DIAGNOSIS AND MANAGEMENT OF PATIENTS WITH UNSTABLE HEMOGLOBINS, HIGH-AFFINITY HEMOGLOBINS, AND METHEMOGLOBINEMIA

Clinical Manifestations of Sickle Cell Trait:
often asymptomatic
Anemia, painful crises rare
uncommon but highly distinctive symptom: painless hematuria often in adolescent males, probably due to papillary necrosis
Isosthenuria more common
Sloughing of papillae w/ urethral obstruction reported, as have isolated cases of massive sickling or sudden death due to exposure to high altitudes or extremes of exercise and dehydration
Avoidance of dehydration or extreme physical stress advised

CLINICAL MANIFESTATIONS OF β THALASSEMIA SYNDROMES

HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN

APLASTIC AND HYPOPLASTIC CRISIS IN PATIENTS WITH HEMOGLOBINOPATHIES

BONE MARROW TRANSPLANTATION, GENE THERAPY, AND MANIPULATION OF HbF

Hb tetramer highly soluble, but individual globin chains insoluble--> unpaired globin precipitates--> inclusions that damage cell & can trigger apoptosis
Normal globin chain synth balanced so that each newly synth α or non-α globin chain will have an available partner w/ which to pair

aa sequences of various globins highly homologous to one another
Each has a highly helical secondary structure
globular tertiary structures--> exterior rich in polar aa that enhance solubility, interior lined w/ nonpolar groups, forming a hydrophobic pocket into which heme inserted
tetrameric quaternary structure of HbA: two αβ dimers
Numerous tight interactions (i.e., α1β1 contacts) hold α and β chains together
complete tetramer held together by interfaces (i.e., α1β2 contacts) betw α-like chain of one dimer and non-α chain of other dimer

Each globin chain: a single heme (a protoporphyrin IX ring complexed w/ a single iron atom in ferrous state (Fe2+))
Each heme can bind a single oxygen molecule

(Fig. 127-1)
Each Hb: a tetramer of globin polypeptide chains: a pair of α-like chains 141 aa long and a pair of β-like chains 146 aa long
HbA (major adult Hb): α2β2
HbF (predominates during most of gestation): α2γ2
HbA2 (minor adult Hb): α2δ2
Embryonic Hbs need not be considered here

(Fig. 127-2)
Hb that have bound some oxygen develop a higher oxygen affinity
This S-shaped oxygen equilibrium curve along which substantial amounts of oxygen loading and unloading can occur over a narrow range of oxygen tensions, physiologically more useful than high-affinity hyperbolic curve of individual monomers

Oxygen affinity modulated by:
Bohr effect: ability of Hb to deliver more oxygen to tissues at low pH; arises from stabilizing action of protons on deoxyhemoglobin, which binds protons more readily than oxyhemoglobin bc latter is a weaker acid--> Hb has a lower oxygen affinity at low pH
2,3-BPG: lowers oxygen affinity
HbA has a reasonably high affinity for 2,3-BPG. HbF does not bind 2,3BPG, so it tends to have a higher oxygen affinity in vivo
Hb also binds NO reversibly--> influences vascular tone, but its clinical relevance remains incompletely understood.

Oxygen acquisition & delivery over a relatively narrow range of oxygen tensions depend on a property inherent in tetrameric arrangement of heme and globin subunits within Hb called cooperativity or heme-heme interaction

RBCs first appearing 6 w after conception: embryonic hemoglobins Hb Portland (ζ2γ2), Hb Gower I (ζ2ε2), and Hb Gower II (α2ε2 )
At 10–11 w: (HbF; α2γ2) becomes predominant
at about 38 w: switch to nearly exclusive synth of (HbA; α2β2)

human hbs encoded in two tightly linked gene clusters:
α-like globin genes on chromosome 16: two α-globin genes and a single copy of the ζ gene
β-like genes on chromosome 11: non-α gene cluster: a single ε gene, Gγ and Aγ fetal globin genes, and adult δ and β genes.

Immediately upstream: typical promoter elements
Sequences in 5′ flanking region of γ and β genes appear to be crucial for correct developmental regulation of these genes,
elements that function like classic enhancers and silencers are in 3′ flanking regions
locus control region (LCR) elements located far upstream appear to control overall level of expression of each cluster
elements achieve their reg effects by interacting w/ transacting TFs; Some of these factors are ubiquitous (e.g., Sp1 and YY1), while others are more or less limited to erythroid cells or hematopoietic cells (e.g., GATA-1, NFE-2, and EKLF).
LCR controlling α-globin gene cluster modulated by a SWI/SNF-like protein called ATRX; appears to influence chromatin remodeling and DNA methylation
association of α thalassemia w/ mental retardation and myelodysplasia in some families appears to be related to mutations in ATRX pathway (pathway also modulates genes specifically expressed during erythropoiesis, ex encode E for heme biosynthesis)
Normal RBC differentiation requires coordinated expression of globin genes w/ genes responsible for heme and iron metabolism.
RBC precursors contain a protein, α-hemoglobinstabilizing protein (AHSP), enhances folding and solubility of α globin, which is otherwise easily denatured, leading to insoluble precipitates: play an important role in thalassemia syndromes and certain unstable hemoglobin disorders
Polymorphic variation in amounts and/or functional capacity of AHSP might explain some of clinical variability seen in patients inheriting identical thalassemia mutations

Solubility & reversible oxygen binding
Both depend most on hydrophilic surface aas, hydrophobic aas lining heme pocket, a key histidine in F helix, and aas forming α1β1 and α1β2 contact points
Mutations in these strategic regions--> alter oxygen affinity or solubility

HbF to HbA transition:
transcription factor Bcl11a plays a pivotal role in its regulation
Small amounts of HbF produced postnatal
A few RBC clones called F cells: progeny of a small pool of immature committed erythroid precursors (BFU-e) that retain ability to produce HbF
Profound erythroid stresses, ex severe hemolytic anemias, bone marrow transplantation, or cancer chemotherapy, cause more of F-potent BFU-e to be recruited--> HbF incr in some pxs w/ sickle cell anemia or thalassemia;
This phenomenon explains ability of hydroxyurea to incr HbF in adults
butyrate and histone deacetylase inhibitors can also activate fetal globin genes partially after birth

five major classes of hemoglobinopathies (Table 127-1)

Structural: mutations alter aa sequence of a globin chain
most clinically relevant variant hemoglobins polymerize abnorm, as in sickle cell anemia, or exhibit altered solubility or oxygen-binding affinity

Thalassemia syndromes: mutations impair production or translation of globin mRNA--> deficient globin chain biosynth
inadequate supply of Hb & imbalances in production of individual globin chains--> premature destruction of erythroblasts and RBC

Thalassemic Hb variants: combine features of thalassemia & of structural hemoglobinopathies

Hereditary persistence of fetal hemoglobin (HPFH): synth of high levels of fetal hemoglobin in adult life

Acquired hemoglobinopathies:
modifications of Hb by toxins (e.g., acquired methemoglobinemia)
clonal abnormalities of Hb synth (e.g., high levels of HbF production in preleukemia and α thalassemia in myeloproliferative disorders).

Hemoglobinopathies esp common in areas malaria endemic
reflects a selective survival advantage for abnormal RBC (a less hospitable environment during obligate RBC stages of parasitic life cycle)
Very young children w/ α thalassemia more susceptible to infection w/ nonlethal P. vivax (Thalassemia favor a natural protection against infection w/ more lethal P. falciparum)

Thalassemias: most common genetic disorders in world

α thalassemia trait (minor) occurs in 1–15% of persons of Mediterranean origin
β Thalassemia has a 10–15% incidence in Mediterranean

Sickle cell disease: most common structural hemoglobinopathy

Hemoglobinopathies: autosomal codominant traits --> compound heterozygotes exhibit composite features of each
ex: sickle β thalassemia exhibit features of β thalassemia and sickle cell anemia;
α chain present in HbA, HbA2, and HbF; α-chain mutations thus cause abnormalities in all three--> α-globin hemoglobinopathies symptomatic in utero and after birth bc normal function of α-globin gene required throughout gestation and adult life
β-globin hemoglobinopathies: asymptomatic until 3–9 m of age, when HbA largely replaced HbF
Prevention or partial reversion of switch should thus be an effective therapeutic strategy for β-chain hemoglobinopathies

Quantitation of Hb profile
HbA2 frequently elevated in β thalassemia trait and depressed in iron deficiency
HbF elevated in HPFH, some β thalassemia syndromes, and occasional periods of erythroid stress or marrow dysplasia

For characterization of sickle cell trait, sickle thalassemia syndromes, or HbSC disease, and for monitoring progress of exchange transfusion therapy to lower % of circulating HbS, quantitation of individual hemoglobins also required

some variants can comigrate w/ HbA or HbS (sickle Hb)--> electrophoretic assessment regarded as incomplete unless functional assays for hemoglobin sickling, solubility, or O2 affinity also performed, as dictated by clinical presentation
best sickling assays involve measurement of degree to which Hb sample becomes insoluble, or gelated, as it is deoxygenated (sickle solubility test)
Unstable Hb detected by their precipitation in isopropanol or after heating to 50°C
High-O2 & low-O2 affinity variants detected by quantitating P50 (partial P of O2 at which Hb sample becomes 50% saturated w/ O2)
Direct tests for % carboxyhemoglobin and methemoglobin, using spectrophotometric techniques

Electrophoresis at pH 8.6 on cellulose acetate membranes simple, inexpensive, reliable for initial screening
Agar gel electrophoresis at pH 6.1 in citrate buffer: often as a complementary method bc each method detects different variants
Some important variants electrophoretically silent: more specialized techniques such as mass spectroscopy (rapidly replacing electrophoresis for initial analysis)

Lab evaluation an adjunct, rather than sole dx aid
Dx best established by recognition of a charac Hx, physical findings, peripheral blood smear morphology, abnormalities of CBC (e.g., profound microcytosis w/ minimal anemia in thalassemia trait)

mutation in β-globin: 6th aa: glutamic acid to valine
HbS polymerizes reversibly when deoxygenated to form a gelatinous network of fibrous polymers that stiffen membrane, incr viscosity, cause dehydration due to K leakage and Ca influx (Fig. 127-3) also sickle shape
Sickled cells lose pliability; altered “sticky” membranes abnormally adherent to endothelium of small venules--> unpredictable episodes of microvascular vasoocclusion & premature RBC destruction (hemolytic anemia) (spleen destroys abnormal RBC)
rigid adherent cells clog small capillaries and venules--> tissue ischemia, acute pain, gradual end-organ damage
Prominent manifestations: episodes of ischemic pain (i.e., painful crises) & ischemic malfunction or frank infarction in spleen, CNS, bones, joints, liver, kidneys, and lungs

Several sickle syndromes occur as result of inheritance of HbS from one parent and another hemoglobinopathy (β thalassemia or HbC (α2β2 6 Glu→Lys)) from other parent.
prototype disease, sickle cell anemia: homozygous state for HbS

Vasoocclusion--> protean manifestations
Intermittent episodes in connective & musculoskeletal--> ischemia manifested by acute pain and tenderness, fever, tachycardia, and anxiety.
These recurrent episodes: painful crises: most common clinical manifestation
frequency, severity vary
Pain can develop almost anywhere in body and may last from a few hrs to 2 w
Repeated crises requiring hospitalization (>3 episodes/yr) correlate w/ reduced survival in adult life, (episodes a. w/ accumulation of chronic end-organ damage)
Provocative factors: infection, fever, excessive exercise, anxiety, abrupt changes in temp, hypoxia, or hypertonic dyes.

Repeated microinfarction can destroy tissues having microvascular beds prone to sickling

splenic function frequently lost within first 18–36 m of life--> susceptibility to infection, partic by pneumococci
Acute venous obstruction of spleen (splenic sequestration crisis), rare occurrence in early childhood, may require emergency transfusion and/or splenectomy to prevent trapping of entire arterial output in obstructed spleen

Occlusion of retinal vessels--> hemorrhage, neovascularization, eventual detachments

Renal papillary necrosis--> isosthenuria
More widespread renal necrosis--> renal failure in adults, a common late cause of death

Bone and joint ischemia--> aseptic necrosis, esp of femoral or humeral heads; chronic arthropathy; unusual susceptibility to osteomyelitis (may be caused by organisms, such as Salmonella, rarely encountered in other settings); hand-foot syndrome caused by painful infarcts of digits and dactylitis

Stroke esp common in children; a small subset tends to suffer repeated episodes; less common in adults & often hemorrhagic

partic painful complication in males: priapism (bc infarction of penile venous outflow tracts); permanent impotence a frequent consequence

Chronic lower leg ulcers probably arise from ischemia & superinfection in distal circulation

Granulocytosis common
white count can fluctuate substantially and unpredictably during and between painful crises, infectious episodes, and other intercurrent illnesses

Acute chest syndrome:
a distinctive manifestation charac by chest pain, tachypnea, fever, cough, arterial oxygen desaturation
can mimic pneumonia, pulmonary emboli, bone marrow infarction and embolism, MI, or in situ lung infarction; Often difficult or impossible to distinguish
thought to reflect in situ sickling within lung--> pain & temporary pulmonary dysfunction
Pulmonary infarction & pneumonia: most frequent underlying or concomitant conditions in pxs w/ this syndrome
Repeated episodes correlate w/ reduced survival
Acutely, reduction in arterial oxygen saturation esp ominous bc promotes sickling on a massive scale
Chronic acute or subacute pulmonary crises--> pulmonary HTN and cor pulmonale, an incr common cause of death as pxs survive longer

Most: hemolytic anemia (hematocrits from 15 to 30%, and significant reticulocytosis)
Anemia once thought to exert protective effects against vasoocclusion (by decr viscosity) but now thought that incr in hematocrit & feedback inhibition of reticulocytosis beneficial, even at expense of incr blood viscosity (maybe bc of adhesive reticulocytes)

Chronic subacute CNS damage in absence of an overt stroke: common phenomenon beginning in early childhood
circulatory dysfunction due to a likely CNS sickle vasculopathy; correlate w/ an array of cognitive and behavioral abnormalities in children and young adults
important to be aware of these often subtle changes bc they can complicate clinical management or be misinterpreted as “difficult px” behaviors :((((((((((

remarkable clinical heterogeneity
Some virtually asymptomatic into or even through adult life
others suffer repeated crises requiring hospitalization from early childhood
sickle thalassemia & sickle-HbE tend to have similar, slightly milder symptoms, perhaps bc of ameliorating effects of production of other Hbs within RBC
Hb SC disease, one of more common variants of sickle cell anemia, frequently marked by lesser degrees of hemolytic anemia & a greater propensity for development of retinopathy and aseptic necrosis of bones; but mostly resemble sickle cell anemia
Some rare Hb variants actually aggravate sickling phenomenon

clinical variability in different pxs inheriting same disease-causing mutation (sickle Hb)--> sickle cell disease focus of efforts to identify modifying genetic polymorphisms in other genes that might account for heterogeneity
interesting patterns ex genes affecting
infl response or cytokine expression appear to be modifying candidates
transcriptional regulation of lymphocytes may also be involved