University of Southern California Dissertations and Theses

Environmental risk factors of Multiple Sclerosis: a twin study

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Environmental risk factors of Multiple Sclerosis: a twin study

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Content ENVIRONMENTAL RISK FACTORS OF MULTIPLE SCLEROSIS:
A TWIN STUDY

by

Khandaker Talat Shamsul Islam

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(EPIDEMIOLOGY)

May 2007

Copyright 2007 Khandaker Talat Shamsul Islam

ii

DEDICATION

I dedicate this dissertation to my father (Dr. Abu-Torab Shamsul Islam) and mother
(Hamida Khatun) who have dedicated their whole life for my education and well-
being. To my brothers, Tanveer Islam and Tareq Islam, who have intellectually
challenged me throughout my life. To my wife, Roksana Karim, who have been a
great friend and companion all along. And to Tajwar Islam Khandaker my son, who
had to sacrifice lot of his childhood years for my personal endeavor.
iii

ACKNOWLEDGEMENTS

I acknowledge all those without whose support I would have never reached
this point of my life. By far the greatest contributors are my parents, Dr. Abu-Torab
Shamsul Islam and Hamida Islam. They sacrificed their whole life for the betterment
of the lives of their three sons. Being the youngest, I was always the trouble one but
my mother persevered to keep me in track. I have no doubts that without her
guidance I would have never reached this far. However, attainment in life is not the
academic degrees or accolades a man collects but rather what he represents. I hope I
can be a person like my parents are. And then there were teachers like Br. Hobart
and Nalini Kanta Sarker, who taught me the meaning of life and the importance of
unselfish love. There are no words or deeds to duly express my gratitude to all the
people who contributed to all that is good in me. So, I would like to use this
opportunity to express my gratitude to all of them - named or unnamed here.
I’d also like to acknowledge the sacrifice my son Tajwar made for this of my
personal achievement. As, I work at home on my dissertation, Tajwar sits by me
quietly reading books and he has been doing it since age three! Hope I can make up
for our lost time.
I would also like to thank Dr. Thomas Mack for teaching me how to think not
only as an epidemiologist but also as a human being. I thank Dr. Frank Gilliland for
iv

teaching me how to think and write like a scientist. I am also indebted to all the
members of my Ph.D. committee for their help in preparing my dissertation.
v

TABLE OF CONTENTS
DEDICATION .............................................................................................................ii

ACKNOWLEDGEMENTS ........................................................................................iii

LIST OF TABLES ......................................................................................................ix

LIST OF FIGURES ....................................................................................................xi

ABSTRACT...............................................................................................................xii

Chapter 1 Overview of Dissertation.............................................................................1
1.1 Introduction........................................................................................................1
1.2 Brief Descriptions of the Chapters.....................................................................6
1.2.1 Chapter1: An Overview of the Dissertation................................................6
1.2.2 Chapter 2: A Clinical Overview of Multiple Sclerosis ...............................6
1.2.3 Chapter 3: A Review of Descriptive Epidemiology of Multiple
Sclerosis ...............................................................................................................6
1.2.4 Chapter 4: A Review of Genetic Susceptibility of Multiple Sclerosis........7
1.2.5 Chapter 5: A Review of Environmental Risk Factors in MS......................7
1.2.6 Chapter 6: Paper I. Differential Concordance of Multiple Sclerosis
among Monozygotic Twins by Latitude of Birthplace: Role of genetic and
non-genetic factors (Islam T: Annals of Neurology 2006; 60(1):56-64).............7
1.2.7 Chapter 7: Paper 2. Activities associated with Sun Exposure in
Childhood Modifies the Risk of Multiple Sclerosis among Monozygotic
Twins (in Press Neurology) .................................................................................8
1.2.8 Chapter 8: Grant Proposal: Role of sun exposure related genes in MS......8

Chapter 2 An Overview of Pathology, Clinical Presentation and Diagnosis
Multiple Sclerosis ........................................................................................................9
2.1 Introduction........................................................................................................9
2.2 Clinical Presentation of Multiple Sclerosis........................................................9
2.2.1 Initial Presentation of MS .........................................................................10
2.2.2 Clinical Classification of Multiple Sclerosis.............................................11
2.3 Pathophysiology of MS....................................................................................13
2.4 Diagnostic Tests...............................................................................................15
2.5 Diagnosis of MS...............................................................................................19
2.6 Pathology of MS ..............................................................................................21
vi

2.6.1 Pathological Classification of MS.............................................................22
2.7 Pathogenesis of MS..........................................................................................26
Chapter 2 References .............................................................................................31

Chapter 3 A Review of Descriptive Epidemiology of Multiple Sclerosis.................39
3.1 Geographic Distribution...................................................................................39
3.2 Racial Distribution ...........................................................................................40
3.3 Migration Studies.............................................................................................41
3.4 Sex and MS ......................................................................................................44
3.5 Age at Onset.....................................................................................................45
3.6 Temporal trend of MS......................................................................................46
3.7 Summary ..........................................................................................................53
Chapter 3 References .............................................................................................55

Chapter 4 Genetic Susceptibility of Multiple Sclerosis .............................................60
4.1 Introduction......................................................................................................60
4.2 Familial Aggregation .......................................................................................61
4.2.1 Familial Recurrence ..................................................................................61
4.2.2 Conjugal Multiple Sclerosis......................................................................66
4.2.3 . Half-Sib Risk...........................................................................................67
4.3 Twin Studies ....................................................................................................69
Chapter 4 References .............................................................................................72

Chapter 5 Environmental Risk Factors of MS ...........................................................75
5.1 Introduction......................................................................................................75
5.2 Infection ...........................................................................................................75
5.3 Sun Exposure ...................................................................................................82
5.4 Dietary Factors.................................................................................................86
5.4.1 Vitamin D..................................................................................................86
5.4.2 Fatty Acids ................................................................................................90
5.5 Sex Hormones ..................................................................................................91
5.5.1 Puberty and MS.........................................................................................95
5.5.2 Pregnancy and MS ....................................................................................95
5.5.3 Oral Contraceptive and MS.......................................................................96
5.5.4 Organic Solvents .......................................................................................97
5.5.5 Smoking ....................................................................................................99
5.6 Summary ........................................................................................................102
Chapter 5 References ...........................................................................................105

Chapter 6 Differential Concordance of Multiple Sclerosis by Latitude of
Birthplace .................................................................................................................119
vii

Chapter 6 Abstract ...............................................................................................119
6.1 Introduction....................................................................................................121
6.2 Methods..........................................................................................................122
6.4 Results............................................................................................................130
6.2.1 Representativeness of cases ....................................................................130
6.2.2 Pattern of Pairwise Concordance ............................................................132
6.2.3 Validity of the Observations ...................................................................136
6.3 6.5 Discussion ................................................................................................140
Chapter 6 References ...........................................................................................144

Chapter 7 Activities Associated with Sun Exposure in Childhood Modifies
Risk of Multiple Sclerosis among Monozygotic Twin (Neurology) .......................148
Chapter 7 Abstract ...............................................................................................148
7.1 Introduction....................................................................................................150
7.2 Method ...........................................................................................................151
7.3 Result .............................................................................................................155
7.4 Discussion ......................................................................................................162
Chapter 7 References ...........................................................................................167

Chapter 8 Grant Proposal- Role of Genes involved in Vitamin D Signaling
and Metabolism Pathway on MS Risk.....................................................................170
Chapter 8 Abstract ...............................................................................................170
8.1.1 Primary hypothesis..................................................................................171
8.1.2 Secondary hypotheses .............................................................................171
8.2 Background and Significance ........................................................................172
8.2.1 Incidence and Prevalence of MS.............................................................172
8.2.2 Sun Exposure and MS.............................................................................173
8.2.3 Vitamin D and MS
3
.................................................................................174
8.2.4 Genes in Vitamin D Signaling Pathway .................................................177
8.2.5 Relevance ................................................................................................182
8.2.6 Cost Effectiveness...................................................................................182
8.2.7 Representativeness ..................................................................................184
8.3 Preliminary Studies ........................................................................................184
8.3.1 Differential Twin Concordance for Multiple Sclerosis by Latitude
of Birthplace
7
...................................................................................................184
8.3.2 Childhood Sun Exposure Influences Risk of MS among MZ Twins
(Accepted for publication in Neurology) .........................................................186
8.3.3 HLA in MS (ongoing research) ..............................................................187
8.4 Research Design and Methods.......................................................................188
8.4.1 Source of Subjects and Study Design .....................................................188
8.4.2 Polymorphism Selection .........................................................................193
viii

8.4.3 Statistical Analysis..................................................................................194
8.4.4 Sample Size and Power...........................................................................199
8.5 Limitations .....................................................................................................203
8.6 Responsiveness to RFA .................................................................................204
8.7 Time Line.......................................................................................................205
8.8 Human Subjects .............................................................................................205
8.8.1 Risks to the Subjects ...............................................................................205
8.12 Vertebrate Animals ......................................................................................209
Chapter 8 References ...........................................................................................210

Bibilography.............................................................................................................220
ix

LIST OF TABLES
Table 2.1. Frequency of Clinical Symptom at Onset.................................................11

Table 2.2: Diagnostic criteria for MS diagnosis (adopted from International
Panel on the Diagnosis of Multiple Sclerosis
38
..........................................................20

Table 2.3: New diagnostic criteria for magnetic resonance imaging
determination of dissemination in space....................................................................20

Table 2.4: New diagnostic criteria for magnetic resonance imaging
determination of dissemination in time......................................................................21

Table 2.5 : Different patterns of Active MS lesions ..................................................23

Table 4.1: Crude sib recurrence rate according to gender of index case,
stratified by parental MS status
1
................................................................................64

Table 4.2: Pairwise MS concordance among MZ and DZ twins ...............................70

Table 5.1: The Role of Sex Hormones on Immunity.................................................92

Table 5.2: Association between oral contraceptive use and MS................................97

Table 6.1: Descriptive Characteristics of Study Population ....................................123

Table 6.2: Median and inter quartile range (IQR) of age at diagnosis and
duration of follow-up since initial diagnosis according to zygosity and
baseline characteristics.............................................................................................127

Table 6.3: Variations in Pairwise Concordance for MS according to zygosity
and baseline characteristics......................................................................................133

Table 6.4: Mean age of MS diagnosis according to zygosity and concordant
status.........................................................................................................................135

Table 6.5: Odds Ratio (OR) and Confidence Limits of MZ Concordance
Estimates for MS according to Birthplace, High Risk Ancestry and Early
Diagnosis of 1 Case.
st a
..............................................................................................136
x

Table 6.6: Odds Ratio and Confidence Limit of Pairwise Concordance (PC)
estimates of Ancestry, Birthplace and Age at Diagnosis for MZ twins pairs
with available objective medical data .
1
....................................................................139

Table 7.1: Baseline characteristics of exposure-discordant and
exposure-concordant MS-discordant MZ twin pairs .
1
.............................................153

Table 7.2: Correlation (Spearmen coefficients) between different measures of
outdoor exposure......................................................................................................157

Table 7.3: Odds ratio and 95% confidence interval* linking MS to various
measures of relative outdoor exposure.....................................................................158

Table 7.4: Odds Ratio (OR) and 95% CI for the association between the Sun
Exposure Index and MS, stratified on known risk factors.......................................160

Table 7.5: Odds Ration(OR) and 95% CI for the association between Sun
Exposure Index and MS, adjusted for possible confounders ...................................161

Table 8.1: Chromosomal location, number of exons, gene size and SNPs
genotyped for the genes under study-put in entire SNP table..................................177

Table 8.2: Characteristics of the MS cases and controls for the three study
populations* .............................................................................................................183

Table 8.3: Odds ratio and 95% CI for the association between the
non-synonymous SNP of VDR at codon 2 (DBP-2G>A), stratified by
HLA-DR2 haplotype................................................................................................196

Table 8.4: Sample size required to detect noted relative risk for given allele
frequency with 0.80 power.......................................................................................201

Table 8.5: Odds ratio (OR) for gene-gene interaction detectable with 80%
power with different allele frequency
1
.....................................................................203
xi

LIST OF FIGURES

Figure 2.1: Schematic Presentation of MS Pathogenesis...........................................28

Figure 3.1: Sex-period specific crude Incidence rate of MS for 3 Finnish
regions (with permission from M.-L. Sumelahtia et.al.I )
51
........................................50

Figure 4.1: The association between familial recurrence risk of MS and
percent of genetic sharing among different relatives and general population. ..........63

Figure 5.1: Association between MS and Different Infections..................................80

Figure 5.2: Association between MS and Smoking.................................................101

Figure 5.3: A schematic presentation of the interplay of environmental and host
factors in MS etiology..............................................................................................104

Figure 6.1: The schematic presentation of the North American Twin registry
Data ..........................................................................................................................126

Figure 7.1: Hierarchical flow of analysis data .........................................................156

Figure 7.2: Distribution of Intra-pair difference* in the Sun Exposure Index in
MS-discordant MZ Pairs..........................................................................................159

Figure 8.1: Pathway diagram of vitamin D metabolism, transport and target
tissue binding. ..........................................................................................................180

xii

ABSTRACT

Multiple sclerosis (MS) is an autoimmune mediated degenerative disease of
the central nervous system that is characterized by focal neurological deficits that are
marked by temporal and spatial variation. It is a debilitating disease affecting the
young adults with a propensity for female and Whites. Beside the latitude gradient,
female sex and familial risk, little is known about the risk factors of MS and even
less regarding its etiology. Increased recurrence risk among 1st degree relatives and
twins of an MS case underscores the importance of genetic susceptibility in MS.
However, the observed of ~20% recurrence risk among co-twin of monozygotic
twins provides a great insight to MS risk as 1) it is 400 times more than the lifetime
risk of general population but 2)80% of the co-twins despite having the same genetic
risk do not get the disease. Thus the genetic susceptibility appears to be a necessary
but not a sufficient risk factor for MS. Identifying environmental risk factors for MS
can greatly reduce the burden of the disease. That have been the focus of my
research. My interest is to know the relationship between environmental and genetic
risk factors and identify specific risk/protective factors of MS. I addressed my
research questions in a large population (~1200) twins with at least one case of MS.
The twins represented MS cases among twins in the North American population
from 1980-1992. Comparing pairwise concordance rate among monozygotic (MZ)
and same-sex dizygotic (DZ) twins by latitude of birthplace I identified the
xiii

importance of gene-environmental interaction in MS risk and showed that the
variation in concordance rate mimics the latitude gradient in MS which can be
explained by environmental and genetic risk factors. Limiting our analysis to MZ
twins we observed that sun exposure during childhood is associated with reduction in
the risk of MS. The protective effect of sun exposure might vary by gender and HLA
status. As the protective effect of sun exposure can be mediated through vitamin D
status, I further plan to investigate the role of genes involved in vitamin D pathway
in MS.
1
Chapter 1 Overview of Dissertation
____________________________________________________________________

1.1 Introduction

Earliest reports and descriptions of a sclerosing disease of the spinal cord and
central nervous system (CNS) that are consistent with Multiple Sclerosis are found in
the medical literature and case reports of mid nineteenth century physicians of
France, Germany, England, Australia, Canada and US. Jean Martin Charcot, a
French physician is one of the earliest to describe the disease and provide an accurate
clinico-pathological association. No consistency in the nomenclature of the disease
existed till 1950s.Charcot and his fellow physicians coined the term that
corresponded to ‘disseminated sclerosis’ in English. Earlier, Australian, British and
US medical literature termed the disease as ‘insular sclerosis’ or ‘lobular and diffuse
sclerosis’. The Germans used terms similar to multiple sclerosis form the beginning.
However, the term became universally accepted since the publication of the
monograph Multiple Sclerosis written by D.McAlpine, N.Compston and C.Lumsden
in 1955
1
. What had been a disease of individual case reporting during the 19th
century became one of the commonest reasons of admissions in neurological wards
in the early 20
th
century.
Since the first identification, multiple sclerosis (MS) has been studied by
various researchers of multiple disciplines with little insight in its etiology. The
advancement of knowledge in genetics and immunology has facilitated the
2
understanding about the disease process and nature of MS. Despite these
advancements the etiology of MS remains essentially unknown. The importance of
genetic susceptibility in the causation of MS is well established. Though genetic
susceptibility might be a required condition for MS, yet it certainly is not sufficient
to cause MS. MS is regarded to be a complex autoimmune disease that is caused by
gene-environmental interaction. Over the last decades some genes have been
identified with putative association with MS risk. The genes can be broadly grouped
into 1) immune related genes, 2) genes common to autoimmune disease and 3) genes
related to Myelin and Oligodendrocytes. Of all the susceptible genes HLA genes has
been most consistently detected in different studies. Other findings have rarely been
replicated by different researchers.
The environmental factors those are essential for onset of MS still remains
largely illusive. Similar to the genetic risk factors, the results on environmental
factors have often been inconsistent. Recent developments in immunology and
cellular neurology along with decades of frustrating search for causative factors of
MS has deviated the interest of the scientific community away from the
epidemiological search for etiological factors of MS. Different drugs that can affect
the immune system have been used to treat MS but the result is often not
satisfactory. The lack of knowledge regarding the causation of MS greatly limits the
development of preventive and curative treatment of Multiple Sclerosis.
3
Over the last few decades MS epidemiology has lagged in comparison to the
advancement in MS immunology or genetics. The challenges of MS epidemiology
are due to the nature of the disease itself. Epidemiological studies are plagued by the
problem of the long latent period between exposure and disease onset, the insidious
onset and the importance of gene-environmental interaction in the disease causation.
Misclassification of both disease and exposure are sources of errors in
epidemiological studies of MS. A cohort study can surely overcome the formal two
problems but given the rarity of MS that approach is not feasible. A well planned
case-control study is still the tool of choice. The former two problems can be
addressed by limiting recall bias. One way to achieve that is to either use some
biological marker that can assess the exposure or acquire data on the same subject
from multiple credible sources and use the verified data for analysis.
Planning case-control studies for MS should also consider the role of gene-
environmental interaction in the causation of MS. MS is unlikely to be due to genetic
factors or environmental factors alone but rather due to exposure to specific exposure
of genetically susceptible individuals. Comparing exposures in MS cases, which are
genetically susceptible to MS, to controls, which might not be genetically susceptible
to MS, will fail to show any association between the exposure and Multiple
Sclerosis. This might not be important in an ethnically homogenous population in
which the MS risk is similar across the population but surely is important in any
study of MS in U.S. and similar countries with heterogeneous population. In US
4
ethnic groups like ”White” encompasses both high risk ethnic groups like
Scandinavians, British and Germans as well as the mid-to-low risk groups such as
Spanish, French and Italians. Case-control studies that only matches on ethnicity will
surely fail to capture this intrinsic difference in MS risk among the case-control
pairs.
A case-control study involving twins can address these methodological
problems in the study of MS epidemiology. By verifying each twin’s responses by
the response form one’s co-twin can limit erroneous reporting and recall bias. Using
questions that verifies exposure within twins can be used as the tool for verification
of the affected twin’s response and thus limit recall bias. Another advantage of using
twins is attainment of increased leverage in risk. In a multifactorial disease like MS,
the array of risk factors (genetic and non-genetic) can be contemplated to form a
bell-shaped curve of risk in the population. Only a small portion of the general
population exposed to the extreme right end of the curve are susceptible to the
disease. However, among relatives of cases the curve is shifted to the right and the
degree of the shift is directly proportional to the degree of closeness to the index
case. The MZ co-twin of the case is exact genetic match and the DZ pairs are 50%
matched on genetic exposure. Comparing the risk factors among MZ discordant pairs
provide the unique scope to identify the environmental exposure that for MS by
controlling for genetic susceptibility. Comparison of level of exposure and timing of
disease among concordant pairs provides information regarding dose effect of the
5
exposure. The DZ pairs can serve as a contrast to compare the effect of an
environmental factor between MZ and DZ pairs to assess the importance of the
genetic factors. If an environmental factor is robust and can cause the disease with
little genetic susceptibility, then it will show similar effects both in MZ and DZ
pairs; whereas if it only acts in presence of the full spectrum of genetic susceptibility
then it will show a substantial effect among MZ but no or little effect among DZ
pairs. This analytical approach has been applied in this dissertation entitled “Role of
Environmental Factors in the Etiology of Multiple Sclerosis: A Twin Study”.
Our objective is to observe association between environmental or other non-
genetic factors that might be associated with MS risk, after controlling for genotype
through zygosity. We have used the North American Twin Registry of patients with
MS for that purpose. This is the largest twin registries of MS in North America. We
have baseline data on 1292 twin pairs with detail questionnaire data on 870 twin
pairs. We intend to tease out evidence for environmental risk factors from this twin
data, identify the risk factors and demonstrate their role in context of gene-
environmental interaction. The identification of environmental factors is important in
finding the specific genetic factors that are associated with MS risk.

6
1.2 Brief Descriptions of the Chapters
1.2.1 Chapter1: An Overview of the Dissertation

In this chapter I have briefly stated the background, importance and objective
of the dissertation. I have also provided a brief overview of the subject matter
covered in the individual chapters of this dissertation.
1.2.2 Chapter 2: A Clinical Overview of Multiple Sclerosis

This chapter describes MS in terms of its natural history, classification,
pathology, pathophysiology and method of diagnosis. The objective of this chapter is
to familiarize the reader with the disease and the disease process to appreciate the
complexity involved in the epidemiological studies of MS.
1.2.3 Chapter 3: A Review of Descriptive Epidemiology of Multiple Sclerosis

A comprehensive review of the existing descriptive epidemiology of MS is
discussed in this chapter. Here I have attempted to describe the differential risk of
MS by geographical location, race/ethnicity, gender, age and other demographic
factors with the hope to understand the inter-relationship between the different
demographic features of MS. Once we try to synthesize a single picture from all
these findings it becomes evident that both environmental and genetic factors plays
important role in MS etiology.

7
1.2.4 Chapter 4: A Review of Genetic Susceptibility of Multiple Sclerosis

This chapter provides a snapshot of the genetic research that is involved in
MS. In this chapter I have presented the existing genetic epidemiology involving
familial aggregations and twin studies that provide inkling in the nature of genetic
susceptibility in MS. However, the role of specific genes on MS risk has not been
discussed.
1.2.5 Chapter 5: A Review of Environmental Risk Factors in MS

This chapter deals with the current knowledge of the role of different
environmental factors in MS. Here I have discussed about the individual risk factors
such as infections, sun exposure, dietary factors, smoking, sex hormone and
chemical exposures.
1.2.6 Chapter 6: Paper I. Differential Concordance of Multiple Sclerosis among
Monozygotic Twins by Latitude of Birthplace: Role of genetic and non-genetic
factors (Islam T: Annals of Neurology 2006; 60(1):56-64)
This is the result from the first data analysis that has been done to address the
environmental issues in MS risk. We observed that risk of concordance varies by
birthplace among MZ twins but not among DZ twins. This signifies the necessity of
genetic susceptibility in MS risk. The abstract was presented as an oral presentation
at the 11
th
International Congress of Twin Studies at the University of Southern
Denmark, Odense, Denmark. The paper has been published in Annals of Neurology.
8
1.2.7 Chapter 7: Paper 2. Activities associated with Sun Exposure in Childhood
Modifies the Risk of Multiple Sclerosis among Monozygotic Twins (in Press
Neurology)
This chapter provides information on the second data analysis. We observed
a strong protective effect of sun exposure among MZ twins. The abstract was
presented at the 131
st
Annual Meeting of American Neurological Association. The
paper has been accepted for publication in Neurology.
1.2.8 Chapter 8: Grant Proposal: Role of sun exposure related genes in MS
Based on my finding of protective effect of sun exposure on risk of MS, I
plan to explore the role of genes involved in vitamin D metabolism and signaling
pathway on MS. This would be addressed by discordant dizygotic pairs and
monozygotic twin-sib pairs. I plan to employ a case-sib design to investigate this
association. As secondary aim, I’d also explore the role of HLA-DR2 haplotype and
childhood UVR exposure as possible modifier of the relationship between the genes
and MS.
9
Chapter 2 An Overview of Pathology, Clinical Presentation and Diagnosis
Multiple Sclerosis
____________________________________________________________________

2.1 Introduction
Multiple sclerosis is the most common demyelinating ailment of the western
world affecting the young adult population
1
. According to a National Multiple
Sclerosis Society, approximately 350,000 Americans suffers from MS. According to
an literature review the prevalence of MS in US was estimated to range from 39 to
173/100,000 with an incidence rate of 5/100,000 in 1996
2
. Using NIEHS
3
data the
prevalence was estimated to be 85/100,000 in US with an evidence of an increasing
trend among female. The prevalence rate of MS among female increased from
90/100,000 in 1982 to 130/100,000 in 1996 where as male rate slightly decreased
from 60/100,000 in 1982 to 40/100,000 in 1996.
Numerous articles are published every year documenting different clinical
aspects of MS. It is neither possible nor relevant to provide a detail account of the
clinical aspect of MS in this chapter. Here I would briefly address the issues that are
pertinent in conducting epidemiological studies of MS.
2.2 Clinical Presentation of Multiple Sclerosis
Multiple sclerosis (MS) is an autoimmune disorder involving the white
matter, specifically the myelin sheath covering of the axons; of the spinal cord and
10
Central Nervous System (CNS) and is clinically characterized by a relapsing-
remitting or progressive course with a pathologic triad of inflammation,
demyelination, and scarring of the CNS (gliosis). Selective destruction of myelin
sheath, localized inflammation and attempts of remyelination by the
oligodendrocytes (OG) marks typical ‘relapsing-remitting’ clinical pattern of
Multiple Sclerosis. The classical lesions of MS are disseminated in time and space;
affecting different parts of the brain at different time.
Symptoms, initial presentations and course of MS are unpredictable and vary
from person to person. The majority of the patients with MS experience an abrupt
presentation of symptoms and signs at onset. Thereafter, the clinical course may be
characterized by acute episodes of worsening (exacerbations or relapses), gradual
progression of disability, or combinations of both.
2.2.1 Initial Presentation of MS
The initial presentation of MS can vary widely between individuals and no
single symptom provides a diagnosis of MS. However, the pathognomonic feature of
MS is transient neurological symptoms due to effects of demyelination of CNS,
disseminated over time. Weakness or diminished dexterity in one or more limbs, a
disturbance of gait, optic neuritis, sensory disturbance, diplopia, Lhermitte’s sign and
ataxia (Table1) are the common initial presentation of MS. After the initial
symptoms the patient can totally recover from the symptom until a different episode
11
occurs after some time. According to Schumacher et.al.
4
, a ‘relapse’ is defined as the
new onset of a demyelinating symptom after complete remission of former symptom
for at least 1 month. According to different studies the rate of relapse can be 0.4-
1.1/year
5-10
. The relapse rate was found to decrease with duration of disease in a
Canadian study
11
where as no such effect was noted in a previous study
5
. With the
progression of the disease the MS patients can show a combination of different
symptoms. In cases where the onset is insidious or has trivial manifestations, the
diagnosis of MS is often delayed for variable length of time.
Table 2.1. Frequency of Clinical Symptom at Onset
Symptom % Symptom %
Sensory Loss 34 Vertigo 4
Weakness 22 Headache 2
Visual symptoms 13 Facial pain 2
Ataxia 11 Fatigue 2
Diplopia 8 Bladder 1

2.2.2 Clinical Classification of Multiple Sclerosis
The clinical course of Multiple sclerosis shows wide range of heterogeneity
similar to its initial presentation. According to a survey conducted by National
Multiple Sclerosis Society (NMSS-USA) MS in 1995, MS can be classified into 5
clinical and 3 severity based sub-groups
12
. The main clinical classifications are
described below.
12
Relapsing-remitting MS (RRMS): RRMS is characterized by clearly defined
relapses of symptoms with full recovery or with sequelae and residual deficit upon
recovery; periods between disease relapses are free of disease progression. Eighty-
five percent of MS cases at initial presentation are of RRMS type. Most of the cases
will overtime progress to secondary progressive MS (SPMS). MRI usually shows
many new and enhancing lesions.
Secondary-progressive MS (SPMS): This form is seen usually after 3 or more years
of RRMS onset. The detrimental effect of RRMS leads to a condition where disease
progresses with or without occasional relapses, minor remissions, and the condition
deteriorates overtime.
Primary-Progressive MS (PPMS): PPMS progresses downhill from onset with
occasional plateaus and temporary minor improvements. This is seen in 10-15% of
cases. Many patients never have a complete relapse. They usually get worse at a
steady rate and might reach a plateau. The progressive trend is often seen in male
older patients.
Progressive-Relapsing MS (PRMS): Approximately 5% MS patients show this
variety where a steadily worsening disease is noted from onset with clear acute
attacks and relapses.
13
Within each group patients might vary in the severity of disease. But two
distinct categories of MS are noted on the basis of severity, namely Benign and
Malignant MS.
Benign MS: In some cases of RRMS, no disability is noted even after 15 years of
disease. It is regarded as a temporal variant of the RRMS.
Malignant MS: In a small proportion of MS cases, young patients with
polysymptomatic onset progress to severe disability, death or both within months to
few years. In some cases death might be due to involvement of the brain-stem
13
.
2.3 Pathophysiology of MS
Understanding the pathophysiology of MS helps us better understand the
disease process and identify the different of aspects of a disease. It should be able to
explain the following issues:
1. The diverse clinical presentation in respect to symptom and severity
2. The characteristics relapsing-remitting nature of the disease.
3. The wide variability in outcome of similar type of brain lesion evidenced
by MRI.
4. The course of disease over years.
14
The main limiting factor in MS is the disruption of the myelin sheath. Myelin
sheath is the fatty tissue covering of the axons. A single Oligodendrocyte is
responsible for maintaining the myelin sheath for 30-40 axons. The myelin sheath is
analogous to the insulators around the electrical wiring. It helps in the conduction
electrical impulses across the neuronal nodes. Demyelination has either negative or
positive effects on axonal conduction. Negative conduction abnormalities consist of
slowed axonal conduction, variable conduction blocks that occur in the presence of
high- but not low-frequency volleys of impulses, or complete conduction block. This
is attributed to the reduced resistance and increased capacitance at nodes due to loss
of myelin
14
. The mechanism of conduction block appears to involve a
hyperpolarization of the resting axon potential due to the exposure of voltage-
dependent potassium channels that are normally buried underneath the myelin
sheath. This change can be detected by evoked potential. Positive conduction
abnormalities include generation of ectopic impulses, spontaneously or after
mechanical deformation, and abnormal "crosstalk" between demyelinated axons.
These problems in the impulse conduction lead to the clinical features of
Multiple Sclerosis. When remyelination occurs following initial demyelination the
conduction problem is temporally corrected and the patient goes into remission. As
the disease persists and different part of the white matter is affected the patient
shows different neurological symptoms after initial remission. Thus the MS patient
15
goes through a period of relapses and remissions. Overtime the Oligodendrocytes are
destroyed and remyelination becomes rare this leads to the progressive stage of the
disease.
2.4 Diagnostic Tests
There is no definitive diagnostic test for MS. The tests are used in association
with clinical presentation to confirm or reach definitive diagnosis. Magnetic resonant
imaging (MRI) is certainly the most important of all the available tests for Multiple
Sclerosis. MRI along with evoked response and immunoassay of cerebrospinal fluid
is used to help in attaining the diagnosis of MS when clinical presentation is not
conclusive.
MRI: Widespread availability of brain and spinal cord MRI has
revolutionized the diagnosis of MS.T-2 weighted MRI showing plaques as areas of
hyperintensities is the standard diagnostic tool for MS. Investigators have defined
criteria to separate MS lesions from other T-2 weighted hyperintense image
15, 16
.
Applying the diagnostic criteria developed by Fazekas et.al.
15
, the sensitivity and
specificity of MRI for MS diagnosis has been found to be 96% and 81%,
respectively
17
. However the classical MRI test is limited in identifying the
underlying pathological features of MS lesions. Different enhancements and types of
MRI are not currently in use routinely or are only used experimentally might help to
detect pathological nature of MS lesions. The use of contrast agent gadolinium-
16
DPTA
18
helps to define new active plaques. Gadolinium-DTPA is usually contained
by intact blood-brain barrier (BBB)
19
so its observation in the brain tissue is a sign of
compromised BBB which correlates with active MS lesion. MRI images with
gadolinium enhancement usually persist for less than 4 weeks
20
. Longitudinal studies
involving repeated MRIs often show that the disease activity in the CNS of MS
patients is much more frequent than observed by clinical presentation
20-22
. The
correlation between MRI measures and clinical status is even stronger with emerging
imaging techniques. Utilizing magnetization transfer imaging, the extent of axonal
loss in MS can be measured
23
. A very low magnetization-transfer ratio in an MS
lesion signifies substantial damage of white matter. The magnetization-transfer MRI
is also used to assess the total burden of MS lesions
24
. A low magnetization-ratio in
areas of apparently normal white matter was found to be a predictor of new lesion
sites
25, 26
.Another MRI technique, diffusion-weighted MRI can also predict new
lesion sites
27
. The molecular movement of water is measured in diffusion-weighted
MRI. A low diffusivity is noted when tissue retains structural integrity
28
. Proton MR
spectroscopy provides 4 different resonance from tetramethylamines; choline
containing phospholipids, creatinine, n-acetyle group and methyl resonance of
lactate
29
. By identifying these different metabolites the pathological events in MS
lesion can be understood. Increased resonance of lactate (inflammatory metabolite),
choline (from cell membrane after myelin breakdown) and decrease in N-
acetylasparate (only in neurons) mark the acute demyelinating phase of MS lesions
30
.
17
The remyelination is marked by normalizing of the N-acetyl state. All these different
types of MRI methods help in quantitative and qualitative assessment of the MS
lesions, whereas, the functional effect of lesions on brain can be assessed by
functional MRI
31
.
No single MRI method will provide the total story regarding the dynamic
phases of MS lesion. However, a combination of MRI analysis if done serially might
provide us with a more detailed picture of the MS lesion. Given the diversity in MS
pathology
32
and pathogenesis
13
it would be extremely beneficial in MS research to
develop MRI strategies that can identify the underlying pathology without any
invasive procedure.
Evoked Potential: The use of evoked potential testing in MS is reflects the effect of
demyelination on conduction of nerve impulse (Denny-Brown Brenner 44). Evoked
potential testing may detect slowed or absent conduction in visual
33
,auditory
34
,
somatosensory
35
, or motor pathways
36
. These tests use computer averaging
techniques to record the electrical response evoked in the nervous system after
repetitive sensory stimuli. One or several evoked responses are abnormal in 80 to
90% of patients with MS. Abnormalities in evoked responses occur with a variety of
neurological disorders that disrupt pathways being measured; thus, they are not
specific to MS. Testing is of diagnostic value when it provides evidence of a sub-
clinical second lesion in a patient who manifests only one abnormality on
neurological examination.
Cerebrospinal Fluid (CSF): CSF abnormalities consist of abnormally increased levels
of intrathecally synthesized IgG, oligoclonal banding, and mononuclear cell
pleocytosis. The ratio of IgG to albumin in the CSF divided by the ratio in the serum
is expressed as the “CSF IgG index". Two or more oligoclonal bands are found in 75
to 90% of patients with MS. Oligoclonal banding may be absent at the onset of MS,
and in individual patients the number of bands present may increase with time. It is
important that paired serum samples be studied to exclude a systemic origin of the
oligoclonal bands.
Other CSF abnormalities also occur but are less specific for MS. In one large
series, CSF mononuclear pleocytosis (>5 cells/ µL) was present in 25% of patients
with MS pleocytosis of >75 cells/ L or a finding of polymorphonuclear leukocytes in
CSF makes the diagnosis of MS unlikely. Pleocytosis is more common in young
patients with relapsing-remitting MS than in older patients with progressive forms of
MS. The total CSF protein content is usually normal or only slightly increased. A
protein elevation >100 mg/dL is rare and should prompt consideration of alternative
diagnosis such as an infection or tumor.
18
19
2.5 Diagnosis of MS
MS is a disease of clinical diagnosis without any pathognomonic sign or
symptom. This has resulted in synthesis of diagnostic criteria on the basis of
combination of clinical features and diagnostic tests. The criteria, overtime, have
incorporated the different available diagnostic tests to help MS diagnosis. The basic
criteria of MS diagnosis are evidence of demyelinating white matter lesions
disseminated in time and space. Documentation of two or more episodes of
symptoms with objective clinical evidence of white matter involvement is sufficient
for MS diagnosis without any requirement of MRI or other diagnostic tests.
Schumecher et.al.
4
provided one of the earliest diagnostic criteria. The ‘Poser
criteria’, formulated by a group of neurologists, became the standard in MS
diagnosis in the 1980’s and 1990’s prior to wide-spread use of MRI in clinical
diagnosis
37
. The availability of MRI has certainly revolutionized MS diagnosis and
has been extensively incorporated in the recent diagnostic criteria for MS developed
by the International Panel on the Diagnosis of Multiple Sclerosis
38
. The diagnostic
criteria recommended by the panel have been presented in Table 2.2. It should be
noted that the MRI evidence also needs to be disseminated in space (Table 2.3) and
time (Table 2.4).

20
Table 2.2: Diagnostic criteria for MS diagnosis (adopted from International Panel on the Diagnosis of
Multiple Sclerosis
38
Clinical Presentation Additional Data Needed for MS Diagnosis
Two or more attacks; objective clinical
evidence of 2 or more lesions
Nonea
Two or more attacks; objective clinical
evidence of 1 lesion
Dissemination in space, demonstrated by MRIb or Two or
more MRI-detected lesions consistent with MS plus
positive CSFc or Await further clinical attack implicating a
different site
One attack; objective clinical evidence of
2 or more lesions
Dissemination in time, demonstrated by MRId or Second
clinical attack
One attack; objective clinical evidence of
1 lesion (monosymptomatic presentation;
clinically isolated syndrome)
Dissemination in space, demonstrated by MRIb or Two or
more MRI-detected lesions consistent with MS plus positive
CSFc and Dissemination in time, demonstrated by MRId or
Second clinical attack
Insidious neurological progression
suggestive of MS
Positive CSFc and Dissemination in space, demonstrated
by1) Nine or more T2 lesions in brain or 2) 2 or more
lesions in spinal cord, or 3) 4-8 brain plus 1 spinal cord
lesion or abnormal VEPe associated with 4-8 brain lesions, or
with fewer than 4 brain lesions plus 1 spinal cord lesion
demonstrated by MRI and Dissemination in time,
demonstrated by MRId or Continued progression for 1 year
a. No additional tests are required; however, if tests [magnetic resonance imaging (MRI), cerebral spinal fluid
(CSF)] are undertaken and are negative, extreme caution should be taken before making a diagnosis of MS.
Alternative diagnoses must be considered. There must be no better explanation for the clinical picture.
b.MRI demonstration of space dissemination must fulfill the criteria derived from Barkhof et al
16
and Tintoré et
al
39
(see Table 2.1).
c.Positive CSF determined by oligoclonal bands detected by established methods (preferably isoelectric
focusing) different from any such bands in serum or by a raised IgG index.
40

d.MRI demonstration of time dissemination must fulfill the criteria listed in Table 2.2
e.Abnormal visual evoked potential of the type seen in MS (delay with a well-preserved wave form).

Table 2.3: New diagnostic criteria for magnetic resonance imaging determination of dissemination in
space
Three out of four of the following
1 1 gadolinium-positive lesion of 9 T2 hyperintense lesions
2 1 infratentorial lesion
3 1 juxtracortical lesion
4 3 periventricular lesions
(1 spinal lesion= 1 brain lesion)

Table 2.4: New diagnostic criteria for magnetic resonance imaging determination of dissemination in
time
1 Gadolinium-enhancing lesion demonstrated in scan done at least 3 months after onset
of a clinical attack at a site different from attack
Or
2 In the absence of gadolinium-enhancing lesion at the 3 month scan, follow-up scan
after an additional 3 months showing a gadolinium-enhancing lesion or new T2 lesion

If criteria indicated are fulfilled, the diagnosis is multiple sclerosis (MS); if the
criteria are partially met, the diagnosis is possible MS ; if the criteria are fully
explored and not met, the diagnosis is not MS.
2.6 Pathology of MS
The term “Multiple Sclerosis” was coined due to the presence of multiple
scarring areas (“plaques”) throughout the white matter of the CNS on microscopic
examination. This characteristic pathological feature of MS represents sharply
demarcated zones of hypocellular areas of white matter characterized by loss of
myelin sheath without any significant damage to the axons
41
. Oligodendrocytes (OG)
may or may not be affected in the early stage of the disease
32
.The OG Precursor cells
though could be found in the plaques at the early stages but are often inactive in the
chronic form of the disease
42
. Extensive remyelination is observed in acute and early
stage of MS but sparsely in chronic MS
41
. Plaques vary in size from 1 or 2 mm to
several centimeters. The size of plaques may appear larger due to merging of
multiple individual lesions. Although selective demyelination with sparing of axon
21
22
cylinders is the hallmark of MS, partial or total axonal destruction, and in extreme
cases cavitations, may also occur
43
.
Though the plaques can be found in any part of the CNS that has myelin
sheath, yet a predilection for optic nerve, periventricular area, brain stem, cerebellum
and spinal cord is observed in MS
41
. The site of lesion is marked by inflammatory
infiltrates and disruption of BBB without destruction of the blood vessels. The
plaques are usually centered on one or more blood vessels. The inflammatory
infiltrate consist of macrophage, different types of T-lymphocytes, B-lymphocytes,
plasma cell and possibly microglia. However, the inflammation is not limited to
areas of demyelination but also present in many parts of the so called ‘normal white
matter’ of a MS affected brain. This leads to the debate whether inflammation is
primary or secondary to demyelination.
2.6.1 Pathological Classification of MS
Despite the marked heterogeneity observed in the clinical presentation, MRI
imaging,
44
lesion pathology
45-47
and response to immunomodulatory treatment
48
, MS
is often considered as a disease with single pathogenic mechanism with varied
clinical presentation. However, In their seminal paper, Lucchinetti et.al.
32
reported
heterogeneity of demyelinating nature of MS lesions. Pathological and
immunological analysis of 51 biopsied and 32 autopsied brain tissues of diagnosed
active MS cases revealed 4 distinct patterns of demyelination, pattern I-IV. The
23
patterns can be broadly classified into two distinctive groups on the basis of tissue of
injury. Destruction of myelin sheath in presence of macrophage and T-cell was the
most prominent feature in pattern I and II where as destruction of oligodendrocytes
(OG) was the hallmark of pattern III and IV. The features of these four patterns are
presented in the Table 2.5.

Table 2.5 : Different patterns of Active MS lesions
Feature Pattern I Pattern II Pattern III Pattern IV
Inflammation
CD3 T cells ++ + + +
Plasma Cells + ++ + +/-
Macrophage + +/- +/- ++
C9neo Absent Present Absent Absent
Demyelination
Perivenous
pattern
Yes Yes No Yes/No
Defined Lesion Edge Yes Yes No Yes
Concentric pattern No No Some No
Oligodendrocyte
Apoptosis - - + -
OG in lesion ++++ ++++ + +
Myelin loss Even Even MAG↑↑ Even
Remyelination
Shadow plaques ++ ++ - -
Clinical Pattern
Acute MS 8.2% 27.4% 27.4% 0%
RR 1.4% 12.3 1.4% 0%
SP 1.4% 8.2 1.4% 0%
PP 1.4% 5.5% 0% 4.1%
C9neo=Activated terminal complement (Adapted from Lucchenetti et.al.
32
)

Pattern I and II were very similar except for the presence of antibody (mainly
IgG) and complement c9neo antigen in the lesions. The most pronounce feature that
distinguished patterns III and IV from the formers were the paucity of OG in the
24
demyelinating lesions and absence of remyelinating shadows. Apoptosis of OG was
observed in pattern III though not in pattern IV. It should be noted that selective loss
of myelin-associated glycoprotein (MAG) occurred only in pattern III.
The authors
32
noted no clear correlation between clinical and pathological
patterns of MS. Pattern II (53.4%) and III (30.1%) was the two most common
patterns observed, where-as pattern IV (4%) was the rarest. Pattern IV was only
observed among cases with primary progressive disease. Despite this heterogeneity
patterns were homogenous within an individual.
Despite the similarity in target tissue, pattern I and pattern II differed on the
basis of mechanism of injury. Activated T-cell and macrophage mediated destruction
of the myelin sheath is observed in pattern I where-as antibody and complement
system mediated inflammation is observed in pattern II. These patterns are similar to
those witnessed in EAE
49, 50
. The signs of oligodendrocyte dystrophy in pattern III
and IV are more likely to be caused by viral
51
or toxic
52
induced injury of the
oligodendrocytes. These dissimilarities among the different patterns points towards
existing multiple pathogenic mechanisms that can result in an MS like conditions.
The authors noted that demyelination patterns were identical for multiple
plaques in individual autopsied brains. As this was a cross-sectional study, it is not
clear from these findings whether the heterogeneity in demyelination patterns remain
25
the same throughout the disease process among MS cases. A recent publication by
Barnett et.al.
13
noted that the possibility of different pathological patterns might co-
exist. They initially studied the brain tissue of a young female (age=14 years), with
RRMS, who died within 24 hours of an acute new symptomatic brainstem lesion.
They noted demyelination similar to Pattern III, as described by Lucchinetti et.al
32
,
in this acute fatal case of MS. Given exceptional findings, they further studied 17
more brain tissues from MS cases. Similar pattern was noted in 6 more cases. These
lesions were similar to the Pattern III in respect to a) zones with apoptotic OG, b)
immunological feature and c) demyelination pattern but differed as they showed
remeylination shadows and active complement activation. Furthermore, these pattern
III type lesions were only a small fraction of the total number of demyelinating
lesions examined for any individual patient.
It is possible that pattern III not only defines a specific pathological pattern of
MS but also represents the features of early MS lesions in many cases of RR MS.
This observation of annihilation of OG at the very early stage of MS lesion prior to
destruction of myelin sheath challenges the existing concept of myelin sheath being
the first target in MS lesions
53, 54
.
These recent findings in MS pathology have immense effect in MS research
and management. A recent publication noted that patients with pattern II pathology
responds better to therapeutic plasma exchange compared to patients with pattern I
26
or III
55
. Similarly the etiology of MS might also differ by pathological patterns. In
the case of epidemiological studies it is virtually impossible to determine the
pathological pattern of MS cases through biopsies. It is essential to develop MRI and
other diagnostic processes to determine the pathological pattern of MS to study the
etiology of MS classified by pathological pattern. Longitudinal studies following
newly diagnosed MS cases over time are essential in the understanding of the
chronological pattern of pathological features of the MS lesions.
2.7 Pathogenesis of MS
Multiple sclerosis is believed to be a disease of multifactorial origin affecting
genetically susceptible people
56
. The predominant hypothesis regarding MS etiology
has been that autoreactive T-cells, formed outside CNS; mount a local inflammatory
cascade once they get access into the CNS, resulting in demyelination. The murine
analogue of MS, experimental autoimmune encephalomyelitis (EAE), is initiated by
immunizing mice with auto-neural antigens or by transfer of activated T-cells against
neural antigen. Their similarity to the true pathogenesis of MS is evidenced by the
successful development of therapies for MS cases based on these models
57, 58
.
Detection of increased frequency of myelin protein reactive CD4
+
cells, possibly
memory T-cells, in the blood of MS patients compared to healthy controls provides
evidence that sensitization to some antigens is also present in MS patients
59
.This
sensitization might be due to exogenous peptide with structural similarity to any of
27
those various neural antigens. Studies have shown that cross reactivity exists
between myelin protein and exogenous peptides
60
.
Figure 2.1 summarizes the important steps in MS pathogenesis. The disease
process comprises of an initial autoimmune mediated inflammatory reaction
61
,
followed by demyelination and simultaneous neurodegeneration and repair
62, 63
.The
autoreactive T-cells, that could have remained dormant for years, might be activated
in the periphery due to some environmental factors; possibly through molecular
mimicracy
64, 65
. The activated T-cells then migrate through the BBB after interacting
with adhesion molecules such as ICAM-1 and VCAM-1
66, 67
. Matrix
metalloproteinases (MMP), expressed by activated T-cell, astrocyte and microglia
68-
71
, can facilitate the migration of the T-cells through the BBB by dissolving the
extracellular matrix surrounding the BBB
72, 73
. The proinflammatory milieu (IFn- γ,
IL-12 and other cytokines from T-cell and APC) also up-regulates the expression of
the adhesion molecules and thus facilitate T-cell migration through the BBB
74
. After
migrating through the BBB, these activated T-cells binds with its putative neural
antigens , i.e. myelin basic protein, myelin-associated glycoprotein, myelin
oligodendrocyte glycoprotein (MOG), proteolipid protein, αB-crystallin,
phosphodiesterases, and S-100 protein, by the T-cell receptor (TCR) and class II
major-histocompatibility-complex (MHC) molecules on local APCs like microglia.
If this binding occurs in presence of proper costimulatory molecules (e.g., CD28 and
CTLA-4) and their ligands (e.g., B7-1 and B7-2) a cascade of proinflammatory
activity ensues.

Figure 2.1: Schematic Presentation of MS Pathogenesis

(Reprinted with permission from: trends of Pharmacological Sciences: Oliver
Neuhaus et.al.
75
)

This in turn further activates local phagocytes like microglia and astrocytes, and
recruits other immune cells. The inflammation mediated demyelination could be
28
29
achieved in multiple ways, such as direct T-cell or macrophage mediated injury,
demyelinating antibody from B-cells, complement deposition, cytokine mediated or
radical mediated such as NO or ROS. These processes can explain the first two of
the four demyelination processes described by Lassman et.al.
76
, i.e. 1) T-cell or
macrophage mediated, 2) complement system mediated, 3) distal
oligodendrogliopathy and oligodendrocyte apoptosis and 4) primary oligodendrocyte
degeneration. However, the mechanism for last two processes remains unknown.
Once the axons are partially or completely denuded of myelin sheath the
axons can no longer effectively conduct neural signals and this leads to the clinical
presentation of MS. Besides demyelination, axonal damage is often evidenced very
early in the disease process
77
, thus the question arises weather axonal damage is a
primary result of macrophage or T-cell mediated injury
78
or is a consequence of
ongoing demyelination process and increased vulnerability of the axons
79
.
Once the initial proinflammatory onslaught is over the local immune system
tries to limit the inflammation and destruction. As macrophages attempt to clear the
myelin debris, an increase in Th2 type cytokine such as IL-10 and TGF-B is noted.
The activity of the remaining OG and OG precursor increases and attempt of
remyelination takes place. The repair of the myelin membrane is further attempted
through resolution of the inflammatory response, spread of sodium channels from the
nodes of Ranvier to cover denuded axon segments and restore conduction, antibody-
30
mediated remyelination. This remyelination is clinically evidenced as the period of
remission.
It needs to be appreciated that heterogeneity exists in the pathogenesis in MS
as is evidenced by its pathological pattern. What we have described above is a
possible scenario but never the only scenario. The recent finding that widespread
destruction of OG occurs even prior to demyelination undermines the concept that
demyelination is the primary process in MS but rather it might be a consequence of
immune response to the primary destruction of OGs. Activated T-cells might come
in contact with neuroantigens outside CNS as neuroantigen can reach the cervical
lymph nodes via lymhatics
80
. The activated T-cells might require less co-stimulatory
signals and serve as APC
81
. Humoral contribution of B-cells can help in the
propagation of the disease
82
. The absence of regulatory cells, such a as CD25
+
CD4
+

and NKCD95, can increase disease susceptibility
83-85
. In short a varied form of
biochemical and auto-immune interaction culminates into multiple sclerosis.

31
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39
Chapter 3 A Review of Descriptive Epidemiology of Multiple Sclerosis

The distribution of disease in time, place and person is the basis of any
epidemiological study. Despite MS being a disease of unknown etiology, a wealth of
information is available regarding its distribution in time, place and person. Its
unique geographical distribution has intrigued researchers from the very beginning
but the factors responsible for the latitude gradient remains essentially unknown.
Detail investigation provides more complex scenario then a simple increase of MS
incidence and prevalence with latitude. A review of the descriptive epidemiology
might provide clues to untangle MS etiology.
3.1 Geographic Distribution

The apparent increase in incidence (IR) and prevalence (PR) rates of MS with
increasing latitude has been observed in both the hemispheres
1,2
. Noting the
geographical distribution of MS from multiple incidence and prevalence studies
across the world, Kurtzke et.al
3
defined 3 risk zones of MS, i.e. ‘High’, ‘Mid’ and
‘Low’. The ‘high’- risk zone (PR ≥30/100,000) includes Northern and central Europe,
Northern US, Canada, Southern Australia and New Zealand. The ‘mid’- risk area
(PR between 5-29/100,000) includes southern US, Southern Europe, Northern
Australia, north Mediterranean basin, South African whites and parts former Soviet
Union. The ‘low’ risk groups (PR<5/100,000) include most of the countries of Asia,
40
Africa, and South and Central America. Japan, which was earlier considered to be a
low MS risk zone (PR=2.5/100,000 in 1975) is now considered to be a member of
‘mid’ risk zone (PR=10.2/100,000 in 2002).
The earlier studies regarding the geographic distribution of MS are difficult
to compare due to methodological issues. Case ascertainment and case definition
varied between studies which made any efficient comparison, essentially impossible.
Many studies since the 1970s, conducted in Europe, Canada, Australia and US used
similar methodology and case definitions making temporal and spatial comparison
feasible. Analyzing the intriguing findings from these studies it becomes apparent
that some environmental factors that are not ubiquitous are associated with the risk
of Multiple Sclerosis. A closer scrutiny reveals a more complex pattern then the
often touted North-South gradient as the risk differed within small regions; higher
prevalence in Sicily but lower in Malta
4
, or same country ;the MS prevalence in
Sardinia is among one of the highest in the world where as the rest of Italy is
considered a ‘mid’ risk zone
5
. These findings do not rescind the notion of gradient
effect in MS but rather emphasizes the complex nature of MS etiology.
3.2 Racial Distribution

One of the highest rates of MS are noted among the Scandinavians and Colts
and the lowest rates among Asians and Africans
6
. The latitude gradient often fails to
describe these differences. This is evidenced by the differences in MS prevalence
41
noted in Japan and Britain. Both of these countries are at similar latitude with similar
health care systems, yet MS prevalence rate in the Great Britain
7-9
is 9-15 folds
greater than the prevalence rate in Japan
3
. In South Africa, MS is almost
unrecognized among the natives
10
where as it is prevalent among the settlers of
European origin
11
. Many ethnic groups, such as Lapps
12
, Native Americans
13
,
Australian Aborigines
14
,Saudis
15
, African Americans
16
, Hispanics
17
and others, show
low risk of MS despite living in regions of ‘mid’ to ‘high’ risk zones. Differential
distribution of MS by race within same geographic area signifies the importance of
genetic susceptibility and/or life style.
Drawing inferences from these racial and geographical distribution, the
genetic risk of MS is attributed to a’Viking origin’
18
.It is postulated that sometime in
the past some mutation occurred in the Nordic population that gave rise to a “MS
Risk” genome and it spread across the globe along the movement of the Vikings. The
higher rates of MS in the northern part of US can be partly explained by the
preponderance of people with North European origin in the northern parts of
US
16
.How-ever similar explanation fails to explain the latitude differences noted in
Japan
19
or Australia
20
.
3.3 Migration Studies
The concept of migration studies is simple: compare the IR and PR of a
disease of the migrants to the non-migrant population of the countries to which they
42
have migrated to and have migrated from. If the migrants maintain the rates of the
place they have migrated from then that’s suggestive of genetic risk of the disease.
On the contrary, if the migrant population attains the rates similar to the place they
have migrated to then that is in favor of an environmental risk of disease. Though
conceptually this seems an easy and straight-forward approach to tease out genetic
an environmental role in disease etiology, in practice this is hard to implement. A
migration study can provide credible data only if the immigrant population is a true
representative of the source population, randomly distributed, large in size, has
demographic data available and the access to medical facility is similar to country of
origin. However, often many of these conditions are hard to meet giving rise to
skewed outcome. This has been evidenced in a study of Vietnamese immigrants in
France
21,22
. Initial analyses showed that the MS risk among children, born of
Vietnamese mothers, who came to France before age 20 was comparable to the MS
risk of the French. But this finding ignores the fact that all of the children had French
father. The genetic admixture could be responsible for this increased risk.
Despite the limitations, some well planned migration studies have provided
important insight in MS etiology. Major works have been done by Dean et al. in
South Africa and Britain and by Alter et al. in Israel
23-27
. One of the major findings
of these studies has been the identification of the phenomenon that people who
migrate after a certain age tend to retain the MS risk similar to that person’s country
of origin, irrespective of the direction of migration form ‘high- low’ or ‘Low’ to
43
‘High’. The study of MS among Army veterans in US by Kurtzke et al.
28
revealed
that death from MS increased by 40% among veterans who were born in the South
but migrated to the North compared to those remained in the South. Those born in
‘high’ risk zone (northern states) but joined the army in the ‘mid’ or ‘low’ (southern
states) risk zone showed gradual decrease in MS risk. Similarly, veterans born in
‘mid’ risk zone showed increased risk if they joined army in ‘high’ risk zone and
decreased risk if they joined army in ‘low’ risk zone.
These studies establish the importance of environmental factors as a possible
modifier of the inherent ethnic susceptibility to Multiple Sclerosis. The ‘critical age’
is hard to discern but evidence shows it should be around ‘teen’ years and possibly
15 years or earlier
25,26,29-32
. The presence of a ‘critical age’ signifies the importance
of time of exposure for the causation of Multiple Sclerosis. It appears that
environmental factors operate during a window period at the early part of childhood.
Further evidence regarding the importance of childhood exposure is provided by
cluster analysis from Norway
33
. Maximum clustering was seen around 13-20 years
of age, similar to earlier findings.
However, findings from a recent migration study contradict the concept of
early ‘critical’ age of MS susceptibility. Hammond et.al.
20
compared the prevalence
rate of MS of UK (UKI) immigrants to Australia to born Australians (AUB).
Information on population distribution and birthplace was available from national
census. The authors estimated the prevalence of MS on June 30
th
, 1981. Cases were
44
initially ascertained through neurologist and then confirmed through interviews and
examinations. The age standardized PRs of AUB and UKI population were
compared, stratified by the geographic areas of Australia that differed in MS risk.
Both AUB and UKI populations showed similar rates for the same geographic areas
and an overall north-south (low to high) gradient. The prevalence rate was 46.3/10
5

(calculated from the region-specific standardized PR) for the UKI population
compared to 36.7/10
5
for the AUB and 99.4/10
5
the population of Southern England.
The PR did not differ by age at migration among the UKI population (Prevalence
OR: 0.96, 95% CI: 0.67-1.37). It appears that people who moved from UK to
Australia attained the risk of AUB population irrespective of age at migration. The
environmental factors that confer a protective effect on MS in Australia seems to
operate well outside the adolescent years.
3.4 Sex and MS
Like many other autoimmune diseases, MS susceptibility is greater among
female compared to male with a female to male ratio ranging between 2:1
34
to
2.7:1
35
. The most common course of MS, relapsing-remitting (RR-MS) is more
common in female than male and the common age of onset is during the 3
rd
and 4
th

decade of life. However, the primary progressive form has no gender bias with an
age of onset around late 5
th
decade
36
. The sex ratio is virtually 1:1 among PP
36
. It
appears that the age of highest risk of MS coincides with the active reproductive life
45
of females where as the increased MS risk among male coincides with the decrease
in testosterone level
37
. The disease pattern shows distinctive disease pattern in the
two sexes. The male sex is associated with unfavorable outcome in respect to
disability in MS
38
. In a study it was observed that the male cases were unable to walk
for more than 10m without rest within 25 years of disease onset where as women
reached that level of disability at 30 years of age. The finding remained unchanged
even when it was restricted to primary progressive course of MS.
3.5 Age at Onset
The age of susceptibility in MS strongly correlates with period of puberty and
active reproductive age. It is extremely rare before age 15, 5% before age 16 with
only 2% before age 10
39
. This study noted that The common age of onset is 20-40
years of age
40
. A recent study reported that MS was diagnosed after age 50
36
in only
4.6% of all MS cases. Of these 50% were of primary progressive type. All patients
older than 54 years of age at the time of diagnosis were of PP type.
The effect of genetic factors on age at onset is unclear. A recent study form
Scandinavia reported modest correlation of age at onset among affected sib pairs
41

(intraclass correlation was 0.28,p-value=0.03) which is suggestive of modest genetic
control on age of onset. Bulman et.al
42
also reported a modest correlation for sib
pairs but a strong correlation (0.90) among MZ concordant pairs suggesting genetic
loading in MS. This finding needs to be scrutinized as the MZ concordance was
46
calculated with only 7 concordant pairs. An intraclass correlation with only 7 twin
pairs is extremely vulnerable to strong association between any single pair at
extreme age points. As the authors did not show the data for the MZ pairs, though
they provided detailed data on all the non twin pairs, the effect of age differences of
the pairs could not be assessed. Another study from Canada, involving 62 sib-pair
did not find any correlation for age at onset among sibs(r= -0.09)
43
. Form the
existing evidence of age at onset the role of genetic loading is not clear.
3.6 Temporal trend of MS

Incidence rate is certainly the best frequency measure of any disease as it is
directly related to increase or decrease of a disease independent of treatment or
survival factors. Due to the problem of long latent period, rarity of the disease and
absence of ‘MS registry’ in most of the countries, temporal incidence data are rare in
MS. Most of the studies provide prevalence rates over time and an increasing trend
of MS is noted from these studies. These results are hard to interpret as increased
prevalence might not be due to increase of the disease itself but rather due to
increased survival or due to improved treatment. Some recent studies in US, Italy
and Scandinavia have employed the same diagnostic procedures over a period of
time to detect the trend in MS incidence rate. We will discuss some of those studies
here.
47
The Mayo clinic maintains a good surveillance system for detecting MS cases
in the Olmstead county of Minnesota. An increasing trend of MS in this region was
reported in an earlier publication
44
. A recent publication noted that the rate has
stabilized over the last two decades
45
. The age sex adjusted (1950 US population)
annual incidence rate of MS in the Olmstead County has been 4.4, 7.9, 6.2 and 7.1
per 10
5
over the period 1955-64, 1965-74, 1974-1984 and 1984-2000, respectively. It
appears the initial increase in MS rates during 1965-74 came down in the next
decade but is on the rise again. The initial increase might be due to the better
ascertainment of MS which is followed by a period of decrease in the rates as an
effect of earlier detection of cases in the previous period. The 1984-2000 period
marks the modern diagnostic era of MS as the Poser criteria of diagnosis was
introduced in the year 1983 and the use of MRI in MS diagnosis became prevalent.
However, the use of Poser criteria and MRI also decreases the false positive cases
over time and thus provides rates that otherwise would have been inflated. The shift
of the age structure of the recent population compared to earlier ones toward an
elderly population also artificially decreases the IR of MS. It was observed that the
age-sex adjusted PR in Dec 1, 2000 was 159.8/10
5
if year 1950 US population was
used as the reference population, where-as the PR was 191.2/10
5
for the same period
if the reference population was year 2000 US population. This substantial difference
in age-adjusted PR was due to the change in age-structure from 1950 to 2000.It
48
seems that the apparent stabilized rate might really be due to increase in MS rates
overtime and further follow-up will be required to observe the true trend.
Scandinavian countries are among the highest incidence and prevalence areas
of MS. Numerous studies have been conducted in these countries providing
invaluable data for MS research. Denmark is the only country with MS registry that
tracks cases since 1948. An increasing trend of MS incidence is observed in
Denmark since 1965
46
using regression method to correct the incidence rate for
reporting delay to the registry. The corrected IR for cases less than 40 years old
increased from 2.5/10
5
in 1965 to 10/10
5
in 1990 and the IR for cases diagnosed after
40 years of age increased from 1.9/10
5
to 4.0/10
5
during the same period.
Studies from other Scandinavian countries also show an increasing trend of
MS. MS registry was established in Oslo during the 1990s. In their first report they
show that the incidence rate for each quinquennium during 1972-1996 period
increased from 3.7/10
5
in the 1972-76 to 8.7/10
5
in the 1992-1996 period
47
. The
increase was more evident in relapsing-remitting type and among females. A recent
publication from Norway also showed increasing temporal trend of MS in Nord-
TrØndelag county
48
. All patients with MS from 1974-2000 was included in the
incidence study. Age adjusted annual incidence rate increased from 3.9/10
5
in 1974-
1978 to 5.1/10
5
in 1994-1998.The incidence rate among women increased more
compared to men, with the highest rate being 10.2/100,000 in 1984-88. Gender
49
stratified analysis show an increase in the rates among females but not among males.
Similar increasing trend was also noted in MØre and Romsdal county
33
.
Population based incidence study in Sweden showed that the crude incidence (1988-
1997) and prevalence (1997) rate was 5.2/10
5
and 154/10
5
at
Västerbotten
49
.Compared to the prevalence rate of 125/10
5
during 1990, a yearly
increase of 2.6% in PR was observed. This increase in the prevalence was attributed
to the increased number of new cases (n=105) compared to the deceased cases
(n=40).The crude incidence rate at Västerbotten for 1990-1997 was two fold greater
compared to the available incidence rate from Göteborg
50
, another Swedish region,
for 1974-1988. Due to use of similar methodology these two rates are eligible for
comparison. The difference in the rates can be due to temporal, spatial or both.
Differential temporal and spatial variation was noted in a recent Finnish study
51
. The
study was conducted in three Finnish districts of Vassa, Seinäjoki and Uusimaa
assessing the MS rate during 1979-93. Using indirect method the standardized
incidence rates (SIR) were utilized to compare the secular trend of MS in the three
districts. An increasing trend was noted in Seinäjoki district, the rates were 1.8, 2.2
and 2.8/10
5
during the periods of 1979-1984, 1985-1988 and 1989-1994,
respectively. During the same periods the SIRs decreased in Vaasa from 1.5 to 0.9
to 0.6/10
5
. and remained stable in Uusimaa ranging from 1.0-0.9/10
5
. The increase in
Seinäjoki was marked among the men and the decrease in Vassa was mainly in
women (Figure 3.1).
Figure 3.1: Sex-period specific crude Incidence rate of MS for 3 Finnish regions (with permission
from M.-L. Sumelahtia et.al.I
51
)

Earlier studies have defined Italy as a ‘mid’ risk zone for MS, however recent
studies
52,53
, evaluating incidence and prevalence rates in different parts of Italy have
shown prevalence rate as high as 69.4/10
5
excluding Sardinia where the PR is even
higher, 143.9/10
5 54
. In Catania (a city in Sicily) an increasing trend was noted during
the period 1975-1994. The average annual incidence rate increased from 1.3/100,000
in 1975-79 to 3.9/100,000 in 1990-94
53
. This increase in MS rate parallels the
decrease in the lag between disease onset and diagnosis. The average lag was
8.6±5.6 years in the first quinquennium (1974-79) and 1.9±2 years in the last
50
51
quinquennium (1990-94). The increase in IR from 1974 to 1994 thus could be due to
earlier diagnosis of MS cases. Similar increase in MS rate with parallel decrease in
lag time was noted in Sardinia
55
. In a recent publication Pugliatti et.al.
55
presented
the natural history of MS in Sassari, a northern province of Sardinia, over a period of
35 years (Jan1,1965-Dec 31,1999). The study is unique given the population
characteristics and findings. The population was estimated to be around 0.4 million
in 2001 with a modest rate of migration (~1.6%).As the migration was mostly within
Sardinia, ethnically the population was essentially homogenous. The population
could be sub-grouped into 7 distinct linguistic and cultural sects. The cases were
ascertained through MS registry and defined using Poser criteria. Age adjusted IR
was calculated by adjusting for 2001 Italian population for each quinquennium
starting from 1965. The IR increased from 1.1/10
5
for 1965-1969 to 6/10
5
(apx.) for
1985-1999.Similar trend was observed for each of the 7 linguistic subgroups though
significant difference in IR existed within those subgroups. This increase in the IR
was marked by the increase of RRMS. The prevalence of RRMS increased from
61% in 1965-1969 to 90% by 1999. This shift in disease pattern was accompanied by
statistically significant increase in the age at onset from 25.7 to 30.6 years, decrease
in lag time between disease onset and diagnosis from 13.0 to 0.9 years over the same
period and no difference in age at onset between RRMS and PPMS. If the increase in
IR was solely due to early diagnosis of MS cases then it should not have been
associated with the observed changes in the disease pattern. It appears that over time
52
some changes in the region has lead to an increased susceptibility to RRMS and the
disease is acquired at an older age then expected. Given the small period of time
and the homogenous nature of the study population, these changes are more likely to
be of environmental origin rather than genetic. It can be postulated that people in
these regions are being exposed to some unknown environmental factors, possibly at
an older age, resulting in acquiring RRMS and at an older age.
These incidence studies along with numerous prevalence studies have shown
an increasing trend of MS, especially in women and RRMS type. Still the question
remains if the increase is due to true increase in disease occurrence or due to better
case ascertainment. The decrease in the lag time between disease onset and diagnosis
certainly provides innuendo of better case ascertainment. Application of Poser’s
diagnostic criteria and MRI certainly has great influence in MS diagnosis. If the use
of these diagnostic tools detect earlier cases than phenomenon similar to ‘feast and
famine’ should be witnessed in the after years. No study has reported a spike like
increase of MS risk following the introduction of either Poser’s diagnostic criteria or
MRI followed by a decline of MS risk. The Finnish study, showing three different
types of trend in three districts using the same methodology, implies that non-
methodological factors play important role in the trend of MS. Two Italian regions,
Sassari
55
and Ferrara
52,56
, are similar for the decreasing trend of lag period between
age at onset and diagnosis yet are different for the MS incidence trend. The rate has
remained stable for 2 decades since 1975 in Ferrara, where as the rate has
53
significantly increased in Sassari. Given these finding, it is highly likely that an
increasing trend in MS is currently in the process. However, it should be recognized
that the increasing trend in MS is not as pronounced as other autoimmune diseases
57
.
This might be due to the fact that 1) MS is a relatively rare disease 2) changes in
diagnostic criteria make it difficult to discern the true temporal effects from
diagnostic bias and 3) long lag time between exposure and disease.

3.7 Summary
The descriptive epidemiology of MS reveals the core essence of MS etiology:
no single pattern can be established unless all the evidences are considered together.
Once the findings from geographic, racial and migration studies are evaluated along
with the age and gender pattern of MS, it impeccably points to a disease that occurs
mainly in genetically susceptible persons who are exposed to certain unknown
exposure in the early part of life. The incidence and prevalent studies provide
sufficient evidence of fluctuation in MS risk over time. The health care system or
change in disease definition fails to explain the secular trends noted in the above
mentioned studies. The secular variation in MS risk signifies the role of
environmental factors in MS risk. Given the propensity of changes in RRMS and
females in most of the studies it appears that the environmental factors mainly affect
RRMS course of the disease and not PPMS.
54
Numerous studies defining the descriptive nature of the disease has purported
a number of etiological hypotheses, most of which remains to be proved or
disproved. Analytical studies are required to test those hypotheses. Well planned
epidemiological studies have the potential to unveil the etiological factors of
Multiple Sclerosis.
55
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21. Delasnerie-Laupretre N, Alperovitch A. Migration and age at onset of
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26. Dean G, Kurtzke JF. On the risk of multiple sclerosis according to age at
immigration to South Africa. Br Med J. 1971;3:725-729.

27. Dean G, McLoughlin H, Brady R et al. Multiple sclerosis among immigrants
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28. Kurtzke JF, Beebe GW, Norman JE, Jr. Epidemiology of multiple sclerosis in
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29. Alter M, Leibowitz U, Speer J. Risk of multiple sclerosis related to age at
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30. Alter M, Okihiro M. When is multiple sclerosis acquired? Neurology.
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33. Midgard R, Riise T, Nyland H. Epidemiologic trends in multiple sclerosis in
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34. Whitacre CC, Reingold SC, O'Looney PA et al. BIOMEDICINE:A Gender
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37. Moretti C, Frajese GV, Guccione L et al. Androgens and body composition in
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38. Confavreux C, Vukusic S, Adeleine P. Early clinical predictors and
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40. Poser S, Raun NE, Poser W. Age at onset, initial symptomatology and the
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42. Bulman DE, Sadovnick AD, Ebers GC. Age of onset in siblings concordant
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43. Warren SA, Warren KG. Influence of gender on susceptibility to multiple
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44. Wynn DR, Rodriguez M, O'Fallon WM, Kurland LT. A reappraisal of the
epidemiology of multiple sclerosis in Olmsted County, Minnesota.
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45. Mayr WT, Pittock SJ, McClelland RL et al. Incidence and prevalence of
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46. Sille Esbjerg NKNK-H. Reporting delay and corrected incidence of multiple
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48. Dahl OP, Aarseth JH, Myhr KM et al. Multiple sclerosis in Nord-Trondelag
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49. Sundstrom P, Nystrom L, Forsgren L. Incidence (1988-97) and prevalence
(1997) of multiple sclerosis in Vasterbotten County in northern Sweden. J
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50. Svenningsson A, Runmarker B, Lycke J, Andersen O. Incidence of MS
during two fifteen-year periods in the Gothenburg region of Sweden. Acta
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51. Sumelahti ML, Tienari PJ, Wikstrom J et al. Regional and temporal variation
in the incidence of multiple sclerosis in Finland 1979-1993.
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52. Granieri E, Malagu S, Casetta I et al. Multiple sclerosis in Italy. A reappraisal
of incidence and prevalence in Ferrara. Arch Neurol. 1996;53:793-798.

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57. Cooper GS, Stroehla BC. The epidemiology of autoimmune diseases.
Autoimmun Rev. 2003;2:119-125.

60
Chapter 4 Genetic Susceptibility of Multiple Sclerosis
____________________________________________________________________
4.1 Introduction

The search for the etiology of multiple sclerosis (MS) has been a battle of
Nature vs. Nurture. In the previous chapter we have discussed the part of the
descriptive epidemiology that emanates the idea of environmental origin of MS. A
review of different aspects of MS epidemiology points towards an interplay between
genetic and environmental factors in the causation of MS. It seems that both genetic
and environmental factors are necessary for the development of MS but nether is
sufficient. Data from monozygotic twin’s best exemplifies the situation. The lifetime
risk of MS for a MZ co-twin of an MS case is 20-30 fold greater than the risk of MS
in general population yet more than 70% of the co-twins of MS cases remain disease
free
1
. If MS was a purely genetic disease than the concordance of MS among MZ
twins would be close to 100% on the other hand if it had no genetic basis than the
risk for the co-twin of MZ case would be slightly higher than the risk in general
population and the co-twins of MZ and DZ cases would be similar due to sharing of
exposure. But in MS the concordance among MZ is 3 to 4 fold of DZ concordance
1
.
Bolstered by the descriptive epidemiology, several studies have been carried
out to detect the gene/s involved in Multiple Sclerosis. Several respectable
researchers across the globe have identified different genes associated with MS but
those results have rarely been replicated. Genes of Major Histocompatibility
61
Complex (MHC) is the only group that has been consistently shown to be associated
with Multiple Sclerosis consistently for over 30 years
2-5
. Recent full genome screen
has revealed other possible sets of genes of interest in Multiple Sclerosis
4,5
. Besides
by using new methods like protein analysis and Reverse RNA analysis the genetic
research in MS has been enhanced. Despite the failure to detect the causative gene/s
for MS, a vast pool of information of epidemiologic sources provides an insight to
the genetic basis of MS.
4.2 Familial Aggregation
4.2.1 Familial Recurrence
Different measurements of familial recurrence or recurrence risk are used in
epidemiological studies to assess the risk of a disease for the family members of the
proband. It is especially important in case of a disease like MS which has a strong
genetic predilection. Familial recurrence risk is the proportion of families of
probands with at least a second case of MS. It is a crude measurement and does not
provide a lifetime risk for the family members of the proband as is provided by the
age adjusted recurrence risk. Studies have reported the recurrence risk of MS among
family members using different measurements and different methodologies making
the comparisons often difficult.
The use of age adjusted recurrence risk is important especially when a
significant portion of a familial group is in an age range where the risk of MS still
62
persists. Sadovnick et al.
6,7
reported from their Canadian study that the crude and
age-adjusted recurrence rates for parents were 2.9% and 2.9% respectively. Where-as
the same rates for children were 0.4% and 2%, respectively. Any further risk of MS
among parents is negligible given their age and that is reflected by the similarity of
crude and age-adjusted rates but the children are in the age range where MS
occurrence is possible and this is reflected by the disparity in the crude and age-
adjusted rates in the two groups.
Familial recurrence rates are available from different clinic or population based
studies in UK
8
, Canada
6,7,9
, Belgium
10
and Italy
11
. All of these studies were
sufficiently robust and methodologically sound to provide stable estimate of the
recurrence risk. The Italian and the British study were based on population based
registry data with 324 and 674 probands, respectively. The Canadian (815 probands)
and the Flanders (674 probands and 21,351 1
st
2
nd
and 3
rd
degree relatives) study
were based on consecutive patients attending MS clinics. Despite the differences in
methodology, geographic region and ethnic population, the rates from these different
studies were comparable. The stepwise increase in the recurrence risk with
increasing levels of genetic sharing supports a polygenic disease etiology (Figure
4.1). The role of environmental factors is also evident from this association. Though
offspring of conjugal MS pairs and MZ twin pairs potentially shares 100% of the
disease genome yet the recurrence risk is almost 40% (20-12/20) greater among the
MZ pairs as MZ pairs are more likely to share similar environment. Similarly, all the
1
st
degree relatives share 25% of the genome yet the risk is greatest among the DZ
pairs and the sibs as they are more likely to share similar environment at a younger
age compared to parent-child pairs.

Figure 4.1: The association between familial recurrence risk of MS and percent of genetic sharing
among different relatives and general population.
0
5
10
15
20
25
General
population
Adoptee 2nd degree parents sibs offspring DZ Both Parent
MS
MZ
Fam ilial R ecurence (% )
0
20
40
60
80
100
120
Probability of sharing com m on 'risk' genom e

The prevalence of the risk genome has been assumed to be 5%.The ‘blue’ line denotes the probability
of sharing same genome and the maroon bars represent the familial recurrence rate.

Increased risk of sibling recurrence has been shown to be associated with
parental MS status in two studies
12,13
. Both of these studies involved MS patients
who attended the Canadian MS clinics. Both studies noted that the recurrence risk
for the sib of an index case increased by at least 2 fold for being female (OR=2.2-
63
64
2.9) or having one parent with MS (OR=2.0-3.5). The recent report included
information on 301 parents and 878 sibs of 301 male index cases and 782 parents
and 2234 sibs of 782 female index cases. The crude sib recurrence rate was 4.9%
(43/878) for sibs of male cases and 3.5 %(79/2234) for sibs of female cases. This
increase recurrence risk among sibs of males, the less affected gender, supports the
multifactorial nature of the disease. Analyzing data according to age of onset for
index case, parental disease status and sex of the sib, highest lifetime risk of 15.9%
was noted for female sib of young cases (age at diagnosis <20 years) with at least
one parental case of MS. Stratifying the sib recurrence risk by parent specific disease
status notes preferentially increased risk among sibs with affected mother compared
to those with no affected mother (Table 4.1). However, total absence of any father-
daughter pair with affected sib is intriguing and raises question weather differential
follow-up had occurred by parent-child pair.

Table 4.1: Crude sib recurrence rate according to gender of index case, stratified by parental MS
status
1
Male Index Case Female Index Case All Index Case
Brothers Sisters Brothers Sisters Brothers Sisters
ALL
None 3.27 3.93 1.53 4.89 2.15 4.64 3.4
Mother 7.02 19.05 5.45 8.18 6.02 12.14 9.1
Father 5.88 5.56 0.00 1.47 1.09 2.33 1.1
1Calculated from Sadovnick et.al.
12

65
Hupperts et al.
14
utilizing a population based MS registry of United Kingdom
and Ireland reported that the pattern of disease does not substantially differ between
different parent-child pairs. The sib recurrence risk of MS among 216 parent-child
concordant pairs was same irrespective of parent-child gender pattern. The
distribution of genders specific concordant parent child pair was essentially same as
that of the Canadian study, i.e. mother-son=19%, mother-daughter=55%, father-
son=9% and father-daughter=18%.Parent-of-origin effect was also neither observed
for age at diagnosis, disability or disease course. However, the sons of the father-son
pairs were twice more likely to suffer from primary progressive course compared to
the child member of any other combination.
No paucity in father-son pair was also observed in a population based
Dannish study. MS cases (n=8,205) was identified through Danish Multiple Sclerosis
Register. Their 1
st
degree relatives (13,316 offspring, 2,710 siblings, 3,556 parents
and 33 twins) were identified through Civil Registration System and followed till
MS diagnosis, emigration, death or December 31
st
, 1997, whichever came first. No
paucity in father-son pair was observed in this cohort rather both sons and daughters
of MS affected father had same increased risk compared to general population (OR:
7.5 95% CL: 5.2-10.8). The findings from this cohort data provide strong evidence in
support of multifactorial nature of MS etiology and limited importance of adult
environment in MS etiology. The familial risk was consistently higher among the
male relatives compared to female and among relatives of male cases compared to
66
female cases. This follows the cannons of mutifactorial disease which states that the
risk of the disease (MS) is higher among relatives of cases of uncommon gender
(Male MS case) in presence of a gender asymmetry in disease risk (i.e., female
preponderance of MS)
15,16
.
4.2.2 Conjugal Multiple Sclerosis
Conjugal MS pairs with children provide another unique situation to study
both the genetic and environmental effect of MS within this fixed family set. Given
the rarity of MS, studies involving conjugal MS pairs are rare. Till date two studies
have been reported, one from UK
17
and another from Canada
18
.
The UK
17
study involved 86 children of 45 conjugal pairs from recorded in a
national register. Five of the 86 offspring had clinically diagnosed MS and another 4
children had history of relapsing neurological dysfunction. The crude recurrence risk
of 5.8% (5/86) for conjugal offspring was approximately 9.7 times of the offspring
recurrence rate (0.6%) in the general population of UK. Of the 45 conjugal pairs, 33
pairs were discordant at the time of first acquaintance. No significant similarities in
disease course, presentation or disability were noted among these 33 pairs. Age of
diagnosis was also similar to that of the general population.
A similar data is also available from the Canadian Collaborative Study
Group
18
. This study involved 26 conjugal pairs identified through the MS clinics in
Canada, along with their 46 offspring. Six of these 49 offspring had MS with a crude
67
risk of 12.2% which is approximately 17 times of the offspring recurrence risk for a
single affected parent family in Canada. The age adjusted risk show an 11 fold
increase in risk for the offspring of concordant MS parents compared to offspring of
discordant MS parents.
This substantial difference in the offspring rates by parental MS concordance
underlies the possible oligogenic or polygenic genetic model of MS. The lack of
influence of spouse’s disease status on course of disease of the 2
nd
MS case of the
conjugal pairs fortifies the findings from the migration studies that the window
period of MS susceptibility must be prior to adulthood.
4.2.3. Half-Sib Risk
The recurrence risk of MS among half-sibs provides a unique opportunity to observe
the ‘parent-of-origin’ effect as well as relative role of gene and environment. Half-
sib data on MS is available only from the Canadian Collaborative Study group
19
.
Using the Canadian MS clinic 1567 index case with at least one maternal or paternal
half-sib was identified. MS status and current age was available on 3,436 half-sibs
and 2,706 full –sibs of these index cases. The observed age-adjusted recurrence risk
was 3.11% for full-sibs and 1.89% for the half-sibs. Observing that the recurrence
risk for half-sibs was not less than half (3.11/1.89=0.61) of the recurrence risk for
full-sibs the authors concluded that the finding does not support an
oligogenic/polygenic risk model of MS. However, this conclusion is not warranted as
68
it can be shown that given the relative commonality of the only known MS
susceptibility allele sequence in the MHC region, i.e. the major HLA-
DRB1*1501
20,21
among people of North European origin (www.allelefrequencies.
net), the joint probability of receiving both the haplotype and a ‘MS’ risk allele
would be 11% in full-sibs 5.4% in half-sibs. On that basis the ratio of half-sib to full-
sib recurrence is estimated at 0.49, well within the observed value of 0.61. Further it
should be noted that once the role of environment and its interaction with gene is
considered the finding rather confirms the presence of polygenic/oligogenic risk
modality.
The other finding from this half-sib study was the significant parent-of-origin
effect (p-value: 0.05).The age-adjusted recurrence risk for maternal half-sibs was
2.35% (95% CI: 1.6-3.1%) compared to 1.31% (95% CI: 0.65-1.96%) for paternal
half-sibs. This parent-of-origin effect bolsters the Canadian group’s earlier finding of
this effect among parent-child concordant pairs
18
.The presence of an asymmetrical
effect of parental disease status on offspring can be due to environmental factors
related to maternal exposure prior to or during pregnancy, in utero condition,
maternal health and diet and breast feeding, and genetic factors like genetic epistasis,
imprinting and mitochondrial genes.
69
4.3 Twin Studies
Classical twin studies provide comparison of concordance between MZ and
DZ pairs to assess the role of genetic factors on disease causation. MZ twins are
genetically identical whereas DZ twins, like full sibs, share only 50% of the genome.
In a disease of genetic etiology, the concordance ratio between MZ and DZ should
be high. A MZ-DZ ratio around 2 is expected for diseases caused by single
autosomal dominant gene as the DZ are expected to share the gene half the time. The
ratio approaches 4 in a recessive genetic model. In oligogenic or polygenic disease
the ratio is expected to be much higher. DZ pairs differ from sib pairs by virtue of
having more common environmental exposure. Thus high concordance ratio for DZ-
sib comparison denotes an environmental effect.
Numerous studies have reported concordance of MS among twins, dating as
far as 1930s. The earlier studies were often small in size, not population based and
employed different diagnostic criteria making it hard to compare across studies (for
further reading please review the classical text book of MS, Multiple Sclerosis by
McAlpine). Since 1978, several twin studies with a population based approach have
reported MS concordance among twins. The findings of some of those studies have
been summarized in Table 4.2. All the studies show a high MZ concordance relative
to the DZs. The MZ and DZ concordance in the French and the Italian studies show a
lower concordance compared to the studies from Northern Europe and Canada. The
MZ-DZ concordance ratio ranges from 2-6.5. Except for the findings of Bobwick et
70
al
22
, no study has reported concordance beyond mid twenties for MZ and single digit
for DZ twins. Among the 2 concordant pairs identified in by Bobwick and
colleagues
22
, only 1 was definite MS where as the other putative concordant co-twin
had a single episode of retrobulber neuritis. Excluding that case yields MZ
concordance of 20%.

Table 4.2: Pairwise MS concordance among MZ and DZ twins
Concordance (%) Author MZ+DZ
pairs
MZ DZ MZ:DZ
Region Method
Bobowick et al
22
5+4 40.0 0.0 NA. US Military registry
Heltberg et al
23
19+28 21.1 3.6 5.9:1 Nether
-lands
Registry of twins and MS
Sadovnick et al
24
26+43 30.8 4.7 6.5:1 Canada MS clinic network
Kinnunen et al
25
7+6 28.6 0.0 NA. Finland Finnish twin Cohort
French MS Study
26
17+37 5.9 2.7 2:1 France Public appeal
Willer et al
1
133+221 18.0 4.1 4.3:1 Canada MS clinic network
Italian
27
198* 14.5 4.0 3.6:1 Italy
*Authors did not provide data by zygosity. The concordance rates for MZ was 22.2% and none for DZ. This is
from published abstract as no article has been published.

The lower rates from France and Italy are suggestive that the concordance
might vary by regional risk of MS. The MZ concordance can vary by region only in
presence of a strong environmental component in MS etiology or genetic
heterogeneity that varies by region. Though genetic heterogeneity is possible but it is
unlikely to differ by region.

71
The focal points of the family and twin studies can be summarized as:
1. MZ concordance risk is more than 100 times of background risk
2. Majority of the MZ twins are discordant (70-80%)
3. The concordance rate is significantly higher in MZs compared to DZs
4. The fall of concordance rate from DZ →1
st
degree →2
nd
degree → 3
rd

degree → background risk is gradual (approximately half for each distance)
(Figure 4.1).
5. The offspring concordance rate of conjugal MS pairs are similar to MZ pairs
6. The adoptee risk is similar to background risk
7. Half-sib risk is about half of full-sib risk
Several conclusions can be drawn from these findings. The importance of the genetic
factors in disease causation can be observed from 1, 4, 6 and 7. Genetic factor
increases susceptibility, even disease causation might be impossible without it
28
, but
that itself is not sufficient to cause disease [2]. The gradient of risk in different
relatives [3, 4, 5 and 7] suggests that MS risk is conferred possibly by multiple genes
that have independent and/or epistatic effect.
72
Chapter 4 References

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recurrence rates in multiple sclerosis. Proc Natl Acad Sci U S A.
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2. Bertrams J, Kuwert E, Liedtke U. HL-A antigens and multiple sclerosis.
Tissue Antigens. 1972;2:405-408.

3. Jersild C, Fog T, Hansen GS et al. Histocompatibility determinants in
multiple sclerosis, with special reference to clinical course. Lancet.
1973;2:1221-1225.

4. Ban M, Sawcer SJ, Heard RN et al. A genome-wide screen for linkage
disequilibrium in Australian HLA-DRB1*1501 positive multiple sclerosis
patients. J Neuroimmunol. 2003;143:60-64.

5. A meta-analysis of whole genome linkage screens in multiple sclerosis. J
Neuroimmunol. 2003;143:39-46.

6. Sadovnick AD, Baird PA. The familial nature of multiple sclerosis: age-
corrected empiric recurrence risks for children and siblings of patients.
Neurology. 1988;38:990-991.

7. Sadovnick AD, Baird PA, Ward RH. Multiple sclerosis: updated risks for
relatives. Am J Med Genet. 1988;29:533-541.

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sclerosis: a geographically based study: 8: familial multiple sclerosis. Brain.
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10. Carton H, Vlietinck R, Debruyne J et al. Risks of multiple sclerosis in
relatives of patients in Flanders, Belgium. J Neurol Neurosurg Psychiatry.
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73
11. Montomoli C, Prokopenko I, Caria A et al. Multiple sclerosis recurrence risk
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12. Sadovnick AD, Yee IM, Ebers GC. Factors influencing sib risks for multiple
sclerosis. Clin Genet. 2000;58:431-435.

13. Sadovnick AD, Yee IM, Ebers GC, Risch NJ. Effect of age at onset and
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14. Hupperts R, Broadley S, Mander A et al. Patterns of disease in concordant
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15. Fraser FC. The multifactorial/threshold concept -- uses and misuses.
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16. Chakraborty R. The inheritance of pyloric stenosis explained by a
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21. Modin H, Olsson W, Hillert J, Masterman T. Modes of action of HLA-DR
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22. Bobowick AR, Kurtzke JF, Brody JA et al. Twin study of multiple sclerosis:
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25. Kinnunen E, Juntunen J, Ketonen L et al. Genetic susceptibility to multiple
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75
Chapter 5 Environmental Risk Factors of MS
____________________________________________________________________
5.1 Introduction
In the light of the available genetic and descriptive data, as we have reviewed
in the earlier chapters, MS etiology can be postulated to be an outcome of a complex
interplay between different environmental and host factors under some specific
conditions. In this chapter I will review environmental factors that are associated
with MS.
5.2 Infection

In the earlier years MS was conceived to be a disease of infectious origin.
However, it is no longer perceived to be an infectious disease though infectious
agents are often investigated as potential risk factors due to their interplay with the
immune system. Till current, no infectious agent has been detected in any MS lesion.
However, this absence does not prove that infectious agents do not play any role in
MS etiology. Several indirect epidemiological evidences support the hypothesis that
infectious agents might play an etiological role in, at least some cases of, MS. The
rationale for considering infectious agents in MS can be summarized as following:
• EBV is common during childhood in regions where MS is rare but common
during puberty in regions where MS is common
1
. This fits the polio model
for MS: MS is caused by some ubiquitous childhood benign infection that is
detrimental to the CNS if acquired at an older age.
• Oligoclonal IgG in CSF: Oligoclonal IgG bands (OIG) are detected in more
than 90% of MS cases
2, 3
. Though the targets of the OIGs are not clear
(Myelin basic proteins and other structural proteins of CNS), a recent study
4

noted that the possible epitopes for the OIG could be Epstein-Barr

Virus
nuclear antigen (EBNA-1) and a heat shock protein B crystalline. Studies
have shown that OIGs in diseases like subacute sclerosing panencephalitis or
cryptococcal meningitis are directed towards causative infectious agents
5, 6
.
• Migration studies: Most of the migration studies have noted that migrants
aged less than 16 years of age at the time of migration tend to acquire the risk
of the place they move to
7-10
. However, some recent studies have observed
that environmental factors might be in play even in adulthood
11
.

Besides molecular mimicry, immune regulation is the other scenario where
infectious agents might play a role. The concept of ‘Hygiene Hypothesis’
12
can be
extended from allergic diseases to autoimmune conditions. According to the
‘Hygiene Hypothesis’ lack of common childhood infections results in Th2 skewing
of the immune system which leads to increased susceptibility of allergic conditions.
However, data reflects that in allergic conditions like asthma and atopy of both Th1
and Th2 type of immunity plays an important role. This lead to the extension of
76
77
‘Hygiene Hypothesis’ to ‘Gatekeeper hypothesis’
13
. According to this hypothesis,
exposure to microbes in early life is necessary for the maturation of acquired
immune system. As body develops active immunity against the exposures it also
propagates the development of proper regulatory T-cell repertoire that checks against
excessive immune activity. Allergic and autoimmune diseases can be perceived as
the two extreme outcome of similar environmental exposure. Initial support to this
role of childhood exposure to infectious disease in MS risk has been propagated
partially by observational findings. One of them being the geographical distribution
of MS (higher prevalence of MS in regions with low rates of childhood infection)
and the migration studies showing the importance of exposure in the early childhood
8, 14, 15
. Further evidence of the role of infectious agents in MS is derived from the
studies detecting viruses and pathogens and their antibodies in the serum and
cerebrospinal fluid (CSF) of MS cases
16-19
. However this association could be
modified by sibship or HLA type
20, 21
. Increased prevalence of viral antibodies,
though to a lesser degree, was also observed in other neurological diseases. These
findings are indicative of an infectious basis of MS rather than infallible proof.
Keeping with this ‘late infection’ effect, it is postulated that MS should be
associated with birth order and family size. First born and children in small sib size
might escape childhood infections at earlier age making them susceptible to late
infection which might increase their risk of MS. No consistent finding is available
from earlier case-control studies . However, the relationship between sib-size and
22-24
78
order and risk for MS might not be a simple relationship as is evidenced from the
findings of a case-control study from Australia . It appears that the protective effect
of sib size and order might be associated with time and dose of exposure
25
. In this
case-control study no association was observed between birth order and MS risk
when considered univariately though a marginally significant inverse association
between sib-size and MS risk was observed (OR: 0.90, 95% CI: 0.81-1.00).
However, a statistically significant inverse association was noted between number of
younger sibs and MS risk (0.77 95%CI: 0.67-0.90) which showed a strong dose
dependent effect. The risk of MS dropped from 0.68 to 0.37 to 0.35 for persons with
1, 2 or 3 or more younger sibs, compared to persons without any younger sib(p-value
for trend:<0.001). Considering exposure to younger sibs in the first 6 years of life
provided even stronger protection against MS. This protective effect of ‘younger
sibs’ was mostly among those who were active in sun exposure activities in the
winter periods (OR: 0.31 95%CI: 0.18-0.55) compared to those who were not (OR:
1.06, 95%CI: 0.36-3.14). It was observed that having younger sibs was inversely
associated with history of infectious mononucleosis and high levels of EBV IgG
(EBV nuclear antigen IgG>300 unit +EBV capsid antigen IgG>300 units) both of
which have been to be described to be associated with MS . The finding of this
study is unique as it shows the interplay between environmental factors in MS.
26-28
Childhood infections, such as mumps, measles, chicken pox, whopping
cough and others, have long been a potential candidate of MS risk factor
29
.
79
Inconsistent findings have drawn a hazy picture of the role of childhood infections in
MS. Comparing results from these different studies are difficult given the use of
different case ascertainment, study design and exposure definitions. Many of these
studies looked at all the available childhood infections but were not penalized for
multiple testing thus it is difficult to be certain whether some of the significant
findings are simply statistical errors or not. However, one pattern that is evident from
these studies, which provide some credulity to the findings, is that most often
association was observed between late infection
30-38
and MS. This finding is
consistent with the late infection hypothesis of MS etiology. Here we will not discuss
the studies conducted prior to 1997 as a lot has been written abut them and the
concerns about those studies have been addressed above (for further review readers
are referred to Granieri et.al.
29
).
In the last decade some well planned population based case-control studies
and nested case-control studies have investigated the role of infectious disease in
MS. Figure1 presents the OR observed in those studies. Three case-control studies
assessing the association between multiple childhood infections and MS observed
association between MS and childhood infections only when it was contacted after
age 15. The association noted between MS diagnosis and history of respiratory tract
infection
39
(RTI) in previous 3-12 months was most likely unrelated to MS etiology
but rather triggering of MS disease process. Exacerbation of MS has been shown to
be associated with RTI
40
.
Figure 5.1: Association between MS and Different Infections
0
0 1 23 4 5 67 8 9 10 11 12
OR
OR
2
3
2
3
Measles
Mumps
2
3
Rubella
2
3
Pox
IM
1
2
EBV Ag
VCA (4)
EBNA (4)
EBNA (5)
EBNA (5) 4-fold ↑
Cpn IgG
RRMS(6)
SSMS(6)
Army(7)
KP(7)
RTI
Dog
Ownership
Disease or
Disease
Marker
1
2

The OR of MS associated with the infectious disease (measles, mumps, pubella, pox and IM) or disease markers
(VCA=Epstein-Barr virus (EBV) viral capsid antigen, EBNA=EBV nuclear antigen, CpnIgG=anti-Chlamydia
pneumoniae antigen) with 95% CI, as observed from some selected recent studies.
[1=Marrie,RA
39
2=Hernan,MA
38
3=Bager,P
41
4=Ascherio,A
27
5=Levin,LI
28
6=Munger,KL (NHS)
42

7=Munger,KL(Army &KPMCP)
43
]

Two nested case-control studies, Nurses Health Study (NHS I and II) and
army cohort, showed strong association between EBV infection and MS
27, 28
. The
IgGs that showed significant association, IgG to EBNA complex and EBNA-1, are
markers of severe or recent primary infection or reactivation of infection. The Nurses
80
81
Health Study I and II by itself, does not provide any information regarding causality
as blood sample was collected after onset of MS. However, in the army cohort
markers of past infection with EBV showed strong association with MS even when
the analysis was restricted to cases where blood sample was collected at least 5 years
before onset of MS. Furthermore, in intra-personal analysis, a 4-fold increase in IgG
levels of EBNA complex and EBNA-1 from first blood sample to a later sample
prior to disease onset, was associated with 3 fold increase risk of MS and the risk
was more than 15 fold if the first blood sample was collected before age 20. These
studies along with earlier findings show the importance of EBV in the etiology of
some cases of MS.
The role of Chlamydia pneumoniae (Cpn) in MS remains unclear. The
interest in Cpn started with the finding that the treatment with antichlamydial agents
improved the clinical conditions of an MS patient with Cpn infection of the CNS
44
.
Since then numerous studies have been conducted to prove an association between
Cpn infection and MS. Some studies observed the presence of Cpn in the CSF of MS
patients
45, 46
where-as others failed to do so
47, 48
. Cpn specific oligoclonal band in the
CSF of MS patients was observed in some studies
49, 50
but not in others
51
.This
differential finding might be due to different laboratory processes, case selection or
time of measurement. Cpn might be associated with specific form of MS or it might
be a secondary infection common in long standing MS. Some evidence show that
Cpn is more likely to be associated with primary progressive form of MS
42, 50
. In the
82
NHS
42
study, women with PPMS were 7.5 times more likely to have positive titer
(1:16) for Cpn specific IgG compared to control but this was not evidenced among a
similar study with young mostly male army cohort
43
. The finding from the NHS
study can be biased as the blood sample was collected 15 years (on average) after
diagnosis of PPMS where as in case of the army cohort blood was collected 4 years
(on average) before diagnosis. So the evidence for Cpn in MS remains vague and
further well designed study is warranted.
Other viruses
52
that have been implicated in MS are human herpesvirus 6
(HHV-6), HLTV-1, coronaviruses and JC viruses. Little epidemiological evidence is
available to substantiate any of these speculations.
5.3 Sun Exposure
The geographical distribution of MS partially conforms to the putative protective
role of the natural ultra violate radiation (UVR) from sun exposure. The annual
average UVR level is inversely related to latitude which is opposite of the
relationship between MS and latitude. The latitudinal difference between Tasmania
(41-43.5
0
S) and North Queensland (10-29
0
S) is at least 12
0
. The UVR level in
Tasmania is 3 UVR index (1 UVR index=25milli Watt-m
-2
) lower compared to
Queensland and the MS prevalence in Tasmania is 6 times greater then that of North
Queensland. This latitudinal gradient of UVR is observed in different parts of the
world as well as in US. The UVR index is approximately 3-5 times higher in the
83
southern part of Europe compared to the northern part. The southern part of Europe
is considered to be mid MS area where as the highest MS rates are noted in the north
Europeans countries. The North-South inverse association is also evident in the US.
The UVR exposure in the southern parts of US is almost 4 folds higher in
comparison to the northern parts of US. A north-south gradient in MS rates has been
noted in the US among WWII veterans
53
and nurses study
54
.
These observations are consistent with the hypothesis that UVR/sun exposure
induced immunosuppression might dampen the auto immune activity in the
susceptible population and thus exert its protective effect in MS. UVR exposure can
exert itsimmunosuppressive effect directly by producing cytokines
55, 56
and indirectly
by producing vitamin D and suppressing melatonin secretion
57
. Animal studies have
shown that UV- radiated keratinocytes produce an array of cytokines including IL-10
and IL-4 through a PGE2 induced pathway
58
. IL-10, a potent immunosuppressor
59
,
alters antigen presentation and IFN- γ secretion by antigen presenting cells (APC) and
thus prevents Th0 to TH1 conversion and TH1 activation
60, 61
. UVR can also reduce
natural killer cell activity and thus affect innate immunity
62
.
Besides the direct effects, UVR can also induce immunosuppression through
modulating melatonin secretion and vitamin D production. T helpers cells with
activated melatonin receptors leads to T cell priming and release of Th1 cytokines
63
.
It has been suggested that UVR suppresses melatonin secretion
57
and increases
84
melanocyte stimulating hormone (MSH)
64
. Formation of Vitamin D, a potent
immunoregulator
65
, at human skin is also dependent on UV exposure.
Epidemiological studies have also demonstrated the putative protective effect
of sun exposure against MS. One of the earliest evidence comes from the WWII US
veteran study
66
. According to the study, the higher MS rate was associated with
latitude at birth place among the veterans, and it attenuated after adjusting for solar
radiation during the winter period. A strong inverse correlation was noted in
Australia between MS and latitude (R
2
=0.80, p-value=0.01)
67
. The same group
reported a strong protective role of sun exposure against MS from a case-control
study conducted in Tasmania
68
. The study evaluated the role of sun exposures during
the childhood (6-15 years of age) among Tasmanians. The odds ratio of MS among
the Tasmanians with 2 or more hours of sun exposure in average was 0.31 (95% CI:
0.16-0.59) compared to those with less than 2 hours of exposure in average. An
occupational study also noted dose dependent protective effect of sun exposure
among workers exposed to sunlight compared to indoor workers
69
.
One concern regarding the potential beneficial effects of UVR is the
carcinogenic effect of UVR. UVR can be considered a complete carcinogen as it
causes both DNA damage and immunosuppression
70
. UVR exposure is directly
related to skin cancers like melanoma and basal cell carcinoma
71
. However, the dose
differential between UVR dose required for biosynthesis of vitamin D, putative
protective effect in MS and the detrimental dose to cause skin cancers needs to be
85
appreciated. A moderately fair person in Boston (42
0
N: a moderate ambient UVR
location) needs only 5 minutes of sun exposure to face, hands and arms thrice a week
during the summer and autumn periods to maintain sufficient vitamin D level;
where-as maximal risk to basal cell carcinoma equates to 14 hours of weekly sun
exposure in Western Australia (29
0
S: a high ambient UVR location)
72
. Protection
from MS appears to be conferred by only 2 hours/week sun exposure in childhood
68
.
This demonstrates how people can derive the beneficial effects of sun exposure
without succumbing to its deleterious effects.
The ecological observation of the negative association between MS and sun
exposure is limited by the absence of individual level data. Sun exposure can greatly
vary within the same population dependent on individual exposure pattern
73
. It has
been observed that personal UVR exposure can vary by as much as 100 fold
depending on behavioral factors at any given latitude with any ambient UVR level.
This limitation is addressed in the case-control and occupational studies. However,
given the lag between exposure, disease onset and collection of information in those
studies, the quantification of exposure is certainly a questionable entity. Furthermore,
given the polygenic nature of MS it is not clear whether the MS cases and non-cases
in those studies had the same baseline risk. Even if the negative association between
sun exposure and MS is accepted to be of etiological importance, question remains
whether this is the result of direct immunoregulatory effect of UVR or is it a
consequence of greater susceptibility to childhood infection among the
86
comparatively immunosuppressed children. Furthermore, how vitamin D intake and
sun exposure interact in MS risk needs to be answered. Future research needs to
address these issues.
5.4 Dietary Factors

Anecdotal evidences links two dietary factors, namely vitamin D and fatty
acids, to MS. As sun exposure is one of the main sources of Vitamin D, thus vitamin
D status in the population is higher near the temperate zones and tapers away from
the equator. A review of previous literature reveals that some ecological studies
showed an association between MS risk and intake of total fat, milk and milk product
though the finding was not consistent
74
. The case-control studies were even less
consistent
74
.
5.4.1 Vitamin D
Vitamin D is considered beneficial in MS due to its immunologic role. Prior
to the 80s, vitamin D was synonymous with calcium and phosphate homeostasis. The
discovery of vitamin D receptor (VDR)
75
and subsequent detection of this receptor in
T-cells
76
instilled the possibility of immunologic role of vitamin D. Further studies
noted that significant amount of VDR is present in CD4 lymphocyte and
macrophages but highest concentration was noted in CD8 cells
77
. Animal studies
revealed the biphasic immunomodulatory effect of D
3
. S.Yang et.al.
78
demonstrated
that t-cell mediated immune reaction is suppressed in vitamin D deficient mice and
87
the same response could be elicited by high doses of 1,25-(OH)
2
D
3
and its analogs.
Various vitamin D supplementation has been shown to prevent the incidence of EAE
in mice
79
.This effects is probably achieved by D3 as it suppresses the production of
inflammatory cytokines like Il-2, IFN- γ and TNF- α
80
, and stimulates anti-
inflammatory cytokines such as TGF β-1 and IL-4
81
. Furthermore, it also inhibits IL-
12 which is an important cytokine for T-cell differentiation. Overall vitamin D
appears to play an important role in T-cell proliferation and differentiation and thus
exert a potent immunomodulatory effect.
In human, the support for the vitamin D hypothesis is mostly limited to the
sun exposure studies. Few epidemiological or clinical studies are available looking
into the association between vitamin D and MS. Recently, a nested case-control
study involving 7 million US army and navy personnel noted an inverse association
between serum 25OH D
3
level and MS risk
82
. The study involved 257 MS cases
diagnosed between 1992 and 2004 and 2 controls from the same cohort matched on
age, sex, race/ethnicity and date of collection of blood sample. Among this cohort,
the blood serum level varied by race and latitude of birth. All models were adjusted
for latitude of birth and stratified by race. Among whites, a 50-nmol/L increase in
serum was associated with a 41% decrease in MS risk (OR: 0.51, 95%CI: 0.36-0.97).
Similar trend was observed for quintiles of serum vitamin D level. The inverse
association did not differ by sex though a stronger effect was observed for vitamin D
88
level prior to age 20. However, no association was observed for vitamin D and MS
among blacks and Hispanics.
A strong protective role of vitamin D in MS incidence was also reported from
a large prospective study involving almost 180,000 participants of the Nurses Health
Study with 173 incident cases of MS
83
. Women in the highest quintile of vitamin D
supplementation had 40% lower risk of MS compared to women in the lowest
quintile. Temporality was not an issue in this study as the association was noted for
baseline vitamin D supplementation that happened years before MS diagnosis.
However, almost 96% of women in the highest vitamin D supplementation quintile
also took multivitamins. It was statistically impossible to separate the effects of
vitamin D from the other vitamins. The inverse association between MS risk and
vitamin D intake was observed even after adjusting for intake of vitamin A and
vitamin C but not for vitamin E, B
1
,B
2
,

B
6
or B
12
. However, the intake amount of the
E and B vitamins increased by 3-5 fold from the lowest to highest quintile of vitamin
D intake where as it was about 2 fold difference for vitamin A or C intake.
Collinearity could have been an issue when logistic models incorporated all these
different vitamins together. The other vitamins also did not show any association
with MS risk univariately in this study and in an earlier study where the NHS I and II
population was explored for association between MS and carotenoids, vitamin C and
vitamin E
84
. In a small (n=11) non-randomized trial decrease in relapse rate was
noted after vitamin D, calcium and magnesium supplementation
85
. Sustained high to
89
normal calcium level might be required to induce therapeutically effective
immunosuppression by vitamin D as evidenced from animal model
86
A six months
double blinded randomized vitamin D supplementation trial among MS patients
(n=39) showed increase in the serum level of TGF- β from baseline for patients
receiving 1,000 I.U. Vitamin D with 800 mg of calcium compared to controls who
received calcium and placebo
87
. However, the vitamin D supplementation group
(n=22) also showed a non-significant higher level of IFN- γ, TNF- α and IL-13 and
lower levels of IL-2 compared to placebo group (n=17). This was a small study of
short duration and did not measure the clinical effect of the supplementation.
However, it shows the role of vitamin D supplementation on the cytokine profile of
MS patients.
Given the biological plausibility and different epidemiological and clinical
evidences, vitamin D can potentially be one of the missing links between genetic
susceptibility and disease expression. A better understanding of the
immunomodulatory pathway of vitamin D is necessary to fully appreciate its role in
MS. Well conducted double blinded randomized clinical trials of adequate power are
required to investigate the protective role of vitamin D among MS patients. To elicit
its role in MS incidence, intervention studies involving highly susceptible groups
need to be considered.
90
5.4.2 Fatty Acids
Besides Vitamin D, fat and its source has often been investigated as a
possible risk factor in MS. Though the ecological studies have often shown an
association between MS and meat and meat products, fish intake and vegetables yet
the findings are not consistent and were not supported by case-control studies (A
detail review is available from Lauer Klaus: Diet and MS :Neurology Vol49(2), Aug
97,ppS55-S61). Ecological studies imply that MS is inversely correlated with high
caloric intake of fat from fish and vegetable. This fits the traditional concept that
omega-6 fatty acid (meat source) is pro-inflammatory and omega-3 fatty acid (fish
and vegetable source) is anti-inflammatory. But this assumption is too simplistic in
reality.
Animal studies have shown that linoleic acid, an Omega-6 fatty acid,
supplementation is beneficial in treatment of EAE
88
and its deficiency results in
exacerbation of symptoms
89
. Feeding of oral gamma-linoleic acid was demonstrated
to markedly decrease the incidence and relapse of EAE and this effect was mediated
through an increase in cell membrane long chain omega-6 fatty acids, PGE
2
production and secretion of TGF- β1
90
. Low levels of linoleic acid and other
polyunsaturated acid has been noted in the brains of MS patients
91
. However, most of
the case-control studies show an increased risk of MS among those who ate more
meat products, a source so Omega-6 fatty acid
74
. A recent study reporting results
from two large cohorts of women nurses (NHS and NHSII) failed to show any
91
association between dietary fat including Omega-3 and Omega-6 polyunsaturated
fatty acid and MS incidence
92
. The available data suggests that fatty acids are
unlikely to play any significant role in the initiation of MS.
5.5 Sex Hormones

The auto-immune diseases (AIDs) are marked by strong gender bias with the
most of the AIDs showing a female propensity. The female-male ratio ranges from
more than 9:1 for diseases like Sjorgen’s syndrome and SLE to 2:1 or 3:1 for disease
like MS and sarcoid. However, AIDs like ulcerative colitis and diabetes mellitus has
no female preponderance. The reason behind this female preponderance of AIDs is
not clear. It still remains largely unknown how sex hormones interact with the
immune system to elicit dimorphic immune responses in the two sexes leading to the
existing gender bias in AIDs.

92
Table 5.1: The Role of Sex Hormones on Immunity
Hormones Effect on Immunity
Stimulator of Macrophage phagocytosis
95, 96
Stimulate IL-1 production
97
Receptors in Thymus
98
Receptors in T-cells
98
Receptors Macrophage
99
Decreases activity of thymus
100
increases CD4+ CD8- (memory helper) cells
101
Extrathymic maturation of T-cells
102
Increased production of IFN γ by murine lymphoid cells
103
Increases IL-1 secretion by macrophages
104
Estrogen
(Dose dependent
biphasic role
93, 94
)
Increased production of IFN and IL-10
93

Receptor synthesized in Thymus
98
Inhibits mitogen induced T-cell proliferation
100
Progesterone
Promotes IL-4 production and development of TH2 cells
105

Androgen receptors in thymocyte
98, 99
Thymus involution
100
Deprivation leads to decrease in CD4- CD8+ cells
106
Castration causes Increase in Pre-B cell
107

Depletion of pre-B cell
108
Decreased production of IFN γ,IL-4 and IL-5
100
Androgen
Inhibits B-cell and T-cell maturation
100

Female mice makes more IFN γ then Males in response to BCG
vaccination
109
Dimorphism in viral effects on CNS
110-113
Gender Differences in
immune response
Testosterone levels directly associated with brain tissue damage in females
with MS whereas in males the tissue damage is related to estradiol levels.
114

The sex hormones; i.e. estrogens, progesterone and androgens, are
synthesized from cholesterol. These hormones have a complex relation with the
immune system. Gender dimorphism is observed for viral infections and CNS injury.
These features have been summarized in Table 5.1. Estrogen receptors has been
detected on CD8
+
cells but not on CD4
+
cells and affects secretion of cytokines like
TNF- αβ and IFN- γ in a dose dependent way
93
.
93
High estrogen levels similar to the levels observed in the peripheral blood
during pregnancy might reduce TNF- αβ and increase IL-10 and thus support an anti-
Th1 immunological milieu. However, at lower levels it results in higher levels of
pro-inflammatory cytokines TNF- αβ and IFN γ. At higher dose Female sex hormones
can also inhibit activation of microglia by inhibiting lipopolysaccharide induced
nitric oxide (NO) production
115
. Testosterone can act directly on the androgen
receptors on CD4
+
to induce anti-inflammatory IL-10 production
116
. Overall, a
stronger immune response is observed among females
117
. The CD4/CD8 ratio is
greater among female compared to male due to an absolute higher level of Cd4
cells
118
.
The murine model of MS, EAE also shows dimorphism by gender
119
. Female
mice are more susceptible and often shows more severe pattern of EAE. The
protective nature of the testosterone was observed by increase of EAE risk among
castrated male mice
120
and decrease in disease severity in female mice implanted
with dihydrotestosterone
121
. Treatment of female EAE affected mice with estrogen
level higher than the normal levels observed during normal menstrual cycle also
causes potent immunosuppression
122
.
Gender differences have been observed in the cytokine profiles and brain
injury patterns. Among RR-MS, the percentage CD3 cells producing TNF α is higher
in males compared to females
123
. Among females a positive correlation was observed
between disability, expressed as EDSS, and IFN- γ (r
2
=0.49, p-value=0.02) and
94
TNF- α (r
2
=0.42, p-value=0.05). However, no correlation was observed between
EDSS and cytokines among males. Tomassini et.al. observed that sex hormones
have sex specific effect on brain damage in MS patients
114
. Testosterone showed a
biphasic pattern among females. The MS female cases showed significantly lower
level of testosterone compared to controls and MS cases with abnormally low levels
of testosterone had more enhancing lesions compared to cases with normal
testosterone level. However, testosterone level showed a positive correlation with
irreversible brain tissue damage and disability. No association was observed between
female sex hormones and disease activity among female cases. However, estradiol
was positively correlated with irreversible tissue damage among male cases. It is
apparent that the sex hormones play different roles in tissue damage in the two sexes.
The biphasic pattern of testosterone among females is paradoxical. If low
testosterone is detrimental leading to more lesions why higher level of testosterone is
associated with irreversible tissue damage? It is possible that the initiation of MS is
not same as propagation of MS. The initiation of MS is dependent on the genetic
susceptibility and immune milieu of the host. As testosterone has an
immunosuppressive role, lack of it might provide an environment that is more
supportive to an autoreactive immune system. However, once the disease process has
started the higher levels of testosterone might be detrimental for the brain tissue due
to the neurotoxic effect of testosterone
124
.
95
5.5.1 Puberty and MS

Puberty is marked by changes in the hormonal levels in both sexes. Puberty
among girls are marked by onset of menstrution
125
. Like other autoimmune diseases
MS is rare before puberty and no sex bias is observed among cases of early onset MS
(before 10 years of age). It has been hypothesized that MS might be associated with
onset of puberty. Considering early puberty leads to longer exposure to sex
hormones, it has been hypothesized that early age at menarche might be a risk factor
of MS. However no consistent association has been observed from epidemiological
studies
30, 126-128
.
5.5.2 Pregnancy and MS

Pregnancy in MS is marked by reduction in relapses during the pregnancy
period, especially in the third trimester with rebound in relapses during the first
postpartal months. This finding has been verified in different ethnic groups and
studies
129
. A recent cohort study, Pregnancy in Multiple Sclerosis (PRIMS) followed
254 women from their 1
st
trimester of pregnancy to a year after delivery
130
. The
investigators reported on the 227 women who had their first live birth during the
study period. Reduction in the relapse rates were observed during the pregnancy
period with marked reduction during the 3
rd
trimester. A rebound in the relapse rate
was observed during the first 3 months after delivery; however the relapse rate came
down to pre-pregnancy levels by the end of 12 months after delivery. Vukusic
96
et.al.
131
followed 161 women of the initial 227 subjects of the same cohort till 24
months after delivery. The relapse rate virtually remained unchanged from 4
th

trimester postpartal onwards (relapse rate =0.5/year). Relapse during the 1
st
trimester
after delivery was associated with the numbers of relapses during pre-pregnancy or
pregnancy period. However, considering the whole period starting from pregnancy to
2 years after delivery, pregnancy seems to have no effect on MS status.
5.5.3 Oral Contraceptive and MS

Given the association between sex hormones and MS, it is conceivable that
exogenous estrogen might have some association with MS. Oral contraceptive (OC)
is a common source of exogenous hormone among women with the highest risk of
MS. Since 993 the association between MS and OC has been evaluated in 4 different
prospective studies (Table 5.2). The findings from the studies are inconsistent as OC
appeared protective in two studies
132, 133
and a marginal risk factor in other two
134, 135
.
Only the GPRD study showed a statistically significant association between MS and
OC use and OC appeared protective in this study. In stratified analysis it appeared
that most protection was associated with current use and when used within 12
months of diagnosis where-as no protective effect was evidenced when used before
12 months of diagnosis. Assimilating the findings from this study along with the
previous studies it seems that OC does not have an etiological association with MS
but rather it might delay the onset of MS symptoms.
97
Table 5.2: Association between oral contraceptive use and MS
Study Result Population
Oxford Family Planning
Association Study
133
OR=0.70
(95% CI: 0.4-1.1)
The Oxford Family Planning Association Study
involved
17,032 White British women, attending family
planning clinics during 1968-74.They were
followed till 1991. The total number of incident
cases of MS during this period was 63.

Royal College General
Practitioners’
Oral Contraceptive Study
134

OR=1.2
(95% CI: 0.80-1.8)
A cohort study involving 46,000 married British
women. It was conducted between 1968 and 1996.
A total of 114 new MS cases were identified in
this cohort.

Nurses Health Study (NHS)
and NHS II
135
OR=1.2
(95% CI: 0.9-1.5)
NHS followed 121,700 nurses from 11 states of
US since 1976.
NHS-II followed 116,671 nurses from 14 states of
US since 1989. 181 new cases of MS were
identified in NHS and 134 in NHS-II.

General Practitioner's Record
Database (GPRD)
132
OR=0.6
(95% CI: 0.4-1.0)
MS cases were diagnosed from Jan 1, 1993 to Dec
31, 2000 from the database. 242 incident MS cases
were identified in this cohort Using a nested-case-
control approach,10 controls were selected for
each case matched on age, practice data and entry
to practice

5.5.4 Organic Solvents

Multiple occupational and case-control studies have shown association
between MS exposure to multiple organic solvent (OS). Landtblom et.al
136

performed a meta-analysis of 13 studies showing a significant association between
MS and OS exposure, with an odds ratio of 1.7 (95% CI:1.1-2.4).Ten of the 13
reviewed studies showed positive association between MS and exposure to OS.
However, many of these studies were methodologically different, small in size and
differed in case and exposure ascertainment.
98
There have been few more studies since 1996 that have explored the
association between MS and OS. One of these studies (Danish study) showed no
assocaition
137
where-as the other two showed an increased risk among those with the
exposure
138, 139
. The Danish study
137
assessed the effect of exposure to OS by
comparing MS rates between Danish male workers exposed to OSs to male workers
without such exposure. However, they had to exclude the cases that had MS at
baseline, which was 40% more among the painters compared to the control group.
The study had potential problem of selection bias as they could only link the MS
registry data to1970 census registry that defined the occupations. In this process they
might have potentially excluded most of the cases that developed MS due to
exposure. Riise et.al.
139
showed 1.9 fold increased propensity of disability
pensioning among workers (painters) exposed to OSs compared to other non exposed
workers in Norway. However, the study does not provide any information regarding
the temporality in exposure and disease onset. The other study from Gothenburg
region in Sweden
138
observed that nurses exposed to anesthetic gases were 2.8-2.9
times more likely to suffer from MS compared to women of Gothenburg region in
Sweden or Denmark. Thirteen anesthetist nurses with MS were identified through
magazine advertisement, of whom 2 were excluded as MS onset was prior to
exposure. To calculate the incidence rate of MS among the anesthetist nurses, the
total number of anesthetist nurses was determined through National Board of Health
and Welfare and the Nurse Union. However, the study result is not adjusted for any
99
other risk factors of MS or does not provide information regarding specific
anesthetic exposure.
It is possible that exposure to OS might be associated with increased risk of
MS but the attributable risk of MS due to exposure to OS would be minimal as the
observed odds ratio for OS ranges 0.8 to 4.0 and only a small fraction of population
are exposed to OS. Understanding the role of OS in MS will provide an
understanding to the mechanism of MS etiology.
5.5.5 Smoking

The studies showing association between personal tobacco smoking and MS
are among the best of the epidemiological studies in MS. Three well planned
prospective studies showed association between MS and personal smoking
(Figure 5.2). The Oxford Family Planning Association Study involved 17,032
White British women, attending family planning clinics during 1968-74, who were
followed till 1991.Inspite of the small sample size with 63 diagnosed cases of MS, a
marginal significant increased risk of MS was observed among women who smoked
more than 15 cigarettes at study entry with a significant trend for increasing numbers
of cigarette smoked. Similar finding was noted from the Royal College General
Practitioners’ Oral Contraceptive Study which was also a cohort study involving
46,000 married British women. However, the most confirming results come from the
two nurses’ cohort of US, NHS I and NHS II. The two cohorts had a total of 237,264
133, 134, 140
133
100
women with 314 diagnosed cases of definite and possible MS. The nurses were first
asked about lifetime MS diagnosis in 1991-1992 questionnaire and the medical
diagnosis was verified through obtaining medical records from the treating
neurologists or the patients internist (in absence of neurologist’s response). The
exposure was assessed through biennial questionnaire since the beginning of the
study. To account for the lag between smoking and MS diagnosis a 4 year lagged
Cox’s proportional hazard model was utilized to assess the association between
smoking and MS. A statistically significant trend was noted between smoking and
MS risk (p-value < 0.01). Temporality was not an issue in this study as exposure was
assessed prior to onset of MS and the finding remained unchanged if exposure was
assessed even before the onset of first symptom. However, no information is
available regarding type of MS and smoking habit.
The mechanism by which smoking can be associated with MS risk is unclear.
It has been postulated that smoking can play an etiological role in MS by acting as an
immunomodulator
141, 142
, a neurotoxic agent
143
, a risk factor for increase
susceptibility to respiratory infection by Chlamydia pneumoniae
144
or a risk factor
for compromised BBB
145
. Whatever the mechanism is, given the increased risk of
MS noted from the different prospective and case-control studies and the high
prevalence of smoking, due importance should be given to the role of smoking in
MS. From the calculation of the attributable risk of MS for women smoking more
that 9 pack-years, it appears that MS can be reduced by 20% in the risk group by
limiting their smoking.
Figure 5.2: Association between MS and Smoking
0
0 123
OR (95% CI)
OFPAS: Ex-smoker
RCGPOCS:>15 cig/day
NHS: Current smoker
NHS:1-9 pack-yr
NHS:10-24 pack-yr
NHS: >24 pack-yr
Cross-sectional: ever smoker

This is a graphical presentation of the odds ratio (OR) and 95% CI for the association between MS and smoking.
The ORs associated with the pack-years from the NHS data shows an increasing trend with increasing pack-years
compared to never smokers.
[OFPAS=Oxford Family Planning Association Study
133
, RCGPOCS=Royal College General Practitioners’ Oral
Contraceptive Study
134
, NHS=Nurses Health Study
146
, Cross-sectional= Norway
147
]

101
102
5.6 Summary

A review of the plausible environmental factors in MS reveals the complexity
and the ambiguity in the existing epidemiological literature of MS. Scrupulous
scrutiny of the findings might shine some light on the intangible risk factors of MS.
It appears that no single factor shows any consistent pattern across similar studies.
Following the principals of Ockham’s razor we often try to avoid explanations that
are based on multiplicity. However, it seems that we need to consider the inter-
relationship between different factors to identify the risk factors of MS. To elucidate
the risk factors we probably need to consider the age of onset, the course of the
disease, sex of the case, and also the presence or absence of different putative risk
factors besides the one under study. It would certainly help if we could characterize
the pathological types
148
by some personal features or non-invasive method. Until
that is achieved, epidemiological studies need to address the aforementioned issues
to minimize measurement errors of the outcome.
An interplay between the environmental and host factors at different stages of
the disease process contributes to the risk of MS (Figure 5.3). Sun exposure, diet
(vitamin D) and exposure to childhood infections probably provide an
immunomodulatory effect that tones down the immune system in a susceptible
person and thus exerts a regulatory control that might provide protection against MS.
Those environmental factors might interact with each other to potentiate their overall
effect. Host factors like ‘sex’ also plays an important role in the homing of the
103
immune response as estrogen skews that immune system towards a Th1 type
response whereas the opposite is true for testosterone. The onset of puberty which is
marked by surge of sex hormones might be the critical period in the lifetime risk of
MS. However, even after production of autoreactive T-cells people are not destined
to have MS unless they are also exposed to stimulatory factors like persistent
infection in adulthood, late exposure to childhood disease, exposure to smoking or
other risk factors. Even after activation of dormant autoreactive T-cells its passage
through blood brain barrier (BBB) depends on different adhesion and matrix
molecules as well as on cytokines and chemokines. The tissue damage could be
modulated by the inflammatory and oxidative conditions. Even after all the damage,
remyelination can play an important role in keeping the disease process in check.
Given the complexity in the disease etiology of MS it is not feasible address
all the issues in any single study. Future research needs to address each of the
possible pathways separately with the aim to identify the possible factors in the
disease causation with a better understanding of each of the pathways.
Figure 5.3: A schematic presentation of the interplay of environmental and host factors in MS etiology.

Black arrows are positive factors and red arrows are negative factors Yellow depicts unknown association

104
105
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119
Chapter 6 Differential Concordance of Multiple Sclerosis by Latitude of
Birthplace
____________________________________________________________________
Chapter 6 Abstract
Objective: Address the inconsistency in the reported concordance of multiple
sclerosis (MS) among twins by zygosity, gender, and latitude.
Method: Medically documented 418 monozygotic (MZ) and 380 like-sex dizygotic
(DZ) pairs were ascertained during 1980-1992 and followed. Study population was
representative of twins with MS. Twins from Canada and adjacent US states (at or
above 420N) were considered “northern”, and ancestry was dichotomized on the
basis of descent from high-risk populations. Diagnosis before median age 29.3 was
considered “early”.
Result: The MZ-DZ concordance ratio was 2.9(95% CI: 1.0-8.9) among men and 2.6
(95% CI 1.5-4.5) among women. The average age at northern diagnosis was
independent of ancestry and 2 years earlier for both MZ (p < 0.02) and DZ (p < 0.01)
patients. Among DZ twins, concordance was independent of all characteristics.
Among MZ twins, concordance was 1.9 times (95% CI 1.2-3.2) greater among
northern twins, 1.9(95% CI: 1.1-3.6) times greater among twins with high-risk
ancestry, and 2.1(95% CI 1.2-3.6) times greater if diagnosis was early. Ancestry and
early diagnosis made independent significant contributions to the differential
concordance by latitude.
120
Interpretation: MS is similarly heritable by gender, and the apparent variation in
MZ concordance by latitude, is influenced by environmental and genetic factors.
This paper was published as an original article in Annals of Neurology Vol. 60(1),
page 56-64 (2006 Jul). The authors of the paper were Talat Islam, W. James
Gauderman, Wendy Cozen, Ann S. Hamilton, Margaret E. Burnett and Thomas M.
Mack.
121
6.1 Introduction
The incidence and prevalence of multiple sclerosis (MS) increases with
distance from the equator in both the northern
1
, and southern
2
hemispheres. Migrant
studies suggest that this gradient can be explained by an environmental determinant
acting in childhood
3
. Infectious agents have long been under investigation
4
, and solar
radiation has been of recent interest.
5
Heredity is also a determinant of MS. Persons of African
6
and Asian
3

heritage suffer lower risk, and persons of Scandinavian and Celtic origin higher risk
7
.
The risk to a first degree relative, cumulatively 3-4%, is 7-40 times that of the
general population
8, 9
and risks to 2
nd
and 3
rd
degree relatives are correspondingly
increased
10
. Etiology is believed polygenic, but other than the HLA-DR2 haplotype,
highly prevalent and predictive of disease in persons of North European origin, the
specific alleles responsible have not been identified
11
.
The pattern of twin concordance for a complex disease may shed light on the
nature of both genetic and environmental determinants. Among twins from northern
populations (mostly female) concordance for MS has been reported to be 20-40%
among monozygotic (MZ), and 3-5% among dizygotic (DZ)
12, 13
pairs, but the sole
observed rate of concordance in male MZ pairs was even lower than that in male DZ
pairs
14
. Especially low rates of MS concordance in MZ twins have been reported
122
from France
15
and Italy
16
. We address these issues of twin concordance by zygosity,
gender and latitude using North American twins.
A prerequisite for the study of MS patterns of occurrence is a system of
representative ascertainment. No population-based MS registries exist other than
those in the relatively small populations of Scandinavia. The only detailed analysis
previously available is based upon the assumption that Canadian patients attending
urban specialty clinics are uniformly representative of all Canadian patients in terms
of age, zygosity, and gender
17
.
The International Twin Study, a continent-wide repository of twins with
cancer and other chronic diseases, was designed to attract the participation of twins
representative of all cases among newspaper readers
18
and pairs so ascertained are
certainly not representative of all twin cases in respect of age, sex, zygosity, or place
of residence. Although the relative participation of concordant pairs is predictably
higher than that of singly affected pairs, this gradient does not appear to vary by
gender or place of residence and this twin repository was utilized in this study.
6.2 Methods

Ascertainment: Twins with MS (or another chronic disease, such as a cancer) were
sought by advertisements in North American newspapers and other periodicals
throughout 1980-92. Ascertainment was designed to capture pairs of twins in whom
123
at least one member had physician-diagnosed MS. No concordant pairs were doubly
ascertained. Pairs identified as discordant for the disease were verified based on the
Table 6.1: Descriptive Characteristics of Study Population
Questionnaire
available*
No Questionnaire
available*
p-value
Category N=568 % 230 %
Male-Male 138 24.3 59 25.7
Female-Female 430 75.7 17073.9
Sex-Pair
Missing n.a. 1 0.4
0.66
Monozygotic 292 51.4 12655.2 Twinship
Dizygotic 276 48.6 10444.8
0.33
Northern States and Canada 276 48.6 85 37.0 Birthplace
Other US States 292 51.4 145 63.0
0.003
1-15 5 0.9 2 0.9
16-20 52 9.2 11 4.8
21-30 239 42.1 88 38.3
31-40 187 32.9 81 35.2
41-50 65 11.4 32 13.9
>50 20 3.5 16 7.0
0.08 Age at
1
st
MS Case
Diagnosis

* Registry data includes information on all twin pairs first interviewed by telephone. Questionnaire data includes
information on the subset for which at least one twin completed a questionnaire.

neurological health of the unaffected co-twin, most often by direct contact. We have
estimated that approximately 27% of the North American twin cases prevalent at any
time during the period were identified. The characteristics of the twin respondents
have been previously described
18
. Each living member of an affected pair was asked
to complete a detailed questionnaire exploring possible predictors of risk, including
the national origins of each grandparent.
Of the 1177 initial respondents with a specific diagnosis of MS, we excluded
28 pairs of uncertain zygosity (Figure 6.1) and 26 pairs born outside North America.
Although 325 unlike-sex DZ pairs were used for some comparisons, this analysis is
124
largely based upon 418 MZ and 380 same sex DZ pairs. A more detailed analysis
with additional variables (described below) is based upon the 292 MZ and 276 DZ
twin pairs from which completed questionnaires were received (Table 6.1).

Latitude: Birthplace was dichotomized into “northern” and “other” categories.
Canadian provinces and states adjacent to Canada at or above 41-42
0
N (AL, OR,
WA, ID, MT, NE, ND,

SD, WY, MI, MN, WI, CT, ME, MA, NH,

NY, RI, VT)
representing the “northern” birthplaces, as defined by Hernan et.al
19
and the more
southerly US states representing the “other” birthplaces.

Ancestry: If one or more grandparents had been born in Scotland, Ireland, Iceland,
Denmark, Norway, or Sweden, the twins were considered to have Celtic or
Scandinavian ancestry. Pairs could not be classified in more detail according to the
number of such grandparents due to small numbers.

Zygosity: Zygosity was assigned according to the twins’ own perception. This
method has been shown to be over 90% accurate
20
, and we have previously used
molecular biology to validate the perceptions of roughly 250 adult twin pairs
21-23
,
confirming self reported zygosity in all but three instances.

125
Age at diagnosis: Diagnoses made before the age of 29.3 years (the median age of
diagnosis among MZ cases) were considered to be “early” (Table 6.2).

Diagnostic Validation: After receiving written consent from each case, the most
recent provider was contacted, and medical records were requested to validate the
diagnosis. Academic MS neurologists (L. Weiner, W. Weiderholt) reviewed the
records of 145 cases who were reported early in study but, like most twin cases, long
after diagnosis. Applying the Schumacher clinical criteria
24
the perceived diagnosis
was confirmed (probable or definite MS) in 141 (97.2%) of these cases. Of the 4
pairs with misdiagnosed cases, 1 single case pair was excluded from the registry and
the other 3 pairs were redefined as discordant pairs.

Follow-up: Periodically, each subject was located (most recently in 2005) and
queried about the basis for the diagnosis and the level of certainty on the part of the
current clinician. Specific questions were asked about the neurological health of the
originally healthy co-twin. As practitioners were identified, with the permission of
each subject, follow-up records, including MRI reports, have been gathered.
As of 2005, 81.2% of the 798 like-sex pairs (83.6% of the males, 80.4% of the
females) had been followed for at least 10 years subsequent to the initial diagnosis
(Table 6.2). The overall median and inter quartile range of follow-up was 26 and 18-
35 years, respectively.
Figure 6.1: The schematic presentation of the North American Twin registry Data

Ascertained Twin Pairs with MS
diagnosis

N=1177

126

Twin pairs without
zygosity N=28

Twin pairs with known zygosity

N=1149

Born outside North America (N=26)

MZ or same-sex DZ pairs

N=798

or unlike-sex DZ pairs (N=325)
Questionnaire returned No questionnaire returned

N=230 N=568

127
Table 6.2: Median and inter quartile range (IQR) of age at diagnosis and duration of follow-up since initial diagnosis according to zygosity
and baseline characteristics.

Age at Diagnosis Duration of follow-up in years
MZ DZ MZ DZ
Characteristics
Median IQR Median IQR Median IQR Median IQR
All twins 29.3 24.4-36.4 30.8 24.4-37.9 26.0 21.0-35.0 25.0 16.0-34.5
Gender

Male-Male 32.0† 25.4-40.0 30.4 23.5-35.6 25.0 21.0-36.0 26.0 16.0-33.0
Female-Female 28.8† 24.3-35.0 31.1 24.5-38.7 26.5 21.0-35.0 25.0 16.0-35.0
Age at Diagnosis ≤29.3 years 24.2† 21.0-26.8 23.5†

20.8-26.1 29.0 23.0-37.0 26.0 17.0-37.0
>29.3 years 36.4† 33.0-41.1 36.7† 32.2-42.0 24.0 19.0-31.0 24.0 14.0-32.0
Birthplace
1
North 27.9† 25.3-37.8 29.5† 23.5-36.3 27.0 21.0-35.0 26.0 16.0-36.0
OtherStates 30.6† 23.5-34.0 31.8† 25.2-38.5 26.0 21.0-35.0 24.0 16.0-33.0
Ancestry
2
Celtic/Scandinavian 28.7 24.4-34.5 29.1 23.4-35.6 23.0 18.0-33.0 28.5 21.5-36.5
Others 29.1 23.9-36.2 30.1 23.5-37.7 27.0 21.0-35.0 25.0 15.5-33.0
† The median values were statistically significantly different between the sub-groups.
1 Canada and the adjacent US states represent North.
2. Twin pairs with at least one grandparent with Celtic or Scandinavian origin.

128
The length of follow-up was similar by zygosity, gender, age at diagnosis, birthplace
and ancestry. Of the 568 pairs who completed questionnaires, 90.8% were followed
for at least 10 years. At least 85% of the pairs in each subgroup (MZ or DZ, male or
female, northern or other, early or late initial diagnosis) were followed for at least 10
years after the diagnosis. Over 93% of the initially unaffected co-twins were
followed beyond the age of 50, and all but one beyond the age of 40.

Adjustments: Over the course of follow-up seventeen originally unaffected twins
were diagnosed with MS, and the physicians of seven cases originally diagnosed as
MS spontaneously revised that initial diagnosis. In addition, we reviewed the
medical records of all cases from the 54 eligible pairs lost to follow-up within 5
years of ascertainment, and identified 6 patients for whom no truly objective
diagnostic evidence was available. Five of the 13 questionable cases thus eliminated
were in single-case pairs deleted from the registry; the remaining 8 were in
concordant pairs re-categorized as single-case pairs.
Primary clinical records of all but two of the second diagnoses in the
remaining concordant pairs were available. Each such patient lived separately from
the co-twin, and consulted a different practitioner following the onset of novel,
specific, distressing, and objectively verifiable symptoms. Review of the available
physicians’ notes indicates that each subsequent diagnosis of MS was made
independent of, and almost always in ignorance of, the diagnosis in the first twin.
129
Statistical Analysis: Comparison of the baseline characteristics of pairs with and
without questionnaire data was performed using chi-square tests. Student’s t-tests
were performed to compare the mean age at diagnosis in different subgroups.
The primary outcome of interest in this analysis was MS concordance assessed
separately for MZ and DZ pairs. Because we preferred a simple comparison between
demographic subgroups, without estimation of heritability, we measured pairwise
concordance (although comparisons based on pairwise and casewise concordance
give similar results). To identify the factors that determine twin concordance we
modeled the probability that a twin pair would become concordant separately in
relation to zygosity, sex, birthplace, ancestry, and age at first diagnosis. Chi-square
statistics were used to assess the significance of these links, to detect differences
between all registry volunteers and the subset of questionnaire respondents, and to
compare the various group-specific estimates of concordance and the proportions of
those diagnosed “early” according to ancestry and region of birth.
Factors modifying the association between concordance and birthplace were
evaluated using an adjusted logistic regression model with concordance as the
dependent variable. The strength of the associations between different covariates was
also tested using regression models. The final model used to assess the strength of
these associations was selected by adding covariates according to the magnitude of
each effect according to univariate analysis; this model was also used to search for
effect modification and confounding. Confounders were confirmed if the main
130
(unadjusted) effect varied by at least 10% after the addition of the putative
confounder to the model. The change in odds ratio after adjustment for each factor in
a single regression model provided an estimate of the effect of that covariate in
explaining the strength of the association. Appropriate interaction terms were added
to the model to assess effect modification.
To evaluate the relative strength of different factors as explanations of MS
concordance, we used a stepwise selection method (logistic regression at a
significance level of 0.05) to identify the most parsimonious model. Once the
parsimonious model was selected we compared each univariate model to the full
model using Pearson goodness-of-fit statistics. The univariate model giving the
highest p-value is considered to be the model with a better fit, relative to that with
the next highest, . Initially we combined MZ and like sex-DZ twins in order to
estimate the relative effect of zygosity. To identify the relative effect of different
environmental and genetic factors within the set of MZ twins, we then limited our
analysis to those pairs. All statistical analyses were performed using SAS version
9.3.

6.4 Results
6.2.1 Representativeness of cases
Among unlike-sex pair respondents, each potentially reported by a person of
either gender, the female to male case ratio was 2.1 (104 male and 221 female cases),
131
consistent with the ratios found in most North American series
26
, The same ratio
calculated on the basis of same-sex fraternal and identical twin cases was higher at
3.0 (Table 6.1), possibly because more females than males chose to participate,
although some case series have produced ratios at least as high
27
. The overall DZ to
MZ twin ratio of 1.7:1 (709:409) is consistent with the ratio of 2.1 expected from the
literature
28-30
, and the DZ to MZ ratio among like-sex pairs of 0.9:1 is consistent with
the ratio of 1.1 expected on the basis of twin prevalence in the population. The
population of that region designated “north” contains about 24% of the continental
population, but, as expected, contributed a higher proportion (44%) of twin cases.
The ratio of northern to other cases was 1.8, comparable to the range of analogous
estimates from military cases of 1.4-2.4
31
, but lower than the only civilian estimate
of 3.1
32
. This apparent deficit could reflect a change in that ratio over time
33
but is
more likely due to the smaller fraction of the northern population served by large-
circulation newspapers. Characteristics of the pairs for whom questionnaire
information was available were similar to the other pairs in respect to zygosity,
gender and age at diagnosis (Table 6.1). Twins born in the north were slightly more
likely to complete the questionnaires than twins born in other states (76.4% vs.
66.8%).
132
6.2.2 Pattern of Pairwise Concordance
By Zygosity: The concordance rate for MS was 13.4% (56/418) among MZ, 5.0%
(19/380) among like-sex DZ twins and 3.7% (12/325) among unlike-sex DZ twins.
The overall MZ to DZ concordance ratio of 3.0 (MZ to like-sex DZ ratio of 2.7)
reflects the heritable nature of MS.
By Gender: Although concordance rates among male MZ and DZ pairs are
somewhat lower than those among females, the MZ to DZ ratio among both males
(2.9) and females (2.6) is still convincingly high in biologic terms (Table 6.3).
By Latitude: Concordance among MZ pairs born in the north was nearly twice as
high as among those born elsewhere (18.6% compared to 9.5%, Table 6.3). This
latitude effect was reasonably consistent by gender among MZ pairs; concordance
rates were 15.5% and 19.3% among northern born MZ male and female twins
respectively, and 9.5% and 9.5% among male and female twins born elsewhere. No
such substantial latitude effect on concordance appeared among either like-sex DZ
pairs (5.4% compared to 4.6%) or unlike-sex DZ pairs (4.0% compared to 3.3%).
Thus the overall impact of northern birthplace among MZ was 18.6/9.5 or 1.96,
whereas that among all DZ twins was 4.4/3.8 or 1.17. Considering the a-priori
established increased risk in the north, we calculated the one-tailed probability that
these two ratios differ at 0.1, indicating that while chance could account for the
observed difference by the conventional criterion, the odds are still 9 to 1 that the
difference is real. The overall like-sex MZ/DZ pairwise concordance ratio was
133
therefore 3.4 (95% CI 1.7- 6.7) among twins born in the north, and 2.1 (95% CI 1.0-
4.4) among those born elsewhere (Table 6.3).

Table 6.3: Variations in Pairwise Concordance for MS according to zygosity and baseline
characteristics.
PC
1
PC
1
Strata
MZ pairs Like-sex DZ pairs
Ratio
(95% CI)
Female-Female 13.9% (45/323) 5.4% (15/277) 2.6 (1.5,4.5)†

Male-Male 11.6% (11/95 ) 3.9% (4/103) 2.9 (1.0,8.9)*
Gender
Ratio (95% CI) 1.2(0.6,2.2) 1.4 (0.5,4.0)
≤29.3 years 18.2% (38/209) 6.1% (10/164) 3.0 (1.5,5.8)†

>29.3 years 8.6% (18/209) 4.2% (9/216) 2.1 (0.9,4.5)‡
Age at
diagnosis
2
Ratio (95% CI) 2.1(1.2,3.6)†

1.5 (0.6,3.5)
North 18.6% (33/177) 5.4% (10/184) 3.4 (1.7,6.7)†

Other States 9.5% (23/241) 4.6% (9/196) 2.1 (1.0,4.4)*
Birthplace
3
Ratio (95% CI) 1.9 (1.2,3.2)†

1.2 (0.5,2.8)
Celtic/Scandinavian 24.4% (11/45 ) 7.1% (4/56) 3.4 (1.2,10.0)*

Others 12.5% (31/247) 5.9% (13/220) 2.1 (1.1,3.9)*

Ancestry
4
Ratio (95% CI) 1.9 (1.1,3.6)*

1.2 (0.4,3.6)
1. The pairwise concordance was calculated as C/(C+D) where C is total number of concordant pairs and D is the
total number of discordant pairs
2. The median age of diagnosis for 1st case of all twins, 29.3 years, was used to distinguish between early and
late diagnosis.
3. Canada and the adjacent US states represent North.
4. Twin pairs with at least one grandparent with Celtic or Scandinavian origin. Information on ancestry was
available from 568 twin pairs with completed questionnaire.

* P-value<0.05 †P-value <0.01 ‡P-value<0.1

By Ancestry: MZ Pairs with Celtic or Scandinavian ancestry were 1.9 (95% CI 1.1,
3.6) times more likely to become concordant than twins without such ancestry (Table
6.3). Again, the ratio was much lower (1.2) among same-sex DZ twins.
By Age at Diagnosis: The diagnoses of 94.6% of the twin cases occurred between
ages 15 and 50 with a range of 8.5-65.5 years. The median age at diagnosis did not
differ by zygosity (Table 6.2). Relative to those born elsewhere, the mean age at
134
diagnosis of northern-born cases from discordant pairs was 2.3 years earlier for MZ
twins and 3.1 years earlier for DZ twins (Table 6.4). The co-twin of an MZ case
diagnosed before age 29.3 was 2.1 times (95% CI 1.2, 3.6) more likely to become
affected than the co-twin of a case diagnosed at a later age. A lesser such tendency
(1.5) was seen when DZ twins were diagnosed early.
The median (mean) lag between the 1
st
and 2
nd
diagnosis was 7.7 (9.3) years,
and was shorter among MZ twins, male twins, and twins diagnosed in later calendar
years (data not shown). Neither the average age at MZ or DZ case diagnosis nor the
length of the interval between MZ concordant cases varied by ancestry. The age at
first diagnosis from concordant pairs did not vary appreciably by latitude (Table 6.4),
and no statistically significant difference in the mean interval between concordant
case diagnoses was observed by latitude (data not shown).
By Factors in Combination: The three “significant” predictors of MZ concordance
(latitude of birth, ancestry, and age at first diagnosis) were mutually associated, and
their relative contributions to the gradient of concordance by latitude were therefore
assessed by the calculation of adjusted odds ratios. Northern cases were more often
diagnosed early (OR 1.8, 95% CI: 1.2, 2.6), and were more often of high-risk
ancestry (OR 2.9, 95% C I: 1.5, 5.6), but early age at diagnosis was not associated
with ancestry. Among twins reporting Celtic or Scandinavian ancestry, concordance
differences by both latitude (northern: 30%, other: 13.3%) and early diagnosis (early:
36%, later 10%) were exaggerated.
135
Table 6.4: Mean age of MS diagnosis according to zygosity and concordant status
A. Stratified by Birthplace
North Other states Zygosity and
Disease Status Mean (SD) Mean (SD)
P-value
1
All MZ Cases 29.7 (8.9) 32.0 (8.5) 0.030
All like-sex DZ Cases 29.9 (8.8) 33.0 (9.7) 0.006
Discordant MZ Cases 29.6 (9.5) 32.0 (8.8) 0.035
Discordant DZ Cases 29.7 (8.9) 32.8 (8.9) 0.008
1. Based on Student’s t-test for the difference in mean age of diagnosis between twins born in ‘North’ (northern
states and Canada) and others states.

B. Stratified by Celtic or Scandinavian Ancestry
Celtic/Scandinavian Others p-value
1
Zygosity and Disease Status
Mean (SD) Mean (SD)
All MZ Cases 30.9 (8.0) 31.0 (8.9) 0.98
All like-sex DZ Cases 31.1 (9.2) 31.4 (9.4) 0.81
Discordant MZ Cases 31.4 (8.6) 30.9 (9.2) 0.77
Discordant DZ Cases 31.3 (7.04) 34.6 (5.4) 0.44
1. Based on Student’s t-test for the difference in mean age of diagnosis between twins with or without
Celtic/Scandinavian ancestry.

The variables predictive of MZ concordance by univariate analysis were then
included in multivariate models. The effects of ancestry and age at diagnosis on
concordance were neither mutually confounded (Table 6.5), nor multiplicative (data
not shown). Together they explained most of the link between PC and northern
birthplace. No statistically significant two-way or three-way effect modification was
detected between early diagnosis, ancestry and birthplace. Although those born in
northern states appeared more likely to be female (OR 1.6, 95% CI 1.0-2.6), female
status was neither a confounder nor an effect modifier for associations between
concordance and ancestry, early diagnosis and birthplace (data not shown).

136
Table 6.5: Odds Ratio (OR) and Confidence Limits of MZ Concordance Estimates for MS according
to Birthplace, High Risk Ancestry and Early Diagnosis of 1
st
Case.
a
Univariate Analysis
b
Multivariate Analysis
OR
(95% CI)

Model 1
c
OR(95% CI)

Model 2
d
OR(95% CI)
Celtic/ Scandinavian Ancestry 2.2 (1.0-4.9)* 2.0 (1.0-5.2)*

2.2 (1.0-4.9)*

Age at Diagnosis ≤29.3 years 2.6 (1.3-5.4)
‡
2.5 (1.2-5.2)
‡
2.6 (1.3-5.4)
‡
Born in Northern States/Canada 1.8 (0.9-3.4)† 1.4 (0.7-2.8) -
a. Analysis was restricted to 292 MZ twin pairs with 40 concordant pairs with no missing data for either of the
three analysis variable.
b. Conditional logistic regression model was fitted to assess the effect of the ancestry, age at diagnosis and
birthplace on MS concordance univariately.
c. Odds ratio and 95% CI from multivariate model where all three variables are adjusted for each other.
d. Odds ratio and 95% CI from the multivariate model measuring the effect of ‘Ancestry’ and ‘Early case
diagnosis’ on MS concordance, with simultaneous adjustment for each other but not place of birth.
‡ p-value ≤ 0.01, *p-value ≤0.05, † p-value ≤0.10

By the Relative Importance of Factors: For MZ and like-sex DZ twins combined the
most parsimonious model selected by the stepwise procedure included both zygosity
and early age at diagnosis. The Pearson goodness-of-fit p-value was 0.005 for a
univariate model with early age of diagnosis and 0.01 for one with zygosity, each
compared to the full model. Among MZ twins, the most parsimonious model
included no factors other than early age at diagnosis.
6.2.3 Validity of the Observations
Potential sources of error include differential ascertainment, follow-up, or diagnosis.
As addressed below, none of these errors could produce the observed differences in
twin concordance with respect to either gender, age at diagnosis or latitude.
137
Differential ascertainment: Responses to newspaper advertisements are certainly
likely to vary by zygosity, gender and place, but since concordance is calculated
within each specific subgroup or by logistic regression, control for these variables is
automatic. An additional difference in ascertainment may depend upon whether one
or both twins are affected, but the psychological factors responsible for this
difference should not be influenced by latitude of birth.
This method of ascertainment simultaneously targeted twins with malignancy
as well as those with MS, permitting ascertainment bias to be assessed by
comparison with population-based cancer rates
18
. We ascertained 52 like-sex twin
pairs concordant for colon cancer, the most common malignancy affecting both
genders. On the basis of relative incidence and population prevalence, one would
expect 38 of these to be female. In fact 35 were, giving no indication of bias in
ascertainment by gender. Among the 681 pairs with breast cancer born in the North,
17.5% of MZ and 7.9% of like-sex DZ pairs ultimately became concordant, whereas
among the 2341 pairs born elsewhere 18.2% of MZ and 8.0% of like-sex DZ pairs
did so. Thus no evidence of differential ascertainment of concordant pairs by either
gender or latitude was evident.
We tested the validity and robustness of our findings by using alternative
study samples in a sensitivity analysis. Since reports have been published on
Canadian twins since 1986, this might have created awareness among the Canadian
twins and their attending physicians. However, when the principle analysis was
138
performed after the exclusion of the 70 pairs born in Canada as well as those cases
ascertained after 1986 (the year of the first Canadian report of concordance), the
results were unchanged (data not shown). Concern might be raised about differential
follow-up and the validity of diagnosis before 15 or after 60. Exclusion of pairs
followed less than 20 years, or of cases diagnosed before 15 or after 60 did not
change the findings (data not shown). When we restricted the analysis to the 244 MZ
pairs for whom objective diagnostic data (MRI, CT or lumbar puncture) had already
become available, each concordance association was strengthened (Table 6.6). The
effect of northern birthplace was strengthened by restricting the analysis to twins
who continued to reside at their place of birth at least until age 15 (OR: 2.14, 95%
CI: 1.1-4.3). Finally, we took the ‘intent to treat’ approach for MS diagnosis by
conducting the analysis on all cases ascertained without the exclusion of
questionable diagnoses or the inclusion of cases who developed MS during the
follow-up period. Again, the conclusions were unchanged (data not shown).

Incomplete follow-up: Most but not all second twins were followed for more than 10
years after the first diagnosis (well after the majority of second cases would be
expected), and the members of each study subgroup (gender, latitude, ancestry, age
at diagnosis) were followed for a roughly identical period (Table 6.2).

139
Table 6.6: Odds Ratio and Confidence Limit of Pairwise Concordance (PC) estimates of Ancestry,
Birthplace and Age at Diagnosis for MZ twins pairs with available objective medical data
1
.
Univariate
Analysis
Multivariate
Analysis
Covariate Strata Pairwise
Concordance
OR(95% CI) OR(95% CI)
Celtic or Scandinavian 28.6% (10/35) Ancestry

Others 16.1% (25/155)
2.1
(0.9-4.9)
†
1.7
(0.9-3.4)
†

≤29.3 26.8% (34/127) Age at diagnosis

>29.3 9.4% (11/117)
3.5
(1.7-7.3) ‡
3.7
(1.8-7.6) ‡

Northern States/Canada 25.7% (27/105) Birthplace

Other States 12.9% (18/139)
2.3
(1.2-4.5)
*
1.3
(0.7-2.3)
1.This analysis was restricted to the 244 MZ twin pairs for whom objective MS diagnostic (MRI, CT and /or
Spinal) data was available.
2.Conditional logistic regression model was utilized to assess the effect of ancestry, age at diagnosis and
birthplace in univariate as well as multivariate models.
†p-value <0.1, *p-value ≤0.05, ‡ p-value ≤0.01

Differential misdiagnosis: Because no comprehensive diagnostic review of a
thousand cases of MS scattered throughout North America is feasible, some errors in
diagnosis are to be expected. This is especially true for cases diagnosed by strictly
clinical means, and most of these were diagnosed before 1990. However, such a
serious diagnosis, if false, is unlikely to be retained through decades of clinic
visitation, and we could find reason to question no more than 1% of the diagnoses..
Although biased over-diagnosis of the identical twin of an MS case might especially
be expected from informed diagnosticians, it also occurred in no more than 1% of
cases. Empirically, nearly all follow-up records have supported the original
diagnosis.
Moreover, there is little reason to expect any residual diagnostic
misclassification to be linked to gender, ancestry, age at first diagnosis, or even
140
latitude. Those few misdiagnoses that have been identified represent both men and
women and persons from all regions. Finally, when a subset of identical twin pairs
with objective diagnostic evidence was separately analyzed, conclusions were
unaltered.
6.36.5 Discussion

Gender: The significant difference in concordance detected between fraternal and
identical twin pairs confirms previous evidence supporting a heritable susceptibility
to MS. Despite the clear evidence of a much higher incidence of MS among women,
we found high MZ/DZ concordance ratios among both females (2.9) and males (2.4),
implying that mechanisms of inheritance are probably identical by gender. The
analogous ratios that had been obtained by screening at Canadian clinics were 9.8
and 0.8
17
, results that are biologically implausible and suggestive of differential
ascertainment.
Latitude: The latitudinal gradient in concordance among identical twins within this
uniformly ascertained and documented sample of affected twin pairs reflects links
between concordance and both ancestry and early diagnosis. The estimated
concordance levels among these MZ (17.0%) and DZ (4.4%) female twins from
northern regions are consistent with those from other northern populations
13, 34, 35
.
Our estimates derived from twins born further south conform to estimates based on
French twins
15
and from the large Italian twin registry
16
.
141
The gradient in concordance according to latitude among MZ pairs is parallel
to and consistent with the geographic gradient in North American MS incidence. No
such gradient was apparent among the 31 concordant DZ pairs, and although chance
might have accounted for this discrepancy, the odds are strongly against it. Such a
zygosity-specific discrepancy probably precludes an explanation based on variable
MS prevalence alone,
Genotype: Scandinavian/Celtic ancestry, i.e. ethnic evidence of a susceptibility
genotype, is an independent and significant predictor of concordance in these MZ,
twins and might explain some of the gradient in twin concordance. The substantial
overall difference between the concordance rate in MZ and like-sex DZ twins is
consistent with the literature in suggesting a polygenic mode of inheritance
36
, and if
the entire susceptibility genotype is represented in proxy by ancestry, it also should
do so equally according to zygosity. However, if only one of several required genetic
components is represented by the ancestry variable, it would be strongly linked to
concordance among MZ twins, who share the entire susceptibility genotype, but
much less strongly linked to DZ twins, who are unlikely to share all polygenic
components.
Environment: Early onset of MS is a characteristic of all northern cases as well as an
independent predictor of MZ concordance, unrelated to ancestry. Early onset
previously has been linked to an increased sibling recurrence risk,
37
to the HLA-
DR15 haplotype among British
38
and Swedish
39
MS cases and to the chemokine
142
receptor 5
40
. However, here it predicted concordance equally well among those with
and without high-risk ancestry, and does so especially among the northern-born. It is
therefore more likely to reflect an environmental correlate of latitude than a genetic
one, as has been concluded from an earlier study
41
. Early onset could represent
either early exposure to a causal factor, such as a virus, or an early environmental
deficit, in protection, such as by exposure to solar flux.
Were any environmental factor to be the sole explanation for the latitudinal
gradient, it also would produce a parallel concordance gradient among DZ twins.
Only in interaction with genetic susceptibility would the gradient appear solely
among concordantly susceptible MZ twins.
Comment: Environmental determinants of twin concordance imply differences in the
commonality of familial environment, i.e. “microenvironmental” variation. Failure to
demonstrate statistical significance between the different recurrence rates of DZ co-
twins and ordinary sibs
17
has led some to categorically deny the existence of such
intra-familial or “micro-environmental” exposure variations. On the contrary, an
observed variation in MZ concordance due to environment directly implies that
environmental exposure varies, at least in intensity, between family members.
Contact with a virus is made more or less likely by the degree of intimacy with the
source. Alternatively, protection by an environmental agent, such as solar flux,
depends upon not only the residential history, but also the degree to which a person,
intentionally or otherwise, adopts or rejects pertinent behavior, such sun-avoidance
42
.
143
Conclusions: Concordance is strongly determined by zygosity in both male and
female twins. When MZ and like-sex DZ twins are combined, both zygosity and
early age at diagnosis are important predictors but the effect of zygosity is greater
than that of early age at diagnosis. Among MZ twins the environmental factor
represented by early age at diagnosis appears to be the strongest predictor of
concordance. Thus the current study provides evidence that MS concordance is
determined by interplay between environmental and genetic factors. In this
circumstance concordantly susceptible identical twin pairs are of great interest, not
only because of the particulars of their genome, but because the timing and intensity
of crucial experiences may shed light on the environmental component of the MS
puzzle, as it has on the etiology of diseases such as breast cancer
44, 45
.
144
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148
Chapter 7 Activities Associated with Sun Exposure in Childhood Modifies Risk
of Multiple Sclerosis among Monozygotic Twin (Neurology)
____________________________________________________________________
Chapter 7 Abstract
Context: Does sun exposure confer protection against Multiple Sclerosis (MS)
irrespective of genetic susceptibility?
Objective: We investigated the effect of sun exposure on risk of MS, comparing
monozygotic (MZ) twin cases to their unaffected co-twins to assess the effect of sun
exposure controlling for genetic susceptibility.
Design: Case-Control twin study.
Setting: Twins with MS were sought through yearly newspaper advertisement
through out North America from 1980-1992, along with other chronic diseases. After
obtaining written permission, providers were asked to provide medical
documentation for each diagnosis, and to update the diagnosis status. Information on
disease status of the affected twins has been updated regularly (till 2005), and fewer
than 4% have been excluded on the basis of misdiagnosis.
Participants: This analysis was restricted to 81 disease and exposure discordant MZ
twin pairs.
Main Outcome Measures: Each twin was asked about pre-disease childhood sun
exposure and outdoor activities in comparison with his/her co-twin. A sun exposure
149
index (SI) was defined as the sum of exposures (9 different sun exposure related
activities) for which an affected twin ranked higher than his/her co-twin. The SI
difference in within each twin pair was calculated by subtracting the SI value of the
affected twin from the SI value of the unaffected twin (range -9 to +9). The data was
analyzed using conditional logistic models.
Results: Relative sun exposure was a strong protective factor against MS within MZ
twin pairs. Lowest risk was noted for those twins who spent more time outdoor ‘in
the sun’ during spring (OR: 0.25, 95% CI: 0.07-0.89) in comparison to the co-twin.
For each unit increase in SI the relative risk of MS decreased by 25%.
Conclusion: After adjusting for genetic susceptibility, early sun avoidance appears
to predict a diagnosis of MS.

This paper is currently in review at Neurology. The authors of the paper are Talat
Islam, W. James Gauderman, Wendy Cozen, and Thomas M. Mack.

150
7.1 Introduction

Multiple sclerosis (MS) is an autoimmune disease of the central nervous
system with a complex etiology. Despite a strong genetic component, limited
concordance (~20%) of MS among monozygotic twins (MZ)
1
, an incidence gradient
according to latitude
2
, and a clear effect of migration
2
, show the importance of
environmental determinants. Because genetic factors fail to fully account for the
latitude gradient, an environmental factor differentially associated with latitude is a
likely candidate. Geographically, the magnitude of solar flux is inversely related to
MS incidence and prevalence; the latter increasing as distance from the equator
increases.
Acheson et.al.
3
hypothesized the possible protective role of sun exposure on
the basis of ecological study of WWII veterans in the 1960s. In a more recent case-
control study conducted in Tasmania, a strong protective role of childhood sun
exposure was observed
4
. These researchers reported that Tasmanians with 2-3 hours
of youthful sun exposure on average per week had nearly a 60% reduction in MS risk
compared to those with less than 2-3 hours. It was suggested that the
immunomodulatory effect of sun exposure was responsible for this apparent
protective effect.
The International Twin Study is a large registry of North American twins
with chronic disease, including MS
5
. This resource provides an opportunity to
151
investigate the association between MS and sun exposures, as reflected by childhood
activities within pairs of MZ twins, only one member of each pair having
symptomatic MS. Such subjects permit us to observe the effect of childhood sun
exposure without confounding by genetic susceptibility.
7.2 Method

Study population: Twins with MS and other chronic diseases were sought through
annual advertisements in periodicals throughout North America from 1980-1992,
along with other chronic diseases. The details of the recruitment process, and a
description of the representativeness of the affected twin pairs have been provided
earlier
5
. In brief, 1149 monozygotic (MZ) and dizygotic (DZ) twin pairs, born in
North America, with at least one documented case of MS were identified. Four
hundred and eighteen (36.4%) respondent pairs were MZ, roughly the proportion
expected. Basic items of information, including address, gender, the date and place
of birth, perceived zygosity, and the date and place of MS diagnosis, were collected.
A detailed questionnaire exploring possible etiologic factors and opinions
about MS etiology was sent to each living member of an affected pair. Completed
questionnaires were received from the members of 292 (70%) MZ pairs, of which 42
pairs were disease-concordant and 250 pairs, were disease-discordant. Of the latter,
193 (77%) pairs were represented by instruments completed by both members.
152
Diagnostic Validation: After obtaining written permission, providers were asked to
provide medical documentation for each diagnosis, and to update the diagnosis
status. Initially, academic MS neurologists (L. Weiner, W. Weiderholt) reviewed the
records of an early sample of cases (both MZ and DZ). Applying the Schumacher
criteria
6
, they judged that 141 of the 145 cases (97%) represented probable MS. (The
remaining 4 were excluded). As follow-up has proceeded to the present, 7
symptomatic twins indicated that their physicians now favored an alternative
diagnosis, and a review of the diagnostic basis of 161 (MZ and DZ) pairs lost to
follow-up identified 6 additional cases for which objective evidence either was
lacking or provided support for an alternative diagnosis. These13 pairs have also
been excluded. Over this period, 17 originally unaffected co-twin developed MS.

Zygosity: The zygosity assignment of each pair was based on the twins’ own
assessment. Twins’ perception of zygosity repeatedly has been shown to be over
90% accurate
7, 8
. We ourselves have assessed the perceptions of more than 150
previous twin pairs using molecular biologic methods
9, 10
, confirming self reported
zygosity in all but one pair.

Exposure Measurement: Information regarding the twins’ outdoor activities in
childhood (prior to diagnosis) was obtained from the completed questionnaires. Each
twin was asked to specify the level of his/her childhood outdoor activity (‘during hot
153
days’, ‘during cold days’, ‘suntanning’, ‘ going to the beach’ and ‘team sports’) and
the time spent outdoors in each season (‘summer’, ’spring’, ’winter’ and ‘fall’), in
comparison to that of the co-twin. Thus the measures of exposure were comparative
in nature rather than quantitative. Informative pairs consisted of only those pairs
discordant for both disease and exposure, and for any given question, only those
pairs who were in agreement about the ranked assessment. Characteristics of the
exposure-discordant identical pairs are compared to those of the exposure-
concordant pairs in Table 7.1.
Table 7.1: Baseline characteristics of exposure-discordant and exposure-concordant MS-discordant
MZ twin pairs
1
.
Exposure-
Discordant pairs
1
Exposure-
Concordant pairs
2
Characteristic
N=79 (%) N =114 (%)
p-value*
Male 13 (16.5) 29 (25.4) 0.13
Born in Canada or an adjacent state 34 (43.0) 43 (37.7) 0.48
North European Ancestry 13 (16.5) 15 (13.2) 0.52
Age of Diagnosis <30 years 38 (46.8) 54 (47.4) 0.94
1. MS-discordant MZ pairs who were discordant for at least one of the 9 (4 seasonal, 2 temperature related, 3
direct) sun exposure related activities.
2. MS-discordant MZ pairs from whom only one questionnaire was completed (59), or who were concordant for
each of the 9 sun exposure related activities (116).
*The P-value according to Chi-square test.

To address the issue of cumulative sun exposure, a sun exposure index (SI)
was calculated for each twin member, the index representing the total number of
responses for which that twin’s exposure exceeded that of the co-twin. There being 9
questions, the possible range of the index was 0 to 9. The pairwise difference in SI
was then calculated by subtracting the SI value for the affected twin from the value
154
for the un-affected co-twin. Thus the range of pairwise difference in SI could range
from -9 to +9.

Other Exposures: The history of childhood infections (chicken pox, red measles,
German measles, hepatitis, polio and mumps) and of infectious mononucleosis (IM)
for each twin was obtained. Those smoking at least 100 cigarettes prior to MS
diagnosis were considered to be smokers, and the age at menarche of each female
twin was recorded. Twins born in Canada and adjacent US states at or above 41-42
0

N were considered to be northern-born and those with at least one grandparent of
Scandinavian or Celtic origin were considered to be of ‘high-risk ancestry’. MS
cases diagnosed before age of 29.3 (median age at diagnosis for MZ twins) years
were considered to have been diagnosed ‘early’.

Analysis: To assess the possible correlation between different measures of sun
exposure, Spearman’s correlation statistics were calculated, separately for affected
and unaffected co-twins. Since the discordant MZ twin pairs represent 1: 1 matched
case-control pairs, tests of association between exposure and disease were performed
by using McNemar’s test as well as conditional logistic regression. All models were
tested for possible confounding and effect modification by personal smoking,
childhood infection, history of infectious mononucleosis and age at menarche.
155
Because age at diagnosis, birthplace, gender and north European ancestry are pair
specific, those were assessed only for effect modification.
The difference in SI value between affected and unaffected twins was tested using
Student’s T test. The magnitude of the link to MS per unit of SI was calculated by
logistic regression models, with assessment of confounding and/or effect
modification. Potential confounders (listed above) were added to the logistic model
one at a time, and assessed on the basis of at least a 10% change in the SI parameter
estimate. Appropriate interaction terms were used to test for effect modification by
those factors. All analyses was performed using SAS 9.1, and unless mentioned
otherwise, all tests were performed using 2-sided alternative hypothesis and a 0.05
level of significance.

7.3 Result

Of the 193 disease-discordant pairs with both twins completing the
questionnaire, the twins in 112 pairs considered each of the 9 exposures to be the
same for each twin. The remaining eighty-one of these pairs were exposure
discordant for at least one of the sun exposure related activities (Figure 7.1). The
demographic features of these exposure discordant pairs did not differ from those of
the 112 exposure concordant pairs. Most twins were female, born in the Northern
states, and of North European ancestry (Table 7.1).
Figure 7.1: Hierarchical flow of analysis data

The closeness within twin members was assessed by whether the twins talked
to each other at least once a month, and most exposure-discordant pairs (87%) had
kept in such close contact with each other, as did most exposure-concordant pairs
(90%). Most cases had been diagnosed at least 10 years prior to study entry, and
most after having attained age 29. Members of only 3 pairs perceived climate or
similar environmental exposure to be a potential risk factor for MS.

156
157
Table 7.2: Correlation (Spearmen coefficients) between different measures of outdoor exposure.
a. Affected MZ twins
Summer Fall Winter Spring Hot
Day
Cold
Day
Suntan Beach Team
sports
Summer 1.00 0.72 0.63 0.69 0.36 0.34 0.05
¶
0.11
¶
0.12
Fall 1.00 0.70 0.72 0.38 0.36 0.03
¶
0.07
¶
0.15
Winter 1.00 0.57 0.39 0.45 0.08
¶
0.10
¶
0.19

Spring 1.00 0.32 0.25 0.05
¶
0.05
¶
0.17
Hot day 1.00 0.66 0.24 0.17 0.16

Cold Day 1.00 0.24 0.14 0.15

Suntan 1.00 0.36 0.26
Beach 1.00 0.29
b: Unaffected MZ co-twins.
Summer Fall Winter Spring Hot
Day
Cold
Day
Suntan Beach Team
sports
Summer 1.00 0.72 0.68 0.74 0.59 0.46 0.22 0.17

0.12
Fall 1.00 0.72 0.87 0.49 0.48 0.18

0.10

0.13
Winter 1.00 0.71 0.52 0.67 0.19

0.16

0.06
¶
Spring 1.00 0.46 0.46 0.17

0.10

0.12
Hot day 1.00 0.72 0.26 0.25 0.04
¶
Cold Day 1.00 0.22 0.19 0.04
¶
Suntan 1.00 0.36 0.26
Beach 1.00 0.28
All correlations were statistically significant except those marked by ¶.

We investigated the correlations between various measures of outdoor
exposure (Table 7.2 a and b). Although the relative frequencies of outdoor exposures
by season were moderately correlated, the relative frequency of specific activities
was largely independent of the seasonal exposure and of each other. More
importantly, the pattern of correlations between different measures was similar
among cases (Table 7.2a) and co-twins (Table 7.2b).
Considering the individual exposures separately, sun exposure during
childhood appeared to be a strong protective factor (Table 7.3); an inverse
association with MS was present for each of the 9 exposure questions. Although the
number of discordant pairs was limited, statistically significant associations were
158
seen for spending more childhood hours outdoors during spring (OR:0. 25; 95% CI:
0.07-0.89), during hot days (OR: 0.40; 95% CI: 0.18-0.91), while sun tanning
(OR:0.38; 95% CI:0.19-0.80) and while at the beach (OR: 0.40;95% CI:0.18-0.91).
Table 7.3: Odds ratio and 95% confidence interval* linking MS to various measures of relative
outdoor exposure.
MZ Twins Exposure Groups
N
†
OR (95%CI) P-Value
Seasonal outdoor exposure
Summer 6/15 0.40 (0.15-1.03) 0.06
Fall 4/7 0.57 (0.17-1.95) 0.37
Winter 5/11 0.45 (0.16-1.31) 0.14
Spring 3/12 0.25 (0.07-0.89) 0.03
Temperature related outdoor exposure
Hot day 8/20 0.40 (0.18-0.91) 0.03
Cold day 9/17 0.53 (0.24-1.19) 0.12
Sun Exposure related activities
Sun tanning 10/25 0.40 (0.19-0.83) 0.01
Beach 8/19 0.42 (0.18-0.96) 0.03
Team sports 8/18 0.44 (0.19-1.02) 0.06
*McNemar’s test was performed to attain the Odds Ratio and 95% confidence interval for the association
between sun exposures related activity and MS among the MZ twins.
†Number of MS-affected twins exposed/Number of control twins exposed.

The histogram in Figure.7.2 shows the distribution of combined pairwise differences
in the index. Under the null hypothesis, the expected mean of SI is zero, and the
calculated mean value of SI of 1 is significantly different from that null value (p-
value from Student’s t-Test=0.0013). Further, the distribution of index differences is
shifted to the right with a substantial right tail. This pattern suggests that cases had
less overall sun exposure in childhood than their unaffected co-twins.

Figure 7.2: Distribution of Intra-pair difference* in the Sun Exposure Index in MS-discordant MZ
Pairs.

*The difference in the Sun Exposure Index (SI) for each MZ pair was calculated by subtracting the SI value of
the affected twin from that of the un-affected co-twin. The skewed distribution to the right with thick right tail
denotes higher SI values for the unaffected twins compared to the affected twins.

Moreover, a statistically significant protective trend in MS risk followed the
increasing within-pair difference (Table 7.4; p-value=0.004). For each unit increase
in the SI difference, the risk of MS decreased by a factor of 0.75. This trend persisted
after stratifying on the potentially modifying characteristics of birthplace, age at
159
160
diagnosis, sex and North European ancestry. No statistically significant interaction
was noted for any of these.
Table 7.4: Odds Ratio (OR) and 95% CI for the association between the Sun Exposure Index and MS,
stratified on known risk factors.
OR 95%CI P-value EM
All MZ twins 0.75 0.62-0.90 0.004
Birth Location 0.29
Southern States 0.63 0.38-0.92 0.04
Northern States and Canada 0.80 0.63-0.98 0.03
Ancestry 0.34
No North European Ancestry 0.96 0.60-1.47 0.83
North European Ancestry 0.71 0.55-0.87 0.004
Age at diagnosis
0.41
>=30 years
0.66 0.42-0.90 0.02
<30 years 0.80 0.62-0.99 0.06
Sex
0.09
Male
1.02 0.68-1.56 0.92
Female
0.69 0.53-0.86 0.003

We could identify no factor confounding the inverse association observed
between childhood sun exposure and MS risk. The association between sun exposure
and MS was neither confounded (Table 7.5) nor modified (data not shown) by
factors such as childhood infection, incidence of infectious mononucleosis, personal
smoking, diet and age at menarche (for female twins).

161
Table 7.5: Odds Ration(OR) and 95% CI for the association between Sun Exposure Index and MS,
adjusted for possible confounders
Adjustment Factors OR 95%CI P-value
Childhood Infection
1
0.76 0.62-0.91 0.007
Infectious Mononucleosis
2
0.76 0.62-0.90 0.004
Smoking
3
0.75 0.60-0.90 0.004
Age of Menarche
4
0.69 0.57-0.85 0.002

*Each factor is separately fitted in the logistic model.
1.History of any childhood infection (chicken pox, red measles, German measles, hepatitis, polio and mumps).
2. History of infectious mononucleosis.
3. History of smoking at least 100 cigarettes prior to MS diagnosis.
4. Age at menarche from the questionnaire response.

We searched for other possible artifacts. To learn whether the results might
have been influenced by the degree of contact between co-twins, we stratified the
twin pairs into two groups based on the above criterion. No difference in the
association between SI and MS was noted between pairs with more (OR: 0.62; 95%
CI 0.43-0.91) or less (OR: 0.86; 95% CI 0.77-1.06) contact. To address the impact of
errors in MS diagnosis, we restricted the analysis to pairs for which detailed medical
records were available (n=56) or pairs who were followed up to the present (n=70).
The ORs from both sub-analyses were similar to the original (available records: OR:
0.75; 95% CI: 0.59-0.93 and complete follow-up: OR: 0.73; 95% CI: 0.58-0.89).
Exclusion of those 3 pairs who considered climate to be a determinant of MS also
did not materially affect the point estimate.

162
7.4 Discussion

To summarize, we found a strong inverse association between the relative
frequency/intensity of childhood activities related to sun exposure and the
appearance of multiple sclerosis within pairs of monozygotic twins, as well as a
linear trend of decreasing MS risk with increasing disparity between the paired sun
exposures. The observed reduction in risk of 50-75% was independent of birthplace
and age at diagnosis.
A similarly strong negative link with childhood (6-15 years) sun exposure
was found in a standard case-control study of prevalent MS cases conducted in
Tasmania
4
. In that study, the odds ratio for MS among children averaging 2 or more
hours of sun exposure per week, compared to less than 2 hours, was 0.31 (95% CI:
0.16-0.59). An earlier study of outdoor and indoor workers also found a dose-
dependent protective effect of sun exposure on MS
11
. However, sun exposure could
be related to both risk and heritable sun tolerance, and neither investigator could
assess subjects with respect to genetic susceptibility. The discordant disease status
among these MZ twins could not reflect inheritance.
The use of unaffected co-twins as controls precludes a genetic interpretation,
and provides other design benefits as well. We simultaneously controlled not only
genome, but age, sex, latitude of birth, skin color, socioeconomic status, and family
history. Because by so doing we could minimize the co-variance from such
163
confounders by design, power was adequate despite the limited number of exposure
discordant pairs.
Although our matched subjects studied here were largely female and
probably better educated than socially representative cases, they are biologically
representative, and generalizability is not seriously diminished. Attention rather
should be focused on the validity of the diagnoses and the meaningfulness of the
comparative exposure measurement. Neither offers a credible explanation for the
results.
The diagnoses were self-reported, and not based on direct neurological
examination, since the cases resided all over North America. Though the validity of
MS diagnosis in this study has been described in earlier publication (Annals of
Neurology) we will address the issues briefly. An early sample of cases was
validated by expert record review, and fewer than 2% cases were considered to be
‘not MS’. Subsequently, cases have been followed and screened for errors by
periodic follow-up and review by telephone interview, questionnaire, and record
review. Virtually all cases have provided the names of their providers. The average
interval between disease onset and inclusion in the study was 10 years and these
twins have been followed for 13 years on average since 1992. Follow-up of roughly
a thousand cases among twins of all kinds for an average of 23 years has turned up
fewer than 20 diagnoses felt to be in doubt by their own physicians, or on the basis
of available records. Residual diagnostic inaccuracy after repeated follow-up of
164
relatively well-educated persons with such a serious diagnosis is unlikely to be
substantial. Furthermore, there is no reason to assume that misdiagnosis would be
non-randomly associated with sun exposure. The exclusion of twin pairs with
incomplete medical records or incomplete follow-up did not change our estimates.
While it is true that responses pertaining to childhood sun exposure might not be
independent if twins were in especially close contact to adulthood. In this regard the
informative pairs appear to be representative of all pairs. In addition, the observed
associations were independent of the degree of close contact. We were unable to
directly quantify the early sun exposure, and the use of subjective comparisons does
not allow us to estimate the level of exposure that might confer protection. However,
conventional studies that make use of questionnaires requesting estimates of time
spent “out in the sun” or “in outdoor activities” are also subject to potential recall
bias and measurement error, especially given a long interval between exposure and
disease, and the variation in weather and behavior. By using the independent
recollections of both twins, we limited both measurement error and recall bias, since
the difference must have been sufficiently notable that both members could recollect
and agree upon it even after many years. Furthermore, at the time subjects completed
their responses, sun exposure was not considered to be an important etiological
factor. Restricting the analyses to pairs in which neither member perceived weather
as a risk factor for MS had no effect on the ORs. Similarly, although confusion
between the sequence of diagnosis and exposure e might be of concern for subjects
165
diagnosed at an early age, the stratified analysis by age at diagnosis did not show any
substantial difference in the ORs.
Sun exposure might induce protection against an autoimmune disease by any
of the several immunosuppressive effects of ultra violet radiation (UVR), especially
UV-B. UVR exposure can exert its immunosuppressive effect directly by producing
cytokines
12, 13
and reducing natural killer cell activity, thus affecting innate
immunity
14
, and indirectly by producing vitamin D and suppressing melatonin
secretion
15
. Animal studies have shown that UV- radiated keratinocytes produce an
array of cytokines including IL-10 and IL-4 through a PGE2 induced pathway
16
. IL-
10, a potent immunosuppressor
17
, alters antigen presentation and IFN- γ secretion by
antigen presenting cells and thus prevents Th0 to TH1 conversion and TH1
activation
18, 19
. UVR can also reduce natural killer cell activity and thus affect innate
immunity
14
. The other potential effect is by means of the induction of vitamin D
production and the effect of that upon Th0-Th1 balance. This effect is probably
achieved by activated vitamin D as it suppresses the production of inflammatory
cytokines like Il-2, IFN- γ and TNF- α
20
, and stimulates anti-inflammatory cytokines
such as TGF β-1 and IL-4
21
.
Reproducibility has always been a major drawback in MS epidemiology. The
strong protective effect of sun exposure on MS risk among MZ twins supports the
similar findings observed by van der Mei et.al
4
. The detection of a similar degree of
reduction of risk in two methodologically different studies conducted in two different
166
populations with wide geographical separation underlines the possible importance of
sun exposure in MS etiology. Studies of the pathway by which sun exposure reduces
MS risk should receive high priority if we are to unravel the mystery of MS etiology.

167
Chapter 7 References

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2. Kurtzke JF. Multiple sclerosis in time and space--geographic clues to cause. J
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variables. Acta Psychiatr Scand. 1960;35(Suppl 147):132-147.

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phenotype, and risk of multiple sclerosis: case-control study. Bmj. Aug 9
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5. Mack TM, Deapen D, Hamilton AS. Representativeness of a roster of
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9. Kumar D, Gemayel NS, Deapen D, et al. North-American twins with IDDM.
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10. Deapen D, Escalante A, Weinrib L, et al. A revised estimate of twin
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11. Freedman DM, Dosemeci M, Alavanja MC. Mortality from multiple sclerosis
and exposure to residential and occupational solar radiation: a case-control
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421.

12. Goettsch W, Garssen J, de Gruijl FR, van Loveren H. UV-B and the immune
system. A review with special emphasis on T cell-mediated immunity.
Thymus. Mar 1993;21(2):93-114.

13. Kripke ML. Ultraviolet radiation and immunology: something new under the
sun--presidential address. Cancer Res. Dec 1 1994;54(23):6102-6105.

14. Sleijffers A, Garssen J, Van Loveren H. Ultraviolet radiation, resistance to
infectious diseases, and vaccination responses. Methods. Sep 2002;28(1):111-
121.

15. Liebmann PM, Wolfler A, Felsner P, Hofer D, Schauenstein K. Melatonin
and the immune system. Int Arch Allergy Immunol. Mar 1997;112(3):203-
211.

16. Shreedhar V, Giese T, Sung VW, Ullrich SE. A cytokine cascade including
prostaglandin E2, IL-4, and IL-10 is responsible for UV-induced systemic
immune suppression. J Immunol. Apr 15 1998;160(8):3783-3789.

17. Shreedhar VK, Pride MW, Sun Y, Kripke ML, Strickland FM. Origin and
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Immunol. Aug 1 1998;161(3):1327-1335.

18. Fiorentino DF, Zlotnik A, Vieira P, et al. IL-10 acts on the antigen-presenting
cell to inhibit cytokine production by Th1 cells. J Immunol. May 15
1991;146(10):3444-3451.

19. Fiorentino DF, Zlotnik A, Mosmann TR, Howard M, O'Garra A. IL-10
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20. Lemire JM. Immunomodulatory role of 1,25-dihydroxyvitamin D3. J Cell
Biochem. May 1992;49(1):26-31.

21. Cantorna MT, Woodward WD, Hayes CE, DeLuca HF. 1,25-
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170
Chapter 8 Grant Proposal- Role of Genes involved in Vitamin D Signaling and
Metabolism Pathway on MS Risk
____________________________________________________________________
Chapter 8 Abstract
Despite a strong genetic component of MS marked by substantial
concordance (~20%) of MS among monozygotic twins (MZ)
1
, environmental
determinants are suggested by the incidence gradient according to latitude
2
. Solar
flux is one latitude-dependent environmental exposure that has been shown to be
associated (inversely) with MS by us
3
as well as other groups
4
. These observations,
along with the importance of sun exposure
5
and latitude of birth
6
on human vitamin
D level and the protective effect of blood vitamin D level against MS
6
led us to
hypothesize that genes associated with Vitamin D metabolism and signaling may
play a role in MS etiology. Previously, we also reported that there is strong evidence
of a gene-environment (GxE) interaction with respect to a latitude effect
7
and
differential risk pattern of sun exposure by HLA genotype.
Thus, our primary specific aim is to analyze data from a matched case-control
within a set of 651 sib-pairs who were discordant for multiple sclerosis (MS) using
previously collected blood samples and genotypic data. The primary hypothesis has
only been addressed partially in three previous studies with mixed results using a
different methodology
8-11
. However, association studies involving multiple genes of
171
the vitamin D pathway and multiple SNPs for each gene (selected by tagSNP
approach) within sib pairs may provide a more valid, less confounded answer to
some of these questions, while controlling for population stratification. We will also
use a pathway analysis approach to assess the overall effect of the genes in this
pathway in respect to MS risk. Furthermore, none of the earlier studies considered
the possible interaction between the vitamin D related genes and sun/ultraviolet
exposure or HLA genotype. We will explore these possible interactions as additional
secondary hypotheses, listed below.
8.1.1 Primary hypothesis

Genes that are involved in vitamin D metabolism and signaling pathway are
associated with multiple sclerosis risk. The analyses will be conducted using results
of an Illumina genotyping assay on 196 single nucleotide polymorphisms of 10
genes related to Vitamin D metabolism and signaling, in a set of 694 multiple
sclerosis cases and their siblings.
8.1.2 Secondary hypotheses
a) The Effect of vitamin D related genes on MS risk varies by HLA-DR2 haplotype.
b) The effect of vitamin D related gene on MS varies by lifetime ultraviolet ray
(UVR) exposure.
c) Vitamin D-related genes interact with each other to modify their effect on MS
risk.
172
d) Develop pathway analysis approach to address the overall effect of the genes on
MS risk.
Recent studies underscore the possible importance of vitamin D and sun
exposure in protection against MS. Detection of association between vitamin D
related genes and MS would provide further evidence regarding the role of vitamin D
in MS. This is of great importance given its preventive and therapeutic implication.
8.2 Background and Significance
8.2.1 Incidence and Prevalence of MS
MS is a common neurological disorder of young adults with a prevalence rate
of 0.9/1000 and incidence rate of 4.2/100,000 in US
12
. Age specific incidence rates
ranged between 9.4 to 18.9 /100,000 person-years for women between 20-45 years of
age as reported from the Nurses Health Study
13
. Within US, similar to the worldwide
pattern, a latitude gradient in MS rates has been observed
13,14
. This latitude gradient
has often been attributed to the preferential settlement of the people with North
European Ancestry in northern latitudes. However, the gradient has attenuated over
the last decades
13,15
, implying the importance of environmental factors. We observed
that the latitude gradient in MS concordance among MZ twins was dependent on
both environmental and genetic factors
7
.
173
8.2.2 Sun Exposure and MS
The geographical distribution of MS is consistent with the hypothesis that
natural UVR from sun exposure is protective. The annual average UVR level is
inversely related to the latitude and MS risk. For example, the difference in average
annual UVR level in Tasmania (41-43.5
0
S) and North Queensland (10-29
0
S) is
2.7kJm
-2
and the MS prevalence in Tasmania is 6 times greater then the MS
prevalence of North Queensland
16
.
Analytic studies have also demonstrated a putative protective effect of sun
exposure against MS. A strong inverse correlation was noted in Australia between
MS and UVR index (R
2
=0.80, p-value=0.01)
16
. The same group reported a strong
protective role of sun exposure against MS from a case-control study conducted in
Tasmania
4
. The study evaluated the role of sun exposure during the childhood (6-15
years of age) among Tasmanians. The odds ratio of MS among the Tasmanians with
2 or more hours of average sun exposure was 0.31 (95% CI: 0.16-0.59) compared to
those with less than 2 hours of exposure. A case-control study in workplace setting
that used death certificates for MS diagnosis noted a dose dependent protective effect
of sun exposure on MS
17
. These findings support the hypothesis that UVR exposure
is strongly protective with respect to MS risk. UVR exposure could exert an
immunosuppressive (protective) effect directly by producing cytokines
18,19
and
natural killer cell activity
20
or through the production of vitamin D
3
. As sun (UVR)
exposure is the main source of the daily requirement of vitamin D
3
21
and vitamin D
3

174
has strong immunomodulatory role
22
, we hypothesize that the protective effect of sun
exposure in MS is mediated through vitamin D
3
.
8.2.3 Vitamin D
3
and MS
The presence of significant number of vitamin D receptor (VDR) in CD4
lymphocytes and macrophages
23
underscores the role of vitamin D in immunity.
Vitamin D supplements have been shown to prevent the incidence of experimental
allergic encephalomyelitis (EAE) in mice (a condition that mimics MS in mice)
24
.
All three subclasses of HLA class II antigens (-DP, -DQ and -DR) are also
downregulated by vitamin D with an overall effect of reduction of antigen dependent
T-cell proliferation
25
. Though the effect of this downregulation on MS pathogenesis
is unclear, polymorphisms of HLA class II antigen have been consistently shown to
be associated with MS risk
26
. It appears that vitamin D is responsible for balanced
cell mediated immunity in response to antigen stimuli
27
.
Few epidemiological or clinical studies have examined the association
between vitamin D and MS. Recently, a nested case-control study involving 7
million US army and navy personnel noted an inverse association between serum
25OH D
3
level and MS risk
6
. Among whites, a 50-nmol/L increase in serum was
associated with a 41% decrease in MS risk (OR: 0.51, 95%CI: 0.36-0.97). Similar
strong protective role of vitamin D in MS incidence was reported from the Nurses
Health Study involving almost 180,000 participants with 173 incident cases of MS
28
.
175
Women in the highest quintile of vitamin D supplement use had 40% lower risk of
MS compared to women in the lowest quintile. The inverse association between MS
risk and vitamin D intake was observed even after adjusting for intake of vitamin A
and vitamin C supplements. Thus, it is pertinent to investigate the possible role of
genes involved in vitamin D pathway in relation to MS risk.
Biosynthesis and molecular activities of vitamin D results from the activity of
a series of enzymes and different protein molecules at different levels
29
. It starts at
skin where UVR exposure acts on 7-dehydrocholesterol to form vitamin D
3
(Figure
1). Vitamin D
3
then undergoes sequential hydroxylation for bio-activation. Vitamin
D transport protein (DBP) binds to gut- and skin-derived vitamin D
3
and transports it
to the liver. In the liver, Vitamin D3 is hydroxylated to 25-hydroxycholecalciferol
(25(OH)D
3
) in presence of the enzyme Vitamin D-25-hydroxylase (CYP27,
CYP2R1). Being bound to DBP, 25(OH)D
3
is then transported to the kidneys where
the enzyme 1-hydroxylase ( CYP27B1) catabolizes 25(OH)D
3
to 1,25(OH)
2
D
3,
the
metabolically most active form of Vitamin D. The enzyme 25-Hydroxyvitamin D
24 hydroxylase (CYP24A1) catabolizes both 25(OH)D
3
and 1,25(OH)
2
D
3
at
position 24 to form 24,25(OH)D
3
and 1,24,25(OH)
2
D
3
, respectively. Both products
undergo further side chain oxidation to form inactive metabolites. These two renal
hydroxylases are regulated in a reciprocal manner to maintain plasma 1,25(OH)
2
D
3

(metabolically active Vitamin D)

level within a narrow range. After transportation to
target tissues, the 1,25(OH)
2
D
3
binds to Vitamin D receptor protein (VDR), and the
176
complex then dimerizes with nuclear receptor retinoic acid receptor (RXR). This
heterodimer complex, 1,25(OH)
2
D
3
/VDR/RXR
,
then binds to the vitamin D receptor
element of target genes to exert the effect of vitamin D on the target gene. In addition
to the biosynthesized vitamin D
3
in skin, the other sources of vitamin D are ingested
Vitamin D
2
from plants and Vitamin D
3
from animal sources. The metabolism of
vitamin D
2
is very similar to the pathway presented above (except for synthesis at
skin). Because vitamin D
3
is the major form of vitamin D in human, we will refer to
it exclusively in the following sections.
It has been observed that the 25-hydroxyvitamin D (25-(OH)D) serum level
varies by dietary vitamin D intake and sunexposure
21
. A change in UVR index from
less than 5 to more than 6 is associated with an increase of serum 25(OH)D serum
level from 71.3 nmol/l to 89.9nmol/L. However, the most active form of vitamin D,
1,25-hydroxyvitamin D(1,25-(OH)D), is more tightly regulated and does not show
any change with sun exposure of dietary intake
30,31
. So, the question arises how can
factors that are associated higher 25-(OH)D can affect MS risk? It should be
considered that extrarenal sites of activation of 25-(OH)D to 1,25-(OH)D includes
macrophage
32
, including microglia
33
. Thus high serum 25-(OH)D can contribute to
enhance immunomodulatory and neuroprotective effects of vitamin D by local
activation of 25(OH)D. Compared to mice fed vitamin D deficient diet, mice fed
vitamin D supplement diet had higher 1,25-(OH)D in CNS but not in serum and
177
fewer signs of EAE
34
. Furthermore, 25(OH)D has been shown to affect innate
immunity
35
and protection against EAE
36
.
8.2.4 Genes in Vitamin D Signaling Pathway
As the enzyme activity, receptor specificity and downstream signaling in the
Vitamin D metabolic pathway is genetically controlled, we hypothesize that
polymorphisms of genes that encode these different enzymes and receptors are
associated with the risk of MS. The genetic location and size of the genes associated
in vitamin D metabolism and signaling pathway is presented in Table 8.1.
Table 8.1: Chromosomal location, number of exons, gene size and SNPs genotyped for the genes
under study-put in entire SNP table
GENE Chromosomal
Location
Number of
Exons
Gene
Size
HapMap
tagSNPs

tagSNP Average
Spacing
CYP27A1 2q33-qter 9 33,311 7 9044.29
CYP2R1 11p15.2 11 14,195 11 4017.64
CYP27B1 12q13.1-q13.3 9 4,859 7 5714.29
CYP24A1 20q13.2-q13.3 12 20,537 42 1203.24
DBP 4q11-q13 13 42,476 13 5575.00
VDR 12q12-q14 11 63,493 38 2460.32
RXRA 9q34.3 10 114023 28 5143.64
RXRB 6p21.3 10 7067 13 3076.92
RXRG 1q22-q23 10 44080 29 2554.45

CYP27A1 encodes the mitochondrial enzyme vitamin D 25-hydroxylase
which catalyzes the hydroxylation of vitamin D3 to 25-hydroxycholecalciferol (25-
(OH)D
3
) in the liver
29
. This is the first step in the bio-activation of vitamin D3. It is
178
also involved in bile acid synthesis
37
. Polymorphism of this gene has been associated
with cerebrotendinous xanthomatosis
38
.
CYP2R1 encodes the microsomal Vitamin D 25-hydroxylase that is also
present in liver. Recent research suggests that this enzyme might be more important
that CYP27A1 in vitamin D metabolism at liver.
39
Polymorphisms of CYP2R1 has
been associated with 25-(OH)D
3
deficiency
39
. A promoter region polymorphism of
CYP2R1 has been associated with serum 25-(OH)D
3
and IgE levels
40
among
families with asthma cases.
The CYP27B1 gene encodes 25-hydroxyvitamin D3-1-alpha-hydroxylase
(1 α-hydroxylase), a P450 enzyme, that is essential in the formation of 1,25 di-
hydroxy D3 (1,25-(OH)
2
D
3
), the most potent form of D3. Any polymorphism of this
gene that would reduce the activity of the enzyme might be involved in MS risk by
lowering the available level of activated D
3
. Polymorphisms of CYP27B1 have been
linked to risk of autoimmune conditions such as Addison's disease
41
, Hashimoto's
thyroiditis
42
, Graves' disease
42
and type 1 diabetes mellitus
42,43
.
CYP24A1 encodes the P450 enzyme, 25 Hydroxyvitamin D 24-hydroxylase,
which plays an important role in the inactivation of activated Vitamin D
3
and thus
provides protection against Vitamin D
3
toxicity and regulates Vitamin D
3
level. It
converts 1,25 (OH)
2
D
3
to the less active form 1,24,25(OH)
3
D
3
that undergoes further
bio-inactivation and also catabolizes 24(R) hydroxylation of 25-(OH)D
3
to
179
24,25(OH)
2
D
3
. A transmission disequilibrium of a CYP24A1 haplotype has been
observed among asthma families
40
. A CYP24A1 haplotype based on 5 single
nucleotide polymorphisms (SNPs) were associated with asthma diagnosis (p =0.001),
total IgE (p =0.001), serum 25(OH)D
3
(p =0.004) and serum 1,25(OH)
2
D
3
(p-
=0.005).
Vitamin D binding protein (DBP) is an alpha-2-globulin with phenotypes
termed group specific components (Gc). DBP is involved with the binding and
transporting of the different vitamin D
3
metabolites. The gene is highly polymorphic,
resulting in more than 120 variants in different populations. However, the common
three variants, i.e. Gc1F, Gc1S, and Gc2, are based on two synonymous SNPs at
codon 416 and 420 of exon 11. One variant, Gc1F, is associated with higher affinity
and more efficient transport of vitamin D metabolites
44
.Thus, functional
polymorphisms in the DBP gene
could
affect the Vitamin D
3
activity by altering its
binding and transport efficiency. Moreover, DBP directly acts on the immune system
by upregulating macrophage activation and chemotaxis of neutrophils (detail review
by Speeckaert et.al.
45
).
DBP polymorphisms have been linked to the risk of many different immune
related conditions. Presence of Lys allele at codon 420 of exon 11 has been
associated with increased risk of Grave’s disease and low 25(OH)D
3
serum levels in
the Polish population
46
. Among French Caucasians it has been observed that Glu
allele of codon 416 of exon 11 is associated with increased risk of type 1 diabetes
47

and IA2 autoantibody
48
; however, no association between DBP and type 1 diabetes
was observed among Germans
49
or Americans of European origin
50
. Furthermore,
polymorphisms of DBP have been shown to be associated with fasting blood sugar
level and glucose tolerance in three different non-Caucasian populations
51,52
. No
association was observed between the three aforementioned polymorphisms of DBP
and MS in one study
8
.
Figure 8.1: Pathway diagram of vitamin D metabolism, transport and target tissue binding.

7-dehydrocholesterol
Vitamin D
3
metabolites
1,25-(OH)
2
D
3
25-(OH)D
3
Vitamin D
3
Catalyst
24-Hydroxylase
(CYP24A1)
1- α Hydroxylase
(CYP27B1)
25-Hydroxylase
(CYP27A1,CYP2R1
)
UVR
Transport
Liver
DBP DBP DBP
Kidney
VDRE
RXR VDR
Target Tissue

Proposed Genes for analysis are presented in bold letters. 1,25 (OH)
2
D
3
in target tissues is represented by ▲.

VDR is a nuclear receptor consisting of 427 amino acids and belongs to the
superfamily of trans-acting transcriptional regulatory factors. In the central nervous
system, it is present on neurons, astrocytes and oligodendrocytes
53,54
. In response to
vitamin D
3
, VDRs can upregulate neurotrophins and inhibits inducible nitric oxide
180
181
synthase and lead to neuroprotection
53,54
. The VDR gene has been shown to be
highly conserved and can be defined into three blocks (A, B, and C). Though high
LD is observed within the blocks, little, if any, LD exists between the blocks
55
. The
‘B’ block contains three of the four commonly studied SNPs of VDR gene (BsmI,
TaqI and ApaI). The other SNP, FokI is located in the region between ‘B’ and ‘C’
blocks. Inconsistent association between these SNPs and conditions, such as
hyperparathyroidism
56,57
, asthma
40,58,59
, rheumatoid arthritis
60-65
, Grave's disease
66-71
,
Type I diabetes
72-76
and MS
8,9,11,61,77,78
have been observed. These inconsistent
findings imply that the SNPs tested were actually in LD with some other functional
SNPs. A recent study noted that the FokI is not in LD with any other common SNPs
and the three common SNPs in block ‘B’ did not capture any information about any
other blocks. Within block ‘B’, these three SNPs could capture only 11 SNPs with
R
2
>0.8 and 15 with R
2
<0.44
55
. Therefore, it appears that these common four SNPs
could capture only limited information about the VDR gene function and other SNPs
are likely to be involved.
Like the VDR, retinoid acid receptors (RXR) also belong to the
superfamily of transcriptional regulators. Three distinct receptors RXR- α, RXR- β
and RXR- γ have been identified. These receptors serve as heterodimeric partners of
activated VDRs. Thus the biological effects of 1,25(OH)
2
D
3
are modulated by RXR
and

aberrant RXR mutation have been shown to result in T
H1
skewing
79,80
.
182
8.2.5 Relevance

MS is the commonest neurodegenerative disease affecting young adults. The
prevalence in US is estimated to be approximately 200,000-350,000
12,81,82
. The
economic and personal cost of MS is immense as it affects mostly during the3rd and
4
th
decade of life. Limiting disability ensues within 10-12 years of onset of relapsing-
remitting type of MS. Without considering the intangible personal suffering, the
annual economic cost of MS is 6.8-11.9 billion dollars
83
. Detection of associations
between MS and vitamin D related genes would provide further credence to the
purported role of vitamin D in MS etiology. Furthermore, it will help to identify
children with high risk and can lead to new treatment and prevention options.
8.2.6 Cost Effectiveness

The population of this proposed study is derived from three well-characterized
populations, namely the 1) International Twin Study (ITS)
7
, 2) California twin study
(CTS)
84
and 2) University of California, San Francisco MS families (UCSFMS)
85
,
totaling 694 case-control pairs (Table 8.2). Detailed questionnaire information on
environmental exposures and family history is available for all of the cases and
controls used in the proposed analysis. Detailed sun exposure information is
available for the participants of the two twin studies. Funding has already been
secured to conduct the genotyping assay (on the Vitamin D genes proposed above),
which will be completed by June of 2007. The amount of information available on
196 SNPs for 694 case-control pairs will be the largest genetic case-control study of
multiple sclerosis to date. Any new study to acquire this information on almost 1,550
individuals will require substantive amount of money and time. Here we are
requesting funds only to support the conduct of the statistical analysis and data
management. The statistical analysis and data interpretation will require extensive
effort from the researchers, because of the density and complexity of the data.
Besides evaluation of main effects of the 196 SNPs of 11 Vitamin D related genes,
we will examine interaction of these SNPs on HLA genotype (previously obtained)
and environmental factors (previously analyzed). Furthermore, considerable time and
effort will be required to develop the pathway analysis approach.
Table 8.2: Characteristics of the MS cases and controls for the three study populations*
Source of Cases and Control
ITS
(case=415)
CTS
Case= (57)
UCSF
(case=222)
Female:Male 2.1

1.7 3.0
Age of Diagnosis (mean±sd) 30.0±7.9 30.4±7.2 32.2±9.8
Age on Dec 31
st
, 2006
MS Case (mean±sd) 62.0±9.0 54.7±8.7 52.9±10.7
MS Control (mean±sd) 63.5±10.0 57.6±7.8 55.3±15.7
Relationship of Control
Sib 249 51
Co-twin 270 46
Other relative 72 38 222
Total Control (948) 591 135 222
* The distributions are presented as mean (SD) for age and frequencies, expressed as percent, for relationship of
controls.

183
184
8.2.7 Representativeness

The representativeness of both ITS population
7
, California Twins
86
and the
UCSFMS
85
families has been reported earlier (detail is provided below). Although
twin MS cases are subjects in this study, their “twin-ness” has no implication with
respect to hypothesis-testing, except that DZ case-twin- unaffected co-twin pairs
have childhood experience in common. Twins are representative of the general
population, and because of the high response rate for both cohorts, we believe that
this set of subjects is highly representative of MS cases in the United States. The risk
of MS to unaffected co-twins of dizygotic (DZ) case-twins is similar to that of
siblings
87
. Furthermore, twinning has no effect on MS status
88
.
8.3 Preliminary Studies
8.3.1 Differential Twin Concordance for Multiple Sclerosis by Latitude of
Birthplace
7

We tested the hypothesis that zygosity, age of onset of the index case, latitude
of birthplace, and Celtic or Scandinavian ancestry were determinants of concordance
in twins (both twins developed MS). Twins born in Canada and adjacent US states,
at or above 41-42 0N, were considered a “northern”, and ancestry was dichotomized
on the basis of known high-risk ancestry (Scandinavian and Celtic). Diagnosis
before the median age of 29.3 was considered “early”. We observed that the MZ-DZ
concordance ratio was 2.9 among males and 2.6 among females. The average age at
185
diagnosis in the north was 2 years earlier for both MZ (p < 0.02) and like-sex DZ (p
< 0.01) patients compared to diagnosis in the south, and was independent of
ancestry. Among MZ twins, concordance was 1.9 (95% CI 1.2-3.2) times greater
among northern-born compared to southern-born twins, 1.9(95% CI: 1.1-3.6) times
greater among twins with high-risk compared to low-risk ancestry, and 2.1(95% CI
1.2-3.6) times greater if diagnosis was early rather than late. Ancestry and early
diagnosis made independent significant contributions to the differential concordance
by latitude. Among DZ twins, concordance was independent of all characteristics.
The concordance rate for the northern twins was only 1.2 times greater than that for
the other twins.
Thus, we concluded that:
a) The latitude gradient exists for MS concordance among MZ twins
b) This latitude gradient can be partially explained by Celtic and
Scandinavian ancestry and earlier age of diagnosis
c) Earlier age at northern diagnosis was due to environmental factors rather
than genetic.
d) Were any environmental factor to be the sole explanation for the
latitudinal gradient, it also would produce a parallel concordance gradient
among DZ twins. Only in interaction with genetic susceptibility would
the gradient appear solely among concordantly susceptible MZ twins who
are completely matched on genes.
186
8.3.2 Childhood Sun Exposure Influences Risk of MS among MZ Twins
(Accepted for publication in Neurology)
Based on our earlier finding, we then wanted to identify the environmental
factors that could explain the latitudinal differences in MS concordance among
identical twins. In that regard, we investigated the association between sun exposure
and MS comparing, disease discordant MZ twins from the ITS with respect to sun
exposure information from the questionnaires they completed. We restricted this
analysis to 79 MS- and exposure- discordant MZ twin pairs. Each twin was asked
during childhood and before any MS diagnosis which twin spent more time outdoors
‘during hot days’, ‘during cold days’, ‘summer’, ’spring’, ’winter’ and ‘fall’,
participated in team sports, and spent more time in ‘sun tanning’ and ‘ going to the
beach’. A sun exposure index (SI) was defined as the sum of differences in the intra-
twin pair sun exposures. The SI difference in within each twin pair was calculated by
subtracting the SI value of the affected twin from the SI value of the unaffected twin
(range -9 to +9). A positive SI indicated that the unaffected co-twin had more sun
exposure than the twin with MS. Each of the nine sun exposure measures appeared to
convey a strong protection against MS within disease discordant MZ twin pairs, with
odds ratios ranging from 0.25 to 0.57. For example, the risk of subsequent MS was
substantially lower (OR: 0.49, 95% CI: 0.19-0.83) for the twin who spent more time
sun tanning. For each unit increase in the SI, the relative risk of MS decreased by
25%. Our finding was very similar to the observed protective effect of sun exposure
187
in relation to MS reported from Tasmania
16
. The demonstration of a similar
reduction of risk from two methodologically different studies conducted in two
widely disparate populations emphasizes the possible importance of sun exposure in
MS etiology. Moreover, our finding notes the importance of sun exposure among
individuals with identical genetic risk for MS.
8.3.3 HLA in MS (ongoing research)
Recently we have genotyped 451 MS cases who were participants of the ITS
along with 641 relative controls for the HLA-DR2 haplotype, in collaboration with
Dr. Jorge Oksenberg at UCSF. Approximately 40% of the population was HLA-DR2
positive. As expected, we observed that cases were more likely to be HLA-DR2
positive than the controls (OR: 1.79, 95%CI: 1.11-2.64). We also observed that the
associated risk of with HLA-DR2 was more apparent for those of northern birthplace
(OR: 2.37, 95%CI: 1.25-4.51) than those with “southern” birthplace (OR: 1.30,
95%CI: 0.73-2.32). This differential HLA-DR2 risk by latitude of birthplace was
apparent even after restricting the analysis to case-control sets without high-risk
Celtic or Scandinavian ancestry. Furthermore, in a sub group analysis (N=38), we
also observed that the protective effect of sun exposure was stronger among people
who were HLA-DR2 positive (with each unit increase in the SI, the relative risk of
MS decreased by 74% compared to those who were HLA-DR2 negative (the
corresponding decrease was 18%). Based on this preliminary result it appears that
188
gene-environmental interaction plays a vital role in MS etiology and can explain
many inconsistent findings. It is also possible that in populations in which HLA-DR2
was less common, we would not see any significant effect of childhood sun
exposure. The likelihood that genes in the Vitamin D pathway interact with
environmental exposure is strong.
8.4 Research Design and Methods
8.4.1 Source of Subjects and Study Design
Subjects were ascertained from three sources: the twin registry developed and
maintained by University of Southern California and the MS family study of
University of California, San Francisco. The characteristics of the three available
populations are presented in table 8.2. The female is to male ratio ranged from 1.7-
3.0. The age of diagnosis was similar in all the population. In all populations, the
current age of the controls and cases were above 50.
International Twin Study:
This is a volunteer twin registry operated out of the University of Southern
California, Department of Preventive Medicine, directed by Prof. Thomas Mack.
Twins with MS and other chronic diseases were ascertained by annual
advertisements in periodicals throughout North America from 1980-1992. The
details of the recruitment process and a description of the representativeness of the
189
twin MS cases have been published and are described above in preliminary studies
7
.
Ascertainment of the original cohort of subjects was designed to capture pairs of
twins in whom at least one member had physician-diagnosed MS. In brief, 1,154
pairs, of known zygosity including 418 MZ pairs and 386 like-sex DZ pairs, with at
least one member of the pair with MS were identified. We have estimated that
approximately 27% of the North American twin cases prevalent at any time during
the period of ascertainment were identified. No concordant pairs were doubly
ascertained. Among the complete set of affected twin volunteers, the ratio of female
to male cases is 3.0, the ratio of affected fraternal to identical twins overall is 1.7,
and among like-sex pairs is 1.0; excepting the slight excess of female volunteers,
each of these ratios is consistent with what would be expected based on the pattern of
cases and of twins in the U.S. The diagnoses of 94.6% of the twin cases occurred
between ages 15 and 50, and roughly two-thirds were diagnosed between 20 and 40.
Basic items of information, including address, gender, the date and place of
birth, perceived zygosity, and the date and place of MS diagnosis, were collected at
the time of recruitment. A detailed 60-page questionnaire exploring possible
etiologic factors and opinions about MS etiology was sent to each living member of
an affected pair. Completed questionnaires were received from 70% of the pairs.
Analysis of the questionnaire data have resulted in the publications described above
in preliminary studies (See Appendix). We were then funded to collect blood from
the cases, their unaffected siblings or co-twins and a related control (siblings, or
190
cousins, when no sibling was available). We received DNA specimens (blood or
buccal) from 958 individuals, representing 416 pairs.
California Twin Study:
This is a population-based registry of California-born twins
86
. California birth
records were linked to the California State Department of Motor Vehicles records to
create a database of over 166,539 twins. Scannable questionnaires (Appendix B)
were sent in several large mailings to 136,156 twins born between 1908 and 1985
and to date 51,609 questionnaires have been returned. The questions on birthplace,
ancestry and outdoor activity are similar to those asked on the International Twin
Study referred to above. At least 1.7 twins of 1000 pairs (40 MZ, 53 DZ, 1 unknown
zygosity) reported a lifetime diagnosis of MS. We used similar protocol described
earlier to collect DNA sample from these twins and controls. We received DNA
specimens (blood or buccal) from 141 individuals, representing 56 pairs with at least
one MS diagnosis.
University California of San Francisco- MS Family Study:
The sib pairs were identified in a large and

well-characterized family-based
cohort of

362 single affected or “singleton” families; these families were required

to
have an affected

proband with either two

living parents or at

least one unaffected
191
sibling
85
. The 205 sib pairs are restricted to those families with at least one
unaffected sib control.

Singleton families were collected

using ascertainment criteria similar to the

multicase families
89
and the annual follow-up of all families continues. Briefly, the
MS cases were identified by reference to the MS registry at UCSF, by physician
referrals, by collaborative arrangements with other MS centers in the USA, and by
advertisements in MS newsletters and at MS functions. The patient's primary
physician or designated representative makes initial contact with the propositus in
each case; individuals expressing a desire to participate are then contacted by letter.
All participants received informed consent as approved by the appropriate IRB. A
brief telephone interview is used to help determine eligibility for the study and to
collect detailed family history information. At the time of blood drawing, the
unaffected sib was interviewed and examined by one of the participating
neurologists.
Diagnostic Confirmation of Diagnosis
International Twin Study
After obtaining written permission, providers were asked to provide medical
documentation for each diagnosis, and to update each diagnostic status. Academic
MS neurologists (L. Weiner, W. Weiderholt, Dept of Neurology, USC) reviewed the
192
records of an early sample of cases (both MZ and DZ). Applying the Schumacher
criteria
90
, they judged that 141 of the 145 cases (97%) had probable MS. (The
remaining 4 were since excluded from all analyses and follow-up). As follow-up has
proceeded to the present, seven symptomatic twins indicated that their physicians
now favored an alternative diagnosis, and a review of the diagnostic basis of 161
(MZ and DZ) pairs lost to subsequent follow-up identified six additional cases for
which objective evidence either was lacking or provided support for an alternative
diagnosis. These 13 pairs have also been excluded. Over this period, 17 originally
unaffected co-twins have developed confirmed MS.

California Twin Registry
The diagnostic confirmation is currently being collected. The collection
procedure is similar to the ITS cases.
University California of San Francisco- MS Family Study:
In

order to limit possible confounding effects of disease heterogeneity

and
misdiagnosis, families in which a primary progressive course

was present in two or
more members were excluded; 69% of patients

had relapsing–remitting multiple
sclerosis at onset. Age of onset was defined

as the first episode of neurological
dysfunction suggestive

of demyelinating disease
91
. To determine

the age of onset,
medical records were reviewed and the patient

was asked to recall his or her initial
neurological symptom,

including visual blurring due to optic neuritis or diplopia,

193
focal weakness, sensory symptoms, incoordination, vertigo, tic

douloureux,
Lhermitte’s symptom or paroxysmal symptoms.

Isolated mild sensory symptoms of
uncertain significance, fatigue

or bladder symptoms alone were not considered as
initial symptoms.

UCSF neurologist confirmed the diagnosis based on review of
medical records.
8.4.2 Polymorphism Selection
The availability of SNP databases and high throughput SNP genotyping
technology has made the characterization of many genes in large populations
feasible. However, the genotyping and analysis of all possible SNPs within each
gene can be costly and inefficient. To reduce the number of SNPs, we have
developed a SNP selection program that combines information on linkage
disequilibrium (LD) with additional information pertaining to each SNP. Specifically
designed to integrate with the Illumina platform, we use genotype information from
either HapMap or user-defined populations and determine bins of SNPs by grouping
SNPs with pairwise LD above a pre-specified threshold. Unique to this process is the
ability to determine bins conditional on LD structure within other ethnic groups and
on SNP frequency across multiple ethnic groups. Final selection of surrogate SNPs
(tagSNPs) to characterize the bin structure is performed conditional on the minor
allele frequency (MAF ≥0.05), the potential for genotyping success, location within a
gene (intron, exon), and known functionality. Additional SNPs were forced in to
194
insure even spacing of SNPs throughout the chromosome and/or based on previous
functional information. To address Illumina requirements, an extensive error
checking procedure is implemented. SNPs that cannot be genotyped successfully are
excluded via the Illumina genotyping score and SNP combinations that may lead to
unstable allele calls via close physical proximity are prioritized and replaced
according to initial selection conditions. While our goal is to capture genetic
information across a gene region with a minimal set of tagSNPs, we aim to do so
without losing SNPs with substantial prior information. In doing so, we hope to not
only characterize the regional association, but also increase our potential to identify
specific disease-causing variants. Applying this method we have identified 196 SNPs
for the 9 genes of interest (Table 8.1).
8.4.3 Statistical Analysis
The approach of our analysis is to test the association of the 196 SNPs of the
nine genes in Table 1 on the risk of MS in case-sib pairs and case-cousin pairs. In
absence of any previous publication, we will either fit a log additive model for the
SNPs or select the best genetic model with the use of lowest value for AIC.
Covariates like ancestry, sex, birth place, age of diagnosis and others will be
considered for inclusion in the model as confounders based on whether their
inclusion changed the SNP effect estimate by more than 10%. Heterogeneity of
associations among subgroups will be assessed by comparing appropriate models
195
with and without interaction terms using a likelihood ratio test. For the vitamin D
related genes, we also plan to evaluate gene-gene (GXG with HLA-DR2 and among
the SNPs) and gene-environmental interaction (GXE) with total childhood UVR
exposure. As long as no correlation exists between the different SNPs (SNPs that are
not at LD with each other), with HLA-DR2 or total childhood UVR exposure, we
can utilize case-case study design to address the GXG and GXE hypothesis. Beside
the gene specific analysis, we plan to implement a Bayesian hierarchical model to
include knowledge regarding the relations among genetic factors and Bayes model
averaging to comprehensively evaluate the set of risk factors that best represents the
underlying complexity between genetic and environmental variation and disease.
Below we have described in detail the statistical approach for our different
hypotheses.
a) Main Effect of SNPs
We will use conditional logistic regression to address the possible
associations between the genes and MS. We will utilize haplotype-based
92
and
principal component-based (Gauderman-currently in review process see Appendix)
analysis techniques to address the overall association between the genes and MS risk.
Besides, we will also analyze the known functional or earlier reported SNPs in
relation to MS. These analyses will not be corrected for multiple comparisons as the
selection of the genes and SNPs are based on a priori hypotheses.
196
b) The Effect of vitamin D related genes on MS risk varies by HLA type (SNP-
HLA)
Using conditional logistic model with appropriate interaction terms, we will
test possible interaction between HLA-DR2 haplotype and any SNP (i.e. A to G
polymorphism of vitamin D binding protein at codon 2), haplotype or principal
component identified through the main effect analysis. In presence of significant
interaction, stratified analysis will be performed and the result will be presented in
tabulated form similar to the mock Table 8.3 below. Based on a result similar to
Table 8.3, our result will be presented as follows. The presence of the variant allele
of DBP-2G>A is associated with a 2.5 fold risk of MS (95%CI:1.23-3.56). However,
the association between DBP-2G>A and MS was modified by HLA-DR2 status
(interaction p-value=0.003). The variant allele of DBP-2G>A was associated with a
5 fold increase of MS among the carriers of HLA-DR2 haplotype, where as the risk
of MS increased only by 1.5 fold the same variant allele among those without the
HLA-DR2 haplotype.

Table 8.3: Odds ratio and 95% CI for the association between the non-synonymous SNP of VDR at
codon 2 (DBP-2G>A), stratified by HLA-DR2 haplotype.
DBP-2G>A
No A-allele A-allele
P-value
HLA-DR2
Negative 1(-) 1.20(0.88-1.53)
Positive 1.50(1.16-1.95) 5.15(2.34-6.12)*
0.003

Main Effect 1(-) 2.54 (1.23-3.56)* 0.01
* P-value ≤0.05
197
c) The effect of vitamin D related gene on MS varies by lifetime UVR exposure
(SNP-UVR)
Based on the residential history since birth, total childhood (before age 13)
UVR exposure will be estimated using GIS based method
84
. This approach has been
shown to provide better assessment of sun exposure then questionnaire method. As,
vitamin D synthesis depends on UVR exposure we will test for possible modifying
effect of the estimated childhood total UVR exposure on the observed genes. UVR
exposure will be dichotomized by the 25
th
percentile value. We will utilize similar
techniques as mentioned above to assess possible effect modifying role of total UVR
exposure at or below the 25
th
percentile value on the association between the vitamin
D related genes and MS.
d) Vitamin D-related genes interact with each other to modify their effect on
MS risk (SNP-SNP)
We will also investigate possible interaction between different SNPs of a
single gene or between SNPs of different genes. However, we cannot test all possible
interactions, as that will require correction for multiple testing to an absurd extent.
Instead, we will utilize the ‘focused interaction testing framework’ FITF method
93
to
test for two and three levels of interactions with weighted correction for numbers of
testing. Initially, we will only test for possible interactions using the SNPs that have
been identified as possible risk factor in earlier analysis. The FITF method will also
198
allow us to include environmental factors, such as total childhood UVR exposure to
test for possible gene-gene-environmental interaction.
e) Develop pathway analysis approach to address the overall effect of the genes
on MS
We have chosen our list of candidate genes and SNPs because they are involved in
vitamin D pathway. Thus, as we branch out from assessing the impact of a single
candidate gene to comprehensively evaluating the factors in pathways (as outlined in
Figure 1), we are faced with the a priori possibility that many interactions, often of
higher-order, will exist between factors. In our regression framework, this
corresponds to the evaluation of numerous interaction terms between variants from
various candidate genes. Following Conti et al.
94
we will incorporate our biological
knowledge regarding the topology of the relations between factors into our Bayes
model averaging approach. Thus, we will use our knowledge regarding the biologic
pathways to assign a hierarchical structured prior. For example, we may believe that
statistical interactions for two candidate genes exist with a higher probability for
CYP27B1 and CYP24A1 metabolism pathway in Figure 1, as there exists a substrate
dependent control between the enzymes coded by these genes compared to any other
two-candidate genes across the pathway. This belief may be represented in differing
baseline probabilities of inclusion for the interaction terms via differing intercepts in
the final model. This hierarchical structure can then extend upwards including
199
interactions with environmental exposures, such as total childhood UVR and other
genetic determinants such as HLA-DR2 status. The overall impact of these
structured priors is to narrow the space of possible models searched via the stochastic
algorithm. Thus, instead of being faced with an impossible number of interacting
terms and possible models, the process is reduced with biological knowledge to an
informed and guided search procedure.
In the process of the stochastic search, the data will serve to update the prior
probability and inform us of the impact of each factor via the posterior probability of
the models selected. Our final inference regarding the importance of each factor and
the models selected must reflect our prior probability structure. To accomplish this,
we will rely on Bayes factors for inference
95
. Bayes factors are the ratio of the
posterior probability odds comparing two hypotheses to the prior probability odds
and can be thought of as a type of marginal likelihood ratio for the comparison of
two hypotheses. Given the prior structure formulated using biological knowledge and
a posterior probability influenced by the data, we will be able to evaluate which
factors, subsets of factors, and complete models describe the real world complexities
for MS susceptibility.
8.4.4 Sample Size and Power
We have available information on 1509 individuals of 694 case-control sets.
We had more than 1 controls for 155 cases and non-sib control for 94 cases. As a
200
conservative approach, all power calculations are based on 1:1 matched case-sib
pairs. We used Quanto
96
for all power calculations for log additive model of the
genes with MS prevalence being 0.9/1000. Detailed regarding the available sample
size and power for each of the hypotheses proposed in the study is described below.

a) The power of the proposed analyses depends on the frequency of the allele
of interest and the expected relative risk (odds ratio). With the minimum allele
frequency (MAF) of 0.05, we have adequate power to detect a relative risk of 1.6 or
above for the primary hypothesis at 5% significance level (Table 8.4). SNPs with
dominant model requires slightly bigger sample size, however recessive models
require much bigger sample size. For example, with 650 case-sib pairs and
MAF=0.05 we have adequate power to detect an OR=2 for both log additive and
dominant model where as for recessive model the OR has to be at least 8.5.
However, for a more common allele (MAF>0.2) we have adequate power to detect
an OR of 2.3 for a SNP with recessive effect.
As 70% (455 out of 650) of the cases are female, we have adequate power to
address the primary hypothesis among females for a gene effect with OR being 2.1
or more (allele frequency=0.05). Similarly, as 358 (55%) of the cases are HLA-DR2
positive, we have adequate power to detect an OR ≥1.9 with MAF=0.05, OR ≥1.6
with MAF ≥0.10 and OR ≥1.5 with MAF ≥0.15 among those with HLA-DR2
haplotype.
201
b) The sample size and power calculation for SNP-HLA hypothesis is based on the
size of the main effects of the SNPs of interest (OR
G
) and HLA-DR2 haplotype
(OR
H
), their MAF and their interaction (OR
GH
). Considering OR
G
=2 and OR
H
=1.8
and a 50% distribution of HLA-DR2 haplotype in the population, we have adequate
power to detect an OR
GH
≥3.2 for MAF=0.05(n=642), OR
GH
≥2.6 for MAF=0.10
(n=638) and OR
GH
≥2.4 for MAF=0.25 (n=650). However, using a case-case
approach, we can detect even smaller OR
GH
with the same sample size. Using case-
case design, we have adequate power to detect an OR ≥1.9 for MAF=0.05 (n=646)
and OR ≥1.6 for MAF=0.25 (n=630).

Table 8.4: Sample size required to detect noted relative risk for given allele frequency with 0.80
power.
Allele Frequency Relative Risk
0.05 0.10 0.15 0.20 0.25 0.30
1.4 0.71 2519 1356 976 793 690 627
1.5 0.67 1685 912 659 538 470 429
1.6 0.63 1221 664 482 395 347 318
1.7 0.59 934 510 373 307 270 248
1.8 0.56 744 408 300 248 219 202
1.9 0.53 610 337 248 206 183 169
2.0 0.50 513 284 210 175 156 145

c) The power and sample size calculation for SNP-UVR hypothesis is similar
to SNP-HLA, except that we have to consider the effect of the lowest quartile of
total childhood UVR exposure on MS (OR
U
) and the effect size of the interaction
between total childhood UVR exposure and SNPs (OR
GU
). As a conservative and
pragmatic approach, we considered the case-sib pair to have same UVR exposure.
202
Considering OR
G
=2 and OR
U
=2, we have adequate power to detect an OR
GU
≥2.3 for
MAF=0.05 (n=647), OR
GU
≥2.0 for MAF=0.10 (n=588) and OR
GU
≥1.8 for
MAF=0.25 (n=562). Using a case-case design, we have adequate power to detect an
OR
GU
≥1.7 for MAF=0.05 (n=535), and OR
GU
≥1.4 for MAF=0.25 (n=568).
d) The power of SNP-SNP analysis depends on the allele frequency and the
effect size (OR) of both the SNPs and their interaction terms (OR
GG
). With a fixed
sample size of 650 case-sib pairs and moderate main effect of both the SNPs
(OR=2.0), we have adequate power to detect an OR
GG
=3.00 when the MAF of both
the SNPs are 0.05 and OR
GG
=1.60 when the MAF of both the SNPs are 0.25 (Table
8.5). Considering bigger main effect of the SNPs or case-case design our power
would improve even more. For example, with the same sample size (n=650) and
main effect of the SNPs (OR=2.0) we have adequate power to detect OR
GG
=1.70
when the MAF of both the SNPs are 0.05.
We did not calculate power for the pathway analysis as it is an exploratory
process and requires multiple information i.e. numbers of terms involved in the
pathway, their interrelationship and effect size that will be available from the
previously noted hypothesis. However, we find that we have sufficient power to
detect the first four hypotheses even after considering moderate main effects, effect
modification and at least 4% data loss.

203
Table 8.5: Odds ratio (OR) for gene-gene interaction detectable with 80% power with different allele
frequency
1
Allele Frequency Allele
Frequency

0.05 0.10 0.15 0.20 0.25
0.05
3.00 2.40 2.20 2.10 2.00
0.1
2.00 1.90 1.80 1.80
0.15
1.75 1.70 1.65
0.2
1.65 1.60
0.25
1.60
1
All power calculation is based on genetic main effects to be 2.0 for both genes for 650 case-sib pairs.

8.5 Limitations
Genetic Overmatching: The major limitation of this study is related to the major
advantage. Because we are using case-sib/cousin, there might be some genetic
overmatching, especially in case of families with multiple cases. However, we have
been able to show very strong associations with very small sample sizes (preliminary
data on HLA-DR2). This is because bias due to population stratification is greatly
reduced, which can actually increase power. Although the more modest sample size
makes chance a more likely explanation, the possibility of alternative explanations
due to unmeasured confounders, including genetics, cultural practices,
socioeconomic status and early childhood factors, is greatly reduced.

Misclassification of exposure: The total childhood UVR exposure will be based on
the residential history provided by the cases at study entry. This is subject to error
due to the time interval. Furthermore, sun exposure related activity that could help to
better categorize childhood sun exposure is available for only the twin study
204
population but not the UCSF population. Thus the estimated total childhood UVR
exposure is subject to misclassification, however this is expected to be non-
differential by UVR exposure.
Self Reported diagnosis: The diagnoses were self-reported, and not based on direct
neurological examination, since the cases resided all over North America. However,
the validity of MS diagnosis in this population has been described in earlier
publications
7
. An early sample of cases was validated by expert record review, and
fewer than 2% cases were considered ‘not MS’. Subsequently, cases have been
followed and screened for errors by periodic follow-up and review by telephone
interview, questionnaire, and record review. Virtually all cases have provided the
names of their providers. The average interval between disease onset and inclusion
in the study was 10 years and these twins have been followed for 13 years on
average since 1992. Follow-up of roughly a thousand cases among twins of all kinds
for an average of 23 years has turned up fewer than 20 diagnoses felt to be in doubt
by their own physicians, or on the basis of available records. Residual diagnostic
inaccuracy after repeated follow-up of relatively well-educated persons with such a
serious diagnosis is unlikely to be substantial.
8.6 Responsiveness to RFA
We propose to conduct a genetic analysis for MS utilizing case-sib/cousin
approach with existing data in an effort that has not been previously funded. The
205
proposal is novel in that we use 9 genes involved in vitamin D metabolism and
signaling pathway many of which have not been studied in any other study. Using
1st and 2
nd
degree relatives as control reduces the possibility of population
stratification and enhances credibility of observed findings.
8.7 Time Line
The study will take 2 years to complete. The assessment of quality of the
genotyped data with the possibility of redoing some of samples that might not work
initially will take 2-3 months. Database setup for genotyped data and linking the
genotyped data with pre-existing questionnaire data will take 1-2 months. The data
from UCSF are stored at UCSF so some time must be spent to identify and transfer
those data. The analysis of the main effects with the development of pathway
analysis will take approximately 8-10 months and 1-2 month to write the main effect
paper. Additional 6-7 months will be required for evaluation of effect modification
by HLA-DR2 pattern and UV exposure pattern by birthplace and childhood
exposure, and writing papers.
8.8 Human Subjects
8.8.1 Risks to the Subjects
Human Subjects Involvement and Characteristics: All of the subjects in this study
are twins from pairs in whom at least one twin was diagnosed with MS, and who
206
registered in the International Twin Study from 1980-1993 in response to
advertisements for twins with cancer and their sib/cousins or sib pairs from UCSF.
The study population consists of 1500 subjects representing 651case-control sets. All
subjects are over 18.

Potential Risks: The majority of the data has already been collected. Risks involve a
breach of confidentiality with respect to the MS diagnosis, but there are no
particularly sensitive questions.
8.8.2 Adequacy of Protections against Risks
The protections described below are adequate to ensure that a breach of
confidentiality is highly unlikely. The protection will be ensured by keeping the
paper copies of the questionnaires in locked file cabinets, issuing anonymous
identification numbers (ID), storing the link between the ID number and name in a
completely separate database on a server that is accessible only to the immediate
group of twin researchers (not even to the PI) and is triply password protected. We
will merge the files to obtain the names of the twins who need to be contacted, but
separate the link again once the data is collected.
8.8.3 Recruitment and Informed Consent
Twins in the International Twin Study were initially recruited into a volunteer
registry by placing advertisements in newspapers and magazines asking for twins
207
with cancer, MS and other chronic diseases around North America. Over 13,000
individuals replied and provided names of themselves and their twins, plus diagnoses
(during the 1980’s and early 1990’s). Subsets of twins were re-contacted for studies
involving their particular disease and we have completed studies on breast cancer,
thyroid cancer, bladder cancer, non-Hodgkin’s lymphoma, multiple myeloma and
Hodgkin’s disease.
We have been contacted by some of the twins who originally sent their
questionnaires in, but only to enquire about the findings. We do plan to send a copy
of the final findings to the twins with MS with a lay summary- the participants really
appreciate this gesture when we have done this in the past.
Protection against Risk
Protection against breach of confidentiality involves storing the hard copies of the
questionnaires in locked file cabinets, limiting access to only the few people directly
involved with the study, and removing the personal identifiers from the database. As
above, after the genotyped data has been cleaned, the link with the personal
identifiers will be removed from the analysis database so that identification is not
possible. The database is stored on a server that is triply password protected. All
personnel working on this study have received formal training in protection of
confidentiality and risks to human subjects. Any published data from the study will
208
be in the form of tabular descriptions of groups of subjects, or in a form which
precludes identification of specific individuals.
8.8.4 Potential Benefits of the Proposed Research to the Subjects and Others
Study participants receive no direct personal benefits from study participation. We
hope that the study will benefit society by contributing to our understanding of MS
and that it will benefit future generations by identifying factors that impact risk by
giving us insight into the etiologic mechanisms of MS.
8.8.5 Importance of the Knowledge to be Gained
It is our hope that this research will help us obtain information needed to by
researchers, patients and clinicians to broaden our knowledge with respect to the
etiologic mechanism of MS in hopes of ultimately preventing the occurrence of this
debilitating disease. The subjects are members of a volunteer twin registry and filled
out and sent the questionnaires in several years ago. In fact, some still contact us
anxious to hear the results of the study.
8.9 Inclusion of Women
Both male and female twins are included in this grant. Approximately 66%
participants of this study is woman.
209
8.10 Inclusion of Minorities
Twins of all ethnic groups were included, however since risk of MS is
highest in whites and since the volunteers in the twin registries were mostly white,
the majority of these subjects are white.
8.11 Inclusion of Children
Although some case-twins were under 18 when they were diagnosed, they
were all over 18 at the time of data collection and participation in this study.
8.12 Vertebrate Animals
None involved.

210
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Abstract (if available)

Abstract Multiple sclerosis (MS) is an autoimmune mediated degenerative disease of the central nervous system that is characterized by focal neurological deficits that are marked by temporal and spatial variation. It is a debilitating disease affecting the young adults with a propensity for female and Whites. Beside the latitude gradient, female sex and familial risk, little is known about the risk factors of MS and even less regarding its etiology. Increased recurrence risk among 1st degree relatives and twins of an MS case underscores the importance of genetic susceptibility in MS. However, the observed of ~20% recurrence risk among co-twin of monozygotic twins provides a great insight to MS risk as 1) it is 400 times more than the lifetime risk of general population but 2)80% of the co-twins despite having the same genetic risk do not get the disease. Thus the genetic susceptibility appears to be a necessary but not a sufficient risk factor for MS. Identifying environmental risk factors for MS can greatly reduce the burden of the disease. That have been the focus of my research. My interest is to know the relationship between environmental and genetic risk factors and identify specific risk/protective factors of MS. I addressed my research questions in a large population (~1200) twins with at least one case of MS. The twins represented MS cases among twins in the North American population from 1980-1992. Comparing pairwise concordance rate among monozygotic (MZ) and same-sex dizygotic (DZ) twins by latitude of birthplace I identified the importance of gene-environmental interaction in MS risk and showed that the variation in concordance rate mimics the latitude gradient in MS which can be explained by environmental and genetic risk factors. Limiting our analysis to MZ twins we observed that sun exposure during childhood is associated with reduction in the risk of MS. The protective effect of sun exposure might vary by gender and HLA status.

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Asset Metadata

Creator Islam, Khandaker Talat Shamsul (author)

Core Title Environmental risk factors of Multiple Sclerosis: a twin study

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Epidemiology

Publication Date 04/02/2007

Defense Date 03/14/2007

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag environmental risk factors,multiple sclerosis,OAI-PMH Harvest,twin study

Language English

Advisor Mack, Thomas M. (committee chair), Cozen, Wendy (committee member), Gauderman, W. James (committee member), Gilliland, Frank D. (committee member), Weiner, Leslie P. (committee member)

Creator Email islam@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m343

Unique identifier UC1154674

Identifier etd-Islam-20070402 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-399781 (legacy record id),usctheses-m343 (legacy record id)

Legacy Identifier etd-Islam-20070402.pdf

Dmrecord 399781

Document Type Dissertation

Rights Islam, Khandaker Talat Shamsul

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu