Thalassemia and Iron Overload Ellis Neufeld, MD, PhD Harvard Medical School April 9, 2016

What is hemoglobin? a a b b Hemoglobin is the oxygen-carrying protein in red blood cells. Oxygen binds to hemoglobin in the lungs and is delivered to the tissues by the circulation. Adult hemoglobin is composed of four polypeptide chains – 2 a and 2 b chains – that are non-covalently bound to each other. Each polypeptide binds one molecule of heme, which contains the oxygen-binding iron atom. a a Heme (Fe) Heme (Fe) b b Heme (Fe) Heme (Fe)

What are hemoglobinopathies? Genetic disorders resulting from qualitative and/or quantitative defects in hemoglobin “Structural” hemoglobinopathies result from qualitatively abnormal hemoglobin (e.g., due to a mutation in a coding region of the alpha or beta globin gene): example, sickle cell disease “Functional” hemoglobinopathies result from quantitatively abnormal hemoglobin (e.g., due to deletions or to a mutation in a regulatory region of the alpha or beta globin gene): example, thalassemia syndromes

Chromosomal loci of a and b globin Developmental expression pattern proceeds along eachlocus:  and  Strong expression requires upstream regulatory elements

What are thalassemia syndromes? Hereditary anemias caused by mutation or deletion of one or more genes responsible for the production of alpha or beta globin chains Alpha thalassemia caused by a defect in alpha globin gene(s) Beta thalassemia caused by a defect in beta globin gene(s) Pathophysiologic consequences are due to both decreased hemoglobin production and imbalanced alpha and beta globin chain synthesis

Beta Thalassemia Mutations Mutation Type Identified Alleles, n Deletions 17 Transcription 22 RNA processing Splice junction Consensus 12 IVS I or IVS II 5 Coding regions Poly A 6 Translation Initiation Non-sense Frameshift 55 Unstable  chains 30 CAP site 1 Unlinked to  locus 3 3’ UTR Insertion The heterogeneity of the beta thalassemia mutations is reflected in the various deletions and mutations associated with the expression of the beta globin gene, and here we see that there are over 200 different types of mutations that lead to defective synthesis of beta globin. In fact, many of these genes are severe enough to cause the complete absence of beta globin synthesis referred as beta 0 production, and some of them are mild to moderate in that they have diminished beta globin production referred to as beta plus. RNA = ribonucleic acid; IVS = intervening sequence; A = adenosine; CAP = catabolite activator (gene) protein; UTR = untranslated region. Reprinted with permission from Weatherall. The Harvey Lectures. 2001;Series 94:1.

Thalassemia: an international health problem Thailand Iran Azerbaijan Italy

Different Beta Thalassemia Mutations are Found Worldwide In fact, here we see that worldwide there are probably only about 21 common mutations that are found in every regional population but, as seen in the highlighted areas of this slide, there are a significant number of milder or beta plus mutations and, in fact, the presence in high frequency of any one milder mutation in a population in combination with the other moderate and severe mutations will result in significant clinical heterogeneity and phenotypic expression. Weatherall. Nature Reviews Genetics. 2001;2:245.

Epidemiology Most common genetic disorder worldwide >240 million people have heterozygous thalassemia (trait) More than two million people with thalassemia intermedia syndromes Hb E/thalassemia 3-gene alpha thalassemia Hundreds of thousands with homozygous or compound heterozygous thalassemia syndromes Approximately 1,000 patients in North America Hemoglobin disorders finally “made it” (2010) onto the WHO top disorders for “burden of disease”

What are the consequences of globin gene dysregulation? Imbalanced expression of alpha and beta chains leads to: Decreased functional hemoglobin, resulting in anemia An excess of alpha or beta chains, causing red blood cell membrane toxicity and cell lysis (hemolysis) Anemia and red cell destruction stimulate increased iron absorption and increased erythropoiesis (if severe enough, the marrow can’t keep up and patients need transfusions)

Clinical variability and phenotypic overlap among β+ thalassemia syndromes Beta thalassemia major b+ thal or b0 thal Transfusion dependent by age 1 usually, sometimes later Prenatal diagnosis Profound microcytosis, hypochromia Extensive multi-organ involvement Beta thalassemia intermedia or “non-transfusion dependent thalassemia (NTDT)” Strong b+ allele(s) may allow transfusion-independence, but may not prevent complications including growth failure, weak bones, iron overload Whether or not to transfuse is, for many patients, a decision to be made, not obvious “yes” or “no”

Bone changes in thalassemia Compression fracture “Thalassemic facies” – avoidable with modern hypertransfusion Rx

Iron overload IT DOESN’T! Hyperabsorption of iron, combined with ineffective red blood cell production (ineffective erythropoiesis), leads to inefficient recycling of iron and excess iron How does the body get rid of excess iron? IT DOESN’T!

End-organ toxicity of chronic iron overload in thalassemia 4/28/2017 End-organ toxicity of chronic iron overload in thalassemia Assessed for 199 patients age 15 yrs Heart disease requiring medication 16% Hepatic failure or cirrhosis 6% Endocrinopathy: Thyroid 14% Parathyroid (“thin bones”) 5% Diabetes 18% HRT for hypogonadism (requiring therapy for sexual characteristics/fertility) 53% Let’s turn now to end-organ toxicity related to iron overload. Cardiac disease continues to be the leading cause of death in young adults with thalassemia major. It is pretty much a tautology to say that severe end-organ toxicity in thalassemia is age related, since it tends to be doe to iron toxicity integrated over years or decades. We will look among the nearly 200 subjects over 15 years of age. A surprisingly large fraction of these patients are on medication for heart disease. 6 percent have significant liver disease. Note the high burden of disease in a young population

Therapeutic options Transfusion with Iron chelation Intravenous or subcutaneous deferoxamine (Desferal) Oral iron chelators: deferasirox (Exjade, Jadenu), deferiprone Novel chelators failed in recent trials due to nephrotoxicity: Stem cell transplantation (curative) Gene therapy – add a new globin gene (first published success 2010 Nature Medicine) 2014 updates: remarkably high gene expression from lenti-viral driven marked beta globin. Eur Hematology Assoc summer 2014 Am Soc Hematol December 2014 Summer 2015: less success if the enotype is beta0 thalassemia Activin receptor 2 trap strategy, reduce ineffective erythropoiesis by ?mechanism

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Mechanisms to alleviate thalassemia Improve chain imbalance Alpha thalassemia trait reduces severity of non-transfused beta thalassemia Gene therapy aims to add gamma or beta globin Gene editing aims to de-repress gamma globin Non-chain imbalance mechanisms: Activin receptor blockade- less ineffective erythropoiesis Increased hepcidin strategies Hepcidin mimetics TMPRS-6 knockdown (RNAi) other

Summary of thalassemia and iron overload Thal major >8 transfusions/year High iron burden due to transfusion End-organ damage due to iron overload Heart, pancreas, pituitary, liver Lifelong chelation therapy Potential deferoxamine toxicity: retina, hearing, pulmonary, renal Thal intermedia or ‘non-transfusion-dependent thalassemia (NTDT) Survive without transfusion Brisk, but ineffective erythropoiesis throughout life; high RBC turnover High iron burden from increased GI absorption and RBC turnover End-organ damage May be similar, but decades later than in thal major