ANKRD55 and DHCR7 Are Novel Multiple Sclerosis Risk Loci

ORIGINAL ARTICLE ANKRD55 and DHCR7 are novel multiple sclerosis risk loci

I Alloza1,14, D Otaegui2,14, A Lopez de Lapuente1, A Antigu¨ edad3, J Varade´ 4,CNu´n˜ez4, R Arroyo5, E Urcelay4, O Fernandez6, L Leyva6, M Fedetz7, G Izquierdo8, M Lucas9, B Oliver-Martos6, A Alcina7, A Saiz10, Y Blanco10, M Comabella11, X Montalban11, J Olascoaga12, F Matesanz7,8,14 and K Vandenbroeck1,13,14

Multiple sclerosis (MS) shares some risk genes with other disorders hallmarked by an autoimmune pathogenesis, most notably IL2RA and CLEC16A. We analyzed 10 single-nucleotide polymorphisms (SNPs) in nine risk genes, which recently emerged from a series of non-MS genome-wide association studies (GWAS), in a Spanish cohort comprising 2895 MS patients and 2942 controls. We identified two SNPs associated with MS. The first SNP, rs6859219, located in ANKRD55 (Chr5), was recently discovered in a meta-analysis of GWAS on rheumatoid arthritis (RA), and emerged from this study with genome-wide significance (odds ratio (OR) ¼ 1.35; P ¼ 2.3 Â 10À9). The second SNP, rs12785878, is located near DHCR7 (Chr11), a genetic determinant of vitamin D insufficiency, and showed a size effect in MS similar to that recently observed in Type 1 diabetes (T1D; OR ¼ 1.10; P ¼ 0.009). ANKRD55 is a gene of unknown function, and is flanked proximally by the IL6ST-- IL31RA gene cluster. However, rs6859219 did not show correlation with a series of haplotype-tagging SNPs covering IL6ST--IL31RA, analyzed in a subset of our dataset (D0o 0.31; r2o 0.011). Our results expand the number of risk genes shared between MS, RA and T1D.

Genes and Immunity (2012) 13, 253--257; doi:10.1038/gene.2011.81; published online 1 December 2011 Keywords: multiple sclerosis; polymorphism; susceptibility; ANKRD55; DHCR7; vitamin D

INTRODUCTION that is, AFF3, ANKRD55, CCR6, CYP2R1, DHCR7, IL2RA, IL10, Multiple sclerosis (MS) is a heterogeneous chronic inflammatory PRDM1 and PXK, all of which emerged recently from GWAS on a disease of the central nervous system driven by a complex number of diseases and conditions different from MS (Table 1). pathogenesis characterized by demyelination and neurodegen- The screen was performed in a Spanish Caucasian cohort eration. Extensive epidemiological data on familial aggregation of comprising 2895 MS patients and 2942 controls, composed of the disease, in conjunction with twin and adoptee studies, argue four geographically separate collections; North of Spain (Basque for a polygenic, multifactorial mechanism.1 Since the advent of Country; 786 MS patients and 718 controls), South of Spain genome-wide association studies (GWAS), swift progress has been (Andalucıá; 1104 MS patients and 1225 controls), East of Spain made in identification of risk loci for MS. Most allelic risk variants (Catalunya; 466 MS patients and 464 controls) and Central identified to date exhibit low penetrance and high frequency of Spain (Madrid; 539 cases and 535 controls). This cohort was the risk allele in unaffected subjects, bringing about modest odds recently used to successfully identify allelic risk variants in the ratios (ORs) that rarely exceed 1.2---exception made for the HLA KIF5A, CD226 and SH2B3 loci, originally discovered in RA and T1D, class II DR*15 haplotype that is identifiable with ORs of 2--3.4.2 as MS risk factors.3 Comparison of data from GWAS on various autoimmune/chronic We provide here a brief overview of the studies that have inflammatory disorders has been shown to be instrumental in enabled the discovery of the nine autoimmune risk genes referred uncovering both disease-specific and shared genetic risk factors. to above. AFF3 emerged originally from a GWAS on T1D, though The latter of these are indicative for intersection of common its association was short of genome-wide significance; rs11676922 pathogenic pathways across a spectrum of diverse conditions, and (or its genetic proxy rs1160542 (D0 ¼ 1, r2 ¼ 1)) located 48 kb 50 are therefore arguably of greater translational interest. The from the AFF3 transcription initiation site was subsequently archetypal, shared autoimmune risk gene is IL2RA that demon- identified and robustly validated as risk locus for RA and juvenile strates susceptibility effects in MS, Type 1 diabetes (T1D), Graves’ idiopathic arthritis.4--7 rs6859219 located in the ANKRD55 gene disease and rheumatoid arthritis (RA), albeit with divergent allelic was identified in a meta-analysis of RA GWAS, and successfully association patterns.1 replicated in the same study.6 An intronic SNP, rs2301436 in In an attempt to identify additional pan-autoimmune loci of FGFR10P, which flanks the CCR6 gene locus was first identified in relevance to MS, we performed a screen of 10 risk single- Crohn’s disease (CD).8 More recently, rs3093023 in the promoter nucleotide polymorphisms (SNPs) located in or nearby nine genes, area of CCR6 at 1.9 kb from the transcription initiation site

1Neurogenomiks Laboratory, Department of Neuroscience, University of the Basque Country UPV/EHU, Leioa, Spain; 2A´ rea de Neurociencias, Inst. Investigacioń Sanitaria Biodonostia, San Sebastiań, Spain; 3Servicio de Neurologıá, Hospital de Basurto, Bilbao, Spain; 4Immunology Department Hospital Clinico San Carlos, Instituto de Investigacioń Sanitaria San Carlos, Madrid, Spain; 5Multiple Sclerosis Unit, Neurology Department H. Clıńico S. Carlos, Instituto de Investigacio´ n Sanitaria San Carlos, Madrid, Spain; 6Servicio de Neurologıá y Laboratorio de Investigacioń, Instituto de Neurociencias Clıńicas del Hospital Regional Universitario Carlos Haya de Ma´laga, Ma´laga, Spain; 7Instituto de Parasitologıá y Biomedicina ‘Lo´ pezNeyra’, CSIC, Granada, Spain; 8Unidad de Esclerosis Mu´ltiple, Hospital Virgen Macarena, Sevilla, Spain; 9Servicio de Biologıá Molecular, Hospital Virgen Macarena, Sevilla, Spain; 10Neurology Service, Hospital Clinic and Institut d’Investigacio´ Biome`dica Pi i Sunyer, Barcelona, Spain; 11Centre d’Esclerosi Mu´ltiple de Catalunya, CEM- Cat, Unitat de Neuroimmunologia Clıńica, Hospital Universitari Vall d’Hebron, Barcelona, Spain; 12Servicio de Neurologıá, Unidad de Esclerosis Mu´ltiple, Hospital Donostia, San Sebastiań, Spain and 13IKERBASQUE, Basque Foundation for Science, Bilbao, Spain; 14These authors contributed equally to this work. Correspondence: Dr K Vandenbroeck, Neurogenomiks Laboratory, Department of Neuroscience, Universidad del Paı´s Vasco (UPV/EHU), Edificio 205, Planta --1, Parque Tecnolo´ gico de Bizkaia, Zamudio 48170, Spain. E-mail: [email protected] Received 23 June 2011; revised 3 October 2011; accepted 31 October 2011; published online 1 December 2011 ANKRD55 and DHCR7 are MS risk loci I Alloza et al 254 Table 1. Confirmed autoimmune loci and risk SNPs tested in the present study for association with MS

Gene SNP Chromosome; Major/ Risk OR (95% CI); P-value Cases/controls Disease or conditionRef chromosome minor allele position allele

a AFF3 rs11676922 Chr2; 100173372 T/A T 1.15 (1.10--1.20); 1.0 Â 10À14 12307/28975 RA6 a ANKRD55 rs6859219 Chr5; 55474337 C/A C 1.18 (1.07--1.28); 9.6 Â 10À12 12307/28975 RA6 a CCR6 rs3093023 Chr6; 167454280 G/A A 1.11 (1.06--1.16); 1.5 Â 10À11 12307/28975 RA6 CYP2R1 rs10741657 Chr11; 14871454 G/A G NA (NA); 3.27 Â 10À20 33996 Vitamin D insufficiency12 b G 1.04 (1.00--1.09); 3.0 Â 10À3 8517/10438 T1D14 (+ 1933 families) DHCR7 rs12785878 Chr11; 70845097 T/G G NA (NA); 2.12 Â 10À27 33996 Vitamin D insufficiency12 b G 1.07 (1.02--1.13); 1.2 Â 10À3 8517/10438 (+1933 T1D14 families) a IL2RA rs706778 Chr10; 6138955 C/T T 1.11 (1.06--1.17); 1.4 Â 10À11 12307/28975 RA6 IL10 rs3024505 Chr1; 205006527 C/T T 1.46 (1.31--1.62); 1.35 Â 10À12 1855/3091 UC19 T 1.26 (1.09--1.45); 1.9 Â 10À3 777/6197 UC20 T 1.24 (1.12--1.37); 2.1 Â 10À5 1689/6197 CD20 T 1.12 (1.07--1.17); 1.6 Â 10À14 22027/29082 CD21 T 1.19 (1.11--1.28); 4.0 Â 10À8 3273/12188 SLE22 C NA (NA); 1.9 Â 10À9 11781/13715 (+4342 T1D23 trios) C 1.32 (1.14--1.51); 1.5 Â 10À4 989/6197 T1D20 PRDM1 rs548234 Chr6; 106674727 T/C C 1.11 (NA); 2.1 Â 10À8 11350/24420 RA24 PRDM1 rs7746082 Chr6; 106541962 G/C C 1.17 (NA); 2.44 Â 10À10 5555/6638 CD8 a PXK rs13315591 Chr3; 58531881 T/C C 1.13 (1.04--1.23); 4.6 Â 10À8 12307/28975 RA6 Abbreviations: CD, Crohn’s disease; CI, confidence interval; MS, multiple sclerosis; NA, data not available; OR, odds ratio; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; SNP, single-nucleotide polymorphism; T1D, type 1 diabetes; UC, ulcerative colitis. aP-values refer to the full combined GWAS (genome- wide association studies) + replication dataset of cases and controls, while ORs (95% CI) are based on the replication dataset only (6768 cases, 8806 controls).6 bP-values refer to the combined dataset, while ORs (95 % CI) are based on the case/control set only (excluding families).14

emerged as risk factor for RA in populations of European ancestry, rs6911490, respectively, are in partial LD (D0 ¼ 0.76, r2 ¼ 0.265), while rs3093024, also located in the CCR6 promoter at 1.5 kb separated by a stretch of 87 kb and are located 50 from PRDM1.8,25 upstream from rs3093023 and in perfect linkage disequilibrium Similarly, the PRDM1 SNPs emerging from the RA and SLE studies, (LD) with it (D0 ¼ 1, r2 ¼ 1) in the European population, was rs548234 and rs6568431, respectively, are 21 kb apart, occur in LD identified as RA risk factor in the Japanese population.6,9 Further (D0 ¼ 0.92, r2 ¼ 0.69), and both are located 30 from PRDM1.22,24 evidence for the generalized involvement of the CCR6 gene region Neither of the UC/CD PRDM1 SNPs occurs in LD with any of the in autoimmune disease risk comes from two independent GWAS RA/SLE SNPs (D0p0.54, r2p0.09), an observation that may be on vitiligo each of which identified strongly associated SNPs indicative for the existence of independent susceptibility effects located 30--175 kb upstream from CCR6.10,11 The CYP2R1 and conferred either by allelic variants at the PRDM1 locus, or at any DHCR7 loci reached genome-wide significance in two GWAS on neighboring locus within reach of these discrete LD patterns. Thus, vitamin D insufficiency.12,13 CYP2R1 rs10741657 and DHCR7 in order to ascertain more complete assessment of the genetic rs12785878 were found to be associated with T1D, a condition diversity at PRDM1, both the RA rs548234 and the CD rs7746082 associated with vitamin D deficiency.14,15 Given the well- SNPs (Table 1) were genotyped. Finally, PXK was disclosed as risk documented candidacy of vitamin D as key factor in the locus in GWAS on SLE and RA, and the SNP identified in the latter pathogenesis of MS, both the SNPs were included in the present screen was included in the present analysis.6,26 association study.16 An intronic SNP in IL2RA, rs12722489, emerged from the first GWAS in MS as one of the first ever non-HLA MS risk polymorphisms to be identified.17 This associa- RESULTS AND DISCUSSION tion was subsequently shown to be conditional to LD with a The overall genotyping success rate amounted to 98.4%. primarily associated SNP, rs2104286, located in the same intron.18 Genotypes of all SNPs in cases and controls were in Hardy-- A new IL2RA SNP, rs706778, that is located in very close vicinity, Weinberg equilibrium (P410À2). Power was calculated for that is at only 96 bp from rs2104286, but nevertheless occurs in detection of genetic effects based on published ORs (Table 2), 2 only partial LD with it (D0 ¼ 0.89, r ¼ 0.19), was identified in a RA and amounted to X77% for seven SNPs but was p43% for three GWAS, and was included in the present analysis to verify whether SNPs. This was either due to the very-low published ORs for the it may constitute a novel marker for association with MS.6 The association of SNPs in the vitamin D pathway genes CYP2R1 and T allele of rs3024505 located 1.04 kb 30 from the polyadenylation DHCR7 with T1D, or to the low risk allele frequency of the PXK site of IL10 has been identified as genetic risk factor in a series of SNP.6,14 Notwithstanding, we identified a moderately significant GWAS on ulcerative colitis (UC), CD and systemic lupus association of DHCR7 rs12785878 with MS in the full cohort 19 --22 erythematosus (SLE). Of note, the alternate C allele of the (OR ¼ 1.10; 95% confidence interval (CI) ¼ 1.02--1.19; PCMH ¼ 0.009) same SNP was found to be associated with enhanced genetic risk (Table 2). The only other, though much stronger, association found for contracting T1D (see Table 1).20,23 The PRDM1 gene region was was that with the ANKRD55 SNP rs6859219 (OR ¼ 1.26; 95% À7 identified as susceptibility locus for CD, UC, SLE and RA through CI ¼ 1.16--1.38; PCMH ¼ 1.9 Â 10 ). Figure 1 shows that this meta-analyses of GWAS coupled to replication exercises in association was detected at nominally significant levels (Po0.05) independent sample sets.8,22,24,25 The most strongly associated in the South, North and East of Spain subsets, but not in the SNPs identified in the CD and UC studies, rs7746082 and Central Spain subset. As a consequence, the Breslow-Day test for

Genes and Immunity (2012) 253 --257 & 2012 Macmillan Publishers Limited ANKRD55 and DHCR7 are MS risk loci I Alloza et al 255 Table 2. Summary of association results in the joint Spanish multiple sclerosis dataset

a Gene SNP Power Cases, total allele Genotyping Risk Merged Merged PCMH OR (95% CI) PBD (%) count/controls, success rates, allele cases, risk controls, risk total allele count cases/controls allele count allele count (%) (%) (%)

AFF3 rs11676922 97 5644/5792 97.5/98.4 A 2834 (50.2) 2832 (48.9) 0.11 1.05 (0.98--1.13) 0.42 ANKRD55 rs6859219 94 5716/5806 98.7/98.7 C 4564 (79.8) 4402 (75.8) 1.9 Â 10À7 1.26 (1.16--1.38) 0.02b CCR6 rs3093023 79 5714/5794 98.7/98.4 G 3273 (57.3) 3279 (56.6) 0.45 1.03 (0.96--1.11) 0.40 CYP2R1 rs10741657 17 5710/5806 98.6/98.7 G 3637 (63.7) 3665 (63.1) 0.43 1.03 (0.95--1.11) 0.51 DHCR7 rs12785878 43 5712/5802 98.6/98.6 G 2113 (37.0) 2017 (34.8) 0.009 1.10 (1.02--1.19) 0.22 IL2RA rs706778 79 5566/5722 96.1/97.2 T 2114 (38.0) 2168 (37.9) 0.94 1.00 (0.93--1.08) 0.18 IL10 rs3024505 94 5710/5808 98.6/98.7 T 790 (13.8) 743 (12.8) 0.10 1.09 (0.98--1.22) 0.93 PRDM1 rs548234 77 5708/5804 98.6/98.6 C 1919 (33.6) 1923 (33.1) 0.67 1.02 (0.94--1.10) 0.28 PRDM1 rs7746082 96 5440/5650 93.9/96.0 G 3914 (71.9) 4032 (71.4) 0.44 1.03 (0.95--1.12) 0.88 PXK rs13315591 41 5706/5800 98.5/98.6 C 377 (6.6) 357 (6.1) 0.30 1.08 (0.93--1.25) 0.13 Abbreviations: CI, conﬁdence interval; OR, odds ratio. aPower was calculated for detection of multiplicative allelic effects at an a level of 0.05 using the ORs for association of each SNP (single-nucleotide polymorphism) with the diseases included in Table 1, based on a sample number of 2895 MS (multiple sclerosis) b patients and 2942 controls (CATS power calculator at http://www.sph.umich.edu/csg/abecasis/CaTS/). PBD was weakly signiﬁcant due to an opposite allelic trend seen in the Central Spain dataset. Upon exclusion of this dataset, the C allele of rs6859219 was associated with MS (C allele frequency of 80.4% in MS À9 patients, and of 75.3% in controls) in the remaining cohort (2356 MS patients, 2407 controls) with PCMH ¼ 2.3 Â 10 ,OR¼ 1.35 (95% CI ¼ 1.22 --1.49), in the absence of stratum-dependent heterogeneity (PBD ¼ 0.66).

were associated with susceptibility to MS, the T allele of IL10 SNP rs3024505, which was recently found to be associated with UC, CD and SLE, displayed a non-significant tendency towards association 19 --22 (OR ¼ 1.09; 95% CI ¼ 0.98--1.22; PCMH ¼ 0.10), and a similar trend was observed for AFF3 rs11676922 (Table 2). ANKRD55(5q11) codes for the ankyrin repeat domain 55 protein of which the function and natural cell types of expression are undocumented. Publicly available human transcriptome data indicate ANKRD55 expression in ovary, testes, endometrium and, likely of higher relevance to autoimmune disease, in resting CD4 þ T cells (http://www.amazonia.transcriptome.eu). Rs6859219 is located in the seventh intron of the ANKRD55 gene, and does not seem to exert functional effects in terms of splicing and transcription factor binding site modification (SNP Function Prediction at http://snpinfo.niehs.nih.gov). The ANKRD55 gene area extending from around intron 6 to exon 9 is characterized by a low degree of LD due to occurrence of a series of recombination hotspots (425cM/Mb) in this region of the gene. Two other candidates in this chromosome area, the IL31RA-- IL6ST genes, though functionally attractive, are located outside the region of LD, at a distance of 158 kb 30 from ANKRD55. In a recent study, we analyzed 13 haplotype-tagging SNPs in the IL6ST-- IL31RA cluster for association with MS in the Spanish--Basque collection, and did not find evidence for association.28 However, the availability of this data for 400 cases and 374 control subjects allowed us to analyze LD patterns between rs6859219 and each of 2 Figure 1. Summary of effect estimate (OR, 95% CI) for (a) the C allele the 13 SNPs, which confirmed absence of correlation (r values of ANKRD55 SNP rs6859219 and (b) the G allele of rs12785878 in ranging between 0.001 and 0.011; D0 values ranging between DHCR7. North of Spain (Basque Country; 786 MS patients and 718 0.06 and 0.31). Pending future independent confirmation, controls), South of Spain (Andalucıá; 1104 MS patients and 1225 ANKRD55, rather than IL6ST--IL31RA, may therefore constitute the controls), East of Spain (Catalunya; 466 MS patients and 464 actual susceptibility gene. The ankyrin repeat is a 33-residue controls) and Central Spain (Madrid; 539 cases and 535 controls). sequence motif involved in protein--protein interaction that is Data for rheumatoid arthritis are taken from Stahl et al.6 and for T1D 14 found in numerous proteins, and accounts for an extraordinary from Cooper et al. variety of biological activities.29 Ankyrin repeat proteins involved in human disease include, among others, p16 in cancer, as well as mutations in the Notch protein associated with the neurological disorder ‘cerebral autosomal dominant arteriopathy with sub- heterogeneity in effect between the population strata was weakly cortical infarcts and leukoencephalopathy’.30,31 Notch signaling is significant at PBD ¼ 0.02. Following removal of the Central Spain involved in immune responses and tissue repair mechanisms subset, the C allele of rs6859219 was associated with MS in the pertinent to MS.32 absence of heterogeneity (OR ¼ 1.35; 95% CI ¼ 1.22--1.49; The second significantly associated gene arising from this study, À9 PCMH ¼ 2.3 Â 10 ; PBD ¼ 0.66). Clearly, the size effect of this albeit with much smaller genetic effect, DHCR7, was recently association positions it within the realm of genome-wide discovered in two GWAS on vitamin D insufficiency.12,13 significance (P ¼ 5 Â 10À7).27 Although none of the other SNPs Rs12785878 is located in the second intron of the NADSYN1 gene,

& 2012 Macmillan Publishers Limited Genes and Immunity (2012) 253 --257 ANKRD55 and DHCR7 are MS risk loci I Alloza et al 256 at a position 8 kb 50 from the transcription initiation site of DHCR7, CONFLICT OF INTEREST and is not predicted to exert functional effects such as The authors declare no conflict of interest. transcription factor binding site and splicing modification (SNP Function Prediction at http://snpinfo.niehs.nih.gov). DHCR7 codes for the enzyme delta-7-sterol reductase (EC 1.3.1.21), which ACKNOWLEDGEMENTS converts 7-dehydrocholesterol (7-DHC) to cholesterol. Vitamin D3 We thank patients with multiple sclerosis and control subjects for making this study is made in the skin when 7-DHC reacts with ultraviolet light. Thus, feasible. Financial support for the study was provided by: the Gobierno Vasco the conversion of 7-DHC by the gene product of DHCR7 removes (reference IT512-10; Convocatoria ‘Grupos de Investigacioń 2010 --2015’), the the substrate 7-DHC from the synthetic pathway for the Ministerio de Ciencia e Innovacioń-FondosFeder (SAF2009-11491), the Fondo de 12,13 production of vitamin D3. The G allele of rs12785878, which Investigacioń Sanitaria FIS (RETICS-REEM RD07/0060, PI081636, PI10/1985, PS09/ is associated with lower levels of 25(OH)D and risk for T1D, 02105), the Junta de Andalucıá (P07-CVI-02551), Ikerbasque, the Basque Foundation enhances susceptibility to MS (Table 2, Figure 1).14 Several studies for Science (Bilbao) and FundaciońIlundain. SNP genotyping services were provided have analyzed vitamin D metabolic pathway genes as determi- by the Spanish ‘Centro Nacional de Genotipado CEGEN-USC, http://www.cegen.org’. nants of risk for MS, generally with negative outcomes.33 --36 Regulation of the MS-associated HLA-DRB1*1501 allele by vitamin D has been reported.37 However, given the only very recent REFERENCES discovery of the functional link between DHCR7 and vitamin D 1 Oksenberg JR, Baranzini SE. Multiple sclerosis genetics -- is the glass half full, or insufficiency, earlier studies have not addressed allelic variation in half empty? Nat Rev Neurol 2010; 6: 429 --437. this gene. Hence, this study is the first to demonstrate a genetic 2 McElroy JP, Oksenberg JR. Multiple sclerosis genetics 2010. NeurolClin 2011; 29: 219 --231. link between an important regulator of vitamin D levels, DHCR7, 3 Alcina A, Vandenbroeck K, Otaegui D, Saiz A, Gonzalez JR, Fernandez O et al. The and risk for MS. 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