Rare Family Cluster of MS Reveals New Clues
Studying five affected siblings, researchers have learned that one version of a tumor suppressor gene may increase the risk of developing MS by 70%
An unexpected potential genetic risk factor for multiple sclerosis (MS) has been identified in a remarkable Chicago family in which an entire generation—four sisters and one brother—is living with MS. The gene, called STK11, is well known as a tumor suppressor in some cancers. Its role in MS is a mystery, but research with animal models suggests that the protein it makes has multiple beneficial roles in protecting neurons in the brain and spinal cord and in myelinating axons throughout the body, according to several talks earlier this month at a meeting of the American Society for Neurochemistry (ASN) in Long Beach, California.
The connection to MS came from a family medical history and two blood samples from the Chicago siblings, in a pilot study led by cell biologist Anne Boullerne, Ph.D., and molecular biologist Douglas Feinstein, Ph.D., both at the University of Illinois, Chicago (UIC). Detailed sequencing of the STK11 gene in two of the sisters revealed that each had two versions, one common and one with a small alteration that seemed to dampen gene activity, as measured by the RNA transcripts. In a pilot study of blood samples from 650 people with relapsing-remitting MS, the same altered allele of the gene was 1.7 times more prevalent in women with MS than in the same number of healthy matched controls.
If the findings are confirmed, the STK11 variation could end up as one of the highest genetic risk factors for MS discovered so far, said Feinstein, a research biologist at the Jesse Brown VA Medical Center and professor of anesthesiology at UIC. The first-known and still highest genetic risk factor for MS comes from certain variations in the human leukocyte antigen complex, which identify and defend the body against invading microbes, and appear to boost the risk of MS by up to 300%. Other published genetic associations with MS are linked with lesser risk increases of about 5% to 50%, he said, while the newly discovered STK11 variant appears to boost the risk by about 70%.
However, even taken together, all the currently known MS risk factors can still account for at most 20% or 25% of MS inheritability, suggesting that there are still many factors (both genetic and environmental) that remain to be discovered.
“It doesn’t cause MS by itself,” Feinstein told MSDF. “About 7% of the total population in [North] America have this gene mutation, so it has to be in conjunction with something else.” That’s more than four times the rate of MS in the United States (135 per 100,000 people) and more than twice that of Canada (291 per 100,000) (MSIF, 2013). MS is considered a genetically complex disease, so more genes may be involved, as well as additional influences, such as infections, obesity, smoking, vitamin D levels, and other environmental agents.
A “weird tumor suppressor” related to myelination?
The STK11 gene makes a protein called LKB1. Only a handful of labs in the world are looking at its role in the central nervous system (CNS), said Feinstein, who co-chaired the ASN session focused on LKB1. Judging by the talks, about half of the labs stumbled across the protein’s CNS effects while looking for something else. In the case of the new risk factor for MS in the Chicago family, a bench scientist made the initial connection outside of the lab while moonlighting as a clinical study coordinator.
The protein’s role in myelination arose for Biplab Dasgupta, Ph.D., a tumor biologist at Cincinnati Children's Hospital in Ohio, when he was following up on reports suggesting that proliferating cancer cells burn a lot of sugar. He turned to LKB1 to explore how cells normally switch from guzzling sugar while they grow to burning oxygen so they can stop dividing and do other important jobs.
“LKB1 is a weird tumor suppressor,” Dasgupta told MSDF. “Loss of this tumor suppressor gives cells a proliferative advantage. But if cells undergo stress, then LKB1 plays a protective role. Not all cancer cells want to lose it.”
When it comes to studying metabolism, few cells in the body are as energy-demanding as myelin-making cells that are differentiating, he said. Myelinating cells wrap neurons and form myelin layers around nerves after birth. In newborn mice, it takes about a month for the neurons to be fully myelinated. Dasgupta’s lab is investigating how the STK11 gene controls myelination of neurons by Schwann cells outside the CNS in mice. (Schwann cells are similar to oligodendrocytes, the glial cells that myelinate axons in the brain and spinal cord.)
Mice missing the STK11/LKB1 gene failed to myelinate the sciatic nerve after birth, Dasgupta reported at the meeting. The problem seemed to come from the mitochondria. Without LKB1, the mitochondria in the underperforming Schwann cells were using less oxygen. The affected mice have a high disability level in their hind legs.
The next big question for Dasgupta is how exactly LKB1 regulates the metabolic shift necessary for mature, functioning Schwann cells. LKB1 can choose from more than a dozen molecular partners to direct a variety of cellular processes. It’s not clear whether the protein works the same way in all types of myelinating cells, such as oligodendrocytes. Feinstein is teaming up with Dasgupta and his mice to test whether and how changes in LKB1 function may be contributing to MS.
“I run a cancer lab,” Dasgupta said in his talk, “but you follow the science where it takes you.” Two other labs—at Stanford University in Palo Alto, California, and Washington University in St. Louis, Missouri—have generated similar mice missing LKB1 in their neurons, he said, but they are explaining the biology underlying the disability in different ways that might account for the lackluster myelination by other potential mechanisms. All three papers are likely to be published together later this year.
LKB1 compensates for neuronal stress
The LKB1 protein also seems to have a cell-death-defying power to compensate for major neuronal stress, also rooted in the mitochondria, discovered Marc Germain, Ph.D., a cell biologist at Université du Québec à Trois-Rivières. He and his colleagues created a mouse model in which they could disable mitochondria in adult neurons to understand why different regions of the brain are vulnerable in different neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis. The idea was that loss of mitochondrial function would lead to cell death. Surprisingly, few neurons died, thanks to the intervention of LKB1 (Germain et al., 2013). “It was very strange,” he told MSDF. It turned out that the intervention of LKB1 compensates—for a while—for the loss of cellular energy and for the slow buildup of molecular garbage in the neurons, which slowly disables and eventually kills them.
“Now I’m more interested in the metabolic aspect of how LKB1 regulates important metabolic functions to occur so that the brain stays functional,” Germain told MSDF. “We showed that if you decrease LKB1, the cells fare worse. The next big question would be: Can you improve survival of these neurons by playing around with the LKB1 pathway? Can we use this as tool?”
Of the 14 known molecular partners of LKB1, the most relevant to MS research is AMP-activated protein kinase (AMPK), a central command center to help cells adapt to changing energy demands. In good times, AMPK can dampen inflammatory gene expression. In bad times, AMPK can shut down energy-draining processes, such as the synthesis of lipids and proteins for myelin, as well as prompt tired cells to generate more mitochondria and switch their fuel source to energy-boosting sugar. AMPK also targets a molecule called mTOR, which somehow directs the differentiation and myelin-making activity of oligodendrocytes.
The speakers seemed to agree that AMPK signaling probably does not explain how LKB1 seems to be important for the developing brain and neuronal survival, but it will be a daunting task to tease out which downstream molecular cascades are at work and what they are doing, Germain told MSDF.
One research team has a head start on another of LKB1’s molecular partners in neurons. LKB1 also organizes proteins inside cells to orient them in one direction or another, known as polarization. Back-to-back papers published in 2007 by two groups, including one led by Maya Shelly, Ph.D., a developmental neurobiologist at Stony Brook University in New York, elucidated that mechanism, which is thought to underlie the first reported link between LKB1 and neuronal development.
In neonatal rats and mice, the protein initiates axon growth from inside the cell, Shelly’s team and another group showed (Shelly et al., 2007; Barnes et al., 2007). Now, Shelly’s lab is investigating how LKB1 and its partner proteins form new neurons in the hippocampus of adult mice, she said in her ASN talk.
An accidental discovery
The story of the protein’s connection with MS started with a chance conversation in April 2012. Boullerne has studied molecular aspects of myelin destruction in animal models and human MS tissue in the lab for more than 20 years, but she has also fostered connections to the hospital MS clinic. In the course of staffing a clinical study unrelated to her research, she escorted a woman with MS to the phlebotomist who was to draw the single blood sample needed for that study.
During the unexpectedly long wait for the blood draw, Boullerne made polite conversation, inquiring about the woman’s family. At first, the answers matched the known epidemiology of MS. The family had immigrated from Puerto Rico to Chicago, where all the children were born, a move to a northern latitude associated with increased risk of MS. The woman then mentioned that her twin sister also had MS. “I thought about the 30% monozygotic twin concordance shown in studies,” Boullerne told MSDF. Then the woman went on to say that the rest of her siblings all had MS. “I was speechless,” Boullerne said.
About 20% of people with MS have an affected relative, such as a sibling, parent, uncle, or cousin, but the woman said that no one else in the extended family had MS—just her, her sisters, and her brother, all now in their late 20s and early 30s. When Boullerne checked the scientific literature later, she could find no other report of five siblings with MS, let alone an entire generation. “She realized her family was extremely rare,” Boullerne said, “and we left it there.”
In medical research, as Boullerne and her team knew well, the study of rare family disease patterns has frequently led to key insights with the potential to help many more people with the same condition. The woman’s family history was not part of the clinical study records, so Boullerne double-checked with the neurologist that all of the siblings had been diagnosed with MS. Yes, she learned, all had some form of MS.
Boullerne said the woman she met was diagnosed with relapsing-remitting MS 7 years ago, but she has no disability, is sharp-witted, and has met with the local MS support group. Her twin is also doing well. Her brother, younger than the twins, is mostly bedridden with progressive MS. In contrast, the third sister, diagnosed the same year as the first, has a disability score of 6 (on the Expanded Disability Status Scale of 1 to 10) and walks with a cane. The fourth and youngest sister had a recent relapse severe enough to send her to the emergency department, but she elects not to receive care or treatment.
Boullerne and her colleagues were eager to learn more about the molecular underpinnings of the MS in the siblings, but they also wanted to proceed carefully with respect for the family. She contacted the woman again in October 2012 to discuss a preliminary genetic screen to search for clues. In planning the scientific strategy, Feinstein consulted Jorge Oksenberg, Ph.D., at the University of California, San Francisco, who has extensive expertise in the genomics of MS. Boullerne soon looped in a bioethicist to address the multiple issues of genetic studies, including designing the detailed protocol, offering genetic counseling, and drafting consent forms that offered individual options on whether each sibling wanted to be informed about incidental findings.
At that stage, the genetic possibilities were theoretically wide open but most likely resided among the 110 genes implicated so far in genome-wide association studies of MS. Then came a major clue. UIC neurologist Elizabeth Hartman, M.D., consulted with an oncologist who had treated the third sister for benign tumors. The oncology team had constructed a family medical tree. The mother had been diagnosed with the rare Peutz-Jeghers syndrome (PJS), a condition usually caused by mutations in one copy of the STK11 gene. The hallmark of PJS is noncancerous growths in the gastrointestinal tract appearing at a young age as well as an increased risk of cancer. The mother and aunts had been treated for breast cancer.
The problematic STK11 gene had not appeared in MS gene studies to date, but those studies are designed to find common gene variants. STK11 is on the borderline of being detectable through the common genome-wide association methods, with the variants found in less than 5% of the European-descent populations tested so far in MS studies. Feinstein sequenced the woman’s STK11 gene and found a variation in an intron, a regulatory area of the sequence not transcribed into the protein. They also discovered a second variation in a different gene that may interact with the STK11 mutation to lead to MS. Boullerne reported back to the woman and is arranging to draw blood samples from all the siblings at the family home, where it would be more convenient for the mostly bedridden brother. The siblings are motivated to participate in the study to help their own children, whose STK11 genes eventually may also be sequenced. So far, only the twins and the third sister have provided blood samples, which confirmed the same STK11 gene changes that may have predisposed them to MS.
In the lab, the team will be investigating the role of LKB1 in oligodendrocytes, astrocytes, and T cells during the progression of mice with experimental autoimmune encephalomyelitis and in novel mice genetically engineered with the STK11 risk gene. Some papers suggest that the loss of LKB1 activates T cells to spew certain inflammatory cytokines, Feinstein noted. These studies have the potential to reveal important details about the genetic predisposition for MS and certain vulnerable molecular pathways. Another mystery looms: What additional factor or factors pushed this generation of siblings into MS?
Although the clinical and bench studies are just beginning, Boullerne has learned one lesson she wants to pass along to other researchers: Record the family history of people with MS in clinical studies, even if the study has nothing to do with genetics.
Key open questions
- What role does the variation in STK11 play in MS? How is the LKB1 signaling pathway affected? What other genetic and environmental factors interact with STK11 and its protein LKB1 to influence the risk or course of MS?
- Do the remaining siblings also have the same variations in their STK11 gene? If so, how did one shared gene result in the apparently wide range in the severity and progression of MS among the siblings?
- How does the LKB1-mitochondria connection influence myelination of nerves in the body? How does LKB1 regulate the metabolic shift necessary for mature functioning Schwann cells? Does LKB1 have an effect on myelination in the CNS, and if so by what mechanism?
- Can alterations in the LKB1 pathway improve the survival of neurons in mice?
- What role does LKB1 play in forming new neurons in mice?
Disclosures and sources of funding
Boullerne and Feinstein have funding from Biogen Idec and the U.S. Department of Veterans Affairs. Dasgupta has funding from the National Institutes of Health, and Cincinnati Children's Hospital, his institution, is licensing intellectual property from some aspects of the study currently under embargo. Germain has funding from the Natural Sciences and Engineering Research Council of Canada.
The text was revised to include additional institutional affiliations of researchers.