Girirajan Lab Seeks To Bridge The Gap Between Fly And Human Genomes
Six hours and 15 minutes: That’s roughly how long a flight from Seattle to State College is. For Dr. Santhosh Girirajan, that was not only a long flight, but also a career-changing one.
It was on this flight that Girirajan read a book that a colleague urged him to read called Time, Love, Memory by Jonathan Weiner. Girirajan was on his way to Penn State to research mice after completing his postdoctoral training at the University of Washington. This book, however, convinced him of the importance of fruit flies as it explored how famous scientist Seymour Benzer founded his career in molecular biology — highlighting Drosophila as an important model organism.
In 2012, when Girirajan arrived in State College following that flight, he had decided that he would research fruit flies in his lab at Penn State after being inspired by Benzer’s journey.
“[It’s] something that I had not really worked with in the past, and this was something which was very, very new for me. [But] I was thinking maybe if I could use a very conserved and basic model system to really test for cellular and neurological phenotypes then that would be fruit flies,” Girirajan said.
The Girirajan Laboratory studies how changes in the genome contribute to common neurodevelopmental disorders such as autism and intellectual disability. The lab uses both Drosophila (fruit flies) and human cell lines to understand the risks caused by genetic mutations and to grasp how gene disruption leads to altered neurodevelopment.
Girirajan and his team are looking into the effects of an autism-associated 16p11.2 deletion in the genome. A recently published study by the lab, titled “Pervasive epistasis modulates neurodevelopmental defects of the autism-associated 16p11.2 deletion,” investigates the interactions between the genes within this specific region of the genome.
The original goal of this study was to identify which of the genes in the 16p11.2 region, a particular area in the genome that has been linked to autism in previous studies, were important in causing these neurodevelopmental problems. Although the team found that all of the genes showed some level of effect when they were disrupted, together their effect was even greater. Matthew Jensen, a graduate student in the lab, detailed the discovery.
“We saw that many different combinations of genes actually cause a less severe phenotype than each individual gene independently on its own. So that’s a type of interaction we call epistatic interaction, when the effects of two genes are not equal to the effects of each gene on its own added together,” Jensen said. “So that explained the deletion a bit more and that also explained why on a human end we see a lot of what we call phenotypic heterogeneity, so meaning that people with the same deletion have very different clinical features.”
To maximize efficiency and maintain relevance to the human species, the Girirajan Laboratory uses Drosophilia. Fruit flies allow the lab to have high throughput because their shorter life cycles allow for quicker results. Additionally, the flies share about 60 percent of their genome with that of humans, which provides the opportunity for serious comparison with human abnormalities.
Dhruba Mayanglambam, the lab’s former postdoctoral scholar, added that Drosophila is a very versatile model organism.
“Another important thing we can do with Drosophila is that we can express or down-regulate the gene of interest in the various tissues. So you can do it in a tissue-specific manner,” Mayanglambam said. “We mostly did our study using the Drosophila eye and nervous system and one important thing about using Drosophila eye is that the phenotype can be easily identified. You can just look at the microscope and can see the phenotype.”
The flies are observed through a plethora of methods, but one particularly useful method is called Flynotyper.
The lab developed the Flynotyper technology to help detect the changes and disorder among the “ommatidial cells” of fly eyes. These ommatidial cells are simply hexagonally-organized structures within the fly eye. The Flynotyper tool allowed the research team to quantify changes to these organized eye structure phenotypes with a “Flynotyper score.”
To understand the effect of the interaction between different genes in the 16p11.2 region of the genome, researchers used one- and two-hit knockdowns, or gene silencers.
For a one-hit knockdown, a single gene would be silenced, while for a two-hit knockdown multiple genes would be silenced together. The two-hit knockdowns were used to compare back to the one-hit knockdowns to see if the increased number of changes in the genome caused for more severe phenotypes. This is known as epistasis.
With a preliminary study like this one, the researchers knew they needed to be clear about how the fly research could be applied to humans.
Cell proliferation is one trait that is examined in the eyes of the flies because it’s linked in several studies to autism defects. The lab isn’t trying to prove that flies with cellular proliferation have autism. Rather, it claims that the cellular defects observed in the flies could be seen in humans too.
A related, more recent study from one of the lab’s graduate students, Lucilla Pizzo, explores gene interactions with the same 16p11.2 deletion on a human level. Pizzo says that the laboratory thinks of the 16p11.2 deletion as a “proof of principle” that certain genetic alterations can cause multiple phenotypes.
“Probands — or the affected [children] — have a higher burden, or a higher number of mutations, affecting genes that are considered to be intolerant to mutations compared to their parents, and that is probably what is modifying the clinical manifestations of 16p11.2 deletion,” Pizzo said. “So basically if a child has the 16p11.2 deletion and has a higher burden of these other rare variants [the secondary mutations], it is more likely to have cognitive deficits [in contrast to having just the 16p11.2 deletion].”
Pizzo said her research aims to demonstrate that when it comes to clinical diagnoses, there is often much more than meets the eye.
“Even when we identify a genetic mutation that we believe is diagnostic, right now, with the power of technology that we have we should take advantage of that and look further into the genetic background to try and interpret how other rare variants could be modulating the phenotype [to give a proper diagnosis],” Pizzo said.
Jensen agrees with Pizzo, as he believes that the lab’s fly study aptly demonstrates the complexity of the human genome as well.
“You can never really just look at one single gene and say this is the causative gene for all the patient symptoms. You have to look at all the genes in the region and on a human end, all of the variants in the genetic background together, and how they influence the various complex disease phenotypes we see,” Jensen said. “It’s not definitive, but I think it’s exciting because I think people haven’t been asking these questions before.”
Girirajan, a former physician, believes his team is doing its best to help bridge the gap between the human and fly genomes, in order to eventually apply the findings in a clinical setting.
“There is no real convergence between human and fly work. However, we are trying to see if we could fill the gap in between human and fly work by adding more validations and adding more sophisticated systems that will actually bring back the relevance to humans…” Girirajan said. “Ultimately the idea is to identify pathways, groups of genes, and their networks, and how we can identify potential therapeutic targets that we can use to treat children with these neurodevelopmental disorders.”
Jensen has a genuine interest in the neurodevelopmental topics he studies, and also understands why the laboratory research he does is so important.
“What’s actually really interesting about things like autism and schizophrenia is that there’s still a lot of uncertainty out there. Definitions are changing for the diseases; some researchers will think that autism and intellectual disability are the same disease, kind of like two sides of the same coin. And the biological mechanisms haven’t yet been established. So in some regards we are way behind cancer,” Jensen said. “At first that kind of scared me a bit, but now it’s really interesting to look at the complexity and to try to actually figure out [the science behind it].”
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