John M. Cunningham, MD, PhD, gives a presentation on DiGeorge Syndrome: Long Distance Genomics and Heroic Therapy.
SPEAKER 1: There are multiple abnormalities that are seen in DiGeorge syndrome. Cardiac abnormalities are seen in about 75% of patients, renal abnormalities in 35% of patients. And probably the most important one just to remember is that behavioral abnormalities occur in up to 50% of patients. But given that both Dr. Wolfe and I are interested in hematopoiesis and immunopoiesis, and we're going to really focus, I think, this afternoon predominantly on immunodeficiency. However, I want to emphasize that you should, as a pediatrician, always think of DiGeorge syndrome when you see either a cardiac defect, some kind of velopharyngeal insufficiency following adnoidectomy, an anterior glottic web, neonatal hypocalcemia. And in young adults, if you've got schizophrenia, 5% or more of patients with schizophrenia actually have a variant of DiGeorge syndrome. You should also think about DiGeorge syndrome, obviously, in the context of dysmorphic features, or even just children with speech delay. For example, Ben Bradley, the ex-editor of the Washington Post, his son was first diagnosed by a speech therapist in the context of he had an occult DiGeorge syndrome. So, as I said, the first part of my talk is going to be about the genetics. Just to remind you that most patients who presented with DiGeorge syndrome-- it's a sporadic mutation that occurs in utero. It occurs in approximately one in 4,000 births. And it involves a deletion of 22q11.21. Interestingly, the deletion only occurs on one chromosome. It's a heterozygous defect. And there is a predominance that the maternal allele is the one that's affected. And there's autosomal inheritance in less than 10% of patients, but it's an autosomal-dominant inheritance. So the diagnosis is made in two ways. Firstly, by fish. Here is fish at the top right. Here is a normal chromosome. These green dots are the telomeres, and these red dots on this chromosome is a marker for TBX1, that Dr. Wolfe mentioned is involved in the middle of the deletion. Whereas the other chromosome, although it has the telomeric dots, it doesn't have the red dots of the gene, suggesting that there's a deletion present. Just to remind you that not all DiGeorge syndrome is on 22q11. 10% of patients have a 10p deletion phenotype, and in those, approximately 30% of them have an immunodeficiency. And then there's CHARGE syndrome, which I'm sure you all remember from med school, which is a mutation in a chromodomain helicase gene. And in those patients, 60% of them have an immune deficiency component to their defect. But they all present as if they are DiGeorge-like syndrome. So I hope you can see this slide, which comes from a New England paper from about four years ago. And here is the proband, who presented with classic DiGeorge syndrome. And it was actually the slide I showed you earlier, but slightly bigger. Here's the TBX genes here. Thank you. Here's the TBX genes. And one of things you must do is you must screen the parents, even if they don't have any defects. And it was specifically interesting. When they looked at the mom, the mom had two TBX genes, so she was normal. But the dad actually had a chromosome that didn't have any TBX on it. You can see here. This one that's blue with no red. But if you look carefully, you'll see that there actually are two red dots here on this chromosome, suggesting that there was a duplication. So what happened there is, remember. It's an autosomal-dominant defect. And so what happened here was that one of the genes or one of the gene regions actually duplicated to protect the father from any defect. He did not have any evidence of DiGeorge, even though both his TBX genes were on one chromosome. This was confirmed by looking at expression of genes in the region. Here's the TBX gene. Here in the region, the three megabase region that's deleted predominantly in standard DiGeorge syndrome. And if you look at the USB-18 gene, which is outside the region, the expression is normal. Whereas only in this dark black bar, which is the patient, the proband, do you see a loss of expression of two genes that are right in the middle of the deleted region. Interestingly, when they went back into the family history and actually typed all the other patients, the father had actually-- this had actually happened in utero when he was born, that he had this duplication occurring. So here is the gene region that we're talking about. It's a three megabase allele. So if you think of chromosome 22, chromosome 22 is about 150 megabases in length. So it's about 1/50 of the whole chromosome is deleted in the situation, as it's an interstitial deletion. 90% of patients actually have this three megabase deletion, and 8% of these patients have a slightly smaller deletion. But the question is, before we started to understand this region, what actually is the gene that is defective here? Because you're losing many, many genes which are involved in very, very important functions. Many of them are actually transcription factors, master regulators of cell proliferation and differentiation, and there are also several proteins that are involved in signaling. So the first thing that people have to do is-- we just go back for a moment-- is how do you diagnose the disease nowadays? So you don't just do fluorescent in situ hybridization and karyotyping. You now go to what's called array comparative genomic hybridization. You take a sample from the patient or a parent. You actually label it with a probe. We label all the DNA with a probe. You apply that to a microwave. You have a control beside this. And what you get is a fluorescent signal after scanning it. And, if I remember rightly, the reds are deletions and the greens are amplifications. And with that, you can actually identify the region that appears to be deleted, as in DiGeorge syndrome. So that's when you order this test, array comparative genomic hybridization, that's what you're ordering. So how do we understand this mechanism? So now I showed you the human chromosome. Now, here is the mouse chromosome. The region is on chromosome 16 in the mouse. And over the last 20 years or so, biologists have used different approaches to delete major parts of this region to try to understand what it's role is in DiGeorge syndrome. When they deleted this region here, upstream of the TBX gene-- here's the TBX gene. They didn't know the TBX gene was important at that stage. The mice were normal. So a smaller deletion didn't cause any problems. When they made major deletions, they actually got cardiac defects, but they were variable in their phenotype. And so they identified a candidate set of genes based on their known function to look at further, and then they knocked out or removed both copies of the TBX1 allele in mice. So before I show you the results of that experiment, I just want to remind you what Dr. Wolfe mentioned earlier. This is the developing embryo. And just to remind you that these branchial arches and pharngeal pouches provide both mesoderm, ectoderm, and ectoderm that form many of the central structures in the body, including the facial structures, cardiac structures, the thymus, et cetera. And I'm not going to go into the details here, but essentially each piece of this region it's controlled by a set of genes on chromosome 22, among others, which actually decides how it migrates from this primitive structure into the fully-formed formed embryo. So here's what happened after they delete this gene TBX1. Here's a normal mouse here, and here's a mouse where both genes were deleted. And there's a distinct change in the facial structure of the mouse. But most importantly, when they dissected the mouse-- here's the heart of the normal mouse with two normal atria. And here's the thymus of the mouse here. I think it's very obvious for everybody, even if you've never seen a mouse before, or seen the inside of a mouse before, that the thymus is completely gone. So this suggested strongly that TBX1 was a critical factor in the Digiorgio syndrome phenotype. Subsequently, many patients who appear to have slightly unusual DiGeorge-like syndromes have been sequence, and several of these patients have been identified that actually have mutations, single-point mutations, rather than deletions of this three megabase region. They have actually been identified as actually having TBX deletions and having a phenocopy of the DiGeorge phenotype. So, let's get back to the first question that I posed. Are there other genes in the deletion involved in the DGS phenotype apart from TBX1? So here's one example of this. Here is DGCR8, which is just upstream of TBX1. And here is it being knocked out in the mouse. And what happens here is they actually have defects in sensorimotor gating and spatial work memory-dependent learning. I read these things out because I'm not a neuroscientist. But these have been implicated as murine mouse variants of what you usually see in patients with schizophrenia. So that suggests that many of the components of the genes that are deleted actually are and part of the phenotype, and it may be that there is interactions between these genes that are important for the generational of the total phenotype. In fact, when you look at TBX1 and you look at what are called epistatic interactions-- that's gene-gene interactions-- there are many other factors that appear to be important that all appeared, interestingly, to be in this region of 22q, which are actually involved in development of the neural crest, and these are shown here on the right. So the take-home message from this part of the talk is that essentially the embryo starts to migrate cells from the branchial arches into the anterior part of the embryo. And a defect in this region of 22q11 can actually result in defect in many particular parts, including the heart, the thymus, and the parathyroid. So, let's switch gears here and talk about therapy for the immunodeficiency state in DiGeorge syndrome. Obviously, as mentioned earlier by Dr. Wolfe, there is a degree of variability in the DiGeorge phenotype. So many of these children present with a partial DiGeorge syndrome with a cardiac defect and sign of pulmonary disease. And they perhaps have low immunobulins, and they have abberant parathyroid function. And they can be treated with antibiotics with immunoglobulin supplementation and calcium and cholecalciferol therapy. What I want to mention to you, which is very important, is that the other presenting phenotype here is not an immunodeficiency state. Because the thymus is either abberant or absent, the baby can actually present with what looks like an Omenn syndrome, with skin rash and immunodeficiency. And so this is a problem of a defect in the balance in the immune state rather than just in the complete immunodeficiency. In fact, approximately 5% of patients with severe DiGeorge syndrome present with autoimmune disease. And this is probably because one of the key cell types that's defective in these patients is what are called T regulatory cells, which are marked by a protein on the surface call CD25, which actually represses the immune system from autoactivation and autoimmune disease generation. However, what we want to talk about in the next 10 minutes or so is what are the cellular therapies for complete DiGeorge syndrome? So what we have to think about is we have to think about collecting the T cell defect. But remember, Dr. Wolfe told you that there's a lack of a thymus. And the thymus is required to educate new T cells. So how do you get around that? The next problem is, how do you select owners for these patients? How do you condition these young children? In other words, do you use chemotherapy or do you not choose chemotherapy? And can we learn lessons from other patients with severe combined immune deficiency? So to remind you what the role of the thymus is-- that 15% to 20% of patients with DGS have an absent thymus. The thymus is sometimes found in aberrant locations, but, as I said, only 1% of them have this thing called complete DiGeorge syndrome. I won't dwell on this, because Dr. Wolfe dwelled on this. But essentially what we have to think about is transplanting cells that either produce a thymus or go through a thymic process like this up here and produce normal cells that can actually give you a T cell-based immunity. If you get it wrong, you get something like this, which is graft-versus-host disease, or autoimmune disease. And if you get it wrong completely, essentially all the cells die. These are the cells in black. So we have to understand what thymic migrants are and how they're marked to understand the next few slides. So here is a child with DiGeorge syndrome who's got complete DiGeorge syndrome. Because this child has no CD44RA cells. These are cells that are the immature educated cells that first show that a child, perhaps, is producing a normal thymic function. After a stem cell transplant-- this is 36 months later-- this is the result. Here, it's a different set of markers. But essentially, there's now nice numbers of CD45RA cells. So that's the goal. That's one way of finding out if the transports actually worked-- is finding out you go from zero 45RA cells to lots of 45RA cells? The second way you look, which has actually been used across about-- what?-- 20 states and in the United States now to screen all patients with immunodeficiencies is to do this assay, which is called a TREC assay, for the T cell receptor excision circle assay. This assay will be utilized for every newborn in Illinois starting July 1, 2014. And essentially what it does is the T cell receptors, before the thymus educates the cells, the T cell receptors are in the germline state. And they excise part of the T cell receptor DNA to make the functional T cell receptor. This this piece of DNA floats around in the blood and tells us that we actually have a normal functioning thymus. If you've got a child with any form of severe combined immune deficiency, including DiGeorge syndrome, you will have no TRECs. And that is the assay that [INAUDIBLE] used to identify the approximately 50 children, probably, in Illinois each year who are born with a severe combined immune deficiency of one type or another. There's a third assay that we use, which looks at the complexity of the T cell response. I won't go into the details. This is called B beta specter typing. But essentially each of these peaks is a response to an antigen or an insult to the child. So what do we know about transparent for skin in general? So this is the education piece that'll be on your boards. Essentially transplants for severe combined immune deficiency-- not DiGeorge syndrome, but other forms, such as IL-2 receptor gamma deficiency-- is really outstanding at this time. If you have a matched sibling or a matched unrelated donor or a mismatched family member who's very close, say a five out of six match, the outcomes are greater than 80%. If you use a half-match father or mother, the results are about 50%. So, in other words, 50% of children can be cured of their SCID with an allergen egg stem cell transplant. So what are we thinking about now in the context of severe combined immune deficiency? This is the algorithm that we use to decide what type of transplant to use. The bottom line is that approximately 70% to 80% overall of children who have severe combined immune deficiency are now cured of their disease with stem cell transplantation. What about in DiGeorge syndrome? So this is a recent study of 17 patients. That's the majority of patients that have been transplanted in Europe in the last five years for DiGeorge syndrome, with complete DiGeorge syndrome. And as you can see, the survival is not as good as with severe combined immune deficiency, with 41% of patients surviving. So in the survivors, though, their mitogen responses normalized. They respond to vaccine. However, they have no evidence of those thymic migrants-- those CD45RA cells, those TRECs. And five of the seven survivors do not require IVIG. So it's a good response, but it's inferior to what we can do with other forms of immunodeficiency. So there's another approach. And the other approach, in the United States, has been and pioneered by Louise Markert, at Duke, and where she has actually decided instead of using allergen egg cells from a parental donor or from a match sibling or a core blood, what about the thymuses that every cardiac surgeon throws away when they do a cardiac procedure? And so at Duke, they have a program where they actually harvest those thymi, and they use them for patients with complete DiGeorge syndrome. So I'm going to tell you the results of those studies. This is how the transplant works. So essentially they take a thymus, harvested by a cardiac surgeon in the operating room. They identify that it's innate. They have a bank of these thymi. They identify a thymus that is a close HLA match. And then they mince it up into single cells and then they sort of just grow it for a couple of days. And then when the baby arrives, they actually take the baby's quadriceps muscle, and a surgeon makes furrows in the quadriceps and inserts thymic tissue into the quadriceps. Now, this is the result of that experiment in a patient that did well. Here is normal thymus up here, and here is a transplanted thymus. This is coming from the muscle of a child who was transplanted about six months prior to this biopsy being taken. So essentially I would defy anybody, really, to see that there's any significant difference between those issues. So what are the results of these studies? Remember, with transplantation, with stem cell transplantation, it's about 40%. In Dr. Markert's hands-- and this has been replicated in studies in Europe-- approximately 65% of patients survived greater than 10 years post-transplant. So this is a significant advance in how we think about treating this severe disease. So So not everybody survives. I think 15 patients died, and the causes of death were fungal disease and viral disease, specifically Aspergillus and CMV viral infections. Three patients died of bleeding. This was a surgical problem early in the procedure. They've now been able to solve that problem. And one patient died of old undefined respiratory failure. From a morbidity point of view, several of these patients still have defective B cell function. So what about those markers that I mentioned a moment ago? So let me just bring you through these markers. So remember, all these children have no T cells whatever. They have less than 50 immaturity T cells in their blood before the procedure. And as you can see, the number of those immature naive T cells increases remarkably after transplant of these thymic cells. So cells sitting in the quadriceps are producing large numbers of immature thymuses, an ectopic thymus. Similarly, when we look at CD45RA-- pre-transplant, no CD45RA. Post-transplant, 41% of the cells are expressing both CD62 ligand and CD45RA, which is these thymic migrants. Similarly, post-transplant, these cells are not just there, but they actually have nice spectra typing. This is a negative control. This is what was happening before the transplant. This is an abberant spectra type, where there's just [INAUDIBLE]. But as you can see from just scanning this, most of these patients had very nice broad responses from a beta spectra typing perspective. I'm not going to dwell on this, but essentially, they also had B cell function, many of these patients-- both responses to soluble CD3 and tetanus toxoid. And that's summarized in this table. But essentially, most of the patients do not have low levels of antibodies, although 10 patients, for various reasons, remain on IVIG. So the summary of transportation for DiGeorge syndrome is the indications are extremely rare. Only 1% of patients with DiGeorge syndrome require a transplant. You can correctness it with either stem cell transplant or thymic transplant, although the results with thymic transparent-- admittedly only in two or three centers in the world-- are much better. There is still significant mortality associated with this procedure, although it has improved significantly over the last 10 years. And I suppose the final question is, given this is such a rare condition, how can these studies inform treatment for more common diseases? So I thought I would end on this slide, which is showing you what we know about educating very immature T cells to be thymic cells. You have to get bone marrow to migrate to the thymus and to be educated. And there are many things now that we're understanding about the thymic endothelial cells and how they may support thymic development. And particularly, there's a new player called Interleukin 22, that appears to be critically important for developing these thymic migrants, which are critical for protection of the child against infection. But I think this is the most interesting slide-- probably I was the most excited about this slide. This is a mouse in which a lymph node in the inguinal area has been identified and has been transplanted with both-- well, in one experiment with mature T cells, and in another experiment with embryonic stem cells. And these cells have been allowed to grow in the mouse. And then they looked at T cell function afterwards. This was an immunodeficient mouse. And what they were able to show was that they were able to develop a thymus in the inguinal lymph node of this mouse. This suggests that-- by the way, they also did a very interesting experiment, as well, where they actually did the same thing under different conditions using a [INAUDIBLE] actually form new pancreas for diabetes therapy. So the role of cellular therapy for these diseases is just starting, and we hope over the next few years that we'll able to cure every child who has complete DiGeorge syndrome.