HEMIS #: 363667Globin gene Haplotype analysis in a Ghanaian population 2009/2010 Globin gene Haplotype analysis in a Ghanaian population 1. Introduction 1.1 Haemoglobin: structure and function The Haemoglobin molecule is made up of four polypeptide chains, each of which has a single haem group consisting of an iron atom located at the centre of a porphyrin ring(Bragg and Perutz, 1952). This molecule is spherical in structure with the Globin chains folded so that the four haem groups lie in surface clefts equal distance and parallel from each other. The molecule is held together in its quaternary structure by bonds between the opposite polar chains and the structure changes as oxygen is taken up by each haem group. The structure of haemoglobin is shown in fig 1.1 below 1
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HEMIS #: 363667Globin gene Haplotype analysis in a Ghanaian population 2009/2010
Globin gene Haplotype analysis in a Ghanaian population
1. Introduction
1.1 Haemoglobin: structure and function
The Haemoglobin molecule is made up of four polypeptide chains, each of which has a
single haem group consisting of an iron atom located at the centre of a porphyrin ring(Bragg
and Perutz, 1952). This molecule is spherical in structure with the Globin chains folded so that
the four haem groups lie in surface clefts equal distance and parallel from each other. The
molecule is held together in its quaternary structure by bonds between the opposite polar
chains and the structure changes as oxygen is taken up by each haem group. The structure
of haemoglobin is shown in fig 1.1 below
Fig 1.1 Quaternary structure of haemoglobin
When an Oxygen molecule binds to the haem group on one of the polypeptide subunit
chains it causes a structural change of the whole haemoglobin molecule and this allows
more oxygen molecules to bind to the three remaining subunit molecules in a summative
fashion from one of the subunits (Ogata and McConnell, 1972).
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HEMIS #: 363667Globin gene Haplotype analysis in a Ghanaian population 2009/2010
Each molecule of haemoglobin can carry up to four molecules of oxygen when fully
oxygenated, with one oxygen molecule binding to each haem group, transporting Oxygen
from the lungs around the body. As blood travels through the arterial system and flows back
to the lungs, haemoglobin picks up Carbon Dioxide from the tissues and carries it to the
lungs. Haemoglobin also acts as a red blood cell buffer and reduces changes in pH within the
red blood cell when they are oxygenated or deoxygenated. Red blood cells contain
approximately 640 million haemoglobin molecules.
1.2 Haemoglobin gene clusters- classification and expression
The genes that code for haemoglobin in humans are found in two clusters on an α or α-like
complex on chromosome 16 and a β complex on chromosome 11 (Manning and Russell.,
2007). The α-like cluster can be found close to the end of chromosome 16 and is made up of
the functional genes α1, α2 and ζ, three pseudo genes ( genes closely resembling functional
genes but contain mutations which render them inactive):ψ ζ, ψα1 and ψα2 and ѳ, which
have no known function in haemoglobin synthesis. The α1 and 2 genes code α Globin chains
from late embryonic stage of life towards the liver and spleen are developed enough to
produce Haemoglobin and by the sixth month of pregnancy bone marrow of becomes the
main site of haemoglobin synthesis (Pallister, 2005). The ζ gene codes for an α like zeta chain
in early embryonic life and is active during the 5th week of gestation and Haemoglobin is
synthesized from erythroblasts in the gestational sac (Kunkel et al., 1955).
The β-like gene cluster is located on the short arm of chromosome 11. (Sutton et al., 1989) It
contains a single pseudo gene (ψβ), and five functional genes (€, Gγ, Aγ, δ and β) which
encode for the €, γ, δ, and β chains respectively. The γ chains are mainly coded in foetal life
by Gγ and Aγ when they combine with α chains to form foetal haemoglobin (Hb F). β Globin
gene synthesis begins after birth(Korf., pg 208., 2007). This shows therefore that locations,
types and rates of synthesis of different Haemoglobins can vary during foetal and adult life.
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The locations of the haemoglobin genes are shown in fig 1.2 below
Fig 1.2 Location of Hb clusters on chromosome 11 and 16
Adult human haemoglobin (Hb A) consists of 2 α-Globin and 2β-globin chains of 141 and 146
amino acid chains respectively and also has a small percentage of Hb A 2 which has 2 δ-
globins and 2 α-globins.
Synthesis of globins is a complex process where transcription produces a messenger RNA
precursor; post transcriptional processing with 5’ end capping and methylation as well as the
various phases of translation, and finally the ionic interaction between the haemoglobin
chains to form mature active haemoglobin(Paul., 1976). Because of this myriad of active
processes and their complex nature, errors can occur during processing causing
haemoglobin disorders. Disorders of haemoglobin are categorised into two kinds; those
causing abnormalities of haemoglobin structure and those causing reduced Globin synthesis
(World Health Organization., 2006)
There are various kinds of structurally abnormal haemoglobins; some are clinically silent
however others have serious clinical implications that affect people who have abnormal
haemoglobin. The mutational mechanisms normally consist of point mutations but in some
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case also include frame-shift mutations, in frame deletions, mis-pairing of homologous
sequences that lead to unequal cross-over of genes and formation of fused genes (Korf., pg
209., 2007)
1.3 Single nucleotide polymorphisms (SNP’s)
Single nucleotide polymorphisms, commonly referred to as “snips” (SNP’s) are the most
prolific type of genetic variation among people. SNP’s show a difference in a singleDNA
nucleotide, where one nucleotide is replaced by another for example, A G, C T and vice
versa in a certain stretch on a DNA strand.
SNP’s occur throughout DNA and are normally exhibited approximately every 300
nucleotides meaning there are around 10 million SNP’s occurring on the human genome.
SNP’s are usually found on DNA in between functional genes however when they occur on a
functional gene or on the regulatory region for a gene, they may cause or have a role in gene
abnormality and disease.
SNP’s usually have no effect on health and development and this explains why they occur so
frequently on the human genome. They do however sometimes provide important insight
into human health. Studies conducted have found that they may affect people’s responses to
drug metabolism (Kudzi et al., 2009), endogenous factors, and the risk of developing
diseases associated with the genome. SNP’s are also useful tools in determining hereditary
passing on of certain disease states such as obesity, diabetes, and cancer (Eftychi et al., 2004,
Herbert et al., 2006,).
1.4.1Haemoglobin Abnormalities
Hb abnormalities are inherited disorders in the Globin chains when the haem group is in the
normal state. They are mostly autosomal recessive abnormalities and common worldwide,
particularly within the malarial regions of Africa, the Mediterranean basin, the Middle East
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HEMIS #: 363667Globin gene Haplotype analysis in a Ghanaian population 2009/2010
and south East Asia (Modell and Darlison, 2008). It is thought that some of the heterozygous
carrier states with abnormal Hb afford protection against malaria (see Section 1.6).
When a haemoglobin abnormality causes a blood disorder it is known as a
haemoglobinopathy and the nature of the Hb abnormality will determine the clinical
significance.
Haemoglobinpathies are described as any blood disorder caused by defects in Globin gene
chain synthesis. Classified into two broad categories; those which cause a reduction in
haemoglobin synthesis (the Thalassaemias), and structural haemoglobinopathies where
haemoglobin variants cause the disorder (Sickle cell disease).
Haemoglobinopathies are the most common single gene disorders in man. They are passed on to
generation to generation and these conditions are more prevalent in populations of African, Arab,
Middle Eastern and Hispanic descent. The most common type of haemoglobinopathies are the
thalassaemias, and sickle cell anaemia.
According to the NHS In the UK, an estimated one in 300 babies of African-Caribbean parents and
one in 60 of West African parents are born with sickle cell disease each year. An estimated 8,000-
10,000 people with sickle cell disease and 600 with β Thalassaemia live in the UK. Approximately 1
in 4 West African, 1 in 10 African-Caribbean, 1 in 50 Asian and 1 in 100 Northern Greek have sickle
cell trait (carrier state). Whilst 1 in 7 Greek, 1 in 10-20 Asian, 1 in 50 African and African-Caribbean
and 1 in 1000 English people have beta thalassaemia trait. Worldwide α thalassaemia carrier states
are commoner than ß thalassaemia carrier states(European haemoglobinopathy registry., 2003)
Sickle cell anaemia and the Thalassaemias exhibit some clinical similarities between the two
conditions. Both are expressed as a direct result of a defect in the Globin chain and patients
suffer chronic haemolysis throughout their life. Patients of both conditions may also need
chronic blood transfusions. Subsequently, patients may be at risk of iron overload that has
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HEMIS #: 363667Globin gene Haplotype analysis in a Ghanaian population 2009/2010
its own clinical implications (Fung et al., 2008). Thalassaemias fall into three major categories;
α-thalassaemia, β-thalassaemia and High Persistence of Foetal Haemoglobin (HPFH). There
are rare thalassaemias which fall outside of these categories, namely δβ and εδβ-
thalassaemia and Hb Lepore (Weatherall, 2001).
1.3.2 Haemoglobin variants
Different kinds of Hb variants exist caused from autosomal recessive pairing of alleles and
are mainly due to substitutions in the β-chain(refer to fig 1.2 ) Rare Hb variants such as
Haemoglobin H and Haemoglobin Barts may also come about from the extension or deletion
of the Globin chain ( Giardine et al., 2007).
The occurrence of abnormal haemoglobins varies significantly between ethnic groups
(Modell and Darlison, 2008). Haemoglobin C occurs in approximately 2-3% of people of West
African descent and most people are heterozygous for it. Homozygosis is rare and has mild
clinical symptoms. Haemoglobin E is one of the more common β globin chain variants and
is relatively common in Southeast Asia (Flatz., 1967). Amino acid substitutions along one
polypeptide chain generally create a high affinity for oxygen. The most prevalent
Haemoglobin disorder caused by an Hb variant is Haemoglobin S which causes the
potentially life threatening disorder known as sickle cell anaemia.
1.3.3 Haemoglobin S and Sickle cell anaemia
One clinical condition that can be caused by having mutations of the parts of haemoglobin
genes is sickle cell anaemia. It is one of the most common hereditary diseases in the world
and mainly affects people whose ancestry is from sub-Saharan Africa although it is also
known to affect Mediterranean, Middle Eastern and Asian populations (Cihan Öner et al.,
1992). The disease shows autosomal recessive inheritance so to have the condition you
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HEMIS #: 363667Globin gene Haplotype analysis in a Ghanaian population 2009/2010
must receive one recessive allele from both parents. The gene mutation causing the
formation of Hb S is: GAG GTG. This mutation results in substitution of the amino acid
valine (Val) for glutamic acid (Glu) in the sixth position of the β- Globin chain (α2 /β2 6val).
Despite being a minor change in the polypeptide sequence, it causes serious implications in
people with this mutation on their Hb gene cluster (Bunn, 1997). Sickle cell anaemia is the
most common haemoglobinopathy and usually occurs between various ethnicities in
populations that are exposed to falciparum malaria and the anopheles mosquito. The
disease was initially classified by Herrick et al in 1910 and Singer and Wells explained the
mutation causing Hb S in 1925.
Hb S in sickle cell patients manifests itself by causing sickling in red blood cells, causing them
to be rigid and take up a crescent shape. Hb S is insoluble and crystallises under low O2
partial pressure (Bunn., 1998, Lonergan et al., 2001, Higgs and Wood, 2008). Sickled Red blood
cells also interfere with oxygen transport as they are less elastic to movement inside
capillaries. Haemoglobin gives up oxygen more readily than normal haemoglobin and the
oxygen dissociation curve is shifted to the right.
Fig1.3 and fig1.4 below show the distortion of red blood cells with Hb S and the shifting of
the Oxygen Dissociation Curve among normal haemoglobins and SCA respectively.
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HEMIS #: 363667Globin gene Haplotype analysis in a Ghanaian population 2009/2010
Fig1.3 Appearance of normal and sickled red blood cells
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Fig1.4 Oxygen Dissociation Curve for normal, sickle cell trait and sickle cell sufferers
1.3.3.2 Complications and clinical implications of sickle cell disease
Red blood cells exhibiting Hb S as explained before are sickled in shape and are inflexible
making them incapable of efficient circulation in small blood vessels, causing them to have a
life span of only 10-20 days as compared to normal healthy Red Blood Cells which live up to
120 days. Anaemia results because of this and other crises occur in sickle cell patients. Sickle
cell disease is a major public health problem in several countries particularly less
economically developed countries (LEDC’s) and gives significant morbidity and mortality in
particular with young children aged 1 to 3 years of age caused by cerebrovascular accidents
and viral infections (Leikin et al., 1989, Athale and Chintu, 1994). Blood transfusions in these
patients is sometimes necessary and can cause iron overload which damages vital organs
such as the heart, liver and spleen (Wood, 2008). Life expectancy in patients with Sickle Cell
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HEMIS #: 363667Globin gene Haplotype analysis in a Ghanaian population 2009/2010
Disease is greatly reduced and patients rarely live past the age of 35. Moreover quality of life
in SCD patients is low as they are constantly dealing with crises and the residual effects of
anaemia and other complications. The crises that manifest themselves in SCD patients
include haemolytic anaemia, Ischaemic pain, susceptibility to bacterial infections and severe
organ dysfunction (Vichinsky et al., 1998, Frempong et al .,1988, Serjeant et al.,). Clinical
complications of SCD occur among homozygous Hb SS, or in those heterozygous for Hb S and
another abnormal haemoglobin e.g. Hb C, β+ thalassaemia or. People with Sickle Cell disease
that are homozygous (Hb SS) exhibit 90-95% Hb S in their erythrocytes (Wood et al., 1980).
Disease severity is determined by the genotype exhibited by the patient and homozygous Hb
SS is the most clinically significant and crises of Sickle cell Disease are classified as being
haemolytic, aplastic or vasco-occlusive. Vasco-occlusive crises are the most common
complication of SCD and they arise from interaction of sickled erythrocytes with White Blood
Cells, Endothelial cells, Platelets and Plasma. Capillaries and microvascular beds become
obstructed and causes problems such as leg ulcers, neuropathic and chronic pain, renal
problems and in some extreme cases stroke (Yale et al., 2000). Ischaemia also results
causing an absolute reduction of oxygen supply to some organs, joints and bones. Ischaemic
injury is recurrent and produces a distinct chronic pain syndrome (McClish et al., 2005,
Shapiro., 1989).
Aplastic crisis may occur in patients with SCA due to parvovirus B19 (B19 virus). Parvovirus
B19 is a DNA virus that infects and destroys erythroid cell progenitors (Setubal et al., 2000).
This crisis is usually preceded by a febrile illness in hereditary haemolytic anaemia’s,
resulting in likely Bone Marrow failure. The crisis is characterised by low Hb concentrations
and a low reticulocyte count (Setubal et al., 2000, Pattison et al., 1981). Carriers, who are
heterozygote for the gene are known as having the sickle cell trait are usually healthy,
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HEMIS #: 363667Globin gene Haplotype analysis in a Ghanaian population 2009/2010
although they can exhibit some symptoms when there is reduced partial pressure of oxygen,
such as vaso-occlusive episodes.
In some sub-Saharan populations the number of people carrying the sickle cell trait can
reach levels of up to 20%. This is because there is some survival benefit because there is a
connection between being a SC carrier and malaria resistance, and in these populations
malaria is one of the biggest killers.
1.4 Malaria and sickle cell disease
Malaria is a disease caused by the parasite Plasmodium falciparum and the disease is
transmitted to people through the bite of the female anopheles mosquito which thrive in
the tropical conditions of sub-Saharan Africa. The mechanism by which HbAS genotype
protects against malaria has been the subject of debate for more than 50 years. While it is
thought that it relates to the physical characteristics of HbAS erythrocytes, a number of
studies (Aidoo et al., 2009, Williams et al., 2005) suggest that sickle cell carriers may also
enhance natural immunity to plasmodium.
Studies show that mortality rate of malaria in people with Sickle cell trait is conclusively
lower than in normal individuals. Experiments involving P.falciparum in vitro have been
conducted to show the growth of the parasite in sickled RBC’s at standard partial pressure of
oxygen and a low O2 atmosphere and at low concentrations an inhibition of plasmodium
growth is shown.(Friedman, 1978) Sickled RBC’s, and those of carriers(Hb AS) provide an
unsuitable environment for the developing life cycle of the malaria parasite and this gives
evidence of the high proportion of heterozygous Hb AS people in malaria endemic areas.
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HEMIS #: 363667Globin gene Haplotype analysis in a Ghanaian population 2009/2010
1.5 β-Globin cluster haplotypes
There are 5 different haplotypes of the βs gene (Hanchard et al., 2007, Nagel and Ranney,
1990) causing SCA and they are named after the geographical region where they were first
identified. They are Senegal, Benin, Bantu, Cameroon and Arab-Indian (Nagel and Ranney
1990). The first four mentioned are mostly found in African populations whereas the Arab-
Indian is found in the Middle East and Asia. The Arab-Indian Haplotype and Senegal
Haplotype exhibit less clinical symptoms than the other haplotypes found in Africa. This has
been reported in studies showing clinical severity among people with different haplotypes
explained by having the post natal expression of Hb F present in people expressing these
haplotypes . The different haplotypes causing SCA can be identified by amplification of their
restriction endonuclease sites on the β-Globin cluster by using RFLP and restriction enzyme
digestion. Approximately 5% to 10% of people exhibit what are referred to as atypical βs
haplotypes (Powers and Hiti, 1993., Rahimi et al., 2007).
Haplotypes for sickle cell anaemia can be studied using techniques that can amplify and
visualise DNA fragments such as southern blotting, polymerase chain reaction,
electrophoresis and restriction enzyme digestion.
1.6Restriction fragment length polymorphism analysis
PCR- RFLP analysis is at present the best method used in the analysis of the blood samples
and is also used for diagnosis of haemoglobinopathies. Polymerase Chain Reaction amplifies
individual alleles on the dried blood samples, and restriction enzyme digestion shows the
researcher which specific allele is present in each sample (Chang et al., 1981 ).
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HEMIS #: 363667Globin gene Haplotype analysis in a Ghanaian population 2009/2010
Restriction enzymes complimentary to the primers used in the amplification cut double
stranded DNA at specific SNP’s. Mutations destroy or alter restriction enzyme sites, allowing
polymorphisms from abnormal genes to be visualised (Sutton et al.,1989).
The Hb S gene is associated to certain DNA structures by specific restriction endonuclease
positions in the β-Globin cluster (Rahimi Z,Karimi M, Hagshenass M, Merat A 2003). PCR
products were treated by suitable enzymes and then Agarose gel electrophoresis will be
used to separate the fragments. The bands are stained with Ethidium bromide and can be