Researchers get help from the tooth fairy

Baby teeth provide biological samples to research autism and other neurological conditions

By Markian Hawryluk, The Bulletin, @markianhawryluk

As a father of a child with autism, Dr. Alysson Muotri knows how difficult and traumatic it can be for some to give a blood or tissue sample.

“I know the pain of having a kid going to the hospital,” he said.

But as a researcher of neurological disorders at the University of California, San Diego, he also desperately needed patient-specific cells to study in his laboratory.

That’s when he enlisted the help of the tooth fairy.

Muotri is one of a handful of researchers across the U.S. who is using discarded baby teeth to jump-start research into autism, Alzheimer’s and other neurological disorders that have proven notoriously difficult to study. By soliciting baby teeth from affected patients, they can access patient-specific cells that can be manipulated and studied in the laboratory, and get a record of what individuals were exposed to dating back to before they were born.

“There’s an interesting story behind every single tooth that arrives in the lab,” he said.

Muotri hit on the idea of using baby teeth after learning about stem cell reprogramming developed by Shinya Yamanaka, who won a 2012 Nobel Prize for his work. Yamanaka invented a process to reprogram skin cells into stem cells that could then be coaxed to form any cell type in the body.

“Immediately, I thought, ‘That’s a great model for autism,’” Muotri said. “Because the problem with autism is we don’t have brain cells to study.”

Researchers had relied on mouse models or samples of brain tissue taken post-mortem. Neither was ideal. Only about 1 percent of treatments that work in mice also work in humans.

“They help us understand basic mechanisms, but when we go to find new drugs or new therapies, curing mice is not the same as curing humans,” Muotri said.

He clearly couldn’t take brain tissue from his patients, but now he had a way to grow the exact same brain tissue from their cells.

The easiest way to get those cells would be to take a blood sample or a tissue biopsy. But that would be difficult to get from children with autism.

“So I thought, ‘Is there any tissue that people would discard?’” he said.

Scientists had tried using mouth swabs, hair cells, even urine as a source of cells, but none of those worked well. Baby teeth, on the other hand, fell out naturally, and if harvested quickly, could provide a viable source of cells for reprogramming.

Muotri’s lab emailed 100 families affected by autism in the San Diego area. They created a Facebook page for what they dubbed the Tooth Fairy Project, and within a couple of years had registered 3,500 families and collected more than 300 teeth.

The project team sends interested parents a collection kit that includes a small tube with a special fluid inside, as well as a questionnaire about the child’s diagnosis and symptoms. When their child loses a tooth, parents can pop it into the tube — after the tooth fairy has conducted her standard transaction — and send it to the lab.

The researchers extract cells from the dental pulp within the tooth and reprogram them to form stem cells. They can then trigger those stem cells to become neurons, growing enough to create a mini-brain in a petri dish. It’s a long and expensive process taking up to a year to complete, but Muotri’s lab is continually working to streamline the process.

Early nibbles of success

The project has already identified at least five gene mutations associated with autism, some of which had not been previously implicated.

Muotri described one case of an autistic boy from Brazil whose family sent in a baby tooth. The researchers could see that the neurons lacked the normal number of synapses. They sequenced the DNA from the tooth and found that the boy had a mutation not previously tied to autism. But researchers knew that the gene was important for creating synapses, the way brain cells communicate with each other. They also knew that a drug, hyperforin, helped promote the creation of synapses. They suggested the family try giving the boy St. John’s Wort extract, which contains hyperforin.

Within a month, independent evaluations from the boy’s school and psychologist confirmed an improvement in his social interactions.

“It’s hard to conclude that this was caused by the St. John’s Wort capsule, because this is just one patient,” Muotri said. “But there are probably people under the autism spectrum that have mutations in their genes or metabolic pathways that we already know and there are drugs out there to help them.”

Muotri recently used the technique to study a rare genetic disorder called Williams syndrome, that affects one in 10,000 people worldwide. The syndrome consists of a range of medical problems including developmental delays. But unlike autism that can result in impaired social functioning or communication, children with Williams syndrome are often overly friendly and have strong language skills.

When Muotri’s team looked at Williams syndrome neurons under a microscope, they saw the brains have many more connections to other neurons than is typical. That might explain why children with Williams syndrome are super-social, gregarious in nature, while children with autism who have fewer neural connections struggle with social interaction, Muotri said.

Because stem cell reprogramming can be used to create any type of cell, baby teeth could provide the basis for researching many other conditions.

“I focus on brain tissue disorders, but I could make heart cells,” Muotri said. “Instead of making a mini-brain, I could make a mini-heart to see if we have some drugs to treat the heart cells.”

Other researchers are looking at new ways to use baby teeth for research. Dr. Lawrence Reiter, a neurology professor at the University of Tennessee, is using stem cells that already exist within dental pulp. Those cells can be transformed into neurons, bone, muscle and other tissue types, with none of the complex reprogramming needed with other types of cells.

“Unfortunately, the biggest challenge may be to convince other scientists that these stem cells can be made into neuronal cultures that accurately represent the human brain at the gene expression level,” Reiter said.

His lab is now exploring some rare genetic conditions, including Prader-Willi syndrome, Angelman syndrome and others. One colleague is using brain cells derived from dental pulp to study the effects of the Zika virus.

Word of mouth

Collecting baby teeth may be a way to include many more individuals in research, Reiter said, particular when advocacy groups get the message out to their members that researchers need a supply of teeth.

“Without a doubt, we would not be able to do these studies without the assistance of parent support groups getting the word out,” he said.

While new stem cell technology has vastly increased the research potential of baby teeth, scientists have been collecting baby teeth for research for decades. In 1958, researchers at Washington University in St. Louis began collecting baby teeth to study links between cancer rates and nuclear fallout from the first U.S. atomic bomb tests. Public health officials in Michigan have mentioned collecting baby teeth in Flint to help them determine what children have been exposed to lead from the city’s drinking water. And a study published earlier this year found that children from war-torn Iraq had higher levels of lead and other heavy metals in their baby teeth that counterparts from more stable parts of the Middle East.

About a decade ago, Cate Quas, a pediatric dentist with Bluefish Dental in Bend, helped sort teeth for a study at the University of California, Irvine, testing whether prenatal exposure to manganese was linked to behavioral issues. She was asked to identify thousands of baby teeth collected from kids in the study to determine where in the mouth they had grown.

“Teeth form from the tip down to the root, so it’s like rings on a tree,” Quas said. “At different times, teeth are at different levels of mineralization.”

Dr. Raymond Palmer, an epidemiologist at the University of Texas Health Science Center in San Antonio, is collecting baby teeth from families of autistic children in the Rio Grande Valley of Texas to determine whether pesticides might be combining with genetic susceptibility to trigger the condition.

“Rarely is something entirely genetic or entirely environmental,” he said. “My problem as an autism researcher has been to identify environmental exposures.”

Testing hair, blood and urine can provide data on recent exposure, but autism researchers need to look much further back in time.

“With something like autism, there are windows, critical periods of neural development that we need to know what you were exposed to,” Palmer said. “You can ask mothers about their exposure and their use of products, but that’s very fallible.”

Baby teeth form in the second trimester of pregnancy and begin to create a record of exposure.

“As they develop that enamel, they just sequester whatever is floating around in there, and it stays in there forever,” Palmer said. “You take that tooth, slice it in half with very sophisticated equipment, and then you can see rings of a tree. You can date what you find inside these teeth.”

Palmer has collected teeth from over a thousand kids in Texas, some from children with autism, some from their unaffected siblings and some from kids who don’t have autism. The researchers are interested not only what compounds kids with autism have in common but also at what stage in their development. They have also been able to scrape the pulp out of the teeth to get DNA samples they can then use to consider both exposure and genetics.

“It’s a one-stop shop for what you’ve been exposed to and for genetic information,” he said. “You’re not asking a question. It’s hard data.”

Biological hard drives

Palmer has been collaborating with Dr. Manish Arora, director of exposure biology at the Icahn School of Medicine at Mount Sinai in New York. Manish, who originally trained as a dentist, grew up in a region of Africa that had experienced civil war and abundant use of pesticides. He became interested in environmental exposure.

One of the major challenges was “trying to figure out what someone was exposed to before they were born,” he said. “On the surface, it seemed like an impossible problem.”

He could ask mothers about potential exposures, but that was subject to recall problems. That’s when his dental training kicked in. Researchers had been grinding up teeth to search for heavy metals or radiation for years, but that would only give them a sense of what compound they had been exposed to, not when.

Arora learned new techniques from nuclear physicists and structural chemists that used lasers to map chemicals at the microscopic level.

“The amazing thing about teeth is they’re made up of this stuff called hydroxyapatite. It’s highly reactive,” he said. “It will trap a lot of different chemicals.”

Every tooth was essentially a record of the environmental exposures that occurred as that tooth was developing, or in Arora’s parlance, a biological hard drive.

Like the strata of earth that contain clues to the lives of the animal and people alive at the time, the layers of teeth record an individual’s various exposures.

“It can capture the information and keep it safe,” he said.

At first, Arora started looking for lead exposures, but has now been able to find organic compounds, pesticides, pharmaceutical drugs, compounds in sunscreens, even the chemical BPA. Manish recently published a paper showing they could find more than 10,000 unique compounds in a tooth.

The biological hard drives were a wealth of information. Arora developed a way to determine when a mother stopped breast-feeding the child. One colleague asked him to analyze a Neanderthal tooth more than 100,000 years.

The analysis was able to show that the Neanderthal’s mother breast-fed him for seven months, then weaned him for another seven. Humans in nonindustrialized societies breast-fed for an average of 2½ months.

Arora is helping researchers study whether supplementing baby formula with iron may be linked to the development of Alzheimer’s disease or Parkinson’s syndrome.

Tooth: Chemical time machine

Arora maintains that using baby teeth could dramatically simplify the research process for rare diseases. Currently, if researchers want to study the link between environmental exposures during pregnancy and a given condition in a meaningful way, they would have to record what pregnant women were exposed to and then wait years to determine whether those children developed the given condition. Because researchers couldn’t know in advance what children would be affected, they’d have to enroll many more pregnant women in the study to ensure they had the numbers to make a meaningful conclusion.

For an uncommon but not rare condition like autism, which affects about 1 percent of live births, researchers would have to enroll some 10,000 mothers to end up with 100 cases of autism. To study ALS, which affects one in 1,000 individuals, they need a million test subjects.

Studying baby teeth is akin to researchers enrolling every mother in America in the clinical study, but only running the diagnostic testing on a sample of those whose children developed autism and a corresponding control group.

“This is really like a chemical time machine to be able to look back and address these conditions that have such a huge impact on society, but they take a long time to occur at low frequency,” Arora said.

Researchers put more weight into a prospective study, one that tracks exposures first and then waits to see what will develop, than a retrospective study, which starts with the patients and then looks back to determine what exposures those patients had in common. Using teeth is way of doing a prospective study retrospectively.

“If you’re doing a prospective study, you collect blood samples every day and you put it in the freezer. You would call that a prospective study even if you analyzed all the blood samples at the end of the study,” he said. “This is like a freezer in your mouth. It’s collecting all the information and keeping it safe for you to analyze it at the end.”

Rather than specializing in one disease, Arora is focusing on the techniques to analyze tooth enamel, and collaborates with various disease experts considering environmental causes of disease.

“What I would like to do is to help a lot of people working across a lot of different diseases,” Arora said. “The method is very versatile and can be applied to so many different things.”

— Reporter: 541-633-2162, mhawryluk@bendbulletin.com

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