Multiple Complications

DURING THE 142 YEARS SINCE FRENCH PHYSICIAN JEAN-MARTIN CHARCOT first described multiple sclerosis, the disease has resisted easy classification. It’s characterized by patchy lesions—scleroses—that develop in the central nervous system when the myelin sheaths meant to protect nerve fibers (and sometimes the fibers themselves) deteriorate. The lesions disrupt signals traveling through the brain, spinal cord and nerves. But MS may progress quite differently in different patients, with some experiencing clearly defined attacks followed by partial or complete remissions and others getting steadily worse as neurological symptoms—numbness, weakness, poor motor control, vision problems—increase. In still others, there is a slow deterioration punctuated by bouts of heightened symptoms.

Although almost all scientists agree that, at bottom, this chronic, disabling condition is an autoimmune disorder, in which the body attacks its own tissues, there appears to be an elaborate mosaic of contributing factors. “There’s evidence of roles for genetics, environmental factors, infections, geography, vitamin D, diet…. It goes on and on,” says neurologist T. Jock Murray, professor emeritus at Canada’s Dalhousie University and author of a 580-page history of the scientific struggle to figure out MS. “It’s far more complex than most diseases, and it’s very difficult to conceptualize in a simple way.”

Yet in 2009, a physician named Paolo Zamboni, affiliated with Italy’s University of Ferrara, put forth a radically straightforward hypothesis to explain the disorder. Zamboni thinks the characteristic lesions of MS are caused by physical obstructions in veins leading out of the brain and spinal cord—a vascular condition known as chronic cerebrospinal venous insufficiency (CCSVI). These blockages, Zamboni says, cause blood refluxes and inflammatory injuries that end up as lesions. “It’s a plumbing analogy,” says Murray. “You have an obstruction that’s causing a problem, so you remove the obstruction and the problem goes away.”

In the studies that underpin this theory, Zamboni used transcranial and extracranial color Doppler sonography—a sophisticated ultrasound imaging technique—to diagnose impaired blood flow in every MS patient he tested. The method found no such blockages in the healthy controls. He found that treating the blockages with a surgical procedure that widens the vessels increased the number of patients who were relapse-free and reduced the rate at which additional lesions formed.

But other research isn’t so black-and-white, and so far most neurologists and hospitals in the United States have been cautious or even skeptical. Preliminary results from one ongoing study show CCSVI in only 56% of MS patients, as well as in 23% of healthy controls and in 43% of patients who have other neurological conditions, suggesting that blocked veins aren’t directly related to MS, even though they may be present in many patients. There have yet to be double-blind studies that confirm a causal link, or placebo-controlled studies to prove the efficacy of removing venous obstructions in MS patients.

Still, Zamboni’s work has attracted considerable attention, particularly among patients and their families. “The simplicity of this idea makes it compelling,” says Murray. Last summer the National Multiple Sclerosis Society and its sister organization in Canada committed more than $2.4 million to seven North American CCSVI research projects.

Recently, though, a surge of research on multiple fronts has begun to produce, if not yet a clear view of exactly what causes the disease, then at least a better understanding of many of the pieces of the mosaic. With advanced neuroimaging, microscopy and genomic sequencing tools facilitating more detailed examination of the characteristics of MS, these are boom times for MS investigations. But with the exception of the work on CCSVI, most of the research isn’t distilling the complexity associated with MS so much as elucidating it.

BEGINNING IN THE 1980S, HARVARD PHYSICIAN AND RESEARCH SCIENTIST David A. Hafler, now chair of the neurology department at Yale, made a name for himself by teasing apart the roles of T cells—immune system components that identify and destroy foreign substances—in MS. Lately, though, he has focused on pinpointing the disease’s genetic basis. In 2007, Hafler and a research group discovered two single nucleotide polymorphisms, or SNPs—genetic variations that make a person more likely to develop a disease—related to MS. “In the past few years alone, we’ve identified about 40 of the perhaps 100 to 150 common variants associated with MS,” Hafler says. “What’s interesting is that the vast majority are related to immune function. When we’ve found them all, we will have understood one of the disease’s fundamental causes.”

Hafler is hardly alone in thinking genetics holds the key to unlocking MS. Familial studies have long shown a strong hereditary component. In the general population, the lifetime risk of developing the disease is about 0.15%. This climbs to as much as 4% for most family members of someone with MS and reaches 25% to 30% for the identical twin of someone who has the disease, which presents a conundrum: Why would the figures for identically genetic twins fall so far short of 100%?

A recent study of twins published in the journal Nature tried to answer this question by probing genetic differences at an unprecedented level of detail. The project, led by neurology professor Sergio Baranzini of the University of California, San Francisco, and geneticist Stephen Kingsmore of the National Center for Genome Resources in Santa Fe, examined identical twins “discordant” for MS—that is, one twin had developed the disease while the other, based on clinical evaluations and MRI scans showing no brain lesions, was found to be healthy. Because identical twins usually develop MS at about the same age, the researchers expected the unaffected twins to remain so.

Although it’s generally assumed that identical twins have identical DNA, tiny disparities have been found in twins who develop from a single egg, and scientists think such variations may be involved in the onset of hereditary diseases. So these researchers spent 18 months peering at discordant twins’ genomes, epigenomes and transcriptomes—combining three levels of genetic analysis not usually performed together.

First the team sequenced the complete genomes of a single pair of discordant twins, checking the sequence of base pairs in each gene an average of 20 to 22 times (a vast undertaking, because the human genome contains some 23,000 genes). Next the researchers turned to the epigenomes of three pairs of discordant twins. The epigenome consists of chemical compounds that can modify the functioning of genes over a person’s lifetime. Twins from a single egg may differ epigenetically, and Baranzini and Kingsmore thought this might explain why sometimes only one gets the disease. They focused on DNA methylation, an epigenetic mechanism that prevents genes from being expressed. “We hypothesized,” says Baranzini, “that the discordance might be explained by variations in the methylation patterns around key genes”—such as those responsible for the proliferation of immune cells involved in MS.

Finally, the researchers drew CD4 T cells—immune cells known to contribute to the development of MS—from each of the same three pairs of twins and examined the cells’ transcriptomes. (A cell’s transcriptome is a set of molecules that record gene activity, a kind of logbook detailing which proteins were synthesized by which genes in the cell.)

Remarkably there were no differences between the discordant twins at any of these three levels of analysis. Thus the study found no incontrovertible evidence for a genetic culprit that pushed some twins into MS and saved the others. But according to Hafler, that doesn’t mean we’ll never find the smoking gun. “There might well be rare mutations in the genome that you wouldn’t pick up at the level at which the researchers were able to examine it,” says Hafler, who considers the researchers’ rigorous three-pronged approach to be the study’s most important contribution. He thinks that using similar methods with still more advanced future technologies could finally solve the riddle of MS. Meanwhile, Baranzini and Kingsmore plan to look for genetic variations in brain cells. That might reveal whether the neurons of MS patients synthesize proteins different from those produced by the neurons of healthy people.

FOR OBSERVERS SUCH AS MURRAY, THE TWINS STUDY UNDERSCORES the idea that environmental triggers must play a significant role in the disease’s development. While Baranzini agrees, he notes that such influences are inherently difficult to identify. “To examine them systematically, you’d have to record every meal, drink and environmental exposure each twin had throughout their lives,” he says. That seems patently impossible—but recent studies have nevertheless done much to show how environmental factors might set off the disease in someone with a genetic predisposition.

One notion that has been around since the 1880s is that there’s an infectious trigger. Studies have linked MS to a slew of familiar infections—including measles, mononucleosis and human herpesvirus 6. One of the most frequently cited, Epstein-Barr virus (which causes mononucleosis), is so common that as many as 95% of American adults have probably been exposed to it. But persistent questions have always confounded this idea. Even taking into account genetic susceptibility, why would MS occur in only a tiny fraction of those exposed to such a common infection? Why are so many viruses associated with MS? And why do tests of spinal fluid and brain lesions from MS patients usually turn up little evidence of viral DNA, suggesting that even if there is an infectious trigger, all traces of the pathogen have vanished?

In May scientists led by Joan Goverman, a professor of immunology at the University of Washington, uncovered some answers. They engineered mice to produce an excess of CD8 T cells, which target myelin basic protein, an essential component in producing the protective nerve cell coating that is destroyed in MS. When the mice were given a virus that pumped out MBP in their bodies, the protein acted like a red flag. CD8 T cells sprang into action, killing first the virus cells and then the mice’s own MBP-producing cells—giving the animals an MS-like disease. These results supported her original hypothesis—that CD8 T cells are just as central as CD4 T cells are in the development of MS. But when she saw mice exposed to an unrelated virus—one that didn’t generate the red flag protein—also getting sick, she had an important insight.

“Because the immune system is such a powerful weapon, it’s important that it tolerate the body’s own tissues,” says Goverman. Seemingly to that end, most of the time people produce T cells that have just one receptor. That means a given T cell will target only a single antigen, or toxic substance, which reduces the risk of T cells setting off a widespread, and potentially destructive, immune response. But sometimes T cells have two receptors. Goverman’s group realized that this was what had happened in their mice. They’d randomly developed a few dual-receptor T cells, in which one receptor recognized a viral protein and the other targeted MBP. If the mice were exposed to a virus that happened to activate the first receptor, the second automatically became active as well—and it eagerly attacked the body’s MBP-producing cells.

Goverman thinks the same phenomenon may occur in humans: Many of us may develop diverse types of dual-receptor T cells, making it possible for any number of viruses to act as triggers. But because most T cells conform to the single-receptor model (it’s still not known why dual receptors are sometimes produced), not many people exposed to a particular virus get MS, even if they have a genetic predisposition. Finally, since the autoimmune response would continue even after the virus itself had been destroyed, it would make sense that MS patients don’t have much viral DNA in their tissues.

“We’ve been waiting for this study,” says Misha Pless, chief of general neurology and neuro-ophthalmology at Massachusetts General Hospital. “It shows in a nice, clean way that a nonspecific viral infection can trigger an autoimmune response in someone who already has a genetic predisposition and has certain T cell populations primed for activation.” So while a pair of identical twins, for example, might have an identical genetic risk of developing MS, it could be that only one will have developed dual-receptor T cells. Then, even if both twins were exposed to the same virus, only one would get the disease.

ONE REASON PLESS WAS SO EXCITED ABOUT GOVERMAN’S STUDY is that it fits into a model of MS that he and many others in the scientific community have championed. Unlike the CCSVI hypothesis, this way of understanding the disease acknowledges its complexity and looks at multiple risk factors that may interact over time. No single factor, or even a partial combination of them, will make someone sick. Instead there has to be a “cascade” of causes. Increasingly scientists who advocate this framework are producing research that helps make sense of seemingly isolated pieces of information.

For years, for example, scientists have been intrigued by an apparent link between MS, sunlight and vitamin D, a compound naturally synthesized in the skin when it’s exposed to ultraviolet rays. Evidence for the connection includes several fascinating facts. The incidence of MS increases dramatically in populations that live far from the sunny equator. MS patients have also been shown to experience more relapses just after seasonal reductions in sunlight, and in one study involving almost 200,000 women, taking a high-dose vitamin D supplement appeared to reduce the risk of MS significantly. But does a lack of vitamin D really trigger MS, and if so, how?

Recently a group of neurologists and geneticists from Canada and the United Kingdom jointly examined the subtle interplay between vitamin D and DNA, and they were able to show that the vitamin interacts directly with a variant of one gene—HLA-DRB1—called HLA-DRB1*1501. People who have that version of the gene—which happens to be the dominant form among northern Europeans—are three times more likely to develop MS than those who don’t have it.

The study, led by Oxford University researcher Sreeram V. Ramagopalan, offered a possible explanation for this increased risk. Ramagopalan and his colleagues showed that vitamin D is essential for the HLA-DRB1*1501 gene variant to function properly. Without vitamin D, a protein known as vitamin D receptor can’t bind to a crucial section of DNA next to the HLA-DRB1*1501 gene, and if that connection isn’t made, the gene can’t turn on and serve its function helping to regulate T cell activity. So for people who have the gene variant, the study suggests, insufficient sunshine could be an important step in triggering the autoimmune reaction that causes MS lesions.

Might other environmental suspects that have been linked to the disease, such as toxins or cigarette smoke, do the same? More research that examines MS from multiple angles may provide answers. What is crucial about Ramagopalan’s study is that it tried to discover how a particular gene functions under different environmental conditions. It’s adding to evidence showing how separate risk factors might pile up until, as Pless says, “a final straw breaks the camel’s back.”

FOR MULTIPLE SCLEROSIS, THE ONWARD MARCH OF SCIENCE hardly seems headed toward simplicity. Most new research appears to add to the number of ideas that must be taken into account. “It’s valuable that science continues to generate multiple theories about MS, because the answer we’re after is not going to be simple,” says Murray. “We’re going to need a global view that integrates each of our incomplete ideas.”

A global view isn’t easy to achieve. Pless recalls the famous 1976 New Yorker cover, Saul Steinberg’s View of the World From 9th Avenue. Manhattan’s buildings, cars and streetlights occupy twice as much space as the dull green rectangle representing the rest of the United States, and the Hudson looms nearly as large as the Pacific. Russia, Japan and China are mere fingernails of land. When thinking about MS, says Pless, researchers have long tended to fall back on this kind of telescoped perspective. The aspect of the disease they consider most important expands until it shuts out the others.

Baranzini, Murray and Hafler all think this attitude is changing. Says Baranzini: “We’re moving away from an approach in which we try to simplify things as much as possible in order to understand them. Now, we’re looking at things from a higher order.” That view could provide a more complete picture of what has long been an extraordinarily puzzling disease.