Epidemiology of Multiple Sclerosis:

From Risk Factors to Prevention

Alberto Ascherio, M.D., Dr.P.H.,1 and Kassandra Munger, M.Sc.2

ABSTRACT

Although genetic susceptibility explains the clustering of multiple sclerosis (MS)

within families and the sharp decline in risk with increasing genetic distance, it cannot fully

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explain the geographical variations in MS frequency and the changes in risk that occur with
migration, which support the action of strong environmental factors. Among these,
vitamin D status, infection with the Epstein-Barr virus, and cigarette smoking are
emerging as the most consistent predictors of MS risk. In this article, we review the
epidemiological data, critically discuss the evidence for causality of these associations, and
briefly discuss the possibility of interventions to reduce MS risk.

KEYWORDS: Multiple sclerosis, epidemiology, vitamin D, infection, environment

In spite of advances in medicine and related EVIDENCE OF ENVIRONMENTAL RISK

sciences, the largest contributions to health and in-
creased life expectancy have historically come from
primary prevention. Is the primary prevention of multi-
ple sclerosis (MS) an attainable goal? We briefly review
here what is known about risk factors for MS and
suggest that we may now have sufficient evidence to
prevent at least some cases of MS and that growing
understanding of the environmental risk factors
for MS may soon lead to more effective prevention
strategies.
Although clinically and pathologically MS is a
complex and heterogeneous disease,1 these distinctions
are difficult to establish in epidemiological studies and
have thus been mostly ignored, except for occasional
attempts to differentiate primary progressive MS from
the more common relapsing-remitting MS. In this
article, therefore, we refer to MS as a single entity,
acknowledging the possibility that there are subtypes
of MS that may differ in their risk factors.
FACTORS OF MULTIPLE SCLEROSIS
MS is a relatively common disease in most of Europe,
the United States, Canada, New Zealand and southern
Australia, but rare in Asia, the tropics, and the sub-
tropics. Within regions of temperate climate, MS in-
cidence and prevalence increase with latitude, both north
and south of the equator.2,3 The lifetime risk of devel-
oping MS in high-risk populations is
1 in 200
for women.4,5 In virtually all populations, women are
affected more commonly than men, with the female-to-
male ratio varying between 1.5 and 2.5, with a trend
toward higher values in the most recent studies.6 The age
at onset follows a relatively constant pattern across
different regions: incidence is low in childhood; rapidly
increases after adolescence, reaching a peak between ages
25 and 35 years (
2 years earlier in women than men);
and then slowly declines.4,7,8
The strongest known risk factor for MS is a
positive family history. Risk of MS is
30 times higher

1
Departments of Nutrition and Epidemiology, 2Department of
Nutrition, Harvard School of Public Health, Boston, Massachusetts.
Address for correspondence and reprint requests: Alberto Ascherio,
M.D., Dr.P.H., Departments of Nutrition and Epidemiology, Harvard
School of Public Health, 655 Huntington Avenue, Boston, MA 02115
(e-mail: aascheri@hsph.harvard.edu).
Multiple Sclerosis and the Spectrum of CNS Inflammatory Demyeli-
nating Diseases; Guest Editor, Claudia F. Lucchinetti, M.D.
Semin Neurol 2008;28:17–28. Copyright # 2008 by Thieme
Medical Publishers, Inc., 333 Seventh Avenue, New York, NY
10001, USA. Tel: +1(212) 584-4662.
DOI 10.1055/s-2007-1019126. ISSN 0271-8235.
17
18 SEMINARS IN NEUROLOGY/VOLUME 28, NUMBER 1 2008

among siblings of affected individuals than in the general
population.9 The results of detailed studies of familial
aggregation, including the investigation of risk among
half-siblings and adopted children, provide convincing
evidence that the aggregation of MS within families is
largely explained by shared genes rather than a shared
environment.10 Consequently, large international con-
sortia have been established to identify the genetic
determinants of MS; although only the HLA-DRB1
locus has so far been firmly linked to MS risk, other
important loci are likely to be discovered through sys-
tematic screening of the whole genome.11
In contrast, support for environmental risk factors
rests primarily on the geographical distribution of MS
and changes in risk among migrants. Important exam-
ples include studies of people who migrated from differ-
ent countries to Israel,12,13 from the United Kingdom to
in the United States provides additional evidence for an
environmental cause.5,19
Sunlight Exposure and Vitamin D
Many geographical and socioeconomic factors display a
latitude gradient that correlates with the prevalence of
MS; therefore, inferring from the migrant data which of
these factors may be etiologically relevant has proven
difficult. An early study among U.S. veterans attempted
to dissect the role of multiple factors, including air
pollution, concentration of minerals in ground water,
measures of annual solar radiation, mean temperature,
annual rainfall, humidity, and altitude. Although each
factor when examined alone correlated with MS inci-
dence, none of these factors remained significantly
related to MS risk after adjustment for latitude.20 This

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South Africa14 and Australia,15 and from U.S. locations
to other U.S. locations (Fig. 1).16 The U.S. migrant
studies are particularly compelling because they are based
on a very large number of MS cases (> 5,000) and
involve a population with relatively complete and uni-
form rates of ascertainment (U.S. veterans). In all these
instances, the incidence of MS in migrants tends to be
intermediate between that of their birthplace and that of
their final residence, and close to the latter when migra-
tion occurs in childhood.17 Although genetic predispo-
sition most likely contributes to geographical variations
in MS incidence,18 it cannot explain the differences in
risk among people of common ancestry who migrate to
areas of high or low MS prevalence.17 Further, the recent
marked decline in the latitude gradient of MS incidence
result suggests that latitude itself, or a closely related
factor, may influence MS risk.
One of the strongest correlates of latitude is the
duration and intensity of sunlight. Strong correlations
between sunlight and MS prevalence were noted in early
ecological studies,21–24 and a link between sunlight
radiation and reduced MS risk was further supported
by the finding of an inverse correlation in Switzerland
between MS prevalence and altitude,25 which is also a
marker of sunlight intensity. Largely because of these
correlations, it was proposed more than 30 years ago that
the higher incidence of MS at higher latitudes could be
due to vitamin D deficiency.26 Exposure to sunlight is
for most people the major source of vitamin D.27 Ultra-
violet B (UVB) radiation (290 to 320 nm) converts

Figure 1 Relative risk of multiple sclerosis (MS) for white male veterans of World War II or the Korean conflict by tier of
residence at birth and at entry into active duty (EAD) (coterminous United States only). Consistent with an environmental
explanation of the latitude gradient within the United States, MS risk decreased among men who moved to lower latitudes
(shaded arrows), and increased among men who moved to higher latitudes (open arrows). (Data from Kurtzke JF, Beebe GW,
Norman JE. Epidemiology of multiple sclerosis in US veterans: III. Migration and the risk of MS. Neurology 1985;35:672–678.)
 EPIDEMIOLOGY OF MULTIPLE SCLEROSIS: FROM RISK FACTORS TO PREVENTION/ASCHERIO, MUNGER
cutaneous 7-dehydrocholesterol to previtamin D3,
which spontaneously isomerizes to vitamin D3. Vitamin
D3 then undergoes a series of hydroxylations, first to 25-
hydroxyvitamin D3 (25(OH)D3), the main circulating
form of the vitamin, and then to 1,25-dihydroxyvitamin
D3 (1,25(OH)2D3), the biologically active hormone.27
However, at latitudes  428 (e.g., Boston, MA) in winter
most UVB radiation is absorbed by the atmosphere; even
prolonged sun exposure is insufficient to generate vita-
min D,28 and vitamin D levels drop.29,30 Although use of
supplements or high consumption of fatty fish, a good
source of vitamin D, or vitamin D–fortified foods
(mostly milk in the United States) may attenuate this
decline, average levels of vitamin D display a strong
latitude gradient. The hypothesis that vitamin D defi-
ciency could increase MS risk could provide a reasonable
explanation for the latitude gradient and change in risk
among migrants. This hypothesis is also supported by
the finding in Norway that MS prevalence is lower in
coastal villages with higher fish consumption than in
inland agricultural communities.31,32
People living in the same area, however, share
many characteristics whose possible contributions to MS
risk cannot be separated in ecological studies. To over-
come this limitation, it is critical to compare the risk of
MS among individuals living in a similar environment,
but exposed to different levels of vitamin D. Numerous
studies have attempted these comparisons, using meas-
ures of sun exposure, vitamin D intake, or direct meas-
urement of vitamin D levels in serum, as described in the
following sections.
Studies Based on Death Certificates or Record
Linkage
In an exploratory investigation based on death certifi-
cates, MS mortality in areas of high and low sunlight was
related to the individual’s usual occupation, classified by
an industrial hygienist as indoor, mixed, or outdoor.33
After adjusting for age, sex, and socioeconomic status,
both outdoor work (odds ratio ¼ 0.53) and residence in
high sunlight areas (odds ratio ¼ 0.74) were associated
with lower MS mortality, consistent with a protective
effect of sunlight exposure. Most importantly, working
outdoors was associated with a significantly lower MS
mortality in areas of high, but not low, sunlight.33 Major
limitations of this study included reliance on death
certificates, which are an inaccurate source for both cause
of death and occupation, and the possible effects of
‘‘reverse causation,’’ that is, individuals with MS could
preferentially choose an indoor rather than an outdoor
occupation.
In a U.K. study using a record-linkage analysis,
the observed rates of skin cancer among individuals with
MS was compared with that expected according to sex-
and age-adjusted population rates in the same districts.34
19
Because the association between sun exposure and risk of
skin cancer is well-established, the hypothesis was that
individuals with MS would have a lower risk of skin
cancer. In fact, skin cancer rates among individuals with
MS was found to be
50% lower than the expected
(p ¼ 0.03). This result provides evidence of reduced sun
exposure among individuals with MS, but it remains
unclear whether this reduced exposure is the cause or the
consequence of MS. The latter possibility seems quite
plausible because individuals with MS may spend less
time outdoors and may reduce their exposure to direct
sunlight because of heat intolerance.
Case-Control Studies
A more direct attempt to measure exposure to sunlight
before the onset of MS was conducted in a few case-
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control studies. In these studies, individuals with MS
were asked to recall the time that they spent in the sun at
different ages before the onset of MS, and their answers
were compared with those of unaffected individuals in
the same population. Although these investigations
should be less affected by the impact of MS on sun
exposure than the studies based on death certificate or
record linkage, bias from differential recall of MS cases
and controls cannot be excluded. Also, participation
rates among controls are often low, and if participation
in the study is related to sun exposure (for example, if
individuals spending more time indoors are more likely
to be contacted and interviewed), bias may result. These
limitations should be considered in interpreting the
results.
A question on exposure to sunlight was included
in two early case-control studies, which were designed to
explore a broad range of environmental factors. In the
first study, conducted in Israel, individuals with MS
(n ¼ 241) reported significantly more time spent out-
doors in the summer during childhood than age- and
sex-matched healthy controls, a result opposite to that
predicted if sunlight exposure was protective (Fig. 2A).35
In the second, including 300 MS cases in Poland, no
association was found between time in the sun before
MS onset and MS risk (Fig. 2B).36
A more targeted attempt to address the possible
role of sun exposure in determining MS risk was made in
a study including 136 MS cases in Tasmania. The
protocol of this study included not only a detailed
assessment of sun exposure at different ages, but also
measurement of actinic skin damage, an indicator of
lifelong exposure to sunlight.37 The correlation between
actinic skin damage and self-reported exposure to sun-
light in childhood provided evidence of the validity of
the questionnaire.38 The relative risks of MS among
individuals who reported on the questionnaire an aver-
age time in the sun of 2 hours or more between the ages
of 6 and 10 years were 0.47 (95% confidence interval
20 SEMINARS IN NEUROLOGY/VOLUME 28, NUMBER 1 2008

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Figure 2 Odds ratios (ORs) and 95% confidence intervals for the relationship of sun exposure in childhood and multiple
sclerosis (MS) in case-control studies. A, B, and C show the OR estimate of the relative risk of developing MS for individuals
with different levels of sun exposure compared with individuals with the lowest levels of exposure. D gives the OR estimate of
the relative risk of developing MS for one unit increase in sun exposure at the ages indicated. Only the results in C and D are
consistent with a lower risk of developing MS among individuals with higher levels of sun exposure. (Data from: [A] Antonovsky
A, Leibowitz U, Smith HA, et al. Epidemiologic study of multiple sclerosis in Israel. Arch Neurol 1965;13:183–193; [B]
Cendrowski W, Wender M, Dominik W, et al. Epidemiological study of multiple sclerosis in western Poland. Eur Neurol
1969;2:90–108; [C] van der Mei IAF, Ponsonby AL, Dwyer T, et al. Past exposure to sun, skin phenotype and risk of multiple
sclerosis: a case-control study. BMJ 2003;327:316–321; [D] Kampman MT, Wilsgaard T, Mellgren SI. Outdoor activities and diet
in childhood and adolescence relate to MS risk above the Arctic Circle. J Neurol 2007;254:471–477.)

[CI]: 0.26 to 0.84) for winter exposure and 0.50 (95%
CI: 0.24 to 1.02) for summer exposure (Fig. 2C). In-
verse, but weaker associations were observed with sun
exposure at older ages. Stronger associations and sig-
nificant trends were found using the interview-based
recall of exposure (p for trend ¼ 0.01, for ages 6 to
15 years) or actinic damage (p for trend < 0.01).
Although differential reporting of sun exposure be-
tween cases and controls, and thus recall bias, cannot
be excluded, this could not explain the inverse associ-
ation with actinic damage, which was objectively meas-
ured. On the other hand, results based on actinic
damage are prone to error due to reverse causation
(a limitation already discussed for the occupation and
record-linkage studies) because actinic damage meas-
ures cumulative exposure to sunlight, including during
the years following MS onset.37
Finally, an interesting study was recently con-
ducted in northern Norway, in counties above the
Arctic Circle (latitudes 66 to 71 N).39 At this latitude,
during winter virtually all vitamin D is provided by
diet; the most important sources are fatty fish and cod-
liver oil, which is rich in vitamin D and commonly
used as a supplement. MS cases (n ¼ 152) and matched
controls (n ¼ 402) were asked to report both the time
spent in outdoor activities at different ages and their
consumption of fish and cod-liver oil supplements in
childhood. Time spent outdoors in the summer was
 EPIDEMIOLOGY OF MULTIPLE SCLEROSIS: FROM RISK FACTORS TO PREVENTION/ASCHERIO, MUNGER 21

associated with a reduced risk of MS, most pronounced
at ages 16 to 20 years (45% lower risk, p for
trend ¼ 0.001) (Fig. 2D). An inverse association was
also found between frequent fish consumption and MS
risk, whereas use of cod-liver oil was inversely associ-
ated with MS risk only among individuals who re-
ported low summer outdoor activities.
Longitudinal Studies
The association between diet and risk of chronic diseases
is difficult to investigate retrospectively because even a
modest difference in recall between cases and controls
can cause a large bias in relative risk estimates.40 Similar
considerations apply to other aspects of lifestyle that
cannot be objectively documented. The probability of
bias is even greater in investigations of disease that have
tudinal studies need to be very large and, not surpris-
ingly, only a few have been conducted.
The only published longitudinal study on dietary
vitamins and MS risk was conducted among participants
in two large cohorts of U.S. nurses, the Nurses Health
Study and the Nurses Health Study II, comprising more
than 200,000 women41 who completed a validated semi-
quantitative food frequency questionnaire. To account
for changes in diet over time, a dietary questionnaire was
administered every 4 years during the follow-up.42,43
Vitamin D intake, from both foods and vitamin supple-
ments, was found to correlate with blood levels of
25(OH)D (available in a subset of 343 women)44 and
to predict a reduced risk of hip fractures,44 thus provid-
ing evidence of its validity. Risk of developing MS
during the follow-up declined with increasing vitamin
D intake. The age-adjusted pooled relative risk (RR)

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an insidious onset, because the disease itself may induce
changes in lifestyle and diet before a diagnosis is made.
Thus, a longitudinal study design with a long period of
follow-up is important to ensure the validity of the
results. Because MS is a relatively rare disease, longi-
comparing women with vitamin D intake of  400 IU/d
to those with none was 0.59 (95% CI ¼ 0.38 to 0.91; p
for trend ¼ 0.006) (Fig. 3A). This RR did not materially
change after further adjustment for risk factors for MS,
including pack-years of smoking and latitude at birth.

Figure 3 Relative risk (RR) of developing multiple sclerosis (MS) comparing (A) individuals who used 400 IU or more of
supplemental vitamin D versus nonusers; and (B) individuals with high-versus-low serum levels of 25(OH) vitamin D. Bars
represent the 95% confidence intervals of the RR estimates. Use of vitamin D supplements and high levels of serum 25(OH)D
are both associated with a reduced risk of developing MS. (Data from: [A] Munger KL, Zhang SM, O’Reilly E, et al. Vitamin D
intake and incidence of multiple sclerosis. Neurology 2004;62:60–65; [B] Munger KL, Levin LI, Hollis BW, Howard NS, Ascherio
A. Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis. JAMA 2006;296:2832–2838.)
22 SEMINARS IN NEUROLOGY/VOLUME 28, NUMBER 1 2008
Because intake of vitamin D was largely from multi-
vitamins, the possibility that the observed association
was due to other micronutrients contained in the multi-
vitamin could not be excluded. However, results of
multivariate analyses combined with biological plausi-
bility suggest that the results are more consistent with a
protective effect of vitamin D itself.
An alternative approach to investigate the relation
between vitamin D and MS risk relies on the use of
biomarkers of vitamin D status. Vitamin D is hydroxy-
lated in the liver to 25(OH)D, the immediate precursor
of 1,25(OH)2D, the active hormonal form of vitamin D.
Serum levels of 1,25(OH)2D are tightly regulated and are
within the normal range even in individuals who are
vitamin D deficient. In contrast, 25(OH)D levels
are sensitive to both vitamin D intake and sun exposure,
and are a marker of vitamin D availability to tissues,
including the immune system. We therefore planned to
examine whether serum levels of 25(OH)D measured in
healthy young adults predict their risk of developing MS.
Because of the low incidence of MS, such a study
required the collection of blood samples from hundreds
of thousands of individuals and a follow-up of several
years. This was made possible by a very large collection of
serum from healthy young adults, which was made
available from the Department of Defense Serum Repo-
sitory (DoDSR). Since 1990, the DoDSR has collected
and stored more than 40 million serum samples left over
from routine HIV and worldwide deployment-related
blood tests. Personnel generally provide one sample at
entry into the military and, on average, every 2 years
thereafter while on active duty. In collaboration with the
Army and Navy Physical Evaluation Boards, we were able
to identify those individuals who developed MS after
providing a blood sample.45 Because of seasonal varia-
tions in sun exposure and thus of 25(OH)D levels, a
single blood sample may be insufficient to determine the
long-term vitamin D status; we restricted the analyses to
individuals with at least two blood samples collected
before MS onset.46 The study included 257 individuals
with MS diagnosed between 1992 and 2004, and two
controls matched by sex, age, race/ethnicity, and time of
collection of the blood samples for each case. This design,
referred to as a ‘‘nested case-control’’ study by epidemi-
ologists, is an efficient equivalent to a fully prospective
study, in which 25(OH)D levels are measured in all
available blood samples. The control group provides
information on the distribution of 25(OH)D levels
among the millions of individuals at risk of developing
MS, at a small fraction of the cost required for measuring
all the samples. The validity of the study rests on the
stability of 25(OH)D levels in stored serum, which was
determined in a pilot study.
The results of this study support the hypothesis of
a protective effect of 25(OH)D.46 Because of the marked
effect of race on 25(OH)D levels (the darker the skin the
lower the amount of vitamin D produced by the same
levels of exposure to sunlight), analyses were conducted
separately in white, African American, and Hispanic
populations. Among white people, MS risk declined
with increasing levels of 25(OH)D—risk was 62% lower
among individuals in the highest quintile (25(OH)D
> 99.2 nmol/L) compared with those in the lowest
quintile (25(OH)D < 63.2 nmol/L) (Fig. 3B). Further,
the reduction in risk of developing MS among individuals
with high 25(OH)D levels was considerably stronger
before the age of 20 years (16 to 19) than at ages 20 or
older. In contrast, no significant association between
25(OH)D levels and MS was found in African Ameri-
cans and Hispanics, but statistical power was low because
of overall low levels of 25(OH)D and small sample size.
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Summary
Overall, the results of epidemiological studies seem to
support a protective role of vitamin D, particularly in
childhood and adolescence. It is worth mentioning,
however, that UV radiation from sunlight has immuno-
suppressive effects that could be independent from the
synthesis of vitamin D and could contribute to MS
prevention.47 These vitamin D–independent effects,
however, do not explain the results of dietary studies.
Further, these results should be interpreted in combina-
tion with those of experimental studies on the effects of
vitamin D. Injection of 1,25(OH) 2D3 can prevent
experimental autoimmune encephalomyelitis (EAE),
an animal model of MS,48,49 whereas vitamin D defi-
ciency accelerates EAE onset.50 Protective effects of
1,25(OH)2D3 have also been observed in animal models
of type 1 diabetes and other autoimmune diseases.51 The
mechanisms underlying these effects are still under
investigation,51–54 but are likely to include induction of
regulatory T cells.55,56
Implications for Prevention
If vitamin D effectively reduces the risk of MS, supple-
mentation in adolescents and young adults could be
effectively used for prevention. Supplements providing
between 1000 and 4000 IU per day of vitamin D would
increase serum 25(OH)D to the levels associated with
MS protection,57–60 without causing hypercalcemia or
other known adverse side effects.59 Although the In-
stitute of Medicine’s current recommendation for ad-
equate intake of vitamin D is only 200 IU/d for adults
younger than 50,61 and the typical amount in multi-
vitamins is 400 IU, there is increasing consensus that
these levels are far below those required for optimal bone
health and the prevention of other vitamin D–related
diseases.62 Because of the high prevalence of low
25(OH)D levels in the United States63 and other coun-
tries with high MS incidence,64 the potential for MS
 EPIDEMIOLOGY OF MULTIPLE SCLEROSIS: FROM RISK FACTORS TO PREVENTION/ASCHERIO, MUNGER
prevention is extremely high. There is therefore an
urgent need to determine in a large randomized trial
whether vitamin D supplements can in fact reduce MS
risk.
HYGIENE HYPOTHESIS AND EPSTEIN-
BARR VIRUS
In 1963, Poskanzer and colleagues, noting the similarity
between the epidemiology of MS with that of polio-
myelitis, proposed that MS, like poliomyelitis, could be
the rare neurological manifestation of a common enteric
infection.65 Risk of MS, as is true for poliomyelitis,
would increase with increasing age at infection with the
responsible microorganism, and would thus be more
common in populations with high levels of hygiene, in
which transmission of infection is delayed. This hypoth-
esis was supported by early findings of increasing MS
incidence with increasing sanitation in Israel,66 and with
increasing socioeconomic status in the United States67
and the U.K.68 A specific microorganism causing MS,
however, could not be found, and the hypothesis has
evolved into a broader ‘‘hygiene hypothesis’’ according to
which multiple infectious exposures in early childhood
would reduce the risk of MS by modulating the immune
response toward helper T cells (Th)2 and regulatory T
cells and attenuating the proinflammatory Th1 cellular
immunity.69,70 Common intestinal helminths, for exam-
ple, induce a predominantly Th2 reaction.71 Interest-
ingly, in a recent study, infection with intestinal
helminths was found to have a striking beneficial effect
in individuals with MS.72,73 The hygiene hypothesis
could explain in part the geography of MS. In the
tropical and subtropical areas where MS frequency is
low, most people live in conditions that favor the trans-
mission of multiple infectious agents. Infections with
common viruses tend to occur in early childhood, and
gastrointestinal infections with bacteria and parasites are
common. In contrast, in temperate zones of high MS
risk, infection with common viruses is often delayed to
late childhood or adolescence, and intestinal parasites are
rare.
An example of variations in age at infection in
different populations is provided by the Epstein-Barr
virus (EBV). In developing countries, almost all children
are infected in the first few years of life, and typically
prevalence of seropositivity is higher than 90% at the age
of 4 years. In contrast, in most developed countries, not
only do many children escape EBV infection until
adolescence, but prevalence of seropositivity to EBV
displays a latitude gradient parallel to that of MS. For
example, in the United States the prevalence of EBV
seropositivity among young adults ranged from more
than 80% in the southeast to just above 50% in the
northwest.74 Unlike intestinal parasites, EBV is not
transmitted by fecal contamination, but by saliva.
23
Thus, the age at infection reflects, in part, cultural
differences (such as sharing of food and food utensils),
and a young age at infection with EBV is not by itself a
marker of a low level of sanitation. Early age at EBV
infection is observed not only in the tropics, but also at all
latitudes in Asia, including highly industrialized nations
such as Japan75 and among Eskimos in Greenland. It is
interesting that the same populations also have a low risk
for MS.76 Notably, age at EBV infection, like MS, also
positively correlates with higher socioeconomic status.74
Because of the correlation between late age at infection
with EBV and MS, the epidemiology of MS is remark-
ably similar to that of infectious mononucleosis,77 which
is a common occurrence of primary EBV infection during
adolescence or later in life.
The similarities between the epidemiology of MS
and that of mononucleosis are striking, and lead to the
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critical question of whether mononucleosis is simply a
marker of a high level of hygiene during childhood or
whether EBV plays a more direct role in the etiology of
MS. The answer to this question rests on the observation
of a paradox. Young adults who are EBV negative are
clearly a subset of the ‘‘high-hygiene’’ set, which, accord-
ing to the hygiene hypothesis, has a high MS risk.
Contrary to this prediction, not only do these individuals
not have a high risk of MS, but, as long as they remain
EBV negative, their risk is about 10 times lower than
that of EBV-infected individuals of the same age
(Fig. 4).78,79 The paradox could be explained if these
individuals, which comprise only a small percent of the
population, carried a rare genetic mutation that confers
resistance to both EBV infection and MS. This inter-
pretation, however, has been virtually ruled out by the
fact that a similar low risk of MS has been found in
EBV-negative children.80,81 These children cannot be
genetically resistant to EBV infection because they
become infected later in life, as can be inferred by the
fact that in the same population prevalence of EBV
positivity increases from
50% in childhood to
95% in
adulthood. After they become infected with EBV, if the
infection results in mononucleosis, their risk of MS
increases two- to threefold above the risk observed
among EBV-positive individuals without a history of
mononucleosis (Fig. 4).79,82,83 These data combined
establish EBV infection as a strong risk factor for MS
and indicate that the increased risk of MS among
individuals raised in a more hygienic environment be-
comes manifest only after EBV infection (EBV variant
of the hygiene hypothesis).79
The mechanisms by which EBV infection in-
creases the risk of MS are still unclear.84 It seems likely
that EBV predisposes to autoimmunity because a pos-
itive association has also been found with systemic lupus
erythematosous.85 In a recent pathological study, strong
evidence has been found of dysregulated EBV infection
in the brain of most MS patients.86 If replicated, this
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Figure 4 Relative risk (RR) of developing multiple sclerosis (MS) according to Epstein-Barr virus (EBV) infection and history of
mononucleosis. Bars represent the 95% confidence intervals of the RR estimates. Compared with individuals who are EBV
positive but have no history of mononucleosis, risk of developing MS is markedly lower among individuals who are EBV negative,
but higher among individuals who are EBV positive and have a history of mononucleosis. (Data from: Thacker EL, Mirzaei F,
Ascherio A. Infectious mononucleosis and risk for multiple sclerosis: a meta-analysis. Ann Neurol 2006;59:499–503; Ascherio A,
Munger KL. Environmental risk factors for multiple sclerosis. Part I: The role of infection. Ann Neurol 2007;61:288–299.)

discovery may lead to a new era in MS research. To
better understand the temporal relation between EBV
infection and MS and the possible role of EBV, we have
investigated longitudinally the antibody responses to
different EBV antigens in healthy adults, comparing
those who eventually developed MS with those who
remained healthy.45,87,88 We found that individuals with
MS experienced an elevation in antibody titers to the
EBV nuclear antigen 1 (EBNA-1) many years before the
onset of neurological symptoms, a result independently
confirmed by others.89 Unexpectedly, this elevation does
not have a clear temporal relationship with the time of
MS onset; rather, the anti-EBNA-1 titers of individuals
who will develop MS increase sometime between 20 and
30 years of age, and then remain elevated.45 EBNA-1 is
a protein consistently expressed in latently infected B
lymphocytes. A decline in anti-EBNA-1 titers is usually
observed in immunosuppression,90 and antibodies to
EBNA-1 tend to correlate with cellular immunity
against EBV.91 In this context, it is interesting that
CD4 þ T cells specific to EBNA-1 are increased in
frequency and recognize a broader range of epitopes in
individuals with MS than in healthy controls.92 Also,
two EBV peptides, one of which was from EBNA-1,
have been recognized as targets of the immune response
in the cerebrospinal fluid (CSF) of MS patients.93
Summary
Overall, there is compelling evidence that EBV infec-
tion is a strong risk factor for MS. It is important to
emphasize, however, that EBV infection by itself cannot
explain some important aspects of MS epidemiology, and
two are worth mentioning. One is the decrease in risk of
MS with migration from high- to low-risk areas, and the
other is the presumed occurrence of an epidemic of MS in
the Faroe Islands.94 As discussed elsewhere, the migra-
tion data and the Faroe epidemics suggest that either
EBV interacts with other infectious or noninfectious
agents to cause MS, or that there are multiple strains of
EBV with different propensity to cause MS.79
Implications for Prevention
In the absence of an effective vaccine, there is not an
obvious and feasible intervention that could contribute
to MS prevention. Results of trials of antiviral drugs in
MS did not support a strong beneficial effect.95,96 The
solid data relating mononucleosis to increased MS risk
suggest that some reduction in MS risk could be
achieved by exposing children to EBV infection early
in life. This conclusion, however, is far from certain,
because, as discussed previously, infection with EBV
early in life correlates with exposure to other infectious
agents that could modulate the immune response to
EBV, and the effect of anticipating age at EBV infection
in the absence of exposure to other infectious agents
remains unknown. Thus, the main implication is that
there is a need to understand the mechanisms that
explain the role of EBV in MS, because otherwise it
may not be possible to translate the epidemiological
evidence into preventive or therapeutic interventions.
 EPIDEMIOLOGY OF MULTIPLE SCLEROSIS: FROM RISK FACTORS TO PREVENTION/ASCHERIO, MUNGER
CIGARETTE SMOKING
Although it does not contribute to explaining the geo-
graphical variations in MS incidence, there is strong and
growing evidence that cigarette smoking is an important
risk factor for MS. An association between smoking and
MS risk was first found in an exploratory case-control
study in Israel more than 40 years ago,35 and it has been
confirmed more recently in case-control investiga-
tions97,98 and in a population-based survey.99 More
robust evidence that smokers have a higher risk of
developing MS than nonsmokers, however, is provided
by the combined results of four longitudinal studies.
Two of these investigations were conducted among
women in the United Kingdom to determine whether
use of oral contraceptives was related to MS risk.100,101
In both, women who regularly smoked were found to
have a higher risk of MS; although, because of the small
number of incident cases of MS (63 of 17,032 in one
study, and 114 of 46,000 in the other), neither study
convincingly demonstrates a significant increase. In the
third investigation, which comprised more than 200,000
women and 315 incident cases of MS, there was a
significant increase in MS risk among smokers, and a
clear dose–response—the age-adjusted risk of MS
among women who reported 25 or more pack-years of
smoking was 70% higher than among those who never
smoked (p < 0.01).102 This result was not materially
changed after further adjustment for ancestry and lat-
itude. Finally, a significant positive association between
smoking and MS risk was found in a case-control study
nested within the General Practice Research Data-
base.103 When the results of these longitudinal studies
are combined, the increasing risk of MS with increasing
exposure to smoking becomes compelling (p < 0.001).104
Although the possibility that this association is due to
some factor that strongly affects both smoking behavior
and MS risk cannot be excluded, there is no evidence
that such a factor exists. Smoking may also adversely
affect the progression of MS. An acute worsening of MS
symptoms immediately following smoking was reported
in a few clinical studies conducted more than 40 years
ago105 and confirmed in later investigations.106 More
recently, a significantly increased risk of transition to a
secondary progressive phase of MS was reported among
smokers with relapsing-remitting MS, as compared with
nonsmokers.103
Several hypotheses have been proposed to explain
the increased risk of MS among smokers. These include
vascular effects, effects on the immune system, increased
production of nitric oxide, increased frequency of respi-
ratory infections, and neurotoxic effects of cyanides and
other components of cigarette smoke.102,103 The obser-
vation that smokers have an increased risk not only of
MS, but also of other autoimmune diseases such as
rheumatoid arthritis, systemic lupus erythematosus,
and Graves’ disease,107 support a systemic effect on the
25
immune system. On the other hand, nicotine or other
components of cigarette smoke have effects on the
integrity and function of the blood-brain barrier, affect
cerebral circulation and signaling pathways in the
CNS,108 and could potentially affect MS risk in multiple
ways. A possible mechanism involves nitric oxide, which
is present in cigarette smoke and also released in the
brain in response to nicotine.109 Peroxynitrites, gener-
ated by the reaction of nitric oxide with superoxide, have
been implicated in the pathogenesis of MS,110 and
elevated CSF levels of nitric oxide metabolites were
reported in individuals with MS and found to be
associated with MS progression.111 Further research on
the mechanisms relating cigarette smoke to MS risk and
progression could be important not only for the imme-
diate purpose of strengthening the evidence of a causal
role of smoking, but also because it could lead to the
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
discovery of new clues toward the prevention and treat-
ment of MS.
Summary
There is strong evidence that smokers have a higher risk
of MS than nonsmokers and suggestive evidence that
smoking may also adversely affect MS progression. The
increase in risk is
70% among heavy smokers.
Implications for Prevention
Smoking cessation is likely to contribute to prevention of
a substantial number of cases of MS. In the United
States,
20% of young adults are regular smokers.112 At
the population level, the risk of MS due to smoking can
be estimated as: [(1.5–1)/1.5]*0.20 ¼ 6%, where 1.5 is
the approximate RR of smoking on MS and 0.20 is the
prevalence of smoking in the population.113 Therefore, if
smoking was eliminated, up to 6% of the MS cases could
be prevented. At the individual level, knowledge that
smoking increases the risk of MS could be a strong
deterrent, particularly for individuals who are at high risk
of MS because of their family history.
ACKNOWLEDGMENTS
We thank Leslie Unger for technical assistance in the
preparation of the manuscript. Dr. Ascherio is recipient
of National Institutes of Health grants R01 NS47467
and R01 NS042194 for the investigation of the epi-
demiology of MS.
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