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Multiple sclerosis
Short report
Low vitamin D and elevated immunoreactivity against Epstein–Barr virus before first clinical manifestation of multiple sclerosis
  1. Bernhard F Décard1,
  2. Nicolas von Ahsen2,
  3. Thomas Grunwald3,
  4. Frank Streit2,
  5. Anke Stroet1,
  6. Petra Niggemeier4,
  7. Volkmar Schottstedt4,
  8. Joachim Riggert5,
  9. Ralf Gold1,
  10. Andrew Chan1
  1. 1Department of Neurology, St. Josef-Hospital, Ruhr-University Bochum, Bochum, Germany
  2. 2Department of Clinical Chemistry, University Medical Center Göttingen, Göttingen, Germany
  3. 3Department of Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
  4. 4German Red Cross Blood Transfusion Service West, Central Laboratory, Hagen, Germany
  5. 5Department of Transfusion Medicine, University of Göttingen, Göttingen, Germany
  1. Correspondence to Dr Andrew Chan, Department of Neurology, St. Josef-Hospital, Ruhr-University Bochum, Gudrunstr. 56, 44791 Bochum, Germany; andrew.chan{at}rub.de

Abstract

Objective Vitamin D deficiency and Epstein–Barr virus (EBV) infection may be associated with the development of multiple sclerosis (MS). We investigated serum 25-hydroxyvitamin D (25-OH-D) levels and anti-EBV immunoreactivity in 25 individuals before the first clinical manifestation of MS.

Patients and methods 56 serum samples of 25 individuals who had donated blood prior to the first clinical MS manifestation (clinically isolated syndrome (CIS)) (four male subjects, 21 female subjects, mean age 31.5 years at time of pre-CIS blood sampling; mean age at disease onset 33.4 years) were available, covering an interval of 7.3 years–2 months (mean 31.5 months) before CIS. In 18 of 25 patients serum samples were also obtained after established diagnosis of MS. Longitudinal age- and gender-matched healthy blood donors (four male subjects, 21 female subjects, 39 samples, mean age 32.5 years) served as controls. Serum 25-OH-D was measured by isotope dilution-liquid chromatography-tandem mass spectrometry. 25-OH-D levels were deconvoluted using published seasonal coefficients from a German population. Immunoglobulin G (IgG) against Epstein–Barr virus nuclear antigen-1 (EBNA1) were assessed using commercially available ELISA.

Results Low 25-OH-D levels were observed during the 24-month pre-CIS interval (47.8 (32.5–77.2) nmol/l, median (IQR); healthy controls: 81.6 (57.7–98.5), p=0.004, however, still higher than after established diagnosis (24.5 (13.7–47.7), p<0.0001 compared with controls). IgG against EBNA1 during the 36-month pre-CIS interval was increased (185.9 (91.2–460.0) IU/ml, median (IQR); healthy controls 63.7 (29.5–121.6), p=0.002).

Conclusions Low vitamin D and remote EBV infection may be associated with clinical MS breakthrough within 2–3 years.

  • MS
  • vitamin D
  • EBV
  • EBNA1
  • Alzheimer's disease
  • multiple sclerosis
  • neuroimmunology
  • rheumatology
  • dementia
  • virology
  • benign intracran hyp
  • clinical neurology
  • genetics
  • immunology
  • neurophysiology
  • neuropsychiatry
  • neurosurgery
  • psychiatry
  • thrombophilia

https://doi.org/10.1136/jnnp-2012-303068

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    Introduction

    Decreased 25-hydroxyvitamin D (25-OH-D) serum concentrations1 and remote Epstein–Barr virus (EBV) infection2 are currently discussed as important risk factors for the development of multiple sclerosis (MS) and clinical disease activity.3 ,4 In this context, higher 25-OH-D serum levels were reported with lower risk to develop MS later in life.5 Likewise, prior to first clinical disease manifestation, elevated immune response especially against Epstein–Barr virus nuclear antigen-1 (EBNA1) was associated with an increased risk for MS.6–9 The aim of this study was to investigate both 25-OH-D serum levels and Immunoglobulin G (IgG) response against EBNA1 before the first clinical MS manifestation in individuals who had donated blood prior to disease onset.

    Patients and methods

    The study was approved by the local ethics committee. Sera from patients who had donated blood (between 1997 and 2004) prior to the first clinical MS manifestation (CIS) were obtained from blood donor centres across Germany after informed consent.10 The date of CIS was retrospectively assessed from medical records. In all, 56 pre-CIS samples from 25 individuals (four male subjects, 21 female subjects, mean age 31.5 years at time of pre-CIS blood donation, mean age at CIS 33.4 years) were available, covering an interval of 7.3 years–2 months (mean 31.5 months) before disease onset. In 18 of 25 patients serum samples could also be obtained after established MS diagnosis (collected 3/2007, mean 32 months after CIS).

    Samples of age- and gender-matched healthy blood donors (four male subjects, 21 female subjects, 39 samples, mean age 32.5 years, Department of Transfusion Medicine, Göttingen) served as controls. Patients with longitudinal samples were matched with a control with the same number of longitudinal samples; for four patients with ≥3 pre-CIS samples, longitudinal controls were only available for selected timepoints.

    Case ascertainment was established by review of medical records, following previously described criteria.10 Overall, 10 cases were consistent with McDonald criteria and 11 cases fulfilled Poser criteria; four cases were classified as probable.

    Serum 25-OH-D was measured by isotope dilution-liquid chromatography-tandem mass spectrometry (Waters Micromass® Quattro Premier System, Milford, Massachusetts, USA) using a commercial calibrator (Chromsystems, Instruments & Chemicals, Munich, Germany). 25-OH-D concentrations were seasonally deconvoluted using published coefficients in an adapted periodic function (25-OH-D (adjusted, month may) = 25-OH-D (month, measured) − 3.3 × cos((month × π)/6) − 6.9 × sin((month × π)/6) + 0.6) from a German population.11 These data pertain to a latitude in the geographic middle of Germany and should thus reflect a correct average. Virus antigen-specific IgG response against EBNA1 was assessed using ELISA (Institut Virion/Serion, Würzburg, Germany). One-way ANOVA test (Kruskal–Wallis) was used to compare cross-sectional 25-OH-D course and EBNA1 IgG response. Results between two timepoints were analysed with Mann–Whitney test. Correlation analysis was performed using Spearman's rank correlation coefficient (GraphPad Software Prism®, La Jolla, California, USA).

    Results

    Cross-sectional analysis revealed a gradual decrease in 25-OH-D levels in pre-CIS samples (figure 1A). Thus, 25-OH-D concentrations after established MS diagnosis were strongly reduced (24.5 (13.7–47.7) nmol/l, median (IQR)) compared with pre-CIS sera (57.1 (37.8–90.3), Mann–Whitney: p=0.0008, Kruskal–Wallis: p<0.05) or healthy controls (81.6 (57.7–98.5), Mann–Whitney: p<0.0001; Kruskal–Wallis: p<0.05).

    Figure 1
    Figure 1

    (A) Cross-sectional 25-hydroxyvitamin D (25-OH-D) levels (nmol/L) were significantly reduced during the two years prior to clinically isolated syndrome (pre-CIS) compared with healthy age- and gender-matched blood donors (healthy controls). Serum 25-OH-D levels in samples obtained after established MS diagnosis (MS) were strongly reduced. (Mann-Whitney Test; *p<0.01; **p<0.005; ***p<0.0001). (B) Increased cross-sectional Epstein-Barr virus nuclear antigen-1 (EBNA1) immunoglobulin G (IgG) response during the pre-CIS phase and after established MS diagnosis (MS) compared with healthy Epstein-Barr virus carriers (healthy controls). (Mann-Whitney Test; *p<0.01). Filled circle in B: one serum was determined as negative for EBNA1. EBNA1 IgG-responses were analysed in 51 and 25-OH-D serum levels were measured in 49 pre-CIS samples of all 25 patients. If more than one sample from a patient was available during a given interval prior to disease onset, the mean value is shown. Results for 25-OH-D and EBNA1 remained in essence unchanged when only cases with available samples after disease manifestation were analyzed or when either the first or last sample of a pre-CIS time interval instead of the mean value was used.

    Furthermore, 25-OH-D concentrations in sera obtained 1–24 months prior to CIS were strongly reduced compared with control samples (p=0.004), but still in tendency lower (47.8 (32.5–77.2)) than in samples collected more than 24 months before CIS (67.4 (31.7–95.4), p=0.38). Median 25-OH-D concentrations in sera obtained more than 24 months before CIS were only 14.2 nmol/l (17.4%) lower compared with healthy controls (p=0.33). Consistent with cross-sectional analyses, intraindividual longitudinal courses from 21 individuals displayed a gradual decline of 25-OH-D concentrations during the pre-CIS phase (figure 2A). Median 25-OH-D concentrations during the 24 months before CIS were 48.1% (1–12 month pre-CIS interval) and 47.1% (13–24 month interval pre-CIS) lower than in healthy controls, respectively (13–24 month pre-CIS vs healthy controls, p=0.006; 1–12 months pre-CIS interval vs healthy controls, p=0.004).

    Figure 2
    Figure 2

    Intraindividual (connecting lines) longitudinal measurements (median: horizontal bars) of 25-hydroxyvitamin D (25-OH-D) concentrations (A) and immunoglobulin G (IgG) responses against Epstein-Barr virus nuclear antigen-1 (EBNA1) (B) prior to clinically isolated syndrome (pre-CIS) and after established MS diagnosis (MS) in comparison with age-and gender-matched blood donors (healthy controls). Intraindividual longitudinal measurements were possible in 21 patients (either pre-CIS course with more than two serum samples (10 patients) or minimum one pre-CIS serum sample and one serum sample after established MS diagnosis (11 patients)). If more than one sample from a patient was available during a given interval prior to disease onset, the mean value is shown. Results for 25-OH-D and EBNA1 remained in essence unchanged when only cases with available samples after disease manifestation were analyzed or when either the first or last sample of a pre-CIS time interval instead of the mean value was used.

    All but one patient and all healthy controls were seropositive for EBNA1 IgG. In cross-sectional analyses, a higher EBNA1 IgG response was observed in pre-CIS (162.5 (79.1–460.0) IU/ml, median (IQR)) and MS (154.8 (79.5–216.6)) serum samples compared with healthy controls (63.7 (29.5–121.6); Mann–Whitney: p<0.005, Kruskal–Wallis: p<0.05). EBNA1 IgG was especially increased during the 3-year interval before CIS (185.9 (91.2–460.0) IU/ml) and after established MS diagnosis (154.8 (79.5–216.6)) compared with healthy EBV carriers (p<0.01, figure 1B). Immunoreactivity against EBNA1 in sera collected more than 36 months prior to CIS (118.8 (26.1–514.6) IU/ml) was higher than in healthy controls; however, with fewer samples (18 samples from eight patients) available for investigation this fell short of statistical significance (p=0.3). Still, longitudinal analyses in 21 individuals also displayed increased median EBNA1 IgG responses during the pre-CIS phase as compared with healthy controls, and again especially 13–36 months before CIS (13–24 month pre-CIS interval vs healthy controls, p=0.009; 25–36 month pre-CIS interval vs healthy control, p=0.02; 37–48 month pre-CIS interval, p=0.06; figure 2B).

    We did not observe interactions between vitamin D status and EBNA1 immunoreactivity either during the pre-CIS phase (Spearman r=0.15; p=0.34) or after established diagnosis (Spearman r=−0.46, p=0.054). Furthermore, we did not find age-dependent differences of 25-OH-D serum levels and EBNA1 IgG response in samples from MS patients or from healthy blood donors. There was no correlation between symptomatology, severity (Expanded Disability Status Scale) or clinical disease course (22/25 CIS/relapsing-remitting MS (RRMS), 2/25 secondary progressive MS (SPMS), 1/25 primary progressive MS (PPMS)) and 25-OH-D serum levels or EBNA1 IgG response (data not shown).

    Discussion

    The present study investigated two potential risk factors for the development of MS in individuals who had donated blood before the first clinical MS manifestation. Cross-sectional analyses revealed a gradual decrease of 25-OH-D levels during the pre-CIS phase towards disease manifestation and established MS diagnosis that was also observed in intraindividual longitudinal analyses. This change was particularly evident during the 24-month interval before CIS. In contrast, IgG response against EBNA1 was increased during the pre-CIS phase compared with healthy matched blood donors, most pronounced during the 36-month interval prior to CIS. 25-OH-D levels and EBNA1 immunoreactivity were not correlated and not associated with type or severity of MS manifestation.

    Whereas our approach offered the opportunity to analyse very rare serum samples longitudinally up to 7 years prior to CIS, methodological drawbacks in addition to limited sample size include recall bias (for example, temporal imprecision of CIS). Whereas we demonstrate changes of 25-OH-D levels in the preclinical phase, we are not able to dissect the determinants of individual vitamin D levels (eg, sunlight exposure, diet).

    Our data are in line with a prospective case-control study among US military personnel, showing that 25-OH-D levels are inversely correlated with the risk of MS later in life.5 In the US study, the inverse correlation was especially pronounced when 25-OH-D levels were analysed before the age of 20, and average 25-OH-D concentrations were stable in the years before MS symptom onset. This is in contrast to our findings where we observed a gradual decline in 25-OH-D levels towards disease manifestation. Although the comparability of absolute vitamin D levels across different studies is limited, 25-OH-D concentrations in the control groups and pre-CIS sera >24 months prior to disease manifestation were similar. This argues against generally lower 25-OH-D-levels in our German cohort as a reason for the particularly low concentrations observed 1–24 months before clinical manifestation and after established MS. However, other confounders in our more heterogeneous cohort in comparison with military personnel with distinct male predominance cannot be ruled out. In addition, reverse causality, that is, a longstanding disease process causing the changes in 25-OH-D and EBNA1 status observed cannot be excluded.

    The results of previous studies suggested that MS risk is related to vitamin D status at different ages, possibly starting in utero and extending through early childhood, adolescence and adult life.12 Our independent data may argue for potential additional mechanisms associated with a 25-OH-D decrease immediately prior to disease manifestation.

    In line with previous prospective MS cohorts we found changes in EBNA1 immunoreactivity with elevated pre-CIS titres during the whole pre-CIS course.6 ,7 Most pronounced alterations were observed approximately 2–3 years prior to clinical disease manifestation. This is similar to other prospective cohorts, which have described changes in seroconversion during an interval of 5.6 years prior to clinical MS-evolution13 and pronounced immunoreactivity against EBNA1 in samples collected <5 years before MS onset.9

    Although we did not observe a direct relationship between 25-OH-D concentrations and EBNA1 immunoreactivity, future prospective studies are needed to investigate potential interactions since vitamin D is a potent modulator of the adaptive immune system.14

    In conclusion, our data from a geographically independent, retrospective cohort add to the growing notion that low 25-OH-D concentrations and indicators of remote EBV-infection may be associated with the risk of developing MS. With the caveat of small sample size, our data suggest the existence of a susceptible pre-CIS period 2–3 years prior to the first clinical disease manifestation. Larger case-control studies are needed to confirm our hypothesis of a well-defined interval of preclinical susceptibility for MS.

    Acknowledgments

    We thank K Hofmann for technical assistance.

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    Footnotes

    • Linked article 303245.

    • BFD and NvA contributed equally to this work.

    • Funding This work was supported by grants from the German Ministry for Education and Research (BMBF, ‘German Competence Network Multiple Sclerosis’ (KKNMS), grant number CONTROL MS, 01GI0914).

    • Competing interests BF Décard has received personal compensation for activities with Teva. N von Ahsen, T Grunwald, F Streit, P Niggemeier, V Schottstedt and J Riggert have no conflicts of interest. A Stroet has received personal compensation for activities with Novartis, Almirall Hermal GmbH and Sanofi. R Gold received personal compensation for activities with Bayer Healthcare, Biogen Idec and Teva Neuroscience and in an editorial capacity from Therapeutic Advances in Neurological Disorders. R Gold has received patent payments from Biogen Idec and research support from the German Ministry for Education and Research (BMBF, ‘German Competence Network Multiple Sclerosis’ (KKNMS), CONTROL MS, 01GI0914), Bayer Healthcare, Biogen Idec, Merck Serono, Novartis and Teva Neuroscience. A Chan received personal compensation as a speaker or consultant for Bayer Schering, Biogen Idec, Merck Serono, Novartis, Sanofi-Aventis and Teva Neuroscience. A Chan received research support from the German Ministry for Education and Research (BMBF, ‘German Competence Network Multiple Sclerosis’ (KKNMS), CONTROL MS, 01GI0914), Bayer Schering, Biogen Idec, Merck Serono and Novartis.

    • Patient consent Obtained.

    • Ethics approval The study was approved by the local ethics committee.

    • Provenance and peer review Not commissioned; externally peer reviewed.

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