Haemoglobin variants
Introduction
In this laboratory practical, we intended to determine the sickle cell disease individuals and distinguish them between individuals carrying the normal haemoglobin variant. This was performed through the implementation of the haemoglobin electrophoresis method that separated the individual’s haemoglobin variants in accordance to their molecular weight.
The haemoglobins are the main cells of human’s blood and are very important in the transportation of oxygen from the lungs to the body’s tissues. They are also involved in the transportation of other waste gases, such as carbon dioxide. A single haemoglobin molecule has the potential to carry up to four oxygen molecules. In fact, the oxygen-binding efficiency of haemoglobin was found to be 1.34 ml per one gram, this is according to the findings of a study that compared the binding-capacity of haemoglobin in healthy and diseases patients (De villota et al., 5545). However, haemoglobin’s oxygen binding efficiency is highly dependent on the type of haemoglobin. For example, HbF (α2γ2) is a physiological haemoglobin that makes up most of foetuses erythrocytes and it has a much greater oxygen-binding capacity due to its molecular structure and charge. A foetal haemoglobin is only normal when it is found in foetal stages of life, otherwise it has the potential to cause a disease if it is found at different stages of life and it is greater than the expected adult amount that is zero to one per cent of the total haemoglobin. Other abnormal haemoglobins can be found in individuals and their existence has been attributed to genetic mutations on the genes they are encoded from. The presence of abnormal haemoglobins can cause very serious diseases that are termed haemoglobinopathies (Wajcman and Moradkhani, 2011).
The types and amount of haemoglobins that can be found in a normal individual are HbA (α2β2), ΗbA2 (α2δ2) and HbF (α2γ2) at an amount of total haemoglobin of 96.5-98.5%, 1.5- 3.5% and 0-1% respectively. On the other hand, genetic mutations have given rise to other abnormal haemoglobins such as HbS (α2βS2) and HbC (α2βC2) that are responsible for sickle cell anaemia and mild chronic haemolytic anaemia. The difference between all these haemoglobin variants is their globin DNA sequences that affects their charge and shape. Moreover, all haemoglobin proteins are tetramers and are composed of four protein subunits. The normal haemoglobin HbA consists of two α2 and two β2 polypeptide subunits. The genetic mutations can happen in both subunits (alpha and beta), however, the majority of the haemoglobin variants are consisted of two normal alpha subunits and two abnormal. Point mutations and deletions of a single nucleotide on the genes encoding the second globin chain result in the production of the βS2 (HbS) and βC2 (HbC) chains. These changes may affect the structure of the haemoglobin, behaviour and its stability (Weatherall et al., 2001).
Results
In this laboratory practical we aimed to distinguish diseased haemoglobin (HbS) between healthy ones. This was achieved through haemoglobin electrophoresis where we recorded the migration distance of the haemoglobin samples and compared them to each other. The results generated are shown on the following figure and table. Figure one is an image of the electrophoresis results that was captured during the practical and table one represents the data generated by figure one but in a tabular form.
Fig. 1 – Haemoglobin electrophoresis-
The above figure is a photographic demonstrations of the individuals’’ haemoglobin proteins after the electrophoresis. The migration distances of all samples have been pointed out on the figure shown as the coloured “arrows”. A ruler has been attached to aid in the determination of the migration distances and the samples’ origin is also clearly marked. The image was provided by Dr Ria Diakogiannaki.
Table 1 – Haemoglobins’ electrophoresis migration distances and their identification-
Cathode (-) Anode (+)
Samples A B C D (Mix 1) E (Mix 2)
Migration distance (mm) 5.5 3.5 4.5 3.5 and 5.5 4.5 and 5.5
Potential variant HbA0 HbA2 HbS HbA2 and HbA0 HbS and HbA0
The above table represents the migration distances determined for all samples measured in millimetres. The samples’ possible haemoglobin identity is also shown on the graph, as well as the direction of the migrating samples.
Discussion
In figure one, where the haemoglobin electrophoresis results are shown, the first three samples were of known haemoglobin content and therefore, their migration distance has been attributed to the haemoglobin type. These are sample A (HbA0) recorded with ~5.5 mm, sample B (HbA2) ~3.5 mm and sample C (HbS) ~4.5 mm migration distances, which was measured from the origin. For the determination of samples D and E that were of unknown content we compared them to the known haemoglobin samples. Significantly, both samples were found to be consisted of two proteins; this is clearly shown on figure one where two distinct bands were formed for both samples. From the comparison of the samples to the known ones, we deduced that sample D consisted of the haemoglobin HbA2 and HBA0 variants, whereas sample D consisted of HbS and HbA0. The haemoglobins in both samples were found to be of the same migration distance as the ones they were compared to. In haemoglobin electrophoresis, the haemoglobin molecules are separated at an alkaline pH, where all protein molecules attain a negative charge and migrate towards the anode. For instance, HbS, which has an extra positive charge compared to HbA0 migrates with a slower rate. Similarly, HbC would migrate towards the anode with a slower rate than HbS due to its mutation that causes the replacement of the negative glutamic acid to lysine (positive charge). Additionally, HbF would have resulted in a band located between HbA0 and HbC due to its net negative charge that is lesser than HbA0 net negative charge.
Sickle cell anaemia is one of the most common haemoglobinopathy, especially between the Mediterranean, the sub-Saharan and the south-east continents. Evidently, this could have been due to peoples’ exposure to malaria, which in the case of heterozygous sickle cell gene, it provides an advantage over the parasitic infection (Weatherall et al., 2001). Moreover, a single nucleotide substitution (Glu>Val) at the sixth position of the β-chain gene results in the abnormal concaved–like structure of the haemoglobin known as “sickle cell” due to valine being a hydrophobic molecule. This structural change renders the cells susceptible to lysis (shortened survival rate) and because of this distinct structure cells clump together and they are very likely to occlude in small capillaries and arteries raising the possibility of experiencing myocardial infractions and strokes (Howard et al., 2013).
The main disorder caused by the breakdown of the erythrocytes due to their defective “sickled” shape is anaemia. The symptoms caused by anaemia range widely, however, they are not experienced by all patients. For example, carriers of the disease (heterozygous) do not constantly express the defective erythrocytes but they might experience a condition named as “sickle cell crisis” when they are exercising or when they are in an environment with much lesser oxygen supply such as a mountain with high altitude. Symptoms experienced are fatigue, pain and swelling due to the impaired transport of oxygen that tissues use to produce energy and the breakdown of the erythrocytes that affects the haematocrit and irritates the nerve endings near the affected areas that results in the swelling and severe pain (Lee et al., 2009).
References
De Villota, E. D., Carmona, M. T. G., Rubio, J. J. and De Andres, S. R. (1981). Equality of the in vivo and in vitro oxygen-binding capacity of haemoglobin in patients with severe respiratory disease. BJA: British Journal of Anaesthesia 53, 1325–1328.
Howard, M. R., Hamilton, P. J. and Britton, R. (2013). Haematology: An illustrated Colour text. 4th ed. Edinburgh: Elsevier Health Sciences 17, 600-630.
Lee, R. G., Wintrobe, M. M., Lee, R. G., Bithell, T. C., Foerster, J., Athens, J. W. and Lukens, J. N. (2009). Wintrobe’s clinical hematology. 12th ed. Philadelphia: Lea & Febiger,U.S IV, 782-790.
Wajcman, H. and Moradkhani, K. (2011). Abnormal haemoglobins: Detection & characterization. 134,.
Weatherall, D. J., Headington and Clegg, J. B. (2001). Inherited haemoglobin disorders: An increasing global health problem. Bulletin of the World Health Organization 79, 704–712.
Cytogenetics: G-banding
Introduction
In this practical session we aimed to produce a karyotype that we would analyse its chromosomes numerical and structural abnormalities for any possible signs of cancer. This was done through chromosomal Giesma staining or G-banding that allowed chromosomes visualisation under the microscope.
The HeLa cell line originates from a patient of cervical adenocarcinoma that died and their cell line is still being used for clinical trials until now. This is because the HeLa cell line had the potential of being long-termed cultured in vitro, while other cells would only survive for a few days. Consequently, this allowed scientists to base their researches around those cells and many discoveries have been found from utilising this unique human epithelial cancer cell line (Rose, 2013). In fact, research scientist Jonas Salk developed a vaccine against polio virus in 1954 by taking advantage the stable growth of the HeLa cells. It has been found that the main cause of cervical cancers is the human papilloma virus (HPV), whose DNA when inserted into a host cell causes the expression of a protein that affects p53 (apoptotic factor) activation. This virus has resulted in the HeLa cells acquiring a very abnormal karyotype that may consist of seventy six to even eighty greatly mutated chromosomes, while a normal karyotype is consisted of forty six chromosomes (Scherer, 1953).
G-banding is the most commonly cytogenetic technique used today for karyotype production that was developed in 1904 by the scientist Gustav Giesma and utilises the Giesma stain. This chromosomal staining technique allows the examination of the chromosomes’ length, banding pattern (stripy look), position of the centromeres and other physically important characteristics between the somatic and sex chromosomes of the human genome. This phenotypic analysis can potentially aid in the diagnosis of numerous genetic disorders such as Down’s syndrome, which is a trisomy in chromosome 23. In a G-banding method, chromosomes are stained differently and this enables their identification and even diagnosis of any chromosomal aberrations. For instance, chromatin regions rich in A-T base content that also appear to be replicating later than their base pair counterparts, are stained darker. While, chromatin regions rich in G-C base content are stained more lightly. This resulting stripy effect of the chromosomes help in the identification of any chromosomal aberrations such as translocations, inverted DNA segments and segment losses (Bickmore, 2001).
Results
In this laboratory practical we aimed to identify chromosomal abnormalities in either the number or structure of the chromosomes that were examined. This was achieved by producing a clear karyotype deriving from the HeLa cell line that implemented the G-banding method that uses Giesma (dye) and trypsin (digestive enzyme) as the main assay reagents. The karyotype that we produced in shown in the following figures where abnormal number of chromosomes is clearly shown.
Fig. 1 – HeLa cell line karyotype-
The figure above is a photographic representation of the HeLa cell line karyotype that was captured during examination under the microscope. The cells have been stained with the Giesma method described as G-banding. The chromosomes count approximately amounts sixty six chromosomes. Despite the chromosomal numerical abnormality, the chromosomes count was very much expected.
Fig. 2 – HeLa cell line karyotype under white and black filter-
The photograph above is the same photograph that was used above on figure one where results of the HeLa cell karyotype using the G-banding method are shown. However, a black and white filter has been applied to allow the contrast between the chromosomes. The areas of dark black and light grey pigmentation on the chromosomes represent different staining and therefore molecular content.
Discussion
Both figures suggest definite numerical abnormalities due to the fact that the chromosomes’ count was sixty six chromosomes. The normal count is twenty three pairs of somatic chromosomes in both males and females and one pair of XY sex chromosomes in males and one pair of XX sex chromosomes in females. A karyotype of sixty six chromosome count highly resembles an example of hyperploidy, which describes a cell bearing a chromosome count greater than the normal. For example, there are twenty spare chromosomes that could either be multiples of the normal euploid chromosome pairs or trisomies (2n+1), which is an extra chromosome of one type in some of the chromosomes. Trisomies can cause numerous developmental disorders depending on the chromosome they happen to be. A trisomy in the sex chromosomes is the Klinefelter syndrome (KS) that is an extra X chromosome in males and causes sterility and other developmental problems (Gardner, 2011).
However, a karyogram would provide further help in the exact identification of what kind of chromosomal numerical abnormality is shown in the figures above. A karyogram is a rearrangement of the chromosomes in accordance to their size and position of their centromere. For the same reason, it is hard to identify any chromosomal aberrations in the photos shown above due to the fact that the chromosomes are neither in order nor resolution wise visible enough to compare their bands (Nicholson and Cimini, 2013).
The results gathered are very consistent to what we expected from examining a HeLa cell line karyotype. The constant culturing of the cells has led to the generation of multiple mutations that have altered the initial and original HeLa cell culture. The HeLa cells are known for their hyperploid chromosome number, numerical abnormalities and their structurally and clonally abnormal chromosomes that are known as HeLa markers. In fact, they are known sites where copies of the HPV18 (Human papilloma virus type 18) reside. All the aforementioned statements are very characteristic anomalies of this unique HeLa cell line and further anomalies would not be surprising. Consequently, the results in both graphs represent a typical hypertriploidy (3n+) (Macville et al., 1999).
References
Bickmore, W. A. (2001). Karyotype Analysis and Chromosome Banding. MRC Human Genetics Unit 1–7.
Gardner, R. J. M. (2011). Chromosome abnormalities and genetic counseling. Oxford: Oxford University Press 2, 257-300.
Macville, M., Schröck, E., Padilla-Nash, H., Keck, C., Ghadimi, M. B., Zimonjic, D.,
Popescu, N. and Ried, T. (1999). Comprehensive and definitive molecular Cytogenetic characterization of HeLa cells by spectral Karyotyping. Cancer Research 59, 141–150.
Nicholson, J. M. and Cimini, D. (2013). Cancer Karyotypes: Survival of the fittest. 3,.
Rose, S. (2013). Henrietta lacks (HeLa) cell genome: Cell line identity and the personal privacy – on biology. Biology.
Scherer, W. F. (1953). Studies on the propagation in vitro of poliomyelitis viruses: Viral multiplication in a stable strain of human malignant epithelial cells (strain HELA) derived from an epidermis carcinoma of the cervix. Journal of Experimental Medicine 97, 695–710.
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