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ROLE OF HEPCIDIN HORMONE IN PATIENTS OF β -THALASSEMIA MAJOR

A thesis

Submitted for Partial Fulfillment of the Requirements

of Master Degree in clinical Pathology

By Abeer Elsayed Ahmed Badawy

M.B.B.CH

Faculty of Medicine-Cairo University

Under Supervision Of

Prof. Dr. Nagelaa Ali Khalifa

Professor of clinical &chemical pathology Faculty of Medicine Zagazig University

Prof. Dr.

Mervat Abdallah Hesham

Professor of Pediatrics Faculty of Medicine Zagazig University

Dr. Ibtessam Ibrahim Ahmed Assist. Professor of Clinical and

Chemical Pathology Faculty of Medicine Zagazig University

Faculty of Medicine Zagazig University

2010

ROLE OF HEPCIDIN HORMONE IN PATIENTS OF β -

THALASSEMIA MAJOR

A thesis

Submitted for Partial Fulfillment of the Requirements of Master Degree in clinical Pathology

By

Abeer Elsayed Ahmed Badawy M. B. B. CH.

Faculty of Medicine - Cairo University

Under Supervision Of

Prof. Dr. Nagelaa Ali Khalifa

Professor of clinical & chemical pathology Faculty of Medicine Zagazig University

Prof. Dr.

Mervat Abdallah Hesham Professor of Pediatrics Faculty of Medicine Zagazig University

Dr. Ibtessam Ibrahim Ahmed

Assist. Professor of Clinical and Chemical Pathology Faculty of Medicine Zagazig University

Faculty of Medicine Zagazig University

2010

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االرحمن االرحيیم بسم هللا

إإنـك أأنـت االعليیـم قالواا سبحانك ال علم لنا إإال ما علمتنـا﴿

﴾االحكيیـم

االعظيیم هللاصدقق

)32( آآيیةسوررةة االبقرةة:

ACKNOWLEDGMENT

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First and foremost, thanks to Allah who helped me to finish this work.

I am honered to express my deepest appreciation and profound gratitude

to Prof. Dr.Nagelaa Ali Khalifa Professor of clinical & chemical pathology,

Faculty of Medicine, Zagazig University, for her kind supervision,

encouragement and constant guidance.

My deepest thanks and gratefulness to Prof. Dr. Mervat Abdallah

Hesham, Professor of Pediatrics, Faculty of Medicine, Zagazig University,

for her continuous support and advice.

I would like to express my deepest sense of gratitude and obligations to

Prof. Dr. Ibtessam Ibrahim Ahmed, Assist.Professor at department of

Clinical and Chemical Pathology, Faculty of Medicine, Zagazig University.

Lastly, I would like to express my deepest thanks to all my colleges in

clinical & chemical pathology department for their help and encouragement.

Dr. Abeer Badawy

2010

LIST OF ABBREVIATIONS

BMP: Bone morphogenetic protein.

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BMT:

Bp:

Dcytb:

DFO:

DMT1:

FEP:

GVHD:

HFE:

HIF:

HIV:

HJV:

HLA:

LCR:

MOD:

NGAL:

NMD:

Nrmap2:

NTBI:

PASP:

PHT:

sTfR:

TfR:

UGTIA:

VSS:

Bone marrow transplantation.

Base pair.

Duodenal cytochrome b.

Desferrioxamine.

Divalent Metal ion Transporter 1.

Free Erythrocyte Porphyrin.

Chronic graft-Versus-Host Disease.

High iron Fe.

Hypoxia-inducible factor.

Human Immune deficiency virus.

Hemojuvelin.

Human leukocyte antigen.

Locus control region.

Multi - organ dysfunctions.

Neutrophil gelatinase-associated lipocalin.

Nonsense- mediated mRNA decay.

Natural resistance macrophage-associated protein 2.

Non-transferrin bound iron.

Pulmonary artery systolic pressure.

Pulmonary hypertension.

Soluble Transferrin receptors.

Transferrin receptors.

Uridine diphosphate-glucoronyl transferase IA.

Volume of distribution at steady state.

LIST OF TABLES

Table 1 Main characteristics of genetic iron overload disorders

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Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

(Deugnier et al., 2008)………………………………………...

The clinical data of the studied groups…………….......

Liver and kidney functions results of the studied groups.........

Complete blood count results of the studied groups…………..

Results of iron study of the studied groups……………………

Hemoglobin Electrophoresis data……………………………...

Hepcidin concentration levels………………………………....

Ratio between hepcidin and Serum Ferritin…………………...

Correlation between hepcidin and other parameters……….....

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LIST OF FIGURES

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Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Fig. 10

Fig. 11

Fig. 12

The β-Globin Gene Cluster on the Short Arm of

Chromosome 11 (Nancy & Livieri, 1999)…………………..

The Normal Structure of the β-Globin Gene and the

Locations and Types of Mutations Resulting in β-

Thalassemia (Nancy & Livieri, 1999)……………………….

Effects of Excess Production of Free α-Globin Chains

(Nancy & Livieri, 1999)……………………………………..

Complications of beta thalassemia…………………………..

Peripheral blood film in Cooley anemia…………………….

Essential roles of iron (Taketani, 2005)……………………..

Normal distribution and storage of body iron (Andrews,

1999)……….………………………………………………..

Major pathways of iron transfer between cells and tissues

(Andrews, 2000)……………………………………………..

Iron absorption (Andrews, 2000)……………………………

Hepatic Iron Burden over Time and the Effect of Various

Hepatic Iron Concentrations in Patients with Thalassemia

Major, Homozygous Hemochromatosis, and Heterozygous

Hemochromatosis (Nancy & Livieri, 1999)………………...

Amino acid sequence and a model of the major form of

human hepcidin. The amino and carboxy termini are labeled

as N and C, The pattern of disulfide linkages between the 8

cysteines is also shown in the amino acid sequence (Ganz,

2003)……………………………………………….………..

Physiology of hepcidin-ferroportin interaction (Rivera et al.,

2005b)……………………………………………………….

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Fig. 13

Fig. 14

Fig. 15

Fig. 16

Fig. 17

Fig. 18

Fig. 19

Fig. 20

Fig. 21

Normal iron homeostasis mediated by an iron-sensing

feedback loop (Wrighting & Andrews, 2008)………………

Hepcidin mRNA expression (Nicolas et al., 2002)………….

Concentration of hepcidine of patients (in circles) and

concentration of hecidin of control (in squares)…………….

The variations of RBCs, HB, MCV, HCT (PCV) for 40

patients (30 patients and 10 controls)……………………….

Hepcidin and Serum Ferritin for 40 patients (30 patients and

10 controls)……………………………………………..

Correlation between hepcidin and Hb………………………

Correlation between hepcidin and HCT…………………….

Correlation between hepcidin and MCV……………………

Correlation between hepcidin and Serium Ferritin………….

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CONTENTS

INTRODUCTION……………………………………………………

AIM OF THE WORK………………………………………………..

LITERATURE REVIEW…………….………………………………

THALASSEMIA………………….………………………………

IRON METABOLISM…………...……………………………….

HEPCIDIN……..…………………………………………………

SUBJECTS AND METHODS………….……………………………

RESULTS………………………………...………………………….

DISCUSSION………………………………………………………..

CONCLUSIONS……………………………….…………………….

RECOMMENDATION…………………………….………………..

SUMMARY…………………………………………….……………

REFERENCES………………………..…………………………...…

.........................................................................................االملخص االعربى

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INTRODUCTION

Introduction  

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INTRODUCTION

The thalassemias are a heterogeneous group of genetic disorders of

haemoglobin synthesis, occurring more frequently in the Mediterranean

region, the Indian subcontinent, Southeast Asia, and West Africa .The

thalassemias are divided according to their severity into major which is

severe and transfusion dependent, intermediate and minor forms of illness.

The β-thalassemias are the most important types of thalassemia because

they are so common and usually produce severe anemia in their

homozygous and compound heterozygous states (Hillman et al., 2005).

In β-thalassemia major, the neonate is well at birth but develops severe

anemia, bone abnormalities, failure to thrive, and life-threatening

complications. In many cases, the first signs are pallor, yellow skin and

scleras in infants ages 3 to 6 months. Later clinical features, in addition to

severe anemia, include splenomegaly or hepatomegaly, with abdominal

enlargement, frequent infections, bleeding tendencies (especially toward

epistaxis), and anorexia (Fucharoen et al., 2000).

Transfusional iron overload is the most important complication of β-

thalassemia and is a major focus of management, which can be prevented

by adequate iron chelation. Extensive iron deposits are associated with

cardiac hypertrophy and dilatation, degeneration of myocardial fibers

(Aessopos et al., 1995; Du et al., 1997).

Hepcidin is a 25-amino-acid iron peptide hormone. Initially identified in

human plasma and urine as an anti-microbial molecule. Hepcidin is the key

regulator of systemic iron homeostasis and a pathogenic factor in anemia of

inflammation and hereditary hemochromatosis. Hepcidin inhibits iron

influx into plasma from duodenal enterocytes that absorb dietary iron, from

Introduction  

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macrophages that recycle iron from senescent erythrocytes and from

hepatocytes that store iron (Park et al., 2001).

Iron–Loading anemias are characterized by ineffective erythropoiesis

and increased intestinal iron absorption. Erythrocyte transfusions further

exacerbate the iron overload. The development of hepcidin- based

diagnostics and therapies for iron-loading anemias may offer more effective

approaches to prevent the toxicity associated with iron overload. The most

common iron- loading anemias are major forms of β-thalassemia

(Papanikolaou et al., 2005).

In the presence of systemic iron overload Patients with thalassemia

major in whom iron overload was more severe and anemia was partially

relieved by transfusions, had urinary hepcidin concentrations that were

higher than in thalassemia intermedia. These findings were interpreted as

supporting the dominant erythropoietic effect of exogenous hepcidin could

prevent the iron overload in iron–Loading anemias (Loreal et al., 2005).

Adamsky and his co-authors (2004) have found that iron overload is

less dominant than anaemia in regulating hepcidin expression in the setting

of the β-thalassemia major mouse model. The decreased expression of

hepcidin may explain the increased absorption of iron in thalassemia.

 

 

 

 

 

 

 

AIM OF THE WORK

Aim  of  THE  work    

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AIM OF THE WORK

The aim of the present work is to measure hepcidin concentration in

patients of β thalassemia major to explain its role in iron metabolism for

those patients who have iron overload.

LITERATURE REVIEW

Literature review

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LITERATURE REVIEW

THALASSEMIA

Definition The thalassemias are a heterogeneous group of genetic disorders of haemoglobin

synthesis, all of which result from a reduced rate of production of one or more of the

globin chains of haemoglobin. The thalassemias are among the most common genetic

disorders worldwide, occurring more frequently in the Mediterranean region, the Indian

subcontinent, Southeast Asia, and West Africa (Weatherall & Clegg, 2001). Brief historical review

Thalassemia was first defined in 1925 when Dr-Thomas B.Cooley

described five young children with severe anemia, splenomegally, and

unusual bone abnormalities and called the disorder erythroblastic or

Mediterranean anemia because of circulating nucleated red blood cells and

because all of his patients were of Italian or Greek ethnicity (Hillman et al.,

2005).

In Europe, Riette 1925 described Italian children with unexplained mild

hypochromic and microcytic anemia in the same year Cooley reported the

severe form of anemia later named after him. In addition, Wintrobe and

coworkers in the United States reported a mild anemia in both parents of a

child with Cooley anemia. This anemia was similar to the one that Riette

described in Italy. Only then was Cooley's severe anemia recognized as the

homozygous form of the mild hypochromic and microcytic anemia that

Riette and Wintrobe described. This severe form was then labeled as

thalassemia major and the mild form as thalassemia minor. The word

thalassemia is a Greek term derived from thalassa, which means "the sea"

(referring to the Mediterranean), and emia, which means "related to blood"

(Yaish, 2007).

Literature review

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Classification of the Thalassemias:

First, a clinical classification, which describes the degree of severity.

Second, the thalssemia can be defined by the particular globin chain that is

synthesized at a reduced rate. Finally, it is now often possible to subclassify

them according to defect in the globin chain synthesis (Cohen et al., 2004).

I. Clinical classification of the Thalassemias

The thalassemias are divided according to their severity into

major,intermediate and minor forms of illness, thalassemia major which is

severe and transfusion dependent , and the symptomless minor forms,

which usually represent the carrier state, or trait. Thalassemia intermedia

describes conditions that associated with a more severe degree of anemia

and splenomegaly than the trait but, not as severe as the major forms to

require regular transfusions (Weatherall & Clegg, 2001).

(a) Thalassemia major

Thalassemia major, the severe form of Thalassemia occurs when a child

inherits two mutated genes, one from each parent. Children born with

Thalassemia major usually develop the symptoms of severe anemia within

the first year of life. They lack the ability to produce normal adult

hemoglobin (HbA, α2β2). Children with Thalassemia major are so

chronically fatigued they fail to thrive and do not grow normally. Left

untreated, this disorder will cause bone deformities and eventually will lead

to death within the first decade of the child’s life (Talwar & Srivastava,

2004).

Literature review

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(b) Thalassemia Intermedia

Thalassemia Intermedia is a mild form of thalassemia that is caused by

the one of the more severe thalassemic genes and one of the milder

thalassemic genes. Children with Thalassemia intermedia start to develop

symptoms later in life than those with Thalassemia major. They are

moderately anemic but a large number of the patients survive without

regular blood transfusions. The severity of Thalassemia intermedia isn't

determined by hemoglobin levels alone; it also depends on how the

individual's feelings,their growth rate and development (Eldor &

Rachmilewitz, 2002).

(c) Thalassemia minor

Thalassemia minor, people with a Thalassemia trait in one gene are

known as carriers or are said to have Thalassemia minor. The only way to

know if they carry the Thalassemia trait is to have a special blood

hemoglobin electrophoresis which can identify the gene. The carriers of

Thalassemia minor show mild hypochromic microcytic anemia (Talwar &

Srivastava, 2004).

Normal Human Hemoglobin:

Function

Hemoglobin main function is carrying oxygen through the body to all of

the organs (Moreno et al., 2004).

Hemoglobin synthesis and structure

Hemoglobin synthesis requires the coordinated production of heme and

globin. Heme is the prosthetic group that mediates reversible binding of

Literature review

7

oxygen by hemoglobin. Globin is the protein that surrounds and protects the

heme molecule (Handin et al., 2003).

The combination of two alpha chains and two gamma chains form

"fetal" hemoglobin (α2γ2 ), termed "hemoglobin F". With the exception of

the first 10 to 12 weeks after conception, fetal hemoglobin is the primary

hemoglobin in the developing fetus. The combination of two alpha chains

and two beta chains form "adult" hemoglobin (α2β2), also called

"hemoglobin A". Although hemoglobin A is called "adult", it becomes the

predominate hemoglobin within about 18 to 24 weeks of birth (Handin et

al., 2003).

The genes that encode the alpha globin chains are on chromosome 16.

Those that encode the non-alpha globin chains are on chromosome 11. The

alpha complex is called the "alpha globin locus", while the non-alpha

complex is called the "beta globin locus" (Handin et al., 2003).

In the first trimester of intrauterine life, ζ, ε, α, and γ chains attain

significant levels and in various combinations form Hb Gower I (ζ2ε2), Hb

Gower II (α2ε2), Hb Portland (ζ2γ2), and fetal hemoglobin (HbF) (α2γ2 136-

G and α2γ2 136-A) .Whereas Hb Gower and Hb Portland soon disappear,

HbF persists and forms the predominant respiratory pigment during

intrauterine life. Before birth, gamma-chain production begins to wane so

that after the age of 6 months postpartum, only small amounts of HbF (<

2%) can be detected in the blood (Wood et al ., 2001).

In early intrauterine life, beta-chain synthesis is maintained at a low

level but gradually increases to significant concentrations by the end of the

third trimester and continues into neonatal and adult life. The synthesis of

delta chains remains at a low level throughout adult life (< 3%). Hence

during normal development, the synthesis of the embryonic hemoglobins

Literature review

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Gower and Portland is succeeded by the synthesis of HbF, which in turn is

replaced by the adult hemoglobins, HbA and HbA2 (Hardison , 2001).

Alpha Globin Locus

Each chromosome 16 has two alpha globin genes that are aligned one

after the other on the chromosome. For practical purposes, the two alph

globin genes (termed α1 and α2) are identical. Since each cell has two

chromosomes 16, a total of four alpha globin genes exist in each cell. Each

of the four genes produces about one-quarter of the alpha globin chains

needed for hemoglobin synthesis. The mechanism of this coordination is

unknown. Promoter elements exist 5' to each alpha globin gene. In addition,

a powerful enhancer region called the locus control region (LCR) is

required for optimal gene expression. (Hoff brand, 2006).

Beta Globin Locus

The genes in the beta globin locus are arranged sequentially from 5' to 3'

beginning with the gene expressed in embryonic development (the first 12

weeks after conception; called episolon). The beta globin locus ends with

the adult beta globin gene. The sequence of the genes is: epsilon, gamma,

delta, and beta. There are two copies of the gamma gene on each

chromosome 11. The others are present in single copies. Therefore, each

cell has two beta globin genes, one on each of the two chromosomes 11 in

the cell. These two beta globin genes express their globin protein in a

quantity that precisely matches that of the four alpha globin genes. The

mechanism of this balanced expression is unknown (Hoffbrand, 2006).

Upstream of the entire β globin complex is the locus control region

(LCR), which is essential for the expression of all the genes in the complex

Literature review

9

(Fig. 1). The general structure of the β globin gene is typical of the other

globin loci. The genomic sequence spans 1600 bp and codes for 146 amino

acids; the transcribed region is contained in three exons separated by two

introns or intervening sequences.The first exon encodes amino acid 1 to 29

together with the first two bases for codon 30, exon 2 encodes part of

residue 30 together with amino acids 31 to 104, and exon 3, amino acids

105 to 146 (Hardison, 2001).

Exon 2 encodes the residues involved in heme binding and αβ dimer

formation, while exons 1 and 3 encode for the non- heme- binding regions

of the β globin chain.The β globin gene promoter includes 3 positive cis-

acting elements: TATA box, CCAAT box and duplicated CACCC motifs.

In addition to these motifs, the region upstream of the β globin promoter

contains two binding motifs for the erythroid transcription factor GATA

1(Marini et al., 2004).

Literature review

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Fig. 1 The β-Globin Gene Cluster on the Short Arm of Chromosome 11

(Nancy & Livieri, 1999).

In Panel A, the β-globin–like genes are arranged in the order during

development. Panel B shows the timing of the normal developmental switching of

human hemoglobin.

Conserved sequences important for gene function are found in the

5’promoter region, at the exon-intron junction, and in the 3 untranslated

regions (3-UTR) at the end of the mRNA sequences. The 5’

untranslated region (UTR) occupies a region of 50 nucleotides between

the CAP site, the start of transcription, and the initiation (ATG) codon.

There are two prominently conserved sequences in the 5’ UTR of the

various globin genes (both α and β). The 3’ UTR constitutes the region

between the termination codon (TAA) and the poly (A) tail. It consists

of 132 nucleotides with one conserved sequence, AATAAA, located 20

nucleotides upstream of the poly (A) tail (Forget et al., 2001).

Literature review

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II. Genetic Classification of the Thalassemias a) Alpha-thalassemia

Alpha thalassemia syndromes can be expressed as α0 and α+. In the α0,

no alpha chains are produced. In the α+, the output of one of the linked pair

of alpha-globin genes is defective, and only some alpha chains are

produced. Within these general categories of the alpha-thalassemia

syndromes, there is considerable genetic and clinical heterogeneity due to

the interaction of the many possible mutations directing globin chain

synthesis (Rimoin et al., 2006).

Silent Carrier (α+-thalassemia carriers):

The loss of one gene diminishes the production of the alpha protein

slightly. This condition is so close to normal that it can be detected only by

specialized laboratory techniques which, until recently, were confined to

research laboratories. A person with this condition is called a "silent carrier"

because of the difficulty of detection (Sabella & Cunningham, 2006).

α0-Thalassemia minor or trait:

The loss of two genes produces a condition with small red blood cells,

and at most a mild hypochromic anemia. People who have this condition

look and feel normal, but thecondition can’t be detected certainly except by

DNA analysis (Sabella & Cunningham, 2006).

Hemoglobin H disease:

Deficiency of 3 α chains leads to production of excess β chains, forms

β4-tetramer and produces a serious hematological problem. Patients with

Literature review

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this condition have a severe anemia, and often require blood transfusions to

survive (Sabella & Cunningham, 2006).

Hemoglobin Bart’s hydrops syndrome:

The loss of all four alpha genes during intrauterine life results in γ4-

tetramers, produces a condition that is incompatible with life. Foetus with

four-gene deletion alpha thalassemia die in utero or shortly after birth.

Rarely, four-gene deletion alpha thalassemia has been detected in utero,

usually in a family where the disorder occurred in an earlier child. In utero

blood transfusions have saved some of these children. These patients

require life-long transfusions and other medical support (Sabella &

Cunningham, 2006).

b) Delta-Thalassemia A hereditary disorder characterized by reduced or absent delta-globin

thus effecting the level of hemoglobin A2 (Bouva et al., 2006).

c) Delta-beta-thalassemia In δβ+ thalassemia, mutational basis is due to extensive deletions of

delta and beta globin structural genes. Abnormal hemoglobin (Hb Lepore)

is produced due to unequal crossing over between mispaired δ and β globin

genes leading to δ and β fusion with segments of δ, β lost (Weatheral &

Clegg, 2000).

d) Beta-thalassemia

Definition:

The β-thalassemias are the most important types of thalassemia because

they are so common and usually produce severe anemia in their

Literature review

13

homozygous and compound heterozygous states. The fact that there are

only two genes for the beta chain of hemoglobin makes beta thalassemia a

bit simpler to understand than alpha thalassemia. (Hillman et al., 2005).

Local Prevalence and geographic distribution of β –thalassemia (in

Egypt)

El-Beshlawy stated that β-thalassemia is the most common chronic

hemolytic anemia in Egypt (85.1%). A carrier rate of 9-10.2% has been

estimated in 1000 normal random subjects from different geographical areas

of Egypt (El-Beshlawy, 1999).

International Prevalence and geographic distribution of β –thalassemia

Worldwide, 15 million people have clinically apparent thalassemic

disorders. People who carry thalassemia in India alone number

approximately 30 million. These facts confirm that thalassemias are among

the most common genetic disorders in humans. β thalassemia is much more

common in Mediterranean countries such as Greece, Italy, and Spain. Many

Mediterranean islands, including Cyprus, Sardinia, and Malta, have a

significantly high incidence of severe β thalassemia, constituting a major

public health problem. For instance, in Cyprus, 1 in 7 individuals carries the

gene, which translates into 1 in 49 marriages between carriers and 1 in 158

newborns expected to have β thalassemia major. As a result, preventive

measures established and enforced by public health authorities have been

very effective in decreasing the incidence among their populations. β

thalassemia is also common in North Africa, the Middle East, India, and

Eastern Europe. Conversely, β thalassemia is more common in Southeast

Asia, India, the Middle East, and Africa (Yaish, 2007).

Literature review

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Classification of β- thalassemia:

It occurs in three clinical forms: major, intermediate, and minor. The

resulting anemia’s severity depends on whether the patient is homozygous

or heterozygous for the thalassemic trait (Rund & Rachmilewitz, 2005).

Molecular basis of β thalassemia

There are two main varieties of β thalassemia alleles; β 0 thalassemia in

which no β globin is produced, and β+ thalassemia in which some β globin

is produced, but less than normal. In contrast to the α thalassemias, the β

thalassemias are rarely caused by deletions. One group of deletions affects

only the β globin gene and ranges in size from 290 bp to > 60 Kb. Of these,

only the 619 bp deletion at the 3’ end of the β gene is common (Thein,

1998).

The other deletions, although extremely rare, are of particular functional

and phenotypic interest because they are associated with unusually high

levels of Hb A2 in heterozygotes. These deletions differ widely in size, but

remove in common a region from positions –125 to +78 relative to the

mRNA cap site in the β promoter which includes the CACCC, CCAAT and

TATA elements (Forget et al., 2001).

The mechanism underlying the markedly elevated levels of Hb A2

appears to be related to the removal of the 5’ promoter region of the β gene.

This may remove competition for the upstream LCR leading to its increased

interaction with the γ and δ genes in cis, enhancing their expression (Wood

et al ., 2001).

There is a disproportionate increase of Hb A2 (α2 δ2) derived from the δ

globin gene cis to the β globin gene deletion. This mechanism may also

Literature review

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explain the moderate increases in Hb F which characterize this group of

deletions and those due to point mutations affecting the promoter region.

Although the increases in Hb F are variable, and modest in heterozygotes,

they are adequate to compensate for the complete absence of β globin in

homozygotes. Two homozygotes for different deletions of this kind have a

mild disease despite the complete absence of Hb A2 (α2 β2) (Craig et al .,

1992).

The vast majority of β thalassemias are caused by point mutations

within the gene or its immediate flanking sequences (Fig. 2). These single

base substitutions, minor insertions or deletions of a few bases are classified

according to the mechanism by which they affect gene regulation:

transcription, RNA processing or RNA translation. Mutations affecting

transcription can involve either the conserved DNA sequences that form the

β globin promoter or the stretch of 50 nucleotides in the 5’UTR. Generally

they result in a mild to minimal deficit of β globin output and can be silent

in carriers (Thein et al ., 2001).

Literature review

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Fig. 2 The Normal Structure of the β-Globin Gene and the Locations

and Types of Mutations Resulting in β-Thalassemia (Nancy & Livieri,

1999).

All β-globin–like genes contain three exons and two introns between

codons 30 and 31 and 104 and 105, respectively. Approximately half of the

β thalassemia alleles affect the different stages of RNA translation and in all

instances, no β globin is produced resulting in β0 thalassemia. Most of these

defects result from the introduction of premature termination codons due to

frameshifts or nonsense mutations and nearly all terminate within exon 1

and 2. Mutations that result in premature termination early in the sequence

(in exons 1 and 2) are associated with minimal steady-state levels of β

mRNA in erythroid cells, due to an accelerated decay of the abnormal

mRNA referred to as nonsense- mediated mRNA decay (NMD)

(Maquat,1995) .

Literature review

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Variants of β thalassemia

Dominantly inherited β thalassemia

In contrast to the common β thalassemia alleles that are prevalent in

malarial regions and inherited typically as Mendelian recessives, some

forms of β thalassemia are dominantly inherited, in that inheritance of a

single β thalassemia allele results in a clinically detectable disease despite a

normal α globin genotype. Heterozygotes have a thalassemia intermedia

phenotype with moderate anemia, splenomegaly and a thalassemic blood

picture. Apart from the usual features of heterozygous β thalassemia, such

as increased levels of HbA2 and the imbalanced α/β globin biosynthesis,

large inclusion bodies similar to those seen in thalassemia major are often

observed in the red cell precursors, hence the original term of inclusion

body thalassemia (Fei et al ., 1989).

Normal Hb A2 β thalassemias

The diagnostic feature of β thalassemia is the hypochromic microcytic

red cells and an elevated level of Hb A2 in heterozygotes, whether β+ or β0.

Normal Hb A2 β thalassemias refer to the forms in which the blood picture

is typical of heterozygous β thalassemia except for the normal levels of Hb

A2. Most cases result from co-inheritance of δ thalassemia (δ0 or δ+) in cis

or trans to the β thalassemia gene, which can be of the β0 or β+ type. One

relatively common form of normal Hb A2 thalassemia is that associated

with Hb Knossos. The mutation activates an alternative splice site reducing

the amount of normal transcript that contains the variant (Trifillis et al .,

1991).

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18

Another fairly common cause of normal Hb A2 β thalassemia phenotype

in the Greek population is the Corfu form of δ β thalassemia. The phenotype

of normal Hb A2 β thalassemia is also seen in heterozygotes for ε γ δ β

thalassemia and overlaps the phenotypes encountered in carriers of α

thalassemia (Trifillis et al ., 1991).

Silent β thalassaemia

The silent β thalassemias cause only a minimal deficit of β globin

production. Heterozygotes do not have any evident hematologic phenotype;

the only abnormality being a mild imbalance of globin chain synthesis.

These mutations have been identified in homozygotes who have a typical β

thalassemia trait phenotype or in the compound heterozygous state with a

severe β thalassemia allele where they cause thalassemia intermedia

(Gonzalez-Redondo et al ., 1989).

β thalassemia trait with unusually high Hb A2

Despite the vast heterogeneity of mutations, the increased levels of Hb

A2 observed in heterozygotes for the different β thalassemia alleles in

different ethnic groups are remarkably uniform, usually 3.5-5.5% and rarely

exceeding 6%. Unusually high levels of Hb A2 over 6.5% seem to

characterize the sub-group of β thalassemias caused by deletions that

remove the regulatory elements in the β promoter (Codrington et al .,

1990).

β thalassemia due to insertion of a transposable element

Transposable elements may occasionally disrupt human genes and result

in their inactivation. The insertion of such an element, a retrotransposon of

the family called L1 has been reported with the phenotype of β +

thalassemia (Divoky et al ., 1996).

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19

β thalassemia due to trans-acting determinants

Population studies have shown that ~1% of the β thalassemias remain

uncharacterized despite extensive sequence analysis, including the flanking

regions of the β globin genes. In several families, linkage studies

demonstrated that the β thalassemia phenotype aggregates independently of

the β globin complex implying that the genetic determinant is trans-acting

(Giordano et al ., 1998).

Somatic deletion of β globin gene

This novel mechanism was recently described in an individual who had

moderately severe thalassemia intermedia despite being constitutionally

heterozygous for β0 thalassemia with a normal genotype. Subsequent

investigations revealed that he had a somatic deletion of a region of

chromosome 11p15 including the β globin complex giving rise to a mosaic

of cells, 50% with one and 50% without any β globin gene. The sum total of

the β globin product is ~25% less than the normally asymptomatic β

thalassemia trait (Badens et al ., 2002).

Clinical features of severe β-thalassemic syndromes

Infants and children affected with β thalassemia have pallor, poor

development, and abdominal enlargement. Hemoglobin electrophoretic

patterns show a variable quantity of HbA2 (0% – 6%) depending on the

genotype of the patient. The anemia is due to a combination of ineffective

erythropoiesis, excessive peripheral red blood cell hemolysis, and

progressive splenomegaly (Weatherall & Clegg, 2000). The latter causes

an increase in plasma volume and a decrease in total red cell mass. The red

Literature review

20

cells are microcytic (mean corpuscular volume <70 fL) with marked

anisochromasia. The bone marrow shows marked erythroid hyperplasia, and

the serum ferritin level is elevated (Wonke, 2001).

In children and young adults, radiologic abnormalities include thinning

of the long bones with sun-ray appearance and dilatation of the marrow

cavities. The skull has a “hair-on-end” appearance because of widening in

the diploic space. Patients with thalassemia have enlarged maxillary sinuses

and tend to have a maxillary overbite. The face gradually assumes a

“mongoloid” appearance. Such changes promote infections in the ears,

nose, and throat. Because of chronic anemia and iron overload,

endocrinopathies such as hypopituitarism, hypothyroidism,

hypoparathyroidism, diabetes mellitus, cardiomyopathy, and testicular or

ovarian failure become common as the child with thalassemia grows older

(Cunningham et al., 2004; Rund & Rachmilewitz, 2005).

Thalassemia can be regarded as a chronic hypercoagulable state.

Venous and arterial thromboembolic phenomena tend to occur more

frequently in thalassemic patients who have undergone splenectomy.

Furthermore, such patients may develop progressive pulmonary arterial

disease due to platelet thrombi in the pulmonary circulation (Eldor &

Rachmilewitz, 2002).

Pathophysiology

Mechanisms of Anemia

Normal hemoglobin, hemoglobin A, is composed of 2 beta and 2 alpha

subunits. In beta thalassemia major, more than 200 mutations have been

described in the beta-globin genes, cause loss of both beta-globin subunits.

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21

This leaves the normally paired alpha subunits unpaired. Unpaired subunits

are catatonic (Handin et al., 2003).

Normally, compensatory mechanisms are present to protect the cell

from the small amounts of unpaired alpha subunits, which may regularly be

present. This significant excess of free α chains caused by the deficiency

of β chains causes destruction of the RBC precursors in the bone marrow

(i.e., ineffective erythropoiesis) (Fig. 3). This ineffective erythropoiesis and

profound hemolysis result in a severe anemia that is usually manifest in

affected individuals by age 6 months (Rimoin et al., 2006).

Fig. 3 Effects of Excess Production of Free α-Globin Chains (Nancy &

Livieri, 1999).

The physiologic response is to attempt to increase red cell production by

expanding the bone marrow space up to 30-fold and/or increase production

of non-beta hemoglobin chains such as A2 (δ) and fetal (γ) hemoglobin.

However, despite these mechanisms, erythropoiesis remains ineffective and

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22

these patients become transfusion-dependent early in life. In fact, the

presence or absence of adequate transfusions significantly impacts the

appearance of these patients and the course of the disease (Sabella &

Cunningham, 2006).

Excess unbound α-globin chains and their degradation products

precipitate in red-cell precursors, causing defective maturation and

ineffective erythropoiesis. Hemolysis Anemia stimulates the synthesis of

erythropoietin, leading to an intense proliferation of the ineffective marrow,

which in turn causes skeletal deformities and a variety of growth and

metabolic abnormalities (Cunningham et al., 2004).

Clinical features of β-thalassemia major

In β-thalassemia major, the neonate is well at birth but develops severe

anemia, bone abnormalities, failure to thrive, and life-threatening

complications. In many cases, the first signs are pallor and yellow skin and

scleras in infants ages 3 to 6 months. Later clinical features, in addition to

severe anemia, include splenomegaly or hepatomegaly, with abdominal

enlargement, frequent infections, bleeding tendencies (especially toward

epistaxis), and anorexia (Fucharoen et al., 2000).

Children with thalassemia major typically have small bodies and large

heads and may also be mentally retarded. Infants may have mongoloid

features because bone marrow hyperactivity has thickened the bone at the

base of the nose. As these children grow older, they become susceptible to

pathologic fractures as a result of expansion of the marrow cavities with

thinning of the long bones. They’re also subject to cardiac arrhythmias,

heart failure, and other complications that result from iron deposits in the

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23

heart and in other tissues from repeated blood transfusions (Fucharoen et

al., 2000).

Complications of β-thalassemia major

Iron overload of tissue is the most important complication of β-

thalassemia and is a major focus of management. Thalassemia major can be

complicated with CCF, hepatic failure, aplastic crisis, intercurrent infection,

growth retardation, delayed puberty, hemosiderosis and hemochromatosis.

Transfusion related infection (HIV, HB, HC), complications related to iron-

chelation therapy, endocrinopathies (diabetes mellitus, hypothyroidism,

hypogonadism), skeletal complications and multiorgan dysfunctions (MOD)

also may found (Fig. 4) (Deugnier et al., 2008).

Fig. 4 Complications of beta thalassemia.

Management of thalassemia and treatment-related complications

(Cunningham et al., 2004).

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24

Hyperbilirubinemia and a propensity to gallstone formation is a

common complication of β thalassemia and is attributed to the rapid turn-

over of the red blood cells, bilirubin being a break-down product of

hemoglobin. Studies have shown that the levels of bilirubin and the

incidence of gallstones in β thalassemia, from trait to major, is related to a

polymorphic variant (seven TA repeats) in the promoter of the uridine

diphosphate-glucoronyltransferase IA (UGTIA) gene, also referred to as

Gilbert’s syndrome (Sampietro et al., 1997).

Cardiac iron overload is the most frequent cause of death from chronic

transfusion therapy. Recurrent pericarditis may be the initial manifestation

of myocardial iron deposition. Ventricular tachycardia and fibrillation or

severe congestive heart failure often proves fatal . Cardiac complications,

such as pulmonary hypertension (PHT), are the leading cause of death in

beta-thalassemia patients. L-Carnitine, due to its role in fatty acid oxidation,

might help control the elevation in pulmonary artery systolic pressure

(PASP) (El-Beshlawy et al., 2008).

Causes of death in patients with Beta - thalassemia major:

The prognosis of patients with homozygous β-thalassemia major has

been improved by transfusion and iron-chelation therapy. Prognosis for

survival without cardiac disease is excellent for patients who receive regular

transfusions and whose serum ferritin concentrations remain below 2500ng

per milliliter with chelation therapy (Borgna-Pignatti et al., 2004).

The most common cause of death in patients with beta thalassemia is

heart failure followed by infection, liver cirrhosis, thrombosis, cancer, and

diabetus (Borgna-pignatti et al., 2004).

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25

Laboratory diagnosis of β-thalassemia major

The CBC count and peripheral blood film examination results are

usually sufficient to suspect the diagnosis. Hb evaluation confirms the

diagnosis in β thalassemia, Hb H disease, and Hb E/β thalassemia.

• In the severe forms of thalassemia, the Hb level ranges from 2-8

g/dL.

• MCV and MCH are significantly low, but, unlike thalassemia trait,

thalassemia major is associated with a markedly elevated RDW,

reflecting the extreme anisocytosis.

• The WBC count is usually elevated in β thalassemia major; this is

due, in part, to miscounting the many nucleated RBCs as leukocytes.

Leukocytosis is usually present, even after excluding the nucleated

RBCs. A shift to the left is also encountered, reflecting the hemolytic

process.

• Elevated reticulocytic count.

• Platelets count is usually normal, unless the spleen is markedly

enlarged.

• Peripheral blood film examination reveals marked hypochromasia

and microcytosis, hypochromic macrocytes that represent the

polychromatophilic cells, nucleated RBCs, basophilic stippling, and

occasional immature leukocytes shown in (Fig. 5)

• In thalassemia major, laboratory results show elevated bilirubin,

urinary and fecal urobilinogen levels (Wonke, 2001).

• Hb electrophoresis and alkali denaturation test reveal an elevated Hb

F fraction, which is distributed heterogeneously in the RBCs of

patients with β thalassemia, Hb H in patients with Hb H disease, and

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26

Hb Bart in newborns with β thalassemia trait. In β -0 β, no Hb A is

usually present; only Hb A2 and Hb F are found (Wonke, 2001).

• Free erythrocyte porphyrin (FEP) tests may be useful in situations in

which the diagnosis of beta thalassemia minor is unclear. FEP level is

normal in patients with the beta thalassemia trait, but it is elevated in

patients with iron deficiency or lead poisoning (Wonke, 2001).

• Decreased hepcidin level in patients with β thalassemia major.

Fig. 5 Peripheral blood film in Cooley anemia.

Iron studies are as follow:

Serum iron level is elevated, with saturation reaching as high as 80%

with decreased total iron binding capacity. The serum ferritin level, which is

frequently used to monitor the status of iron overload, is also elevated.

However, an assessment using serum ferritin levels may underestimate the

iron concentration in the liver of a transfusion-independent patient with

thalassemia. Increased levels of transferrin receptors (TfR) and soluble TfR

(sTfR). Complete RBC phenotype, hepatitis screen, folic acid level, and

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27

human leukocyte antigen (HLA) typing are recommended before initiation

of blood transfusion therapy (Hillman et al., 2005).

Management of B-Thalassemia Major

1- Regular blood transfusion and chelation therapy:

Regular transfusion therapy to maintain hemoglobin levels of at least 9

to 10 g per deciliter allows for improved growth and development and also

reduces hepatosplenomegaly due to extramedullary hematopoiesis as well

as bone deformities (Cunningham et al., 2004 & Old et al., 2001).

Choice of the scheme for blood transfusion in thalassemia major:

(a) Intermediate schemes: mean Hb 9-10 gm/dl are acceptable in terms

of daily living.

(b) Hypertransfusion schemes: mean Hb 10 gm/dl or greater, improve

the quality of life without accelerating the lethal complication of iron

overload

(c) Supertransfusion program: Maintaince of mean hemoglobin level at

11-12 gm/dl Hb level is not allowed to drop below 12 gm/dl and is

raised regularly to 14 by transfusion every 2-3 weeks

supertransfusion permits an excellent quality of life (Cohen et al.,

2004).

Prevention of Secondary Complications

The most common secondary complications are those related to

transfusional iron overload, which can be prevented by adequate iron

chelation. After ten to 12 transfusions, chelation therapy is initiated with

desferrioxamine B (DFO) administered five to seven days a week by 12-

hour continuous subcutaneous infusion via a portable pump. Recommended

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28

dosage depends on the individual's age and the serum ferritin concentration.

Young children start with 20-30 mg/kg/day, increasing up to 40 mg/kg/day

after age five to six years. The maximum dose is 50 mg/kg/day after growth

is completed. The dose may be reduced if serum ferritin concentration is

low. By maintaining the total body iron stores below critical values (i.e.,

hepatic iron concentration <7.0 mg per gram of dry weight liver tissue),

desferrioxamine B therapy prevents the secondary effects of iron overload,

resulting in a consistent decrease in morbidity and mortality (Borgna-

Pignatti et al., 2004).

The survival of individuals who have been well transfused and treated

with appropriate chelation extends beyond age 30 years. Offbrand et al

(2003); said that iron-chelation therapy is largely responsible for doubling

the life expectancy of patients with thalassemia major but Deferoxamine

continues to be the most common iron-chelating agent in use, but it has

several limitations: the need for parenteral administration (which is painful

and reduces compliance), side effects, and cost (which is prohibitive in

underdeveloped countries) (Borgna-Pignatti et al., 2004).

Much effort has been invested in the development of new orally active

chelators. Deferiprone, an orally administered chelator, was initially thought

to be an inadequate chelator that might worsen hepatic fibrosis. However,

cumulative worldwide experience indicates that the drug is safe and

effective. Long-term administration of deferiprone does not appear to be

associated with liver damage (Wanless et al., 2003). Adverse effects of

deferiprone include arthralgia, nausea and other gastrointestinal symptoms,

fluctuating liver enzyme levels, leukopenia, and rarely agranulocytosis and

zinc deficiency. Most of these effects can be monitored and controlled

(Ceci et al., 2002)..

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29

Overly vigorous chelation is associated with deferoxamine-induced

bone dysplasia, which can slow growth velocity in children and may be

only partially reversible (Rund & Rachmilewitz, 2005).

Deferasirox recently became available for clinical use in patients with

thalassemia. It is effective in adults and children and has a defined safety

profile that is clinically manageable with appropriate monitoring. The most

common treatment-related adverse events are gastrointestinal disorders,

skin rash, and a mild, non-progressive increase in serum creatinine

concentration. Post-marketing experience and several phase IV studies will

further evaluate the safety and efficacy of deferasirox. New strategies of

chelation using a combination of desferrioxamine and deferiprone have

been effective in individuals with severe iron overload; toxicity was

manageable (Wu et al., 2004; Tanner et al., 2007).

2- Bone marrow transplantation

Bone marrow transplantation (BMT) from an HLA-identical sib

represents an alternative to traditional transfusion and chelation therapy. If

BMT is successful, iron overload may be reduced by repeated phlebotomy,

thus eliminating the need for iron chelation. The outcome of BMT is related

to the pretransplantation clinical conditions, specifically the presence of

hepatomegaly, extent of liver fibrosis, and magnitude of iron accumulation.

In children who lack the above risk factors, disease-free survival is over

90% (Gaziev & Lucarelli, 2003).

A lower survival rate of approximately 60% is reported in individuals

with all three risk factors. Chronic graft-versus-host disease (GVHD) of

variable severity may occur in 5%-8% of individuals. BMT from unrelated

donors has been carried out on a limited number of individuals with β-

thalassemia. Provided that selection of the donor is based on stringent

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30

criteria of HLA compatibility and that individuals have limited iron

overload, results are comparable to those obtained when the donor is a

compatible sib. However, because of the limited number of individuals

enrolled, further studies are needed to confirm these preliminary findings

(La Nasa et al., 2005).

3- Cord blood transplantation.

Cord blood transplantation from a related donor offers a good

probability of a successful cure and is associated with a low risk of GVHD

(Locatelli et al., 2003; Walters et al., 2005). For couples who have already

had a child with thalassemia and who undertake prenatal diagnosis in a

subsequent pregnancy, prenatal identification of HLA compatibility

between the affected child and an unaffected fetus allows collection of

placental blood at delivery and the option of cord blood transplantation to

cure the affected child. On the other hand, in case of an affected fetus and a

previous normal child, the couple may decide to continue the pregnancy and

pursue BMT later, using the normal child as the donor (Orofino et al.,

2003).

4- Hematopoietic Stem-Cell Transplantation

Although hematopoietic stem-cell transplantation is the only available

curative approach for thalassemia, it has been limited by the high cost and

the scarcity of HLA-matched, related donors. The past several years have

brought progress in the realms of conditioning regimens, donor

identification and selection, and the development of alternative sources of

hematopoietic stem cells (Talwar & Srivastava, 2004).

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31

5- Splenectomy

Splenectomy is usually not needed if regular transfusion therapy is

followed. If the child already has a big spleen ( his transfusion requirement

increases to more than times of normal or more than 200 ml packed red

cells or over 400 ml of whole blood per kg), splenectomy is indicated .

Portal vein thrombosis is a recognized complication after splenectomy due

to hypercoagulable state in thalassemia (Wonke, 2001).

6- Diet and vitamins:

No strict regulations regarding diet can be recommended. However,

food rich in iron e.g., meat, liver, kidney and green leafy vegetables should

be avoided.Diet should include food high in phosphorus or phytates e.g.,

cereals bread, milk, soya beans, roasted peas, etc. to inhibit iron absorption.

Similarly tea can be taken along within an hour after meals to reduce iron

absorption (Wonke, 2001).

7- Vitamin C

Ascorbate repletion (daily dose not to exceed 100-150 mg) increases the

amount of iron removed after DFO administration. Vitamin C facilitates

iron chelation with DFO and should be supplemented in patients receiving

DFO (5 mg/kg/d maximum of 200 mg/d). However, in unchelated patients a

low vitamin C status is beneficial (Wonke, 2001).

8- Folic acid

Folic acid (5 mg per week) should be given to patients receiving on or

irregular transfusion. This is because of relative folate deficiency due to

increasesd folate consumption. However,patients receiving regular blood

transfusions ordinarily do not require folic acid unless actual deficiency

state exist (Wonke, 2001).

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32

Other therapies

1- Combination therapy and Induction of fetal hemoglobin synthesis

New chelation strategies, including the combination or alternate

treatment with the available chelators, are under investigation. Induction of

fetal hemoglobin synthesis can reduce the severity of β-thalassemia by

improving the imbalance between alpha and non-alpha globin chains.

Several pharmacologic compounds including 5-azacytidine, decytabine, and

butyrate derivatives have had encouraging results in clinical trials. These

agents induce Hb F by different mechanisms that are not yet well defined.

Their potential in the management of β-thalassemia syndromes is under

investigation (Pace & Zein, 2006).

2- Hydroxyurea treatment

The efficacy of hydroxyurea treatment in individuals with thalassemia is

still unclear. Hydroxyurea is used in persons with thalassemia intermedia to

reduce extramedullary masses, to increase hemoglobin levels, and, in some

cases, to improve leg ulcers. A good response, correlated with particular

polymorphisms in the beta-globin cluster (i.e., C > T at -158 G gamma), has

been reported in individuals with transfusion dependence. However,

controlled and randomized studies are warranted to establish the role of

hydroxyurea in the management of thalassemia syndromes (Bradai et al.,

2003 ).

3- Correction of molecular defects

The possibility of correction of the molecular defect in hematopoietic

stem cells by transfer of a normal gene via a suitable vector or by

homologous recombination is being actively investigated (Sorrentino &

Niehuis, 2001).

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33

Initial efforts at gene therapy were directed against diseases of the β -

globin gene. This therapeutic strategy involves the insertion of a normally

functioning β -globin into the patient's autologous hematopoietic stem cells

(Puthenveetil & Malik 2004).

The major problems with this type of gene therapy have been related to

vector construction. The genetic elements of the vector that are necessary

for appropriate regulation of the inserted gene have been defined .However,

the therapeutic gene must be inserted into a hematopoietic stem cell and

must be expressed at high levels, over an extended period, in an erythroid-

specific manner. In addition, the vector must be safe from recombination or

mutagenesis. Oncoretroviral and adenoviral vectors have been found to be

unsuitable for various reasons) (Puthenveetil &Malik 2004).

Recombinant human erythropoietin was shown to provide the benefit of

increasing "thalassemic erythropoiesis" without raising fetal hemoglobin.

The effect appeared to be dose-dependent and was observed primarily in

patients with thalassemia intermedia who had undergone splenectomy

(Persons et al., 2003).

Recently, long-acting darbepoetin alfa was shown to increase

hemoglobin levels substantially in patients with hemoglobin E β–

thalassemia disease. Two important obstacles to the use of recombinant

human erythropoietin are its relatively high cost and its subcutaneous

administration route, which restrict its use in developing countries.

Appropriate clinical protocols are needed to delineate the role of

recombinant human erythropoietin (alone or in combination with the

aforementioned drugs) in the treatment of thalassemia (Persons et al.,

2003).

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34

IRON METABOLISM

Iron is an essential nutrient that is required for the oxygen-carrying

capacity of hemoglobin. Failure to incorporate adequate iron into heme

results in impaired erythrocyte maturation, leading to microcytic,

hypochromic anemia. Therefore, circulating factors that modulate iron

availability are of major importance in erythropoiesis. Normally, the total

body iron endowment is maintained within a tight range between 3 and 5 g

(Andrews, 2000).

Systemic iron is distributed among erythrocyte precursors in the bone

marrow, tissue macrophages, liver, and all other tissues, with the largest

amount found in circulating erythrocytes. Homeostasis is maintained by

regulating the levels of plasma iron. Hepcidin, a circulating peptide

hormone, has recently emerged as a key modulator of plasma iron

concentration and thus, a central regulator of iron homeostasis (Nemeth,

2004).

The cross-talk which has taken place in recent years between clinicians

and scientists has resulted in a greater understanding of iron metabolism

with the discovery of new iron-related genes including the hepcidin gene

which plays a critical role in regulating systemic iron homeostasis.

Consequently, the distinction between (a) genetic iron-overload disorders

including haemochromatosis related to mutations in the HFE, hemojuvelin,

transferrin receptor 2 and hepcidin genes and (b) non-haemochromatotic

conditions related to mutations in the ferroportin, ceruloplasmin, transferrin

and di-metal transporter 1 genes, and (c) acquired iron-overload syndromes

has become easier (Loreal et al., 2005).

Iron is one of the most common elements in nature and a transition

metal , iron is involved in electron transport and maintenance of the

Literature review

35

respiratory chain , it is required for the functioning of proteins involved in (

oxidative energy production, oxygen transport, mitochondrial respiration

and inactivation of harmful oxygen radicals) , it is essential for the synthesis

of hemoglobin and myoglobin , it plays an important role in detoxification

of the reactive species and it is rate limiting in DNA synthesis (Fig. 6)

(Taketani , 2005).

In a normal balanced state, 1–2 mg of iron enters and leaves the body

every day. Dietary iron is absorbed by duodenal enterocytes and circulates

in the plasma bound to transferrin, the main iron transport protein. Most of

the circulating iron is used by the bone marrow to generate hemoglobin for

red blood cells, while around 10–15% is utilized by muscle fibers to

generate myoglobin. Iron released by tissue breakdown is absorbed and

recycled. Excess iron is stored by parenchymal cells in the liver and

reticuloendothelial macrophages. Traces of iron are lost each day by

sloughing of mucosal cells, loss of epithelial cells and any blood loss. Since

the human body has not evolved a mechanism to clear excess iron, disorders

of iron balance, such as iron overload, are among the most common

diseases in humans (Andrews, 1999).

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36

Fig. 6 Essential roles of iron (Taketani, 2005).

In a normal state, once iron has been absorbed it is complexed with

transferrin . Because of the important role of iron in metabolism, the human

body has many mechanisms to absorb, transfer and store iron, but none to

excrete excess iron Although serum ferritin levels are indicative of body

iron levels, a number of conditions can alter the correlation between serum

ferritin levels and body iron stores. Acute and chronic inflammation and

infections can greatly influence levels; ascorbate levels and increased

erythropoiesis can also affect circulating ferritin levels (Fig. 7) (Fleming

and Bacon, 2005).

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37

Fig. 7 Normal distribution and storage of body iron (Andrews, 1999).

Iron Metabolism and Homeostasis

Iron metabolism

Nearly all circulating iron is bound by the abundant serum glycoprotein

transferrin. Transferrin can carry one (monoferric transferrin) or two

(holotransferrin) atoms of iron per protein molecule. Erythroid precursors

are the primary consumers of circulating transferrin-bound iron. During

differentiation, changing rates of hemoglobin production correlate with

variations in the cell surface complement of transferrin receptor-1 (TFR1).

Fluctuations in TFR1 expression control the amount of transferrin-bound

iron entering into erythroid cells (Chan &Gerhardt, 1992).

The process by which transferrin delivers iron to these cells is called the

transferrin cycle. Upon binding to TFR1 at the cell surface, transferrin and

its iron are endocytosed. These endosomal compartments are actively

Literature review

38

acidified by proton pumps. Acidification facilitates iron release from

transferrin because low pH decreases the affinity of the protein for iron.

Iron then leaves the endosome through divalent metal ion transporter 1

(DMT1, also known as Nramp2 and SLC11A2) to become available for

heme biosynthesis (Canonne-Hergaux et al., 2001).

The insertion of iron into protoporphyrin IX, the final step in heme

biosynthesis, occurs in the mitochondrion. Mitoferrin, a protein mutated in

anemic zebra fish, was recently shown to act as a mitochondrial iron

importer necessary for heme biosynthesis . However, another group has

postulated that iron is transferred from endosomes directly into

mitochondria through direct membrane contacts between the organelles.

The fates of TFR1 and apotransferrin are more certain—they are recycled to

the cell surface and circulation, respectively, where they repeat the cycle

(Sheftel, 2007).

The amount of circulating, transferrin-bound iron is determined by three

coordinated processes: macrophage iron recycling, duodenal iron

absorption, and hepatic iron storage. When iron is administered

therapeutically, it is assimilated by one or more of these three tissues, which

play critical roles in iron metabolism and the maintenance of iron

homeostasis (Fig. 8) (Andrews, 2000).

1. Macrophage iron recycling Normal human erythrocytes have a finite life span of approximately 4

months. Tissue macrophages remove senescent and damaged erythrocytes

from circulation and breakdown hemoglobin to recycle iron, supplying most

of the requirement for new erythropoiesis. The process by which

macrophages distinguish aged and damaged erythrocytes is not fully

Literature review

39

understood, but it is likely that morphological changes in the erythrocyte

membrane facilitate recognition and uptake by macrophages (Knutson &

Wessling-Resnick, 2003).

Binding of erythrocytes to the macrophage cell surface initiates

phagocytosis and lysosome-mediated degradation of the erythrocyte

membrane. Heme oxygenases catalyze the oxidation of heme to biliverdin,

free iron, and carbon monoxide (Yachie, 1999).

Fig. 8 Major pathways of iron transfer between cells and tissues

(Andrews, 2000).

Similar to the transferrin cycle, liberated iron may be pumped from the

phagosome into the cytoplasm by DMT1, though this has not been

definitively established. Heme-derived iron can be utilized by the cell,

sequestered within the multimeric iron storage protein ferritin or exported

into the plasma (Jabado et al., 2002).

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40

The transmembrane transporter ferroportin is activated in macrophages

after erythrophagocytosis (Canonne-Hergaux et al., 2006). Ferroportin is

the only cellular iron exporter that has been identified in vertebrates

(Abboud & Haile, 2000). It is expressed in cells of the extraembryonic

visceral endoderm that provide nourishment to the developing embryo, in

the intestinal epithelium and in spleen and liver macrophages that recycle

iron (Donovan et al., 2005).

Tissue macrophages contain large amounts of iron, apparently because

they are unable to export it to the circulation. These results support the idea

that most iron liberated from heme in macrophages is mobilized through

ferroportin-mediated iron export to be utilized for erythropoiesis. Because

ferroportin transports ferrous iron, iron must be oxidized to its ferric form in

order to bind circulating transferrin (Harris, 1999).

A circulating ferroxidase, ceruloplasmin, is thought to carry out the

oxidation of iron exported from macrophages also ceruloplasmin appears to

be necessary to keep ferroportin on the cell surface. For both of these

reasons, it is not surprising that ceruloplasmin deficiency (a

ceruloplasminemia) leads to tissue iron loading with low transferrin

saturation and, often, mild anemia (De Domenico, 2007).

2. Duodenal iron absorption

In contrast to some other metals, there is no regulated mechanism for

iron excretion through the liver or kidneys. Macrophage-mediated iron

recycling alone cannot sustain erythropoiesis over the long term. Early in

life, the overall iron endowment must be increased to support growth. Later,

small obligatory iron losses from bleeding and exfoliation of skin and

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41

mucosal cells would lead to negative iron balance if not offset by

continuous iron intake. Thus, iron balance is achieved through regulated

dietary iron absorption (Andrews, 2000).

Dietary iron absorption occurs in the most proximal part of the

duodenum, the first section of the small intestine (Fig. 9). There, acidity

from stomach acid aids in the absorption of both the heme iron, primarily

derived from hemoglobin and myoglobin in meats and the inorganic iron,

from other food sources (Qiu et al., 2007).

Inorganic iron in the intestinal lumen is primarily in its ferric, oxidized

form. In order for iron to be absorbed, it must be reduced to the ferrous

form. Reduction of iron can be carried out by an enterocyte apical

membrane protein duodenal cytochrome b (Dcytb) (McKie et al., 2001).

The fact that the same iron transporter is used for cellular iron uptake

both in transferrin cycle endosomes and at the apical surface of the

intestinal epithelium is somewhat surprising. These two membranes are

quite different and must be reached through distinct targeting signals.

However, both sites are within a low-pH milieu, important because DMT1

uses cotransport of protons to move iron across the membrane (Sacher et

al., 2001; Xu et al., 2004).

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42

Fig. 9 Iron absorption (Andrews, 2000).

Depending on body iron needs, intracellular iron can be stored within

the enterocyte or exported into the plasma. Net absorption requires transfer

across the basolateral surface of the epithelium—iron retained within

enterocytes is lost from the body when those cells senesce and are shed into

the gut lumen. It appears that the transmembrane transporter ferroportin is

responsible for most if not all basolateral iron transfer (Donovan et al.,

2005).

A ferroxidase activity acts in concert with ferroportin-mediated export.

This can be supplied by the enterocyte-associated multicopper oxidase,

hephaestin, or by its circulating homologue, ceruloplasmin (Vulpe et al.,

1999).

3. Hepatocyte iron storage

Hepatocytes serve many important functions including detoxification of

the blood production of proteins that aid in host defense and storage of

essential nutrients such as glucose and iron. Excess circulating iron in both

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43

transferrin-bound and nontransferrin-bound forms can be taken up by

hepatocytes (Trenor et al., 2000).

Similarly, DMT1 cannot account for all hepatocyte iron uptake because

DMT1 knockout mice and human patients carrying loss-of-function

mutations in DMT1 accumulate iron in the liver (Iolascon et al., 2006).

Other hepatic iron importers must exist to participate in efficient iron

storage. Hepatocyte membrane proteins such as megalin, TFR2, CD163,

and L-type calcium channels are candidate iron importers that employ a

variety of molecular mechanisms to bring iron into cells (Borregaard &

Cowland, 2006).

Although hepatocyte iron uptake is not fully understood, it is clear that

once inside the cell, iron can be utilized in cellular processes or sequestered

in ferritin. When iron loss or demand is too great to rely solely on recycling

and absorption, iron is mobilized from hepatic storage to sustain

erythropoiesis. Ferroportin and ceruloplasmin are believed to be involved in

the process of iron export from hepatocytes, but this has not been shown

directly (Kozyraki et al., 2001; Kristiansen et al., 2001).

4. Systemic iron homeostasis

Systemic iron homeostasis maintains body iron content within tolerable

limits and dictates iron distribution. As the largest consumer of iron, the

erythron is particularly sensitive to iron insufficiency. When body iron

stores are depleted, iron deficiency anemia ensues. Rarely, iron deficiency

anemia can be caused by genetic lesions that interfere with intestinal iron

absorption, erythroid iron assimilation, or both. The best characterized of

these are mutations preventing the production of transferrin or inactivating

DMT1 (Iolascon et al., 2006; Mims et al., 2004).

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44

Far more commonly, deficiency results from an imbalance between

increased iron requirements associated with growth and blood loss and iron

acquisition from the diet. In this case, iron deficiency anemia is

characterized by low plasma iron, decreased iron stores, and the

accumulation of iron-free protoporphyrins. Iron-deficient erythrocytes are

small (microcytic), pale (hypochromic), and relatively fragile. Oxygen-

carrying capability caused by iron deficiency has measurable effects on

quality of life, associated with symptoms including fatigue and tachycardia

(Beutler et al., 2000).

Iron replacement therapy, through dietary supplementation, intravenous

iron, or transfusion of iron-rich erythrocytes, is the only treatment for iron

deficiency anemia. Although essential for several important cellular

functions, iron’s capacity to donate and accept electrons makes the metal

toxic. Several proteins such as transferrin, ferritin, lactoferrin, lipocalin, and

myoglobin exist to bind iron in a variety of contexts. Binding to these

proteins renders iron less able to react with its environment (Britton et al.,

2002).

Thus, a safe upper limit of body iron for each individual is set by the

capacity of iron-binding proteins to sequester iron. Once this capacity is

reached, free iron accumulates within the plasma and cells. The

accumulation of excess iron is undesirable because ferrous iron reacts with

hydrogen peroxide to produce hydroxyl radicals. These radicals damage

macromolecules resulting in cellular and tissue dysfunction and organ

failure associated with iron overload disorders (Britton et al., 2002).

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Iron Overload

The human body can only excrete one to two milligrams of iron. With

every unit of blood, about 200 to 250 milligrams of iron are transfused,

therefore iron overload will occur. Generally, iron overload can be

classified as primary (hereditary) or secondary (acquired), depending on

whether it results from a primary defect in the regulation of iron balance or

secondary to other genetic or acquired disorders or their treatment:

Primary iron overload results from abnormally regulated iron

absorption: Hereditary hemochromatosis is an example; caused by a

missense mutation on the C282Y gene. This mutation results in the

incorrect processing of regulatory receptors in the intestine (Porter, 2001).

Secondary iron overload can result as the indirect effect of a condition

or occur as a result of its treatment:

Ineffective erythropoiesis and anemias requiring repeated blood

transfusions such as (beta-thalassemia major, thalassemia intermediate,

myelodysplastic syndromes and sickle cell disease) (Porter, 2001).

When iron homeostasis is in balance, iron is absorbed from the diet

(gut) at a rate equivalent to 1–2 mg/day. After absorption from the

duodenum, iron enters the plasma where it is complexed with transferrin.

Transferrin-bound iron in the plasma is the main pool supplying iron to the

erythron, which cycles 20–30 mg of iron each day, as well as to hepatocytes

and other parenchyma, which cycle around 10% of this amount (Anderson

et al., 2007).

When transferrin becomes completely saturated during conditions of

iron overload, iron circulates in the bloodstream as extracellular non-

transferrin-bound iron (NTBI). In the case of transfusion the

reticuloendothelial system loses the capacity or gets filled up and has no

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46

ability to store more iron. It is bound to transferrin and when the capacity of

transferrin is saturated, it becomes the non-transferrin bound iron (NTBI)

(Anderson et al., 2007).

As iron loading progresses, the capacity of serum transferrin, the main

transport protein of iron, to bind and detoxify iron may be exceeded and a

non–transferrin- bound fraction of plasma iron may promote the generation

of free hydroxyl radicals, propagators of oxygen-related damage. The body

maintains a number of antioxidant mechanisms against damage induced by

free radicals, including superoxide dismutases, catalase, and glutathione

(Hershko et al., 1998).

In the absence of chelating therapy the accumulation of iron results in

progressive dysfunction of the heart, liver, and endocrine glands. Within the

heart, changes associated with chronic anemia are usually present in

patients who are not receiving transfusions and are aggravated by iron

deposition. In response to iron loading, human myocytes in vitro increase

the transport of non–transferrin-bound iron possibly thereby aggravating

cardiac iron loading (Parkes et al., 1993).

Extensive iron deposits are associated with cardiac hypertrophy and

dilatation, degeneration of myocardial fibers. In patients who are receiving

transfusions but not chelating therapy, symptomatic cardiac disease has

been reported within 10 years after the start of transfusions and may be

aggravated by myocarditis and pulmonary hypertension (Aessopos et al.,

1995; Du et al., 1997).

The survival of patients with β-thalassemia is determined by the

magnitude of iron loading within the heart. Iron-induced liver disease is a

common cause of death in older patients and is often aggravated by

infection with hepatitis C virus. Within two years after the start of

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47

transfusions, collagen formation and portal fibrosis have been reported; in

the absence of chelating therapy, cirrhosis may develop in the first decade

of life. The risk of hepatic fibrosis is augmented at body iron burdens

corresponding to hepatic iron concentrations of more than 7 mg per gram of

liver, dry weight (Fig. 10) (Niederau et al., 1996).

Fig. 10 Hepatic Iron Burden over Time and the Effect of Various

Hepatic Iron Concentrations in Patients with Thalassemia Major,

Homozygous Hemochromatosis, and Heterozygous Hemochromatosis

(Nancy & Livieri, 1999).

The transport of non–transferrin-bound iron is increased, possibly

aggravating iron loading in vivo. Iron loading within the anterior pituitary is

the primary cause of disturbed sexual maturation, early secondary

amenorrhea occurs in approximately one quarter of female patients over the

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48

age of 15 years. Even in the modern era of iron-chelating therapy, diabetes

mellitus is observed in about 5 percent of adults (Olivieri et al., 1998).

As the iron burden increases and iron-related liver dysfunction

progresses, hyperinsulinemia occurs as a result of reduced extraction of

insulin by the liver, leading to exhaustion of beta cells and reduced

circulating insulin concentrations. Studies reporting reduced serum

concentrations of trypsin and lipase suggest that the exocrine pancreas is

also damaged by iron loading. Over the long term, iron deposition also

damages the thyroid, parathyroid, adrenal glands and may provoke

pulmonary hypertension, right ventricular dilatation, and restrictive lung

disease. Bone density is markedly reduced in patients with β-thalassemia

(Tai et al., 1996; Olivieri et al., 1998).

Iron Chelation Therapy

Indications for chelation therapy

(i) Transfusions of 2 units/month persisting for at least one year.

(ii) Ferritin level of 1000 ng/ml.

(iii) Patients in which transplant is imminent.

(iv) Consider earlier chelation therapy in patients with compromised

organ function who experience increased transfusion burden

(Franchini & Veneri 2004).

Treatment Options

Three products are available worldwide:

1) Deferoxamine: Deferoxamine is indicated for the treatment of acute iron intoxication

and of chronic iron overload due to transfusion-dependent anemias.

Deferoxamine chelates iron by forming a stable complex that prevents the

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49

iron from entering into further chemical reactions. It readily chelates iron

from ferritin and hemosiderin but not readily from transferrin; it does not

combine with the iron from cytochromes and hemoglobin. Deferoxamine

does not cause any demonstrable increase in the excretion of electrolytes or

trace metals. Theoretically, 100 parts by weight of Deferoxamine is capable

of binding approximately 8.5 parts by weight of ferric iron (Borgna-

Pignatti et al, 2004).

Deferoxamine is metabolized principally by plasma enzymes, but the

pathways have not yet been defined. The chelate is readily soluble in water

and passes easily through the kidney, giving the urine a characteristic

reddish color. Some is also excreted in the feces via the bile. Long-term

therapy with deferoxamine slows accumulation of hepatic iron and retards

or eliminates progression of hepatic fibrosis. Iron mobilization with

deferoxamine is relatively poor in patients under the age of 3 years with

relatively little iron overload. The drug should ordinarily not be given to

such patients unless significant iron mobilization (e.g., 1 mg or more of iron

per day) can be demonstrated (Borgna-Pignatti et al, 2004).

2) Deferiprone: Promising chelating compounds are the 3-hydroxypyrid- 4-ones which

form strong, highly stable and water soluble 3:1 complexes with the Fe3+-

ion at physiological pH both in vitro and in vivo. The binding constants are

high in comparison with those of desferrioxamine and transferrin, the

physiological transport protein for Iron. They are capable of mobilizing iron

from transferrin, ferritin, hemosiderin, hepatocytes and macrophages .The

affinity for divalent metal ions is low (Franchini & Veneri 2004).

The first representative of this group which has been tested in humans is

deferiprone. This compound has shown very little toxicity in animal studies.

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50

After oral ingestion, deferiprone is rapidly absorbed, metabolised in the

liver and excreted in the urine as glucuronide (at least 90% of the absorbed

dose), as iron complex or as unchanged drug .Relatively little is known

about the pharmacodynamics of deferiprone (Franchini & Veneri 2004).

3) Deferasirox: Exjade (deferasirox) is an orally active chelator that is selective for iron

(as Fe3+). It is a tridentate ligand that binds iron with high affinity in a 2:1

ratio. Although deferasirox has very low affinity for zinc and copper there

are variable decreases in the serum concentration of these trace metals after

the administration of deferasirox. The clinical significance of these

decreases is uncertain. Deferasirox is highly (~99%) protein bound almost

exclusively to serum albumin. The percentage of deferasirox confined to the

blood cells was 5% in humans. The volume of distribution at steady state

(Vss) of deferasirox is 14.37 ± 2.69 L in adults. Deferasirox and metabolites

are primarily (84% of the dose) excreted in the feces. Renal excretion of

deferasirox and metabolites is minimal (8% of the administered dose). The

mean elimination half-life (t1/2) ranged from 8-16 hours following oral

administration (Cappellini et al., 2006).

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51

HEPCIDIN

Hepcidin is the key regulator of systemic iron homeostasis and a

pathogenic factor in anemia of inflammation and hereditary

hemochromatosis. Hepcidin inhibits iron influx into plasma from duodenal

enterocytes that absorb dietary iron, from macrophages that recycle iron

from senescent erythrocytes and from hepatocytes that store iron. Hepcidin

acts by binding to the cellular iron exporter ferroportin and causing its

internalization and degradation. Hepcidin production is increased by iron

excess and inflammation and decreased by anemia and hypoxia, however,

the molecular mechanisms of hepcidin regulation by iron, oxygen and

anemia are still unclear. Iron-loading anemias are disorders in which

hepcidin is regulated by opposing influences of ineffective erythropoiesis

and concomitant iron overload (Pigeon et al., 2001).

Hepcidin peptide:

Hepcidin is a 25-amino-acid iron peptide hormone. Initially identified in

human plasma and urine as an anti-microbial molecule. located on

chromosome 19q13.1, encodes a precursor protein preprohepcidin of 84

amino acids (aa). During its export from the cytoplasm, this full-length pre-

prohepcidin undergoes enzymatic cleavage, resulting in the export of a 64

aa prohepcidin peptide into the ER lumen. Serum prohepcidin levels have

been widely used to evaluate iron overload in clinical and preclinical studies

(Park et al., 2001).

Bioactive hepcidin indeed bears structural similarity to disulfide-rich

antimicrobial peptides. Hepcidin is synthesized in the liver as a propeptide

and has a characteristic furin cleavage site immediately N-terminal to the

25-amino-acid peptide (Park et al., 2001).

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52

The molecule is a simple hairpin whose 2 arms are linked across by

disulfide bridges in a ladderlike configuration. One highly unusual feature

of the molecule is the presence of disulfide linkage between 2 adjacent

cysteines near the turn of the hairpin. Compared with most disulfide bonds,

disulfide bonds formed between adjacent cysteines are stressed and could

have a greater chemical reactivity. Like other antimicrobial peptides,

hepcidin displays spatial separation of its positively charged hydrophilic

side chains from the hydrophobic ones, a characteristic of peptides that

disrupt bacterial membranes (Fig. 11) (Detivaud et al., 2005).

Fig. 11 Amino acid sequence and a model of the major form of human hepcidin.

The amino and carboxy termini are labeled as N and C, The pattern of disulfide

linkages between the 8 cysteines is also shown in the amino acid sequence (Ganz,

2003).

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53

Molecular regulation of hepcidin

The expression of hepcidin is regulated by many genes as HFE,

Hemojuvelin (HJV) and growth differentiation factor 15 (GDF15)

(Papanikolaou et al., 2004) .

HFE, Human hemochromatosis protein an MHC-class 1-like molecule,

is highly expressed in crypt cells (enterocyte, macrophage and hepatocyte).

The HFE gene is located on short arm of chromosome 6 at location 6p21.3.

It seems to enhance transferrin-bound iron uptake from the plasma into

crypt cells via TfR1, and may also inhibit the release of iron from the cell

via ferroportin. It regulates hepcidin expression, mechanism is uncertain but

it may participate in a signaling complex with TfR2 and interacts with TfR1

& β-2-microglobulin (Munoz et al.,2009).

Hemojuvelin (HJV) is a membrane-bound and soluble protein in

mammals that is responsible for the iron overload condition known as

juvenile hemochromatosis in humans, a severe form of hemochromatosis.

The hemojuvelin protein is encoded by the HFE2 gene. Mutations in HJV

are responsible for the vast majority of juvenile hemochromatosis patients.

Hemojuvelin is highly expressed in skeletal muscle and heart, and to a

lesser extent in the liver. One insight into the pathogenesis of juvenile

hemochromatosis is that patients have low to undetectable urinary hepcidin

levels, suggesting that hemojuvelin is a positive regulator of hepcidin, the

central iron regulatory hormone (Papanikolaou et al., 2004).

For many years the signal transduction pathways that regulate systemic

iron homeostasis have been unknown. However, a study by Babitt et al

2006 suggested that hemojuvelin interacts with bone morphogenetic protein

(BMP), possibly as a co-receptor, and may signal via the SMAD pathway to

regulate hepcidin expression (Zhang et al., 2008).

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54

Recently, the iron and erythropoiesis-controlled growth differentiation

factor 15 (GDF15) has been shown to inhibit the expression of hepcidin in

β-thalassaemia patients, thereby increasing iron absorption despite iron

overload. (Tamary et al., 2008).

Mechanism of hepcidin action

Hepcidin causes a decrease in serum iron. Injection of hepcidin agonist

into mice results in hypoferremia already within 1 hour, and a similar effect

was seen with acute induction of hepcidin expression in tetracycline-

inducible transgenic mice. The hypoferremia develops because hepcidin

blocks the supply of iron into plasma while the relatively small plasma iron

pool is rapidly used up by erythrocyte precursors. Hepcidin blocks iron

flows from macrophages recycling iron, from stores in the liver and from

enterocytes absorbing dietary iron (Viatte et al., 2005).

The molecular mechanism is based on hepcidin's interaction with

ferroportin. Ferroportin is the only known cellular iron exporter in

vertebrates, and is expressed in all the tissues handling major iron flows as

reticulo-endothelial macrophages, hepatocytes and duodenal enterocytes.

Hepcidin binds to ferroportin and causes its internalization and degradation

in lysosomes, thus effectively blocking the export of iron from the cells

(Nemeth et al., 2004).

In vitro, the internalization of ferroportin occurs less than 1 hour after

addition of hepcidin, consistent with the kinetics of hypoferremia observed

in vivo. Likewise, injection of radiolabeled hepcidin in mice resulted in

equally rapid accumulation of radioactive hepcidin in ferroportin-rich

organs (spleen, duodenum and liver), providing further support for the key

role of hepcidin-ferroportin interactions in the regulation of iron transport

(Fig. 12) (Rivera et al., 2005b).

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55

Fig. 12 Physiology of hepcidin-ferroportin interaction (Rivera et al.,

2005b).

• Ferroportin = iron export protein.

• Circulating hepcidin.

• Hepcidin binds to ferroportin.

• Internalization, then ferroportin degradation.

• Degraded ferroportin.

• Decreased iron release due to decreased ferroportin.

Hepcidin maintains iron homeostasis through a physical interaction with

ferroportin has led to a plausible model for the normal maintenance of iron

homeostasis (Fig. 13) and the disruption of homeostasis in human disease

(Zoller &Cox 2005).

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56

Fig. 13 Normal iron homeostasis mediated by an iron-sensing feedback

loop (Wrighting & Andrews, 2008).

Hepcidin is the principal regulator of extracellular iron concentration

Hepcidin is increased by iron loading and this provides the homeostatic

loop to maintain normal extracellular concentrations of iron. A rise in

plasma iron (e.g. after a meal or an iron supplement) leads to increased

hepcidin production. In turn, elevated hepcidin reduces the concentration of

ferroportin molecules on the cell surface and inhibits the entry of iron into

plasma, thus allowing the iron concentration to return to normal levels.

Conversely, in iron deficiency, hepcidin production decreases, allowing a

greater export of iron through ferroportin into plasma; this results in an

appropriate rise in circulating iron (Delaby et al.,2005).

Chronic alterations of hepcidin expression result in systemic disorders

of iron metabolism and maldistribution of iron in the body. Homozygous

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57

disruption of the hepcidin gene in humans or mice leads to severe iron

overload. Conversely, overexpression of hepcidin in transgenic mice

resulted in severe microcytic, hypochromic anemia. Mice with tumor

xenografts engineered to overexpress hepcidin also developed hypoferremia

and anemia, with iron sequestration in the stores (Rivera et al., 2005a).

Similarly, overproduction of hepcidin by liver tumors in patients with

type 1a glycogen storage disease caused iron-refractory anemia which

resolved only after resection of the tumor, or after liver transplantation.

Altogether, these studies confirm the role of hepcidin as the negative

regulator of iron absorption, recycling and release from stores (Weinstein et

al.,2002).

Regulation of hepcidin synthesis; implications for the disorders of iron

metabolism

As an iron-regulatory hormone, hepcidin synthesis is increased by iron

loading and inflammation and is decreased by anemia and hypoxia. Except

for inflammation, the molecular pathways underlying regulation of hepcidin

are not well understood. Dysregulation of hepcidin synthesis, however,

appears to be the key factor in the pathogenesis of a spectrum of iron

disorders, with hepcidin deficiency causing iron overload and elevated

hepcidin mediating anemia of inflammation (Loreal et al.,2005).

Regulation by inflammation

Hepcidin synthesis is markedly induced by infection and inflammation.

In animal models, injection of turpentine, lipopolysaccharide, or Freund's

adjuvant increased hepatic hepcidin mRNA expression, and in humans,

infusion of lipopolysaccharide resulted in a rapid increase in urinary

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hepcidin. These effects are mediated by inflammatory cytokines including

interleukin (IL)-6 and IL-1. IL-6 is sufficient for hepcidin induction since

direct treatment of primary hepatocytes with IL-6 resulted in rapid

upregulation of hepcidin mRNA and infusion of human volunteers with IL-

6 resulted in increased urinary hepcidin excretion within just 2 hours after

infusion (Nemeth et al.,2004 ; Nemeth et al.,2003).

Hepcidin as a mediator of anemia of inflammation

Hepcidin increase was associated with hypoferremia in all the

inflammatory models. Increased hepcidin appears to be the key factor in the

development of anemia of inflammation. Hypoferremia and anemia of

inflammation have likely developed during evolution as a host defense

strategy against infection, limiting the growth of invading microbes.

However, the same strategy has become maladaptive with the increasing

incidence of non-infectious diseases associated with excessive cytokine

production, including rheumatologic diseases, inflammatory bowel disease,

multiple myeloma and other malignancies (Rivera et al., 2005a).

Anemia of inflammation is characterized by decreased serum iron and

impaired mobilization of iron from stores, evident from the presence of iron

in bone-marrow macrophages and increased ferritin levels. These are the

very features observed in mouse models with increased hepcidin.

Intraperitoneal injection of synthetic hepcidin resulted in hypoferremia

within 1 hour, and chronic over expression of hepcidin in tumors resulted in

anemia and hypoferremia despite increased liver iron stores (Rivera et al.,

2005b).

Furthermore, patients with infection or inflammatory disorders have

elevated urinary excretion of hepcidin compared to healthy controls. Thus,

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59

the molecular pathway from inflammation to anemia centers on the elevated

plasma hepcidin which causes the internalization and degradation of

ferroportin in macrophages, hepatocytes and duodenal enterocytes,

sequestering iron in these cells and blocking iron flows into plasma

(Nemeth et al.,2003).

As the bone marrow continues to utilize iron for hemoglobin synthesis,

the small plasma iron compartment becomes rapidly depleted causing

hypoferremia. Persistent hypoferremia, as in chronic inflammation, leads to

iron-restricted erythropoiesis and anemia. However, it still remains to be

established whether the increase in hepcidin is the essential factor in the

development of this disorder, since inflammation may contribute to anemia

by alternative hepcidin-independent mechanisms including decreased

erythropoietin production, blunted response to erythropoietin and shortened

erythrocyte lifespan (Nemeth et al., 2003).

Regulation of hepcidin by anemia and hypoxia

Inadequate delivery of oxygen to tissues, which occurs in anemia or

hypoxemia, would be expected to result in homeostatic decrease in hepcidin

synthesis. The decrease in hepcidin levels would in turn allow increased

iron mobilization from macrophages and hepatocytes, and increased iron

absorption from the diet, making more iron available for erythrocyte

production. Indeed, hepcidin was shown to be suppressed by anemia and

hypoxia; however, the molecular pathways that regulate this response are

still unclear. Though anemia may act by causing liver hypoxia, it is also

possible that the pathways of hepcidin regulation by oxygen and by

anemia/erythropoiesis are independent (Nicolas et al., 2002).

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60

Fig. 14 Hepcidin mRNA expression (Nicolas et al., 2002).

Exposure to hypoxia decreased hepcidin mRNA expression (Fig. 14). In

general, cellular oxygen sensing and the related transcriptional control are

largely mediated by the hypoxia-inducible factor (HIF). However, unlike

most of the target genes that are transcriptionally activated by HIF, hepcidin

expression is negatively regulated by hypoxia. In addition, except for

human hepcidin promoter, the promoters in other mammals do not contain

the consensus binding sites for HIF, and direct involvement of HIF in

transcriptional regulation of hepcidin remains to be explored (Nicolas et al.,

2002).

Hepcidin mRNA increased by dietary iron and mice injected with

bacterial LPS (indirect effect mediated by IL-6). Decreased by Hypoxemia

and increased erythroid iron demand (phlebotomy or hemolysis).

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Hepcidin and anemia of chronic disease

Hepcidin appears to block iron uptake in the duodenum and release

from RE macrophages, thereby decreasing delivery of iron to RBC

precursors. A hepcidin antagonist might benefit patients with ACD but

might be contraindicated in patients with infections . Elevated urinary

hepcidin levels might be useful to diagnose ACD (Nemeth et al., 2003).

Role of Hepcidin in Iron –Loading Anemias

Iron–Loading anemias are characterized by ineffective erythropoiesis

and increased intestinal iron absorption. Erythrocyte transfusions further

exacerbate the iron overload, the development of hepcidin- based

diagnostics and therapies for iron-loading anemias may offer more effective

approaches to prevent the toxicity associated with iron overload. The most

common iron- loading anemias are the intermediate and major forms of β-

thalassemia, but other rare anemias also complicated by iron loading,

including congenital dyserythropoietic anemia, X- Linked sideroblastic

anemia and anemia associated with divalent metal ion transporter 1

(DMT1) mutations (Papanikolaou et al., 2005) .

Role of Hepcidin in β-thalassemia major

In the presence of systemic iron overload, patients with thalassemia

major in whom iron overload was more severe and anemia was partially

relieved by transfusions, had urinary hepcidin concentrations that were

higher than in thalassemia intermedia. These findings were interpreted as

supporting the dominant erythropoietic effect of exogenous hepcidin could

prevent the iron overload in iron–Loading anemias (Loreal et al., 2005).

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62

Iron overload of tissue is the most important complication of β –

thalassemia major and is a major focus of management. In patients who are

not receiving transfusions, abnormally regulated iron absorption results in

increases in body iron burden ranging from 2 to 5 g per year, depending on

the severity of erythroid expansion. Regular transfusions may double this

rate of iron accumulation (Loreal et al., 2005).

Elevated levels of hepcidin in the bloodstream effectively shut off iron

absorption by disabling the iron exporter ferroportin. Conversely, low levels

of circulating hepcidin allow ferroportin to export iron into the bloodstream.

Aberrations in hepcidin expression result in disorders of iron deficiency and

iron overload. It is clear that erythroid precursors communicate their iron

needs to the liver to influence the production of hepcidin and thus the

amount of iron (Loreal et al., 2005).

In a mouse model of beta-thalassaemia, Weizer-Stern and his co-

authors (2006) observed that the liver expressed relatively low levels of

hepcidin, which is a key factor in the regulation of iron absorption by the

gut and of iron recycling by the reticuloendothelial system. It was

hypothesised that, despite the overt iron overload, a putative plasma factor

found in beta-thalassaemia might suppress liver hepcidin expression. Sera

from beta-thalassaemia patients were compared with those of healthy

individuals regarding their capacity to induce changes in the expression of

key genes of iron metabolism in human HepG2 hepatoma cells. Sera from

beta-thalassaemia major patients induced a major decrease in hepcidin

(HAMP) expression. A significant correlation was found between the

degree of downregulation of HAMP induced by beta-thalassaemia major

sera.

Literature review

63

Adamsky and his co-authors (2004) have found that iron overload is

less dominant than anaemia in regulating hepcidin expression in the setting

of the β-thalassemia major mouse model. The decreased expression of

hepcidin may explain the increased absorption of iron in thalassemia.

Recently, decreased expression of hepcidin was found in hereditary

haemochromatosis in association with elevated levels of nontransferrin

bound iron. The elevated expression of NGAL, an alternative iron delivery

vehicle, supports the role of nontranferrin bound iron in the abnormal iron

regulation in thalassemia. The decreased HFE expression level is similar to

the finding in hereditary haemochromatosis (Bridle et al., 2003).

Regulation of hepcidin by iron and the lessons from hereditary

hemochromatosis

The only clues about molecules involved in the pathway of hepcidin

regulation by iron come from mutations causing hereditary

hemochromatosis. In addition to juvenile hemochromatosis caused by

inactivating mutations in the hepcidin gene itself, it appears that hepcidin

deficiency is the unifying cause of most types of hereditary

hemochromatosis. Measurements of urinary hepcidin excretion or hepatic

mRNA expression showed that patients and animal models with

homozygous disruption of HFE, transferrin receptor 2 (TfR2) and

hemojuvelin (HJV) all had hepcidin levels inappropriately low for the

systemic iron load (Muckenthaler et al., 2003; Ahmad et al., 2002).

While the precise function of the three molecules is not known, they

likely participate in the sensing of iron or the consequent signal transduction

that regulates hepcidin synthesis and release. Importantly, the degree of

hepcidin deficiency appears to correlate with the severity of the disease. The

Literature review

64

most severe form, juvenile hemochromatosis, is caused by mutations in

either the hepcidin or HJV gene and these are phenotypically

undistinguishable. Patients with HJV mutations have very low or

undetectable urinary hepcidin suggesting that HJV is the key regulator of

hepcidin (Papanikolaou et al., 2004).

Genetic iron-overload disorders may be divided into haemochromatotic

and non-haemochromatotic forms according to patho-physiological and

phenotypic criteria (Table 1) (Pietrangelo, 2007). Haemochromatosis refers

to hereditary iron-overload disorders characterized by normal

erythropoiesis, increased transferrin saturation and parenchymal distribution

of iron deposition, and related to an inaccurate production and/or regulation

and/or activity of hepcidin (Pietrangelo, 2007).

Hepcidin and Erythropoeisis

Hepcidin expression is regulated in response to bone marrow needs

Iron absorption is increased in patients with congenital anemias

characterized by ineffective erythropoiesis. Clinically, increased intestinal

iron absorption compounds the effects of transfusional iron overload in

patients with thalassemia syndromes, sideroblastic anemia, or congenital

dyserythropoietic anemias (Adamsky et al., 2004).

Finch (1994) proposed the existence of an erythroid regulator of

systemic iron homeostasis. The erythron, composed of developing erythroid

cells in the bone marrow and circulating erythrocytes, utilizes about 80% of

the iron found in the plasma. Anemia results from the inability of the

erythroid compartment to receive its full complement of iron. The putative

erythroid regulator communicates the iron needs of the erythron to

influence changes in intestinal iron absorption (Breda et al., 2005).

Literature review

65

Table 1 Main characteristics of genetic iron overload disorders

(Deugnier et al., 2008).

Genetic iron

overload disease Gene

Chromos

ome

Transmis

sion

Onse

t

Clinical

expressio

n

Haemochromatotic

HFE HFE 6p21.3 Recessive Late

Articular

and

hepatic

Hemojuvelin HJV 1p21 Recessive Early

Cardiac

and

endocrine

Hepcidin HAMP 19q13.1 Recessive Early

Cardiac

and

endocrine

Transferrin

receptor 2 TfR2 7q22 Recessive Late Hepatic

Ferroportin disease

type B

SLC40A

1 2q32 Dominant Late

Articular

and

hepatic

Nonhaemochromat

otic Ferroportin

disease type A

SLC40A

1 2q32 Dominant Late Rare

A (hypo)

ceruloplasminemia

Cerulopl

asmin 3q23-q25 Recessive Late

Neurolog

ical

A (hypo)

transferrinemia

Transfer

rin 3q21 Recessive Early

Hematolo

gical

Literature review

66

Hepcidin is an effective inhibitor of iron absorption, the erythroid

regulator includes a mechanism to decrease hepcidin production.

Accordingly, low hepcidin levels have been reported with thalassemia and

other disorders with ineffective erythropoiesis. In these disorders, decreased

hepcidin expression leads to relief of inhibition of ferroportin, resulting in

increased iron release from recycling macrophages and absorptive

enterocytes, increasing availability of iron for erythropoiesis. However, the

iron cannot be effectively utilized by the erythron, leading to accumulation

and tissue iron overload in the face of anemia (Gardenghi et al., 2007;

Jenkins et al., 2007).

Stimulation of erythropoiesis with phenylhydrazine resulted in hepcidin

suppression as expected, but the simultaneous inhibition of erythropoiesis

by irradiation prevented hepcidin suppression despite severe anemia. In

addition, irradiation prevented hepcidin suppression after erythropoietin

administration, ruling out the direct effect of erythropoietin on hepcidin

synthesis (Breda et al., 2005).

SUBJECTS AND METHODS

Subjects and methods  

67

SUBJECTS AND METHODS

This study was carried out in pediatric and clinical pathology

departments of Zagazig University Hospitals in the period from March 2009

to April 2010. The study included 40 children divided into two groups:

I- Group (I): healthy controls group

This group included (10) apparently healthy subjects aged between 4-11

years with a male to female ratio of 1.5:1 (6 males and 4 females).

II- Group (II): patients group

The studied group included 30 β-thalassemic major children patients

aged between 4-10 years with a male to female ratio of 1.5:1 (18 males and

12 females). They were selected from patients attending pediatric

hematology unit and already diagnosed as β-thalassemia major.

All study members were subjected to the following:

a) Full history taking to collect data from children or mothers: Personal

history, diagnosis, blood transfusion, iron chelation and history of

complications (hepatic, renal, cardiac and endocrinal complications).

b) Clinical examination included:

1) General examination

2) Local examination

• Head and neck examination for mongoloid face, earthy face of poorly

chelated patient.

• Cardiac examination to assess cardiac complications.

• Abdominal examination to assess spleen and liver state

(splenomegally, splenectomy or hepatomegally).

Subjects and methods  

68

• Abnormalities of long bones and skull.

Laboratory investigations:

A-Routine:

(1) C.B.C by (SYSMEX SF 3000).

(2) Serum iron (colorimetric) (Huebers et al., 1987).

(3) Serum ferritin (Cobas E 411).

(4) TIBC( colorimetric ) (Finch & Huebers, 1986)

(5) Hemoglobin electrophoresis (Valeri et al., 1965).

(6) Liver function tests, kidney function tests and serum Alkaline

phosphatase test were done by (Selectra XL).

B- Special investigations

Hepcidin hormone:

Hepcidin was measured by Enzyme Linked Immunosorbant Assay in

the serum (Park et al., 2001). The Kit was supplied from DRG

International,Inc.,USA.

The principle:

The DRG Hepcidin ELISA Kit is a solid phase enzyme-linked

immunosorbent assay, based on the principle of competitive binding. The

microtiter wells are coated with a monoclonal antibody directed towards the

antigenic site of the bioactive Hepcidin 25 molecule. Endogenous Hepcidin

of a patient sample competes with the added Hepcidin-biotin conjugate for

binding to the coated antibody. After incubation the unbound conjugate is

washed off. An incubation with a streptavidin-peroxidase enzyme complex

Subjects and methods  

69

and a second wash step follows. The addition of substrate solution results in

a colour development which is stopped after a short incubation The

intensity of colour developed is reverse proportional to the concentration of

Hepcidin in the patient sample.

The reagents:

The kit reagents include:

(a) Standard: concentrations of synthetic pepide Hepcidin.

(b) Control low and high.

(c) Assay Buffer.

(d) Enzyme Complex contains : Streptavidin peroxidase.

(e) Substrate Solution contains:Tetramethylbenzidine(TMB).

(f) Stop Solution contains: H2SO4.

(g) Wash Solution (40X concentrated).

Reagent Preparation:

• All reagents and required number of strips were brought to room

temperature prior to use.

• The lyophilized contents of the standard vials were reconstituted with

0.5 mL Aqua dest.

• The lyophilized content of the control was reconstituted with 0.5 mL

Aqua dest. And was left to stand for 10 minutes in minimum.

• Deionized water was added to the 40X concentrated Wash Solution.

30 mL of concentrated Wash Solution was diluted with 1170 mL

deionized water to a final volume of 1200 mL.

Subjects and methods  

70

Specimen collection

Specimens have been prepared by collecting blood by clean

venipuncture, allowed to clot, and serum has been separated by

centrifugation at 2500 x g for 10 min at 4oC.Specimens were frozen until

use at -20oC.

Assay Procedure

• The desired numbers of micro-titer wells were secured in the holder.

• 10 µl of Sample Buffer was dispensed into each well.

• 20 µl of each Standard, Control and Sample with new disposable tips

were dispensed into appropriate wells.

• Incubation for 30 minutes at room temperature on a plate shaker at ≈

500 rpm was done.

• 150 µL of Assay Buffer and 100 µL of Enzyme Conjugate were

added to each well.

• Incubation for 180 minutes at room temperature on a plate shaker at ≈

500 rpm was done.

• The contents of the wells were briskly shaked out then the wells

were Rinsed 5 times with diluted wash solution (400 µL per well)

and were Striked sharply on absorbent paper to remove residual

droplets .

• 100 µL of Enzyme Complex was dispensed into each well.

• Incubation for 45 minutes at room temperature was done.

• The contents of the wells were briskly shaked out then the wells were

Rinsed 5 times with diluted wash solution (400 µL per well) and

were Striked sharply on absorbent paper to remove residual droplets.

• 100 µL of substrate solution was added to each well.

Subjects and methods  

71

• Incubate for 30 minutes at room temperature was done.

• The enzymatic reaction was stopped by adding 100 µl of Stop

Solution to each well.

After adding the Stop Solution, the OD at 450±10 nm was red with a micro-

titer plate reader within 10 minutes.

Calculation of Results

Calculations were done as follows: The average absorbance values for

each set of standards, controls and patient samples were calculated. A

standard curve was constructed by plotting the mean absorbance obtained

from each standard against its concentration. The mean absorbance value

for each sample was used to determine the corresponding concentration .

As the value of iron absorbance increases, the concentration of hepcidin

in serum decreases and vice versa (i.e., reverse proportion relationship). The

concentration of hepcidin in serum was calculated using the curve fitting

equation and shown in Fig. 15.

Subjects and methods  

72

Fig. 15 Concentration of hepcidin of patients (in circles) and

concentration of hecidin of control (in squares).

Statistical analysis Data were entered checked and analyzed Epi-Info version 6 and SPP for

windows version 11 (Dean et al., 1994). SPSS windows version 11 was

used for data analysis as follows:

A- Descriptive statistics:

In which data were summarized using:

1) The arithmetic mean ( X ) as an average describing the central

tendency of observations was applied.

2) The standard deviation (SD) as a measure of dispersion of the results around the

mean was calculated. B- Comparison of means:

The comparison was done using the student "t" test for comparison of

means of two independent groups.

Subjects and methods  

73

C- Correlation study:

Correlation between variables was done using correlation coefficient

"r".

This test detects if the change in one variable was accompanied by a

corresponding change in the other variable or not. The value of r usually

lies between -1 and +1, where positive indicate a tendency for X and Y to

increase together while negative values indicate a tendency for X to

increase with decrease of Y and vice versa. The significance of ‘r’ was

obtained from the ‘t’ distribution with (n-2) degrees of freedom where n is

number of observation in each group .

D- Level of significance:

For all above mentioned statistical tests done, the threshold of

significance is fixed at 5% level (P value), where:

a) P-value > 0.05 indicates non- significant results.

b) P-value < 0.05 indicates significant results.

c) P-value < 0.001 indicates highly significant results.

RESULTS

Results

74

RESULTS

This study included 40 subjects divided into 10 normal control subjects (Group

I) and 30 β thalassemia major patients (Group II). The age of the normal subjects

in Group I ranged from 5 to 11 years and that of the patients in Group II ranged

from 4 to 10 years as represented in Table 2:

Table 2: Clinical data of the studied groups.

I

N = 10

II

N = 30 t p

Age (years)

Range

X ± sD

5– 11

7.8 ± 1.87

4 – 10

6.97 ± 1.9

1.2 0.23

Gender N % N % X2 P

Male 6 60.0 18 60.0 0.0 1.0

Female 4 40.0 12 40.0

Results

75

Table 3: Liver and kidney functions results of the studied groups.

I II

t P X ± SD (Range) X ± SD (Range)

Total serum Bilirubin (mg/dl)

0.61± 0.15 (0.4- 0.9)

1.7± 0.27 (1.3- 2.5) 12.0 0.001**

Direct Bilirubin (mg/dl)

0.15± 0.07 (0.1- 0.3)

0.55± 0.15 (0.3- 0.9)

7.9 0.001**

Total protein (g/dl) 7.1± 0.35 (6.2- 7.6)

6.8± 0.62 (5.4- 7.9) 1.33 0.18

S. Albumin (g/dl) 4.2± 0.3 (3.9- 4.8) 3.8± 0.9 (2.5- 5.1) 1.27 0.2 SGPT (U/L) 36.5± 3.6 (30- 40) 77.8± 6.3 (60- 89) 19.6 0.001** SGOT (U/L) 29.4± 3.6 (23- 34) 67.7± 5.5 (60- 80) 20.3 0.001**

ALP (U/L) 41.6 ± 7.8 (30- 50) 89.36 ± 6.2 (80- 99)

19.65 0.001**

Bl. Urea (mg/dl) 31.4 ± 3.1 (25-

35.5) 28.6 ± 5.5 (18- 36) 1.48 0.14

S. Creatinine (mg/dl)

0.78 ± 0.29 (0.1- 1.2)

0.86 ± 0.27 (0.2- 1.3)

0.79 0.56

BUN (mg/dl)

10.0 ± 1.16 (8- 11.5) 9.2 ± 1.3 (6.5- 12) 1.72 0.08

Table (3) showed that there was a highly significant elevation of Total

Bilirubin , Direct Bilirubinin , SGPT, SGOT and Alkaline phosphatase in patient

group compared to control group (P < 0.001). However there was no significant

difference for Total protein , S. Albumin , blood urea, S. Creatinine and BUN

between the two groups.

Results

76

Table 4 Complete blood count results of the studied groups.

I II

t P X ± SD (Range) X ± SD (Range)

WBC (103/cm) 5.2 ± 0.95 (4.5 - 6) 7.8 ± 3.3 (4 – 12.5) 2.48 0.016* RBCs (106/cm) 5.0 ± 0.13 (4.9 – 5.3) 2.9 ± 0.4 (2.3 – 3.8) 16.2 0.001**

HCT (%) 42.8 ± 1.98 (39.2 – 44.5) 18.7 ± 2.5 (15.2 – 24.6) 27.4 0.001** HB (g/dl) 10.8±0.4 (10.0 – 11.2) 7.4±0.6 (6.4 – 8.7) 16.4 0.001** MCV(fl) 84.9±3.1 (80 – 89) 63.7±2.4 (60.2 – 69.1) 22.49 0.001** MCH (Pg) 21.4 ± 0.6 (20 – 22.2) 25.6±4.2 (19.1 – 36.5) 3.09 0.003* MCHC(g/dl) 25.3 ± 1.3 (23.1 – 27.8) 40.2±6.5 (30.2 – 55.3) 7.12 0.001** RDW(%) 13.29 ± 0.5 (12.5- 14) 13.79± 1.6 (11.5- 16.9) 0.96 0.65 PLT(103/cm 290 ± 21.6 (250- 320) 245.3± 59.6 (180- 526) 1.3 0.25 PDW(%) 15.2 ± 0.96 (14- 16.6) 12.85± 1.0 (11.2- 14.7) 6.24 0.001** MPV(fl) 9.2 ± 0.9 (8- 10.5) 9.11± 1.2 (7.9- 13.9) 0.31 0.75

Table (4) and Fig. 16 showed that there was a highly significant reduction in

group II compared to group I as regard RBCs, HCT, HB, MCV, MCHC and PDW

(P < 0.001). However there was a significant elevation for WBC in group II

compared to group I (P < 0.003) . No significant difference for RDW, PLT and

MPV tests.

Fig. 16 Variations of RBCs, HB, MCV, HCT (PCV) for 40 patients (30 patients and 10 controls).

Results

77

Table 5: Results of iron study of the studied groups.

I II Mann –

Whitney U test X ± sD( Range) X ± sD( Range) Serum Iron (µg/dL)

100.5 ± 19.643 (80-150)

257.033 ± 16.866 (200-278)

p = 2.737×10-6 < 0.001**

Serum Ferritin (ng/ml)

157.0 ± 51.001 (100-250)

1442.9 ± 522.185 (597-2500)

p = 2.996×10-6 < 0.001**

TIBC(µg/dL) 274.4± 61.925 (110-330)

139.5±16.706 (120-180)

p = 1.8537×10-4 < 0.001**

Table (5) showed that there was a highly significant difference between group I

and group II for Serum Iron, Serum Ferritin and TIBC, (P < 0.001).

Table 6: Hemoglobin Electrophoresis data of the studied groups.

I II

t P X ± SD (Range) Median

X ± SD (Range) Median

HBA (%) 96.95± 0.55 (96.3- 97.9) 96.95

50.1± 26.6 (0.0- 86) 57

8.63

0.001**

HBA2

(%)

2.6± 0.4 (2- 3.2) 2.65

3.45± 1.0 (1- 6) 3

5.52 0.003**

HBF (%) 0.45± 0.2 (0.1- 0.8) 0.45

46.1± 2.7 (11- 99) 40

22.04 0.001**

Results

78

Table (6) showed that there was a highly significant difference between the two

groups for HBA, HBA2 and HBF, (P < 0.001).

Table 7: Hepcidin concentration levels of the studied groups.

I II M P Hepcidin (ng/ml) X ± SD

210.9 ± 12.8 66.2 ± 95.6 0.001**

Range (194.347 – 231.65) (-390.657– 147.47) 8.98 Median 209.89 80.351 21.98

Table (7) and Fig.17 showed that there was a highly significant reduction of

hepcidin in Group (II) compared to Group (I) (p < 0.001)

Fig. 17 Hepcidin and Serum Ferritin for 40 patients (30 patients and 10

controls).

Results

79

Table 8: Ratio between hepcidin and Serum Ferritin of the studied groups..

I II Mann–Whitney U test

X ± SD 1.455 ± 0.396 0.063 ± 0.061 P = 3.015×10-6 <

0.001** Range 1.017 0.379 median 1.444 0.074

Table (8) showed that there was highly significant increase as regard hepcidin /

Serum Ferritin ratio in Group (I) compared to Group (II). The hepcidin / Serum

Ferritin ratio in the patients group was markedly reduced.

Table 9: Correlation between hepcidin and other parameters of the studied

groups.

r P Significance Hb (g/dl) 0.68 < 0.001** HS HCT(%) 0.56 < 0.001** HS Mcv (fl) 0.61 < 0.001** HS Fe (ng/ml) -0.72 < 0.001** HS S.Iron (µg/dL) -0.63 < 0.001** HS TIBC(µg/dL) 0.21 < 0.001** HS

Table (9) and Fig. 18-21 showed that there was a positive correlation between

hepcidin in one side and Hb, Hct and Mcv in the other side (p<0.001), (Figs. 18-

20). While there was a negative (i.e., inverse) correlation between hepcidin and

(ferritin &Serum Iron) (P< 0.001) (see Fig. 21). There was a positive correlation

correlation between hepcidin in one side and TIBC (p<0.001) in the other side.

Results

80

Fig. 18 Correlation between hepcidin and Hb.

Fig. 19 Correlation between hepcidin and HCT.

Results

81

Fig. 20 Correlation between hepcidin and MCV.

Fig. 21 Correlation between hepcidin and Serium Ferritin.

 

 

 

 

 

 

 

DISCUSSION

Discussion  

 82  

DISCUSSION

β-thalassemia is the most common chronic hemolytic anemia in Egypt

(85.1%). A carrier rate of 9-10.2% has been estimated in 1000 normal

random subjects from different geographical areas of Egypt. β-thalassemia

is much more common in Mediterranean countries constituting a major

public health problem (El-Beshlawy, 1999).

Finch (1994) proposed the existence of an erythroid regulator of

systemic iron homeostasis. The erythron, composed of developing erythroid

cells in the bone marrow and circulating erythrocytes, utilizes about 80% of

the iron found in the plasma. Anemia results from the inability of the

erythroid compartment to receive its full complement of iron. The putative

erythroid regulator communicates the iron needs of the erythron to

influence changes in intestinal iron absorption (Breda et al., 2005).

Iron absorption is increased in patients with congenital anemias

characterized by ineffective erythropoiesis. Clinically, increased intestinal

iron absorption compounds the effects of transfusional iron overload in

patients with thalassemia syndromes (Adamsky et al., 2004).

The most common secondary complications are those related to

transfusional iron overload, which can be prevented by adequate iron

chelation. Iron-loading anemias are disorders in which hepcidin is regulated

by opposing influences of ineffective erythropoiesis and concomitant iron

overload (Pigeon et al., 2001).

Hepcidin is an effective inhibitor of iron absorption; the erythroid

regulator includes a mechanism to decrease hepcidin production.

Accordingly, low hepcidin levels have been reported with thalassemia and

other disorders with ineffective erythropoiesis. In these disorders, decreased

hepcidin expression leads to relief of inhibition of ferroportin, resulting in

Discussion  

 83  

increased iron release from recycling macrophages and absorptive

enterocytes, increasing availability of iron for erythropoiesis. However, the

iron cannot be effectively utilized by the erythron, leading to accumulation

and tissue iron overload in the face of anemia (Gardenghi et al., 2007 and

Jenkins et al., 2007).

This study aim was to measure hepcidin concentration in patients of β

thalassemic major to explain its role in iron metabolism for these patients

who have iron overload.

This study revealed a statistically significant elevation of total Bilirubin

and Direct Bilirubin (TB-DB) in the patient group compared to the control

group, on the other hand there was no significant difference in the Total

protein and S. Albumin. There was a statistically significant elevation in the

patient group as regard SGPT, SGOT and Alkaline phosphatase, compared

to the control group.

Hyperbillirubinemia is a result of chronic Hemolysis and ineffective

erythropoiesis. Which together cause the anemia that occurs in thalassemia.

The relative contributions of these two pathologic processes differ in

various forms of thalassemia. The bone marrow of patients with thalassemia

contains five to six times the number of erythroid precursors as does the

bone marrow of healthy controls with 15 times the number of apoptotic

cells in the polychromatophilic and orthochromic stages (Centis et al.,

2000;Mathias et al., 2000).

Ineffective erythropoiesis the major cause of accelerated apoptosis, is

caused by excess -chain deposition in erythroid precursors. Although the

exact mechanism is not known, a death-receptor–mediated pathway seems

to be involved with Fas–Fas ligand interactions (De Maria et al., 1999).

Discussion  

 84  

In normal erythropoiesis, apoptotic mechanisms seem to play a

regulatory role and are required for normal erythroid maturation (Testa,

2004). Accelerated apoptosis is associated with a rise in extracellular

exposure of phosphatidylserine, an important signal for removal by

activated macrophages, whose numbers are increased in thalassemic bone

marrow erythrocytes , resulting in their accelerated peripheral destruction (

Angelucci et al., 2002).

In this work there was significant reduction in the patient group as

regard RBCs, HCT, HB, MCH, , MCV and MCHC. On the other hand, the

WBC count was elevated in β thalassemia major patient group.

Leukocytosis is usually present, even after excluding the nucleated

RBCs. A shift to the left is also encountered, reflecting the hemolytic

process. The anemia is due to a combination of ineffective erythropoiesis,

excessive peripheral red blood cell hemolysis, and progressive

splenomegaly. The latter causes an increase in plasma volume and a

decrease in total red cell mass. The red cells are microcytic (mean

corpuscular volume <70 fL) with marked anisochromasia. Hypochromic

microcytic anemia lead to MCV and MCH are significantly low, low

MCHc, low HCT and low Hb (Wonke, 2001).

In this study there was a highly significant difference between the two

groups for HBA, HBA2 and HBF, as HbA is the major hemoglobin found

in adults and children. Hb A2 and HbF are found in small quantities in

adult life but in β thalassemia major there was elevated HbF and Hb A2 but

HbA is in small quantities (Rachmilewitz &Schrier et al., 2001).

This study showed that there was a highly significant elevation of

Serum Iron and Serum Ferritin in the patient group compared to the control

Discussion  

 85  

group. This was accompanied by subsequent reduction of TIBC in the

patient group.

Oxidation of α-globin subunits leads to the formation of hemichromes,

it bind to or modify various components of the mature red-cell membrane,

such as protein band 3, protein 4.1, ankyrin, and spectrin. After

precipitation of hemichromes, heme disintegrates, and toxic non–

transferrin-bound iron species are released. The resulting free iron catalyzes

the formation of reactive oxygen species (Rachmilewitz &Schrier et al.,

2001).

Iron-dependent oxidation of membrane proteins and formation of red-

cell "senescence" antigens such as phosphatidylserine (Kuypers & Jong.

2004) cause thalassemic red cells to be rigid and deformed and to aggregate,

resulting in premature cell removal (Tavazzi et al., 2001).

Ineffective erythropoiesis and hepatosplenomegaly together result in

Hypochromic microcytic anemia which in turn increases iron absorption

plus transfusional iron overload both lead to increased levels of iron, ferritin

and decreased TIBC (Porter, 2001).

The most common secondary complications are those related to

transfusional iron overload, which can be prevented by adequate iron

chelation. Iron-loading anemias are disorders in which hepcidin is regulated

by opposing influences of ineffective erythropoiesis and concomitant iron

overload (Pigeon et al., 2001).

The results of the present study revealed reduction of Hepcidin level in

patient group compared to the control group this reduction was statistically

significant. On the other hand, the ratio between hepcidin concentration and

Serum Ferritin was highly reduced in patient group compared to the control

group. This reduction was statistically significant.

Discussion  

 86  

The results showed that there was a positive correlation between

hepcidin and Hb, PCV and MCV (i.e., as the value of hepcidin increases,

the values of the Hb, PCV and MCV increase and vice versa) while there

was a negative (i.e., inverse) correlation between hepcidin and (ferritin

&Serum Iron) (i.e., as the values of hepcidin increase, the values of ferritin

and Serum Iron decrease and vice versa).

Patients of β thalassemia major have decreased concentrations of

hepcidin due to opposing influences of ineffective erythropoiesis and

concomitant iron overload. This agreed with Wrighting and Andrews

2008 who reported that the erythroid regulator includes a mechanism to

decrease hepcidin production. Accordingly, low hepcidin levels have been

reported in patients with thalassemia and other disorders with ineffective

erythropoiesis.

Wrighting and Andrews 2008 reported also that hepcidin expression

was downregulated in a hepatocytic cell line after treatment with

thalassemic sera.

This result was in agreement with Rund and Rachmilewitz 2005 who

reported that Hepcidin levels were found to be low in patients with

thalassemia intermedia and thalassemia major. Furthermore, serum from

patients with thalassemia inhibited hepcidin messenger RNA expression in

the HepG2 cell line, which suggests the presence of a humoral factor that

downregulates hepcidin.

Zimmermann et al 2008 reported that hepcidin concentrations should

be high in iron-loaded persons with β -thalassemia; however, hepcidin

concentrations are low in these persons, unless they have recently received a

transfusion. Production of growth differentiation factor 15 (GDF-15) by the

Discussion  

 87  

expanded erythroid compartment contributes to iron overload in thalassemia

by inhibiting hepcidin gene expression.

Nemeth and Ganz 2006 reported that when the ratio of urinary

hepcidin to serum ferritin was analyzed as an index of appropriateness of

hepcidin response to iron load, this ratio was still greatly decreased in

thalassemia major patients when compared to normal subjects, indicating

the continued regulation of hepcidin by a suppressive factor.

Pak et al 2006 reported that patients with β -thalassemia would be

expected to have high hepcidin levels. To the contrary, patients with β-

thalassemia have almost uniformly low urinary hepcidin. These and other

clinical observations in iron-loading anemias would argue that

erythropoiesis is able to suppress hepcidin production even in the face of

severe iron overload.

Papanikolaou et al 2005 reported that hepcidin was measured in 8

patients with thalassemia major and 7 with thalassemia intermedia. Patients

with thalassemia had very low urinary hepcidin levels, despite high serum

ferritin levels that reflected systemic iron overload. Several patients with

thalassemia had no detectable hepcidin.

The current study didn't agree with Origa et al 2007 who reported that

hepcidin levels were elevated in thalassemia major, due to transfusions that

reduce erythropoietic drive and deliver a large iron load, resulting in

relatively higher hepcidin levels. In the presence of higher hepcidin levels,

dietary iron absorption is moderated and macrophages retain iron,

contributing to higher serum ferritin.

This result agreed with Brissot et al 2008 who reported that hepcidin

deficiency in thalassemia major due to ineffective erythropoiesis leads to

growth differentiation factor15 (GDF15) overexpression by the

Discussion  

 88  

erythroblasts, which inhibits hepcidin expression. This could explain why

(hepatocytic) iron overload can develop in thalassaemia in the absence of

transfusions, and why hepcidin expression is relatively low in this disease

despite transfusional iron excess (which should, by itself, lead to marked

increased hepcidin expression).

Kemna et al 2008 reported that erythropoietin that stimulates

erythropoietic activity has been shown to down-regulate liver hepcidin

expression. However, in the absence of erythropoietic activity, hepcidin

expression is no longer suppressed. The strong inverse association between

erythropoietic drive and hepcidin production was also observed in several

patients with congenital chronic anemias, which are characterized by low

urinary hepcidin levels. The dotblot method was used to observe

low/normal hepcidin levels for the degree of iron load in thalassemic

patients.

Kattamis et al 2006 reported that urinary hepcidin was found to be

suppressed in patients with thalassemia major. Tissue hypoxia triggers the

production of EPO, which results in pronounced erythroid proliferation

accompanied by increased sTfR levels. Hypoxia and yet-undefined signals

from the robust erythroid activity down-regulate hepcidin production.

Nemeth and Ganz 2006 reported that Patients with chronic anemias

with hemolysis or dyserythropoiesis, such as thalassemia syndromes suffer

from iron overload. Measurements of urinary hepcidin in these patients

indicated that hepcidin levels were severely decreased, despite systemic iron

overload reflected by the patients’ elevated serum ferritin levels. Even in

regularly transfused thalassemia patients, hepcidin levels were

inappropriately low given the patients’ iron load, as indicated by the

Discussion  

 89  

decreased ratio of urinary hepcidin to serum ferritin, used as an index of

appropriateness of hepcidin response to iron load.

Toledano et al 2008 reported that Several diseases with chronic iron

overload such as hereditary hemochromatosis and β-thalassemia major are

characterized by low hepcidin expression in the liver. The low hepatic

hepcidin in these patients is probably responsible for the intestinal

absorption of iron.

Tanno et al 2007 also reported that Serum from thalassemia patients

suppressed hepcidin mRNA expression in primary human hepatocytes, and

depletion of GDF15 reversed hepcidin suppression. These results suggest

that GDF15 overexpression arising from an expanded erythroid

compartment contributes to iron overload in thalassemia syndromes by

inhibiting hepcidin expression.

 

Conclusions  

90

CONCLUSIONS

• Transfusional iron overload is the most common secondary

complications in β thalassemia major.

• Hepcidin is a central regulator of iron homeostasis.

• Hepcidin concentration was decreased in the thalassemic group

although elevated iron levels.

• Patients of β thalassemia major have decreased concentrations of

hepcidin due to opposing influences of ineffective erythropoiesis and

concomitant iron overload.

 

Recommendation  

91

RECOMMENDATION

● Further study is recommended on large scale including the

development of hepcidin- based diagnostics and therapies for iron-loading

anemias that may offer more effective approaches to prevent the toxicity

associated with iron overload.

● Further study is recommended to screen patients with β thalassemia

who develop secondary iron overload to detect hepcidin level as in the

future, therapeutic use of hepcidin and hepcidin agonists may help to

restore normal iron homeostasis.

● Further study is recommended for evaluating and measuring hepcidin

regulators as growth differentiation factor15 (GDF15) , Hemojuvelin (HJV)

and others in cases of ineffective erythropoiesis and their effects on

hepcidin expression.

 

 

 

 

 

 

 

 

 

 

SUMMARY

Summary  

92  

SUMMARY

The thalassemias are a heterogeneous group of genetic disorders of

haemoglobin synthesis , all of which result from a reduced rate of

production of one or more of the globin chains of haemoglobin. The

thalassemias are among the most common genetic disorders worldwide,

occurring more frequently in the Mediterranean region, the Indian

subcontinent, Southeast Asia, and West Africa.

The most common secondary complications are those related to

transfusional iron overload, which can be prevented by adequate iron

chelation.

The survival of individuals who have been well transfused and treated

with appropriate chelation extends beyond age 30 years. Iron-chelation

therapy is largely responsible for doubling the life expectancy of patients

with thalassemia major.

Systemic iron is distributed among erythrocyte precursors in the bone

marrow, tissue macrophages, liver, and all other tissues, with the largest

amount found in circulating erythrocytes. Homeostasis is maintained by

regulating the levels of plasma iron. Hepcidin, a circulating peptide

hormone, has recently emerged as a key modulator of plasma iron

concentration, and, thus, a central regulator of iron homeostasis.

Hepcidin binds to ferroportin and causes its internalization and

degradation in lysosomes, thus effectively blocking the export of iron from

the cells.

This study was conducted to measure hepcidin concentration in patients

of β thalassemic major to explain its rule in iron metabolism for these

patients who have iron overload.

Summary  

93  

This study was carried out in pediatric and clinical pathology

department of Zagazig University Hospitals and included 30 β thalassemic

major children and 10 apparently healthy children as a control group.

All the studied groups were subjected to:

• Full history taking and thorough clinical examinations .

• Laboratory investigations: C.B.C, Serum iron, Serum ferritin, TIBC,

Hemoglobin electrophoresis, Liver function tests, kidney function

tests and Hepcidin hormone was measured by Enzyme Linked

Immunosorbant Assay in the serum.

From the results, it was found that:

• There was a highly significant difference between the two groups for

Total Bilirubin and Direct Bilirubin .

• There was high significant difference between the two groups as

regard SGPT, SGOT and Alkaline phosphatase.

• There was a highly significant difference between the two groups as

regards RBCs, HCT, HB, MCV, MCHC and PDW. Also there was a

significant difference for WBC and MCH.

• There was a highly significant difference between the two groups for

Serum Iron, Serum Ferritin and TIBC.

• There was a highly significant difference between the two groups for

HBA, HBA2 and HBF

• There was a highly significant increase in the control group

compared to patient group for Hepcidin . Hepcidin concentration was

decreased in the patients group although elevated iron levels

Summary  

94  

compared to the control group who have normal iron levels and

increased hepcidin concentration .

• The ratio between hepcidin and Serum Ferritin in the patients group

was lower than that in the control group.

• There was a positive correlation between hepcidin in one side and

Hb, Hct, and Mcv in the other side. Also there was a negative

correlation between hepcidin and serum ferritin.

 

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االملخص االعربى

االثالسيیميیا( االتي تسبب تكسر كريیاتت االدمم االحمرااء) من أأهھھھم أأمرااضض االدمم االوررااثيیة ااالنحالليیة

االشائعة على مستوىى االعالم بشكل عامم ووعلى مستوىى منطقة االبحر ااألبيیض االمتوسط بشكل خاصص

-لمتوسط بيیتايیميیا هھھھو مرضض اانيیميیا االبحر ااالبيیض ااس.ااهھھھم نوعع من االثال االكبرىى االتى تؤثر على اانتاجج

حيیث اانن االحديید وواالبرووتيیناتت االتى تعد ااساسيیة لحيیاةة ااالنسانن,ووبالتالى تنتج ااالنيیميیا من نقص االحديید.

تنظيیم ووااستقراارر مستوىى االحديید وواالمحافظة على هھھھذاا ااالستقراارر مهھم لصحة ااالنسانن.

-أأعرااضض اانيیميیا االبحر ااالبيیض االمتوسط بيیتا : فقر االدمم٬، االشحوبب, االخمولل ٬،قلة االكبرىى هھھھي

االحركة٬، االعصبيیة االشديیدةة٬، فقداانن االشهھيیة وو تأخر االنمو٬، قد يیعاني االطفل من بعض هھھھذهه ااألعرااضض أأوو

كلهھا. ووعند االفحص االسريیريي يیالحظ االطبيیب شحوبب االوجهھ ووتضخم بعض ااالعضاء كالطحالل

فالل فيیحدثث برووزز في عظامم وواالكبد٬، ووفي االسنوااتت االعشر ااألوولى للطفل تتغيیر مالمح بعض ااألطط

االوجهھ وواالفك ووكبر حجم االبطن ووبعض االتغيیيیر في االهھيیكل االعظمي.

يیحتاجج االمصابب إإلى عمليیاتت نقل ددمم للمحافظة على هھھھيیموجلوبيین االدمم بدررجة تسمح للمصابب

بمماررسة حيیاتهھ االطبيیعيیهھ٬، وولكن بسبب نقل االدمم االمتكررر تترسب كميیاتت كبيیرةة من االحديید في جسم

بة االتلف بكل عضو يیتركز بهھ االحديید٬، فعلى سبيیل االمثالل يیتسبب االحديید االزاائد بمرضض االمصابب مسب

االسكريي في سن صغيیرةة ووفشل عضلة االقلب ووتضخم االكبد وواالطحالل وواالعقم ووفشل االنمو وواالبلوغغ لذلك

يیحتاجج االمريیض ووبسن صغيیرةة إإلى أأددوويیة لتغسل االدمم من االحديید االمترسب مثل االديیسفراالل.

وويیتم االتحكم فى ااستقراارر مستوىى االحديید بشكل ااساسى بوااسطة االهھيیبسيیديین االذىى يیصنع فى االكبد.

حيیث تعد االخاليیا االكبديیة االمصدرر االخلوىى ااالساسى للهھيیبسيیديین.وويیتم ااخرااجج االحديید االخلوىى االى

االدمم٬، ووذذلك عبر االفيیرووبرووتيین. وواالخاليیا االمصدررةة االبالززما فى حالة اانخفاضض تركيیز االهھيیبسيیديین فى

لهھذاا االحديید هھھھى االخاليیا االطالئيیة لالثنى عشر٬، وواالخاليیا االبلعميیة وواالخاليیا االكبديیة.

ااما عند ااررتفاعع تركيیز االهھيیبسيیديین فانة يیرتبط بالفيیرووبرووتيین٬، حيیث يیتم ااستبطانن االفيیرووبرووتيین

االى ددااخل االخليیة ووتدميیرةة. ووكنتيیجة لذلك فانن ااخرااجج االحديید االخلوىى االى االبالززما يیقل بيینما يیترااكم

االحديید ددااخل االخليیة فى صوررةة االفريیتيین.

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ستوىى االحديید وويیقل نتيیجة لفقر االدمم وونقص ااألكسجيین. وويیزدداادد تصنيیع االهھيیبسيیديین نتيیجة لزيیاددةة م

االدمم االمصاحب لاللتهھابب يیقل مستوىى االحديید فى االبالززما مما يیحد من عمليیة تصنيیع رووفى حالة فق

كريیاتت االدمم االحمرااء.

تم ااجرااء هھھھذهه االدررااسهھ في االعيیاددةة االخاررجيیة لوحدةة االدمم بقسم ااألططفالل بمستشفى االزقاززيیق

-ن مريیض بانيیميیا االبحر ااالبيیض االمتوسط بيیتااالجامعي علي ثالثيی االكبرىى ووعشرةة أأططفالل ااصحاء

كمجموعة ضابطة.

معرفة ددووررهھھھرمونن االهھيیبسيیديین فى مرضى اانيیميیا االبحر ااالبيیض االمتوسط هھھھو هھھھدفف االدررااسة

-بيیتا االكبرىى حيیت ااززدديیادد نسبة االحديید.

ووااالصحاء بالنسبة لعد كرااتت ووقد ااظظهھرتت االنتائج ووجودد تبايین ملحوظظ بيین مجموعتى االمرضى

االدمم االحمرااء٬، نسبة االهھيیموجلوبيین وو متوسط حجم كرااتت االدمم االحمرااء. وولقد ووجد اايیضا ووجودد تبايین

ملحوظظ بيین االمجموعتيین بالنسبة لمستوىى االحديید وواالفريیتيین فى االدمم وواالقدررةة االكليیة التحادد االحديید.

فى هھھھؤالء االمرضى ووعلى االعكس ااظظهھرتت االنتائج اايیضا اانن االهھيیبسيیديین يیوجد بتركيیز ااقل

بتركيیز ااكبر فى ااالصحاء حيیث نسبة االحديید ااالقل.

اايیضا ووجد اانن نسبة االهھيیبسيیديین االى االفريیتيین فى االمرضى ااقل من نسبتهھا فى ااالصحاء.

ووهھھھذاا يیتفق مع اانن االهھيیبسيیديین مثبط فعالل المتصاصص االحديید٬، وواانن منظم االمولدةة للكريیاتت

تاجج االهھيیبسيیديین.ووبالتالى ووجدتت االتركيیزااتت االقليیلة مع االثالسيیميیا وواامرااضض االحمرااء لديیة االيیة لنقص اان

ااخرىى مصاحبة بالتكويین االغيیر فعالل لكريیاتت االدمم االحمرااء.فى مثل هھھھذةة ااالمرااضض فانن نقص

االهھيیبسيیديین يیؤددىى االى ااتاحة االفيیرووبرووتيین٬،فيیزيید ااخرااجج االحديید من االخاليیا االطالئيیة لالثنى عشر٬،

مما يیتيیح فرصة ااكبر من تصنيیع كريیاتت االدمم االحمرااء.وواالخاليیا االبلعميیة٬،