The Mount Sinai Children’s Environmental Health Center, in partnership with Autism Speaks, is holding a one day workshop, Exploring the Environmental Causes of Autism and Learning Disabilities, at the New York Academy of Medicine on Wednesday, December 8, 2010. The goal of the workshop will be to develop new strategies for discovery of environmental risk factors for autism, autism spectrum disorder (ASD) and other neurodevelopmental disorders in children.
The workshop will help identify opportunities to study gene/environment interactions in autism, and help guide research priorities for the newly formed Autism and Learning Disabilities Discovery and Prevention Program at Mount Sinai. Scientists from National Institute of Environmental Health Sciences, Centers for Disease Control and Prevention, National Institute of Child Health and Human Development and leading academic institutions from around the world will present recent research and engage in discussions to identify gaps and opportunities, especially in the area of environmental causation by toxic chemicals.
Autism Speaks has partnered with SAGE Labs, a division of Sigma Life Science, to create and validate the first line of rat models of autism. Previous rodent models of ASD, which are important for understanding the biological basis of autism and drug discovery, have used mice. Such models allow scientists to examine the downstream effects of genetic mutations on brain development and behavior. For studies of behavior, learning and cognition, the rat is the animal of choice due to its complex behavioral repertoire. With these new genetically modified rats, the richness of previous behavioral and physiological research can be leveraged and applied directly to autism. These new rat models will now be validated in the laboratories of several scientists who will be studying brain development and behavior of the animals.
Edward Weinstein, Ph.D., the Director of SAGE labs, answers some questions about the new model animals and how they will benefit autism research.
1) Generally speaking, why do we need a rat model of autism when mouse models exist?
Dr. Weinstein: Researchers have been able to create genetically modified mice for the past 25 years, so a large number of mouse models have been created, addressing all sorts of human diseases and disorders. But mice are not always the best model system for every condition. For example, researchers who study ocular disease greatly prefer the rabbit. And the rat is an excellent model for toxicology, cardiovascular, and cognitive research. In the past, researchers would create the best genetically modified mouse model possible to answer their scientific questions. Now they can create the most relevant genetically modified model in whatever species best models the condition they are studying.
2) Why can’t the same technology that one uses to make a genetically modified mouse work with rats?
Dr. Weinstein: Genetically modified mice, also sometimes called knockout mice, are created by making changes to the DNA in a mouse embryonic stem cell. Researchers tried unsuccessfully for decades to develop rat models using the same technology. SAGE labs has developed a new technology, called zinc finger nuclease technology, which allows us to create knockout rats without the use of embryonic stem cells.
3) Can you describe how the Sigma technology works in a manner accessible to our community?
Dr. Weinstein: The technology is actually fairly straightforward. We create an enzyme called a zinc finger nuclease (ZFN), which has been especially constructed to target a specific gene. We can then inject that ZFN into a one-cell rat embryo where it will bind the targeted gene and cause a break in the DNA. This effectively “knocks out”, or inactivates, the gene. The one-cell embryo is then transferred into a surrogate rat and 21 days later you have a genetically modified rat that is born. This genetically modified rat will be exactly like every other rat, with the exception of the inactivation of that one specific gene that was targeted. Part of the beauty of this technology is that it not only works for creation of genetically modified rats, but also mice and rabbits. It should work in virtually any animal model system.
4) What do you see are the next steps in using genetically modified rats for autism research?
Dr. Weinstein: We have worked to create a suite of genetically modified rats that we hope will be useful for autism research. The real challenging work, however, comes in the validation of the models. Testing these rats to see if there really are behavioral differences in them will be key to determining their usefulness.
In addition, autism research is still at the stage where new genetic links are continuously being uncovered. We hope to continue to serve the research community by expanding our platform of autism models as rapidly as possible.
5) Genetics is only one component of the autism puzzle. Research has identified environmental agents that either alone or in combination with a genetic vulnerability lead to the altered neurodevelopment we see in autism. Can environmental and gene-environment interaction questions be posed using these new rats?
Dr. Weinstein: We get kind of hung-up when we say “genetic model.” We absolutely can do these experiments. Using the genetically modified rats, we can carefully time exposures both in utero and during early post-natal life. Gene-environment interactions are extremely important to understand and we hope that researchers will seek answers to those questions with these new autism model rats.
6) Why did you partner with Autism Speaks for your foray into rat models of genes important in autism?
Dr. Weinstein: We have a lot of expertise in the use of Zinc Finger Nucleases and the creation of genetically modified animals. However, we don’t have the in-depth expertise or background in autism that is necessary for determining which are the best models to create. Working with Autism Speaks has been critical from a scientific aspect. Also, members of Autism Speaks are so well connected to the research community, you know that when you get scientific advice from Autism Speaks, you are getting advice from an entire field of researchers.
We look forward to sharing the results of the behavioral and physiological evaluations of these new rats as scientists begin working with them.
About one in every 10,000 babies is born with two few or too many genes on chromosome 15. The likelihood that these babies will be on the autism spectrum is as high as 80%, making these rare genetic events a target for autism research.
Gene targets. Of the estimated 30,000 genes that make up the human genome, between 700-900 genes lie on chromosome 15. Specific segments of this chromosome are associated with autism, narrowing the number of genes of interest to fewer than 30. These genes are located on the long arm of chromosome 15, called q. The most studied region so far is known as 15q11-13, where 11-13 describes an exact physical location on the chromosome (the region in red in the picture). Sometimes babies will have extra copies within this area, beyond the normal two parental 15q11-13 regions, resulting in 3, 4, 5, or even 6 extra copies of all the genes in the duplicated region. In other cases, portions of this region are deleted, leaving no copies of these genes.
How do chromosome copy variations occur? Autism Speaks interviewed a key researcher, Dr. Carolyn Schanen, in an article ‘Understanding the Role of Chromosome 15 in Autism’, that explains these events in detail (link to e-Speaks). The basic concept is that, during cell division, the DNA sequences of our chromosomes literally break apart and recombine; during this process, sometimes there is an aberrant duplication of genetic material. Some forms of 15q11-13 duplications are inherited and some occur in the child and are not found in the parents. Sorting out all of the possibilities is one of the goals of the research of 15q duplication and deletions.
Overlapping syndromes in 15q11-13. Deletions of genes in the 15q11-13 region are associated with Angelman’s Syndrome or Prader-Willi Syndrome, whereas duplication of such genes results in a different syndrome (15q11-13 Duplication Syndrome). A syndrome is named when common physical features or behaviors are observed. Prader-Willi occurs in about 1 in every 10,000 births and is characterized by hypotonia, hyperphagia (obsessive eating = characteristic weight gain) and diagnosis of an Autism Spectrum Disorder (ASD) in 25-45% of individuals. The occurrence of Angleman Syndrome is about 1/12,000 and is characterized by sleep disturbance, ataxia (unstable walking), frequent laughter, excitable personality and hand flapping movements. ASD is also linked with this syndrome.
Babies with 15q11-13 duplications typically have hypotonia (low muscle tone), minor facial differences (nose, eyes, ears), intellectual disability and are diagnosed with autism spectrum disorder. Seizures can be present in the newborn or develop later resulting in epilepsy that is hard to control, and sometimes lethal.
What are these genes doing? A gene’s job is to be a faithful blueprint of information, such as the code for the production of a protein. The level of production of any particular protein is carefully regulated by the cell and irregularities in the levels of proteins can impair cell function, or even lead to cell death.
For example, the product of gene UBE3A in the 15q11-13 region is involved in targeting proteins to be broken down (degraded) within cells and deficits are linked to brain pathology in both Angelman’s Syndrome and autism. Another gene in the region, called ABPA2, makes a protein crucial in getting neurotransmitters out of neurons. And the CHRNA7 gene is an acetylcholine receptor that mediates fast signal transmission at synapses. The general idea is that too little of these gene products would be linked to low muscle tone and ataxia. A new report from an international group shows that deletion of genes at 15q13.3 is linked to epilepsy. Three genes in the 15q11-13 region make protein subunits that need to aggregate with other subunits to form the receptor for the brain’s main inhibitory neurotransmitter, GABA. The extra copies are thought to create an unstable receptor and the lack on inhibition leads to excessive cell firing (seizures). Researchers are just beginning to measure gene products in brain cells of those with 15q duplications.
Changes in the brain. A special project evolved to thoroughly examine postmortem brain tissue of young adults and children with 15q duplications. In 2006, sudden deaths in otherwise healthy individuals with 15q duplications created a concern about mortality risk in these families (Isodicentric 15q Exchange Advocacy and Support– IDEAS) posted a Physician Advisory on their site at to provide information to concerned parents. The group also encouraged families to request an autopsy in the event of death as well as brain donation to the Autism Speaks’ Autism Tissue Program (ATP). Since then there have been 10 brain donations and in collaboration with the New York Institute for Basic Research, this tissue has been made available for research. The preliminary results show a disorganization of cell production and placement. The arrow in this picture of a brain section of a preadolescent boy shows a whole extra row of cells that are not typically seen in a region of the hippocampus termed the dentate gyrus, an area of the brain associated with memory and attention functions. Disturbances of brain architecture like this are linked to alterations in brain cognitive function, and often to seizure activity.
Many more changes have been observed that will be described as the research continues. The goal is to link anatomical changes to specific types of gene copy variations and behavioral characteristics linked to autism that include social deficits, communication deficits, ritualistic behaviors, mental retardation, aggression, anxiety, epilepsy, sensory abnormalities, sleep disorder as well as unique abilities. These behaviors are generated by the brain and it will take time, effort and funds to keep up with the discovery of syndromes identified by chromosome copy variations (an abnormal number of chromosomes is called aneuploidy) and the particular characteristics of those with the disorders and the patterns of brain changes seen by researchers.
This understanding of how genes contribute to physical features and behavioral characteristics requires ongoing support by dedicated families who are partnering with scientists to help better understand their child’s disorder. With increased understanding of the underlying biological processes, we may someday be able to develop treatments that can significantly reduce the impairments associated with these conditions. Contribution of biomaterial resources like DNA and brain donation is vital to this effort.
Additional reading. An excellent open access article by another of the science advisors, Agatino Battaglia, is http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613132. Brain research papers by investigators focused on autism and related disorders are posted on www.atpportal.org.
Autism Speaks’ Autism Tissue Program supports specialized neuropathology research by providing approved scientists access to the most rare and necessary of resources, post mortem human brain tissue. We wish to recognize the commitment and generosity by our ATP donor families. More information can be found at www.autismtissueprogram.org or call 877-333-0999 for information or to initiate a brain donation.
By Geri Dawson, Chief Science Officer, Autism Speaks
Science moves so slowly and is so labor-intensive that we don’t often have moments to celebrate an achievement or breakthrough that has resulted from our investments. With this week’s announcement of Phase 2 results from the Autism Genome Project, we are celebrating such an achievement.
Several years ago, when I was a professor at the University of Washington, I remember a phone call from Andy Shih, Ph.D. (Autism Speaks VP, Scientific Affairs) who asked if he could take my colleague, Jerry Schellenberg, and me out to breakfast. Over coffee, Andy described to us an idea he had: Would we be willing to collaborate with other scientists around the world and add the genetic data we have been collecting to a combined database? While each of us at that time had been working independently to try to discover autism risk genes, we knew that ultimately we would need much larger samples to deal with the significant heterogeneity that exists in autism spectrum disorder. After a lot of discussion and questions, Andy convinced us that this would be a worthwhile effort and thus we became part of what became known as the “Autism Genome Project,” or the AGP. Eventually, Andy talked with over 50 groups worldwide and cajoled each of them to join the effort. What ensued was a series of monthly conference calls, complex negotiations and agreements that Andy helped broker, the creation of a combined database, and yearly meetings during which the goals for analysis and future data collection would be discussed. Today, the AGP is considered a driving force in autism genetic research.
Meanwhile, Clara Lajonchere, Ph.D. (Autism Speaks VP, Clinical Programs) was spearheading an effort to create a database of multiplex families called the Autism Genetic Resource Exchange (AGRE). She was leaving most of us collecting similar samples in the dust as she quickly assembled the largest private genetic individual data base that exists. Her ability to form partnerships with families, engaging them in the process of scientific discovery, was a model for us all. Not surprisingly, Clara readily agreed to join the AGP since AGRE’s basic premise was “collaboration and data sharing.”
Fast forward to this week when the AGP published the largest and most comprehensive study of copy number variations (CNV) – small deletions or duplications in our genome that can disrupt gene function – in autism families. By comparing CNVs found in 1,000 individuals with autism with those from 1,300 individuals without autism, the AGP reported the following:
- Several novel ASD genes were discovered, and many genes previously implicated by other studies were confirmed. Some of these genes are involved with communication between neurons, while others help regulate cell growth and how they respond to environmental stimuli.
- It was confirmed that autism risk genes are rare variants in our genome that occur very infrequently or not at all in the general population, and each person with ASD may have a unique risk gene or set of risk genes. Some of these genes are “highly penetrant” meaning that, if you carry this risk gene, you very likely will develop ASD, whereas other only raise the risk for ASD and need to combine with other genetic and/or environmental risk factors to cause ASD. Some of these are inherited, but many appear “de novo” meaning that they only exist in the child and not the parents.
- In the not-so-distant future, we will start to see more comprehensive genetic testing being conducted in the clinic to provide parents with information about whether their child may be at risk for ASD, so they can watch for signs or better understand the cause of their child’s ASD. It will be important to consider carefully what tests are appropriate and interpret them in a manner that is responsible and helpful for parents.
- Although the fact that so many rare genes can be related to risk for autism seems to form an overwhelmingly complex picture of autism, there is a path forward: These genes appear to cluster around specific biochemical pathways in the brain and, thus, point to new directions for developing drugs that could potentially help recover function of these pathways. This is good news for families.
Most of all, I see the publication of this report as a celebration of the fruitful partnership between the families and the scientific community. While Autism Speaks staff like Andy and Clara helped create and implement unique and productive scientific endeavors like the AGP, ultimately, it is the families who contributed their time and literally a part of themselves that is helping us put together this puzzle called autism piece by piece.
This is a guest post by Alycia Halladay, Ph.D. Dr. Halladay is Autism Speaks’ Director, Research for Enivronmental Services.
Instead of focusing on just genetics or just environmental factors, autism researchers have been studying gene-environment interactions as possible risk factors of the disorder. A next series of posts will begin to try and explain why this is an important concept, and how it is changing the way scientists think about causes.
Why is this concept important?
First, in the context of risk factors, if only the separate contributions of genetics and environmental influences is calculated without considering the interaction, the proportion of the disorder that is attributable to both is underestimated. For example, environmental factors may play an important role in the development of some diseases. However, in others, the effect is only seen in susceptible individuals. Studies that examine gene-environment interactions can do the following (taken from Hunter, 2005)
- Obtain a better estimate of the risk associated with genetic and environmental risk factors
- Strengthen the association between environmental risk factors and disease
- Help researchers understand the biological mechanism of disease
- Determine which environmental factors produce risk
- Lead to new prevention and therapeutic strategies
What does it mean?
As most people know, genetics typically refers to the stable sequence of nucleotides on DNA strands in every cell of the human body. The nucleotides are translated to amino acids, which in turn create proteins. The amino acid sequence determines how proteins are configured, which may affect their function. Put in an oversimplistic fashion, these proteins are what affect cell function. Some of the genetic code is inherited from both parents, and will be conferred to their children; another, more recently studied type of genetics, called epigenetics, refers to a change in protein synthesis that is not due to alterations in the DNA code. In other words, the DNA code stays the same but the way it is expressed changes. These concepts will also be discussed in a later chapter. With regards to the term “environment”, this is a term that can refer to many “non-genetic” influences on biology and behavior. Typically when they hear “environment” people think of one of the hundreds of thousands of potential chemicals and toxins that are present in food, air and water. However, environment can also include some demographic characteristics like socioeconomic status, nutritional status and education, as well as medical procedures and illnesses, and exposure to vitamins, pharmaceuticals and/or alternative medicines. It can even refer to exposures that we may not be thinking about every day, like UV sunlight, cosmetics, food additives, and ventilation in the home. While most people think of gene-environment interactions as an environmental risk factor producing more profound effects in a susceptible individuals, some genes may offer protection against deleterious environmental effects. Other genes may promote healthy development and their effects stifled, or even enhanced, in different environments. These concepts will be explored further in a different chapter.
How are these interactions determined and studied?
The best way to determine whether an interaction exists in a human population is an epidemiologic study. One of the biggest challenges is the need for large samples, or many individuals to enroll and participate. Typically, self-report measures are obtained from all participants and family members, and DNA and other biologicals are included to study DNA/RNA and level of exposure. If other measures are available such as medical records, these are also collected throughout the study. Genetic and environmental factors, and their interaction, can be studied retrospectively (after the disease has developed) or prospectively (prior to when the disease appears). Each design has strengths and weaknesses, and in many cases both approaches are taken to identify and then replicate findings. Other study designs include case-control vs. case-case. Case-control refers to studying both individuals with and without the disease. Case-case refers to studying cases (in this case individuals with autism only) both with and without different exposure levels and/or genotypes.
In honor of the anniversary of Autism Speaks’ founding on Feb 25, for the next 25 days we will be sharing stories about the many significant scientific advances that have occurred during our first five years together. Our ninth item, Copy Number Variations, is from Autism Speaks’ Top 10 Autism Research Events of 2007.
Three studies published in 2007 pointed autism researchers toward the importance not only of mutations within the DNA code of specific genes, but also of variations in the number of copies of genes, known as Copy Number Variations (CNV). Created by submicroscopic deletions or duplications of DNA sequences, recent advances have only now allowed CNV to become routinely detectable, establishing an entirely new avenue of genetic research.
Armed with the latest technology and three different collections of patient DNA, including Autism Speaks’ Autism Genetic Resource Exchange (AGRE), researchers at Cold Spring Harbor scanned the genome for the presence of CNV in autism. In February 2007 they reported that not only do individuals with autism have more CNV than individuals without autism, but that CNV in autism occur more often as “de novo” or spontaneous mutations (mutations not found in the DNA of either parent). They also found that these spontaneous CNV appear to be more common in families with only one child with autism (simplex) than those with multiple affected children (multiplex).
With their data suggesting that genetic mechanisms may be different in different types of autism, the researchers then carefully studied the inheritance pattern of autism in the many families in Autism Speaks’ AGRE and IAN databases. In July they published that they could fit the data to a model in which there are at least two distinct ways that genes may play a role in the development of autism: spontaneous CNV might help to explain autism in simplex families, whereas inherited gene mutations may be at the root of autism spectrum disorders in a greater portion of multiplex families. Such a model is significant because although autism has been thought to have a strong genetic component, so far it has not been shown to be as clearly inherited as other simple genetic disorders.
The team from Cold Spring Harbor has provided a new theory of autism risk that stands to influence how future autism genetic research is conceptualized. Although their results require replication, it also now leads to the question of what causes this increase in spontaneous CNV, opening the door to an exploration of the interplay between genetics and environmental factors. Possible risk factors include age of the parents, specific toxicological factors and accumulated exposures, as well as genetic predisposition.
Update since this story was first run: Improvement in DNA technology since the publication of this first major autism CNV study has meant that new insights into the role of CNV are actively being pursued. The largest and most comprehensive autism CNV study to date was published in April 2009 in the top research journal, Nature. The new study focused on inherited rather than spontaneous CNV, with the researchers finding CNV variations in genes important for neural development [for more details, see http://www.autismspeaks.org/science/science_news/top_ten_autism_research_events_2009_cnv.php]. Researchers in autism are continuing to lead geneticists in other fields with an intense focus on CNV and early recognition of their importance as potential disease risk factors.
In honor of the anniversary of Autism Speaks’ founding on Feb 25, for the next 25 days we will be sharing stories about the many significant scientific advances that have occurred during our first five years together. Our eighth item, Creation of Neuroligin-3 Mutant Mouse, is from Autism Speaks’ Top 10 Autism Research Events of 2007.
Animal models have long been employed to replicate some of the behavioral and biochemical characteristics of autism. The models are chosen for study either because they have behaviors reminiscent of autism, or because they have received genetic or environmental manipulations believed to be linked, directly or indirectly, to autism.
Yet, only with the recent progress of detailed genetic studies in developmental disorders have these models been based on the actual genetic differences found in humans with autism. Some of these newer models for autism include mouse models of medical genetic syndromes that show overlap with autism, e.g., Fragile X syndrome, Rett syndrome and Tuberous Sclerosis. However, no model existed that contained the precise genetic defect found in anyone whose autism is not caused by one of these other genetic syndromes. This changed in October 2007, when researchers in Texas reported they had succeeded in replacing the mouse neuroligin-3 gene with a human version containing the exact mutation discovered in 2004 to be the cause of autism in a Swedish family with two affected brothers. Excitingly, the initial exploratory studies have found the “humanized neuroligin-3″ mouse has several unusual behaviors, including deficits in some social behaviors and an increased ability for spatial learning in a swimming test.
This mouse provided the research community with a strong new tool to directly assess the neurobiology, behavioral deficits and, conceivably soon enough, treatment approaches for autism. Such models are a vital part of the drug discovery process because measurement of changes in their behaviors can be used as surrogate markers for preclinical evaluation of new therapeutics.
Since this story was first run: Genetic studies continue to provide new opportunities for the generation of animal models of autism, including many related to the function of the neuroligins. In 2009 the same group of researchers carried out a behavioral characterization of mice lacking the neurexin-1alpha gene, which creates proteins that serve as binding partners for the neuroligins. Published in the Proceedings of the National Academies of Science, the scientists have now discovered that the neurexin-1alpha mice have abnormal brain physiology and increased repetitive behaviors.
In honor of the anniversary of Autism Speaks’ founding on Feb 25, for the next 25 days we will be sharing stories about the many significant scientific advances that have occurred during our first five years together. Our fourth item, Convergence on PTEN Signaling Pathway, is from Autism Speaks’ Top 10 Autism Research Events of 2007.
Model systems for studying the complex and fundamentally human challenge of autism will be vitally important to solving autism because they allow researchers to study the underlying biology in a manner that is not possible in humans. Finding an animal model system with similar behavioral tendencies as humans allows researchers to study which biochemical pathways break down in autism and, most importantly, how they can be treated. In 2007, researchers added an important new model system to their arsenal, the “PTEN conditional knockout” mouse.
PTEN is a gene that encodes for a protein involved in several critical signaling pathways inside cells, including metabolism, growth and survival. To carry out its cellular duties, PTEN interacts with several other important proteins in a biochemical signaling cascade. Other proteins in this signaling pathway have previously been tied to developmental disorders such as Tuberous Sclerosis and Neurofibromatosis. In 2005, researchers found that within a small subset of individuals with autism and macrocephaly (large heads) 17% had mutations in the PTEN gene. This raised the possibility that disrupting PTEN activity, and the signaling pathways within which it functions, may result in some forms of autism. This year researchers succeeded in using complex genetic manipulations to shutdown the mouse version of the gene (PTEN) in the brain of young mice. Surprisingly, not only did these animals grow larger brains, the mice also displayed abnormal social behaviors and seizures, both of which can be features of autism.
These results provided important data supporting the emerging relevance of cellular signaling pathways to autistic behaviors, and are now focusing some researchers on specific molecules that could potentially become targets for cell-based therapeutics.
Update since this story was first run: Having discovered that disrupting the PTEN signaling pathway leads to autism-associated signs, in 2009 the researchers went on to further show that manipulation of the PTEN signaling pathway can serve as a successful treatment strategy. Making use of these same mice, they found that treatment with a pharmacological inhibitor of the PTEN pathway improved the autism-like pathology of the animals, including unusual social behaviors and seizures. Published in the Journal of Neuroscience, the newest data strengthen the case for focusing on this signaling pathway as a viable target for novel autism therapeutics.