Based on the article: The thalassemias and related disorders

please discuss all the factors that can affect oxygen
affinity to hemoglobin and its transport through the human
body.

Article: The thalassemias and related
disorders.

Abstract:

The thalassemias, sickle cell disease, and other
hemoglobinopathies represent a major group of inherited disorders
of hemoglobin synthesis. The abnormal hemoglobins were reviewed in
the July 2006 issue of Baylor University Medical Center
Proceedings. Because of immigration patterns and population flow,
these disorders are becoming increasingly more prevalent in the
USA. In this article, the clinical aspects of the more common
thalassemia syndromes are reviewed. For most symptomatic patients
with thalassemia, there is no definite cure; only supportive
management of the anemia is possible. A very limited number of
patients with thalassemia may be cured by bone marrow
transplantation from HLA-identical donors. Other tentative
approaches to management include stimulation of fetal hemoglobin
synthesis and attempts at somatic cell gene therapy. Prevention of
disease transmission by carrier screening programs along with
prenatal diagnosis remain of paramount importance in the reduction
of these diseases worldwide. The thalassemias are a group of
anemias that result from inherited defects in the production of
hemoglobin. The thalassemias are among the most common genetic
disorders worldwide, occurring more frequently in the Mediterranean
region (1), the Indian subcontinent, Southeast Asia, and West
Africa (2). Ineffective bone marrow erythropoiesis and excessive
red blood cell hemolysis together account for the anemia. Since
reticulocytes manufacture equimolecular quantities of alpha and
beta chains, mature erythrocytes contain essentially equimolecular
amounts of each chain (3). Patients with thalassemia do not produce
enough hemoglobin (Hb) A (α2β2) because their cells cannot
manufacture either the alpha or beta polypeptide chain of human
hemoglobin. Alpha-thalassemia depresses only the production of the
alpha chains, and beta-thalassemia depresses only the production of
the beta chains. Clinically, both alpha- and beta-thalassemia may
occur in the major (homozygous), intermediate, and minor
(heterozygous) genetic forms and also can interact with the
presence of abnormal hemoglobins in the same individual (4—6). To
explain the nature of the thalassemia syndromes, it is necessary to
outline the interplay of the various polypeptide chains of
hemoglobin during normal human development. In the first trimester
of intrauterine life, zeta, epsilon, alpha, and gamma chains attain
significant levels and in various combinations form Hb Gower I
(ζ2∊2), Hb Gower II (α2∊2), Hb Portland (ζ2γ2), and fetal
hemoglobin (HbF) (α2γ2 136-G and α2γ2 136-A) (7). Whereas Hb Gower
and Hb Portland soon disappear, HbF persists and forms the
predominant respiratory pigment during intrauterine life. Before
birth, gamma-chain production begins to wane so that after the age
of 6 months postpartum, only small amounts of HbF (<2%) can be
detected in the blood (8). In early intrauterine life, beta-chain
synthesis is maintained at a low level but gradually increases to
significant concentrations by the end of the third trimester and
continues into neonatal and adult life. The synthesis of delta
chains remains at a low level throughout adult life (<3%). Hence
during normal development, the synthesis of the embryonic
hemoglobins Gower and Portland is succeeded by the synthesis of
HbF, which in turn is replaced by the adult hemoglobins, HbA and
HbA2 (Figure). Figure Figure Developmental changes in human globin
chain production, sites of erythropoiesis, and cell morphology.
Reprinted with permission from Wood WG, 1993 (9). Clinically the
thalassemia syndromes are heterogeneous due to the many possible
mutations affecting the human globin chain loci. These mutations
include those of gene deletions, as well as globin chain
initiation, translation, and termination (10, 11).

BRIEF HISTORICAL REVIEW

By the beginning of the 20th century, European clinicians had
become aware of an anemia syndrome in infancy associated with
enlargement of the spleen (12). In the American literature the
first clinical description of thalassemia is attributed to the
Detroit pediatricians Thomas B. Cooley and Pearl Lee (13). The
actual term thalassemia was coined by George Whipple (14, 15). How
this term arose remains obscure, although it is reported that early
patients were mostly of Mediterranean origin. During the 1960s a
genetic basis of the thalassemia diseases was proposed, linking
them to unbalanced globin chain synthesis (16, 17). The stage was
set for further progress. Simpler methodology was developed that
made it possible for routine laboratories to analyze levels of
hemoglobin A2 and confirm the diagnosis of thalassemia (18). Other
observations on the alterations of hemoglobin patterns in patients
with thalassemia led to the discovery of HbH (β4) (19) and Hb Barts
(γ4) (20), which later became established markers of
alpha-thalassemia. At Johns Hopkins University, David Weatherall
and associates labeled reticulocytes of thalassemic patients with
radioactive amino acids in vitro and were able to demonstrate that
in patients with alpha- and beta-thalassemia, alpha- or beta-chain
production was defective because of unbalanced globin chain
synthesis (21). At that point, it became necessary to determine
whether protein synthesis was abnormal at the level of the
structural gene or of globin chain synthesis. A series of
experiments revealed a quantitative or qualitative deficiency of
specific messenger RNA in many thalassemia syndromes as well as
defects in the translation of the messenger RNA to protein (22).
This latter stage requires ribosomal units that can initiate
(promote or enhance), elongate, and terminate the globin chain
(23). Hence a clearer picture of the genetic control of human
hemoglobins had emerged. It became clear that several structural
loci, i.e., alpha, beta, gamma, and delta, were responsible for the
production of their respective globin chains. ALPHA-THALASSEMIA
Each human diploid cell contains four copies of the alpha-globin
gene, located on chromo-some 16. Whereas alpha-thalassemia is
usually caused by one or more deletions of the alpha-globin chain
loci, not all alpha-thalassemias are due to gene deletions (24).
Clinically there are four alpha-thalassemia syndromes: silent
carrier, alpha-thalassemia trait, HbH disease, and hydrops fetalis
syndrome. These occur because of inheritance of molecular mutations
affecting the output of one, two, three, or four of the
alpha-globin genes (Table). Table Table Alpha-thalassemia syndromes
According to written convention, the alpha-thalassemia syndromes
can be expressed as α0 and α+. In the α0, no alpha chains are
produced. In the α+, the output of one of the linked pair of
alpha-globin genes is defective, and only some alpha chains are
produced. Within these general categories of the alpha-thalassemia
syndromes, there is considerable genetic and clinical heterogeneity
due to the interaction of the many possible mutations directing
globin chain synthesis. Since alpha chains are present in both
fetal and adult hemoglobins, a deficiency of alpha-chain synthesis
affects hemoglobin production in fetal as well as in adult life. A
reduced rate of alpha-chain synthesis in fetal life results in the
formation of gamma-chain tetramers (Hb Barts). In adult life, a
deficiency of alpha chains results in the formation of beta-chain
tetramers (HbH) as well as a deficiency in the formation of HbA2
(α2δ2). The anemia that ensues is also due to shortened red cell
survival: beta-chain tetramers (HbH) can precipitate and form
inclusion bodies that damage the red cell membrane. Since Hb Barts
(γ4) is more stable than HbH (β4), it does not readily form
inclusion bodies. Nevertheless, both Hb Barts and HbH show no
heme-heme interaction and have high oxygen affinities.
Consequently, they are extremely poor oxygen carriers (25). In
response to the oxygen deprivation caused by the anemia, the
dyserythropoietic marrow expands, leading to extramedullary
erythropoiesis in the bone, liver, and spleen. This erythropoiesis
gives rise to skeletal deformities and bony fractures,
megaloblastic anemia due to folate deficiency, and hyperuricemia
with gout.

GLOBIN CHAIN TERMINATOR CODON MUTATIONS

The term codon represents the sequence of three bases that
determines the specificity of an amino acid to be included in a
polypeptide chain. Several mutations that give rise to elongated
chains have been described in the terminator codon of both the
alpha-and beta-chain genes. The first elongated globin was
discovered in a Chinese family in Jamaica and was called Hb
Constant Spring (Hb CS). The alpha chain in Hb CS has 172 instead
of 141 amino acids (26). Also, there is a marked reduction of
alpha-globin RNA in the red cells of homozygous Hb CS individuals
(27). Similar mutations that give rise to elongated beta-chain
globin have been described. The Hb TAK beta chain consists of 157
amino acids instead of 146 (28). BETA-THALASSEMIA The beta-gene
cluster region resides on chromosome 11. The beta-thalassemias can
be divided into several varieties. In β0 thalassemia, there is a
total absence of beta-chain production. In β+ thalassemia, there is
a partial deficiency of beta-chain production. Hypochromia and
microcytosis characterize all forms of beta-thalassemia. Because
the synthesis of beta chains is almost completely inhibited in
thalassemia major, a severe anemia begins at about 3 to 6 months of
age, the time when gamma-chain synthesis normally decreases. The
anemia produces a stress situation in the bone marrow. This leads
to the continuation of HbF synthesis but at a rate far below what
is necessary for adequate compensation of the anemia. The HbF
produced is unevenly distributed in the red cells and accounts for
the anisochromasia. Large numbers of imperfect red cells are
destroyed in the bone marrow, giving rise to ineffective
erythropoiesis, which is such a prominent feature of the disease.
Accelerated apoptosis, the major cause of ineffective
erythropoiesis, is caused by excess alpha chain deposited in the
erythroid precursors (29). The hemoglobin pattern in patients with
homozygous thalassemia (beta-thalassemia major) consists of a
variable increase in HbF, which then accounts for 8% to 90% of the
total hemoglobin concentration. The terms beta-thalassemia
intermedia, beta-thalassemia minor, and beta-thalassemia trait or
carrier are used to reflect the decreasing clinical severity of the
anemia. DELTA-BETA-THALASSEMIA In δβ+ thalassemia, an abnormal
hemoglobin, Hb Lepore, is produced (30). Hb Lepore has a normal
alpha chain combined with a nonalpha chain that consists of the
N-terminal residue of the delta chain fused with the C-terminal
residue of the beta chain. Many different varieties of Hb Lepore
have been described in which the transition from delta to beta
amino acid sequences occurs at different points (25). Essentially
the Lepore nonalpha chain is a delta-beta fusion chain. This
hemoglobin has little clinical importance yet is of great genetic
interest. The occurrence of Hb Lepore serves to establish the
concept of neighboring delta- and beta-chain loci on the same gene,
with the delta locus leading the beta locus (31). Individuals who
carry such genes have up to 25% Hb Lepore in their circulation as
well as increased levels of HbF (5%–70%). The Lepore hemoglobins
have been found sporadically in most racial groups and rarely give
rise to significant anemia. HEMOGLOBIN E BETA-THALASSEMIA Compound
heterozygotes for HbE and beta-thalassemia are extremely common in
Thailand and Southeast Asia. Patients with these disorders can
suffer from a moderate to severe anemia and require regular
transfusion (2). Today HbE thalassemia is considered one of the
most common and most important hemoglobinopathies in the world.
This condition is becoming more prevalent in the USA as a result of
Asian immigration.

HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN

Hereditary persistence of fetal hemoglobin (HPFH)is a state in
which the pattern of hemoglobin production of the unborn child
continues into adult life. The switching on of the beta-and
delta-chain loci does not take place, and the adult continues
producing gamma chains. No deleterious effects are apparent, even
when 100% of the hemoglobin synthesized is HbF (α2γ2). More than 40
examples of the heterozygote state of HPFH have been described to
date. In these individuals, about 15% to 30% of the hemoglobin is
HbF. HPFH becomes clinically important when it is inherited
together with beta-thalassemia or the sickle-cell gene. In such
cases, HPFN’s increased output of HbF ameliorates the degree of the
anemia and acts as a protective agent against sickling. Increased
levels of HbF may also occur in various disorders such as juvenile
chronic myeloid leukemia, Blackfan-Diamond anemia, Fanconi anemia,
and paroxysmal nocturnal hemoglobinuria. Many of the conditions
resulting in increased HbF in adult life appear to involve an
increased erythropoietic drive, which results in a higher
proportion of erythroid progenitor cells activating their inherent
ability to synthesize some amounts of HbF. Treatment of sickle cell
anemia with hydroxyurea may increase fetal hemoglobin over 20%,
ameliorating the anemia with improvement in the clinical
manifestations of the disease. An excellent review of HbF increases
in adult life has been published (9). CLINICAL FEATURES OF SEVERE
THALASSEMIC SYNDROMES Infants and children affected with
thalassemia have pallor, poor development, and abdominal
enlargement. Hemoglobin electrophoretic patterns show a variable
quantity of HbA2 (0%– 6%) depending on the genotype of the patient.
The anemia is due to a combination of ineffective erythropoiesis,
excessive peripheral red blood cell hemolysis, and progressive
splenomegaly (25). The latter causes an increase in plasma volume
and a decrease in total red cell mass. The reticulocyte count is
usually <1%. The red cells are microcytic (mean corpuscular
volume <70 fL) with marked anisochromasia. The bone marrow shows
marked erythroid hyperplasia, and the serum ferritin level is
elevated. For diagnostic purposes the parents’ hematologic status
should be evaluated. In children and young adults, radiologic
abnormalities include thinning of the long bones with sun-ray
appearance and dilation of the marrow cavities. The skull has a
“hair-on-end” appearance because of widening in the diploic space.
Patients with thalassemia have enlarged maxillary sinuses and tend
to have a maxillary overbite. The face gradually assumes a
“mongoloid” appearance. Such changes promote infections in the
ears, nose, and throat. Because of chronic anemia and iron
overload, endocrinopathies such as hypopituitarism, hypothyroidism,
hypoparathyroidism, diabetes mellitus, cardiomyopathy, and
testicular or ovarian failure become common as the child with
thalassemia grows older (32, 33). Thalassemia can be regarded as a
chronic hypercoagulable state (34). Venous and arterial
thromboembolic phenomena tend to occur more frequently in
thalassemic patients who have undergone splenectomy. Furthermore,
such patients may develop progressive pulmonary arterial disease
due to platelet thrombi in the pulmonary circulation. The reasons
for the pro-coagulant effect in thalassemia remain obscure,
although it has been proposed that erythrocyte membrane
abnormalities such as phosphatidylserine formation on the surface
of thalassemic red cells activate the coagulation system.
THERAPEUTIC MEASURES Chronic anemia in thalassemic patients is
managed with blood transfusion, iron chelation, and splenectomy in
cases of hypersplenism. In recent years several different
transfusion regimens have been proposed to promote normal growth,
decrease cardiac load, and lessen iron deposition in tissues. More
recently, clinical experience suggests that such criteria may be
achieved by maintaining a hemoglobin level of 9 to 10 g/dL
throughout life (35). The transfusions are given once a month using
washed, filtered, or frozen red cells in order to reduce noxious
reactions to foreign cells and plasma proteins. A few medical
centers specializing in the treatment of thalassemic patients use
younger red cell populations, “neocytes,” for transfusion (36) with
the aim of reducing transfusion frequency. Prevention of
hemosiderosis Hemosiderosis in thalassemia is secondary to frequent
transfusions and increased iron absorption associated with
ineffective erythropoiesis. One unit of packed red cells contains
about 250 mg of iron, and some patients receive >100 units of
packed red blood cells. Because no mechanisms exist for increasing
iron excretion (except for phlebotomy), thalassemic patients are
set up for developing hemosiderosis. Iron deposition is ubiquitous
and occurs in the skin, endocrine organs, liver, spleen, and heart.
Cardiac toxicity and pump failure is a frequent cause of death.
Hence, iron overload causes most of the mortality and morbidity
associated with thalassemia (37). Nontransferrin-bound iron
(low-molecular-weight iron) is a highly toxic state of iron formed
when the iron-binding capacity of transferrin has been exceeded
(38). Hence, long-term transfusion programs should be accompanied
by therapy with iron-chelating agents within the first 3 years of
life (39). At present deferoxamine is widely used for iron
chelation. However, because it is poorly absorbed from the
gastrointestinal tract, it must be administered intravenously or
subcutaneously via a metering pump. Deferoxamine gives rise to
neurosensory toxicity in about 20% of cases, causing high-frequency
hearing loss. Color and night blindness have also been reported.
Physicians prescribing deferoxamine should perform auditory and
ophthalmic examinations biannually. Ferritin levels below 2500
mg/mL are associated with improved survival (40). However, ferritin
levels tend to be unreliable in the presence of liver disease, and
then liver biopsy may be indicated. It is important to try to
maintain serum ferritin levels below 1500 mg/mL. Ascorbic acid
supplementation in small doses (≤100 mg/ day) will increase iron
chelation with deferoxamine. However, it can generate free radicals
with resultant cardiac toxicity. Finally, the oral iron chelator
deferasirox (Exjade) is now widely used and well tolerated except
for mild gastrointestinal toxicity. This drug is well absorbed with
hepatobiliary excretion of chelated iron into the gut rather than
the urinary excretion noted with deferoxamine. Stimulating HbF
production Individuals with HPFH demonstrate that preventing or
reversing the switch from fetal to adult hemoglobin would provide
efficacious therapy for thalassemia and various other
hemoglobinopathies. It has been observed that some patients
recovering from cytotoxic therapy have reactivated HbF synthesis.
Several therapeutic agents such as erythropoietin, hydroxyurea,
cytarabine, and butyrate analogs have produced an increase in HbF
synthesis in the thalassemic patients by stimulating the
HbF-producing progenitor cell population. Bone marrow
transplantation To date, over 1000 bone marrow transplants have
been performed in thalassemic patients at medical centers of
excellence (43). Disease-free survival of 80% to 90% was achieved
in patients who had adequate iron chelation and did not suffer from
liver failure or hepatomegaly. In patients without these good
prognostic features, disease-free survival rates dropped to 50%.
Somatic gene therapy Human globin genes have been transferred into
mouse cells (44). Successful application of gene transfer for the
treatment of thalassemia is experimental and will require that the
newly introduced genes do not alter the growth properties of the
bone marrow cells by the recombinant retroviral genome (45). Design
of vectors that ensure adequate production of globin messenger RNA
to correct the deficiency in thalassemic red cells requires more
experimentation. Prevention In countries with a high incidence of
thalassemia, it is vitally important to offer prospective genetic
counseling and to warn carriers about the risks of intramarriage.
To date, attempts at this approach have been relatively
unsuccessful. Hence, considerable efforts have been directed
towards prenatal diagnosis programs. As carrier states of the
thalassemias are readily identifiable, affected fetuses can be
diagnosed. Recent efforts have been directed to early diagnosis by
fetal DNA analysis (46) carried out on amniotic fluid cells or by
chorionic villus sampling. Also, the development of oligonucleotide
probes to detect individual mutations has markedly increased the
accuracy rate of prenatal diagnosis. The harvesting of fetal cells
from the maternal circulation is being explored for this purpose .
Acknowledge I am deeply grateful to the late Dr. Max Perutz and
Professor Herman Lehmann who first stimulated my interest in
hemoglobinopathies and to various commanders in the Royal Air Force
and Special Air Service for much assistance. I would also like to
thank Kathy Cypert (née Martin) for her patient and determined
secretarial fortitude.