Biallelic loss-of-function variants in SAXO6, a microtubule inner protein of photoreceptor cilia, cause late-onset retinal dystrophy by destabilizing axonemal microtubules.
0:00Welcome to Base by Base, the papercast that brings genomics to you wherever you are. Thanks for listening, and don't forget to follow and rate us in your podcast app. Imagine yourselves as having um, having these tiny, highly specialized antennas projecting right from their surfaces.
0:15Yeah, Vacilia. Right, cilia, and they are absolutely essential for sensing the world around you. Now, what happens if the microscopic scaffolding inside those antennas starts to fail? And, you know, not when you are a child, but slowly, almost imperceptibly, much later in adulthood.
0:32Exactly. We actually know quite a lot about the surface of these cellular antennas. I mean, science has mapped out the proteins that build their outer walls in really incredible detail. But what really happens when the internal support beams, uh, the proteins hidden entirely away inside these microscopic tubes begin to collapse.
0:49How could a single missing microscopic support beam lead to a gradual, progressive form of blindness that is exactly what we are exploring in today's deep dive into the hidden architecture of human sight?
1:02It is such a compelling question because it, well, it forces us to look beyond the obvious. When we think about cellular structures or really even human-made architecture, we naturally focus on the visible exterior.
1:14Right, the stuff we can easily see. Exactly. But the integrity of that architecture frequently relies on hidden internal components that are, quite frankly, incredibly difficult to isolate and study. When those internal components are compromised, the downstream effects on human health can be profound, yet they remain practically invisible to our standard diagnostic tools.
1:36Today, we celebrate the work of Abigail Armoye, Carlo Revolta, and their extensive team of collaborators, spanning institutions like the Institute of Molecular and Clinical Ophthalmology, Basil, and the University of Basil, who have advanced our understanding of the genetic and structural causes of inherited retinal diseases.
1:54To really grasp the significance of their findings, we need to understand the clinical landscape these researchers are navigating. They're investigating a category of conditions known as inherited retinal diseases or IRDs.
2:06These are genetic disorders, right? Right. Yes. Genetic disorders that specifically affect the survival and the function of your photo receptors. Those are the highly specialized neurons in the back of your eyes that actually detect light and translate it into a signal your brain can understand.
2:22And this research specifically touches on 2 prevalent subtypes of these diseases which manifest in, um, in pretty devastating ways for patients. The 1st is retinitis pigmentosa. Right. For someone living with this condition, the onset is usually insidious.
2:37You typically experience progressive night blindness first. Then slowly over time, you begin to lose your peripheral vision. It creates a sort of tunnel vision effect. Yeah, and eventually, the disease progresses to affect the central vision as well, leading to total blindness.
2:53The 2nd subtype they focused on is con rod dystrophy, which presents quite differently. In this condition, instead of losing the peripheral vision first, the cone cells in your eye are the primary casualty early on.
3:04And your cone cells are the ones that handle color vision, right? Along with sharp central visual acuity. Exactly. So for a patient with cone rod dystrophy, they are losing their ability to read, to recognize faces, or to see fine details right in the center of their visual field.
3:21Plus, it causes a severe painful sensitivity to light, which is known clinically as photophobia. That is the daily reality for these patients. Now, if we pan out and look at the bigger picture of genetic medicine, we encounter a massive hurdle in treating these conditions.
3:38It is known in the field as the missing heritability gap. The missing heritability gap. Right. Over the years, scientists have linked more than 500 different genes to various forms of inherited retinal diseases, and incredibly, between 17 and 25% of those genes are directly related to the function of those tiny cellular antennas we mentioned.
3:57The celia, which really highlights just how vulnerable the celia in your eyes are to genetic defects. I mean, if even one protein is built in correctly, The whole antenna can just fail. It can, but here is the heartbreaking part for families dealing with these diseases.
4:11Despite our incredibly advanced genetic sequencing technologies today, a huge gap remains. Depending on the specific study or population, anywhere from 20% to 50% of patients with these conditions still lack a clear genetic diagnosis.
4:27We really need to pause and think about the human element of that statistic. Imagine sitting in a doctor's office, losing your site and agreeing to undergo comprehensive genetic testing. You wait weeks for the results, hoping for an answer, a target, maybe a pathway to a clinical trial.
4:45And the results come back completely negative. Exactly. The doctors essentially tell you that you have a genetic disease, but they cannot find the gene responsible. There are hidden genetic culprits out there that our standard diagnostic lenses simply haven't been able to bring into focus.
4:59So, to figure out where these hidden culprits might be operating. We have to look much, much closer at the biology of the eye itself. We need to talk about the photoreceptor cilium. You could think of it as a highly modified version of a standard cellular antenna.
5:13In most cells, a normal primary pilium just sits there, acting as a non-moving sensor for the cells environment. But in your eye, this structure is specialized to an extreme degree. It really is a marvel of biological engineering.
5:26This specialized pilium acts as the vital connection between 2 main compartments of the photoreceptor neuron. On one side, you have the inner segment. Which is like the factory. Yes, you can conceptualize the intersegment as the cell's bustling biosynthetic factory.
5:42It is where all the necessary proteins, energy molecules, and lipids are continuously manufactured. And on the other side is the outer segment, which is where the actual magic of vision happens. The outer segment is packed with these stacked membranous disks that house all the light sensing machinery.
5:58So the cellular antenna, the cilium, acts as the only physical bridge connecting the factory to the light sensors. And that physical bridge is incredibly narrow. It is a microscopic hallway about 1,100 nanometers long and only 300 nanometers wide.
6:12Every single molecule required to build and maintain those light sensors over your entire lifestan has to be safely transported through that tiny corridor. And the structural integrity of that bridge is maintained by an arrangement of microtubules.
6:25These are basically tiny hollow protein tubes that form a cellular skeleton. For a very long time, researchers have focused intensely on the proteins that attach to the outside of these tubes. Because that is where most of the genetic discoveries have been made historically.
6:39Right. But the team in this paper decided to focus on a protein that was, until now, a bit of a mystery in human disease, a protein known as MDM1. MDM1 stands for mouse, double minute one nuclear protein. Historically, it was mainly known in the scientific community as a protein that hangs around the cell's centrosome.
6:59And the centrosome, along with structures called centrioles, basically acts as the main organizational scaffolding center for a cell, right? Yes, MDM one was known to suppress the duplication of these centrioles.
7:10We did have some clues from animal models, though. Scientists knew that if you knocked out this protein and mice, it caused their retina to degenerate. But mice are not humans. Its true role in the human eye, and crucially, whether a defect in this protein actually caused blindness in humans was totally, completely unproven.
7:28To solve this mystery, the research team had to employ a highly rigorous multi-layered methodology. They couldn't just guess they needed hard data. So they started with genetic sleuthing. They gathered extended cohorts of individuals diagnosed with late onset recessive retinal dystrophy.
7:46Importantly, these were patients who previously fell into that missing heritability gap. They had the disease, but no clear genetic diagnosis. To find the needle in the haystack, they utilized whole XM sequencing, and in some more complex cases, whole genome sequencing.
8:02For those listening who might wonder about the difference, Holixum sequencing focuses specifically on the protein coding regions of your DNA, the instructions that actually build the cell's machinery. Which represents about one to 2% of your total DNA.
8:15Exactly. Whole genome sequencing, on the other hand, looks at almost everything, including the vast regulatory regions between the genes. By meticulously sifting through terabytes of genetic data from these undiagnosed families and comparing their DNA to healthy populations, they finally found a matching anomaly in the MDM1 gene.
8:33But here is the critical limitation of modern genomics. Finding a mutation in a sequence of DNA doesn't tell you how the resulting protein actually functions or fails to function inside the three-dimensional architecture of the human eye.
8:48Genetics gave them the what, but they still needed the how and the where. To figure that out, they needed to look at the cellular skeleton itself. But the structural beams inside these cellular antennas are so incomprehensibly small that standard laboratory microscopes simply cannot see them clearly.
9:06This is where we run into a fundamental law of physics called the diffraction limit of light. Even with the best traditional light microscopes, structures that are closer together than about 200 nanometers just blur into a single glowing blob.
9:18You cannot distinguish the individual parts. So how do you see the minute nanoscale details of a structure as delicate as a cilium? Instead of trying to build an impossibly perfect magnifying glass, the researchers use a brilliantly innovative technique called iterative ultrastructure expansion microscopy.
9:36They change the sample itself. I am obsessed with how they did this. It sounds like science fiction. Can you explain how it works? I like to think of it like drawing a highly complex, detailed picture on a deflated balloon.
9:48When that balloon is completely deflated, the lines of your drawing are squished tightly together. If you look at it, you can't tell what the drawing is. It just looks like a dense smudge of ink. But if you take a deep breath and inflate the balloon, the rubber expands uniformly in all directions.
10:04Suddenly the lines separate and all those intricate details become perfectly clear. So they essentially inflated a human tissue. Essentially, yes. They embedded human retinal tissue into a specialized water absorbent polymer gel.
10:18Then, through a series of chemical reactions, they forced that gel to swell and expand. Wow. They physically enlarged the retinal tissue by a factor of about 10. By inflating the biological balloon, they pulled the densely packed proteins apart just enough to bypass that physical limit of light.
10:35This allowed them to clearly see the nanoscale scaffolding of the cilia using a standard convocal microscope. That is just incredible. But seeing the structure still wasn't enough. They didn't stop with imaging.
10:46No, they needed biochemical proof of how this specific protein was physically interacting with the cellular skeleton. They needed to see the molecular handshake. So they used a technique called cross-linking mass spectrometry, and to get enough cilia to actually run this test, they had to source them from somewhere highly unusual.
11:05They needed a massive volume of modal cilia, the kind that actively beat and move. So they isolated 1000s of moving cilia from the epithelial cells of bovine tracheas. Cow windpipes. They use the microscopic hairs inside the windpipes of cows to solve human blindness.
11:22Science is wild. It is a perfect example of how highly conserved these cellular structures are across different mammalian species. The fundamental building blocks of a cow's airway cilia are incredibly similar to the sensory cilia in a human eye.
11:36Once they extracted these bovine cilia, they applied a chemical cross linker. What does a cross-linker actually do in this context? You can visualize the chemical cross-linker as a pair of microscopic molecular handcuffs.
11:48If 2 proteins are sitting right next to each other physically touching or interacting, this chemical binds them permanently together. It freezes them in place. Then by taking this frozen complex and running it through a mass spectrometer, which analyzes the mass and charge of molecules to determine their exact composition, they could map the precise amino acid level connection.
12:10They were searching for the exact point of contact between the MDM1 protein and the microtubial structures. Let's unpack the results. What did this combination of genetic sleuthing, balloon expanding microscopy, and cow windpipe biochemistry actually reveal?
12:25Here is where it gets incredibly rewarding for those patients who spent years living in the dark regarding their diagnosis. The team identified biologic loss of function variants in the MDM1 gene across 6 subjects from 5 unrelated families.
12:38And we should clarify what biolic loss of function means for the listener. We inherit 2 copies of most genes, one from each parent. Biolek means that both copies of the gene the patient inherited carried a mutation.
12:49And loss of function means those mutations were severe enough that the body could not produce any working version of the protein at all. The essential building material was completely missing. Because this study provided such definitive proof linking the gene, not just to centrioles, but to a crucial structural role in the cellular antenna itself.
13:08The scientific community actually officially renamed the gene. It was changed from MDM1 to SAC those six. That stands for stabilizer of Xonimal Microtubial 6. Renaming a gene is a significant event in biology.
13:22It reflects a fundamental shift in our understanding of its true biological function, moving away from how it was initially and perhaps narrowly discovered in mice. And what's truly fascinating here is where the researchers finally located Saxo 6 using that expansion microscopy.
13:37It isn't just floating randomly around in the cell fluid, and it isn't attached to the outside of the sillier skeleton like most known disease causing proteins. Saxo6 is a microtubule inner protein. In the field, we call them MIPs.
13:49It is an inside job. The microtubules that make up the skeleton of the cellular antenna are long, hollow tubes. The researchers proved that SACAXO6 laves completely hidden inside the lumen, the empty interspace of these ciliary microtubules.
14:05That perfectly explains why it was so incredibly elusive for so long. If your genetic investigations and microscopic imaging are only looking at the outside of the cellular scaffolding. You are never going to see the support beams running through the dark interior.
14:18And the structural nuance they uncovered by looking inside is striking. The ciliary skeleton extends from the inner segment factory straight through the connecting hallway and distally into the light sensing outer segment.
14:29Right. In the rod photo receptors, the ones responsible for your night and peripheral vision, the researchers noticed an asymmetry. The skeleton of a standard celium is generally made of 9 pairs of microtubules arrange in a perfect circle.
14:43But in rod cells, SAXO6 didn't line every single microtubule evenly. It only collocalized with 6 to 7 of those microtubial doublets. But in the cone cells, the ones responsible for color and sharp central vision, it was completely different.
14:57In the cones, SXO6 bound to all 9 doublets in the circle. That structural asymmetry in the rods versus the total reinforced symmetry in the cones is a massive clue. It's just a highly specialized way. These different cells manage the physical stress of keeping those delicate outer segments stable over the course of an entire human lifespan.
15:18That physical stress is not trivial. Our eyes are constantly moving, and the proteins in those outer segments are constantly being renewed. The scaffolding has to be incredibly resilient. And the cross-linking mass spectrometry data provided the final molecular confirmation of this resilience.
15:34The researchers found the exact molecular handshake. Saxo6 binds directly to alpha tubulum. Alpha tubulin is one half of the fundamental two part building block that constructs the entire microtubial wall.
15:45And the chemical handcuffs prove just how tight this connection is. The cross-link data shows, but the physical distance between these two interacting proteins is an incredibly tight 30 angstroms are less.
15:56To put that scale into perspective for you. 30 Angstroms is roughly the width of a standard DNA double helix. It is an unbelievably tight intimate connection, hidden deep inside the dark core of the microtubial structure.
16:10If we synthesize all this data and look at the broader implications for human health, this study is a landmark. It links sex of proteins to mendili meaning inherited genetic conditions for the very 1st time.
16:23It provides definitive proof that mutations and microtubial inner proteins can directly cause human inherited retinal diseases. This firmly establishes the condition as what we classify as a 1st order seliopathy.
16:35The primary defect causing the blindness is intimately, structurally tied to the architectural integrity of the cilium itself. As I was reading through the paper, there was a clinical observation that really made me pause.
16:46Obviously, the primary symptom for all these patients in the study was progressive blindness. But the clinical data showed that 5 out of the 6 patients also had type 2 diabetes. Now, my 1st thought playing the skeptic was, isn't type 2 diabetes incredibly common in the aging population anyway?
17:00How do we know this isn't just a total statistical coincidence that these specific older patients happen to have both? It is the exact right question to ask, and the researchers rigorously addressed it.
17:11They ran the statistical probabilities comparing this specific small cohort to the general population. The statistical enrichment for type 2 diabetes in this specific group yielded a P value of .0002. Which, in plain English, means there is roughly a one in 1000 chance that this is just a random coincidence.
17:30That is a very strong statistical signal. A very strong signal that raises profound implications. Are we looking at a localized eye disease, or are we looking at a syndromic sillyopathy? Syndromic sillyopathies?
17:42Are conditions that affect multiple systems in the body simultaneously? A classic example is Bardep Beetle syndrome, which often presents with both retinal degeneration and systemic metabolic issues like severe obesity and diabetes.
17:54Because, as the researchers demonstrated with the cow tissue, Saxo 6 isn't just in the eyes, it is found in Cilia throughout the entire body, including the airways. Correct. Since this internal support beam is utilized in various tissues, it is entirely plausible that losing it affects metabolic signaling pathways elsewhere.
18:14such as in the pancreas or adipose tissue. However, the study rightly exercises caution and notes this as a critical area requiring much further large scale clinical investigation before definitive systemic claims can be made.
18:27We also have to acknowledge the study's broader impacts on genetic screening, as well as its current limitations. For the fuel of genomics as a whole. This fundamentally redefines how we approach those patients, trapped in the missing heritability gap.
18:40If a patient comes in with progressive blindness, and their genetic sequencing comes back negative for all the known genes that build the outside of the cellular antenna, doctors and researchers now know they have to look inside the support beams.
18:53This opens up a whole new category of hidden structural proteins to investigate. As for limitations, the scientific journey is never fully complete. While the researchers successfully proved that Saxo 6 resides completely inside the microtubule and binds tightly to alpha tubulin, We still lack a complete three-dimensional model of the interaction.
19:11For their structural analysis, using ultrahigh resolution techniques, such as cryoelectron microscopy, will be necessary. We need to see exactly how 606 bridges the individual building blocks of the tubulent wall.
19:24Does a single Saxo6 protein span across multiple units to reinforce the structure. How exactly does its physical presence absorb or regulate the inner stability of the microtubule when the cell is under mechanical stress?
19:37The dynamic mechanics of this internal support system still need to be fully mapped. The discovery of SACO 6 variants reveals that the microscopic internal scaffolding of our retinal cilia is absolutely essential for long term vision.
19:50When these hidden inner support proteins are lost due to a genetic mutation. The photoreceptor's delicate architectural structure, eventually collapses under the weight of time, leading to late onset blindness.
20:02It serves as a powerful reminder that what happens on the very inside of a microscopic tube entirely hidden from view can ultimately determine whether a complex sensory neuron survives or degenerates over the decades of a human life.
20:15If our cellular antennas require such intricate hidden internal scaffolding to function over decades, what other structural microskeletons in our bodies are quietly holding our senses together, just waiting to be mapped?
20:29What does this mean for the future of genetic medicine, and how we look for the hidden causes of human disease? It is going to be fascinating to see where this leads the field next. This episode was based on an open access article under the CCBY4 license.
20:44You can find a direct link to the paper and the license in our episode description. If you enjoyed this, follow or subscribe in your podcast app and leave a 5 star rating. If you'd like to support our work, use the donation link in the description.
20:55Now, stay with us for an original track created especially for this episode and inspired by the article you've just heard about. Thanks for listening and join us next time as we explore more science, base by base.
