Thalassemia A to Z Tim R. Randolph, PhD, MT(ASCP) Chair and Associate Professor Department of Clinical Laboratory Science Doisy College of Health Sciences Saint Louis University

Thalassemia Thalassemia Definition Normal Globin genes Genetic disorder characterized by absent or deficient synthesis of one or more of the normal globin genes. (Quantitative) Normal Globin genes Chromosome 16 (alpha-like) Alpha Zeta Chromosome 11 (beta-like) Beta Delta Gamma Epsilon

Molecular Mechanisms of Thalassemia

Thalassemia Pathophysiology Mechanism Microcytic Hypochromic anemia Mutated globin gene > decreased globin chain synthesis > decreased Hb synthesis of Hbs that contain that chain > reduced Hb concentration in RBCs > Anemia Microcytic Hypochromic anemia Anemia > decreased oxygen carrying capacity > increase in number of RBC divisions > smaller RBCs > lower MCV > higher MCHC (still low) > microcytosis precedes hypochromia

Thalassemia Thalassemia Two Major Types of Thalassemia Alpha Thalassemia Beta Thalassemia Since between 95-97% of normal adult Hb is HbA (alpha2/beta2), mutations in the alpha and/or beta genes has the greatest impact on Hb concentration and oxygen carrying capacity

Alpha-Thalassemia Alpha Thalassemia Etiology Gene Deletion (Light Switch) Chromatid Crossover Normal purpose to increase genetic diversity in race Disease causing when malfunctions Point mutation Most still result in total abrogation of gene expression Short stretch deletions Most also result in total abrogation of gene expression Alpha2 genes produce 2-3 times the product as alpha1

Alpha-Thalassemia Alpha-thalassemia Affected Individuals Mediterranean's Greeks, Italians, Sicilians Orientals Southeast Asia African Blacks Milder version 1 deleted alpha gene on each chromosome Both are alpha1 deletions

Alpha-Thalassemia Basic Genetics Inheritance pattern Four genotypes Homozygous Hemoglobin H Disease Heterozygous Silent Carrier

Inheritance Pattern of alpha-Thalassemia – Ch. 16

Alpha-Thalassemia Major (Hydrops fetalis) Homozygous (Major – Hydrops fetalis - a0-Thal) Affected alleles 4 Affected individuals Mediterranean descent Southeast Asians, Greeks, Italians Pathogenesis No alpha chains Can’t make any normal hemoglobins (HbA, HbA2, HbF) Incompatible with life

Alpha-Thalassemia HbH Disease Hemoglobin H Disease Affected alleles (3) Affected individuals Mediterranean descent Southeast Asians Greeks, Italians Pathogenesis One normal gene > makes about 40% normal a genes > reduced HbA, A2, F > increase in abnormal Hbs (Barts, H) > abnormal Hbs have increased O2 affinity > spleen sequestration > anemia

Alpha-Thalassemia HbH Disease CBC Microcytic/hypochromic anemia Low RBC, Hb, Hct Microcytic (MCV <80fL) & Hypochromic (MCHC <32g/dL) Description of blood smear Microcytic/hypochromic Marked poikilocytosis Tget, Tdrp, Ell, Spch Polychromasia (Retic = 5-10%) NRBCs

Alpha-Thalassemia HbH Disease

Alpha-Thalassemia HbH Disease Lab findings Heinz Body test ( 1% Brilliant Cresyl Blue) Hemoglobins present (Electrophoresis/HPLC) Increased Barts (g4) at birth = 25% Increased HbH (b4) = 2-40% in adults Decreased HbA2(1.5%), HbF (1% at birth), A (rest)

Alpha-Thalassemia Alpha-Thal Minor (a-Thal 1) Heterozygous (Minor) Affected alleles (2) Affected individuals Orientals (Deletion of both a genes on one chromosome) Mediterraneans African Blacks (deletion of both a1 gene on both chromsomes) Pathogenesis Reduced alpha chain production (75%) > insufficient reduction to produce symptoms > most are asymptomatic

Alpha-Thalassemia Alpha-Thal Minor (a-Thal 1) Lab Findings CBC RBC, Hb (10-12 g/dL), Hct Mild anemia or low end of reference range MCV = 60-70fL MCH = 20-25pg MCHC = Normal Description of blood smear Mildly microcytic/normochromic Slight anisocytosis Poikiolocytosis (Tget)

Microcytic Hypochromic Anemia Thalassemia

Alpha-Thalassemia Alpha-Thal Minor (a-Thal 1) Hemoglobins present At birth Hb Barts = 5% (2% if African American) Normal in adults Lab findings Some inclusion Bodies with Brilliant Cresyl Blue

Alpha-Thalassemia Alpha-Thal Minor (a-Thal 1) Silent Carrier Affected alleles (1) Affected individuals Orientals 30% of African Americans Pathogenesis Three normal a genes > normal production > asymptomatic Description of blood smear (Few Tget) Hemoglobins present (Normal)

Beta-Thalassemia Beta-thalassemia Etiology Basic Genetics Inheritance Pattern Two genotypes Beta-thal major Beta-thal minor 5 Types of Mutations (dimmer switch)

Normal Transcription

Beta-Thal Point Mutations Promoter Mutation (Start Site) RNA polymerase has decreased binding Mutated Stop Codon Extended mRNA and extended protein Nonsense mutation Causes added stop codon or frame shift Splice Site Mutation Lengthens the mRNA and the protein

Beta-Thal Promoter Mutation

Beta-Thal Mutated Stop Codon

Beta-Thal Non-sense Mutation Normal mRNA/Stop (UAA, UAG,UGA) AUA-GAU-UUC-CGA-AUC-CCA-UAA-UCA Add Stop Codon AUA-GAU-UUC-UGA-AUC-CCA-UCA-UCA Frame Shift Mutation AUA-GAU-UUC-GA-AUC-CCA-UCA-UCA Result Short mRNA>>Short protein>>Degradation Altered mRNA>>Longer or shorter>>Degradation

Beta-Thal Transcription/Translation

Beta-Thal Splice Site Mutation

Beta-Thal Intermedia

Microcytic Hypochromic Anemia Thalassemia Beta-thalassemia Major Affected Alleles 2 Affected Individuals Mediterraneans Italy, Greece, Algeria, Saudi Arabia Orientals Southeast Asia African Blacks (Milder) Pathogenesis

Microcytic Hypochromic Anemia Thalassemia Beta-thalassemia major Description of blood smear Hemoglobins present Black form Lab tests

Microcytic Hypochromic Anemia Thalassemia

Thalassemia (Iron Panel)   DISEASE SERUM FERRITIN (NG/ML) IRON (UG/DL) IRON-BIND CAPACITY TRANS. SATUR. (%) TISSUE STORES Normal  Iron Deficiency Anemia  Sideroblastic Anemia  Anemia of Chronic Disease  Thalassemia 25-250 Male 10-120 female i h N-h 65-185 250-440 20-45 i-N 2+ - 3+

Beta-Thal Lab Diagnosis Peripheral Blood Microcytic Hypochromic Anemia Target Cells Hemoglobin Electrophoresis i in b-containing hemoglobins Hb A h in non-b-containing hemoglobins Hb A2, F

Microcytic Hypochromic Anemia Thalassemia Beta-thalassemia minor Affected alleles 1 Affected individuals Mediterranean (Mod) African Black (Mild) Pathogenesis Moderate to mild to asymptomatic Description of blood smear

Microcytic Hypochromic Anemia Thalassemia

Microcytic Hypochromoc Anemia Beta-Thalassemia Minor Hemoglobins present HbA2 = 3.5-7% HbF = 1-2% HbA = remainder Other lab tests Normal Differentiation alpha-thal/beta-thal from IDA Hb electrophoresis Beta-thal has hHbA2 Iron studies

Microcytic Hypochromic Anemia Thalassemia Beta-thalassemia Intermedia Patient Population Severe B+ Gene Clinical Classification Intermediate Symptoms Other Thalassemias Delta/Beta-thalassemia 100% HbF Lepore Thalassemia Delta/beta fusion Mechanism of Formation

Microcytic Hypochromic Anemia Thalassemia Other Thalassemias Alpha-thal trait/Beta-thal trait Gamma/Beta-thalassemia Delta0-thalassemia Hereditary Persistence of Fetal Hemoglobin Etiology – suppressor gene mutation Affected individuals – Black-Greek-Swiss Three types Two genotypes Homozygote – mild anemia with 100%F Heterozygous – asymptomatic with 20-30% HbF DNA Probes

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