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1. INTRODUCTION 1.1: Structure of Hemoglobin

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

and Sharma 2001).

1.2: Globin gene clusters:

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.

1.3: α globin genes cluster:

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

the telomere (Flint et al 1997)(Fig.1.1).

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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).

1.4: Normal Structural Variation

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

and Higgs et al 1993).

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

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gene, cluster restriction fragment length polymorphisim is produced by a large number of

single base – polymorphic sites (Higgs et al 1986)

1.5: β – globin gene cluster

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

consist of the embryonic ε gene (Baralle et al 1980), duplicated γ – globin gene

(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

and Clegg 2001a) it contains four elements, HS1 to HS 4.

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

single – point RFLP (Jaffrey 1987 and Antonarakis et al 1982).

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Globin gene structure, function and regulation

Globin gene contains three trans regions called the exons which are separated by two

introns or intervening sequences (IVS) of variable length. Transcription begins at Cap

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

synthesis (Dierks 1983).

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

and terminates gene transcription (Richard, L.et al 1993).

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

between the exons and introns which are

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( ) AG/ GT ( ) AGT at the 5’ end and ( ) N ( ) AG /G at the 3’ end of intron. GT and

AG dinucleotides are maintained in all cases, mutation in these sequences frequently

leads to thalassemia as reported by (Weatherall 2001b).

1.6: Globin gene expression

Hemoglobin synthesis is heterogeneous at all stages of development. It is confined to

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)

(Peschle et al 1985). α - globin gene expression remains constant throughout life.

Whereas the protein products of α1 and α2 genes are identical, however the steady state

level of α2 – mRNA predominates over the α1 – mRNA by approximately 3:1 (Liebhaber

et al 1986). β- gene is expressed in yolk sac cells and stays at a steady level throughout

development. Presence of a β gene downstream may be important in suppressing γ gene

expression (Behringer et al 1990 and Enver et al 1990). γ gene expression is

incompletely switched off in adults (Lloyd et al . 1992; Roberets et al 1997a and

Stamatoyanopoulos et al 1997). A sindicated in a study CH haplotypes are useful genetic

determinants for beta-thalassemia major and intermedia patients, while the 3'HS1 (+179

C →T) mutation may have functional consequences in gamma-globin genes expression

(Papachatzopoulou et al 2007). It has been reported recently that cAMP-dependent

pathway, the activity of which is augmented by multiple cytokines, plays a role in

regulating HBG expression in beta-thalassaemia (Bailey et al 2007).

C T

AG

CT

CT

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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,

position independent expression of like globin genes (Grosveld et al 1987). This

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

interaction with a common LCR.

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,

AAUAAA is conserved in the 3’untranslated region of the RNA approximately10 – 30

nucleotides upstream of where the initial transcript is cut and the poly (A) additional tract

is added. In only θ genes this signal sequence is AGUAA.

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1.8: Regulation of globin – gene function

Regulatory sequence for the globin genes include the promoters series of enhancer

elements on a master regulatory region

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

Weisman1984). Low levels of transcription of the δ – globin is partially due to the

modification of CCAAT sequence to CCAAC. Many erythroid specific genes have in

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

promoters of δ and α - genes (Donz et al 1996).

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

transcription (Fig: 1.2).

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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

of Aγ enhancers loss is observed (Liu et al 1998) while deletion of enhancer to 3’ to gene

significantly reduces the expression of the β gene in this system (Liu et al 1997). A 30 to

100 kb 3’ to the β cluster, additional enhancer sequence are identified 3’ deletion of 3’

breakpoint causes Hereditary Persistence of Fetal Hemoglobin (Feing E.A & Forget

1989, Anagnou et al 1995)

1.8.3: β - globin locus control region

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

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GATA – 1, FOG (Friend of GATA – 1), NF – E 2, EKLF SSP (Weatherall 2001b)

(Fig: 1.2).

1.8.5: RNA processing

Primary RNA transcription of globin gene has a half life of about 5 minutes and requires

further processing including capping and sequencing (Fig: 1.2).

1.8.6: Capping

Addition of 7 – methylguanosine residue at 5’ end of mRNA is known as capping. It

prevents the exonneucleulytic degradation of nascent transcript and in ribosomal binding

to mRNA during translation (Weatherall 2001b).

1.8.7: Poly(A) addition

3’ end of the primary transcript is cleaved at a specific point that is usually 10 – 25

nucleotides downstream of a highly conserved AAVAA motif. This is followed by the

addition of 200 – 300bp long tract of poly (A) residue. Addition of Poly (A) ensures the

stability of mRNA (Weatherall 2001b).

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

(Collins and Weisman 1984) (Fig: 1.2).

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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

a nucleophilic attack by 2’OH group on an A residue 10 – 60 nucleotides upstream of 3’

acceptor site. This forms a 2’ – 5’ phosphodiester bond to produce a ‘lariant’ structure

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

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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)

Codons (Hoffbrand A.2005).

1.8.10: Switching over of globin gene

Developmental hemoglobin switching involves sequential globin gene activations and

repressions that are incompletely understood. As an adaptation of changing oxygen

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

insights on the control of gene loci by remote regulatory elements (Stamatoyannopoulos.

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

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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

and developmental – specific expression of the individual globin genes is governed by

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

tissue-restricted and ubiquitous transcription factors. Sever β - thalassemia usually

manifest as a result of the decline in the synthesis of fetal hemoglobin (α2γ2) during the

first year of life (Olivieri,1999).As the developmental expression is thought to rely on

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

genes are autonomously silenced at the appropriate developmental stage, expression of

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

globin genes is complex, involving chromatin remodeling as well as interactions among

multiple trans-acting factors. These include GATA-1, NF-E2, SSP/NF-E4, erythroid

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

EKLF activates a contransfected β-globin promoter but is unable to activate the

endogenous, chromosomally located β-globin gene (Robert et al 2001).

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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 &

Bradford 1932). Originally called thalassemic anemia, it was abbreviated to thalassemia

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 –

greppi – Micheli, or haemolytic jaundice with decreased red-cell fragility. By 1949 it

became apparent that thalassemia was not a single disease but a complex syndrome

characterized by wide phenotypic diversity. First genetic evidence of Cooley’s anemia

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).

Inherited disorders of haemoglobin are the commonest monogenic diseases. It is

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

thalassaemias (Weatherall 2000a).

1.10: Thalassemias:

Thalassemia are defined as a group of inherited hematological disorders characterized by

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

other polypeptide chains resulting by decreased intracellular hemoglobin content

(hypochromia) and small size of red cells (microcytosis). Because of continued normal

production of unaffected globin chain the imbalanced globin – chain synthesis leads to

the accumulation of unstable aggregates of these unpaired globin chains leading to

oxidative membrane damage and premature destruction of erythrcytes in the peripheral

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

the red blood cells.

1.11: Genetic classification

Depending on the type of the effected globin chain, thalassemias can be classified into

α-,β-, γδβ thalssemia. (Table -1).

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

production is particularly mild (Weatherall 2001b).

1.11.1.1: Molecular Pathology of β- Thalassemia

Majority of β – thalassemias are caused by point mutations. Almost 200 β – thalassemia

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

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have its own set of common mutations (Kazazian et al, 1990). These mutations affect the

gene expression by a variety of mechanisms (Table1.1).

Table -1.1: Thalassemias and related disorders (Weatherall 2001b). α- thalassemia

αo

α+

Deletion (-α ) Non deletion (-αT)

β thalassemia βo β+ Normal Hb A2 Type 1 (Silent )

Type 2 δβ thalassemia (δβ)+ (δβ)o

(Aγδβ)o γ Thalassemia δ thalassemia δo δ+ εγδβ Thalassemia HPFH Deletion (δβ)o, (A γδβ)o Non deletion Linked to β – globin genes Gγβ+, A γ β+ Unlinked to β – globin genes

1.11.1.2: Deletion restricted to the 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

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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

beta thalassaemia intermedia is caused by a new single nucleotide deletion in exon 2 of

the beta globin gene: Hb Morgantown (beta91 CTG→CG) (Luo et al 2005).

1.11.1.3: Mutations affecting β – globin gene transcription

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

polymerase and lower the level of mRNA to 10 to 25 % of normal; this is compatible

with relatively mild phenotype of β+ thalassemia (Treisman et al 1983)

1.11.1.4: Transcriptional mutations:

Several different base substitutions have been found that involve the conserved

sequences upstream from β – globin gene (Weatherall 2000 and Huisman et al 1997).

Despite of considerable variability in the clinical severity associated with mutations of

this type in, the phenotype is β+ thalassemia.

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

one of the promoter elements is characterized by “Silent” β thalassemia (Gonzalez –

Redando et al 1989). A→ C substitution at the CAP site (+1) even in homozygous state

may show the clinical features of β – thalassemia trait (Wong et al 1987).

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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

in their associated phenotypes (Orkin et al 1982). C → T substitution at position 5 of

IVS – I produces abnormal spliced RNA and causes sever β+ - thalassemia phenotype

(Orkin et al 1982) T→ C substitution at position 6, commonly found in

Mediterranean region produces a very mild form of β+ thalassemia (Tamgagnini

et al 1983) G- C substitution at position 5 has been found in Melanesia and causes

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

phenotype effects. G →A substitution at position 110 of IVS – I is the most

common form of β – thalassemia in Mediterranean region. It causes 10 percent

slicing and results in sever β+ thalassemia (Spiritz et al 1980, Busslinger et al

1981). Mutation at 116 in IVS -I produces a new acceptor site and causes βo

thalassemia phenotype (Weatherall et.al. 1985). Activation of donor sites within

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

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mutations can activate this site so that it is utilized during RNA processing within

production of abnormal mRNA (Weatherall et al 1985).

Amino acid substitution like A→ G in codon 19, G → A in codon 26, and G→T in codon

27 mutations produces hemoglobins Malay, Hemoglobin E and Knossos. At least 38

mutations result in abnormal RNA splicing and cause thalassemia ranging in severity

from β+ to βo – thalassemia (Baysal and Carver 1995). Some mutations involve poly

adenylation signal site AAUAAAA in the 3’ untranslated region of β – globin mRNA

(Orkins et al 1985) like T→ C substitution in this sequence leads to reduction in the

transcription length of normal β -globin mRNA and result in sever β+ thalassemia

phenotype (Orkin et al 1985).

1.11.1.6: Mutations causing Abnormal Translation of Messenger

RNA

Chain termination mutation

Some substitutions of the base change an amino acid codon into nonsense codon and

result in termination of chain and prevent translation of mRNA resulting in βo

thalassemia. Many such mutations have been described (Huisman 1997). These include

codon 36 mutation which is common in the Mediterranean region (Treeartin et al 1981,

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

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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

codons 41 and 42 are common Asian Indian mutations (Kazazian et al 1984).

Combination of frameshift 41/42 and βo-thalassemia or Hb E produced mild to moderate

symptoms with thalassemia intermedia phenotype and severe symptoms with thalassemia

major phenotype (Laosombat et al 2001).

Unstable β - globin variants

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

rise to a blood dyscrasia ranging from dominantly inherited β – thalassemia to a

hemolytic anemia. Unstable hemoglobin may produce thalassaemia intermedia phenotype

(Dash et al 2006).

Hemoglobinopathies caused by unstable beta-chain variants have a dominant

thalassemia-like phenotype in which carriers have the clinical expression of thalassemia

Intermedia. Highly unstable alpha-globin variants, on the other hand become

phenotypically apparent only when they interact with other alpha-thalassemia mutations

(Traeger-Synodinos et al 2000).

Thalassemia syndromes and unstable hemoglobins traditionally represent two

phenotypically separate disorders of hemoglobin molecule. Highly unstable hemoglobin

variants, often have phenotypic characteristics associated with ineffective erythropoiesis

(thalassemias) as well as peripheral hemolysis (unstable hemoglobins). Many highly

unstable beta chain variants cause a dominant thalassemia-like phenotype in which

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heterozygotes for such mutations have a clinical expression similar to thalassemia

intermedia. Phenotypic expression of highly unstable alpha-globin variants is usually less

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

classified as nondeletional alpha-thalassemia determinants (Traeger-Synodinos et al

1999). Heterozygous genotype for a novel 6 bp (TGGTCT) deletion of beta-globin gene

involves codons 33-35. This deletion results in the removal of two valine residues from

beta-globin chain at position 33/34.

(B15/B16) and the substitution of the tyrosine residue at position 35 (C1) by an aspartic

acid (beta 33-35 [B15-C1] Val-Val-Tyr→0-0-Asp). This abnormal haemoglobin is

called Hb Dresden and is found to be exquisitely unstable.

Mediterranean beta-thalassemia (thal) mutation, IVS-I-110 (G→A), in trans position to a

beta-globin gene mutation at codon 107 (GGC→>GAC), gives rise to a rare unstable

beta chain variant Hb Lulu Island or beta107 (G9) Gly→Asp (Papassotiriou I et al

2006).

Silent β thalassemia

A number of extremely mild β – thalassemia alleles are either silent or almost

unidentifiable in heterozygotes. Some involve the region of the promoter boxes of

β – globin genes, others involve the CAP sites or the 5’ or 3’ untranslated regions

(Weatherall 2000b, Huisman 1997).

β-thalassaemia heterozygotes with C → T substitution at nucleotide position -101 from

the Cap site, in the distal CACCC box of the beta-globin gene promoter is the most

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common silent beta-thalassaemia mutation in the Mediterranean population

(Maragoudaki et al 1999). These alleles usually cause thalassemia Intermedia.

Dominantly inherited β thalassemia

Mutations inherited in the dominant fashion causing symptomatic β – thalassemia have

been identified (Thein 1992). Many of them involve exon III of β globin gene and include

frame shift premature termination mutation, and complex rearrangements leading to

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

β – globin chain (Kazazian et al 1986). In the heterozygous state dominant thalassemia

mutations form hyper – unstable haemoglobin variants that precipitate in the erythroid

cells and cause thalassemia Intermedia (Thein 1992).

β – Thalassemia due to unknown mutation

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)

and polyadenylation site (AATAAA→AAAAAA)] cause thalassemia intermedia in a

Tunisian patient (Jacquette A et al 2004).

Variant forms of β thalassemia

Several forms of β – thalassemia in heterozygous state with normal Hemoglobin A2 are

identified (Weatherall et al 2000b). Some of them are due to “silent” β – thalassemia

alleles others reflect the coinheritance of β and δ Thalassemia gene.

22

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

thalassemia is a rare form of δβ thalassemia in the Sardinian population. Homozygous

state for nondeletion δβº thalassemia, which produced a symptomless clinical phenotype

with a peculiar Hb pattern has been reported (Galanello et al 2002).

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

thalassemia is shown in the table 1.2.

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

in β thalassemia. Production of high levels of γ chain leads to relatively a mild degree of

globin chain imbalance and hence these conditions are much milder than the β

thalassemia. Gγδβ thalassemia is characterized by the deletion of Aγ genes, and synthesis

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.

23

Table 1.2: The main groups of disorder of δ and β chain production

Condition Homozygote Heterozygote

Gγ Aγδβ Thalassemia Thal Intermedia 100% HbF

Thal minor Hb F 5 – 20%; HbA2 1.5 -2 % α /non- α1.3 – 1.8 /1

Gγδβ Thalassemia As above As above

δβ Lpore Thalassemia Thal major or intermedia 80% HbF ; 20% HbLepore

Thal minor Hb Lepore8 – 20%

Aγ Gγ HPFH Thal minor 100% Hb F

Normal blood picture, 15 – 30 % HbF; 1.5 – 2 % HbA2

Other forms of δβ thalassemia are Hb Lepore disorders. These disorders are

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

deficiency of δ and β chains.

24

Gγ Aγδβ 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

synthesis ratios of approximately 3 are observed.

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.

Hb F levels are 5 – 20% range and A2 is normal or slightly reduces.

Gγ δβ Thalassemia Clinical and hematological finding are same as Gγ AγHPFH, although homozygotes are

slightly more severely affected. These conditions can be differentiated by chemical

analysis of HbF which shows only the presence of Gγchains.

Sardinian δβ Thalassemia Clinical and hematological findings are similar to other forms of δβ thalassemia.

However they have HbF that is mainly nearly all Aγ type.

INTERACTIONS OF δβ THALASSEMIA AND LEPORE HEMOGLOBIN Compound heterozygous state of δβ thalassemias and Lepore hemoglobin with βO or β+

thalassemia results in the phenotype of thalassemia Intermedia.

25

γ δβ THALASSEMIA This is a rare form of thalassemia, not found in homozygous state. Adults heterozygotes

have hematologic changes similar to those of β thalassemia heterozygotes. The condition

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

analysis (Weatherall .1983)

1.11.2: Incidence and population genetic:

Initially thalassemia was thought to be confined to Mediterranean, African and Asian

ancestry but sporadic cases have been reported in many ethnic groups. It is now thought

that malaria has played a role in the propagation of thalassemia genes.

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

of β - Thalassemia remain un characterized. This is the first study of beta-thal mutations

from Iraq (Al-Allawi et al 2006).

1.11.3: Thalassemia Intermedia

1.11.3.1: Definition

Thalassemia Intermedia is the term used to describe the clinical and hematological

findings in patients with β - Thalassemia. Although not transfusion dependent, these

26

patients manifest a more sever degree of anemia than that found in heterozygous carriers

for α - or δ thalassemia (Weatherall 1996). Beta(0)-thalassaemia intermedia (beta(0)-TI)

describes patients who lack beta-globin synthesis yet manifest a non-transfusion-

dependent form of beta-thalassaemia (Chang et al 2001).

The proportion of patients with homozygous thalassemia intermedia is strikingly different

among different ethnic groups. About 10% of patients of Mediterranean ethnicity who are

homozygous can be classified as intermediates. In contrast, more than 70% of African-

American patients who are homozygous may be classified as such. This difference

reflects the kinds of thalassemia mutations, especially so-called “mild mutations,” that

are prevalent in these ethnic groups (Pearson et al 1996)

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’.

In 1940 , Silvestroni and Bianco describeded anemia microcitica costituzionale

(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

27

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

themselves or with those for structural hemoglobin variants.

Thalassemia Intermedia entails considerable clinical and genetic heterogeneity. Anemia

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

families (Weatherall 2001b).

1.11.3.3: Clinical Presentation

Thalassemia Intermedia present with extraordinary diverse clinical spectrum from almost

complete health to a condition characterized by severe growth retardation and skeletal

deformities requiring transfusion therapy. Typical features of patients with thalassemia

are skeletal changes, particularly in the skull and in the malar bones. Patients develop

oro-facial deformities and subsequently demonstrated clinical and laboratory

abnormalities consistent with thalassemia intermedia (Ficarra et al 1987). Skeletal

abnormalities in beta-thalassemia are widening of medullary spaces, rarefaction of bone

trabeculae, thinning of cortical bone, and perpendicular periosteal spiculation. Premature

epiphyseal fusion (PEF) is found though more rarely (Colavita et al 1987).

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

28

pulmonary hypertension. Bone deformities, extramedullary hematopoiesis (EMH), and

spleen and liver enlargement are the consequences of hypoxia and enhanced

erythropoiesis. Afebrile, EMH-related pleuritis represents a potentially life-threatening

complication in thalassemia (Aessopos 2006). Two main factors determine cardiac

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

that leads to an increased pulmonary vascular resistance and an increased systemic

vascular stiffness (Aessopos et al 2007a).

The clinical picture of TI patients who have not received transfusions or have

occasionally received transfusions is dominated by the consequences of chronic

hemolytic anemia, tissue hypoxia, and their compensatory reactions, such as bone

deformities and fractures, extramedullary hemopoiesis, spleen and liver enlargement,

hypercoagulability, and pulmonary hypertension. These complications, especially the

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

with regular transfusion-chelation therapy showed suppression of the anemia-related

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

further improvement on Survival (Aessopos 2007b).

Mild anemia is one of the hematological findings in heterozygous β thalassemia. Patients

with β– thalassemia Intermedia have hemoglobin level usually below 9 – 10 g/dl

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

29

transfused to avoid these distressing complications. Osteoporosis and osteopenia are

frequent complications of thalassemia major (TM) and thalassemia intermedia (TI)

(Origa et al 2005). Fractures are frequent among the aging patients with β - TM (Vogiatzi

et al 2006) and TI. Spinal cord compression due to extramedullary hematopoiesis is a

rare complication of thalassemia (Saghafi et al 2005).

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

complications like hypersplenism or folic acid deficiency, nutritional deficiency or inter

current infection which may exacerbate anemia.

Thalassemia intermedia patients usually do not require blood transfusion however all

patients show variable degrees of erythropoietic marrow expansion to compensate for

anemia. This is the major cause of complications in untransfused individuals

(Camaschella et.al.1996). Kinetic studies clearly separate cases that, or will be, clinically

intermediate, showing a higher medullary uptake of radioactive iron, a less ineffective

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

chelation and splenectomy (Najean Y et al 1985).

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

contributed to the patients' tophaceous gout (Kumar and Gruber 2003).

30

Hypocholesterolemia accompanies anemias with high-erythropoietic activity. We suggest

that the high-erythropoitic activity-associated hypocholesterolemia is due to increased

cholesterol requirements by the proliferating erythoid cells (Shalev et al 2006). Patients

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).

Pulmonary hypertension (PHT) is part of the cardiopulmonary complications of

thalassemia lack of systematic treatment in TI leads to a cascade of reactions that

compensates for chronic anemia but at the same time allow the development of PHT

(Aessopos and Farmakis 2005).

Low transfusion regimen may cause a decrease in serum concentration of EPO, which is

independent of the level of hemoglobin (Dore et al 1993).

Venous thromboembolic events such as pulmonary embolism, deep venous thrombosis

patients with and portal vein thrombosis have been observed in adult thalassemia

patients, mainly in beta-thalassemia intermedia. Clinical findings are consistent with

alterations that indicate a state of activation of the haemostatic mechanisms in

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

the increased turnover has been suppressed by hypertransfusion. Thrombotic tendencies

in patients with low Heparin co factor II levels in the presence of haemolysis might in

principle decreased by blood transfusion (O'Driscoll et al 1995).

Some occular abnormalities like degeneration of retinal pigment epithelium, lenticular

opacities, vascular abnormalities, and angioid streaks have been reported in TI

31

(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

significant age-related frequency in hemoglobin disorders, especially beta-thalassemia

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

manifestation of PXE has been reported (Aessopos et al 2008).

1.11.3.4: Clinical Grading

Clinical phenotype of homozygous beta thalassemia varies in severity from mild

thalassemia intermedia to the severe thalassemia major (Galanello et al 2002). Three

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.

32

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

started after 10 years.

1.11.3. 5: genotypes

Based on the molecular genetics five different genotypes are identified in

thalassaemia intermedia patients:

Group I: homozygosity for mild mutations Group II: combinations of mild/severe mutations Group III: homozygosity or double heterozygosity for severe mutations Group IV: heterozygosity -87/IVS1-6 Group V: IVS1-6/CD 6-A This is of particular importance for genotype-phenotype correlation, carrier

detection, genetic counseling and prenatal diagnosis (Rigoli et al 2003).

1.11.3.6: Age at presentation

One of the most useful indicators of thalassemia Intermedia is the age at presentation.

Mean age of presentation is 13.1 months with a range of 2 to 36 months (Kattimis et al

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

33

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

CD39 C → T nonsense mutation.

It was suggested that bone marrow erythroblast may mature further and generate more

hemoglobin. These patients may have hyperbilirubinemia due to increased catabolism of

hemoglobin (Berdoukas 1993).

Thalassemia Intermedia shows diverse spectrum of clinical heterogeneity where patient

may be anemic in early child hood while others may not be diagnosed until adolescence

or later age (Ahern et al 1975). Spleen may be enlarged, occasionally massively

(Farhangi Sass and Bank 1970).

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

al 1964). Chronic hypersplenism is common (Rapaport et al 1957) and gallstones

frequently form (Erlandson et al 1964). Unconjugated hyperbilirubunema resulting from

ineffective erythropoiesis is common (Weatherall and Clegg 1981). Bone

demineralization in adult thalassaemic patients due to the contribution of growth

hormone and insulin-like growth factor I at different skeletal sites has been reported by

Scacchi et al (2008).

Other complications include diabetes mellitus secondary to iron overload (Erland et al

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

34

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

hepatomegaly (Celada 1982) hypopituitarism and prophyria (Bannarman et al 1967).

Tumor – like masses of bone marrow are common (Knoblich 1960). Folate deficiency

may worsen anemia and megaloblastic erythropoiesis (Erlandson et al 1964).

1.11.3.7: Molecular basis of β- Thalassemia Intermedia phenotype

Thalassemia intermedia is a clinical entity characterized by moderate, non-transfusional

anemia and hepatosplenomegaly. This phenotype can result from different genetic

combinations and is sometimes present in patients with only one parent showing the

thalasemia minor phenotype (Martinez-Lopez J et al 1998). Many Interactions and

mutations can give rise to this phenotype (table 1.3).

1.11.3.8: Interactions of silent β thalassemias

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

(Gonzalez – Redondo et al 1988).

β 5’ untranslated region (UTR ) + 10 (- T)

This mutation with compound heterozygous state with β CD 39 C → T is characterized

by a 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

35

state with several beta thalassemia alleles including IVSI – I G → A and IVSI – 110 G →

A (Ho et al 1996) mutations TI is usually very mild.

IVS 2 844 C → G

In homozygous form, this mutation (Murru et al; 1991) causes extremely mild form of

β - thalassemia Intermedia. Interaction with splice mutation β IVS2 – 745 C → G also

causes mild form of thalassemia Intermedia (Rosatelli et al 1994). Splicing mutations are

common causes of beta-thalassemia. Some mutations permit normal splicing as well as

aberrant splicing. Reduced level of normal beta-globin synthesis produces a mild disease

(thalassemia intermedia) (Vadolas et al 2006).

β CAP + 1 A → C

Observed in Asian Indians, this mutation can cause thalassemia Intermedia of varying

severity (Wong et al 1987).

β Termination codon + 6 C → G

With IVSI – I (G → A) causes moderately sever thalassemia Intermedia (Maragaudaki et

al 1998).

Codon 104(-G)

Codon 104(-G), a heterozygous frameshift mutation in exon 2 of HBB, resulted in a

dominantly inherited beta0-phenotype with mild anemia in a German kindred, and

thalassemia intermedia in the index patient. A co-inherited a gene triplication, long-term

transfusion therapy, and ineffective erythropoiesis were confounding factors (Lahr et al

2007) (table 1.4).

36

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

association with haplotype VIII (beta-87/VIII) or V (beta-87/V). β beta-87/VIII showed a

configuration of rare polymorphisms in the 5' sub-haplotype. This has been reported to

exert an increasing effect on Hb F synthesis (De Angioletti et al 2004, Rosatelli et al

1989).

β - 30 T → A

This is common in Turkish, Macedonian and Tunisian population and causes mild form

of thalassemia intermedia (Fei et al 1988)

β - 29 A→ G

This is common in Africans and Afro – Americans and produces fairly mild thalassemia

Intermedia (Weatherall 2001b).

37

Table 1.3: Thalassemia intermedia (Weatherall D.J.2001)

1. Mild defects in β – globin production Homozygous mild β+ thalassemia Compound heterozygous for severe βo or β+ and mild β+ thalassemia Interactions of βo with ‘silent’ or ‘mild’ β thalassemia Homozygous for ‘silent’ β thalassemia

2. Reduced globin imbalance due to co-inheritance of α and β thalassemia Homozygous or compound heterozygous βo or β+ thalassemia with two or three α gene Homozygous or compound heterozygous severe βo or β+ thalassemia with non – deletion α2 – gene mutation Homozygous or compound heterozygous severe β+ thalassemia with one or two α – gene deletions

3. Sever β thalassemia with increased capacity for γ – chain synthesis homozygous or compound heterozygous βo or β+ thalassemia with heterocellular HPFH

Homozygous or compound heterozygous βo or β+ Thalassemia with a particular β – globin RFLP haplotype Mechanism unknown 4. Deletion forms of δβ thalassemia and HPFH

Homozygous (δβ)o or (A γ δβ) thalassemia Compound heterozygous for βo or β+ and (δβ)o or (A γ δβ) thalassemia Homozygosity for Hb Lepore (some cases) Compound heterozygosity for (δβ)o, G γ β+ or A γ β+ HPFH and βo or

β+thalassemia Compound heterozygosity for (δβ)o thalassemia and (δβ)o HPFH

5. Compound heterozygousity for β or δβ thalassemia and β- chain structural variants Hbs S/, C/ E/β or δβ thalassemia Many other rare interactions

6. Other β thalassemia alleles or interactions Dominant β thalassemia β thalassemia Trait associated with αααα – gene arrangements Highly unstable β – globin chain variants

38

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.

Mut

atio

ns

-101

(C→

T)

-92

(C→

T)

-88(

C→

T)

-87(

G→

C)

-87(

C→

T)

-86(

C→

A)

-30

(T→

C)

-29

(A→

G)

5’U

T R

+10

- T

5’U

T R

+22

(G-A

)

5’ U

TR +

33 (C

-G)

CA

P+1

(A→

C)

CD

19 (A

→G

)

CD

26(G

→A

)

CD

27 (

G→

T)

IVSI

-6 (

T→C

)

IVS

2-84

4 (→

G)

β te

rm +

6 (C

-G)

AA

TAA

A→

A

ATA

AG

AA

TAA

A→

A

AC

AA

A

AA

TAA

A→

A-A

AA

CD8 - AA STI TI TI

CD8/9+G STI

CD15 G-A TI

IVSI-I G-A TI TI TI

IVSI-5 G-C TI TI/STI

IVSI-5 G-T MTI

IVSI-110 G-A MTI TI STI TI MTI MTI TI STI

CD36/37-T MTI

39

Mut

atio

ns

-101

(C

→T)

-92

(C→

T)

-88

(C→

T)

-87

(G→

G)

-87

(C→

T)

-86

(C→

A)

-30

(T→

C)

-29

(A

→G

)

5’U

T R

+10

-T

5’U

T R

+22

(G-

A)

5’ U

TR +

33 (C

-G

)

CA

P+1

(A→

C)

CD

19 (A

→G

)

CD

26 (

G→

A)

CD

27 (

G→

T)

IVSI

-6 (

T→C

)

IVS

2-84

4 (C

→G

)

β Te

rm +

6 (C

-G)

AA

TAA

A→

A

ATA

AG

AA

TAA

A→

A

AC

AA

A

AA

TAA

A→

A- A

AA

CD39C-T TI STI TI TI MTI STI

CD41-C MTI

CD41/42 -TTCT MTI /STI

CD44-C MTI STI

IVS2-1 G-A MTI TI MTI STI

IVS2-654 C-T TI

IVS2-745 C -G MTI TI MTI

IVS2-745 C-G

IVS2-748 C-A

IVS2-849 A-G

40

β Codon 19AAC → AGC ; β 19 Asn → Ser, Hemoglobin Malay

This mutation is found amongst Southeast Asians of Malaysian origin. In homozygous

state, it causes mild form of thalassemia Intermedia (Yang et al 1989).

β codon 27 (GCC → TCC ; β 27 Ala →Ser; Hb Knossos )

A codon 27 change activates an alternative splice site, resulting in a slight reduction in

the quantity of normal β – globin messenger RNA. More over δ – globin in cis position

has a deletion of a single A in codon 59 leading to premature termination at codon 60. It

is found in Mediterranean and adjacent regions (Weatherall 2001b). Compound

heterozygous form with IVSI – 6 T → C, Codon 8 – AA, IVSI →110 G→A, IVSI – I G

→ A and IVS2 – I G → A results in mild to moderate thalassemia intermedia.

An A→G transition at the usual intervening sequence 2 (IVS2) acceptor splice site has

been described. Functional analysis of transcripts produced by this mutant gene in a

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

normal beta-globin mRNA (Atweh GF et al 1985).

β IVS I → 6 T → C

Common in Mediterranean populations, a splice mutation results in mild to moderate

form of thalassemia Intermedia (Weatherall 2001b).

β 87 C → T

In compound heterozygous form with allele IVSI – 110 G → A demonstrate moderately

severe form of thalassemia Intermedia (Weatherall 2001b). PolyA; AATAA →

41

AATAAG, found in Kurdish Jewish families, poly A; AATAAA→ AATGAA

demonstrates mild form of Thalassemia Intermedia, where as poly (A): AATAAA →

AACAA in combination with IVS2-1 G → A causes moderately severe form of

thalassemia Intermedia. Poly A: AATAAA → AATAGA observed in Malaysia is

associated with an extremely mild interaction with HbE and there fore is probably a mild

β – thalassemia allele (Weatherall 2001b).

G →T CHANGE IN CODON 121

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

was the result of a spontaneous mutation that occurred during spermatogenesis in a

paternal germ cell (Kazazian Jr. et al 1986).

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)

Hb Knossos (beta 27 (B9) Ala→Ser) is a hemoglobin variant that can cause beta (+)

thalassemia intermedia syndrome (Baklouti et al 1986). Hb Knossos is characterized by

reduced synthesis and by interaction with beta-thalassemia, in which the double

heterozygotes display typical features of thalassemia intermedia. Hb Lepore and Knossos

(beta 27 Ala→Ser) have been found to be associated with beta-thalassemia intermedia

42

picture with total absence of Hb A2. This indicates that beta Knossos gene is most

probably flanked by a delta (0)-thalassemia gene. Hb Knossos, representing 70% of total

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).

BETA + THALASSAEMIA--PORTUGUESE TYPE Clinically, the homozygotes range from asymptomatic to thalassaemia intermedia and

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

common types of beta thalassaemia as regards red cell morphology, haemoglobin

analysis and globin chain synthesis. Globin gene mapping excluded the presence of alpha

thalassaemia in these patients and demonstrated no abnormalities in the beta-like globin

gene cluster. Restriction enzyme site polymorphisms around the beta gene cluster are

identical on both chromosomes in all of the homozygotes, confirming their homogeneity

(Tamagnini et al 1983).

HEMOGLOBIN MISSISSIPPI (HBMS: BETA 44SER→CYS)

Hemoglobin Mississippi has anomalous properties that include disulfide linkages with

normal beta-, delta-, gamma-, and alpha-chains and formation of high molecular weight

multimers. HbMS-beta +-thalassemia may result from proteolytic digestion of HbMS, as

well as excessive alpha-chains characteristic of beta +-thalassemia. This causes

excessive of cellular damage that results in the phenotype of thalassemia intermedia

(Steinberg et al 1987).

43

Hb Dhofar

Hb Dhofar is a variant haemoglobin (beta(29 (GGC-GGT) gly-gly), beta(58 (CCT-CGT)

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

severe beta(+) thalassaemia allele resulting in a thalassaemia intermedia phenotype (Daar

et al 2008)

1.11.3.9: INSERTION/FRAMESHIFT MUTATION This mutation is characteraized by an insertion of eight nucleotides into exon 2 of beta-

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

morphological changes, significant reticulocytosis, and marked increase in the proportion

of fetal hemoglobin (Williamson et al 1997).

1.11.3.10: MICROSATELLITES AND THEIR FUNCTIONAL PRELEVANCE. Short tandem repeats are abundantly present within the genome. They are commonly

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

could be involved in controlling gene expression. (TG)n (CG)m dinucleotide repeat

polymorphisms in the two gamma-globin gene IVS2s has been observed and in vivo and

in vitro data demonstrates a possible contribution of the gamma-gene IVS2s polymorphic

44

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

(−α/αα) are often indistinguishable from normal (Weatherall 1981).

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

by defective mRNA synthesis processing or translation (Kan 1985).

There are less common, “nondeletional” forms of α+-thalassemia caused by mutations

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

of excess production of γ chains of fetal hemoglobin (α2γ2) (Allen et al 1997).

In the South West Pacific region, the striking geographical correlation between the

frequency of α+-thalassemia and the endemicity of Plasmodium falciparum suggests that

this hemoglobinopathy provides a selective advantage against malaria in the South West

45

Pacific region, the striking geographical correlation between the frequency of α+-

thalassemia and the endemicity of Plasmodium falciparum suggests that this

hemoglobinopathy provides a selective advantage against malaria. Paradoxically, α+-

thalassemia increases the incidence of contracting mild malaria in the first 2 years of life

(Sammy Wambua et al 2006). The mechanism whereby α+-thalassemia protects against

malaria may have an immunological basis. Parasitised α+-thalassaemic erythrocytes

bound greater levels of antibody from malaria endemic sera and were more readily

phagocytosed by blood monocytes compared with control cells. A greater frequency of

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

the community control children was more common in homozygous α+-thalassemia.

Increased reticulocyte count in α+-thalassemia may underlie the increased susceptibility

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

frequency of exposure to both Plasmodium falciparum and Plasmodium vivax (Anuja

Premawardhena et al 2004).

1.12.1: Molecular Pathology of Alpha thalassemia

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

46

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

homologous regions I, II and III (Higgs et al 1989, Higgs et al 1984).

The chromosome with a hybrid α2/ α1 gene (-α 3.7 deletion) results from reciprocal

recombination between Z segments, which are 3.7 kb apart, whereas recombination

between homologous X segments, which are 4.2 kb apart , results in a single α 1 gene ,

thus deleting the entire α 2 gene ( - α 4.2 deletions).

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

second most common α - thal – 2 is the - α 4.2 determinant which is found at high

frequencies in South china, mainly in the Guangxi (58%) and Jiangxi (29%) (Baysal &

Huisman 1994).

Other less common α - thal – 2 determinants are the - α 2.7 and -α 3..5 deletions. The

former deletes only the α - 1 – globin gene, leaving the α 2 locus intact, while in the

- α 3.5 type, the deletion extends from the 5’ end of the α1 gene to the 5’ end of the θ 1

gene (Baysal and Huisman 1994).

Deletion of proline at alpha37(C2) is predicted to result in severe instability of the variant

hemoglobin, which on interaction with a synthesis-deficient alpha-thalassemia mutation

causes a relatively severe dyserythropoietic anemia, representing an alternative

phenotype associated with highly unstable alpha-chain variants (Traeger-Synodinos et al

2000).

47

According to the output of α - chain, α - thalassemias are classified into αo –

thalassemia with no output of α- globin chains, α- thalassemia – 1, and α + - thalassemia

with a reduced α- chain out put α-thalassemia – 2 (Weatherall 2001b). α - thalassemias

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

nondeletional α-thalassemia (αT α or ααT) (Reviewed in Higgs et al 1989).

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

as Polynesia mainly Melanesia and remote parts of India α - thalassemia - 2 is inherited

by more than 50 % of all individuals (Baysal and Huisman 1994).

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,

1960: Higgs et al 1989). Sever determinants (- -) only occur at high frequency in

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

Barts or hydrops fetalis syndrome is observed in 1:300 hospital births (Thumasathit et al ,

1968) and may account for up to 26% of prenatal deaths (Cong & Shong 1982).

48

Alpha-Thalassemia mutations are one of the most common mutations in Man, and they

cause Hb H disease and Hb Barts hydrops fetalis. Hb H disease is not necessarily a

benign disorder as has been generally thought (Chui 2005).

1.12.2: Heterozygous β thalassemia with ααα/αα

This interaction result in a variable phenotype displaying a spectrum from β thalassemia

trait alone, mild type of TI to sever TI. Additional alpha-genes may increase the severity

of heterozygous beta-thalassemia. Co-existence of -alpha3.7 mutations with homozygous

beta-thalassemia may convert a transfusion-dependent thalassemia major to a non

transfusion-dependent thalassemia intermedia (Al Qaddoumi et al 2006). Alpha globin

gene triplication is an important genetic determinant underlying thalassemia intermedia in

North Indians (Panigrahi et al 2006a). However presence of extra copies of alpha-globin

gene has been shown to worsen the degree of anemia in beta-thalassemia heterozygotes.

Therefore presence of triplicated alpha-globin genes should always be considered in

apparent beta-thalassemia carriers who were more symptomatic than expected (Ma et al

2001, Oggiano et al 1992 and Beris et al 1991).

49

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 chromosomes crossing over toproduce the - α3.7, α α α anti3.7 and - α4.2, α anti4.2 haplotypes are also shown (Higgs et al, 1989).

50

1.12.3: Homozygous β thalassemia with ααα/ααα

A triplicated α- gene arrangement behaving as an α - thalassemia alleles was reported by

Higgs and Pressley (1980) from Saudi Arabia.

1.12.4: Heterzygous β thalassemia with ααα/ααα

Homozygous state for the triplicated α -globin arrangement in association with

heterozygous β thalassemia has been observed in the Mediterranean population only.

Patients heterozygous for βo – or sever β+ - thalassemia mutations had clinical picture of

moderate to sever form of thalassemia intermedia (Galanello et al 1983).

1.12.5: Heterozygous β – thalassemia associated with αααα/αα

Heterozygous β thalassemia with inheritance of six α - globin genes in the homozygous

inheritance with the triplicated α globin gene or heterozygous inheritance of the

quadruplicated and normal α - globin – gene complement has been reported (Thompson

et al 1989). This genotype is associated with mild to moderate form of thalassemia

intermedia. Individuals heterozygous for codon 39 CAG-to-TAG mutation are also

heterozygous for a triplicate alpha-globin gene locus (alpha alpha alpha anti 3.7). Thus

compound heterozygous condition of a beta39 C-to-T mutation and triplicate alpha-

globin gene increases alpha:beta-globin chain imbalance and accounts for the presence of

beta-thalassemia intermedia (Rhodes et al 1999, De Angioletti M et al 1992, Advani et al

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

intermedia (Thein et al 1984).

51

1.12.6: Dominant β thalassemia associated with ααα/αα

Severe effect of this particular β- thalassemia allele together with the usual degree of

imbalanced globin synthesis results in severe form of TI. In dominant β thalassemia

associated with ααα/αα interaction (Thein et al 1990). Beta-thalassemia heterozygotes

conjuncted with alpha-globin gene triplication was the major cause of beta-thalassemia

intermedia in a Korean family (Chen et al 2000). However combination of a triplicated

alpha globin locus with heterozygous beta-thalassaemia produces a clinical phenotype of

thalassaemia intermedia (Camaschella et al 1987).

1.12.7: Phenoptype effect of Interactions of α and β thalassemia

Relative excess of alpha- over beta-globin chains in the erythroid precursors is the major

pathophysiological feature of homozygous beta-thalassemia. Its clinical picture is usually

characterized by a transfusion-dependent dyserythropoietic anemia (thalassemia major).

However, some patients present with moderate anemia that does not require regular blood

transfusions (thalassemia intermedia). Molecular heterogeneity of beta-thalassemia

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-

thalassemia when inherited in combination (Kulozik et al 1993).

Excess alpha-globin chains play a major role in the pathophysiology of homozygous

beta-thalassaemia. In beta-thalassaemia carriers a minor effect of alpha-globin chain

excess is reflected in a minimal or mild anaemia without clinical symptoms. Factors that

increase alpha-chain excess in heterozygotes are expected to accentuate the severity of

the clinical and haematological phenotype. Homozygous alpha-gene triplication interacts

52

with a severe beta-thalassaemia mutation to cause an alpha-chain excess equivalent to

that observed in homozygous beta-thalassaemia intermedia. In heterozygotes for severe

beta-thalassaemia mutations with one additional alpha-globin gene, the alpha-chain

excess causes a more pronounced degree of anaemia than is seen in beta-thalassaemia

heterozygotes (Traeger-Synodinos et al 1996).

Major determinants of disease severity in beta thalassaemia, the beta thalassaemia

mutations, with co-inheritance of alpha thalassaemia trait and high HbF determinants act

as ameliorating factors. Presence of an alpha thalassaemia deletion significantly reduces

initial disease severity, although the effect on pubertal development is less clear

(Gringras et al. 1994) Co inheritance of different α - thalassemia alleles modifies clinical

picture of sever β – thalassemia. Significant amelioration in homozygous or compound

heterozygous state (Weatherall et al 1981) is observed when there is coinheritance of one

or more α - thalassemia determinants. How ever co – inheritance of one or more α -

thalassemia alleles less effectively ameliorate thalassemia (Weatherall 2001).

1.12.8: β – thalassemia trait modified by the co – existence of α

thalassemia

Co existence of α thalassemia is important in the context of thalassemia Intermedia.

Significant effect on red cell indices are noted by the co – inheritance of β – thalassemia

with α+ thalassemia (-α / - α ). β – thalassemia with homozygosity for α+ thalassemia

and heterozygosity for αo Thalassemia can result in normal or almost normal

hematological picture with an elevated hemoglobin A2 level. It is assumed that in

heterozygotes the compensatory increase in β - chain production from the β locus in

53

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

βo heterozygous for the deletional forms of α+ thalassemia. It appears that there is a

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

compensating the defective output of their partner (Weatherall 2001b).

Frameshift mutation in exon 3 of the beta-globin gene, a single nucleotide deletion (-C)

in between codons 140/141 (GCC/CTG→GCC/TG), was found in an 8-year-old

Argentinean girl with clinical picture of thalassemia intermedia. It leads to a beta-chain

that is elongated to 156 amino acids [(141)Trp-Pro-Thr-Ser-Ile-Thr-Lys-Leu-Ala-Phe-

Leu-Leu-Ser-Asn-Phe-(156)Tyr-COOH]. The resulting hemoglobin was named

Hemoglobin Florida (Weinstein et al 2006).

1.12.9: Co-inheritance of homozygous or compound heterozygous β – thalassemia gene with different varieties of α - thalassemia Inheritance of one or more α- thalassemia alleles might reduce the severity of

homozygous or compound heterozygous β - thalassemia. In a study on moderately severe

β- thalassemia intermedia with homozygous or compound heterozygous inheritance of

β- thalassemia 3 were heterozygous for α+ thalassemia deletion (-α/αα) while two were

heterozygous for non- deletion α+ thalassemia alleles (Weatherall et al 1981). A genetic

combination of silent beta-thalassaemia, high Hb A2 beta-thalassaemia and single alpha

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

54

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

intermedia phenotype has also been reported by Harteveld et al (2008)

βo – Thalassemia

In Cypriot, Sardinian and Asian patients that are homozygotes for βo thalassemia with co

inheritance of two α - globin genes loss, (-α / - α) or non deletional form of α+

thalassemia involving α2 – globin genes more likely to have clinical phenotype of

thalassemia Intermedia than homozygous βo – thalassemia patients with co inheritance of

single α- globin gene are deletion usually have thalassemia major with slightly late onset

clinical presentation.

β+ - thalassemia

Homozygous or compound heterozygous β+ thalassemia with co inheritance of two

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

α thalassemia allele produce contrasting effects. This is because non – deletional

α thalassemia alleles causes greater reduction in α - chain production (Thein et al 1988,

Galaenello et al 1989). Co – inheritance of α thalassemia varies in different population.

In Italian patients co inheritance of deletional forms of α+ thalassemia causes thalassemia

Intermedia (Camaschella and Cappelini 1995) whereas mild form of β - thalassemia

rarely occurs in Israel. This reflects a low frequency α - thalassemia in this population.

Co inheritance of heterozygous state for α+ thalassemia with compound heterozygous

state for IVSI- 6 T→ C / IVSI-110 G → A respectively produces mild and moderately

55

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.

Co inheritance of single missing α gene with heterozygosity for IVSI – 5G →C / Fr 8 – 9

+ 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

trait (Waye et al 1996) Point mutation in the polyadenylation signal (AATAAA-

AATAAG) is known to cause a moderately severe beta-thalassaemia phenotype (Rund et

al 1993).

Reasonable correlation exists between the mildness of phenotype and homozygous βo,

severe β+ thalassemia and homozygous α+ thalassemia. However single α - globin gene

has minimal phenotype effect on homozygosity of βo thalassemia.Whereas effect on

compound heterozygous β+ or βo or homozygous β+ has unpredictable effect due to loss

of single α- globin gene. Role of α thalassemia in the generation of thalassemia

Intermedia is dependent upon its relative frequency in the population (Kuloziki et al

1993).

1.12.10: Homozygous β+ or βo thalassemia associated with compound heterozygosity for α+ and αo thalassemia Loukopoulos et al (1978) described the occurrence of moderately severe form of β

thalassemia intermedia by the combination of β thalassemia homozygous with HbH

disease. Furbetta et al (1979) described the association of βo thalassemia homozygous

with HbH disease. Extremely high hemoglobin A2 in these patients as reported may be

56

due to the high levels of δ - chain production, which normally do not reach the peripheral

blood in homozygous β thalassemia.

1.12.11: Pathophysiology

Globin imbalance is central to importance in the severity of HbH disease and

heterozygous αo, α+ and β+ thalassemia show similar degree of anemia and

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

degree of ineffective eryhtropoisis and myeloid / erythroid ratio of 0.55 (Loukopoulos et

al 1978). Membrane association of abnormal or partially oxidized alpha-globin chains has

a more deleterious effect on the membrane skeleton than do beta-globin chains.

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

al. 1995). Hyperactivity of macrophages to chronic hemolysis causes an increase in

cytokines, such as IL-8, found in thalassemia syndromes (Dore et al 1995). Severity of

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

57

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

lysosomal fragility due to increased iron storage (Frigerio et al 1984). Iron-dependent

oxidative reactions in beta-thalassaemic erythrocyte membranes are involved in

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

intermedia erythrocytes, perhaps as a consequence of their elevated intracellular

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%

decrease in titratable SH groups indicating a change in lipid-to-protein ratio. The

difference being due, in part, to increased oxidative stress (Kahane and Rachmilewitz

1976)

Oxidative stress causes the attachment of haemichrome to membrane, thus aggregation of

band 3 and erythrophagocytosis removes RBC (Cappellini et al 1999). Membrane

association of abnormal or partially oxidized alpha-globin chains has a more deleterious

effect on the membrane skeleton than do beta-globin chains (Yuan et al. 1995). Loss of

phospholipid asymmetry, and consequently exposure of phosphatidylserine (PS), is

58

thought to play an important role in the cell pathology. Thus hemichrome binding to band

3, hemichrome-mediated oxidation of band-3 cytoplasmic domains, generation of high-

molecular-weight band-3 aggregates, and enhanced opsonization by anti-band-3

antibodies is a possible sequence of events leading to phagocytic removal of erythrocytes

in thalassemia (Mannu et al 1995, Scott et al 1992,Schrier et al 1989).

Anemia in thalassemias is caused by a combination of ineffective erythropoiesis

(intramedullary hemolysis) and a decreased survival of adult RBCs in the peripheral

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

macrophages. Presence of PS-exposing subpopulations of thalassemic RBC are most

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

in beta-thalassemia patients targets hepatic hepcidin transcript levels independently of

iron metabolism genes controlling hepcidin expression (Camberlein et al 2008).

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

decreased response of TM platelets to aggregating agents suggests in vivo platelet

activation in thalassemic patients (Eldor et al 1989).

59

In transfusion-dependent patients, increased values of serum transferrin correspond to

lesion worsening (Sergiacomi et al 1993). Abnormal metabolism of iron with its

deposition in the tissues and impairment of the vessels of the microcirculatory bed results

in osteoarthropathy in beta-thalassemia (Musaev et al 1991) Extramedullary hemopoiesis

in the lower spinal column may cause low back pain (Gouliamos et al 1991, Papavasiliou

et al 1990, Sakai et al 1990).

Pseudo-Gaucher cells were reported in a bone marrow biopsy of hemoglobin E disease

and a spleen from thalassemia intermedia patients (Sharma et al 2007).

Evidence for the existence of a chronic hypercoagulable state observed in patients with

beta thalassemia intermedia, suggests the expression of a procoagulant surface by

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

not related to iron overload or hepatic dysfunction (Bhattacharyya et al 2007). Protein Z,

thrombin-antithrombin complexes, Protein C concentration (PC:Ag) and activity

(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

thrombin-antithrombin complex levels were significantly increased and related to PC

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

be the dominant mechanism implicated in the lipid abnormalities of TI patients

(Amendola 2007).

60

1.12.12: Role of Hb F in generating thalassemia Intermedia

Co-inheritance of alpha-thalassaemia, certain variants of the beta-like globin gene cluster

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

major factor in the generation of the phenotype of β - thalassemia Intermedia. Parents of

Thalassemia Intermedia patients had elevated HbA2 levels, but co – existence of an

α - thalassemia gene seemed unlikely (Weatherall et al 1981).

Clinical manifestations of beta-thalassemia (beta-thal) intermedia phenotypes are

influenced by the persistence of fetal hemoglobin (HbF) and by several polymorphisms

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,

but not in wild-type subjects (Guida et al 2006).

Mild form of β+ thalassemia Hereditary persistent of fetal hemoglobin appeared to be

segregating (Knox – Macaulay et al 1973). Detailed studies of parents or relatives did not

always evidence for a second determinant of this type (Weatherall 1981).

1.12.13: DETERMINANTS WITH IN β - GLOBIN GENE

CLUSTER

Production of Hb F is related to a particular RFLP haplotype (Labie et al 1985a, Thein et

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).

In Asian Indians strong association of - + - ++ with β thalassemia Intermedia was found .

61

In Asians it was found in 14 out of 28 β - globin chromosomes from thalssemia

intermedia patients and mainly 10 out of 84 chromosomes from thalassemia major.

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

haplotype has a likely relationship.

1.12.14: Xmn- I polymorphism

This haplotype is in linkage desequilibrium with Xmn –I polymorphism ( C →T - 158 )

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

play an important role in underlying the mild form of homozygous βo thalassemia in

Afro – Asian population and less in Italians. In Sardinia majority of the patients showed

thalassemia intermedia who were also homozygous or heterozygous for the

Mediterranean haplotype IX which contains - + - + + 5’ subhaplotype, Frameshift (- 1bp)

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

limited effect (Galanello et al 1989).

In Asian Indians a strong association of Gγ Xmn-I (+) polymorphism with the βo-

thalassemia IVSI – I G → T mutation is observed (Ho et al 1998a). Homozygoutes for

this mutation have a slightly milder disease than other forms of βo thalassemia. Many of

62

them become transfusion dependent only in later life. It is believed that the XmnI (+)

polymorphism in the promoter region of the Gγ gene is associated with an increased

capacity for fetal hemoglobin production. In conditions of erythroid “stress”

homozygocity for this polymorphism may sometimes be associated with a milder clinical

course in homozygous or compound heterozygous β thalassemia. However there may be

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

occasional cases of γ – globin gene triplication or quadruplication (Yang et al. 1986)

there is no definite evidence that regions other than Gγ – 158 C → T XmnI

polymorphism play a major role in elevating γ – chain synthesis in β thalassemia

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).

63

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

content in beta thal intermedia patients (Mastropietro et al 2002).

1.13: CORFU δβO THALASSEMIA

The Corfu delta betao thalassemia clinically resembles thalassemia intermedia. In the

homozygous state there is a complete absence of hemoglobin (Hb) A and Hb A2 and a

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).

1.14: Deletion form of β thalassemia

Usually high levels of HbA2 and significantly increased levels of HbF in heterozygotes

have been associated to at least 17 different β thalassemia mutations particularly if they

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

64

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

functional beta-globin gene. It extends to a region of frequent loss of heterozygosity

called LOH11A, which is located close to the beta-globin locus. Thus, Loss of

heterozygosity can be a cause of non-malignant genetic disease (Badens et al 2002).

1.15: Point Mutation of the β- Globin gene

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

Gγ XmnI (+) polymorphism (Gonzalez- Redando et al 1988). Heterozygotes for the

promoter gene mutations tend to have a slightly higher level of fetal hemoglobin than

those for other forms of β thalassemia.

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

function. The Hematologic phenotype and molecular structure of the rearranged

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

gene region (Kulozik et al 1992). Interaction of hereditary persistence of fetal

hemoglobin HPFH-6 with beta-thalassemia causes thalassemia Intermedia. Much milder

65

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).

1.17: Homozygous or compound heterozygous β thalassemia with

heterocellular HPFH

Hereditary persistence of fetal hemoglobin (HPFH) is a condition whereby a

continuously active gamma-globin gene expression leads to elevated fetal hemoglobin

(Hb F) levels in adult (Stamatoyannopoulos and Grosveld. 2001). HbF can be raised in

certain forms of heterocellular HPFH with β thalassemia or Sickle cell gene. In

heterozygous state either alone or together with β thalassemia the augmentation of fetal

haemoglobin production is extremely small. Yet when it interacts in severely affected

homozygous or compound heterozygous state determinants the fetal hemoglobin by

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).

1.18: Heterozygous β thalassemia with thalassemia intermedia

phenotype

There are three main mechanisms whereby β – thalassemia heterozygous can be

associated to thalassemia intermedia.

1) β-thalassemia complicated by the inheritance of more than the usual number of

α - globin genes.

66

2) β-globin gene mutations producing products of unusual properties and giving rise

to heterozygous phenotypes now called dominant β thalassemia.

3) Both duplicated and quadruplicated α - globin gene arrangements have little

effect on normal individuals but when inherited with a single β – thalassemia

allele sufficient globin imbalance produces β- thalassemia which is more severe

than heterozygous β – thalassemia.

1.19: Dominant forms of β thalassemia

This group of β thalassemia is transmitted in an autosomal dominant fashion. It is

associated with clinical features and inclusions in the normoblasts and peripheral red cells

after splenectomy (Weatherall et al 1973).

1.19.1: Mechanism of dominant β thalassemia phenotype.

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

reflecting their heme biding properties and molecular instability. In heterozygotes,

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

these conditions are characterized by ineffective erythropoiesis.

67

1.19.2: Thalassaemia-like carriers not linked to beta-globin

gene cluster

This genetic determinant appears haematologically heterogeneous, displaying either a

silent beta-thalassaemia-like phenotype or a typical beta-thalassaemia carrier-like

phenotype in different families. Compound heterozygosity for both beta-thalassaemia-

like determinant and typical beta-thalassaemia allele result either in thalassaemia

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

transcription factors erythroid, Kruppel-like factor and nuclear factor (erythroid-derived

2) were normal. Beta-globin mRNA levels determined by real-time polymerase chain

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

unknown transcriptional regulator(s) of the beta-globin gene. The knowledge of these

rare beta-thalassaemia-like determinants have implications for clinical as well as prenatal

diagnosis of beta-thalassaemia (Faa et al 2006).

1.20: Haemoglobin Lepore

Homozygous Haemoglobin Lepore (Hb Lepore) can also cause thalassemia intermedia

(Pasangna et al 2005).

68

1.21: Hemoglobinopathies

Although over 400 variants of structural haemoglobin have been identified, only,

haemoglobins S, C, and E, reach high frequencies in certain populations (Weatherall

2000b). Structural hemoglobin variants typically are caused by a point mutation in a

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

with frequency. Homozygous Hb C and Hb S produce significant clinical manifestations,

whereas Hb E and Hb D homozygotes may be mildly symptomatic. Although

heterozygotes for these variants are typically asymptomatic, their diagnosis may be

important for genetic counseling.Thalassemia, in contrast, results from quantitative

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-

thalassemias. Severity of clinical manifestations in these disorders relates to the amount

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

microcytosis. Thalassemia intermedia (i.e., Hb H disease) is typified by a moderate

variably compensated hemolytic anemia that may present with clinical symptoms during

a period of physiologic stress such as infection, pregnancy, or surgery (Clarke and

Higgins 2000).

Hemoglobin E (beta26Glu → Lys) is the most common hemoglobin (Hb) variant in

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

country (Kishore et al 2007).

69

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

E beta-thalassemia is an important cause of childhood chronic disease in Southeast Asia.

It is characterized by the presence of hemoglobin E and F and amount of hemoglobin E

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

acts as a mild beta+thalassemia. However, a remarkable variability in the clinical

expression ranging from a mild form of thalassemia intermedia to transfusion-dependent

conditions is observed. Severe hemoglobin E beta-thalassemia may have clinical features

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

(Fucharoen and Winichagoon 2000).

Hemoglobin E is a beta chain variant that has its most significant interaction with

thalassaemia. The compound heterozygous state, thus produced, can result in a

thalassaemia intermedia/major phenotype with affected individuals being transfusion

dependent (Kakkar 2005). Haemoglobin E beta thalassemia (HbE beta thalassemia) has a

remarkable variability in clinical expression ranging from a mild form of thalassemia

intermedia to a transfusion dependent condition (Tyagi et al 2004). Patients with Hb beta

+ [IVS 1-5 (G→C)] clinically present as beta-thalassaemia intermedia and remain

asymptomatic in the absence of blood transfusions. Children who receive blood

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,

70

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

ought to be included in the differential diagnosis of beta-thalassemia major or intermedia

in subjects of Southeast Asian descent who have HbA on hemoglobin electrophoresis.

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

(Morle et al 1984). Retrospective serological analysis has shown a relatively high

frequency of exposure to both Plasmodium falciparum and P. vivax (Anuja

Premawardhena1 et al 2004).

1.22:EF Bart's disease EF Bart's disease is an uncommon form of thalassaemia intermedia resulting from the

co-inheritance of alpha-thalassaemia and haemoglobin E in the same subject. DNA

mapping and haemoglobin electrophoresis indicate that there are four genotypes

involving 5 abnormal globin genes, responsible for this thalassaemia syndrome

(Fucharoen et al 1988)

1.23: Hb Vicksburg

A new hemoglobin variant, Hb Vicksburg, produces the phenotype of thalassemia

intermedia in patients who are doubly heterozygous for this variant and

betao-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

71

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

cis to the structural mutation (Adams and Steinberg 1981).

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

patients. Postpartum splenectomy might be necessary in some patients (Nassar et al

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

haemolytic anaemia following transfusional, alloimmunisation and portal vein

thrombosis. Therefore all thalassemic patients should be tested for various blood antigen

systems so that transfusion reactions can be avoided (Bianconcini et al 1993).

1.25: Genotype phenotype relationship

Prediction of beta-thalassaemia major phenotype from beta-genotype is generally

relatively straightforward. However, despite the ability to accurately define the beta-

thalassaemia mutations, prediction of a beta-thalassaemia intermedia phenotype from the

genotype sometimes remains problematic this has important implications in genetic

counseling and prenatal diagnosis. The clinical expression of beta-thalassemia

intermedia is variable and complications are more frequent than in the minor form.

Thromboembolism risk increase after splenectomy (Pierre 2006).

72

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

beta-thalassemia mutation; (3) cotransmission of delta-thalassemia which may normalize

the raised HbA2 typical of heterozygous beta-thalassemia and (4) presence of a silent

mutation which can be defined only by imbalanced beta-globin chain synthesis. A

number of molecular mechanisms can produce non transfusion dependent thalassemia

syndromes referred to as thalassemia intermedia. The most common are homozygosity

for mild beta-thalassemia mutations, coinheritance of alpha thalassemia with

homozygous beta-thalassemia or genetic determinants that ensure continuous production

of HbF in adult life or the presence of heterozygosity for hyperunstable globin variants

(Cao A et al 1994). Molecular basis of thalassemia intermedia, are very heterogeneous,

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-

thalassemia allele associated with a high residual beta-globin chain production.

Coinheritance of alpha-thalassemia or genetic determinants that increase the gamma-

chain production may cause mild thalassemia. Less frequent mechanisms are double

heterozygosity for beta-thalassemia and triplicated alpha genes, and the presence of a

hyperunstable hemoglobin variant. For significant number of βo 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

73

levels of RBC as well as HbA2 with significantly higher levels of Hb H. Clinical

variability was remarkable ranging from totally asymptomatic conditions to severe

thalassemia Intermedia characterized by marked hemolytic crises, liver and spleen

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).

Alpha-thalassemia is phenotypically and genotypically heterogeneous. DNA analysis is

invaluable as it provides a specific diagnosis and enables reliable genetic counseling.

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

may lead to a thalassemia-intermedia-like picture which could be misdiagnosed as beta-

thalassemia (Oron-Karni et al 2000). The severity correlated inversely with the number

of functional alpha-globin genes.

1.26: Geographical distribution

Thalassemia Intermedia is observed in Indians and Pakistanis (Weatherall et al 1981b),

Arabs (Cividalli et al 1978; Weatherall et al 1981a), Britons (Knox – Macaulay et al

1973), Algerians (Godet et al 1977) and Italians (Bianco et al 1977).

74

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

prevalence of 37.5 and 27.5% respectively in Pakistani immigrants. Of the 10 common

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

homozygous beta-thalassemia (total 50 cases). In heterozygous beta-thalassemia, alpha

alpha alpha anti-3.7 triplication was the predominant factor (Panigrahi I et al 2006b).

Unstable hemoglobin should be suspected in a patient with thalassaemia Intermedia

phenotype even if both parents are haematologically normal (Dash et al 2006).

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

thalassaemia intermedia/major phenotype with affected individuals being transfusion

dependent (Kakkar 2005). Alpha globin gene triplication is an important genetic

75

determinant underlying thalassemia intermedia in North Indians. Patients with alpha-

triplication may develop jaundice with marked increase in serum bilirubin following

antecedent aggravating factors (Panigrahi et al 2006a). Most common

hemoglobinopathies observed in 1015 cases were sickle cell trait (29.8%), sickle cell

disease (7.5%), sickle cell-beta-thalassemia (1.7%), beta-thalassemia trait (18.2%),

thalassemia major (5.3%), thalassemia intermedia (0.9%), Hb E trait (0.9%), Hb E

disease (0.3%), E-beta-thalassemia (0.7%), Hb D trait (0.2%) and SD 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%).Transfusion

dependent beta-thalassemia syndrome was prevalent in Brahmin, Karan, Khandyat, Teli,

etc. Most of the cases belonged to Anugul district, followed by Khurda, Nayagarh,

Phulbani, Cuttack, Jajpur, Dhenkanal, Ganjam, Keonjhar, Mayurbhanj, etc. The

heterogeneous population harbors almost all major hemoglobinopathies in general castes,

scheduled castes and tribes that belong to Coastal and South-Western regions of Orissa

(Balgir 2005).

Nucleotide -88 (C→T) promoter mutation is a common beta-thalassemia mutation in the

Jat Sikhs of Punjab, India (Garewal et al 2005). Hb Hofu [beta 126(H4)Val→Glu]- βo-

thalassemia [codons 8/9 (+G)] combination were identified in a Thalassemia Intermedia

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

76

βo 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

and a frameshift codons 47- 48 (+ATCT) in Punjabis (Garewal et al 1994).

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

co-inheritance of one or more alpha-globin gene deletions (-alpha 3.7) and the presence of

the XmnI polymorphism were associated with less severe disease in Indians (Nadkarni et

al 2001). Nine different types were found, of which six were associated with betao, 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

co-inheritance of a high Hb F determinant associated with the - + - + + 5' beta haplotype

and the inheritance of a mild beta-thalassaemia mutation are the major ameliorating

factors of disease severity in Asian Indians (Thein et al 1988). Coexistence of a novel

beta-globin gene deletion (codons 81-87) with codon 30 (G→C) mutation was also

identified in an Indian patient with βo-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

intermedia in North Indians (Panigrahi et al 2006a) In heterozygous beta-thalassemia,

77

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

Shengxue Granules is that it can up-regulate the expression levels of alpha-hemoglobin

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

decrease the hemolysis (Liu 2006).

Unstable Hemoglobin Hemolytic anaemia due to unstable hemoglobins arising from spontaneous mutation has

been reported (Dash et al 2006)

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

(29.8%), sickle cell disease (7.5%), sickle cell-beta-thalassemia (1.7%), beta-thalassemia

trait (18.2%), thalassemia major (5.3%), thalassemia intermedia (0.9%), Hb E trait

(0.9%), Hb E disease (0.3%), E-beta-thalassemia (0.7%), Hb D trait (0.2%) and SD

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%).

Heterogeneous population harbors almost all major hemoglobinopathies in general castes,

scheduled castes and tribes that belonging to Coastal and South-Western regions of

Orissa (Balgir 2005).

78

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

the clinical picture of thalassaemia Intermedia (Wainscoat et al 1984).

1.28:Thalassemia Intermedia In China

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.

β0 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

to the Gγ-globin gene or by the number of alpha-globin genes present (Antonarakis et al

1988). Codons 41/42 (-CTTT) beta0-thalassemia and nt - 28 (A → G)beta(+)-thalassemia

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).

Hb Westmead is a common alpha-globin gene mutation in this population apart from Hb

Constant Spring and Hb Quong Sze. (Wong et al 2004).

(G)gamma((A)gammadeltabeta)(o)-deletion and beta-thalassemia (cd 41-42), Hb H

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disease (genotype -alpha(3.7)/-(SEA) have also been identified. Patients

possessing(G)gamma((A)gamma delta beta)(o)/beta-thalassemia were not transfusion

dependent. This could be due to co-inheritance of alpha-thalassemia-2 (genotype-alpha

(3.7)/alphaalpha) in children together with their compound heterozygous condition (Tan

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.

1997). Another mutation (GAA-TAA) in codon 121 that produces a thalassemia

intermedia phenotype with inclusions in erythrocytes has also been identified in Hong

Kong (Wakamatsu et al 1994). The molecular defects of beta- thalassemia intermedia in

Guangdong Province were highly heterogeneous and its spectrum was different from the

reports from other provinces of China (Zhang L et al 2007).

Alpha Thalassemia

Marked genetic and clinical variability of Hb H syndrome is due to the molecular

heterogeneity of alpha-thalassemia (thal). The hallmark is the presence of excess beta

chains forming Hb H (β tetramer). Classical Hb H disease in Chinese presents as "alpha-

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

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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).

A rare thalassemia intermedia case caused by co-existence of Hb H disease (--(SEA)/-

alpha(4.2)) and beta-thalassemia major (beta (CD17A)>T/beta (IVS2-654C)>T) has also

been indentified. The practice of prenatal diagnosis in this case may also provide

reference for diagnosis of similar cases (Li et al 2008).

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

alpha2-globin gene, are the most prevalent nondeletion alalpha-thalassemias in Southeast

Asia. Hematological findings confirm the mild thalassemia intermedia phenotypes for

pure homozygous Hb CS and homozygous Hb CS with Hb E heterozygote and Hb E

homozygote (Singsanan et al 2007).

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1.29: Thalassemia Intermedia In Japan

Thalassemia is relatively rare in northeast Asia including Japan where malaria is

uncommon. However, thalassemia in Japan has peculiar mutation spectrum and

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

Southeast Asia (Hattori 2002).

In one study that was carried out in 100,000 samples 29 contained electrophoretically

detectable abnormalities in the heterozygous state condition. 12 had beta-thalassemia, 3

alpha-thalassemia, 1 delta-thalassemia. Among 45 carriers of beta-thalassemia from 12

families, 5 were noted to have thalassemia intermedia (Imamura et al 1980).

1.30: Thalassemia Intermedia In Lebanon

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

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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

increased production of HbF, which shows strong linkage to particular mutations

(IVSII-1, cd8 and cd30).

1.31: Thalassemia Intermedia In Thailand

Haemoglobin (Hb) Hope [beta136(H14)Gly→Asp(GGT→ GAT)] is one of the unstable

haemoglobin variants of the beta-globin chain, which is demonstrated in people of

various ethnic backgrounds. This hemoglobin was reported in a Thai female patient with

clinical thalassaemia intermedia since childhood. Homozygous Hb Hope without

abnormal alpha globin chain involvement, heterozygous Hb Hope in association with

-alpha(3.7) mutation, in clinically silent individuals were identified (Sura et al 2007).

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

alpha-thalassemia 1 (SEA type) with the Hb Constant Spring followed by deletion 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-

thalassemia, Hb Pakse, Hb CS. (Boonsa et al 2004).

An unusual form of thalassemia intermedia caused by interaction of the hemoglobin

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).

1.32: Thalassemia Intermedia In Brazil

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

83

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

mutation at codon 39 (C → T), leading to severe forms of betao-thal. However, the

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)

15.1%, IVS-I-5 (G → C) 9.3%, IVS-I-110 (G → A) 8.2%, codon 39 (C → T) 3.5%, and

codon 30 (AGG → AGC) 1.1% are amongst the common mutation (Araujo et al 2003).

1.33: Thalassemia Intermedia in Greece

A major factor modifying the clinical expression of homozygous high-HbA2

beta-thalassemia in Greece is the inheritance of mild beta-thalassemia mutations.

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

cases in one study) in thalassemia intermedia phenotype (Kanavakis et al 1995). In

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

situations where beta chain synthesis in trans is eliminated (Jankovic et al 1991).

84

Homozygous beta ++ has also been found to be associated with thalassemia Intermedia

(Kattamis et al 1982).

1.34: Thalassemia In Pakistan Pakistan has a population of more than 160 million people with an overall carrier

frequency of approximately 5.6% for beta-thalassemia. Punjab is the largest province of

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

(Baig et al 2006). It is observed that 58% of the siblings of beta-thalassaemia major

children in Hazara division of Pakistan had beta-thalassaemia trait (Anjum et al 2001).

Serum ferritin estimation in carriers of beta- thalassaemia trait is important (Qureshi et al

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

between the percentages of microcytic hypochromic cells as a screening test is an easy,

reliable and sensitive index which can be used for mass screening of beta thalassaemia

trait particularly in a population where iron deficiency is also prevalent (Saleem et al

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

85

Indians in 1991 (Varawalla et al 1991). Two other mutations IVS-1 nt.5 (G-C) and codon

8-9 (+G) mutations were identified in 1995 (Khan et al 1995).

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)

mutation is a rare polymorphism on an ancestral allele in which betao 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

characterized. The complete spectrum comprises of 19 mutations. There are important

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

(52.2%). A novel 17 bp deletion involving Cd126-13 was also identified (Ahmed et al

86

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

codons 41/42 (-CTTT) are older mutations as determined by multiple haplotype

associations and widespread geographical distribution. Microepidemiology of

beta-thalassaemia in this region reflects considerable ethnic diversity, gene flow from

population migration and natural selection by malaria infection (Varawalla et al 1992).

Spectrum of mutations is heterogeneous in this population and 19 different mutations in

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

Gujarat (Thein et al 1984).

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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

alpha genes (Khan et al 2003).

Level of Hb Bart's is directly related to the inheritance of alpha-thalassaemia gene. Hb

electrophoresis for Hb Bart's in cord blood is a very simple method of determining out

the prevalence of alpha-thalassaemia gene in a given population. 2.4% of the general

population was found carrier of alpha-thalassaemia gene of these. 75% had alpha-

thalassaemia-2 gene and 25% were carriers of alpha-thalassaemia-1 gene (Rehman et al

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

alphao thalassaemia (Hassall et al 1998). The common beta-thalassemia lesions in the

Mediterranean region and Asia are caused by defective mRNA synthesis, processing, or

translation (Kan 1985).

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A rare alpha2-globin chain variant, Hb Sallanches [alpha104(G11) Cys→Tyr], in a

Pakistani family having three homozygous patients with transfusion-dependent Hb H

disease were identified. This variant, previously reported in a French patient and a West

Indian homozygous patient with Hb H disease, is due to a mutation at codon 104

(TGC→TAC) (Khan 2000).

δβ-Thalassaemia

Delta beta-thalassaemia is a rare disorder in Pakistan. Although the clinical picture is

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

postnatal period. Haematological and genetic features of δβ-thalassaemia in Pakistani

patients were characterized. In the study all heterozygotes and 4/6 homozygotes were

asymptomatic. One homozygote had thalassaemia intermedia while another had

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)

(Ahmed and Anwar 2006b).

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

delta beta-thalassemia. All subjects had the Asian-Indian Inv/Del (GγAγδβ)o that

89

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

the Asian-Indian Inv/Del (GγAγδβ)o degrees. This finding could explain the unusually

well-preserved capacity to produce fetal hemoglobin in delta beta-thalassemia (Ahmed

and Anwar 2005b).

Hemoglobinopathies Hemoglobin D-Iran (Hb D-Iran, beta 22 Glu→Gln) is a beta-chain variant that was first

described in 1973. Hb D-Iran in combination with normal Hb A (Hb D-Iran trait) is a

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

homozygous Hb D-Iran in an infant of Pakistani descent has been described, hematologic

values, hemoglobin electrophoresis, peripheral blood smear, and clinical course suggest

that homozygous Hb D-Iran is a relatively benign condition with mild microcytic anemia,

poikilocytosis, and minimal hemolysis (Thornburg CD et al 2002). D-Los Angeles (alpha

2 beta 3 121 (glutamine→glycine) and thalassemia trait has also been detected in a

Pakistani family (Dawod et al 1988).

1.35: Treatment

Treatment of β -thalassemia depends on the clinical severity and ranges from no

treatment, in cases of β-thalassemia traits or some mild thalassemia Intermedia , to

frequent transfusions with chelation therapy and augmentation of fetal-hemoglobin

synthesis, in cases of thalassemia major or intermedia (Robin et al 2006). Pleurodesis

90

seems to be an effective and safe therapeutic option for exudative effusions, while

transfusion-chelation therapy combined with hydroxyurea may be helpful in suppressing

increased erythropoiesis (Aessopos 2006a). HU appears suitable for the treatment of leg

ulcers unresponsive to conventional treatment in patients with thalassaemia intermedia

(Gamberini 2004). Due to genetic heterogeneity of beta-thalassemia (beta-thal) patients,

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

for 96 h preferentially enhanced (G) gamma-and (A)gamma-globin mRNA (Hydroxyurea

2006). In addition to acting in synergy with the XmnI polymorphism, alpha deletions

may be an independent factor predicting good response to HU in thalassemia intermedia

(Panigrahi 2005). Hydroxyurea may be an alternative to transfusions for TI patients as

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

consequences. Growth retardation in regularly transfused thalassemic children has been

observed. Hypothyroidism is unlikely to be the sole cause of growth retardation; it may

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

overloaded due to improper iron chelation because of socioeconomic reasons. To

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,

L1)FP in Pakistani thalassaemic patients were studied. Deferiprone (l1, 1, 2- dimethyl-3-

91

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

increased compliance (Ayyub et al 2005). Studies of the standardized, 3D, 16-segments

map of the circumferential distribution of T2* values, of cardiovascular magnetic

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

evaluated. Cardiovascular magnetic resonance can be a suitable guide to cardiac

management in TI, as well as in TM (Ramazzotti et al 2008)

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

and anti-Jka. Rate of red cell alloimmunization in beta-thalassaemia major is relatively

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

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"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

antibodies was found to be an important complication. Patients who develop this

complication should be tested for presence of underlying antibodies and considered for

immunosuppressive treatment (Bhatti et al 2004).

Transmission rate of hepatitis C virus (HCV) infection through household contacts is to

be very low but may play a role in HCV spread by infected persons (Akhtar et al 2004).

In HCV seropositive thalassaemic children 20.5%were positive for anti-HCV antibodies.

HCV genotype 3a and 3b were found in 89% and 11% respectively (Akhtar et al 2002).

Transfusion transmitted hepatitis G virus in polytransfused β- thalassemia major children

was found in 21%, however no causal relationship between HGV and hepatitis was

observed (Moatter et al 1999).

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,

β-thalassaemia major and certain haematological malignancies. Allogeneic peripheral

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

is present in up to a quarter of adult beta-thalassemia Patients (Vidergor et al 2007).

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

93

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

families so that an affected child is a marker of a high risk group.

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

healthy children used prenatal diagnosis. Testing of extended families is therefore a

feasible way of deploying DNA-based genetic screening in communities in which

consanguinous marriage is common (Ahmed et al 2002). Frequency of consanguineous

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

measurement of MW seems to be an objective, simple test for discriminating between

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

thalassaemia intermedia (Sbyrakis et al 1987). Evaluate quality of life (QOL) in

transfusion-independent patients with thalassemia (non-Tx) compared with that in

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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

concern of overall health status or indications of recent deterioration in health. In contrast

with previous beliefs, transfusion-independent thalassemia patients also suffer serious

impairment in QOL (Pakbaz 2 005).

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

extensively studied. This includes transfusion, chelation (intravenous and oral),

antioxidants and various inducers of fetal hemoglobin (hydroxyurea, erythropoietin,

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

independent anemia in the TM patients (Chaisiripoomkere et al 1999 and de

Montalembert et al 1989). Continuous Increase in Erythropoietic Activity despite the

Improvement in Bone Mineral Density by Zoledronic Acid in Patients with Thalassemia

Intermedia-Induced Osteoporosis(Voskaridou et al 2008). Rgular chelation therapy with

deferoxamine is effective in patients with secondary hemochromatosis. Self-administered

deferoxamine subcutaneously once or twice a day seems to be the most practical method

to protect against the progression of hemochromatosis (Kobayashi et al 1996 and de

Montalembert et al 1989). Recently, much effort has been focused on various inducers of

95

fetal hemoglobin (HbF) such as recombinant human erythropoietin (rHuEPO), especially

in beta-thalassemia intermedia. HbF values registered a slight, non-significant increase.

rHuEPO treatment has a beneficial effect in transfusion-dependent beta-thalassemia

patients. Although a slight increase in HbF levels was observed, other possible

mechanisms are probably involved (Wong et al 2004). Viral-mediated globin gene

transfer in hematopoietic stem cells effectively treats a severe hemoglobin disorder (May

et al 2002). Administration of recombinant human erythropoietin (rHuEpo) in

combination with iron and folic acid may ameliorate blood indices as an alternative

choice to blood transfusion (Lialios et al 2000, Bourantas et al 1997, Leroy-Viard et al

1991 and Chaidos et al 2004). Thus recombinant erythropoietin seems to be an effective

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

the level of haemoglobin F (HbF) by pharmacological agents has been proposed to

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,

has been shown to enhance HbF synthesis (Zeng et al 1995).

Hydroxyurea stimulating fetal hemoglobin (Hb) production. can eliminate transfusion

needs in children with beta-thalassemia major. This could be particularly useful in

countries where supply of blood and chelating agents are limited (Bradai et al 2003, de

Paula et al 2003, Cario et al 2002 and Cao et al 2005). Hydroxyurea may be an

alternative to transfusions for TI patients as well as for TM patients in countries that have

limited blood supplies (Bradai 2007).

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Butyrate derivatives can stimulate fetal hemoglobin in patients with intermediate

thalassemia Intermedia (Domenica Cappellini et al 2000) Reduced serum or erythrocyte

Mg levels have been reported in human beta thalassemia. These deficiencies may play a

role in the cellular abnormalities characteristic of this disorder. Dietary Mg

supplementation improves some of the cellular function abnormalities of β thalassemia

intermedia (De Franceschi et al 1998).

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.

Reprogramming of differentiation in intact, mature, adult white blood cells in response to

inclusion of monoclonal antibody CR3/43 has been described. This form of retrograde

development has been termed "retrodifferentiation", with the ability to re-express a

variety of stem cell markers in a heterogeneous population of white blood cells. This

form of reprogramming, or reontogeny, to a more pluripotent stem cell state ought to

recapitulate early hematopoiesis and facilitate expression of a fetal and/or adult program

of hemoglobin synthesis or regeneration on infusion and subsequent redifferentiation.

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

from differentiated cells to regenerate transiently an otherwise genetically degenerate

tissue such as thalassemic blood (Abuljadayel et al 2006).

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1.36: Gene therapy in Thalassemia Intermedia

Gene therapy approaches to these disorders envision stem cell targeted gene transfer,

autologous transplantation of gene-corrected stem cells, and functional, phenotypically

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

the hemoglobin disorders (Arthur et al 2003).

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

immunodeficiency virus-based lentiviral (LV) vectors were shown to transmit human

beta-globin gene and a large LCR element, resulting in correction of beta-thalassemia

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 Religion is believed to have a significant impact on individuals from minority ethnic

groups when making decisions about prenatal genetic screening, prenatal diagnosis and

termination of pregnancy. They generally considered religion and faith as an important

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

diagnosis is feasible and acceptable in a Muslim country such as Pakistan (Ahmed et al

2000) Pakistani women's attitudes towards prenatal diagnosis and termination of

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 Thalassemia is usually diagnosed in infancy and is characterized by ineffective

erythropoeisis, bone marrow expansion and rapid destruction of erythrocytes. The

curative treatment available for this disease is either blood transfusion and chelation or

bone-marrow transplantation which is too expensive. Therefore, the only option is to

prevent the birth of an affected child by Carrier testing and prenatal diagnosis. Carrier

screening refers to identification of heterozygotes (carriers). In obstetrics, screening

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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

made retrospectively, following the birth of an affected child or prospectively. Carrier

detection is based on the full blood counts and Hb electrophoresis and estimation of Hb

A2 and F levels followed by definitive diagnosis (mutation detection) by DNA analysis

which is guided by the results of hematological parameters. Prenatal diagnosis is one of

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

possible. It is offered in the I, II and III trimester of pregnancy accomplished by mutation

analysis on PCR-amplified DNA from chorionic villi sampling, amniocentesis and fetal

cordocentesis.

Sample selection in prenatal diagnosis: First trimester: Chorionic Villi Sampling Second trimester (15-19 weeks): Amniocentesis Third trimester (>22 weeks): 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

Parameters α–thalassemia (αº-thal)

β–thalassemia (Heterozygous)

δβ–thalassemia

Red Blood Cell Count

Milder hematological changes

High

Milder hematological Changes

Mean Corpuscular Volume (MCV)

Reduced

Reduced (60-70fl)

70fl

Mean Corpuscular Hemoglobin (MCH)

Reduced

Reduced (19-23pg)

24pg

Red Blood Cell Morphology

Microcytosis, Hypochromia

Microcytosis, Hypochromia

Microcytosis, Hypochromia

Hb concentration

Slight Reduction

Less than Normal (2g/dl)

Normal or slightly reduced

HbA2

Low to Normal (1.5-2.5%)

Elevated (4-6%)

Normal or slightly reduced

HbF

---

Elevated (2 - 1-3%)

Increased (5-20%)

Osmotic fragility

Decrease

Decrease

---

α/β-globin biosynthetic ratios

0.7

1.5 – 2.5

1.5

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Table 1.6: Clinical and Hematological features of the β- Thalassemia Intermedia Syndromes (Richard et al 1993) Features Major Intermedia Minor Minima

Severity of manifestations ++++ ++ +, ± ± , 0

Genetics Homozygotes, Double Heterozygotes

Homozygotes, double heterozygotes, rarely heterozygotes

Heterozygotes Heterozygotes

Splenomegaly ++++ ++ , +++ +. 0 0

Jaundice +++ ++.+ 0 0

Skeletal changes ++++, ++ +,0 +,0 0

Hb,g/dl <7 7 – 10 >10 Normal

Hypochromia ++++ +++ ++ +

Microcytosis +++ ++ + 0

Target cells 10 – 35% ++ + ±

Basophilic stippling ++ + + 0, +

Reticulocyte (%) 5 – 15 3 – 10 2 – 5 1 – 2

Nucleated red cells +++ +, 0 0 0

±: little or no abnormality, +: mild abnormality, ++++: prominent abnormality

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Table 1.7 : Hemoglobin fractions in the genotypic variants of β - Thaalassemia syndromes. (Richard et al 1993)

Genotype Hb A (%) Hb A2 (%) Hb F (%)

Other Hemoglobins

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

(δβ) lepore/(δβ) lepore 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

β0/(δβ) Lepore 0 Decreased Increased Hb Lepore (10%)

β+/(δβ) Lepore Present Decreased Increased Hb Lepore (10%)

β0/β Present > 3.2 1.5 - 12 None

(δβ)0/(δβ)0 0 0 100 None

(δβ)0/(δβ) lepore 0 0 92 Hb Lepore (8%0)

α / β Present Increased Normal or ↑ed ± HbH

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