by Nancy Fliesler
Matthew Huddleston, surrounded by an assortment of toys, picks up an airplane and repetitively crashes it into the wall. "That's what I call a crash landing!" he says, mimicking a character from "Star Wars." Lindsay Jackson, a research assistant from the Developmental Medicine Center (DMC), sits beside him and tries to join the game, but Matthew doesn't respond to her overtures.
This isn't idle play. Seven-year-old Matthew, diagnosed with Asperger's Disorder, is a study subject. Jackson is part of the Autism Care and Research Program at Children's Hospital Boston, which is seeking the genetic causes of autistic spectrum disorders, or ASDs.
The effort is unique in drawing researchers from diverse clinical fields: genetics, genomics, developmental medicine, neurology, neuroscience, cognitive science and bioinformatics. Plans are underway to add other Boston institutions. At the end, it's hoped, will come a better biological understanding of the ASDs, tests to diagnose them early and more effective medical treatments. All have so far been elusive.
Matthew's mother, Maureen, waits nearby. "Anything we can do to help new families coming up behind us, we feel we should do," she says.
Autism was first described, in 1943, as children's "inability to relate themselves in the ordinary way to people and situations." The past two decades have seen a surge in ASD diagnoses. Some attribute this to greater clinical recognition; others blame environmental factors. The Centers for Disease Control and Prevention estimates that two to six children per 1,000 are affected.
Today, several ASDs are recognized, including Autistic Disorder, defined by social and communication difficulties and restricted, stereotyped or repetitive behaviors, interests or activities; Asperger's Disorder, similar to autism but with no significant delay in language or cognitive development; and PDD-NOS (Pervasive Developmental Disorder-Not Otherwise Specified), involving social impairment plus either impaired communication skills or stereotyped behaviors, interests or activities.
For decades, autism was blamed
on emotionally distant "refrigerator mothers." Then, in 1977, a study of twins provided evidence for a genetic basis: when one twin had autism, the other twin was much more likely to also have autism if he or she was identical rather than fraternal.
Nearly 30 years later, despite extensive research, no specific causative gene has been found for any of the ASDs. There are several challenges. First, each recognized ASD probably encompasses a variety of sub-disorders, involving different genes. Second, ASDs are commonly believed to require changes in more than one gene. Third, there's the likely role of environmental factors: even identical twins don't always share autism.
The studies to date have lacked the size and statistical power to overcome these challenges. And few have integrated behavioral, genetic, cognitive science and neuroscience perspectives on autism in the same group of children. That integration is the goal of Children's Autism Care and Research Program.
Matthew, a triplet, was different from his siblings even as a newborn. "He had a frustration, a demanding of attention that my other kids didn't have," says Maureen. "On the playground, if he finds the tire swing is broken, he will have a monumental biting, scratching tantrum. I try to pre-teach him every situation that might come up to avoid any possible scene."
Tiny points of light
As Matthew plays, Jackson makes notes on a form known as the Autism Diagnostic Observation Schedule. The point isn't how well Matthew performs a task—telling a story, describing the action in a picture—but how he does it. Is his language spontaneous and expressive, or does he use stereotyped phrases, like the line from "Star Wars"? Does he use gestures? Does he pick up on social cues, like when Jackson mentions it's her birthday, or does he fall silent or change the subject?
These observations will help establish Matthew's phenotype, a rigorous, standardized description of his behavior and affect. Many previous autism studies did not phenotype subjects rigorously, or did not involve homogenous groups, diminishing the value of their findings.
Phenotyping also involves a visit with genetic counselor Heather Peters, who takes a thorough medical history of child and family, and an examination by geneticist Ingrid Holm, MD, who looks for subtle physical features that might indicate a genetic syndrome. Next, a special 3D camera takes a picture of Matthew's head and computes a variety of measurements: subtle things like distance between the eyes might become clues in the search for autism genes.
In addition to the DMC, headed by Leonard Rappaport, MD, behavioral assessments will take place in Children's Department of Neurology, under the direction of David Urion, MD, and at collaborating institutions. "Behavior is the only way we diagnose ASDs, so we all need to be on the same page," says DMC clinical psychologist Ellen Hanson, PhD.
Lou Kunkel, PhD, Zak Kohane, PhD, and Mike Greenberg, PhD, tend to finish each other's sentences. When they exchange ideas, the excitement is palpable. Each is a leader in his field: Kunkel, director of Children's Program in Genomics, is one of the world's leading geneticists; Kohane, director of the hospital's Informatics Program, is known worldwide for his use of bioinformatics to interpret genetic information; and Greenberg, director of Children's Neurobiology Program, is internationally recognized for his research on genetic regulation of the developing nervous system.
They've come together to study ASDs at an opportune time. In the past five years, the entire human genome sequence has become available, millions of markers have been identified to expedite the search for genes, and technologies for sequencing genes to find mutations have vastly improved. "The technology has moved ahead," says Kohane, "but that puts an even greater premium on the ability to synthesize the information."
The synthesis will begin with the 10 or more chromosomal areas that have already been linked with autism. Somewhere under those "linkage peaks" might lie one or more causative genes. But each peak can contain up to several hundred genes, and sequencing all of them in large numbers of children, with and without ASDs, would be prohibitively expensive. Kunkel, Kohane and Greenberg are using several tactics to home in on the likeliest suspects.
White blood cells from children like Matthew may provide the first clues. They will be tested for RNA, a cousin of DNA whose presence indicates that a gene is being "expressed"—turned on—and actively functioning. The investigators believe that gene expression in white blood cells may reflect what's happening in the brain.
In Kunkel's Genomics Laboratory, tiny microarrays or "gene chips" are used to analyze the activity of 30,000 genes at once. On a computer screen, a grid of glowing lights indicates genes expressed at varying intensities (above). With computational help from Kohane's team, Kunkel's group will compare gene expression patterns between children with and without ASDs. Are some genes turned on that shouldn't be? Does a gene that should be on show only weak activity? And do any genes with altered activity fall under a known chromosomal linkage peak? If so, they could be important.
In a complex disorder like autism, Kohane notes, alterations in gene expression can be quite subtle. It may not be a suspect gene itself that's markedly altered, but a gene it interacts with, or even an entire biological pathway. Kohane's team will use sophisticated computer algorithms, some borrowed from artificial intelligence, to plot difficult-to-spot relationships among whole groups of genes.
The closest attention will be paid to genes and pathways involved in brain function. And the top candidates will be those involved in the development of synapses.
As a baby is exposed to stimuli and experiences—his mother's voice, a colorful toy—new connections, or synapses, form between his brain cells. As he develops, the important synapses are strengthened while others wither away. Synapse formation and refinement are integral to proper brain development.
Greenberg, who studies synapses in animals, believes that children with ASDs have altered synapse development, perhaps because their brains have trouble processing incoming signals. "The brain develops in concert with the external environment," he says. "We've been finding that experience shapes synaptic connections by regulating a genetic program that affects synaptic maturation and refinement. Subtle variations in this program might underlie autism."
Greenberg's lab has already identified several genes involved in synapse formation. Kunkel and Kohane are cross-checking them against the gene expression studies and the autism linkage peaks, looking for an auspicious match. On the flip side, as candidate genes emerge from Kunkel and Kohane's work, Greenberg's lab will examine their effects in synapses, using rats as a model.
"We can use a 'gene gun' and shoot in any gene they want to test, or knock down expression of a particular gene and see the effect," Greenberg says. "Does the synapse go away? Does it function differently? Do you get more synapses?"
To date, virtually all candidate autism genes that have been sequenced code for proteins. Yet surprisingly, more than 95 percent of the human genome doesn't code for proteins at all. These vast chromosomal regions, once termed "junk DNA," are now known to include functioning stretches of code that are inherited virtually intact over generations. Increasingly, scientists believe they may harbor disease-causing mutations. (See Research rundown)
Filling in the family tree
Kunkel, Kohane and Greenberg suspect that these previously unrecognized parts of the genome might contain fresh clues to autism, and perhaps explain why a causative gene hasn't been found. "Maybe people haven't looked in quite the right way," Kunkel says. "We don't want to leave any stone unturned."
At a meeting in Kunkel's office, discussion heats up about tiny RNA molecules called microRNAs. First identified in the worm genome in 1993, they appear to function both in the nervous system and in early development.
"We have evidence that microRNAs are functioning at synapses. They turn off or suppress translation of the message from a gene. This fine-tuning of the message may alter cognitive function in a subtle way," Greenberg says.
"We can see if your microRNAs are the same as those we're finding under the linkage peaks," suggests Kunkel. "Then we can sequence them and see if there are variants in kids with autism."
In a later phase of the research, families' saliva samples will be collected for a type of DNA analysis called transmission disequilibrium testing, or TDT. To make the collection as large as possible—ideally thousands strong—Children's is working with institutions around Boston and beyond. The analysis is expected to yield new linkage peaks and, with luck, actual genes involved in the different ASDs.
TDT searches for genes shared between parent and child more often than would occur by chance. Everyone inherits two copies of each of their genes, one from mom, one from dad. In turn, they pass just one copy, or allele, to their own child, so that the child has a 50/50 chance of inheriting one or the other allele. But if an allele is involved in an ASD, and you're looking just at families with ASDs, you'd expect that allele to be shared between parent and child more than half of the time.
Once such a blip is picked up, Kunkel's team will search for a variant allele using markers called single nucleotide polymorphisms (SNPs)—variations in the chemical "spelling" of DNA that are already mapped to specific chromosome locations. SNPs usually don't cause disease themselves, but if they reside close enough to a disease gene, they'll be inherited along with it. If a certain SNP shows up in families with ASDs, but not in unaffected families, an autism gene might be nearby. More SNPs are then used to narrow the search.
When the search can be narrowed no more, Kunkel's team will sequence selected genes—and other elements like microRNAs—at that chromosomal location.
A roughly parallel process is underway in a completely different group of patients. Children's Chief of Genetics Christopher Walsh, MD, PhD, is studying large Middle Eastern families in which the parents are related. Twenty of these families have two or more siblings diagnosed with an ASD.
In traditional Arab societies, marriage between cousins (typically between a man and his father's brother's daughter), promotes family stability. But over generations, it increases the likelihood that offspring will inherit rare mutations, especially in isolated populations descending from a small number of "founders." Add a high birth rate and large family size (averaging six children per mother), and you have an ideal population for studying genetic disorders.
"To map a gene for autism in American families, averaging two to three kids per family, you would need to pool many families," says Walsh. "But if one family has a problem because of a gene on chromosome 1, and another because of a gene on chromosome 13, pooling them may give you conflicting information. In larger families, one family alone may be enough to definitively localize a gene."
Using a technique called homozygosity mapping, Walsh compares the genomes of affected and unaffected family members. He's looking for recessive mutations—those that cause disease only when a child inherits two copies.
A familiar face
"We check each set of chromosomes from beginning to end, looking for one place where the child has two identical pieces of DNA on both chromosomes," Walsh explains. "Eventually we find a spot where all affected children have two identical chunks of DNA, and where unaffected children have something different."
So far, four such spots have been found in three separate families. Walsh's team has begun sequencing the genes in these chromosomal areas; if they find a disabling mutation, the same gene will be sequenced in the Boston children. Likewise, the studies by Kunkel, Kohane and Greenberg may help narrow Walsh's search.
While these genetic studies are going on, Chuck Nelson, PhD, director of the DMC's research laboratory, will study what happens in the brains of children with ASDs. Children, aged 2 to 12, will be fitted with small sensors, covering their scalps like flowery shower caps (see photo above). As they're presented sensory stimuli or perform cognitive tasks, the sensors will detect bursts of electrical activity. Children over 6 will also have a functional MRI, a type of brain scan that pinpoints areas where the brain is working hardest by measuring blood flow and oxygen consumption. Functional MRI studies will also be done by Children's Neurology and Radiology departments.
Nelson, an internationally known cognitive neuroscientist, hypothesizes that patterns of brain activity will differ between children with and without ASDs. "If I look at you, I know exactly what area lights up in your brain," Nelson says. "But in a child with autism, it could be a completely different area."
Specifically, he expects that children with ASDs will show impaired recognition and processing of faces and speech, cognitive tasks that have a social component. Visual tests will show children a series of objects, comparing their responses to faces with those to other objects. The faces may be shown upside down, blurred or displaying different emotions. In auditory tests, children will hear words they know versus words they don't, spoken by their mothers versus complete strangers; again, responses will be compared. Older children will also undergo memory testing (memories in autistic children are thought to be either poor or highly enriched).
The researchers will then synthesize these findings with the genetic information. "It may be that the genes that are altered in children with autism are involved in specific cognitive deficits," Nelson speculates.
Matthew Huddleston has many strengths: he's smart, creative and engaging. "I'm good at swimming, I'm good at collecting, I'm good at making things you've never seen before," he says. "Like strawberry French fries with milk dip." But his mother hopes that the Children's study can take aim at his deficits.
"He doesn't understand tone of voice or subtleties of body language," she says. "People don't even realize he's talking to them because he doesn't look them in the eye. When other kids start spontaneous play, Matthew doesn't get what they're playing or how to play it. His biggest struggle is trying to make connections with other kids."
The Children's researchers are also looking for connections. Reaching across academic boundaries, they are probing autism from every possible angle. With luck, the knowledge that emerges will help children like Matthew connect with the world.
If you have any questions regarding this article or the study please contact the study coordinators, Lindsay.Jackson@childrens.harvard.edu (617) 355-3076 or Elizabeth.Baroni@childrens.harvard.edu (617) 355-0526
To learn more about supporting Children's Hospital Boston's autism research, contact Donna Richardson in the Children's Hospital Trust at (617) 355-2061 or