Lecture: The Fundamentals of Genotyping & Phenotyping for Inherited Retinal Diseases (IRD)

Emerging clinical trials and treatment for Inherited Retinal Diseases (IRD) are providing hope to patients and families where previously no options were available. To gain access to such interventions, one must obtain a correct genotype. To get to that genotype, care phenotyping will lead to appropriate testing. During this live webinar, Dr. Levin will discuss key tips to deeply phenotype patients and then select and use genotypic testing to arrive at the most accurate gene based diagnosis. Questions received from registration and during the webinar will also be discussed live.

Lecturer: Dr. Alex Levin, Chief, Pediatric Ophthalmology and Ocular Genetics, Flaum Eye Institute, Rochester, NY, USA


DR LEVIN: Hello, everyone. So nice to see you and have the opportunity to give this talk. It’s a great pleasure to be doing this on behalf of Orbis, that wonderful educator through their Cybersight platform, which I urge you all to go to. There is so much in Cybersight for you to enjoy, from surgical videos to teaching and ophthalmology, every discipline of ophthalmology. It’s a great place for information, and I hope this is of some added use to all of you. So let’s talk about the fundamentals of phenotyping and genotyping for inherited retinal diseases. Now, the goal here, the goal with every patient, is to take a phenotype — in this case, retinitis pigmentosa — and convert that to a genotype. Why do we bother? Well, it’s fun. It’s exciting. There’s an element of discovery and academic advancement and understanding what a patient’s underlying genetic defect is. There’s an element of curiosity. I would like to know what’s causing this problem. And it’s way cool to do this kind of work in rare diseases. But there are more practical advantages, specifically for the patient. Number one, it relieves uncertainty. Many of these patients are walking around, not knowing what they have. Wondering. What is their problem? What does it mean to them? And we can therefore counsel them appropriately. What’s their reproductive risk of having another child who’s affected? What if they want to get married? What’s their chance of marrying a carrier? And also identifying carriers, who may in fact have reproductive risk. It also helps us prognosticate for the family. Instead of just being RP, now you’re this specific kind of RP. We can better help to know what the future may hold. And getting a diagnosis opens up a whole world of support for the families. Not only Dr. Google and all the information they can get there, but support groups and other appropriate sources. Excuse me. Another reason we do all this is the prospect of treatment. And we all know that treatment is coming. Treatment is here. We have the approved RP65 treatment. Luxturna. We have many clinical trials going on for a number of inherited retinal dystrophies. Many of our patients are anxious to get into these trials. And there’s also the hope that there’s preventative things that patients can do. To help slow the progress of their disease. And in a sense, prevent them from going astray and becoming ineligible for a trial. So all of this is important for us. And this is why we go forward with this problem. Lastly, there’s the issue of early detection. Early detection is sometimes the best route to getting into a treatment trial. And getting into perhaps processes that can slow a disease. So how do we get there? So here is question number one. These are audience response questions. My question to you is: What would you order for that patient? I’ll show you one more time. There it is. It’s all I wanted to tell you. What would you order? A large free retina panel? Whole exome sequencing? Whole genome sequencing, or a sequencing of the KIF11 gene? So please respond on your screen that just came up. We’ll wait a few seconds for the answers to show. You might answer what you think you would do and what you would practically do as well. So here’s the answer. We have a large group doing large gene panels, whole exome sequencing, whole sequencing, and 25% who said KIF11. That’s really good. I’ll tell you why it’s really good. I can tell you that from that one slide, I see this. I’m gonna order KIF11. I’m not very smart. I’m actually really dumb. But I see these things every day in clinic. I’ll get one positive result. The patient likely has microcephaly, I’ll do an IVFA, peripheral non-perfusion, prevent further vision loss through laser treatment. I can tell you the inheritance pattern. All of that for a single gene test. That was pattern recognition. I must say I’m surprised that 25% of you got that right away. That’s pretty darn good. But I can’t teach you pattern recognition. That’s something that only you guys can do. Through time and experience. What if you ordered a large gene panel, whole exome sequencing or whole gene sequencing? What if you did that? Maybe that was an expression of not recognizing what it is. But what would happen? First of all, would you do genetic counseling? An absolute prerequisite for every genetic test, formal genetic counseling before and after treatment. But you would get a lot of noise. You would come back with all these — what we call variants of unknown significance. You would have what we call a false genome rate. And that’s the cost, as I’ll show you, to the patient. It’s not really free. There’s a cost. A downstream cost. There’s an injury to the patient from all of this information, whole genome. Whole exome. And in fact, you might not even get the gene. Maybe KIF11 wasn’t included on your panel. Maybe it wasn’t tested correctly if it is. And if you did whole genome or whole exome, you would have to have the right information to give to the lab so they filter out the right genes to report back to you. So you may have gotten a negative test. So how do we get to this goal? That’s where I want to backtrack. Most of us don’t have that opportunity to just say: Boom, that’s KIF11. I know that. And that’s where we get to the most critical part of the process. Phenotype, phenotype, phenotype, phenotype. Deep phenotyping, as some people have called it. And that can lead to a genotype through a focused gene hypothesis. To cut down the noise, to cut down the false genome rate, and get us closer to where we’re trying to go. We do phenotyping through history, physical exam, family history, and ocular examination. And what I would like to do is go through each of these. To give you some hints and clues to get you closer to a genetic diagnosis. And history, history, history. That is really the key. Be a Sherlock Holmes. Try and find the answer. And I will tell you that 90% of the time, the answer lies in history. And the prelude to the examination and testing. So let’s try here. Which of the following questions should you ask in RP? This is a fairly easy question. Would you ask about kidney problems? Hearing problems? Polydactyly? Or all of the above? We’ll wait for all those answers to come in. And the answer is: Very good. All of them should be asked for. In every patient with retinitis pigmentosa. Why do I say that? Let’s take a look. When you ask a history for a patient with retinal dystrophy, start with a simple question. Why are you here? Was the finding picked up on an incidental finding on an optometric examination? Is there visual loss? You want to take it from the beginning. I always ask the patient: When did you first start having an eye problem? Go back to the beginning. And they say: Well, I was in grade 2. I got glasses. Then around grade 10, glasses weren’t fixing my problem. I went to this doctor and that doctor. What did you see? Sometimes they’ve seen someone very good and you trust their recommendation, their diagnosis. What prior genetic testing was done? Get a feel for their journey. What happens along the way? And ask them then what their current symptoms are. Is it a night vision problem? More than a day vision problem? Do they have photophobia? That might suggest more involvement of the macula or the cones? Is it blurry vision? Was it fast? Was it slow? Has the problem been stable? Let me give you some examples. If you want to find out about a patient’s night vision, many patients, especially with RP, don’t even realize they have a problem. It happens so slowly. So incrementally slowly, and then children don’t even know they’re different from their peers, because they’ve never known anything different. Ask: Can you see the stars at night? When you go to a movie theater, back in the pre-pandemic days, can you find your seat in the movies? Does a child sleep with a nightlight? And then ask why. Some children sleep with a nightlight just because they like to. But some do it because they wouldn’t be able to find their way to the washroom, because they’re blind at night. Does the child play night games? Does the child like to go play hide and seek, or when the sun goes down, is this a child that’s going in the house? Look for these little clues. When you’re trying to assess for color vision, is the patient a night owl? Achromatopsia patients, especially the more severe ones, the parents will say… Ugh, the kid is up all night. I can’t get them to go to sleep. When the lights are out, he’s so much more comfortable. He’s there under his covers with his laptop, looking at the screen. And then you can ask more specific questions with color. For example, in blue cone monochromatism, patients have a little bit of nystagmus, decreased vision, X linked pattern of inheritance. Ask if they’ve seen the Wizard of Oz. Patients with this disorder can’t tell when it went from black and white to color. It all looks the same to them. I had a patient who said whenever there’s a Western on television he has to ask his wife if it’s in black and white or color. Ask if they can see traffic lights better at night. Another sign of blue cone monochromatism. Ask about the pace of onset. Certain disorders are known for their fast pace. The batten disorders. Occult macular dystrophy can happen very quickly. CAR/MAR/AIR, they all come on very quickly. Stargardt can sometimes come on very quickly. That’s very helpful, as opposed to the slow cadence of retinitis pigmentosa. And when you have macular involvement, it can help lead you towards Bardet-Biedl syndrome. Certain things would be unusual in RP, to have macular involvement. What was the age of onset? Early onset disorders like Leber congenital amaurosis, ECORD Early Childhood Onset Retinal Dystrophy and SECORD, Severe Early Childhood Onset Retinal Dystrophy — these are kids who had problems before they were ten years old. Did the child ever drive? Did he always need help in school? Did she always have problems navigating? Was her problem identified early? Was there nystagmus? Often a sign of early onset. Versus late onset. Like late onset retinal dystrophy, which can get you down to a single gene. So it’s important to ask these questions. And stability is important. Things like congenital stationary night blindness and achromatopsia don’t change much over time. I see patients who come in with retinitis pigmentosa who think they’re going blind and I’m happy to tell them they have achromatopsia instead of a cone dystrophy and they’re not gonna get worse. The slow versus fast progression is also a very important question to ask. So as you can see, we’re honing in on history that can lead us to a category of problems, if not a single gene. Then you can back off and start to ask about the medical history. Start at the very beginning. Ask about the pregnancy. Were there any problems? Was there any exposures of interest? Was the patient sick? During her pregnancy? How was the pregnancy conceived? Was it by in vitro fertilization? And how was the birth? Did the child go home with the parent? Or was the child in the neonatal intensive care unit, and if she was, why? What things happened? What went on in the nursery? Subsequently, were there any early surgeries or admissions related to that stay? How can that all be important? Well, here is a patient with peripheral non-perfusion. Guess what? There’s a long list of things that could do this. And you can see just through history, as you go down this list, you can see… Was there shaken baby? Was this child admitted with subdural hemorrhage? Incontinentia pigmenti? All of these things will help you. Look at the bottom, which should be at the top. Retinopathy of prematurity. Knowing that the patient was premature and in the NICU can collapse this diagnosis down very quickly to a single non-genetic cause in this case. When you ask a past medical history, I encourage you to use five different lines of questioning. You say to the patient: Are you healthy? Oh yeah. I’m perfectly fine. Then you ask: Do you have any medical problems? They say no. No medical problems. Then you ask: What medications are you taking? They say well, I’m on digoxin. I’m on Keppra. It’s like… You just told me you were healthy. Why are you on all these medications? It’s another way to get the patient to respond. And for every medication you list on the chart, you must have an answer. Why are they taking this medication? What is it for? In fact, you might see an antidepressant on there. Many of our patients with inherited retinal dystrophy suffer from depression. You might ask: Is your depression related to your vision? And that will get you to an important part of their management. You want to know what vitamins are. Are they unfortunately taking large dose vitamin A, as many patients are, which can make you worse in some patients? Are they on herbal supplements? Are they wasting their money taking things that have no proven efficacy? You can help them with that. Have they had surgery? Make a list of the surgeries. These may be relevant. The patient doesn’t know that surgery to remove a finger, to repair a renal defect, is important to their retinal dystrophy. Only if you elicit these problems can you make the connections. Have they ever been admitted? The patient says I’m perfectly fine. I’m on no medications. Have you ever had surgery? Yeah, I had heart surgery when I was a baby. You said you were fine. You have to keep digging. The fifth way: Are there any parts of your body that weren’t formed right? These are all ways of teasing out that past medical history to get the answers that you seek. Certain questions become important. Things that suggest a ciliopathy: Obesity, polydactyly. When you’re asking obesity, look at the parents. Are they obese as well? Look at the type of obesity. Is it truncal, like we see in Bardet-Biedl, or total obesity? Something different. Is there a history of renal or liver dysfunction? If there’s liver dysfunction, is the patient alcoholic? Hearing. Did the hearing have onset early? Did they have cochlear implants? Or was it later in life? Is there developmental delay? And then again, go back to the NICU history. Is there another reason for developmental delay? Is there gonadal dysfunction? Either reproductive immotility or other problems, hypospadias, things that would tip you off that maybe there’s something in the ciliopathy realm. These questions suggest mitochondrial disorder in the family. First of all, maternal transmission of the problem without any paternal transmission. But think about: Stroke, encephalopathy, myoclonus, ataxia. Psychiatric disorders in the family. Cardiomyopathy can be a sign of ciliopathies. GI dysmotility and other things. Diabetes. There’s a disorder, maternally inherited diabetes and deafness. You want to put these things together and it will begin to get you a picture to narrow your gene differential. There are some odd questions that we’re asking. Does the child have sleep problems? In particular, nightmares or night terrors? That’s a characteristic of the Batten disorders. Can the patient feel their own pulse? Interesting question. Can they button their buttons on a shirt? Patients with posterior column defects and mutation in FLCVR1 gene have retinitis pigmentosa and a loss of vibratory sensation and can’t do those things. Can’t feel their own pulse. I’ve had patients in the office where I say can you feel your pulse? And show them now. Can they determine their hat size? I’ve got a small head. Microcephaly. That was the KIF 11 and some other disorders. Lastly, what about the back of their head? Patient with high myopia. Here’s one. You can see right here there’s a little area of alopecia. And this is a sign of Knobloch syndrome, a cause of retinal dystrophy associated with high myopia. There can be an encephalocele there, and this can help you understand why the patient has the myopic retina that they do. You also want to ask about medications. Right? Which of these drugs mimics Stargardt disease? Let’s let you answer that. Pentosan. Chloroquine. Thorazine. Or vitamin A. Which ones do you think mimics Stargardt disease? It’s a little tricky. And the studio audience says… Ah, I got you this time. It’s actually Pentosan. Elmiron. Vitamin A makes Stargardt’s worse. It doesn’t mimic. That would be a night blindness thing that you would get from vitamin A deficiency. Hypervitaminosis really doesn’t cause a retinal dystrophy. So what we see is that this helps us. Pentosan is a drug that can look like a maculopathy. Macular dystrophy, just like Stargardt. Ask if the patient is on chemotherapy. Sometimes patients are shy to tell you if they’re on chemotherapy. Antipsychotics cause retinopathy. And chloroquine has a distinct picture without the flecks and subretinal lipofuscin we would see in Stargardts. So you need to specifically ask: Have you ever taken these drugs? We then get the family history. When you’re asking family history, remember, it’s just a story. And when things “run in a family”, we want to note those. Sometimes we have to examine family members, in order to get to our diagnosis on the proband. But you want to know: Did anybody have the same problem? Are there any vision problems? And really anything that runs in the family. And you have to dig deep to understand better what they mean by someone with a vision problem. So, for example, you have to ask questions: Were there miscarriages? It’s a sensitive question. A hard question to ask. But in particular, were they male miscarriages? That’s a sign of X-linked dominant disorder. You want evidence of a constricted gene pool, which will raise your concern about the possibility of autosomal recessive disease. Is there known consanguinity, where the parents are known to be cousins or related. And in some countries, that’s very common. And that is okay. But you need to know those geographies. So you have to ask. How did the parents meet? Well, if they met at University, it’s unlikely that they’re related. But if they met at church… Maybe they share geography. I had a patient once from a country in Africa. I said: Are you and your wife related? They said no. I said: Where are they from? She’s from this village. I’m from that village. I said… Well, how close are the villages? Well, you cross a little foot bridge over the stream to get to the other village. That’s a constricted gene pool. Are they from the same religion? Especially if it’s a religion that is not common in that country. And then you want to know about relationships in the family. Who is adopted. Who is married to who. Who had kids with who. And sometimes when you get this information, you get surprises. Kids who didn’t know they were adopted. And so on and so forth. And when you’re doing the family history, you need to see which problems segregate with which individuals. So I saw a family the other day. The father has RP and deafness. He’s married to a woman with proven Waardenburg syndrome who is deaf. They have a daughter who has Waardenburg syndrome but not Usher syndrome. You want to see which things go with which individuals. Especially in a Deaf community, you may have multiple Deaf people in a family without the RP. Who has what? Also ask about cancer. Not only because cancer genetics is an advancing science where we can help families. But for example, if you’ve got pigment spots in the retina, congenital hypertrophy of the retinal pigment epithelium, it may be Gardner syndrome. You ask… Did anyone in the family have colon cancer? How did you know that? My father had a colectomy. Some people have problems with driving and low vision. Oh, it’s a problem, but if I put my glasses on, I can see fine. You have to explore the family history to get an idea of what’s actually happening. And then physical exam. This thing that’s pictured here, you may not be familiar with. You put it in your ears, hold it to patients’ chest, and you can hear their heart. A stethoscope. Well, if you can’t do a full physical exam of a patient, you need to get someone who has. Because oftentimes, as I’ll show you, there are things which can give you the clue to your very own patient. Maybe the patient had an earlier problem. This is a photograph that a patient shows me from when the child was a baby. This was the rash of incontinentia pigmenti, which would explain the peripheral non-perfusion or falciform retinal fold due to that. Take the mask off in the pandemic. If you’ll notice, this woman has a bifid uvula. She came in for a retinopathy. She’s got radial perivascular retinal lattice, retinal detachment in one eye. She has Stickler Syndrome. She may not have a cleft palate associated with this disorder. That’s the only sign of clefting that she has. Here’s a boy who came in to see me. Look how he wears his hair. If you didn’t look closely, you would notice there’s a hearing aid there. He has a cone dystrophy. He has this. I said to the mom: Could you have him take off his shoes? And mom said to me: Is this the eye doctor? What’s going on? And sure enough… He has what’s called Bowes lines on the toes, the nails. And he has amelogenesis imperfecta on his teeth. And this tells us that he has Heimler syndrome, a retinal dystrophy due to a peroxisomal gene. You would only need to test two genes in this patient. Sometimes you need to check really carefully. Look at that circle there. A little scar on the edge of this patient’s hand, where she had prior finger removed. She forgot to even tell you. You have to look. But guess what? I can tell you, that patient doesn’t have Bardet-Biedl. Because if you look at Bardet-Biedl, their hands look different. They have tapered fingers. And you can see this patient is overweight as well. These details matter. And if you don’t seek them, I can tell you, you won’t find them. Then… And only then… After you’ve done your history, asked history all those different ways, got your family history, did your physical exam, then and only then can you do an eye exam. And that’s where things begin to fall into place. Look at the pupils. Do the pupils dilate when you give them light? The paradoxical pupil, characteristic of CSNB and other advanced retinal dystrophies? Are the eye movements abnormal? Is there ophthalmoplegia or ptosis that might suggest a mitochondrial etiology? What is the patient’s vision? Of course. Slit lamp examination. What’s the cause of the vision defect? The patient may be 2200 from a cataract and have a normal retina. What does the vitreous look like at the slit lamp, when you’re evaluating a patient for possible vitreoretinopathy like Stickler syndrome or Wagner syndrome? And please, please, don’t forget to refract your patients. I’ve seen patients who come to me, told they’re going blind from retinal dystrophy, and they just have pathologic myopia. And is their myopia axial? Or is it lenticular? Is the lens central or is it becoming loose? And what is their best corrected vision? You know, a patient comes to you with 20/200 vision, if you can give them glasses and make them 20/50 even with a retinal dystrophy, you’re gonna be a hero. Don’t forget this important step of the exam. And finally, we’re doing inherited retinal dystrophies. Look how far we had to come to get to the retina exam. And if you need to, don’t be afraid to do an examination under anesthesia or sedation, to get a good look at the retina and to get the appropriate diagnostic tests to help you understand what’s there. One thing that’s very helpful is photography. Especially in young kids, who can sit, for example, at an Optos, or wide field, non-mydriatic camera. Oftentimes when you see that still shot, you see things that you don’t see in real life. But also, photography is wonderful. But you must do an in-person live examination. Can’t tell you how many patients I see where I thought there was optic atrophy, but in real life, there’s not. Or the photographic artifact tells you something that isn’t there. Use it, but use it with a grain of salt. And then we get to the supportive diagnostic studies. The OCT usually of the macula, but sometimes the nerve fiber layer can be helpful, when you’re wondering: Is it an optic neuropathy? Or is it a retinal disease? Fundus autofluorescence. Color vision testing. Something beyond. Usually a D15 or D100. Goldmann visual field. That’s the standard of care rather than a Humphrey. There’s so much more information you get from a Goldmann. Multifocal if they can see well enough and a ffERG would be helpful. Dark adaptation. 3 lead VEP. That can be helpful for diagnosing albinism or related disorders. And sometimes IVFA can be useful as well, as I showed you. So now we phenotype, phenotype, phenotype, phenotype. How do we get to a genotype? Well, that’s gonna be putting the puzzle pieces together. So here is the patient. It’s got some intraretinal cystoid spaces. Don’t call it CME. You don’t know if it’s CME. That would be a leaking intraretinal cystoid space. Don’t call them cysts. It’s not epithelial line. Is it leaking or non-leaking? Let’s put it together. We don’t need a fluorescein often to be sure. But let’s see. 8-year-old boy. He’s got day vision. Blurry vision. Medically entirely healthy. Full field ERG shows a depressed b wave. Fundus autofluorescence is normal except for an abnormal loss of normal foveal hypofluorescence. D15 normal. Goldmann visual field normal. What’s he have? Here is his pattern of inheritance. His maternal grandfather had the same disorder. Or had a disorder with a retinal detachment. He’s got juvenile X-linked retinoschisis. You can test one gene and get your answer. What if I took the same thing, gave it to a 30-year-old patient with night vision problems, slow progression of this disease. Otherwise entirely healthy. Only other affected family member. And look down here. He’s got paraarteriolar sparing that can be seen in this test. Either fluorescein or fundus autofluorescence. One gene. CRB1. You don’t need a whole panel. You don’t need whole genome sequencing if you do this process of phenotype, phenotype, phenotype. How about if we take a 20-year-old male? RP, sporadic, and the mom said she had something wrong on her exam. It looked funny in there. The guy is entirely well. His vision is fairly poor at this age. Well, hm. Male RP. The most common gene with early onset RP is gonna be RPGR. Well, guess what? Here’s mom. You have to examine the family member. And she shows this pattern of the X-linked recessive carrier, that radial pattern of changes in autofluorescence due to lionization. One gene. RPGR. And this is a gene that has to be tested correctly. It’s not included on many panels, and even if it is, they don’t test the open reading frame of the gene, which is where many mutations lie. So the big panel would be less effective. Not to mention all the downstream effects. You can get to one gene by looking at this study. Here’s a patient I just saw recently. Comes in with 20/200 vision in each eye. Only affected family member. Otherwise entirely healthy. She had normal ERG, FAF, OCT, D15, even her exam was normal. Here’s her Goldmann visual field. What does she have? Nothing! I tricked her into seeing 20/20 through the phoropter. She doesn’t need any gene test. So this is to show you the value of doing the appropriate history and exam. There were other indicators that she might be faking on the history. And do tests as needed. Rather than go right to a gene test. As many people do. A large panel with information is typical. Again, the goal. Phenotype, phenotype, phenotype. Gets you to a genotype. Well, it’s easy. Right? No. It’s actually hard. It takes a lot of work and a lot of time. If you have a diagnosis, do the test for that specific diagnosis. But regardless of the test, remember, pre- and post test genetic counseling is essential. I don’t have time to go into that. But there’s many things that have to be told to the patient before they get a test and interpretation afterwards. If you almost have a diagnosis, like… I’m pretty sure maybe it’s maculopathy, or maybe there’s hearing loss… You know what? ABCA4 is the most common gene mutation. USH2A, can cause isolated RP. Go for the money. As a famous bank robber, Willie Sutton said, when asked why he robbed banks. He said: That’s where the money is. In your country, there may be particular genes that are more commonly the cause of associated problems. Test those first. It if you have almost a diagnosis, based on the retina pattern, is it a maculopathy? A cone distribution? Smaller panels? Is it autosomal recessive or X-linked recessive RP? Smaller panel. Or the disease type. Are you looking at a vitreoretinopathy, retinal non-perfusion? You can get a smaller panel to help avoid that false genome rate. The basic principles when choosing a test are to narrow your gene hypothesis using the methods I’ve shown as much as possible. But also, you have to understand the test. If you don’t understand how to interpret a test, as I’m gonna show you, don’t use it. This isn’t just a hemoglobin level, where it’s pretty easy to interpret. Then again, you have to know: Is the MCV high? Is it macrocytic anemia, et cetera, et cetera. Keep going. And we can find an answer for a gene 80% of the time. But you say: Oh, it’s free and it’s so easy. I want to do a large free panel. It ain’t free! Companies are paying for these panels, because they want to get patients who will get their treatments that cost hundreds of thousands of dollars. You can do better than that. Many of these free panels are done without any in-person genetic counseling. They have the false genome rate, with unintended consequences, as I’ll show you, and you still have to interpret the tests, as I’ll show you. You get the wrong diagnosis. And if you get the wrong diagnosis, that leads to inappropriate treatment. Or enrollment in the wrong clinical trial. If you’re doing gene therapy for the wrong gene, it ain’t gonna work. The wrong counseling. You counsel recessive when it’s a dominant disorder or vice versa. And unnecessary screening. People who are being screened because they’re at risk when they’re really not. Let me show you an unfortunate case. Six month old boy, presented with nystagmus. He was entirely healthy. Normal prenatal, neonatal exam. He had a complete eye examination that was normal except for nystagmus. The family history was interesting. Revealed… Not really much in the way of clues to the inheritance pattern. The parents were very intent on having a normal baby. They had prenatal carrier screening testing. Mom has a gene for familial chloride diarrhea. Dad had the DHDD5 gene, the second most common cause of retinitis pigmentosa in Ashkenazi Jews. And they go… Can my child get these? These are all recessive disorders. There’s only one copy of each gene that’s found to be abnormal. The other parent would have to have the same one and the child would have to get both. So likely the risk is very, very low. Well… They went to a big academic children’s hospital after the child had the nystagmus. And they did whole exome sequencing. And guess what? They found sequence variations in USH2A, WFS1, for Wolfram syndrome. Does this child have RP, or even worse, Usher syndrome? It doesn’t cause nystagmus. The kid’s had two hearing tests, and they’re normal. But it may be too young to know. Wolfram syndrome, rare recessive or dominant neurodegenerative disease? Very rare. Doesn’t present with nystagmus. Hearing test normal, negative family history, no DI/DM. None of the signs. But it’s too early to tell. These parents are worried. So every time you get a test… All of these tests… I just put it here to show the amount. Not to go through them. But we look for… Is the gene mutation in a place of evolutionary concern? What is the biologic significance? What do in silico computer models show? Has it been reported in the database? And so on and so forth. We have to test the parents as well to make sure each has one copy, as opposed to the child having both abnormalities on the same chromosome, which not be as important for the recessive disease. We do for the same with the Wolfram syndrome. And guess what we came up with? The child just has nystagmus. The child just has sequence variations. Usher, no. RP, no. Wolfram no. Can we be 100% sure? Maybe not. This is the impact on the parents. The poor parents, who are gonna wonder every day. I’m still in touch with them. They just can’t get it out of the back of their minds. That this testing gone astray has led them down a road, where they don’t know where they’re going. When you’re choosing a test, you can go to this website. Clinical tests. NIH. And this is a site that will tell you what tests are available, where to send for what genes, and so on and so forth. If money is an issue, research contacts and collaborations can be helpful. You can get research testing for free. And then it can be confirmed with a clinical laboratory. There are ways to get all the tests that you want done. You can email me! There are challenges. When you have an unknown diagnosis. When you don’t have the knowledge to interpret a test. When you don’t feel in control over the testing, and therefore you order free panel just for convenience. Cost and access I realize are a problem in every country. If you don’t know a diagnosis, and more than one organ system is involved, start with a karyotype or a microarray, looking for pieces of chromosome that are extra or deleted. Taking out more than one gene. Maybe the problem is similar to something else. And that could help you think… Oh, maybe it’s this. I can get a candidate gene that I can test, that’s close to this. Or a panel, as I said, for the disease category. Maybe it’s two diagnoses. Maybe I test for the hearing separately from the RP. There are different ways to tease out the diagnosis. But if you have an unknown diagnosis, all the tests that you’ve done in a focused way are negative. And you have no theory. You just have a patient with RP, sporadic RP. That is when we can turn to something like large panel whole exome sequencing. Customized panel exome slice, or if it’s mitochondrial, whole mitome. That’s when you do those things, or even whole genome sequencing. I would say with whole genome sequencing, we use them more when there’s really a chance we can do something now for a patient. Especially life saving, where we can do rapid genome. To find an answer. Just do it first? Too much information! There are 56 mandatory disclosure actionable genes, like fibrillin-1 for Marfan. But what about the rest? Who wants to know if they’re at risk for Alzheimer’s disease? And it’s costly. And you have to involve the family. There are data management issues. You get the results back that are dependent on what you put in. Phenotype, phenotype, phenotype. And it ain’t perfect. We don’t have the answer for everything. Even these tests can be normal. Due to epigenetics and environmental phenomena. And some things we just don’t have an answer for. But we get an answer about 80% of the time. Genetic testing is a process. It requires genetic counseling. It’s complex and it’s difficult. But if you phenotype, phenotype, phenotype, phenotype, you will get to a genotype more accurately. In a tiered fashion, starting specifically, and then moving out as needed for a given patient. This is a formula that works. And I encourage you to use it based on the tips you’ve heard here today. If we make a diagnosis, well… Then we’ve got knowledge for the patient. And ourselves. They can get the support. The patients have some feeling of control. And let me tell you: Almost every patient would rather have a bad diagnosis than live a life of uncertainty. Genetic counseling can be done. We can identify those at risk in the family. And now we give some idea of the future. And the future is bright. I tell all my retinal dystrophy patients, especially the young ones, you will have treatment. I’m certain of that. There’s so much going on. Is it going to be gene-based? Gene therapy or stem cell, say, with CRISPR to correct the defect, give the cell back to the patient, their own stem cells? That’s where you need to have a gene diagnosis. Or will it be gene agnostic? Medical manipulation? Gene therapy? Stem cell? These are all coming down. Patients will get treated. Especially young patients. Within their lifetime. Will treatment restore all of their vision? Maybe not. For RP. But maybe we’ll keep them from getting worse or accentuate their vision in visual field. All of these things are real and very possible. And should drive you to be very careful in phenotyping, and then in genotyping. The first step to “you will have treatment!” Is to get a genotype. That’s why this work is so important for our patients and for ourselves. I’m gonna stop there. Purposely left some time, as instructed, for questions. I think I left 15 minutes. I’m gonna stop my share. I’m gonna do one thing, though, that I forgot. If you let me, while I’m still sharing… I forgot to put this in. Please feel free to contact me any time with questions that aren’t covered here. There’s my email address. I’ll leave that there for you to copy down. And it’ll be on the slide set. I’m always happy to answer your questions, to help you out with a case, help you figure out a way to get testing, et cetera, et cetera. Feel free to reach out. I’m gonna go to the Q and A here. Feel free to put your questions in. Into the Q and A on your screen. Question one. In the first case, what would you do next if you did not get a positive response from a single gene evaluation? That is a fabulous question. What does a negative gene test mean? Well, a negative gene test can mean many different things. It could be that you got the wrong diagnosis. And therefore you tested the wrong gene. Rethink your diagnosis. Phenotype, phenotype, phenotype. It could be that you have the right diagnosis. You tested the right gene. It was correctly negative. But there’s another gene, either known or yet to be discovered, for that syndrome. That’s when we would expand our panel to look for another gene, if you’re doing a research investigation. Another possibility is that you have the right diagnosis in the general sphere of things. So for example, a microcephalic patient with that type of retinopathy could be one of the pseudochorioretinitis syndromes that also have peripheral retinal non-perfusion. Or maybe if there was not microcephaly, I look at other FEVR genes. Exudative vitreoretinopathy. Follow the line. Expand back in your puzzle to get a pathway for treatment for diagnosis to get you where you need to go. Do the right tests. And think it out as you go. The next question. Is there a way to support and establish a genetic testing service for retinoblastoma in Nigeria? That’s a very timely question. One of our ophthalmology fellows is Onochie Ike Okoye from Nigeria, the only ocular geneticist in all of Africa, going back to Nigeria, his home country. And he’ll have the opportunity to work with you, to help you set up genetic testing services there. Retinoblastoma is a single gene test. There are some complications in that. It’s not the easiest test in some ways. But setting up testing is also not as important as trying to set up pathways to get testing. What do I mean by that? You don’t have to have a lab. I don’t have a lab for every single gene in the world. Many tests get sent out from my center. You need to link up with an ocular geneticist like Dr. Okoye. When he’s back in Nigeria, who now knows the people to connect with, to send blood to, who will accept blood specimens, and the places and the pathways. I know that in some countries, getting blood out of the country is an issue. And for those countries, there are ways to use existing facilities to get genetic testing done. And not every person in the world is going to have access to testing. That’s a problem we’re trying to solve by working with Orbis to bring genetic testing to other countries. And we’re working on that now. Piloting a project in Thailand and the Philippines. But… If you have the pathways, you don’t have to set up the tests yourself. How do you deal with VUS in Pakistan on retinal gene panel test done in the USA? Yes. VUS, which means… Variant of unknown significance… Is the biggest problem with large gene panels. Every person listening to me right now has VUSes in their genes for retinal disease. We all have them. The question is: How do you evaluate a VUS? And that’s where ocular genetics knowledge comes into play. I quickly showed you those slides that have all the different things that we go through. There are many databases and ways to analyze a given gene change for its biological significance, likelihood of pathogenicity. And unfortunately you can’t trust the lab. The lab will tell you one thing, and we disagree. We think it’s something else. You have to do your own homework on every test you get back. And you can do that, once you learn how to manipulate the databases or whatever. You can send me an email. I’ll help you find it out. But having ocular geneticists or access to them is very helpful. Another thing is testing family members. If a patient has two mutations, let’s say, that come back in the USH2A gene, they’re not causing disease if they’re on the same chromosome. If you test the parents, and see that each one parent carries one, that’s helpful. Right? Doesn’t prove it’s causing the disease. But that testing is also another way to unravel the VUS. Sometimes it’s a VUS, but the phenotype is so darn convincing that that helps you turn that VUS into a pathologic change. So it’s a lot of work sometimes to analyze those variants. What is the role of coenzyme Q in Kearns Sayre and vitamin A in RP? A little bit off-topic. But I’ll just say there’s many things called the mitochondrial cocktails that people recommend sometimes for mitochondrial disease. I’m not sure that we’ve got good evidence that they do anything. Coenzyme Q being one of the ingredients of those cocktails. To tell you the truth, mitochondrial specialists… They recommend these all the time. I’m not sure there’s any good evidence that they make a difference. Vitamin A in RP — based on studies that were very poorly done a long time ago, there are some genotypes of RP where vitamin A can make you worse. In particular A4. So one has to be careful. We do not recommend vitamin A for our patients. Can the ERG guide us to narrow the number of genes? Absolutely. Thank you for that question. The full field ERG is gonna tell you: Is this a rod disorder alone? Rod-cone? Cone-rod? Or simply a cone disorder? Right there, you’ve got four categories of disease. Right? And that will help you narrow your gene hypothesis. So it’s very, very helpful. Of course, if you have a full field ERG that’s normal, and you have some subtle changes under the fovea in your OCT, is this blue cone monochromatism? RP1L1, occult macular dystrophy that has a normal field ERG? Even a normal ERG can help you. Great question being asked about artificial intelligence to help detect certain phenotypes. Especially for facial dysmorphic features. I must say that AI has been disappointing in this realm. Because AI can’t do the phenotype, phenotype, phenotype. There’s a limit to what it can do. AI can recognize certain patterns. But we really are far away from putting that into use. There is a thing called FACE2, the number 2, gene for facial dysmorphism. I find it very disappointing. It can give you some suggestions of what a patient has. It uses AI. But I think we’ve got a long way to go, before AI is gonna really help us in these dystrophies. It’s gonna have to be paired very carefully with phenotyping, and all the forms of phenotyping that I spoke about. What is the phenotype diagnosis of Q1, please? So patients with KIF11, it’s a dominant disorder, usually with microcephaly and peripheral non-perfusion. The thing that was so characteristic in that was that patch of silvery, glassy-like chorioretinal changes below the macula. Below the inferior arcade. That’s a very characteristic finding in KIF11 and should make you think of that disorder when you see it, along with other findings. Thank you for this question. Would I have taken that patient or that picture and ordered a genetic test? No, I would not. I would have done phenotype, phenotype, phenotype, phenotype. I would have asked for family history, hat size, past medical history. I would have done the necessary tests. I probably would have done a fluorescein angiography before I even did the gene test. But I could have waited ’til afterwards, to prove that my theory was correct, before I put the kid through that. But maybe on clinical exam, there was evidence of peripheral non-perfusion. And by the way, that reminds me: Never never never rely on your clinical exam to rule out peripheral non-perfusion. Do a fluorescein. If doing ERG, OCT, AFG, which I assume is autofluorescence, and we establish the type of retinopathy, when do you recommend genetic testing? After I’ve completed my phenotype. I do all those steps, and when I have a clear view of where I’m headed on this pathway, that’s when I do the genotype. In some countries, there’s a current practice. A patient comes in. They’ve got a retinopathy, order a big panel. And they work backwards from that. That is not gonna help you. That is just gonna get you pigeonholed in the wrong directions and the wrong way. You want to phenotype first. And genotype later. It looks like we’re out of time and that’s our last question. I want to thank you all very, very much for spending this hour with me. Again, you’ve got my email. [email protected] Always happy to help. Thank you for attending. Have a great day.

Last Updated: October 31, 2022

2 thoughts on “Lecture: The Fundamentals of Genotyping & Phenotyping for Inherited Retinal Diseases (IRD)”

  1. Thank you Sir for a detailed Lecture! wonderful Take home message that do not rush in for genotype without proper phenotyping with detailed exam starting with proper history and arriving at a diagnosis. Genotype is a commitment towards the patient for continuous guidance on treatment and Genetic counselling! Thanks again!


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