1. Sickle Cell Anemia: The Whole Story
Tim R. Randolph, PhD, MT(ASCP), CLS(NCA)
Department of Clinical Laboratory Science
Doisy College of Health Sciences
Saint Louis University

2. Sickle Cell Anemia Objectives
By the end of this session the participants will be able to:
Describe the molecular biology associated with the genetic mutation and protein polymerization
Discuss abnormal lab findings associated with sickle cell disease and trait
Discuss symptoms and treatment for patients with sickle cell disease and trait

3. Thalassemias vs Hemoglobinopathies
Genetic mutation (435)
Globin gene(s)
Lack of gene expression
Decreased normal Hb
Anemia
Low RBC, Hb, Hct, MCH, & MCHC
Increased RBC divisions
Smaller RBC
Low MCV
Increases Hb/RBC
Normalization of MCH and MCHC
Hemoglobinopathies
Genetic mutation (1065)
Globin gene(s)
Normal gene expression
Release of abnormal Hb
Low oxygen saturation
Hb polymerization
Vaso-occlusion
Hemolysis
Anemia
Normal MCV, MCH, & MCHC

4. Sickle Cell Anemia Hemoglobin S Definition Etiology
A genetic point mutation resulting in one nucleotide change and a single amino acid substitution that changes the Hgb molecule from Hgb A to Hgb S
Etiology
Single nucleotide substitution (A >>T) resulting in a change in one codon (GAG>>GTG) at the 6th position on the beta gene and the substitution of a valine (+0) for a glutamic acid (-1) at the 6th position of the beta chain - (a2b2GLU>>VAL)

5. Sickle Cell Anemia

6. Sickle Cell Anemia NORMAL 0 1 2 3 4 5 6 MET VAL HIS LEU THR PRO GLU
MET VAL HIS LEU THR PRO GLU
ATG GTG CAC CTG ACT CCT GAG
SICKLE
ATG GTG CAC CTG ACT CCT GTG
MET VAL HIS LEU THR PRO VAL

8. Sickle Cell Anemia

9. Deoxyhemoglobin S Polymer Structure
C) Hydrophobic pocket
for 6b Val
A) Deoxyhemoglobin S
14-stranded polymer
(electron micrograph)
B) Paired strands of
deoxyhemoglobin S
(crystal structure)
D) Charge and size prevent
6b Glu from binding.
Dykes, Nature 1978; JMB 1979
Crepeau, PNAS 1981
Wishner, JMB 1975

10. Sickle cell Anemia

11. Sickle Cell Anemia

13. Sickle Cell Anemia Hemoglobin S Basic Genetics
Simple Mendelian genetics
Two beta genes
Two genotypes
Homozygous
HbSS = Sickle Cell Anemia/Disease (SCD)
Heterozygous
HbAS = Sickle Cell Trait (SCT)

14. Sickle Cell Anemia

15. Sickle Cell Anemia Hemoglobin S Homozygous Affected alleles2
Affected individuals
African Blacks
Pathogenesis
Nucleotide substitution (A>T) >> amino acid substitution (GLU>VAL) >> +1 charge >> DeoxyHgb >> structural change >> polymerization >> Sickle shaped RBCs >> vaso-occlusion >> Splenic removal >> Anemia, stroke, MI, organ infarcts, increased infections

16. Sickle Cell Anemia Clinical Features Begin at 6 months of age
Pain in fingers and toes (vaso-occlusion)
Other vaso-occlusive crises
Triggered by acidosis, hypoxia, dehydration, infection and fever, and exposure to extreme cold
Bones, lungs, liver, spleen, penis, eyes, central nervous system (CNS), and urinary tract for 4-5 days to weeks
Acute stroke occur approximately 600/100,000 patients per year
Silent infarcts occur at a rate of 20% of HbSS children
Enlarged spleen
Rapid trapping and dramatic anemia
Increased bilirubin with jaundice & increased retics
Functional hyposplenism (Howell-Jolly, Pappenheimer)
Frequent infections

17. Sickle Cell Anemia Diagnosis Microscopic Solubility Electrophoresis
Normo/normo anemia, Sickle cells, Target Cells, NRBC/Poly/retic
Solubility
Turbid (insoluble)
Electrophoresis
Band at S (homozygote) (>80%)
No band at A
Hgb F = 1-20%, Hgb A2 = 2-5%

18. Sickle Cell Anemia

22. Sickle Cell Anemia

24. Sickle Cell Anemia Hemoglobin S Homozygous Treatment Prophylactic
Immunizations (pneumococcus)
Antibiotics (penicillin G) (daily till age 5 or 6)
Hydroxyurea, Butyrates, 5-Azacytadine (AzaC)(HbF)
Crisis Management
Pain management (analgesics/narcotics)
IV fluids
Oxygen
Anemia Management
Transfusions

25. Sickle Cell Anemia Hemoglobin S Homozygous Prognosis Sequelae
Good if followed closely by a physician
Many are reaching retirement age
Sequelae
Acute chest syndrome (25% of deaths)
Stroke
Silent stroke
Priapism
Gallstones
Renal failure

26. Sickle Cell Anemia Mechanism of Hydroxyuria
Generates Nitric Oxide (NO)
Activates NO/cGMP signaling pathway
Upregulate g-globin gene expression

27. Sickle Cell Anemia Mechanism of Butyrate Epigenetic modified DNA
Acetylation of histones
Acetylation of lysine residues on histones
Removes positive charges
Reduces affinity between histones and DNA
Opens chromatin
RNA polymerase & transcription factors bind promoter easier
Acetylation enhances transcription & activates genes
Histone deacetylation represses transcription & silences genes
Butyrate acetylates genes

28. Sickle cell Anemia

30. Sickle Cell Anemia Mechanism of 5-Azacytidine & Decitabine
Epigenetic modified DNA
Hypomethylation of promoters
Methylation of cytosine nucleotides in promoters silences genes
Drugs are cytidine analogs > incorporate into DNA > covalently bind DNMT > deplete DNMT > hypomethylation
Hypomethylation of promoters activates genes

32. Sickle Cell Anemia Hemoglobin S Heterozygous Affected alleles - (1)
Affected individuals - Same as homozygote
Pathogenesis - Same but rarely occurs
Description of blood smear - Target cells
Laboratory tests
Solubility - Positive
Hemoglobin electrophoresis
Band at A and S with a little at F and A2

36. Sickle Cell Anemia Sickle Cell Trait
Evidence that carriers are not totally asymptomatic
Adverse events can occur under extreme stress
Military boot camp
HS, college and pro athletes
Recreational athletes
Runners
Rhabdomyolysis
Some evidence of minor ongoing joint & muscle pain

37. Sickle Cell Anemia Summary of sickle cell
Point mutation causes a single amino acid change
Alters deoxyHgb structure causing polymerization & sickle cell formation
Sickle cells cause vaso-occlusion leading to stroke, micro-infarctions and organ death
Hemolytic anemia causes splenomegaly and hyposplenism leading to increased infections
Treated with immunizations, antibiotic, gamma chain activators and transfusions

40. Thank You
Questions?????