MUTATIONAL ANALYSIS OF HBB GENE LINKED WITH

BETA THALESSEMIA IN POPULATION OF DISTRICT

MANSEHRA
Introduction
Thalassaemia is a common globally prevalent monogenic disorder with an incidence of around
70,000 new cases per annum. (Weatherall,2010)

Thalassemia is life-long disabling heritable disorder; need repetitive blood transfusion

procedure. Thalassemia is among the most common genetic blood disorders worldwide .[2
Arana et al., 2002 ] In Pakistan, 50,000 to 100,000 patients are suffering from thalassemia and
every year 5,000 babies are born with thalassemia. In Pakistan, 5% of the total population
suffers from thalassemia. It was found noteworthy harmful impact of thalassemia in areas of
physical and mental health.[3 Indaratnaet al.,2010] They confront with number of side effects
predominantly exceding level iron in body vital organs and heart, liver etc .[4. Azarkeivan et
al.,2008] Many studies have investigated the link of between hope and quality of life. Hope has
been linked to wellness and illness experiences and recovery . [5. Duggleby et al., 2012 , 6
Chaturvedi et al., 2007].It is thought to be necessary for survival of people with chronic
diseases. Studies suggest that higher levels of hope are related to better outcomes in
academics, athletic competitions and health .[7 Indaratna et al., 2010] Hope has been studied
by various disciplines such as social sciences, psychiatry, theology and nursing. Hope is a central
concept of positive psychology.[8 Bryant and Cvengeros, 2004 ,9 Boehm et al.,2011 ]

Thalassemias or Mediterranean anemia comes from the Greek words “Thalassa” meaning sea,
and “Emia” meaning blood, was described in 1925 by a physician who studied Italian children
with severe anemia, early childhood death and huge abdominal organs, and caused by impaired
synthesis of one or more globin chains of the hemoglobin (consisting of 4 polypeptide chains),
resulting in much less oxygen being bound to the hemoglobin molecules and transported
through the body [10 Shirzadfar et al., 2018 ,11 Uthman, 2007]. Depending on which
polypeptide chains are affected, the thalassemias are named α, β, γ or δ thalassemia. It is the β
chain that is most frequently affected, so that this disorder is called β-thalassemia. β-
thalassemia major is a homozygous form in which both alleles are severely mutated so that β
chain synthesis is stopped completely, whereas β-thalassemia minor is a heterozygous form
resulting in an approximately 20% reduction of polypeptide synthesis. To compensate this
reduction, more HbA2 and HbF are produced: in β-thalassemia major it is more HbF, and in β-
thalassemia minor primarily HbA2. In 1946, the cause of thalassemia was found to be an
abnormal hemoglobin structure. The body reacts by destroying red blood cells, causing anemia.
To compensate for this deficiency, the body tries to make more red blood cells faster, resulting
in other thalassemia complications such as bone disorders, spleen enlargement, and heart
problems. In the 1960s, doctors discovered a new way to treat thalassemia, and began
replacing fresh blood instead of patient blood every month. This method was most commonly
used for patients with thalassemia major and is still used to treat the diseases. But, after each
passing blood transfusion, the body encountered an increased amount of iron that could not
be removed naturally. As a result, most patients with thalassemia died for the same reason. The
researchers later discovered the drug to remove excess iron from the body by treatment with a
drug called deferoxamine [12 Brittenham et al., 1994]. This drug prevented iron-inducted heart
disease and helped patients live much longer. Recently, two oral drugs have dramatically
improved the quality of life of patients with iron overload from transfusions for thalassemia.
Furthermore, In Iran as a premarital screening, the man’s red cell indices are checked first, if
cell hemoglobin <27pg or red cell volume <80fl, the woman is tested at last. When both have
this feature, their hemoglobin A2 concentrations are measured. If both have a concentration
above 3.5% of hemoglobin A2, (diagnostic of thalassemia trait) they are referred to the local
designated health post for genetic counseling [13 Samavat and Modell, 2004]. People with
thalassemia minor, if malaria is diagnosed, their likelihood of death is lower than that of others.
Therefore, thalassemia minor has a great advantage. Of course, malaria treatment does not
eliminate thalassemia. In this research, the natural structure of the hemoglobin gene and the
various types of thalassemic disease will be described, then the signs and symptoms of the
disease, and finally the treatment of the disease will be explained.

HUMAN HEMOGLOBIN: STRUCTURE AND FUNCTION

Human hemoglobin consists of a tetramer of globin polypeptide chains: A pair of α-like chains
with 141 amino acids and a pair of β-like chains with 146 amino acids.[14 Marengo-Rowe,2006]
Each globin polypeptide chain enfolds a single heme moiety, which can bind a single oxygen
molecule. Thus one molecule of hemoglobin can bind and transport four molecules of oxygen.
Unpaired polypeptide chains of hemoglobin are insoluble and tend to form inclusions, which
can damage red blood cells (RBC). However, the tetramer formed by α and β chains are soluble
and prevent any cell damage. Normal globin production is regulated so that any new chain
formed will have a partner to pair. In thalassemia syndromes, this regulation is impaired
resulting in overproduction of either α or β chain and underproduction of other. This mismatch
results in accumulation of unpaired chains and hence insolubility and precipitation of such
globin chains. The major adult hemoglobin A (HbA) has two α and two β chains (α2 β2), minor
adult hemoglobin has two α and two δ chains (α2 δ2) and fetal hemoglobin has two α and two
γ chains (α2 γ2).
Genetics of human hemoglobin
The human hemoglobins are encoded in two gene clusters: α-like globin genes present on
chromosome 11 and β-like globin genes on chromosome 16. Normally an individual inherits two
β-globin genes and 2-α globin genes from each parent i.e., normal adult hemoglobin is α2
β2.Depending upon whether the genetic defects or deletion lies in transmission of α or β globin
chain gene, thalassemias are classified into α and β-thalassemias. [15 Trent,2006] Thus, patient
with α-thalassemias have impaired production of α chains whereas patient with β-thalassemias
have impaired production of β chains. Each of two main types of thalassemias may occur as
heterozygous (minor) or homozygous state (major). The former is generally asymptomatic while
the latter is severe congenital hemolytic anemia.

Normal structure and expression of globin gene clusters

Human hemoglobin is a heterotetramer protein, compose of two alpha and two beta subunits
as shown in Figure 1. Each subunit contains a heme group, an iron containing compound that
binds to oxygen [16 Maton et al.,1993]. The synthesis of hemoglobin is controlled by two
developmentally regulated multigene clusters: the alpha-like globin cluster on chromosome 16
and the beta-like 9.5 chromosome 11. In healthy persons, the synthesis of alpha and beta
globin chains is finely balanced during terminal erythroid differentiation but the mechanism of
balanced expression is unknown [17 Steensma et al., 2005,18 Lehmann and Carrell,1968)].

FIG.1 Hemoglobin structure

Epidemiology
Beta thalassemia is prevalent in countries around the Mediterranean, the Middle East, parts of
Central Asia, India, southern China and the northern states Africa and South America. The
prevalence of the most carriers is seen in Cyprus (14%), Saardinia (10%) and Southeast regions
of Dinia (10%) and Southeast Asia. A high frequency of beta thalassemia gene in these areas
due to natural selection against malaria is Plasmodium falciparum (19 Flint et al., 1998). The
immigrant populations and marriage between different ethnic groups causes thalassemia to be
common between all countries, even countries in northern Europe where thalassemia did not
previously there. It is estimated that about 1.5 percent of the world population are thalassemia
carriers, with about 60 000 people marked the birth annually; the majority of other are in
developing countries (20 Vichinsky,2005). According to the Thalassemia International
Federation assessment, only about 200 000 patients with thalassemia major are alive and
registered and regularly receive treatment in all over world (21 Taher et al,2013). Our country
has a large number of cases is affected beta-thalassemia major; its prevalence is various in
different geographical areas. The highest prevalence of β-thalassemia has been reported
around the Caspian Sea and the Persian Gulf by more than 10%. The prevalence of this disorder
in other areas is between 8-4% (22 Rahim and Abromand, 2008).

Pathophysiology
Hemoglobin (Hb) is the molecule that carries and transports oxygen all through the body.
Normal human hemoglobin is a tetramer formed by two pairs of globin chains attached to
heme. The hemoglobin type is determined by the combination of tetra-globin chains (α, β, δ,
and γ chains). Each globin chain is structurally different and thus has different oxygen affinity,
electrical charge, and electrophoretic mobility. Normal adult hemoglobins are expressed as A2,
A and F (fetal). Ninety-five to ninety-eight percent of adult hemoglobin is A the major
hemoglobin, which consists of two α- and two β-chains (α2, β2). Hemoglobin A2 (α2, δ2), the
remainder of hemoglobin in adults is a minor component (less than 3.3%), and 1% or less of F
(α2, γ2) (23Nathan & Oski, 1993.), the gamma hemoglobin (Hb-F) is the predominant
hemoglobin found only during fetal development. The equal production of α and non-α (β, δ, γ)
globin chains is necessary for normal red blood cell (RBC) function. The failure in hemoglobin
synthesis is a main cause of microcytosis and anemia in many population groups around the
world. Hb variants are characterized by the gene mutation of the globin chains form
hemoglobin (i.e., the replacement of different amino acids at a certain position). Thalassemia
occurs when there is decreased or absent production of one of the types of globin chains (most
commonly either α or β), that cause insufficeient amount of normal structure globin chains.
This results in an imbalance between α- and β-chains and causes the clinical features of
thalassemia (24Nathan & Gunn, 1966), it can be separated into two major types such as α-
thalassemia and β- thalassemia.

Fig.2. Red blood cell morphology is altered in patients with all forms of thalassemia.
Hypochromic microcytes and target cells are the main features in asymptomatic individuals.
Patients with more severe forms of thalassemia have the anisocytosis and poikilocytosis,
hypochromic microcytic, target cells, ovalocytes, occasional fragmented red blood cells.

The absence or decreased of normal production of α-globin chains results in a relative excess
of γ-globin chains in the fetus and newborn, and β-globin chains in children and adults. When
globin chains are not produced in equal amounts, any excess chains accumulate and precipitate
damaging the RBC and accelerating its destruction. The absence of normal production of α-
chains results in a relative excess of γ-globin chains in the fetus and newborn, and β-globin
chains in children and adults. Further, the β-globin chains are capable of forming soluble
tetramers (β-4, or Hb-H); yet this form of hemoglobin is unstable and tends to precipitate
within the cell forming insoluble inclusions (Heinz bodies) that damage the red cell membrane.
α-Thalassemia is generally less severe because the excess unpaired β-chains that accumulate
are less damaging to RBCs than the unpaired α chains. Furthermore, diminished
hemoglobinization of individual red blood cells results in damage to erythrocyte precursors and
ineffective erythropoiesis in the bone marrow, as well as hypochromia and microcytosis of
circulating red blood cells. (Fig 1) In β-thalassemia, reduced amount (β+) or absence (β0) of β-
globin chains excess α-chains accumulate in the RBC and precipitate because they are highly
insoluble. These precipitated globin chains occur in both erythroid precursors in the bone
marrow and circulating RBCs. The destruction of precursor RBCs results in ineffective
erythropoiesis, increased erythropoietin, and proliferation of the bone marrow. This expanded
bone marrow (up 25 to 30 times normal) can result in the characteristic bony abnormalities of
β-thalassemia if the process is not prevented by transfusion therapy. Prolonged and severe
anemia and increased erythropoietic drive also result in hepatosplenomegaly and
extramedullary erythropoiesis, leading to their premature death and hence to ineffective
erythropoiesis. The degree of globin chain reduction is determined by the nature of the
mutation at the β-globin gene located on chromosome 11. Peripheral hemolysis contributing to
anemia is more prominent in thalassemia major than in thalassemia intermedia, and occurs
when insoluble α-globin chains induce membrane damage to the peripheral erythrocytes.
Genes that regulate both synthesis and structure of different globins are organized into 2
separate clusters. The α-globin genes are encoded on chromosome 16 and the γ, δ, and βglobin
genes are encoded on chromosome 11 as demonstrated in Fig 2. Each individual normally
carries a linked pair of α-globin genes, 2 from the paternal chromosome, and 2 from the
maternal chromosome. Therefore, each diploid human cell has four copies of the αglobin gene.
The four α-thalassemia syndromes thus reflect the disease state produced by deletion or no-
function of one, two, three, or all four of the α-globin genes (25Higgs et al., 1989) (Table 1).
The silent carrier state of α-thalassemia represents a mutation of one copy of the α-globin gene
and results in no hematologic abnormalities.
Fig.3. Schematic represent of the globin gene loci. The upper panel shows the α-globin locus
that resides on chromosome 16. Each of the four alpha globin genes contributed to the
synthesis of the α-globin protein. The lower panel shows the β-globin locus that resides on
chromosome 11. The two γ-globin genes are active during fetal growth and produce
hemoglobin F. The "adult" gene, beta, takes over after birth

Molecular basis and classification

The thalassemia syndromes are one of the most thoroughly studied diseases at the molecular
level. Consequently, some explanation for the clinical heterogeneity seen in patients can be
explained at the molecular level.

α-Thalassemias
The major clinical syndromes resulting from α-thalassemia were first recognized in the mid
1950s and early 1960s through the association of the abnormal hemoglobins (Hb-H and Hb
Bart’s) with hypochromic microcytic anemia in the absence of iron deficiency (26Minnich et al.,
1954, 27Rigas et al., 1955), 28Lie-Injo & Jo, 1960). α-Thalassemia is divided into deletional and
non deletional types (29Bain 2006). There are at least 40 different deletions. The size of the
deletion is important and affects the clinical phenotype of hydrops fetalis. Over 95% of α
thalassemia is caused by large deletions involving one or both of the α-globin genes. The
αglobin gene cluster occurs on the short arm of chromosome 16, band 16 p 13.3 and includes
the α-globin genes as well as the embryonic genes (as two identical α-globin genes (α1 and α2)
that are aligned one after the other on the chromosome). Common α-thalassemia deletions
that spare the embryonic gene allow for the production of functional embryonic hemoglobin
early in gestation. In contrast, the large deletions (severe) lack the benefit of embryonic
hemoglobin. Non-deletion mutations may have a more severe phenotype than most of the
deletional mutations. The most common non-deletional α-thalassemia mutation is Hemoglobin
Constant Spring; this mutation of the stop codon results in 31 amino acids being added to α
chain. Depending on the production of α-globin chains, α-thalassemia determinants can be
classified into two groups: α° and α+. In α°-thalassemia the production of α-chains by the
affected chromosome is completely abolished; α+-thalassemia is defined by the variable
amounts of α polypeptide chains which can still be expressed in cis to the thalassaemic cluster.
This nomenclature, which describes α-thalassemias in terms of α-globin chain
expression/haplotype, has replaced the previous classification of these defects into severe (α-
thalassaemia-1) and mild (α-thalassaemia-2) forms (30Weatherail & Clegg, 1981). In the
passed , genetics of these syndromes were more confusing. This was b.comecause the adult
carriers of α-thalassemia do not produce large amounts of either Hb-H or Hb Bart’s. Although
the relatives of the affected individuals do not have a readily defined phenotype, it was
eventually shown that the offspring of individuals with Hb-H disease have raised levels of Hb
Bart’s (γ4) in the neonatal period (31Na-Nakorn et al., 1969), and the parents of individuals
with Hb-H disease and the Hb Bart’s hydrops fetalis syndrome have mildly hypochromic,
microcytic red cell indices (32Ali, 1969); sometimes Hb-H inclusions could be demonstrated in
occasional red cells (33McNiel JR, 1968). By 1969 it had been shown that HbH disease results
from the inheritance of α-thalassemia-1 x α-thalassemia-2 and the Hb Bart’s hydrops fetalis
syndrome results from α-thalassemia-1 x α-thalassemia 1) (NaNakorn et al., 1969,34 Pootrakul
et al., 1967). The structural organization of the α-globin genes revealed by blot hybridization
analysis (35Orkin, 1978), Normal individual have two α-genes on each chromosome 16 or four
copies of the α-globin gene (αα/αα) and carriers for α-thalassemia have either three (−α/αα) or
two (− −/αα) α genes. Thus, the most frequently encountered genotype of Hb-H disease is − −/−
α and Hb Bart’s hydrops fetalis is − −/− − (Orkin, 1978, 36Orkin et al., 1979, 37Phillips 3d et al.,
1980). Thus by 1980 the molecular genetics of α-thalassemia was understood. The four
αthalassemia syndromes thus reflect the disease state produced by deletion or nonfunction of
one, two, three, or all four of the α-globin genes (25Higgs et at., 1989). α-Thalassemia trait
occurs with deletion or nonfunction of two α-globin genes. The two genes are deleted from the
same chromosome (cis-deletion) or one gene is lost from each chromosome 16 (trans deletion).
The cis-deletion is most common in Asian and Mediterranean populations, whereas individuals
of African descent usually have the trans-deletion (25Higgs et al., 1989). Both varieties of α-
thalassemia trait produce an asymptomatic, mild anemia associated with microcytosis.
Hemoglobin H (Hb-H) disease, a three-gene deletion, usually results from inheritance of the cis
α-thalassemia trait from one parent and the one gene deletion from the other parent.
Therefore, this abnormality is rare in the black population because the cisdeletion is
uncommon. Hydrops fetalis results from deletion of all four α-globin genes and generally causes
death in utero because no physiologically useful hemoglobin is produced beyond the embryonic
stage. Although the α-thalassemia syndromes also are of varying clinical severity, these
differences cannot be explained by the number of deleted or nonfunctional genes. One of the
most frequent α-thalassemia mutations is the _ _SEA deletion, which deletes both α-globin
genes but spares the embryonic gene. Homozygosity for this deletion (_ _SEA) is the most
common cause of hydrops fetalis (38Chui & Waye 1998). The sparing of the embryonic gene
allows enough functional embryonic hemoglobin (Hemoglobin Portland 1 and Hemoglobin
Portland 2) to allow gestation to continue and the phenotype of hydrops fetalis to develop. In
contrast, other common α-thalassemia mutations (_ _FIL, _ _THAI) also lack the entire
embryonic α-globin cluster, and therefore do not produce the functional embryonic
Hemoglobin Portland. These embryos may terminate unnoticed early in gestation (Chui & Waye
1998). Over 5% of individuals in the Philippines are carriers for the _ _SEA or _ _FIL mutation.
Hydrops fetalis, while most common in Southeast Asia, is found worldwide among many ethnic
groups; _ _MED is a common α0-thalassemia mutation in Mediterranean regions, particularly
Greece and Cyprus. It has resulted in hydrops fetalis. Non-deletional α-thalassemia is found
throughout the world. Up to 8% of Southeast Asians are carriers of Hemoglobin Constant
Spring. In the Middle East, Hemoglobin αTSaudiα is a common α-thalassemia non-deletional
mutation. It is a mutation of the polyadenylation signal sequence of the α 2 gene, resulting in
decreased expression of structurally normal α chains. Hemoglobin Koya Dora, another
structural non-deletional mutation, is found in India. Other structural mutations, such as
hemoglobin Quong Sze found in Southeast Asia, are highly unstable and result in defects in the
hem pocket (Skordis, 2006, Leung et al., 2002).

α Thalassemia trait
α-Thalassaemia trait is usually caused either by the interaction of the normal haplotype with a
α°- or a α+-thalassaemia determinant or by the homozygosity for two α+ haplotypes. Much less
frequently this phenotype can be the result of compound heterozygosity for a deletional α+-
thalassaemia and a α+ determinant caused by a point mutation or even homozygosity for the
latter kind of determinant. Depending on the nature and localization of the mutation, the
phenotype of the trait can thus range from the silent cartier to individuals showing very
pronounced haematological abnormalities. Patients with α-thalassemia trait have microcytosis,
hypochromia, and mild anemia. Small amounts of hemoglobin Bart’s (a tetramer of γ chains:
γ4) may be noted on a newborn screen. Individuals with this disorder are asymptomatic and
do not require transfusions or any other treatment. The diagnosis of α-thalassemia trait is
considered when the patient has the appropriate RBC abnormalities, when iron deficiency and
β-thalassemia trait have been excluded, and when family studies (CBC, hemoglobin profile, and
review of the peripheral smear) are consistent with the diagnosis (Nathan & Oski 1993). To
make the diagnosis with complete certainty requires characterization of gene deletions with
restriction endonuclease mapping or globin chain synthesis studies showing a decreased (α:β
ratio. However, this confirmation rarely is indicated clinically.

Hemoglobin H diseases
Hemoglobin H (Hb-H) disease is the most severe non-fatal form of α-thalassemia syndrome,
mostly caused by molecular defects of the α-globin genes in which α-globin expression is
decreased, causes a moderate anemia with hypochromia, microcytosis, and red cell
fragmentation. Two common genotypes lead to the phenotype of Hb-H disease are
αthalassemia-1/α-thalassemia-2 and Hb Constant Spring/α-thalassemia-1. Both genotypes are
equally common but Hb Constant Spring/α-thalassemia-1 is more severe than
α-thalassemia1/α-thalassemia-2 (Fucharoen et al., 1988). Compound heterozygotes for α0- and
α+thalassemia (--/-α) with only one functional α-globin gene have a severe imbalance in globin
chain synthesis with a two- to five-fold excess of β-globin chains synthesis (Nathan & Oski
1993.). Newborns have large amounts of Bart’s Hb. (25Higgs et al., 1989). When the switch
from γ- to β-globin chain production occurs, Hb Bart’s (γ4) switches to Hb-H (β4) and the typical
picture of Hb-H disease results. The excess β-globin chains precipitate and form a characteristic
abnormal hemoglobin; hemoglobin H (Hb-H) or β-globin tetramer (β4). This causes a phenotype
of mild to moderate chronic hemolytic anemia named Hb-H disease characterized by readily
detectable Hb-H inclusion bodies in the peripheral blood cells. Hb-H is unable to transport
oxygen at physiologic conditions; therefore, patients have a more severe deficit in oxygen
carrying capacity than would be expected from their measured hemoglobin level (Nathan &
Oski 1993). Increased red cell destruction occurs because Hb-H containing cells are sensitive to
oxidative stress. Thus the complications of the disease are related to hemolysis and include
jaundice, hepatosplenomegaly, gallstones, and leg ulcers. Most affected individuals have a mild
disorder with an Hb concentration of 7-10 g/dL and require only symptomatic care with
occasional transfusions. Therefore, iron overload is rarely a problem in these patients. The
clinical phenotypes of Hb-H disease found in nondeletional α-thalassemia (--/αTα) are often
more severe than those caused by α+-thalassemia resulting from simple deletion (--/α). Recent
molecular analysis of more than 500 thalassemia carriers at the Department of Pediatrics, Siriraj
Hospital, Thailand, revealed that the frequency of deletional α-thalassemia is significantly
higher compared with non-deletional mutations (mainly Hb CS and Pakse) in Thai population
(15%-20% vs 1%-2%, respectively). However, the number of symptomatic patients with Hb-H
disease due to non-deletional mutations appeared to be higher than those with deletional Hb-H
(60% vs 40% from 350 Hb-H disease patients), as shown in Table 1, suggesting that non-
deletional Hb-H patients have more significant clinical symptoms and require more medical
attention (Fucharoen & Viprakasit, 2009). A striking clinical feature of Hb-H disease is the
sudden drop in the Hb concentration with associated symptoms of acute anemia during
episodes of pyrexia (Chinprasertsuk et al., 1994). It has been postulated that fever either alone
or together with oxidative substances released in the process of infection, induces the unstable
Hb-H to precipitate in the red cells as inclusion bodies. These red cells then either hemolyze or
are rapidly destroyed by the reticuloendothelial cells. Blood transfusions should be given
together with treatment for infections. Body temperature should be normalized as quickly as
possible in order to reduce induction of Hb-H precipitation within the red cells. Hb Constant
Spring is detected in addition to Hb-A and H in patients with Hb Constant Spring α-thalassemia-
1. They are slightly more severe than classical Hb-H disease with lower Hb concentrations,
larger spleens, higher levels of Hb-H and more red cells containing inclusion bodies (Fucharoen
et al., 1988).

Hemoglobin Bart’s Hydrops fetalis syndrome

Hydrops fetalis, the most severe form of α-thalassemia, occurs in infants whose parents both
have α-thalassemia syndrome (25Higgs et al., 1989). As discussed previously, these infants have
deletion of all four α-globin genes and produce only Hb Bart’s, Hb-H, and small amounts of
embryonic hemoglobins. Therefore, they have very little physiologically useful hemoglobin and
are hydropic secondary to severe anemia. These infants are usually stillborn or die shortly after
delivery (Chui & Waye, 1998, Leung et al., 2008, Lorey et al., 2001, Michlitsch et al., 2009,
Weatherall 2008) Advances in perinatal care and recognition of surviving homozygous
thalassemia newborns have precipitated studies of long-term survivors with this disorder.
Recently, the Newborn Screening Program of California reported 8 surviving α-thalassemia
major newborns along with 500 Hemoglobin H babies (Michlitsch et al., 2009). In southern
China, the prevalence of α0-thalassemia trait is 8.5% and 0.23% of births had homozygous
αthalassemia (Chui & Waye, 1998). In addition to China and Southeast Asia, Bart’s hydrops
fetalis is now being recognized in Greece, Turkey, Cyprus, India, Sardinia, and other parts of the
world (Chui & Waye 1998, Yang & Li, 2009, Suwanrath-Kengpol et al., 2005)

Clinical and haematological examinations reveal severely anaemic infants with variable
hemoglobin levels (3-10g/dl) and marked anisopoikilocytosis with large hypochromic red cells
and with the presence of numerous erythroblasts. The analysis of the hemolysate shows, in the
hydrops caused by the deletion of four α genes, about 80% Hb Bart's (γ4) and 20% Hb Portland
1 (ζ2 γ2) with very small amounts, if any, of Hb Portland 2 (ζ2 β2) and HbH (β4) (Kutlar et al.,
1989). Lower levels of Hb Portland 1 have been observed in genetic compounds for the SEA
deletion and the large Fil deletion which also eliminates the ζ gene (Kutlar et al., 1989). In the
rare cases of Hb-H disease with hydrops fetalis, in addition to Hb Bart's and the embryonic Hb
Portland 1 and 2, variable amounts of HbH, HbF and HbA can also be detected (Chan et al.,
1997). The lack of hem-hem interaction or Bohr effect and binds oxygen irreversibly tightly and
high oxygen affinity of Hb Bart's make this γ tetramer unsuitable for the delivery of oxygen to
the tissues. The ensuing hypoxia is the cause of fetal hydrops and intrauterine death caused by
massive organomegaly, severe albuminemia, and heart failure. This leads to gross body edema,
growth failure.

The blood film of neonate with hemoglobin Bart’s hydrops fetalis showing anisocytosis,
poikilocytosis and numerous nucleated red blood cells (NRBC).

β-Thalassemias
The β-thalassemias are widespread throughout the Mediterranean region, Africa, the Middle
East, the Indian subcontinent and Burma, Southeast Asia including southern China, the Malay
Peninsula, and Indonesia. Estimates of gene frequencies range from 3 to 10 percent in some
areas. (Weatherall, 1994) Within each population at risk for β-thalassemia a small number of
common mutations are found, as well as rarer ones; each mutation is in strong linkage
disequilibrium with specific arrangements of restriction-fragment length polymorphisms, or
haplotypes, within the β-globin cluster. A limited number of haplotypes are found in each
population, so that 80 percent of the mutations are associated with only 20 different
haplotypes. This observation has helped demonstrate the independent origin of βthalassemia
in several populations (Flint et al., 1993). There is evidence that the high frequency of β-
thalassemia throughout the tropics reflects an advantage of heterozygotes against. Plasmodium
falciparum malaria, (Weatherall, 1987) as has already been demonstrated in α-thalassemia
(Allen et al., 1997). The β-globin gene cluster is located on chromosome 11 and is not
duplicated like the αglobin genes. Therefore, each diploid cell contains only two β-globin genes.
Mutations are described that affect every step in the process of gene expression from
transcription and translation to post-translational stability of the β-globin chain (Kazazian Jr.,
1990) The variable clinical severity of the β-thalassemia syndromes depends on how
significantly these different mutations affect β-globin synthesis. Although over ninety such
mutations are known, a given mutation generally is found in one ethnic group and not another.
Nearly 200 different mutations have been described in patients with β-thalassemia and related
disorders. Although most are small nucleotide substitutions within the cluster, deletions may
also cause β-thalassemia (Weatherall 1994). All the mutations result in either the absence of
the synthesis of β-globin chains (β0-thalassemia) or a reduction in synthesis (β+thalassemia)
(Fig. 2). Mutations in or close to the conserved promoter sequences and in the 5' untranslated
region down-regulate transcription, usually resulting in mild β+ thalassemia. Transcription is
also affected by deletions in the 5' region, which completely inactivate transcription and result
in β0 -thalassemia. Both splicing of the messenger RNA (mRNA) precursor and ineffective
cleavage of the mRNA transcript are result in βthalassemia. In some mutations, no normal
message is produced, whereas other mutations only slightly reduce the amount of normally
spliced mRNA. Mutations within invariant dinucleotides at intron–exon junctions, critical to the
removal of intervening sequences and the splicing of exons to produce functional mRNA, result
in β0 -thalassemia. Mutations in highly conserved nucleotides flanking these sequences, or in
“cryptic” splice sites, which resemble a donor or acceptor splice site, result in severe as well as
mild β+-thalassemia. Substitutions or small deletions affecting the conserved AATAAA sequence
in the 3' untranslated region result in ineffective cleavage of the mRNA transcript and cause
mild β+thalassemia. Mutations that interfere with translation involve the initiation, elongation,
or termination of globin-chain production and result in β0 -thalassemia. Approximately half of
all β-thalassemia mutations interfere with translation; these include frame-shift or nonsense
mutations, which introduce premature termination codons and result in β0-thalassemia. A
more recently identified family of mutations, usually involving exon 3, results in the production
of unstable globin chains of varying lengths that, together with a relative excess of α-globin
chains, precipitate in red-cell precursors and lead to ineffective erythropoiesis, even in the
heterozygous state. This is the molecular basis for dominantly inherited (β+) thalassemia. In
addition, missense mutations, resulting in the synthesis of unstable β-globin chains, cause
β-thalassemia. β-thalassemia includes three main forms: Thalassemia Major, variably referred
to as Cooley's Anemia and Mediterranean Anemia, Thalassemia Intermedia and Thalassemia
Minor also called " β-thalassemia carrier", "β-thalassemia trait" or "heterozygous β-
thalassemia". According to Thalassemia International Federation, only about 200,000 patients
with thalassemia major are alive and registered as receiving regular treatment around the
world (Thalassemia International Federation: Guidelines for the clinical management of
thalassemia 2nd edition. 2008 [http://www.thalassemia. org.cy]). The most common
combination of β-thalassemia with abnormal Hb or structural Hb variant with thalassemic
properties is β-thalassemia/Hb-E which is most prevalent in Southeast Asia where the carrier
frequency is around 50%.

Hereditary transmission
The beta thalassemias are inherited in an autosomal recessive manner. The parents of an
affected child are obligate heterozygotes and carry a single copy of a disease causing beta
globin gene mutation. At conception, each child of heterozygotes parents has 25% chance of
being affected, 50% chance of being an asymptomatic carrier, and 25% chance of being
unaffected and not carrier.(2 Galanello and Origa ,2010)

β-thalassemia trait
Carriers of thalassemia, individuals with this disorder are heterozygous for a mutation that
affects β-globin synthesis (Kazazian Jr., 1990). They are mildly anemic with hypochromic,
microcytic RBCs. Targeting and elliptocytosis are often seen. As with α-thalassemia trait, one
must exclude iron deficiency to make the diagnosis. In general, patients with βthalassemia trait
have a lower mean corpuscular volume (MCV) and a higher red cell count for the degree of
anemia than seen in iron deficiency. Thus, the Mentzer index (MCV/RBC) is useful as a
screening test to differentiate thalassemia from iron deficiency. If the Mentzer index is < 13,
thalassemia is more likely; if > 13, iron deficiency is more common. Hb electrophoresis is
normal with iron deficiency, but with β-thalassemia trait the hemoglobin A2, (Hb A2) is often
e1evated. Globin chain synthesis studies show an excess of α-chains (Nathan & Oski 1993).
These patients need no treatment, but should receive genetic counseling regarding the
potential for having a child with β-thalassemia major or a combination of β-thalassemia trait
and sickle hemoglobin (Sβ-thal). When both parents are carriers there is a 25% risk at each
pregnancy of having children with homozygous thalassemia. Within the first months of life,
adult hemoglobin containing 2 pairs of α and βchain (Hb-A: α2β2) physiologically replaces fetal
hemoglobin (HbF: α2γ2).In β–thalassemia, deficient of production structurally normal β–chain
lead to anemia, largely as a consequence of ineffective hemopoiesis (59Olivieri 1999, Nathan &
Gunn, 1966).

Thalassemia intermedia
These β-thalassemia patients who clinically are between the extremes of thalassemia trait and
thalassemia major (Nathan & Oski 1993), have milder anemia and by definition do not require
or only occasionally require transfusion. The regular transfusion therapy is not required initially.
These patients usually maintain a hemoglobin level of 7 g/dL without transfusions. At the
severe end of the clinical spectrum, patients present between the ages of 2 and 6 years and
although they are capable of surviving without regular blood transfusion, growth and
development are retarded. At the other end of the spectrum are patients who are completely
asymptomatic until adult life with only mild anemia. Therefore, pregnant or older patients are
less able to tolerate the anemia and may need transfusion support. Hypertrophy of erythroid
marrow with the possibility of extramedullary erythropoiesis, a compensatory mechanism of
bone marrow to overcome chronic anemia, is common. Its consequences are characteristic
deformities of the bone and face, osteoporosis with pathologic fractures of long bones and
formation of erythropoietic masses that primarily affect the spleen, liver, lymph nodes, chest
and spine. Enlargement of the spleen is also a consequence of its major role in clearing
damaged red cells from the bloodstream.

β-Thalassemia major (Cooley’s anemia)

A Detroit pediatrician, Thomas Cooley, first described this disorder in 1925 after noticing
similarities in the appearance and clinical findings in several anemic children of Greek and
Italian immigrants (60Cooley et al., 1927). Prior to the advent of routine transfusion therapy,
βthalassemia major patients did not survive beyond the first few years of life. Survival is now
improved with hyper transfusion regimens, iron chelation therapy, and bone marrow
transplantation. Serious thalassemia is associated with iron overload, tissue damage, and
increased risk of cardiovascular complications. β-Thalassemias are the most important among
the thalassemia syndromes with an average trait prevalence of 7% in Greece, 15% among
Cypriots, and 4.8% in Thailand (65Weatherall, 1998, 62Weatherall & Clegg, 2001). Clinical
presentation of thalassemia major occurs between 6 and 24 months. Affected infants fail to
thrive and become progressively pale. Feeding problems, diarrhea, irritability, recurrent bouts
of fever, and progressive enlargement of the abdomen caused by spleen and liver enlargement
may occur. In some developing countries, where due to the lack of resources patients are
untreated or poorly transfused, the clinical picture of thalassemia major is characterized by
growth retardation, pallor, jaundice, poor musculature, genu valgum, hepatosplenomegaly, leg
ulcers, development of masses from extra medullary hematopoiesis, and skeletal changes
resulting from expansion of the bone marrow. Skeletal changes include deformities in the long
bones of the legs and typical craniofacial changes (bossing of the skull, prominent malar
eminence, depression of the bridge of the nose, tendency to a mongoloid slant of the eye, and
hypertrophy of the maxillae, which tends to expose the upper teeth). In β-thalassemia major,
severity of anemia requires initiation of blood tranfusions during infancy. If a regular
transfusion program that maintains a minimum Hb concentration of 9.5 to 10.5 g/dL is initiated,
growth and development tends to be normal up to 10 to12 years (Thalassemia International
Federation: Guidelines for the clinical management of thalassemia 2nd edition. 2008
(http://www.thalassemia.org.cy)). Transfused patients may develop complications related to
iron overload. Complications of iron overload in children include growth retardation and failure
or delay of sexual maturation. Later iron overload related complications include involvement of
the heart (dilated myocardiopathy or rarely arrythmias), liver (fibrosis and cirrhosis), and
endocrine glands (diabetes mellitus, hypogonadism and insufficiency of the parathyroid,
thyroid, pituitary, and, less commonly, adrenal glands) (64Borgna-Pignatti & Galanello, 2004).

Classification of beta thalassemia

1. Beta thalassemia major

2. Beta thalassemia intermedia

3. Beta thalassemia minor

Beta thalassemia major
Thalassemia major clinical symptoms appear between 6 and 24 month after birth. Babies with
thalassemia major will suffer nutritional problems, diarrhea, irritability, frequent bouts of fever,
abdominal distention and a progressive increase in the size of spleen and liver (20
Papakonstantinou et al., 2015). So skeletal changes including changes in the long bones of the
leg and craniofacial changes is seen in patients. In fact a regular blood transfusions can maintain
hemoglobin levels in the range of 5.9 to 5.10 grams per deciliter in patients and grow up is close
to normal until about 10 to 12 years (21 Taher et al.,2013). The patients, who receive blood,
may involve complications of iron overload in the tissues. Complications of iron overload in
children include delayed growth and sexual maturation. The next complications of iron
overload including heart damage Myocardiopathy and rarely, arrhythmia, liver (fibrosis and
cirrhosis), endocrine glands (diabetes), hypogonadism, parathyroid and thyroid insufficiency
(22 Trivedi and Sagare,2014). Complications consequent contamination blood transfusion cause
transmit hepatitis B virus that and C, human immunodeficiency virus. The risk of hepatocellular
carcinoma in patients with infectious liver and iron overload are greater eventually these
patients die due to cirrhosis of the heart muscle caused by iron deposition in the heart (23
Salama et al., 2015, 24 Elalfy et al.,2013).

Beta-thalassemia intermedia
Symptoms of people beside thalassemia major are determined later, they have milder anemia
and do not need to regular blood transfusion. At one end of the spectrum of clinical symptoms
will be displayed between the ages of 2 to 6 years in these patients (10 Pagon et al.,
2013).Although, they can survive without regular blood transfusions. But suffer from a lack of
sufficient growth. At the other end of the spectrum of disease, does not show specific
symptoms except mild anemia into adulthood. In general, patients with thalassemia
intermedia, hypertrophy and increased erythroid mass and the possibility of extra medullary
hematopoiesis are common for overcome the chronic anemia (25 Galanello and Origa,2012).
Extra medullary erythropoiesis may cause neurological damage such as pressure on the spinal
cord and vertebral column. Foot ulcers and incidence of thrombosis are more common in
thalassemia intermedia compared to the thalassemia major, especially after splenectomy (26
Keikhaei et al.,2013).

Beta-thalassemia minor
 Thalassemia minor carriers except for mild anemia are clinically asymptomatic, this type of
thalassemia is heterozygous state that only one allele of theβ gene defect on chromosome 11
and the other allele is normal. The disease can be inherited by β 0 gene (nonsynthetic) and β+
gene (synthesis reduced) (12 Origa,2017).

Dominant beta-thalassemia

In contrast with the classical recessive forms of beta-thalassemia, which lead to a reduced
production of normal beta globin chains, some rare mutations result in the synthesis of
extremely unstable beta globin variants which precipitate in erythroid precursors causing
ineffective erythropoiesis. These mutations are associated with a clinically detectable
thalassemia phenotype in the heterozygote and are therefore referred to as dominant beta-
thalassemias [13 Weatherall,1998). The presence of hyper-unstable Hb should be suspected in
any individual with thalassemia intermedia when both parents are hematologically normal, or
in families with a pattern of autosomal dominant transmission of the thalassemia intermedia
phenotype. Beta globin gene sequencing establishes the diagnosis.

β-thalassemia associated with other Hb anomalies

Hemoglobin E (Hb-E)
The most common combination of β-thalassemia with abnormal Hb or structural Hb variant
with thalassemic properties is Hb-E/β-thalassemia which is most prevalent in an area stretching
from northern India and Bangladesh, through Laos, Carmbodia, Thailand, Vietnam, Malaysia,
the Philippines, and Indonesia where the carrier frequency is around 50%. Hb-E is caused by a
mutation of the 26th amino acid of a normal β-chain, glutamine, is replace by lysine. This
mutation also activates a cryptic synthesis of the β-globin chain and leads to a thalassemic
phenotype. Furthermore, the hemoglobin E gene, which can interact with β-thalassemic alleles
and cause a broad phenotypic spectrum, reaches a frequency of up to 50% in Thailand
(65Weatherall 1998). These Hb-E/beta-thalassemias may be identified to three categories
depending on the severity of symptoms:

Mild Hb-E/β-thalassemia
It is observed in about 15% of all cases in Southeast Asia. This group of patients maintains Hb
levels between 9 and 12 g/dl and usually does not develop clinically significant problems. No
treatment is required.
Moderately severe Hb-E/β-thalassemia
The majority of Hb-E/β-thalassemia cases fall into this category. The Hb levels remain at 6-7
g/dl and the clinical symptoms are similar to thalassemia intermedia. Transfusions are not
required unless infections precipitate further anemia. Iron overload may occur.

Severe Hb-E/β-thalassemia
The Hb level can be as low as 4-5 g/dl. Patients in this group manifest symptoms similar to
thalassemia major and are treated as thalassemia major patients.

Patients with HbC/beta-thalassemia may live free of symptoms and be diagnosed during routine tests.
When present, clinical manifestations are anemia and enlargement of the spleen. Blood transfusions are
seldom required. Microcytosis and hypochromia are found in every case. The blood film shows
distinctive Hb C crystals with straight parallel edges, target cells, and irregularly contracted cells with
features of thalassemia such as microcytosis.

The association of hereditary persistence of fetal Hb (HPFH) with beta-thalassemia mitigates the
clinical manifestations which vary from normal to thalassemia intermedia.

Individuals with HbS/beta-thalassemia have a clinical course similar to that of Hb SS.

Thalassemia in Pakistan
Beta thalassemia In Pakistan, is one of the most common genetic disorder with a carrier rate of
1.4–8.0% (average 5.0%) in different parts across the country with a total frequency of <5.0%
[12 Ahmed et al.,2000 ,13 Alwan et al.,1997].

Pakistan has a population of approximately 180 million people and β-thalassemia has an overall
carrier frequency of more than 5% in Pakistan and there are approximately nine million carriers
ofβ-thalassemia in the country (Hafeez et al,2007)Approximately 40,000 cases of transfusion
dependent children with thalassemia major are presently registered and each year nearly 5000
affected children are born nationwide.The associated risk factors are high frequency of the
gene, consanguineous marriages, increased birth rate, and large population size (Alwan and
Modell 1997). It is reported that Pakistani children rank highest among WHO’s member states
of Eastern Mediterranean Region (EMRO) in the list of transfusion dependent Thalassemia
disorders (Alwan and Modell 1997). In a recent study it is was reported that in the Pakistani
population Beta Thalassemia is more prevalent in Punjabis (60.7 %) as compared with Saraikis
(25.5 %) (Ahmed 1998; Ahmed et al. 2002; Khan et al. 1995). Another study investigated the
ethnic and geographical ratio of beta Thalassemia distribution in Pakistan and found that the
prevalence of the disorder was higher in Sindhis (48 %), followed by Punjabis (21 %), Balochis
(19 %) and Pathans (12 %) ethnic groups (Abdullah et al. 2011). Ishaq et al. (2012) reported
inadequate knowledge about prenatal diagnosis and premarital screening among parents of
children with Thalassemia; however, they did not specifically identify the areas which need
improvement, and they did not discuss false conceptions about the disease due to socio
cultural and religious factors that influence decision making about birth planning (Gill and
Modell 1998).

Regardless of being the population Bat high risk^ for Thalassemia major, evidence suggests that
Pakistan is possess poor knowledge of the disease (GillandModell1998). This lack of awareness
is influenced by societal factors, literacy rate, cultural boundaries and certain religious
preferences held by the people. This apparent lack of knowledge and awareness along with
psychosocial and cultural issues act as barriers for the prevention and disclosure of the disease
(Chattopadhyay 2006). Parental screening helps couples to determine their carrier status and
assess the risk of having an affected pregnancy/birth (Atkin et al. 1998) but unfortunately
genetic testing is still not a common practice, and diagnoses are not made until parents are
faced with serious complications.

Previously, there have been few studies investigating the spectrum of beta thalassemia
mutations in various regions and ethnic groups of Pakistan. The present study was carried out
to determine the prevalence of beta-thalassemia mutation in distict Mansehra, and to identify
any genetic determinant that causes variable phenotypes. Furthermore, this study provides
valuable information that can be utilized in thalassemia prevention programs through carrier
screening, genetic counseling and prenatal diagnosis (PND) to control affected birth in the
population.

Occurrence of thalassemia
Exact data about the prevalence of hemoglobin disorders is not available in Pakistan but its
vertical transmission can be prevented by proper screening and counselling in families of
Thalassemia patients. Although spread of Thalassemia is difficult to prevent at this time in
Pakistan because of unawareness, lack of education, remote health counselling facilities. But this
can overcome with a program of health education, testing for the trait, genetic counselling and
easy accesses to prenatal diagnosis can provide families with full medical information to help
them have healthy children. Young people need to learn about their carrier status early enough to
consider all available options, including marriage and undertaking a pregnancy. Pakistan has a
population of approximately 180 million people and b-thalassemia has an overall carrier
frequency of more than 5% in Pakistan and there are approximately nine million carriers of b-
thalassemia in the country [21 Hafeez et al.,2007]. Approximately 40,000 cases of transfusion
dependent children with thalassemia major are presently registered and each year nearly 5000
affected children are born nationwide. Thalassemia is particularly prevailing in areas in which
malaria is found to be endemic in past and present, but the exact mechanism is still not known. It
is thought that in areas where malaria was prevalent, humans underwent a small genetic change
in their DNA which gave them an advantage over others making them more resistant to the
malaria infection. This is because important changes occurred in the RBC environment following
this genetic change that did not allow the parasite to survive and multiply, causing illness and
ultimately death. In Pakistan, the number of registered thalassemia children in different
thalassemia centers is around 22,000 whereas a similar number of children are living in villages
and are not registered with any thalassemia center. The occurrence of thalassemia carriers is 5-
8% in various racial groups which translate into 7 to 10 million individuals. The average life
expectancy of β-thalassemia patients in Pakistan is 10-12 years. Studies have suggested that
poverty, consanguinity and unawareness about the disease are the prominent factors in increasing
the prevalence of this particular genetic disorder.

Signs and symptoms of thalassemia

Iron overload:
The most common complications related to patients on regular transfusion are iron overload.
People with thalassemia can get an overload of iron in their bodies, and too much iron can
result in damage to the heart, liver, and endocrine system.

Infection:
People with thalassemia have an increased risk of infection and this happen is so dangerous for
organs of the body.

Bone deformities:
In this disease, the natural development of the body is affected. Consequently, it may be
observed in patients with thalassemia. In most cases, skull bone is seen.The bones of the face
and the skull become thicker, and in addition to skeletal malformations.

Enlarged spleen:
Enlargement of the spleen has more infectious, viral and bacterial causes, and is secondarily
due to bugs in the blood flow and liver failures. That is, if the liver becomes inflamed, it will
squeeze the spleen. Thalassemia is one of the diseases that lead to enlargement of the spleen.

Symptoms like anemia:

For example, Shortness of breath, Cold hands and feet, pale skin, Irritability, Dark urine and
Fatigue

MANAGEMENT
The management of thalassemia is guided by the severity of anemia, suppression of excessive
erythropoiesis and prevention of excess iron overload.

Blood transfusion
Severe anemia with hemoglobin <7 g% for more than 2 weeks is widely accepted as an
indication to start blood transfusion.[13 Toman et al.,2011]. The goal should be aimed to
maintain a pre pre transfusional Hb level of 9 to 10 g/dl and a post-transfusion Hb level of 13 to
14 g/dl. Such regime generally prevents growth impairment, organ damage and bone
deformities. Care should be taken to avoid faster transfusion exceeding 5 ml/kg/h and amount
of transfused RBC should not exceed 15 to 20 ml/kg/day. The frequency of transfusion is usually
every 2 to 4 weeks.

Patients with thalassemia intermedia may survive without chronic transfusion but the
development of hypersplenism may require splenectomy in such patients. Vaccination against
Streptococcus pneumoniae, Hemophilus influenzae and Neisseria meningitidis may be required
in such individuals. These individuals may develop iron overload from increased gastrointestinal
absorption of iron even without transfusion and therefore chelation therapy is started when
the serum ferritin concentration exceeds 300 ng/ml.

Carriers of α-thalassemia generally do not need treatment because their anemia is very mild.
Prophylactic iron should never be given to carriers of α-thalassemia as they are at risk of
developing iron overload. Individuals with HbH disease may require blood transfusions and
splenectomy. Oxidative drugs should be avoided in these patients.

Transfusional hemsiderosis
Thalassemia patients on chronic transfusion are susceptible for acquiring bloodborne infections
apart from developing iron overload. Patients who receive more than 100 units of packed RBCs
usually develop hemosiderosis. Serum ferritin, liver biopsy and imaging modalities like magnetic
resonance imaging and superconducting quantum-interference device can measure iron
overload in the body. Complications arising from iron overload are cirrhosis, endocrine
dysfunction[14 Giardina and Forget,2008. ,15. Prakash and Aggarwal ,2012] (glucose
intolerance, hypogonadism, hypothyroidism, hypoparathyroidism) and cardiomyopathy. To
prevent these complications iron chelating agents like desferoxamine (parenteral use) and
deferasirox (oral use) should be used early when starting transfusion therapy.

Bone marrow and cord blood transplantation

Bone marrow transplantation (BMT) remains the only definitive cure currently available for
patients with thalassemia. However the major limitation of allogenic BMT [16 Angelucci and
Baronciani ,2008] is the lack of an human leukocyte antigen identical sibling donor. Cord blood
transplantation is another option where with a low risk of graft versus host disease.

Gene therapy
Gene therapy for thalassemia is not very successful and the future of this therapy will depend
on efficiency of gene delivery and various other factors such as viral titers, non-oncogenic
insertion, the variable expression of globin genes and the variable contributions of the β-
thalassemia phenotype.[17]( Breda et.al.,2010)
Objectives

Chapter 2

MATERIALS AND METHODS

Sample Collection
Ethical Approval and Consent Procedure
The proposed study and consent procedure were approved by the institutional bioethical
committee of Hazara University, Mansehra. The description of the study was explained to
each subject who was willingly offering themselves for studies and appropriate informed
consent for taking blood samples was obtained from participants. Information about the co-
morbidities, ethnicity, occupation, level of education was recorded.

Setting and Study Population

Blood samples were collected from different thalassemic centers of Mansehra, the study was
conducted at center of Human Genetics, Hazara University, Mansehra, KP Province, Pakistan.
Different demographic parameter including sex, age, clinical symptom and family history of
beta-thalassemia with important pathological parameters were collected by direct interviews
with the parents of affected children. A signed consent form was obtained from the
participants. In this study, 70samples (acquired from families) were mined to find out the
incidence of b-thalassemia mutations in the population of Mansehra.

Duration of Sampling
In a duration of two months survey, 70patients suffering from beta thalassemia were selected.

Blood Sampling
About 5ml of blood were collected in EDTA tubes from individuals suffering from
thalassemia. The collected blood samples were stored in deep freezer at -20 °C for further
experimentation.

DNA Extraction
For the determination of different mutations of beta thalassemia, human genomic DNA was
extracted from whole blood samples using a standard protocol of DNA extraction, i.e the
phenol-chloroform method with little modification was used for the DNA extraction from the
leukocytes. The protocol optimized by Goode et al., 2011 was modified for the extraction of
DNA from blood. First of all, the EDTA tubes were thawed in order to melt the frozen blood. A
volume of 200 µl of the blood from each EDTA tube was shifted to 2ml labeled Eppendorf tube.
About 200µl of dilution buffer (98ml water, 0.5g sodium chloride, 2ml 50X TAE), 300 µl lysis
buffer (20mM of EDTA, 20mM of tris base and 4% of SDS with PH maintaining 8.0), 5 µl BME
(beta marceptoethanol) and 6.0 µl of proteinase K was added to the blood sample. The
Eppendorf tubes were vortexed in order to mix the lysis solution with the blood. The samples
were incubated for 3 hours and vortexed after every 30 minutes. After incubation, 400µl phenol
chloroform isoamyl alcohol was added and centrifuged at 8000rpm for 18 minutes. Three layers
were generated after centrifugation that consisted of upper layer of deoxyribonucleic acid
(DNA), central layer of proteins and terminal layer of phenol. The upper layer or the
supernatant was gently transferred to fresh marked tubes. Equal quantity of chilled isopropanol
was added to each tube. For about an hour samples were kept at -20˚C.Centrifugation of
samples were done for 10 minutes at 8000 revolutions per minute (rpm), to get the DNA pellet.
The supernatant was discarded and the pellet was washed with 500µl of 70% ethanol. The
samples were centrifuged at 8000rpm for 5 minutes and the tubes were kept inverted until it
got completely dry. After drying, addition of 40µl of double distilled water to the pellet was
done and the samples were preserved at room temperature for shorter period of time so that
the DNA got dissolved in the water.

Gel Electrophoresis
For examination of quality and quantity of DNA, the procedure of gel Electrophoresis was
adopted. About 1ml of Tris acetate buffer and 49ml of purified water was added in the flask for
the preparation of gel. About 0.5g of agarose was added to the flask and stirred well and
heated to get the agarose dissolved. For cooling, boiled solution was kept at room temperature.
About 15μl of EtBr was added to it. Combs were fixed in the gel tray and solution was poured.
For solidification of gel, it was kept at room temperature. Abstraction of combs caused
development of wells in the gel. About 300ml of 1X TAE running buffer was added in a gel tank.
In the gel tank, the gel tray containing the gel was completely dipped in running buffer. About
3μl of Deoxyribonucleic acid blended with 2μl bromophenol loading dye was carefully laden
into the wells. Electrophoresis was done for 25 minutes at 100 voltage current. The gel tray was
removed from the tank and examined through UV transilluminator. Confirmation of DNA was
done and photographed.

PRIMER SELECTION
ARMS-Primers and sequences
Primers Sequence(5'-3') PCR Product bp
IVS - I-5 (G – C ) Mutant 5-CTCCTTAAACCTGTCTTGTAACCT TGTTAG-3 285bp
Normal 5-CTCCTTAAACCTGTCTTGTAACCTTGTTAC-3
FSC 8-9 (+G) Mutant 5-CCTTGCCCCACAGGGCAGTAACGGCACACC-3 225bp
Normal 5-CCTTGCCCCACAGGGCAGTAACGGCACACT-3

ARMS PCR
ARMS PCR is a PCR-based method, which uses allele-specific priming. In this method, an
oligonucleotide primer with a triple end complementary to the sequence of a specific mutation,
coupled with a common primer is used in one PCR reaction. In parallel, a corresponding normal
primer coupled with a common primer is used in another PCR reaction. The presence of an
amplified product in the first reaction indicates the presence of the mutation while its absence
suggests presence of the normal DNA sequence at that specific site. In the second reaction the
presence of an amplified product suggests presence of a normal DNA sequence at that specific
site while its absence suggests presence of the mutation (8 Newton et al.,1989 ) (Figure 1).
Figure 1 shows ARMS-PCR turn on a 2% agarose gel. Presence of an amplified mutant the ARMS
primer product indicates presence of the mutant allele. All samples contain an internal control
band. Sample 1 contains an amplified product in the normal primer but lacks it in the mutant
primer; hence, implying a normal individual. Sample 2 contains an amplified product in both the
normal and mutant primers; assigning the individual of minor type. Sample 3 contains an
amplified product only in the mutant primer; hence, it is the sample of an individual who is of
major type.

PCR CODITION FOR ARMS

PCR Reagents
The ratio of the reagents used for the ARMS PCR are mentioned

S.NO PCR Reagents Reaction Volume

1 Common C 0.25µL
2 Forward-Primer(N) 0.25µL
3 Reverse-primer(M) 0.25µL
4 Template DNA 2µL
5 Taq DNA polymerase 7.5µL
6 ddH2O 5µL
Final Volume 15µL

A total of 20 µL of final PCR reaction volume was used for this purpose. The reaction volume
was composed of 0.5 micrograms of the DNA template, 0.01 µg of each of the four primers (2
control primers, 1 common primer, and 1 mutant/ normal ARMS primer for the normal/ mutant
reaction), 0.5 unit Taq DNA polymerase, and 0.2 mM of each dNTP in a solution of 10 mM tris-
C1, 50 mM MgCl2, and 1 mM spermidine.

Condition for PCR

TEMPARATURE PROFILE USED FOR ARMS PCR ANALYSIS
The PCR tubes containing the reaction mixture and template DNA were shifted to thermocycler
and specific conditions were applied which are shown in figure
 35CYCLES

95˚C 94˚C

5min 45s 72˚C 72˚C

68˚C 45s 10min

40s

10˚C HOLD

PCR conditions were 94ºC for 5 minutes (pre-denaturation), following 35 cycles of

denaturation: 94ºC for 45 seconds, 68ºC for 40seconds (annealing) and extension at 72ºC for
45seconds with a terminal elongation step at 72˚C for 10 min and then 10 ºC on hold.

Observation of PCR Amplified DNA

The preparation of 2.0% agarose gel involved melting of 2.0 grams of agarose in 49ml of
distilled water and 1ml of 1X TAE buffer in oven for about 2 minutes. When all the particles of
agarose got completely dissolved and the solution became transparent, it was kept cool down
to 45˚C or till borne by hand then 10μl of EtBr was added. The cooled solution was dispensed in
the gel casting tray and affixation of combs were done for formation of wells in the gel and kept
it for 5 minutes to solidify. After solidification the combs were removed from the gel and gel
was shifted to gel tank in the running buffer. PCR products were blended with 2μl bromophenol
loading dye and loaded into the wells. Gel electrophoresis was done for approximately 25
minutes at 100 volts in 1X TAE running buffer. The amplified product were visualized under UV
light, the size of band was compared with the 10kb (+) DNA ladder and photographed.

Electrophoresis conditions
For electrophoresis, 100 Volts for 25 minutes was used for separation of targeted amplicons.
100bp or 50bp DNA was used as size ladders for estimating the size of amplicon. The amplified
PCR product was visualized under ultraviolet (UV) light and pictures taken using a digital
camera.
 Chapter 3
RESULTS
Survey of different hospitals and sample collection
A consent form was filled from patients with thalassemia characters to collect different
demographic information like name, age, time of the disease inception, prescription and
address. All the individuals were briefed about the study and consent forms were got signed.

Extraction of genomic DNA

The genomic DNA was extracted from blood samples by using modified phenol chloroform
method (Chapter 2) in the Centre for Human Genetics, Hazara University, Mansehra.

Gel Electrophoresis of genomic DNA

The quality and quantity of Extracted DNA was confirmed on 1% agarose gel and the results are
shown clearly in the figure 3.1.

Fig.3.1. Agarose gel profile for extracted genomic DNA of thalassemia patients.

Gel Electrophoresis of PCR product

Blood groups among thalassemic patient
Blood group Male Female Total Percentage
A+
A-
B+
B-
AB+
AB-
O+
O-
TOTAL

Data regarding consanguinity of parents.

Consanguineous marriages No. of cases %ages
of parents
First cousin marriages 30
Second cousin marriages 10
Total
Non-consanguineous 4
marriages
Data not available
ARMS-PCR Screening:-
DNA was extracted from seventy thalassemic patient’s samples. ARMS-PCR technique was
employed to screen seven studied types of β-thalassaemia mutations using a specific set of
primers for each mutation. In all successful ARMS-PCR reactions, products for normal,
heterozygous and /or homozygous cases for each type of the studied mutations were observed.
For normal cases, the ARMS-PCR products was found within the normal primer reactions while
in positive diagnosed patients, the ARMS-PCR products was found within both normal and
mutant reactions in heterozygous cases and only within mutant primer reactions in
homozygous cases.

ARMS-PCR products were observed as shown in table 6 and the representative figures

Chapter 4
Discussion
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