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Wednesday, September 15, 2010

All you need to know about the Usher Syndrome and Related Disorders Conference, Part III: Genes, Proteins, and Networks

by Jennifer Phillips, Ph.D.

We’re slowly progressing through the wealth of interesting talks at the Conference last May, and even though I didn’t actually attend the Valencia meeting, I have enough familiarity with the dense molecular topics therein to give an adequate summary and their relevance to the Usher community. My plan is to cover the Usher gene overviews along with a few studies of particular players in Part III, and recap the talks that dealt with the topics of new genes and related genetic disorders in Part IV.

Prologue: It should come as no surprise to the readers here that Usher syndrome has multiple genetic causes—that is, mutations in one of twelve (so far) distinct chromosomal regions known as loci result in deafblindness in humans. Even though there are important differences in the onset, and precise symptoms of Usher syndrome, there are obviously enough commonalities to treat all cases of Usher as one general disorder. As such, one of the main research goals for those of us who study these molecules is the two-pronged investigation of

a) what they have in common, i.e. why defects in all these different genes cause a very specific type of sensorineural deafness and retinal degeneration, and
b) what makes each gene different, i.e. why mutations in some genes cause more severe forms of Usher than others.

So with that in mind, let’s get started on the summaries…

The Usher protein network in the inner ear
Hannie Kremer, Radboud University Nijmegen, The Netherlands
Figure 1: A—an electron microscope scan of the actin bundle, made up of organized stereocilia, projecting from a single hair cell. B—a cartoon of these structures showing a passive mode (gray, background) and an active mode (green, foreground), in which the stereocilia bend in unison in response to an incoming sound wave. C. A cartoon showing a magnified view of the tips of two individual stereocilia (green) tethered together by the Usher protein, Cadherin 23.

The above figure shows the organization of a specialized structure found in mechanosensory hair cells. The hair bundle is the structure that give ‘hair cells’ their name, although it’s not really made of hair, but actually comprised of tubes of actin that protrude in the same direction from the tops of these cells and are connected at the tips so that they will move as a unit in response to vibration, causing the hair cell to signal the auditory neurons that a sound has been perceived. Mice with mutated or deleted Usher genes have clear and specific defects in the formation of stereocilia, the actin bundles that project from the tops of the mechanosensory hair cells:

Figure 2: from Lefevre et al, 2008. The far left panel shows several hair cells (outer hair cells (OHC) and inner hair cells (IHC) )from the cochlea of a mouse without an Usher mutation. The rest of the panels are images from this same region of the cochlea from mice with defects in Usher genes. From left to right: wild-type, Myo7a, Ush1c, Cdh23, Pcdh15, Ush1g.


The observed defects in the length, width, orientation and abundance of stereocilia in the Usher mice show a clear requirement for Usher proteins in the proper formation of these structures. As all of this growth and development takes place before birth in humans, it’s no surprise that the hearing loss in most Usher cases is congenital. But what exactly makes the stereocilia so disorganized? We know from biochemical studies that many of the Usher proteins can physically interact with each other, and we know from antibody localization studies that many of the Usher proteins localize to particular regions of the hair bundles, so detailed studies of these in combination with the mouse Usher mutants provides evidence (alluded to in figure 1C) that the Usher proteins work together in a complex to guide the organized growth of the actin bundles by linking the individual stereocilia together. In addition to this proposed function of an Usher complex, individual Usher proteins appear to play a role in the outgrowth of the actin structures. Finally, there is some, less-well understood evidence that some Usher proteins may also be involved in events at the other end of the hair cell—the part that communicates with the auditory neurons.

Why you care

Understanding when and how the Usher proteins are required for normal cellular development and function is critical for the discovery of targeted therapies. The more we can find out about what these molecules are doing in there, the better chance we have of identifying ways to compensate for defective proteins in Usher patients.

The Usher proteins in the retina
Uwe Wolfrum, Johannes Gutenberg University of Mainz, Germany


Figure 3: Usher protein localization in subcellular regions of the photoreceptor cell. Abbreviations from top to bottom: BM: basement membrane; RPE: retinal pigmented epithelial cell; OS: outer segment; CC: connecting cilium; IS: inner segment; N: nucleus; S: synapse; N: neuron.
adapted from Reiners et al, 2006.

One of the confounding things about Usher syndrome is the asynchronous timing of the hearing and vision defects. We have good evidence from the mouse models that the congenital hearing loss is due to developmental defects, but the vision loss is progressive, which would suggest a problem with function rather than with formation. In all the human and animal models we currently have access to, there doesn’t appear to be anything wrong with the structure of the retina, but something is causing photoreceptors to die, slowly, over a course of months or years. Again, researchers can seek answers by looking at the protein localization within the retina, and from knowing that these proteins have the potential to physically interact with each other to form multi-molecular complexes. Because it’s the photoreceptor cells that degenerate, most of the protein localization studies have focused on this cell type. Similar to the colocalization pattern seen in hair cells, many Usher proteins conglomerate in distinct functional regions of the photoreceptor cells. One exciting finding is that many of these proteins (indicated by colored boxes in figure 3) localize in the region of the connecting cilium. The connecting cilium in photoreceptors serves as a conduit for moving proteins and other molecular cargo that are synthesized in the cell body, or inner segment, up to the outer segment where they’re required to perform various functions. The abundance of Usher proteins at this particular location has prompted researchers to examine whether any of them might interact with known factors in molecular trafficking, and indeed, some do. Although the evidence is still being gathered, it’s tantalizing to imagine that defects in cargo loading or transport, or indeed, in the structure of the conduit itself, might be responsible for the reduced function and ultimate degeneration of photoreceptors. This is a very hot area of research and new ways of assessing these functions are being developed in numerous labs worldwide. Additionally, and not unlike the situation in hair cells, there is some evidence that these proteins localize to and function somehow in the synapse, through which the photoreceptor signals to adjacent neurons when stimulated.
Why you care

Again, understanding the where, when, and how of Usher protein activity is essential to finding therapeutic ways to overcome defects in these processes. These are incredibly complex cells, and there is strong evidence that Usher proteins perform multiple functions within them, so there is still a great deal to be learned, but every new bit of data adds to the big picture that will someday reveal new ways to address or prevent this progressive vision loss.

Usher scaffold proteins provide complementary functions in retina and inner ear
Monte Westerfield, University of Oregon, Eugene, Oregon, USA

Most of the Usher gene studies using animal models have been conducted in mice or rats, but a few of us are using the zebrafish to address these questions. Zebrafish have hair cells and retinas very similar those found in humans and these animals provide a great system in which to study the consequences of targeted Usher gene or protein disruption. In the above summaries, I alluded to the fact that Usher proteins display the ability to physically interact in a complex. This is achieved through the presence of particular protein interaction domains, some of which provide a place to stick, and others of which are capable of binding to that sticky place, rather like the hook and loop surfaces found in Velcro. Several Usher proteins contain multiple sticking places, called PDZ domains, along their length, and are thought to be required inside a particular cell (e.g. a photoreceptor or a hair cell) to serve as a scaffold or an organizing structure for localizing other Usher proteins that may function across cell boundaries, or, in the case of the links between stereocilia depicted in Figure 1C, way up the page, between membranes of structures from the same cell. Interestingly, although there are characteristics that make PDZ domains identifiable, there appears to be a great deal of specificity in which PDZ domains certain Usher proteins with PDZ-binding capabilities choose to interact with. In other words, even though there are a couple of different Usher proteins, namely Ush1c and Ush2d, that look very similar with respect to the presence and placement of PDZ domains, they don’t appear to have very much functional overlap. Is this because they’re not in the same place at the same time, or are there other regulatory factors that influence which of these proteins is the more suitable ‘scaffold’ in a given cellular situation? We are pursuing these questions, as well as ones of general function of all the involved PDZ proteins. Zebrafish in which these PDZ proteins have been disrupted appear to have similar functions in hair cells to what has been shown in mice, with respect to the development and organization of stereocilia. We can also detect functional problems at the behavioral and cellular level. In the retina, we see specific defects in synaptic transmission between photoreceptors and retinal neurons when we disable one of the PDZ domain containing proteins (Ush1c) and we’ve also discovered that Ush1c and Ush2d proteins are all present in another retinal cell type called the Müller glia. Müller glial cells have a variety of functions related to maintaining the health, development and function of retinal neurons, including photoreceptors, so the possibility that these cells could somehow be involved in the defects observed in Usher syndrome is intriguing.

Why you care

Investigating Usher gene function in organisms other than Mammals adds a wealth of additional experimental techniques with which to understand the role of Usher proteins at various developmental and functional time points in the ear and the retina. In addition to the general benefit of obtaining more information about the complex workings of these molecules, the zebrafish system also lends itself to high-throughput testing of various genetic and molecular therapies.

Thursday, September 2, 2010

A Low Cost Solution to a Difficult Problem

by Mark Dunning

Jennifer wrote about vitamin A in her last post and gave her opinion on whether or not the studies done to date were enough to warrant prescribing vitamin A in supplement form to patients. My first thought on reading her post was ‘Hmm. I respectfully disagree with her.’ I know the studies and came to a different conclusion. My daughter takes vitamin A supplements under the supervision of a physician. Please note the last part of the sentence. You should not take my advice on this. Nor, I know she would agree, should you take Jennifer’s advice. You should take that of your physician.

That brings me to my second thought. ‘Why the heck didn’t any doctor ever explain their opposition to vitamin A supplementation to me as well as Jennifer did?’ I’ve talked to a lot of physicians that don’t prescribe vitamin A, some who are strident opponents of prescribing it, and none of them explained their reasoning as clearly as Jennifer. On the one hand I’m proud of my blog mate for her thoughtful review but I’m also terribly concerned about it. Like I said, you shouldn’t take the advice of Jennifer or me. But if you’re not getting lucid advice from your physician, what do you do?

Looking back at our decision, the reason we chose to use vitamin A supplementation was because one doctor took the time, much like Jennifer, to go through the studies with us and explain why he thought these studies supported using supplementation. We spoke to other doctors that did not agree with supplementation, but none of them went through the studies that supported their position. In fact, most of them didn’t mention any other studies done at all. They were just worried vitamin A wasn’t safe in large doses.

Now being worried that vitamin A isn’t safe is a justifiable concern, but they came to that conclusion somehow. They didn’t just pull it out of the air. Why couldn’t they, like Jennifer, tell me how they came to that conclusion? This left us with the opinion that they either 1) didn’t know about the studies and were just winging it or 2) didn’t really have anything to support their opinion. But if you read Jennifer’s post, you can see that there certainly is an argument to be made against it. So how come, in my experience, was only one side able to make their case?

All of this brings us back to another of Jennifer’s recent postings. We have to educate physicians on Usher syndrome and the treatments that are currently available, or might soon be, so they can advise patients accurately and appropriately. But we also desperately need to train them on how to appropriately communicate with patients and that is the focus of this post.

I have a little experience in this area. I have been fortunate enough to be asked to speak on a number of occasions to doctors, nurses, professional staff, and medical school students at Harvard Medical School, Children’s Hospital Boston, and M.I.T. These are some of the most prestigious institutions in the country. I’m not pointing that out to brag (OK, maybe a little) but rather to illuminate a fact which I will get to in just a bit. First, here are the things I usually tell them:

We live in a Google world. As soon as you hint that you, Mr. Doctor, suspect Usher syndrome as a diagnosis that family is going to go home and Google it. Even if you don’t breathe the term Usher syndrome, they are going to go home and Google all of the symptoms they saw you scribble down and come up with Usher syndrome on their own. Be prepared before you meet with them and give them ‘safe’ web sites for information on Usher syndrome (like this and this) so you can be confident that they are finding out the information from accurate, credible sources instead of reading it from some crackpot blog. Oh, wait, that didn’t come out right...

Make sure the family understands when they will receive the results of tests. This is a personal bugaboo for me. I didn’t go for the ABR test of my daughter’s hearing because I thought it was like a blood test. I didn’t realize we would get the results right then. So I learned about my daughter’s deafness over the phone from my sobbing wife. On the flip side, there is no need to get a family all worked up over having blood drawn for a genetic test when the results will not be known for weeks. It’s just a blood draw, nothing more.

Schedule a time to meet with the family to discuss the test results when you schedule the test. In other words, don’t wait until you get the test results to call the family and schedule a time to talk. Why? See below.

Never give the test results over the phone. Look, I know a negative test for Usher can be given over the phone, but you really need to talk in person with someone if it is a positive result. Heck, given the sensitivity of some of these genetic tests (50% accuracy) even a negative test result might not eliminate Usher completely. This brings me back to point number 3. If you call up a family and say ‘Hi, I have your test results. I’d like you to come in to discuss them’ the family is going to read in to that. It must be bad. Why wouldn’t they just tell us over the phone? So solve the problem up front and tell them before they have the test that they will have to come in to get the results, good or bad, because you never give results over the phone.

Never tell a family their loved one ‘failed’ a test. This one drives me batty because I know it’s used with the newborn hearing screen all the time. Right when mom and dad are basking in the promise of their newborn child, they are told their baby is a failure. No, it’s not. You don’t fail a hearing screen or an ABR or a blood test. The results are positive or they indicate that we should do more testing. This isn’t calculus. You’re not a failure if you have Usher syndrome or hearing loss.

After you give the diagnosis, schedule a follow-up appointment with the family. When a family gets the diagnosis they go numb. That first appointment is not the time to discuss how the myosin-7a protein chain operates. Even if you are speaking to a family of genetic engineers, they are not going to hear you or be able to absorb all you are telling them. Answer every question they have but encourage them to ask the same question again later if they need clarification because they will. Then schedule a follow-up appointment with them for a week or so later when they’ve had time to digest the diagnosis and clear their heads. That’s when they will have the best questions and it’s a time when they will remember the answers. This follow-up appointment, by the way, could be by phone. Whatever method will most encourage the family to show up.

Tell them what they might do next. Just because there are no more tests to be done and a diagnosis is made isn’t the end of the line. Sure, in the case of Usher syndrome there are no certain treatments, but don’t shrug and tell them to come back next year to see if their vision has deteriorated. Give them options (like vitamin A) along with your opinion on it. Tell them where to find the studies, the supporters and the opponents. Tell them about research under way or where to find out information about it, like the Usher Coalition site or the Foundation Fighting Blindness. It’s alright to give them hope for the future.

Tell the family what that future might hold. This isn’t ‘you’ll meet a tall dark stranger’ stuff. I mean tell them the truth and not just about the diagnosis. In fact, the stuff beyond the diagnosis is more important (I’ll explain why in a bit). You can and should tell them, Ms. Doctor, about the normal prognosis for someone with RP, and with hearing loss, both with an implant or hearing aid and without, and about the vestibular component. But you should also tell them that most people with Usher syndrome who want to go to college, get married, have kids, and have a challenging career do just that. Again, it’s alright to give them hope.

Physicians are responsible for physical well being. They work with the body. But the diagnosis of Usher syndrome is not traumatic for families only because it may portend a life of deaf-blindness in the future. It is traumatic for families because they interpret deaf-blindness to mean a life of isolation and limitation. It’s not just the physical results of Usher syndrome that are frightening, it’s the potential social and psychological results. Physicians need to recognize this and address it as part of delivering the diagnosis.

Now the list above, to me, seems like pretty obvious stuff. The first time I presented it I thought I was going to get a ‘no duh!’ type response. I didn’t. In fact, I’ve heard from a number of people that after attending the talk they fundamentally changed their approach to some of their processes. If these prestigious institutions that I mentioned and those that attend them or work for them find this illuminating enough to change their way of doing things, we’ve got a big problem. If they aren’t teaching doctors this stuff, no one is. These are the most forward thinking institutions who, to their credit, are willing to not only listen to some dumb parent but act on what he says. I shudder to think what is going on in the rest of the world.

So back to Jennifer’s point about educating physicians. One of the problems is how to do it at a reasonable cost. We could, for instance, hire a blimp to drop Usher syndrome flyers on the next AMA convention. I don’t think that’s our most cost effective approach. More to the point who is the ‘we’ that might write up the flyers and hire said blimp?

I think that gets lost in all of these discussions. We all fall in to the ‘someone should do something’ trap sometimes. We see pictures of floods in New Orleans or unemployment statistics or polar bears swimming where there once was ice and say ‘someone should do something.’ But who, exactly, is the ‘someone’ who is going to do something to help educate physicians about Usher syndrome?

Well, I have seen that someone and he (or she) looks a lot like you. You see that list above? Talk to your doctor about it. Make sure he or she is thinking about it. Send them a link to this post. Or the next time you see them tell them that you read this fascinating article by this brilliant author who suggested that you should never give test results over the phone and ask them for their thoughts. Put the bug in their ear. Make them think about it. They might not agree with all of it, but don’t assume they are thinking about it. They weren’t thinking about it in the group I met at the places I’ve spoken, I can tell you that, and they are just about the best in the world.

The point to all of this is that there is a low cost way to educate the physicians that are diagnosing Usher syndrome that can began immediately. It’s you.