diybio session at open science summit 2010

So, I wanted to thank the organizers for the opportunity to speak. My name is Hugh Rei.. and thsi is my daughter, Vietrice who was bron six years ago. And .. what's most striking is the fact that she has muscle weakness. it's the fact that she has a number of different findings warrants the name syndrome and the syndrome does not have a name and anything else. And, uh, this is the way that I have been able to put it together clinically. She has a phenotype with syndromic features in blue and these are all, some of these are complicated names. In essence, she shares features of other syndromes and what's interesting about this to me is that these syndromes- MFS, beals, LDS, they are all related. There's a common theme to why the clinical features overlap even though the genes are completely different. Some of these are dominant alleles, represents mutations in the fibril tube gene, MFS is in myofibrin 1, and then there's TGF-beta1 and 2. And it turns uot that all of these were thought to be allelic to be arrising from the same gene with different variants, 20 years ago, until they were clinically teased uot and shown to be different, different not only in the genetic origins, but also in the way that they march through in their natural history.

Vee doesn't have any of these because those genes in her are normal. Um, so, I set out to try to figure this out, and this was a major clue for me. The reason I took her back to Baltimore to see Dr. Bquisick, what she eneded was a very careful physical exam that documented every little thing that made her different, and make note of things that are within normal range, and this was a result of exercise and this put me in the biochemical neighborhood of what might be going wrong. To make a long story short, if I could have the next slide, I looked at some genes and.. I looked at a number of different genes that shared some similarity, and where I was taking a guess as to what might be going on, I looked very much like what a myostatin receptor activator looked like, so I sequenced those genes. Everything that I looked at, was within normal limits. And so I decided that maybe the better approach would be to go genomic rather than picking off one gene at a time. Look at as many genes as possible. I looked around for someone to help me do this, and Illumina was kind enough to do this- the transcriptome (your RNA). The reason I wanted to do the trio- mother, father and daughter, I could use the mother and father as a filter for variants. There are 3M variants per genome, there might be 10M, but there are no fewer than 3M depending on how you define variant, but new mutations occur rarely, and for Vee there might be 300 new mutations, that is to say new mutations that occurred in her somatic cell that we don't have as parents, so they would have occurred in the germ cells and they are represented in her somatic cells. I have reasons by virtue of these other three conditions that she might have a new mutation. We were just looking at the transcriptome, and we expected 1%, and we found 1 new mutation that her parents didn't have, but it didn't seem to explain the condition. So, then I set about to look using different models of genetics, because there's a dominant model. Maybe what she had was not autosomal recessive, where she has inhereited an allele from each parents (being homozygous). I used three different criteria: parents had to be homozygous carriers, there had to be a zyogote too. There were 938 instances of this. The expression of the gene of teh parents had to be different, because if itw as the same it wouldn't be effected. These are the sequecnes, tyou can't read the sequences. The stretches of the RNA that is turned into DNA, and that sequence leaves out one instanceo fa single T that is inserted in this gene. Both of her parents had this insertion, and Vee was a homozygote and it effected the RNA levels by a mechanism of RNA decay that causes the RNA transcript to get chewed up because it's out of frame and had a nonsense variant in it. And the consequence of that insertion throwing off the gene is that we chop of the c-terminals of the protein. So you can see in the parents, whcih are on the bottom, mom and dad are heterozygotes, we had three Ts, in some cases, four Ts, and Vee has all four Ts in every instance and there are hundreds of these. She is a bona-fide, homozygote for this sort of alelle and has the effect of choppipng off this part of the protein, the factor A domain, and it's lost.

the other reason this was interesting to me was that this was known to be involved in TGF-beta singaling, and I was looking for something in that pathway. She's heterozygote, she had decreased RNA for that one particular transcript, and this protein was n TGF-beta signaling, it was of high-interest to me. I took the next step, to take the muscle cells in culture, and to knock-down this particular gene to see what influenced, to knocking down this particular gene and seeing what happens on the normal differentiation of these cells in culture. This is open science, all of this work has been done by a very long list of volunteers so some of the population genetics looking at allele frequency- Alen Beggs at Harvard, And this was at San ALamno, and then some analysis at MIT, and at Illumina, etc., all of the data is available, but it's not publsihed, it's available for those who are interested. $700 in total has been spent on this whole project, mainly for primers and thingsss that I usedd for early stages in the project. I am reasonably optimistic that I and all of us will figure out what's going on in here. I don't think this is an idle effort. This insertion allele which I carry is rare but it's in every population around the world and there are probably other similar kids who have a problem with their muscles, and the mechansim here is of interest because it's something that could be remedied with existing medicines as opposed to muscular dystrophy where the muscular cells are truly degenerating and it's an irreversible process. These are some of the people that have helped me out.

Jay Flatley

Gary Schroth

Irina Khrebutkova

Steffen Durinck

Shujun Luo

Kevin Gunderson

Ray McCauley

Chris Burge

Vincent Butty

John Cleary

Roni Cohen

Marc Vidal

Tong Hao

Andy Fire

Amy Chen

Nick Okanskinski

Julie Yu

Alan Little ford

Se-Jin Lee

Nazli MacDonald

John Sotos

Alan Beggs

David Clapham

Hal Dietz

David Valle

Victor McKusick

People like to work on projects where there's some connection with the project. It's been very gratifying, so it's nice. So in terms of management, which is the reason you want a diagnosis, it's not just because you want to know, but because you have something yuo expect, systems you need to monitor; all those other diseases have devastating vascular disease with early death- the exception is, where that is rare, but the other two are terrible diseases quite honestly however they are coming under more management. Losarten is a common .. almost off patent.. byu the grace and providence it down-regulates TGF-beta signaling,w hich is overactive in ... and it has an amazing effect on patients. This is going to change hteir lives. Vee is on this medicine, she's at risk for vascular conditions, but she doesn't have any evidence of thsoe complications .. yet. I have been trying to find other Vees, and Alan is helping me on that. So far she is doing okay, so I'll leave it at that.