Fishing Expeditions and Autism: A Big Catch for Genetic Research?

[Guest guest]

http://www.psychiatrictimes.com/display/article/10168/1364835?pageNumber=2

January 1, 2009

Psychiatric Times. Vol. 26 No. 1

Molecules of the Mind

Fishing Expeditions and Autism: A Big Catch for Genetic Research?

J. Medina, PhD

Dr Medina is a developmental molecular biologist and private consultant, with research interests in the genetics of psychiatric disorders.

I am a fan of the television show Deadliest Catch—a documentary series that follows the travails of deep-sea fishermen in the Bering Sea. (Actually, it is mostly about deep crab fishing.) Living in Seattle, I have actually seen some of the boats filmed on the show.

The variety of equipment the fishermen use to capture sea life is extraordinary. Trawlers and purse seiners—boats that use long-line nets and gill nets—make it possible to catch thousands of fish at a time. I am constantly struck by the comparison between these large, industrial efforts and the "weekend" fishermen that Seattle also has by the thousands. The amateurs use simple fishing poles to catch one fish at a time. Where the Deadliest Catch boats are based, you can often see both styles side by side. I mention these 2 contrasting styles of fish harvesting because there is a comparison that I would like to make in this month's column and in the next.

It is not much of a stretch to say that isolating the genes responsible for complex behavioral disorders can seem like fishing expeditions (complete with analogous net comparisons). There are giant efforts that deploy the molecular equivalent of purse seiners designed to snag large groups of genes that share a potential involvement in whichever presenting behavior is under study. These efforts can be contrasted with technologies that use the equivalent of small fishing poles, the goal of which is not to catch large, glittering groups of nucleotides but single genes, one at a time.

In this column and the next, we will tackle one of the most slippery issues in the behavioral sciences: the genetic basis of autism. We will closely examine 2 sets of genetic "fishing" techniques that each attempt to isolate sequences associated with the disorder. This month's column will describe the success of the genetic equivalent of Deadliest Catch nets—large genetic screens that are capable of isolating many genes at one time. Next month, I will focus on research that is more reminiscent of our weekend fishermen with fishing pole–like techniques that can isolate single sequences.

My description of one of these larger fishing techniques for genes will begin with some comments on the diagnostic categories of autism. I will show just how hard it is to come up with behavioral profiles that are sufficiently robust to withstand the cold mathematical scrutiny of the behavioral genetics laboratory. I will then briefly describe some of the details of a technique called homozygous mapping and some of the surprising recent success using the technique with Eurasian and Middle Eastern families.

Diagnostic difficulties

One of the biggest difficulties in characterizing autism at the molecular level is its complexity: autism is impossible to characterize in monolithic, overarching diagnostic terms. Symptoms can include social deficits, communication problems, and obsessive-compulsive and repetitive behaviors. Many patients with autism cannot detect changes in the affective state of another person or predict a person's interior motivational states based on specific visual cues (canonical Theory of Mind tests). And many of these behavioral symptoms are accompanied by GI complaints, seizures, epilepsy, and sleep disorders. Do these variations in symptoms describe specific disease states, each with their own unique genetic etiologies?

We do not currently know. Autism is usually classified as a severe form of 1 of the 5 so-called pervasive developmental disorders (PDDs). Children who display milder symptoms may have an autism spectrum dis­order (ASD). Asperger syndrome is often separated from classic autism because there is usually no delay in language development. These terms are frequently used interchangeably, unfortunately, which reflects the fluid nature of the diagnostic observations. One of my favorite categories decries a form of diagnostic surrender: PDD, not otherwise specified.

(Just so you know, terms like these drive behavioral geneticists nuts!)

But there are genes

All this has not stopped us from researching the phenomenon, of course. And the results after years of looking are clear: there is a tantalizing and substantial genetic component to the disorder regardless of how it is classified.

Initial studies that confirmed a genetic role came from the traditional family and twin heritability studies, some of which are now decades old. Some of the best recent work comes from assessing sibling recurrence risk. Usually described as a percentage, sibling recurrence risk is the formal probability that a younger sibling of a child with autism will also have the disorder. When autism is defined narrowly, the normal rate in unrelated populations is about 1 affected child per 500 (0.002%). When you look at sibling recurrence risk, the rate rises to about 1 in 6 (15%). Thus, there is ample reason to pursue genetic research in this area.

But that is where the easy stuff ends. Although many genes over the years have been nominated as the source of the behavioral anomalies, few studies have been successfully replicated. Specific chromosomal inversions, large deletions, chromo- somal translocations, and changes in copy number of individual genes (Figure) have been observed as risk factors for autism. In fact, you can practically name any type of mutation and find that it has been associated in the past decade, at least to some extent, with some part of the autism spectrum.

It is now clear that multiple genes expressed in specific combinations are involved differently in creating specific autistic behavioral profiles. It is also clear that wide nucleotide variations within these candidate genes exist that are undoubtedly more capable of predicting discrete autistic behaviors than others.

Looking at first cousins

Given the large number of potential genes in autism, one might expect that the research method of choice would include the deployment of large, Deadliest Catch–like gene fishing protocols. Recent progress has indeed been made using one of these larger screening technologies, a technique called homozygous mapping. What follows is a brief description of the technique.

Human genetic disorders that have complex, multigene origins have 2 overall causes. The first arises from the random roll of the meiotic dice—presenting cases that show no pattern of previous inheritance. In rare cases, heritable forms of what appears to be the same disease also exist. Mutations in these patients clearly show a pattern that can be transferred from one generation to the next. Homozygous mapping is capable of identifying these rare, heritable (invariably recessive) disease forms. The technology takes advantage of the presence of consanguineous families . . . which should probably be explained before we go further.

For decades, molecular biologists have known about the great power of studying persons whose parental lineages share a close, common ancestor. The probability of their offspring exhibiting an autosomal recessive trait is much greater than in the general population. (Recall that autosomal recessive conditions are traits that are expressed when the subject has 2 identical copies of a particular gene in a nonsex chromosomal background.) Homozygous mapping employs such populations and can be divided into 2 steps:

1. Subjects who carry a specific, well-defined disorder are identified. Accomplishing this first step, which requires the researchers to decide on a specific set of diagnostic criteria, is one of the hardest parts of the entire procedure.

2. Once identified, subjects are screened for nucleotide sequences that they share in common and are homozygous for both chromosomes. The assumption is that these regions are donated from both paternal and maternal lineages who themselves shared a recent common ancestor. That is a reasonable supposition if you are studying closely related persons, such as first cousins.

Although admittedly a tough technique to execute properly, homozygous mapping has proved to be successful in isolating gene sequences that mediate rare diseases related to neural development. Until very recently, however, it had not been tried on such complex challenges as autism. When it was, it proved to be invaluable in the autism-screening procedures.

The data

Researchers first had to find consanguineous families with autistic children. They established a collaborative network called the Homozygosity Mapping Collaborative for Autism (HMCA) in the Middle East and throughout Eurasia.1 The reason for this geographic localization has to do with statistical access. It is quite common in the Middle East for cousins to marry each other. Since the families tend to be large, the researchers reasoned they would most likely find persons that met both their genetic and behavioral criteria. They hit pay dirt. The researchers were able to find 88 consanguineous families with autistic children.

The investigators next scanned the genomes of all participants at high resolution. They were looking for a wide variety of chromosomal aberrations, such as inversions, deletions, duplications, and something called copy-number variations.

After exhaustive screening, the researchers found that 6 chromosomal regions in the HMCA sample had inherited, homozygous deletions. These deletions varied in size from a low of 18 kilobases to more than 880 kilobases.

Exactly what genes were on these important chromosomal regions, and how might their characterization increase our understanding of autism? To discover what happened next, we need to switch fishing protocols. We are going to tie up our large fishing trawlers, which is what homozygous mapping is, and inspect the catch. Once inspected, the next steps will then involve breaking out our much smaller fishing poles, putting some bait on the end, and casting our lines back into the genomic waters.

As you see in the Figure, a large number of genes were netted in this experiment. I will describe exactly what was in the catch and how this increases our understanding of autism next month.

http://www.psychiatrictimes.com/display/article/10168/1387605

March 11, 2009 Psychiatric Times. Vol. 26 No. 3

Molecules of the Mind

Fishing for Genetic Links in Autism

J. Medina, PhD

Dr Medina is a developmental molecular biologist and private consultant, with research interests in the genetics of psychiatric disorders.

This is the second installment in a 2-part series that addresses approaches to understanding the molecular underpinnings of autism.

In my January column ("Fishing Expeditions and Autism: A Big Catch for Genetic Research?" Psychiatric Times, January 2009, page 12), I described the great difficulties research­ers face characterizing the genetic basis of the disease. Complexities range from trying to establish a stable diagnostic profile to making sense of the few isolated mutations that show clear associations (either with disease or syndrome variants).

Using the metaphor of a fishing net, I discussed 2 overall research strategies that geneticists commonly use to catch these elusive sequences of interest. One strategy is to cast nets that act like large purse seiners to collect many sequences in a single (and usually quite expensive) effort. The other strategy is akin to dropping a single fishing line into the genetic waters to see if anything "bites." In Part 1, I described one particularly successful strategy that snagged a large number of useful sequences. Here, the focus narrows: I will not describe the isolation of many sequences, but rather only one. Our "catch" is called MeCP2, a gene whose mutations can give rise to a wide spectrum of related postnatal neurodevelopmental disorders—including autism spectrum disorders.

I will start with some background regions about gene regulation, move to the biological functions of MeCP2, and then focus on studies in animal models that provide tantalizing hints about the origins of autistic behavior. My goal is to show that research prog­­ress in autism is a continuum of efforts, ranging from large projects with lots of identifiable sequences to small projects that focus on the properties of single genes.

Gene typologies and their regulation

There is a lot of heavy-duty molecular biology behind MeCP2. Getting the clearest view requires us to review 4 pieces of background information. Feel free to skip to the section "MeCP2 and Rett syndrome" if Class II genes and CpG islands are working parts of your vocabulary.

Gene classes. As you recall from your undergraduate days, genes are broken down into 3 classes. Class I genes encode the information necessary to make ribosomal RNAs. Class II genes encode the information to make mitochondrial RNA, and these genes are in the distinct minority (only about 2% of activatable sequences). Class III genes encode transfer RNAs.

Class II genes can be broken down into 2 functional parts. The first part includes the nucleotides that are necessary to encode the protein, which are called the "structural sequences." The second part, which often lies in front of the gene, is called the "promoter." Promoters act like tiny on-off switches that either allow or block the manufacture of the cognate message.

And how is that message made? The enzyme complex that creates the message is called "RNA polymerase II." Not all genes are transcriptionally active at the same time, and some never become activated at all (eg, neurons do not do the same job as, say, skeletal muscles, and have very different activation profiles). Understanding how the RNA polymerase II complex knows which gene to turn on in a complex cellular environment has been a focus of intense investigation for decades.

There are many mechanisms to help this complex decide which gene to turn on. RNA polymerase II enzyme can be shown which genes it needs to turn on and which are supposed to be left alone. There are escort-like proteins that physically bind to the RNA polymerase II and guide the complex to its proper genetic destination. Another mechanism involves proteins that bind to the gene to be transcribed rather than to the RNA polymerase II. These proteins then act like homing beacons, guiding a wandering RNA polymerase II to its proper nucleotide destination. There are also repressor proteins that work in a similar but opposite fashion. They render a gene that could be activated into a repressed, transcriptionally inert state.

Histone protein complexes. Histones are groups of proteins around which DNA wraps, like twine around a ball. There are many of these wound-up balls along a chromosome that function like physical barriers. If the DNA that harbors a class II gene is wrapped around the histone complex, RNA polymerase II can have a very difficult time binding to it, and the gene is rendered silent. If the histone complex is bulldozed out of the way, the class II gene becomes available for activation.

Chromatin is a com­bination of DNA and histones: this mass of molecules can form surprisingly complex, higher-ordered structures. The structures are so specific that antibodies capable of binding to them can be created. The structures can then be individually isolat­ed intact through a protocol called "chro­matin immunoprecipitation."

Methyl groups and methylation reactions. Methylation reactions involve adding a methyl group (CH3) to certain nucleotides in the double helix. If the methylation occurs on or near a class II gene, it can be rendered inactive.

How does this work? The methyl groups studded along the length of a gene often attract repressor proteins that perform the actual silencing function. Such repressor proteins are attracted to a given segment of DNA via sequences on the DNA called "CpGs" (sometimes referred to as CpG islands), which are short regions of DNA enriched for cytosine and guanine nucleotides (the "p" refers to the phosphodiester bond between cytosine and guanine). CpG islands often cluster at the promoter sites within the class II gene. CREB. The last piece of background information is the biology of the CREB protein (which stands for the tongue-twisting name "cyclic AMP response element binding"). The characterization of CREB function is one of the great research achievements in all of molecular neurobiology. The reason? CREB is involved in activating the genes that take part in learning, and it does so in virtually every animal ever tested (including humans). It specifically activates gene sequences involved in establishing memory formation by binding to their promoter regions and transcriptionally activating the gene. CREB is a classic activator. Understanding the genes to which CREB normally binds has resulted in the isolation of many sequences in­volved in human learning. Understanding CREB bi­ology remains a subject of intense interest and plays a powerful role in our story.

MeCP2 and Rett syndrome

With this admittedly lengthy background information, we can return to the biology of the MeCP2 protein and its role in autistic spectrum disorders. The MeCP2 gene was first characterized by researchers who were interested in Rett syndrome. An X-linked dominant disease, Rett syn­- drome affects 1 in 10,000 persons and is found mainly in females. Symptoms usually present within the first 6 to 18 months of life and include motor and speech difficulties, sei­zures, increasing cognitive impairment, and growth retardation. About half of those affected eventually become non­ambulatory and many have chronic GI disorders.

Several types of mutations have been characterized, from deletions and insertions to subtle point mutations (changes in single base pairs). Most germane to our story, MeCP2 mutations were eventually associated with certain autism spectrum disorders. While this association hardly represents the overarching genetic explanation for even a single autistic category, the finding was important. The function of MeCP2 is well known, and has been established in animal models that carry MeCP2 mutations. Having such a well-characterized ally could be useful in the understanding of autism.

MeCP2 is expressed in all the body's cells, but some of its highest concentration of activities is in the CNS. The hypothalamus, at least in laboratory animals, is particularly robust. The MeCP2 binds to DNA that has been previously methylated. Indeed, MeCP2 literally means "methyl CpG binding protein 2." This binding functions as a gene-silencing mechanism and, until recently, MeCP2 was considered to be a canonical repressor protein. As we shall see, this job description turned out to be overly simplistic