Basics of Genetics: An Introduction to PFIC as a set of Genetic Disease of Cholestasis

SPEAKERS

Dr. Chunyue Yin, Walter Perez, Dr. Akihiro Asai, Emily Ventura, Tara Kearns

Walter Perez  00:04

Well, good morning, everybody. And welcome this morning. I hope that you’re all doing amazing. And

Emily Ventura  00:17

That was a beautiful start, I’m gonna have you hang on just one second.

Walter Perez  00:24

Could you give me the thumbs up?

Emily Ventura  00:27

 I will. The joys of a virtual conference. All right, take it away.

Walter Perez  00:41

Well, good morning, everybody and welcome this morning. I hope that you’re doing great and looking forward to not just a great session, but a great weekend. Hey, as we start, I’m reminded of a story. Have you guys ever heard of the two hunters and the grizzly bear? I heard it this past week and it goes something like this. 

There’s two hunters out in the woods, and they come in contact with a giant grizzly bear. They’re frozen in time looking at each other, knowing not knowing what to do. And one hunter goes to the next and says, “What do we do?” The other one says, “We should run.” And the other hunter returns and says, “I don’t think we can outrun this bear.” Another friend, with all his wisdom says, “Well, I don’t have to run the grizzly bear. I just have to run you.” 

And that’s just a little bit of light humor for you this morning. But you know what it got me thinking….. running. Are you joining us this morning running from this past year? Are you running from maybe some challenges that the pandemic brought or running from maybe even the challenges that PFIC itself is bringing to you?

 Well, here we are to the Family PFIC Conference 2021 and I hope you slow down for a moment and come together, join us as we’re about to network, connect. We’re about to learn together. Hopefully, we can contact each other and encourage and maybe even give each other some hope. So stop running. And just hold up for a minute and just join us. 

Walter Perez  02:14

My name is Walter Perez. And I have the privilege of sitting on the PFIC board and the privilege of being the president of the board. I live in Edmonton, Alberta, Canada, and I have a four year old child Armando who actually has PFIC, PFIC 2. And that’s what brings me into this journey, joining the PFIC Network. And I’m excited. I’m excited that you have chosen to spend a couple of days with us. And, and just maybe just checking us out or maybe there’s a session out there that is really catching your attention.

 Because you know what? The PFIC Network, it is doing things right now. It is exciting to see where we’re at. If, if you look at this conference itself, it’s been a couple of years since we’ve got together. And regardless of maybe some challenges that have taken place, we’ve been able to come together virtually, and bring it to you. And so I’m glad that you stopped by because you’re gonna get a glimpse of what is going on in the Network, you know, whether that’s checking out our website, which is I believe a state of the art website with a lot of resources and information that I believe is evolving not only daily, but weekly and monthly.

 So there’s always something going on there. It’s come a long way, whether you’re looking at resources, or even some of the committees that are involved, like the the Parent Advisory Board, that meets on a monthly basis, or, you know, we have a medical board and a Medical Advisory Board that has a huge contribution. And even as we look at some future, things that are evolving, whether that’s the global PFIC Committee, which brings global leaders together to not just encourage or share resources, but just to stay connected on a global level. 

There is so much going on right now for the PFIC Network. And I am glad that you’re here to be part of it. I’m reminded of a quote that says, “If you want to go far, go alone.”…or sorry…”If you want to go fast, go alone. But if you want to go far go together.” And this is a prime example of what’s going on in the PFIC Network. Together, we are doing so much. There’s groups of volunteers and group of people that are coming together to make this happen. 

And so for that, I’m reminded of specific, three individuals that have been a huge part of the PFIC Network. We have Melanie, Tara and Emily who are co founders and came together with vision and you’re about to hear from two individuals. Emily, our executive director, and Tara, who is our vice president of the board. And let me pause for a moment and say, if you have not had a chance to meet them, or had a chance to connect with them, do so.

 Because you’re going to be surprised, and you’re going to be impacted by what, but what they do, but just their heart and vision for you, families that are coming in contact with the PFIC Network. They just have…they are amazing leaders and just want to add value. And so I’m very thankful that I have the privilege of working alongside them. And please, get around them, listen to their hearts. Without further ado, let me introduce to you, Tara, as she’ll share for a moment. Tara over to you.

Tara Kearns  05:46

Thank you so much, Walter. That was really kind of you to say. Thank you for getting us started this morning. And I just welcome everybody to the PFIC Conference. Thank you so much for taking time out of your day to join us. I’m so excited to get started. And Walter really, you know, he really hit on it very well. We have just come such a long way. 

So my name is Tara Kearns. And like he said, I’m the vice president of the board and one of the cofounders. It’s so wild to hear where we are today. Just a few short years ago, Emily, Melanie and I were, you know, meeting virtually for the very first time. We met in what started as a Facebook group. Some of you patients and caregivers are part of that Facebook group. And at the time, there was a website, that’s all there was. PFIC.org. And it hadn’t been updated in a while. And, you know, it was started by a mom who just wanted to get information out and she was reaching a time in her life where she wasn’t able to upkeep it and wanted to hand it over. 

So Melanie, Emily, and I all volunteered. We were all strangers to each other. And one of the very first times we met, we met on Facebook Messenger, we realized that there was a chat feature on Facebook Messenger and that’s the very first time that we met. So it feels like a lifetime ago, but really, it was about five years ago. And up until that time, I had never got to meet face to face with another family. I had never met another child that had PFIC. And I can’t tell you the impact and the power that those first moments brought. It was just so moving. 

And so in the early days, Melanie, Emily and I, we would have…. we can’t even call them board meetings. They were really times where we would get together and we would come up with ideas, right? We would talk about our struggles through PFIC. We would talk about our dreams for PFIC. And we said, “Let’s just rehab this website. Just do it. Let’s update the website. Let’s get some more information out there.” And that was really our goal, was to just help monitor the website.

 Well, for many of you other PFIC caregivers, you know that we can’t do anything on a small level. Everything is is constantly learning more and trying to grow and trying to understand. So we all have that in common. I think when it comes to our children, we’re all deeply motivated for answers. So there were many nights that Melanie, Emily and I talked about our dreams for PFIC. We wanted to build awareness. And we wanted to become more than just a website that families could land on.

We dreamed about networking and creating relationships, and helping people find hope. That didn’t… we didn’t feel like existed when our children were diagnosed. We wanted to connect to a medical community and we wanted to talk about gene therapy and we wanted to talk about drug trials and changing policy. We wanted to do so many things.

 And just five short years ago, it really felt like pipe dreams, right? But we were determined. So what started as a website quickly turned into an application for nonprofit status. And that’s how the PFIC Network was born. Just three moms trying to make a way and trying to make a voice and a name for this very rare disease. So that’s where we started and so it’s so awesome to see how far we’ve come. Two years ago, at our first PFIC family conference, it was the first time that I met, in person, any other PFIC families. That’s when we met Walter, who’s now the president of our board. That’s when I met Emily, who’s our executive director, and many other PFIC ambassadors and board members

. At the time, we were all just strangers, but very quickly realized that we had been lifetime friends and didn’t even know it. So I just cannot thank you guys enough for joining us on this journey. We have already grown so much. And I can’t see I can’t wait to see where the next five years take us. We all have passion and commitment.

 And we couldn’t have done it without the leadership of Emily, who, again, was one of our co founders, and who has now been appointed as our executive director of the PFIC Network. So I would like to turn it over to Emily, and she’s going to share some words with you in a welcome. And so thank you again, guys for joining us.

Emily Ventura  11:12

Wow, thanks, Tara. And thanks, Walter. That was an amazing introduction to you guys, and our wonderful organization. My name is Emily Ventura, I have the privilege of serving as Executive Director for the PFIC Network. I have the privilege of being a part of the PFIC community, as a parent and caregiver. And I have the privilege to know and meet, you know, members of the community worldwide, both patient/caregiver community and the scientific community. I think that Walter and Tara outlined it beautifully. 

You know, our organization started small. It remains small, however, we have accomplished a lot. We have a fairly solid foundation in all areas of our mission, support, education, advocacy and research, and we plan to continue to grow that foundation. You know, we’re gonna continue to offer the same support services that are there and available for everybody. And we’ve learned to access, you know, the amazing resources that are out there. 

Translation services, and, you know, outreach, you know, opportunities, sending educational materials to the wonderful providers that take care of our patients. You know, we plan to continue to do those, and we plan to do more. As we move forward, I’m really excited, you know, to say that we’re planning to put together our very first research funding opportunity that should be coming out by the end of this year. This is a patient led initiative. 

So we’re working towards bringing the patient voice to research. The scientific community wants to know, they want to know what we need. So, you know, our goal here going forward is to identify the needs of our community, bring those needs to the scientific community, and bridge that gap. Find ways that we can work together to find those solutions. So please take our patient priorities in research survey. It’s here on the platform. 

We hope to hear your voice. We hope to bring your voice to the scientific community. And without further ado, you know, I would like to turn it over to the wonderful presenters who are going to start us off, from Cincinnati Children’s Medical Center. Just a few of the wonderful providers, researchers who are working hard towards finding answers for the PFIC community.

 Dr. Yin and Dr. Asai are going to start us off. Dr. Balistreri had unfortunately had an emergency come up so Dr. Asai is going to take his place as part of his team. 

Emily Ventura  13:52

Just a brief note, before we get started, a few housekeeping things. If any of our members are using the translation software for the first time, you can look in the left hand corner of your screen top left hand corner, there should be a button that says “Live”. You can click that button, and then you can go in it’ll open up another tab. On that tab is where you can select your language and that’ll set up a closed captioning service. It is AI based so it is a translation software. It may not be perfect, but hopefully it’ll help to kind of bridge that language gap.

 A few disclaimers. These webinars are set up for information and educational purposes only. They’re not set up to give medical advice. If you have any questions specifically related to your case, please consult your medical team. Our Zoom is not a HIPAA compliant compliance platform, so if you are asking questions, please leave all patient identifiers, names and birthdays and such out. And this session is being recorded for future use.

 If the presenters give permission, we will be able to use them on our website, but they will be recorded and used in the platform and they’ll be available for the next few weeks for the conference. If you have any questions, you can ask them throughout the chat, throughout the session in the chat, and we will address all the questions at the end of the session. Without further ado, I’m going to stop and Dr. Asai, I’m going to turn it over to you. So thank you very much.

Dr. Akihiro Asai  15:34

Can you hear me? 

Emily Ventura  15:50

Yes, loud and clear.

Dr. Akihiro Asai  15:51

 Okay. Let me put this in Screen Mode. You see presentation?

Emily Ventura  15:58

Yes. 

Dr. Akihiro Asai  15:59

So thank you, everyone for giving me this opportunity. Unfortunately, Dr. Balistreri having emergency and he asked me to present his slide set last night. So he he wanted to apologize for for missing this opportunity. And but I’m taking…I’ll do my best to present this slide. Wonderful slide from Dr. Balistreri. My name is Akihiro Asai, Aki Asai. I’m a partner of Dr. Balistreri. I’m one of the liver doctor, pediatric liver doctor in Cincinnati Children’s Hospital. And we work together and we share some patients, and we see liver patients in our hospital together. 

So today we have three big pictures, goals for this talk. One is this “Put the PFIC and other genetic disease in the history of medicine”, because Dr. B, Dr. Balistreri,  has a long history and experience and he wanted to put the how this disease was conceptualized and what’s the progress we are making for this disease. And for to understand PFIC, we will need to learn about the bile, bile acid and hepatocyte, which is the liver cells. And also, we want to put some new progression about this 2021 version of diagnostic process for neonatal cholestasis. 

So for those three topics, we hope to cover this talk. So we start with this history. In the past, about 40 years ago, we have named this disease name, neonatal cholestasis. This is very vague name. It’s mainly saying newborn baby has jaundice. Nothing else, because we did not understand any mechanism into it. So back then, we had, when the baby was born with the jaundice, abnormal jaundice, not the breast milk jaundice. It’s the persistent disease jaundice. We only had four possibilities. One is this neonatal hepatitis, which is alone, just having that. And the biliary atresia, that’s a very different disease category. 

That’s actually a devastating disease also. And others are miscellaneous, and the other is a virus, which is the infection through pregnancy from the mom. The majority of the disease, we did not know. So we just put they just put the name “neonatal cholestasis”. So this, since 1970, it’s been a long years to figure this out. Back then we are calling neonatal hepatitis, neonatal cholestasis, or giant cell hepatitis, because when we do a biopsy for those babies, we see…sorry, it’s behind this comments, but there is a big hepatocyte, the liver cell. We call that…that’s why we call it “giant cell” with inflammation around. And we didn’t know what’s going on. We did not know the cause. So we calling that idiopathic or sporadic. 

And sometimes it clusters in the same family, so we call it “familial”. But basically we were just describing what’s going on. We did not know anything about why this disease is happening. This is Dr. B’s comment, “The beginning of wisdom is to call things by the right name”. So we have to really put the right name to to put our thought into the focus. So back then, 2000…sorry, 1978, 1977, the expert at the time, from worldwide get together and they are writing this article. They are trying to put the right name for this disease instead of neonatal hepatitis. So we put, at the time, we were trying to coin the name. And I think it was the consensus at the time, they started using the term for PFIC. So there was some big background for this. 

Back in 2000…1977, it was we were calling progressive familial, intrahepatic cholestasis. So we tried to describe as detailed as possible to put the name into it. This was the beginning of name for the PFIC. So kind of fast forward. So bottom line, right now, what we know is, we know that it is a genetic disease, and that the gene defect is causing the liver disease. The liver disease, the intrahepatic cholestasis, meaning that nothing wrong outside the liver, it’s within the liver that is causing the cholestasis. 

And we are now, what we are doing right now is each gene makes molecule in the hepatocyte and those molecule has some function. And if you lose those function, you have disease. So we kind of tried to connect all, at the molecular level of finding to the disease. So that now, this is current, so this now 2021, now when we have a baby with jaundice, disease jaudice, we have this many possible disease. At the time was 65. And now it’s get to the 25%. The others, we were finding the cause of it, cause of the those other disease. 

So the big pie 65% of those unknown disease, it’s now 25%. And with this 25%, we started identifying PFIC, Alagille syndrome and bile acid synthetic defect. Those are all related to the bile acid metabolism or bile acid pathway. So we’re making the progress in terms of identifying the disease. And within this 25%, PFIC is one of them. So this is actually one of the histological comments from back in 1930. This Dr. Beach was commenting, “There is a disease.”. 

He didn’t know anything about PFIC, but he was predicting there’s a disease like a PFIC that exists in the babies, because he was saying there is a jaundiced baby without any obstruction of bile flow, so it must be a process of making the bile and then excreting the bile out of the liver.  So this was his prediction about the PFIC. So let’s move on.

Dr. Akihiro Asai  23:20

 What….to understand the PFIC, we will have to learn about bile and bile acid in the liver cells. So let’s do this quickly. Later on to me, sorry, Dr. Yin and think Dr. Thompson will go over those details. I’m just giving the big, big picture for now. So this is when you cut the small piece of liver and put it in the microscope, this is what we see. So those are the anatomy, the microscopic anatomy of the liver. And those red ones are arteries. 

Blue is veins. It’s a portal vein, so it’s coming from the intestine to the liver. This is green one is the bile duct. So the blood runs through this white area and it goes draining to this central blue part. This is the draining vein. And then between those irrigation systems, this is the pink one, those are the cord or we call the liver cell parenchyma. So basically the blood coming from the heart, coming from the intestine washes through the liver between those sinusoidal space and give it nutrition, give it oxygen and take out all the waste and then goes to the draining vein.

 And then if you look close, what is going on in this blue green part. So those are the one liver cell we call hepatocyte. And this one makes bile. This one produces bile and dumping into this green area that we call bile canaliculi. It’s a small tiny draining pipe between the cells. And those green tiny tubes get together and they make a bile duct. And that’s the dumping mechanism of those waste, or bile goes to the intestine. So just more close up, this is like a high magnification pictures. This one hepatocyte and the second hepatocyte facing together, and they make bile acid. This bile acid, it’s important ingredient of bile. If you don’t have bile acid, you cannot really make bile.

 So bile acid is the most important component of the bile, which is made from cholesterol within the hepatocyte. So the hepatocyte, the liver cell, makes cholesterol which makes bile acid from cholesterol. And then this bile acid needs to excrete, meaning that get out of the hepatocyte. It goes into the bile canaliculus, which is the draining small tiny tube.

 Ahen they get collected and make a flow to the intestine. So after we get this bile into the intestine, they run through the intestine and then get absorbed back to the hepatocyte. They have a circulation from the hepatocyte to the intestine and intestine to the hepatocyte. So this is called enterohepatic circulation. 

Dr. Akihiro Asai  26:29

So what is bile? I think it’s…we are not gonna go deep into detail about it, but it’s made from cholesterol. So it’s a  biochemical ingredient your liver makes. And with with this bile acid, your liver makes bile flow. Without bile acid, you cannot really have a flow. So there is actually two bile acids. One is a good one. Most of the bile acids are good one for you but there is some bad ones. I’m just oversimplifying it. But this is probably important to see the balance between them. So if you want to have a more good bile acid in your body, and in your intestine, and then less bad bile acid, you need the balance for this. So what can go wrong in this system?

 While it’s a lot of steps, so each step has the error, because each step was processed by the protein that your gene makes. So if you have a problem in the genes, you cannot make those proteins and enzymes. So each step of those could go wrong. And it actually is going wrong in a patient with PFIC. So I’m gonna go quick for this. There is a each step. So you have to make bile acid. But if you can’t, you have a disease called these disorders of bile acid synthesis defect. If you have a defect in terms of excreting those bile acids, you have a transport issue. And you have… if you cannot really make those small, tiny bile ducts, you have a problem…that’s the Alagille syndrome

. And you have other concerns and sometimes medications, sometimes infection can do the same thing. So this….I think we can skip this. So there is each step of this bile synthesis and the excretion, there is each gene for each step.

 And Dr. Yin will go over for those later on. But I’m kind of fast forward. So if you make bile acid, it has to get out. And then to do that, you need to have this gene called BSEP. And for BSEP to function, you need to have this gene called FIC 1 and MDR3. So you need those three genes functioning very well in the canalicular membrane for the bile acid to get out. And then when it comes back, you need to have those genes to uptake. So each step has a possible mutations and problem.

So as we know, PFIC 1 is this FIC 1 made by this gene. If you have a mutation in this gene, you can have PFIC 1. Same thing with the PFIC 2, same thing with the PFIC 3. Those detail will come later in the presentation. So also we just have seen that not just that process of bile acid, you have to have a tight seal of this bile canalicula. So you need to have a tight junction here between the cell. If you have a mutation of these proteins, you have a not so tight junction, so you can have a leak of those bile acid or bile. It leaks between the cells and goes into the blood and that causes the problem. 

So this is Dr. B’s picture that… this story, going back to the history. So it started with these three genes that are causing PFIC Types 1, 2, and 3. Now this research progressed, we start seeing more and more genes involved in this disease. And it goes to a certain categories. So that’s why he’s making these branch pictures. And we’re going to go quick, over it. So there is another gene called FXR related to those genes. And there is an other set of genes that can, that is important for the bile acid metabolism. 

If you have mutation, you have the PFIC, and you have the other group of gene that’s important for vesicle trafficking. And if you miss those genes, you can have a PFIC. There is a some more new genes that keep getting found and causing the disease. So I think this number, this list of genes will go more and more as we our research progresses. 

Dr. Akihiro Asai  31:13

So in this setting of those new diseases, new genes coming up, how are we making a diagnosis? So this is 2021 version of in the clinic, how to make that diagnosis. In this situation, first, we start with patients. So we see the patient. We found the cholestasis. We found that this is not the usual jaundice. It’s disease, it is cholestasis. And then we run the blot test, this biochemical test. It’s basically the blot test. We start seeing more and more concern for PFIC and we do liver biopsy still. 

It’s important to see the actual event going on in the liver, so we do a biopsy. And then we stain some of those proteins that we talked about before. We have this technique called immunohistochemistry to identify those genes in the liver. And then now we have those molecular genetic testing. We have new…this is new, and then it’s getting more and more sophisticated and more and more available and faster. 

So we integrate those genetic testing, actually early in the phase. This is the comparison from old to new diagnosis method. So the old, we were doing the biopsies and tests, everything and at the end, we do genetic tests to confirm. If it’s correct, then good. If it’s not, then we don’t have anything to do. But now, because we have a next generation sequencing technology, which is basically much faster, much efficient genetic sequencing tests, we run this genetic tests in all the phases of the diagnostic process. So we are now calling sequence first. 

At this point, what we’re doing in this sequencing is basically we are reading the DNA patterns. So DNA has four different patterns and like letters in the DNA, we read each one of those sequences and we target for those genes that are related and then trying to find out. Or if we nowadays, we can just read all the genes at one time, which is the whole exome or whole genome sequencing, that we just basically screen everything out at one time. For this diagnostic clinic part, Dr. Yin will go in detail a little bit later.

 So for those those new approach of 2021, what can we do? So we can actually find the genes that’s causing the disease. That’s the main purpose of this. But at the same time, we’re now finding a new type of leakage, which is the VOUS (variant of unknown significance), meaning that it’s new. No one has seen this type of mutation. No one has really put it into a context of clinical disease, but it’s there. So it’s new, and we have to do more investigation for those.

 And also, we have completely new genes that are not related to the bile acid metabolism but now, patient has the PFIC and we found this mutation. And this new gene may be this gene is important for the bile acid metabolism. So those are the ones we are working on. So that’s the experimental model system to finding out what’s happening in a patient and the information we get from gene sequencing. We are connecting those two together by the research. 

Dr. Akihiro Asai  34:52

So we have this test. In the past was six…. it was with 25, 27 genes. But now we have a 66 genes to sequence at one time. And in the past, it was this EGL Genetics. But now they have a shift in the business model. So now we are sending this to a Prevention Genetics, that’s the company. If you need more information about this test, this is the you can type basically “Prevention Genetics” and then “cholestasis gene panel”. That’s what we can run. So those are the one they will test in this company. And then the red ones are the ones we just quickly talked about. So there is some more and more genes in this list and then more to come, actually. 

There are the new genes that have been identified in the past three years and those are the ones that will be integrated to those tests soon. So this is kind of an overview of what happened in the past and what we are doing right now. And then this will…we will continue to improve making a diagnosis, trying to connect the new findings, and trying to understand more the bile acid metabolism so that we can understand more, what’s going on in the baby patient. And hopefully, we can find the treatment options, better treatment options in the future.

 So this is basically, first we make the diagnosis. So Dr. B always says, “Every patient deserves a diagnosis.” And then this will allow us to go to a personal approach, because each one has a different type of mutation. And based on the mutation, we will have a better functional classification of those mutations and we will be able to tailor treatment and hopefully cure for those patients in the future. Thank you very much. I’m handing this to Dr. Yin.

Dr. Chunyue Yin  37:06

So thank you Aki for the talk. And I will just continue with this topic and fill some holes you left haha. Some explanation to the genetic testing in specific. So thank you. All right. Okay, so let me start the presentation right here. So hi, everybody. My name is Chunyue Yin. I am a researcher, Assistant Professor working in the Division of Gastroenterology Hepatology, and Nutrition at Cincinnati Children’s Hospital. 

My own research is to study the mechanism of PFIC disease, mainly using animal models. And the goal is to develop new and better diagnostic and therapeutic strategies. And I am mainly focusing on PFIC 2. I also work with clinicians like Aki and Alex and Dr. B and also human geneticists to work on identifying new genes and new mutations that cause PFIC. So today, I am going to give a brief introduction of the genetic testing of PFIC.

 And then I will talk about how genetics works and what genetic testing results mean. And then what the testing can and cannot do, and then the challenges we are facing. So this is kind of a Biology 101. 

Dr. Chunyue Yin  38:50

We all know our body is made up of trillions of cells, and each cell contains a complete set of a person’s genetic information. It is the DNA that is packed in these thread-like structures called chromosomes. The DNA contains the instructions that guide how a cell should function. And it is encoded within a sequence of units represented by the letters you see here: ATGC. And these units are called bases. Sections of the DNA are called genes. And then the genes make proteins to help the cells to carry out their functions. 

And then the basic unit of the proteins are called amino acids. So next….oops. So when there are changes in the DNA sequence of single genes, you have mutations. And PFIC is actually a group of childhood cholestatic liver diseases with similar clinical features, but each PFIC type is caused by mutations in different genes. I am listing the most common type of PFIC here and I will go into a little bit the mechanism here. And I know Dr. Richard Thompson is going to go much depth then today’s my talk. So, as Aki talked about, like this is a diagram of how hepatocyte cells look like. You have two hepatocytes side by side and then in the middle, you have this structure called bile canaliculus. 

And this is where the bile flows get excreted from the hepatocyte into the bile ducts. And this excretion process is regulated by a bunch of bile acid transporters on the canaliculus. So you have this FIC 1 protein that is important for the structure of bile canaliculus. And then, this ABCB11 gene encodes bile salt export pump, so it is the actual pump that kicks out the bile acid. And then this ABCB4 gene encodes a protein called MDR3. 

As Aki mentioned, some of our bile acids are actually toxic, and you don’t want it to just float around in your bloodstream and cause damage. So the function of MDR3 protein is to pack the bile acids in a structure called micelle so that it is protected and separate from the outside world. So if you don’t have this protein, the toxic bile acid is gonna go everywhere and damage cells. If you have mutations in these three transporters, obviously, there will be liver damage, and there’ll be problem in the bile excretion from the hepatocytes. Aki also mentioned the tight junctions. So it is the function of the tight junction is to seal off the bile canaliculus, so the bile will not leak out. 

If you have mutations in proteins that make the tight junction, then you will have leaky tight junction. So the bile may go into the bloodstream, and then to places that it’s not supposed to. So mutations in this tight junction protein 2 is what we have here for PFIC 4.

 And then more recently, there are other proteins and mutations discovered to be connected to PFIC. So FXR is a protein that regulates the expression of these transporters. So if you have bad FXR protein then these transporters are not expressed at the correct level, so you have problems. And then this is PFIC 5. So MYO5B is a protein regulate where these transporters are located. So if there’s a mutation in MYO5B, these transporters will not get to the bile canaliculus, so they cannot function properly. And in this is what we call PFIC 6. So.

 So the mutations we found in PFIC patients, they are all… they have difference. And they are two main classes. One is called point mutation. This means there’s a change in one single base in the DNA. So I put a example here. Let’s say a normal DNA sequence is ATGC and then for this point mutation, this G changed to A. The second big group is called frameshift mutation. It is mainly caused by either the loss or gain of bases. So again, normal is ATGC. In this deletion mutation, you have T and G deleted, and the insertion you have a T and C inserted in between T and G in a normal sequence.

 And then duplication is you have a you know, it’s pretty straightforward. It is a duplication of the normal sequence. So the consequence of these different mutations can vary too. So I put a example here. This is the protein of bile salt export pump, the protein that is impaired in PFIC 2. So you can see it has very complicated structures and different components.

 So this is what a normal BSEP protein should look like. Some of the mutations  that they actually do not cause any change to the protein itself. So we call them silent mutations. So they are harmless, they are fine. And then some mutations actually result in a much shorter protein, or sometimes it doesn’t even make any protein. And this time, you can see like this protein mutant protein stop right here. So it loses all these complicated structures that should be present in normal protein. So, this type we call nonsense.

 This is probably the most severe type because you just completely lose the protein there. And then there’s another group is called missense mutation. So, it has changed the protein a little bit. Sometimes it’s just one or two amino acids. So, that’s, you know, that can, if the amino acid is so important, it is the key for the protein function, then it’s very severe. But sometimes it may just impair the protein a little bit. So the protein still has some function, it’s just not at the same level as the normal protein.

Dr. Chunyue Yin  46:40

In human, we have 23 pairs of chromosomes. So all the chromosomes are in pairs, because one is from the dad and the other is from mom. Likewise, we have two copies of each gene, one from dad and one from mom. And this is really important, when we think about the genetics of PFIC. Usually PFIC disease is, oops, is actually caused by what we call autosomal recessive inheritance. That means you have to have both copies of the gene defective to develop a disease. 

So when we look at this diagram here, in PFIC families, both parents dad and mom, are usually carriers, which means they have one copy of the normal gene, and then one copy of the mutant gene or defective gene. And then their children 25% chance you will have a child that who is completely normal. He or she doesn’t inherit any of the affected gene. And then 50% chance they will be carriers, just like the parents, so they have one normal gene and then one copy of the defective genes. So they are usually healthy, they don’t have liver disease, but they will pass on that mutation to their next generations.

 And then unfortunately, 25% chance the children… that the child will have two copies of the affected genes, and then they will develop PFIC. So I also want to emphasize, it’s important to keep in mind that the both parents are carriers…. they carry each carry one copy of the affected gene, but the exact mutation in the affected gene can be the same or can be different. 

Dr. Chunyue Yin  48:59

So let’s see how this works. Now I am just going to talk about what genetic testing is. It’s a type of test to identify changes in genes, sometimes in chromosomes and the proteins. And then the results of a genetic test can confirm or rule out a suspected genetic condition and then can help determine if a person’s chance of developing a disease or passing a genetic disorder. Like I said, because different PFIC types, they share some similar clinical features, the benefit of doing a genetic testing is we can figure out exactly which type of PFIC the patient has.

 And because you know, the different proteins are affected in different types of PFIC, it is beneficial to know exactly which proteins is affected and we can develop the specific type of treatments for the patients. So as Aki said, there are two types of genetic testing. One is called DNA sequencing panel or gene panel. So what these companies and hospitals will do is collect the DNA from patients and then just sequence specific genes.

 And see, these genes are known to cause PFIC or other liver disease and if you find mutations in these genes, okay, you know, the patient likely have that disease. But when the test does not find any mutations in the known genes, it will be beneficial to do a whole exome sequencing or whole genome sequencing. 

The difference is this whole exome, whole genome sequencing will actually sequence all the genes in the body, not just a few selected genes. So you get a much bigger, comprehensive picture of you know, what can go wrong in the DNA sequence, and then maybe we can get a diagnosis from this strategy. So I put a sample of genetic testing result here. If, you know, you get a positive result, you get some answers there. And then these are the components of the genetic testing results. So it will tell you which gene is mutated.

 So I put an example here, this is ABCB11. That’s the gene that is mutated in PFIC 2, so that’s is listed here. And then AR here means this PFIC 2 is autosomal recessive. And it will list actual nucleotide change. So in this case, C base at this position changed to a T. And then the consequence of this nucleotide change is a protein amino acid change. And then the zygosity says homozygous. That means they detect two copies of this same mutation in this patient. And the type of mutation this lab determined is pathogenic. It means it’s likely to cause impaired protein function. So there’s another possibility that the report may show two different mutations.

 That means the patient has two copies of mutant genes, but the exact mutations are different. That’s why it’s called heterozygous. So has one copy of this mutation, and then one copy of the other mutation, and both mutations are pathogenic. So overall, you know, this patient doesn’t have a normal ABCB11 gene. Both copies are the affected. So this is a situation you can call, okay, this kids, this kid probably has PFIC 2. 

Because pathogenic mutations are the ones that have been previously reported in PFIC patients and then its function has been studied and validated by researchers. So we know it severely impairs the function of the protein. And they are usually nonsense missense mutations that result in complete loss or severe impairment of the protein. Sometimes you will see a “likely pathogenic.”, this term. That means based on the change in the amino acid, it is very likely that this mutation will impair the protein function, but it has not been previously reported, or has not been formally validated studied by researchers. 

It is important to keep in mind that doctors cannot use just a positive genetic testing result to predict the course or severity of the disease. You still need a liver biopsy or other lab tests to really evaluate the status of the disease. The reason why is there are many other factors that can affect the disease other than the mutations in these genes that are associated with PFIC.

 First of all, is a person’s genetic makeup. So you and I, although 99.9% of our DNA is either the same, but it is that .1% that makes us different human beings. And that .1% difference also will have an impact on you know how your liver can tolerate the new mutations and and how you respond to drug treatments. And also environmentally, what you eat, do you exercise a lot, are you exposed to a virus or toxins can have an impact of your disease severity and progression. That’s why sometimes you have two patients with exactly the same mutations in PFIC genes, but they present very different liver injury.

And also for just one person, you….you can have different stages of liver disease, and then it it is because of the environment changes, or you have a life changing events, like you get pregnant or you’re under a lot of stress, all these things can alter your liver disease presentation here. So that’s why it is really important to still do the liver biopsy lab tests to monitor your disease.

Dr. Chunyue Yin  56:39

Then there’s this this frustrating part of genetic testing. So sometimes you get negative results. That means they cannot find any mutations in the known genes. But it is possible just the technology is not good enough to detect all the genetic changes. So you need a further testing to confirm a negative result. So a negative result doesn’t mean you don’t have the disease, I’ll put it that way. Unfortunately, 50% of patients with cholestasis do not have mutation in the known genes, so they do not have a molecular diagnosis. And then the third possibility is inconclusive results.

 So I still put it this example here. If you look at the type of the mutation, it’s called VOUS that’s variants of uncertain significance. And then in this case, this patient has two copies of this VOUS mutations. And also, in other cases, you may have, you know, one copy of mutation that is pathogenic and te other is VOUS. All this cannot lead to a definitive positive diagnosis because VOUS is usually is predicted to change amino acid but it is not clear to what extent it will affect the protein function and to cause liver disease. So one thing that is helpful is to do genetic testing on your other family members healthy or with a liver disease. 

If VOUS is presented in all the affected members, but absent in the unaffected members, it is likely disease causing. But this is all just kind of indirect evidence. We still need research to be done in the lab to study the VOUS and confirm it is actually disease causing. So with all these problems, the only way to kind of concrete is to do research. So we work in a lab try to develop new tools to diagnose and identify new genetic causes of PFIC. 

And then as we collect more different mutations, and then the patient data, we can hopefully form a correlation like these type of mutation will cause more severe disease or less severe disease. And then the ultimate goal is to develop effective treatments so that we don’t have to go through the transplantation routes. So I think that’s all I have on my slides. And then I will be happy to answer questions if you guys have any. And I can answer questions for Aki too. I will try my best. So all right.

Emily Ventura  59:51

Awesome. Thank you so much, Dr. Yin. That was fantastic presentation. I can’t get my video turned on at the moment. I may ping Jeff to try to work on that in the background. But until then I’ll I’ll keep talking to you. We do have some really good questions. A lot of that was…. I haven’t heard much of that information, so really, thanks for breaking that down for us. 

A few questions about the genetics panel. So the first question we had was if if a patient had testing performed through the EGL panel, should they also get the newer version or should they also resubmit testing through the new panel?

Dr. Chunyue Yin  1:00:35

Of course, if you get a result, you don’t have to do that. And then actually, cost wise, these gene panels are actually more expensive than the whole exome whole genome sequencing. I… first, of course, I would talk to the doctors and your genetic consultants. But I mean, if you do a panel of 66 genes and then the newer one is 88 genes, I think it’s probably….. for me, if I picked an option, I would just go for the whole exome whole genome sequencing.

Emily Ventura  1:01:12

Okay, so to clarify… the link that was in Dr. Asai’s presentation, that’s not the that’s not for whole exome sequencing? That’s just the genetics panel for those 66 genes that you have identified so far?

Dr. Chunyue Yin  1:01:28

Right

Emily Ventura  1:01:28

That’s not an all inclusive, that’s what’s accessible now. 

Dr. Chunyue Yin  1:01:32

That’s correct. 

Emily Ventura  1:01:33

Right. Okay. Could you actually very quickly give a, and you likely addressed it, but just to kind of hone in on this, because it’s kind of complex, a very quick comparison between a whole exome sequencing and the genetics panel, just a quick breakdown between the two? 

Dr. Chunyue Yin  1:01:53

Okay, so the gene panels, they will only sequence whatever number of genes on that panel. So the newest version is 88 genes. These genes are all have been connected to liver disease, not just PFIC. So, for the whole exome sequencing is you actually sequence every single gene in your body. So it’s not 88 genes, it’s actually millions of genes. And then there’s also called whole genome sequencing. So in addition to sequence the genes themselves, they also sequence the part of DNA that doesn’t make a protein.

 So there are some we call, like regulatory sequence exists in these DNA. They are an important regulator of you know, how much a gene should be expressed and where it should be expressed. So if you have mutations in these regulatory sequence, they will also affect the protein. So again, whole genome is the kind of the biggest, broadest because it’s really sequence every single nucleotide in the genome. 

Emily Ventura  1:03:21

Thank you for that. There’s so many out there. It’s very complicated, but we appreciate the breakdown.

Dr. Chunyue Yin  1:03:26

Yeah, I was because I work on this whole genome or whole exome sequencing results…. very, like if you find pathogenic mutations in them, great. You get an answer. But if you don’t, and then you find these genes, they have mutations, and they can be important for liver function, but then you need to do research to see if they really are the reason the patient developed liver disease. You know, so that’s, that gets really, really complicated. That’s why the, your lab cannot give you a definitive answer, that’s usually the case.

Emily Ventura  1:04:07

I see. Okay. Another question from the audience. There’s a few questions related to this topic. How common is whole genome sequencing as part of the diagnostic process? And is there still value for whole genome sequencing after diagnosis has been made through panel testing?

Dr. Chunyue Yin  1:04:26

So I, I will say whole genome whole exome sequencing for it’s, for us researchers, it’s probably more valuable because we want to discover new genes. And then that’s the only way like if we have a, you know, an overall picture of what’s happening to your genome, we are more likely to identify things that are not reported before. But for patients you need to find a good lab, not only just sequence your DNA, but also can like have connections to the hospital and geneticists to really have a way to interpret this genetic results

. Otherwise, it’s just giant data that is not very informative. So the whole genome whole exome sequencing is only used when there’s nothing found in the panel testing. So you go panel testing. If you find something, so probably there’s no need to do these whole genome or whole exome sequencing anymore.

Emily Ventura  1:05:42

Okay, so step one, panel sequencing. 

Dr. Chunyue Yin  1:05:46

Yep

Emily Ventura  1:05:47

And then if a diagnosis…if it’s no diagnosis, then whole genome sequencing

Dr. Chunyue Yin  1:05:54

Right. 

Emily Ventura  1:05:55

And then hopefully, something is identified… if working with a laboratory that is trained to identify. Correct?

Dr. Chunyue Yin  1:06:04

Yep. And also you want, like, reputable laboratory provide high quality sequencing, because that can vary a lot. 

Emily Ventura  1:06:16

Okay, thank you. Another question from the audience, again, related to this topic. So now that genetic testing is more readily available, is it recommended that siblings, who maybe do not express PFIC disease, but you know, could be carriers or…I mean, you know, is it recommended that siblings get tested? And is that happening more often that you know of?

Dr. Chunyue Yin  1:06:43

So, if there’s patient in the family, and then that patient has a clear genetic diagnosis, then the healthy siblings can just sequence that particular gene to see if they have the same mutations, carry the same mutations. You don’t have to do the whole genome exome sequencing. So that’s, that’s kind of overkill haha.

Emily Ventura  1:07:15

Yeah, okay. Okay, but it would be relevant to kind of screen like family members?

Dr. Chunyue Yin  1:07:20

Exactly. Yeah. 

Emily Ventura  1:07:22

Okay, that was gonna follow into one of my personal questions. If, you know, things have evolved to where we’re identifying, or you’re identifying as carriers of the disease are starting to show symptoms? Or if that’s, you know, a valuable part of the, you know, should that become standard for us families? You know, should we be thinking about ourselves and siblings as carriers? Have we shown a correlation between that yet in the literature?

Dr. Chunyue Yin  1:07:50

So, so far, I will say majority of the cases, PFIC carriers do not develop liver disease. But it’s possible that environmental trigger, like pregnancy, or, you know, a big change in your lifestyle, when you eat, your metabolism level. Or maybe you like, take Tylenol, Tylenol or other like drugs that affect liver function, then your liver will react more to that than a normal not carrier human being. So I would not overthinking it, but when you start, like to notice you have liver issues, you know why.  Because you are a carrier, you are more suceptible to all these triggers, then, just like other people that do not have any mutations. 

Emily Ventura  1:08:57

Okay. Well, that’s helpful to hear to from an accessibility standpoint. I know from my personal experience, you know, I’ve been I had a panel done just for research. This was 10 years ago, but you know, it’s not it doesn’t seem standard that family members get tested. And, you know, when we, you know, our clinicians may decide that that would be okay, but it’s not necessarily covered. Testing isn’t covered for family members yet. So I think that’s something that’s constantly on our minds, you know, the accessibility, because now it seems to be more available.

Dr. Chunyue Yin  1:09:37

Yeah and the cost is way down now. 

Emily Ventura  1:09:41

Okay. Yeah, that’s good to know. So that’s kind of hopeful for for the future. Maybe we’ll be able to access testing more frequently. Thank you. Another question from the audience. So once a patient has a genetic diagnosis, how often should they have a biopsy?

Dr. Chunyue Yin  1:10:03

So, biopsy is invasive, and it has its risk. So it’s usually the standard is you keep track of your blood test results. And then if your AST ALT bilirubin levels go up and change a lot, then that’s time to do a biopsy. But if they are stable, and you probably don’t want to go through a biopsy. And then now there are more advanced imaging technique. It can monitor fibrosis through imaging without you know, really going to the liver physically. So these are other options.

Emily Ventura  1:10:48

Okay, would it be safe to say, you know, once a patient has a diagnosis, the biopsy would be moreso looked at to assess severity of the disease if there’s signs of progression?

Dr. Chunyue Yin  1:11:05

Yes. That’s correct. And then also, like, let’s say you have a missense mutation, so you still make a protein, but there’s something wrong with it. Sometimes you can get a biopsy and then do some, you know, staining on them and also, just even look at the protein itself. You can get a sense of like, what is really the consequence of this mutation. And then that can help the physician understand, like, the mechanism of the liver injury, and then how to manage the patients. So it’s helpful not just for diagnosis, but also for treatment information.

Emily Ventura  1:11:56

Okay. I see. Thank you. I have one last question before we wrap up. So I still hear the term idiopathic neonatal hepatitis. And I refer back to Dr. Asai’s earlier presentation today. It seems that term, you know, PFIC fell into that category, you know, 30 years ago, but as, as, you know, the evolution of understanding you know, kind of moves forward PFIC seems to have…. is falling outside of that category. But is it still possible that a patient kind of would lump into that category that idiopathic neonatal hepatitis, you know, to not rule out PFIC as part of that?

Dr. Chunyue Yin  1:12:45

So, I, I mean, I can confirm with Aki later, but my understanding is that idiopathic neonatal hepatitis is the classed as you don’t have a clear molecular diagnosis. If you have one, then you get out of that group, and then you have your own disease type there.

Emily Ventura  1:13:12

Okay, but perhaps the patient who, you know, they still potentially could have PFIC  in one of the genes that maybe wasn’t on the panel or whatever, that they could still be in that category, it just hasn’t been identified yet?

Dr. Chunyue Yin  1:13:23

 That’s right. 

Emily Ventura  1:13:24

Okay. One last question before we go, if transplanted, do you no longer have the gene to pass on to your offspring years later or is that still a possibility?

Dr. Chunyue Yin  1:13:35

Unfortunately, transplant is just swapping out the liver. Your genetic information is still the same. You are still the carrier of the mutation. So you know, what you pass down to your child offsprings is the genetic information in the germ cells, and then transplantation will not change anything to the germ cells. So you are still a carrier even after transplantation.

Emily Ventura  1:14:08

Okay, that makes a lot of sense. Thank you very much. And we are just at time, so I’m going to wrap us up. But if anybody has any lingering questions that they felt like they didn’t get to ask, you can send us a message or hold on to them. I believe Dr. Yin and or a member of your team will be available in the panel q&a tomorrow. So you can ask them then as well. The panel q&a is at 2:15 tomorrow. So thank you very much for wonderful, wonderful presentation. I really appreciate both you and Dr. Asai joining us for the first one this morning. And our next presentation is in 15 minutes. So see everybody then. Thanks Dr. Yin.