Pharmacogenomic study in patients with multiple sclerosis

Objectives:Weaimed 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-b 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-b responders (n5 416) and nonresponders (n5 414) according to clinical criteria. In a second or validation phase, the most significant polymorphisms associated with IFN-b response were genotyped in an independent validation cohort of 555 patients with MS (281 IFN-b responders and 274 nonresponders). Results: Seven single nucleotide polymorphisms (SNPs) were selected from the screening phase for further validation: rs832032 (GABRR3; p 5 0.0006), rs6597 (STUB1; p 5 0.019), rs3747517 (IFIH1; p5 0.010), rs2277302 (PELI3; p5 0.017), rs10958713 (IKBKB; p5 0.003), rs2834202 (IFNAR1; p5 0.030), and rs4422395 (CXCL1; p5 0.017). None of these SNPs were significantly associated with IFN-b response when genotyped in an independent cohort of patients. Combined analysis of these SNPs in all patients with MS (N 5 1,385) revealed 2 polymorphisms associated with IFN-b response: rs2277302 (PELI3; p 5 0.008) and rs832032 (GABRR3; p 5 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-b. Nevertheless, additional genetic and functional studies of PELI3 and GABRR3 are warranted. Neurol Neuroimmunol Neuroinflamm 2015;2:e154; doi: 10.1212/NXI.0000000000000154 GLOSSARY EDSS 5 Expanded Disability Status Scale; GABA 5 g-aminobutyric acid; IFN 5 interferon; MS 5 multiple sclerosis; SNP 5 single nucleotide polymorphism; TLR 5 toll-like receptor. Interferon b (IFN-b) is one of the most widely prescribed disease-modifying therapies for patients with relapsing-remitting multiple sclerosis (MS) and has demonstrated positive effects on reducing disease activity. However, IFN-b is only partially effective, and a significant proportion of patients with MS do not respond to this treatment. Despite From the Servei de Neurologia-Neuroimmunologia (M.F.B., S.M., J.R., X.M., M.C.), Centre d’Esclerosi Múltiple de Catalunya (Cemcat), Institut de Recerca Vall d’Hebron (VHIR), Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain; Institute of Evolutionary Biology (UPF-CSIC) (C.M.-S., X.F., A.N.), PRBB, Barcelona, Spain; National Institute for Bioinformatics (C.M.-S., X.F., A.N.), Universitat Pompeu Fabra, Barcelona, Spain; Servicio de Neurología (L.L., O.F.), Instituto de Neurociencias Clínicas, Hospital Regional Universitario de Málaga, IBIMA, Málaga, Spain; Department of Neurology (U.K.Z.), University of Rostock, Rostock, Germany; Department of Neurology (J.K.), Multiple Sclerosis Centre Amsterdam, Vrije University Medical Centre, Amsterdam, the Netherlands; Pole des neurosciences et INSERM U1043 (D.B.), Université de Toulouse III, Hopital Purpan, Toulouse, France; Neuroinmunología (J.A.G.-M., A.J.S.), Hospital Universitario Puerta de Hierro, Madrid, Spain; Servicio de Neurología (E.U., R.A.-L.), Hospital Clínico San Carlos, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain; Department of Neurology and Immunology (L.M.V., J.C.A.-C.), Hospital Ramón y Cajal, IRYCIS, Madrid, Spain; Department of Neurology (J.L.-S.), John Hunter Hospital, Newcastle, Australia; Hunter Medical Research Institute (J.L.-S.), University Newcastle, Australia; University Newcastle (J.L.-S.), Callaghan Campus, Australia; Neurogenomiks Group (K.V.), Universidad del País Vasco (UPV/EHU), Leioa, Spain; IKERBASQUE (K.V.), Basque Foundation for Science, Bilbao, Spain; Achucarro Basque Center for Neuroscience (K.V.), Zamudio, Spain; Servicio de Neurología (A.R.-A.), Hospital de Basurto, Bilbao, Spain; Clinic of Neurology (J.S.D.), Clinical Centre of Serbia (CCS), Faculty of Medicine, University of Belgrade, Serbia; Laboratory of Genetics of Neurological Complex Disorders and Department of Neuro-rehabilitation (F.M.B.), Institute of Experimental Neurology (INSPE), Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy; Department of Neurology (A.C.), St. Josef-Hospital, Ruhr-University Bochum, Germany; Department of Neurology (J.O.), School of Medicine, University of California, San Francisco; and Institució Catalana de Recerca i Estudis Avançats (ICREA) (A.N.), Barcelona, Spain. Funding information and disclosures are provided at the end of the article. Go to Neurology.org/nn for full disclosure forms. The Article Processing Charge was paid by the authors. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially. Neurology.org/nn © 2015 American Academy of Neurology 1 a 2015 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. many efforts to identify allelic variants influencing the response to IFN-b, to date there is a lack of genetic biomarkers reliably associated with therapeutic response. A previous IFN-b transcriptomic study conducted by our group (Cemcat) implicated genes belonging to the type I IFN pathway and genes related to the toll-like receptor (TLR) pathway in the clinical response to IFN-b. The 2 genome-wide pharmacogenomic studies published thus far in relation to the response to IFN-b reported the involvement of type I IFN– responsive genes and the overrepresentation of genes coding for glutamate and g-aminobutyric acid (GABA) receptors among the polymorphisms that best discriminated between IFN-b responders and nonresponders, suggesting a relationship between neuronal excitation and response to IFN-b. Building on these observations, in the present study we aimed to genotype an extensive panel of polymorphisms located in genes related to the type I IFN and TLR pathways and genes coding for GABA and glutamate receptors in a multicenter cohort of IFN-b responders and nonresponders in order to investigate their potential association with the response to this treatment. METHODS Definition of response to IFN-b therapy. Patients with relapse-onset MS treated with IFN-b 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. Responders had no relapses during the follow-up period. 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-b neutralizing antibody status was not available for patients included in the study. Single nucleotide polymorphism selection. 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. Study design. In a first or screening phase of the study (phase I), 384 SNPs were genotyped in 830 patients withMS classified according to their response to IFN-b. 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). Overall genotyping success rate was 95%. 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-b). Genotyping was performed using TaqMan assays at the Cemcat (Barcelona, Spain). Genotyping success rate was 97%. A summary of demographic and baseline clinical characteristics of all patient cohorts is shown in table 1. Statistical analysis. Data processing, missingness, HardyWeinberg analysis, and allele frequency comparisons between responders and nonresponders to IFN-b were performed with SNPator (www.snpator.org). Possible stratification owing to different population origin was assessed using the CochranMantel-Haenszel test implemented in PLINK (http://pngu. mgh.harvard.edu/;purcell/plink/). Standard protocol approvals, registrations, and patient consents. The study was approved by the corresponding local ethics committee, and all patients gave their informed consent. RESULTS In the first phase of the study, a total of 384 SNPs were genotyped in 830 patients withMS classified according to their clinical response to IFN-b 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 IFNb were selected for further validation (table 2): (1) rs832032 (GABRR3; p 5 0.0006), (2) rs6597 (STUB1; p 5 0.019), (3) rs3747517 (IFIH1; p5 0.010), (4) rs2277302 (PELI3; p 5 0.017), (5) rs10958713 (IKBKB; p 5 0.003), (6) rs2834202 (IFNAR1; p 5 0.030), and (7) rs4422395 (CXCL1; p 5 0.017). 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-b (figure e-1). Combined analysis in the whole cohort of patients with MS (N 5 1,385; table 2) revealed 2 SNPs associated with the response to IFN-b 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. 2 Neurology: Neuroimmunology & Neuroinflammation a 2015 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. Table 1 Summary of demographic and baseline clinical characteristics for all the cohorts of patients with MS (responders and nonresponders to IFN-b treatment) Phase Sets N Female/male (% female) Age, y Disease duration, y EDSS IFN-b (1/2/3) R NR R NR R NR R NR R NR R NR I Toulouse 67 68 53/14 (80.1) 47/21 (69.1) 28.9 (8.0) 27.9 (8.7) 4.5 (5.4) 4.8 (5.8) 2.0 (1.2) 1.7 (1.3) 15/39/13 16/36/16 Serbia 24 24 17/7 (70.8) 15/9 (62.5) 31.9 (6.4) 33.3 (7.8) 3.6 (3.5) 6.1 (4.9) 0.9 (0.8) 2.3 (0.9) 12/0/12 14/0/10 Bochum 4 4 2/2 (50.0) 2/2 (50.0) 37.8 (4.6) 36.0 (9.8) 3.8 (3.8) 4.3 (3.6) 2.1 (1.1) 3.9 (2.5) 0/1/3 1/1/2 Rostock 43 43 36/7 (83.7) 36/7 (83.7) 40.9 (9.9) 38.3 (11.4) 5.0 (4.8) 4.6 (5.8) 1.8 (1.6) 3.0 (1.7) 12/9/22 6/6/31 Newcastle 32 32 25/7 (78.1) 25/7 (78.1) 42.0 (11.1) 35.2 (12.2) 6.3 (9.1) 6.0 (5.6) — — 16/2/14 16/2/14 San Francisco 2 2 2/0 (100) 2/0 (100) 40.5 (6.4) 41.5 (6.4) 1.9 (0.9) 3.7 (4.1) 1.0 (1.4) 1.5 (0.7) 1/1/0 1/1/0 Milan 6 6 4/2 (66.7) 3/3 (50.0) 39.6 (8.8) 40.8 (15.8) 3.7 (5.1) 6.3 (7.2) 1.5 (0.8) 2.5 (1.8) 4/2/0 4/2/0 Madrid PH 20 20 12/8 (60.0) 14/6 (70.0) 37.7 (8.3) 32.7 (9.8) 4.6 (2.7) 7.2 (2.8) — — 10/5/5 10/8/2 Madrid SC 29 30 21/8 (72.4) 15/15 (50.0) 33.4 (7.4) 34.1 (7.7) 5.3 (4.8) 5.1 (6.5) 2.0 (1.6) 2.1 (1.3) 12/7/10 12/5/13 Malaga 32 32 23/9 (71.9) 23/9 (71.9) 40.0 (9.5) 44.0 (10.8) 16.0 (4.0) 15.0 (7.6) 1.0 (1.3) 2.0 (1.4) 10/17/5 9/16/7 Bilbao 28 25 21/7 (75.0) 18/7 (72.0) 36.5 (9.8) 34.0 (8.4) 8.0 (7.4) 8.1 (8.2) 2.2 (2.0) 2.5 (1.5) 13/3/12 11/5/9 Barcelona 129 128 103/26 (79.8) 99/29 (77.3) 40.8 (8.8) 40.9 (10.3) 13.7 (7.2) 13.3 (7.3) 2.4 (1.8) 5.3 (2.1) 38/37/54 34/36/58 All phase I 416 414 319/97 (76.7) 299/115 (72.2) 37.5 (4.1) 36.5 (4.6) 6.4 (7.0) 7.0 (3.5) 1.7 (0.5) 2.7 (1.1) 143/123/150 134/118/162 II Madrid RC 33 41 27/6 (81.8) 29/12 (70.7) 38.0 (9.4) 35.9 (9.2) 6.2 (6.1) 3.8 (5.2) 1.6 (0.8) 1.8 (1.3) 21/4/8 22/13/6 Malaga 129 114 82/47 (63.6) 75/39 (65.8) 45.5 (11.1) 46.8 (11.1) 16.3 (7.9) 17.3 (7.6) 1.4 (1.5) 1.7 (1.4) 48/27/48 41/38/3 Rostock 43 43 36/7 (83.7) 36/7 (83.7) 37.7 (10.2) 35.5 (11.1) 3.0 (0.9) 3.1 (0.9) 1.8 (1.3) 2.2 (1.1) 9/17/17 6/7/30 Amsterdam 76 76 55/21 (72.3) 50/24 (67.6) 35.5 (8.3) 35.0 (9.4) 4.0 (4.2) 4.2 (4.0) 2.5 (1.3) 3.1 (1.4) 24/27/25 18/25/31 All phase II 281 274 200/81 (71.2) 190/84 (6.9) 40.6 (11.0) 40.14 (11.8) 9.8 (8.7) 9.9 (8.8) 1.8 (1.4) 2.2 (1.5) 102/75/98 87/83/70 Both phases All cohorts 698 688 519/178 (74.5) 489/199 (71.1) 38.6 (10.6) 37.7 (11.6) 8.9 (8.0) 8.7 (7.9) 1.8 (1.5) 2.7 (2.1) 245/198/248 221/201/232 Abbreviations: EDSS 5 Expanded Disability Status Scale; IFN 5 interferon; Madrid PH 5 Hospital Universitario Puerta de Hierro, Madrid; Madrid RC 5 Hospital Ramón y Cajal, Madrid; Madrid SC 5 Hospital Clínico San Carlos; NR 5 nonresponder; R 5 responder. Unless otherwise indicated, data are expressed as mean (SD). a IFN-b (1a intramuscular/1b subcutaneous/1a subcutaneous). N eulogy:N eroim m unlogy & N euinflam m aion