Hemoglobinopathies- Part II Nemer El Mouallem MD VCU Health system 05/30/2018

Hemoglobinopathies - part 2 Structural mutants resulting in a thalassemic like phenotype: Hb Lepore Constant Spring Hb E Decreased solubility Crystallization Hb C disease Unstable hemoglobins Congenital Heinz body hemolytic anemia Hb Koln M hemoglobins (familial cyanosis/congenital methemoglobinemia) Abnormal oxygen affinity mutations High affinity hemoglobin (familial erythrocytosis, Hb Chesapeake) Low affinity hemoglobin (familial cyanosis, Hb Kansas)

Reminder- Human Globin Genes

Hemoglobin Lepore Condition affecting globin chain synthesis Fusion of β and δ chains Decreased synthesis of beta like globins Homozygotes Beta thal major phenotype (more commonly intermedia) Hb electrophoresis: 8-30% Hb Lepore, 70-92% HbF Heterozygotes: beta thal minor phenotype

Hemglobin constant spring Non- deletional form of alpha thalassemia Mutation in stop codon of α2 globin decreased synthesis of normal α globin and increased synthesis of abnormal chain with extra 31 amino acids Homozygous constant spring: HbH phenotype: intermittent transfusions needed Near normal MCV

Hemoglobin E Beta-thalassemia like hemoglobinopathy β codon 26: Glu  Lys leading to alternative splicing  decreased mRNA production of normal β globin- similar to β+ Second most common hemoglobin variant: 30 million worldwide, mostly in SE Asia RBC cytoplasm: precipitated alpha chains, increased oxidant stress

Hemoglobin E trait Heterozygotes (hemoglobin E trait, HbAE) not usually anemic, but may have minimal degrees of microcytosis and hypochromia. Hemoglobin analysis shows approximately 30 percent HbE, 1 percent HbF, and 70 percent HbA. ●Homozygotes (hemoglobin E disease, HbEE) minimal anemia along with hypochromia, target cells, and prominent microcytosis. Hemoglobin analysis shows >90 percent HbE and no Hb A, with the remainder being HbF.

Hemoglobin E

Hemoglobin E HbE/β0 thalassemia Phenotype similar to severe thalassemia intermedia HbE 60-85%, HbF 15-40% Mild to moderate microcytic hemolytic anemia Ineffective erythropoiesis and iron loading

Hereditary persistence of fetal hemoglobin Causes Deletions involving β and δ genes Inactivation of HbF suppressor by decreased expression of KLF1 transcription factor Up-regulation of γ chain synthesis Almost 100% HbF in homozygotes Clinically silent

Hemoglobin C Beta globin chain mutation Less soluble than HbA  forms hexagonal crystal Presence induces red cell dehydration  High MCHC HbC trait: HbAC; benign carrier trait HbC disease: HbCC; mild hemolysis and splenomegly, minimal to moderate anemia Prevalent in Atlantic West Africa HbSC variant

Unstable Hemoglobin diseases Congenital Heinz body anemia Rare autosomal dominant mutation Leads to defective binding of heme to globin About 200 variants  heterogenous phenotype Heinz bodies, oxidative membrane damage, hemoylsis

Unstable hemoglobin diseases Normocellular to microcytic hemolytic anemias Heinz bodies detected by new methylene blue or bromocresol green RBC stability testing : heat stability test or isopropanol stability test Molecular testing for mutation analysis

Unstable Hemoglobin Diseases

Unstable Hemoglobin Diseases Avoid oxidants Transfusions as needed Splenectomy in severe cases

Hemoglobin M disorders Typically autosomal dominant: can be a mutation in the α, β, or γ globin chains Single amino acid substitution in the heme pocket globin chain Hereditary methemoglobinemia and cyanosis Amino acid substitutions in heme pocket leading to Fe oxidation: Fe2+ to Fe3+

Hemoglobin M disorders Clinical Manifestations: Asymptomatic cyanosis Slate grey/brownish skin No dyspnea Normal life expectancy

Hemoglobin M disorders Diagnosis Abnormal SpO2 Hb electrophoresis, Hb spectrophotometric absorbance Methemoglobin <30% Definitive diagnosis is made by DNA sequencing Cyanosis not reversible with methylene blue or Vit C Treatment: Major issue is misdiagnosis and unnecessary treatments Cytochrome B5 or CB5 reductase deficiency variants

Acquired forms of Methemoglobinemia

Abnormal oxygen affinity mutations Hemoglobin O2 Dissociation Curve

Abnormal oxygen affinity mutations

Hemoglobins with high O2 affinity Low p50 (left shift), inadequate tissue oxygenation Autosomal Dominant Familial erythrocytosis Alpha or beta chain can be affected Example: Hb Chesapeake

Hemoglobins with high O2 affinity Diagnosis: Hb electrophoresis, HPLC PCR or gene sequencing Treatment: Polycythemia mild, phlebotomy not needed

Hemoglobins with low O2 affinity Much less common High p50 (right shift in dissociation curve) Asymptomatic cyanosis, though oxygenation to tissues is adequate No treatment required Example: Hb Kansas

Screening Prenatal screening In the US, per ACOG: -CBC with RBC indices to all pregnant women -Hemoglobin electrophoresis or HPLC for low MCV or MCH OR based on ancestry: African, Mediterranean, Middle Eastern, Southeast Asian, or West Indian descent

Screening

Examples Which hemoglobinopathy is this? HbA 20% HbA2 5% HbF 75%

Examples Which hemoglobinopathy is this? HbA 71% HbE 28% HbF 1%

Example Which hemoglobinopathy is this? HbA 90% HbA2 7% HbF 3%

Examples Which hemoglobinopathy is this? HbA 0% HbE 70% HbF 30%

Example Which hemoglobinopathy is this? HbA 0% HbA2 4% Hb F 96%