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INTRODUCTION 1.1: Structure of Hemoglobin (PDFDrive)
INTRODUCTION 1.1: Structure of Hemoglobin (PDFDrive)
INTRODUCTION 1.1: Structure of Hemoglobin (PDFDrive)
1. INTRODUCTION
Hemoglobin contains two pairs of unlike polypeptide chains, one chain of each pair is α
or α - like chain while the other is non α (β, γ or δ ) chain. α chains of all human
hemoglobins are alike. Non - α chains include β chains of normal adult hemoglobin
(α2 β2), γ chains of fetal hemoglobin (HbF) α2 γ2 and δ chains of hemoglobin A2 (Helen
Genes that regulate the synthesis and structure of different globins are organized in two
separate clusters, the α or α - like globin genes and β and β allied globin genes.
α - like globin genes are encoded on chromosome 16 and are found in the order
5’ - ζ - ψζ - ψα2 - ψα1 - α2 - α1-θ - 3’. The α Globin gene cluster occupies a region of
70 kilobases close to the short arm of chromosome 16 band p13.3. The CAP site of ζ
gene is designated 0. α2 gene lies 20 kb away from the ζ - gene on the centromeric
side, a further 3.7 kb away. 40kb upstream of the ζ globin gene lies HS (Hypersensitive
sites) which is the α globin gene regulatory elements. In addition to the three functional
α - like genes, the cluster also (Bunn 1986) contains three pseudogenes (ψζ,ψα2 and
ψα1) and gene θ (Clegg 1987). α globin gene cluster lies between 170 and 430 kb from
1
Fig: 1.1. Organization of human α and β- globin gene clusters on chromosome 16
and 11.The genetic control of various embryonic, fetal and adult haemoglobins is
also shown. Hatched boxes in the individual genes represent 3’ and 5’ untrasnlated
regions, black boxes represent exons and the white boxes represent intervening
sequences (IVS) (Weatherall 1987).
From the 16p telomeric repeats there are four polymorphic, subtelomeric alleles in which
α - globin genes lie 170kb (A), 245kb (D), 350 kb (B) and 430kb (C) (Wilkie et al 1991a
As a result of unequal genetic exchange, normal persons may have 4,5 or 6 α - globin
genes (Goossens et al. 1980; Higgs et al, 1980: Galanello et al . 1983; Gu et al 1987) and
2,4,5 or 6 ζ like genes (Winichagoon et al 1982; Felice et al . 1986; Trent et al 1986 and
Titus et al 1988).
Around the α - globin locus 10 variable number of tandom repeats (VNTRs) have been
identified (Flint et al 1997). They produce highly polymorphic segments of cluster and
can be used as genetic markers throughout the genome and have been used to produce
individual – specific finger prints (Jeffreys 1987 and Fowler et al 1988). In α - globin
2
gene, cluster restriction fragment length polymorphisim is produced by a large number of
β – globin gene cluster is on the short arm of chromosome 11 (Fritsch et al 1980; Spritz
et al 1980, Baralle et al 1980, Slightom et al 1980). The β - like globin gene cluster
(Slightom et al 1980) and δ (Spritz et al 1980) and β – globin genes (Lawn et al: 1980).
The two γ – globin genes are identical except at codon 136, where the Gγ gene contains a
glycine and the Aγ gene an alanine residue unequally expressed during fetal development
(Alter 1979). γ genes are duplicated one codes for glycine Gγ and the other for alanine
Aγ (Fig. 1.1).
The two fetal γ genes lie 15 and 20 kb downstream from the embryonic ε gene, while the
δ and β genes are 35 and 43 kb further downstream (Weatherall and Clegg 2001b) Locus
control region (LCR) is the regulatory region that is essential for the expression of all the
genes in the complex. It is present upstream of the ε gene and spans ∼ 15kb. (Weatherall
β, γ, δ or ε chains have 146 amino acid; valine and histidine are at the beginning of β
genes while Tyr β 145 and His β 146 at the c- terminal residues. δ chain differs from β
chain only 10 residues γ chains differ by 39 residues. β gene cluster contains a series of
3
Globin gene structure, function and regulation
Globin gene contains three trans regions called the exons which are separated by two
site, immediately after this is the promoter region that consists of 100 base– pairs. Three
short sequences within this region bind RNA polymerase that catalyzes messenger RNA
Two sequences are important for the initiation of gene transcription, these are called
TATA box and CAT box. Mutations involving these sequences reduce enzyme binding
and thereby limit mRNA transcription. AATAAA is the sequence present downstream
from the third exon, it tiggers the enzyme process that cuts mRNA at an appropriate point
Two other promoter elements, CCAT box and CACC homology α box on the upstream
from CAP site are also required for optimal transcription (Weatheral and Clegg 2001b).
From the Cap site the first exon encompasses ∼ 50 bp of 5’ untranslated sequences (UTR)
and codons for amino acids 1- 31 in α and 1- 29 together with two bases of codon 30 in
the β- globin gene. Exons 2 encode amino acids 32 – 99, the portions of the globin
polypeptide that is involved in haem binding and amino acids 31 – 104 that is α1 β2
( α2β1) contacts.
Remaining amino acids 100 – 141 for α, 105 – 146 for β and 3’ untranslated region of
100 bp are encoded on exon 3. The IVSI intron varies in length from one allele to
another. Intervening sequences are removed from the initial transcript and the exon
sequences are joined with mRNA. This process is dependent on sequences at the border
4
A
(C) AG/ GT ( G ) AGT at the 5’ end and ( C ) N ( C ) AG /G at the 3’ end of intron. GT and
T T T
yolk sac in the embryo, where hemoglobin Gower 1 (ζ2ε2) Gower 2 (α2ε2) and Portland
(ζ2γ2) are produced at 5 weeks gestation the ζ/ζ + α chains synthesis ratio is 0.82 ± 0.04.
By the 6th week of gestation it declines to 0.03 during development there is transition to
fetal hemoglobin (HbF, α2γ2) to adult hemoglobin HbA1 (α2β2) and HbA2 (α2δ2)
Whereas the protein products of α1 and α2 genes are identical, however the steady state
et al 1986). β- gene is expressed in yolk sac cells and stays at a steady level throughout
determinants for beta-thalassemia major and intermedia patients, while the 3'HS1 (+179
5
1.7: Temporal Control:
Expression of globin genes is sequential during fetal development. In the early embryo
erythropoiesis mainly takes place in the yolk sac. It shifts to the liver in fetal life and then
to the bone marrow in the late prenatal and postnatal life (Weatherall and Clegg 1981).
More than 20kb 5’ to the ε globin gene, cis – activating erythroid specific DNAase – I
Hypersensitive sites are present (Tuan et al 1985). This region confers a high level,
cis – acting sequences responsible for this effect are called Locus control region (LCR).
Orkin (1982) and Behringer et al (1990) postulated that temporal regulation of β –like
globin genes results from competition between embryonic fetal and adult globin genes for
It was described that trans- acting factors like GATA binding protein, synthesized in the
yolk sac, fetal liver and bone marrow may bind to a DNA sequence motif (T/A) GATA
(A/G) present in ε, γ and β-globin promoters for the order by expression of the respective
genes (Orkin 1982) expression. GATA – 1 and GATA -2 have been shown to be essential
(Shivdasani and Orkin 1996) for the transcriptional control of erythroid specific gene.
LCR for α - globin cluster has been suggested in the sequences upstream from the ζ-
globin gene (Higgs et al, 1990). Addition of a poly (A) tract at the 3’ end of the mRNA is
involved in the processing and stability of mRNA. A poly (A) additional signal,
nucleotides upstream of where the initial transcript is cut and the poly (A) additional tract
6
1.8: Regulation of globin – gene function
Regulatory sequence for the globin genes include the promoters series of enhancer
1.8.1: Promotors:
DNA sequences present upstream of transcriptional start sites where the transcription
complex including RNA polymerase binds are called promotors. The first 5’ untranslated
sequences is the TATA and CCAAT homology boxes found 30 and 70 bp upstream of
mRNA CAP site (Anagnou et a. 1985; Myers et al 1986; de Boer et al, 1988. and
Antoniou and Grosveld 1990) and are critical for correct siting of initiation and high level
of transcription CCAAT site is duplicated in two γ – globin genes, both are necessary for
maximum rates of initiation. 90pb upstream from the initiation site the GGGGYG (Y: a
pirimidin nucleotide) or the invertal type “CRCCC” (R: a purine nucleotide) (Collins and
addition CACCC box in the promoter upstream of the CCAAT box. CACCC box
homologies are found in most of the β – gene promoters but are not found in the
More distal regions of the promoters of these genes includes GATA – 1 & NF – E2 for
erythroid transcription factors and site for the ubiquitous factors YY1, Sp1 and Oct – 1.
Though not fully understood, these factors are necessary for maximum rate of
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1.8.2: Enhancers
In addition to the promoter sequences more distal sequences are also present. These
sequences increase the level of gene transcription and are called “enhancers”. These
enhancers may lie 5’ or 3’ to the gene or within the gene itself. These include the
regulatory region of α - cluster HS – 40 and elements β LCR HS2. A small region of 800
bp lying 3’ of the Aγ gene (Bodine & Ley 1987; Purucker et al .1990; Balta et al 1994)
and two segments of β – globin gene, one in the large intervening sequences and one 3’ to
the gene (Behringer et al 1987; Kollias et al 1987) have enhancing properties. No effect
significantly reduces the expression of the β gene in this system (Liu et al 1997). A 30 to
breakpoint causes Hereditary Persistence of Fetal Hemoglobin (Feing E.A & Forget
Control of β - globin gene resides in the locus control region (LCR) which consists of
five DNase – hypersensitive sites that lie upstream of β - globin genes (Talbot et al 1989;
Collins et al 1990).
1.8.4: Transcription
The multi protein complex required for transcription of globin genes includes an enzyme
RNA polymerase II. Which transcribes DNA into a mRNA copy. Some of the
transcription factors involved in the regulation of erythropoiesis and globin synthesis are
8
GATA – 1, FOG (Friend of GATA – 1), NF – E 2, EKLF SSP (Weatherall 2001b)
(Fig: 1.2).
Primary RNA transcription of globin gene has a half life of about 5 minutes and requires
1.8.6: Capping
addition of 200 – 300bp long tract of poly (A) residue. Addition of Poly (A) ensures the
1.8.8: Translation
Transcription of the globin gene is initiated at the “Cap Site” which is located 50bp
upstream of the initiation codon (AUG). As transcription proceeds, exons and introns are
included and extends well beyond the highly conserved 3’ AATAA polyadenylation site
9
Fig 1.2: Typical mammalian gene and steps entailed in its transcription and translation.
Exons are shown in black and introns (intervening sequence, IVS) unshaded. Regions of
gene which code for untranslated portions of messenger RNA are indicated as NC (non-
coding regions). Position of 5' regulatory boxes are indicated (Weatherall 1987).
1.8.9: Splicing
Removal of intervening sequences is carried out initially by cleaving of 5’ splice site after
containing the intron and the 3’ exon. 3’ OH group now attacks 3’ splice site and joins
two exons and releases free lariant intron (Weatherall 2001b). The intron sequences are
thus excised and the donor and the acceptor sites of exons are sealed. Donor sites are
10
identified by the nucleotides GT at the 5’ end of intron and acceptor sites by AG at the 3’
end. In addition to these dinucleotides, nucleotide sequences adjacent to them called the
consensus sites, are required for accurate and efficient splicing (Mount 1982) (Fig: 1.2).
After the processing of primary transcript to mRNA it is exported from nucleus to the
cytoplasm. Amino acids are transported to mRNA template on carriers called transfer
RNAs. The order of amino acids in a globin chain is determined by a triplet code. tRNA
carries amino acid to the template and finds the position. mRNA is translated from 5’ to
the 3’ end. There are specific initiation (AUG) and termination (UAA, UAG, UGA)
requirements, different hemoglobins, all composed of two different pairs of globin chains
each attached to a heme moiety, are synthesized in embryo, fetus and adult (Wood and
Weatherall 1983). Molecular investigations of the last 20 years have delineated the two
basic mechanisms that control globin gene activity during development – autonomous
silencing and gene competition. Studies of hemoglobin switching have provided major
2005). The β - like genes undergo two switches (embryonic → fetal → adult). At 6
months after birth, HbF comprises less than 5% of the total hemoglobin and continues to
fall until reaching the adult level of < 1% at 2 years of age. It is at this stage that
mutations affecting the β gene become clinically apparent. The switch from fetal (γ) to
11
adult (β) hemoglobin production is not complete since small amounts of β expression
persist in adult life. The residual amount of fetal hemoglobin (α2γ2) is presenting a sub-
set of erythrocytes called F cells which also contain adult (α2β2) hemoglobin. The tissue
the direct physical interactions between the globin promoters and the β - LCR (Carter et
al .2002 and Tolhuis et al. 2002), the interaction is mediated through binding of
manifest as a result of the decline in the synthesis of fetal hemoglobin (α2γ2) during the
mechanisms of gene slicing and gene silencing and gene competition, mediated by the
different transcription factors in embryonic, fetal and adult cells, the ξ- and γ- globin
the adult β globin gene depends on lack of competition from the γ gene for the LCR
sequences (Wood, 2001). Previous studies have shown that developmental regulation of
Krüppel-like factor (EKLF), and other proteins. For example, EKLF, a positive regulator
specific for the adult β-globin promoter, requires posttranslational modification and/or
interaction with other factors to mediate a hemoglobin switch; in K562 cells, transected
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1.9: Historic Background
Cooley and Lee in 1925 described a severe form of anemia with spleenomegaly and bone
changes (Cooley and Lee 1925). In 1932 George H published a comprehensive account
of the pathologic changes in this disease in Whipple and William L.Bradford (Whipple &
from θαλασσα, “the sea” by Whipple. Fernando Rietti of Ferrara described a mild form
of hemolytic jaundice in which the red cells showed increased osmotic resistance (Rietti
1925). Similar descriptions were published subsequently by other Italian workers (Greppi
1928 and Micheli et al 1935). Thus the condition was known as La Malattia di Rietti –
became apparent that thalassemia was not a single disease but a complex syndrome
was determined by Caminopetros (1938), Neel (1950) and Bianco et al (1952) alluded
was that this was a homozygous state of a recessive trait resulting in decreased
intracellular hemoglobin content (Hypochromia) and small sized red cells (Microcytosis).
estimated that about 7 % of world's population are the carriers of thalassemic gene. These
disorders fall into two groups: the structural variants of haemoglobin and the
1.10: Thalassemias:
early onset of anemia resulting from reduced rate of synthesis of one or more globin
chains caused by globin chain mutations (Low 2005). Thalassemias are the commonest
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monogenic syndromes (Thein 1992), characterized by decreased synthesis of one or the
(hypochromia) and small size of red cells (microcytosis). Because of continued normal
production of unaffected globin chain the imbalanced globin – chain synthesis leads to
circulation and also at earlier stages of maturation in the bone marrow (Forget and Olvieri
2003). This decreases the hemoglobin level in the blood and oxygen carrying capacity of
Depending on the type of the effected globin chain, thalassemias can be classified into
1.11.1: β - Thalassemia
Two main types are described; βo Thalssemia in which no β globin chain is produced and
β+ thalassemia in which some β- globin chains are produced. Less sever forms of β
thalassemia are sometimes designated β++ to indicate that the defect in β- chain
alleles have now been characterized (Weatheral 2001b). Studies on the molecular
genetics of thalassemia in various ethnic groups have shown that each group tends to
14
have its own set of common mutations (Kazazian et al, 1990). These mutations affect the
α- thalassemia
o
α
+
α
Deletion (-α )
Non deletion (-αT)
β thalassemia
o
β
β+
Normal Hb A2
Type 1 (Silent )
Type 2
δβ thalassemia
(δβ)+
o
(δβ)
o
(Aγδβ)
γ Thalassemia
δ thalassemia
o
δ
δ+
εγδβ Thalassemia
HPFH
Deletion
o
(δβ)o, (A γδβ)
Non deletion
Linked to β – globin genes
Gγβ+, A γ β+
Unlinked to β – globin genes
Fourteen deletions affecting only the β – gene have been described. Of these the 619 – bp
deletion is common, and is restricted to Sindhi and Punjabi population of India and
15
Pakistan accounting for about 20% of β – thalassemia alleles (Thein et al 1984 Varawalla
et al 1991). These deletions vary widely in size, but remove always a region from
position – 125 to +78 relative to the mRNA CAP site in the β promoter, which includes
the CACCC, CCAAT and TATA elements (Weatherall 2001b). Dominantly inherited
Mutations affecting beta globin transcription are Promoter Mutations. A group of 19 such
mutations have been described which are single base substitutions in the conserved DNA
sequences that form the β – globin promoter. These mutations reduce the binding of RNA
Several different base substitutions have been found that involve the conserved
sequences upstream from β – globin gene (Weatherall 2000 and Huisman et al 1997).
Several of these mutations are close to CCAT box as exemplified by C-T substitution at
position -88 and – 87 (Orkin et al 1984, Orkin et al 1982).While the others lie within the
ATA box homology (Ponez 1983). C→ T substitution at position – 101 which involves
Redando et al 1989). A→ C substitution at the CAP site (+1) even in homozygous state
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1.11.1.5: RNA – Processing mutations
Ends of exons and introns are marked by the presence of dinucleotide, GT at the 5’
(donor site) and AG at the 3’ (receptor site). Single – base changes that involve either of
these splice junctions abolish normal RNA splicing and cause βo – thalassemia (Huisman
et al 1997).
Single – base substitution with in the consensus sequence of the IVS – 1 donor site
surrounding the invariant dinucleotide at the splice junctions show remarkable variability
IVS – I produces abnormal spliced RNA and causes sever β+ - thalassemia phenotype
β thalassemia in New Guinea (Hill et al 1988). Mutations creating new splice sites
within either introns or exons affect RNA processing and cause variable
1981). Mutation at 116 in IVS -I produces a new acceptor site and causes βo
exons may result in abnormal splicing. Within exon 1 there is a cryptic donor site in the
region of codons 24 through 27. G T dinucleotide sites are present at this site. Several
17
mutations can activate this site so that it is utilized during RNA processing within
Amino acid substitution like A→ G in codon 19, G → A in codon 26, and G→T in codon
mutations result in abnormal RNA splicing and cause thalassemia ranging in severity
o
from β+ to β – thalassemia (Baysal and Carver 1995). Some mutations involve poly
RNA
Some substitutions of the base change an amino acid codon into nonsense codon and
thalassemia. Many such mutations have been described (Huisman 1997). These include
Rosatelli et al 1987) and codon 17 mutation that is common in southeast Asia (Chang and
Kan. 1979).
Insertion or deletion of one two or four nucleotides in the coding region of β – globin
gene disrupts the normal reading frame. Translation of mRNA takes place in the addition
18
of anomalous amino acids until a termination codon is reached in the new reading frame.
Several frameshift mutations of this type have been described (Huisman et al 1997).
Insertion of one nucleotide between codons 8 and 9, and deletion of four nucleotides in
symptoms with thalassemia intermedia phenotype and severe symptoms with thalassemia
In spite of being highly unstable, some β – globin chain variants are capable of forming
tetramers that precipitate in the red cells precursors or in the mature erythrocyte and give
(Dash et al 2006).
phenotypically apparent only when they interact with other alpha-thalassemia mutations
(Traeger-Synodinos et al 2000).
19
heterozygotes for such mutations have a clinical expression similar to thalassemia
severe, due mainly to a gene dosage effect. They are often characterized only on
interaction with other alpha-thalassemia mutations. It is for this reason that they are
involves codons 33-35. This deletion results in the removal of two valine residues from
(B15/B16) and the substitution of the tyrosine residue at position 35 (C1) by an aspartic
beta-globin gene mutation at codon 107 (GGC→>GAC), gives rise to a rare unstable
2006).
Silent β thalassemia
β – globin genes, others involve the CAP sites or the 5’ or 3’ untranslated regions
the Cap site, in the distal CACCC box of the beta-globin gene promoter is the most
20
common silent beta-thalassaemia mutation in the Mediterranean population
been identified (Thein 1992). Many of them involve exon III of β globin gene and include
elongation of β globin gene and highly unstable β – globin gene products (Higgs et al
1986).27 such mutations have been identified (Baysal and Carver 1995). The most
common of this type is a GAA → TAA change at codon 121 leading to truncated
mutations form hyper – unstable haemoglobin variants that precipitate in the erythroid
Some typical β – thalassemias are observed with out any detectable mutation in the β –
globin gene or its immediate flanking regions (Semenza et al 1984, Kazazian et al 1990)
Compound heterozygosity for two new mutations in the beta-globin gene [codon 9 (+TA)
21
δβ thalassemia
The δβ–thalassemia may be divided into δβ+ and δβº based on the residual output of the
δ- and β-chains from the affected chromosome. δβ+ thalassemia is due to the presence of
two different mutations within the same β-like gene cluster. δβº Thalassemia on the other
hand are due to large deletions involving the εγδβ-gene cluster. Nondeletion δβº
state for nondeletion δβº thalassemia, which produced a symptomless clinical phenotype
DUCTION
Inherited disorders of δ and β chain synthesis are of two main types, the δβ thalassemia
and the Hb Lepore syndromes. A classification and description of the main forms of δβ
Many of the δβ thalassemia are produced by the deletions of δ and β globin genes. These
are associated with persistent synthesis of γ chain at a much higher level than is observed
globin chain imbalance and hence these conditions are much milder than the β
of Gγ chains only. If the production of Aγ is preserved in the mutation than both type of
γ chains are produced and the condition is called Gγ Aγδβ thalassemia. At the molecular
level these disorders are very heterogeneous and require the determination of underlying
defects.
22
Table 1.2: The main groups of disorder of δ and β chain production
Thal minor
G A Thal Intermedia
γ γδβ Thalassemia Hb F 5 – 20%; HbA2 1.5 -2 %
100% HbF
α /non- α1.3 – 1.8 /1
G
γδβ Thalassemia As above As above
characterized by δβ fusion genes directing the synthesis of δβ chain fusion. These are
produced by the unequal crossing over of δ and β globin genes. Depending on the exact
position of crossing over resulting in the δβ fusion several different forms of hemoglobin
Lepore have been found. Most common being Hb Lepore Boston found commonly in
parts of Italy and Yugoslavia. Hemoglobin Lepore produce much more sever clinical
phenotype than δβ thalassemia because of less output of γ chains to compensate for the
23
G A
γ γδβ Thalassemia
Homozygous forms of this conditions have a mild anemia with hemoglobin values in the
8 – 11 g/dl range. Red cells have typical thalassemic changes with low MCV and MCH
values. Hb A and A2 are absent and Hemoglobin F is 100%. Marked imbalance with α/ γ
Hetrozygous forms are similar to β thalassemia heterozygotes although red cell changes
are less marked. It contains both Gγ and Aγ chains in heterozygous and homozygous state.
G
γ δβ Thalassemia
Clinical and hematological finding are same as Gγ AγHPFH, although homozygotes are
Sardinian δβ Thalassemia
24
γ δβ THALASSEMIA
This is a rare form of thalassemia, not found in homozygous state. Adults heterozygotes
is associated with a hemolytic anemia, jaundice in newborn period. α and non α chain
production imbalances are seen at birth and adult life. Diagnosis require globin gene
ancestry but sporadic cases have been reported in many ethnic groups. It is now thought
The eight most frequent mutations encountered in Iraqi population are IVS-II-1 (G→A),
codon 44 (-C), codon 5 (-CT), IVS-I-1 (G→A), codon 39 (C→T), IVS-I-6 (T→C),
codons 8/9 (+G) and IVS-I-5 (G→C). These mutations accounted for 81.7% of the
thalassemic defects in this population. The less frequent mutations are codon 8 (-AA),
IVS-I-110 (G →A), codon 30 (G→C) and codon 22 (-7 bp).Genetic abnormality in 11.5
1.11.3.1: Definition
Thalassemia Intermedia is the term used to describe the clinical and hematological
25
patients manifest a more sever degree of anemia than that found in heterozygous carriers
among different ethnic groups. About 10% of patients of Mediterranean ethnicity who are
American patients who are homozygous may be classified as such. This difference
reflects the kinds of thalassemia mutations, especially so-called “mild mutations,” that
1.11.3.2: History
La – Malattia di Rietti – greppi – Micheli (Hemolytic jaundice with reduced red cell
fragility). This condition was characterized by much more sever anemia, jaundice and
splenomegaly.
In 1940 Wintrobe (1940) described a milder form of thalassemia. In Italy many patients
with Mediterranean anemia of intermediate severity have been reported (Marmont and
Bianchi 1948) Chini and Valeri (1949) called this condition ‘Mediterranean haemopathic
syndromes’.
(Silvestroni & Bianco 1944 – 45). A mild form of haemolytic jaundice in which red cells
showed increased osmotic resistance was reported in 1925 by Fernando Rietti of Ferrara
(Rietti 1925). Many description were published shortly afterwards by other Italian
26
workers including Greppi (1928) and Micheli et al (1935). This form of anemia was
described by Rietti, Greppi and Michli and was reviewed by Chini and Valeri (1949).
The earliest reports of milder forms of thalassemia called it thalassemia Intermedia. The
term thalassemia intermedia appeared in the literature in the 1950s (Sturgeon et al 1995).
The term thalassemia intermedia is a useful descriptive title for clinical phenotype that
can result from the interaction of many different thalassemia alleles, either among
is variable and may patients have splenomegaly.They are clinically as well as genetically
variable. Parents may have mild thalassemia or a single gene may be inherited in the
Thalassemia Intermedia present with extraordinary diverse clinical spectrum from almost
are skeletal changes, particularly in the skull and in the malar bones. Patients develop
Thalassemia intermedia have a later clinical onset and a milder anemia than thalassemia
major, characterized by high output state, left ventricle remodeling, and age-related
27
pulmonary hypertension. Bone deformities, extramedullary hematopoiesis (EMH), and
spleen and liver enlargement are the consequences of hypoxia and enhanced
disease in this form. One is the high output state that results from chronic tissue hypoxia
and from hypoxia-induced compensatory reactions. The other is the vascular involvement
The clinical picture of TI patients who have not received transfusions or have
hemolytic anemia, tissue hypoxia, and their compensatory reactions, such as bone
latter two, are getting more frequent and severe over the years. Nowadays, although TI
patients have almost no changes in the course of the disease, well-treated TM patients
disorders in parallel to prolongation of life. The new oral iron chelators and the magnetic
resonance imaging application for early detection of heart iron load are promising for
particularly if there is associated splenomegaly. At the other end of the spectrum, there
are patients with miserable childhood gross skeletal deformities. They are periodically
28
transfused to avoid these distressing complications. Osteoporosis and osteopenia are
(Origa et al 2005). Fractures are frequent among the aging patients with β - TM (Vogiatzi
Another group has hemoglobin values between 6 and 9 g/dl. They grow reasonably well
and reach adult life. They may become transfusion dependent if they develop
Thalassemia intermedia patients usually do not require blood transfusion however all
(Camaschella et.al.1996). Kinetic studies clearly separate cases that, or will be, clinically
erythropoiesis than that seen in Cooley's disease and a greater peripheral haemolysis. In
our study, no overlap was seen between the two groups. Iron kinetic studies are then of
prognostic interest and may help in therapeutic decisions, transfusion regimen, iron
One case in English literature of crystal proven gout in thalassemia intermedia was
reported that indicates the relative rarity of gout in this clinical setting despite evidence of
urate over production in one report. Long survival and renal insufficiency may have
29
Hypocholesterolemia accompanies anemias with high-erythropoietic activity. We suggest
with thalassemia may suffer from a sensory polyneuropathy especially as they grow
older, this is particularly so and if they are not optimally treated (Sawaya et al 2006).
compensates for chronic anemia but at the same time allow the development of PHT
Low transfusion regimen may cause a decrease in serum concentration of EPO, which is
patients with and portal vein thrombosis have been observed in adult thalassemia
thalassemias. These alterations are related to high platelet counts due to splenectomy
and/or liver dysfunction (Cappellini et al 2005, Ciceri et al 2000). Low plasma heparin
cofactor II levels are related to increased red cell turnover and can be normalized once
in patients with low Heparin co factor II levels in the presence of haemolysis might in
30
(Gartaganis et al 1989, Aessopos et al 1989). An acquired diffuse elastic tissue defect
that resembles inherited pseudoxanthoma elasticum (PXE) has been noticed with a
and has been held responsible for a number of complications observed in these cases,
some of which are quite severe. Patients with beta-thalassemia intermedia, who presented
with severe visual acuity impairment associated with angioid streaks, the typical ocular
grades of the disease are described by Ho et al (1998) these are mild, moderate and
severe.
Severe:
If the transfusion started before the age of 4 years, it is usually required every 3 and 4
months. The patients with the transfusion requirement between these extremes were
classified as “severe”.
Moderate:
Severe if transfusion was started at the age of 4 years or above and frequency between 6
– 16 weeks.
31
Mild
Patients in the “mild” group maintain their hemoglobin at 7.5 g/dl or higher with out
transfusion. They are transfused less than once every 2 years if transfusion is started
before the age of 10 years transfusion interval is less than 6 months if transfusion is
1.11.3. 5: genotypes
One of the most useful indicators of thalassemia Intermedia is the age at presentation.
1975). In a study 11% of patients presented in the first year while 30% presented in the
second year and 59% presented after the age of 2 years. However, the socioeconomic
environment and other factors influence the age at presentation (Modell and Berdaukas
32
1984). Thalassemia Intermedia with βo thalassemia homozygousity may also present late
(Cao 1988). Those who were transfusion dependent presented at mean age of 8.5 ± 9.1
months, while transfusion independent patients presented at mean age of 17.4 ± 11.8
months. All these patients maintained their hemoglobin and were homozygous for the
It was suggested that bone marrow erythroblast may mature further and generate more
may be anemic in early child hood while others may not be diagnosed until adolescence
Regular transfusions are usually not required. Growth and development are usually
normal and skeletal changes of β- thalassemia major types are rarely seen (Erlandson et
hormone and insulin-like growth factor I at different skeletal sites has been reported by
Scacchi et al (2008).
1964) and leg ulcers (Weatherall and Clegg 1981), Hyperuricaemia (March et al 1952)
gout (Weatherall and Clegg 1981) . Pancytopenia (Schiliro et al 1983) has been reported
33
but uncommon. Anemia may worsen in pregnancy (Walker et al 1969). Iron overload can
develop even in patients who have not been transfused, this may cause heart failure
Tumor – like masses of bone marrow are common (Knoblich 1960). Folate deficiency
anemia and hepatosplenomegaly. This phenotype can result from different genetic
combinations and is sometimes present in patients with only one parent showing the
β - 10 C → T
This mutation which occurs in distal CACC box, to down regulate globin gene
transcription very slightly and causes extremely mild form of thalassemia intermedia
β CAP + 33 C → G
This is almost silent in the heterozygous state although the HbA2 level may be slightly
elevated and the α/β - globin synthesis ratio varies from 1.5 to 1.8. in the heterozygous
34
state with several beta thalassemia alleles including IVSI – I G → A and IVSI – 110 G →
IVS 2 844 C → G
In homozygous form, this mutation (Murru et al; 1991) causes extremely mild form of
causes mild form of thalassemia Intermedia (Rosatelli et al 1994). Splicing mutations are
aberrant splicing. Reduced level of normal beta-globin synthesis produces a mild disease
β CAP + 1 A → C
Observed in Asian Indians, this mutation can cause thalassemia Intermedia of varying
β Termination codon + 6 C → G
al 1998).
Codon 104(-G)
35
β - 88 C → T
This mutation decreases the rate of transcription. It is common among Africans and Afro
– Americans and may cause mild form of Thalassemia Intermedia (Weatherall 2001b).
Compound heterozygote for two beta-globin gene promoter mutations, like nucleotide
(nt)-88 C→T mutation from the cap site, and two-nucleotide (AA) deletion between nt -
29 and -26 within the TATA box of the beta-globin gene causes TI phenotype (Basran et
al 2006).
β - 87 C → G
This mutation reduces the binding of transcription factors to β- globin gene (Treisman et
al 1983) and produces thalassemia intermedia (weatherall 2001b). -87 were found in
configuration of rare polymorphisms in the 5' sub-haplotype. This has been reported to
1989).
β - 30 T → A
This is common in Turkish, Macedonian and Tunisian population and causes mild form
β - 29 A→ G
This is common in Africans and Afro – Americans and produces fairly mild thalassemia
36
Table 1.3: Thalassemia intermedia (Weatherall D.J.2001)
37
Table-1.4: Some interactions of silent, mild and severe β Thalassemia alleles as the basis of thalassemia intermedia (TI). Where
data is available the severity is classified as mild (MTI) or severe (STI). TI indicates a variable phenotype, or insufficient
information.Data from Huisman et al. (1997). Ho et al (1998) and references cited in text.
AATAAA→A-AAA
5’ UTR +33 (C-G)
5’UT R +22 (G-A)
β term +6 (C-G)
CAP+1 (A→C)
IVSI-6 (T→C)
5’UT R +10- T
CD27 (G→T)
CD19 (A→G)
CD26(G→A)
AATAAA→
AATAAA→
-101(C→T)
Mutations
-29 (A→G)
-92 (C→T)
-30 (T→C)
-87(G→C)
-86(C→A)
AACAAA
-88(C→T)
-87(C→T)
AATAAG
CD8 - AA STI TI TI
CD8/9+G STI
CD15 G-A TI
IVSI-I G-A TI TI TI
CD36/37-T MTI
38
CD44-C
CD41-C
CD39C-T
Mutations
IVS2-1 G-A
IVS2-654 C-T
IVS2-748 C-A
IVS2-745 C-G
IVS2-849 A-G
IVS2-745 C -G
CD41/42 -TTCT
TI
-101 (C→T)
MTI
MTI
MTI TI
-92 (C→T)
STI
-88 (C→T)
-87 (G→G)
TI
-87 (C→T)
-86 (C→A)
TI
-30 (T→C)
TI
-29 (A→G)
5’UT R +10 -T
5’UT R +22 (G-
MTI
39
A)
5’ UTR +33 (C-
MTI
G)
CAP+1 (A→C)
/STI
MTI
CD19 (A→G)
CD26 (G→A)
TI
CD27 (G→T)
IVSI-6 (T→C)
STI
STI
MTI
IVS 2-844
MTI
(C→G)
β Term +6 (C-G)
AATAAA→
AATAAG
AATAAA→
AACAAA
STI
AATAAA→
A- AAA
β Codon 19AAC → AGC ; β 19 Asn → Ser, Hemoglobin Malay
the quantity of normal β – globin messenger RNA. More over δ – globin in cis position
heterozygous form with IVSI – 6 T → C, Codon 8 – AA, IVSI →110 G→A, IVSI – I G
An A→G transition at the usual intervening sequence 2 (IVS2) acceptor splice site has
transient expression vector indicates that the mutation inactivates the normal acceptor
splice site and results in some utilization of a cryptic splice site near position 580 of
IVS2. This mutation would be expected to produce a beta-globin gene which results in no
β IVS I → 6 T → C
β 87 C → T
40
AATAAG, found in Kurdish Jewish families, poly A; AATAAA→ AATGAA
associated with an extremely mild interaction with HbE and there fore is probably a mild
A child with severe beta – thalassemia intermedia has been described, born to a Greek-
Cypriot with hematological findings of beta-thalassemia trait, and a Polish father who is
hematologically normal. G→T change in codon 121 of the beta-globin gene in the child
β -101 C → T SUBSTITUTION
This mutation was observed in patients with mild thalassaemia intermedia, `her
haemoglobin levels were around 9.5 g/dl and haemoglobin F levels < 25% (Maragoudaki
et al 1999).
Hb Knossos (beta 27 (B9) Ala→Ser) is a hemoglobin variant that can cause beta (+)
41
picture with total absence of Hb A2. This indicates that beta Knossos gene is most
hemoglobin in this study, displayed decreased affinity for oxygen (P50 = 35 mm Hg), a
fact presumably accounting for the relatively good tolerance of the condition (Morle et al
1984).
they are characterized by low levels of HbF (less than 20%) indicating only a mild deficit
in beta globin production. Heterozygotes are indistinguishable from those with the more
analysis and globin chain synthesis. Globin gene mapping excluded the presence of alpha
gene cluster. Restriction enzyme site polymorphisms around the beta gene cluster are
(Tamagnini et al 1983).
Hemoglobin Mississippi has anomalous properties that include disulfide linkages with
normal beta-, delta-, gamma-, and alpha-chains and formation of high molecular weight
(Steinberg et al 1987).
42
Hb Dhofar
pro-arg)) associated with a thalassaemic phenotype and unique to the Sultanate of Oman.
Clinical and haematological data suggest that this mutation behaves like a moderately
et al 2008)
globin gene. As a result of the shift in the protein reading frame, this gene codes for an
elongated beta-globin chain (159 amino acids) with an abnormal amino acid sequence
beyond residue beta 99. Patients with this mutation present with a mild form of beta-
thalassemia intermedia with moderate anemia, evidence of iron overload, severe red cell
used as polymorphic markers but their potential functional role is poorly understood.
Several of these microsatellites have been described within the beta-globin locus, some
polymorphisms in the two gamma-globin gene IVS2s has been observed and in vivo and
43
microsatellites to the variable Hb F synthesis in major haemoglobinopathies
(Lapoumeroulie et al 1999).
1.12: α -Thalassemia
There are two α-globin genes on each chromosome 16 (αα/αα) and, in α+-thalassemia,
one gene of the pair is deleted (−α). Clinical effects are modest as reduced α-globin
chain synthesis in homozygotes (−α/−α) causes only mild anemia (average hemoglobin
level 1–2 g/dl lower than normal), hemoglobin level and red cell indices in heterozygotes
Mutations affecting almost every stage of globin gene expression has been described. The
only lesion not yet characterized is the one affecting enhancing sequences, although such
sequences have not yet been identified in the globin gene system. Clinically important
alpha-thalassemias are the deletion types that occur at a much higher frequency than the
nondeletion lesions. In contrast, apart from one deletion beta-thalassemia lesion found in
Pakistan, clinically significant beta-thalassemia lesions are not caused by gene deletion.
The common beta-thalassemia lesions in the Mediterranean region and Asia are caused
that reduce the output of one or the other α-globin genes. Both variants are associated
with increased levels of the γ4 tetramer (Hb Bart’s) in the neonatal period as a reflection
In the South West Pacific region, the striking geographical correlation between the
this hemoglobinopathy provides a selective advantage against malaria in the South West
44
Pacific region, the striking geographical correlation between the frequency of α+-
thalassemia increases the incidence of contracting mild malaria in the first 2 years of life
bound greater levels of antibody from malaria endemic sera and were more readily
malaria has been found in young children with thalassemia than normals children both in
Papua New Guinea and Vanuatu. In the latter study infestation with, Plasmodium vivax
was increased particularly in children aged <30 months. It was proposed that this increase
may act as a natural vaccine against Plasmodium falciparum. These findings were
confirmed by S. J. Allen and his colleagues in their study Plasmodium vivax infection in
to Plasmodium vivax infection because this parasite only infects reticulocytes (Allen et
al 1997). Although it has been recognized for many years that symptomless carriers are
more resistant to malaria. Retrospective serological analysis has shown a relatively high
Premawardhena et al 2004).
α-2 and α-1 genes are embedded within two highly homologous 4 – kb duplicated
segments. The sequence homology of these regions has been conserved throughout
45
evolution by gene conversion and unequal crossover events (Fig1.3). They are further
subdivided into smaller homologous segments X,Y and Z separated by the non
3.7
The chromosome with a hybrid α2/ α1 gene (-α deletion) results from reciprocal
between homologous X segments, which are 4.2 kb apart , results in a single α 1 gene ,
Seven types of α - thal- 2 determinants have been reported to date. The - α3.7 deletion is
the most common and is divided into three type I, II and III, depending on the precise
location of the homologous recombination between the Z boxed (Higgs et al 1984). The
4.2
second most common α - thal – 2 is the - α determinant which is found at high
frequencies in South china, mainly in the Guangxi (58%) and Jiangxi (29%) (Baysal &
Huisman 1994).
2.7 3..5
Other less common α - thal – 2 determinants are the - α and -α deletions. The
former deletes only the α - 1 – globin gene, leaving the α 2 locus intact, while in the
3.5
-α type, the deletion extends from the 5’ end of the α1 gene to the 5’ end of the θ 1
2000).
46
o
According to the output of α - chain, α - thalassemias are classified into α –
+
thalassemia with no output of α- globin chains, α- thalassemia – 1, and α - thalassemia
are most frequently due to deletion of the genes and less frequently it results from
mutations involving one or some nucleotides within the structural gene, so called
α - thalassemia – 2 occurs more frequently than any other type of thalassemia and has a
frequency of upto 30% in certain parts of Africa (While in other parts of the world such
Individuals who inherit two or three functional α-genes (-α/αα, -α/-α or - - /α α) have α
thalassemia trait with a mild hypochromic microcytic anemia (Higgs et al 1989). Those
who inherit one α gene (- - / - α) have HbH disease, a moderately sever hemolytic
anemia with a variable clinical course (Wasi et al , 1974). Those who inherit no α genes
(--/--) develop sever intra – utrine anemia which in the absence of intensive neonatal care
and life – long transfusion (Bianchi et al 1986) results in death at or around the time of
birth, a condition known as the Hb Barts (γ4) hydrops fetalis syndrome (Lie – Injo & Hie,
Southeast Asia, 3.45% to 5.3% of the population are carriers (Hundrieser et al 1988). In
Mediterranean basin less than 1% of the population are carriers (velati et al 1986). Hb
1968) and may account for up to 26% of prenatal deaths (Cong & Shong 1982).
47
Alpha-Thalassemia mutations are one of the most common mutations in Man, and they
trait alone, mild type of TI to sever TI. Additional alpha-genes may increase the severity
gene has been shown to worsen the degree of anemia in beta-thalassemia heterozygotes.
apparent beta-thalassemia carriers who were more symptomatic than expected (Ma et al
48
Fig: 1.3: The duplicated XYZ box arrangement containing the α genes. Nonhomologus
regions (I,II and III) are indicated. The extent of each deletion is indicated by the solid
blocks and the limits of the breakpoints are represented by solid lines. Misaligend
anti3.7 anti4.2
chromosomes crossing over toproduce the - α3.7, α α α and - α4.2, α
haplotypes are also shown (Higgs et al, 1989).
49
1.12.3: Homozygous β thalassemia with ααα/ααα
quadruplicated and normal α - globin – gene complement has been reported (Thompson
globin gene increases alpha:beta-globin chain imbalance and accounts for the presence of
1992). The greater degree of globin chain imbalance resulting from two additional alpha
chain genes is the likely mechanism for clinically severe phenotype of thalassaemia
50
1.12.6: Dominant β thalassemia associated with ααα/αα
Severe effect of this particular β- thalassemia allele together with the usual degree of
conjuncted with alpha-globin gene triplication was the major cause of beta-thalassemia
Relative excess of alpha- over beta-globin chains in the erythroid precursors is the major
However, some patients present with moderate anemia that does not require regular blood
mutations and alpha- and gamma-globin gene expression play an important role in
modifying the clinical phenotype. Thus genetic factors do not significantly alter the
clinical phenotype when present alone but ameliorate the course of homozygous beta-
excess is reflected in a minimal or mild anaemia without clinical symptoms. Factors that
51
with a severe beta-thalassaemia mutation to cause an alpha-chain excess equivalent to
mutations, with co-inheritance of alpha thalassaemia trait and high HbF determinants act
initial disease severity, although the effect on pubertal development is less clear
thalassemia
Significant effect on red cell indices are noted by the co – inheritance of β – thalassemia
52
transposition to β chain - thalassemia gene locus is mediated at the transcriptional or
translational levels. These changes are observed in hemoglobinization of the red cells in
major increase in β and α globin production from the normal loci. It may however be
appreciated that α and β globin gene loci do not achieve their full capacity for
Frameshift mutation in exon 3 of the beta-globin gene, a single nucleotide deletion (-C)
β- thalassemia 3 were heterozygous for α+ thalassemia deletion (-α/αα) while two were
globin gene deletion may cause mild thalassaemia intermedia. Reduced alpha globin
chain output results in a more balanced globin chains synthesis which in turn accounts for
53
the mild clinical phenotype (Galanello et al 1984). simple heterozygosity for the common
beta degrees -thalassemia mutation beta39 (C→T), both presenting with a thalassemia
βo – Thalassemia
In Cypriot, Sardinian and Asian patients that are homozygotes for βo thalassemia with co
single α- globin gene are deletion usually have thalassemia major with slightly late onset
clinical presentation.
β+ - thalassemia
α - globin genes and some times with single α globin gene deletion is associated with
milder clinical phenotype. Inheritance of single α-globin gene deletion and non deletional
rarely occurs in Israel. This reflects a low frequency α - thalassemia in this population.
54
severe phenotypes respectively. Co inheritance of heterozygous state for α+ thalassemia
with homozygousity for IVSI – 110 G→A mutation causes sever form of thalassemia
intermedia.
+ G are associated with extremely mild phenotype (Ho et al 1998). Mutation of the beta-
globin gene initiation codon (ATG→AAG) which should give rise to beta (0)-thalassemia
al 1993).
Reasonable correlation exists between the mildness of phenotype and homozygous βo,
1993).
with HbH disease. Extremely high hemoglobin A2 in these patients as reported may be
55
due to the high levels of δ - chain production, which normally do not reach the peripheral
1.12.11: Pathophysiology
morphological changes of their red cells and almost the same abnormalities of membrane
function showing the same rate of potassium leak but they show different red cell
survival time. Normal red cell survival is demonstrated in individuals with balanced
globin synthesis but those having unbalanced globin synthesis and HbH disease had
shortened red cell survival (Knox – Macauly et al 1972). This is because excess α -
chains are more injurious to erythroid precursors and their progeny than excess β chains.
Homozygous β thalassemia together with HbH genotype may show grossly hypochromic
red cells with severe degree of potassium leak, almost normal red cell survival, mild
Accumulation either of unmatched alpha or beta globin chains in turn causes the
intramedullary and peripheral hemolysis that leads to varying degree of anemia (Yuan et
the clinical course in βO-thalassemia does not correlate with an imbalance between alpha
and gamma chain synthesis in the peripheral blood but is determined by the synthetic
56
ratio in the bone marrow cells where the bulk of hemoglobin synthesis takes place
(Cividalli et al 1978).
Iron overload is an important factor in cellular damage. Studies have shown an enhanced
premature cell removal and anaemia. Membrane-bound free iron significantly correlates
with bound haemichromes suggesting a causal relation.It is how ever poorly related to
serum non-transferrin iron which seems to contribute little to the damage from outside
the cells. Spleen plays an important role in the removal of cells with more membrane iron
(Tavazzi D et al 2001).
Since the only other route of glucose metabolism in erythrocytes is the pentose phosphate
pathway (PPP), these results indicate that PPP is more active in beta-thalassaemia
oxidative state (Ting et al 1994). Red blood cell membranes from patients with beta-
thalassemia major and intermedia had an average of 25% less sialic acid and a 50%
difference being due, in part, to increased oxidative stress (Kahane and Rachmilewitz
1976)
effect on the membrane skeleton than do beta-globin chains (Yuan et al. 1995). Loss of
57
thought to play an important role in the cell pathology. Thus hemichrome binding to band
blood. This premature destruction of the thalassemic RBC could in part be due to a loss
of phospholipid asymmetry, because cells that expose PS are recognized and removed by
likely important, because they could provide a surface for enhancing hemostasis reported,
and mediate the rapid removal of these RBCs from the circulation (Kuypers et al 1998).
Many factors such as iron overload, liver injury, hormonal disturbances and aging affects
lipids and LP pattern in patients with major and intermedia form of beta-thalassaemia
(Papanastasiou et al 1996). It was suggested in a study carried out in France that Anemia
Since beta-TI RBCs show essentially normal levels of [Ca2+]i and normal Ca influx,
their high total Ca content should not be associated with any of the deleterious effects
observed in vitro with increased levels of [Ca2+]i (Bookchin et al 1988). The short
platelet life span in addition to reported increased circulating platelet aggregates and
58
In transfusion-dependent patients, increased values of serum transferrin correspond to
deposition in the tissues and impairment of the vessels of the microcirculatory bed results
in the lower spinal column may cause low back pain (Gouliamos et al 1991, Papavasiliou
Evidence for the existence of a chronic hypercoagulable state observed in patients with
thalassemia intermedia red blood cells may be the major underlying factor giving rise to
platelet and coagulation inhibitor abnormalities in these patients. These alterations were
(PC:Act) were measured. PZ, PC:Ag and PC:Act were significantly lower in thalassemia
major and thalassemia intermedia subjects than in 30 healthy controls (p < 0.001), while
levels but not to PZ levels. PZ and PC levels are reduced in thalassemia but only PC has
an effect on the thalassemia hypercoagulable state (Del Vecchio et al 2007). Lipid profile
in TI patients is not influenced by age, sex, liver injury, hemoglobin or ferritin levels; the
higher erythroid bone marrow activity with the enhanced cholesterol consumption could
(Amendola 2007).
59
1.12.12: Role of Hb F in generating thalassemia Intermedia
and elevated fetal haemoglobin (HbF) production are all associated with beta(0)-TI
(Chang 2001). Variation in the ability to produce HbF in the postnatal period might be a
located in the promoters of A- and beta-globin genes. The -158GgammaT and the (AT)9
(T)5 alleles were found to be associated with increased levels of HbF in beta-thal carriers,
segregating (Knox – Macaulay et al 1973). Detailed studies of parents or relatives did not
CLUSTER
al 1987) 5’ haplotypes Hind, IIE , Hind III Gγ, Hind III Aγ, Hind II ψβ, and Hind II 3’
ψβ related HbF production in thalassemia Intermedia was determined (table 13. 6 p 566).
60
In Asians it was found in 14 out of 28 β - globin chromosomes from thalssemia
Relatively mild clinical course was shown with homozygosity for this haplotype.Where
as in thalassemia major patient’s homozygosity of this haplotype was associated with late
presentation. Absolute level of HbF and heterozygousity and homozygocity for this
and has been associated with increased expression of Gγ gene in Sickle cell anemia or
β thalassemia of diverse origin (Gilman & Huisman 1985; Harano et al 1985; Labie et al
1985a). Thus homozygosity and heterozygosity for Gγ Xmn I (+) polymorphism might
Afro – Asian population and less in Italians. In Sardinia majority of the patients showed
at codon 6 has been associated with this haplotype in this population (Galanello et al
1989). Homozygous state for Gγ Xmn – I (+) site ameliorates the clinical picture of
homozygous β – thalassemia mutation whereas heterozygousity for this state has more
In Asian Indians a strong association of Gγ Xmn-I (+) polymorphism with the βo-
this mutation have a slightly milder disease than other forms of βo thalassemia. Many of
61
them become transfusion dependent only in later life. It is believed that the XmnI (+)
homozygocity for this polymorphism may sometimes be associated with a milder clinical
another region in the β –globin gene cluster that is related to an increased propensity for
fetal hemoglobin production. Phenotypic heterogeneity among patients who carry this
polymorphism is not clear. In sickle cell anemia, different chromosomes carrying Xmn I
site change are associated with widely different levels of fetal hemoglobin production
(Weatherall 2001b). Apart from the deletional and nondeletional forms of HPFH and
intermedia (Weatherall 2001b). Xmn I polymorphism was found in association with this
prevalent mutation and was detected in the homozygous state in majority of the of the
patients homozygous for the IVS-II-1 (G → A) mutation in a study carried out in Iran
(Karimi et al 2002).
C→ T change at position -158 in the promoter of the Gγ gene creates an XmnI cleavage
site (Labie et al 1985b). This substitution causes a high proportion of Gγ chains in HbF.
HbF levels were found to be highest with Xmn – I +/+ individuals. Xmn I +/+ or I
genotype have been associated with increased HbF levels in Yugoslav patients also
(Efremov et al 1987).
62
β –thalassemia Mutations and hemoglobin F production
In rare forms partial or complete deletion of β globin gene involving β – globin gene
promoters may cause elevated HbF level to a degree to produce a mild phenotype.
Severity of beta-thal intermedia and the increased Hb F level are strictly dependent upon
the type of beta-globin gene mutation. No relation is found between Hb F synthesis and
Epo secretion. Mutation Gγ -158 C →T, which is common among patients with beta-thal
intermedia and very rare in thal major patients does not seem, to influence the Hb F
O
1.13: CORFU δβ THALASSEMIA
o
The Corfu delta beta thalassemia clinically resembles thalassemia intermedia. In the
high level of Hb F.β globin gene contains G→A mutation at IVS 1 position 5. Mutation
in the beta globin gene is not the sole cause of the absence of Hb A in Corfu delta beta
zero thalassemia it is more likely that deletion of a 7.2 kilobase (kb) segment containing
part of the delta globin gene and sequences upstream which contains sequences necessary
for the normal activation of the beta globin gene are at fault (Kulozik et al 1988).
Usually high levels of HbA2 and significantly increased levels of HbF in heterozygotes
involve the β – globin – gene promoter region. It is thought that the absence of β –globin
gene promoters causes upregulation of δ and γ gene by freeing certain particular DNA
63
binding proteins which may be rate limiting (Huisman et al 1997). Del 619 pb at 3’ end
encountered in Northern India gives rise to a mosaic of cells with either one or no
called LOH11A, which is located close to the beta-globin locus. Thus, Loss of
γ chain production may be increased in the presence of even very mild β- thalassemia
allele. Fetal hemoglobin production is also increased with mutation on chromosome with
promoter gene mutations tend to have a slightly higher level of fetal hemoglobin than
1.16: HPFH
The most common forms of hereditary persistence of fetal hemoglobin synthesis (HPFH)
and delta betao -thalassemia result from simple deletions of the beta-globin gene cluster
or point mutations in the gamma-globin gene promoters or deletions of 11.5 kb and 1.6
kb or an inversion of 7.6 kb. Larger deletions remove both the delta-and the beta-globin
genes with 3' flanking sequences. While the smaller deletions affect DNA of unknown
beta-globin gene cluster are consistent with a competitive relationship between fetal and
adult globin genes and/or with translocation of enhancer sequences into gamma-globin
64
phenotype with with Hb E IVS1→5 G-C mutation and G insertion between codons 8/9
and the beta (E)-gene have also been illustrated (Fucharoen S et al 2002).
heterocellular HPFH
(Hb F) levels in adult (Stamatoyannopoulos and Grosveld. 2001). HbF can be raised in
heterozygous state either alone or together with β thalassemia the augmentation of fetal
several g/dl (Weatherall 2001b). Genetic determinant(s) of high HbF in the absence of
HPFH is linked to intergenic haplotype T and does not disrupt intergenic transcription
(Papachatzopoulou et al 2006).
phenotype
α - globin genes.
65
2) β-globin gene mutations producing products of unusual properties and giving rise
associated with clinical features and inclusions in the normoblasts and peripheral red cells
Heterozygous forms of β thalassemia are mild while these are more severe. Nonsense or
frameshift mutations that produce truncated β chains upto 72 residues in length are
usually associated with a mild phenotype which produces long unstable products
mRNAs associated with these mutation are not transported to the cytoplasm and hence no
gene product is produced. mRNA with mutations in exon 3 are transported and translated
normally. Lack of helix H expose one of the batches of helix G and also of helix E and
F.This leads to aggregate and hence precipitates of β chain products in the form of
inclusion are present in the progenitors of these patients (Thein et al 1990). Therefore
66
1.19.2: Thalassaemia-like carriers not linked to beta-globin
gene cluster
intermedia or thalassaemia major. By linkage analysis both the silent and the typical beta-
like determinants are not linked to the beta-globin cluster. Sequence analysis of the
hypersensitive site cores of locus control region and of the genes coding for the
reaction were reduced in both types of beta-like carriers. These results indicate the
existence of causative genetic determinants that are not yet molecularly defined. They
most likely, result from either a reduction or loss of function of a gene that codes for
Homozygous Haemoglobin Lepore (Hb Lepore) can also cause thalassemia intermedia
(Pasangna et al 2005).
67
1.21: Hemoglobinopathies
Although over 400 variants of structural haemoglobin have been identified, only,
globin gene that produces a single amino acid substitution in a globin chain.Although
most of them are of limited clinical significance, a few subtypes have been identified
heterozygotes for these variants are typically asymptomatic, their diagnosis may be
reduction in globin chain synthesis. Those with diminished beta-globin chains are termed
beta-thalassemias while those with decreased alpha-chain production are called alpha-
of globin chain produced and the stability of residual chains present in excess. The
thalassemia minor syndromes are characterized clinically by mild anemia with persistent
variably compensated hemolytic anemia that may present with clinical symptoms during
Higgins 2000).
Southeast Asia and the second most prevalent worldwide. However in India, it is
prevalent in Bengal and the north-eastern region, but relatively rare in the rest of the
68
Milder phenotype of HbE/beta-thalassaemia cannot be attributed to co-precipitation of
HbE and excess free alpha-globin chains (Wickramasinghe, Lee et al 1997). Hemoglobin
ranges from 35% to 75%. These patients are generally classified as having thalassemia
intermedia because they have inherited a beta-thalassemia allele and hemoglobin E which
of thalassemia major. Phenotypes of thalassemia major can be predicted from the early
onset of clinical symptoms and the requirement of regular blood transfusion from infancy
Hemoglobin E is a beta chain variant that has its most significant interaction with
dependent (Kakkar 2005). Haemoglobin E beta thalassemia (HbE beta thalassemia) has a
transfusion develop progressive iron loading with age. Serum ferritin and serum alanine
transaminase levels are significantly raised in patients who receive blood transfusions. In
the presence of blood transfusion but with out adequate iron chelation therapy,
69
splenectomy becomes an inevitable procedure at some stage of the disease (George and
Wong 1993)
Since Hb Malay migrates with HbA on electrophoresis and chromatography, this variant
The possible presence of this mutation should also be considered for genetic counseling
in couples at risk (Ma 2000). Hb O-Arab (beta 121 Glu→Lys) and a beta
zero-thalassaemia trait has also been found to be associated with thalassemia Intermedia
Premawardhena1 et al 2004).
mapping and haemoglobin electrophoresis indicate that there are four genotypes
(Fucharoen et al 1988)
1.23: Hb Vicksburg
intermedia in patients who are doubly heterozygous for this variant and
o
beta -thalassemia. Structural analysis of Hb Vicksburg demonstrate deletion of leucine at
beta 75 (E19). Hb Vicksburg is expected to comprise the major portion of the hemolysate
70
in the patients because of the presence of beta 0-thalassemia on the trans chromosome,
but it comprises only 7.6%. Thus, Hb Vicksburg is synthesized at a rate lower than that
expected on the basis of gene dosage. Deletion of beta 75, for a number of reasons, is not
expected to lead to diminished synthesis of the variant. The most plausible explanation
for the low output of Hb Vicksburg is that a mutation for beta +thalassemia is present in
1.24: Pregnancy
Intrauterine growth restriction (IUGR) complicates more than half of the pregnancies
with TI. Red cell transfusion is needed in most cases even in non-transfusion-dependent
2006). How ever in one case pregnancy could not follow its normal course and was
interrupted during the 24th week because of intrauterine death of the fetus due to
thrombosis. Therefore all thalassemic patients should be tested for various blood antigen
relatively straightforward. However, despite the ability to accurately define the beta-
intermedia is variable and complications are more frequent than in the minor form.
71
Classical phenotype of heterozygous beta-thalassemia may be modified by a number of
environmental and genetic interacting factors. Some of these are (1) coinheritance of
alpha-thalassemia which may normalize the red blood cell indices; (2) presence of a mild
the raised HbA2 typical of heterozygous beta-thalassemia and (4) presence of a silent
of HbF in adult life or the presence of heterozygosity for hyperunstable globin variants
but in general any factor that is capable of reducing the globin-chain imbalance results in
a milder form of thalassemia. These factors include the presence of a silent or mild beta-
chain production may cause mild thalassemia. Less frequent mechanisms are double
heterozygosity for beta-thalassemia and triplicated alpha genes, and the presence of a
o
hyperunstable hemoglobin variant. For significant number of β thalassemia
homozygotes with a thalassemia intermedia phenotype the modifying factors have not
been defined. In contrast, there are simple beta-thalassemia carriers who, for unknown
reasons, have an unusually severe clinical phenotype (Galanello and Cao 1998) In a
patients with nondeletion genotype the analysis of hematological values revealed lower
72
levels of RBC as well as HbA2 with significantly higher levels of Hb H. Clinical
enlargement and the necessity for frequent transfusions. The genotype do not justify the
gravity of the phenotype in every case, differences in clinical manifestations are notable
and are not easily explainable in subjects apparently having the same genotype (Mirabile
et al 2000). Individuals heterozygous for beta +33 C-G mutation alone are clinically and
hematologically silent with normal red blood cell indices and normal levels of
hemoglobin (Hb) A2. A direct relationship between genotypic and phenotypic severity is
clearly demonstrated in these cases with obvious implications for prenatal diagnosis (Ho
et al 1996).
Clinical presentation of individuals carrying two or more alpha-globin lesions was highly
variable. In general, the severity correlates inversely with the number of functional alpha-
globin genes. In some cases, impairment of two alpha-globin genes by point mutations
thalassemia (Oron-Karni et al 2000). The severity correlated inversely with the number
73
1.27: Thalassemia Intermedia In India
Pattern of mutations in Uttar Pradesh differed from those in other Indian states and in
families who migrated from Pakistan. Frequency of IVS-I-5 (G→C) and 619 bp deletion
mutations was 64.3 and 2.5% respectively in families from Uttar Pradesh compared to a
Asian Indian mutations, eight were observed in subjects studied from different parts of
India (Agarwal et al 2000). In another study carried out by Verma et al 91.8% of the
subjects had one of the five commonest mutations [IVS-I-5 (G→C), 34.1%; 619-bp
deletion, 21.0%; IVS-I-1 (G→T) 15.8%; codons 8/9 (+G), 12.1%, and codons
41/42 (-CTTT), 8.7%. 5.9% of the subjects had a less common mutation(Verma et al
1997, Agarwa et al 1994) In a study carried out by Panigrahi et al, the possible molecular
basis was (i) co-existent α-deletions (n=16/50), (ii) homozygous XmnI polymorphism
(n=17/50), (iii) both factors (n=3/50), and (iv) milder beta-alleles (n=9/50) in
alpha alpha anti-3.7 triplication was the predominant factor (Panigrahi I et al 2006b).
Hemoglobin E is very common in north-east India with relatively fewer reports from rest
of the country. Reports of hemoglobin E in the Punjabi population are even rarer.
Hemoglobin E is a beta chain variant that has its most clinically significant interaction
with thalassaemia. The compound heterozygous state thus produced can result in a
74
determinant underlying thalassemia intermedia in North Indians. Patients with alpha-
triplication may develop jaundice with marked increase in serum bilirubin following
hemoglobinopathies observed in 1015 cases were sickle cell trait (29.8%), sickle cell
Sickle cell disorders with high level of fetal hemoglobin were common in general castes
etc. Most of the cases belonged to Anugul district, followed by Khurda, Nayagarh,
scheduled castes and tribes that belong to Coastal and South-Western regions of Orissa
(Balgir 2005).
patient (Pande et al 1995). The six severe and common Indian mutations seen in
Thalassemia Intermedia patients are IVS 1-5 (G→C), 619 bp deletion, IVS 1-1 (G→T),
codons 8/9 (+G), codon 15 (G→A), codons 41/42 (-CTTT). Majority of the severe and
mild TI group, IVS 1-1 (G→T), codon 30 (G→C), capsite +1 (A→C), poly A (T→C),
-28(A→G), and -88 (C→T). Four mild mutations in combination with other severe β+ or
75
o
β mutations resulted in a very variable clinical presentation. This indicates that, in
majority of Indian patients beta genotype cannot predict the phenotype (Colah et al
2004). Other mutations found in India are frameshift codon 55 (+A) in Maharashtrans
It was observed that the nature of beta-thalassemia mutations was not very different
between the beta-thalassemia major and beta-thalassemia intermedia groups. How ever
3.7
co-inheritance of one or more alpha-globin gene deletions (-alpha ) and the presence of
the XmnI polymorphism were associated with less severe disease in Indians (Nadkarni et
o,
al 2001). Nine different types were found, of which six were associated with beta one
with severe beta+ and two with mild beta+ thalassaemia. Comparison of the beta-globin
gene cluster haplotypes, alpha globin genotypes and beta gene mutations of the
thalassaemia major group with the thalassaemia intermedia group suggests that the
and the inheritance of a mild beta-thalassaemia mutation are the major ameliorating
beta-globin gene deletion (codons 81-87) with codon 30 (G→C) mutation was also
o
identified in an Indian patient with β -thalassemia (Shaji 2002). A 10329 base pair
deletion which results in the loss of 5' beta promoter region and the entire beta-globin
gene is associated with unusually high levels of Hb A2 in the heterozygous state (Craig et
al 1992). Hb Lepore has also been reported in the Indian population (Shaji et al 2003).
Alpha Thalassemia
alpha globin gene triplication is an important genetic determinant underlying thalassemia
76
alpha alpha alpha anti-3.7 triplication was the predominant factor (Panigrahi I et al
2006b). One of the possible molecular mechanism of the effects caused by Yisui
stabilizing protein (AHSP) and erythroid transcription factor GATA-1 mRNAs, enhance
the protein synthesis of AHSP which can bind the relative excess free alpha-globin,
prevent the formation of alpha -globin-cytotoxic precipitates in red blood cells and
Unstable Hemoglobin
Hemolytic anaemia due to unstable hemoglobins arising from spontaneous mutation has
Hemoglobin E
Hemoglobin E is very common in north-east India (Kakkar 2005). In one study most
common hemoglobinopathies observed amongst 1015 cases were: sickle cell trait
disease (0.2%). Sickle cell disorders with high level of fetal hemoglobin were common in
general castes (0.3-20.7%), scheduled castes (0-8.9%) and scheduled tribals (0-5.5%).
scheduled castes and tribes that belonging to Coastal and South-Western regions of
77
δβ thalassemia
Deletion within the beta globin gene complex starts 3 kilobases from the 3' end of the
Aγ gene. The deletion removes the delta and the beta globin genes and continues to a
variable extent in the 3' direction. Heterozygotes for this deletion have about 25% Hb F
with a G gamma:A gamma ratio of 70:30. Iinteraction with beta+ thalassaemia results in
Seven different mutations have been identified and the A to G substitutions in the TATA
box of beta-globin gene account for 42% of these mutant beta-globin genes. Most
patients have a beta(+) thalassaemia and one copy of TATA box mutation.
0
β thalassaemia intermedia the mild phenotype may be explained either by the presence
of the - + - + + 5' beta-globin gene cluster haplotype which contains the XmnI site -158 nt
mutations together with concurrent (SEA) alpha-thalassemia (SEA) deletion has been
identified in China. A novel mutation of -73(A→T) in the CCAAT box of the beta-
globin gene has been identified in a patient with the mild beta-thalassemia intermedia
(Chen et al 2007).
78
disease (genotype -alpha(3.7)/-(SEA) have also been identified. Patients
Jin Ai et al 2003). In a study carried out in Hong Kong 5.0 percent of the subjects were
carriers of alpha-thalassemia of whom 4.5 percent were carriers of the Southeast Asian
type of deletion in which both alpha-globin genes on the same chromosome 16 are
deleted. 3.4 percent were carriers of either beta-thalassemia or a mutation that codes for
hemoglobin E. 0.3 percent were carriers of both alpha- and beta-thalassemias (Lau et al.
intermedia phenotype with inclusions in erythrocytes has also been identified in Hong
Guangdong Province were highly heterogeneous and its spectrum was different from the
Alpha Thalassemia
thalassemia intermedia" and is due to a double heterozygosity for two deletional forms of
alpha-thal, alpha-thal-1 and alpha-thal-2. Majority of cases with alpha-thal-1 defect have
a deletion of at least 18.1 kb starting 3' to the zeta 1 gene which includes the psi alpha
and the two alpha genes; it is similar to that described in Thais. However deletion of the
entire zeta-alpha gene cluster (zeta-alpha-thal-1) has also been described. In alpha-thal-2
79
defects, the rightward deletion (alpha -3.7 kb, all type I defects) is more common than the
leftward deletion (alpha - 4.2 kb); one of the latter is associated with Hb Q. A few alpha-
thal defects belong to the nondeletion type, the most common being Hb Constant Spring
(CS). This anomaly, when coinherited with alpha-thal-1, produces Hb H-CS disease
which is associated with marked anemia and splenomegaly due to the instability of alpha-
CS chain. Hb Quong Sze produces alpha-thal-2 phenotype because of the unstable alpha-
Quong Sze chain. Classical Hb H disease and Hb New York (NY) [alpha
113(G15)Val→Glu] with severe anemia, requiring frequent blood transfusions due to the
deleterious effect of increased alpha-NY chain turnover has also been described (Chan et
al 1988).
been indentified. The practice of prenatal diagnosis in this case may also provide
Hemoglobin Constant Spring (Hb CS) and Hb Paksé, two abnormal Hbs characterized
by elongated alpha-globin chains resulting from mutations of the termination codon in the
Asia. Hematological findings confirm the mild thalassemia intermedia phenotypes for
80
1.29: Thalassemia Intermedia In Japan
characteristics. Most β-thal patients in Japan are heteorozygotes and have thal minor
phenotype. They are many a times misdiagnosed as iron deficiency anemia. Thirty-four
mutations of β-thal have thus far identified, ten of which accounts for 80% of beta-thal
carriers. Among them 60% are native to Japanese while 40% are probably from abroad.
One exception is homozygosity for -31G-A which leads to thal Intermedia. More than
half of the patients with alpha-thal are of Southeast Asian type, but mutations in the
remaining patients seem to be unique to the patients of Japanese descend. Thus Japanese
thalls have dual origin. Frequency of beta-thal is 1 in 600 to approximately 1 in 1,000 and
that of alpha (+)-thal (- alpha/) is 1 in 400, alpha-thal trait (- alpha/- alpha) is therefore
extremely rare. Another alpha-thal trait (- -/alpha alpha) is one fifth of beta-thal.
Seventeen families of HbH disease (- -/- alpha) were found. Many of them are related to
In one study that was carried out in 100,000 samples 29 contained electrophoretically
Three factors: mild beta-globin gene mutations, deletions in the alpha-globin gene and
the presence of a polymorphism for the enzyme Xmn I in the Ggamma-promoter region
81
have been observed.: 68% of patients have a mild beta+ mutation (IVSI-6, cd29, -88 or
-87), while 26% of patients are positive for the Xmn I polymorphism associated with
various ethnic backgrounds. This hemoglobin was reported in a Thai female patient with
Phenotypes with globin genotypes in patients with HbH disease in northeast Thailand
was studied. It was found that most prevalent molecular defect was interaction of
three alpha-globin genes with the SEA type alpha-thalassemia 1 and the 3.7- or 4.2-kb
deletion of alpha-thalassemia 2 (14 of 52 patients) and the interaction of the SEA alpha-
Constant Spring (Hb CS), homozygous Hb E and alpha degrees -thalassemia found in
two unrelated pregnant Thai women has been reported (Fucharoen et al 2007).
Main hereditary hemoglobin disorders of clinical significance in Brazil are sickle cell
disease and beta-thalassemia. Sickle gene was introduced by the slave trade whereas
82
beta-thal was introduced later due to a massive migration (mostly by the Italians)
between 1870 and 1953 to the southeast Brazil. Molecular studies performed in the
southeast of the country showed a marked prevalence (47 – 54%) of the nonsense
northeast region of the country has a different demographic history characterized by the
insignificant Italian migration. Owing to this and since the majority of cases of beta-thal
in Pernambuco (a state located in the northeast of the country) have mild or intermediate
clinical and laboratory features a different spectrum of beta-thal mutations in this region
is likely as reported by the fact. 33.3% had beta-thal intermedia, 33.3% had HbS/beta-thal
and 23% were beta-thal trait individuals. IVS-I-6 (T →C) 62.8%, IVS-I-1 (G →A)
codon 30 (AGG → AGC) 1.1% are amongst the common mutation (Araujo et al 2003).
Although there is not always a complete correlation of genotype with clinical phenotype,
the inheritance of two mild beta-thalassemia alleles results in almost all cases (11 of 12
another study carried out in Greek patient two mutations, IVS-I-1 (G→A) and a C→G
mutation at position 6 3' to the terminating codon (term + 6) were identified suggesting
that C→G mutation in this untranslated region of the beta-globin gene causes a slight
decrease in the stability of the mRNA which becomes clinically important only in
83
Homozygous beta ++ has also been found to be associated with thalassemia Intermedia
(Kattamis et al 1982).
the country with more than 50% of the population. The state of beta-thalassemia is
alarming as consanguinity rate is very high (>81%) and the literacy rate is low in South
Punjab. A thalassemia prevention program is the need of the hour in this part of Pakistan
1995). Two forms of hypochromic microcytic anaemia i.e. iron deficiency and
beta-thalassaemia trait are common in Pakistan. Iron deficiency was found in 9% while
beta-thalassaemia was seen in 3% in the study carried out by Afroz et al (1998). Ratio
reliable and sensitive index which can be used for mass screening of beta thalassaemia
1995). Thalassemia trait is found in 11% of the Pakistani population (Molla et al 1992).
Khattak MF and Saleem M. reported the incidence to be 5.4%. In Pathans the incidence
was 7.96% while in Punjabis it was 3.26% prevalence rate (Khattak and Saleem 1992)
most frequent mutations were IVS-1 position 5 (G-C), codons 8/9 (+G), IVS-1 position 1
(G-T), codons 41/42 (-CTTT) and the 619 bp deletion at the 3' end of the gene. Mutations
at IVS-2 position 1 (G-A) and codon 30 (G-C), previously were described in Asian
84
Indians in 1991 (Varawalla et al 1991). Two other mutations IVS-1 nt.5 (G-C) and codon
Five most common mutations identified in Pakistan are IVS1-5 (G-C), IVS1-1 (G-T), Fr
41-42 (-TTCT) Fr 8-9 (+G) and deletion 619 bp (Khateeb et al 2000). Molecular basis of
beta-thalassemia in Thailand, Pakistan, Sri Lanka, Mauritius, Syria, and India, has been
studied. The results confirm and extend earlier findings for Thailand, Pakistan, India,
Mauritius and Syria. Two novel mutations were identified, codon 55 (-A) and IVS-I-129
(A→C), both found in Sri Lankan patients. Two beta-thalassemia mutations were found
to coexist in one beta-globin gene: Sri Lankan patients homozygous for the betao codon
16 (-C) frameshift were also homozygous for the beta+ codon 10 (C →A) mutation.
Studies of Sri Lankan, Pakistani, and Indian carriers suggest that codon 10 (C →A)
o
mutation is a rare polymorphism on an ancestral allele in which beta codon 16 (-C)
mutation has arisen. Each country was found to have only a few common mutations
accounting for 70% or more of the beta-thalassemia alleles (Old et al 2001). For
β - thalassemia a gene over 4000 homozygotes are born each year in Pakistan.
β-thalassaemia alleles from five major ethnic groups of Pakistan have been
ethnic and regional differences in the prevalence of mutations. The five most common
mutations, IVSI-5 (G-C) (37.3%), Fr 8-9 (+G) (25.9%), del 619 (7.0%), Fr 41-42 (-
TTCT) (6.7%) and IVSI-1 (G-T) (5.4%), constitute 82.3% of the total thalassemia
population in Pakistan. Fr 8-9 (+G) is the most common mutation in Northern Pakistan
(41.3%), whereas IVSI-5 (G-C) is the most frequent mutation in Southern Pakistan
85
1997). The IVS-I-5 (G→C) Asian Indian mutation was the most frequent mutation
reported from United Arab Emirates (el-Kalla and Mathews 1993) Determination of
beta-globin gene haplotypes in North-west Pakistan, Gujarat, Punjab and Sindh suggest
that high frequency alleles i.e. intervening sequence 1 (IVS-1) nucleotide 5 (G-C) and
beta-thalassaemia in this region reflects considerable ethnic diversity, gene flow from
all ethnic groups are identified. The four most common mutations, IVS-I-5 (G→C)
(37.7%), codons 8/9 (+G) (21.1%), 619 bp deletion (12.4%), and IVS-I-1 (G→T) (9.5%),
account for 80.7% of the alleles. There are differences between ethnic groups and also
between provinces. Amongst the four provinces of Pakistan, IVS-I-5 (G→C) mutation is
more prevalent in Sindh and Balochistan, bordering India in the south and Iran in the
southwest, while codons 8/9 (+G) mutation is more common in Punjab and the North
West Frontier Province, bordering India in the northeast and Afghanistan, respectively.
619 bp deletion is (46%) in Gujratis and Memons residing in the Province of Sindh,
neighboring the Indian Gujrat (Khan and Riazuddin. 1998). β-thalassemia mutation
codon 45(-T) has also been identified in Pakistan (El-Kalla and Mathews 1997).
Population and genetic studies suggest the origin for the Indian deletion beta thalassaemia
(600 bp deletion involving the 3' end) in people from Sindh and the adjacent area of
86
Alpha-Thalassaemia
Frequency of –alpha (3.7) allele was found to be 8.3%. Ethnic differences were
statistically significant for Pashtoons vs. Balochis and Pashtoons vs. Sindhis. In this
group, 24.6% of the patients had one or two alpha genes deleted. Prevalence –alpha (4.2)
was found to be 0.2% while that of alpha alpha alpha (anti3.7) allele was 0.9%.
The –alpha (4.2) allele was found only in Sindhis, while alpha alpha alpha (anti3.7) was
present in Punjabis, Sindhis and Balochis. 22.4% of the patients were with triplicated
electrophoresis for Hb Bart's in cord blood is a very simple method of determining out
population was found carrier of alpha-thalassaemia gene of these. 75% had alpha-
1991). Prevalence and molecular basis of alpha thalassaemia in the British South Asian
population was determined. Of the 266 subjects in whom gene mapping was performed,
28 had a single alpha+ thalassaemia deletion and one was homozygous for this deletion
(gene frequency 0.056). Half of the heterozygotes had normal mean cell haemoglobin
(MCH) values. 16 subjects had probable non-deletional alpha+ thalassaemia none had
Mediterranean region and Asia are caused by defective mRNA synthesis, processing, or
87
A rare alpha2-globin chain variant, Hb Sallanches [alpha104(G11) Cys→Tyr], in a
disease were identified. This variant, previously reported in a French patient and a West
δβ-Thalassaemia
very mild its combination with beta-thalassaemia trait can produce a sever transfusion
dependent thalassaemia. DNA based diagnosis is possible in the prenatal as well as the
patients were characterized. In the study all heterozygotes and 4/6 homozygotes were
transfusion dependent anaemia. Mean Hb, TRBC, MCV, MCH, Hb-F and Hb-A2 in delta
beta-thalassaemia heterozygotes were 11.6 g/dl, 5.37 x 1012/L, 70.9 fl, and 21.7 pg, 14%
and 2.6% respectively. Similar values in the four untransfused homozygotes were 10.6
g/dl, 5.34 x 1012/L, 69.2 fl, and 20.8 pg, 100% and 0% respectively. Mutation analysis
revealed that all 13 individuals had the same Inv/Del (G)gamma(Agammadelta beta)
XmnI Ggamma-polymorphism
The XmnI Ggamma-polymorphism (C-T polymorphism at position -158 to the Ggamma-
globin gene) was studied in 13 individuals from six unrelated Pakistani families with
o
delta beta-thalassemia. All subjects had the Asian-Indian Inv/Del (GγAγδβ) that
88
included six heterozygotes, six homozygotes, and one compound heterozygote of delta
beta- and beta-thalassemia. All seven delta beta-thalassemia heterozygotes (including one
compound heterozygote) had -/+ genotype, all six homozygotes had +/+ genotype. The
results strongly suggest a tight linkage between the XmnI Ggamma-polymorphism and
o
the Asian-Indian Inv/Del (GγAγδβ) degrees. This finding could explain the unusually
Hemoglobinopathies
Hemoglobin D-Iran (Hb D-Iran, beta 22 Glu→Gln) is a beta-chain variant that was first
benign condition. Hb D-Iran has also been described in combination with sickle
hemoglobin and beta thalassemia, but never as a homozygous mutation. One case of
values, hemoglobin electrophoresis, peripheral blood smear, and clinical course suggest
that homozygous Hb D-Iran is a relatively benign condition with mild microcytic anemia,
2 beta 3 121 (glutamine→glycine) and thalassemia trait has also been detected in a
1.35: Treatment
89
seems to be an effective and safe therapeutic option for exudative effusions, while
increased erythropoiesis (Aessopos 2006a). HU appears suitable for the treatment of leg
several efforts have been undertaken to determine the efficacy of hydroxyurea treatment.
The results indicated that erythroid progenitor cells treated with 30 mumol/l hydroxyurea
2006). In addition to acting in synergy with the XmnI polymorphism, alpha deletions
well as for TM patients in countries that have limited blood supplies (Bradai M et al
2007)
The treatment of thalassemia mainly includes blood transfusion which has its own
however have a potentiating or permissive role. Strong association of transfused iron load
and decreased thyroid function stresses the need for intensive chelation therapy (Jain et al
1995). It was observed that Pakistani thalassaemic patients are significantly iron
overcome this problem iron chelaters were used. In a study carried out at Armed forces
institute of Pathology, Rawalpindi the efficacy and adverse effects of Deferiprone (DFP,
90
hydroxypyrid-4-one) is a bidentate oral iron chelater that binds iron in a 3:1 ratio. It also
was concluded that DFP was well tolerated and caused fewer side effects. It had much
better patient compliance and was effective in lowering serum ferritin level in previously
most poorly chelated patients. It also has the potential advantage of economy and
resonance (CMR) in thalassemia major (TM) and thalassemia intermedia (TI) patients
and of electrocardiogram (ECG) changes associated with TM, have been carried out.
Similarly, the segment-dependent correction map of the T2* values and the artifactual
variations in normal subjects and the T2* correction map to correct segmental
measurements in patients with different levels of myocardial iron burden have been
Exposure to hepatitis C virus (HCV) and its effect on ALT levels was studied in
transfusion dependent cases of thalassaemia major. 60% cases were anti HCV positive
and also showed raised Alanine Transaminase (ALT) levels. Of 14 anti HCV negative,
Hepatitis B Surface Antigen (HBs Ag) negative patients, seven showed raised ALT,
indicating the possible chances of acute viraemia (Bhatti et al 1995). Another hazard of
transfusion is the Red cell immunization. Red cell alloantibodies were detected in 4.97%
patients, and belonged mainly to Rh system, with one example each of anti-K, anti-Jsb
low in our setup and may be related to red cell homogeneity between the donor and
recipient population. Routine pre-transfusion matching of blood, other than ABO and Rh
91
"D" antigens is not recommended because of low rate of red cell alloimmunization, and
high costs associated with such testing. Hyperhaemolysis, due to acquired red cell
complication should be tested for presence of underlying antibodies and considered for
be very low but may play a role in HCV spread by infected persons (Akhtar et al 2004).
HCV genotype 3a and 3b were found in 89% and 11% respectively (Akhtar et al 2002).
was found in 21%, however no causal relationship between HGV and hepatitis was
Stem cell transplantation facility became available in Pakistan in 1999. Since then both
allogeneic and autologous procedures have been carried out for severe aplastic anaemia,
blood stem cell transplantation is also feasible and life saving in otherwise fatal disorders.
This could be carried out effectively in Pakistan (Farzana et al 2003). Allogeneic BMT is
the only curative therapy for beta-Thalassaemia patients. Success rate can be increased if
patients are selected carefully and transplanted at an early age (Hashmi et al 2004). GHD
Acute GvHD developed in 68% of the patients with in beta-Thalassaemia who underwent
allogeneic stem cell transplantation. Morbidity and mortality due to severe acute and
chronic GvHD remains high despite standard prophylaxis against GvHD. New strategies
92
are needed to prevent and treat GvHD (Hashmi et al 2005). In developing countries
where consanguinous marriages are common gene variants are trapped within extended
In one of the studies carried out in such families, 31 percent of the persons with an index
case were carriers; carriers had a 25 percent risk of producing an affected child. 8 percent
of the married couples consisted of two carriers. Carriers married to carriers with two or
more healthy children avoided further pregnancy and most such couples with one or no
marriages among British Pakistanis was found to be 55 percent (Darr and Modell 1988).
Therapy of thalassemia has in the past been confined to transfusion and chelation. In
order to develop an objective test for discriminating between patients with thalassaemia
intermedia requiring blood transfusion and those not likely to require transfusion, the
medullary width (MW) in the midpoint of the second left metacarpal and the bone mass
were measured. Changes were visible radiologically and was concluded, therefore, the
patients requiring or not requiring blood transfusions. It was also observed that bone
deformities were reversible if transfusions were instituted using MW(greater than 0.5 cm)
regardless of the age or the haemoglobin concentration. This test may help clinicians to
decide about the optimal time for institution of regular transfusion in patients with
93
transfused patients (Tx) and to identify the factors that affect QOL in thalassemia. The
most commonly reported affected domains were feelings such as anxiety, depression, and
Recently, novel modes of therapy have been developed for thalassemia based on the
pathophysiology and molecular pathology of the disease, both of which have been
butyrates, hemin). Most of the newer therapies are suitable primarily for thalassemia
intermedia patients (Rund and Rachmilewitz 2000, Hoppe et al 1999, Bachir and
Galacteros 1994 and Karimi et al 2006). Therapeutic approaches for homozygous beta-
thalassemia entail blood transfusions and iron chelation therapy with deferoxamine or
deferiprone for preventing tissue hemosiderosis. Multiple transfusions may modulate the
response of serum EPO to the degree of anemia, resulting in increased EPO levels and
deferoxamine subcutaneously once or twice a day seems to be the most practical method
Montalembert et al 1989). Recently, much effort has been focused on various inducers of
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fetal hemoglobin (HbF) such as recombinant human erythropoietin (rHuEPO), especially
patients. Although a slight increase in HbF levels was observed, other possible
transfer in hematopoietic stem cells effectively treats a severe hemoglobin disorder (May
combination with iron and folic acid may ameliorate blood indices as an alternative
treatment for anaemia in beta-thalassemia intermedia but long term randomized trials are
needed especially for patients with beta thalassemia major (Nisli et al 1996). Increasing
ameliorate the severity of the disease by improving the balance in globin chain synthesis.
Hydroxyurea, as an effective agent with low toxicity for activating gamma-globin gene,
countries where supply of blood and chelating agents are limited (Bradai et al 2003, de
alternative to transfusions for TI patients as well as for TM patients in countries that have
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Butyrate derivatives can stimulate fetal hemoglobin in patients with intermediate
Mg levels have been reported in human beta thalassemia. These deficiencies may play a
Fetal hemoglobin switching agents have been proposed to treat genetic blood disorders,
such as sickle cell anemia and beta-thalassemia, in an effort to compensate for the
dysfunctional form of the beta-globin chain in adult hemoglobin. The rationale behind
this approach is to pair the excess normal alpha-globin chain with the alternative fetal
gamma-chain to promote red blood cell survival and ameliorate the anemia.
inclusion of monoclonal antibody CR3/43 has been described. This form of retrograde
variety of stem cell markers in a heterogeneous population of white blood cells. This
recapitulate early hematopoiesis and facilitate expression of a fetal and/or adult program
This novel clinical procedure may profoundly modify the devastating course of many
genetic disorders in an autologous setting, thus paving the way to harnessing pluripotency
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1.36: Gene therapy in Thalassemia Intermedia
Gene therapy approaches to these disorders envision stem cell targeted gene transfer,
corrective globin gene expression in developing erythroid cells has been adopted.
Lentiviral vector systems potentially appear to afford adequately efficient gene transfer
into stem cells and are capable, with appropriate genetic engineering, of transferring a
globin gene with the regulatory elements required to achieve high-level, erythroid-
specific expression. Results obtained in use of lentiviral vectors to insert a γ-globin gene
into murine stem cells with phenotypic correction of the thalassemia phenotype are
uncertain and suggest evaluation of the risk of gene therapy strategies for the treatment of
Gene transfer for beta-thalassemia requires gene transfer into hematopoietic stem cells
using integrating vectors that direct regulated expression of beta globin at therapeutic
levels. Among integrating vectors, oncoretroviral vectors carrying the human beta-globin
gene and portions of the locus control region (LCR) have suffered from problems of
vector instability, low titers and variable expression. In recent studies, human
intermedia in mice (Malik & Arumugam 2005). Thus lentiviral vectors are very
promising drugs for the treatment of β-thalassemia intermedia and β-thalassemia major
(Stefano et al 2003).
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1.37: Prenatal diagnosis
groups when making decisions about prenatal genetic screening, prenatal diagnosis and
factor in the decision-making process, but the perceived severity of the condition would
play a more important role (Ahmed et al 2006c) Two renowned Islamic scholars ruled
that a pregnancy could be terminated if the fetus was affected by a serious genetic
disorder, and if termination was done before 120 days (17 weeks) of gestation. Prenatal
diagnosis of beta-thalassaemia was introduced in Pakistan in May 1994. During the first 3
½ years of the service 300 couples availed the test. This study demonstrates that prenatal
pregnancy are influenced by various factors, and therefore their religion should not be
taken as a proxy for their attitudes either for or against termination of pregnancy (Ahmed
et al 2006a)
1.38: Diagnosis
curative treatment available for this disease is either blood transfusion and chelation or
prevent the birth of an affected child by Carrier testing and prenatal diagnosis. Carrier
98
provides the prospective parents with information about whether their children could
inherit a genetic disorder so that they could consider reproductive alternatives. It can be
detection is based on the full blood counts and Hb electrophoresis and estimation of Hb
the most effective and direct approaches for prevention of thalassemia. The object of
prenatal diagnosis is to provide an accurate and rapid result as early in the pregnancy as
analysis on PCR-amplified DNA from chorionic villi sampling, amniocentesis and fetal
cordocentesis.
α+-thalassemias are characterized by red cells which are often not microcytic,
HbA2 and HbF levels are always normal therefore reliable diagnosis can only be
achieved by DNA analysis.
Silent β-thalassemias are characterized by normal MCV, MCH, HbA2, HbF,and total
haemoglobin, and is solely defined by the slight imbalance in the α/β-globin
synthesis ratios.(Table 1.5 and 1.6 and 1.7)
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Table 1.5: Laboratory parameters of alpha , Beta and delta beta thalassemia
α–thalassemia (αº-
β–thalassemia δβ–thalassemia
Parameters thal)
(Heterozygous)
α/β-globin biosynthetic
0.7 1.5 – 2.5 1.5
ratios
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Table 1.6: Clinical and Hematological features of the β- Thalassemia Intermedia Syndromes (Richard et al 1993)
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Table 1.7 : Hemoglobin fractions in the genotypic variants of β - Thaalassemia
syndromes. (Richard et al 1993)
Normal
β/β 97 2.5 – 3.2 <1 None
Thalassemia major
β0/β0 0 1.0 – 5.9 >94 Free α - chain
β+/β+, Mediterranean Present 2.4 – 8.7 20 - 90 Free α - chain
β0/β+ Present 0.6 – 3.4 >75 None
lepore lepore
(δβ) /(δβ) 0 0 70 - 92 Hb Lepore (8 – 30 %)
Thalassemia Intermedia
β+/β+, Black Present 5.4 – 10.0 30 - 73 None
β0/(δβ)0 0 0.3 – 2.4 60 - 99 None
β+/(δβ)0 20 - 30 Present Decreased None
Lepore
β0/(δβ) 0 Decreased Increased Hb Lepore (10%)
Lepore
β+/(δβ) Present Decreased Increased Hb Lepore (10%)
β0/β Present > 3.2 1.5 - 12 None
(δβ)0/(δβ)0 0 0 100 None
lepore
(δβ)0/(δβ) 0 0 92 Hb Lepore (8%0)
Normal or
α/β Present Increased ± HbH
↑ed
Thalassemia Minor
β+/β > 90 3.5 – 8.0 1-2 None
β0/β >90 3.5 – 8.0 1-2 None
(δβ)0/ β < 90 2.5 – 3.0 5 - 20 None
(δβ) lepore / β Present 1.2 – 2.6 1-3 Hb Lepore(5 – 15%)
(γδβ)0/β Present 2.5 – 3.2 < 1 -2 None
Thalassemia Minima
βsilent / β 97 < 3.2 <1 None
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