All About Multiple Sclerosis

More MS news articles for April 2004

Multiple sclerosis

http://www.jci.org/cgi/content/full/113/6/788

J. Clin. Invest. 113:788-794 (2004).
David A. Hafler
Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA. The Broad Institute, Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts, USA

Abstract

Multiple sclerosis is a complex genetic disease associated withinflammation in the CNS white matter thought to be mediatedby autoreactive T cells. Clonal expansion of B cells, theirantibody products, and T cells, hallmarks of inflammation inthe CNS, are found in MS. This review discusses new methodsto define the molecular pathology of human disease with high-throughputexamination of germline DNA haplotypes, RNA expression, andprotein structures that will allow the generation of a new seriesof hypotheses that can be tested to develop better understandingof and therapies for this disease.

Historical perspective

A French neurologist at the Salpetrière in Paris, JeanMartin Charcot, first described multiple sclerosis (MS) in 1868,noting the accumulation of inflammatory cells in a perivasculardistribution within the brain and spinal cord white matter ofpatients with intermittent episodes of neurologic dysfunction(1-3). This led to the term sclérose en plaques disseminées,or multiple sclerosis. The more recent observation in 1948 byElvin Kabat of increases in oligoclonal immunoglobulin in thecerebrospinal fluid of patients with MS provided further evidenceof an inflammatory nature to the disease (4, 5). In the pasthalf-century, several large population–based MS twin studiesdemonstrated a strong genetic basis to this clinical-pathologicentity (6-13). Lastly, the demonstration of an autoimmune, attimes demyelinating, disease in mammals with immunization ofCNS myelin (experimental autoimmune encephalomyelitis, or EAE),first made by Thomas Rivers at the Rockefeller Institute in1933 with the repeated injection of rabbit brain and spinalcord into primates (14), has led to the generally accepted hypothesisthat MS is secondary to an autoimmune response to self-antigensin a genetically susceptible host (see box, What we know aboutMS). It should be pointed out that although the inflammationfound in the CNS of patients with MS is thought to representan autoimmune response, this is based on negative experimentswhere investigators have not been able to consistently isolatea microbial agent from the tissue of diseased patients. Nevertheless,primary viral infections in the CNS may induce an autoimmuneresponse (15), and the recurring lesson from the EAE model isthat the minimal requirement for inducing inflammatory, autoimmuneCNS demyelinating disease is the activation of myelin-reactiveT cells in the peripheral immune system (16, 17).

Advances in immunology have provided clinicians with powerfultools to better understand the underlying causes of MS, leadingto new therapeutic advances. The future calls for extendingthe original observations of Charcot and Kabat by defining themolecular pathology of MS at the level of DNA haplotype structure,CNS and peripheral mRNA and protein expression, leading to thegeneration of a new series of disease-related hypotheses.

Pathology

 Gross examination of brain tissue of individuals with MS revealsmultiple sharply demarcated plaques in the CNS white matterwith a predilection to the optic nerves and white matter tractsof the periventricular regions, brain stem, and spinal cord.As was recognized early on and so elegantly investigated inmore recent studies, substantial axonal injury with axonal transectionsis abundant throughout active MS lesions (18).

The inflammatory cell profile of active lesions is characterizedby perivascular infiltration of oligoclonal T cells (19) consistingof CD4+/CD8 a/ß (20, 21) and g/d (22) T cells and monocyteswith occasional B cells and infrequent plasma cells (23). Lymphocytesmay be found in normal-appearing white matter beyond the marginof active demyelination (24). Macrophages are most prominentin the center of the plaques and are seen to contain myelindebris, while oligodendrocyte counts are reduced. In chronic-activelesions, the inflammatory cell infiltrate is less prominentand may be largely restricted to the rim of the plaque, suggestingthe presence of ongoing inflammatory activity along the lesionedge. Recently four pathologic categories of the disease weredefined on the basis of myelin protein loss, the geography andextension of plaques, the patterns of oligodendrocyte destruction,and the immunopathological evidence of complement activation.Two patterns (I and II) showed close similarities to T cell–mediatedor T cell plus antibody–mediated autoimmune encephalomyelitis,respectively. The other patterns (III and IV) were highly suggestiveof a vasculopathy or primary oligodendrocyte dystrophy, reminiscentof virus- or toxin-induced demyelination rather than autoimmunity(25). It was of interest that the pattern of pathology tendedto be the same in multiple lesions from any single individualwith MS.

Natural history

MS, like other presumed autoimmune diseases, is more commonin females and often first manifests clinical symptoms duringyoung adulthood. At its onset, MS can be clinically categorizedas either relapsing-remitting MS (RRMS, observed in 85–90%of patients) or primary progressive MS (PPMS). Relapses or "attacks"typically present subacutely, with symptoms developing overhours to several days, persisting for several days or weeks,and then gradually dissipating. The attacks are likely causedby the traffic of activated, myelin-reactive T cells into theCNS, causing acute inflammation with associated edema. The abilityof high dose steroids to so quickly abrogate MS symptoms suggeststhat the acute edema and its subsequent resolution underliethe clinical relapse and remission, respectively. Studies inacute disseminated encephalomyelitis (ADEM) in humans (26) andEAE in rodents (27) suggest that immunologically, acute attacksare self-limited by regulatory T cells.

The outcome in patients with RRMS is variable; untreated, approximately50% of all MS patients require the use of a walking aid withinten years after clinical onset (28), although the consequenceson prognosis of newer treatment regiments are not as yet clear.Increased attack frequency and poor recovery from attacks inthe first years of clinical disease predict a more rapid deterioration.Multiple MRI lesions, particularly those that gadolinium enhanceon the first MRI scan, also predict a more severe subsequentcourse. Early on in the disease, there are frequent gadolinium-enhancedMRI lesions, consistent with acute fluxes of activated, autoreactiveT cells into the CNS causing a breakdown of the blood-brainbarrier which may be associated with clinical events. However,with time, the extent of recovery from attacks is often decreased,and baseline neurological disability accrues. Ultimately, approximately40% of relapsing-remitting patients stop having attacks anddevelop what may be a progressive neurodegenerative secondarydisorder related to the chronic CNS inflammation, known as secondaryprogressive multiple sclerosis (SPMS) (29). The evolution tothis secondary progressive form of the disease is associatedwith significantly fewer gadolinium-enhanced lesions and a decreasein brain parenchymal volume (30, 31). Similarly, while earlierRRMS is sensitive to immunosuppression (32), as times goes on,responsiveness to immunotherapy decreases and may in fact disappearin later forms of SPMS. Thus, rather than conceiving MS as firsta relapsing-remitting and then a secondary progressive disease,it could be hypothesized that MS is a continuum where thereare acute inflammatory events early on with secondary inductionof a neurodegenerative process refractory to immunologic intervention.This hypothesis awaits experimental verification where earlyimmunotherapy prevents the onset of secondary progressive disease.Such critical investigations require new models of investigationusing natural history studies that can be performed over decades.

The primary progressive form of MS is characterized from theonset by the absence of acute attacks and instead involves agradual clinical decline. Clinically, this form of the diseaseis associated with a lack of response to any form of immunotherapy(32). This leads to the notion that PPMS may in fact be a verydifferent disease as compared to RRMS. A recent commentary pointsout the similarities between PPMS and human T lymphotropic virustype I–associated myelopathy (15), where there is a progressivedecline in neurologic function from the disease onset.

Diagnosis

In the absence of a specific immune-based assay, the diagnosisof MS continues to be predicated on the clinical history andneurological exam; that is, finding multiple lesions in timeand space in the CNS. The use of MRI has had a major impacton allowing the early and more precise diagnosis of the disease(33). In a recent prospective study, patients experiencing theirfirst episode suggestive of CNS demyelination and having MRIevidence of at least three typical lesions were followed foran average of 42 months. Within that period, a significant proportionof patients developed an additional relapse, thus qualifyingfor the diagnosis of clinically definite MS. If there were noMRI lesions, the probability of developing MS was substantiallyless. More than half of those developing MS experienced theadditional relapse within one year of their first episode (34).Thus, it seems reasonable to label the first attack of whatappears to be MS as "singular sclerosis" and to explain to patientsthat there is a high likelihood of developing MS. This indicatesto the patient that you have an understanding of the underlyingproblem but that the prognosis is not as yet clear, allowingpatients who never have another attack to be saved from carryinga diagnosis of MS.

ADEM is a monophasic demyelinating illness that can presentwith clinical, imaging, and laboratory manifestations indistinguishablefrom an acute MS attack (35). However, typical ADEM is seenin pediatric populations and has more of an explosive courseassociated with alterations in mental status, and a post-viralor post-vaccination history is often elicited. This diseaseis associated with significant responses to myelin proteins,indicated both by T cell and antibody measurements (26, 36).It is usually not difficult to distinguish this disease entityfrom "singular sclerosis," and the laboratory measurement ofcirculating high affinity antimyelin basic protein antibodiesin ADEM but not MS may aid in the diagnosis. It should be notedthat there are many reports demonstrating low affinity antimyelinautoantibodies by ELISA in sera and CSF of patients with MS,though their role either in the disease’s pathogenesisor in predicting outcome is still not well defined. However,high affinity antimyelin basic protein or myelin oligodendrocyteantibodies appear to be more difficult to detect in the serumor CSF of patients with MS while antimyelin oligodendrocyteantibodies can be found in MS CNS plaque tissue (37).

MRI is the optimal imaging modality for MS. From a diagnosticstandpoint, it is important to keep in mind that the typicalappearance of multiple lesions on MRIs is not specific for MS.In the appropriate clinical setting, however, this appearanceprovides an important ancillary diagnostic tool that may establishthe multifocality of CNS involvement. MRI has also been usedto assess MS disease activity, disease burden, and the dynamicevolution in these parameters over time (38). MRI is 4–10times more sensitive than the clinical evaluation in capturingCNS lesions (39), and serial studies have unequivocally demonstratedthat clinically apparent changes reflect only a minor componentof disease activity. Lesions in the cerebrum are much more likelyto be clinically silent, as compared to lesions in the brainstemor spinal cord.

Therapy

Therapies for MS have emerged over the last two decades withthe demonstration of efficacy of three classes of immunomodulatingtherapies that impact the course of early MS: immunosuppressivedrugs such as mitoxantrone and cyclophosphamide; ß-IFNs;and an MHC-binding protein that engages the T cell receptor(TCR), glatiramer acetate (GA). The underlying pathology ofMS as an inflammatory CNS disease was instrumental in leadingto the drug treatments presently used. While these drug therapieswere not prospectively designed based on a detailed understandingof the disease’s pathophysiology, examination of thesedrug’s mechanisms of action has provided insight intothe etiology of MS. Newer therapies in clinical trials are basedon a more rational understanding of the disease, and these willbe discussed in more detail.

Disease mechanisms

Immunopathophysiology of MS.

It is often asked whether EAE, briefly discussed above, is reallyMS. Our laboratory investigates the pathophysiology of MS bydirectly studying patients with the disease and does not directlyinvestigate mouse models. Some of our colleagues will even notein good humor that MS is a superb model of EAE! (V. Kuchrooand S. Miller, personal communication.) The truth is, we areand we will always be appropriately limited with human experimentation.Experimental models, be they Newtonian physics or rodent modelsof autoimmunity, are just that — models. They do not representtruth (try to investigate the velocity of a sub-atomic particleapproaching the speed of light using Newtonian physics), andare only as useful as the question one asks of the model. Forexample, it was shown almost a decade ago that the a4ß7integrin, VLA-4, was critical for T cell traffic into the CNSof mice with EAE (40). This resulted in a highly successfulphase II trial of anti–VLA-4 in patients with RRMS (41),which is now in phase III investigations. This is an excellentexample of how the EAE model, if used to ask the correct question,might be highly useful in developing therapies for MS.

A second critical lesson from the EAE model is that of epitopespreading, first observed by Eli Sercarz (42). With the injectionof a single myelin protein epitope into mice with subsequentdevelopment of EAE, it was observed that T cells became activatedagainst other epitopes of the same protein; this was followedby T cell activation in response to other myelin proteins thatbecome capable of adoptively transferring the disease to naivemice. The epitope spreading requires costimulation with B7/CD28,suggesting that with tissue damage in the CNS an adjuvant iscreated in the CNS with the expression of high amounts of B7.1costimulatory molecules associated with antigen release (43).Moreover, we have recently observed that a transgenic mouseexpressing DR2 (DRB1*1501) and a TCR (Ob1A12) cloned from theblood of a patient with MS recognizing an immunodominant myelinbasic protein peptide p85–99 (MBP p85–99) spontaneouslydeveloped EAE with epitope spreading to a number of antigensimplicated in MS including a-ß crystalline, and proteolipidapoprotein (D. Altman, V. Kuchroo, and D. Hafler, unpublishedobservations). As we have observed high expression of B7.1 costimulatorymolecules in the CNS white matter of patients with MS (44),and as most patients exhibit T cell reactivity to a number ofmyelin antigens (45), it is likely that by the time a patientdevelops clinical MS there has been epitope spreading with reactivityto multiple myelin epitopes. However, the presence of clonallyexpanded T cells in the CSF and brain tissue of patients withthe disease raises the issue that there may be clonal reactivityto just a few myelin antigens. Single cell cloning of T cellsfrom the inflamed CNS tissue screened against combinatorialand protein libraries may allow new insights into the pathophysiologyof MS.

Data from a number of laboratories combined with experimentaldata from the EAE model where myelin antigen is injected withadjuvant into mammals indicate that there are autoreactive Tcells recognizing myelin antigens in the circulation of mammals.It appears that the activation of these T cells is the criticalevent in inducing autoimmune disease (Figure 1). We and othersfirst demonstrated over a decade ago that T cell clones isolatedfrom the blood of patients with MS frequently exhibit exquisitespecificity for the immunodominant p85–99 epitope of myelinbasic protein (45-47). However, while the TCR appears to behighly specific in recognizing this peptide, altering the peptideligand can change the TCR conformation to yield a higher degreeof T cell cross-reactivity (48). Using combinatorial chemistry,even a greater degree of cross-reactivity could be demonstrated,and a number of viral epitopes were identified that could triggerautoreactive T cell clones in a manner that would not be predictedby simple algorithms (49). Indeed, one MBP-reactive T cell clonerecognized an epitope of an entirely different self-protein,the myelin oligodendrocyte glycoprotein. Hence, a significantdegree of functional degeneracy exists in the recognition ofantigens by T cells. This is consistent with the hypothesisthat MS is triggered by autoreactive T cells activated by microbialantigens cross-reactive with myelin (50). The high frequencyof activated, myelin-reactive T cells in the circulation andCSF of patients with MS (51, 52) is consistent with the hypothesisthat the disease is initiated by a microbial infection.


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 circulation 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) Regulation of autoimmune responses. Naturally occurring mechanisms may exist to regulate autoimmune responses including the induction of autoreactive Th2 (IL-4, IL-5, IL-13), Th3 (TGF-ß), or Tr1 (IL-10) cytokine–secreting T cells that migrate to the CNS and downregulate (red arrow) inflammatory Th1 autoreactive T cells (green arrow). Therapies may attempt to induce Th2 (Copaxone, altered peptide ligands), Th3 (mucosal antigen), or Tr1 (ß-IFNs, steroids). ThP, precursor T cell.

Novel therapeutics

Peptides bound to MHC as therapeutic options.

It was recognized almost a decade ago that the strength of signaldelivered through the TCR determines which cytokines are secretedby the T cell (53). The cell apparently measures affinity inpart by timing the engagement between the TCR and the peptide/MHCcomplex. With longer engagement, a qualitatively different TCRcomplex has time to form, and the extent of z chain phosphorylationincreases correspondingly (54). Altered peptide ligands (APLs),which bind with low affinity to the TCR, weaken this signal.The ability of APLs to change the cytokine program of a T cellfrom a Th1 to a Th2 response was exploited first by Kuchrooand coworkers as a therapy for autoimmune disease (55). Usingthe EAE model of MS, these authors showed that APLs can activateIL-4 secretion by both encephalitogenic T cells and naive Tcell clones that cross-react with self-antigens.

Injection of APLs is of clear therapeutic value in treatingdifferent models of EAE (56, 57), and autoreactive human T cellclones can also be induced to secrete the anti-inflammatorycytokines IL-4 and TGF-ß after TCR engagement by APLs(58, 59). However, it was noted that while APLs can induce Th2cytokine secretion of MBP-reactive T cells isolated from theperipheral blood T cell of patients with MS, they can also inducea heteroclitic response in some patients, activating these MBP-reactiveT cells against the patient’s own tissues (60). Thesedata provide a strong rationale for the therapeutic use of APLsin patients with autoimmune disease. However, they also raisethe issue that in some instances, highly degenerate TCRs canrecognize APLs as self-antigens.

A recently published phase II clinical trial testing an alteredMBP p85–99 peptide confirms both of these conclusions.At the higher peptide dosage tested, two of seven MS patientsdeveloped remarkably high frequencies of myelin basic protein–reactiveT cells, and these responses were likely associated with significantincreases in MRI-detectable lesions (61) and perhaps even diseaseexacerbations. In contrast, patients treated with lower dosesof the APL showed no such disease flare-ups and may have indeedexhibited some degree of immune deviation towards increasesin IL-4 secretion of MBP-reactive T cells (61, 62). Thus, APLsrepresent a classic double-edged sword. In our outbred population,given the high degree of degeneracy in the immune system, itis unclear whether it is possible to find APLs of self-peptidesthat pose no risk of cross-reactivity with self.

An alternative approach to the use of a single APL is the administrationof peptide mixtures that contain many different antigen specificities.Random copolymers that contain amino acids commonly used asMHC anchors and TCR contact residues have been proposed as possible"universal APLs." GA (Copaxone) is a random sequence polypeptideconsisting of four amino acids (alanine (A), lysine (K), glutamate(E), and tyrosine (Y) at a final molar ratio of A:K:E:Y of 4.5:3.6:1.5:1)with an average length of 40–100 amino acids (63). Directlylabeled GA binds efficiently to different murine H-2 I-A molecules,as well as to their human counterparts, the MHC class II DRmolecules, but does not bind MHC class II DQ or MHC class Imolecules in vitro (64). In phase III clinical trials, GA subcutaneouslyadministered to patients with RRMS decreases the rate of exacerbationsand prevents the appearance of new lesions detectable by MRI(65, 66). This represents perhaps the first successful use ofan agent that ameliorates autoimmune disease by altering signalsthrough the TCR.

A "universal antigen" containing multiple epitopes would beexpected to induce proliferation of naive T cells isolated fromthe circulation, due to its expected high degree of cross-reactivitywith other peptide antigens. Indeed, GA induces strong MHC classII DR-restricted proliferative responses in T cells isolatedfrom MS patients or from healthy controls (64). In most patients,daily injection with GA causes a striking loss of responsivenessto this random polypeptide antigen, accompanied by greater secretionof IL-5 and IL-13 by CD4+ T cells, indicating a shift towarda Th2 response (67-70). In addition, the surviving GA-reactiveT cells exhibit a high degree of degeneracy, as measured bytheir ability to cross-react with a large variety of peptidesrepresented in a combinatorial library (68).

Thus, in vivo administration of GA induces highly cross-reactiveCD4+ T cells that are immune-deviated to secrete Th2 cytokines.We have proposed that GA-induced migration of highly cross-reactiveTh2 (and perhaps Th3) cells to sites of inflammation allowstheir highly degenerate TCRs to contact self-antigens, whichthey recognize as weak agonists, much like APLs. These T cellsthen apparently secrete suppressive, Th2/Th3 cytokines, thusrestricting local inflammation. Thus, knowledge of the stronggenetic association for MHC in patients with MS has indirectlyled to a number of therapeutic trials and new insights intothe disease.

Cytokines and costimulatory signals.

ß-IFN has similarly had a major impact on the treatmentof RRMS, though whether it can prevent the transition to SPMSis still not as yet known. The mechanism of action of ß-IFNis also not as yet clear, and likely involves alterations ofa number of different pathways including induction of IL-10and inhibition of T cell traffic by blocking metalloproteinases(71). Clinical trials that block the common IL-12 and IL-23p40 chain are about to begin, as are efforts to block costimulatorysignals provided by B7-CD28 interactions with CTLA-4 Ig.

What remains unknown: the genetic basis of MS

In summary of over a century of research on MS, the scientificcommunity has demonstrated that MS is a complex genetic inflammatorydisease of the CNS white matter accompanied by T cell, B cell,and macrophage infiltration; the antigenic target of these immunecells is not certain, but are likely to be common myelin antigensshown to be encephalitogenic in the EAE model. To date, theMHC gene region is the only area of the human genome clearlyassociated with the disease, though the precise genes in thatregion responsible for MS are not as yet known. In the sameway that Charcot and then others defined the key features ofMS by simply examining brain pathology and observing inflammation,it is critical to redefine the molecular pathology of inflammatoryhuman disease in terms of germline DNA sequence based on thehaplotype map, transcription products by RNA microarrays (72),and translation products by tandem mass spectrometry. The combinationof such approaches will likely generate a new series of hypothesesthat can be examined by both animal and in vitro models of humandisease. As MS is a complex genetic disease, understanding whichcombinations of genes provide the multitude of perhaps relativelyminor risk factors which in the population as a whole provideprotection from microbial disease but together, in unfortunaterandom combinations, result in human autoimmune disease is acentral goal of present research efforts.

New approaches to understanding the genetic basis of MS

Approximately 15–20% of MS patients have a family historyof MS, but large extended pedigrees are uncommon, with mostMS families having no more than two or three affected individuals.Studies in twins (6-10, 12, 13) and conjugal pairs (73) indicatethat much of this familial clustering is the result of sharedgenetic risk factors, while studies of migrants (74) and apparentepidemics (75) indicate a clear role for environmental factors.Detailed population-based studies of familial recurrence risk(76-78) have provided estimates for familial clustering with ls, the ratio of the risk of disease in the siblings of an affectedindividual compared with the general population equal to approximately20–40 (79, 80). It has become clear that this representsa complex genetic disease with no clear mode of inheritance.

Genetic diseases may fundamentally be divided into two types.First are the "gene disruptions," where there is a gene mutationor deletion, which exhibits high penetrance, and where thereis the emergence of a clear clinical phenotype. Sickle cellanemia and muscular dystrophy are two such examples with mutationsof the hemoglobin and dystrophin genes, respectively. In thesediseases, linkage studies, i.e., linking rather large segmentsof the human genome identified by so-called microsatellite markersamong family members with the disease, followed by positionalcloning of the disease gene, have been a powerful tool in humangenetics. Such studies in families with multiple sib pairs withMS have been less successful. Specifically, to date, the onlyconfirmed genetic feature to emerge from these efforts is theassociation and linkage of the disease with alleles and haplotypesfrom the MHC on chromosome 6p21 (81-86). In the mid 1990s, wholegenome screens for linkage (87-89) were published. While theseinvestigations have continued to accumulate whole genome linkagedata and almost all of these screens have found more regionsof potential linkage than would be expected by chance alone,no other clearly statistically significant region has emergedby linkage investigations.

The other types of genetic diseases are more complex; an alternativehypothesis emerging from the linkage studies is that MS, asa common disease, is caused by common allelic variants eachwith only subtle but important variations in function. Put anotherway, crude theoretical modeling of human population historysuggested that variants which have a high population frequencyas a whole, and are likely to be responsible for complex traits(the common disease–common variant hypothesis), will generallybe very old and therefore accompanied by rather little linkagedisequilibrium (90). Quantitatively, this may translate to dozensof gene regions each with risk factors of less than x1.1–x1.4but which in concert lead to major risk for disease development.It may be postulated that as populations emerged out of Africa30,000 to 50,000 years ago, exposure to new microbes resultedin what are thought to be major population bottlenecks, withsurvival of individuals with allelic variants allowing for resistanceto the novel infectious event. These combinations of differentgenes providing resistance to the population, when randomlycoming together, result in a hyper-responsive immune system,with subsequent autoimmune diseases the price an individualmay pay for protection of the general population. Organ specificitymay have emerged because each infectious agent evolved witha population bottleneck would select for a single "MHC restricting"element and subsequent antigen specificity.

Identifying the common allelic variants that may underlie suchcommon diseases requires a different approach from linkage studies.One method might be to actually sequence the whole genome amonga group of 5,000 patients with MS as compared to an equal numberof healthy controls. While this would be the most sensitiveapproach, as all variants would be identified, at this stageof technology it would be impossible to even consider. It couldbe argued that as there appear to be only about 10 million variant,single nucleotide polymorphisms (SNPs) in the population, wecould just examine those in the patients with disease comparedto control subjects. This would also be far beyond present technologies.The possible emerging solution is both elegant and simple, andis based on a recent observation that was in fact suggestedby studies of the MHC region over a decade ago. The discoveryis that genetic variants tend to occur together in what arecalled "haplotype blocks." That is, recent investigations (91)have shown that recombination is not uniformly distributed alongchromosomes, as previously assumed, but rather is concentratedin hot spots that are on average some 20 to 40kb apart (haplotypeblocks). It has also been shown that in Europeans and Americansof European descent there is very little haplotype diversitywithin these genomic haplotype blocks (92). Again, this extensivelinkage disequilibrium is most probably the consequence of asevere population bottleneck affecting Europeans some 30,000to 50,000 years ago (93). The European population is thus idealfor screening for association of allelic variants with disease,since very few SNP markers from each of these linkage disequilibriumblocks will be required to screen the entire genome (94). Itis expected that there will be approximately 100,000 such haplotypeblocks. Assuming that three SNPs are required to interrogatefully the haplotype diversity associated with each block, thewhole genome could be screened using approximately 300,000 SNPs(~10% of all SNPs). This approach has been used by Rioux, Daly,Lander, and coworkers to identify the IBD5 locus in a previouslyidentified linkage peak in patients with inflammatory boweldisease (95).

A whole genome association scan, while attractive, is only beginningto be feasible as the cost of genotyping continues to decrease.It is also possible that such an approach may fail because MSmay be the result of more than the one genetic syndrome thatit is generally believed to be or that hundreds or even thousandsof genes, each representing only a fractional risk factor, areassociated with the occurrence of MS. Epistatic effects of geneswill also complicate the analysis. Nevertheless, large, properlypowered experiments will definitively answer the question asto issues of disease heterogeneity and relative risk factors,and will prevent the wasting of resources on underpowered investigationsthat may provide no definitive answers.

The formation of international consortiums, which allow significantcollections of patients, combined with high-throughput genotypingwill be critical in performing whole genome scans based on thehaplotype map. These collaborative efforts, although using manyresources, will be necessary in providing a true road map forrational drug discovery. In this regard, the International MSGenetic Consortium was created two years ago by institutionsaround the globe including the University of Cambridge, theUniversity of California at San Francisco, Duke University,Vanderbilt University, Harvard Medical School, the MassachusettsInstitute of Technology, and the Brigham and Women’s Hospital.These new partnerships in medical science requiring collaborationsacross scientific disciplines and medical institutions willchallenge the fabric of funding, authorships, and scientificcredit that have traditionally defined academic success. Finally,unlike "gene knockout diseases" which require gene therapy thathas been difficult to achieve clinically, elucidation of specificpathways will likely require only minor modification of allelicgene functions. Studies in the EAE model have indicated thatmodification of only a few gene loci are required to eliminatedisease risk. Thus, pharmacologic targeting of relatively fewpathways (with proper safeguards for privacy) in populationsscreened for disease risk may be the ultimate treatment forboth the inflammatory and degenerative components of MS.

Acknowledgments

The author wishes to thank all of the present and past laboratorymembers and colleagues for so many of the ideas and conceptsused in this article. In particular, I would like to acknowledgeAmit Bar-Or, Kevin O’Connor, John Rioux, and Phil De Jagerwho provided specific assistance, and my long-term partnershipswith Howard Weiner, Samia Khoury, and Vijay Kuchroo.

Footnotes

Nonstandard abbreviations used: acute disseminated encephalomyelitis(ADEM); altered peptide ligand (APL); experimental autoimmuneencephalomyelitis (EAE); glatiramer acetate (GA); myelin basicprotein peptide p85–99 (MBP p85–99); primary progressivemultiple sclerosis (PPMS); relapsing-remitting multiple sclerosis(RRMS); single nucleotide polymorphism (SNP); secondary progressivemultiple sclerosis (SPMS); T cell receptor (TCR).

Conflict of interest: The author has declared that no conflictof interest exists.

References

  1. Charcot, J. 1868. Comptes rendus des séances et mémoires lus à la société de Biologie..
  2. Charcot, J. 1868. Histologie de la sclérose en plaque. Gazette des Hôpitaux. 41:554-566.
  3. Charcot, J. 1877. Lectures on the diseases of the nervous system. The New Sydenham Society. London, United Kingdom. 157–222..
  4. Kabat, E.A., Glusman, M., and Knaub, V. 1948. Quantitative estimation of the albumin and gamma globulin in normal and pathologic cerebrospinal fluid by immunochemical methods. Am. J. Med. 4:653-662.
  5. Kabat, E.A., Freedman, D.A., Murray, J.P., and Knaub, V. 1950. A study of the crystalline albumin, gamma globulin and the total protein in the cerebrospinal fluid of one hundred cases of multiple sclerosis and other diseases. Am. J. Med. Sci. 219:55-64.
  6. Mackay, R.P., and Myrianthopoulos, N.C. 1966. Multiple sclerosis in twins and their relatives. Arch. Neurol. 15:449-462.[Medline]
  7. Williams, A. et al. 1980. Multiple sclerosis in twins. Neurology.30:1139-1147.[Abstract]
  8. Ebers, G.C. et al. 1986. A population-based study of multiple sclerosis in twins. N. Engl. J. Med. 315:1638-1642.[Abstract]
  9. Heltberg, A., and Holm, N. 1982. Concordance in twins and recurrence in sibships in MS. Lancet. 1:1068.
  10. Kinnunen, E., Koskenvuo, M., Kaprio, J., and Aho, K. 1987. Multiple sclerosis in a nationwide series of twins. Neurology. 37:1627-1629.[Abstract]
  11. Utz, U. et al. 1993. Skewed T-cell receptor repertoire in genetically identical twins correlates with multiple sclerosis. Nature. 364:243-247.[Medline]
  12. Mumford, C. et al. 1992. The UK study of MS in twins. J. Neurol.239:62.
  13. French Research Group on Multiple Sclerosis. MS in 54 twinships: concordance rate is independent of zygosity. Ann. Neurol. 32:724-727.
  14. Rivers, T.M., Sprunt, D.H., and Berry, G.P. 1933. Observations on attempts to produce acute disseminated encephalomyelitis in monkeys. J. Exp. Med. 58:39-53.
  15. Hafler, D.A. 1999. The distinction blurs between an autoimmune versus microbial hypothesis in multiple sclerosis. J. Clin. Invest. 104:527-529.[Free Full Text]
  16. Ben-Nun, A., Wekerle, H., and Cohen, I.R. 1981. The rapid isolation of clonable antigen-specific T lymphocyte lines capable of mediating autoimmune encephalomyelitis. Eur. J. Immunol. 11:195-199.[Medline]
  17. Goverman, J. et al. 1993. Transgenic mice that express a myelin basic protein-specific T cell receptor develop spontaneous autoimmunity. Cell. 72:551-560.[Medline]
  18. Trapp, B.D. et al. 1998. Axonal transection in the lesions of multiple sclerosis. N. Engl. J. Med. 338:278-285.[Abstract/Free Full Text]
  19. Wucherpfennig, K.W. et al. 1992. T cell receptor V alpha-V beta repertoire and cytokine gene expression in active multiple sclerosis lesions. J. Exp. Med. 175:993-1002.[Abstract]
  20. Traugott, U., Reinherz, E.L., and Raine, C.S. 1983. Multiple sclerosis: distribution of T cell subsets within active chronic lesions. Science.219:308-310.[Medline]
  21. Hauser, S.L. et al. 1986. Immunohistochemical analysis of the cellular infiltrate in multiple sclerosis lesions. Ann. Neurol. 19:578-587.[Medline]
  22. Wucherpfennig, K.W. et al. 1992. Gamma delta T-cell receptor repertoire in acute multiple sclerosis lesions. Proc. Natl. Acad. Sci. U. S. A.89:4588-4592.[Abstract]
  23. Prineas, J.W., and Wright, R.G. 1978. Macrophages, lymphocytes, and plasma cells in the perivascular compartment in chronic multiple sclerosis. Lab. Invest. 38:409-421.[Medline]
  24. Prineas, J. 1975. Pathology of the early lesion in multiple sclerosis. Hum. Pathol. 6:531-554.[Medline]
  25. Lucchinetti, C.F., Bruck, W., Rodriguez, M., and Lassmann, H. 1996. Distinct patterns of multiple sclerosis pathology indicates heterogeneity on pathogenesis. Brain Pathol. 6:259-274.[Medline]
  26. Pohl-Koppe, A., Burchett, S.K., Thiele, E.A., and Hafler, D.A. 1998. Myelin basic protein reactive Th2 T cells are found in acute disseminated encephalomyelitis. J. Neuroimmunol. 91:19-27.[Medline]
  27. Khoury, S.J., Hancock, W.W., and Weiner, H.L. 1992. Oral tolerance to myelin basic protein and natural recovery from experimental autoimmune encephalomyelitis are associated with downregulation of inflammatory cytokines and differential upregulation of transforming growth factor beta, interleukin 4, and prostaglandin E expression in the brain. J. Exp. Med. 176:1355-1364.[Abstract]
  28. Weinshenker, B.G. 1994. Natural history of multiple sclerosis. Ann. Neurol. 36(Suppl):S6-S11.[Medline]
  29. Confavreux, C., Vukusic, S., Moreau, T., and Adeleine, P. 2000. Relapses and progression of disability in multiple sclerosis. N. Engl. J. Med.343:1430-1438.[Abstract/Free Full Text]
  30. Khoury, S.J. et al. 1994. Longitudinal MRI imaging in multiple sclerosis: correlation between disability and lesion burden. Neurology. 44:2120-2124.[Abstract]
  31. Filippi, M. et al. 1995. Correlations between changes in disability and T2-weighted brain MRI activity in multiple sclerosis: a follow-up study. Neurology.45:255-260.[Abstract]
  32. Hohol, M.J. et al. 1999. Treatment of progressive multiple sclerosis with pulse cyclophosphamide/methylprednisolone: response to therapy is linked to the duration of progressive disease. Mult. Scler. 5:403-409.[Medline]
  33. McDonald, W.I. et al. 2001. Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the Diagnosis of Multiple Sclerosis. Ann. Neurol. 50:121-127.[Medline]
  34. Achiron, A., and Barak, Y. 2000. Multiple sclerosis — from probable to definite diagnosis: a 7-year prospective study. Arch Neurol. 57:974-979.[Abstract/Free Full Text]
  35. Griffin, D.E. 1990. Monophasic autoimmune inflammatory diseases of the CNS and PNS. Res. Publ. Assoc. Res. Nerv. Ment. Dis. 68:91-104.[Medline]
  36. O’Connor, K.C. et al. 2003. Myelin basic protein-reactive autoantibodies in the serum and cerebrospinal fluid of multiple sclerosis patients are characterized by low-affinity interactions. J. Neuroimmunol. 136:140-148.[Medline]
  37. Genain, C.P., Cannella, B., Hauser, S.L., and Raine, C.S. 1999. Identification of autoantibodies associated with myelin damage in multiple sclerosis. Nat. Med. 5:170-175.[Medline]
  38. Bourdette, D., Antel, J., McFarland, H., and Montgomery, E. 1999. Monitoring relapsing remitting MS patients. J. Neuroimmunol. 98:16-21.[Medline]
  39. Barkhof, F. et al. 1992. Relapsing-remitting multiple sclerosis: sequential enhanced MR imaging vs clinical findings in determining disease activity. AJR Am. J. Roentgenol. 159:1041-1047.[Abstract]
  40. Yednock, T.A. et al. 1992. Prevention of experimental autoimmune encephalomyelitis by antibodies against alpha 4 beta 1 integrin. Nature. 356:63-66.[Medline]
  41. Miller, D.H. et al. 2003. A controlled trial of natalizumab for relapsing multiple sclerosis. N. Engl. J. Med. 348:15-23.[Abstract/Free Full Text]
  42. Lehmann, P.V., Forsthuber, T., Miller, A., and Sercarz, E.E. 1992. Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen. Nature.358:155-157.[Medline]
  43. Miller, S.D. et al. 1995. Blockade of CD28/B7-1 interaction prevents epitope spreading and clinical relapses of murine EAE. Immunity. 3:739-745.[Medline]
  44. Windhagen, A. et al. 1995. Expression of costimulatory molecules B7-1 (CD80), B7-2 (CD86), and interleukin 12 cytokine in multiple sclerosis lesions. J. Exp. Med. 182:1985-1996.[Abstract]
  45. Ota, K. et al. 1990. T-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis. Nature. 346:183-187.[Medline]
  46. Pette, M. et al. 1990. Myelin basic protein-specific T lymphocyte lines from MS patients and healthy individuals. Neurology. 40:1770-1776.[Abstract]
  47. Martin, R. et al. 1990. Fine specificity and HLA restriction of myelin basic protein-specific cytotoxic T cell lines from multiple sclerosis patients and healthy individuals. J. Immunol. 145:540-548.[Abstract/Free Full Text]
  48. Ausubel, L.J., Kwan, C.K., Sette, A., Kuchroo, V., and Hafler, D.A. 1996. Complementary mutations in an antigenic peptide allow for crossreactivity of autoreactive T-cell clones. Proc. Natl. Acad. Sci. U. S. A. 93:15317-15322.[Abstract/Free Full Text]
  49. Hemmer, B. et al. 1997. Identification of high potency microbial and self ligands for a human autoreactive class II-restricted T cell clone. J. Exp. Med. 185:1651-1659.[Abstract/Free Full Text]
  50. Wucherpfennig, K.W., and Strominger, J.L. 1995. Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell. 80:695-705.[Medline]
  51. Scholz, C., Patton, K.T., Anderson, D.E., Freeman, G.J., and Hafler, D.A. 1998. Expansion of autoreactive T cells in multiple sclerosis is independent of exogenous B7 costimulation. J. Immunol. 160:1532-1538.[Abstract/Free Full Text]
  52. Lovett-Racke, A.E. et al. 1998. Decreased dependence of myelin basic protein-reactive T cells on CD28-mediated costimulation in multiple sclerosis patients. A marker of activated/memory T cells. J. Clin. Invest. 101:725-730.[Abstract/Free Full Text]
  53. Evavold, B.D., and Allen, P.M. 1991. Separation of IL-4 production from Th cell proliferation by an altered T cell receptor ligand. Science.252:1308-1310.[Medline]
  54. Sloan-Lancaster, J., Shaw, A.S., Rothbard, J.B., and Allen, P.M. 1994. Partial T cell signaling: altered phospho-zeta and lack of zap70 recruitment in APL-induced T cell anergy. Cell. 79:913-922.[Medline]
  55. Nicholson, L.B., Murtaza, A., Hafler, B.P., Sette, A., and Kuchroo, V.K. 1997. A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple myelin antigens. Proc. Natl. Acad. Sci. U. S. A. 94:9279-9284.[Abstract/Free Full Text]
  56. Nicholson, L.B., Greer, J.M., Sobel, R.A., Lees, M.B., and Kuchroo, V.K. 1995. An altered peptide ligand mediates immune deviation and prevents autoimmune encephalomyelitis. Immunity. 3:397-405.[Medline]
  57. Brocke, S. et al. 1996. Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein. Nature. 379:343-346.[Medline]
  58. Windhagen, A. et al. 1995. Modulation of cytokine patterns of human autoreactive T cell clones by a single amino acid substitution of their peptide ligand. Immunity.2:373-380.[Medline]
  59. Ausubel, L.J., Krieger, J.I., and Hafler, D.A. 1997. Changes in cytokine secretion induced by altered peptide ligands of myelin basic protein peptide 85-99. J. Immunol. 159:2502-2512.[Abstract]
  60. Ausubel, L.J., Bieganowska, K.D., and Hafler, D.A. 1999. Cross-reactivity of T-cell clones specific for altered peptide ligands of myelin basic protein. Cell. Immunol. 193:99-107.[Medline]
  61. Bielekova, B. et al. 2000. Encephalitogenic potential of the myelin basic protein peptide (amino acids 83-99) in multiple sclerosis: results of a phase II clinical trial with an altered peptide ligand. Nat. Med.6:1167-1175.[Medline]
  62. Kappos, L. et al. 2000. Induction of a non-encephalitogenic type 2 T helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in a placebo-controlled, randomized phase II trial. The Altered Peptide Ligand in Relapsing MS Study Group. Nat. Med. 6:1176-1182.[Medline]
  63. Bornstein, M.B. et al. 1987. A pilot trial of Cop 1 in exacerbating-remitting multiple sclerosis. N. Engl. J. Med. 41:533-539.
  64. Fridkis-Hareli, M., and Strominger, J.L. 1998. Promiscuous binding of synthetic copolymer 1 to purified HLA-DR molecules. J. Immunol. 160:4386-4397.[Abstract/Free Full Text]
  65. Johnson, K.P. et al. 1998. Extended use of glatiramer acetate (Copaxone) is well tolerated and maintains its clinical effect on multiple sclerosis relapse rate and degree of disability. Copolymer 1 Multiple Sclerosis Study Group. Neurology. 50:701-708.[Abstract]
  66. Filippi, M. et al. 2001. Glatiramer acetate reduces the proportion of new MS lesions evolving into "black holes." Neurology. 57:731-733.[Abstract/Free Full Text]
  67. Duda, P.W., Krieger, J.I., Schmied, M.C., Balentine, C., and Hafler, D.A. 2000. Human and murine CD4 T cell reactivity to a complex antigen: recognition of the synthetic random polypeptide glatiramer acetate. J. Immunol.165:7300-7307.[Abstract/Free Full Text]
  68. Duda, P.W., Schmied, M.C., Cook, S.L., Krieger, J.I., and Hafler, D.A. 2000. Glatiramer acetate (Copaxone) induces degenerate, Th2-polarized immune responses in patients with multiple sclerosis. J. Clin. Invest.105:967-976.[Abstract/Free Full Text]
  69. Qin, Y., Zhang, D.Q., Prat, A., Pouly, S., and Antel, J. 2000. Characterization of T cell lines derived from glatiramer-acetate–treated multiple sclerosis patients. J. Neuroimmunol. 108:201-206.[Medline]
  70. Dabbert, D. et al. 2000. Glatiramer acetate (copolymer-1)-specific, human T cell lines: cytokine profile and suppression of T cell lines reactive against myelin basic protein. Neurosci. Lett. 289:205-208.[Medline]
  71. Stuve, O. et al. 1996. Interferon beta-1b decreases the migration of T lymphocytes in vitro: effects on matrix metalloproteinase-9. Ann. Neurol.40:853-863.[Medline]
  72. Dyment, D.A., and Ebers, G.C. 2002. An array of sunshine in multiple sclerosis. N. Engl. J. Med. 347:1445-1447.[Medline]
  73. Robertson, N.P. et al. 1997. Offspring recurrence rates and clinical characteristics of conjugal multiple sclerosis. Lancet. 349:1587-1590.[Medline]
  74. Dean, G., McLoughlin, H., Brady, R., Adelstein, A.M., and Tallett-Williams, J. 1976. Multiple sclerosis among immigrants in greater London. Br. Med. J. 1:861-864.[Medline]
  75. Kurtzke, J.F., Gudmundsson, K.R., and Bergmann, S. 1982. MS in Iceland. 1. Evidence of a post-war epidemic. Neurology. 32:143-150.[Abstract]
  76. Carton, H. et al. 1997. Risks of multiple sclerosis in relatives of patients in Flanders, Belgium. J. Neurol. Neurosurg. Psychiatr. 62:329-333.[Abstract]
  77. Robertson, N.P., Clayton, D., Fraser, M., Deans, J., and Compston, D.A. 1996. Clinical concordance in sibling pairs with multiple sclerosis. Neurology.47:347-352.[Abstract]
  78. Sadovnick, A., Baird, P., and Ward, R. 1988. Multiple sclerosis: updated risks for relatives. Am. J. Med. Genet. 39:533-541.
  79. Risch, N. 1990. Linkage strategies for genetically complex traits. II. The power of affected relative pairs. Am. J. Hum. Genet. 46:229-241.[Medline]
  80. Sawcer, S., and Goodfellow, P.N. 1998. Inheritance of susceptibility to multiple sclerosis. Curr. Opin. Immunol. 10:697-703.[Medline]
  81. Allen, M. et al. 1994. Association of susceptibility to multiple sclerosis in Sweden with HLA class II DRB1 and DQB1 alleles. Hum. Immunol.39:41-48.[Medline]
  82. Coraddu, F. et al. 1998. HLA associations with multiple sclerosis in the Canary Islands. J. Neuroimmunol. 87:130-135.[Medline]
  83. Haegert, D.G. et al. 1993. HLA-DQA1 and -DQB1 associations with multiple sclerosis in Sardinia and French Canada: evidence for immunogenetically distinct patient groups. Neurology. 43:548-552.[Abstract]
  84. Hauser, S.L. et al. 1989. Extended major histocompatibility complex haplotypes in patients with multiple sclerosis. Neurology. 39:275-277.[Abstract]
  85. Kellar-Wood, H.F. et al. 1995. Multiple sclerosis and the HLA-D region: linkage and association studies. J. Neuroimmunol. 58:183-190.[Medline]
  86. Olerup, O., Hillert, J., and Fredrikson, S. 1990. The HLA-D region-associated MS-susceptibility genes may be located telomeric to the HLA-DP subregion. Tissue Antigens. 36:37-39.[Medline]
  87. Haines, J.L. et al. 1996. A complete genomic screen for multiple sclerosis underscores a role for the major histocompatability complex. The Multiple Sclerosis Genetics Group. Nat. Genet. 13:469-471.[Medline]
  88. Sawcer, S. et al. 1996. A genome screen in multiple sclerosis reveals susceptibility loci on chromosome 6p21 and 17q22. Nat. Genet. 13:464-468.[Medline]
  89. Ebers, G.C. et al. 1996. A full genome search in multiple sclerosis. Nat. Genet. 13:472-476.[Medline]
  90. Kruglyak, L. 1999. Prospects for whole-genome linkage disequilibrium mapping of common disease genes. Nat. Genet. 22:139-144.[Medline]
  91. Daly, M.J., Rioux, J.D., Schaffner, S.F., Hudson, T.J., and Lander, E.S. 2001. High-resolution haplotype structure in the human genome. Nat. Genet. 29:229-232.[Medline]
  92. Gabriel, S.B. et al. 2002. The structure of haplotype blocks in the human genome. Science. 296:2225-2229.[Medline]
  93. Reich, D.E. et al. 2001. Linkage disequilibrium in the human genome. Nature.411:199-204.[Medline]
  94. Johnson, G.C. et al. 2001. Haplotype tagging for the identification of common disease genes. Nat. Genet. 29:233-237.[Medline]
  95. Rioux, J.D. et al. 2001. Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn disease. Nat. Genet. 29:223-228.[Medline]
  96. Viglietta, V., Baecher-Allan, C., and Hafler, D. 2004. Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J. Exp. Med. In press..


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