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

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 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 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)

Sickle Cell Anemia

Sickle Cell Anemia NORMAL 0 1 2 3 4 5 6 MET VAL HIS LEU THR PRO GLU 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 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

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 Homozygous Affected 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 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

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%

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 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

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

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

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?????