TABLE OF CONTENT
TITTLE PAGE…………………………………………………………………………………i
APPROVAL PAGE……………………………………………………………………………..ii
CERTIFICATION……………………………………………………………………………..iii DEDICATION………………………………………………………………………………….iv ACKNOWLEDGEMENT…………………………………………………………………….…v
TABLE OF CONTENT……………………………………………………………………….vi
LIST OF TABLES……………………………………………………………………………viii
LIST OF FIGURES…………………………………………………………………………….ix ABSTRACT………………………………………………………………………………………x
CHAPTER ONE; INTRODUCTION……………………………………………………..….1
1.0 BACKGROUND…………………………………………………………………………….1
1.1 STATEMENT OF PROBLEM……………………………………………………………….6
1.2 AIMS AND OBJECTIVES…………………………………………………………………..7
CHAPTER TWO; LITERATURE REVIEW……………………………………………..…..8
2.0 MOLECULAR PATHOGENESIS OF SICKLE CELL DISEASE………………………….8
2.1 PHENOTYPIC MANIFESTATIONS OF SICKLE DISEASE………………………….…10
2.2 VARIANTS OF SICKLE DISEASE AND THEIR GEOGRAPHICAL DISTRIBUTION…12
2.3 SICKLE CELL DISEASE AND ANEMIA………………………………………………….14
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2.4 SICKLE CELL DISEASE AND OXIDATIVE STRESS………………………………..14
2.5 SICKLE CELL DISEASE AND G6PD DEFICIENCY…………………………………..16
2.6 TREATMENT OF SICKLE CELL DISEASE………………………………………..…..17
CHAPTER THREE; METHODOLOGY……………………………………………….….20
3.1 REAGENTS AND APPARATUS/EQUIPMENT FOR LABORATORY ANALYSIS…..20
3.2 STUDY POPULATION AND DESIGN……………………………………………….…21
3.3 DATA COLLECTION…………………………………………………………………….21
3.4 BLOOD COLLECTION…………………………………………………………………..21
3.5 LABORATORY ANALYSIS……………………………………………………………..22
3.6 STATISTICAL ANALYSIS ……………………………………………………………..23
CHAPTER FOUR; RESULTS…………………………………………………………..…..24
CHAPTER FIVE; DISCUSSION ……………………………………………………..….38 CONCLUSION………………………………………………………………………….…..41 REFERENCE…………………………………………………………………………..……42
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LIST OF TABLES
TABLE 1: Gender Distribution of Study Population………………………………………….…26
TABLE 2: Demographic and Clinical Characteristics among Patients………………………..…31
TABLE 3: Relationship between Anemia and Genotype Status of Patients……………………..32
TABLE 4: Relationship between G6PD Deficiency and Genotype Status of Patients………..…33
TABLE 5: Interaction between Genotype Status and G6PD Deficiency on SOD and Anemia Indices in Patients………………………………………………………………………………34
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LIST OF FIGURES
FIGURE 1: Frequency of Crisis in Sickle Cell Anemia Participants………………………….27
FIGURE 2: Level of Crisis among Sickle Cell Anemia Participants………………………….28
FIGURE 3: Level of Hospitalization among Sickle Cell Anemia Participants…………………29
FIGURE 4: Frequency of Blood Transfusion among Sickle Cell Anemia Participants…………30
FIGURE 5: Interaction between Genotype and G6PD Status on SOD Level……………….….35
FIGURE 6: Interaction between Genotype and G6PD Status on Hemoglobin Level……….….36
FIGURE 7: Interaction between Genotype and G6PD Status on Hematocrit Level…………….37
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ABSTRACT
The relationship between anemia, SOD (superoxide dismutase) and G6PD (glucose-6-phosphate) in SCD (sickle cell disease) patients were determined for the better understanding of their pathophysiology. Hemolytic anemia is one of the most common complications of sickle cell. It can be influenced by different factors, malaria infections, oxidative stress, dehydration, environmental stress and much more. SOD are enzymes that help with the regulation of oxidative stress and G6PD deficiency together with SCA are at high frequencies in malaria epidemic regions. Both SCA and G6PD deficiency patients suffer from anemia and can be said to coexist in some individuals. This study aims to find out the relationship between anemia, SOD and G6PD in SCD patients, if they coexist together or influence each other’s manifestations. A total of 70 patients were used including AA and AS genotypes as control. Anemia indices including hemoglobin (Hb) concentration, hematocrit were tested for. The presence of SOD and G6PD deficiency were tested for in all the patients. G6PD non deficient SS patients were 32.9% and 7.1% were deficient. The hemoglobin (Hb) concentration and hematocrit in SS patients were normal 6.76mg/dL which normally ranges from 5.0-10.0mg/dL. The SOD levels in SS were also at normal levels (2.45 10-1 U). There was no relationship between anemia, SOD and G6PD deficiency in sickle patients. These parameters can exist independently but cannot influence their prevalence as there was no interaction between G6PD deficiency and sickle cell to influence SOD level and anemia indices. However, anemia is associated with sickle cell disease.
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CHAPTER ONE
INTRODUCTION
1.0 BACKGROUND
Sickle cell disease (SCD) is a group of genetic disorder that is inherited in an autosomal recessive manner due to the homozygous or heterozygous state of the mutation. It is caused by a single base mutation in the β-globin gene of hemoglobin, where GAT is replaced by GTT in the 6th codon of exon 1 resulting to valine instead of glutamic acid on the sixth position in chromosome 11. In the normal adult hemoglobin (HbA), there are 2 α-globin chains and 2 β-globin chains that form a globin tetramer. They are stabilized by intramolecular points of contact, without any interaction between them. When they bind or release oxygen they retain their normal shape but in the mutated β-globin there is a hydrophobic interaction between the adjacent valine amino acids which align into polymers and distort the shape of the red blood cells. These polymers, which are poorly soluble, distort the normal shape of the red blood cells, changing it to a sickle or crescent shape which prevents the normal flow of blood in the blood vessels (microcirculation) and increasing its adhesion to the endothelium of the vessels. This leads to vaso-occlusive crisis and hemolytic anemia which are the hallmark of the disease. SCD is a systemic pleiotropic disease that affects almost all the organs of the body or causes tissue infarction and a good number of other clinical manifestations throughout the affected individual’s life as a result of the polymerization of the beta hemoglobin under deoxygenated, acidic or dehydrated conditions and hypoxia. Sickled RBCs are more readily destroyed or are broken down prematurely by the reticulo-endothelial system due to their rigidity makes them filtered by the spleen. Most of the clinical manifestations are protean in nature and vary in frequency and severity among patients. SCD is a hemoglobinopathy in which the single base substitution mutation in the β-globin chain can result to either hemoglobin S, C, β+ or β ͦ thalassemia, D, E or OArab and are all known as hemoglobin variants but when they are
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combined with HbS they are known as SCD variants. Individuals, who are affected with sickle cell anemia which is one of the variants of SCD, have two copies of the mutated gene (HbSS). Other heterozygote individuals have one copy of the Hb S and other variant which could be Hb C, Hb β+ or β ͦ thalassemia. The mutation in HbSS is Glu6Val, in HbSC is Glu6Lys, the mutation in hemoglobin D glutamine replaces glutamic acid at position 121 of the gene and the mutations that cause the Sβ+ or the Sβº are deletions or additions of a single base substitute or more in the HBB gene (Serjeant 2013; Ashley-Koch et al., 2000; Heiman and Greist, 2010; Bunn, 1997; Booth et al., 2010; Al-Jafar et al., 2016; Kaur et al., 2013; Ballas, 2002; Ballas et al 2010; Wild and Bain, 2006 and Emecheba et al., 2017). Carrier individuals have one copy of the mutated hemoglobin and normal hemoglobin (Hb AS) and are said to have sickle cell trait. They are also said to be protected from malaria infection, resulting to the high frequency of the Hb S variants in malaria epidemic regions. Not all the variants of the mutated hemoglobin are detrimental, a concept known as genetic polymorphism. Millions of people worldwide are affected with this disease with the highest population in Africa but it can also be seen in Sub-Saharan Africa, Eastern Saudi Arabia, Central India Mediterranean, Afro-Caribbean, South and Central American, Arab and East Indian. Some of these variants are frequent in some of these populations than others. The two commonest hemoglobin variants in Nigeria are HbS and HbC. HbS is distributed fairly well in Nigeria but HbC is commonly seen among the westerns (Yoruba) and decreases from the west eastwards. It was estimated in Nigeria around 1982 that 30,000 infants are born each year with SCD as it is seen as the country with the highest affected individuals with the trait ranging from 20-30%. As of recent it is estimated that >40 million individuals are carriers, >150,000 infants are born each year with the disease and about 1 million survive past childhood (Galadanci et al., 2013; Emecheba et al., 2017 and Grosse et al., 2011).
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According to Robbins, (2014) the major cause of the symptoms in patients with SCD is the sickling of the red blood cells. The clinical or phenotypic manifestations of SCD are grouped into three, which include hemolytic anemia, pain episodes or crisis and severe organ damage. The sickled cells are unable to deliver oxygen to tissues in the body and this leads to tissue or organ damage. They also die faster than normal cells which lead to anemia, a blood condition that the red blood cells are lower than normal and it is a major symptom in patients with SCD. Due to their inflexibility they are unable to pass through small capillaries, causing blockage in the blood vessels leading to severe vaso-occlusive crisis. Other signs and symptoms of sickle cell disease which vary from person to person and can change over time include; acute pain (sickle cell or vaso-occlusive crisis), frequent infections, pulmonary complications, leg ulcers, priapism, brain complications (clinical stroke and silent stroke), eye problem, retarded growth and puberty, kidney problem (nocturnal enuresis), gallstones, liver complications (intrahepatic cholestasis), joint complications (avasualar or aseptic necrosis) and metal health. Lack of a large, readily accessible population for clinical studies has contributed to the absence of standard definitions and diagnostic criteria for the numerous complications of SCD and inadequate understanding of SCD pathophysiology (Ballas et al., 2010). Most of these complications found in SCD patients can be triggered by a lot of factors such as malaria infections, stress, temperature change (favorably warmth) and dehydration. As discoveries are been made, new body of evidence has shown that oxidative stress is a significant pathway sickle cell complications and morbidity as it enhances the sickling phenomenon of the cells. These could all contribute to the heterogeneous phenotypic expression of the disease. Oxidative stress is an imbalanced redox status caused by over production of oxidants (reactive oxygen specie) and depletion of antioxidants. This excess oxidant state leads to release of heme, auto-oxidation of
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hemoglobin, uncoupling of nitric oxide synthase activity leading to a decrease in NO. It has been observed that the antioxidant defense systems in SCD are ineffective in neutralizing the excess oxidant specie. Normal erythrocytes counter oxidative stress using self-sustaining activities of antioxidant defense enzymes such as superoxide dismutase (SOD) which is a key enzyme in dismuting super radicals into hydrogen peroxide. The activity of this enzyme is seen to be higher in SCA patients.
Anemia is a medical condition where the red blood cells or hemoglobin level in the body is lower than the normal level. Sickle cell disorders are associated with variable degrees of anemia depending on genotype, with the most severe decrease in hemoglobin level seen in sickle cell anemia and the least severe in hemoglobin S-β+ thalassemia. Normal red blood cells live for 120 days while the sickle cells live for 10-30 days as a result of continuous breakage of the cells. When the body is short of red blood cells, the tissues do not receive adequate amount of oxygen and this leads to fatigue or weakness. Severe anemia episodes may result from a variety of causes, including hyperhemolysis, acute splenic sequestration, and aplastic crises (Ballas et al., 2010). Although chronic hemolytic anemia is a major feature of sickle cell disorders, a marked drop in hemoglobin with an increased hemolytic rate is referred to as hyperhemolysis. Hemolytic anemia varies intensively among the genotypes of sickle cell disease and it may be the driving force behind some complications of sickle cell disease because of its effects on Nitric oxide (NO) bioavailability which its decrease is associated with pulmonary hypertension, priapism, leg ulceration, and possibly with non-hemorrhagic stroke (Kato et al., 2007).
Another clinical symptom that can be associated with SCD is G6PD deficiency. Glucose-6-phosphate dehydrogenase deficiency is a genetic disorder that results to an inadequate production of G6PD enzyme. This enzyme helps to regulate many biochemical processes in the body
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including the proper and normal functioning of the red blood cells. This deficiency causes the red blood cells to break prematurely called hemolysis leading to a common medical problem called hemolytic anemia. This anemia could lead to paleness, jaundice, fatigue, rapid heart rate and so on. In individuals with this deficiency, hemolytic anemia can be triggered by bacterial or viral infections, antibiotics or antimalaria drugs, favism which is caused by eating fava beans. This deficiency occurs exclusively in males. This deficiency results from mutations in the G6PD gene. This gene provides the instruction for producing the enzyme which is involved with the chemical reactions that prevent reactive oxygen species from accumulating to toxic levels in the body. With these mutations occurring, the production or structure of the enzyme is altered leading to an accumulation of the reactive oxygen species and would be harmful to the red blood cells. This gene is found on the X-chromosome and since males have only one copy of this chromosome they are more affected then females that have 2 copies of the chromosome and it is very rare for the mutation to occur on both genes. G6PD deficiency just like SCD is prevalent where malaria is epidemic and very common among the black population with a protective role against malaria. The presence of the G6PD deficiency can lead to an increase in the severity of crisis in SCD patients. Studies have also shown that this deficiency is prevalent in SCD patients more than the general population but this could be otherwise in some other population. The coexistence of this relationship can lead to hemolytic anemia, acute splenic sequestration and vaso-occlusive crisis. Patients usually are asymptomatic, these disorders do not alter the hemoglobin (Hb) levels and RBC count in stable conditions (Genetic home reference, 2018; Benkerrou, et al., 2013; Memon, et al., 2016, Firempong, et al., 2016 and Al-Nood, 2011). Over the years, measures like prenatal screening, parent education, better medical care, immunization and the use of penicillin prophylaxis have been used to increase the life expectancy
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of affected individual, having the three basic therapeutics modalities as hydroxyurea, blood transfusion and bone marrow transplant. World Health Organization (WHO) has promoted several national screening programs with the goal of informing reproductive choice in other to reduce severely affected infants (Kaur et al., 2013). This study is designed to access the relationship between sickle cell disease and the factors that trigger their complications. Hemolytic anemia is the most common clinical manifestation found in each of the single genotype of this disorder. Oxidative stress is known to increase the anemia rate in SCD patients and leads to vaso-occlusive crisis and any other clinical complications and how it affects G6PD patients is quite unclear but antimalaria drugs can trigger hemolytic crisis. Individuals that have only G6PD deficiency tend to have hemolytic anemia as the main clinical symptom, and the relationship between SCD and G6PD is not definite for all population but they are common in black population and have a protective role against malaria. Thus, it will be of interest to evaluate the influence of oxidative stress, anemia and G6PD deficiency on SCD patients. 1.1 STATEMENT OF PROBLEM Nigeria is said to have the highest number of SCD cases, having the two most common variants as SS and SC. Despite the high burden of SCD in Nigeria, it has been difficult to improve the care and management of diseases. Most of the new treatments, therapies, and creation of awareness is lacking or is not widely available especially in the rural regions. The pathophysiology of the diseases, to an extent is not really understood resulting from lack of assessable data which also leads to the inability of providing a permanent cure for the disease. Studying the various factors that triggers their crisis and the degree of their phenotypic manifestations would give a better understanding of the disease
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pathophysiology and more data would be available in order to provide better improved treatment of the disease. 1.2 AIMS AND OBJECTIVES
This study intends to assess the relationship between anemia, SOD and G6PD deficiency and how they increases the vaso-occlusive crisis in SCD patients visiting ESUTH and UNTH in Enugu State, Nigeria
The objectives of this study are to determine:
Screen patients for sickle cell anemia based on their genotype status-using questionnaire.
Assess hemoglobin concentration and hematocrit level in patients.
Quantify the level of superoxide dismutase (SOD) activity in patients.
To determine the absence or presence of G6PD deficiency in SCD
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