

In view of the fact that this study was designed as anxploratory approach toward identification of IFN-�

Likelihood ratio(�2 statistic [1 df]) P value

6.25 .0125.40 .020

esponse genes and given the restrictions imposed by

ttaiftrtItsIosags

hInplupfhwpScgIbtdiab

rbatiIarDMBsvg

he maximum number of response-classified MS pa-ients available for inclusion in the study, we have notpplied any correction factors. Therefore there is annherent risk that the associations we have found aroserom type I statistical errors. Ideally, these results needo be replicated by other groups or in distinct cohorts ofesponse-classified MS patients. At any rate, the iden-ification of LMP7, previously shown to constitute anFN-� response modifier in chronic hepatitis infec-ion,45 as an IFN-� response modifier in MS in ourtudy is reassuring. Indeed, the sharing of the type IFN receptor by IFN-� and IFN-�, as well as of manyf their target genes through the common ISRE tran-cription pathway, gives credence to the hypothesis thatt least some response genes should be shared by theeneric group of type I IFNs and should operate irre-pective of disease treated.

Other genes previously associated with IFN responseeterogeneity include the PKR, eIF-2�, iNOS, andRF-1 genes.49 Although included in the screen, we didot detect the polymorphisms previously reported. Re-orted polymorphisms within these genes may haveow frequencies in the Irish population, making themndetectable within this study. Detection of polymor-hisms within our pooled DNA required a minor allelerequency of 20%. Therefore the possibility of notaving detected potentially relevant polymorphismsith a minor allele frequency lower than 20% isresent. Genes showing less pronounced differences inNP allele frequencies between the pools should beonsidered by typing individuals. Because numerousenes may contribute collectively to the response toFN, some of these weaker markers may neverthelesse important in the construction of an accurate predic-ive response model. Validation in other IFN-treatediseases (such as cancers and hepatitis B and C viruses)s also required because the potentiality of population-nd disease-specific IFN response genes cannot as yete excluded.In conclusion, this work has highlighted a number of

enes that have been shown to be significantly associ-ted with response heterogeneity of MS patients toIFN-� and has also highlighted SNPs within otherenes that should, in the future, be examined for aotential role in response heterogeneity.

We thank Drs Knight and Savage for their assistance in perform-ng pyrosequencing.

Drs Vandenbroeck, Graham, and Hawkins were supported byrant RRG11.5/RSG1726 (“The Genetic Epidemiology of Multipleclerosis in Northern Ireland”) from the Northern Ireland HPSS R&Dffice (Belfast, Ireland). Drs Cunningham, Patterson, and O’Rourke

eceipt of nondirected research funding from Biogen-Idec (Cam-ridge, Mass) and Ares-Serono (Geneva, Switzerland). He has beenmember of advisory boards for Ares-Serono and Teva Pharmaceu-

icals (Mijdrecht, The Netherlands). Dr Hutchinson has received ands currently receiving nondirected research funding from Biogen-dec, Schering (Kenilworth, NJ), and Ares-Serono. He is a medicaldvisory board member for a phase III trial of natalizumab and haseceived funding for advice to Biogen-Idec in relation to natalizumab.r Droogan has received honoraria as an advisor to Biogen-Idec. DrcDonnell has attended medical conferences with the support ofiogen-Idec and Ares-Serono. The Queen’s University of Belfast has

ubmitted a British patent application (No. 0427691.1; primary in-estigators Drs Vandenbroeck and Cunningham) on pharmaco-enomic markers.

eferences1. Interferon beta-1b in the treatment of multiple sclerosis:

final outcome of the randomized controlled trial. TheIFN� Multiple Sclerosis Study Group and the Universityof British Columbia MS/MRI Analysis Group. Neurol-ogy 1995;45:1227-85.

2. Brosnan CF, Raine CS. Mechanism of immune injury inmultiple sclerosis. Brain Pathol 1996;6:243-57.

3. Neighbour PA, Miller AE, Bloom BR. Interferon re-sponses of leukocytes in multiple sclerosis. Neurology1981;31:561-6.

4. Jacobs L, O’Malley JA, Freeman A, Reese P. Intrathecalinterferon as treatment of multiple sclerosis. A plannedmulticenter study. Arch Neurol 1983;40:683-6.

5. The Once Weekly Interferon for MS Study Group. Evi-dence of interferon beta-1a dose response in relapsing-remitting MS: the OWIMS Study. Neurology 1999;53:679-86.

6. Francis D. Use of drugs to alter disease course. In:Thompson A, McDonald I, editors. Key advances in theeffective management of multiple sclerosis. London:Royal Society of Medicine Press; 1999. p. 31-5.

7. Grigoriadis N. Interferon � treatment in the relapse-remitting multiple sclerosis. Clin Neurol Neurosurg2002;104:251-8.

8. Antonelli G, Dianzani F. Development of antibodies tointerferon beta in patients: technical and biological as-pects. Eur Cytokine Netw 1999;10:413-22.

9. Giovannoni G, Munschauer FE, Deisenhammer F. Neu-tralising antibodies to interferon beta during the treatmentof multiple sclerosis. J Neurol Neurosurg Psychiatry2002;73:465-9.

0. Kerr IM, Stark GR. The control of interferon-induciblegene expression. FEBS Lett 1991;285:194-8.

1. Levy DE, Kessler DS, Pine R, Reich N, Darnell JE Jr.Interferon-induced nuclear factors that bind a shared pro-moter element correlate with positive and negative tran-scriptional control. Genes Dev 1988;2:383-93.

2. Williams BRG. Transcriptional regulation of interferon-stimulated genes. Eur J Biochem 1991;200:1-11.

3. Platanias LC, Fish EN. Signaling pathways activated by

4. Wang IM, Blanco JCG, Tsai SY, Tsai MJ, Ozato K.Interferon regulatory factors and TFIIB cooperativelyregulate interferon-responsive promoter activity in vivoand in vitro. Mol Cell Biol 1996;16:6313-24.

5. Poser CM, Paty DW, Scheinberg L, McDonald WI,Davis FA, Ebers GC, et al. New diagnostic criteria formultiple sclerosis: guidelines for research protocols. AnnNeurol 1983;13:227-31.

6. Waubant E, Vukusic S, Gignoux L, Durand-Dubief F,Achiti I, Blanc S, et al. Clinical characteristics of re-sponders to interferon therapy for relapsing MS. Neurol-ogy 2003;61:184-9.

7. Rudick RA, Lee JC, Simon J, Ransohoff RM, Fisher E.Defining interferon beta response status in multiple scle-rosis patients. Ann Neurol 2004;56:548-55.

8. Antonelli G, Bagnato F, Pozzilli C, Simeoni E, Bas-tianelli S, Currenti M, et al. Development of neutralizingantibodies in patients with relapsing-remitting multiplesclerosis treated with IFN-beta1a. J Interferon CytokineRes 1998;18:345-50.

9. Bansal A, van den Boom D, Kammerer S, Honisch C,Adam G, Cantor CR, et al. Association testing by DNApooling: an effective initial screen. Proc Natl Acad SciU S A 2002;99:16871-4.

0. Mooney SD, Altman RB. MutDB: Annotating humanvariation with functionally relevant data. Bioinformatics2003;19:1858-60.

1. Mooney Lab. MutDB. Available from: URL:http://mutdb.org. Accessed Nov 8, 2005.

2. Riva A, Kohane IS. A Web-based tool to retrieve humangenome polymorphisms from public databases. In: Pro-ceedings of the American Medical Informatics Associa-tion 2001 annual conference; Nov 3-7, 2001; WashingtonDC.

3. Hubbard T, Andrews D, Caccamo M, Cameron G, ChenY, Clamp M, et al. Ensembl 2005. Nucleic Acids Res2005;33:D447-53.

4. Rebhan M, Chalifa-Caspi V, Prilusky J, Lancet D. Gene-Cards: encyclopedia for genes, proteins and diseases.Rehovot (Israel): Bioinformatics Unit and Genome Cen-ter, Weizmann Institute of Science; 1997.

5. GeneCards. Available from: URL:http://www.genecards.org. Accessed Nov 8, 2005.

6. Matys V, Fricke E, Geffers R, Gößling E, Haubrock M,Hehl R, et al. TRANSFAC: transcriptional regulation,from patterns to profiles. Nucleic Acids Res 2003;31:374-8.

7. Transfac. Databases. Available from: URL:http://www.biobase.de/pages/products/databases.html. Accessed Nov 8,2005.

8. Transcription regulatory regions database (TRRD).Available from: URL:www.bionet.nsc.ru/trrd/. AccessedNov 8, 2005.

9. Kel A, Goessling E. MATCH. Available from: URL:

0. Werner T. Computer-assisted analysis of transcriptioncontrol regions. Matinspector and other programs. Meth-ods Mol Biol 2000;132:337-49.

1. TESS: Transcription element search software. Availablefrom: URL:www.cbil.upenn.edu/tess. Accessed Nov 8,2005.

2. Prestridge DS. SIGNAL SCAN: A computer programthat scans DNA sequences for eukaryotic transcriptionalelements. CABIOS 1991;7:203-6.

3. Ewing B, Hillier L, Wendl MC, Green P. Base-calling ofautomated sequencer traces using phred. I. Accuracyassessment. Genome Res 1998;8:175-85.

4. Ewing B, Green P. Base-calling of automated sequencertraces using phred. II. Error probabilities. Genome Res1998;8:186-94.

5. Breen G, Harold D, Ralston S, Shaw D, St Clair D.Determining SNP allele frequencies in DNA pools. Bio-techniques 2000;28:464-70.

6. Devlin B, Roeder K. Genomic control for associationstudies. Biometrics 1999;55:997-1004.

7. Sham PC, Curtis D. Monte Carlo tests for associationsbetween disease and alleles at highly polymorphic loci.Ann Hum Genet 1995;59:97-105.

8. Weir BS. Genetic data analysis II: methods for discretepopulation genetic data. Sunderland (MA): Sinauer As-sociates; 1996.

9. McDonnell GV, Kirk CW, Middleton D, Droogan AG,Hawkins SA, Patterson CC, et al. Genetic associationstudies of tumour necrosis factor alpha and beta andtumour necrosis factor receptor 1 and 2 polymorphismsacross the clinical spectrum of multiple sclerosis. J Neu-rol 1999;246:1051-8.

0. Bluyssen HA, Vlietstra RJ, Van der Made A, Trapman J.The interferon-stimulated gene 54 K promoter containstwo adjacent functional interferon-stimulated responseelements of different strength, which act synergisticallyfor maximal interferon-alpha inducibility. Eur J Biochem1994;220:395-402.

1. Xu Z, Williams BR. Genomic features of human PKR:alternative splicing and a polymorphic CGG repeat in the5’-untranslated region. J Interferon Cytokine Res 1998;18:609-16.

2. Sriram U, Barcellos LF, Villoslada P, Rio J, BaranziniSE, Caillier S, et al. Pharmacogenomic analysis of inter-feron receptor polymorphisms in multiple sclerosis.Genes Immun 2003;4:147-52.

3. Muldoon J, Uriel A, Khoo S, Ollier WE, Hajeer AH.Novel IFN-alpha receptor promoter polymorphisms.Genes Immun 2001;2:159-60.

4. Leyva L, Fernandez O, Fedetz M, Blanco E, FernandezVE, Oliver B, et al. IFNAR1 and IFNAR2 polymor-phisms confer susceptibility to multiple sclerosis but notto interferon-beta treatment response. J Neuroimmunol2005;163:165-71.

5. Sugimoto Y, Kuzushita N, Takehara T, Kanto T, Tatsumi

the low molecular mass polypeptide 7 gene influences theinterferon response in patients with chronic hepatitis C. JViral Hepat 2002;9:377-84.

6. Gilli F, Marnetto F, Caldano M, Sala A, Malucchi S, DiSapio A, et al. Biological responsiveness to first injec-tions of interferon-beta in patients with multiple sclero-sis. J Neuroimmunol 2005;158:195-203.

7. Gilli F, Sala A, Bancone C, Salacone P, Gallo M, Gaia E,et al. Evaluation of IFNalpha bioavailability by MxAmRNA in HCV patients. J Immunol Methods 2002;262:187-90.

8. Hijikata M, Mishiro S, Miyamoto C, Furuichi Y, HashimotoM, Ohta Y. Genetic polymorphism of the MxA gene pro-moter and interferon responsiveness of hepatitis C patients:revisited by analyzing two SNP sites (�123 and �88) invivo and in vitro. Intervirology 2001;44:379-82.

9. King JK, Yeh SH, Lin MW, Liu CJ, Lai MY, Kao JH, etal. Genetic polymorphisms in interferon pathway andresponse to interferon treatment in hepatitis B patients: a

0. Nakanishi H. Microglial functions and proteases. MolNeurobiol 2003;27:163-76.

1. Beck H, Schwarz G, Schroter CJ, Deeg M, Baier D,Stevanovic S, et al. Cathepsin S and an asparagine-specific endoprotease dominate the proteolytic process-ing of human myelin basic protein in vitro. Eur J Immu-nol 2001;31:3726-36.

2. Driessen C, Bryant RA, Lennon-Dumenil AM, Vil-ladangos JA, Bryant PW, Shi GP, et al. Cathepsin Scontrols the trafficking and maturation of MHC class IImolecules in dendritic cells. J Cell Biol 1999;147:775-90.

3. Hong J, Zang YC, Hutton G, Rivera VM, Zhang JZ.Gene expression profiling of relevant biomarkers fortreatment evaluation in multiple sclerosis. J Neuroimmu-nol 2004;152:126-39.

4. Koike F, Satoh J, Miyake S, Yamamoto T, Kawai M,Kikuchi S, et al. Microarray analysis identifies interferonbeta-regulated genes in multiple sclerosis. J Neuroimmu-

CLINICAL PHARMACOLOGY & THERAPEUTICS2005;78(6):635-46 Pharmacogenomics of IFN-� response in MS 646.e1

ppendix 1. Gene list

No. Gene name Abbreviation

Effect on regulation oftranscription by type I

IFN (� or �[or both]) (dependent

on cell type)Database

accession no.

References (concerning IFNtranscriptional effects, plus

ISRE information)

1 IFN-�-inducible T-cell �chemoattractant

BR1, ITAC, SCYB11,H-174

Up-regulated AF113846 Rani et al1 (1999)Rani et al2 (1996)

2 CC chemokine receptor 3 CCR3 Up-regulated AF2473603 Fas ligand FASL Up-regulated AF035584 Der et al3 (1998) Frigerio et

al4 (2000)4 Guanylate-binding protein GBP Up-regulated MutDB Weinstock-Guttman et al5

(2003)5 IFN-induced gene 6-16 H6-16 Up-regulated Y00828 Koike et al6 (2003) Der et

al3 (1998) Whyatt et al7

(1993) Porter et al8

(1988)6 IFN-induced gene 9-27,

Leu13 antigen,IFN-induced protein 17

H9-27, IFI17,IFITM1

Up-regulated J04164 Sturzebecher et al9 (2003)Der et al3 (1998)Weinstock-Guttman et al5

(2003)7 Interleukin 1�-converting

enzyme/caspase 1ICE Up-regulated L27475

8 IFN-� IFNB Up-regulated J00218 Maniatis et al10 (1992)9 IFN-induced 14-kd

transmembrane proteinIFM3, 1-8U Up-regulated X57352 Sturzebecher et al9 (2003)

Ji et al11 (2003)10 Interleukin 1� IL-1� AY13707911 Interleukin 4 IL-4 Up-regulated AF395008 Li-Weber et al12 (1994)12 Interleukin 5 IL-5 Down-regulated AF353265 Sturzebecher et al9 (2003)

McRae et al13 (1997)13 Interleukin 7 receptor IL-7r Up-regulated AY44970914 Interleukin 10 IL-10 Up-regulated U16720,

AF418271Feng et al14 (2002)

15 Interleukin 13 IL-13 Down-regulated AF377331 McRae et al13 (1997)16 Inducible nitric oxide

synthaseINOS/NOS2A Down-regulated NM_000625 Guthikonda et al15 (1998)

Levy et al16 (1988) Xieet al17 (1993)

17 IP-10 chemokine(inflammatory protein)

IP10, CXCL10 Up-regulated AC112719 Sturzebecher et al9 (2003)Tomura and Narumi18

(1999) Neville et al19

(1997)18 IP-30 chemokine

(inflammatory protein)(30 kd)

IP30, IFI-30, GILT Up-regulated MutDB Sturzebecher et al9 (2003)Der et al3 (1998)

19 CXCL 11gene/chemokine IP-9

IP9, CXCL11 Down-regulated Y15221

20 IFN regulatory factor 1 IRF-1 Up-regulated MutDB Sturzebecher et al9 (2003)Buttmann et al20 (2004)

21 IFN regulatory factor 2 IRF-2 Up-regulated AC099343 Der et al3 (1998) Harada etal21 (1994)

22 IFN-stimulated gene 12 ISG12 Down-regulated AJ296088,AL079302

Martensen et al22 (2001)

23 IFN-stimulated gene 15 ISG15, IFI-15K Up-regulated M31020,M20483

Koike et al6 (2003) Meraroet al23 (2002) Gariglio et

CLINICAL PHARMACOLOGY & THERAPEUTICS646.e2 Cunningham et al DECEMBER 2005

ppendix 1—Cont’d

accession no.

ISRE information)

24 IFN-stimulated gene 20 ISG20 Up-regulated NT_033276 Gongora et al25 (2000)25 IFN-stimulated gene 54 ISG54, IFI-54K,

IFIT2, IFI54,G10P2

Up-regulated X07557 Der et al3 (1998) de Veer etal26 (1998) Whatelet etal27 (1988) Bluyssen etal28 (1994)

26 IFN-inducible gene IFI-56(56 kd)

ISG56, IFI56 Up-regulated X06559 Koike et al6 (2003) Der etal3 (1998) Whatelet etal29 (1987) Whatelet etal27 (1988)

27 2�-5� Oligoadenylatesynthase

OAS Up-regulated X06560 Der et al3 (1998)Floyd-Smith et al30

(1999)28 Platelet endothelial cell

adhesion molecule 1PECAM-1 Up-regulated X96848 Whatelet et al29 (1987)

29 Double-strandedRNA-dependent proteinkinase

PKR Up-regulated AH008429 Der et al3 (1998)Weinstock-Guttman et al5

(2003) Tanaka andSamuel31 (1994)

30 Cyclooxygenase 2 PTGS2 Up-regulated AF27695331 Regulated on activation,

normal T cell-expressedchemokine

RANTES Up-regulated AF088219 Casola et al32 (2002)Cremer et al33 (2002)

32 Secretory leukocyteprotease inhibitor

SLP1 Up-regulated X04502

33 TNF-relatedapoptosis-inducingligand

TRAIL Up-regulated AC007051 Sato et al34 (2001)

34 Vascular cell adhesionmolecule 1

VCAM1, CD106 Up-regulated M73255 Neish et al35 (1995)

35 Sp100 gene promoter Sp100 Down-regulated X95472 Grotzinger et al36 (1996)36 RNA-specific adenosine

deaminaseADAR1 Up-regulated AF084518,

AL606500Der et al3 (1998) George

and Samuel37 (1999)37 TNF receptor p75/80,

CD120bTNFp75/80, CD120b Not reported U53483 Santee and Owen-Schaub38

(1996)38 SPRR2A gene encoding

small proline-richprotein

SPRR2A X53064

39 Polymericimmunoglobulinreceptor

Not reported Y08254 Piskurich et al39 (1997)

40 Cytochrome b558heavy-chain gene

GP91-PHOX, CYBB Up-regulated AL627245 Kumatori et al40 (2002)

41 Complement componentC2

C2 Down-regulated X77331

42 Prepro-orexin gene,preprohypocretin

PPORX Up-regulated AF118885 Waleh et al41 (2001)

43 CD40 ligand gene CD40L D3179344 Interleukin 3 IL-3 Down-regulated AF365976 Koike et al6 (2003)

accession no.

ISRE information)

46 Transported, ABC MHC2

TAP 2, RING 11 X66401

47 IFN regulatory factor 7H IRF-7H Up-regulated AF277159 Sturzebecher et al9 (2003)Ji et al11 (2003) Lu etal41a (2000)

48 Complement factor 1precursor

IF AF005095

49 Tryptophanyl-tRNAsynthase, WRS gene

IFP-53 Down-regulated X82107,X71411

Strehlow et al42 (1993)

50 Survival motor neuronprotein

SMN Up-regulated Baron-Delage et al43 (2000)

51 IFN-�1 IFN-a J00210 Ragg and Weissman44

(1983) Lopez et al45

(1997)52 Complement factor B Bf, GBG, CFAB,

PBF2MutDB,

NT_007592Huang et al46 (2001)

53 IFN-� IFN-y MutDB54 Human mimecan gene AF112465 Tasheva and Conrad47

(2003) Tasheva48 (2002)55 MHC class 1 (HLA-A) HLA-A MutDB Gobin et al49 (1999)56 MHC class 1 (HLA-B7) HLA-B7 Up-regulated D83956 Johnson and Pober50 (1994)

Gobin et al49 (1999)57 HLA-E HLA-E MutDB58 HLA-F HLA-F MutDB Gobin et al49 (1999)59 Low-molecular mass

polypeptide 7LMP-7 Up-regulated X87344 Der et al3 (1998)

60 Human TNFreceptor-associatedfactor 6

TRAF6 Down-regulated MutDB Der et al3 (1998)

61 Tumor necrosis factor B TNFB M1644162 Tumor necrosis factor A TNFA M19441 Ji et al11 (2003)63 IFN regulatory factor 3 IRF3 AF11218164 Biliary glycoprotein C-CAM-1, BGP-1,

CD66, CD66AX67277 Chen et al51 (1996)

65 IFN-induced protein p78 Mx1, Mx78, MxA Up-regulated AF135187 Sturzebecher et al9 (2003)Ji et al11 (2003) Der etal3 (1998)Weinstock-Guttman et al5

(2003) Ronni et al52

(1998)66 Thymosin � AJ29515867 FMS-related tyrosine

kinase 3FLT3 U29874

68 Cyclin-dependent kinaseinhibitor 1A

p21, Cip1 AF497972

69 Myeloid cell nucleardifferentiation antigen

MNDA Up-regulated AP002534 Ji et al11 (2003) Landolfo etal53 (1998) Kao et al54

(1996)

accession no.

ISRE information)

70 IFN-�-inducible protein IFI-16 Up-regulated M63838 Koike et al6 (2003) Der etal3 (1998) Landolfo etal53 (1998) Kao et al54

(1996)71 Suppressor of cytokine

signaling proteinSOCS-1 Not reported Z46490 Schluter et al55 (2000)

72 Signal transducer andactivator oftranscription 1

STAT 1 Up-regulated AH008323 Ji et al11 (2003) Der et al3

(1998)Weinstock-Guttman et al5

(2003) Wong et al56

(2002)73 Signal transducer and

activator oftranscription 2

STAT 2 Up-regulated U18671 Ji et al11 (2003)

74 c-myc Promoter bindingprotein

C-myc, IRLB X00364 Alexandrova et al57 (1990)Turpaev et al58 (1991)

75 IgE switch region S epsilon Up-regulated X56797 Mills et al59 (1990)76 Cathepsin S CTSS Up-regulated NT_032962 Ji et al11 (2003) Shi et al60

(1994) Storm van’sGravesande et al61 (2002)

77 HLA-DMA (majorhistocompatibilitycomplex, class II, DM�)

DMA, D6S222E,HLADM, RING6

X87344

78 Protamine 3 PRM3 Z4694079 Mannose-binding lectin MBP1 AL58385580 IFN-�/-� receptor IFNAR X60459 Weinstock-Guttman et al5

(2003)81 MHC class 1 (HLA-G) HLA-G Up-regulated AB088083 Sturzebecher et al9 (2003)

Gobin et al49 (1999)Lefebvre et al62 (2001)

82 IFN-inducible gene IFI-60(60 kd),tetratricopeptide protein

IFI-60, IFIT4,CIG-49, RIG-G

Up-regulated MutDB Koike et al6 (2003) de Veeret al26 (1998)

83 Toll-like receptor TLR-4 AF172169,AH009665

84 Promyelocytic leukemia PML gene Up-regulated X91752 Der et al3 (1998)85 Interleukin 22 IL-22 AF38751986 TGF-�-stimulated clone TSC22 Up-regulated AF256226 Koike et al6 (2003)87 Synapsin II gene SYN2 X8985188 DNA-directed RNA

polymerases III 39-kdpolypeptide

RPC39 Up-regulated AL121893 Koike et al6 (2003)

89 A kinase (PRKA) anchorprotein 4

AKAP4 Down-regulated MutDB Koike et al6 (2003)

90 Homo sapiens guaninenucleotide-bindingprotein (G protein), �

GNA13 Down-regulated MutDB Koike et al6 (2003)

accession no.

ISRE information)

91 Toll-like receptor 7 TLR-7 Up-regulated NM_01656292 Mitogen-inducible gene 6

proteinMig-6, GENE-33 NM_018948 Koike et al6 (2003)

93 Human synapsin I(SYN1) gene

SYN1 Down-regulated M55301

94 Intercellular adhesionmolecule 1

ICAM-1 AY225514

95 Interleukin 7 IL7 Up-regulated AY449709 Koike et al6 (2003)96 MHC class 1 (HLA-C) HLA-C MutDB,

AB088111Gobin et al49 (1999)

97 Eukaryotic translationinitiation factor (eIF)2A

eIF-2� Not reported MutDB

98 PMA-responsive gene APR Up-regulated Der et al3 (1998)99 Interleukin 8 IL8 Up-regulated M28130 Ji et al11 (2003)

100 Preprotachykinin PPTA, TAC1 Not reported AC004140.1

ppendix 2. PCR and sequencing primers

Gene Primer name Primer sequence (5�¡3�) Fragment sizeAnnealing temperature,

BR-1 BR1-FWD CCA AAG TAG TCC GAC AAT TA 459 55.0°C, 3.0 mmol/LBR1-REV ACA TTA GAA AAG GAA CTT ATA GG

CCR3 CCR3-FWD TTA GTT GGT TCC ACG GCA ATG CT 920 62.5°C, 2.0 mmol/LCCR3-REV GAT AAG TTC TTG CCC GGA TAACCR3-intF CAG GTC TTT AGG AGG CCG AGA CTCCR3-intRV AGT CTC GGC CTC CTA AAG ACC TG

FasL FASL-PCRFW GCC TGG GCA ACA TAG CAA GTC 1385 60.0°C, 2.5 mmol/LFASL-PCRRV ACC AAG GCA ACC AGA ACC ATG AAFASL-SEQ-inF1 CGG CAG GTC AGG GTA AAT GFASL-SEQ-inR1 GGA CGG GAC CCT GTT GCT GFASL-SEQ-inF2 TGG GTA GCA CAG CGA CAG CFASL-SEQ-inR2 TCT GAG GGG AGA GAC CAT G

GBP GBP1-PCR-FW AAA CCT AAT GGG CAT TTT GTT AGAC

1009 58.0°C, 2.0 mmol/L

GBP1-PCR-RV CAG TGA AGC AGT GGG GTT GAA TGBP1-SEQ-F-IN GGA CAG CAT GGC CTT AGG TGBP1-SEQ-Rv-IN ACT TAA GGC CAT GCT GTC C

H6-16 H6-16-FWD GGT CTT ACC ATA TGC CCA TGA CCCA

906 60.0°C, 2.0 mmol/L

H6-16-REV TGA CAG TGT TAA GGC CAC AGA TCGC

H6-16-SEQ-inF1 GGG GGA GCT GGT GAT CAG GH6-16-SEQ-inR1 CCT GAT CAC CAG CTC CCC CH6-16-SEQ-inF2 GTT TAC TCG CTG CTG TGC CH6-16-SEQ-inR2 GGC ACA GCA GCG AGT AAA C

H9-27 9-27 FWD TTT CCT CCC ACC CTG CCA TAA GT 444 60.0°C, 2.5 mmol/L

ICE ICE-FWD CAA GGT CAA ATA ACT GCA TTC CG 889 64.8°C, 1.5 mmol/LICE-REV CTG TAC CCT CTG TCA GAC TTTICE-INT-FWD CCC TCC TAC CCT GAT CTA TICE-INT-RV ATA GAT CAG GGT AGG AGG G

IFNB IFNB-F ATT CTC AGG TCG TTT GCT T 485 55.0°C, 2.0 mmol/LIFNB-RV GCC ACA GGA GCT TCT GAC ACT

H1-8U 1-8U-FWD GCC CCT CCT TCC TCT CTA TCT T 754 61.5°C, 1.5 mmol/L1-8U-REV AAA CAC GTG CAC TTT ATT GAA TGC

C1-8U-INT-FWD CCT GCT GCC TGG GCT TCA TAG1-8U-INT-REV CTA TGA AGC CCA GGC AGC AGG

IL1B IL1B-PCR-F CTT CCA CTT TGT CCC ACA TAT AC 859 62.5°C, 2.0 mmol/LIL1B-PCR-RV AGA GGA GAC CTG CTC ATG TTA CTIL1B-SEQ-F-IN GTC TCT CTG CCT CTT TGT GIL1B-SEQ-RV-IN CAC AAA GAG GCA GAG AGA C

IL-4 IL4-FWD CAT TAC AAC AAA TTC GGA CA 564 55°C, 2.0 mmol/LIL4-REV AGA TGG TGC CAG ATA GGT ACT CA

IL-5 IL5-FWD GCC CAA GGA TGG AGT CAA GG 842 65°C, 2.0 mmol/LIL5-REV TCA TTG GCT ATC AGC AGA GTT CGA

TBetaine/DMSO

IL5-intF ATA GTA GAA CAT AGC CGA TCT GAAA

IL5-intRV TTT CAG ATC GGC TAT GTT CTA CTA TIL-10 IL10-FWD CTG TGA CCT AGG AAC ACG C 537 55°C, 3.0 mmol/L

IL10-RV CGG AGA TCT CGA AGC ATG TTAIL-13 IL13-FWD TCC AGG TAC TCA GGG TTG TCA CA 571 55°C, 2.0 mmol/L

IL13-RV ATG ACC TCA TCT TGG GAA TCA CIP-10 IP-10-FWD TCT CAT CTC TGT CCG CAT TC 914 61.0°C, 2.0 mmol/L

IP-10-REV TCA AAG CAG GCC AGT CCT ATT ACIP-10-INTER-F GCC ATT TTC CCT CCC TAA TTCIP-10-INTER-RV GAA TTA GGG AGG GAA AAT GGC

IFI30 IFI30-PCRFW GCC CCA CCT CAC CTC CCC TAA A 691 65.0°C, 2.5 mmol/LIFI30-PCRRV AGT ACA CAG TAG GCG CTC AGG GAA

CIFI30-SEQ-inF TGT CGC CAC TTC TGC TGT T

IP-9 IP-9-FWD GGT GTC AAA TAA TAT AGG CCT TA 827 55°C, 2.0 mmol/LIP-9-REV GCC AGA TAT TCT AGC CAA ATC AAIP-9-INT-FWD TGA GTG TGA AGG GCA TGG CIP-9-INT-REV GCC ATG CCC TTC ACA CTC A

IRF-1 IRF1-FWD TGT CAG GAA GGC GTA GAA TG 1380 60.0°C, 2.0 mmol/LIRF1-RV TTA AAA ACC AGA GGA TGT GTA GGG

AIRF1-SEQ-inF1 TGC CCA GGT ACT GTT CAA CIRF1-SEQ-inR1 ATT TGT TCG ATT GGA GCC CIRF1-SEQ-inF2 GGG CTC CAA TCG AAC AAA TIRF1-SEQ-inR2 GTT GAA CAG TAC CTG GGC A

IRF-2 IRF2-FWD CAT TTT CTT ACC ACA CGC GTT TT 572 58.0°C, 2.0 mmol/LIRF2-RV GAT GAG TTC TGG AGC CTG TGC GAC

CISG12 ISG12-FWD TCT AGC ACA GTG GGC CTT CAG TTG

AGA1160 68°C/94°C, 2.5

mmol/L

ISG12-RV TGG GGT GAG GGA GGG AGG GCT GTTG

ISG12-SEQ-inF1 GGT GGA GCT GCT TAA AAG GISG12-SEQ-inR1 CCT TTT AAG CAG CTC CAC CISG12-SEQ-inF2 CAT GCC TGG GCG GGG TGT TISG12-SEQ-inR2 AAC ACC CGG CCC AGG CAT G

ISG15 ISG-15-F GCC CTA AAC CGA GTG TTG TTA TCT C 992 68°C/94°C, 3.5mmol/L

ISG-15-R GCC TTC AGC TCT GAC ACC GAC ATISG-15-INT-FW CCA CAG CCC ACA GCC CAC AISG-15-INT-RV TGT GGG CTG TGG GCT GTG G

ISG20 ISG-20-F TGT TGC AAT CCA CTC CAC GAA CG 1160 68°C/94°C, 1.5mmol/L

ISG-20-R GGG AAG TGG GAG AGG GGT TAT TISG-20-INT-FW GCA AGA GGC CAG CTT CCA CISG-20-INT-RV GTG GAA GCT GGC CTC TTG C

ISG-54 ISG54-FWD ACA AGT CCC CAA CTT AAT CT 513 55.0°C, 1.5 mmol/LISG54-REV ACA AAG GTA GAG GCA TAG TAA GG

IFI56 IFI56-FWD GCA ACC AAA AGC CTT GTT ACT CAAT

860 55.0°C, 2.0 mmol/L

IFI56-REV CAT TCC TTC CCC ATA TCA GACIFI56-INT-FWD CAG AAC GGC TGC CTA ATT TIFI56-INT-REV AAA TTA GGC AGC CGT TCT G

OAS 2�5OAS-FWD ACT GTG AAA GGA TTT CAT CAA CAAC

605 61.5°C, 1.5 mmol/L

2�5OAS-RV TCT CTC ATT CTC AAT CTC AAC TCT TPECAM-1 PECAM-FWD CCT ACTT CCT CTG CTA TTC TCG 863 60.0°C, 2.0 mmol/L

PECAM-REV AAG CCA GGT AGA AAC ATC TGT TACA

Betaine/DMSO

PECAM-int-FWD ATT ACC TGA CCA GCG CCA CAGPECAM-int-REV CTG TGG CGC TGG TCA GGT AAT

PKR PKR-FWD AGC CCC CGA CTT GAACTG AAT CCT G 553 55.0°C, 2.0 mmol/LPKR-REV CCC CCT GCC CTG CTC ACC TGC

PTGS2 PTGS2-FWD TCC CAT CCA AGG CGA TCA GT 721 65.0°C, 1.5 mmol/LPTGS2-REV AAT ATC CAC GGA GTT CTT TCG GAC TPTGS2-int-FWD CTC CAC AGC CAG ACG CCC TCAPTGS2-int-REV TGA GGG CGT CTG GCT GTG GAG

RANTES RANTES-FWD TGG AAG GTA AAA CTA AGG ATG TC 870 61.0°C, 2.0 mmol/LRANTES-REV AGC CAC ATA CCC TCT CAT AAGRANTES-int-FWD TTG ACC ACC ACA GCC CCT GRANTES-int-REV CAG GGG CTG TGG TGG TCA A

SPLI SPLI-FWD TTC CAG GTC CGG GTA G 490 55.0°C, 2.0 mmol/LSPLI-REV GAC CCT GCA GCC CAG ATT AGA

TRAIL TRAIL-PCR-F AAA GGA TAG TGA CAG CGA GAC AT 877 2.0 mmol/LTRAIL-PCR-R CAC GAT CAG CAC GCA GGT BetaineTRAIL-SEQ-F-IN GAA GAG AGA AAT GGG CTT GTRAIL-SEQ-R-IN TCA TGG CCT GGC TGT CAT A

VCAM-1 VCAM-1-PCR-F AAA TCA ATT CACA TGG CAT AG 844 64.0°C, 2.5 mmol/LVCAM-1-PCR-R GAT TCA TTG TCAG CGT AGA TGVCAM-1-SEQ-F-IN GTA TCT GCA TCGG GCC TCA CTVCAM-1-SEQ-R-IN AGT GAG GCC CGA TGCA GAT AC

Sp100 Sp100-FWDSp100-REV

GGG CAG AGC AGC TAA GGA CAAGGA TTA AAG TGA AGC CTT AGG CA

535 55.0°C, 1.5 mmol/L

ADAR1 ADAR1-FWD CGG AGG GGT TCG ACT TGT AA 540 62.5°C, 1.5 mmol/LADAR1-REV GGA AGG TGG CAG TGA CGG TGT Betaine/DMSO

TNFr75/80 TNFr75/80-FWD ATG CTG GAG TGG TCG GGT CGG 566 65.0°C, 2.0 mmol/LTNFR75/80-REV CTG TCA CGA CGC GGG TAA GAG Betaine/DMSO

SPRR-2A SPRR2A-PCR-F TGA GTC AGG AGA GCT TAA CCC AG 1160 65.0°C, 1.5 mmol/LSPRR2A-PCR-R GGG TGG GGA AGG TGT CAC ASPRR2A-SEQ-F-IN AAT TCT CCC TGA GCA GCC CTT TGSPRR2A-SEQ-R-IN CAA AGG GCT GCT CAG GGA GAA TT

PolyIG POLY-IG-FWD GGG CTA TAT CCT CTA CCT G 1049 57.0°C, 1.5 mmol/LPOLY-IG-REV GAC TTG CTA GGT GCC ATA CTT TAPOLY-IG-int-F CGG GAC GAT GGC TCA GGT APOLY-IG-int-R TAC CTG AGC CAT CGT CCC G

GP91 GP91PHOX-FWD GCT TAA AAA TTG TGA TCA AA 555 55.0°C, 2.0 mmol/LGP91PHOX-REV GAA GAA TGG ACT CAG TCT GTT

C2 C2-PCR-F TTA TTC CTT TGA GTA ACC AAC CC 851 55.0°C, 2.0 mmol/LC2-PCR-R ACA GGA CCC CTG ACG TTC CCC2-SEQ-F-IN GAG ACA GGG CAA AGG TTT CACC2-SEQ-R-IN GTG AAA CCT TTG CCC TGT CTC

PPHORX PPORX-FWD ATC TCC AGG GAG CAG ATA GAC AG 500 63.0°C, 2.0 mmol/LPPORX-REV ACA GCA CCC AAC CCA ATG T

CD40L CD40L-PCR-F CTC TTA ACT GCA GCC TAT TT 1023 62.5°C, 2.5 mmol/LCD40L-PCR-R GGC TTG TGG TTC ATC TTA CCT TGCD40L-SEQ-F-IN CCG CTT CGT ATT AGT AAG ACD40L-SEQ-R-IN GTT GCA GAA CAC ACT TCC T

IL-3 IL-3-PCR-F TGA TGG CAG ATG AGA TCC C 828 55.0°C, 2.0 mmol/LIL-3-PCR-R CAA TCC CAC TGA GAT AGG TGA TAIL-3-SEQ-F-IN CAG ACA ACG CCC TTG AAG ACAIL-3-SEQ-R-IN TGT CTT CAA GGG AGT TGT CTG

TAP01 TAP01-PCR-F CAC CAG CCT CGC GTG CCT 901 55.0°C, 2.0 mmol/LTAP01-PCR-R GGA GCG GGA CAC CTA GAG CTATAP01-SEQ-F-IN AGG GAG AGG CGA GAA GGG TGTTAP01-SEQ-R-IN ACA CCC TTC TCG CCT CTC CCT

TAP02 TAP02-PCR-F CCC AGA GTA ACC GCC ACT AAA GG 778 58.0°C, 1.5 mmol/LTAP02-PCR-R CCA CAG TAA AGC CGC GTC CTAP02-SEQ-F-IN AGC CCG GAC CAC TTA GCT CTAP02-SEQ-R-IN GAG CTA AGT GGT CCG GGC T

IRF-7H IRF-7H-FWD GCC GGG CGT GGT GGT TCA T 1189 62.5°C, 2.5 mmol/LIRF-7H-RV GCC ACT GCA GCC CCT CAT AGC MercaptoethanolIRF-7H-SEQ-inF1 GCC CTC CAC TCC TCC CTA CTCIRF-7H-SEQ-inR1 GAG TAG GGA GGA GTG GAG GGCIRF-7H-SEQ-inF2 GGC CTG GAC ACT GGT TCA ACAIRF-7H-SEQ-inR2 TGT TGA ACC AGT GTC CAG GCC

CF1 CF-1-FWD CCA GCC AGC CTC TTG TGA CAG TA 1532 55.0°C, 2.0 mmol/LCF-1-RV TGG CAT TGT TGT AAC TGA ACT ATG TCF-1-SEQ-inF1 CCT AAG TGT ATG CCC TTC CTGCF-1-SEQ-inR1 CAG GAA GGG CAT ACA CTT AGGCF-1-SEQ-inF2 ATT TGC CTT GAG TGT GTT ACC TCCCF-1-SEQ-inR2 GAA ATT ATA TTG GAA GCA ATT

IFP53 IFP53-PCR-F TCC CAC TGG CAC CCT TCA TCG TC 978 60.5°C, 2.0 mmol/L

IFP53-SEQ-F-IN AGG CAA CCC ACC CCT GAT TIFP53-SEQ-R-IN AAT CAG GGG TGG GTT GCC T

NOS2A NOS2A-PCR-F CCT CTC AAC TTC TCC CTA ATGTAG T

925 61.5°C, 2.0 mmol/L

NOS2A-PCR-R CCT CAG TTT TCG ACT CGC TANOS2A-SEQ-F-IN CCA CTC CGC TCC AGT CTT GNOS2A-SEQ-R-IN AGG CCC TGG CAG TCA CAG T

IFNa IFN-A-FWD GGA ACA AGA TGG GGA AGA CAA TA 468 60.0°C, 1.5 mmol/LIFN-A-REV GCC TTC TGG AAC TGG TTG CC

Bf BF-FWD GAA ATC CTC AGG GCT CCT ACC AG 1016 60.0°C, 1.5 mmol/LBF-REV TGT CTG CAC AGG GTA CGG GTA GAA

GBF-INTER-FW-1 TGG GTG GTT TGG GCT TAC TBF-INTER-FW-2 CTC TGC CTG ATG CCC TTT ABF-INTER-RV-1 AGT AAG CCC AAA CCA CCC ABF-INTER-RV-2 TAA AGG GCA TCA GGC AGA G

IFNg IFN-GAM-FWD GGC TTG TAT TGT ATT TCTA CTG G 899 57.0°C, 2.0 mmol/LIFN-GAM-REV TGA CAG CCT ATC AGA GAT GCT ACIFN-GAM-INT-F CCC AAA TGC CAC AAA ACC TIFN-GAM-INT-R AGG TTT TGT GGC ATT TGG G

MIME MIME-FWD CGC AGG CAA GAA TAG GGT 1070 57.0°C, 2.5 mmol/LMIME-REV ATC CTC ATA ATC TTG GCT AAA TAMIME-int-F AGA CAG GAT CTT GGG ACT TTAMIME-int-R TAA AGT CCC AAG ATC CTG TCT

HLA-A HLA-A-FWD AGGCACAACTTTTCCGGATT 988 61.0°C, 2.0 mmol/LHLA-A-REV TATTCCGTGTCTCCTGGTCCCAATAHLA-A-INTER-F CCCAGTTCTCACTCCCATTGHLA-A-INTER-RV CAATGGGAGTGAGAACTGGG

HLA-B HLA-B-FWD CCGTGCTCAGTTTCCCTACACAA 1007 62.0°C, 2.5 mmol/LHLA-B-REV GATGGGGAGTCGTGACCTGHLA-B-INTFWD CATGGCGCCCCGAACCGTCHLA-B-INTREV GACGGTTCGGGGCGCCATG

HLA-E HLA-E-FWD GGAGGGCAATGGCACGATCTT 975 60.0°C, 2.0 mmol/LHLA-E-REV CGGCCTCGCTCTGATTGTAHLA-E-INTER-F ATCCGGACTCAAGAAGTTCHLA-E-INTER-RV GAA CTT CTT GAG TCC GGA T

HLA-F HLA-F-FWD ATGGGAGGCAGAAAGTTCAATCAAG 930 64.0°C, 2.0 mmol/LHLA-F-REV GTTCACTCACCAGCCTCGCTCTHLA-F-INTER-F CCGCAGTTCCCAGGTTCTAHLA-F-INTER-RV TAGAACCTGGGAACTGCGG

LMP7 LMP7-PCR-F ATT TGC CTT GAG TGT GTT ACC TCC 1593 63.2°C, 2.0 mmol/LLMP7-PCR-RV AGA TCG CAT AGA GAA ACT GTA GTG

TCCLMP7-SEQ-F-IN GCC AGG CGG GAA CAG AGG GLMP7-SEQ-RV-IN TCG TGT CAT CTA AAG GCG GLMP7-SEQ-F-IN-2 CAG TTG GCC CAG GAC CTG TLMP7-SEQ-RV-IN-2 AAT CCC GCC TAC TGT TCT G

TRAF6 TRAF6-FWD GAA AAA TCG TGT GCT AAG TAC TG 637 55.0°C, 2.5 mmol/LTRAF6-REV GGT GGG TCA AAC TCT ACA TCA TTRAF6-int-RV CAC ACA CTC ACA CCC CAC A

TNFa TNFa-FWD AAACACAGGCCTCAGGACTCAAC 886 60.0°C, 1.5 mmol/L

TNFa-INTER-F GAAGTTTTCCGCTGGTTGATNFa-INTER-RV TCAACCAGCGGAAAACTTC

TNFb TNFb-FWD AGATGGGCTGGAAAGTCCGTATA 1005 60.0°C, 1.5 mmol/LTNFb-REV CCCCAGAAGGAGGAGGTGTAGTNFb-INTER-F CTCCTGCCCCATCTCCTTGTNFb-INTER-RV CAAGGAGATGGGGCAGGAG

IRF3 IRF3-FWD CGG TCT CCT CCA CTG AAC TCG TA 1245 65.0°C, 2.0 mmol/LIRF3-RV CCA GGT CCA GCT GCG ACAIRF3-SEQ-inF1 GTG GCG TCC GAG AGC GAG GTA GCIRF3-SEQ-inR1 GCT ACC TCG CTC TCG GAC GCC ACIRF3-SEQ-inF2 ACT GGC GGA ATT GAG GGA GTG GAIRF3-SEQ-inR2 TCC ACT CCC TCA ATT CCG CCA GT

BILIARY BILIARY-FWD GCC ACC CAC CTC CCT CTA TCA 1050 65.0°C, 2.5 mmol/LBILIARY-REV CCT TAA TTG TCA CGC TGA CTTBILIARY-int-FWD CAG AGC TTC CTG GAC AAA CCCBILIARY-int-REV GGG TTT GTC CAG GAA GCT CTG

MX1 MX1-FWD ACT ATG TTG GCC AAG CTC GC 1024 65.0°C, 1.5 mmol/LMX1-REV AGA AAA GGG CAG CCT CGA ATGMX1-int-FWD CGG CCT GGA GGG ATA TTC TTGMX1-int-REV CAA GAA TAT CCC TCC AGG CCG

TB4 TB4-FWD TTG TGA CGC GGG CGA TGA AGC 683 65.0°C, 2.5 mmol/LTB4-REV TTC TAA AGA TGG GGG TGG GTC GGTB4-int-FWD CCG CCC TCT TTG GTC CAC ATB4-int-REV TGT GGA CCA AAG AGG GCG G

FLT3 FLT3-PCRFW CAA GAA CAA ATG AAG AGG ACG TG 415 60.0°C, 2.0 mmol/LFLT3-PCRRV CAG CAG CAG GAG GAG ATA GGT

CDKN1A CDKN1A-F TCTGTCTGCCTTGCTGCATGGAC 1002 58.5°C, 2.5 mmol/LCDKN1A-R GCCTGCCTCCTCCCAACTCATCCDKN1A-INT-FW TAGCTTGCCCTTCAGTTGCCDKN1A-INT-RV GCAACTGAAGGGCAAGCTA

MNDA MNDA-FWD ATC ATT TTC CCC TAT TAG CTG T 1442 57.0°C, 2.5 mmol/LMNDA-REV CCC AAA CCC AGT CAC CAA GTC ATCMNDA-INTER-FW-1 AGA CCC ACC CCT ATC CAA GMNDA-INTER-RV-1 GTC TCA GGC AAT TAT TCT CTAMNDA-INTER-FWD-2 CTG ATA GGC GTG GCT TCA AMNDA-INTER-RV-2 GAT TGT AGA TGG CCT TCT TACMNDA-INTER-FWD-3 AGA GAA TAA TTG CCT GAG ACC

IFI-16 IFI16-FWD CAC CCG AAA AAT GTT AAC TGT ACCC

1109 58.0°C, 2.5 mmol/L

IFI16-REV CCT TCC CCT CCA CTC CCC TAT ACCIFI16-int-FWD AAT CAC AAG GGG ACA ATC AIFI16-int-REV TGA TTG TCC CCT TGT GAT T

SOCS-1 SOCS1-PCR-F GGCAGAAAGTGGAACCCGAGGTA 992 60.0°C, 2.0 mmol/LSOCS1-PCR-RV CGGGAGAGACAAAGCGGTGAG Betaine/DMSOSOCS1-SEQ-F-IN GAGGAGGGAGGGGAGTCCASOCS1-SEQ-RV-IN TGGACTCCCCTCCCTCCTC

STAT1 STAT1-FWD TAC CTG GGT TCG CTT GGC TAA TA 1825 60.0°C, 2.5 mmol/LSTAT1-RV CCT AAG TTG GAA AAT AAT CGG TGT

CAGSTAT1-SEQ-inF1 ATT TGT GGA GTA GGC GTGSTAT1-SEQ-inR1 TAA TTG AGT TTG GGG ATG AA

STAT1-SEQ-inR2 TCA CAA GAC TCT CCC AAA ASTAT1-SEQ-inF3 TTC ATC CCC AAA CTC AAT TASTAT1-SEQ-inR3 CAC GCC TAC TCC ACA AAT

STAT 2 STAT2-FWD CCA GTC GAG CAT TTC CTT TGA GT 441 55.0°C, 2.5 mmol/LSTAT2-REV AAG ATT CTG CAG CAT TTC CCA C

CMYC CMYC-FWD CGC AGT TGC ATC TCC GTA TTG 918 57.5°C, 1.25 mmol/LCMYC-REV GGG CAG CAG CTC GAA TTT CCMYC-int-FWD GCC GAT TTC GAT TCC TCT GCMYC-int-REV TGG ACA GTC CCC TGC TAT C

IgE IGE-PCR-F TGC CAG CTC CAA GCG GGT CAC A 880 62.5°C, 3.0 mmol/LIGE-PCR-RV CAC CAG CCA GCC CTG CCA GTC GGIGE-SEQ-F-IN GTC CAG GAA CCC GAC AGA GIGE-SEQ-RV-IN CTC TGT CGG GTT CCT GGA C

CTSS CTSS-PCR-F TCC ACT TTG TCC CCA AGA CCA TAG 1265 64.0°C, 1.5 mmol/LCTSS-PCR-RV CTA CCT TTT CCT TGT ATT GTT TGC

CAT AGCTSS-SEQ-F-IN TGT CGT TCA ATT TGA CTC TTCCTSS-SEQ-RV-IN CTT TCC AGG ATT TGC CAA CCTSS-SEQ-F-IN-2 GTT GGC AAA TCC TGG AAA GCTSS-SEQ-RV-IN-2 GAA GAG GCA AAT TGA ACG ACA

DMA DMA-PCR-F TGA TGT TAA ACC CTA CGC TTC T 1060 60.0°C, 2.0 mmol/LDMA-PCR-RV CAG GCC ACT GTA TTG ACT AGA GADMA-SEQ-F-IN TAT GTG TGG TTG GTA AAC GATDMA-SEQ-RV-IN ATC TTG AGT GGA GGA GTG AAA

PRM3 PRM3-PCR-F ACC TGC ACC TCC TAT GTT CAAATG A

1136 55.0°C, 2.0 mmol/L

PRM3-PCR-RV TTT TCA TGG AGG ATT CGT GGPRM3-SEQ-F-IN GGC CTC TGG TTG GTG GTG TPRM3-SEQ-RV-IN TAG TCC TCA TTT GCC CTG GPRM3-SEQ-F-IN-2 CCA GGG CAA ATG AGG ACT APRM3-SEQ-RV-IN-2 ACA CCA CCA ACC AGA GGC C

MBP MBP-FWD AGG GAA ACT TGG AGG CTT AGA CC 1027 60.0°C, 2.5 mmol/LMBP-REV CCT GGG AAG CCG TTG ATGMBP-int-F AGA TGG ACC CGA AGA GGA CAMBP-int-R CTT TCG GTG GCA GTG AGA AC

IFNAR01 IFNAR01-PCRFW TCT CGC CCC TCA GCC AAG TC 834 58.0°C, 2.5 mmol/LIFNAR01-PCRRV CAG CTG CGT GCC CTA CCT CCIFNAR01-SEQ-inF GAG GCC TGC GAT TTC TAAIFNAR01-SEQ-inR TTA GAA ATC GCA GGC CTC

HLA-G HlaG-PCRFW CGG AAA CTT AGG GCT ACG GAA TG 801 65.0°C, 2.5 mmol/LHlaG-PCRRV CGG GTG GGT GAG CGA GGA CTTHlaG-SEQ-inF CCG GGA TGA AAA GTG AAHlaG-SEQ-inR TTC ACT TTT CAT CCC GG

IFIT4 IFIT4-FWD CTG CCT CCT CTT ACC CAT CTTACC T

1242 65.0°C, 2.5 mmol/L

IFIT4-RV TCT GAG AGT CTG CCC AAG TGA TAGT

IFIT4-SEQ-inF1 AGG AAT TGG CTC ACC TGA TIFIT4-SEQ-inR1 CAG GAT GCC CAG ATA TTT GIFIT4-SEQ-inF2 CAA ATA TCT GGG CAT CCT GIFIT4-SEQ-inR2 ATC AGG TGA GCC AAT TCC T

TLR4 TLR4-PCRFW GAG GTA TGT AAG GTA GAA TGA GGTC

886 59.5°C, 2.0 mmol/LBetaine/DMSO

TLR4-PCRRV GAA GGA TAC AGA GAA GAT GAG CATLR4-SEQ-inF TGC TAA GGT TGC CGC TTT CTLR4-SEQ-inR GAA AGC GGC AAC CTT AGC A

PML PML-PCRFW GCC CCC ACC ACC TCA GAT CCA C 1088 68°C/94°C, 2.5mmol/L

PML-PCRRV AAC CCC ATC ATC CCC TAA CCC AA 2-Step PCRPML-SQ-inF GGG CTG CGT GTG GCT CAT CPML-SQ-inR GAT GAG CCA CAC GCA GCC C

IL22 IL22-PCRFW AAT TAG ATT AGC CAA GAC AGT TATT

933 55.0°C. 2.5 mmol/L

IL22-PCRRV ACG AGA AAG AGC AGG ATT GAG AIL22-SEQ-inF GGG CTC CTG TGG TGG TTA GIL22-SEQ-inR CTA ACC ACC ACA GGA GCC CTSC22-PCRFW CCA GGC CAG CCC ACC CCA CGA G

TSC22 TSC22-PCRRV AAT GCC AGC CCC TCT CTT ACTAAC G

800 64.0°C. 2.0 mmol/LAcetamide

TSC22-SQ-inF TGC ATG AAA TCC CAA TGG TTSC22-SQ-inR ACC ATT GGG ATT TCA TGC A

SYN2 SYN2-PCRFW GGA GGG GGA GAG AAG TAC ATA CTGT

1012 68°C/94°C. 2.0mmol/L

SYN2-PCRRV GGC GAT GAA GCT GCT GTC CG 2-Step PCRSYN2-SQ-inF CAG CTG GGA CAC GGC TCT TSYN2-SQ-inR AAG AGC CGT GTC CCA GCT G

RPC39 RPC39-PCRFW CCA TAG TCG CAT TTC CAT TTAAGT G

1180 55.0°C, 2.5 mmol/L

RPC39-PCRRV AGG CTG GGA AGG AAC AAG TGG GTRPC39-SQ-F01 TCG GGA CCC CTA ATG CAT CRPC39-SQ-F02 GTA GCG GCG CCT GTA AGT G

AKAP4 AKAP4-PCRFW GCG GAG TTA GAG ATG CAA T 901 55.0°C, 2.5 mmol/LAKAP4-PCRRV CAT ACC CCT ACT AGA GAA TCA GAAKAP4-S-F01 TCC TTA GAG CCC TCC ATC TAKAP4-S-R02 AGA TGG AGG GCT CTA AGG A

GNA13 GNA13-FW AGG AGC AGT GTG GAG AAG GTA GA 803 55.0°C, 2.5 mmol/LGNA13-RV CCG GTT ATT GAC GAT TGTGNA13-S-F01 ATT GCC CAG GAT ATT CTC AGNA13-S-F02 TGA GAA TAT CCT GGG CAA T

TLR7 TLR7-FW ACT CCT TGG GGC TAG ATG GT 954 58.0°C, 2.75 mmol/LTLR7-RV CTG GGG AGA AAA TGC AGA AA AcetamideTLR7-S-F01 GTT AAA AGT GCT CTC CCT GAA AGTLR7-S-Rv01 CTT TCA GGG AGA GCA CTT TTA AC

MIG6 MIG6-FW TTT GCA CTG ATC CCT TAC TAA TA 988 54.5°C, 2.0 mmol/LMIG6-RV ACT TAA TTC TCA CAG CTC GTMIG6-S-F01 TGT TTG CTT GCT TGG CTA TTCMIG6-S-Rv01 GAA TAG CCA AGC AAG CAA ACA

SYN1 SYN1-FW GGT TCC CAT ATC CCA CCT CT 863 58.5°C, 2.0 mmol/LSYN1-RV TGG TCC TAA AAC CCA CTT GCSYN1-S-F01 TGC CCA TGT GTG TTA CTC TGSYN1-S-Rv01 CAG AGT AAC ACA CAT GGG CA

ICAM ICAM-1-FW CTT CCT TTT TCT GGG AGC TGT A 1000 62.0°C, 2.5 mmol/L

ncabdt

ICAM-1-S-F01 GAA ACG GGA GGC GTG GAICAM-1-S-Rv01 TCC ACG CCT CCC GTT TC

IL7r IL7r-FW GCT CTG CCA TTG TTG CAT AA 857 63.0°C, 2.0 mmol/LIL7r-RV CCC CCA CAC TTA CTG TGC TTIL7r-S-F01 CAG GAG TTC GAG ACA AGC CTIL7r-S-Rv01 AGG CTT GTC TCG AAC TCC TG

HLA-C HLA-C-FW GAA GAG CAT TGG GAC TGC AT 960 61.0°C, 2.0 mmol/LHLA-C-RV TGG GTG ACT GGG GAC TTT AGHLA-C-S-F01 CCG AGG TAA GGT AAG GCA AAGHLA-C-S-Rv01 CTT TGC CTT ACC TTA CCT CGG

eIF2a eIF2a-FW GTG ACT TGT ACA GAA CTT TGC 595 60.0°C, 1.5 mmol/LeIF2a-RV GCA ATG AAC AGG AAA GGT GGeIF2a-F02 TGC TTG CTA GTT TGT TTC CCA C 563 60.0°C, 1.5 mmol/LeIF2a-Rv02 GCC ATG TAC ATC ACA GGT TTA CTG

APR APR-FW CAT GAG GGG ACT CCT TTCA AA 888 62.5°C, 3.0 mmol/LAPR-RV GAC AAG GAG CAT TTC GAA CC Betaine/DMSOAPR-S-F01 GCA CAT TGT ATA TGA TTC GGTAPR-S-Rv01 ACC GAA TCA TAT ACA ATG TGC

IL8 IL8-FW CCT CAA GTC TTA GGT TGG TTG G 894 60.0°C, 2.0 mmol/LIL8-RV GGG CAC ATG TCT TCA CAT AGA AIL8-S-F01 GGA TAA AGA GCA TGA AGC AACIL8-S-Rv01 GTT GCT TCA TGC TCT TTA TCC

TAC1 TAC1-FW GAT CAG TGC CCC GAT AA 765 58.0°C, 2.0 mmol/LTAC1-RV TGA CAT CTT TGG TGG AGT AGTTAC1-S-F01 GGA TAA TGG CGT GGA TAT G

Given for each gene fragment is the PCR primer pair, as well as any internal sequencing primers required for sequencing (indicated with either “int” or “seq” in primerame). Fragment sizes are given along with varying PCR components and the annealing temperature of each. All PCR reactions were performed in 20-L volumes, withomposition as discussed in the Methods section, with the exception of magnesium chloride and enhancers if required, listed here for each reaction. The lengths of thennealing and extension steps were 30 seconds and 30 seconds per 500 base pairs, respectively. A minimal extension of 30 seconds for fragments with less than 500ase pairs was applied. The time of extension reactions has been applied by use of 30 seconds per 500 base pairs as a rule. Designs of all primers were based on currentatabase gene sequences. For sequences, please refer to Table I for database and accession numbers. Where used, the enhancers have been used at fixed concentrationshroughout (betaine, 1.0 mol/L; dimethylsulfoxide, 5%; acetamide, 5%; and mercaptoethanol, 0.5 mmol/L).

PCR, Polymerase chain reaction.

ppendix 3. Genotyping primers and probes used in individual typing

Polymorphic site PCR primersDetection probes and method of

genotyping

DMA (FP-SBE) (F) 5� TGG TGG GAA GTC TTA TGA FP-SBE allele-specific reporters(R) 5� TTA ATG ATA GAA AGC AGA GTA GA (F) 5� CAT TTA CCA ACC ACA

CAT APCR condition: 55°C, 1.5-mmol/L MgCl2

DMA (TaqMan;AppliedBiosystems,Foster City,Calif)

(F) 5� TCTTATGAGGCAGTGGAAACAACAG TaqMan probes with respective labels

(R) 5� CAAAGCATCACTGATATCGTTTACCA VIC (F) 5�CCACACATAGCAGCTG

FAM (F) 5�CCACACATAACAGCTG

genotyping

PKR CCGn Repeat (F) 5� [HEX] AGG GTT CCT GGC CGT GCAGG

Fragment sizing performed by use ofcapillary electrophoresis with anABI 3100 analyzer and an internalsize standard

(R) 5� CCG CGC TCC CTC GGC TGCPCR condition: 57°C, 2.0-mmol/L MgCl2

IFNAR �408 (C/T) (F) 5� TCT CGC CCC TCA GCC AAG TC Digestion of amplicon with BstNIproduces fragments of 204, 124,106, and 96 base pairs

(R) 5� CCT TGA CCT TCA CAG GAT CGPCR condition: 60°C, 1.5-mmol/L MgCl2

IFNAR GTn Repeat (F) 5� [FAM] ATA GGC CGG AAA GAG TGAGGA A

Fragment sizing performed by use ofcapillary electrophoresis with anABI 3100 analyzer and an internalsize standard

(R) 5� CCG CAG ATC CCA CCA GTTPCR condition: 62°C, 1.5-mmol/L MgCl2

IFNAR rs1012334 (F) 5� AAA GAA CAA AAA TTC CCT TAA AC FP-SBE allele-specific reporters(R) 5� GTT AAA TCA GTG ACC ATA TCA CC (F) 5� AAT CTA AGA CTT AGT

TGC CTT TTTPCR condition: 56°C, 1.5-mmol/L MgCl2 (R) 5� TGT GAA TAC AAG AAA

GAA CAA AAA TSBE, (F) probe at 61°C and 22 cycles

IP9 rs6825045 (F) 5� GGA AAC TTC TTT AGG CAC TC FP-SBE allele-specific reporters(R) 5� TCC ATT TAA TTG TCG GAC TAC (F) 5� TTT CTG TAA ATA ACT

GAA TTT TCPCR condition: 50°C, 1.5-mmol/L MgCl2 (R) 5� CCC CTC ATA CGC AGT

CCA GASBE, (F) probe at 59°C and 22 cycles

ISG12 rs3814821 (F) 5� CTT GTG ACT TCA TTC TGC GTA G FP-SBE allele-specific reporters(R) 5� GCA CTG CCC CTT GAC AC (F) 5� TTT TGT ATA GAT ATG

TGA AGG ATG AAPCR condition: 55°C, 1.5-mmol/L MgCl2 (R) 5� TTT TTA GTC CTT TCA

CCC ACCSBE, (R) probe at 61°C and 22

cyclesISG56 3-base pair

element(F) 5� TTA GTT TCA CTT TCC CCT TTC G FP-SBE allele-specific reporters

(F) NA(R) 5� TTC AAA ACC TTT AGA CAT TAT

GGC A(R) 5’27 ACA GCA GCC AAT GGT

GTA APCR condition: 55°C, 2.5-mmol/L MgCl2 SBE, (R) probe at 56°C and 22

cyclesLMP7 rs2071543 (F) 5� CGG ACA GAT CTC TGG GTG CT Digestion of amplicon with BsmI

produces fragments of 304, 174,and 130

(R) 5� TCT CGG GGA CTG AAG GCT APCR condition: 56°C, 1.0-mmol/L MgCl2

CTSS rs1136774rs3754212

(F) 5� TCC TTG TTG CTTT TGA AAT CT Pyrosequencing allele-specific duplexprobe

(R) 5� TTC TCC CAT TTG GTC TTC AA (F) 5� ACC AGC AGT TGC TCC

genotyping

MxA rs2071430�123nt

(F) 5� TCA GCA GGC CTG GGT TCG GG Pyrosequencing allele-specific

(F) 5� CGA GGC AAG TGC TG(R) 5� CTC ACA GAC CCT GTG CTG AG (F) 5� GCT AGG TTT CGT TTC TGPCR condition: 60.0°C, 1.5-mmol/L MgCl2 Typing of SNPs performed in

multiplexIFP53 rs2234518 (F) 5� GAA GGG GAA GCA GAA AAG TAA

TGFP-SBE allele-specific reporters

(R) 5� AAT GAA ACC CTC TTG AGA ATC G (F) 5� GCG GGG CAT TTA CAGTTT C

(R) 5� AGT CGT GGG CGG TGGAAA G

PCR condition: 55°C, 1.5-mmol/L MgCl2 SBE, (R) probe at 57°C and 22cycles

IFP53 rs7143006 (F) 5� CAG GGT GGG GAG AAC GA FP-SBE allele-specific reporters(R) 5� CCT GGC AAA GAT GGC AAT A (F) 5� GGG GAA GAG TAT TAA

AGA CCA TTPCR condition: 55°C, 1.5-mmol/L MgCl2 (R) 5’27 GAG TGC CCT GCC CAG

CCA GSBE, (F) probe at 60°C and 22 cycles

int, in, IN, Internal fragment primers; FWD, F, forward primers; REV, RV, R, reverse primers; FP-SBE, fluorescent polarization-single-base extension.Where internal primers are numbered, the order of the primers across the fragment is indicated.

ppendix 4. Genes, ISRE locations, and detected polymorphic markers (within pooled DNA)

GeneApproximate ISRE location (in relation

to ATG or TI*)

Location of polymorphicsites identified throughpooled DNA screening

Peak distortions betweenpools: Response versusnonresponse or cases

versus controls(susceptibility)

ITAC �204 CAAAACAGAAACT �192 NDM vNACCR3 *�748 TGAAACTCAAACT �735 NDM NAFASL 1147 GGAAACTGAGGCAG 1134

861 TGAAAATGAAAAC 849843 GAAATACAAAGC 832172 GAAAGAGAAAGA 160

NDM NA

GBP *�129 TGAAACTGAAAGT NDM NAH6-16 *�110 GGAAAATGAAACT NAH9-27 �172 GGAAATAGAAACT �159 NDM NAICE �439 TGTTTCTATTTCTT �425 NDM NAIFNB �165 GAAAACTGAAAGG �153

�152 GAGAAGTGAAAGT �139�141 GTGGGAAATTCCT �130

NDM NA

1-8U *�35 GAAACCGAAACT �24 NDM NAIL-1� �1363 GAGAAACTGATAACT �1349 �1065 (ATG) C/T,

rs3087258 �773(ATG) A/T, novelSNP

NA (S) Case (60:40),control (80:20)

IL-4 �259 GGTTTCATTTTC �248 �33 (ATG) C/T,rs2070874

IL-5 �501 GCAGTTTCCATTTC �488 NDM NA

to ATG or TI*)

IL-7r *�261 GAAATGGAAGAGT �249 NDM NAIL-10 �207 ttGAAAACTAAGTtt �197 �162 (ATG) C/T novel

IL-13 �206 TCTTTCCTTTATG �194 NDM NAINOS *�1489 AGAAAGTGACAAG *�624

TGGAAGGGAAAGG *�101GGAAACCAAAAAA

NDM NA

IP10 277 GGTTTCACTTTC 289 NDM NAIP30 313 CGTTTCGCTTTCGCAT 328 NDM NAIP9 �601 GAAACACAGGGAAACT �586 �418 (ATG) G/A novel

SNP(R) NAb compared with

Res/NR (60:40compared with 80:20)

IRF-1 Regions below 70% homology, includedbecause of function in IFN signaling

NDM NA

IRF-2 *�258 GAAAATGAAATTGAC NDM NAISG12 Regions between 60%-70% homology

to ISRE, within �1250 to �600 (TI)�1092 (ATG) C/T,

rs3814821(R) NR compared with

Res/NA (60:40compared with 80:20)

ISG15 *�113 GGAAACCGAAACT �100 NDM NAISG20 �638 GAAAGGAAACAAA �626 �2 (ATG) A/G alleles,

rs3200942(R) NAb compared with

Res/NR (75:25compared with �80:�20)

ISG54 *�123 AATTTCACTTTCT �100*�99 AGTTTCACTTTCC �86

NDM NA

ISG56 �209 TAGTTTCACTTTCC �194 �149 (ATG), a novel3-base pair repeatelement

(R) NR compared withRes/NAb (70:30compared with NDM)

OAS *�85 AGGAAACGAAACC � 72 NDM NAPECAM-1 �288 aGAAACTAAACAAa �277 NDM NAPKR �6301 GGAAAACGAAACT �6289 Indications within pools

for presence ofmicrosatellite as aresult of unreadabledata, introducedwithin the pools indifferent positionswithin 2 base pairs

(R) Res compared withNR/NAb, alleledifference notcalculated fromsequencing data

PTGS2 *�49 GGCTTGGTTTTC �37 �397 (ATG), T/A novelSNP

(S) Case (70:30), control(80:20)

RANTES *�123 GAAAACTGAAATA *�117GGAAAAGAAAACT *�111CAAAACGGAAAAG

306 (ATG) A/G,rs2280789

(S) Case (60:40), control(80:20)

SLP1 � 172 taGGAAAGATAAACAaa �160 NDM NATRAIL �751 CCTTTGCCTTTCT �739 �103

TCATTCGCTTTCA �91NDM NA

to ATG or TI*)

VCAM1 �136 ACTTTCTATTTCA �124 �186 (ATG) C/T,rs3917027 �109(ATG) C/T,rs3783608 �79(ATG) T/G, novelSNP

NA NA NA

Sp100 (�)370, 130 base pairs from TI site �221 (ATG) C/T,rs2017675

(R) NAb compared withRes/NR (60:40compared with 80:20)

ADAR1 (�)170 base pairs from TI site NDM NATNFp75/80 �113 GCTTTCGCTTTCA �100 NDM NASPRR2A �842 TAGTTTCACTTCCTG �828 �750 (ATG) A/G,

rs447187NA

Polymericimmunoglobulinreceptor

*�140 CAAAGGAAACC �128*�108 CAAAATGAAAGG �96*8 AGTTTCAGTTTT 20

NDM NA

GP91-PHOX �106 aGAAAAGGAAACCg �93 NDM NAC2 �536 aAGTTTATCTTCAa �525

�195 aGGTTTCACCCTTc �184NDM NA

PPORX �180 ACTTGCCTTTGTCT �167 NDM NACD40L �374 CTTTCCTTGAAAACT �361 NDM NAIL-3 147 CCTTTGCCTTTGCT 160 NDM NATAP 1 �198 cGATTTCGCTTTCcc �186 NDM NATAP 2 �573 gGGGAAGCGAAAGCg �561 NDM NAIRF-7H �929 CAAAAGCGGAAACTC �916

�372 agGTTTCGCTTTCCcg �361NDM NA

Complementfactor 1

�1298 CAAAAGGTAACC �1286�1016 GTTTTCTCTCAAG �1004�233 AGTTCCAATTGGT �221

24 (ATG) T/C SNP26 (ATG) T/G SNP40 (ATG) C/A SNP42 (ATG) G/A SNP60 (ATG) T/G SNP67 (ATG) T/A SNP

(S) Case (80:20), control(70:30) weak (R)associations (S) Case(70:30), control (80:20) (S) Case (60:40),control (80:20) weak (R) associations(S) Case (70:30),control (80:20) (R)Res versus NR/NAb(85:15 compared with�90:�10) (S) Case(85:15), control (70:30)

IFP-53 *�337 AGTTTCNCTTTCC �331 (TI) A/G,rs2234518 29 (TI)T/A, rs7143006

(R) Res compared withNR/NAb (�10:�90compared with 20:80)(R) NAb comparedwith NR/Res (80:20compared with �10:�90)

to ATG or TI*)

iNOS/NOS2A *�1489 AGAAAGTGACAAG *�624TGGAAGGGAAAGG *�410GGAAACCAAAAAA *�101GAAGTGAAACC

NDM NA

IFN-a �153 GAAATGGAAAG �142 NDM NABf �272 TGCAGTTTCTGTTTCCTT

�25525 (ATG) A/T,

rs4151667 180(ATG) C/T,rs4151667 181(ATG) A/G, novelSNP

IFN-y �284 GTGAAGTAAAAGTGCC �268 NDM NAMimecan gene �656 tCTTTCAGTTTTC �668 �297

GAAACTTAAAAT �285NDM NA

HLA-A � 182 AGTTTCTTTTCTC �170 �164 (ATG) C/G,rs2735113 �309(ATG) G/T, novelSNP

HLA-B7 �179 GAGTTTCACTTCTTC �164 �412 (ATG) rs1131214�410 (ATG)rs3177921 �409(ATG) rs1131212

(S) Case (60:40), control(80:20) (S) Case (70:30), control (80:20)(R) Res versus NR/NAb (85:15 comparedwith �90:�10)

HLA-E �183 AGTTTCCCGTTC �172 NDM NAHLA-F �188 AGTTTCTCTTTCT �176 �222 (ATG) rs1362126 (S) Case (70:30), control

(80:20) (R) Res versusNR/NAb (minor) (83:17 compared with�90:�10)

LMP-7 �819 GGAAACCTCAGAACAGT�802

rs2071543 (R) Res versus NR/NAb

TRAF6 �425 GGAAATGGAAAT �413 NDM NATNFB Regions below 70% homology, included

because of literature (concerning IFNeffects)

�91 (ATG) G/C alleles,novel �752 (ATG)A/G alleles, novel

(R) NAb versus Res/NR(85:15 compared with�90:�10) (R) Resversus NR/NAb (�90:�10 compared with80:20)

TNFA Regions below 70% homology, includedbecause of literature (concerning IFNeffects)

�488(ATG) A/G,rs1800629-61 (ATG)A/G alleles, novel

(S) Case (70:30), control(80:20) (S) Case (70:30), control (80:20)

IRF3 �1038 GGAAACCCAAAA �1026 NDM NABGP-1 (�)230 base pairs from TI site NDM NA

to ATG or TI*)

MxA *�102 GGTTTCGTTTCTG �90 �88 (TI) G/T,rs2071430 �123 (TI)C/A

(R) Res compared withNR/NAb (60:40compared with 80:20)(R) Res comparedwith NR/NAb (80:20compared with �90:�10)

Thymosin � �432 taGGAAATGGAAACgc �419�172 aGAAAGAGGAAGCTcg�160 32 aGAAATTCGATAAGtc44 48 tcGAAACTGAAGAAGa60

NDM NA

FLT3 �877 GAAACCGGGAAAGAC �891 NDM NAP21 �626 GGAAACTGAGACCT �612 �577 (ATG) T/A

alleles, novel(S) Case (75:25), control

(85:15)MNDA �49 TGAAATAGAAAGC �36 101

AGCTTTCATTTCT 114NDM NA

IFI-16 �4825 GAAACGAAAGCTA �4813�4390 GAAAAGTGAAAACT�4377 �4025 ttTTTTCAGTTTCaca�4013

NDM NA

SOCS-1 �161 GGAAAGAGAAACC �149 NDM NASTAT 1 �679 ctGAACTGAAAAATcaa �668

�338 GGGAAAAGAGAGAGC�324

NDM NA

STAT 2 �351 catTTTCAGATTTCagag �339 NDM NAC-myc �626 gaGAAAGAAGAAAAgct �613 NDM NAS epsilon �421 GAGAGAAAAGGGAACT NDM NACathepsin S �1151 ATGAAACTGAAATGA �961 (ATG) C/T,

rs3754212 �958(ATG) A/G,rs1136774 536(ATG) A/G, novel14 (ATG) T/C,novel

(R) NR compared withNAb/Res (60:40compared with 85:15)(R) NAb comparedwith Res/NR (55:45compared with 80:25)(S) Case (50:50),control (80:20) (S)Case (80:20), control(�90:�20) weak(R) associations

DMA Regions below 70% homology, includedbecause of literature (concerning IFNeffects)

228 (ATG) C/T, novelSNP

(S) Case (100:0), control(60:40)

PRM3 �764 TAAAAGACAAAGTC �750�480 TTTCACACTTTC �469

�795 (ATG) C/G,novel SNP

MBP1 �754 agTTTTCTAATTGCCag �741�703 caGAAAGTAGAGAGGT�691 �399gaGAAAGAGGAAGCTc �386

NDM NA

to ATG or TI*)

IFNAR Included based on function in relation tosignaling

�408 (ATG) C/T SNPGTn repeat rs1012334(literature)

(R) Res versus NR/NAb(R) Res versus NR/NAb

HLA-G 755 GGAAAGTGAAACT 743389 TGAAAAGTGAAGG 377

�200 (ATG) C/T,rs1631950

IFI-60 �102 AGTTTCACTTTCC �90 NDM NATLR-4 177 GCTTTCACTTCCTCTCA 160

�65 gaGACCAGAAAGCTggg �75NDM NA

PML gene 561 tcTTTTCTGTTTCaa 551 25ccGAGAATCGAAACTaa 13

NDM NA

IL-22 251 ctGGAAATTAGATAAtt 239 NDM NATSC22 �110 cgTTTTCTGTTTCTC �97 61

TTTCAATTTCTTTTCT 74 252tcGAAATGTTAAAA 264

111 (ATG) G/T,rs3736887 148(ATG) A/G,rs7997320

SYN2 �741 TTTCTCCCCTTTCC �728 �880 (ATG) A/T,rs6802933

RPC39 805 GGGAATCAAGAAA 793 �48GAAATAGAAAACA �60

NDM NA

AKAP4 �208 AGTTTCTATTTTC �220 NDM NAGNA13 �560 agGGAAACAGAAAGT �548 NDM NATLR-7 51 GGAAACAACTGAACC 66 �119 (ATG) G/T, novel

Mig-6 403 GGAAACCTGAACT 415 NDM NASYN1 �400 GGAAACAGGAAGC �387 NDM NAICAM-1 456 GAAACTGAGACT 445 445 (ATG) T/C SNP

within ISRENA

IL7r NDM NAHLA-C 361 CAAAAAGGAAACT 379

857 GAAGTGAAACTC 875NDM NA

eIF-2a Literature-based NDM NAAPR Literature-based NDM NAIL-8 59 CAAAAAAGAAACAT 77 NDM NATAC1 �1502 CATTTCAGCTTTC �1490 NDM NA

The term nondetectable mutation is used because, with the detection limit, some rare polymorphisms may not be detected and in some cases the pools may not beepresentative of the frequency of some polymorphisms within the population. This screen has been performed as an initial screen for the identification of polymorphicarkers located in or close to ISRE-containing genes.ISRE, Interferon-stimulated response element; TI, transcription initiation; NDM, nondetectable mutation; NA, not applicable; (S), allele peak differences found

etween cases and controls; (R), allele peak differences found between response status pools; NAb; high neutralizing antibody titer pool; Res, positive responder pool;R, nonresponder pool.*Position from TI site.

ppendix References1. Rani MRS, Gauzzi C, Pellegrini S, Fish EN, Wei T,

Ransohoff RM. Induction of beta-R1/I-TAC byinterferon-beta requires catalytically active TYK2.J Biol Chem 1999;274:1891-7.

2. Rani MR, Foster GR, Leung S, Leaman D, Stark GR,Ransohoff RM. Characterization of beta-R1, a gene thatis selectively induced by interferon beta (IFN-beta) com-pared with IFN-alpha. J Biol Chem 1996;271:22878-84.

3. Der SD, Zhou A, Williams BR, Silverman RH. Identi-fication of genes differentially regulated by interferonalpha, beta, or gamma using oligonucleotide arrays. ProcNatl Acad Sci U S A 1998;95:15623-8.

4. Frigerio S, Silei V, Ciusani E, Massa G, Lauro GM,Salmaggi A. Modulation of fas-ligand (Fas-L) on humanmicroglial cells: an in vitro study. J Neuroimmunol2000;105:109-14.

5. Weinstock-Guttman B, Badgett D, Patrick K, HartrichL, Santos R, Hall D, et al. Genomic effects of IFN-betain multiple sclerosis patients. J Immunol 2003;171:2694-702.

6. Koike F, Satoh J, Miyake S, Yamamoto T, Kawai M,Kikuchi S, et al. Microarray analysis identifies interferonbeta-regulated genes in multiple sclerosis. J Neuroim-munol 2003;139:109-18.

7. Whyatt LM, Duwel A, Smith AG, Rathjen PD. Theresponsiveness of embryonic stem cells to alpha andbeta interferons provides the basis of an inducible ex-pression system for analysis of developmental controlgenes. Mol Cell Biol 1993;13:7971-6.

8. Porter AC, Chernajovsky Y, Dale TC, Gilbert CS, StarkGR, Kerr IM. Interferon response element of the humangene 6-16. EMBO J 1996;7:85-92.

9. Sturzebecher S, Wandinger KP, Rosenwald A, Sathya-moorthy M, Tzou A, Mattar P, et al. Expression profilingidentifies responder and non-responder phenotypes tointerferon-beta in multiple sclerosis. Brain 2003;126:1419-29.

0. Maniatis T, Whittemore L-A, Du W, Fan C-M, Keller A,Palombella VJ, et al. Positive and negative control ofhuman interferon-� gene expression. Harvey Lect 1992;44:1193-220.

1. Ji X, Cheung R, Cooper S, Li Q, Greenberg HB, He XS.Interferon alfa regulated gene expression in patientsinitiating interferon treatment for chronic hepatitis C.Hepatology 2003;37:610-21.

2. Li-Weber M, Davydov IV, Krafft H, Krammer PH. Therole of NF-Y and IRF-2 in the regulation of human IL-4gene expression. J Immunol 1994;153:4122-33.

3. McRae BL, Picker LJ, van Seventer GA. Human recom-binant interferon-beta influences T helper subset differ-entiation by regulating cytokine secretion pattern andexpression of homing receptors. Eur J Immunol 1997;27:2650-6.

4. Feng X, Yau D, Holbrook C, Reder AT. Type I inter-ferons inhibit interleukin-10 production in activated hu-

tions for Th1-mediated diseases. J Interferon CytokineRes 2002;22:311-9.

5. Guthikonda P, Baker J, Mattson DH. Interferon-beta-1-b(IFN-B) decreases induced nitric oxide (NO) productionby a human astrocytoma cell line. J Neuroimmunol1998;82:133-9.

6. Levy DE, Kessler DS, Pine R, Reich N, Darnell JE.Interferon-induced nuclear factors that bind a sharedpromoter element correlate with positive and negativetranscriptional control. Genes Dev 1988;2:383-93.

7. Xie Q, Whisnant R, Nathan C. Promoter of the mousegene encoding calcium-independent nitric oxide syn-thase confers inducibility by interferon gamma and bac-terial lipopolysaccharide. J Exp Med 1993;177:1779-84.

8. Tomura K, Narumi S. Differential induction of inter-feron (IFN)-inducible protein 10 following differentia-tion of a monocyte, macrophage cell lineage is related tothe changes of nuclear proteins bound to IFN stimulusresponse element and kappaB sites. Int J Mol Med1999;3:477-84.

9. Neville LF, Mathiak G, Bagasra O. The immunobiologyof interferon-gamma inducible protein 10 kD (IP-10): anovel, pleiotropic member of the C-X-C chemokinesuperfamily. Cytokine Growth Factor Rev 1997;8:207-19.

0. Buttmann M, Merzyn C, Rieckmann P. Interferon-betainduces transient systemic IP-10/CXCL10 chemokinerelease in patients with multiple sclerosis. J Neuroim-munol 2004;156:195-203.

1. Harada H, Takahashi E-I, Itoh S, Harada K, Hori TA,Taniguchi T. Structure and regulation of the humaninterferon regulatory factor 1 (IRF-1) and IRF-2 genes:implications for a gene network in the interferon system.Mol Cell Biol 1994;14:1500-9.

2. Martensen PM, Segaard T, Gjermandsen IM, Butten-schon HN, Rossing AB, Bonnevie-Nielsen V, et al. Theinterferon alpha induced protein ISG12 is localized tothe nuclear membrane. Eur J Biochem 2001;268:5947-54.

3. Meraro D, Gleit-Kielmanowicz M, Hauser H, Levi BZ.IFN-stimulated gene 15 is synergistically activatedthrough interactions between the myelocyte/lymphocyte-specific transcription factors, PU.1, IFNregulatory factor-8/IFN consensus sequence bindingprotein, and IFN regulatory factor-4: characterization ofa new subtype of IFN-stimulated response element.J Immunol 2002;168:6224-31.

4. Gariglio M, Gaboli M, Mana C, Ying GG, Gribaudo G,Cavallo R, et al. Characterization of nuclear factorsinvolved in 202 gene induction by interferon-alpha inmurine leukemia cells. Eur J Biochem 1994;221:731-9.

5. Gongora C, Degols G, Espert L, Hua TD, Mechti N. Aunique ISRE, in the TATA-less human Isg20 promoter,confers IRF-1-mediated responsiveness to both inter-feron type I and type II. Nucleic Acids Res 2000;28:

6. de Veer MJ, Sim H, Whisstock JC, Devenish RJ, RalphSJ. IFI60/ISG60/IFIT4, a new member of the humanIFI54/IFIT2 family of interferon-stimulated genes.Genomics 1998;54:267-77.

7. Whatelet MG, Clauss IM, Content J, Huez GA. TheIFI-56K and IFI-54K interferon-inducible human genesbelong to the same gene family. FEBS Lett 1988;231:164-71.

8. Bluyssen HA, Vlietstra RJ, van der Made A, Trapman J.The interferon-stimulated gene 54 K promoter containstwo adjacent functional interferon-stimulated responseelements of different strength, which act synergisticallyfor maximal interferon-alpha inducibility. Eur J Bio-chem 1994;220:395-402.

9. Whatelet MG, Clauss IM, Nols CB, Content J, HuezGA. New inducers revealed by the promoter sequenceanalysis of two interferon-activated human genes. EurJ Biochem 1987;169:313-21.

0. Floyd-Smith G, Wang Q, Sen GC. Transcriptional in-duction of the p69 isoform of 2’,5’-oligoadenylate syn-thetase by interferon-beta and interferon-gamma in-volves three regulatory elements and interferon-stimulated gene factor 3. Exp Cell Res 1999;246:138-47.

1. Tanaka H, Samuel CE. Mechanism of interferon action:structure of the mouse PKR gene encoding theinterferon-inducible RNA-dependent protein kinase.Proc Natl Acad Sci U S A 1994;91:7995-9.

2. Casola A, Henderson A, Liu T, Garofalo RP, BrasierAR. Regulation of RANTES promoter activation in al-veolar epithelial cells after cytokine stimulation. Am JPhysiol Lung Cell Mol Physiol 2002;283:L1280-90.

3. Cremer I, Ghysdael J, Vieillard V. A non-classicalISRE/ISGF3 pathway mediates induction of RANTESgene transcription by type I IFNs. FEBS Lett 2002;511:41-5.

4. Sato K, Hida S, Takayanagi H, Yokochi T, Kayagaki N,Takeda K, et al. Antiviral response by natural killer cellsthrough TRAIL gene induction by IFN-alpha/beta. EurJ Immunol 2001;31:3138-46.

5. Neish AS, Read MA, Thanos D, Pine R, Maniatis T,Collins T. Endothelial interferon regulatory factor 1cooperates with NF-kappa B as a transcriptional activa-tor of vascular cell adhesion molecule 1. Mol Cell Biol1995;15:2558-69.

6. Grotzinger T, Jensen K, Will H. The interferon (IFN)-stimulated gene Sp100 promoter contains an IFN-gamma activation site and an imperfect IFN-stimulatedresponse element which mediate type I IFN inducibility.J Biol Chem 1996;271:25253-60.

7. George CX, Samuel CE. Characterization of the 5’-flanking region of the human RNA-specific adenosinedeaminase ADAR1 gene and identification of aninterferon-inducible ADAR1 promoter. Gene 1999;229:

8. Santee SM, Owen-Schaub LB. Human tumor necrosisfactor receptor p75/80 (CD120b) gene structure andpromoter characterization. J Biol Chem 1996;271:21151-9.

9. Piskurich JF, Youngman KR, Phillips KM, HempenPM, Blanchard MH, France JA, et al. Transcriptionalregulation of the human polymeric immunoglobulin re-ceptor gene by interferon-gamma. Mol Immunol 1997;34:75-91.

0. Kumatori A, Yang D, Suzuki S, Nakamura M. Cooper-ation of STAT-1 and IRF-1 in interferon-gamma-induced transcription of the gp91(phox) gene. J BiolChem 2002;277:9103-11.

1. Waleh NS, Apte-Deshpande A, Terao A, Ding J, KilduffTS. Modulation of the promoter region of prepro-hypocretin by alpha-interferon. Gene 2001;262:123-8.

1a. Lu R, Au WC, Yeow WS, Hageman N, Pitha PM.Regulation of the promoter activity of interferon regu-latory factor-7 gene. Activation by interferon and silenc-ing by hypermethylation. J Biol Chem 2000;275:31805-12.

2. Strehlow I, Seegert D, Frick C, Bange FC, Schindler C,Bottger EC, et al. The gene encoding IFP 53/tryptophanyl-tRNA synthetase is regulated by thegamma-interferon activation factor. J Biol Chem 1993;268:16590-5.

3. Baron-Delage S, Abadie A, Echaniz-Laguna A, Melki J,Beretta L. Interferons and IRF-1 induce expression ofthe survival motor neuron (SMN) genes. Mol Med 2000;6:957-68.

4. Ragg H, Weissman C. Not more than 117 base pairs of5’-flanking sequence are required for inducible expres-sion of a human IFN-alpha gene. Nature 1983;303:439-42.

5. Lopez S, Reeves R, Island ML, Bandu MT, Christeff N,Doly J, et al. Silencer activity in the interferon-A genepromoters. J Biol Chem 1997;272:22788-99.

6. Huang Y, Krein PM, Winston BW. Characterization ofIFN-gamma regulation of the complement factor B genein macrophages. Eur J Immunol 2001;31:3676-86.

7. Tasheva ES, Conrad GW. Interferon-gamma regulationof the human mimecan promoter. Mol Vis 2003;9:277-87.

8. Tasheva ES. Analysis of the promoter region of humanmimecan gene. Biochim Biophys Acta 2002;1575:123-9.

9. Gobin SJ, van Zutphen M, Woltman AM, van den ElsenPJ. Transactivation of classical and nonclassical HLAclass I genes through the IFN-stimulated response ele-ment. J Immunol 1999;163:1428-34.

0. Johnson DR, Pober JS. HLA class I heavy-chain genepromoter elements mediating synergy between tumornecrosis factor and interferons. Mol Cell Biol 1994;14:1322-32.

1. Chen CJ, Lin TT, Shively JE. Role of interferon regu-

(cell CAM-1) by interferon-gamma. J Biol Chem 1996;271:28181-8.

2. Ronni T, Matikainen S, Lehtonen A, Palvimo J, Dellis J,Van Eylen F, et al. The proximal interferon-stimulatedresponse elements are essential for interferon respon-siveness: a promoter analysis of the antiviral MxA gene.J Interferon Cytokine Res 1998;18:773-81.

3. Landolfo S, Gariglio M, Gribaudo G, Lembo D. The Ifi200 genes: an emerging family of IFN-inducible genes.Biochimie 1998;80:721-8.

4. Kao WY, Dworkin LL, Briggs JA, Briggs RC. Charac-terization of the human myeloid cell nuclear differenti-ation antigen gene promoter. Biochim Biophys Acta1996;1308:201-4.

5. Schluter G, Boinska D, Nieman-Seyde S-C. Evidencefor translational repression of the SOCS-1 major openreading frame by an upstream open reading frame. Bio-chem Biophys Res Commun 2000;268:255-61.

6. Wong LH, Sim H, Chatterjee-Kishore M, HatzinisiriouI, Devenish RJ, Stark G, et al. Isolation and character-ization of a human STAT1 gene regulatory element.Inducibility by interferon (IFN) types I and II and role ofIFN regulatory factor-1. J Biol Chem 2002;277:19408-17.

7. Alexandrova NM, Itkes AV, Imamova LR, Chernov BK,Tulchinsky EM, Ulyanov NB, et al. Human c-myc gene

feron response sequence (IRS). FEBS Lett 1990;265:67-70.

8. Turpaev KT, Itkes AV, Alexandrova NM, PokrovskayaOV, Imamova LR, Chernov BK, et al. Binding of pro-teins of HeLa S3 cell extract to oligonucleotides con-taining the consensus interferon-response sequence(IRS) and to the IRS-containing fragment of the humanc-myc gene. Eur J Biochem 1991;200:107-11.

9. Mills FC, Brooker JS, Camerini-Otero RD. Sequencesof human immunoglobulin switch regions: implicationsfor recombination and transcription. Nucleic Acids Res1990;18:7305-16.

0. Shi G-P, Webb AC, Foster KE, Knoll JH, Lemere CA,Munger JS, et al. Human cathepsin S: chromosomallocalization, gene structure, and tissue distribution.J Biol Chem 1994;269:11530-6.

1. Storm van’s Gravesande K, Layne MD, Ye Q, Le L,Baron RM, Perrella MA, et al. IFN regulatory factor-1regulates IFN-gamma-dependent cathepsin S expres-sion. J Immunol 2002;168:4488-94.

2. Lefebvre S, Berrih-Aknin S, Adrian F, Moreau P, PoeaS, Gourand L, et al. A specific interferon (IFN)-stimulated response element of the distal HLA-G pro-moter binds IFN-regulatory factor 1 and mediates en-hancement of this nonclassical class I gene by IFN-beta.

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