Genetics and Genomics|Autism

By Michelle Pflumm, PhD

Donnie T. had no interest in going out for ice cream or visiting the candy store. What he really loved to do was watch tops spin. “He was happiest when he was left alone,” his physician Leo Kanner, MD, of Johns Hopkins Hospital wrote in 1938, “almost never cried to go to his mother, [and] never seemed to notice his father's homecomings.”

We now understand that the “fascinating peculiarities” Kanner saw in 5-year-old Donnie, which he called “autism,” are likely due to miswiring or missing neural connections in the brain. What lies behind these changes, however, continues to fascinate researchers to this day.

A complex genetic disease

In the late 1970s, researchers began to appreciate the strong role of genetics in autism. Studies of twins showed that, if one twin has autism, the second twin is much more likely to be affected if the twins are identical (essentially 100 percent genetically identical) than if they are fraternal (25 percent genetically identical). Autism also is associated with several rare genetic diseases, further suggesting genetic mechanisms.

These studies raised hopes that by understanding the genetic cause of autism, it might be possible to treat the disease. But finding specific genes has proven to be much more complicated. Autism is defined by a set of distinct behavioral symptoms that can be extremely variable, that can be triggered by multiple genetic changes and environmental factors, and that may even represent more than one disease.

“You can have a kid who has an IQ of 60 and has seizures and a kid who has an IQ of 140 who is at MIT and they both have autism,” saysCharles Nelson, PhD, Research Director of the Developmental Medicine Center at Boston Children's Hospital. “You've got to deal with that somehow.”

The hunt for genes begins

In the early 1960s, when karyotyping became available, researchers inspected the chromosomes of people with autism, seeking deleted or duplicated regions that might contain the critical genes. They largely came up empty-handed.

Some experts, however, suspected that deletions and duplications were there, but were simply too small to be detected by this technique. With the introduction of chromosomal microarray analysis in 2005, however, researchers could detect deleted or duplicated regions at roughly 100-fold greater resolution.

Using chromosomal microarray analysis, the Genetics Diagnostic Laboratory of Boston Children's Hospital, led by director Bai-Lin Wu, PhD, noticed that several patients with a diagnosis of autism spectrum disorders had missing or extra sections of chromosomes 15 and 16. Deletions or duplications of these chromosome regions--15q on chromosome 15 and 16p on chromosome 16--occur in about 1 percent of autistic children.

These regions are now under active investigation to zero in on possible causative genes within them.
“I don't think we're going to find one genetic cause that explains 50 percent of autism,” says David T. Miller, MD, PhD, of the Division of Genetics and the Department of Laboratory Medicine, a coauthor on these studies. “It's going to be an incremental process. Even if it's 1 percent at a time, that's still progress.”

The most comprehensive clinical study to date, a collaborative effort between the Genetic Diagnostic Laboratory at Children's and the Boston-based Autism Consortium, detected potentially significant deletions and duplications in 18 percent of the 933 patients tested, with about 7 percent being clearly associated with the disease. This represents a big improvement over traditional karyotyping.

“A lot of genetic studies found chromosomal deletions and duplications mainly for technical reasons,” says Christopher Walsh, MD, PhD, chief of the Division of Genetics. “With the new technology we now have, they are easy to see. Point mutations [changes in a single base pair or ‘letter’ of the gene sequence] are probably going to be more common than deletions or duplications, but they are much more difficult to find.”

Looking to the Middle East

The big problem is that most of the genetic changes associated with autism are rare. Most of them also occur spontaneously, rather than being inherited, and they are often unique. Only by testing thousands and thousands of people can even deletions be tied to the disease.

To find these rare genetic changes, Walsh's team has been using a classic genetics approach: homozygosity mapping. The idea: Take a look at extremely large families that have a closely shared common ancestry (often because of marriage between cousins) that “enriches” them for rare susceptibility genes. Then, compare their chromosomes side by side to identify small fragments of the genome that are shared between family members with the disease. These areas can then be further explored with DNA sequencing, to find specific point mutations, or simply by chromosomal microarray analysis to find deletions or duplications associated with the disease.

沃尔什说：“当您有一个足够大的家庭，有三到四个受影响的人时，您可以在基因组中确定一个或两个基因所必须存在的地方。”

Walsh's team recruited nearly 100 families with autism in parts of the world where marriage between cousins is common, such as the Middle East. Coupled to chromosomal microarray analysis, the team zeroed in on six likely susceptibility genes in five out of the first 78 families studied. All the genes are close to or located within the chromosome deletions. Many of them appear to be involved in wiring up circuits in the brain.

“The new genes confirm the theory that a lot of autism seems to have to do with connections and with the changes in these connections that enable learning,” says Walsh.

现在，沃尔什（Walsh）团队正在测序其他家庭中与自闭症相关的染色体区域，以期识别额外的敏感性基因和点突变。将来，沃尔什（Walsh）希望以共同的族裔血统将这种类型的分析扩展到美国的家庭，以找到可能有助于该疾病的其他基因。

Making more connections

有超过100个基因与孤独症和计数less possible combinations of them, it is increasingly clear that identifying the key missing or miswired brain connections in autistic children will be difficult indeed. And add to that the growing consensus that autism isn't one disease, but many, each involving different sets of genes. That's where computational power can come to bear, to crunch the genetic data and look for patterns.

“Autism is a multifaceted, polygenic disease,” says Dennis Wall, PhD, director of the Computational Biology Initiative at Harvard Medical School and a researcher with the Children's Hospital Informatics Program (CHIP). “In order to disentangle that forest, we feel it is extremely important to implement network biology approaches to pull together connections we already know about and use them to develop a more comprehensive picture of the disease.”

沃尔正在使用他所谓的交叉疾病方法，查看与自闭症共享症状的其他行为障碍，例如共济失调（肌肉协调丧失）癫痫（癫痫发作）（癫痫发作）和严重的抑郁症，从而可能共享与风险相关的基因。

沃尔说：“在许多其他疾病中，遗传原因得到了更好的理解。”“我们可以将有关这些疾病的信息用作发射台，以预测自闭症中的新基因。”

沃尔和他的团队确定了13种疾​​病，共有127个先前确定的自闭症相关基因中的66种。通过包括与这66个基因相互作用或紧密共同表达的其他基因，它们将列表扩展到了334个可能的敏感性基因的网络。

但是，这334个基因实际上与自闭症有关吗？沃尔和他的团队查看了加州大学戴维斯分校M.I.N.D.标识的基因列表。在自闭症患者的血液中失调的研究所。他们发现，这些基因中的289个（87％）在自闭症患者的基因表达上显示出显着差异，这表明它们确实是有望与风险相关的基因。

Now, in collaboration with Children's Director of Informatics Isaac Kohane, MD, PhD, and Director of Genomics Louis Kunkel, PhD, Wall and his team are refining their predictions of autism susceptibility genes. By further fine-tuning the underlying networks of genes disrupted in autism, Wall hopes to better understand the variability of the disease, and ultimately identify key genes to target in the disease.

A promising future

With a growing list of genes identified, autism experts are beginning to define sub-types of the disease. By coupling the power of bioinformatics with next-generation microarray and gene-sequencing technologies, new therapeutic targets may eventually emerge in this puzzling disease.

沃尔什说：“与其看起来像一个大灰色区域一样，我们开始看到它的子合成剂在更尖锐的救济中。”“我们越了解这些条件，就越能定制每个患有该疾病的孩子的治疗。”

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