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Clinical Presentation and of Beta in the - Webinar Transcript

Tim Davis: Okay. On behalf of the APHL Workgroup, I would like to welcome you to this webinar on screening in the US. My name is Tim Davis and I'm the work group chair. The topics of today's webinar are on defining beta thalassemia, clinical presentations and why newborn screening is important. Our first speaker is Dr. Maria del Pilar Aguinaga. Dr. Aguinaga is Professor of the Department of and Gynecology and Internal at the Meharry Medical College and Adjunct Professor in the Department of Medicine, Division of at Vanderbilt University.

Tim Davis: She's the director of the Meharry Sickle Cell Center. Dr. Aguinaga's research interests are in sickle cell disease in women's health and her clinical interests lie in the diagnosis of disorders, for which she is consulted nationally and internationally. She has numerous publications in the field of . Her research work has received funding from the NHLBI, NICHD, [Pfizer 00:01:16], OMH and the state of Tennessee Department of Health. She directs the state of Tennessee newborn screening hemoglobinopathy and confirmatory and Reference Laboratory housed at the Meharry sickle cell center.

Tim Davis: Our Second Speaker is Dr. Michael Bender. Dr. Bender has a broad background in and long-term commitment to both basic science and clinical medicine with them dovetailing in the study of Hemoglobin genes and disorders thereof. Integrated with his basic science work, he has been a long-term commitment to the care and education of patients with hemoglobinopathies. He has run a program for children with hemoglobinopathies in Washington State for over 20 years and has had funding from the Health Resources and Services Administration and the Washington State Department of Health to develop a collaborative to improve clinical care and provide community outreach for families affected by sickle cell. He provides consultation to the Washington state newborn screening program on hemoglobinopathies and is in northwest resource for community .

Tim Davis: Our final speaker is Dr. Kathryn Hassell. Dr. Hassell is a professor of medicine in the division of hematology at the University of Colorado Denver where she directs the Colorado Sickle Cell Treatment and research center. She provides care for adults living with the thrombotic disorders, hemoglobinopathies and other nonmalignant hematological disorders. Dr. Hassell is also the supervisor of the hemoglobinopathies newborn screening follow up programs for the state of Colorado and Wyoming. She is a consultant to the Colorado State Department of Health for laboratory testing for hemoglobinopathies and is the medical

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director for hemoglobinopathies testing in the special chemistry section of the University of Colorado Hospital Clinical Laboratory. Dr. Hassell Conducts Clinical Research and has held numerous leadership roles in national projects. Our first speaker is Dr. Aguinaga. Dr. Aguinaga you're up.

Dr. Aguinaga: Good afternoon. Thank you Tim for the kind introduction. I'm Dr. Aguinaga and it is a pleasure for me to participate in this webinar, sponsored by the Association of Laboratories in their efforts to come to a consensus about how to diagnose the beta thalassemia disorders in the newborn period. I am going to give you today a general folder view of the thalassemia Syndrome. We're going to look at the definition for thalassemia and hemoglobinopathies, the worldwide distribution, and certainly if we talk about beta thalassemia, we have to cover .

Dr. Aguinaga: We will look at the incidence and prevalence ability on gene expression and globin chain synthesis, the molecular defects that can lead to beta thalassemia, the different types of beta thalassemia, how to diagnose these syndromes and some interesting cases that we found in the laboratory showing beta thal trait or beta thalassemia, the clinical manifestations and the current treatment. The beta hemoglobinopathies like beta thalassemia and sickle cell disease are the most common monogenic diseases worldwide and constitute a growing and serious public health problem in many nations around the world. If we look at the hemoglobin molecule, it is a tetrameric composed of two alpha chains and two non-alpha chain, each with the heme group responsible for binding in the pheresis state to form a functional summable hemoglobin with buffering and a special oxygen binding characteristic.

Dr. Aguinaga: The thalassemia are hereditary disorders which result from or deletions in the globin gene locals that lead to decrease or no production of a particular globin chain. There are quantitative disorders of hemoglobin synthesis. In general are ultra-formal recessive disorders, whereas the hemoglobinopathies are qualitative, the structural disorders of hemoglobin synthesis. The thalassemias and the hemoglobinopathies are inherited hemolytic and they're characterized by abnormalities of hemoglobin resulting in crystallization red cell membrane damage leading to its increased destruction, such that cannot match the destruction of the red blood cells.

Dr. Aguinaga: The word thalassemia is derived from the Greek language Thalassa meaning sea. And it was applied originally to these disorders because of the high frequency of their occurrence in individuals living around the . The thalassemias are world health problem and they're distributed widely. Severe thalassemia was first described in North America in 1925 by Thomas B Cooley [inaudible 00:06:30] based on observations of Italian children, and it was published in the transactions of the American Pediatric Society briefly describing

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the Kennecott Syndrome that later on became known as Cooley's anemia or beta thal major. If we look at the world map, we will see in the Asia Pacific basing in Southeast Asia there is strong distribution of the thalassemia, in the , in the Mediterranean area, contracts in South America and North America.

Dr. Aguinaga: If we look at specific color distribution of the thalassemias along with hemoglobinopathies in Asia Pacific region, we see that they are conscious with a very high incidence of hemoglobin E and beta thalassemia for example, Vietnam, Thailand, Malaysia, Indonesia and so on. Hemoglobin E is caused by a in codon 26, a G to A Substitution, having change for lysine. And it's one of the most important beta globin variants in Asia. At least 20,000,000 people are hemoglobin E trait worldwide and nearly 1,000,000 are at risk for hemoglobin E beta thalassemia. The hemoglobin E itself process a reduced beta globin gene expression similar to beta plus thalassemia.

Dr. Aguinaga: If we look at the incidence and prevalence is estimated that about one out of every 100,000 thousand babies are born each year with thalassemia. About 1.5% of the global population are carriers for beta thal and this translates to 80 to 90,000,000 people as carriers for beta thalassemia. In regions in northern Italy we can have 1% or 6.5% carriers of beta thalassemia. In some regions of South East Asia you could have 50 to 70%. In the European Union, it still including England since have not Brexit yet, they have about one out of 10,000 born with thalassemia. In the United States, the incidence is not really known, but it is estimated at about 1000 people are affected with beta thal major and of course is the United States, we have the combination compounded the recital for sickle beta thalassemia with an incidence of one in 1667 African-American birth.

Dr. Aguinaga: The thalassemia major, it is estimated that there are close to 23,000 people born with this disorder in combination of E beta thalassemia, about 19,000 mainly in Southeast Asia and then we have other like sickle beta thalassemia. There are between 300 to 400,000 babies born with a serious of hemoglobin disorder each year. And according to an article by Weatherall in 2012 it is estimated that 95% of thalassemia birth are in Asia, and Middle Eastern regions and by the year 2025 they will be about 900,000 clinically significant thalassemia births per year. This is a tremendous increase in thalassemia birth in just a few years and because of migration patterns, this may become a very important problem to diagnose [inaudible 00:10:02] in the United States.

Dr. Aguinaga: If we look at the structure of the globin genes which are fairly concerting nature, we see that the way these genes lie on the genome, they got also the way they got to express during ontogeny. We have in , the beta globin gene cluster and chromosome 16, the beta globin gene cluster. First we have the expression of the embryo RNA chains like epsilon and theta giving rise to the

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embryo RNA hemoglobin like Gower and Portland and then the special of the gamma globin genes leading to the synthesis of hemoglobin F and then adult . If we look here, we have hemoglobin F formed by alpha (2) gamma (2), hemoglobin A or normal adult hemoglobin formed by alpha (2) beta (2), formed by alpha (2), delta (2). In a normal adult, most of the hemoglobin will be A, a small percentage for A (2) and a minimum percentage for .

Dr. Aguinaga: Now during the RNA processing, after transcription has taken place, the RNA is going to be a splice. And here we have the exons which have the [inaudible 00:11:22] region for the hemoglobin molecule. And these exons are separated by intervening sequences or interrupting sequences also cold introns that are responsible for the splicing of the mRNA. In some forms of beta thalassemia, we find mutations in these regions. Some other times in the five prime upside mutations in [inaudible 00:11:45] regions that they're going to lead to a decreased availability of the beta globin gene.

Dr. Aguinaga: What happens in the ? In the bone marrow, we have erythroblast and when we have mutations that affect beta globin genes and chromosome 11 we're going to have a defective synthesis of the beta globin units and therefore there's going to be an accumulation of alpha globin chains that cannot pair with beta globin chains because of the decreased availability and these alpha globin chains are going to aggregate leading to intramedullary premature death of the erythroid precursor like program cell death, apoptosis and leading to ineffective erythropoiesis an extramedullary a short and survival leading to of the cell.

Dr. Aguinaga: Then molecular defects that can render the thalassemia can be of two types. You can have deletion which mainly is seen in the alpha thalassemias or a lot of mutations. Non-deletional types are commonly seen in the beta thalassemia, but you could also have deletional types beta thalassemia, because the imbalance produce at the decreased availability of the particular the globin chain, there is an accumulation of the other chain leading to the from the unpaired chain with a subsequent thalassemia pathophysiology. If we look at the molecular these days, they can have promoter region mutations, chain termination mutation for example, codon 39 within the structure gene producing [inaudible 00:13:20], beta zero thalassemia or is splice junction mutation, for example, ideas one 110 or mutations that may cause new splicing junction which activates cryptic splice or RNA cleavage defects or deletion of defect from the beta and delta genes or sometimes HPFH, Hereditary Persistence of Fetal Hemoglobin that can be either by [inaudible 00:13:44] in the promoter regions or deletion of pipes. And there are still unknown mechanisms that can lead to the thalassemia.

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Dr. Aguinaga: When they say complete or partial lack of beta globin and there is a decreased availability the alpha Globin cannot form this stable molecules, which is not the case when you have Alpha thalassemia because beta can form a stable tetramers like Bart's, well, hemoglobin is not a stable, but you can detect them at birth. And with the premature death of the red blood cells, hemolysis leads to the . We can have beta thalassemia types like beta zero thalassemia or beta plus thalassemia. In beta zero there is a complete absence of hemoglobin A and there is a complete lack of beta globin chain production. In beta thalassemia plus there is production of a small amount of functional beta chain.

Dr. Aguinaga: The classical syndromes of beta thalassemia can be classified into four. We have the silent carrier state which is the mildest form of beta thalassemia, almost undetectable. Then we have beta thalassemia minor, which is heterozygous disorder resulting in a mild hypochromic microcytic hemolytic anemia or beta thalassemia intermediate which is between the two, minor and major and could have occasional transfusions and the beta thal major which is a homozygous disorder resulting in severe transfusion dependent hemolytic anemia.

Dr. Aguinaga: The silent carrier state for beta thalassemia, you have beta globin gene mutations that will affect this slightly, the production of the beta globin chains. The patients have nearly a normal alpha globin chain ratio as a beta globin chain ratio and really not hematological abnormalities. And they can present with normal levels of hemoglobin A2.

Dr. Aguinaga: In beta thalassemia minor, we'll have in a hetero cycle to state one normal beta globin gene and one with a beta thal mutation and they will lead to production of reduced betaglobin chain. This produces a microcytic hypochromic red blood cell with mild, asymptomatic hemolytic anemia, unless there is a stress such as , infection or some type of nutritional deficiency like folic acid deficiency. We will see all the peripheral blood smears target cells. There will be increased hemoglobin A2 levels more than 3.5% percent and sometimes you can have elevated levels of fetal hemoglobin and the hemoglobin levels usually is between 10 and 13 grams per deciliter with normal or elevated red blood cell count.

Dr. Aguinaga: In beta thalassemia intermedia, this might be either heterozygous for mutations, causing mild decrease in beta chain production or they may be homozygous beta plus, beta plus metaphors for his supper causing a severe more serious reduction in beta chain production. The hemoglobin is lower, like more than seven grams per deciliter without transfusion increased hemoglobin A2 increased fetal hemoglobin and the peripheral blood smear which show microcytic, hypochromic red blood cells.

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Dr. Aguinaga: In beta thal intermediate, you will see anemia, , increased levels, total hemolysis and the anemia will worsen under stressors like I mentioned before, and they may become transfusion dependent as adults. They tend to develop and usually they survive into adulthood.

Dr. Aguinaga: In beta thal major or Cooley's anemia or beta zero thalassemia, there is absolute absence of hemoglobin A. This is a severe microcytic, , and usually they're detected early in childhood because of failure to thrive in the infants. They present with , variable degrees of jaundice, abdominal enlargement and in some cases, marked hepatosplenomegaly and the severe anemia will lead to bone changes. And without intervention, death will occur in the first decade of life.

Dr. Aguinaga: This is a picture of severe beta thalassemia with a massive hepatosplenomegaly in this child. And in the next picture we can see, there is the bossing of the skull and dental deformities in these babies.

Dr. Aguinaga: In beta thal major, we have a hemoglobin level between four to 8%. Will you go back? To beta thal? Yes. Between four to eight grams per deciliter, we have the characteristic changes in the skull, like I just showed long bones, hand bones, wasting of the limbs, protrusion of the upper teeth and mongoloid facial features. Physical growth and developmental delay. The peripheral blood smear will show hypochromic, microcytic red blood cells, extreme , , targets cells, teardrop cells, , on numerous nucleated red blood cells because of ineffective erythropoiesis.

Dr. Aguinaga: In beta thal major, also the red blood cell is very small, 50 to 60 centiliters retic counts are increased and most of the hemoglobin present is fetal hemoglobin, with an increase in hemoglobin A2 as a child develops.

Dr. Aguinaga: The clinical diagnosis will you come from severe anemia starting in the first year of life requiring blood transfusions and because of the anemia, the bone marrow increases blood production. And this is a picture of thalassemia intermedia showing microcytic hypochromic cell.

Dr. Aguinaga: In the next slide we have some HPLC chromatogram. We have a beta EDC and on the left you'll see about 5% peak, with hemoglobin in it and about 10% fetal hemoglobin. On the right on the IEF, you see the main hemoglobin in E band and it's very faint fetal band. In the one underneath that we have our beta zero beta E thalassemia where you will see about 51% of hemoglobin E, 43% of hemoglobin F and you're going to take both rounds on the IES.

Dr. Aguinaga: There are also certain like hemoglobin Malay, hemoglobin [inaudible 00:20:10], hemoglobin Lepore that can produce beta thalassemia

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. So in these some hemoglobinopathies can render thalassemia. And hemoglobin E is very common, a normal hemoglobin in Southeast Asia and when paired with a beta zero thalassemia mutation can produce severe transfusion dependent E beta zero thalassemia as the one we saw in the slide before.

Dr. Aguinaga: Then, this is from our lab. We have a newborn with hemoglobin F sickle and A This is a sickle beta plus thalassemia. You will see a major peak for hemoglobin F, for hemoglobin S and a minor peak for hemoglobin A. When this baby grows up, we will have in the next slide a sickle beta plus with a major round for S and then for A on increased hemoglobin A2. And this is another case from the lab, which is beta thal trait where you have A2 increase about 4.2% fetal about 6.8%, and the rest hemoglobin A. It's hard to diagnose at birth. You're seeing hemoglobin A2 levels because newborns have very little hemoglobin A2. And of course that changes after they get six months of age.

Dr. Aguinaga: For beta thal major treatment, usually they require transfusion, beginning around one year of age and they continue throughout life. And because of the excessive number of transfusions, they develop transfusional , and if you don't chelate their iron, this patient will develop cardiac disease and can die. And of course there are other dangers in continuous transfusion besides iron overload, which is the development of alloimmunization, developing antibodies through the transfused red blood cells and certainly the risk of transfusion transmitted diseases.

Dr. Aguinaga: So, in the management of the thalassemia, first you have to establish a clinical diagnosis and then you can look at the genetic definition of this type of thalassemia. Family counseling, and education are key components of the management. chelation, sometimes a splenectomy when it's really necessary. A cure is a stem cell transplantation.

Dr. Aguinaga: The repeated blood transfusion will correct the anemia, and unfortunately this will lead to accumulation of iron in the organs, particular the heart, liver and pancreas which can lead to organ failure and death. And then the use of iron chelating drugs has really increased survival and quality of life of these patients. Now they got living until the late forties, early fifties with over 90% of adults in full time activities. The ultimate cure is bone marrow transplant. But there are challenges in the proportional with matched sibling donor as a limiting factor. And certainly recently, a genetic engineering using and the development of new drugs . So, there are very promising therapies for Beta thalassemia.

Dr. Aguinaga: I would like to thank my colleagues from the Meharry Sickle Cell Center and the Tennessee Department of Health and I would also like to acknowledge funding

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from TDH, the National Sorority of Phi Delta Kappa and the Nashville Duffers Golf League. Thank you very much.

Tim Davis: Thanks Dr. Aguinaga. Welcome Dr. Bender.

Dr. Bender: Good morning. Thank you all for being present. Next slide please. So goals today are first to share some of the arguments for newborn screening for beta thalassemia, but also to share some potential barriers to the acceptance of universal newborn screening for beta thalassemia. I think it's important to be aware of the need for data as to whether there are better outcomes for newborns, for infants that receipt that have beta thalassemia detected by newborn screening as opposed to picked up later. And to review the rationale for screening some presumptive homozygous hemoglobin variants to the compound heterozygous state. For example, EE versus E beta zero thalassemia, which Dr. Aguinaga mentioned in her talk. And then the share some ideas on communication in newborn screening results to providers.

Dr. Bender: Next slide please. And this is just to briefly summarize what Dr. Aguinaga shared about some of the potential dangers and irreversible complications of beta thalassemia including growth delays, those dramatic bony abnormalities and massive organ [inaudible 00:25:02], cardiac abnormalities, endocrine abnormalities, and ultimately death. Next slide, please.

Dr. Bender: We know that as clinicians we can do a great job of preventing or minimizing complications of thalassemia if we diagnose it early. And if there's access to high quality specialty care. This is an oversimplification, but with transfusions, iron chelation and aggressive multidisciplinary care, outcomes keep improving. We can also cure an increasing number of patients with stem cell transplant or gene therapy. We can provide huge benefit if patients are diagnosed and if access to care is available. So why is it not an obvious slam dunk to do universal newborn screening for a beta thalassemia? And a lot of it's about timing. Next slide.

Dr. Bender: So this is a classic plot of hemoglobin switching through development. Next please. So a couple of years ago we talked about alpha thalassemia, which is dramatically different because you have a full level of alpha expression early in embryonic or fetal development. So at birth, alpha genes are fully expressed, so alpha globin abnormalities actually present in utero. Next please. In contrast is with discussing last slide. Data expression is low at birth as the adult beta gene and slowly increases over the first year or two of life. Next, please. Thus, if there's abnormalities of beta genes such as beta thalassemia, there's not a sudden uniform presentation at birth, but it occurs ... Next please, over the first couple of years of life and it can occur anywhere in that period. Next, please.

Dr. Bender: So newborns, when they're born, they've got a lot of fetal hemoglobin, so they're absolutely fine, this is if they have beta thalassemia and I'm going to

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focus on transfusion dependent beta thalassemia, the thal majors that was discussed. In the first months of life, fetal hemoglobin falls, and while hemoglobin A normally rises to compensate for that, in severe beta thalassemia that's not the case. So, what's this do for the newborn? Well, it's not a dramatic onset of problems typically. You have to think about the steps of pathophysiology that are occurring. First, there's abnormalities if hemoglobin synthesis that affects erythropheresis where ineffective erythropheresis increasing anemia, increasing iron accumulation that affects marrow spaces. It's a complex process which is slowly developing over weeks to months or years depending on the severity.

Dr. Bender: But eventually kids of anemia delay growth. They can present with irritability, pallor or just poor feeding. They can present with them a large liver and, or and have tremendous metabolic stress. There's the iron overload, endocrine abnormalities, thinning of cortical bone and boney abnormalities and ultimately cardiac failure and death. Again between the first month, to years of life. So, one of the difficulties is that the presentation is highly variable. It can be very slow and protean until very late, at which time people can present in failure and collapse and die. And so it's hard to paint a uniform picture of at what age beta thalassemia must be diagnosed to intervene safely. Some patients get transfused at a few months of life, other after a year or so. Next slide please.

Dr. Bender: So what happens if not diagnosed early enough? Well, the ultimate concern would be death. But then there's those irreversible bony changes that were shown by Dr. Aguinaga. And then organ damage, some of which can be irreversible. They can have tremendous impact on growth and development. And then there's the sequelae from inappropriate care. Often people are diagnosed with a microcytic, hypochromic anemia and then treated inappropriately with iron. Patients don't respond to iron because they're not iron deficient, which often can lead to accusations of families not following therapy. Sometimes babies are just transfused empirically because they're anemic, which is helpful, but if they're not diagnosed with thalassemia, they just give regular blood.

Dr. Bender: And for thalassemia patients, cost for recurrent transfusions and some other reasons, we do some extended phenotypic matching to prevent the development of allo-antibodies. And then we may miss the opportunity to do genetic counseling of parents before they have subsequent children. Next slide, please. So when to diagnose, when is it essential? And it's obvious that it's important to diagnose before severe or irreversible sequelae. So why isn't this a newborn screening for the beta thal such a slam dunk? Well, let's first look at sickle cell disease where it was obvious. If you look at the little plot there, that's in the 1970s showing survival. And in the 1970s about 20% of children with sickle cell died before they turn six years of age, often of pneumococcal

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infection, often they died before they were definitively diagnosed as having sickle cell.

Dr. Bender: And in these septic deaths with pneumococcus, death can often occur in hours after an infant or toddler presents with fever. So, most infants and toddlers have fevers, but no one rushes to the emergency room. So diagnosing early made a huge difference. In beta thalassemia, it is usually not this dramatic. The caveat here is if people don't have adequate access to healthcare in the US, in developing countries in adoption medicine, then there may be a very prolonged delay in diagnosis and people can be diagnosed when they're in cardiac failure and die. Now, some argue newborn screening is not essential in the United States as thalassemia will be diagnosed and treated before it's too late. Essentially, the idea is that it will be caught by the safety net of US medical care. And my question is really, will this happen? And is there data for this? Next slide please.

Dr. Bender: So to diagnose thalassemia in infancy, if you don't do newborn screening, what happens? How we do this? So, unfortunately we don't know details of how most children of beta thalassemia present. I can share some scenarios that we see, but everyone's going to be different. Now they convincingly argue for universal newborn screening, it helps to know the exact natural history of the presentation of thalassemia.

Dr. Bender: At what age do children present? At what complications when they present? How sick are they? Do they have a mild anemia, growth failure? Do they have irreversible abnormalities or in cardiovascular collapse? And we don't know this. What workups have they had prior to diagnosis? And how many visits and to whom before they're diagnosed? Was it at first encounter they noticed the anemia and fully work it up and refer appropriately? Or like many are there numerous workups evaluations and referrals? And where they were misdiagnoses is underway to definitive diagnosis. And through this, what's the cost to the patient, the family and the healthcare system? Next slide, please.

Dr. Bender: So as a pediatrician you have to ask, how do children with beta thalassemia present? And primary care providers are stuck in a very tough situation because they see hundreds of children that present pale or irritable or not feeding well or not growing well. So how do you tease out those rare events to those with beta thalassemia compared to just other issues? So a question is when to work up such a child, with some pallor. The workup tends to be variable, so what workup did you and how aggressively do you work it up? If someone presents with a large spleen or liver, it's the same questions. And then what follow up does the child or family get? What access to medical care do families have? And it's highly variable.

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Dr. Bender: So do you just do some blood work? Do you do some imaging? As a specialist, these are minor, but for primary care, this is a big deal. Do you refer to hematology? You don't refer every child that looks a little pale. So what do people do? Well, often they get a CBC found as microcytic, hypochromic anemia. Then what happens? Do they just give iron? Do they check lead levels? Do they think it's GI blood loss from a milk enteropathy? It's highly variable. And sorting this out delays the diagnosis and can lead to inappropriate interventions. Meanwhile, many clinics can do point of care hemoglobin checks. So, they'll just do that as a screening. So, this process can be slow of first checking a hemoglobin, then some blood work, bringing him back, kids get lost for follow up. Meanwhile, the baby has a following hemoglobin. Again, the idea is some say there's a safety net to catch these kids before trouble occurs. Next slide please.

Dr. Bender: So the big question is how sick does an infant need to be fully worked up and appropriately referred? And this is very tough. Next please, so what are the risks of delayed diagnosis and Dr. Aguinaga went over this, but it can be delays in receiving definitive treatment, whether it's presenting with life threatening anemia, the growth delays, fatigue, ineffective erythropoiesis with the bony changes. It can lead to the misdiagnosis or inappropriate treatment. As I mentioned, recurrent courses of iron being given an appropriately worsening iron overload, unnecessarily studies conflict between providers and stress with parents in terms of accusations of not giving iron appropriately, the inappropriate transfusions I mentioned and then the counseling issues. Next slide please.

Dr. Bender: So what are the approaches to this? One is education and awareness of providers. And this is ideal, but it's not always realistic due to the scale of this needle in a haystack problem. And I'll show you some iron deficiency screening data and this requires access to healthcare. There's newborn screening, which is not perfect, it will miss some, it'll overcall some, but it'll catch many. I go by, it's the better is the enemy of the good approach. And then it also overcomes issues of access, making it more equitable. And then there's the safety net possibility waiting to have the diagnosis figured out, which can be topped with a variable presentation in these rare events where there's no single presentation. Next slide please. So we need good access to healthcare, and then we also need to know how people present, but we're not sure.

Dr. Bender: We're really counting on people being diagnosed appropriately. So I'm going to pull a little slight a hand here since with that data for beta thalassemia, but instead talk about iron deficiency anemia. And in 2010 there were clear guidelines from the AAP to screen children for Hemoglobin abnormalities at 12 months of age. But the screening test is not particularly sensitive or specific. Screening includes a complex history including socioeconomic status, heritage,

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diet, depending on results, treating with iron and needing to follow up. The big concern is that kids don't get follow-up as recommended.

Dr. Bender: Next slide please. So, this was a study of about 5,000 kids and the bottom line was these are children picked up in general pediatric clinics for being anemic by hemoglobin and no more than 25% of the infants that were screened positive, ended up with a full CBC within six months. So inappropriate follow-up. And no more than 11% had correction. So really a real concern about follow-up. Next slide, or next.

Dr. Bender: So, there's no data, but if this inefficient diagnosis of general medicine is not leading to effective intervention, how smooth are things going for people with thalassemia? So let's do newborn screening. Next slide. So briefly what to do in terms of dealing with homozygous variants versus compound hets. And this is tough because each state lab varies in their technologies and their algorithms. So let's take someone with SS and newborn screening. What do they have? It could be SS or sickle beta zero thalassemia or maybe a severe beta plus thalassemia. It doesn't matter too much because all these initially will be treated pretty much the same. But what about other variances was brought out such as hemoglobin E. How do you distinguish EE and E beta zero thalassemia? EE is quite mild. E Beta zero thalassemia, some are potentially transfusion dependent at a young age. So what's the messaging, how to deal with this. Next please.

Dr. Bender: So again, the approach varies from state to state. In Washington led by Tim Davis, we do DNA testing and I strongly recommend this. There's many possible DNA assays, but I'm going to mention or focus on ours, allele specific real-time PCR with a normal to normal, and a mutant allele also known as Taqman assay because most state labs are set up to do this. So I'm going to show an example for sickle cell testing, but it's the same approach for hemoglobin E. And again, it's not perfect and in this caveat, but it's a wonderful help. Next please.

Dr. Bender: So this is a normal child with FA, and you see an amplification with the A or normal primers. Next please. In FAS, you see amplification with both S and A primers. Next. Now take the child with FS, you only get amplification in this case with the S primers, and this is usually SS. Next please. So, when you do a protein assay and all you get is sickle or FS, with no detectable hemoglobin A, and you do DNA testing and you only get S DNA, most likely it's SS. Next.

Dr. Bender: But when you do DNA testing, sometimes you don't see hemoglobin A, but you get amplification of a normal gene. What are the options here? Next. So that could be sickle beta zero thalassemia or sickle beta plus thalassemia with a low amount of hemoglobin A, so it's complex. Next. So, the same approach can be

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used for sorting out EE versus E beta thalassemia or other variance. And it's very important to do this. Next.

Dr. Bender: And I just want to finish here with some caveats. It's rare, but sometimes you only see S protein and only S DNA but it's not FS. It could also be hemoglobin S with the deletion of the beta gene or S with a variant which alters binding of the PCR primers.

Dr. Bender: Next. And here if it's sickle beta thalassemia could be severe, next, or it could be sickle HPFH which is mild. Next, and the sickle variant we don't know the phenotype, so it's tough. Next, I just want to finish with communication. Messaging for thalassemia in newborn screening isn't as easy as the sickle hemoglobinopathies with a limited number of mutations in a high conference. It's a significant sickle hemoglobinopathy. In beta thalassemia, there's hundreds of mutations with a full spectrum of disease, there's a compound heterozygous as we'll be described in the next talk. It's harder drawing lines as to what's clinically significant and there's just a huge variation for a lot of the conditions. Last slide please.

Dr. Bender: And so messaging. It needs to include statements about the degree of certainty of a clinically significant form, which depends on the technology and lab algorithm used and cutoffs used to report. It depends on the caveats and limitations of testing. It needs to message about the potential severity and the need to be seen by a hematologist and then the potential role for genetic counseling. And last, thank you very much for your time.

Tim Davis: Thanks Dr. Bender and welcome Dr. Hassell.

Dr. Hassell: Thank you very much and good afternoon to everyone. I have two [inaudible 00:43:54] before I begin. The first is [inaudible 00:43:55] acknowledging that they helped us to conduct other survey and I'm sharing the results of our collective work. And the second is although I was introduced and do have a hand in our state and our hospital's a testing for hemoglobinopathies, I would not have nearly the expertise that you just heard described by my colleagues as regards on the technology. So mine is to give you the 10,000 foot view of important principles that we learned about the status of newborn screening for beta thalassemia in the US and have some thoughts about that, but the group of us together collectively, those with most expertise will answer some of the highly technical question. Next slide please.

Dr. Hassell: So appreciating that [inaudible 00:44:39] and the clinical background that you've heard, other working group and APHL start to understand the landscape, if you will, of newborn screening programs approaches to beta thalassemia. And as you've heard described, there's a significant challenge that is to say that all babies at birth may appear to be well, with beta thalassemia major at that point

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because they have absent or a little bit of A. And the challenge is in the newborn period to understand who will never go on to make anymore A and have beta zero thalassemia such that they will incur all the clinical complications that you've heard, which might be more effectively addressed with early detection. And what about those individuals who carry a beta zero or beta plus thalassemia gene that might impact their hemoglobin health, if they will, and create a hemoglobinopathy?

Dr. Hassell: And recognition of this is very challenging based on the change in hemoglobin production that you saw Dr. Bender describe. So, our purpose with this survey was to gather information and ultimately to develop training webinar along with recommendations on screening and reporting of beta thalassemia. Next slide. So the survey was disseminated electronically from end of August until early October of 2018. There were 46 respondents [inaudible 00:45:56] program. The caveat is, some people said, “Well we use a different state lab or we use a different follow-up program to ask them what we do.” So this is again a 10,000 foot summary view with some of the nuance details outlet to the side for the moment.

Dr. Hassell: And we first explored three basic areas, did prove my colleagues described. The first is do labs and programs seek to detect and report possible beta thalassemia major? But you heard described in most detail. Secondly, when a variant is present, but there's no hemoglobin A, so F and S as the examples Dr. Bender just spoke about initially, does this mean that the child actually has F and S utero genes or do they have S and beta zero thalassemia? And to his point in one, I'll emphasize that it doesn't matter in sickle cell disease. But in hemoglobin EE versus E beta zero, there is a clinically significant difference in the phenotype and onset of difficulties.

Dr. Hassell: And then the third area was when there's a gene that makes some hemoglobin A, a beta plus T in combination with the variant. And here the challenge is distinguishing between a person who has a single say S gene, but the other gene is normal sickle cell trait. Or what if that other gene isn't normal, what makes A just not enough? And that would be a sickling disorder called sickle beta cell thalassemia. So the survey sought to explore as best we could devise these three areas. Next slide please.

Dr. Hassell: An initial question engaged the respondents in how did they do newborn screening for hemoglobinopathy? You can see depicted on the slide, most programs utilize in one order or another, Isoelectric focusing or high performance liquid chromatography, HPLC. A few programs used only one or the other and some reflex to DNA. And some them simply said, “Well we send it somewhere else.” Next slide please.

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Dr. Hassell: So, the initial question was, does your newborn screening program and this could refer to the lab or I suppose in the follow up component as well. Does the report of the newborn screen comment on any type of beta thalassemia? And so 82% of the programs that responded said, “Yes, we comment about beta thalassemia.” So the first area of interest for those programs with 38 of them has said they did so. We asked, “Do you report quality possible beta thalassemia major?” And recognizing we're a screening program, we aren't making diagnoses, but we are giving advice to a clinician in a follow-up program that safety net as Bender alluded to, the bids may be an infant potentially affected by significant Hemoglobinopathy and 28 of the 38 programs said, “Yes, we report this.” Whatever reporting meant to them and agreed to the same impossible beta thalassemia major.

Dr. Hassell: Next slide. But we were then interested to learn from the laboratory perspective, how did they determine that they should make this report and specifically do you have a cutoff for the percentage of hemoglobin A that for their program would discern between an infant who hasn't made enough A even at this early age, and thus raises the concern of the potential presence of beta thalassemia major. And 16 programs out of the 28 who said they report this, said they had a cut off and so we asked, “What is it?” And so as you can see highlighted the two most common responses were either using a threshold of less than 1% or less than 3%. And one program and said less than two and a half percent. Interestingly, one program at least stated as they responded to the survey that if the hemoglobin A was less than 20% at birth, that they might likely as it is possible beta thalassemia major.

Dr. Hassell: And then other programs where they used the combination of testing between IEF and HPLC, they commented that there was no visual A presuming, looking at an isoelectric focusing gel. If there was no cutoff, but they said, “Yeah, we do an assessment to make that report.” The comments were, if the only reportable band is F, although it wasn't characterized what reportable match, if there's an absence of A, one presumes literally nothing is visualized on the gel. Or some programs said, “We look at qualitative outcomes in collaboration with our consultant PP hematologists or others.” Next slide please.

Dr. Hassell: So, what do we know about, what can be done at the newborn screening level to try and detect a child potentially effected by beta thalassemia major? And the largest amount of data come out of the UK. And so I want to share with you the newborn screening program experience in the UK. So in 2003 in the UK ... First let me put the context. So, the UK does universal screening for Hemoglobinopathy and they have a fairly tight connection in that country between the newborn screening last and the follow-up program, the centers that would actually treat hemoglobinopathy including beta thalassemia.

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Dr. Hassell: Well in 2003, they recommended a cutoff of less than 1.5%, done by HPLC most usually. There is purely, they acknowledge in their [inaudible 00:51:18], than expert opinion. They didn't have any data, they just said, “This seems pretty low even for a newborn.” And so we think this is a place where we had a trigger, a consideration of beta thalassemia major. So, someone later presented in 2013, 10 years later, they reported on some work that they did to see how is this fairing.

Dr. Hassell: And so the initial work, looking and assessing hemoglobin A levels on newborn screening. So there were three cohorts of children that they looked at. They looked at all 4.6 million babies that were screened in 2005 to 2012. They looked at a subset between 2004 and 2012 and looked prospectively at how many infants were identified with hemoglobin A less than a threshold, and then they did single subset look back. So, they looked at all the infants that were clinically ultimately discovered to have beta thalassemia major and looked back at their newborn screen to see if they could tell what the hemoglobin A values were at birth on the newborn screen. So, trying to evaluate that threshold if you go. And so there were 51 babies in the lookback and 119 babies prospectively identify who had this 1.5% or lower hemoglobin A at the time of newborn screening.

Dr. Hassell: And they found no false negative, No babies were missed, at least as they knew it through their connection to the system of care in the UK. There were 15 false positive. Some were because hereditary persistence of fetal hemoglobin was present really robustly making fetal, not just as a natural consequence of hemoglobin in the normal individual. But the most usual thing was that the babies were at younger gestational age. You saw that curve, that's switched really start as you get into the late third trimester and as the child comes in at newborn screen before that there's going to be a lower percentage of hemoglobin A and that was the most usual thing they found with the so called false positives, that it's being worried about beta thal major when it wasn't the case. Next slide please.

Dr. Hassell: They did graph from a subset of 30,000 on transfused babies. Just a reminder who's blood is it? They need to be untransfused or it's not their hemoglobin A. But looking at a subset of 30,000 you can see the Blue Line is the fifth percentile of all those kids and the 1.5% threshold is well below that. So it made some sense even as they looked at their experience in those 10 years that this would be an acceptable threshold. Next slide please.

Dr. Hassell: A subsequent study was done with a prospective now country ride validation study, including all of the centers doing newborn screening. And they did a study to see when babies were identified as having less than 1.5% hemoglobin A, they look to see what happened.

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Dr. Hassell: Now the diagram actually shows that they went looking at less than 3% and as you'll see if you follow there on the left side of the diagram, as you're looking at the slide, there was one false negative and that child was clinically decided to certain to have beta thalassemia major or a classical case of beta thal major, and the hemoglobin A was 1.7% at birth. But they really focused on those who had less than 1.5, there are 81 cases. DNA confirmation was done in 36 cases and DNA wasn't done in 46, 9 were left to follow them up. But what they found was that there were six cases that were falsely positive. That is this approach, unduly raised the alarm about some [inaudible 00:54:57] beta thalassemia major when in fact that was not the case. And all of them were less than 32 weeks gestation. Next slide please.

Dr. Hassell: So this experience from the UK using this threshold, I'm suggesting that if one is using HPLC or electrophoresis, other labs which I think globin chain balances that had HPLC, but there's a 98 to 99% specificity and sensitivity and the positive predictive value is especially if you exclude infants who are less than 32 weeks of age. At gestation rather that the positive predictive value is upwards of 95%. They did look to see if particular mutations that cause beta thalassemia major were more likely to be associated with a slightly higher, slightly lower percentage of hemoglobin A and that was, there was no correlation in the [inaudible 00:55:49] that they did. Next slide please.

Dr. Hassell: We also updated from the New York newborn screening program which was presented in New Orleans Symposium in 2017. And this information was courteously provided by Chris Dawley and this is the work of Bennett and colleagues. Hopefully coming to publication soon. But this is taken from their poster. And the point I wanted to make is that if in the program they used an approach whereby HPLC the hemoglobin A was less than 2.5% of birth and then they followed this for those infants with Sanger sequencing. That if you use a threshold of 2.5%, that there were no false positives, and there were no false negative. And there were some instance where testing only detected to have the heterozygous and there were some who had no mutation identified. But they posit that this would be an acceptable threshold as well for trying to capture individuals who may be at risk for having beta thalassemia major as a screening program approach. Next slide please.

Dr. Hassell: The secondary research to understand is, okay, let's say you only have one of those beta zero genes, but you also have a variance. Dr. Bender covered this thoroughly. So I will be brief. So we did ask the program, "Do you report this infant potentially has, may have, think about E beta zero thal explicitly? Not just FNLA but explicitly do you report G-beta zero thalassemia as what's been possibly detected at newborn screening?" And then you can see about half the programs are that they specify that a C-beta zero, E-beta zero, F-beta zero. And so we were interested if you do this, how do you distinguish between E and E or E and beta zero. And if you report, this is homozygous C Diseases. No beta zero

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involved, this is E Plus P equals to your E Plus E F Plus F. Do you comment just as a caveat or maybe there is a beta zero balance? So next slide please.

Dr. Hassell: Let's see what the country does. When distinguishing between homozygous and variant beta zero thalassemia and I think Dr. Bender [inaudible 00:58:09] on with what seems to be clinically most important, is that EE, which is likely to be mild or E-beta zero? Because the result is FE no way. And so programs, even as they might've said, they use the language of E-beta zero, most programs says “We don't distinguish because we can't.”

Dr. Hassell: Interestingly some program says that if there's barely or no visible A and isoelectric focusing and they have lots of 1% A on HPLC, AE but then they have virtually no way this for their program was read as homozygous, E plus E. Some programs as you've heard described in Washington, I use DNA, others send to our reference lab or perhaps DNA is done and I'll point out this last one, I think it was probably just either by us or otherwise miss categorize because they comment as varied is more than A. But A is presence in 5% that doesn't make any sense for a beta zero thalassemia. We'll talk about that in a minute. Next slide please.

Dr. Hassell: We then asked if you report hemoglobin EE as a possible condition present in this infant. If you stay that beta zero could also be in the differential considerations and most programs said, “No.” If they're confident on reporting E plus E then they don't comment about the beta zero being a possibility. Although one program said, “Guy got us thinking.” Five programs says, “Yes. We always say that the possibilities for either of these and other answers included, we've hung out the act sheets where this was discussed. We send letters in fact sheets or we just lift frankly this could be a CDE disease that's present.” Next slide please.

Dr. Hassell: And so you appreciate here the biologic challenge of newborn screening. So baby as you've heard described with E plus E is only going to make E. There shouldn't be any A on the newborn screen. If there's FE beta zero thalassemia, there may be virtually no A presence. So that absence of A actually it doesn't help you sort out what the genes may be providing. Next slide please. And so most literature that are looking for help suggest that DNA analysis is necessary to distinguish to be homozygous variant and a variant with a beta thalassemia gene present in the newborn period.

Dr. Hassell: [inaudible 01:00:36] my colleagues clinically comment that later in life there's , HbA2 is high. There's a persistent elevation of fetal hemoglobin name with a beta thalassemias. And so later in life [inaudible 01:00:47], but for newborn screening lasting programs, it can be very challenging to try and infer what the are by finding an FE no A without honestly looking at the DNA. Next slide please.

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Dr. Hassell: The final way, where beta thalassemia throws a monkey wrench into all of our newborn screening efforts for hemoglobinopathy is when one gene is a variant CDEF and the other one is a beta thalassemia because if the other gene is normal, that's a trait which is often clinically formless or nearly so, and that's something that requires attention, whereas the variant plus beta thalassemia not making quite enough A to counteract that variant hemoglobin, especially this nationally hemoglobinopathy, those are two very different things. And so we asked programs, “Do you report this C-beta thalassemia is expected. This is E-beta plus. This is importantly, I would argue S-beta plus thalassemia?” And the three quarters of the program said, “Yeah, we distinguish this.” And so we asked, “How do you distinguish between that decided to C gene, that other gene is beta thalassemia rather than normal. How do you tell it that second gene is doing?” Next slide please.

Dr. Hassell: So, programs especially that use IEF as the principal, the mainstay for their training comments, but an HPLC too that the visual variant band is stronger or the quantity is greater than A, that's aging. If it's normal, usually wins in the production game and there's more A than variant in a trait. And so if there's let's say this must be a beta thalassemia present with the variant. Others are more commonly interestingly, programs use a quantification. So, many programs use as a variant is twice as much as the A. They felt pretty confident that this was a variance beta thalassemia. This was S-beta plus thalassemia not sickle cell trait.

Dr. Hassell: Other programs use a relative percentages as the variant is 3% greater than hemoglobin A. And they use the example of was 6% and A is 3% and others commented that they only needed the 1% difference. That there's 5% S and 4% A that this would get a possible sickle beta plus thalassemia report out of the lab. Other split the ratio but the principle of the same twice as much variant or 30% more variant than was present with A. And then labs that could have commented that they would reflect to DNA that sorted out. And then some programs just, “So we don't try, we let the consultants do it.”

Dr. Hassell: And I think that's an interesting thought given the relatively common finding of sickle cell traits versus sickle beta plus thalassemia as a common potential scenario in the US. Next slide please. There are some data about this issue that I could find. So based on California's experience and they're published work that the variant being twice as great as hemoglobin A and makes sense and resonated with the experience and they're very large newborn screening program. A recently published French study demonstrated that the ratio is having twice as much as a variant and specifically S and C having twice as much as those as compared to A was very consistently, specifically 100% of the time for their data could distinguish between sickle cell trait and sickle beta plus thalassemia or C trait and C-beta plus thalassemia. I think and I couldn't find any detailed or data to this issue.

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Dr. Hassell: As you've heard hemoglobin E is under produced and so this thalassemia, it's trickier I think when as hemoglobin E because you're not going to get as much production as you might as robust S or robust C. But in any case, these are what the literature, data support in terms of using a quantified approach and an algorithm to distinguishing between trait and a variant in the presence of beta plus thalassemia. Next slide please.

Dr. Hassell: We then asked respondents, “To whom do you report results when you think if your report, then there's 30 programs to do.” When there's a beta thalassemia in the mix with a variant. And you can see it reflects, I think always difficulty in writing survey questions because I hope they all tell the submitter. And maybe that's the at some case or newborn screening, perhaps not follow-up program. I bet they report stuff all the time to the newborn screening follow-up program. But in any case, you can see there's a variety of individuals who receive information about the presence of a variance with beta thalassemia present and beta plus hemoglobin zero. Next slide please.

Dr. Hassell: And then we did inquire as to whether or not molecular testing is used for beta thalassemia. Eight programs of 21% of the respondents said “Yes.” And when we asked what methods do we use, you can see, and we were characterized by our colleagues from Washington State, Taqman and the Real Time PCR allelic discrimination. And then our one program volunteered its specific mutations for which they interrogate a sample to look for the presence of beta thalassemia. Next slide please.

Dr. Hassell: Finally, we asked, “Does your newborn screening programs provide follow-up recommendations for patient retesting and follow up?" And the vast majority of programs do sometimes that [inaudible 01:06:32] mailing actually for communications to the follow-up program to the submitted or the physician or their consultant in terms of their follow-up specialty care. Next slide please.

Dr. Hassell: So it was a very interesting thing to see whether the paucity of data is available and newly evolving in this arena. When we're talking about the various serious condition of Cooley's anemia or possible beta thalassemia major, that intent is never going to make hemoglobin A, beyond the little bit that you see at birth. 60% of the programs report that in some of them discern which instance they're going to report by using cutoffs, which very much usually I have determined by HPLC or the lack of visible A relying predominantly on isoelectric focusing and a few programs are using DNA to go the next step in terms of tertiary levels of testing.

Dr. Hassell: And there are some data I would submit that support a quantitative cutoff perhaps at 1.5 or 2.5% recognizing this as mostly generated by using specifically the HPLC, the Bio-Rad and hemoglobin techniques that aren't commonly in the field. Next slide please.

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Dr. Hassell: As regards to [inaudible 01:07:54] pardon me, distinguishing that SE no A, is that two SS or is that an E and a beta zero which as Dr. Bender has noted, that's probably clinically an important thing to distinguish as we can. 40% of the programs say that they're reporting E-beta zero style, for example, a variable of beta zero thalassemia. But it's difficult to understanding exactly how that's being distinguished unless DNA is being used. Because as we talked about, it can be very difficult to sort out that FTE MLA. And what's the underlying genetics might actually be based on at least analyzing the proteins or lack zero in the newborn screening program. Next slide, please.

Dr. Hassell: And then finally, clearly since our goal in newborn screening for hemoglobin off seasons to identify hemoglobinopathy, but these are conditions distinguishing between a trait and a hemoglobin disease is important. And 55% of the programs responded, they report that they think this is a variant beta plus thalassemia versus the trait. And that's often a ratio is used as you can see. And this is as I said, clinically important to distinguish. The follow-up was very different and there are some data to support the ratios that are commonly used, but what was lacking is the reality in our day to day lives and the newborn screening programs.

Dr. Hassell: Okay. So it's not two to one, it's 1.8 to one, it's 1.5 to one. So it's easy to pick very discrete ratios. But unfortunately and their data don't always fall neatly on one side or the other. Next slide. And so I think we're looking for some guidelines and APHL and the CDC in 2015, put out a monograph about newborn screening for hemoglobinopathies. And they described the things we've discussed in principle, but given their acknowledgement that laboratory methodologies very not everyone uses HPLC or they may have different versions and now I'm [inaudible 01:10:02] used sometimes it's IEF and many capillary like race is coming on board, they give guidance in a general sense. But the specifics that might help standardize programs were not forthcoming because I think we don't know what they are yet.

Dr. Hassell: The UK was a little more affirm, given us a unified and arguably smaller system with standardized approaches to screening across the country and they said if you have an hemoglobin A versus 1.5 this is what they would call F only and the baby should be referred for further evaluation for possible beta thalassemia major. If they have and I picked FS, FE might be more clinically relevant if they have a finding where there's no appearance, they are very little parent A they refer to a diagnostic algorithm. They send them off through their follow-up program to a diagnostic center to sort out between the two.

Dr. Hassell: As Dr. Bender said for sickle cell anemia, that doesn't matter very much but it may matter in terms of the intensity of follow for EE versus the beta zero thal. And then in terms of distinguishing and they explicitly comments in the UK guidelines about sickle cell trait from sickle beta plus thalassemia, they say

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usually in their experience that the S is 120% greater than the A, so their standard in their program is to call that sickle beta plus thalassemia, and if you need to sort it out her that they would recommend DNA usually done through their follow-up or clinically specialty centers and or to rely on parental testing to see if they can sort it out as well. So, while there's evolving information and lots of thoughtful approaches, not really [inaudible 01:11:45] per se and that's the because there is a lot of grayness in this area given the challenges of beta thalassemia and it's a manifestation in the newborn period as opposed to later in life. Next slide please.

Dr. Hassell: So, see I have just a minute and so I want to make a point and emphasize what I just said with this. I put it as a picture, in Belize along the east west highway I walked down there, headed there this Saturday to do some medical missionary work. And so there's this thing called the sleeping giant in Belize. And so as the first day I drove this side I said, "I see the sleeping giant." I know it's there. I know it's a reality. If you look just to the left and right well you can see the big nose and then the chin, and then follow down the neckline. And then a belly and maybe he's got his legs folded. And I said, "There you go, there's the sleeping giant." And another non Belizean said, "Yup. I know the sleeping giant is there and I can see it, it makes sense to me. I can see everything."

Dr. Hassell: And they let me go for three or four years of telling other non-Belizeans about this until they finally handed over to me and said, "Dr. Hassell, that's not the sleeping giant. No matter what it looks like." Actually that's further west, west to Guatemala most tourists and others don't ever see it. And so the point is, we know thalassemia is out there, beta thalassemia is out there, and collectively we do things that make sense. But as Dr. Bender in particular called for, we need a growing expansion of the available data to know that what we're looking at is what we're really looking at is we tackle this problem in the newborn screening. And with that I'm going to stop and thank everyone for their attention to my presentation today.

Tim Davis: Thanks Dr. Hassell.

Careema Yusuf: Great. Thank you to everybody. We now have an opportunity for the audience to ask questions of the speakers. Please feel free to press star seven to unmute your line or if you'd rather type your question in the chat box that's located in the lower left hand corner, you can do that. And I'm happy to read out your question for you.

Dr. Bender: This is Dr. Bender and I just wanted to reinforce what Dr. Hassell was saying about ratios of variants to hemoglobin A, but it gets even more complicated because there's an assumption that the affinity for Alpha chains is the same for various variants and we know that's not true. So some variants actually have a

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higher affinity for Alpha. And so looking at the ratio of eight, a variant can really pull you at times.

Careema Yusuf: Thank you. Again it's star seven to unmute your line. More if you'd like, you can type your question in the chat box in the left hand corner of your screen. Okay. One last call for questions, star seven to unmute your line or you can type your question in the chat box on the left hand corner. All right. I do have a question. The parson says, “I just want to put into context why our quantitative cutoff is higher than 1.5. It's actually 2.5.” I think that's what it says. Early on we actually discovered two, I guess two cases that were above the 1.5% cutoff. And this person is from the New York program? Yes. Two cases. They found two cases above the 1.5%, so they're now using 2.5 as their cutoff.

Dr. Hassell: Yeah, and this Kathy Hassell, and I get to the New York and you guys do awesome work. I was very impressed with the information on the poster and look forward to the publication. My apologies if I missed it, recent publication. The UK programs did miss the one infant at 1.7% and I do think, well they've adopted the 1.5% there's certainly room for discussion. And permitting, if you will, it's an age old question of newborn screening, right. And allowing for some, if you will, false positives gathering more infants, had identified you don't really have the condition to capture those relatively few. And I completely understand why the New York program picked the 2.5 and I [inaudible 01:16:46] about, if we pick a number where that should be.

Careema Yusuf: Thank you. Any other questions? Again, you can press star seven to unmute your line or you can type it, no question in the checkbook.

Speaker 6: Hello?

Careema Yusuf: Hello.

Speaker 6: Yeah, is this cutoff that was being discussed, is it only for thal major or is it for the intermedia or is it set only thalassemia major?

Dr. Hassell: So this is Kathy Hassell and my colleagues may also weigh in of course, I did. It was specifically to identify transfusion dependence thalassemia major that as might imagine there many fewer data and they are considerably murkier when one looks at the intermedia. And certainly anything beyond into the thal minor territory as we heard our first presenter discuss because variability and how much A is produced by those individuals, is quite high because some of them may go on to only have thal minor and some, intermedia and there's so many different combinations of mutations that might lead to that may impact the level of hemoglobin A. So I would say the UK has adopted and perhaps the programs in our country that I do look for thal major, it is targeting that transfusion dependence thalassemia major.

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Careema Yusuf: Great. Thank you. Any other questions? So I have a question in the chat box saying, should we embark on a national study involving all the state public health programs to come to consensus on when and how to test for beta thalassemia in the US? I guess I'd ask that of the speakers. Any thoughts?

Dr. Bender: This is Bender, I think that's a wonderful question and it's an important one. And I think it's really breaking it up into a couple of things. One is the diagnostics, which Dr. Hassell was talking about, but also we really need to obtain a better idea of the natural history of presentation for the various forms of beta thalassemia in the US. And that's going to be a very large and complex process. But I think that would be very important and wonderful to do.

Dr. Hassell: This is Kathy Hassell. I concur. I think that's a practicality financial and restores the investments, recycles investment all always come into play for these questions, but I do think there's an opportunity with well-defined and circumscribe goals to do this and Careema I don't know if you're going to announce about the Round Table that's going to take place maybe you could provide more details about that in the upcoming meeting, but it may end up being more ad hoc than very formal. It may end up with collaboration to APHL for whom as I declared, I can't speak but would encourage, I think it's an area of newborn screening for hemoglobinopathy that is potentially quite right for at least a consensus development.

Careema Yusuf: Thank you Kathy. And yes, thank you for reminding me for the opportunity to advertise the Round Table that will be held at the Newborn Screening Testing Symposium in April, it is going to be at 7:00 AM on Tuesday, April 9th. The Round Table will talk about the status of beta thalassemia in newborn screening, cover what we've covered in this Webinar as well as have a discussion. And I think the question that was posed would be a great question to deliberate over at this round table.

Dr. Aguinaga: Yeah, it will be great ... This is Dr. Aguinaga, to do a retrospective study with diagnose cases or beta thal intermedia, with thal major, seeing that they have a DNA analysis available and when was he diagnosed? That's also important because some may be diagnosed at birth if it's a beta thal major, some maybe diagnosed later on in life when they present clinical symptoms. So, I think from the all the data that the set up will be a wonderful retrospective study that we could do and come up with an indicator, so when and how to test for beta thal in the US. Thank you.

Careema Yusuf: Thank you. Again, you can press star seven to unmute your line or you can type your question in the chat box. Okay. I'm hearing none and don't see any additional in the chat box. I'd like to thank everyone for their time this afternoon. Just a quick reminder that PACE credits are being offered for this Webinar, you will receive a follow-up email in a couple of days about that. And

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the slides will be available. We have recorded this Webinar and we will send out a link as well to the slides and the Webinar in the coming week. Thank you again and have a great rest of the afternoon.

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