Title Overview: Hereditary Hematological Disorders of red cell shape. Disorders Red cell Enzyme disorders Disorders of Hemoglobin Inherited bleeding disorders- platelet disorders, coagulation factor Anthea Greenway MBBS FRACP FRCPA Visiting Associate deficiencies Division of Pediatric Hematology-Oncology Duke University Health Service Inherited Thrombophilia Hereditary Disorders of red cell Disorders of red cell shape (cytoskeleton): cytoskeleton: • Mutations of 5 proteins connect cytoskeleton of red cell to red cell membrane • Hereditary Spherocytosis- sphere – Spectrin (composed of alpha, beta heterodimers) –Ankyrin • Hereditary Elliptocytosis-ellipse, elongated forms – Pallidin (band 4.2) – Band 4.1 (protein 4.1) • Hereditary Pyropoikilocytosis-bizarre red cell forms – Band 3 protein (the anion exchanger, AE1) – RhAG (the Rh-associated glycoprotein) Normal red blood cell- discoid, with membrane flexibility Hereditary Spherocytosis: Clinical features: • Most common hereditary hemolytic disorder (red cell • Neonatal jaundice- severe (phototherapy), +/- anaemia membrane) • Hemolytic anemia- moderate in 60-75% cases • Mutations of one of 5 genes (chromosome 8) for • Severe hemolytic anaemia in 5% (AR, parents ASx) cytoskeletal proteins, overall effect is spectrin • fatigue, jaundice, dark urine deficiency, severity dependant on spectrin deficiency • SplenomegalSplenomegaly • 200-300:million births, most common in Northern • Chronic complications- growth impairment, gallstones European countries • Often follows clinical course of affected family • Underestimate as mild forms not clinically significant members • 75% AD, remainder AR or new mutations • Severe anemia with concurrent parvovirus infection- (subsequently AD inheritance) red cell aplasia 1 Investigations: Treatment: • Blood film- spherocytes, increased reticulocytes • Hematinics- Folic Acid supplementation • Elevated bilirubin, LDH • Blood Transfusion as needed • Osmotic fragility • Splenectomy- indications: frequent transfusion, poor growth, massive splenomegaly with risk • Flow cytometry rupture (lifestyle limitations) • Gene tests not required • Cholecystectomy • Further studies: SDS page- detects molecular • Monitor growth and development defect of Red cell membrane proteins in specific • Education and Genetic Counseling- genogram, families (not required) likely inheritance and risk to future offspring Hereditary Elliptocytosis/ Pyropoikilocytosis/ Red Cell Enzymopathies: SE Asian Ovalocytosis: • Most forms AD, except HPP which is AR • Red cell enzyme pathways responsible for • Alpha spectrin (65% of HE), Beta Spectrin(30%), protein 4.1 energy production and prevention of damage to (5%) • HE- 2.5 to 5: 10,000 US red cell- glycolytic pathway (PK deficiency), redox potential (G6PD deficiency) • No Nucleus- space efficiency- O2 carrying • Africa/SE Asia up to 30% of population (protective against capacity malaria) • Severe hemolysis associated with homozygous/ compound • Limited lifespan 120 days in N red cells, reduced heterozygous forms in membrane disorders, enzyme disorders and • Clinical Spectrum- asymptomatic to life threatening hemolysis haemoglobinopathies/thalassaemia. G6PD Deficiency: G6PD Deficiency: • Glucose-6-phosphate dehydrogenase enzyme-essential to • Red cell unable to overcome counter oxidant stress to red blood cells oxidant stress- drugs, • Most common red cell enzymopathy 400 million cases infections worldwide • Clinical findings: • Gene located on X chromosome- X linked disorder asymptomatic, episodic • Affected hemi zygote males, carrier hemizygous females (silent), hemolysis to severe chronic affected hemizygous females due to unequal lionization hemolysis • 12% African American men, 20% AA women hemizygous, 1% • Severity depends on degree homozygous, 35% Greek/Mediterranean, 70% Kurdish Jews of enzyme deficiency • Majority of patients • 10 distinct enzyme variants-almost all point mutations, rare asymptomatic in absence of deletions oxidant stress-drugs, foods • G6PD B+ Caucasian/wild type, G6PD A- AA/African form, G6PD (fava beans), fever/illness, Mediterranean form chemicals-naphthalene 2 Drugs/Chemicals causing oxidant stress Diagnosis/Treatment: in G6PD deficiency: • Diagnosis: Classical Clinical features-ethnicity, family history • Acetanilid Diphenhydramine • Acetaminophen • Dapsone Isoniazid • Aminopyrine • Furazolidone L-DOPA • Ascorbic acid (except in very high doses) • Blood film- bite and blister cells, anaemia, reticulocytosis, • Methylene blue Menadione elevated LDH • Nalidixic acid Paraaminobenzoic acid • Aspirin • Quantitative enzyme level-falsely elevated with reticulocytosis • Naphthalene (mothballs, henna) • Chloramphenicol Phenacetin • Chloroquine • Treatment: Avoid Oxidant drugs/ chemicals/ foods - Fava Beans • Niridazole Phenytoin • Colchicine • Nitrofurantoin Probenecid (especially in early spring) • Phenazopyridine Procainamide • Phenylhydrazine Pyrimethamine » Careful observation with fever, other triggers • Primaquine Quinidine • Sulfacetamide Quinine » Transfusion as required • Sulfamethoxazole Streptomycin » Genetic Counseling- female carriers, male • Sulfanilamide Sulfamethoxpyridazine • Sulfapyridine Sulfisoxazole offspring, screen siblings/extended family, monitor • Thiazosulfone Trimethoprim • Toluidine blue Tripelennamine neonates • Trinitrotoluene Vitamin K • Data from Beutler, E, Blood 1994; 84:3613. Other Red cell Enzyme Disorders: Thalassaemia/ Hemoglobinopathies • Rare- classified as non-spherocytes hemolysis • PK deficiency- homozygosity for mutant PK gene, results in reduced enzyme levels •Most AR • Clinical features- hemolysis, splenomegaly • Blood film- no spherocytes, reticulocytes, normal osmotic fragility • Diagnosis: enzyme level • Treatment- supportive (folic acid), transfusion, splenectomy Hb A- Adult Hemoglobin Globin gene clusters in man: Hemoglobin Synthesis • Gene for beta globin is epsilon gamma delta beta Chromosome 11 on chromosome 11 G A • Gene for alpha globin is 5’ 3’ on chromosome 16 Chromosome 16 • Adult Hemoglobin (Hb A) is α2β2 Hb F Hb A2 Hb A • Fetal Hemoglobin (Hb F) is α2γ2 • Hemoglobin A is α2δ2 Figure drawn by Dr. Ross Hardison, which can be found 2 at: <http://globin.cse.psu.edu>. 3 Globin Chain Synthesis: Diagnosis • HEP, HPLC or isoelectric focusing used to identify variant hemoglobin's • Separates variant hemoglobin's based on differences in charge • Sickle solubility testing detects only HbS, so should rarely be used Hemoglobin Electrophoresis on cellulose acetate at pH 8.6 Sickle Cell Disease (SCD) History of Sickle Cell Disease • SCD refers to a group of disorders • First described in 1910 by James Herrick characterized by a predominance of HbS • SCD is most common in persons of African, • SCD affects 1 in 375 African American live Mediterranean, Arabic, and Indian descent births • Individuals with sickle cell trait with resistance to malarial infection • 1 out of 10 African Americans with trait • In the mid 1970’s the National Sickle Cell Anemia Act • Includes: HbSS, HbSC led to the Cooperative Study of Sickle Cell Disease HbS/β−thalassemia, HbS/Other (CSSCD) which prospectively followed over 3500 infants with sickle cell disease to determine the natural history of the disease Pathophysiology Normal Blood Smear • Mutation at sixth position of beta globin chain changes glu → val • With deoxygenation, the Hb S • HEP: AF molecule polymerizes within the • Hgb 10.5 – 13.5 gm/dL RBC leading to characteristic • MCV 72 – 100 fL shape changes • Retics < 1.5% • Sickled erythrocytes are rigid and obstruct small blood vessels • Smear: normal • Sickled RBCs have a shorter half life than normal RBCs 4 HbSS Disease HbSC Disease • HEP: SF • HEP: SC • Hgb 6.5 – 8.5 gm/dL • Hgb 9.0 – 12.0 gm/dL • MCV 80 – 100 fL • MCV 60 – 80 fL • Retics 5 – 15% • Retics 3 – 5% • Smear: sickled cells, • Smear: microcytosis, NRBCs, polychromasia hypochromia, target cells Inheritance of Sickle Cell Disease Newborn Screening in NC • Inherited in an autosomal • Universal screening since 1994, targeted from 1986 recessive fashion • Once abnormal screen is detected, family, local • All 50 States, DC, Virgin physician, and state counselor are notified Islands, and Puerto Rico have universal screening- • Confirmatory testing and family studies done HEP • If diagnosis is confirmed, referral to Sickle Cell • Sickledex test (sickle Center; tracking ensured by state counselors solubility) false negative if • GOAL: Education, comprehensive care, and Hb S% low, poor test initiation of penicillin prophylaxis by 2-3 months of age Clinical Manifestations Diagnosis of Sickle Cell Disease of Sickle Cell Disease HEP in MCV HbA Hb F One Other Sickle Cell Variants 2 Increased susceptibility to infections/ Asplenia NB fL % % parent parent Hemolysis – “break down of red cells” Hb SS (SCA) FS N or ↑ < 3.6 < 25 AS AS Anemia A, ↑ A , ↑ F, Jaundice,,g gallstones Sickle β0thalassemia FS ↓ > 3.6 < 25 AS 2 ↓ MCV Acute vaso-occlusive events A, ↑ A , Painful events, pneumonia Sickle β+thalassemia FS, FSA ↓ > 3.6 < 25 AS 2 ↓ MCV Stroke, splenic sequestration, priapism Chronic organ damage HbSC Disease FSC ↓ NA < 15 AS AC Spleen, kidneys Sickle Cell Trait FAS N < 3.6 < 1.5 AS A Lung, brain, eyes, hips Hemoglobin's reported in order of quantity. Fetal hemoglobin is significantly reduced by 6-12 months of age. 5 Increased Susceptibility Gallstones to Infections • Develop functional asplenia due to • Chronic hemolysis results repeated infarcts within the spleen in formation of pigmented • Leads to increased risk of sepsis, (bilirubin)