When a gene goes awry

When Mustafa K. Khokha, M.D., first saw the Drewniak baby in the summer of 2012, he was worried. The baby boy, first treated at Norwalk Hospital, had a high fever and severe diarrhea that was getting worse. Clinicians at Yale-New Haven Hospital’s Pediatric Intensive Care Unit were frantically trying to keep up with fluid losses. Infection, the most likely culprit, had been ruled out. Khokha, associate professor of pediatrics (critical care) and of genetics, wondered whether an underlying genetic cause would explain the baby’s condition, and called Richard Lifton, M.D., Ph.D., chair and Sterling Professor of Genetics. Genome sequencing, Lifton felt, might yield clues to the infant’s ailment. Both Lifton and Khokha thought that the baby might have a new genetic mutation—a de novo mutation—since both of his parents were healthy.

They used high-throughput exome sequencing to analyze the 21,000 protein-coding genes in the genomes of the baby and both his parents. Meanwhile, Neil D. Romberg, M.D., assistant professor of pediatrics (immunology), examined the baby for a rash. Romberg recognized it as the first sign of a massive immune response, and lab tests indicated that the baby had widespread inflammation. “Inflammation in the absence of infection is usually a genetic problem,” said Romberg.

The Yale Center for Genome Analysis rushed the sequencing and analysis. Completed in just a few days—a heroic effort—test results arrived the day after the 3-week-old baby died. The analysis found no de novo mutations that explained his illness.

A few days after the funeral, Erik Drewniak was still reeling from the loss of his infant son when he came down with a fever. He didn’t think it was anything to worry about. All his life he had had fevers that spiked as high as 106 degrees. But this time the fever persisted and he was having difficulty breathing. Concerned about pneumonia, he went to the emergency room. At Norwalk Hospital, Drewniak’s condition deteriorated. He went into respiratory failure and was put into a medically induced coma so that he could be intubated. Soon afterward, he was in intensive care at Yale-New Haven Hospital where tests showed he was suffering from inflammation and hemorrhaging of the bowels, lungs, and brain. His condition was serious, but he improved and went home nine weeks later.

A clue in a random mutation

With every sequencing of a patient’s genome, new, previously unseen protein-altering variants in genes emerge. These random mutations are due to variation in the human population. When Romberg suggested that the baby might have had an autoinflammatory disease, Lifton invited him to his lab to look at a list of variants. Romberg recognized a mutation in NLRC4, a gene involved in the innate immunity pathway—the first line of defense in the body’s response to infection—that could contribute to disease, although it had never been shown to have such an effect. That same day, while gathering information about the baby from Norwalk Hospital, Lifton’s research coordinator learned that Erik Drewniak was hospitalized with a severe illness. For Lifton, that was an “aha” moment.

“We recognized at that point that the variant in NLRC4, which was shared between the father and the baby, might link their diseases,” he said. It turned out that Drewniak and his infant son shared a genetic mutation causing an illness that had never before been described.

Advances in genomic sequencing technology, combined with clinical expertise and biochemical analysis, allowed Yale doctors to identify the mutation, home in on the pathway it affected, and devise a personalized therapeutic strategy that saved Drewniak’s life. Using whole-exome sequencing to diagnose diseases is increasingly proving to be both useful and practical. Five years ago it would have cost about $10,000 to do the exome sequencing performed on the Drewniak family; today it costs about $500. Not surprisingly, this service is rapidly expanding. Last year, Yale sequenced about 500 patients; this year about 1,000 will be sequenced. The Drewniaks’ experience is just one example of the ways in which personalized medicine, which is being supported by President Obama’s recently announced Precision Medicine Initiative, is gaining ground.

Passing on a gene

To confirm that the Drewniaks’ illnesses were caused by the newly identified mutation, the Yale team sequenced other family members, including Erik’s parents and two children who are half-siblings of his infant son. While his parents and daughter do not have the NLRC4 mutation, the sequencing showed that his son, now 7, does. Like his brother, he was hospitalized as an infant, although in his case it was due to kidney failure. Like his father—who was also hospitalized as a baby for a fever and severe diarrhea—he suffers from periodic high fevers. The mutation was in fact de novo, but it first occurred in Erik Drewniak, who passed it on to two of his children. His young son has a 50/50 chance of passing it on to his offspring.

Once a genetic mutation emerged as the likely cause of disease in three Drewniak family members, the next step was to understand how a single change in this particular protein code could wreak such havoc. “Exome sequencing by itself is not the solution,” said Khokha. “It’s just the first step.” Romberg enlisted Barbara I. Kazmierczak, Ph.D., M.D., associate professor of medicine (infectious diseases) and of microbial pathogenesis, who had been studying NLRC4 in mice. NLRC4 belongs to a group of intracellular proteins that react when bacterial toxins breach the cell wall. Kazmierczak and Romberg’s research showed that the mutated NLRC4 protein activates a powerful inflammatory pathway that sent the Drewniaks’ immune systems into overdrive, even though no bacterial infection was present. “Their system is always on when it should be off,” said Kazmierczak.

Today, Erik Drewniak is healthy, as he has been for much of his life. “It didn’t affect me when I was growing up,” he says of his illness. He played in bands and was on the tennis team in high school, missing only an occasional event because of a fever. His son is also relatively healthy. “He does kung fu and other typical kid stuff,” said Drewniak. But the mutation they share causes a constant state of inflammation in their bodies that can become dangerous.

When Erik Drewniak was hospitalized in 2012, Nikolai A. Podoltsev, M.D., Ph.D., assistant professor of medicine (hematology), treated him for what appeared to be hemophagocytic lymphohistiocytosis (HLH), another disease in which the immune system becomes overstimulated. Drewniak’s condition, while different, closely resembles HLH and responded to powerful anti-inflammatory medication and immunosuppressants, which are used to treat HLH.

Understanding the disease

Now that Podoltsev and Romberg (who treats Drewniak’s son) know the mechanism of this disease, they are better equipped to treat it in the future. “Of course it’s easier to manage him going forward understanding what we’re dealing with,” said Podoltsev. The Drewniaks’ care involves managing the inflammation and monitoring symptoms. The mutation causes production of high amounts of cytokines—in this case interleukin 1 beta—that cue the immune system to start reacting to an infection, so interleukin 1 inhibitors could be useful if either of them experiences a flare-up.

Beyond the therapeutic implications of these discoveries, the ability to pinpoint the cause of mysterious illnesses is invaluable. “When a baby dies of diarrhea, it’s very disturbing because it doesn’t happen in this country,” said Khokha. “It feels like a failure.” Being able to tell his parents why their baby died provided some solace. Fortunately for families like the Drewniaks, diagnosing rare genetic illnesses is becoming more common. “We’ve gotten to the point where genetic sequencing is fast enough and cheap enough and we’ve done enough of it, that this is no longer science fiction,” said Romberg.

“It’s clear there will be many more cases like this one,” said Lifton. Of the 21,000 protein-coding genes in the human genome, he said, scientists know what happens when about 3,000 of them are mutated. “When I’m asked what remains to be done, the answer is practically everything.” Researchers are beginning to tackle this undertaking. The Yale Center for Mendelian Genomics is one of three national centers created by the National Institutes of Health to try to understand the genetic causes of diseases like the one in the Drewniak family. The center has identified about 250 new disease genes since it was established in 2012.

Meanwhile, Khokha and his colleagues often see children with birth defects or unexplained illnesses that they are unable to diagnose. A new program, Pediatric MAP, supported by the Yale School of Medicine, the Yale Center for Clinical Investigation, and Yale-New Haven Hospital will allow them to carry out the research that was done for the Drewniaks on a broader scale. The hope is that by formalizing the program, Yale doctors will be able to provide answers for these families by identifying genetic mutations and unraveling their biology with the ultimate goal of developing new therapeutic approaches.

For Drewniak, the knowledge of what caused his infant son’s death is empowering. “Genetics is something you can latch on to as a definite explanation,” said Drewniak. “It’s not the environment, it’s not something you did or didn’t do, it’s genetics. It made me understand not just him, but all three of us. Everything just came together and now it all makes sense.”

Jill Max is a freelance writer in Trumbull, Conn.