Sickle Cell Anaemia and its Molecular Diagnosis

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Table of Contents

Abstract
Introduction
Pathogenesis of sickle cell anaemia
Classical methods for sickle cell anaemia diagnosis
DNA-based methods for sickle cell anaemia diagnosis
Conclusion
Reference List

Abstract

Genetic diseases can result from several environmental factors; however, they primarily result from inherited genes. Genetic diseases are known to occur as a result of an abnormality in an inherited gene. Sickle cell anaemia is one of the most common genetic diseases. This report discusses its pathogenesis and presents classical methods available for its diagnosis. The classical methods identified in this report are haemoglobin electrophoresis, isoelectric focusing, and high-performance liquid chromatography. Finally, the document discusses DNA-based methods used in sickle cell anaemia. The two methods presented in this report are Restriction Fragment Length Polymorphisms and Polymerase Chain Reaction.

Introduction

Any disease that occurs as a result of an abnormality in one’s genome is classified as a genetic disease. Genetic diseases are acquired through heredity, or gene mutation. Sickle cell anemia is one such genetic disease caused by genetic inheritance¹. Sickle cell anemia is inherited as an autosomal recessive condition meaning that the gene that contains the disorder can be passed from the parent to either male or female child. However, both parents must have sickle cell genes for the inheriting child to have two sickle cell genes^2. The disease results from mutations that take place in the DNA sequence of a single gene and is therefore classified as a monogenetic disorder. Thus, it is a disorder of the blood and occurs as a result of inherited abnormal hemoglobin. The analogous nature of hemoglobin may cause red blood cells to rip apart. This leads to a reduction in the number of red blood cells as hemolysis continues, resulting into anaemia. The disease is the most widespread among the inherited blood anaemias and majorly affects Africans and African Americans.

Pathogenesis of sickle cell anaemia

The β-globin chain of hemoglobin undergoes the process of mutation and this causes the replacement of valine, a type of a hydrophobic amino acid, by hydrophilic amino acids³. The β-globin is located at the diminutive side of chromosome 11⁴. In this case, haemoglobin S (HbS) is formed by an alliance between “2 mutant β-globin subdivisions and 2 wild-type α-globin subdivisions forms”⁴.in case of low oxygen, the nonexistence of a polar amino acid at the sixth position of the β-globin chain increases the polymerisation of non-covalent haemoglobin. This deforms red blood cells into a sickle shape, and as a result, reduces their elasticity. This reduction in red blood cell elasticity is most important in the pathophysiology of sickle-cell anaemia.

The elasticity of normal red blood cells facilitates their deformation thereby enabling their passage through capillaries. However, in sickle-cell anaemia, low-oxygen condition enhances red blood cell sickling. Continuous sickling damages the cell membrane. The condition becomes worse since the cells can no longer regain their original shape even when normal oxygen condition is returned. Consequently, the red blood cells attain rigidity which is difficult to deform while passing through thin capillaries. This causes vessel occlusion, as well as, ischaemia. Ischemia is a restriction within the blood supply which leads to dysfunction of tissue or vasoconstriction. A high number of white blood cells may result in the formation of cytokines, and this may cause sickle cell anaemia.

In general, the pathogenesis of sickle cell anaemia is by haemolysis, which is the rupture of cells within the spleen as a result of their distorted shape. Even though the bone marrow will always produce more red blood cells to compensate for the ruptured cells, the rate of rapture is higher than that of production making it hard to balance or restore the situation.

The significant decrease in the Haemoglobin A in human beings is caused by the fact that sickle celled red blood cells can only survive for 10 to 20 days compared with the 90 to 120 days that normal red blood cells last. Normally, an individual has Haemoglobin A which has two β and α chains, Haemoglobin A2 (also with two β and α chains), and Haemoglobin F with two α and γ chains. Haemoglobin A comprise of 96-97% of normal haemoglobin. This means that haemolysis reduces the amount of haemoglobin in the body significantly.

Classical methods for sickle cell anaemia diagnosis

Haemoglobin electrophoresis

Haemoglobin electrophoresis is a test used to detect various forms of haemoglobin. The test applies gel electrophoresis principle that helps to disintegrate haemoglobin into its various components. It is used to detect abnormal HbS levels in the red blood cells and other abnormal haemoglobin-related disorders. According to the Southern⁵ this technique uses electrical current in separating normal and abnormal haemoglobins within the blood using cellulose acetate. Various haemoglobins have varied charges, and these charges determine the rate of movement of haemoglobins depending on whether the gel is acidic or alkaline. The procedure involves staining of cellulose acetate gel with Ponceau red, and the Electrophoresis run at 150-200 volts. The amount of every type of haemoglobin within the current is measured and an abnormal type of haemoglobin found in the blood implies that a disease/disorder is present. In this case, HbS means that the sickle cell anaemia is present.

Isoelectric focusing (IEF)

IEF is used to analyse whole blood samples and haemolysates as well. The technique gives reliable separation of HbA from HbF and other variant hemoglobins such as HbS, HbC, HbD-Punjab, HbE and HbO-Arab⁶. The technique can be semi-automated to allow the screening of large number of blood samples. For example, it can be used to separate HbA into A₀, A₁, A(αmet), A(βmet), as well as, A(αβmet).

High-performance liquid chromatography (HPLC)

HPLC can be used to quantify haemoglobins HbS, HbA₂, and HbF for detection, conditional identification, and the quantification of several variant haemoglobins. This method provides precise quantification of HbA_{2 as} opposed to isoelectric focusing.

It is used to separate HbA, HbA₂, HbF, HbS, HbC, Hb-D-Punjab and G-Philadelphia from one another⁷. It allows for the detection of haemoglobin variants which were previously unidentified by other methods.

DNA-based methods for sickle cell anaemia diagnosis

Restriction Fragment Length Polymorphisms

Mutation which causes sickle cell anaemia coincidentally lives in the recognition sites of some restriction enzymes which are; Bsu II, Cvn I, and Mst II. A restriction enzyme identifies and separates double-stranded DNA from a specific nucleotide sequence⁸.This sequence is called restriction site, and exists within or around any particular gene. Those with sickle cell mutation do not have the restriction cell site found in people who have normal β-globin genes. Restriction Fragment Length Polymorphisms has been used to directly test for the existence of an abnormal gene in an at-risk fetus. This method applies a radioactively labeled β-globin probe in diagnosing sickle cell anaemia. Gene mutation that causes the disorder coincides with a Cvn I site. However, chromosomes which have sickle mutation do not have the site which chromosomes that have normal β-globin genes possess. This is digested with Cvn I and hybridization to produce β-globin probe⁹. Thereafter, an analysis can now be done. DNA of persons with sickle cell anaemia produces a 1.3 kb fragment, and that from parents (both mother and father) who have normal β-globin genes produce a 1.1 kb fragment¹⁰.Carriers have 1.3-kb and 1.1-kb fragments.

Polymerase Chain Reaction (PCR)

This is an in-vitro technique used to make several copies of specific DNA sequences. In this method, sickle cell anaemia is diagnosed on a PCR-amplified DNA through direct visualization done under ultraviolet light¹¹. Sickle cell mutation is first amplified under a large complex genomic DNA. Molecular diagnosis of the disease necessitates the detection of one distorted nucleotide in a single gene within the genome. As a result, the region of the genome that contains the altered nucleotide has to be amplified. Amplified DNA that has β-globin gene codon 6 is digested with Cvn I. It is then electrophoresed with agaorose gel¹². Smaller restriction fragments migrate through agarose gel electrophoresis faster as compared to larger restriction fragments. The sixth codon DNA is then labeled with Ethidium Bromide before visualization under UV light. When placed in ultraviolet light, digested DNA appears as a smear. The sickle mutation gets rid of the recognition sequence of Cvn I enzyme. As a result, HbS allele can now be visualized as a 340-bp, while HbA allele as 200 or as 140-bp bands¹³. In addition, a 100 base pair of constant band is present for every person tested.

Polymerase Chain Reaction (PCR) method is faster and more efficient since it does not require performing of Southern blotting, autoradiography or hybridization, all of which uses labeled probe, compared to Restriction Fragment Length Polymorphisms¹⁴. Besides, it can be applied to any genetic disease where mutation that causes the disease forms or eliminates restriction site.

Conclusion

Sickle cell anaemia is passed down through lineages or family tree with abnormally shaped red blood cells or an abnormal haemoglobin type known as haemoglobin S (HbS). HbS distorts the shape of red blood cells particularly when they are exposed to conditions of low oxygen making them become sickle shape. They become rigid and therefore unable to deliver sufficient oxygen to an individual’s body tissues. Besides, they also become unable to pass through constricted blood capillaries and undergo haemolysis, thus affecting blood flow in the body. The tests commonly carried out to diagnose and monitor patients who suffer from sickle cell anaemia include haemoglobin electrophoresis, isoelectric focusing and high-performance liquid chromatography. Several DNA-based methods have also been developed to perform diagnosis for sickle cell anaemia. Restriction Fragment Length Polymorphisms and Polymerase Chain Reaction are some of the most accurate DNA-based methods used to diagnosis the disease.

Reference List

Arnold, JL. Sickle cell anaemia. Medscape Reference. 2011. Web.

Cobb, BD, Waterfall, CM. Single tube genotyping of sickle cell anaemia using PCR-based SNP analysis. Oxford Journals, 2001; 29 (23): e119.

Consensus conference. Newborn screening for sickle cell disease and other hemoglobinopathies. JAMA.1987; 258:1205-1209.

Erlich, HA, Gelfand, DH, Saiki, RK. Specific DNA amplification. Nature, 1988; 331: 461.

Ober C. Molecular genetics. Illinois: Global Library of Women. 2008. Web.

Southern, EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology, 1975; 98: 503.

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