In this report, a protocol for the preparation and analysis of DNA which permits more rapid and sensitive antenatal detection of sickle cell anaemia. MST II digested DNA was used to analyse by agarose gel electrophoresis. The 1.2-kb βA and 1.4-kb βS MST II fragments are well separated. Sickle cell disease is due to the substitution of valine for glutamic acid at position 6 of the beta globin chain. This substitution allows haemoglobin S molecules to polymerize when deoxygenated. The polymerized haemoglobin S distorts the erythrocyte into the characteristic 'sickle' shape. The sickling disorders are a group of inherited diseases of the haemoglobin molecule characterized by chronic haemolytic anaemia. It is important to identify couple at risk for an affected pregnancy. In addition, prenatal diagnosis aims to provide an accurate, rapid result as early in pregnancy as possible. Prerequisites include the characterisation of their disease causing mutations, obtaining foetal material promptly and safely and investigating the genotype of the foetal DNA based on the parental mutations.
1.0 Introduction:
Sickle cell disease is the most common genetic disorder to affect Blacks (Honig, 1996; Konotey, 1992; Sergeant, 1985). For most affected children, the parents are usually unaware of the presence of the disease (Bainbridge et al., 1985). In developed countries, newborn screening programmes have been established to ensure early diagnosis and thus early enrolment into a comprehensive healthcare programme (Githens et al., 1990; Vichinsky et al., 1988). As in developing countries, sickle cell disease is prevalent, newborn screening for sickle cell disease is needed. In contrast, with advances in management, pregnant women with sickle cell disease now survive to have children (Ntim et al., 2006). However, the pregnancies of women with severe sickle haemoglobinopathies are associated with a high incidence of maternal and perinatal morbidity and mortality. In this report, sickle cell gene analysis was performed by using agarose gel electrophoresis of digested genetic sequences (Atkin et al., 1998).
Human beta globin genes include embryonic (ε)-globin genes, fetal (γ)-globin genes and human adult (β)-globin genes, which are found on the short arm of human chromosome 11 (Ley et al., 1984). Sickle cell anaemia, a human genetic disease, is the result of single base pair (adenine to thymine) change in the beta globin gene. Amino acid substitutions is the replacement of one of the three bases in the RNA adenine, A; guanine, G; cytosine,C; uracil, U. Sickle cell anaemia is corresponding to the sixth amino acid residue, in which changing of glutamic acid to valine in the beta globin protein (Steinberg, 1999), at the same time abolishes an MST II restriction site which spans codons 5 to 7.
A mutation in a single base in the DNA sequence of a gene affects the hybridization of an oligonucleotide complementary to the region of the mutation (Conner et al., 1983). The beta globin genes arranged 5' to 3' in order of expression during development.
Figure : The representation of the globin gene loci, it shows the beta globin locus. The two gamma globin genes are active during fetal growth and produce hemoglobin F; the "adult" gene, beta, takes over after birth.
The replacement of A by T at the 17th nucleotide of the gene for the beta chain of haemoglobin changes the codon GAG for glutamic acid to GTG which encodes valine. Thus the sixth amino acid chain becomes valine instead of glutamic acid. It gives rise to sickle cell disease in homozygotes because the modified chain has a tendency to crystallise at low oxygen concentration. In the case of sickle cell haemoglobin, the replacement of a negatively charged glutamic acid in the standard HbA beta-globin by a neutral valine in HbS results in a protein with a slightly reduced negative charge. In homozygous individuals, the HbA tetramer electrophoresis as a single fast band, and the HbS tetramer as a single slow band. Heterozygous individual comprises with both forms of the tetramer, and therefore runs as two bands.
Figure 2: Amino acid sequence of normal adult haemoglobin with glutamic acid in position 6 and mutant adult haemoglobin with valine in position 6.
Sickle cell disease is inherited in an autosomal recessive manner. Homozygosity for the sickle mutation (HbSS) is responsible for the most common and most severe variant of sickle cell disease (Platt et al., 1994). If one parent has sickle cell anaemia (SS) and the other has sickle cell trait, there is a 50% chance of a child being affected with sickle cell trait (AS). When both parents have sickle cell trait (AS), a child has a 25% chance of being affected sickle cell disease (SS) (Koch et al., 2000). HbAS is heterozygote for the βs globin with no symptoms of the disease.
The sequence of the standard βA allele happens to correspond to an MST II restriction site, which is altered in the βS allele. The beta globin gene region includes two flanking MST II sites. Restriction enzyme MST II was showed by Yuet Wai Kan and Andrees Dozy in 1978. This enzyme cuts normal beta globin DNA at a particular site, it will not recognize (and therefore will not cut) DNA that contains the sickle cell mutation. MST II recognized the sequence CCTNAGG (where N = any nucleotide). Sickle cell disease is due to a single point mutation in the beta globin gene on chromosome 11 that changes CCTGAGG to CCTGTGG. Therefore, the A to T mutation that causes sickle cell anaemia also causes the loss of the recognition site for the restriction enzyme MST II (Wilson et al., 1982).
Figure 3: Restriction Fragment Length Polymorphism (RFLP) resulting from beta globin gene mutation.
In the normal cell, the sequence corresponding to 5th to 7th amino acids of the beta globin peptide is CCTGAGGAG, which can be recognized by the restriction enzyme Mst II. In the sickle cell, one base is mutated from A to T, making the site unrecognizable by Mst II. Thus, Mst II will generate 0.2 kb and 1.2 kb fragments in the normal cell, but generate 1.4 kb fragment in the sickle cell. These different fragments can be detected by southern blotting.
A restriction fragment length polymorphism (RFLP) is defined by an enzyme, which cuts the double stranded DNA at a particular sequence of bases, a probe, a labelled, complementary segment of DNA that will anneal to a portion of the digested sample, and a set of variable fragment length bands that appear on Southern blot (William, 2005). The use of RFLP analysis is to diagnose genetic disease and identification of disease carriers. Restriction enzyme and genetic disease are linked if a polymorphic region is close to the area responsible for a disease. The polymorphic region which is capable of being cut with a restriction enzyme is known within the gene responsible for a disease (Saiki et al., 1985).
Figure 4: The sickle cell mutation destroys an MST II site and generates a disease-specific RFLP.
The MST II restriction nuclease recognizes the sequence CCTNAGG where N = A, C, G or T. A restriction site for MST II is found in the normal βA-globin allele but is destroyed by the sickle cell mutation. The nearest flanking MST II sites are located, respectively, 1.2 kb upstream in the 5′-flanking region of the β-globin gene and 0.2 kb downstream at the 3′ end of the first intron. Conservation of these flanking sites results in the βA-associated (1.2 kb + 0.2 kb) MST II RFLP and the sickle cell-associated 1.4 kb MST II RFLP.
Case study:
A 23-year-old married woman (Patient B) of Turkish descent attended the routine antenatal screening. Patient B's full blood count and blood film results were, haemoglobin 10.3 g/ dl, red blood cell count 3.5 x 1012/ L, MCH 25pg, blood film normal. She had further tests, Haemoglobin S solubility test showed turbidity, positive solubility, indicated presence of haemoglobin S; citrate agar electrophoresis at pH6.0 showed haemoglobin S and haemoglobin A, indicated she is a sickle cell trait, genotype βAβS, heterozygous state. Patient B is suggested to take another test, sickle cell gene analysis with her partner (sample F) and unborn child (sample E).
2.0 Materials and Method:
2.1 Material
2.1.1 Equipment needed
250ml flask
Marker pen
Microwave
Electrophoresis chamber
Gel bed
Transfer pipette
Micropipette
2.1.2 Solutions
Agarose gel, Buffer
2.1.3 Specimen
DNA samples
2.2 Methods
2.2.1 Preparing the gel bed:
First, a clean and dry casting tray was closed off in the open ends by using a tape. A ¾ inch wide tape was used to extend over the sides and bottom edge of the bed. The extended edges of tape were folded back onto the side and the bottom. To form a good seal, the contact point was pressed firmly. Second, a well formed template, also known as a comb was placed in the first set of notches at the end of the bed. It is important that the comb sits firmly and evenly across the bed.
2.2.2 Casting agarose gels
First, the agarose gel was prepared by dissolving 0.8g of agarose into 2ml of concentrated 50x buffer and 98ml distilled water by using a 250ml flask. This solution was then mixed properly. The volume in the flask was marked with a marker pen to indicate the level of the solution on the outside of the flask. The mixture was agitated to disperse clumps of agarose powder. The flask was covered with plastic wrap to minimize evaporation. Next, this mixture was heated in a microwave to dissolve the agarose powder. The mixture was heated on high for 1 minute. Afterwards, the mixture was swirled and heated on high in bursts of 25 seconds until all the agarose was completely dissolved. The final solution appeared transparent without any undissolved particles. Then, the agarose solution was cooled to 55°C with careful swirling to promote even dissipation of heat. If detectable evaporation has occurred, add distilled water to bring the solution up to the original volume as marked on the flask. After that, the cooled agarose solution was poured into the bed. It is important the bed is on a level surface. The gel was allowed to solidify completely after approximately 20 minutes. It became firm and cool to the touch.
2.2.3 Preparing the gel for electrophoresis
The tape was removed carefully and slowly from the gel bed, after the gel was completely solidified. The comb was removed slowly by pulling straight up. It is to prevent tearing the sample wells. After that, the gel was placed into the electrophoresis chamber; it was properly oriented, centred, and placed level on the platform. Next, 50x buffer was diluted in distilled water to make 1 litre of 1x buffer. Next, the electrophoresis apparatus chamber was filled with 1x buffer which completely covered the gel. The next stage was to load the DNA sample in wells and conduct electrophoresis.
2.2.4 Loading the samples
DNA samples' volumes were checked, as sometimes a small amount of DNA sample cling to the walls of the tubes. The DNA samples were incubated before pipetted into wells. It is important that the entire volume of DNA sample is at the bottom of the tubes before starting to load the gel. After that, the DNA samples in tubes A to F were loaded into the wells in order. 25µl of DNA sample was used for each well. A micropipette was used to pipette the sample into the well.
A sickle cell gene sample
B sickle cell trait (carrier) sample
C normal gene sample
D mother's DNA sample
E unborn child's DNA sample
F father's DNA sample
After the DNA samples were loaded, the next step was to carefully snap the cover down onto the electrode terminals. It is important that the negative and positive colour coded indicators on the cover and apparatus changer are properly oriented. The plug of the black wire was inserted into the black input of the power source (negative input). The plug of the red wire was inserted into the red input of the power source (positive input). The power source was set at the required voltage and conducted electrophoresis for the length of time determined by tutor (e.g. 125 V for 30 minutes). Next, the current was checked to see if it flows properly. You should see bubbles forming on the two platinum electrodes. After the electrophoresis was completed, power was turned off and the power source was unplugged. The leads were disconnected and the cover was removed. The gel was removed from the bed for staining with Methylene Blue.
2.2.6 Staining the gel
After electrophoresis, the gel was placed on a flat surface and was moistened with several drops of electrophoresis buffer. The ethidium bromide staining card adhesive was removed and placed on the surface of the gel and stained for 15 minutes. An empty beaker was kept on top of the gel with the card surface to maintain a good contact between gel surface and the staining card. Finally, the card was then removed from the gel surface and was rinsed with electrophoresis buffer. It was examined under Ultra violet Transilluminator.
3.0 Results
Figure 5: Migration of DNA fragments in agarose gel viewed under ultra violet transilluminator.
DNA sample
(Genotype)
Result
Control A
HbSS
Sickle cell disease
Control B
HbSA
Sickle cell trait
Control C
HbAA
Normal
Sample D
HbSA
Sickle cell trait
Sample E
HbSS
Sickle cell disease
Sample F
HbSA
Sickle cell trait
Results have shown that Control A is sickle cell disease, genotype HbSS; control B is sickle cell trait, genotype HbSA; control C is normal, genotype HbAA. Sample D, which is patient B, the mother, is a sickle cell trait, her genotype HbSA. Sample E is the unborn child, the result shown that he/ she has sickle cell anaemia, genotype HbSS. Sample F, the father, is a sickle cell trait, genotype HbSA.
4.0 Discussion
Patient B is a mild anaemia patient regarding the case study above, low haemoglobin level, low blood cell count and low MCH. Haemoglobin S solubility test showed turbidity, positive solubility in patient B, and citrate agar electrophoresis test indicated the presence of sickle cell haemoglobin S and haemoglobin A. Patient B is a sickle cell trait pregnant woman. Sickle cell gene analysis has been suggested to identify the risk of passing on the disorder to the unborn child. Based on the migration of DNA fragments in agarose gel electrophoresis, one band was presented on S band, control A and sample E has the same migration DNA fragment, therefore sample E, the unborn child is sickle cell disease. In control B, three bands were presented, both HbS and HbA; this indicated that control B is a sickle cell trait, genotype HbSA in which sample D (Patient B) and sample F (father) has the same migration DNA fragments. The unborn child has inherited both sickle cell genes from each parent.
In women with sickle cell disease, the entire pregnancy is a high risk period that warrants close monitoring. With improved medical care and the use of prophylactic penicillin, the frequency of sickle crises in pregnancy has decreased significantly; but they may still occur and constitute an obstetric emergency (Grossetti et al., 2009). Repeated transfusions of red blood cells can greatly decrease disease severity and hematopoietic stem cell transplantation can cure sickle cell disease. However, exchange transfusion may be indicated in women with a serious obstetric or haematological complication (Elsayegh and Shapiro, 2007).
The diagnosis of sickle cell disease is established by demonstrating the presence of significant quantities of HbS by isoelectric focusing (IEF), cellulose acetate electrophoresis, high performance liquid chromatography (HPLC) or DNA analysis. In this report, we discussed about the sickle cell gene analysis by using agarose gel electrophoresis. This method is the easiest way of separating and analyzing DNA. The purpose of the gel is to quantify the particular band of the given DNA. The DNA is visualised in the gel by addition of ethidium bromide under ultra violet lamp. It gives fluoresces reddish-orange colour in the presence of DNA (Lewis et al., 2006).DNA is negative charge nucleic acid molecules, it moves through an agarose matrix with an electric field (electrophoresis) to positive electrode. DNA molecules of different lengths separate, the shortest moving farthest. When DNA is treated with restriction enzymes, the DNA is cut into fragments of various sizes. These fragments can be separated in a gel on the bases on their electric charge and size.
Hydroxyurea is a treatment for sickle cell disease which is contraindicated in pregnancy. This treatment is only for adult. The efficacy of hydroxyurea in the treatment of sickle cell disease is generally attributed to its ability to increase foetal haemoglobin (Lanzkron et al., 2008). This may prevent the cells from becoming rigid and clogging the blood vessels. Besides, hydroxyurea is a cytotoxic agent which has the potential to cause life threatening cytopenia. In addition, this drug should not be used in patients likely to become pregnant. Therefore, each sickle cell anaemia patient must be evaluated carefully before hydroxyurea therapy is begun, and careful monitoring must continue while the patient is on this agent. If hydroxyurea therapy has any beneficial effects, they last only as long as the patient continues to take the prescribed dose but it is not a cure (Charache and Terrin, 1995).
Obstetric units have adopted prophylactic transfusion regimens to pregnant woman who is sickle cell anaemia. The basis for exchange transfusion is to decrease the concentration of haemoglobin S, thus increasing the overall oxygen carrying capacity of the blood, which in turn reduces the changes of sickling and hence tissue damage. However, the disadvantages of exchange transfusions include transfusion reaction, alloimmunisation and exposure to infection. Infants are typically started on a course of penicillin that extends from infancy to age six. This treatment is meant to ward off potentially fatal infections. Bone marrow transplant has been shown to cure sickle cell anaemia in severely affected children. Besides, there are indications for a bone marrow transplant such as stroke, recurrent acute chest syndrome, and chronic unrelieved pain. Bone marrow transplant tends to be the most successful in children.
5.0 Conclusion:
In conclusion, it is important to determine the genotype of an individual. The newborn screening for sickle cell disease and an improved awareness for antenatal screening will increase the detection of sickle cell disease in the population. This will allow the appropriate care and management to reduce the morbidity and mortality of sickle cell disease.
6.0 References
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