Globin Synthesis and Abnormal Hemoglobin

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

each globin chain has a covalently-bound heme group
reduced iron (ferrous) component can reversibly bind gas (O₂, CO₂, CO)
RBC longevity: dependent on stability of hemoglobin tetramer

Globin gene organization and developmental expression

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

Regulation of globin gene expresion and the thalassemias

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

RNA Processing

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

Protein stability/multimer formation

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.

Detection: based upon altered mobility during electrophoresis under native or denaturing conditions. A detectable mutation must result in an altered charge:mass ratio. However up to 45% of globin mutations do not result in altered charge:mass ratio. Can then use direct DNA sequence analysis

Clinically significant hemoglobinopathies

(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.