The Upside of Genetic Mutations

Last week I attended a scientific meeting held in Stresa, Italy and organized by a parent group, Pro Rett Ricerca. One of the most well received talks of the speaking program was presented by Monica Justice, PhD of Baylor College of Medicine, who discussed data collected from her RSRT-funded project. What follows is an excerpt from a recent conversation between the two Monicas following the Italian meeting.

"My hope is that we find a molecule(s) that we can manipulate to reverse the symptoms in these girls. Reverse is a strong word but given the extraordinary work of Adrian Bird, it's what I'm striving for."
—Monica Justice, PhD

MC: Dr. Justice, it was great to see you in Italy. I thought it might be helpful to give our readers some insight into your project. But first I’d like to start with you, the soul behind this impressive undertaking. How did you end up in science?

MJ: My grandfather was a vet and I had an uncle who was a physician. I have always had a deep love for animals so as a child and young adult I wanted to be a vet. My father thought that was not an appropriate career for a woman and he and my uncle encouraged me towards the medical field. Early on, however, I realized that my true passion was in basic research. I went to graduate school thinking I would focus on immunology and microbiology but my very first class would change my career path forever. My professor was switching into the mouse genetics field and invited me to join his lab. I loved mouse genetics from the very start and knew immediately this was exactly where I wanted to be.

MC: Most lay readers of this blog will not have realized that mouse genetics as a field exists. Can you elaborate on this specialty?

MJ: When I entered the field most mouse genetics was being carried out in a few labs, The Jackson Laboratories being the primary one in the US, and research centered around a few mouse mutations that primarily altered mouse coat color. I think the perception from the science community at large was that we weren’t doing particularly important work. That perception changed with the introduction of very powerful research tools. One such tool was the ability to alter genes in mouse embryonic stem cells to engineer DNA mutations at will. The second was the ability to use a strong mutagen, called N-ethyl-N-nitrosurea or ENU, to do forward genetics – more on that later. This was also the time when molecular biology was exploding. Rather quickly the mouse became THE model organism of choice. I’ve ridden that wave since my graduate student days. Today nearly every institution that is doing cutting edge research has a mouse genetics core. I suspect there are now about 2000 true mouse geneticists worldwide. Nearly every person who works on human disease now works with mouse models.

MC: Please tell our readers the basics behind your Rett project.

MJ: Our Rett project is based on two discoveries: 1) that you could make the symptoms of Mecp2 mutant mice better if you introduced brain derived neurotrophic factor (BDNF) and 2) Adrian Bird’s finding that you could actually reverse very severe symptoms in the mice by reintroducing the gene. Because of those two findings I believe that Rett symptoms can be altered by other genetic mutations. I felt strongly that the genetic approach that I was familiar with would be an ideal strategy to try and identify suppressors of the symptoms of Mecp2 knockout (ko) mice.

So let me tell you a bit about the screen. I use a powerful mutagen, ENU, that induces mutations in mouse sperm at a very high rate. We give the mutagen to wildtype (normal) male mice and then mate them to female Mecp2 knockout mice. A certain percentage of their offspring will have no Mecp2 and a sporadic mutation somewhere in their genome. We then analyze the mice very closely and look for any that appear healthier than your typical Mecp2 ko mouse. For example, a Mecp2 male ko mouse is dead by 6-14 weeks. If a mouse in our screen lives much longer than that, we hypothesize that there is a mutation in another gene that suppresses the ill effects of having no Mecp2. We currently have mice that are over a year old and still do not show signs of Rett.

MC: How many mice has your project generated?

MJ: We have used about 10,000 mice at this point and envision needing 5,000 more to find and understand the current genes of interest. To reach saturation for our screen, meaning that we are confident that the mutagen has generated mutations in every gene that could potentially be a suppressor, we would need to screen through five times the number of males that we have done thus far. Statistically, I estimate that there are 25-50 suppressor genes that we would expect to find were we to hit saturation.

MC: What kind of precedents are there for success using ENU modifier screens?

MJ: The first successful modifier screens were done in bacteria and yeast. The technique gained momentum in the late 1980’s early 1990’s when Gerry Rubin carried out a modifier screen in the fruit fly for genes that would interfere with a particular pathway. Dr. Rubin is a famous scientist who now is the Director of the Howard Hughes Medical Institute Janelia Farm Research Campus. Historically, people thought that fruit flies were the only organism that you could do this with. It’s clear now that the mouse is an equally powerful organism. My graduate mentor, Vernon Bode, did a screen in mice for PKU modifiers, which was finished by Bill Dove at the University of Wisconsin Madison. And an Australian group that works on diseases of the blood did an ENU mouse screen looking for genes that influence platelet counts. Each of these screens was very successful.

MC: Is there data to show that modifier genes are the rule or the exception in disease?

MJ: That’s a very interesting question. I work in the Department of Human and Molecular Genetics at Baylor. What I see from many of my colleagues’ work is that genetic modification of disease is the rule and not the exception.

MC: Can you envision a situation where you find modifiers in the Mecp2 ko mice but those genes are not implicated in the human disorder?

MJ: I do not think that we will find modifiers that are mouse specific only. I believe that because the mouse model for Rett Syndrome is amazingly similar to the human disease. Also, DNA methylation (which is critical to MECP2) and some of the possible functions of the MECP2 gene are highly conserved between species. So it’s very likely that the MECP2 gene in people and in mice is doing the same thing.

MC: What do you foresee as the best possible outcome?

MJ: I foresee finding a molecule that would help forge neuronal connections, and help these connections be maintained and molded.
Furthermore, whatever molecules we find that suppress Rett symptoms may also give us important biochemical information on other genes that may interact with Mecp2.

MC: Did being at Baylor, a Rett hotspot, impact your decision of taking on this Rett project?

MJ: I have been very much aware of Rett since I moved to Baylor in 1998, a year before Huda Zoghbi identified the gene. I have been on the student committee of some of Dr. Zoghbi’s students, which kept me up- to- date on the ongoing work. MECP2 is a transcription factor and I’ve always been interested in transcriptional regulation. What really brought me into the project was when you called me up with a proposition.

MC: When I was the Director of Research at the Rett Syndrome Research Foundation (several years before the merger with IRSA to form IRSF) I piggybacked an early morning think tank during the RSRF Rett Syndrome Symposium in Chicago. About two dozen creative thinkers were kind enough to drag themselves out of bed and brainstorm with me about potential key experiments that could significantly move the field forward. At the top of that list was an ENU mouse modifier screen. The group also gave me a list of potential people who could undertake such a laborious and intense project…it was a small list and your name was on it. As you well know, RSRF then organized a workshop at the Mouse Genetics meeting, which took place in Charleston, SC in 2006. Those discussions led to the funding of your project.

MJ: I was so thrilled when you called. There was a time when I would have turned down this project. But the timing of your call was perfect. The project appealed to me very strongly as a geneticist but also as a compassionate person who wants to make a difference in the life of others.

Your readers should also know that this is a project that the NIH would never have funded. It was too risky and too “out there”. I knew this was a viable technique and I was confident with the expertise of my lab with regards to mouse breeding, husbandry and handling that would move this project along quickly so I am very grateful to have had the funding to pursue this. People should be aware of how much private foundations such as RSRT move the field forward by funding high risk, but high impact projects.

MC: Speaking of compassionate person, at the end of your talk in Italy you got a little emotional. I was quite touched by that. Can you tell our readers what was going through your mind?

MJ: Yes, I got a little “verklempt” and I got teased quite a bit at dinner for that. I take this project very seriously because I feel that what we are doing could have an impact on people’s lives – an impact that perhaps wouldn’t happen without the screen – I guess as I was up there in front of scientists and the organizers of this meeting who have children suffering from Rett – the importance of our efforts hit me hard.

MC: Are you able to give our readers a hint at the data that your project has thus far yielded?

MJ: The project is at an exciting point. We are close to identifying our first suppressor gene and we have a few more potential genes that we are pursuing as well. Once they are identified we will begin experiments to confirm that they are indeed interacting with Mecp2m, first in the mice and then in people. I also think the modifiers we have so far are just the tip of the iceberg. So we have a lot more screening ahead of us.

I love this project, it’s fun, it’s exciting, and each new piece of data that we identify brings us closer to our goal.

MC: On behalf of families everywhere who love a child with Rett Syndrome we wish you Godspeed. We look forward to hearing about future progress. Thank you also to Pro Rett Ricerca and especially Rita Negri and Laura Rassetti for their tireless work to organize this meeting.

MJ: It was my pleasure and honor to attend the meeting in this most beautiful area of Italy, near where you were born.