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Fortunate encounter

Image: Jon Han

Mary Armanios still remembers meeting the patient who set the direction of her career. David Matushik, 21, had been diagnosed at the Hospital of the University of Pennsylvania with aplastic anemia, a potentially fatal failure of his bone marrow to produce blood cells. In fall 2003, he came to a Johns Hopkins outpatient clinic for a second opinion. "You could spot him in the waiting room," Armanios recalls. Matushik was not much past adolescence, but a shock of gray hair sprang from his forehead. He was unnervingly skinny; Armanios later learned that he had tried eating a 6,000-calorie diet to gain weight with no success. He had liver fibrosis and osteoporosis and would soon develop idiopathic pulmonary fibrosis, diseases that rarely strike before middle age. Because of the anemia, he required frequent transfusions. With too few white blood cells, his immune system had become a punching bag and he suffered constant infections.

By training, Armanios was an oncologist and pediatrician. (She is now an associate professor of oncology at the Sidney Kimmel Comprehensive Cancer Center.) She would not normally have seen a patient like Matushik. But a colleague in Hematology had done a chromosome analysis and noticed that one of Matushik's genes was reversed, and he asked Armanios to take a look. This abnormal gene is named TERT, and it provides instructions for making one component of a critical enzyme called telomerase. In regard to Matushik's illness, the abnormality turned out to be incidental. But Armanios was studying a population of lab mice that had been genetically engineered to lack telomerase, and, like Matushik, they were becoming old before their time. Intrigued by a possible connection, Armanios invited Matushik for a visit. Once she talked with him, she realized that besides the genetic abnormality, he had an assortment of other symptoms similar to what she had observed in her mice.

Every time a cell divides, its chromosomes divide and the telomeres capping them shorten. When the protective caps become too short, the cell dies.

If Matushik's condition was genetic, then some of his relatives should exhibit similar symptoms, Armanios reasoned. She began contacting family members. With their permission, she traveled to Delaware and interviewed and collected blood from as many of Matushik's relatives as she could. She found a family history riddled with sickness. Three months after Armanios first met Matushik, his father died of liver disease at 49. An aunt and uncle both suffered pulmonary fibrosis, and both would die in their 50s. Eventually, Armanios pieced together a genetic history, tracing through three generations the same abnormality in the TERT gene and its consequences. Family members in all three generations, including David, had a rare inherited disorder called dyskeratosis congenita. They also exhibited a shortening of their telomeres, the caps at the end of each strand of DNA that protect chromosomes and regulate cellular aging. The Matushiks' symptoms seemed to start earlier and become more severe with each subsequent generation, a pattern known as genetic anticipation.

What Armanios learned resulted in a paper published in the Proceedings of the National Academy of Sciences that for the first time linked dyskeratosis congenita, mutations in the TERT gene, and shortened telomeres. For Armanios it was only the beginning—the start of a decade of work that has produced new understanding of the role of telomere syndromes in diseases of the bone marrow, lungs, liver, and gastrointestinal tract, and links between abnormal telomeres and gene mutations. She has established the Telomere Clinic at the Kimmel Cancer Center, to help patients like David Matushik manage their conditions. He died in 2013, but Armanios believes the research findings helped extend his life by 10 years past what his prognosis had been on their initial meeting, and the research he sparked may lead to new understanding of diseases that annually kill tens of thousands of people.

Prior to meeting David Matushik in 2003, Armanios had joined Carol Greider's laboratory as a research fellow. In the early 1980s, Greider, now a Nobel laureate and professor of molecular biology and genetics at the School of Medicine, had been a young scientist at the University of California, Berkeley, trying to solve a central mystery about telomeres. Every time a cell divides, its chromosomes divide and the telomeres capping them shorten. When the protective caps become too short, the cell dies. This is a natural process. But cells have to divide a lot to develop and sustain a healthy body, so scientists knew there had to be a mechanism for repairing telomeres each time they divide. Greider identified a molecule called telomerase that rebuilds the telomeres at the end of a dividing chromosome. She shared the 2009 Nobel Prize in physiology or medicine for the discovery.

Around the turn of the millennium, scientists at UC Berkeley and Imperial College London discovered independently that people with dyskeratosis congenita often have a mutated form of a protein called dyskerin. The researchers soon realized that dyskerin was part of the telomerase enzyme. This was the first time anyone had discovered a connection between a telomerase-related gene and a clinical condition. DKC patients frequently develop aplastic anemia, and people with aplastic anemia often die from infections that the body would normally fight off easily. The only known treatment is a bone marrow transplant, but doctors had for some time noted that patients suffering DKC in addition to the anemia almost invariably died after transplantation, usually from lung disease.

Armanios was taking care of the Greider lab's telomerase-free mice when she met David Matushik. Greider had told her about the connection between telomerase and DKC, but a disease so obscure that it didn't even appear in medical textbooks did not excite her. "I said, 'I've never heard of that; I don't want to study that,'" she recalls. But a seriously ill patient with abnormalities in one of his telomerase genes—now that was interesting. Armanios recognized that even though Matushik lacked the classic DKC symptoms, he had the same underlying condition. That told her DKC needed to be reclassified. It was not, as doctors had thought for almost a century, a disease of the skin, nails, and tongue. It was in fact a disease of the genes—specifically the TERT telomerase gene.

In the 2005 paper, Armanios observed that many DKC patients develop idiopathic pulmonary fibrosis. Matushik had been diagnosed with the lung disease in 2004, soon after he met Armanios. His grandmother, aunt, and uncle also had suffered from it. Pulmonary fibrosis causes lung tissue to thicken and develop honeycomb-shaped scars. Patients cough incessantly and their breath becomes raspy; under a stethoscope it sounds like Velcro being ripped apart. Eventually sufferers can no longer breathe at all, and unless they receive a new lung, they die.

Pulmonary fibrosis advocates assert that IPF kills 30,000 to 40,000 yearly. Yet the disease is little known to the public, and research funding devoted to it is a small fraction of that available for many equally or less deadly conditions. The term "idiopathic" means "cause unknown." Armanios suspected that some cases of IPF could be traced to faulty telomere genes. The pattern of genetic anticipation she had seen in the Matushiks resembled what she had observed in mice and led her to think that telomerase and short telomeres may be important in IPF genetics.

Armanios was still a fellow in Greider's lab, but convinced she was on to something she reached out to Vanderbilt University's Jim Loyd, one of the nation's premier IPF researchers. She asked him for DNA samples of familial IPF patients, which she intended to screen for telomerase mutations. She found that 8 percent of the patients in Loyd's database had telomerase gene mutations. Eight percent is a small fraction, but far larger than would be expected by chance. Armanios recalls calling Loyd's colleague John Phillips: "I told him, 'I found mutations in your families,' and he said, 'Who are you?'" Evidently Loyd's team had thought it was so unlikely that Armanios would find a genetic cause for IPF that he had forgotten he had even given her samples.

Armanios and Loyd, along with Greider and others, published a paper in The New England Journal of Medicine in 2007 laying out their findings. It was only the second time in more than a century of research that anyone had identified a cause for idiopathic pulmonary fibrosis. Loyd says the link to telomerase is "the most important advance ever for understanding IPF."

After completing a second two-year fellowship in Greider's lab, Armanios in 2007 set up her own lab down the hall in the Preclinical Teaching Building at the Johns Hopkins medical campus in East Baltimore, and she began piecing together a story about how telomerase gene mutations could cause the diverse set of conditions that researchers kept uncovering. The thinking ran like this: DKC affects nails and skin—high-turnover tissues that the body has to replace often. To replace tissues, our bodies need a special class of cells­—stem cells. Although each cell in your body has a complete copy of your entire genome, most cells use only the genes needed to do one specialized task—be a white blood cell, for example, or a skin cell or a lung cell. Stem cells, by contrast, are generalists that retain the ability to differentiate into many specialized tissue types depending on what the body needs.

Since stem cells must typically divide multiple times in the process of differentiating, the ability to maintain telomere length is critical. If the telomeres shorten too quickly, the stem cells will die before the differentiation is complete. Scientists have long known that stem cells in bone marrow are essential for producing the highly specialized white blood cells of the immune system, so a connection between telomerase and aplastic anemia seemed natural. By the same token, telomerase should be important in the gut, another organ with a high turnover in cells. Indeed, Matushik had digestive problems and an inability to keep on weight. But what of Matushik's problems with his lungs and liver? Those are not high-turnover organs. Nevertheless, experts now agree there is reason to think both need to replenish their cells: They are on the front lines of exposure to environmental toxins. Toxins damage lung, liver, and gut cells and those cells need to be replaced—again, by stem cells.

Armanios set up a registry and began to collect samples from as many people as she could with telomerase gene mutations. That has allowed her and others to identify six telomere genes involved in families with pulmonary fibrosis, and she suspects there are at least as many remaining to be discovered. If any of these genes has a mutation, telomerase will not work properly, telomeres will shorten, and people will get sick. Researchers have found that around 15 percent of familial IPF sufferers have telomerase mutations. This finding is the first major chink in the armor of a disease that had previously repelled nearly all attempts to understand it. Ultimately the condition called IPF will probably be replaced by more precise diagnoses that reflect actual mechanisms of disease, predicts Johns Hopkins geneticist David Valle. "A fog of pseudoknowledge has been lifted to expose real biology."

In 2011, Armanios and some of her colleagues noticed that in one of their families with telomere syndromes, two sisters had developed at early ages another devastating lung disease: emphysema. The first patient they studied was a 55-year-old woman who had been diagnosed at age 44. Her sister was diagnosed even younger, at 34, with both emphysema and pulmonary fibrosis; she died at 46. In an unrelated experiment, the researchers found that lab mice with short telomeres exposed to cigarette smoke often developed emphysema. So Armanios began a study of an NIH database that contained the genomes of 292 emphysema patients. She and her team reported earlier this year that around 1 percent had mutations in the TERT gene—as with IPF, a fraction that is small yet far larger than would be expected by chance.

Emphysema damages air sacs in the lungs and reduces the amount of oxygen that makes it from the lungs into the blood. Along with chronic bronchitis it forms chronic obstructive pulmonary disease, COPD, the third-leading cause of death in the United States. Because it is so strongly associated with smoking, people often assume that emphysema is the victim's fault, but there is more to the disease than exposure to tobacco smoke. Women get emphysema far more than men; no one knows why. And about 1 percent of emphysema sufferers have a mutation in a gene for a protein called alpha-1 antitrypsin. In just the last four years, thanks largely to Armanios' work, TERT has joined alpha-1 antitrypsin as the only known genetic risk factors for emphysema. Armanios expects that as researchers test the full set of telomerase-related genes, the fraction of emphysema cases that can be traced to them will increase.

She also suspects that telomeres play a role in susceptibility to emphysema even for people without genetic mutations. One of Greider's major early discoveries was that telomeres shorten as people age. For example, an average person starts with around 1,600 of the nucleotide units that form telomeres for the chromosomes in white blood cells; by age 80 this number will have fallen by almost half. Autopsies show that anyone who lives long enough will begin to develop pulmonary fibrosis—Armanios calls the disease "the hair graying of the lung"—but this isn't usually clinically relevant because most people die of something else first. People in the bottom 1 percent of the telomere length distribution may be at increased risk of the same conditions that people with telomerase mutations get routinely. Since telomere length is rarely measured, that risk is almost never recognized. But Armanios thinks telomere length could explain much of why some people smoke like a chimney for decades and die at an advanced age of something other than lung disease, while others smoke much less yet end up on oxygen and die young.

That hypothesis "makes perfect sense," says Norman Sharpless, chair of the Lineberger Comprehensive Cancer Center at the University of North Carolina at Chapel Hill. Sharpless edits the journal in which Armanios' paper was published. He says the initial finding was based on a relatively small sample size and needs to be replicated by other researchers, but if it holds up, "it could go on to be one of the most important discoveries in telomere biology ever."

Last November, Armanios got a call from a geneticist at the University of South Florida. He told her, "I might have a patient for you." That patient turned out to be Bethany Matushik, David's half sister, who had come in to get a genetic workup and learn what the future held for her as a carrier of risky genes. Armanios invited Bethany to visit her at Johns Hopkins and told her what scientists had learned about telomere diseases and how Bethany could keep herself healthy. (Inspired, Bethany says she hopes to attend Johns Hopkins and study genetics.)

In the years since he had influenced the direction of Armanios' career, David had graduated from the University of Delaware and helped found and run Green Delaware Recycling, an organization that advised businesses in Delaware on sustainable practices. But his telomeres continued to deteriorate and his lung problems were probably exacerbated by smoking, which Armanios thinks he may have taken up to cope with the stress of chronic illness. He continued to get sick and ultimately died in 2013 of a staph infection.

"A fog of pseudoknowledge has been lifted to expose real biology."
David Valle

Nevertheless, Armanios says, his case shows how the basic research she and Greider do can translate into patient therapies. Armed with their novel understanding of telomere syndromes, Armanios recommended that Matushik receive a bone marrow transplant but without the common immunosuppressant drug Busulfan, which is known to be toxic to the lungs. Matushik had the operation at the University of Pennsylvania, and his condition improved markedly. "If he had not had this [telomere syndrome] diagnosis, he would have been transplanted with a regimen that would have killed him within a year, she says. "But he lived 10 years."

At the Kimmel Cancer Center's Telomere Clinic, Armanios often gets one request a day for a screening or consultation; inquiries come from around the world. She and Greider and their team have also established use of an assay to determine telomere length based on a blood sample, which could help clinicians recommend, for example, specific screenings for adverse effects of medications for patients with short or long telomeres. She continues to meet with families. Over a weekend in early June, Armanios, along with several of her graduate students and a genetic counselor, drove to another state to meet a large family they had found with telomere syndromes. Like David Matushik, many in the family had gone gray at an early age, and many had died young from IPF and other diseases. The news that their mysterious medical history finally had an explanation was not welcomed by all, Armanios says. Older family members in particular said they didn't want to know what the researchers found. But the younger generation? They wanted to know everything.

Johns Hopkins Magazine thanks the Matushik family for permitting the use of their names and medical histories.

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