What Causes Genetic Copy Number Variations?
                                                                                       A discussion with Lucas Argueso, PhD

"It is known that certain environmental exposures can accelerate the pace of mutation, and this is my main interest, discovering and characterizing agents ... that may speed up ... mutagenesis."

Links:

Homepage at Colorado State University

Commentary in Environmental and Molecular Mutagenesis: Contrasting mechanisms of de novo copy number mutagenesis suggest the existence of different classes of environmental copy number mutagens

"We now know that autism is strongly associated with mutations that are new to a family, also referred to as de novo mutations."

"Some CNVs have a peculiar feature in that nearly identical mutations can form independently in completely unrelated individuals."

"Those CNVs are then classified as recurrent.... There may be mutagens out there that trigger recurrent CNVs."

"Mutagenic forces are present in nature, however, excessive exposure to specific mutagenic agents (sunlight, smoke, etc.) does accelerate the pace of mutation."

"About 8% of autism cases are associated with a de novo CNV, not present in the parents. Most of the de novo CNVs associated with autism are classified as recurrent."

"I am not aware of anyone else who like us, is specifically looking for environmental mutagens that can increase recurrent CNVs, the main class of CNVs associated with autism."

Lucas Argueso, PhD, is Assistant Professor and Boettcher Investigator, Department of Environmental & Radiological Health Sciences, at Colorado State University. Dr. Argueso grew up in Brazil, and has a Ph.D. in Genetics and Development from Cornell University. His research interests include chromosomal rearrangements and phenotypic consequences of altered genome architecture; the effects of environmental exposure on copy number variation (CNV) and chromosome structure; and molecular mechanisms of DNA double-strand break repair.

Cognizant of the literature demonstrating that novel copy number variations are playing a role in the etiology of a subset of autism cases, Dr. Argueso has been asking about the mechanisms and exposures that could possibly lie at the root of these genomic disruptions.

Interviewed by Jill Escher, December 2015

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Tell me about your background, Dr. Argueso: how did you come to study mutagenesis? And how did your thinking drift to autism?

I have been interested in all mechanisms of genetic variation since very early in my career. I researched DNA base mutations as a graduate student and much larger chromosome structure mutations as a postdoc. Both types of mutations appear naturally at a basal rate in each new generation, in people and in all organisms—it is just part of being alive.

However, it is known that certain environmental exposures can accelerate the pace of mutation, and this is my main interest, discovering and characterizing agents present in the environment that may speed up the mutagenesis process.

We now know that autism is strongly associated with mutations that are new to a family, also referred to as de novo mutations. This means that the parents do not have the mutation, but their affected child is the first one in the family to have it. The first de novo mutations discovered in autism were changes in chromosome structure that lead to gene copy number variation, or CNVs. This happened around the mid 2000’s, precisely when I was studying the fundamental biological mechanisms controlling this class of mutation in a yeast model cell system. The discovery of the important role of de novo CNVs in autism made me redirect my research to the question of whether environmental exposures could somehow cause CNVs, particularly those found in autistic children.

What are the different types of mutations seen in the germline?

Mutations are generally arranged in two major classes: Variations in isolated bases in the DNA sequence, or Single Nucleotide Polymorphims (SNPs); and structural variations (SVs) in chromosome structure. SNPs are very small, local changes in chromosomes, but they can be sufficient to alter or eliminate the function of a gene, thus conferring an altered trait and in some cases genetic disease. SVs are large changes in the structure of chromosomes and can alter the function of the genes in that region, sometimes many genes are affected by a single SV.

CNVs are one specific type of SV in which a large segment of a chromosome is either deleted or duplicated. When this happens the overall dosage of the genes in that region of the chromosome falls out of balance relative to the two copies that each gene typically is found in the general population. An individual carrying additional or fewer copies of a certain gene may have traits or disease consequences caused by this dosage imbalance.

What forms do CNVs take?

There are two major classes of CNVs: recurrent and non-recurrent CNVs. Mutations, both SNPs and SVs, are formed by accident and are typically randomly distributed in the genome. For this reason, most de novo mutations tend to be unique to the individual where they first appear and are non-recurrent.

Some CNVs however, have a peculiar feature in that nearly identical mutations can form independently in completely unrelated individuals. An autistic child from Germany and another autistic child from the US may be born with almost exactly the same autism-causing CNV, even though their parents and no one else in their families has that CNV. Those CNVs are then classified as recurrent because they can appear identically in independent families.

We and others believe that the differences in the two CNV classes suggest that the mechanisms of mutagenesis must also be quite different, and therefore there may be mutagens out there that trigger recurrent CNVs but do not trigger non-recurrent CNVs, and vice versa.

Are CNV’s natural, or are they induced by mutagenic forces, or both?

Both. CNVs and all other types of mutations occur naturally because of mistakes that occur during the replication of the genome, or exposure to natural products that can damage the DNA. A low rate of new mutations in each generation is actually essential for evolution, and is the reason why organisms are able to successfully adapt to new environments. Mutagenic forces are present in nature, however, excessive exposure to specific mutagenic agents (sunlight, smoke, etc.) does accelerate the pace of mutation and can lead to negative effects, including cancer and genetic diseases in the subsequent generations.

Are there specific human phenotypes associated with CNVs?

Yes, many, and we’ve only started discovering them. There are many well characterized human genomic disorders such as a form of Charcot-Marie-Tooth, Smith-Magenis, Potocki-Lupski, and CNVs associated with autism, such as duplications or deletions of the 16p11.2 region of human genome. Each of these and others have very distinct features, and each person carrying the CNVs may have different manifestations of the disorders.

However, CNVs are also associated with many human features that are not linked to disease. They simply help make healthy people and human populations different from each other. One of the most interesting examples is the variation in copy number of the gene that encodes the enzyme amylase, needed to digest starch. It turns out that populations from Asia where rice is a staple of the diet evolved many copies of this gene, while certain populations from Africa that have meat-rich diets have a lesser need for this enzyme and also have fewer copies of the same gene.

What percentage of autism cases appear to be associated with germline CNVs? Are these de novo mutations or inherited from the parents and earlier ancestors?

One recent comprehensive study estimated, taking into account many different studies, from different populations, that about 8% of autism cases are associated with a de novo CNV, not present in the parents. Most of the de novo CNVs associated with autism are classified as recurrent. It is also the case that some CNVs associated with autism are already present in one of the parents, but do not cause significant symptoms that would lead to a diagnosis. An interesting theory has been developed in recent years that proposes that in some cases the combined effect of two CNVs is needed to trigger stronger autism symptoms: one CNV that had already been present in the family, and another is a de novo CNV that is new to the affected child.

How predictive are CNVs of autism or other disorders?

It depends on the region of genome where the CNV is detected. CNVs in some regions have very strong effects and are highly predictive of a disease outcome, regardless of the composition of the rest of the genome, or genetic background.

In other cases the effect is weaker and not necessarily deterministic. Some of these CNVs can be asymptomatic in some individuals, or cause weak symptoms that do not reach the level that would prompt a firm diagnosis. In other individuals with a different genetic background the same CNV may have a stronger effect that does cause clear diagnosable symptoms.

Could human sperm be tested for CNV’s?

Yes, but not without destroying it in the process. It is possible to extract DNA from a single sperm cell and use it for genetic tests, but then that sperm cell is no longer usable.

Interestingly, in the case of female reproductive cells, it is now possible to infer whether CNVs are present or not, using methods that preserve the viability of the egg cell for later in vitro fertilization and implantation. This procedure has actually been recently used and healthy, mutation free babies were born after the mother’s eggs were harvested, screened for the absence of CNVs and other chromosome abnormalities, then fertilized with the father’s sperm, implanted, and carried to full term gestation.

My ASD kids had microarrays performed but no CNVs were detected. And no other frank mutations. What else in their genomes should we be looking for?

There are a number of plausible possibilities. There could be epigenetic changes that do not change the sequence or structure of chromosomes, but that change the function of genes important to ASD. There also could be specific existing common variants already present in the parents that when combined, lead to ASD.

There could also be rare CNVs or base mutations that are not picked up by conventional clinical microarray tests, but that might be revealed by whole genome analyses. One of the challenges with many clinically available tests is that they are designed find the most frequent and best understood sources of genetic disorders. In the case of ASD, where multiple genetic causes exist and many of which are rare or even unique to a child, it is extremely difficult to design tests that can pick up every possibility. Also, epigenetic alterations and combinations of common variants are not routinely analyzed, leaving a number of possibilities not investigated.

Finally, there is also the possibility that no genetic or epigenetic mutation is present, and the symptoms may be derived from an exposure during embryonic development or early childhood that somehow perturbed the normal development without necessarily affecting the genome. Likely all of those scenarios may play larger or smaller roles in each case.

Are other researchers looking into the sources of de novo mutations seen in autism, or are you pretty much alone in this quest?

There are many colleagues looking at sources of de novo DNA base mutations in humans, leading to autism and all kinds of other genetic disorders. There are some, but fewer, of us looking at sources of de novo CNVs. Hopefully we are not alone, but within this smaller group, I am not aware of anyone else who like us, is specifically looking for environmental mutagens that can increase recurrent CNVs, the main class of CNVs associated with autism.

What are we learning about the mechanisms behind CNVs? Or any particular exposures that might precipate them?

It is still very early in this research. We do know that a particular cellular pathway, non-allelic homologous recombination, is how recurrent CNVs are formed. We are currently developing lab tests designed to identify mutagenic exposures that activate this pathway. We are planning to use these tests in a broad search of chemical compounds to find such recurrent CNV mutagens, and hopefully we will find some candidates in the next couple of years.

How can studying yeast as a model organism lead to discoveries about autism?

It turns out that model organisms like yeast, fruit flies, worms, or mice, are extremely useful tools to accelerate discoveries in problems directly relevant to human health. Many of the basic processes that govern the workings of a human cell are highly conserved in other organisms. Relatively simple model organisms like yeast are much more amenable to basic lab studies, which in turn illuminate paths for investigation in much more complicated human cells.

A great example of this is how the pathways that control the cell division cycle were first discovered and understood in the yeast model. Years later researchers learned that when the same cell cycle control pathways are perturbed in human cells, those cells give rise to cancerous tumors. It was research done in yeast that cleared the way to figure out some of the most important mechanisms of cancer formation in humans, and this work ended up earning yeast researchers the Nobel Prize for Medicine.

At the fundamental cellular level the mechanisms of CNV formation in yeast are very similar to the mechanisms of CNV formation in humans, so the yeast model offers a great way to help us learn the basics more quickly, and then translate those discoveries to humans, for example, to identify mutagens that can cause CNVs in yeast, and then protect humans from getting exposed to those compounds, therefore preventing CNVs that can cause autism.

This is fascinating and important information, Dr. Argueso. It's wonderful to see a mutagenesis researcher starting to connect the dots between the molecular histories of germ cells and pathogenesis in offspring. I am truly grateful for your work and inquiring mind.

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