Many common human diseases are often devastating in their effects, due to mutations in single genes. Sickle cell disease is an inherited disorder of haemoglobin synthesis. It is due to a single nucleotide change in β-globin gene leading to the substitution of Valine for Glutamic acid at position 6 of the β-globin chain. Clinical manifestation arises from the tendency of the haemoglobin S to polymerise and deform red blood cells into the characteristic sickle shape.
The aim of this report was to establish which sickle cell β-globin genes were present in the mother's, father's and the unborn child's DNA sample whilst comparing with three control DNA samples. The DNA samples had then been digested with Mst II restriction enzyme and ran under gel electrophoresis. Results were analysed according to the bands produced.
The mother's DNA and the father's DNA, both produced three bands of one βS allele and the two βA allele. The results suggest both are carriers for heterozygous sickle cell β-globin gene (AS). Sickle cell trait is a benign condition that has no haematological manifestations and is associated with normal growth and life expectancy.
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The unborn child's DNA sample produced one band of the βS allele. The result suggests the unborn child is homozygous for sickle cell β-globin gene (SS). Sickle cell anaemia is a common genetic condition inherited from both carrier parents. Due to short life span of sickle red cell, severe haemolytic anaemia would develop within the first few months after birth. Lifelong treatment and monitoring is needed for sickle cell patients that include antibiotics, pain management and blood transfusions.
Diagnosis is important in order to determine the genes present and the possible treatments available, in order to stop the complications of the disease.
A human cell has many complex interactions regulating and expressing human genes. Alterations in gene structure, function and the process involved in protein synthesis may influence a person health. Changes in gene structure are called mutations, which permanently change the sequence of DNA, nature and type of protein made (Clancy, 2008). Some gene mutations have no significant effect on the protein product, while others cause partial or complete changes. The impact of the mutation is determined by how a protein is altered and its importance of body functioning (Smeltzer et al, 2009) (Primrose et al 2001).
Many human diseases often have devastating effects due to gene mutation that include; deletion (loss), insertion (addition), duplication (multiplication) or rearrangement (translocation) of a longer DNA segment (Lodish et al, 2007). However in this report we are focusing on the single missense mutation in the β-globin gene of haemoglobin that causes sickle cell anaemia. But first we need to know the normal haemoglobin structure and how it changes due to this mutation and the consequence related.
Haemoglobin (Hb) is the main constituent of Red Blood Cells (RBCs), making up over 98% of the protein content. The primary function of haemoglobin is to transport oxygen from the lungs to the tissue cells of the body and to carry carbon dioxide (CO2) back to be expelled (Estridge and Walters, 2000). Haemoglobin is a tetrameric protein, composed of two parts; haem group and globin chains. The haem portion contains iron, whilst the globin portion contains four polypeptide chains held together by interactions between peptide chains (Figure 1) (Kent, 2000).
Figure : The quaternary structure of normal haemoglobin (Kent, 2000).
The structure of human haemoglobin changes during development to several different globin chains (Figure 2). By the 12th week of gestation, embryonic haemoglobin is replaced by fetal haemoglobin, Hb F (α2γ2). This is the predominant haemoglobin of fetal life and is slowly replaced after birth by adult haemoglobins, Hb A (α2β2) and Hb A2 (α2δ2). Hb A is the major haemoglobin found in adults and children. Hb A2 and Hb F are found in small quantities in adult life (Provan, 2007) (Frenette and Atweh, 2007).
Haemoglobin synthesis is directed by controlling genes which are activated or deactivated at certain stages of human life, resulting in different globin synthesis at different ages. The genes controlling α-globin chains are located on chromosome 16, while genes for β-globin chains are located on chromosome 11 (Sarnaik, 2005). However mutations of α-globin genes affect haemoglobin production in both fetal and adult life because the α-globin chains are shared by both fetal and adult haemoglobins. Mutations due to defective β-globin production only manifest after birth when Hb A replaces Hb F (Provan, 2007).
Always on Time
Marked to Standard
Figure : The genetic control of normal human haemoglobin (Hb) during development (Provan, 2007).
There are more than 750 genetically determined variants of haemoglobin, however many are harmless while some have serious clinical effects. Haemoglobinopathies are a group of disorders that cause changes in the type or amount of the haemoglobin that is produced (Lewis et al, 2006).The three main categories of haemoglobinopathies are;
Production of structurally abnormal globin chains (sickle cell diseases such as haemoglobin S).
Production of structurally normal, but decreased amounts of globin chains (thalassemias).
Failure to switch globin chain synthesis from Hb F to Hb A (hereditary persistence of fetal haemoglobin) (Bain, 2001) (Lewis et al, 2006).
Sickle cell disease (SCD) is a hereditary disorder of haemoglobin synthesis. Haemoglobin S (Hb S) is a variant of normal haemoglobin, containing abnormal β-globin chain (Lovell et al, 2006). SCD is frequently found in the Afro-Caribbean populations and sporadically throughout the Mediterranean region, India and the Middle East (Jeremiah, 2006) (Okpala et al, 2002).The most common SCD consist of homozygous state for sickle cell gene known as sickle cell anaemia (SS), both genes are abnormal. The heterozygous state is known as sickle cell trait (AS). Only one chromosome carries the gene and produces about 40% is abnormal Hb S, and 60% normal Hb A (Provan, 2007) (Hoffbrand et al, 2001).
This abnormal β-globin chain in Hb S is produced by a single base change mutation from an A to T in the sixth codon of exon 1 in the β-globin gene on chromosome 11 (Lovell et al, 2006). As a result of this mutation, the hydrophobic neutral amino acid Valine takes the place of hydrophilic polar Glutamic acid in the β-globin chain (Lodish et al, 2007).
Figure : Sickled and normal red blood cells. The misshaped red blood cell on the left is caused by a missense mutation and an incorrect amino acid in β-globin chain of the haemoglobin (Clancy, 2008).
This substitution creates a hydrophobic spot on the outside of the abnormal haemoglobin structure and sticks to the hydrophobic region of an adjacent abnormal haemoglobin molecules β-globin chain (Lovell et al, 2006). Haemoglobin S is extremely insoluble and forms crystals when exposed to low oxygen tension. Deoxygenated sickle haemoglobin polymerise only occurs after oxygen is unloaded and transferred to cells in the body (Sarnaik, 2005). The haemoglobin molecules containing mutant β-globin chains come out of solution when returning to the lungs. The insoluble haemoglobin molecules then stick together and forms long fibres, each consisting of seven intertwined double strands with cross-linking inside the cell (Hoffbrand et al, 2001). These fibres distort and harden the membrane of the red blood cell, twisting the cell into a characteristic sickle shape (Figure 3) (Figure 4) (Cummings, 2006) (Clancy, 2008).
Figure : The result of the mutation in the β-globin chain predisposes the haemoglobin molecule to polymerise (clump together) in low oxygen tension, into rigid fibres causing sickling (distortion) of erythrocytes that contain the abnormal haemoglobin (Hoffbrand et al, 2005).
The polymerisation of Hb S in the circulating red cells is influenced by the oxygenation status, the intercellular haemoglobin concentration and the presence of non-sickle haemoglobins. Acidosis, hypoxia and dehydration promote polymer formation by reducing the oxygen affinity of haemoglobin (Kumar and Clark, 2005). In sickle cell trait the presence of Hb A within the red cells inhibits polymerisation by diluting Hb S. The inhibitory effect of Hb F on polymerisation of Hb S is due to the greater amino acid difference between the βS-and γ-globin chains (Hoffbrand et al, 2005).
The sickling of erythrocytes leads to increased rigidity, loss of deformability, increased adhesiveness to endothelial cells and red cell membrane damage (Sarnaik, 2005). All of this adversely affects the flow of properties of the red cells through the microvasculature causing severe pain and damage to tissues and various organs (Hoffbrand et al, 2001) (Lodish et al, 2007). The lowered number of red blood cells reduces the oxygen-carrying capacity of the blood and results in haemolytic anaemia. (Cummings, 2006) (Provan, 2007).
The definitive diagnosis of SCD requires DNA analysis. Prenatal fetal DNA for diagnosis can be isolated from chorionic villus cells or by amniocentesis in the first and second trimester (Pace, 2007) (Embury, 1995). The identification of disease carrier is done by direct detection of the βS mutation with RFLP (restriction fragment length polymorphisms) analysis, using Mst II endonuclease digestion (Chang and Kan, 1982) (Tantravahi and Wheeler, 2003). The mutation of SCD destroys the recognition site for Mst II within the β-globin gene (Figure 5) (Strachan and Read, 1999). Results produced by southern blot analysis and gel electrophoresis are used in detecting the βS and βA alleles respectively (Pace, 2007).
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Figure 5: Prenatal diagnosis for sickle cell anaemia using RFLP of MstII. The sickle cell mutation destroys an MstII site and generates a disease-specific RFLP (Strachan and Read, 1999).
The aim of this report was to establish which sickle cell β-globin genes were present in the mother's, father's and the unborn child's DNA sample whilst comparing with three control DNA samples. The DNA to be tested was extracted from the mother's and father's white blood cells. The DNA sample of the unborn child was obtained from amniocentesis. The three control DNA samples were normal, sickle cell trait and sickle cell anaemia. All the DNA samples had then been digested with Mst II restriction enzyme. The DNA samples were then run under gel electrophoresis and analysed to according to the bands produced, which would reveal the sickle cell β-globin genes present in the samples. Diagnosis is important in order to determine the genes present and the possible treatments available in order to stop the complications of the disease.
Materials and Methods:
Concentrated (50X) TBE buffer
DNA samples of mother, father and unborn child (digested with Mst II enzyme)
Control samples of normal, sickle cell trait and sickle cell anaemia
Ethidium Bromide card
250ml conical flask
Small plastic container
Gel bed (casting tray)
Well-former template (comb)
Visible Gel Visualisation System.
Preparing the gel bed
The open ends of a clean dry gel bed (casting tray) was closed off using a masking tape. The ¾ inch wide tape was extended over the sides and bottom edge of the bed. The extended edges of the tape were then folded back onto the sides and bottom edge of the bed. The contact points were pressed firmly and evenly across the bed. A well-former template (comb) was placed in the first set of notch at the end of the bed. The comb was checked to make sure that it sat firmly and evenly across the bed.
Casting agarose gel
A 250ml conical flask was used to prepare the gel solution. The following components were added to the flask; 0.8g of agarose, 2ml of concentrated (50X) TBE buffer and 98ml of distilled water. The total volume of 100ml in the flask was marked with a marker pen. The mixture in the flask was swirled to disperse the clumps of agarose powder. The mixture was then heated on a microwave to dissolve the agarose powder. This was done in order to make sure the final solution appears clear without any undissolved particles. The flask was covered with plastic wrap to minimize evaporation and the mixture was heated on High for 1 minute. The mixture was then swirled and re-heated again on High in bursts of 25 seconds until all the agarose was completely dissolved. The agarose solution was then left to cool to 55°C with careful swirling to promote even dissipation of heat. If detectable evaporation had occurred, then distilled water was added to bring the solution up to the original volume of 100ml marked on the flask. Once the gel was slightly cooled, the interface of the gel bed seal with tape needed to be checked, to prevent the agarose solution from leaking. This was done using a transfer pipette
and a small amount of cooled agarose was deposited to both inside ends of the bed. Wait approximately 1 minute for the agarose solution to solidify. The gel bed was placed on a flat levelled surface and then the cooled agarose solution from the flask was poured onto the bed. The gel was allowed to completely solidify until it became firm and cool to touch after approximately 20 minutes.
Preparing the gel for Electrophoresis
After the gel was completely solidified, the tape was removed from the gel bed carefully and slowly. The comb was then removed by slowly pulling it straight up. This was done carefully and evenly to prevent tearing the sample wells. The gel bed was placed into the gel electrophoresis chamber, properly oriented, centred and levelled on the platform. Then the 50X TBE buffer was diluted in distilled water to make 500ml of 1X TBE buffer. Therefore 10ml of TBE buffer was added 490ml of distilled water. The electrophoresis apparatus chamber was filled with 1X TBE buffer, whilst making sure the gel is completely covered in the buffer.
Loading the samples
The volume of the DNA samples pre-digested with Mst II enzyme were checked, to make sure the entire volume of the sample is at the bottom of the tube before starting to load the gel. Using a pipette, 25µl of DNA samples in tube A-F were loaded into the wells in consecutive order of;
Sickle cell gene sample
Sickle cell trait (carrier) sample
Normal gene sample
Mother's (Patient B's) DNA sample
Unborn child's DNA sample
Father's DNA sample
Running the Gel
After the DNA samples were loaded, the cover was carefully snapped down onto the electrode terminals. The negative and the positive colour-coded indicators on the cover and the apparatus chambers should be properly oriented. The plug of the black wire was inserted into the black input of the power source (negative output). The plug of the red input was inserted into the red input of the power source (positive input). The power source was set at the required voltage of 125V and the electrophoresis was conducted for the length of time of 30 minutes. The current flowing was checked to see if it is flowing properly, by looking at the bubbles formed on the two platinum electrodes. After the electrophoresis was completed, the power was turned off. The power source was unplugged and the leads were disconnected and removed from the cover.
Staining the gel
After electrophoresis the gel was placed on a flat surface. The gel was then moistened with several drops of electrophoresis buffer. Whilst wearing gloves, the adhesive side was removed and the Ethidium Bromide card was placed on the well-moistened gel. Finger was firmly run over the entire surface of the card several times. The gel containing the Ethidium Bromide card was placed into a piece of plastic wrap. Then the gel casting tray and a small empty beaker was placed on top. This would ensure that the card maintains a good contact with the gel surface. The Ethidium Bromide card was allowed to be in contact with the gel for 10 minutes. After 10 minutes the Ethidium Bromide card was removed, and the gel was transferred to a small plastic container. The surface of the gel was rinsed with buffer and then examined on a Visible Gel Visualisation System.
Direction of migration
Unknown Patient Samples
Figure 6: Photograph of gene identification by gel electrophoresis of the three control samples (A,B,C) and three unknown patient samples (D,E,F,).
The control sample results produced the following results; Sample A produced one band (βS allele). Sample B produced three bands (βS allele and two βA alleles) and Sample C produced 2 bands (two βA alleles). The unknown patient samples, whose gene's needed to be established, produced the following results; Sample D produced three bands (βS allele and two βA alleles). Sample E produced one band (βS allele) and Sample F produced three bands (βS allele and two βA alleles).
The principle of gel electrophoresis is to separate DNA according to their different electrophoretic mobilities. The smaller fragments of DNA will travel further and more quickly through the agarose gel towards the positive electrode than larger fragments, once they have been digested by the restriction enzyme MstII. (Chang and Kan, 1982).
In sickle cell anaemia, the sickle cell allele has a single-nucleotide substitution that converts an adenine base to a thymine base in the second position of the sixth codon of this gene. This changes the normal β-globin gene (CCT-GAG-G) into a sickle cell β-globin gene (CCT-GTG-G). The mutation also causes the loss of recognition site for the restriction enzyme Mst II, and so the sickle cell gene sequence cannot be cut. Therefore the homozygous sickle cell β-globin gene (SS) produces one large band (Cummings, 2006). However the sequence for normal β-globin gene (CCT-GAG-G) corresponds to an Mst II restriction site (CCTNAGG, where N = any nucleotide), and therefore it cuts this sequence into two different sized bands (Cummings, 2006).
The bases of these principles are used to establish which sickle cell β-globin genes were present in the mother's, father's and the unborn child's DNA sample whilst comparing with three control DNA samples. Where sample A was of sickle cell gene and produced one band (βS alleles). Sample B was of sickle cell trait and produced three bands (one βS alleles and two βA alleles). Sample C was of normal gene and produced 2 bands (two βA alleles) (Figure 6).
Mother's and Father's DNA sample results;
Sample D contained the mother's DNA and Sample F contained the father's DNA, both produced three bands (Figure 6). Whereby the βS allele band stayed near the origin and the two βA allele bands migrated further towards the cathode. Comparing the allele migration with that of the control sample B, the results suggests both are carriers for heterozygous sickle cell β-globin gene (AS).
Sickle cell trait is a benign condition with normal appearance of red cells on a blood film. The individual has inherited a normal β globin gene and a βS globin gene, producing more of dominant Hb A and Hb S (Provan, 2007). Individuals are not anaemic, have no clinical abnormalities and under physiologic conditions have a normal life expectancy (Provan, 2007) (Hoffbrand et al, 2005) (National Institutes of Health, 2004).
Haematuria is the most common symptom and is thought to be caused by minor infarcts on the renal papillae, where red cells at susceptible to sickling (Derebail, 2010). Red cells do not sickle unless oxygen saturation is <40%. Painful crises and splenic infarction have been reported in severe hypoxia, such as unpressurised aircraft or under anaesthesia and would require prompt oxygen therapy (Tiernan, 1999) (Frietsch, 2001). Many individuals will have decreased ability to concentrate their urine.
Women with sickle-cell trait need additional care if general anaesthetic is used during labour. There may be an increased incidence of urinary tract infection during pregnancy. Also if adequate oxygen level is not maintained then there is increased risk of pre-eclampsia and tissue infarction (Warrell, 2005). (Hoffbrand et al, 2001).
When both parents have sickle-cell trait (AS), their child has a 25% chance of being normal, 25% chance of having sickle-cell anaemia, 50% chance of having sickle cell trait (Frenette, and Atweh, 2007)
Unborn child's DNA sample results;
Sample E contained the unborn child's DNA sample and it produced one band only (Figure 6). This band was of the βS allele which stayed near the origin. Comparing the allele migration with control sample A, the results unfortunately suggests the unborn child has the sickle cell gene. The unborn child has a homozygous sickle cell β-globin gene (SS) and will suffer from sickle cell anaemia.
Sickle cell anaemia is a common genetic condition due to a haemoglobin disorder. The inheritance of the mutant haemoglobin genes was from both sickle cell trait parents (Cummings, 2006). The child would appear normal at birth, due to the predominant haemoglobin of fetal life, however the mutation due to defective β-globin production only manifest when Hb A replaces Hb F after birth causing severe anaemia to develop within the first few months. The child would also have low haemoglobin concentration, high reticulocyte count and the blood film would show sickled erythrocytes (Provan, 2007).
As a consequence of the disease, the child would suffer from painful crises, due to sickle cells blocking blood flow through vessels. This results in tissue damage that causes abdominal pain episodes, stroke, priapism, damage to spleen, kidney and liver aswel. Damage to spleen can cause overwhelmed bacterial infections especially in young children (Smeltzer et al, 2009). Between 6 and 18 months of age affected child would present with painful swelling of the hands/ feet (hand-foot syndrome).
More serious and life threatening crises include the sequestration of red cells into the lung or spleen (Provan, 2007). Vaso-occlusions leads to membrane damage causing shorter red cell life to span (15 days instead of 120 days), resulting in a haemolytic anaemia (Sarnaik, 2005).
Lifelong treatment and monitoring is needed for sickle cell patients that include antibiotics, pain management and blood transfusions (Provan, 2007).To prevent serious infections, by 2-3 months of age the child should receive daily doses prophylactic penicillin daily, this is continued until at least 5 years of age (Pass el al, 2000) (Steinberg, 1999). Painful crises should be managed with adequate analgesics, hydration and oxygen. Hydroxyurea is a drug, when given daily it reduces episode painful crises and acute chest syndrome. People taking the medicine also need fewer blood transfusions and have fewer hospital visits (Wang et al, 2001) (Wiles and Howard, 2009). Blood transfusions are used to treat severe anaemia and sickle cell complications. A sudden worsening of anaemia due to an infection or enlargement of the spleen is a common reason (Josephson et al 2007).
There are various ongoing researches on gene therapy, bone marrow transplants and new medicines for sickle cell anaemia. These should provide better treatments for sickle cell anaemia and way to predict the severity of the disease (Frenette and Atweh, 2007) (Persons, 2009).
It can be concluded that gel electrophoresis separates DNA according to their different electrophoretic mobilities. When digested with MstII, the smaller fragments of DNA will travel further and more quickly through the agarose gel towards the positive electrode than larger fragments.
The mother's DNA and the father's DNA, both produced three bands of one βS allele and the two βA allele. The results suggest both are carriers for heterozygous sickle cell β-globin gene (AS). Sickle cell trait is a benign condition that has no haematological manifestations and is associated with normal growth and life expectancy. Haematuria is the most common symptom however under severe hypoxia, they may experience painful crises and would require oxygen therapy.
The unborn child's DNA sample produced one band of the βS allele. The result unfortunately suggests the unborn child is homozygous for sickle cell β-globin gene (SS). Sickle cell anaemia is a common genetic condition inherited from both carrier parents. Due to the short life span of sickle red cell, severe haemolytic anaemia would develop within the first few months after birth. The child would also suffer from painful crises, due to sickle cells blocking blood flow through vessels. Painful crises should be managed with adequate analgesics, hydration and oxygen. Lifelong treatment and monitoring is needed for sickle cell patients that include antibiotics, pain management and blood transfusions.