Hemoglobinopathies- Part I Nemer El Mouallem MD VCU Health system 05/23/2018

Hemoglobin A structure Tetramer -2 pairs of globin chains -Alpha-beta dimers aggregate to form tetramers Heme -Complex of Fe2+ and protoporphyrin -Covalently bound to each globin molecule -Reversibly binds one O2 molecule

Normal Hemoglobin found in adults HbA : α2β2 97% HbA2: α2δ2 3% HbF: α2γ2 <1%

Human Globin Genes

Thalassemias Most common single gene Mendelian diseases Disorders of the synthesis or structure of hemoglobin Almost 1500 variants described

Thalassemias and Malaria

Hemoglobinopathies vs Thalassemias Hemoglobinopathies: amino acid substitutions leading to an abnormal globin structure . Examples: HbS HbE HbC Thalassemias: reduced amounts or absence of a structurally normal globin chain -Alpha thalassemias -Beta thalassemias

Hemoglobinopathies vs Thalassemias Interaction among the two are common: Hemoglobin S/beta thalassemia Hemoglobin S/alpha thalassemia Hemoglobin E/beta thalassemia

Diagnostics Hb electrophoresis Cellulose acetate (alkaline): ID’s HbA, HbF, HbS, HbD, HbE, HbC, HbO, HbH Citrate agar (acidic): distinguishes Hb C from Hb E or Hb O HPLC (High performance liquid chromatography) Retention time, peak characteristics influenced by single aa substitutions Accurately identifies 75% of Hb variants Molecular biology PCR, gene sequencing

Thalassemia mutations Alpha thalassemias Mostly caused by gene deletions Clinically expressed in fetus and at birth Beta thalassemias Mostly caused by point mutations Expressed several months after birth when switch from γ to β globin

Beta thalassemias Decreased synthesis of beta globin genes  excess of alpha globin chains Alpha globins aggregate to form insoluble inclusions in erythroid precusors Intramedullary death of erythroid precursors: ineffective erythropoeisis

Beta thalassemias Unpaired and unstable alpha globin chains and excess Fe cause: Membrane lipid oxidation Membrane protein damage Decreased RBC deformability Removal from circulation  Membrane damage leads to polysaccharide exposure and hypercoagulability

Beta thalassemias High degree of erythropoietic activity coupled with ineffective erythropoiesis  Death of erythroid precursors in the bone marrow Hemolytic panel is positive though retic level not as high as expected These phenomena are more evident in thalassemia major and intermedia

Beta thalassemias β0 thal mutations: critical point mutation or deletion leading to no expression of beta globin Β+ thal mutations : partial expression of beta globin chain

Beta thalassemias

Beta Thalassemias

Beta thalassemias

Beta thalassemias

Beta Thalassemia

Beta Thalassemia major Clinical Features Hematologic: severe microcytic anemia, splenomegaly, extramedullary hematopoiesis Skeletal changes: expanded marrow cavity, thalassemic facies, osteopenia, thin cortex Growth Retardation Thrombembolism

Beta Thalassemia major Cardiopulmonary: Myocardial Fe overload with arrhythmia, CHF, hemolytic pulmonary hypertension Liver: Hepatic iron overload  fibrosis, cirrhosis. Pigmented gallstones Endocrinopathies: DM, hypoparathyroidism, hypogonadism and delayed puberty Transfusion related: infection, alloimmunization

Thalassemia major- iron overload Each unit of blood contains ~ 225 mg of Fe

Thalassemia major- iron overload

Beta thalassemia major Treatments Comprehensive care centers, multi-speciality, strong social services Hypertransfusion beginning 2nd or 3rd year: Maintain Hb > 9-10.5 Splenectomy for increasing transfusion requirement Iron chelation starting after age 3 : keep liver iron < 5 mg/g Consider: stem cell transplantation, hydroxyurea, prenatal diagnosis, gentic counseling

Iron Chelators Deferoxamine: Given by prolonged infusion Deferasirox: once daily PO dosing , can remove cardiac iron Deferiprone: Orally active, removes cardiac iron, limited use in US Iron chelator related toxicities: growth delay, skin reactions, bone marrow suppression, renal/hepatic damage, Yersinia infections, agranulocytosis (Deferiprone)

Alpha Thalassemias Decreased synthesis of alpha globin genes, excess of beta or beta like globin chains Potential formation of abnormal hemoglobins Hemoglobin barts: γ4 Hemoglobin H: β4

Alpha Thalassemias α+: deletion of a single gene on chromosome 16 α0: deletion of both genes on chromosome 16 Point mutations are less common, but phenotype more severe

Alpha Thalassemias

Alpha Thalassemias

Alpha Thalassemias Silent Carrier 3 of 4 alpha genes present +/- mild anemia Low MCV

Alpha Thalassemias Alpha thalassemia trait 2 of 4 alpha genes are present Homozygous α+ or heterozygous α0 Clinical features: +/- mild anemias, MCV <78, Hb Barts 2-10% in newborns Do not confuse with Fe deficiency anemia. Diagnosis of exclusion, confirm with molecular testing

Alpha Thalassemias Hemoglobin H disease β4 1 of 4 alpha genes present 20-40% Hb Barts in newborn and 5-40% HbH in adults -visualized by brilliant cresyl blue -Hb electrophoresis -HPLC

Alpha thalassemias HbH disease- Clinical presentation: Hemolytic anemia of varying degrees Microcytosis Splenomegaly Ineffective erythropoiesis Fe loading Treatment: Transfusions for symptomatic anemia or severe anemia Hb <7 + iron chelation

Alpha thalassemias Hemoglobin Bart’s : Hydrops Fetalis Intrauterine treatments: transfusions, stem cell transplant, improved perinatal care After birth complications: Growth retardation, neurodevelopmental delays, lifelong transfusions, associated congential abnormalities Genetic counseling and screening is important

Alpha Thalassemias Fetus complications in absence of all alpha globin genes

Alpha Thalassemia- RBC indices

Atypical alpha thalassemias Alpha thalassemia- mental retardation syndromes ATR-16: alpha thal retardation associated with Ch 16 X-linked mutations in ATRX Alpha thalassemia- MDS : acquired alpha thalassemia in MDS