Looking into blindness

Chris -   If you have a patient with an inherited form of blindness and you want to know what gene is causing the condition and how it contributes to the disease process, wouldn't it be nice if you could turn some skin cells into some of the affected retinal photoreceptors in order to be able to unpick the pathology?

Budd -   My name is Budd Tucker.  I'm an Assistant professor at the University of Iowa in Iowa City.  Retinitis pigmentosa is one of the many forms of an inherited retinal degenerative disease.  So, patients with retinitis pigmentosa present with reduced vision.  They will often have poor night vision from very early in life.  As the disease progresses, there is cell death in the outer retina and these cells are the light-sensitive photoreceptor cells.  So, once those cells die being a part of the central nervous system then you essentially go blind.  And when there's a complete loss of those cells then there's no vision observed at all.  So, the question is, how do we treat someone with retinitis pigmentosa or retinal degenerative diseases like this?  There is good evidence now that transfer of genes, so, wild type full length genes can be used to restore vision or prevent vision loss in people with genetic disease.  So, in order to do that, you would need to know what the disease causing genes are and in turn, you would need to know what the disease causing mutations are and how that disease would normally progress.

Chris -   So, do we not know what the mutation is that is causing retinitis pigmentosa cases?

Budd -   See that's the exact problem we're talking about because retinitis pigmentosa currently can be caused by as many as 60 different genes and hundreds of mutations in those genes.  That's what we've currently identified.  Well, a year and a half ago, we identified a completely novel retinitis pigmentosa gene.  We believe that because of the cases that are still out there with unidentified genes and we know they have RP or retinitis pigmentosa, that there's going to be in total probably 100 different genes which were responsible for causing this disease.

Chris -   Is the issue then not just one of genetic sequencing?  We know the person appears to have the disease.  We take some DNA from them, we sequence their genome and we identify where in their RP gene things have gone wrong.

Budd -   Right, so that is the issue.  Why does that not work?  Because for this patient, we knew he had retinitis pigmentosa, but we could not find his disease-causing gene.  We could not find the mutations in his disease-causing gene.  So, one of two things were true.  He either had mutations in a disease-causing gene that we already knew.  We just couldn't find those mutations because they were in non-coding space or this was an entirely new gene that we needed yet to find.

Chris -   So, how did you approach that?

Budd -   So, the way we approach that, we did rounds and rounds of exome sequencing, just as you said.  So, we sequence this entire individual's whole coding region, three to four times.  We could not find two disease-causing mutations in any retinitis pigmentosa genes.  So, the way we thought to approach it then was to start with the person's retina.  Well, how do we do that?  It's very difficult to get a retinal biopsy from someone.  It's like getting a brain biopsy.  It's part of your central nervous system.  So, the goal would be to make retina from this individual.  Then what we could do is look at the transcript in the retina.  So, is the transcript and in turn protein expressed.

Chris -   So you're saying, you'd take some other cell source in the body and reprogramme that cell source to recapitulate the retina and then you can quiz that tissue and ask, what is going wrong in here to generate this RP phenotype in this person.

Budd -   Right, precisely.  We do that from taking a biopsy from the skin and we reprogramme that using four transcription factors back into stem cells called induced pluripotent stem cells or iPS cells and then we used a protocol that we have developed to take these cells and drive them into a photoreceptor or retinal and then photoreceptor lineage.

Chris -   So, you turn these cells into the sorts of cells that are affected by the disease in the retina by effectively mimicking the environment that they would grow up in so they turn into that particular cell type.  How do you then ask, why in this individual, they get the disease?

Budd -   So, what we did in this case is we took the transcript, isolated RNA and then we did simple PCR up and down some of the suspected genes.  When we did that, we found one mutation that we had already identified in whole exome sequencing in the DNA which was just a single point mutation.  Then we found another mutation which actually was in a non-coding region, which causes single base pair switch which was in a cryptic splice site.  So then what happened was that a large piece of the non-coding DNA got stuck in-between coding regions and started to code.  So, that caused an insertion of a stop-coder.

Chris -   Do you know why that abnormal protein triggers the disease in this person?

Budd -   We think we do.  So, this gene usherin encodes for a protein called usherin which is a very large extracellular matrix protein.  Now, this very large extracellular matrix protein is tethered to the photoreceptor.  It's actually expressed at the base of the connecting cilium of the photoreceptor.  So, the photoreceptor has a bridge or connecting cilium which is vital, which connects the inner portion of the photoreceptor to the outer portion.  The outer portion is the structure that really holds the photo pigments and things like this - the machinery required for detection of light.  Yet, all of those components for the most part are made in the cell body.  So, they need to be trafficked across this bridge.  Usherin is responsible for something within that structure.  We're not quite sure exactly what it is at this point.  We do know what the protein, where the protein is expressed.  Interestingly, when you transplant this individual cell into a mouse model - a retinal disease, this individual - I mean, these human photoreceptor cells go on to develop normal looking photoreceptor cells.  They make outer segment like processes and they have normal - what's looks like normal - connection cilium.  So, it doesn't appear to be stimulating an early developmental defect in photoreceptor development in this individual.  Clinically, this individual did not lose his vision or start to lose significant vision until he's mid to late 40s.  So, it seems like what's happening here was that the mutation in this large gene is over time inducing stress of the endoplasmic reticulum, so, something known as ER stress.  So, it suggests that this mutation is causing a protein misfolding response, gums up the endoplasmic reticulum and over time, the cells succumb to this ER stress and die.

Chris -   Obvious question, but did you go back to the original patient, armed with your mutations you've found and check that they were actually what was wrong with the patient.  They weren't just mutations that cropped in the reprogramming process to make those stem cells into retinal cells.

Budd -   Absolutely.  So, we went back and we sequence through this gene, this individual gene from his blood and we found that mutation in his gene.