Risk Factors in Early Multiple Sclerosis
Conditions: Multiple Sclerosis, Magnetic Resonance Imaging, Biomarkers, Clinically Isolated Syndrome, Radiologically Isolated Syndrome
Sponsor: University of Colorado, Denver

RISEMS 
 
 
 
 
COMIRB # 17-1884  
 Version 21Apr2021 
Risk factors in Early Multiple Sclerosis 
Enrique Alvarez, MD, PhD 
Neurology 
John Corboy, MD 
 
Neurology 
Jill Norris, PhD 
 
Epidemiology 
 
I. 
Hypotheses   
Multiple sclerosis (MS) is a progressive disabling disease of the central nervous system (CNS) which is 
associated with substantial personal, financial and societal costs. There now are 15 approved disease 
modifying therapies (DMTs) available, and data supports that early intervention is associated with best 
outcomes. The ultimate cause of MS remains unknown, but increased risk is associated with a host of 
genetic and environmental factors. First degree relatives (FDRs) of MS patients have a 10-20 fold 
increased risk of being diagnosed with MS themselves. But diagnosis, still, is delayed for many, in part due 
to lack of a specific biomarker of disease activity, and also due to vagueness of early symptoms in many.  
More recent studies, have clearly outlined a prodrome in individuals ultimately diagnosed with MS, prior to 
overt clinical symptoms typical of MS. Thus, it would be extremely helpful if we could develop a risk 
stratification algorithm that outlined those with both high and low risk of developing MS as early as possible 
in the disease process. The central hypothesis of this protocol is that it is possible, using FDRs of patients 
with MS and assessing a variety of both known and unknown risk factors for MS, to define a risk algorithm 
for earliest signs of development of MS. In addition, while starting with a relatively limited cross-sectional 
analysis, we hypothesize that development of a larger, longitudinal study will allow us to improve sensitivity 
by simple accrual of larger numbers of participants over time with the endpoint of early signs of MS on MRI 
scans, but also improve sensitivity by adding additional markers of MS risk as they become available.  
II. Specific Aims, among asymptomatic first degree relatives (FDRs), aged 18-30, of multiple sclerosis 
(MS) patients: 
1:  Determine the prevalence of brain MRI lesions disseminated in space (DIS), consistent with MS  
2:  Gather data on potential risk factors or early signs related to MS development, including markers for: 
genes, immunological function, environmental factors, neuroaxonal damage, Vitamin D levels, lipid 
metabolism, activity levels, mood abnormalities, and cognitive function. From this, develop a risk factor 
score, incorporating all relevant potential markers of increased risk of DIS.    
3:  To use this pilot, cross-sectional study as a base for development of a long-term, longitudinal, multi-
center study to determine genetic and environmental risks for pre-symptomatic MS  
4: To create and maintain a biobank of specimens for future analysis as other potential biomarkers become 
available.  
 
III. 
Background and Significance   
MS is a progressive, inflammatory and degenerative condition of the CNS with mean clinical onset at age 
301. MS is the most common CNS inflammatory disorder, with a prevalence of about 1/500 women and 
1/1000 men in Colorado. It is a major cause of disability in young adults, and financial and psychosocial 
costs to the patient and society are great2. Diagnosis remains challenging, as early symptoms may be 
vague and transient, and there is no highly sensitive and specific biomarker or diagnostic test3. But there 
are now 15 disease modifying therapies (DMTs) available, all of which are most effective in those who are 
younger and relatively newly diagnosed4. Delays in treatment are associated with greater accumulation of 
clinical symptoms and magnetic resonance imaging (MRI) scan abnormalities5-11. Thus, there is a 
substantial need to develop early detection techniques for MS, so as to treat as early as possible. 
But what is early? While clinical symptoms are the first overt indication of MS, evidence is rapidly 
expanding that there is a prodrome12, or pre-clinical phase, prior to first symptoms and then diagnosis of 
MS. A clinical prodrome may be defined as the time between the onset of decline in a baseline level of 
functioning until formal diagnostic criteria are met. In the Nurses Health Studies I and II, the 341 women 
diagnosed with MS after entry into the study had lower levels of physical activity four to six years prior to 
MS diagnosis compared to age-matched control women13. This prodromal period may be associated with 

increased health care use and costs, as seen in a recent Canadian study of the five years preceding the 
diagnosis of MS14. In an Argentinian case-control study15, diminished cognitive performance in school was 
reported in individuals who developed a first attack consistent with MS, so-called clinically isolated 
syndrome (CIS), up to 10 years after leaving school. MRI scans also detect many asymptomatic lesions in 
MS, especially early in the course16,17. Similarly, individuals destined to develop MS have early signs of 
anxiety and depression several years prior to diagnosis of MS18. The majority of patients with CIS have 
already developed MRI evidence consistent with demyelination at some prior, indeterminate time point, i.e. 
the first scans typically show multiple areas of abnormality that have not been associated with any clinical 
symptoms. In CIS clinical trials, the average number of asymptomatic lesions on first brain MRI scan after a 
first clinical attack has exceeded 175-11. In addition, a small percentage of people undergo MRI scans for 
reasons seemingly unrelated to MS, yet the scans reveal abnormalities highly consistent with MS.  This is 
referred to as radiologically isolated syndrome (RIS)19-21, and there is now a clinical trial underway to 
determine if very early treatment is beneficial in these patients (NCT02739542). Both CIS and RIS are 
predictive of new clinical disease activity consistent with MS. In a study of RIS, over a quarter of 
participants had cognitive impairment similar to that seen in MS21. RIS, CIS and early onset RRMS are all 
associated with diminished brain volumes compared to age matched controls22-24, suggesting 
neurodegeneration occurs very early, even in the prodromal phase.  
Damage to the neuronal structures of the CNS can also be seen early in MS. Presumed damage to 
neural tissue has been associated with elevated levels of the neuronal cytoskeleton, Neurofilament light 
(NfL), in CIS and early MS patients, and are independently associated with relapses, disability 
progression, and MRI markers of brain lesion volume and atrophy25-29. Similarly, evaluation of lipids may 
be important in early MS. MS is a demyelinating disorder of the CNS, and about 25% of total body 
cholesterol is located in the brain, most incorporated in the myelin sheaths wrapping around axons30.  
Comorbidities linked to dyslipidemia are associated with increased risk for development of MS30 and 
disability progression31-33. After CIS, elevated total cholesterol (TC) level has been associated with Gad-
enhancing lesions34, and TC and LDL levels have been associated with development of new and newly 
enlarging T2 lesions on brain MRI35. Four years after CIS, and with use of interferon, there are rapid 
decreases in LDL, HDL, and TC, which then are followed by a slow return to baseline levels. Greater 
percent decrease in HDL at 3 months is associated with less brain atrophy over four years36. Thus, 
abnormal lipid profiles appear to be associated with damage to the CNS, even early in the course of MS. 
Neither NfL nor lipid profiles have been evaluated as early markers CNS damage in pre-symptomatic 
individuals at high risk of developing MS. 
The preponderance of evidence is consistent with the concept that MS results from the imposition of one or 
more environmental insults in individuals with a genetic background that predisposes them to MS1.  
Epidemiological studies have identified both genetic37-41 (over 200 genes have been identified) and 
environmental risk factors (exposure to Epstein-Barr virus42, obesity43-48, vitamin D deficiency49-54, and 
smoking55-60) associated with the development of MS, and/or more progressive disease over time, but the 
triggers at onset remain unknown. Finally, asymptomatic individuals with parents or siblings with MS (first 
degree relatives - FDRs), have a 10-20 fold increased lifetime risk of developing MS compared to those 
without a relative with MS61. This translates to about 4-5% of girls/women and 2-3% of boys/men who might 
be ultimately diagnosed with MS in their lifetimes. Diagnosis in multiple family members enhances this risk 
further. In addition, a variety of cross-sectional MRI studies62,63 have identified that about 10% of 
asymptomatic FDRs of MS patients have brain MRI lesions that could be demyelinating in nature, including 
fulfilling criteria for lesions disseminated in space (DIS)63-66. Thus, given that the median age of onset of 
relapsing MS is about 30 in multiple studies, we might expect that by age 30, 2-5% of FDR might have 
developed MS, but up to 5-10% may have developed MRI signs that place them at risk of developing MS, 
i.e. RIS.  
In the recent Genes and Environment in Multiple Sclerosis (GEMS) study67 of 65 female FDRs of MS 
patients, those with a combined genetic (based on 64 known single nucleotide polymorphisms, SNPs, in 
2012) and environmental (female sex, prior history of mononucleosis and smoking) high risk score were 
compared to FDRs with a lower score for developing MS. Those with a high risk score showed a trend 
toward higher BMI and weight, but there were no differences in age, height, cigarette smoking exposure, 

25-hydroxyvitamin D level, or history of infectious mononucleosis or migraine. Five (four in high risk, one in 
low risk groups) of the 65 women had MRI evidence of lesions disseminated in space as seen in MS.  
Those in the high risk group had relatively impaired quantitative vibratory sensation testing, but there were 
no differences in quantitative brain volume measurements or retinal nerve fiber layer (RNFL) thickness on 
optical coherence tomography. The overall conclusion of this cross-sectional study was that high risk 
individuals showed early subclinical risk signs of MS, noting the study was underpowered to detect a 
difference in neuroimaging outcomes between higher-risk and lower-risk individuals.  
Type I diabetes (T1D) shares many clinical and immunological features with MS68, including that FDRs of 
T1D are at high risk of developing T1D. In our institution, the long-term Diabetes Autoimmunity Study in the 
Young (DAISY) has sought to understand specific risk factors associated with this T1D development69-72. 
John Cambier and associates have identified loss of B cell anergy in some FDRs’ of T1D patients73. Also 
here at the University of Colorado, Anschutz Campus, our colleague, Dave Wagner, and associates have 
identified a unique, pathogenic T cell population in T1D, an expansion of CD40 on CD4+ cells74.  
Subsequently, we have shown a similar expansion of CD40 expression in MS patients of all ages 
compared to controls75. Studies looking at CD40 expression in FDRs of T1D patients are underway. Thus, 
it is possible both of these immunological biomarkers, B cell anergy and T cell overexpression of CD40, 
may be present in FDRs of MS patients. 
Innovation – The GEMS study described above is one approach to identification of early MS, but it had 
several limitations. It used a pre-defined risk score, including a SNP analysis of just 64 SNPs, when more 
recent analysis suggests over 200 may be relevant. It employed a long, detailed, and expensive brain MRI, 
but did not include spine or FLAIR* analysis. The cost limited participation. GEMS did not include potential 
immunological markers, markers of CNS damage (such as NFL or lipids), activity levels, or a detailed 
environmental screen. Due to privacy issues, they were unable to verify the MS diagnosis in FDRs. Finally, 
it was limited to 100 patients, of which only 65 (females only) were analyzed. Other studies (as yet 
unpublished) designed to look at risk factor algorithms for development of MS are employing larger 
numbers of patients, but minimal, if any, MRI analyses, no or minimal immunological or CNS damage 
markers, and no activity levels. Most importantly, they are using full clinical diagnosis of MS as the 
outcome measure, greatly limiting the number of “positive” patients, and biasing toward a much later time 
in the disease process. Thus, our study has great innovative value for several reasons: 
a. We will define pre-symptomatic MS in 300 FDRs using stringent MRI criteria, including FLAIR* 
sequences109-113, thus significantly increasing sensitivity and detection in the prodromal phase. 
Proband MS patients will be defined from our own population of MS patients known to fulfill formal 
diagnostic criteria66 for MS.  
b. We will analyze genetic, immunological, vitamin, lipid, viral, and CNS damage markers; activity 
levels76; and a mood and cognitive screen, with the goal of defining an expanded risk stratification 
scheme, not a pre-defined one  
c. We will include a detailed environmental screen77-84, employing approaches very similar to the 
DAISY study, and Dr. Norris, lead epidemiologist in DAISY, will participate as a Co-PI. 
d. We will compare those FDRs with and without brain MRI abnormalities to each other, and to non-
FDR controls (blood and environmental screen, not MRI) 
e. We will create a biobank of blood, both plasma and PBMCs, including pre-processed serum/plasma 
and RNA, allowing us to add to the risk stratification model over time.  
 
IV. 
Preliminary Studies  
The background for the entire study is as listed above under Significance. We have extensive experience 
in blood and tissue banking, as Dr. Corboy has been director of the Rocky Mountain MS Center Tissue 
Bank, which has collected over 500 autopsied brains and spinal cords for distribution to researchers in the 
US and abroad. Dr. Alvarez is head of our MS Center’s Biobank of CSF and blood specimens. In addition, 
references 73-75 outline results with T1D and MS patients with potential immunological abnormalities 
relevant to FDRs of MS patients. Similarly the DAISY study references speak to the capacity to do detailed 
environmental screening in a very similar population of young patients and the expertise of Dr. Norris. Drs. 

Cambier and Wagner have already developed the lab tests described. NfL (Quanterix) and lipid studies are 
commercially available and we have an agreement with Quanterix to perform the studies as in references 
25-29. We also have performed over 50 clinical trials in MS, and have significant experience with large 
recruitments, including over 450 for our Biobank and several hundred for a study looking at development of 
a JC Virus serology test (the STRATIFY-1, and STRATIFY-2 studies85,86), for which we were one of the 
largest recruiters internationally. 
V. 
Research Methods 
Approach - The concept in this pilot study is to perform a cross-sectional analysis of the FDRs of MS 
patients. FDRs were explicitly chosen as they have enhanced risk of MS, thus enriching for abnormal, pre-
symptomatic MRI scans. Broadly, we will perform an abbreviated, “screening”, brain MRI in 300 
neurologically asymptomatic FDRs; draw blood for genetic, immunological, vitamin, lipid, viral, and CNS 
damage markers; and do an environmental screen, with mood and cognitive screening tests. Activity levels 
will be monitored with step analysis with the Godin Leisure-Time Exercise Questionnaire (GLTEQ)87, which 
has been validated in both adults88 and children89 with MS. FDRs with abnormal brain/spine MRIs fulfilling 
DIS64-66 will then be compared to age-and sex-matched FDRs not having MRIs fulfilling DIS, and to age-
and sex-matched non-FDRs who do not carry a diagnosis of MS or have symptoms suggestive of MS (no 
MRI for the latter group, but all other measures taken). The long-term goal is to create a platform for a 
longitudinal study, likely at multiple sites, with the capacity to interrogate stored samples of DNA, RNA, 
plasma and PBMCs with associated environmental screening and the screening MRI scans as described 
above. As part of this, we also have had preliminary discussions regarding partnering with Savvysherpa for 
development of a long-term activity level monitoring system specifically for MS.  
A.  Outcome Measure(s):  Individuals will be defined as having pre-symptomatic MS if they have 
brain/spine MRI abnormalities fulfilling DIS as defined by the Swanton64-66 criteria. Importantly, we will also 
obtain sequences which allow for determination of whole brain volume measures. The following MRI 
sequences will be obtained: 
1. 
Whole brain T1-weighted images acquired using 3-Dimensional fast spoiled-gradient recalled 
acquisitions (T1-3D-FSPGR) with 1 mm slice thickness and isotropic voxel dimensions. 
2. 
Whole brain 3-Dimensional Double Inversion Recovery (DIR) acquisition with 1.5 mm slice 
thickness. 
3. 
Whole brain T2-weighted  Fluid Attenuated Inversion Recovery (FLAIR ) images with 1 mm slice thickness 
and isotropic voxel dimensions will be combined with 
4. 
Whole brain T2*-weighted segmented echo-planar imaging (segEPI) images with <=1 mm slice thickness and 
isotropic voxel dimensions to obtain FLAIR* images. 
The sequences were specifically chosen to: a) screen brain with the greatest specificity (FLAIR*); b) allow 
for later volumetric analysis of the brain; and c) use the most sensitive technique for cortical and subcortical 
brain lesions (3D-DIR). 
 
B. Description of Population to be Enrolled:  The population will be a total of 300 women and men, of 
all races and ethnicities, between the ages of 18-30, inclusive, who have no symptoms of MS, but have 
at least one FDR with documented MS fulfilling McDonald 2017 criteria. Up to 1000 will be screened for 
inclusion, but some will be excluded due to inability to undergo a brain/spine MRI or have blood drawn, 
lack of interest after discussion of study requirements, or symptoms concerning for MS (the latter will be 
referred to a qualified neurologist for evaluation). FDRs with one or more siblings/parents with MS will 
be included, and more than one FDR of a proband MS patient will be allowed. A control group (based 
on # of FDRs with positive MRIs - we anticipate 15-30) of age- and sex-matched controls who have no 
known family members with MS (out to first cousins and grandparents) will have blood drawn and fill 
out the environmental questionnaire, but will not undergo a brain MRI. The controls will be matched 
1:1:1 for those FDRs with positive MRI scans suggesting pre-symptomatic MS and those age-and sex-
matched FDRs who do NOT have positive MRI scans suggesting pre-symptomatic MS. The ages were 
specifically chosen so as to have a good opportunity to collect an adequate number of MRI-positive, 
asymptomatic participants prior to the known median age of MS clinical onset (30 years), but not so 
young as to lower the likelihood of detecting asymptomatic brain MRI lesions.   

1. FDR Inclusion Criteria 
a. Male and female 
b. All races and ethnicities 
c. Ages 18-30 
d. Must have a parent, sibling or child with MS fulfilling McDonald 2017 criteria 
e. No symptoms suggestive of MS on formal screen 
f. Ability to undergo a non-contrast MRI and venipuncture, and perform an 
environmental screen, mood screen and cognitive test 
2. FDR Exclusion Criteria 
a. Symptoms suggestive of MS on formal screen 
b. Prior diagnosis of autoimmune disease that may be associated with CNS 
dysfunction and MRI lesions, e.g. Sjogren’s  
3. Non-FDR Inclusion Criteria 
a. Male and female 
b. All races and ethnicities 
c. Ages 18-30 
d. Must NOT have a FDR (parent, sibling, child)  or second degree relative 
(grandparent, aunt or uncle) with MS fulfilling McDonald 2017 criteria 
e. No symptoms suggestive of MS on formal screen 
f. Ability to undergo venipuncture and perform an environmental screen.  
4. Non-FDR Exclusion Criteria 
a. Symptoms suggestive of MS on formal screen 
b. Prior diagnosis of autoimmune disease that may be associated with CNS 
dysfunction and MRI lesions, e.g. Sjogren’s  
 
FDR participants will be enrolled from among the FDRs of MS patients seen at the Rocky Mountain 
Multiple Sclerosis Center at the University of Colorado in Aurora, Colorado (RRMSC at CU). Our center 
has approximately 3500 unique patients, of whom nearly all have asymptomatic FDRs, many in the 
appropriate age range. Documented MS proband patients will be made aware of the study via 
interactions in the office, our newsletter, and IRB-approved information given to them for their FDRs.  
FDRs who are then interested will initiate contact with the research group. If this does not provide 
adequate enrollment, we will recruit by local advertisement through known offices of neurologists in the 
Denver and surrounding regions. Age- and sex-matched non-FDR controls will be recruited from the 
friends of FDRs who are positive on the screening MRI, or will be recruited from local recruitment in the 
department of Neurology offices, from those non-MS conditions. FDRs and controls will be screened on 
the phone for basic demographic criteria (e.g. age, location where they live) and a checklist of basic MS 
symptoms, and to be certain they do not have previously undiagnosed MS or other 
autoimmune/neoplastic disorders. Those with concerns for MS at screening will be referred to a 
qualified neurologist for assessment. 
 
      C. Study Design and Research Methods:  
1. Prescreen FDRs by phone for ability to participate and lack of MS symptoms, using a standardized 
script to exclude those with overt MS symptoms. Proband MS patients will have documented MS by 
McDonald 2017 criteria66.   
2. Qualified FDRs will be scheduled for a study visit after completing a pre-screening questionnaire by 
phone. If they are not excluded by pre-screen, and wish to participate, a study visit will be 
scheduled. A waiver of documentation of consent will be obtained over the phone so as to instruct 
participants to fast from midnight prior to the first study visit. This will also allow them to fill out the 
environmental questionnaire on-line before their visit and at their convenience. The environmental 
survey must be completed at least three days prior to their visit. If not, the study visit will be 
canceled and rescheduled once its complete. 
3. A study visit will include (See Table of activities below):  
a. Read, ask questions regarding, and sign the consent. 
b. Document completion of environmental questionnaire.   
c. Obtain a measured height and weight, and compute a Body Mass Index (BMI) 
d. Perform a non-contrast brain MRI (per section V.A)  

e. Perform a fasting Blood draw consisting of no more than 100 ccs of blood. 
i.   Blood drawn at the primary study visit will be a maximum of 100 ccs to be 
processed in the following fashion. Participants will not be made aware of their blood 
sample results 
1. 30 ccs for immediate cell sort by David Wagner, PhD into T and B 
lymphocytes, for CD40 expression studies, requiring primary analysis with 
fresh cells, and done on all samples. Also for HLA analysis. 
2. 20 ccs processed for delayed analysis of SNPs, viral serologies (Herpes 
Simplex Virus 1 and 2, Cytomegalovirus, Varicella-Zoster Virus, and Epstein-
Barr Virus), vitamin D levels, and neuroaxonal (NfL - Quanterix) and myelin 
damage (lipids), which all can be performed on stored samples.  
3. 50 ccs processed and stored for delayed analysis of plasma and PBMCs, 
RNA and DNA, for studies not yet determined 
 
f. Perform the Perceived Stress Scale and the Neuro-QoL screen for anxiety and depression;  
i. Participants screening for concerns for suicidality on the mood screen will be referred 
to an emergency room for evaluation.  
g. Perform a Symbol Digit Modality Test, a screen for cognitive function. The latter will be done 
in the written format.  
i. Participants will not be advised of Cognitive screen results. 
h. As a measure of physical activity, we will use Godin Leisure-Time Exercise Questionnaire 
(GLTEQ)87, which measures mild, moderate and strenuous physical activity during a typical 
seven day period. This has been validated in MS88,89, and is scored either into three levels of 
activity (sedentary, moderately active, or active), or may be used as a continuous measure.   
i. 
As a dietary assessment, we will use the Harvard 2007 Booklet FFQ114, which is a semi-
qualitative food frequency questionnaire used to assess intake of various nutrients over the 
past year. This has been robustly validated in a variety of populations115-118, and nutrient 
intakes are computed by assigning a daily frequency weight. 
4. MRIs will be read by a board-certified neuroradiologist (Dr. David Rubinstein) with extensive 
experience in reading brain MRIs in general, and MS MRIs in particular. All FDRs will receive a 
follow-up contact concerning the results of their MRI within one week. FDRs with a brain MRI 
fulfilling criteria for DIS will be referred to a qualified neurologist to determine if they should be 
treated or monitored more closely. Individuals with abnormal brain MRI scans not suggestive of or 
fulfilling DIS criteria, but having non-specific brain lesions (as can be seen with migraine and many 
other conditions), also will be referred to a qualified neurologist for evaluation as needed. Those 
FDRs with unexpected abnormalities (e.g. severe sinus disease, pineal cyst, pituitary abnormality, 
cavernous angioma, etc.) will be referred as necessary to a qualified physician.     
5. FDRs with a positive brain MRI showing lesions DIS will be asked to return within four months for a 
repeat blood draw of no greater than 100 cc of blood, to be compared to age-and sex-matched 
FDRs without a positive brain MRI (who will also be asked to return within 4 months for the repeat 
blood draw) and age-and sex-matched non-FDRs, both in a 1:1 ratio. The former will be identified 
from those already in the study, and the latter will be recruited from age- and sex-matched friends 
of the FDRs with positive brain MRIs. The non-FDRs will be prescreened and perform all the study 
procedures as the FDRs on the first study visit, with the exception of the MRI.  
a. This blood draw will be for B cell anergy and cytokine studies in Dr. Cambier’s lab, and 
repeat CD40 studies on non-FDR controls (i.e. not already done by Dr. Wagner’s lab) which 
require fresh cells, but done only on this subset of participants. The rest of the blood will be 
processed and stored as described in C3ei.3 above for potential future use.   
6. Data will be stored in a HIPAA-compliant database. Analysis will be done via case-cohort approach 
with the roughly 15-30 MRI-positive participants, controls as noted, and looking at the markers 
individually and together.   
 
 
 
 
 

 
 
 
 
 
STUDY PROCEDURES 
 
Visit 1 
Visit 2 (within 4 months after V1) 
FDRs 
 
 
 
 
MRI positive 
Vitals 
MRI 
Blood draw 
Environmental Questionnaire 
NeuroQOL 
SDMT 
Activity Questionnaire 
Diet questionnaire 
Blood draw 
 
MRI negative 
Controls  
Vitals 
MRI 
Blood draw 
Environmental Questionnaire 
NeuroQOL 
SDMT 
Activity Questionnaire 
Diet questionnaire 
Blood draw 
 
MRI negative  
Non-Controls 
Vitals 
MRI 
Blood draw 
Environmental Questionnaire 
NeuroQOL 
SDMT 
Activity Questionnaire 
Diet questionnaire 
 
NonFDR 
Controls 
 
N/A 
Vitals 
Blood draw 
Environmental Questionnaire 
NeuroQOL 
SDMT 
Activity Questionnaire 
Diet questionnaire 
 
 
D. Description, Risks and Justification of Procedures and Data Collection Tools:   
1. Blood Draws: The risks of phlebotomy are small and include temporary discomfort from the needle 
stick, bruising and rarely infection. The amount of blood drawn at each time point for this study is 
well under the established recommended maximum for adults. 
2. MRI: The most common side effect of having an MRI is flashing lights in the eyes. This is caused by 
the magnetic waves and is not harmful.  Some people also experience warmth and reddening of the 
skin. This usually goes away in a few minutes. This is a NON-contrast scan. In addition, there is an 
emotional risk of identifying abnormal MRI lesions in individuals with no symptoms concerning for 
MS, and an as-yet unknown risk of developing symptom concerning for, or disability related to, MS.  
This potential risk should be offset by the potential benefit of earliest possible intervention for a 
potentially disabling disorder.  
3. Confidentiality:  There is a potential for loss of confidentiality. A unique study number will be 
assigned to each subject at inclusion, immediately after informed consent has been obtained. This 
number will serve as the subject’s identifier. Link between study number and subject’s identity will 
be kept separately from study data on a secure server accessible only to study personnel. Data will 
be stored in a HIPAA-compliant database.  
 

E. Potential Scientific Problems:   
The largest potential hurdle would be inadequate numbers of FDRs with positive brain MRIs. That said, 
we view this cross-sectional study as a proof of concept that patients can be recruited, and that we can 
develop a biobank of specimens to be interrogated in the future. The long-term goal is creation of a 
longitudinal, multi-center study similar to the DAISY study of children at increased risk for T1D. Thus, 
we have no illusion that we will settle this issue with a relatively small cross-sectional study, but plan to 
use this study to lay the groundwork for a larger, longitudinal study, especially recruiting at other 
national/international sites.  
F.   Data Analysis Plan:  Statistical Approach 
a. Based on previous studies62,63, we estimate that 5-10% of the 300 FDRs (15-30) in our pilot study 
will exhibit an MRI endpoint. In order to test for associations with this endpoint to obtain valuable 
preliminary data, we will perform a nested case control study. We will match two controls per case 
based on age and sex: one group will be FDRs not fulfilling DIS on brain MRI; the second will be 
non-FDRs who are age- and sex-matched to FDRs who do, and do not, fulfill DIS on MRI scans. 
The non-FDRs will perform the environmental screen and undergo venipuncture for blood analysis 
identical to the other two groups, but will not have a MRI scan.  
b. Aim 1. Descriptive statistics 
c. Aim 2. Association analyses; with individual factors and in a multiple risk factor analysis. In 
previous studies37-60, HLA type, Vitamin D levels, Epstein Barr Virus exposure, smoking and early 
adult obesity have conferred a maximum odds ratio of 2-3  (many SNPs confer an odds ratio of 
1.1-1.4) of developing MS when studied individually. In the GEMS67 study, participants were 
stratified into high and low risk categories based on a deep phenotyping of 64 SNPs, and 
environmental screen (including sex). Comparative analyses were performed on the high and low 
risk groups for both neurological exam and MRI measures. In this study, we plan to use the known 
risk factors, and add potential biomarkers (e.g. NfL, lipid levels) to define the risk of DIS in this 
population. For the power analysis we made the assumption that no single marker would exhibit an 
odds ratio greater than 2 for increased risk of DIS, and that all markers are additive. In this early 
proof of concept phase, with an estimated case sample size of 30 with 60 age- and sex-matched 
controls, our priori power analysis suggests an expected power ranging from 20-33% for risk 
factors such as smoking, obesity, and vitamin D deficiency. Using frequency and odds ratio from 
the GEMS67 study (OR = 2.9) we expect a power of 64% to detect an effect of HLA type risk. While 
this study will be statistically underpowered, and estimates will be imprecise, we expect that some 
results will have clinical significance. We plan to use these results and stored samples as the 
groundwork for larger, longitudinal, multi-site studies. These future studies will allow us to enroll 
samples sizes of greater than 500, necessary to reach 80-90% power. In reality, it is possible the 
effect some factors may depend on the levels of other factors90. Example hypothesis that we will 
test: FDRs with DIS lesions are more likely to have been exposed to EBV as indicated by elevated 
EBV serology than FDRs of the same age and sex without DIS lesions. By extension, we will then 
define a risk score combining factors that are discriminatory, e.g. Vitamin D level, smoking, 
obesity, elevated NfL levels, impaired B cell anergy, and HLA type may each have a roughly 2 fold 
increased risk, whereas those with four of these factors have a six-fold increased risk of DIS on 
MRI.  
Given the matched case-control design, we will use multivariable logistic regression, allowing us 
to adjust for confounders, to estimate and test risk factors controlled for others, and to gain 
greater precision.  The clustering effects of matching can be incorporated using conditional 
logistic regression, or repeated measures methods such as GEE. 
d. Aims 3 and 4 do not have statistical implications.  
G. Summarize Knowledge to be Gained:   
This is a highly innovative research protocol which seeks to duplicate the approach of the successful 
DAISY study, but in a MS population. We aim to define a set of environmental, genetic, lipid, 
neuroaxonal/myelin damage or immunological markers of earliest disease activity in MS.  From this, we 
ultimately hope to develop a risk stratification algorithm for development of MS. This will allow for earliest 
possible intervention for this disabling disease. The purpose of Aim 4 will be to enhance the risk profile 
over time, as other potential, highly sensitive, precise biomarkers become available. One example is 

microRNA (miRNA), which are a class of small (~21-25 nucleotides), noncoding RNAs. These are very 
important in posttranscriptional regulatory processes91. MiRNA’s may directly and indirectly promote 
neurodegenerative action through a variety of pathways. MiR-155, known to be upregulated in MS, and 
silences CD47, a protein involved in phagocytic signaling processes that block macrophage activation92. 
MiRNA’s may contribute to disease progression by driving differentiation of Th1 and Th1792-95, known 
pathogenic T-cell subsets96,97. MiRNA may have potential as a tool for relapse prediction98-100, 
distinguishing different subtypes of MS101-105 (i.e. RRMS vs. PPMS vs. SPMS), and determine disease 
progression104,105. MiRNA’s may have potential as therapeutic targets for controlling disease activity98,99. 
Untreated MS patients display elevated levels of miR-126 relative to healthy controls. After these patients 
underwent DMT treatment, levels of miR-126 decreased to similar amounts observed in controls98. Non-
invasiveness, easy analysis, and high-stability make miRNA a desirable biomarker for disease 
prediction106-108. However, one miRNA sampled from various compartments (plasma, serum, lymphocytes) 
yields significantly different results, contributing to the complexity of comparing results across studies92,107. 
Additionally, RNA extraction, miRNA measurement, and analyzation do not have a standardized protocol, 
resulting in heterogeneous results across different studies. As novel, consistent, and standardized 
techniques become available, miRNA expression studies with longitudinal design at a large site with control 
comparators may allow for insight on miRNA’s ability to predict development of MS, relapses, and even 
disease subtype.  
 
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