Environmental Factors Associated with Disease Progression after the First Demyelinating Event: Results from the Multi-Center SET Study

Objectives To investigate the associations of environmental MS risk factors with clinical and MRI measures of progression in high-risk clinically isolated syndromes (CIS) after the first demyelinating event. Methods We analyzed 211 CIS patients (age: 28.9±7.8 years) enrolled in the SET study, a multi-center study of high-risk CIS patients. Pre-treatment samples were analyzed for IgG antibodies against cytomegalovirus (anti-CMV), Epstein Barr virus (EBV) early nuclear antigen-1 (EBNA-1), viral capsid antigen (VCA), early antigen-diffuse (EA-D), 25 hydroxy-vitamin D3 and cotinine levels and HLA DRB1*1501 status. The inclusion criteria required evaluation within 4 months of the initial demyelinating event, 2 or more brain MRI lesions and the presence of two or more oligoclonal bands in cerebrospinal fluid. All patients were treated with interferon-beta. Clinical and MRI assessments were obtained at baseline, 6, 12, and 24 months. Results The time to first relapse decreased and the number of relapses increased with anti-CMV IgG positivity. Smoking was associated with increased number and volume of contrast-enhancing lesions (CEL) during the 2-year period. The cumulative number of CEL and T2 lesions during the 2-year period was greater for individuals in the highest quartile of anti-EBV VCA IgG antibodies. The percent loss of brain volume was increased for those in the highest quartile of with anti-EBV VCA IgG antibodies. Conclusions Relapses in CIS patients were associated with CMV positivity whereas anti-EBV VCA positivity was associated with progression on MRI measures, including accumulation of CEL and T2 lesions and development of brain atrophy.

the corresponding current image. The result was then smoothed with a Gaussian kernel of 0.5 mm. Cross-sectional regions of interest (ROIs) were overlaid on the subtraction image to facilitate the identification of new and enlarging T2-lesions.
Global and Tissue-Specific Atrophy Measures: For baseline analyses, the SIENAX cross-sectional software tool was used (version 2.6), with corrections for T1hypointensity misclassification using an in-house developed in-painting program.
Normalized whole brain volume (WBV), normalized gray matter volume (NGMV) and normalized white matter volume (NWMV) were measured as previously described 3 .
For longitudinal changes of the WBV, we applied the SIENA method 4 to calculate the percentage brain volume change (PBVC). To quantify longitudinal GM and WM volume changes, we used a modified hybrid of FMRIB's SIENA and SIENAX tools. We used a brain-and skull-constrained co-registration technique to place both baseline and followup images into a joint space halfway between the two. Next, we combined baseline and follow-up intracranial volume masks via union, and valid voxel masks via intersection, ensuring that the same imaging volume was analyzed at both time points. Finally, we segmented the resulting images with a modified longitudinal version (L-FAST) of FMRIB's FAST tissue segmentation tool 5 that uses a 4-dimensional joint hidden random Markov field to prevent misclassification between time points when longitudinal intensity changes are lacking (or minimal). Total tissue volume was calculated for both baseline and follow-up for each tissue compartment from partial volume maps. The reproducibility of this analysis is similar to the SIENA method 4 .

Data Analysis
SPSS (IBM Inc., Armonk, NY, version 19.0) statistical program was used for all statistical analyses. In view of the multiple testing, a conservative p-value of ≤ 0.01 was used to assess significance; p-values ≤ 0.05 were considered to be trends.

Data Transformations:
The homozygous and heterozygous rs3135005 genotypes associated with HLA DRB1*1501 positive allele were categorized as HLA DRB1*1501 positive and the remaining homozygous rs3135005 genotype was categorized as HLA DRB1*1501 negative. Non-smokers were defined as those with cotinine ≤ 10 ng/ml and active smokers those with > 10 ng/ml. Subjects were considered anti-CMV positive if the relative concentration was greater than unity and anti-CMV negative if the relative concentration was unity or less.
The occurrence of anti-EBV EBNA-1 (100%) and VCA (99.5%) positivity was nearly ubiquitous in our study sample (Table 1). Because there are no established MS-relevant clinical standards for anti-EBNA-1 and anti-VCA levels, the anti-EBNA-1 and anti-VCA relative concentrations were categorized into quartiles, which are easier to interpret, using the observed quartile thresholds. Indicator variables for subjects in the highest quartiles of anti-EBNA-1 and anti-VCA levels were then obtained.
The raw 25(OH)VD 3 levels were logarithm transformed and deseasonalized using sinusoidal regression 6 . The form for the regression equation for 25(OH)VD 3 was: The T represents the month of blood draw (January = 1 to December = 12 scale with

Effects of Risk Factor Combinations
The initial risk factor combination was constituted with the anti-CMV positivity-anti-EBV VCA highest quartile pair because anti-CMV positivity was strongly associated with clinical measures, e.g., as number of relapses and time to relapse, whereas anti-EBV VCA in the highest quartile was associated with lesional and brain atrophy measures.  Figure S2 summarizes a subset of these results.
We also conducted additional regression analyses for PBVC that included both main Based on these analyses, we surmise that the anti-CMV positivity-anti-EBV VCA highest quartile combination is a parsimonious explanatory predictor because it exhibits stronger associations with more clinical and MRI variables.