Sickle Cell Anemia:

The Whole Story

Tim R. Randolph, PhD, MT(ASCP), CLS(NCA)

Department of Biomedical Laboratory Science

Doisy College of Health Sciences

Saint Louis University

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

Thalassemias vs Hemoglobinopathies

Thalassemia– 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

Sickle Cell Anemia

Hemoglobin S

– DefinitionA 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

– EtiologySingle 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)

Sickle Cell Anemia

Sickle Cell Anemia

NORMAL

0 1 2 3 4 5 6

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

Sickle Cell Anemia

Deoxyhemoglobin S Polymer Structure

Charge and size prevent

6b Glu from binding.

Hydrophobic pocket for 6b Val

Paired strands of deoxy Hb S

(crystal structure)

Wishner, JMB 1975

Dykes, Nature 1978;

JMB 1979

Crepeau, PNAS 1981

Deoxyhemoglobin S

14-stranded polymer

(electron micrograph)

Sickle cell Anemia

Sickle Cell Anemia

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)

Sickle Cell Anemia

Sickle Cell Anemia

Hemoglobin S

– HomozygousAffected alleles– 2

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

Sickle Cell Anemia

Clinical Features– Begin at 6 months of age

– Pain in fingers and toes (vaso-occlusion)

– Other vaso-occlusive crisesTriggered 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 spleenRapid trapping and dramatic anemia

Increased bilirubin with jaundice & increased retics

Functional hyposplenism (Howell-Jolly, Pappenheimer)

– Frequent infections

Sickle Cell Anemia

Diagnosis

– MicroscopicNormo/normo anemia, Sickle cells, Target Cells, NRBC/Poly/retic

– SolubilityTurbid (insoluble)

– ElectrophoresisBand at S (homozygote) (>80%)

No band at A

Hgb F = 1-20%, Hgb A2 = 2-5%

Sickle Cell Anemia

Sickle Cell Anemia

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

Sickle Cell Anemia

Hemoglobin S

– Homozygous

Prognosis

– 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

Sickle Cell Anemia

Mechanism of

Hydroxyuria

– Generates Nitric Oxide

(NO)

– Activates NO/cGMP

signaling pathway

– Upregulate g-globin

gene expression

Sickle Cell AnemiaMechanism 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

Sickle cell Anemia

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

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

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

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

Thank You

Questions?????