Nicholas Volker gets excited when he opens a new toy that his mother, Amylynne Santiago Volker, brought him in June at Children's Hospital of Wisconsin. Nicholas, who has a serious intestinal disease, was feeling much better after developing a blood infection two days earlier.
James Verbsky is drawn to pediatrics by its ethical simplicity - the notion that you do anything to save a child. Yet as the doctor talks with colleagues at Children's Hospital of Wisconsin in the summer of 2009, he finds himself doubting their proposal for 4-year-old Nicholas Volker. They want to sequence Nicholas' DNA.
The boy in the Batman cape has a mysterious disease marked by painful holes leading from his intestine to his skin. Fecal matter leaks through the holes. In 2 1/2 years, Nicholas has made more than 100 trips to the operating room.
Verbsky, a 39-year-old immune specialist, has run every test he can think of to pin down the source of Nicholas' disease. So have the other doctors. They are running out of options.
Some favor a bone marrow transplant based on the theory that Nicholas' immune system has gone out of control and is destroying healthy cells. But the procedure is risky and the doctors are not certain it will help. How can they be certain? They don't understand the problem they're trying to fix.
Verbsky's colleagues hope the answer can be found by reading the boy's genetic script. But the hospital and the Medical College of Wisconsin have never sequenced all of a patient's genes and were not planning to until 2014.
To do so for Nicholas could take months, cost a small fortune and still leave more questions than answers.
Every human carries thousands of variations in the genetic script, the long chain of chemical bases that makes us who we are. These differences - the base adenine in one position instead of guanine - determine everything from harmless diversity (brown hair vs. blond) to the defects that cause disease. Although some differences are harmful, most are not. Some are even beneficial.
They could find 20,000 of these differences in Nicholas' genome, Verbsky worries, so many that they will never determine which of them caused his illness.
"I was skeptical when they said they were going to sequence him," he would later say. "I laughed, to be totally honest."
The board in Nicholas' room at Children's Hospital shows his alias, "Batman." He sometimes wears a Batman cape.
Soon after the July Fourth weekend, 10 doctors and scientists meet at the Medical College to discuss sequencing Nicholas. The man who called the meeting, Howard Jacob, head of the Human and Molecular Genetics Center, has never met Nicholas. He has been weighing whether to sequence him for a little more than a week, ever since the boy's pediatrician, Alan Mayer, suggested the idea.
Although Jacob can find no published reports of a patient being sequenced to diagnose a disease, when he reads the spirit in the room he finds a willingness to try.
Mike Tschannen (pronounced Shannon), the research associate who runs the college's sequencing lab, looks at the question this way: "If we choose not to do this, why are we here? This is the entire reason we're here."
The scientists decide not to read Nicholas' entire genome, a massive undertaking that could cost up to $2 million. Instead, they plan to target a little more than 1%, the exons. Part of every gene, exons carry the instructions for making proteins. It is the failure to make proteins correctly that causes many diseases.
While zooming in on this section of the genome cuts the cost substantially, the estimate is not trivial: $75,000. Donors will have to be found.
The Medical College, a relatively small player in genomics with just one sequencing machine, will be attempting something that large centers with dozens of machines have not done.
As they leave the conference room that day, one of the most optimistic is Liz Worthey, a senior research scientist from Scotland. Since the moment she learned of the project, she has felt: We can do it. Now, she will be called upon to make sense of the thousands of variations they expect to find in Nicholas' genetic sequence.
The project will be different from anything Worthey has ever done. As a researcher, she is accustomed to thinking that her work may affect thousands of people, years in the future. This time she may help a single child.
This time she does not have the luxury of years.
"We need urgent prayers now please," Nicholas' mother, Amylynne Santiago Volker, writes in her online journal.
It is July 14, 2009. She has received worrisome news.
Two of the doctors, David Margolis, a bone marrow transplant specialist, and James Casper, a blood expert, are recommending giving Nicholas very high doses of chemotherapy, a treatment that has proved successful in children with Crohn's disease. Unable to reach a definitive explanation for Nicholas' illness, the doctors have settled on Crohn's as a working diagnosis.
If he survives the chemotherapy - and the doctors believe he will - the hope is that the drugs will wipe out his immune system, allowing it to reset and develop normally this time.
Amylynne cannot get past the "if." Too many times in the last few years, she has been warned that Nicholas might not live out the night.
She leans heavily on her faith. Yet she cannot ward off the fear that something will go wrong.
At this point, DNA sequencing is not her focus. Doctors are asking that she decide on the chemotherapy plan soon, before Nicholas becomes too sick for treatment.
"What do I choose?" she writes. "How do I make the right decision? What if I wait? What if I don't wait? Either way I could compromise my son's life."
Nicholas, 5, has his height checked by a nurse before getting the new feeding tube. At 4 he weighed less than 20 pounds, well below the 35-pound average.
The sequencing of Nicholas' DNA begins with money and paperwork and blood.
In addition to its scientific challenges, the project straddles a sensitive regulatory border. Is it research for the greater good, or treatment for a single patient?
Children's Hospital has a rule prohibiting staff from raising money to pay for the care of one patient. But Howard Jacob says that's not what he's doing. He is raising money to run a pilot test of sequencing because the technology has the potential to help many patients.
The interpretation differs when pediatrician Mayer consults the hospital's institutional review board. The board oversees human subject research, but this is not research, says chairman Paul Scott. Reading Nicholas' DNA is an attempt to help one patient; it is nothing more than the practice of medicine.
In fact, sequencing is both: a way to help Nicholas, and to test a technology that could help others. But before the project can proceed, money must be raised, a task for which Jacob is well-positioned. He speaks to business groups frequently and co-founded a start-up company, PhysioGenix, which helps drug developers test their compounds.
Once Jacob begins making calls, it does not take him long to raise the money. In addition, the company that makes the sequencing machine decides to collaborate with the Medical College team by performing the first run for free.
Amylynne Volker is hopeful, though she has been warned that sequencing may not reveal an answer. Nicholas has undergone many tests already: at least nine focusing on individual genes and 35 examining his immune system. And still, no diagnosis.
Before sequencing can begin, Amylynne must sign a stack of releases, so many pages she does not read most of them. Then medical staff go to her son for blood, a more efficient source of DNA than saliva. All they need is a teaspoon.
If the disease is genetic, the clues lie packed inside the nuclei of Nicholas' white blood cells, the portion of his blood sample that contains DNA. First, scientists must remove the red blood cells, platelets and plasma.
Although there is a machine that can extract DNA, the individual strands emerge in better shape when the job is done by hand. Mike Tschannen, who runs the sequencing lab, knows the hands he wants working with Nicholas' blood. He goes to Gwen Shadley, a research technologist at the Medical College who has a talent for pulling good DNA from samples.
Tschannen does not tell her whom the blood comes from, whether the person is alive or dead. Only this: Handle it with tender loving care. It's a very special sample.
Shadley first uses a solution to burst the red blood cells, then a centrifuge to separate out the plasma, platelets and cell debris. At the bottom of the test tube are the white blood cells. She adds detergents to break open those cells, releasing the DNA inside. Then she separates out the other cell contents - proteins, sugars, fats. What's left gets poured into a test tube of alcohol solution. She places the tube on a machine that rocks gently back and forth.
Tiny strands of DNA like fine white thread drift through the solution and clump together, all visible to the naked eye.
"It's absolutely gorgeous to see," Shadley says. "I was born and raised on a farm. It's almost like watching a birth."
Within 24 hours Nicholas' blood is pared down to DNA, his genetic secrets reduced to a clear, ordinary-looking liquid.
Tschannen places a large drop of the liquid inside a sealed container surrounded by dry ice and sends it by overnight mail to the pharmaceutical giant Roche.
The company's 454 Life Sciences division is performing the first sequencing run. Technicians begin by breaking the long strands of DNA with their 3.2 billion chemical base pairs into shorter, readable stretches. Using pressurized nitrogen gas, they shear Nicholas' DNA into segments of roughly 500 to 800 bases.
The segments are loaded onto a special chip the size of a microscope slide that captures only the exons. Whatever does not stick to the chip - the non-exon portion - is washed away.
Nicholas' exons are attached to tiny beads and spread over a sequencing plate the size of a standard Post-it note. The plate is then loaded into the machine.
The sequencing machine works by reading individual segments, then reassembling them into the complete string.
One by one each of the four bases - adenine, thymine, guanine, cytosine, or A, T, G, C - washes over the plate containing Nicholas' exons. A light flashes each time the base encounters a match. A matches T; G matches C.
A camera photographs the pattern of light flashes.
A computer translates the photos into Nicholas' sequence.
Amylynne Santiago Volker carries her groggy son, Nicholas, back from the operating room in April after he had a new gastrointestinal feeding tube installed at Children's Hospital of Wisconsin. Nicholas suffers from a rare genetic defect that devastates his digestive system. All of his food comes from the tube.
Meanwhile, Worthey and research scientist Stan Laulederkind get an early sense of the best candidates to cause an illness like Nicholas'. They scan the medical literature for papers that tie specific genes to any of the boy's symptoms. Whenever a pathway - a group of interacting genes - is linked to a symptom, they include all of the genes.
By August, a month or so into the sequencing project, their finished list contains more than 2,000 suspects. Doctors are confident one is the gene they're hunting. Yet they know the machine will produce a much longer list of possibilities.
To prepare for the deluge, Worthey and David Dimmock, a pediatric genetics specialist at Children's and the Medical College, devise a strategy to narrow down the thousands of differences they expect to find between Nicholas' sequence and what is considered normal. To this point relatively few human genomes have been read, so "normal" has yet to be fully established.
The Medical College team is guided by two assumptions: The crucial difference in Nicholas' DNA, the one at the root of his disease, must sabotage an important process in the body and must have been undiscovered until now, since Nicholas' disease does not appear in medical literature.
To find it they must eliminate the differences that do not have dire consequences. Some produce the same amino acid as the normal sequence. Others change an amino acid but do not disrupt a vital function in the body.
Existing tools can analyze some effects of these differences. But scientists have nothing that can perform the broad analysis Worthey and her colleagues require.
So she designs her own tool. Working with a group of software developers at the Medical College, she pulls together new and existing algorithms and data from different sources to create a program that can tackle a case of Nicholas' complexity. She calls the program Carpe Novo, Latin for "seize the new." Carpe Novo is still being developed in August when the results arrive from the first read of Nicholas' DNA.
James Verbsky's initial fear that they would find 20,000 variations was not far off. In all, Nicholas has 16,124.
That's the size of the haystack they're searching.
Late in August, Nicholas is receiving high doses of chemotherapy, the treatment doctors have recommended. The treatment Amylynne has been dreading. His fever hovers around 104. He vomits up to 20 times in a single day.
When Nicholas looks into a hand mirror he sees his head, bald from the chemo. He shrugs and walks away. He wants to go home.
Finally, in early September, his pediatrician calls with the first good news in months.
"NIC IS IN REMISSION," Amylynne writes in her journal. "PRAISE GOD!!!!"
After 250 consecutive days in the hospital, he returns home to Monona, a Madison suburb. He enjoys the simple pleasures of jumping in leaf piles, riding the bus to 4-year-old kindergarten, and trick-or-treating with Batgirl (his mom) and Wonder Woman and Robingirl (two of his sisters). He can even eat - as long as he adds just one new food a week, Mayer says.
One day that fall, Amylynne writes in her journal, "Now he is dancing in front of the television, eating some kind of sticky treat."
Somewhere in a pool of 16,000 variations in Nicholas' genetic script lurks the cause of his disease, if only Worthey and Dimmock can find it.
Dimmock, an Englishman, discovered his passion for pediatrics in the resiliency of the children he cared for a decade ago at a tin-roofed hospital in Uganda. He would treat them for malaria one day, and watch them race across the hospital grounds the next.
For more than a year, Worthey and Dimmock have worked together on research using sequencing to understand the genetics of liver failure and the genes that cause mitochondrial disease. But Dimmock has not lived solely in the research world with its slow progress toward conclusions. He screens babies for metabolic diseases and sees children born with genetic conditions. He knows what it means to need a diagnosis yesterday.
Although their roles blend at times, Worthey is the data-miner, Dimmock, the clinician. She gets computers to pry information from Nicholas' lengthy genetic script; he compares potential mutations with the boy's clinical profile.
Based on the results from the first sequencing run, they list variations in 32 genes that appear promising.
Two raise particular interest: a gene called CLECL1 and another called XIAP, both involved in regulating the immune system. CLECL1 was among the more interesting suspects on Worthey's list of 2,000-plus genes; XIAP did not make the list.
Not all genes are captured in one sequencing run, so they must repeat the process several times. Each run is like a slide superimposed over the previous slides, adding depth and resolution to the picture. After the first run at Roche, Nicholas' DNA passes through the sequencing machine at the Medical College four times.
To this point, Mike Tschannen has used the Medical College machine solely for research, sequencing rats and bacteria and examining a few specific areas of human biology. Now, he sees a chance to show that a small lab with one machine "can do science that may change the world."
In an eight-day span at the end of September, Tschannen works 92 hours sequencing Nicholas' DNA three times. In early October, he performs one final run.
Multiple copies of each short segment of DNA pass through the machine on each run. After five runs, each segment of Nicholas' DNA has been read an average of 34 times, enough to reduce significantly the possibility that a mutation could be missed.
Worthey and Dimmock filter Nicholas' variations with the software tool and search a database of genetic differences. They discover that many of his variations, including CLECL1, are common and can be eliminated.
Their list drops to eight.
Worthey examines what each gene does and conducts a more thorough literature search. She zeros in on two genes, then discovers that one, GSTM1, is commonly altered in people who are perfectly healthy.
That leaves one prime suspect: XIAP.
Since the previous literature search two months ago, a new article has appeared in the Proceedings of the National Academy of Sciences linking XIAP to a pathway involved in inflammatory bowel disease. Several of Nicholas' symptoms resemble that disease.
It is now November 2009. Worthey scans the publicly available human genome sequences. Then she goes further, asking researchers with unpublished genome data to look for this variation in XIAP. In all she is able to check about 2,000 human genomes.
Not a single one has the variation.
It must be Nicholas' mutation.
Amylynne Santiago Volker comforts Nicholas at Children's Hospital of Wisconsin in April after he got a new gastrointestinal feeding tube inserted. Nicholas has had more than 100 trips to the operating room in his short life.
For the first time, his disease begins to make sense. If Worthey and Dimmock are correct, the holes in Nicholas' intestine, the ravaged colon, all of it stems from a single misplaced base in the long chain of his DNA.
On the X chromosome, on the gene XIAP, the rest of humanity has the sequence thymine-guanine-thymine.
Nicholas has thymine-adenine-thymine. In the single-letter shorthand scientists use, he has what amounts to a typo, an A instead of a G.
The bases in this sequence make an amino acid, the 203rd in a chain of almost 500. That amino acid is supposed to be cysteine, and has been in all humans examined to this point.
But in Nicholas, the one-letter change produces an entirely different amino acid, tyrosine.
His tyrosine is part of a long chain that makes a protein, also called XIAP. This protein has two important jobs: it blocks a process that makes cells die and it helps prevent the immune system from attacking our intestine.
In Nicholas, however, the protein is made incorrectly. In his body, the immune system is at war with his intestine.
Since the human genome is composed of more than 3 billion base pairs, Nicholas' mutation represents the smallest possible error in a vast blueprint. Imagine one letter out of place in the 55 million-word Encyclopaedia Britannica online edition.
Even this image does not do justice to Nicholas' terrible luck. Not only is his misspelling unique among the human genomes examined, it is unique among the animal genomes Worthey checks. Fruit flies, rats, mice, cows, chickens, chimpanzees - every organism she can find makes cysteine at this position.
To Worthey, the extreme rarity of his mutation across the species carries an unmistakable message.
"If all of those organisms have (cysteine) at that position, then clearly it's important because over all that time it has never been allowed to change," she says, "(If it did) something bad obviously happened to stop that line from evolving any further. So everything has a cysteine."
On a Friday afternoon in mid-November, Amylynne's cell phone rings. It is Mayer, Nicholas' pediatrician.
Has anybody contacted you from the hospital? he asks.
Nicholas and Amylynne have been home for six weeks. Nicholas, now 5, has been going to school, playing, eating.
Mayer says the doctors are excited. They have found the mutation. Maybe.
From the beginning, Mayer has felt he would know the right mutation when he saw it. After reading the paper on XIAP, he is confident they have their culprit. Still, he is cautious with Amylynne.
Her mind skims over the new information.
All right, she says, what disease? How many years does Nic have to live? Tell me the bad news first.
Mayer explains that a mutation on Nicholas' X chromosome has caused the illness in his gut. But there's more. The same mutation has also caused a second extremely rare disease called XLP. Only boys get this second disease, which leaves them unable to fight off one of the most common human viruses, Epstein-Barr.
Most die before the age of 10. The only cure is a bone marrow transplant.
A few days later, Amylynne meets with Dimmock, the genetics specialist. He explains what the doctors have found, and why it leads them to believe Nicholas needs a bone marrow transplant, a risky procedure that had been under consideration before the sequencing. First, Nicholas' blood must be sent to a federally approved clinical lab to confirm the mutation.
Dimmock tells Amylynne that he would like to test her blood to determine whether she has the same mutation. Some mutations are not inherited but occur as the egg forms.
The doctor explains that what they find may have implications for her children and other relatives. They may learn what the likelihood is that they have the mutation and could pass it to their own children.
Dimmock goes through the consents that must be signed, and asks a series of questions.
Are you Nicholas' mother?
Really? Amylynne says. You're asking me if I'm the mom?
Is Sean Nicholas' father?
Yes and Yes.
Although the questions seem odd and uncomfortable, the need to ask them is not far-fetched. Doctors have learned that a proportion of tests for genetic diseases inadvertently disclose unsettling information, for example, that Dad isn't actually Dad. DNA tells our secrets.
Finally, Amylynne must answer two questions: Does she want her own DNA tested, and does she want to know the results?
Yes and Yes.
Feeling the effects of chemotherapy, Nicholas Volker breaks down while a photographer (not pictured) from a nonprofit tries to take his portrait in July. His mother and sisters Mariah and Kristen look on.
In early December 2009,the boy's disease reawakens.
Nicholas loses weight. He becomes lethargic. The Volkers worry about swine flu or mononucleosis. They take Nicholas to Children's, hoping he won't have to spend another Christmas there.
Amylynne has moved into a room at the Ronald McDonald House near the hospital. She buries her head in a pillow.
"This must be what hell is like," she writes in her journal.
Nicholas' ileum, the final section of his small intestine, is full of pus and ulcers. Amylynne worries he'll never eat again.
A few days before Christmas, Nicholas erupts.
"I want my food back. Give me my food back," he screams, an outburst Amylynne records in her journal. "I don't want to get better, I want to be sick and have my food."
It falls to the skeptic to prove that sequencing has worked.
Despite his early reservations about the technique, James Verbsky feels a rush of excitement when he learns a mutation has been found and confirmed by the clinical lab. The fact that the mutation is unprecedented, that evolution appears to have kept it from taking hold in other species, tells him that in all likelihood they have found the culprit.
Still, the immune specialist knows the scientists need more evidence before they can say that this defect caused Nicholas' disease. They must show the mutation prevents the XIAP protein from doing its job.
To do this, Verbsky designs two tests. In one, they stimulate Nicholas' cells, adding a product made by bacteria to see whether the cells will recognize it and respond, as they should, by releasing a protein. Three times they perform the test. Each time other human cells release the protein.
Nicholas' cells do not.
In the second test, Verbsky and his team try to determine whether Nicholas' XIAP protein is curbing a process that causes cells to die. Once again Nicholas' cells differ from other human cells. More of his cells die. His protein isn't saving them as it should.
Now, Verbsky believes, they have the evidence. The single-letter mutation prevents Nicholas' protein from performing its jobs; that is why his gut doesn't work. The answer, so logical yet unexpected, prompts the scientist to reconsider the future of the test he once doubted.
To this point, doctors facing a mysterious condition would often single out suspicious genes and test them one by one. Each genetic test could take two to three months and cost up to $3,000.
Sequencing now gives medicine the chance to dispense with piecemeal methods and examine all of the genes at once. While it has cost roughly $75,000 to sequence and sift through Nicholas' DNA, the price is plummeting and should reach $1,000 or less in a few years.
"In five years," Verbsky says, "this is what we will do. I have no doubt."
Early in 2010, Amylynne returns for a second meeting with Dimmock. The tests on her DNA and immune system are complete.
The doctor begins with a preamble, one he always gives before genetic testing and again when the results come back. We have no say in the genes we pass to our children. There are benefits to reading our DNA, but they are balanced by the potential harm of what the DNA tells us.
Then the preamble is over.
You carry the mutation, he says.
Like all females, Amylynne has two X chromosomes. One has the normal gene; the other has the mutation. The normal X appears to compensate for the bad X. That's why she is not afflicted with her son's disease.
Like all males, Nicholas has only one X chromosome. It has the mutation.
Amylynne's eyes fill with tears.
The doctor is saying she did not get the disease.
She just passed it to her son