patients with relapse - onset ms treated with ifn- and with a follow - up of at least 2 years were classified as responders and nonresponders . responders were patients with no relapses and no increase in the expanded disability status scale ( edss ) score over the follow - up period . nonresponders were patients having 1 or more relapses during the follow - up period and an increase in the edss score of at least 1 point confirmed at 6 months . we further classified the response of a subgroup of patients according to the number of relapses during the first 2 years of treatment without considering edss progression . two different criteria were considered for nonresponders : ( 1 ) 1 or more relapses during the 2-year follow - up ( criteria 1 ) , and ( 2 ) 2 or more relapses over the follow - up period ( for this criterion , patients with only 1 relapse were not considered in the analysis ; criteria 2 ) . information on the ifn- neutralizing antibody status was not available for patients included in the study . genes related to the type i ifn and tlr pathways and genes coding for gaba and glutamate receptors were selected through bibliographical searches , and a total of 384 biologically informative single nucleotide polymorphisms ( snps ) were selected for genotyping using select your snps , an online tool for selecting tagsnps from a roster of candidate genes . in a first or screening phase of the study ( phase i ) , 384 snps were genotyped in 830 patients with ms classified according to their response to ifn-. there were 416 responders and 414 nonresponders . genomic dna samples were obtained using standard methods and genotyping was performed using an illumina goldengate assay at progenika biopharma sa ( bizkaia , spain ) . the full list of snps and probes is provided as table e-1 at neurology.org/nn . in a second or validation phase of the study ( phase ii ) , the most significant snps obtained in phase i were genotyped in an independent cohort of 555 patients with ms ( 281 responders and 274 nonresponders to ifn- ) . a summary of demographic and baseline clinical characteristics of all patient cohorts is shown in table 1 . summary of demographic and baseline clinical characteristics for all the cohorts of patients with ms ( responders and nonresponders to ifn- treatment ) data processing , missingness , hardy - weinberg analysis , and allele frequency comparisons between responders and nonresponders to ifn- were performed with snpator ( www.snpator.org ) . possible stratification owing to different population origin was assessed using the cochran - mantel - haenszel test implemented in plink ( http://pngu.mgh.harvard.edu/purcell/plink/ ) . the study was approved by the corresponding local ethics committee , and all patients gave their informed consent . patients with relapse - onset ms treated with ifn- and with a follow - up of at least 2 years were classified as responders and nonresponders . responders were patients with no relapses and no increase in the expanded disability status scale ( edss ) score over the follow - up period . nonresponders were patients having 1 or more relapses during the follow - up period and an increase in the edss score of at least 1 point confirmed at 6 months . we further classified the response of a subgroup of patients according to the number of relapses during the first 2 years of treatment without considering edss progression . two different criteria were considered for nonresponders : ( 1 ) 1 or more relapses during the 2-year follow - up ( criteria 1 ) , and ( 2 ) 2 or more relapses over the follow - up period ( for this criterion , patients with only 1 relapse were not considered in the analysis ; criteria 2 ) . information on the ifn- neutralizing antibody status was not available for patients included in the study . genes related to the type i ifn and tlr pathways and genes coding for gaba and glutamate receptors were selected through bibliographical searches , and a total of 384 biologically informative single nucleotide polymorphisms ( snps ) were selected for genotyping using select your snps , an online tool for selecting tagsnps from a roster of candidate genes . in a first or screening phase of the study ( phase i ) , 384 snps were genotyped in 830 patients with ms classified according to their response to ifn-. there were 416 responders and 414 nonresponders . genomic dna samples were obtained using standard methods and genotyping was performed using an illumina goldengate assay at progenika biopharma sa ( bizkaia , spain ) . the full list of snps and probes is provided as table e-1 at neurology.org/nn . in a second or validation phase of the study ( phase ii ) , the most significant snps obtained in phase i were genotyped in an independent cohort of 555 patients with ms ( 281 responders and 274 nonresponders to ifn- ) . a summary of demographic and baseline clinical characteristics of all patient cohorts is shown in table 1 . summary of demographic and baseline clinical characteristics for all the cohorts of patients with ms ( responders and nonresponders to ifn- treatment ) data processing , missingness , hardy - weinberg analysis , and allele frequency comparisons between responders and nonresponders to ifn- were performed with snpator ( www.snpator.org ) . possible stratification owing to different population origin was assessed using the cochran - mantel - haenszel test implemented in plink ( http://pngu.mgh.harvard.edu/purcell/plink/ ) . the study was approved by the corresponding local ethics committee , and all patients gave their informed consent . in the first phase of the study , a total of 384 snps were genotyped in 830 patients with ms classified according to their clinical response to ifn- treatment . the results of the allelic association analysis for all the study cohorts ( combined and stratified by ms center ) are shown in table e-2 . the most significant snps associated with the response to ifn were selected for further validation ( table 2 ) : ( 1 ) rs832032 ( gabrr3 ; p = 0.0006 ) , ( 2 ) rs6597 ( stub1 ; p = 0.019 ) , ( 3 ) rs3747517 ( ifih1 ; p = 0.010 ) , ( 4 ) rs2277302 ( peli3 ; p = 0.017 ) , ( 5 ) rs10958713 ( ikbkb ; p = 0.003 ) , ( 6 ) rs2834202 ( ifnar1 ; p = 0.030 ) , and ( 7 ) rs4422395 ( cxcl1 ; p = 0.017 ) . summary of results of allele association analysis in a second phase of the study , these 7 snps were genotyped in an independent cohort of 555 patients with ms classified according to the same clinical criteria as in phase i. as depicted in table 2 , allelic analysis in this validation cohort showed a lack of statistically significant associations between selected polymorphisms from phase i and the response to ifn- ( figure e-1 ) . combined analysis in the whole cohort of patients with ms ( n = 1,385 ; table 2 ) revealed 2 snps associated with the response to ifn- with p values below 0.01 : rs2277302 , a synonymous polymorphism located in the peli3 gene ; and rs832032 , a polymorphism located in the gabrr3 gene that results in a premature stop codon and protein truncation . a classification of the response to ifn- based only on the number of relapses during the first 2 years of treatment was performed in a subgroup of 526 patients with ms from the screening cohort . it did not result in stronger associations with the response to ifn- relative to the combined classification including relapses and edss progression ( table e-3 for criteria 1 ; table e-4 for criteria 2 ) . previous pharmacogenomic studies have suggested a role for genes responsive to type i ifns , genes related to the tlr pathway , and genes coding for neurotransmitter receptors in the response to ifn- treatment in patients with ms . prompted by these observations , we genotyped an extensive panel of polymorphisms positioned in genes within these pathways in a large multicenter cohort the largest thus far published in the field  of responders and nonresponders to ifn-. classification of patients with ms into ifn- responders and nonresponders was conducted after 2 years of follow - up according to common stringent criteria based on the presence of relapses and progression of the edss score . these clinical criteria were similar to those used in previous studies . despite the statistically significant associations with the response to ifn- observed for some of the snps genotyped in the screening phase of the study , none of these associations remained significant following genotyping in an independent cohort of responders and nonresponders to ifn-. while the reasons for this lack of association observed between selected polymorphisms from the screening phase and the response to ifn- are manifold , a lack of statistical power to detect statistically significant associations in the validation cohort can not totally be ruled out . in fact , combined analysis in the whole cohort of 1,385 patients with ms revealed significant associations for 2 polymorphisms , rs832032 and rs2277302 . rs832032 is located in the gabrr3 gene and encodes a subunit of the gaba(c ) receptor . it is interesting that this polymorphism results in a premature stop codon and subsequent protein truncation . at present , there is no evidence in the literature of a functional relationship between gaba(c ) receptors and ifn- mechanism of action . rs2277302 is a synonymous snp located in the peli3 gene , which codes for an e3 ubiquitin protein ligase involved in tlr signaling . in a recent publication , peli3-deficient mice expressed increased levels of endogenous ifn- in response to tlr3 activation , and an autoregulation mechanism was proposed in which peli3 inhibited type i ifns by targeting ifn regulatory factor 7 . these findings are in agreement with previous data from our group revealing increased baseline levels of endogenous ifn- in peripheral blood cells from ifn- nonresponders . overall , results from this study do not support an association between polymorphisms positioned in genes belonging to the type i ifn or tlr pathways or genes coding for gaba or glutamate receptors and the clinical response to ifn-. findings with rs832032 and rs2277302 polymorphisms warrant further studies exploring the role of gabrr3 and peli3 in the response to ifn- in larger cohorts of patients with ms . m.f . bustamante : study concept and design , manuscript drafting and revision , genotyping , analysis and interpretation of the data . c. morcillo - surez : manuscript drafting and revision , statistical analysis , analysis and interpretation of the data x. farr manuscript revision , statistical analysis supervision , analysis and interpretation of the data . a. navarro : manuscript revision , statistical analysis supervision , analysis and interpretation of the data . x. montalban : manuscript revision , contribution of patients and clinical data , study concept and design , analysis and interpretation of the data . m. comabella : manuscript drafting and revision , study concept and design , analysis and interpretation of the data , study supervision . s. malhotra received research suport from fis , agncia de gesti d'ajuts universitaris i de recerca , and marie curie initial training network . j. rio served on the advisory boards of biogen - idec , genzyme , and novartis and received speaker honoraria from schering - bayer , serono , biogen , and teva . o. fernndez served on the advisory boards for biogen - idec , bayer - schering , merck - serono , teva , novartis , actelion , almirall , and allergan ; has received travel funding and/or speaker honoraria from biogen - idec , bayer - schering , merck - serono , teva , novartis , actelion , almirall , and allergan ; and received research support from biogen - idec , bayer - schering , merck - serono , teva , novartis , actelion , almirall , and allegan . zettl received speaker honoraria and travel grants from bayer , aventis , teva , merck - serono , and biogen - idec . j. killestein served on the advisory boards for novartis , merck - serono , biogen - idec , genzyme , and teva ; received speaker honoraria from biogen , novartis , teva , and merck - serono ; has consulted for novartis , merck - serono , biogen , and teva ; and received research support from schering ag , biogen , merck - serono , teva , genzyme , and novartis . d. brassat received travel funding and/or speaker honoraria from biogen , sanofi , genzyme , teva , merck - serono , bayer , and almirall ; served on the scientific advisory board for chugai ; and received research support from french ministry of health , french multiple sclerosis society , and european union . garca - merino is on the scientific advisory board for biogen , novartis , roche , genzyme , and merck ; received travel funding and/or speaker honoraria from biogen , novartis , almirall , genzyme , roche , and merck ; and received research support from novartis , biogen , and fis . r. alvarez - lafuente is on the scientific advisory board for hhv-6 foundation ; received travel funding from novartis , merck , biogen , bayer , teva , and sanofi ; is on the editorial board for isrn infectious diseases , austin virology and retro virology , and austin journal of multiple sclerosis & neuroimmunology ; is on the speakers ' bureau for novartis , merck , and biogen ; and received research support from novartis , merck , bayer , teva , isciii - feder , isciii , retics - reem , fundacion mutua madrilena , fundacion lair , fundacion salud , and fundacion genzyme . villar received travel funding and/or speaker honoraria from biogen , merck - serono , novartis , genzyme , teva , bayer , and roche ; holds a patent for biomarker for selecting good responders to ifn beta in multiple sclerosis ; has consulted for bayer and biogen ; and received research support from genzyme , merck - serono , spanish ministry of economy and competitiveness , and ms research . alvarez - cermeo is on the scientific advisory board for biogen - idec , novartis , genzyme , roche , and merck - serono ; received speaker honoraria from biogen - idec , novartis , genzyme , and merck - serono ; and received research support from biogen - idec , novartis , merck - serono , carlos iii institute , and spanish ministry of economy . j. lechner - scott served on the scientific advisory board for bayer , biogen , novartis , and teva ; received travel funding and speaker honoraria from biogen , novartis , and merck - serono ; and received research support from biogen , novartis , hunter medical institute , university of newcastle australia , and john hunter charitable trust . k. vandenbroeck received royalties from taylor & francis group and received research support from ministerio de economa y competitividad ( mineco)convocatoria proyectos de investigacin , funciones intracelulares de tipo - no - citoquina de las subunidades de la familia il-12 , ayudas para grupos de investigacin del sistema universitario vasco  gobierno vasco , neurogenomiks group . drulovic served on the scientific advisory board for novartis , merck , and bayer ; received travel funding and/or speaker honoraria from novartis , merck , medis , and bayer - schering ; and received research support from ministry of education and science of republic of serbia . f. martinelli boneschi received travel funding and speaker honoraria from genzyme , teva , and biogen ; is on the speakers ' bureau for biogen , teva , and genzyme ; and received research support from teva , biogen , genzyme , gw - pharma , and ministero della salute , fondazione cariplo , foundazione italiana sclerosi multipla . a. chan served on the scientific advisory board for bayer - schering , biogen , genzyme , merck - serono , novartis pharma , sanofi - aventis , teva , and ucb ; received speaker honoraria from allmirall , bayer - schering , biogen , genzyme , merck - serono , novartis , sanofi - aventis , and teva ; holds a patent for proteomic profile of nmo ; has consulted for bayer - schering , biogen , genzyme , merck - serono , novartis , sanofi - aventis , and teva ; received research support from biogen , novartis , genzyme , german ministry for education and research , and ruhr university bochum ; and was an expert consultant for sanofi - aventis . j. oksenberg is on the editorial board for genes & immunity and received research support from nih and national multiple sclerosis society . a. navarro is on the editorial board for bioinformatics , evolution , and biology letters and received research support from red espaola de esclerosis mltiple ( reem ) , spanish instituto de salud carlos iii instituto nacional de bioinformtica , and direccin general de investigacin cientfica y tcnica ( dgicyt ) , spanish government . x. montalban served on the scientific advisory board for novartis , teva , merck , biogen , bayer , almirall , actelion , genzyme , octapharma , receptos , roche , sanofi , and trophos ; received travel funding from novartis , teva , merck , biogen , bayer , almirall , acetelion , genzyme , octapharma , receptos , roche , sanofi , and trophos ; is on the editorial board for multiple sclerosis , journal of neurology , the international ms journal , revista neurologia , and therapeutic advances in neurological disorders ; has consulted for novartis , teva , merck , biogen , bayer , almirall , acetelion , genzyme , octapharma , receptos , roche , sanofi , and trophos ; and received research support from multiple sclerosis foundation of barcelona . m. comabella received speaker honoraria from bayer - schering , merck - serono , teva , sanofi - aventis , genzyme , and novartis ; is on the editorial board for journal of neuroimmunology , multiple sclerosis journal , and plos one ; and received research support from red espaola de esclerosis mltiple ( reem ) and fondo de investigacin sanitaria .
objectives : we aimed to investigate the association between polymorphisms located in type i interferon ( ifn)-induced genes , genes belonging to the toll - like receptor ( tlr ) pathway , and genes encoding neurotransmitter receptors and the response to ifn- treatment in patients with multiple sclerosis ( ms).methods : in a first or screening phase of the study , 384 polymorphisms were genotyped in 830 patients with ms classified into ifn- responders ( n = 416 ) and nonresponders ( n = 414 ) according to clinical criteria . in a second or validation phase 
 , the most significant polymorphisms associated with ifn- response were genotyped in an independent validation cohort of 555 patients with ms ( 281 ifn- responders and 274 nonresponders).results : seven single nucleotide polymorphisms ( snps ) were selected from the screening phase for further validation : rs832032 ( gabrr3 ; p = 0.0006 ) , rs6597 ( stub1 ; p = 0.019 ) , rs3747517 ( ifih1 ; p = 0.010 ) , rs2277302 ( peli3 ; p = 0.017 ) , rs10958713 ( ikbkb ; p = 0.003 ) , rs2834202 ( ifnar1 ; p = 0.030 ) , and rs4422395 ( cxcl1 ; p = 0.017 ) . 
 none of these snps were significantly associated with ifn- response when genotyped in an independent cohort of patients . 
 combined analysis of these snps in all patients with ms ( n = 1,385 ) revealed 2 polymorphisms associated with ifn- response : rs2277302 ( peli3 ; p = 0.008 ) and rs832032 ( gabrr3 ; p = 0.006).conclusions : these findings do not support an association between polymorphisms located in genes related to the type i ifn or tlr pathways or genes encoding neurotransmitter receptors and the clinical response to ifn-. nevertheless , additional genetic and functional studies of peli3 and gabrr3 are warranted .