1. 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

2. 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

4. Molecular Mechanisms of Thalassemia

5. 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

7. 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

8. 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

10. 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

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

12. Inheritance Pattern of alpha-Thalassemia – Ch. 16

13. 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

14. 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

15. 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

16. Alpha-Thalassemia HbH Disease

17. 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)

18. 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

19. 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)

20. Microcytic Hypochromic Anemia Thalassemia

21. 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

22. 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)

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

26. Normal Transcription

27. 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

28. Beta-Thal Promoter Mutation

29. Beta-Thal Mutated Stop Codon

30. 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

31. Beta-Thal Transcription/Translation

32. Beta-Thal Splice Site Mutation

34. Beta-Thal Intermedia

35. 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

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

38. Microcytic Hypochromic Anemia Thalassemia

39. 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
Male female i h
N-h
65-185
20-45
i-N

40. 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

41. 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

42. Microcytic Hypochromic Anemia Thalassemia

43. 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

44. 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

46. 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

48. Thank You
Questions???