Genes and Immunity (2010) 11, 660–664 & 2010 Macmillan Publishers Limited All rights reserved 1466-4879/10 www.nature.com/gene ORIGINAL ARTICLE A non-synonymous SNP within membrane metalloendopeptidase-like 1 (MMEL1) is associated with multiple sclerosis M Ban1,15, JL McCauley2,15, R Zuvich3,15, A Baker1, L Bergamaschi4, M Cox5, A Kemppinen1, S D’Alfonso4, FR Guerini6, J Lechner-Scott5, F Dudbridge7, J Wason7, NP Robertson8, PL De Jager9,10, DA Hafler11, LF Barcellos12, AJ Ivinson13, D Sexton3, JR Oksenberg14, SL Hauser14, MA Pericak-Vance2, J Haines3, A Compston1 and S Sawcer1 1Department of Clinical Neuroscience, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK; 2Miami Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL, USA; 3Center for Human Genetics Research, Vanderbilt University Medical Center, Nashville, TN, USA; 4Department of Medical Sciences and Interdisciplinary Research Center of Autoimmune Diseases, University of Eastern Piedmont, Novara, Italy; 5University of Newcastle, John Hunter Hospital, Newcastle, New South Wales, Australia; 6Laboratory of Molecular Medicine and Biotechnology, Don C Gnocchi Foundation IRCCS, S Maria Nascente, Milan, Italy; 7MRC Biostatistics Unit, Cambridge, UK; 8Department of Neurology, University Hospital of Wales, Heath Park, Cardiff, UK; 9Program in Translational NeuroPsychiatric Genomics, Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; 10Broad Institute, Cambridge, MA, USA; 11Department of Neurology, Yale University School of Medicine, New Haven, CT, USA; 12Division of Epidemiology, School of Public Health, University of California at Berkeley, Berkeley, CA, USA; 13Harvard NeuroDiscovery Center, Harvard Medical School, Boston, MA, USA and 14Department of Neurology, University of California San Francisco, San Francisco, CA, USA Several single-nucleotide polymorphism (SNP) genome-wide association studies (GWASs) have been completed in multiple sclerosis (MS). Follow-up studies of the variants with the most promising rankings, especially when supplemented by informed candidate gene selection, have proven to be extremely successful. In this study we report the results of a multi-stage replication analysis of the putatively associated SNPs identified in the Wellcome Trust Case Control Consortium non-synonymous SNP (nsSNP) screen. In total, the replication sample consisted of 3444 patients and 2595 controls. A combined analysis of the nsSNP screen and replication data provides evidence implicating a novel additional locus, rs3748816 in membrane metalloendopeptidase-like 1 (MMEL1; odds ratio ¼ 1.16, P ¼ 3.54 Â 10À6) in MS susceptibility. Genes and Immunity (2010) 11, 660–664; doi:10.1038/gene.2010.36; published online 24 June 2010 Keywords: multiple sclerosis; MMEL1; genetics Introduction genome-wide association studies (GWASs) have been performed in MS: four based on indirectly screening Multiple sclerosis (MS) is considered to be an auto- common variation through linkage disequilibrium (LD)4–7 immune disease of the central nervous system that is and the fifth based on directly typing non-synonymous characterized pathologically by inflammation, demyeli- coding SNPs (nsSNPs).8 It is this last study, performed as 1 nation and axonal degeneration. Epidemiological part of the Wellcome Trust Case Control Consortium evidence suggests that it results from unknown (WTCCC), that has provided the foundation for the work environmental factors acting on genetically susceptible reported in this study. 2,3 individuals. Individually, none of the five MS GWASs thus far In the past 2 years, the application of high-throughput completed succeeded in identifying statistically unequi- genotyping methodologies has resulted in rapid progress vocal association at the screening stage (outside the in the identification of relevant susceptibility genes in expected association with the major histocompatibility MS. To date, five single-nucleotide polymorphism (SNP) complex), although each provides an invaluable ranking of the variants tested. The extent to which the higher Correspondence: Dr M Ban, Department of Clinical Neuroscience, ranges of such rankings are enriched for genuine University of Cambridge, Addenbrooke’s Hospital, Box 165, Hills associations depends upon the power of the study and Road, Cambridge CB2 2QQ, UK. the prior odds that the variants tested are causative, or E-mail: [email protected] are in tight LD with such rankings. Follow-up studies of 15These authors contributed equally to this work. Received 29 January 2010; revised 3 May 2010; accepted 11 May the variants with the most promising rankings, especially 2010; published online 24 June 2010 when supplemented by informed candidate gene Association of MMEL1 with multiple sclerosis M Ban et al 661 selection, have proven to be extremely successful in MS. For example, by extending the typing of a modest number of the most promising variants identified in the WTCCC nsSNP screen, we have already identified association with the tyrosine kinase 2 gene (TYK2) and MS.9 Similar highly focused replication efforts based on GWAS results have been equally successful in other complex genetic diseases such as coeliac disease,10 type I diabetes11 and rheumatoid arthritis (RA).12 In this article we report our most recent efforts to mine more deeply into the ranking generated in the nsSNP screen.8 Results Figure 1 Odds ratio for the rs3744816(T) allele in MMEL1. The area A two-stage replication effort was used in this study. In of the box is proportional to the number of cases included in the the first stage, 1664 of the most promising markers study. The error bars indicate the 95% confidence intervals. The identified in the nsSNP screen were typed in an population-specific odds ratios are shown for reference. independent set of 1038 cases and 1379 controls. These new data were combined with the original data from the nsSNP screen to provide a refined ranking. The results of methionine (ATG) at amino acid position 509. This this extension analysis are provided in Supplementary change is predicted to be ‘probably damaging’ based Table S1. In stage 2 of our replication effort, we chose the on the program polymorphism phenotyping (PolyPhen: 13 most promising SNPs identified from the extension http://genetics.bwh.harvard.edu/pph). MMEL1, which analysis for further investigation in 2406 cases and 1216 is also known as Neprilysin 2 (NEP2), belongs to the M13 controls. Five SNPs were found to have P 10À4; o family of metalloendopeptidases. Unlike other members however, as rs601338 and rs602662 are in tight LD we of this family that are ubiquitously expressed, MMEL1 only considered rs602662 further (the more strongly expression is limited to the testis and central nervous associated). Of the remaining 331 SNPs with a P 0.05, o system. In rodents, the largest isoform is predominantly we chose further 9 SNPs that were within candidate expressed in testis, cleavage of this isoform producing a genes. As one of these SNPs (rs12912505) failed secreted soluble enzyme. The second isoform, which is manufacture, in total we genotyped 12 SNPs. 23 amino acids shorter, is a membrane-associated protein The results of the 12 SNPs analysed in both replication highly expressed in the central nervous system.13 Little stages are shown in Table 2 along with the combined data exist though as to the exact function of the MMEL1 analysis of all available data from the nsSNP screen, gene. As with other members of the metalloendopepti- stage 1 and stage 2 replication efforts. The population- dase family, MMEL1 seems to be involved in neuropep- specific results for the top 12 SNPs are shown in tide degradation,13 and a role in Alzheimer’s disease has Supplementary Table S2. Using the Breslow–Day test, also been suggested as it is involved in the degradation some evidence for heterogeneity was shown for one SNP of b-amyloid.14 (rs379707). A random effects model was therefore The associated SNP lies within a region of extensive LD applied for the analysis of this SNP, although it did not on chromosome 1 encompassing the MMEL1 gene and alter the odds ratio (data not shown). In the combined just extending to the tumour necrosis factor receptor super- analysis of all available data, the most significant family 14 gene (TNFRSF14). A SNP within TNFRSF14 association was with rs3748816, which lies in the (rs2234167) that lies on the boundary of this LD block was membrane metalloendopeptidase-like 1 gene (MMEL1) typed in the first replication stage but did not show any (P ¼ 3.54 Â 10À6, odds ratio ¼ 1.16, 95% confidence evidence for significance in the combined analysis of the interval 1.09–1.24). The modest power in the second original nsSNP screen and stage 1 replication data stage limits the extent to which the evidence for (P ¼ 0.2514). The LD between rs3748816 and rs2234167, association is increased by this typing. However, the as determined from the 4858 individuals who were typed result for rs3748816 remains nominally significant even if for both markers in stage 1 of the study, is low (D’ ¼ 1, corrected for the number of tests performed in the r2 ¼ 0.083). Considerable work will be required to fine original screen. A detailed plot of the effect size of this map this signal to ascertain whether the signal is because variant within MMEL1 by the country of ascertainment is of association with TNFRSF14 or MMEL1. shown in Figure 1. Two recent GWASs in RA also identified an association with the MMEL1 gene.12,15 The SNPs tested in the RA screens (rs3890745 and rs10910099) were not analysed in Discussion the current study. Both SNPs though are in strong LD By systematically extending the analysis of the putatively with rs3748816 (r2 ¼ 0.962 and 0.924, respectively, based associated SNPs identified in our nsSNP GWAS, we on HapMap data) with the major allele increasing provide suggestive evidence implicating an additional susceptibility in both of the RA screens and in our study. locus (rs3748816 in MMEL1) involved in MS suscept- Growing evidence suggests that there is an overlap in ibility.