Overview – Hemoglobin (Hgb) tetrameric protein comprised of 2 a-like globin chains and 2 b-like globin chains.

• b-globin genes: Chromosome 11, localized to a "single" domain
• Insulators: specific sequences at each end of domain restrict activity of activator or repressor DNA-binding proteins
• cis-acting locus control region (LCR) : responsible for general control of domain
• a globin genes: chromosome 16
• Embryonic globin: e and x ; confined to the first 10 weeks of gestation
• Hgb F (fetal): (a 2g 2) synthesis from birth to about 36 weeks than switches to adult hemoglobin production
• Hgb A (Adult): (a 2b 2) 98% of adult hemoglobin; synthesis begins at 36 weeks gestation
• Hgb A₂ : (a 2d 2) : Memorization aid: remember ADult for a d .
• KEY CONCEPT: any defect in the transcription, translation, expression of Globin chains can result in a blood anemia

• Efficiency of globin gene transcription is dependent on the following processes:
• Transcription: controlled by trans-acting DNA binding proteins that bind specific sequences (promoters and enhancers)
• Promoter: DNA sequence located immediately upstream of (5’ to) transcriptional start site which serves as a binding site for RNA POL II; activity of promoter is orientation-specific (needs to be located immediately upstream to work)
• Enhancers: independent of orientation (may be located at the 5’ or 3’ end) and may be exerted over a long distance (1000’s of base pairs)
• LCR: enhancer element and binds erythroid-specific GATA-1 and AP-1 transcriptional activator proteins
• Chromatin Structure: structure must be "open" facilitating easy access of trans-acting proteins to DNA sequences and thus permissive for transcription. Specifically:
• Degree of cytidine methylation at CG sequences within the gene: Ý methylation Þ less efficient transcription
• Acetylation of core histone proteins: Ý acetylation Þ more efficient transcription

• Capping: a methyl-guanosine "cap" is placed on the 5’ terminus (origin) of the RNA strand. Thes m7GTP cap protects the nascent RNA from degradation and functions in facilitating spicing, nuclear transport, and subsequent translation
• Splicing: excision of DNA; a and b globin genes: contain 3 exons and 2 introns
• Exons: transcribed DNA, joined by splicing to generate mature RNA
• Introns: non-coding, internening sequences; are removed from the nascent RNA strand during transcription
• Correct splicing: dependent upon recognition of splice donor site (at 5’ exon/intron boundary) and splice acceptor site (at 3’ intron/exon boundary)
• Splicesome: splicing apparatus that is facilitated by "splice enhancer" sequences in nearby DNA;
• Aberrantly-spliced mRNA: generally contains premature termination codons Þ results in a and b thalassemia
• RNA cleavage and polyadenylation: Cleavage of the 3’ terminus and subsequent polyadenylation" important for mRNA stability and efficiency of translation

Translation – initiation, elongation, termination

• Initiation: initiator complex with the 40S ribosomal subunit, binding to 5’ terminus of the capped mRNA, binding of the 60S ribosomal subunit and identification of the translational start site (UAG and AUG)
• Elongation: rapid sequential addition of amino acids as encoded by the nRNA to the growing peptide chain utilizing t-RNA
• Termination: occurs when a UAG, UGA, or UAA codon is read in frame
• Mutation of UAA termination codon Þ a - and b - thalassemia
• Hb constant Spring: T-C (U-C) mutation in termination codon of a 2 gene and is associated with ß globin mRNA stability

• Dimerization: principle controlling step in hemoglobin assembly: once a and b globin peptides are synthesized they quickly bind heme and associate to form a highly stable a b dimer. Dimers then reversibly form a 2b 2 tetramers.
• Association results from different net surface charges (a globin – positive; b globin – negative)
• ß in the rate and stability of a b dimerization resulting form amino acid substitutions, chain terminations, or elongations change the net electrostatic charge of one globin chain Þ rapid degradation of globin chain.
• a tetramers are highly unstable (compared to b tetramers) Þ leads to failure of erythrocyte maturatoin and hemolysis. - accounts for ß clinical severity of a -thalassemia–when a deficient, b tetramers are stable enough for RBC to mature.
• Other structural defects in thalassemia: unequal recombination within the globin genes
• Hb Lepore and Hb Anti-Lepore: crossover between the b globin gene of Ch 11 and d globin gene of Ch 11

Abnormal Hemoglobin – Over 500 structural Hb variants have been described. More b than a chain mutations.

• (1) Sickle Syndromes (hemoglobins S, C, O, and D)
• HbS: An AÞ T transversion within codon 6 of the b -globin chain results in the substitution of valine for glutamic acid Þ leads to the synthesis of Hb S tetramers. Hb S tetramers polymerize when in deoxy confirmation
• Most common hemoglobin mutation in the world. Gene frequency: 4% among African Americans
• Homozygous mutation (Hb S–S): 100% sickling of cells; Heterozygous mutation (Hb S) 40% cells sickled: therefore Clinicaly normal
• Hb D; HbO: incapable of polymerizing by themselves but can co-polymerize with HbS tetramers
• Double heterozygous individuals (Hb SD or Hb SO) are the same as HbSS; gives a negative "sickle prep" result
• Hb C: 2^nd most common hemoglobin. Exhibit mild to moderate hemolytic anemia characterized by splenomegaly and target cells on peripheral blood smear
• Heterozygotes (Hb AC): asymptomatic, exhibit targe cell formation on peripheral smear
• Double heterozygosity (Hb SC): exhibit mild hemolytic anemia and vaso-occlusive complications: eye, avascular necrosis of femoral head. Peripheral smear: target cells, few sickled cells
• (2) Unstable hemoglobins – Congenital Heinz Body hemolytic Anemia (CHBA)
• Mutation: over 100 unstable hemoglobin variants has been described. > 75% are b chain mutations
• Process: mutation encoding amino acid substitution in vicinity of the heme-binding pocket Þ perturbs heme binding or disrupts hydrophobic interior of the moleculeÞ altered globin molecule denatures, aggregates, precipitates in aqueous cytosol. Denatured aggregates visible as "Heinz bodies" Þ ß deformability contributes to RBC destruction in spleen.
• Heinz body: reduces RBC deformability; release of intracellular heme contributes to cell membrane damage through lipid peroxidation and protein crosslinking
• Many mutations are charge-neutral and the mutant Hbs thus co-migrates with HbA
• Genetic: autosomal dominant
• Family history: anemia, early cholelithiasis
• Presentation: present early in childhood with anemia, jaundice, splenomegaly
• Hemolysis may be accelerated by fever or ingestion of oxidant-type drug.
• "aplastic crises" : erythroid hypoplasia associated with acute infection (parvovirus) with profound anemia and reticulocytopenia
• Diagnosis: supravital staining to detect Heinz bodies or electrophoresis
• Treatment: supportive. Folate, avoid oxidant drugs, treat fevers promptly, transfusion during aplastic crises
• (3) Hemoglobins with abnormal Oxygen Affinity
• Pathogenesis: mutation in hemoglobin either Ý or ß the affinity of hemoglobin with oxygen
• Oxyhemoglobin dissociation curve: sigmoid shaped curve
• Cooperativity: partial O₂ saturation of a hemoglobin tetramer markedly Ý O₂ affinity of the remaining heme groups. Permits release of a significant amount of oxygen over a relatiely small drop in O₂ tension (thus maxing O₂ delivery)
• p50 – O₂ tension at which hemoglobin is 50% saturated; reflects oxygen affinity. Influenced by: pH, 2,3 dPG
• Common to all normal embryonic, fetal, and adult hemoglobin species
• Genetics: autosomal dominant
• Diagnosis: O₂ affinity testing, hemoglobin electrophoresis, spectral analysis (rule out methemoglobinemia or corboxyhemoglobin)
• Presentation: Ý affinity: asymptomatic, mild erythrocytosis (because of impaired O₂ delivery); ß affinity: asymptomatic cyanosis
• (4) Methoxyhemoglobin and methemoglobinemia (Familial Cyanosis)
• Methoxyhemoglobin: hemoglobin containing ferric (Fe⁺³) rather than ferrous (Fe⁺²) heme
• Heme is maintained in ferrous state via interaction with amino acids (histadine) in heme-binding pocket of globin chain
• Cytochrome b5 reductase (methemoglobin reductase): NADH-dependent enzyme involved in physiological reduction of ferric heme resulting from auto-oxidation and exposure-related oxidation
• Oxidation of heme: ß O₂ delivery in 2 ways:
• Ferric heme cannot bind oxygen Þ carrying capacity is diminished
• Oxidation of 1 heme group of hgb tetramer confers Ý O₂ affinity on remaining heme groups Þ ß O₂ loading in tissues
• Mechanism:
• M hemoglobins: Globin gene mutation (histidine Þ tyrosine), autosomal dominant
• Congenital methemogobinemia: reduce the activity of methemoglobin reductase, autosomal recessive
• Heterozygous condition at risk for toxic methemoglobinemia with exposure to drugs
• Oxidizing compounds: antimalarials, nitrates, phenazopyridine, analine dyes, sulfa drugs (dapsone), nitroprusside, acetaminphen
• Clinical manifestations: cyanosis
• Diagnosis: + family history, of h/o oxidant exposure, Ý conc of methoxyhemoglobin, electrophoresis, cytochrome b5 reductase
• Treatment: unnecessary unless methoxyhemoglobin > 40% Þ remove toxic exposure, methylene blue (ß methoxyhemoglobin via activity of NADPH-flavin reductase)
• (4) Structurally Variant Hemoglobins associated with Thalassemia phenotypes
• Mechanism: abnormality of the globin gene Þ REDUCED accumulation of globin chain
• Mutation: Hb E: GÞ A transition at codon 25 Þ ß production of full-length mRNA
• Homozygous (Hb E): no clinical symptoms, mild microcytic anemia target cells on peripheral smear
• Heterozygote (Hb AE): minimal anemia
• Hb constant spring and Hb Lepore: discussed above.