FALL 2009
Into a single cell.
Into the dark nucleus.
Into the twenty-three matched pairs of chromosomes. There lies Nic’s essence—and his secret.
Scientists are finally ready to put their best technology to the test, to peer into the boy’s twenty-one thousand or so genes and read the script. This is the power that next-generation sequencing machines have given the team investigating Nic’s disease. It is an awesome, unprecedented power. Yet it is not enough.
To return to the image of a camera scanning the genetic script, scientists are now, in effect, bypassing more than 98 percent of the letters to focus only on the protein-making exome. But that still means the camera must glide over more than 35 million letters. The camera is scanning the genetic landscape for differences between Nic’s script and the reference genome, our best approximation of normal. There will be thousands and thousands of differences. All along, the problem has been that doctors do not understand the nature of the enemy, so they don’t know what to look for; they know only that the disease is something they’ve never seen before. Scientists will face much the same problem when they comb through the thousands of differences between Nic’s sequence and the normal sequence: which of all those differences is the mutation, the terrible flaw in his molecular machinery?
As Howard Jacob’s team prepares to mine Nic’s exome for answers, the boy’s doctors take the extreme approach to his disease that has been discussed before, the approach that frightens Amylynne. They destroy her son’s immune system using a powerful chemotherapy drug called Cytoxan. Then they allow Nic’s stem cells to rebuild his immune system from scratch. The procedure is not the same as the transplant they have talked of giving Nic. A transplant of bone marrow from someone else would give the child an entirely different immune system. This procedure, known formally as immune ablation, is more like rebooting a computer. Still, it is agonizing, and not a step the doctors or Amylynne take lightly. The child’s fever hovers around 104 degrees. He vomits as many as twenty times in a single day. One day, he looks into a handheld mirror and sees that all of his hair is gone. He shrugs and looks away.
By early September Nic is recovering, and his immune system has successfully reconstructed itself. He is able to go home. He gets to jump in leaf piles and go trick-or-treating. He can even eat some of the candy. In an effort to keep the disease at bay, the doctor allows him to eat just one new food each week. Mayer tells Amylynne that Nic’s disease is in remission, but they both understand what that means; Nic’s disease has been in remission before.
Around this time, Liz Worthey begins work on the new program to make sense of all the variations and oddities that the researchers will find in Nic’s sequence. Working with Medical College software developers, she begins designing an entirely new tool to assist in the effort to pin down the origin of Nic’s disease.
The tool Worthey and her colleagues construct must help them to bypass the vast number of genetic variations that do not have dire consequences.
The scientists will be looking at the exons, the portions of the genome that determine the proteins our bodies need to function properly. As they search through Nic’s genes for a mutation capable of causing his terrible disease, the scientists will encounter three types of variations. Some changes in the script still allow our bodies to make the correct amino acid. Other variations alter an amino acid without disrupting the overall chain of amino acids that forms a protein. So even though one of the amino acids is different, we still make the right protein in the right amount—imagine this as the equivalent of rewording a few paragraphs in a book but allowing the meaning to stay the same.
Then there is a third kind of variation, the kind that scientists believe is causing Nic’s disease. This kind prevents the manufacture of an important protein and leaves a vital bodily process in disarray. In such cases, the typo results in a more serious problem. Key sections of the book are suddenly missing, or a critical chapter ends midsentence. The mistake omits information the reader needs to make sense of the story.
Scientists keep lists of known genetic variations and how they affect different species. One database called dbSNP describes single-letter variations and what is known about each of them. But single-letter changes are only one type of variation. There are a range of others—for instance, there are portions of the genome where the same letters repeat, perhaps ten times in a row in some people, twenty times in others. Databases likewise exist for those types of variations. Each database is continually updated as scientists discover new variations and new information about known variations. The problem is that there is no master list, no single tool Worthey and her colleagues can consult as they scan Nic’s script. They will be forced to plow through a number of different databases and to continually assess the harmfulness of each genetic difference they discover. That will make reading Nic’s script into a much longer and costlier task.
What Worthey needs is a single, one-step tool that pulls together all current knowledge of genetic variations, a tool that will eliminate all of the inconsequential differences in Nic’s script.
Over the course of several months, Worthey and her colleagues build that tool. They make a software program that combines new and existing algorithms, as well as data about genetic variations that scientists have encountered previously, all in order to flag changes in the script that might mean something. It is a vastly more powerful analytic tool than any previously available to geneticists.
Worthey calls the program Carpe Novo, Latin for “seize the new.”
When the results arrive from Roche’s first sequencing of Nic’s DNA, Worthey has an early version of Carpe Novo already working, though she’s still adding new information. The sequencing, too, is just a first draft; it likely contains mistakes and errors that will be corrected in the next few sequencing runs Jacob’s lab will conduct. Each new run is like a slide superimposed over the previous slides, adding resolution and depth, bringing the picture into clearer focus. For now Worthey has more than enough to start, and she doesn’t waste any time. They need to act fast. The numbers are already daunting.
James Verbsky’s pessimistic guess that they could end up looking at twenty thousand genetic differences proves to be close to the actual mark. The Roche sequencing comes back with 16,124 differences between Nic’s script and that of the reference genome. That’s the size of the haystack they are searching.
Worthey’s program will act as a kind of filter, narrowing down the daunting list of 16,124 suspects. Once Carpe Novo has reduced the field of suspects, it will then be up to Worthey and others to act as medical detectives, checking the remaining genetic variations to see whether they can be linked to Nic’s symptoms.
Fortunately, Worthey is not alone on the case. She works closely with David Dimmock, the pediatric genetics specialist who joined Children’s Hospital and the Medical College in July 2008. For more than a year, Dimmock and Worthey have worked together on research that has used sequencing to understand the genetics of liver failure and the genes that cause mitochondrial disease.
A young, fair-haired Englishman, Dimmock brings to his work a deep compassion and a maturity that comes from years of confronting human suffering. More than a decade earlier, Dimmock was working in a tin-roofed hospital in Uganda caring for patients with malaria, HIV, tuberculosis, cholera, and dysentery. Most were children. Every day two, three, four children would die at the hospital.
After his time in Uganda, Dimmock spent portions of the next three years journeying to Tajikistan and Afghanistan, where he worked with medical teams. Tajikistan was at the tail end of a bloody civil war, and he learned how grateful a doctor can become for the small things: sharp needles, clean gloves, and the ability to drive through the countryside in safety.
He also gained an awareness of the limits a doctor must accept. “There are definitely things you are powerless to do,” he says. “That recognition that you can’t win every time does change your willingness to recognize when you are in a futile situation. There’s nothing quite like the death of a child. And if you’re seeing it two or three times a day, that doesn’t make it any less horrific.”
In other words, he has spent years absorbing the two opposing principles at play in the treatment of Nic Volker: the knowledge that medical care can reach a point of futility and the knowledge that few things in life are more terrible than the death of a child. This time, in order to save a child, the team is redefining the point of futility, pushing past the threshold of what is possible in medicine.
Once Worthey has put the initial sequencing data through Carpe Novo, she and Dimmock discuss the findings. The two scientists bring different backgrounds and strengths to the task. As a researcher, Worthey has honed her patience and focus, learning to work for long periods on studies that may bear fruit only after many years, affecting thousands of patients she never sees. As a doctor, Dimmock has not confined himself to the slow-moving research world. He sees children come in with terrible genetic conditions. He has some idea of what it means to be the parent of such a child and to need a diagnosis—yesterday. Although their roles blend sometimes, Worthey is the data miner; Dimmock, the clinician. She uses computers to pry information from the millions of letters in Nic’s exome. Dimmock tests her insights against Nic’s symptoms, looking at how potential mutations might cause the specific problems inside the child’s body. After they have combed through the initial results, new data comes in and they plow through it, bent on narrowing down the field of suspect genes.
At the end of September, Mike Tschannen performs three more sequencing runs with Nic’s DNA. He works ninety-two hours in eight days. He has taken to heart what Jacob told him: this effort could prove monumental for everyone. Scientifically, what they are doing will likely be counted a success if the equipment works and if they get an accurate picture of Nic’s genetic script. If sequencing then allows them to solve the mystery of the boy’s disease, it will be an even greater achievement. But Tschannen has developed his own idea about what’s at stake. When he thinks of Nic and his family, Tschannen knows they have a very different definition of success from that of the scientific community. The work will not be a success to the Volkers unless it leads, not only to an understanding of Nic’s disease, but to a treatment. Tschannen adopts the family’s view: if Nic dies, they have failed.
In early October, he sequences Nic’s DNA for the fifth and final time. At this point, each segment has been read an average of thirty-four times, enough to reduce significantly any chance that a mutation has somehow escaped detection.
This last sequencing run allows Worthey and Dimmock to whittle the initial field of more than 16,000 genetic differences down to thirty-two suspects. These are the genes that appear promising. They meet with others involved in the sequencing project in a large conference room at the Medical College and discuss the early results. Dimmock and Worthey describe the thirty-two suspects, focusing on two in particular that have piqued their interest. One is a gene called CLECL1, which is involved in regulating the immune system. Worthey had flagged CLECL1 in her previous list of two thousand–plus potential suspects.
The other gene that intrigues Worthey and Dimmock is XIAP. This gene influences the immune system, though it had not even made Worthey’s top two thousand list. In the conference room, there is skepticism about the gene, especially among the immunologists. If XIAP could cause such havoc, it would have been implicated previously in inflammatory bowel diseases. But the immunologists have pored through the medical literature. XIAP has no such track record.
Worthey continues to update Carpe Novo as the team works, and as she does, the software program provides new insights about the most promising genetic differences. She finds that some of the thirty-two suspects can be eliminated. Among those discarded is the once-promising CLECL1. The new database shows that Nic’s CLECL1 variation is actually relatively common. If CLECL1 were causing his disease, doctors would have seen many people with Nic’s illness. But all of the searches and conversations with colleagues around the country have failed to find a single case like Nic’s.
By eliminating CLECL1 and others, Worthey and Dimmock have now gone from an original list of more than 16,000 differences down to 32, down to 8. There is only one sequencing run remaining when Worthey prepares to meet again with the immunologists, including Verbsky, to take stock. In preparation, she compiles a full report on the final eight suspects.
She thinks of this as her “hot list.”
Although the list does not include CLECL1, it includes another intriguing suspect called GSTM1. This gene has passed through all of the different filters Worthey has designed to screen out irrelevant differences in Nic’s script. GSTM1 makes a protein that is involved in pumping chemicals through the membranes of cells. This process is critical in helping to clear harmful chemicals from the body. A mutation in this gene could be deadly if, for instance, you ate fish contaminated with mercury and could not flush the mercury from your cells.
There’s just one problem: the doctors know Nic isn’t sick because of a failure in this process. He is sick because no matter what he eats, something keeps boring holes through his intestine. It is that symptom, linked to the family of illnesses known as inflammatory bowel disease, that brings Worthey back to XIAP.
All along she has had a suspicion that this gene, an immune regulator no one took too seriously, might be the culprit. The fact that XIAP has flown under the radar might be a sign. They are looking for something unusual. Perhaps this gene has not appeared in their literature searches because doctors have found very few people with mutations in the gene. And perhaps they have found few mutations because there is something important about the gene, something so crucial that a mutation is almost impossible to survive. But in order to convince herself, let alone Nic’s doctors, she must have more than a hunch.
So she looks more closely at how the change in Nic’s XIAP sequence, though small—just a single misplaced letter—would gain significance as it cascades through his body. The first consequence is that this tiny change causes a different amino acid to be made. While amino acids can differ in some places on a protein, the amino acid in this particular position is always the same—in everyone except Nic.
The amino acid is part of a long chain of several hundred amino acids that makes a protein, also known as XIAP. Worthey comes to believe that this one link in the chain, the one position where Nic differs from the norm, is critical to the form and structure of the protein XIAP.
She has looked closely at Nic’s XIAP gene. She wants to be confident when she presents her findings to the others. On November 16, 2009, at 8:41 a.m., she emails members of the team working on Nic’s sequence. “Good morning. I would like to schedule a meeting to go through the findings from the exomic genome sequencing of Dr. Mayer’s patient,” she begins. “We have identified a variant in XIAP.”
For Mayer and the other doctors, this is the first word that the sequencers may have found an answer in Nic’s genetic script. As soon as he receives the email, Mayer begins searching for background on the gene. He finds something interesting. Only a few months ago, Worthey and Stan Laulederkind searched the medical literature and discovered nothing linking XIAP to Nic’s symptoms. That’s why the gene was not among their two thousand–plus suspects.
But since then, a new paper has appeared. In August, Proceedings of the National Academy of Sciences published a paper in which researchers from the University of California, Santa Barbara, linked the XIAP protein—the one affected by Nic’s mutation—to a pathway involved in inflammatory bowel disease.
All at once, Mayer sees the significance. Worthey has found an extremely rare change in Nic’s XIAP gene. That change alters the protein XIAP. And now, in this paper, scientists are suggesting for the first time that the protein XIAP appears to play a role in diseases similar to Nic’s.
Hours after Worthey’s email, Mayer sends one of his own to members of the team. He attaches a copy of the published paper and an accompanying review. The paper, he explains, shows that XIAP is important in sensing microbes in the intestine. Our immune system is set up to identify bad microbes in the intestine and eliminate them. Problems in XIAP appear to throw off the immune system’s ability to distinguish between good and bad microbes, meaning that the system may attack the intestine when it should not. The gene even appears to have an important structural role, helping to hold together the scaffolding for the protein; if the gene malfunctions, the scaffolding collapses, undermining the protein. Piece by piece they are assembling a case; XIAP’s fingerprint is all over Nic’s disease.
Worthey continues performing due diligence, going back over Nic’s entire exome, running through various scenarios involving the eight suspect genes.
Her confidence grows. She and Dimmock study the effect this change in the XIAP gene would have on the XIAP protein. They find that Nic’s unusual sequence results in reduced production of the protein. The boy’s white blood cells wind up with just 60 percent of the XIAP protein needed. Dimmock and Worthey now have strong reason to believe that the change in Nic’s sequence is preventing his XIAP gene from functioning as it should.
Dimmock still harbors a nagging doubt. He believes the mutation is to blame for the problem with Nic’s immune system, the failure of his natural killer cells to perform as they should. But the inflammatory bowel disease, the ulcers and holes in the intestine, might be unrelated. He just can’t be sure.
Worthey feels more certain about XIAP. She makes the gene her prime suspect. Because it is her prime suspect, she places the gene last on the list she presents to the immunologists at the meeting. She gets all of her data in order, laying out a clear path that she hopes will lead them to the same conclusion she has reached: it has to be XIAP.
Her decision to place XIAP last on the list, however, has an unforeseen consequence. At the meeting, the immunologists plow through the other suspects so methodically that they run out of time and finish before they have even begun to discuss XIAP. To Worthey’s disappointment, she only has time to tell the doctors that her top pick is the one they didn’t get to.
Still, her frustration diminishes when Mayer talks with her after the meeting. He has reviewed Worthey’s findings and the recent journal paper, and he understands what they mean.