Science in medicine

Multiple sclerosis
David A. Hafler

Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Womens Hospital and Harvard Medical School, Boston, Massachusetts, USA.
The Broad Institute, Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts, USA.

Multiple sclerosis is a complex genetic disease associated with inflammation in the CNS white matter
thought to be mediated by autoreactive T cells. Clonal expansion of B cells, their antibody products,
and T cells, hallmarks of inflammation in the CNS, are found in MS. This review discusses new meth-
ods to define the molecular pathology of human disease with high-throughput examination of germline
DNA haplotypes, RNA expression, and protein structures that will allow the generation of a new series
of hypotheses that can be tested to develop better understanding of and therapies for this disease.

Historical perspective Pathology

A French neurologist at the Salpetrire in Paris, Jean Martin Char- Gross examination of brain tissue of individuals with MS reveals
cot, first described multiple sclerosis (MS) in 1868, noting the accu- multiple sharply demarcated plaques in the CNS white matter with
mulation of inflammatory cells in a perivascular distribution with- a predilection to the optic nerves and white matter tracts of the
in the brain and spinal cord white matter of patients with intermittent periventricular regions, brain stem, and spinal cord. As was recog-
episodes of neurologic dysfunction (13). This led to the term sclrose nized early on and so elegantly investigated in more recent studies,
en plaques dissemines, or multiple sclerosis. The more recent obser- substantial axonal injury with axonal transections is abundant
vation in 1948 by Elvin Kabat of increases in oligoclonal throughout active MS lesions (18).
immunoglobulin in the cerebrospinal fluid of patients with MS pro- The inflammatory cell profile of active lesions is characterized by
vided further evidence of an inflammatory nature to the disease (4, perivascular infiltration of oligoclonal T cells (19) consisting of
5). In the past half-century, several large populationbased MS twin CD4+/CD8 / (20, 21) and / (22) T cells and monocytes with
studies demonstrated a strong genetic basis to this clinical-patho- occasional B cells and infrequent plasma cells (23). Lymphocytes
logic entity (613). Lastly, the demonstration of an autoimmune, at may be found in normal-appearing white matter beyond the mar-
times demyelinating, disease in mammals with immunization of gin of active demyelination (24). Macrophages are most prominent
CNS myelin (experimental autoimmune encephalomyelitis, or in the center of the plaques and are seen to contain myelin debris,
EAE), first made by Thomas Rivers at the Rockefeller Institute in while oligodendrocyte counts are reduced. In chronic-active lesions,
1933 with the repeated injection of rabbit brain and spinal cord the inflammatory cell infiltrate is less prominent and may be large-
into primates (14), has led to the generally accepted hypothesis that ly restricted to the rim of the plaque, suggesting the presence of
MS is secondary to an autoimmune response to self-antigens in a ongoing inflammatory activity along the lesion edge. Recently four
genetically susceptible host (see box, What we know about MS). It pathologic categories of the disease were defined on the basis of
should be pointed out that although the inflammation found in myelin protein loss, the geography and extension of plaques, the
the CNS of patients with MS is thought to represent an autoim- patterns of oligodendrocyte destruction, and the immunopatho-
mune response, this is based on negative experiments where inves- logical evidence of complement activation. Two patterns (I and II)
tigators have not been able to consistently isolate a microbial agent showed close similarities to T cellmediated or T cell plus anti-
from the tissue of diseased patients. Nevertheless, primary viral bodymediated autoimmune encephalomyelitis, respectively. The
infections in the CNS may induce an autoimmune response (15), other patterns (III and IV) were highly suggestive of a vasculopathy
and the recurring lesson from the EAE model is that the minimal or primary oligodendrocyte dystrophy, reminiscent of virus- or
requirement for inducing inflammatory, autoimmune CNS toxin-induced demyelination rather than autoimmunity (25). It was
demyelinating disease is the activation of myelin-reactive T cells in of interest that the pattern of pathology tended to be the same in
the peripheral immune system (16, 17). multiple lesions from any single individual with MS.
Advances in immunology have provided clinicians with powerful
tools to better understand the underlying causes of MS, leading to Natural history
new therapeutic advances. The future calls for extending the origi- MS, like other presumed autoimmune diseases, is more common in
nal observations of Charcot and Kabat by defining the molecular females and often first manifests clinical symptoms during young
pathology of MS at the level of DNA haplotype structure, CNS and adulthood. At its onset, MS can be clinically categorized as either
peripheral mRNA and protein expression, leading to the generation relapsing-remitting MS (RRMS, observed in 8590% of patients) or
of a new series of disease-related hypotheses. primary progressive MS (PPMS). Relapses or attacks typically pre-
sent subacutely, with symptoms developing over hours to several
Nonstandard abbreviations used: acute disseminated encephalomyelitis (ADEM); days, persisting for several days or weeks, and then gradually dissi-
altered peptide ligand (APL); experimental autoimmune encephalomyelitis (EAE); pating. The attacks are likely caused by the traffic of activated,
glatiramer acetate (GA); myelin basic protein peptide p8599 (MBP p8599); primary
myelin-reactive T cells into the CNS, causing acute inflammation
progressive multiple sclerosis (PPMS); relapsing-remitting multiple sclerosis (RRMS);
single nucleotide polymorphism (SNP); secondary progressive multiple sclerosis with associated edema. The ability of high dose steroids to so quick-
(SPMS); T cell receptor (TCR). ly abrogate MS symptoms suggests that the acute edema and its
Conflict of interest: The author has declared that no conflict of interest exists. subsequent resolution underlie the clinical relapse and remission,
Citation for this article: J. Clin. Invest. 113:788794 (2004). doi:10.1172/JCI200421357. respectively. Studies in acute disseminated encephalomyelitis

788 The Journal of Clinical Investigation http://www.jci.org Volume 113 Number 6 March 2004
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(ADEM) in humans (26) and EAE in rodents (27) suggest that exam; that is, finding multiple lesions in time and space in the CNS.
immunologically, acute attacks are self-limited by regulatory T cells. The use of MRI has had a major impact on allowing the early and
The outcome in patients with RRMS is variable; untreated, approx- more precise diagnosis of the disease (33). In a recent prospective
imately 50% of all MS patients require the use of a walking aid within study, patients experiencing their first episode suggestive of CNS
ten years after clinical onset (28), although the consequences on prog- demyelination and having MRI evidence of at least three typical
nosis of newer treatment regiments are not as yet clear. Increased lesions were followed for an average of 42 months. Within that peri-
attack frequency and poor recovery from attacks in the first years of od, a significant proportion of patients developed an additional
clinical disease predict a more rapid deterioration. Multiple MRI relapse, thus qualifying for the diagnosis of clinically definite MS. If
lesions, particularly those that gadolinium enhance on the first MRI there were no MRI lesions, the probability of developing MS was sub-
scan, also predict a more severe subsequent course. Early on in the dis- stantially less. More than half of those developing MS experienced
ease, there are frequent gadolinium-enhanced MRI lesions, consistent the additional relapse within one year of their first episode (34). Thus,
with acute fluxes of activated, autoreactive T cells into the CNS caus- it seems reasonable to label the first attack of what appears to be MS
ing a breakdown of the blood-brain barrier which may be associated as singular sclerosis and to explain to patients that there is a high
with clinical events. However, with time, the extent of recovery from likelihood of developing MS. This indicates to the patient that you
attacks is often decreased, and baseline neurological disability accrues. have an understanding of the underlying problem but that the prog-
Ultimately, approximately 40% of relapsing-remitting patients stop nosis is not as yet clear, allowing patients who never have another
having attacks and develop what may be a progressive neurodegener- attack to be saved from carrying a diagnosis of MS.
ative secondary disorder related to the chronic CNS inflammation, ADEM is a monophasic demyelinating illness that can present
known as secondary progressive multiple sclerosis (SPMS) (29). The with clinical, imaging, and laboratory manifestations indistin-
evolution to this secondary progressive form of the disease is associ- guishable from an acute MS attack (35). However, typical ADEM is
ated with significantly fewer gadolinium-enhanced lesions and a seen in pediatric populations and has more of an explosive course
decrease in brain parenchymal volume (30, 31). Similarly, while earli- associated with alterations in mental status, and a post-viral or post-
er RRMS is sensitive to immunosuppression (32), as times goes on, vaccination history is often elicited. This disease is associated with
responsiveness to immunotherapy decreases and may in fact disap- significant responses to myelin proteins, indicated both by T cell and
pear in later forms of SPMS. Thus, rather than conceiving MS as first antibody measurements (26, 36). It is usually not difficult to distin-
a relapsing-remitting and then a secondary progressive disease, it guish this disease entity from singular sclerosis, and the laborato-
could be hypothesized that MS is a continuum where there are acute ry measurement of circulating high affinity antimyelin basic protein
inflammatory events early on with secondary induction of a neu- antibodies in ADEM but not MS may aid in the diagnosis. It should
rodegenerative process refractory to immunologic intervention. This be noted that there are many reports demonstrating low affinity
hypothesis awaits experimental verification where early immunother- antimyelin autoantibodies by ELISA in sera and CSF of patients with
apy prevents the onset of secondary progressive disease. Such critical MS, though their role either in the diseases pathogenesis or in pre-
investigations require new models of investigation using natural his- dicting outcome is still not well defined. However, high affinity
tory studies that can be performed over decades. antimyelin basic protein or myelin oligodendrocyte antibodies
The primary progressive form of MS is characterized from the appear to be more difficult to detect in the serum or CSF of patients
onset by the absence of acute attacks and instead involves a gradual with MS while antimyelin oligodendrocyte antibodies can be found
clinical decline. Clinically, this form of the disease is associated with in MS CNS plaque tissue (37).
a lack of response to any form of immunotherapy (32). This leads to MRI is the optimal imaging modality for MS. From a diagnostic
the notion that PPMS may in fact be a very different disease as com- standpoint, it is important to keep in mind that the typical appear-
pared to RRMS. A recent commentary points out the similarities ance of multiple lesions on MRIs is not specific for MS. In the appro-
between PPMS and human T lymphotropic virus type Iassociated priate clinical setting, however, this appearance provides an impor-
myelopathy (15), where there is a progressive decline in neurologic tant ancillary diagnostic tool that may establish the multifocality of
function from the disease onset. CNS involvement. MRI has also been used to assess MS disease activ-
ity, disease burden, and the dynamic evolution in these parameters
Diagnosis over time (38). MRI is 410 times more sensitive than the clinical
In the absence of a specific immune-based assay, the diagnosis of MS evaluation in capturing CNS lesions (39), and serial studies have
continues to be predicated on the clinical history and neurological unequivocally demonstrated that clinically apparent changes reflect

What we know about MS

Inflammation in CNS white matter Different pathological subtypes. Early T cell infiltration. Later CD4 and CD8 T cells
with loss of myelin and axons.
Increase in IgG in CSF CSF Ig is oligoclonal. B and T cells in CNS and CSF are also oligoclonal.
Complex genetic disease s of 2040. High degree of inheritability. So far, only MHC region on chromosome 6 clearly
associated with MS.
Antibody autoreactivity Variable results among different studies. Agreement on presence of autoantibodies in MS plaque tissue.
No high affinity autoantibodies in serum.
T cell autoreactivity Autoreactivity to self-myelin antigens in patients with MS and normal subjects. In patients with MS,
modest increase in frequency of myelin-reactive, activated T cells.

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Figure 1
Working hypothesis as to the cause of MS. (I) In a genetically susceptible host, common microbes both activate the APCs through toll receptors and
contain protein sequences cross-reactive with self myelin antigens. This leads to what can be defined as the minimal requirement for inducing an
autoimmune, inflammatory CNS disease in mammals. (II) Underlying immunoregulatory defects, such as decreases of regulatory T cells in the cir-
culation of patients with MS, allow the further pathologic activation of autoreactive T cells (96). (III) Activated myelin-reactive T cells migrate into the
CNS and recognize antigen presented by microglia, local APCs. Th1 cytokines are secreted and an inflammatory cascade is initiated. (IV) Regula-
tion of autoimmune responses. Naturally occurring mechanisms may exist to regulate autoimmune responses including the induction of autoreac-
tive Th2 (IL-4, IL-5, IL-13), Th3 (TGF-), or Tr1 (IL-10) cytokinesecreting T cells that migrate to the CNS and downregulate (red arrow) inflammato-
ry Th1 autoreactive T cells (green arrow). Therapies may attempt to induce Th2 (Copaxone, altered peptide ligands), Th3 (mucosal antigen), or Tr1
(-IFNs, steroids). ThP, precursor T cell.

only a minor component of disease activity. Lesions in the cerebrum Disease mechanisms
are much more likely to be clinically silent, as compared to lesions Immunopathophysiology of MS. It is often asked whether EAE, briefly
in the brainstem or spinal cord. discussed above, is really MS. Our laboratory investigates the
pathophysiology of MS by directly studying patients with the dis-
Therapy ease and does not directly investigate mouse models. Some of our
Therapies for MS have emerged over the last two decades with the colleagues will even note in good humor that MS is a superb
demonstration of efficacy of three classes of immunomodulating model of EAE! (V. Kuchroo and S. Miller, personal communica-
therapies that impact the course of early MS: immunosuppressive tion.) The truth is, we are and we will always be appropriately lim-
drugs such as mitoxantrone and cyclophosphamide; -IFNs; and an ited with human experimentation. Experimental models, be they
MHC-binding protein that engages the T cell receptor (TCR), glati- Newtonian physics or rodent models of autoimmunity, are just
ramer acetate (GA). The underlying pathology of MS as an inflam- that models. They do not represent truth (try to investigate the
matory CNS disease was instrumental in leading to the drug treat- velocity of a sub-atomic particle approaching the speed of light
ments presently used. While these drug therapies were not using Newtonian physics), and are only as useful as the question
prospectively designed based on a detailed understanding of the dis- one asks of the model. For example, it was shown almost a decade
eases pathophysiology, examination of these drugs mechanisms of ago that the 47 integrin, VLA-4, was critical for T cell traffic
action has provided insight into the etiology of MS. Newer therapies into the CNS of mice with EAE (40). This resulted in a highly suc-
in clinical trials are based on a more rational understanding of the cessful phase II trial of antiVLA-4 in patients with RRMS (41),
disease, and these will be discussed in more detail. which is now in phase III investigations. This is an excellent exam-

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 science in medicine

ple of how the EAE model, if used to ask the correct question, engagement, a qualitatively different TCR complex has time to form,
might be highly useful in developing therapies for MS. and the extent of chain phosphorylation increases corresponding-
A second critical lesson from the EAE model is that of epitope spread- ly (54). Altered peptide ligands (APLs), which bind with low affinity
ing, first observed by Eli Sercarz (42). With the injection of a single to the TCR, weaken this signal. The ability of APLs to change the
myelin protein epitope into mice with subsequent development of cytokine program of a T cell from a Th1 to a Th2 response was
EAE, it was observed that T cells became activated against other epi- exploited first by Kuchroo and coworkers as a therapy for autoim-
topes of the same protein; this was followed by T cell activation in mune disease (55). Using the EAE model of MS, these authors
response to other myelin proteins that become capable of adoptively showed that APLs can activate IL-4 secretion by both encephalito-
transferring the disease to naive mice. The epitope spreading requires genic T cells and naive T cell clones that cross-react with self-antigens.
costimulation with B7/CD28, suggesting that with tissue damage in Injection of APLs is of clear therapeutic value in treating different
the CNS an adjuvant is created in the CNS with the expression of high models of EAE (56, 57), and autoreactive human T cell clones can
amounts of B7.1 costimulatory molecules associated with antigen also be induced to secrete the anti-inflammatory cytokines IL-4 and
release (43). Moreover, we have recently observed that a transgenic TGF- after TCR engagement by APLs (58, 59). However, it was
mouse expressing DR2 (DRB1*1501) and a TCR (Ob1A12) cloned noted that while APLs can induce Th2 cytokine secretion of MBP-
from the blood of a patient with MS recognizing an immunodominant reactive T cells isolated from the peripheral blood T cell of patients
myelin basic protein peptide p8599 (MBP p8599) spontaneously with MS, they can also induce a heteroclitic response in some
developed EAE with epitope spreading to a number of antigens impli- patients, activating these MBP-reactive T cells against the patients
cated in MS including - crystalline, and proteolipid apoprotein (D. own tissues (60). These data provide a strong rationale for the ther-
Altman, V. Kuchroo, and D. Hafler, unpublished observations). As we apeutic use of APLs in patients with autoimmune disease. However,
have observed high expression of B7.1 costimulatory molecules in the they also raise the issue that in some instances, highly degenerate
CNS white matter of patients with MS (44), and as most patients exhib- TCRs can recognize APLs as self-antigens.
it T cell reactivity to a number of myelin antigens (45), it is likely that A recently published phase II clinical trial testing an altered MBP
by the time a patient develops clinical MS there has been epitope p8599 peptide confirms both of these conclusions. At the higher
spreading with reactivity to multiple myelin epitopes. However, the peptide dosage tested, two of seven MS patients developed remark-
presence of clonally expanded T cells in the CSF and brain tissue of ably high frequencies of myelin basic proteinreactive T cells, and
patients with the disease raises the issue that there may be clonal reac- these responses were likely associated with significant increases in
tivity to just a few myelin antigens. Single cell cloning of T cells from MRI-detectable lesions (61) and perhaps even disease exacerbations.
the inflamed CNS tissue screened against combinatorial and protein In contrast, patients treated with lower doses of the APL showed no
libraries may allow new insights into the pathophysiology of MS. such disease flare-ups and may have indeed exhibited some degree of
Data from a number of laboratories combined with experimental immune deviation towards increases in IL-4 secretion of MBP-reac-
data from the EAE model where myelin antigen is injected with adju- tive T cells (61, 62). Thus, APLs represent a classic double-edged
vant into mammals indicate that there are autoreactive T cells recog- sword. In our outbred population, given the high degree of degener-
nizing myelin antigens in the circulation of mammals. It appears that acy in the immune system, it is unclear whether it is possible to find
the activation of these T cells is the critical event in inducing autoim- APLs of self-peptides that pose no risk of cross-reactivity with self.
mune disease (Figure 1). We and others first demonstrated over a An alternative approach to the use of a single APL is the administra-
decade ago that T cell clones isolated from the blood of patients with tion of peptide mixtures that contain many different antigen speci-
MS frequently exhibit exquisite specificity for the immunodominant ficities. Random copolymers that contain amino acids commonly used
p8599 epitope of myelin basic protein (4547). However, while the as MHC anchors and TCR contact residues have been proposed as pos-
TCR appears to be highly specific in recognizing this peptide, altering sible universal APLs. GA (Copaxone) is a random sequence polypep-
the peptide ligand can change the TCR conformation to yield a high- tide consisting of four amino acids (alanine (A), lysine (K), glutamate
er degree of T cell cross-reactivity (48). Using combinatorial chemistry, (E), and tyrosine (Y) at a final molar ratio of A:K:E:Y of 4.5:3.6:1.5:1)
even a greater degree of cross-reactivity could be demonstrated, and a with an average length of 40100 amino acids (63). Directly labeled GA
number of viral epitopes were identified that could trigger autoreac- binds efficiently to different murine H-2 I-A molecules, as well as to
tive T cell clones in a manner that would not be predicted by simple their human counterparts, the MHC class II DR molecules, but does
algorithms (49). Indeed, one MBP-reactive T cell clone recognized an not bind MHC class II DQ or MHC class I molecules in vitro (64). In
epitope of an entirely different self-protein, the myelin oligodendro- phase III clinical trials, GA subcutaneously administered to patients
cyte glycoprotein. Hence, a significant degree of functional degenera- with RRMS decreases the rate of exacerbations and prevents the
cy exists in the recognition of antigens by T cells. This is consistent appearance of new lesions detectable by MRI (65, 66). This represents
with the hypothesis that MS is triggered by autoreactive T cells acti- perhaps the first successful use of an agent that ameliorates autoim-
vated by microbial antigens cross-reactive with myelin (50). The high mune disease by altering signals through the TCR.
frequency of activated, myelin-reactive T cells in the circulation and A universal antigen containing multiple epitopes would be
CSF of patients with MS (51, 52) is consistent with the hypothesis that expected to induce proliferation of naive T cells isolated from the cir-
the disease is initiated by a microbial infection. culation, due to its expected high degree of cross-reactivity with
other peptide antigens. Indeed, GA induces strong MHC class II DR-
Novel therapeutics restricted proliferative responses in T cells isolated from MS patients
Peptides bound to MHC as therapeutic options. It was recognized almost a or from healthy controls (64). In most patients, daily injection with
decade ago that the strength of signal delivered through the TCR GA causes a striking loss of responsiveness to this random polypep-
determines which cytokines are secreted by the T cell (53). The cell tide antigen, accompanied by greater secretion of IL-5 and IL-13 by
apparently measures affinity in part by timing the engagement CD4+ T cells, indicating a shift toward a Th2 response (6770). In
between the TCR and the peptide/MHC complex. With longer addition, the surviving GA-reactive T cells exhibit a high degree of

The Journal of Clinical Investigation http://www.jci.org Volume 113 Number 6 March 2004 791
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degeneracy, as measured by their ability to cross-react with a large Genetic diseases may fundamentally be divided into two types. First
variety of peptides represented in a combinatorial library (68). are the gene disruptions, where there is a gene mutation or deletion,
Thus, in vivo administration of GA induces highly cross-reactive which exhibits high penetrance, and where there is the emergence of
CD4+ T cells that are immune-deviated to secrete Th2 cytokines. We a clear clinical phenotype. Sickle cell anemia and muscular dystrophy
have proposed that GA-induced migration of highly cross-reactive are two such examples with mutations of the hemoglobin and dys-
Th2 (and perhaps Th3) cells to sites of inflammation allows their trophin genes, respectively. In these diseases, linkage studies, i.e., link-
highly degenerate TCRs to contact self-antigens, which they recog- ing rather large segments of the human genome identified by so-called
nize as weak agonists, much like APLs. These T cells then apparent- microsatellite markers among family members with the disease, fol-
ly secrete suppressive, Th2/Th3 cytokines, thus restricting local lowed by positional cloning of the disease gene, have been a powerful
inflammation. Thus, knowledge of the strong genetic association tool in human genetics. Such studies in families with multiple sib
for MHC in patients with MS has indirectly led to a number of ther- pairs with MS have been less successful. Specifically, to date, the only
apeutic trials and new insights into the disease. confirmed genetic feature to emerge from these efforts is the associa-
Cytokines and costimulatory signals. -IFN has similarly had a major tion and linkage of the disease with alleles and haplotypes from the
impact on the treatment of RRMS, though whether it can prevent MHC on chromosome 6p21 (8186). In the mid 1990s, whole genome
the transition to SPMS is still not as yet known. The mechanism of screens for linkage (8789) were published. While these investigations
action of -IFN is also not as yet clear, and likely involves alterations have continued to accumulate whole genome linkage data and almost
of a number of different pathways including induction of IL-10 and all of these screens have found more regions of potential linkage than
inhibition of T cell traffic by blocking metalloproteinases (71). Clin- would be expected by chance alone, no other clearly statistically sig-
ical trials that block the common IL-12 and IL-23 p40 chain are nificant region has emerged by linkage investigations.
about to begin, as are efforts to block costimulatory signals provid- The other types of genetic diseases are more complex; an alternative
ed by B7-CD28 interactions with CTLA-4 Ig. hypothesis emerging from the linkage studies is that MS, as a common
disease, is caused by common allelic variants each with only subtle but
What remains unknown: the genetic basis of MS important variations in function. Put another way, crude theoretical
In summary of over a century of research on MS, the scientific com- modeling of human population history suggested that variants which
munity has demonstrated that MS is a complex genetic inflammato- have a high population frequency as a whole, and are likely to be
ry disease of the CNS white matter accompanied by T cell, B cell, and responsible for complex traits (the common diseasecommon variant
macrophage infiltration; the antigenic target of these immune cells is hypothesis), will generally be very old and therefore accompanied by
not certain, but are likely to be common myelin antigens shown to be rather little linkage disequilibrium (90). Quantitatively, this may trans-
encephalitogenic in the EAE model. To date, the MHC gene region is late to dozens of gene regions each with risk factors of less than
the only area of the human genome clearly associated with the disease, 1.11.4 but which in concert lead to major risk for disease develop-
though the precise genes in that region responsible for MS are not as ment. It may be postulated that as populations emerged out of Africa
yet known. In the same way that Charcot and then others defined the 30,000 to 50,000 years ago, exposure to new microbes resulted in what
key features of MS by simply examining brain pathology and observ- are thought to be major population bottlenecks, with survival of indi-
ing inflammation, it is critical to redefine the molecular pathology of viduals with allelic variants allowing for resistance to the novel infec-
inflammatory human disease in terms of germline DNA sequence tious event. These combinations of different genes providing resistance
based on the haplotype map, transcription products by RNA microar- to the population, when randomly coming together, result in a hyper-
rays (72), and translation products by tandem mass spectrometry. The responsive immune system, with subsequent autoimmune diseases the
combination of such approaches will likely generate a new series of price an individual may pay for protection of the general population.
hypotheses that can be examined by both animal and in vitro models Organ specificity may have emerged because each infectious agent
of human disease. As MS is a complex genetic disease, understanding evolved with a population bottleneck would select for a single MHC
which combinations of genes provide the multitude of perhaps rela- restricting element and subsequent antigen specificity.
tively minor risk factors which in the population as a whole provide Identifying the common allelic variants that may underlie such com-
protection from microbial disease but together, in unfortunate ran- mon diseases requires a different approach from linkage studies. One
dom combinations, result in human autoimmune disease is a central method might be to actually sequence the whole genome among a group
goal of present research efforts. of 5,000 patients with MS as compared to an equal number of healthy
controls. While this would be the most sensitive approach, as all variants
New approaches to understanding the genetic basis of MS would be identified, at this stage of technology it would be impossible to
Approximately 1520% of MS patients have a family history of MS, even consider. It could be argued that as there appear to be only about 10
but large extended pedigrees are uncommon, with most MS fami- million variant, single nucleotide polymorphisms (SNPs) in the popula-
lies having no more than two or three affected individuals. Stud- tion, we could just examine those in the patients with disease compared
ies in twins (610, 12, 13) and conjugal pairs (73) indicate that to control subjects. This would also be far beyond present technologies.
much of this familial clustering is the result of shared genetic risk The possible emerging solution is both elegant and simple, and is based
factors, while studies of migrants (74) and apparent epidemics (75) on a recent observation that was in fact suggested by studies of the MHC
indicate a clear role for environmental factors. Detailed popula- region over a decade ago. The discovery is that genetic variants tend to
tion-based studies of familial recurrence risk (7678) have provid- occur together in what are called haplotype blocks. That is, recent inves-
ed estimates for familial clustering with s, the ratio of the risk of tigations (91) have shown that recombination is not uniformly dis-
disease in the siblings of an affected individual compared with the tributed along chromosomes, as previously assumed, but rather is con-
general population equal to approximately 2040 (79, 80). It has centrated in hot spots that are on average some 20 to 40kb apart
become clear that this represents a complex genetic disease with (haplotype blocks). It has also been shown that in Europeans and Amer-
no clear mode of inheritance. icans of European descent there is very little haplotype diversity within

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 science in medicine

these genomic haplotype blocks (92). Again, this extensive linkage dise- created two years ago by institutions around the globe including the
quilibrium is most probably the consequence of a severe population bot- University of Cambridge, the University of California at San Francisco,
tleneck affecting Europeans some 30,000 to 50,000 years ago (93). The Duke University, Vanderbilt University, Harvard Medical School, the
European population is thus ideal for screening for association of allel- Massachusetts Institute of Technology, and the Brigham and Womens
ic variants with disease, since very few SNP markers from each of these Hospital. These new partnerships in medical science requiring collab-
linkage disequilibrium blocks will be required to screen the entire orations across scientific disciplines and medical institutions will chal-
genome (94). It is expected that there will be approximately 100,000 such lenge the fabric of funding, authorships, and scientific credit that have
haplotype blocks. Assuming that three SNPs are required to interrogate traditionally defined academic success. Finally, unlike gene knockout
fully the haplotype diversity associated with each block, the whole diseases which require gene therapy that has been difficult to achieve
genome could be screened using approximately 300,000 SNPs (10% of clinically, elucidation of specific pathways will likely require only minor
all SNPs). This approach has been used by Rioux, Daly, Lander, and modification of allelic gene functions. Studies in the EAE model have
coworkers to identify the IBD5 locus in a previously identified linkage indicated that modification of only a few gene loci are required to elim-
peak in patients with inflammatory bowel disease (95). inate disease risk. Thus, pharmacologic targeting of relatively few path-
A whole genome association scan, while attractive, is only begin- ways (with proper safeguards for privacy) in populations screened for
ning to be feasible as the cost of genotyping continues to decrease. It disease risk may be the ultimate treatment for both the inflammatory
is also possible that such an approach may fail because MS may be and degenerative components of MS.
the result of more than the one genetic syndrome that it is generally
believed to be or that hundreds or even thousands of genes, each rep- Acknowledgments
resenting only a fractional risk factor, are associated with the occur- The author wishes to thank all of the present and past laboratory
rence of MS. Epistatic effects of genes will also complicate the analy- members and colleagues for so many of the ideas and concepts used
sis. Nevertheless, large, properly powered experiments will definitively in this article. In particular, I would like to acknowledge Amit Bar-
answer the question as to issues of disease heterogeneity and relative Or, Kevin OConnor, John Rioux, and Phil De Jager who provided
risk factors, and will prevent the wasting of resources on underpow- specific assistance, and my long-term partnerships with Howard
ered investigations that may provide no definitive answers. Weiner, Samia Khoury, and Vijay Kuchroo.
The formation of international consortiums, which allow significant
collections of patients, combined with high-throughput genotyping Address correspondence to: David A. Hafler, Jack, Sadie and David
will be critical in performing whole genome scans based on the haplo- Breakstone Professor of Neurology (Neuroscience), NRB, 77 Avenue
type map. These collaborative efforts, although using many resources, Louis Pasteur, Harvard Medical School, Boston, Massachusetts
will be necessary in providing a true road map for rational drug dis- 02115, USA. Phone: (617) 525-5330; Fax: (617) 525-5333; E-mail:
covery. In this regard, the International MS Genetic Consortium was [email protected].

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