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Home , Hereditary spherocytosis, Inclusion bodies, Sickle cell trait, Spherocytosis, Thalassemia

Title Overview:

Hereditary Hematological Disorders of red shape. Disorders Red cell Enzyme disorders Disorders of Inherited bleeding disorders- platelet disorders, coagulation factor Anthea Greenway MBBS FRACP FRCPA Visiting Associate deficiencies Division of Pediatric -Oncology Duke University Health Service Inherited Thrombophilia

Hereditary Disorders of red cell Disorders of red cell shape (): cytoskeleton:

• of 5 connect cytoskeleton of red cell to red cell membrane • Hereditary - sphere – (composed of alpha, beta heterodimers) – • Hereditary Elliptocytosis-ellipse, elongated forms – Pallidin (band 4.2) – Band 4.1 ( 4.1) • Hereditary Pyropoikilocytosis-bizarre red cell forms – Band 3 protein (the anion exchanger, AE1) – RhAG (the Rh-associated glycoprotein)

Normal red cell- discoid, with membrane flexibility

Hereditary Spherocytosis: Clinical features:

• Most common hereditary hemolytic disorder (red cell • Neonatal - severe (phototherapy), +/- anaemia membrane) • Hemolytic - moderate in 60-75% cases • Mutations of one of 5 (chromosome 8) for • Severe hemolytic anaemia in 5% (AR, parents ASx) cytoskeletal proteins, overall effect is spectrin • , jaundice, dark urine deficiency, severity dependant on spectrin deficiency • SplenomegalSplenomegaly • 200-300:million births, most common in Northern • Chronic complications- growth impairment, 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 - (subsequently AD inheritance) red cell aplasia

1 Investigations: Treatment:

• - spherocytes, increased • Hematinics- Folic Acid supplementation • Elevated , LDH • as needed • Osmotic fragility • - indications: frequent transfusion, poor growth, massive with risk • rupture (lifestyle limitations) • tests not required • • Further studies: SDS page- detects molecular • Monitor growth and development defect of Red cell membrane proteins in specific • Education and - 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 • /SE Asia up to 30% of population (protective against capacity ) • Severe 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 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, , • 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/

• 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 on chromosome 11 G A

• Gene for alpha globin is 5’ 3’ on Chromosome 16 • Adult Hemoglobin (Hb A) is α2β2 Hb F Hb A2 Hb A • Fetal Hemoglobin (Hb F) is α2γ2 • is α2δ2 Figure drawn by Dr. Ross Hardison, which can be found 2 at: .

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

• 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 with resistance to malarial infection • 1 out of 10 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−, 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

• 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: , NRBCs, polychromasia hypochromia, target cells

Inheritance of Sickle Cell Disease 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/ 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, 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 in formation of pigmented • Leads to increased risk of , (bilirubin) gallstones ppyarticularly with • Occur in 1/3 sickle cell pneumoniae patients by adulthood • Immunizations with HIB, Prevenar, • Symptoms: abdominal Pneumovax, Meningococcal pain, nausea, vomiting (Menactra) • Laparoscopic • Penicillin prophylaxis cholecystectomy if – 3 years for HbSC symptomatic – 5 years for HbSS, HbS/β0thalassemia

Acute Chest Syndrome Splenic Sequestration

• New pulmonary infiltrate with • Most common in young children fever, dyspnea, chest pain, (< 2 years of age) hypoxia, increased WBC • Anemia, thrombocytopenia and • Lower lobes most commonly splenomegaly involved; 1/3 bilateral • May cause hypovolemic shock and death if occurs acutely • May have associated pleural • Usually require PRBC effusions transfusions • May be caused by infection, • 50% recurrence rate sickling, fat embolism, • Splenectomy for severe or atelectasis recurrent events

Stroke

• Occurs in 5 – 10% of • Osteonecrosis of bone in children with SCA areas with limited • Thrombotic or infarctive collateral circulation event involvinggg large • Femoral, humeral heads intracranial arteries most commonly involved • Present with weakness, • May occur at any age; up aphasia, seizures, LOC to 50% of adults affected • Often results in permanent • Occurs in all genotypes neurological damage and of sickle cell disease long-term disability

6 Genetic Counseling in SCA: Thalassaemia

• Partner testing to determine present of Hb S, or • A group of disorders characterized by a deficiency in disease causing variant such as Hb C, beta the synthesis of globin chains thalassaemia – quantitative abnormality (reduced rate of synthesis) • CBC, blood film, Hb electrophoresis/IEF/HPLC – Compared with Haemoglobinopathies –inherited disorder resulting in production of abnormal haemoglobin such as • Gene testing not required Hb E (common in SE Asia) or SCA • alpha or beta Thalassaemia • Most common in persons of Mediterranean, Arabic, Indian, Asian descent • Severity ranges from asymptomatic to transfusion dependent anemia

Alpha Thalassaemia Alpha Thalassaemia Carrier:

• 4 alpha (α) globin genes- α1 and α2 • Single gene deletion on one chromosome • Most commonly deletion of 1-4 of α globin • Clinically silent genes • Normal RBC parameters and electrophoresis, • Silent Carrier State – 1 α globin gene is can be missed missing • Alpha Thalassaemia Trait – 2 α globin genes • Diagnosis requires DNA analysis, no abnormal missing (Cis or Trans) globin chain produced so IEF/HEP normal • Hemoglobin H Disease – 3 α globin genes • Molecular testing: targeted mutational analysis missing by PCR for common mutations, full gene • Hydrops Fetalis – 4 α globin genes missing sequencing

Alpha Thalassaemia Trait Hemoglobin H Disease

• Mild microcytosis • Genotype α-/-- α/- – MCV 65 – 75 fL • Hemoglobin H = β4 • Mild anemia – Unstable hemoglobin leads to α/- – Hgb 10.5 – 12 gm/dL increased hemolysis • Inheritance may be either αα/-- • Chronic Chromosome 16 (cis) or α-/α- (trans) – Hemoglobin 7-10 gm/dL – 30% African Americans have αα/α- – MCV 55-65 fL , 2-3% α-/α- • Splenomegaly – may result in – Cis form such more common in SE need for splenectomy asia/FIL/MED/THAI, large deletions affecting both genes • May need transfusions with • Can be confused with illness/pregnancy deficiency anemia • Clinical severity depends- • May have Hb H or Barts on deletional and non-deletional newborn screen forms Heinz bodies

7 Genetic Counseling for Alpha Hemoglobin Barts Thalassaemia:

• Genotype --/-- • At risk population- African American, other-SEA, • Causes hydrops fetalis or premature infant death in utero, all middle eastern, Indian postnatal haemoglobins contain α chains, therefore • Known family history incompatible with life • Suspicion on red cell indices e.g. microcytosis • Massive heppgyatomegaly due to extramedullar y hemato poiesis • Hemoglobin electrophoresis may be unhelpful for • Hgb 4-10 gm/dL carrier and trait as no abnormal Hb produced in adults • Significant morbidity in mother during pregnancy • and partner screening: PCR, gene • Hemoglobin Barts elevated in all newborns with alpha sequencing Thalassaemia mutations (carriers/trait and HbH) • Counseling depending on potential outcome of couple – Tetramers of γ chains = γ4 • Prenatal diagnosis- CVS/amniocentesis –molecular testing

Alpha Thal- trans Alpha Thal- Cis/Silent carrier

Alpha Thal- Cis/Cis Beta Thalassaemia:

• Impaired production of β chains, relative excess of α chains, unstable, precipitation • β chain production begins after birth, predominant from 6/12 aggyge, therefore not clinically significant before this, where Hb F (α2 /γ2) predominates • Over 100 mutations known to affect β globin gene, non-deletional- splice mutations, nonsense/frame shift mutations, promoter region mutations

8 Beta Thal inheritance Beta Thalassaemia Minor

• Beta Thalassaemia minor occurs •AR when one of the β globin genes is defective – Complete absence of the beta globin protein βo – Reduced synthesis of the beta globin protein β+ • Relative excess of α chains • Mild •HEP: Hb AF with • Thalassaemia intermedia: elevated A2 (> 3.5%) moderately severe but does not need regular transfusion;may occur •Hb 9.5-12 gm/dL with βo/β+ or β+/β+ •MCV 60-75 fL

Complications in Beta Thalassaemia Major Beta Thalassaemia Major • Homozygous βo/βo, no β chains • Skeletal changes due

• HEP: HbF and HbA2, No Hb A to extramedullary • Ineffective erythropoiesis hematopoiesis • Severe microcytic hypochromic • Hyperbilirubinemia anemia and gallstones • Transfusion dependent • Splenomegaly requiring splenectomy • Chronic transfusions lead to if untreated • Poor growth • Patients also hyperabsorb • Endocrine dysfunction dietary iron • Cardiac dysfunction

Bone Marrow Transplantation Genetic Counseling in β Thalassaemia:

• Goal is “cure” of SCD or thal •AR • Only 25% of patients have • Both parents carriers- β Thalassaemia minor HLA-matched sibling (may be silent, microcytosis only or confused • Require high-dose chemotherapy and radiation with iron deficiency) therapy as preparative • Compound heterozygosity with other regimen; sterility likely haemoglobinopathies e.g. Hb E/β, Hb S/β, Hb • Currently reserved for patients with significant C/β thal also possible complications such as stroke, • Gene testing-PCR/sequencing required for recurrent episodes of ACS, or pain or those with HLA prenatal diagnosis matched siblings

9 Inherited Bleeding Disorders: Inherited Platelet disorders:

• Platelet disorders • Coagulation Deficiencies •Rare • Skin and mucous membrane bleeding • Joint and muscle bleeding • Quantitative and Qualitative defects described (mouth/ nose/ genitourinary/ heavy • Bleeding with trauma but also periods) spontaneously more common • Normal platelet number 150- • Bleeding with minor trauma- cuts depending on defect 400,000 • Immediate and generally milder • Delayed and severe bleeding post • Platelet count is normal from birth, bleeding with surgery operatively can detect from birth • Mild-moderate Bruising/petechiae • Large, palpable bruises, no petechiae • Testing- CBC and film (characteristic blood film changes- size of platelets, colour granules, • Rarely haemathroses/muscle and soft • Examples- Haemophilia A and B, inclusions ect), platelet function bleeds Factor deficiencies- XI, V, VII studies, flow cytometry for GP on platelet surface, gene sequencing, • Examples- TAR, Bernard-Soulier other-electron microscopy syndrome, Von Willebrand’s disease

Quantitative Platelet Deficits TAR Syndrome

• Severe thrombocytopenia (15-30, 000) with absence of bilateral radii • Thrombocytopenia with absent radii (TAR) – Can have GI, other skeletal, and cardiac abnormalities, as well – Always have thumbs (compared with Fanconi’s anaemia) • Amegakaryocytic thrombocytopenia • Autosomal recessive inheritance – Very rare • X-linked thrombocytopenia • Most common cause of death is bleeding – Intracranial • Wiskott-Aldrich syndrome • Etiology unclear – Poor maturation of megakaryocytes? • May-Hegglin Anomaly • Treatment: – Supportive • Other – Thrombocytopenia usually improves after 2 years of age – – BMT for recalcitrant cases • Prognosis – Trisomies 13 and 18 – 50% survive to age 3 – Not a pre-malignant condition –Trisomy 21

TAR Syndrome Wiskott-Aldrich Syndrome

• Syndrome consisting of eczema, immunodeficiency, and thrombocytopenia • Etiology: – Mutation in WAS protein • Xp11.22-23 • Expressed only in hematopoietic cells – Affects cellular architecture • Impacts cell signaling and protein shuttling – WASp may function as a bridging protein to the cell cytoskeleton • X-linked inheritance – 4 in 1 million male births • Labs: – Thrombocytopenia with small platelets (4-5 fL) – Low number of T-cells • B-cell numbers are preserved – Low IgM levels – Poor response to vaccines (esp. polysaccharides) – Mutational analysis • Die from infection, bleeding, or malignancy – Most die in teens without BMT • Treatment: – Supportive care – Steroids and IVIG can improve platelet counts – Splenectomy –BMT

10 X-linked thrombocytopenia May-Hegglin Anomaly

• Macrothrombocytopenia and leukocyte • Thrombocytopenia without eczema or immune inclusions • Etiology: deficiency – Mutation in the MYH9 gene – Decreased production of non-muscle • Etiology: – Leads to defective megakaryocyte maturation – MttiMutation i n th thWASe WASp gene – Other MYH9 defects: Sebastian, Fechtner, Ebstein’s syndromes • Autosomal dominant disorder – X-linked inheritance – 180 cases reported – Gene Sequencing/Linkage analysis • Thought to be a less-severe phenotype of WAS • Labs: – Platelets: 40-80K, up to 30 fL – WBC: Cytoplasmic on Wright staining • Treatment – Supportive care – Steroids not effective – Most patients do well • Prognosis-overall good, bleeding variable

Amegakaryocytic thrombocytopenia Qualitative Platelet Defects

• Severe thrombocytopenia (plts ,20,000) and absent plt precursors in BM without physical abnormalities • Glanzmann thrombasthenia – Type I: severe, early onset of thrombocytopenia and failure – Type II: slowly increasing platelet count in first year of life, then bone marrow • Bernard-Soulier syndrome failure in preschool years. • Etiology: • ADP storage pool defect – Mutatio n in c-mplp , t he t hro mbopoet in r eceptor. – Type I: total loss of the receptor • Alpha-granule storage deficiency – Type II: mutation in the extracellular domain of the TPO receptor • Some residual function • AR, rare condition • Treatment: supportive, BMT • Often not detected till adulthood, not syndromic • Prognosis: – Poor without BMT – Pre-malignant condition

Glanzmann Thrombasthenia Bernard-Soulier Syndrome

• Severe bleeding syndrome • Severe bleeding syndrome • Etiology: • Etiology: – Mutation in GPIIb/IIIa – Mutation in GP Ib/IX/V – Responsible for platelet aggregation – Responsible for platelet adhesion • Autosomal recessive inheritance • Autosomal recessive inheritance • Clinical characteristics • Clinical characteristics – Easy bruising – Easy bruising – Mucocutaneous bleeding – Mucocutaneous bleeding – Severe hemorrhage after surgery or trauma – Severe hemorrhage after surgery or trauma – Spont aneous bl ee ding uncommon – Spont aneous bl ee ding uncommon – Bleeding tendencies variable-even in same patient • Labs: • Labs: – Platelets: mildly low (have shortened half-life) – Platelets: normal to high – Platelet size: large – Platelet size: normal – Prolonged bleeding time – Prolonged bleeding time – Platelet aggregation: to everything but ristocetin – Platelet aggregation: only to ristocetin • Treatment: • Treatment: – Supportive care – Supportive care • Patients can develop to GP Ib/IX/V • Patients can develop antibodies to GP IIb/IIIa – Some patients respond to DDAVP – Some patients respond to DDAVP – Anti-fibrinolytic response variable – RFVIIa –BMT –BMT

11 ADP Storage Pool Defect Alpha-Granule Deficiency

• Mild to moderate bleeding disorder • Mild bleeding disorder • Etiology: – Absence of ADP or ATP in dense granules (or absence of the granules themselves) • Etiology • Autosomal recessive inheritance – Failure to develop or maintain alpha granules • Clinical characteristics: – “Grey platelet syndrome” – Mild to moderate bleeding • Autosomal recessive inheritance – Epistaxis – Menorrhagia • Clinical characteristics – Hemorrhage after surgery or trauma – Relatively mild course • LbLabs: – Easy bruising – Platelets: normal – Platelet size: normal – Excess bleeding after surgery or trauma – Prolonged bleeding time • Labs: – Platelet aggregation: no second wave – Platelets: mild to moderate thrombocytopenia • Treatment: – Supportive care – Platelet size: large, washed out – Most (75%) patients respond to DDAVP – Prolonged bleeding time – Anti-fibrinolytics • Treatment: • Associated conditions: – Hermansky-Pudlak syndrome – Some patients respond to DDAVP – Chediak-Higashi syndrome – Anti-fibrinolytics

Questions ?

Resources: http://www.genetests.org http://www.cooleysanemia.org

Pediatric Hematology-Oncology: Dr Courtney Thornburg Dr Anthea Greenway [email protected]

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