10 Hemoglobinopathies + Associated Anemias.pdf

Hemoglobinopathies and Associated Anemias 202410 Michael M. Yakubovskyy, MD, PhD 1 The Place of Hemoglobinopathy-Associated Anemias in the Working Classification Learning Objectives LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory...

Hemoglobinopathies and Associated Anemias 202410 Michael M. Yakubovskyy, MD, PhD 1 The Place of Hemoglobinopathy-Associated Anemias in the Working Classification Learning Objectives LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) LO2. Discuss the underlying mutation and pathogenesis of HbC disease, identify the RBCs with HbC inclusion, explain the clinical behavior of HbSC disease LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺-thalassemia 3 Sickle Cell Disease 4 LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) Sickle Cell Disease (SCD): General • A normocytic hemolytic anemia with extravascular hemolysis - NB: A small proportion of sickled RBCs undergo intravascular hemolysis • Demographics: African Americans • Genetics - Autosomal recessive disorder - Point mutation (Glu —> Val at amino acid 6) in β-globin (HBB) gene on chr. 11 • Major steps in pathogenesis 1. Polymerization of deoxygenated HbS 2. Distortion of RBC shape (sickling) 3. Extravascular hemolysis (spleen), microvascular obstruction, and ischemic tissue damage (various organs) S (=Hb⍺2ß 2) 5 LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) Sickle Cell Disease: Variants • Sickle cell anemia (SCA): both HBB genes are mutated (homozygosity) - Hb electrophoresis: 90% of HbS, 8% of HbF, and 2% of HbA2 (no HbA) • NB: the terms “sickle cell disease” and “sickle cell anemia” can be used interchangeably • Sickle cell trait: single HBB gene is mutated (heterozygosity) - Hb electrophoresis: <50% of HbS, >50% of HbA, and ≈2% of HbA2 - ≈10% of African Americans are affected - As a rule, there is no sickling, and patients are asymptomatic 6 LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) Formation of Sickle Polymers • Left: deoxygenation of HbS leads to the formation of a double strand of aggregated HbS molecules • Middle: seven double strands aggregate to form a 14-strand helical polymer • Right: Deoxygenation and polymerization of HbS induce deformation into sickle and other abnormal shapes 7 LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) Few Relevant Details of Sickling With reoxygenation, sickle cells may acquire a normal shape, but with repetitive deoxygenation and polymerization of 8 HbS, sickling becomes irreversible LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) Factors Influencing Sickling Enhancing Factors 1 Hypoxia (hypoxemia) Dehydration —> ↑HbS 2 concentration Acidosis —> ↑O2 release —> 3 RBC deoxygenation ↓O2 tension 4 - At high altitude - In the renal medulla Preventing Factors HbF with higher than HbA affinity to O2 1 - No sickling in infants < 6 months 2 Hydroxyurea —> ↑HbF synthesis HbS/HbC heterozygosity (HbSC disease) —> milder sickling if 3 compared with SCA - HbC/HbSC diseases are delivered later 9 LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) SCA: Clinical Correlates • Mild-to-moderate-to-severe normocytic normochromic anemia - HCT: 20% - 30% Hb: 50 - 110 g/L Reticulocytosis (CRC: 3 - 15%) —> normal MCV Irreversible sickled RBCs • Chronic clinical course - Anemia (pallor, fatigue, dyspnea, etc.) - Hyperbilirubinemia (UCB > CB) • Jaundice • Pigmented gallstones - Erythroid hyperplasia in BM —> bone changes • Cortical thinning • “Crew-cut” or “hair-on-end” appearance on skull x-ray • Crisis (four types, see the next slide) 10 LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) SCA: Crises • • • • Vaso-occlusive (pain) crises Sequestration crisis Aplastic crisis Hyperhemolytic crisis 11 LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) Vaso-occlusive Crisis • Vaso-occlusion (blockage of microcirculation by sickled cells) —> vaso-occlusive (pain) crises: hypoxic ischemia and infarctions with severe pain • Common sites/manifestations of vaso-occlusion - Dactylitis (hand-foot syndrome): bone infarcts with swelling of hands and feet in infants - Acute chest syndrome: lung infarctions, chest pain, fever, and cough; a common cause - - of death in adulthood Repetitive splenic infarcts —> fibrosis and shrinkage (autosplenectomy)—> risk of infection with encapsulate bacteria (Streptococcus pneumoniae and Haemophilus influenzae): common cause death in children Priapism: continuous painful erection Other manifestations • • • • Aseptic necrosis of femoral head Infarctions in the renal medulla Ischemic stroke Retinopathy —> loss of visual acuity —> blindness 12 LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) SCA: Capillary Occlusion by Sickle Cells, Liver 13 https://commons.wikimedia.org/wiki/File:Sickle_cell_di sease_and_cirrhosis_-_very_high_mag.jpg Nephron, CC BY-SA 3.0 <https://creativecommons.org/licenses/by-sa/3.0>, via Wikimedia Commons LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) Other Types of Crisis • Sequestration crisis - Children with intact spleen - Massive entrapment of sickle red cells —> rapid splenic - enlargement, hypovolemia, and shock May be fatal • Aplastic crisis - Infection of red cell progenitors by parvovirus B19 —> transient cessation of erythropoiesis —> sudden worsening of anemia with reduction in reticulocyte count (CRC) • Hyperhemolytic crisis - An acute drop in hemoglobin with high reticulocyte count (CRC) 14 LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) SCA: Speen Pathology • Mechanism: repetitive splenic infarcts —> fibrosis and shrinkage (autosplenectomy) • Sequelae - Risk of infection with encapsulate bacteria (Streptococcus pneumoniae and Haemophilus influenzae); common cause death in children - Risk of Salmonella parathyphi osteomyelitis - Howell-Jolly bodies (nuclear remnants the RBCs) on PBS 15 LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) SCA: Splenic Infarct and Howell-Jolly Bodies (Arrows) https://openi.nlm.nih.gov/detailedresult?img=PMC3829791_tm_6p02f2&query=&req=4 16 LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) SCA: PBS and BM • PBS - Nucleated RBCs - Polychromasia with reticulocytes - Sickle cells • Occasional shistocytes due to intravascular hemolysis - Howell-Jolly bodies (with hyposplenism and autosplenectomy) • BM: erythroid hyperplasia • Biochemistry - ↑ serum bilirubin (UCB > CB) - ↑ urine urobilinogen - ↓ serum haptoglobin due to intravascular hemolysis 17 SCA: Nucleated RBCs, Polychromasia, Sickle Cells https://commons.wikimedia.org/wiki/File:Polychromasia.jpg Prof. Osaro Erhabor, CC0, via Wikimedia Commons 18 LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) SCA: Special Tests • High performance liquid chromatography (HPLC): the best test (Access Medicine: Harrison, Willams, etc.) • Sickling/metabisulphite test: the test used in review books - Uptodate: “There is no clinical situation in which a sodium metabisulfite test (Sickledex) is clinically indicated in the screening or management of sickle cell disorders (trait or disease)” • https://www.uptodate.com/contents/diagnosis-of-sickle-celldisorders?search=sickle cell anemia&topicRef=7145&source=see_link#H11 • Hb electrophoresis: a standard test • Newborn screening (“heel stick”): identification of HbS - Family genetic study 19 LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) SCA: Hb Electrophoresis 20 LO1. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of sickle cell disease (anemia and trait) SC Trait, SCA, and HbCS Disease: High Performance Liquid Chromatography 21 HbC Disease 22 LO2. Discuss the underlying mutation and pathogenesis of HbC disease, identify the RBCs with HbC inclusion, explain the clinical behavior of HbSC disease HbC-Associated Conditions • Demographics: African Americans • Genetics - Autosomal recessive disorder - Point mutation (Glu —> Lys at amino acid 6) in β-globin gene • Variants - HbC disease (in homozygotes): a mild hemolytic anemia with extravascular hemolysis and splenomegaly • MCV: 70 - 75 fL —> microcytic anemia • CRC: 3 - 4% - HbC trait (in heterozygotes): asymptomatic - HbSC disease: similar to SCA, but with a milder clinical course + 0 - HbC-ß /ß -thalassemia 23 LO2. Discuss the underlying mutation and pathogenesis of HbC disease, identify the RBCs with HbC inclusion, explain the clinical behavior of HbSC disease HbC: PBS • HbC disease - HbC crystals, particularly after splenectomy - Damage of RBC membrane by HbC crystals + K - —> loss of and water —> spherocytes (with decreased osmotic fragility) Target cells with ↑MCHC Reticulocytes • HbSC disease - Sickle cells - HbC crystals - Target cells 24 https://commons.wikimedia.org/wiki/File:Blood_film_of_Hemoglobin_SC_disease.jpg Spicy, CC BY-SA 4.0 <https://creativecommons.org/licenses/by-sa/4.0>, via Wikimedia Commons Thalassemias 25 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia Thalassemias (Thal) • Inherited disorders characterized by - Decreased production of α- or β-globin chains with following - Anemia, hypoxia, and extravascular hemolysis related to imbalance in synthesis of globin chains • Demographics: Mediterranean, African, and Asian regions endemic for malaria - As with HbS, globin mutations may be protective against falciparum malaria • RBC biology in thalassemias - Heme synthesis is not affected - Deficiency of α- or β-globin chain—> excess of the unaffected globin chain • α-thalassemia: decreased/absent α-globin chains with excess of β-globin chains • β-thalassemia: decreased/absent β-globin chains with excess of α-globin chains 26 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia ß-Thal: ß-Globin Gene Mutations (Deletion: 0 ß, Point Mutation: + ß) Legend (common mutation sites): PR (promoter site27 mutations); SPL (splicing mutations); chain termin LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia β-Thal: Mechanisms of Anemia • Reduction/absence of β-globin —> inadequate HbA formation — > production of hypo-hemoglobinized red cells that are - Small in size (microcytic) and - Pale (hypochromic) • An excess in unpaired α-chains —> formation of insoluble hemoglobin precipitates —> - Damage of RBC membrane —> splenic sequestration —> - extravascular hemolysis Damage of erythroid precursor membrane and their apoptosis —> ineffective erythropoiesis • 70% - 80% of erythroid precursors die in the BM 28 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺-thalassemia ß-Thal: Key Events in Pathogenesis • Excess in and precipitation of α-chains —> destruction of erythroid precursors —> anemia • Erythropoietin release —> marrow expansion - Massive erythroid hyperplasia in BM —> - erythroferrone synthesis Impaired bone growth and skeletal deformities Extensive extramedullary hematopoiesis —> hepato- and splenomegaly • Repetitive blood transfusions and erythroferrone release (see the next slide) —> iron overload—> secondary hemochromatosis 29 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺-thalassemia β-Thal: BM Erythropoiesis Hemochromatosis 1. 2. 3. 4. 5. 6. 7. Destruction of erythroid precursors Erythropoietin release Hyperplasia of erythroid precursors Erythroferrone release Inhibition of hepcidin synthesis Increased iron absorption Secondary hemochromatosis 30 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia β-Thal: Genetic Foundation of Clinical Classification • Clinical classification is based on the severity of anemia (minor/trait, intermedia, and severe), which in turn depends on the 0 + gene defect (β or β ) and gene dosage (homo- or and heterozygous) - β-thalassemia minor/trait: a patient inherits only one - 0 β + β or allele; the condition is is asymptomatic or mildly symptomatic 0 + β-thalassemia intermedia: variable inheritance of β or β alleles; anemia is severe, but not so bad as with ß-thal major 0 + β-thalassemia major: patients have two β or two β alleles —> severe anemia 31 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia ß-Thal: Clinico-Genetic Correlates Classification Type ß-thal minor (trait) ß-thal intermedia Genotype • Heterozygous ß-thal • β0/β + • β /β • Variable genotype 0 β /β + β /β • From and + + 0 + 0 0 • To β /β and β /β (but not β /β ) Manifestations Asymptomatic with mild or no anemia Severe, but does not require regular blood transfusions • Homozygous β-thal ß-thal major Severe, requires regular blood transfusion 0 0 β /β • + + • β /β 0 + • β /β 32 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia ß-Thal Minor: PBS • RBCs: small (microcytic) and pale (hypochromic), but regular in shape • Target cells: cells with an increased surface area-tovolume ratio and central accumulation of the cytoplasm • Basophilic stippling • Fragmented RBCs 33 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia ß-Thal Major: PBS • • • • • • RBCs: microcytic, hypochromic Target cells Basophilic stippling Fragmented RBCs + Poikilocytosis: variation in cell shape • Anisocytosis: variation in cell size • Nucleated RBCs (normoblasts) 34 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia ß-Thal Intermedia: PBS • RBCs abnormalities are similar to ß-thal major, but less severe - Severity is difficult to estimate looking at a single image 35 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia ß-Thal: Hb Spectrum (%) HbA(α2β2) HbA2(α2δ2) HbF(α2γ2) Normal Adult 96 3 1 ß-Thal Minor 80-85 5 10-15 5-10 8-10 85-90 ß-Thal Interm. ß-Thal Major 36 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia ß-Thal (Major > Intermedia > Minor): Visceral Pathology • Extramedullary hematopoiesis —> - Bones: crew-cut skull, chipmunk face - Hepato- and splenomegaly • Extravascular hemolysis —> jaundice • Secondary hemochromatosis: “bronze diabetes” • Pigmented gallstones 37 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia HB Electrophoresis (ß-Thal, Sickle Cell Trait, and HbSC Disease + ß heterozygous 1. (trait): HbA/HbA2 2. Normal adult: HbA + 3. ß homozygous (ß-thal intermedia/ major): HbA/HbF 10.Sickle cell trait: HbA/HbS 11.HbSC disease: HbS/HbC 38 https://pubmed.ncbi.nlm.nih.gov/22089618/#&gid=article-figures&pid=fig-1-uid-0 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia α-Thal: Genetics • α-thalassemia: deletion of one or more α-globin genes 39 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia α-Thal: Genetic Foundation of Clinical Classification • The severity of the disease is proportional to the number of missing α-globin genes • The loss of a single α-globin gene (-/α α/α)—> silent carrier state • The loss of two α-globin genes (αα/-- or α/- α/-)—> α-thal trait with manifestations similar to ß-thal minor - (α/α -/-) genotype is common in Asia and is more severe then α/- α/- • The loss of three α-globin genes —> relative excess on β-globin chains—> formation of stable β4 tetramers (HbH) —> HbH disease: extravascular hemolysis and anemia similar to ß-thal intermedia - Common in Asians • Deletion of all four α-globin genes may be lethal in utero because RBCs have virtually no oxygen-delivering capacity. An excess in γ-globin chains —> formation of γ4 tetramers (Hb Barts) —> hydrops fetalis 40 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia α-Thal: HbH Disease • • • • • (α/- -/-) —> β4 tetramers (HbH) PBS: similar to ß-thal intermedia Clinical manifestations: similar to ß-thal intermedia Hb electrophoresis: HbH Supravital stain: HbH aggregates (similar to Heinz bodies) 41 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia α-Thal: Hb Barts Disease • (-/- -/-) = no ⍺-globin genes —> excess of γ-globin —> formation of γ-globin tetramers (Hb Barts) • High affinity to Hb Barts to oxygen —> no oxygen delivery to tissues st nd • Survival of an embryo/fetus in the 1 and 2 trimesters is possible due to synthesis of ζ2γ2 (Hb Portland) that is capable to deliver oxygen to peripheral tissues • Trimester 3: rapid fetal growth, decline of Hb Portland production — > an increase in tissue hypoxia —> anoxia —> hydrops fetalis: severe pallor, generalized edema and massive hepatosplenomegaly —> death in utero or soon after delivery - Survival is possible only with intrauterine blood transfusion 42 LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia α-Thal: Newborn Infant with Hydrops Fetalis https://commons.wikimedia.org/wiki/File:Newborn_infant_with_severe_hemolytic_disease_(erythroblastosis_foetalis)_resulting_in_hydrops_foetalis.png 43 Benkerroum Zineb, Lachiri Boutaina, Lazrak Ikram, Moussaoui Rahali Driss, Dehayni Mohammed, CC BY 4.0 <https://creativecommons.org/licenses/by/4.0>, via Wikimedia Commons LO3. Describe the etiology, pathogenesis, morphological changes, clinical presentation, complications, laboratory data, treatment, and prognosis of β- and ⍺thalassemia Summary of Lab findings in Thalassemias (α and β •) RBC count: normal-to-increased (very characteristic) • MCV: low • PBS - Microcytic hypochromic RBCs Target cells Polychromatophilic RBCs Fragmented RBCs Nucleated RBCs (normoblasts) - β-thal minor: moderate increase in HbA2 and HbF β-thal major: moderate increase in HbA2 and severe increase on Hb F α-thal trait: normal HbA and HbA2 HbH disease: increased HbH (β4) 67 Hb Barts disease: increased Hb Barts (γ4) • Hb electrophoresis 44 67 The End 45 Slide 1 Hemoglobinopathies - Genetics (hemolytic anemias) Hemoglobin gene diseases Dr. Larsen Turningpoint id: Larsen 1 Slide 2 Correlate changes in fetal expression pattern of hemoglobin (Hb) with onset of Hb diseases Explain molecular mechanisms causing disease and modulating disease severity in the different Hb diseases Recognize inheritance pattern of the Hb diseases and apply that knowledge to family counseling (including risk calculation) Learning Objectives List and explain the clinical presentation, etiology, pathogenesis, diagnosis, laboratory data, complications and treatments of the Hb diseases Interpret results from electrophoretic and other DNA/protein methods applied to Hb diseases List and explain the interaction of thalassemia mutations with structural Hb variants Reading: Emery’s elements of medical genetics (15th ed.) pp 154-162 Sample questions in Canvas Lectures presenting relevant material include Hemoglobin, Risk Calculation, DNA Technology. We will assume you known this material. 2 Slide 3 Overview • Introduction • Diseases involving changed protein sequence • Diseases involving protein truncation or reduced expression • Mixed forms (some concepts visited more than once) Clinico-Pathologic Correlations of these diseases will be presented by Dr. Yakubovskyy next hour 3 Slide 4 Tetramers • All hemoglobin proteins (should) consist of 2 α-like + 2 β-like subunits in one tetrameric protein • If production of β-hemoglobin is reduced 10%, so will production of αhemoglobin • However, if production of one is reduced more than 50% - then surplus of the other type will be seen • Remember, Hb is found in RBC, with normal lifespan of about 120 days  constant, lifelong production • RBC lifespan usually reduced in Hb diseases 4 You will see at later slides that the surplus of the other type of hemoglobin chain o�en is at the center of the molecular mechanism of disease. According to the companies that make review courses for NBME step exams, molecular mechanisms are important! Slide 5 Routine identification • Protein electrophoresis under two different conditions together identifies most common variants • Cellulose acetate at pH 8.6 • Citrate-agar at acidic pH • Or iso-electric focusing can do the same in one gel • More advanced problems are sent to specialty labs. [Document on protein electrophoresis in Canvas] 5 Please use �me to understand the electrophoresis of hemoglobins and what the expected electrophoresis patern would be for genotypes in each disease. Slide 6 Hemoglobin Gene Expression 6 This slide shows you when hemoglobin molecules are produced. No�ce that on any given point in �me, the majority of RBCs that are present were produced weeks to months earlier. Also, No�ce the �ming of expression switch from gamma to beta. Slide 7 Caption and info • • • • • • HbGower(1) = ζ2ε2 (zeta2-epsilon2) HbGower(2) = α2ε2 (alpha2-epsilon2) HbPortland = ζ2γ2 (zeta2-gamma2) HbF = α2 γ2 (alpha2-gamma2) HbA = α2 β2 (alpha2-beta2) HbA2 = α2 δ2 (alpha2-delta2) C E O D G Lepore Portland • Expression switch involves epigenetics (methylation) of genes no longer needed • Hemoglobin types Application Barts A F S A2 H Adult Newborn 7 You need to be cognizant of which chains are found in each of HbF, HbA, HbA2 – and from later in the lecture, HbS, HbC, HbE, HbH, Hb Barts proteins. Epigene�cs is a term you heard about in FFM1 and you will hear more about in Reproduc�ve. For now, remember that it does not refer to changing the DNA sequence but to changing access to the DNA and therefore altera�on of the expression patern of proteins. Notes about the electrophoresis: here are presented the expected patern in an adult and in a newborn. The adult produces a lot of beta chain which combines with alpha to become HbA, and small amounts of delta which together with alpha becomes HbA2. The thin line to the le� in the adult therefore indicates this lower amount of HbA2, and the much heavier band under HbA indicates the α2 β2 . In the newborn as expected, the majority of the protein produces is HbF and about 20% HbA. HbA2 is not yet present at that age. Slide 8 Hemoglobin Gene Clusters • All α-like genes in one cluster, all β-like in another (on different chromosomes) • Locus control region (LCR) upstream (5’) of it all • Progression so that more downstream genes are more heavily expressed later in development 8 Alpha hemoglobin genes on 16pter-16p13.3 Beta hemoglobin genes on 11p15.5 The chromosomal loca�on will not be tested! The Greek leter Psi used in front of some of the genes listed above indicates a pseudogene = an inac�ve copy of a gene. Slide 9 Variants and alleles • More than 1000 variants described, most neutral • OMIM lists 522 alleles of the β-hemoglobin gene • Variants are Hb with changed amino acid content (usually from point mutation) – and those causing disease are pathological variants • Alleles include both those causing variants and those with reduced expression • Each variant is caused by a different allele at the DNA/gene level • Heterozygote frequency can be very high in some populations 9 Slide 10 Diseases due to changed Hb proteins Mutations in beta-hemoglobin: Sickle Cell Disease and Hemoglobin C disease These are autosomal recessive 10 Slide 11 Hemoglobin S: Sickle Cell Anemia N: S: • All carriers of this anemia have exactly the same mutation • Mutation happened independently at least twice • HbS: tetramer of α2βS2 11 Glutamic acid to valine (charged to uncharged) in codon #6 Note that you need to be fluent in going back and forth between the different designa�ons for the same molecule, HbS: tetramer of α2βS2 --- HbSS is o�en used to designate a homozygote for Sickle Cell. Regarding how many �mes the muta�on has happened (independently) – this is the text book version. Some recent reports claim that the muta�on really only happened once. Slide 12 Electrophoretic pattern of Sickle Cell carrier and homozygote A2 C E O S D G Lepore F A Portland Application Barts H N. Adult Heterozygote Sickle cell homozygote 12 This is electrophoresis of na�ve protein tetramers. Slide 13 13 High occurrence of Sickle Cell alleles in historical malaria areas Slide 14 Why is HbS common? • In areas with Plasmodium falciparum malaria Genotype HbAA Survival HbAS Medium Higher than HbAA HbSS Low • This is known as “heterozygote advantage” • High frequency of Thalassaemia in the Mediterranean and east Asia probably have the same cause • As does some enzyme deficiencies taught by biochemistry 14 Frequently used nota�on: HbAA = homozygote normal HbAS = heterozygote HbSS = homozygote for sickle cell alleles What is the mechanism of heterozygote advantage? Here we will describe one hypothesis which builds on the fact that sickling of a red blood cell happens when the hemoglobin becomes anoxic. In a heterozygote for sickle cell disease, the oxygen levels inside the RBC usually does not become low enough that sickling happens. Infec�on with malaria changes this, because one early stage of infec�on is for the parasite to enter a RBC and start dividing. This is a drain on the oxygen of the cell, it become more anoxic than usual, and sickling is likely to happen. The sickled cell is likely to be recognized as foreign by macrophages and therefore, parasite load is reduced in the body of the person. With reduced parasite load comes improved survival. Slide 15 Sickle cell disease vs Sickle cell anemia • “Sickle cell anemia” = = = disease found in homozygote • “Sickle cell disease”: two usages 1. Disease found in homozygote 2. Descriptor for everyone who has any sickled cells = = = homozygotes + heterozygotes • Heterozygotes most frequently described with “Sickle cell trait” 15 The two usages of Sickle Cell Disease are in current use in different laboratories. Slide 16 Traits Modifying Severity of HbSS • HbF (persistence of fetal Hb) • α-thalassaemia trait or silent carrier (lower Hb concentration) • HbC • Why? Sickling is similar to precipitation. The larger the concentration of HbS is, the more likely the patient experience sickling. • Dr. Yakubovskyy will explain some environmental factors involved, some of these also depends on the concentration. 16 These are all expected to improve the clinical picture. Please see individual sec�ons for more detail Slide 17 Molecular testing for HbS • Restriction enzyme difference (MstII) and allele specific oligonucleotide (ASO) hybridization presented during FFM1 (DNA technology). 17 Therefore, I will assume this known. Slide 18 Hemoglobin C Heterozygote advantage • Glu6Lys • Mutation happened only once in western sub-Saharan Africa • HbC crystals seen in blood analysis • HbAC and HbCC both significantly lower risk of Malaria death • Significantly less pathology than HbAS and HbSS, respectively • HbSC gives a somewhat milder sickle cell disease compared to HbSS but worse than HbAS • HbSC is example of compound heterozygote 18 Glutamic acid to Lysine: nega�ve charge to posi�ve charge. No�ce the codon number if 6, same loca�on as in HbS Compound heterozygote: person carrying two different disease causing alleles in the same gene. Slide 19 Diseases with diminished production of Hb Thalassemia (American English) Thalassaemia (British English) 19 Slide 20 THALASSEMIA Autosomal Recessive Disorder Absence or decreased synthesis of α OR ß chains QUANTITATIVE HEMO Beta-Thalassemia GLOBIN ABNORMALITIES Alpha-Thalassemia Diminished beta chain synthesis Diminished alpha chain synthesis Common in Afro-Americans, Greeks, Italians, East Indians and East Asians. Common in South-East Asians and AfroAmericans • Fetal Hb – Predominant in fetus, trace in adults HbF • Adult Hb – 95-97% adult blood – HbA - 2ß2.. • Adult Hb 2 - 1-4% adult blood – HbA2 - 2 Fetal Hb - Predominant in fetus, trace in adults - HbF Adult Hb - 95-97% adult blood - HbA - 2 2 Adult Hb 2 - 1-4% adult blood - HbA2 - 2 2 2. 2 2 2. 20 2 From NIH: Beta thalassemia occurs most frequently in people from Mediterranean countries, North Africa, the Middle East, India, Central Asia, and Southeast Asia. Slide 21 -Thalassemia • Single base substitutions including frameshifts • Rare: large deletions or insertions • Estimates: about 60,000 people with symptomatic β-Thal born each year, less then 300,000 alive 21 Most people with β-Thalassaemia do not survive very long! No�ce that the more common type of muta�on is different in beta- vs alpha-thalassemia. Slide 22 Genetics • β-Hb production from the thalassemia allele can be none (β0) or reduced (β+, sometimes milder versions are stated as β++) while normal alleles can be designated βN or βWT. • Homozygous β0 β0 or compound heterozygote β0β+ usually have βthalassaemia major • Homozygous β+β+ usually have β-thalassaemia intermedia • The heterozygotes with one normal allele (β0 βN, β+βN) all have βthalassaemia minor (= carrier) • See also last section 22 Genotype-phenotype correla�on is incomplete: no�ce the use of the word usually indica�ng varia�on among pa�ents in each group Slide 23 Mechanism -Thalassemia • Severely reduced production of β-Hb chains: • α-Hb chains in surplus • Cannot produce homo-tetramers • Precipitation inside cells • Centers for oxidative damage of membrane proteins and lipids • Increased destruction of RBC precursors in bone marrow (therefore ineffective RBC formation) • Shortened RBC lifespan in circulation • In contrast to sickle cell animia, this is a more constant problem. 23 For molecular mechanism of cell damage, focus on the chain that is in surplus. Slide 24 Alpha-Thalassemia 24 Slide 25 -Thalassemia alleles • α-Thalassemia carriers: malaria areas of Africa and Asia (especially south-east Asia), rare in the Mediterranean • Allele -thalassemia 1: usually found in people of Asian descent only • Allele -thalassemia 2: African and Asian descent 1 2 25 The drawing shows a normal copy of the gene as a blue box and a deleted copy is illustrated with crossing out. Reminder: you have seen already that a normal chromosome would have two copies producing alpha-Hb, so in a person with two copies of the chromosome (expected) missing one copy lowers produc�on by 25%. E.g., 1/3 of people from Thailand are carriers of one of these two alleles Slide 26 -Thalassemia Deletions on Chromosome 16, • • • • • A B C D E A: silent carrier B, C: α-Thalassemia trait (mild anemia) D: hemoglobin H disease, moderately severe anemia E: hemoglobin Barts disease, hydrops fetalis Notice that HbXYZ disease and HbXYZ protein are very different from each other! 26 Hemoglobin H disease and Hemoglobin Barts disease is rare in people of African descent but more common in people from SE Asia because of the distribu�on of the two types of alleles (see previous slide) Slide 27 Hb-Tetramers • Reminder: production of alpha chains and beta-like chains are balanced down to 50% • Deletion of one or two alpha genes • Deletion of three alpha chains = HbH disease  surplus of beta-like chains • In fetus/newborn mainly gamma-4 = HbBarts protein • In adult mainly beta-4 = HbH protein • Deletion of four alpha chains = HbBarts disease  surplus of beta-like chains • In fetus gamma-4 = HbBarts protein 27 Slide 28 a - Thal normal : B-Thal or ↓ ↑ HbAz : HbAz 4HbF + HbE + wont , iron deficiency 84 B4 Application 1 S D G Lepore Barts A Portland F H EzUz N Adult - . 2 3 newborn WI 4 Adult 5 A2 C E O HbEs present e WI age 2 HbH disease & HDH ↓ HbBarts - AbH andE - HDA Adult +b Barts disease 28 Please hear the explana�on in class or on Panopto for what this slide is used for. Similar ques�ons might appear on the exam. Slide 29 Mechanisms alpha-thalassemia • Imbalance of Hb with β- or γ-Hb chains not bound to α-chains • β- and γ-Hb chains can form homo-tetramers • Therefore, less precipitation than in β-thalassemia • Shortened RBC lifespan and aborted production just like in β-thalassemia 29 The bullet sta�ng “less precipita�on” is under the assump�on of equal % reduc�on in produc�on Slide 30 Comparison of Thalassemias Disease Chain in surplus Homo-tetramers Precipitation α-thalassemia β-chain (and γ-chain) Yes Yes, but less of the surplus β-thalassemia α-chain No Yes, all surplus 30 No�ce that the table is for severe thalassemia, not for carrier or “trait” Slide 31 Mixed processes Where both content and amount of protein changes 31 Slide 32 HbE • The HbE is a substitution GAG-to-AAG in codon 26 leading to Glu26Lys • This substitution also leads to altered splicing (slow removal of intron 1, partial use of alternative splice donor around codon 26)  some products are non-functional  reduced concentration of Hb • HbE is common in India and South-east Asia, up to 30% allele frequency in some areas • Most common Hb variant in California • Heterozygotes HbAE some protection against P. falciparum malaria 32 No�ce again change of charge of the amino acid. What is the consequence for electrophoresis? Also no�ce the codon number, where is this rela�ve to the sickle cell muta�on? This is a mixed process because HbE both changes an amino acid and reduces produc�on. Slide 33 HbE phenotypes • HbE/HbA heterozygotes near normal MCV • HbE/HbE homozygotes frequently benign (mild hypochromic, microcytic anemia) • Can be classified as mild β+ thalassemia (Hb > 10 g/dL) 33 Slide 34 HbE/ -thalassemia compound heterozygotes • Mild (15%): 9-12 g/dL Hb – rarely need treatment except when infections hit • Intermediate (most): 6-7 g/dL Hb – similar to β-Thal intermedia • Severe (rest – up to ½ in some reports): 4-5 g/dL Hb – treat as β-Thal major • What causes the variation? • β0 vs β+ • Persistence of HbF to varying degrees: in many E/β0 with no HbA, 40-60% of Hb is HbE, the rest HbF (and a little HbA2) • Possibly difference in splicing efficiency and other modifier genes? 34 No�ce that genotype does not directly predict severity of phenotype. Again no�ce that to understand this, you need to know which subunits are found in the different types of hemoglobin tetramers. Beta-thalassemia intermedia is essen�ally those who have reduc�on beyond 50% and therefore show symptoms but who does not have a severe enough reduc�on that they need frequent transfusions. Slide 35 HbS/ -Thalassemia compound heterozygotes • Symptoms will be Sickle Cell disease, not β-Thalassemia • Sickle cell anemia for Sβ0 (or slightly milder due to lower concentration of Hb) • Milder for Sβ+ 35 Reason for the outcome of this compound heterozygote: β-Thalassemia would be the expected outcome when less than 50% of normal beta-chain is produced. In this case there is 50% produc�on of beta chain. However, the fact that the beta-chain produced is of the beta-S type with no produc�on of normal beta-chain means that the person will have sickle cell anemia. Slide 36 Persistent HbF • Fetal hemoglobin is <1% in most adults. (Why?) • Some people synthesize high amounts of HbF – No significant clinical problem • Genetic variation at position –158 of the G gamma globin gene • In people with HbSS, HbF constitute 2-20% • Hydroxyuria is used experimentally to increase HbF production in βthalassemia 36 The second bullet: the high amounts of HbF in people with inherited persistent HbF will happen even if they have no hemoglobinopathy alleles. Hydroxyuria mechanism of ac�on may be (hypothesis!) a selec�ve destruc�on of the fastest dividing RBC precursers – it so happens that HbF producing cells (F-cells) are dividing at a slower pace.