Epstein–Barr virus candidate genes and multiple sclerosis

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Epstein-Barr virus candidate genes and multiple sclerosis Kelly Claire Simon ScD, Hollie Schmidt, Sara Loud, Alberto Ascherio

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S2211-0348(14)00337-X http://dx.doi.org/10.1016/j.msard.2014.10.003 MSARD223

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Multiple Sclerosis and Related Disorders

Received date: 3 August 2013 Revised date: 12 May 2014 Accepted date: 28 October 2014 Cite this article as: Kelly Claire Simon ScD, Hollie Schmidt, Sara Loud, Alberto Ascherio, Epstein-Barr virus candidate genes and multiple sclerosis, Multiple Sclerosis and Related Disorders, http://dx.doi.org/10.1016/j.msard.2014.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Epstein-Barr virus candidate genes and multiple sclerosis

Kelly Claire Simon, ScD1,2, Hollie Schmidt, MS3 , Sara Loud, MS3 , Alberto Ascherio, MD, DrPH1,2,4

1-Department of Nutrition, Harvard School of Public Health, 655 Huntington Ave, Boston, MA, USA 2-Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Ave, Boston, MA, USA 3-Accelerated Cure Project, 460 Totten Pond Road, Waltham, MA, USA Waltham, MA, USA 4-Department of Epidemiology, Harvard School of Public Health, 677 Huntington Ave, Boston, MA, USA

Corresponding author: Kelly Claire Simon 665 Huntington Ave Department of Nutrition Boston, MA 02215 Fax: 617-432-1973 [email protected]

Keywords: Multiple sclerosis Single nucleotide polymorphisms Epstein-Barr virus Infectious mononucleosis Gene Epidemiology

Abstract Background: Previous infection with Epstein-Barr virus (EBV) and a history of infectious mononucleosis (IM) have been previously associated with an increased risk of multiple sclerosis (MS). Whether there are common genetic factors that may partially explain these associations has not been thoroughly explored. Objective: To investigate whether select polymorphisms in genes associated with IM and susceptibility are related to MS risk, a self-reported history of IM or antibody titer against Epstein-Barr virus nuclear antigen 1 (anti-EBNA1). Methods: A case-control study including 1213 MS cases and 454 controls enrolled in the Accelerated Cure Project for MS (ACP) Repository. Select polymorphisms in HLA-A, SH2D1A and IL15RA and antiEBNA1 Ab titers were measured using stored blood samples provided by participants. Generalized linear models were used to assess the associations between select polymorphisms and odds of MS, odds of IM or anti-EBNA1 Ab titers. Results: No significant associations were observed between the selected polymorphisms and odds of MS, odds of IM or anti-EBNA1 Ab titer. Conclusion: It is unlikely that any of the studied polymorphisms contribute to the explaining the association between anti-EBNA1 Ab titer or history of IM and MS.

1.1 Introduction Previous infection with Epstein-Barr virus (EBV) and high antibody titers to Epstein-Barr nuclear antigen (EBNA) are well established multiple sclerosis (MS) risk factors. (Ascherio and Munger, 2007) It is clear, however, that there is a strong genetic component to MS susceptibility, including the indisputable contribution of HLA alleles (Lincoln et al. , 2009), and more modest effects of several other genes. (De Jager et al. , 2009) Except for one recent study considering variants in the MHC region, (Ramagopalan et al. , 2011) whether shared genetic susceptibility may explain part of the observed association between IM or anti-EBNA Ab titers and MS risk has been little investigated. We, therefore, sought to determine if candidate genes, selected for their potential role in EBV infection or development of IM, were associated with MS, anti-EBNA1 Ab titers, or IM among over 1200 individuals with MS participating in the ongoing Accelerated Cure Project for MS Repository study. 1.2 Methods 1.2.1 Study Population This study includes participants in the Accelerated Cure Project for Multiple Sclerosis (ACP) Repository. Full details of the study and all relevant documentation can be found at www.acceleratedcure.org. Enrollment in the study began in May 2006 and subjects have been recruited at 10 MS specialty clinic sites. MS patients are identified from the patient base or recruited from the surrounding communities at each collection site and, if interested, are screened against the exclusion/inclusion criteria. Inclusion criteria include 1) at least one demyelinating event characteristic of MS, optic neuritis (ON), neuromyelitis optica (NMO), transverse myelitis (TM) or acute disseminated encephalomyelitis (ADEM) (demyelinating event is defined as a symptom or constellation of symptoms referable to a disruption of the CNS white matter or myelin within gray matter. Characteristic syndromes include hemibody sensory or motor symptoms, mono-sensory symptoms, monoparesis, brainstem syndrome or cerebellar

syndrome, lasting at least 24 hours and not acute in onset), 2) at least 18 years old and able to give informed consent or less than 18 years old with parental permission and ability to give assent and 3) individuals > 18 years willing and able to provide up to 110 mL blood via venipuncture or less than 18 years willing and able to give 50mL. Case exclusion criteria include 1) individuals with clinical or radiological evidence of stroke, meningitis, neoplastic, peripheral nervous system or primary muscle disease, or other well-characterized and defined diseases of the nervous system excepting those under study (see inclusion criteria 1), 2) individuals with a history of blood borne pathogens, 3) individuals with a history of allogeneic bone marrow transplant and 4) Individuals < 37lbs. Controls, unrelated and related, are referred by the cases. Controls include related and unrelated individuals who have not experienced any CNS demyelinating events characteristic of MS, TM, ADEM, NMO or ON and have not been diagnosed with any demyelinating disease. Inclusion criteria 2) and 3) and all exclusion criteria for cases apply for controls. At the time of our receipt of samples, the study population included 1617 participants for whom for whom we had biological samples for serum analysis (1213 MS, 454 controls). This study was approved by appropriate ethical review boards. 1.2.2 History of infectious mononucleosis Demographic, personal habits, individual and family medical history information is collected via a case report form (CRF) administered by the study staff. This includes a question on whether the participant had infectious mononucleosis (IM) and, if so, at what age. 1.2.3 Anti-EBNA1 Ab titers Each ACP Repository participant provided blood samples for use in research. Serum was processed into aliquots at the clinical sites, stored at -70C to -80C at the site, and then shipped frozen to the repository’s central lab facility for storage at -80C (SeraCare Bioservices, Inc., Gaithersburg, MD). DNA was extracted from whole blood at the central lab facility. IgG antibody titer levels against the Epstein-

Barr virus nuclear antigen 1 (anti-EBNA1) were measured in the laboratory of Dr. Nadar Rifai at the Children’s Hospital Boston using a commercially available ELISA which was modified for quantitative analysis relative to known standards. 1.2.4 EBV/IM related SNPs Genes were chosen on the basis of previous literature, and included SH2D1A, IL15RA, and HLA-A. SH2D1A was the primary gene of interest because rare mutations cause X-linked lymphoproliferative disorder (XLP) which is a rare, often fatal susceptibility to EBV infection/IM. (Bassiri et al. , 2008) We chose tag SNPs to capture the variation in the coding region of this gene. Using Hap Map Rel. 22 data from the CEPH population (Utah residents with ancestry from northern and western Europe), (International HapMap Consortium et al. , 2007) tag SNPs were chosen using the pairwise tagging function in Haploview (Barrett et al. , 2005). We genotyped a single rare loss of function variant in IL15RA because a report had shown that EBV-associated IM results in a seemingly permanent deficit in IL15RA expression. (Sauce et al. , 2006)Finally, two SNPs in HLA-A (RS2530388 and RS6457110) were genotyped because of a previous report that they were associated with odds of IM. (McAulay et al. , 2007) Genotyping was performed at the Harvard-Partners Center for Genetics and Genomics using the OpenArrayTM SNP Genotyping Platform (BioTrove, Woburn, MA). Blinded concordance of duplicate quality control samples was 100%. 1.2.5 Statistical analysis Generalized linear models were used to assess the association between individual SNPs and odds of MS, anti-EBNA1 Ab titer or odds of IM adjusted for age (5 year age groups), ethnicity, race (Latino/Hispanic versus not), study site and gender. Because SH2D1A is an X-chromosome gene, we also conducted sensitivity analyses only among women for these SNPs and this did not materially change results.

This study was approved by the appropriate ethical review boards. 1.3 Results 1.3.1 Odds of MS The genotype distributions in the MS cases and controls and OR for MS are shown in Table 1. As expected, the ‘A’ allele of the HLA-A*0201 tagging SNP, rs6457110, was associated with decreased odds of MS (OR=0.86 (95% CI: 0.72, 1.02)) similar to previous reports (International Multiple Sclerosis Genetics Consortium et al. , 2011) None of the polymorphisms in SH2D1A were associated with odds of MS (Table 1). Results were similar upon further adjustment for HLA-DR15, self-reported history of IM and anti-EBNA1 Ab titer.

1.3.2 Odds of IM None of the identified polymorphisms showed association with history of IM after correction for multiple comparisons (Table 2), although the number of controls was small and power was only moderate to detect modest effect sizes. Among controls, the association for the HLA-A polymorphism, rs6457110, was in the same direction for the ‘A’ allele (OR=0.86; p=0.52), albeit attenuated compared to the original report (OR=0.65). (McAulay, 2007) We also observed a marginally significant association between SH2D1A-RS2049995 and IM among MS patients (uncorrected p=0.046), though this was likely due to chance, and significance would not withstand multiple comparison correction. 1.3.3 Odds of higher anti-EBNA1 Ab titer In regards to anti-EBNA1 IgG Ab titers, no SNPs were significantly associated with titers either in healthy controls or MS patients (Table 3).

1.4 Discussion In this investigation, we did not find significant associations between polymorphisms in HLA-A, SH2D1A or IL15RA and MS prevalence or EBV infection, as measured by a history of IM and anti-EBNA1 Ab titers after correction for multiple comparisons. We observed an uncorrected significant increase in odds of IM among MS patients, however, this was likely due to chance and did not persist after multiple comparison correction. In regards to HLA-A, which has been previously associated with MS and IM, although non-significant, the observed association with rs6457110 was similar to expected based on previous literature(International Multiple Sclerosis Genetics Consortium, 2011) , with the minor ‘A’ allele being more commonly observed in healthy subjects. This allele was also associated with a nonsignificant decreased odds of IM and, in contrast, a non-significantly higher anti-EBNA1 Ab titer in controls. It does not appear, however, that any susceptibility to IM conferred by this polymorphism explains the association with MS as models simultaneously adjusted for IM and rs6457110 genotype yielded similar point estimates to models in which each was modeled without the other suggesting little evidence of confounding by genetic susceptibility. There are clear limitations to our analysis. First, we limited ourselves to potential biological candidate genes and did not conduct an exhaustive analysis of variants in this region. We either chose SNPs based on previous literature or, in the case of SH2D1A, provided tagging coverage. This approach relies on the presence of important common previously characterized variants and the possibility clearly exists for a role of rare mutations or yet to be discovered mutations. Given the strong association between EBV and MS and the still elusive biological relationship, further work is needed to understand susceptibility not only to MS, but also to these risk factors which have health implications beyond MS.

1.5 Conclusion In conclusion, we did not find evidence for a strong association between our investigated polymorphisms and history of IM, anti-EBNA Ab titers or odds of MS, suggesting these polymorphisms are unlikely to explain the observed association between these factors and MS.

Acknowledgments The authors thank Leslie Unger for technical assistance and manuscript preparation and Mindy Collins to assistance with sample selection and shipping. The authors also thank the participants in the Accelerated Cure Project for MS who make this research possible. Funding Acknowledgment This work was supported by a grant from the ACP (#20091101). The authors thank the individuals who have generously participated in the ACP Repository to make this work possible. We also thank Mindy Collins for her assistance in coordinating the biological sample selection and acquisition.

Conflict of Interest The Authors declare that there are no conflicts of interest.

References

Ascherio A, Munger KL. Environmental risk factors for multiple sclerosis. Part I: The role of infection. Ann Neurol. 2007;61:288-99. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005;21:263-5. Bassiri H, Janice Yeo WC, Rothman J, Koretzky GA, Nichols KE. X-linked lymphoproliferative disease (XLP): a model of impaired anti-viral, anti-tumor and humoral immune responses. Immunologic research. 2008;42:145-59. De Jager PL, Jia X, Wang J, de Bakker PI, Ottoboni L, Aggarwal NT, et al. Meta-analysis of genome scans and replication identify CD6, IRF8 and TNFRSF1A as new multiple sclerosis susceptibility loci. Nat Genet. 2009;41:776-82. International HapMap Consortium, Frazer KA, Ballinger DG, Cox DR, Hinds DA, Stuve LL, et al. A second generation human haplotype map of over 3.1 million SNPs. Nature. 2007;449:851-61. International Multiple Sclerosis Genetics Consortium, Wellcome Trust Case Control Consortium, Sawcer S, Hellenthal G, Pirinen M, Spencer CC, et al. Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis. Nature. 2011;476:214-9. Lincoln MR, Ramagopalan SV, Chao MJ, Herrera BM, Deluca GC, Orton SM, et al. Epistasis among HLADRB1, HLA-DQA1, and HLA-DQB1 loci determines multiple sclerosis susceptibility. Proc Natl Acad Sci U S A. 2009;106:7542-7. McAulay KA, Higgins CD, Macsween KF, Lake A, Jarrett RF, Robertson FL, et al. HLA class I polymorphisms are associated with development of infectious mononucleosis upon primary EBV infection. J Clin Invest. 2007;117:3042-8.

Ramagopalan SV, Meier UC, Conacher M, Ebers GC, Giovannoni G, Crawford DH, et al. Role of the HLA System in the Association Between Multiple Sclerosis and Infectious Mononucleosis. Arch Neurol. 2011;68:469-72. Sauce D, Larsen M, Curnow SJ, Leese AM, Moss PA, Hislop AD, et al. EBV-associated mononucleosis leads to long-term global deficit in T cell responsiveness to IL-15. Blood. 2006;108:11-8.

Table 1. Association between EBV-related candidate SNPs and MS risk Genotypes SNP

Cases (%) Controls(%)

HLA-A:RS2530388 HLA-A:RS6457110 SH2D1A-RS2049995 SH2D1A-RS2239481*

TT/TA/AA AA/AG/GG AA/AG/GG

49/41/10 42/44/14 67/29/4 72/26/2

47/44/9 35/51/14 67/28/5 75/21/4

OR for MS 0.98 (0.81, 1.17) 0.86 (0.72, 1.02) 0.98 (0.81, 1.18) 0.96 (0.79, 1.18)

SH2D1A-RS4465087 SH2D1A-RS5956646 SH2D1A-RS5958470 SH2D1A-RS6649202 IL15RA-RS4749858

AA/AG/GG CC/CG/GG AA/AG/GG TT/TC/CC GG/GC/CC

66/33/1 32/52/17 34/49/18 37/46/18 96/4/0

66/33/1 36/48/17 38/46/16 39/46/15 95/5/0

0.98 (0.78, 1.23) 0.94 (0.81, 1.1) 1.00 (0.86, 1.16) 0.94 (0.81, 1.1) 0.73 (0.42, 1.27)

TT/TA/AA

X chromosome gene-genotype frequencies among women only Adjusted for age at interview/blood draw, study site, race/ethnicity and gender

pvalueuncorrected 0.78 0.08 0.81 0.73 0.87 0.42 0.96 0.45 0.27

Table 2. Association between EBV related candidate SNPs and a history of IM among MS patients and healthy controls

SNP HLA-A:RS2530388 HLA-A:RS6457110 SH2D1A-RS2049995 SH2D1A-RS2239481 SH2D1A-RS4465087 SH2D1A-RS5956646 SH2D1A-RS5958470 SH2D1A-RS6649202 IL15RA-RS4749858

Healthy individuals OR for mono** 1.4 (0.9, 2.2) 0.9 (0.59, 1.4) 1.2 (0.7, 1.8) 0.7 (0.4, 1.3) 1.4 (0.8, 2.3) 0.9 (0.6, 1.3) 0.8 (0.6, 1.3) 0.9 (0.6, 1.3) NA*

p-value 0.16 0.52 0.53 0.30 0.24 0.49 0.41 0.50

MS patients OR for mono** 1.0 (0.8, 1.2) 0.9 (0.8, 1.1) 0.8 (0.6, 1.0) 1.0 (0.8, 1.2) 1.1 (0.9, 1.2) 1.0 (0.8, 1.2) 1.0 (0.8, 1.2) 1.1 (0.9, 1.3) 0.9 (0.4, 1.7)

p-value 0.86 0.52 0.046 0.87 0.40 0.76 0.97 0.27 0.65

Includes 315 MS cases with IM and 750 cases without a history of IM and 50 healthy controls with a history of IM and 360 healthy controls without such a history. Adjusted for age at interview/blood draw, study site, race/ethnicity and gender *Because of the rare minor allele frequency, the adjusted OR in healthy controls could not be estimated. **Additive model for increasing number of minor alleles

Table 3. Association between EBV related candidate SNPs and change in anti-EBNA1 Ab titer relative to mean titer among MS patients and healthy individuals

Genotypes SNP HLAA:RS2530388 HLAA:RS6457110 SH2D1ARS2049995 SH2D1ARS2239481 SH2D1ARS4465087 SH2D1ARS5956646 SH2D1ARS5958470 SH2D1ARS6649202 IL15RARS4749858

Healthy individuals Genotype specific means

MS patients p-value

Genotype specific means

p-value

TT/TA/AA

3.9/3.8/3.7

0.55

4.0/4.0/4.0

0.27

TT/TA/AA

3.8/3.9/4.0

0.12

4.0/4.0/4.0

0.64

AA/AG/GG

3.8/3.9/3.9

0.41

4.0/4.0/4.0

0.39

AA/AG/GG

3.8/3.8/3.7

0.60

4.0/4.0/4.0

0.78

AA/AG/GG

3.8/3.9/4.0

0.42

4.0/4.0/3.9

0.43

CC/CG/GG

3.7/3.8/3.9

0.13

4.0/4.0/4.0

0.31

AA/AG/GG

3.8/3.8/3.9

0.24

4.0/4.0/4.0

0.50

TT/TC/CC

3.8/3.9/3.9

0.66

4.0/4.1/4.0

0.29

GG/GC/CC

3.8/3.7/NA

0.60

4.0/4.0/4.7

0.44

*Adjusted for age at interview/blood draw, study site, race/ethnicity and gender

highlights -

Previous infection with EBV and history of IM have been associated with increased MS risk. We sought to determine if select polymorphisms potentially associated with EBV or IM were associated with MS. None of the studied polymorphisms appeared to contribute to explaining the association between EBV or IM and MS.