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Hemoglobinopathies Dr Pupak Derakhshandeh, PhD Ass Prof of Medical Science of Tehran University 1 Hemoglobinopathies Disorders of Hemoglobin 2 Disorders of Hemoglobin 5 % of world population: carrier for genes, important disorders of hemoglobin 3 Structure and function of hemoglobin Oxygen carrier In vertebrate: red blood cells Four subunits: 2α- and 2-chains 4 Hemoglobin 5 Each Subunits Globin: Polypeptide chain Heme : Prosthetic group (Iron-Containing pigment) Heme + Oxygene Oxygene transporting 6 Normal adult hemoglobin HbA: 2 α globin chain (141 AA) 2 globin chain (146 AA) α22 Equal length 7 Normal adult hemoglobin HbA2: 2 α globin chain 2 d globin chain a 2d 2 8 Normal adult hemoglobin HbF: 2 α globin chain 2 γ globin chain α2γ2 9 Normal adult hemoglobin 10 Globin genes synthesis Lessons from the thalasemia. Nature Reviews, Genetics, volume2, 2001 11 Thalassemia Onset: Childhood Hypo chromic / Microcytic anemia Low level of MCV / MCH Mean corpuscular volume (MCV) Mean corpuscular hemoglobin (MCH) -Thal: Elevated HbA2 (α2d2) HbF (α2γ2) α-Thal: Normal HbA2, HbF 12 13 Thalassemia Minor 14 Thalassemia major 15 Thalassemia major 16 Thalassemia Minor • Thalassemia minor is an inherited form of hemolytic anemia that is less severe than thalassemia major. • This blood smear from an individual with thalassemia shows small (microcytic), pale (hypochromic), variously-shaped red blood cells. • These small red blood cells (RBCs) are able to carry less oxygen than normal RBCs . 17 Hematological values & a-Thalassemia a a a a s Molecular diagnosis of hemoglobin disorders, Clin. Lab. Haem. 2004, 26, 159–176 18 Thalassemia Major 19 Thalassemia major an inherited form of hemolytic anemia red blood cell (hemoglobin) abnormalities the most severe form of anemia the oxygen depletion in the body becomes apparent within the first 6 months of life 20 If untreated, death usually results within a few years Note the small, pale (hypochromic), abnormally-shaped red blood cells associated with thalassemia major The darker cells likely represent normal RBCs from a blood transfusion 21 Diesease Autosomal recessive Deficiency: Synthesis of α/globin Origin: Mediteranean, African, Iranian, Indian, Southeast Asian Resistant to malaria 22 Prevalence of -Thalassemia ~ 1.5 % in Africans and African Americans ~ 30 % in Sardinia 23 Pathogenesis of -Thalassemia In adequate Hb production Reduced MCV/MCH Unbalanced accumulation of a globin subunits Ineffective Erythrocyt 200 different mutations In Iran over 70 mutations ! 24 Prenatal diagnosis I. ARMS-PCR (22 common mut.) II. PCR-RFLP (9 inf. RFLPs) III. RDB (60 mut.) IV. Sequencing 25 ARMS-PCR 26 PCR-RFLP 1 2 3 M 4 5 6 7 27 globin mutations 1. Transcriptional mutations (+) In promotor regulatory elements -101(silent) -92 (silent) -88 -30 28 globin mutations 2. RNA-Processing (º) Splice junction IVSI-1 Cd30 IVSI-2 IVSI-3’ end del 25bp IvsI-130 Consensus splice sites (º/ +) IVSI-5 IVSI-6 IVSII-844 29 globin mutations Cryptic splice sites in Introns (+) IVSI-110 IVSII-745 Cryptic splice sites in exons Cd 26 (HbE) Cd 121 (HbD panjab/O Arab) 30 -Thalassemia major Onset: 6 months Severe hemolytic anemia Hb level< 7 g/dl Skin: pale Growth retardation don’t eat or sleep well Hepatosplenomegaly Bone marrow expansion: Make more red cells Expantion in face and skull Spleen: destroy of young red cell 80% of untreated patients: † by 5 y. Treatment: Cardiac/Hepatic: † by 30 y. Transfusion +Chelation > 30y. 31 a-Thalassemia 32 Peripheral Blood Smear (1) Normochrome Normocyte MCV a MCH a 33 Peripheral Blood Smear (2) Hypochrome Microcyte MCV MCH 34 Defected globin chains 35 Prevalence of α-Thalassemia 0.01 % in non malarial areas ig. UK, Japan ~ 49 % in Southwest Pacific Islands 36 37 α globin mutations Deletions: 80-85 % of αThalassemia Del: 3.7 kb (most frequent) Del: 4.2 kb α2 InsI-5bp deletion (αHph1α) α2 InCd T>C (αNco1α) αº Variant: --MED --CAL --SEA 38 a-Thalassemia Trait -a/aa – Hemoglobin is with in the reference range. – Reticulocyte count is within the reference range. – Mean corpuscular volume (MCV) is 75-85 fL. – Mean corpuscular hemoglobin (MCH) is 26 pg. 39 a-Thalassemia Alpha1 thalassemia minor (--/aa) – Hemoglobin is within the reference range. – Reticulocyte count is within the reference range. – MCV is 65-75 fL. – MCH is 22 pg. 40 Hemoglobin H disease Peripheral smear from a patient with hemoglobin H disease showing target cells, microcytosis and hypochromia. Morphological abnormalities are similar to those observed in beta thalassemia. In alpha2 thalassemia (silent trait) only mild microcytosis is 41 observed. HbH disease • Hemoglobin H disease – – – – – Hemoglobin is 7-10 g/dL. Reticulocyte count is 5-10%. MCV is 55-65 fL. MCH is 20 pg. The peripheral blood smear shows small misshapen red cells, hypochromia, microcytosis, and targeting. – Brilliant cresyl blue stain demonstrates hemoglobin H inclusion bodies. 42 HbH disease Functional α globin : 1 α: globin ratio : 0.3 Hb level: 7-9 g/dl Genotype: --/-α HbH Inclusion (Heinz body): Many Moderate anemia Hepatosplenomegaly Galstones, infection, folic acid deficiency 43 Hydrops fetalis – Hemoglobin is 4-10 g/dL. – MCV is 110-120 fL. – The peripheral blood smear shows severe hypochromia, and nucleated red blood cells. 44 Hydrops fetalis Functional α globin : 0 α: globin ratio : 0.0 Genotype: --/-HbH Inclusion (Heinz body): Present Severe anemia Heart defect/fatal in utero/ shortly after birth 45 haemoglobinopathies • Reduced synthesis of globin chains (Thalassaemia) • Synthesis of a structurally abnormal Hb variant Thalassaemia β-thalassemia ( over 200 point mutations) a-thalassemia (over 80 deletions & point mutations) 46 a-globin genes cluster Chromosome16p13.3 Exon I Intron I Blood,Vol 91, No 7 (April 1), 1998: pp 2213-2222 Exon II Intron II Exon III 47 Alpha-Thalassemia inheritance Autosomal recessive 48 a-Thalassemia mutations 1) alpha-globin gene deletions approximately 90% of mutations 2) alpha-globin point mutations approximately 10% of mutations 49 a-thalassemias phenotype + a or a-thalassemia 2 - non-functional one a-globin gene (-a) o a or a-thalassemia 1 - non-functional both a-globin genes (--) 50 + a -Thalassemia Rightward Leftward Archives of Iranian Medicine, Vol 4, No 4, October 2001 51 a0-Thalassemia ~20 kb ~23 kb ~30 kb > 300 kb Southeast Asia Philippines Thailand Chinese Mediterranean Greece, Turkey Blood,Vol 91, No 7 (April 1), 1998: pp 2213-2222 (cd51a1) 52 Outcome of deletions a-globin genes a-globin genes affected production a-globin chains Genotype 0 100% aa/aa Normal, healthy 1 75% -a/aa Silent carrier, healthy 2 50% aa/- or -a/-a Carrier, a-thalassaemia trait, Mild hypochromic microcytic anemia 3 25% --/-a Hemoglobin H disease, mild to severe hemolytic anemia - -/- - Hemoglobin Bart's, hydrops fetalis 53 4 0% Outcome Haemoglobinopathies and clotting disorders. Vol. 36, No. 10, October 2007 HbH Disease 54 Method detection a-Thalassemia mutations Salting out DNA extraction - human whole blood PCR-Based Strategies - Gap-PCR - Multiplex Gap-PCR DNA Sequencing 55 Gap-PCR 56 Molecular diagnosis of hemoglobin disorders, Clin. Lab. Haem. 2004, 26, 159–176 Primers Multiplex PCR (2) Chong SS, et al. Single tube multiplex-PCR screen for common deletional determinants Of a-thalassemia. Blood 2000;95:360–362. 57 Primers DNA Sequencing Name M13S 1 13 2 S6 3 S8 Sequence 5'-3' 5´ - CGA CGT TGT AAA ACG ACG GCC AGT CGC CAG CCA ATG AGC GCC - 3´ 5´ - TCC ATT GTT GGC ACA TTC CG - 3´ 5´ - TGT CCA CGC CCA TGC TGG CAC - 3´ American Journal of Hematology 74:99–103 (2003) 58 Hba1 Sequence 59 Hba2 Sequence 60 Gap-PCR 61 MED Mutation 1. Positive control 2. Marker 200bp 3. Negative control Cycle Temperature Time 1 94 5 minute 94 30 second 60 30 second 72 120 second 72 7 minute 35 1 62 Multiplex PCR 63 Multiplex PCR 1) Smart taq DNA Polymerase 2) AMS Buffer 3) DMSO (5-10%) 4) Mgcl2 (50 mM) Cycle Temperature Time 1 94 5 minute 94 60 second 60 60 second 72 120 second 72 7 minute (2 mM) 5) dNTP (10mM) (0.2 mM) 35 6) Primers (10pm) (0.3 µl) 7) ddH20 8) Template 1 64 Result 65 Multiplex PCR (1) 66 Multiplex PCR (2) 67 Multiplex PCR (3) 68 Multiplex PCR (4) 69 Patient samples Male Female RBC : 5.81 MCV :75.9 MCH :25.6 HbA2 :2.6 RBC : 5.41 MCV :75.8 MCH :25.0 HbA2 :2.7 70 Patient sample HbH -a3.7/-(a)20.5 71 Mutations in Khorasan province 65 (57.5%) 41 (36.3%) Deletion Without Deletion Point mutation 6 (6.2%) 72 Sequencing 950 bp PCR Product a1 S8 Primer a2 S6 Primer Khorasan province - a2: 7 sample (4 point mutation) - a1: 7 sample (2 point mutation) Khoozestan province - a2: 4 sample (4 point mutation) - a1: 7 sample ( all normal) 73 Mutant Seq. AAAAAA Normal Seq. AATAAA 74 a2 IVSII-55 Normal CGC>CTC 75 a2 IVS II-24 Normal TCT>TTT 76 Khorasan & Khoozestan province HbA MCV MCH 2 RBC Hb Hct aa/aa 5.53 + 0.51 13.52 + 1.45 41.73 + 3.61 73.96 + 6.09 24.48 + 2.23 2.96 + 1.21 --MED/aa 0 0 0 0 0 0 3.7 -a /aa 5.68 + 0.43 14.07 + 1.14 42.98 + 3.49 75.45 + 5.23 25.31 + 1.25 2.66 + 0.5 -a3.7/-a3.7 5.74 + 0.56 13.1 + 0.94 41.25 + 3.97 71.9 + 2.89 22.9 + 1.54 2.83 + 1.57 5.81 + 0.64 13.97 + 1.57 42.23 + 3.75 71.2 + 2.38 23.47 + 0.76 2.37 + 0.55 -a /aa 5.73 + 0.7 14.33 + 1.52 75.9 + 4.49 24.95 + 0.59 2.28 + 0.56 -a20.5/aa 0 0 0 0 077 3.7 4.2 -a /-a 4.2 43.45 + 2.78 0 Relationship of MCV to mutations 78 Relationship of MCH to mutations 79 Treatment Blood transfusion (3-4 weeks for life) Iron accumulation in body Remove the iron: Desferal: Infused under the skin (8-12 h/6 times a week) Bone marrow transplantation A sib brother or sister HLA matched 80 Treatment • Avoid iron supplementation. It contributes to iron overload • Administer folate supplementation to provide adequate amounts of the vitamin for increased utilization resulting from the hemolytic process and high bone marrow turnover rate. • Provide prompt attention to infection, especially in children who have had a splenectomy. • Administer blood transfusions only if necessary. • If chronic transfusion is needed (hemoglobin H disease), iron chelation therapy should be 81 considered to avoid iron overloading. Surgical Care Hemoglobin H disease – Perform a splenectomy if transfusion requirements are increasing. – Surgical or orthodontic correction may be necessary to correct skeletal deformities of the skull and maxilla due to erythroid hyperplasia. 82 Sickle Cell disorder 83 Sickle Cell disorder Stuck the red cell in the vessels In children: Spleen, chest, wrists,ankles In adults: hips and shoulders Anemia (Hb 7-8 g/dl) Infections (take antibiotics) Painful crises (6-18 months) Swollen and inflamed (hand/food syndrome) 84 What are the Complications? • • • • • • • • • pain episodes increased infections bone damage yellow eyes or jaundice early gallstones lung blockage kidney damage and loss of body water in urine painful erections in men (priapism) blood blockage in the spleen or liver (sequestration) • eye damage • low red blood cell counts (anemia) • delayed growth 85 The combination of hemoglobinopathies Doesn't cause any health problem: α+ Thalassemia / α+ Thalassemia (-a/-a) HbH disease: αº Thalassemia / α+ Thalassemia (--/-a) Hydrops fetalis: αº Thalassemia / αº Thalassemia (--/--) 86 Doesn't cause any health problem α+ / º Thalassemia/Thalassemia α+ / º Thalassemia / HbC α+ / º Thalassemia / HbD α+ / º Thalassemia / HbE α+ / º Thalassemia / HbO Arab α+ / º Thalassemia / HbS 87 Thalassemia / Thalassemia Caused severe health problem! 88 Other combinations HbC / Thalassemia (no problem) HbD / Thalassemia (no problem) HbE / Thalassemia (serious anemia) Hbs / Thalassemia (intermediate-severe) HPFH* / Thalassemia (no problem) *Heriditary persistance of fetal hemoglobin 89 Doesn't cause any health problem HbC / HbC HbC / D, E, O Arab, HPFH HbD / HbD HbD / C, E, O Arab, HPFH HbE / HbE HbE / C, D, O Arab, HPFH 90 Doesn't cause any health problem HbO Arab / HbO Arab HbO Arab/ C, E, D, HPFH HPFH / HPFH HbH / Thalassemia ! Thalassemia major/α+/º Thalassemia! Thalassemia major / HbC, D 91 serious anemia HbH / α+/º Thalassemia HbS / Thalassemia HbS / HbC HbS / HbD HbS / HbE HbS / O Arab 92 Prenatal Diagnosis (PND) 93 ThankS for Your Attention 94