|Year : 2018 | Volume
| Issue : 2 | Page : 98-103
Fetal hemoglobin gene expression in patients with sickle cell disease in North Central Nigeria
Amos Dangana1, Idris Abdullahi Nasir2, Jessy Thomas Medugu3, Felix O Emelike4, BO Oluwatayo5, Abubakar Shehu Haruna6
1 Department of Medical Laboratory Services, University of Abuja Teaching Hospital, Abuja, Nigeria
2 Department of Medical Microbiology and Parasitology, College of Health Sciences, University of Ilorin, Ilorin, Nigeria
3 Department of Medical Laboratory Science, University of Maiduguri, Maiduguri, Nigeria
4 Department of Medical Laboratory Science (Haematology Unit), Ambrose Alli University, Edo State, Nigeria
5 Department of Haematology, College of Veterinary and Medical Laboratory Science, Vom, Plateau State, Nigeria
6 Department of Family Medicine, University of Abuja Teaching Hospital, Abuja, Nigeria
|Date of Web Publication||2-May-2018|
Mr. Idris Abdullahi Nasir
Department of Medical Laboratory Services, University of Abuja Teaching Hospital, PMB 228 Gwagwalada, FCT Abuja
Source of Support: None, Conflict of Interest: None
BACKGROUND: Fetal hemoglobin (HbF) plays a dominant role in ameliorating morbidity and mortality associated with sickle cell disease (SCD).
OBJECTIVE: This study evaluated the distribution pattern of HbF and total Hb concentration among 75 participants with homozygous sickle cell trait (HbSHbS) as test cases and 71 with homozygous normal trait (HbAHbA) as controls.
MATERIALS AND METHODS: Ethylenediaminetetraacetic acid-anticoagulated blood samples were collected from all participants. They were tested for HbF and HbA fractions using high-performance liquid chromatography, while total Hb concentration was determined by cynomethemoglobin technique.
RESULTS: Participants with HbSHbS genotype showed mean ± standard error of mean (SEM) of HbF levels of 6.5 ± 0.8%, HbA1 showed mean ± SEM of 2.6 ± 0.3%, and HbA2 showed mean ± SEM of 4.9 ± 0.1%. Those with HbAHbA (control participants) genotype showed mean ± SEM of HbF levels of 0.5 ± 0.04%, HbA1 showed mean ± SEM of 87.3 ± 0.4%, and HbA2 showed mean ± SEM of 3.2 ± 0.1%, while the mean ± SEM Hb concentration of test cases was 6.5 ± 0.16 g/dl and that of controls was 12.32 ± 0.13 g/dl. The total Hb concentration of sickle cell patients was significantly lower than that of nonsickle cell patients. There was a positive correlation of Hb concentration (g/dl) with HbSHbS gene expression.
CONCLUSION: Findings from this study revealed that the lesser episodes of sickle cell crisis, the lower the HbF expression and higher the total Hb concentration. Hence, it is recommended that the determination of HbF, HbA1, and HbA2 levels be considered in conjunction with other routine complete blood count and hematology tests in the diagnosis and clinical management of SCD.
Keywords: Fetal hemoglobin, HbC, HbS, hemoglobinopathy, sickle cell crisis
|How to cite this article:|
Dangana A, Nasir IA, Medugu JT, Emelike FO, Oluwatayo B O, Haruna AS. Fetal hemoglobin gene expression in patients with sickle cell disease in North Central Nigeria. Int J Health Allied Sci 2018;7:98-103
|How to cite this URL:|
Dangana A, Nasir IA, Medugu JT, Emelike FO, Oluwatayo B O, Haruna AS. Fetal hemoglobin gene expression in patients with sickle cell disease in North Central Nigeria. Int J Health Allied Sci [serial online] 2018 [cited 2022 May 17];7:98-103. Available from: https://www.ijhas.in/text.asp?2018/7/2/98/231686
| Introduction|| |
Fetal hemoglobin (HbF, α2y2) is the predominant Hb in fetal life. The globin chains of HbF are coded by y-gene of β-globin clusters on chromosome 11 in humans. After birth, HbF is gradually replaced by adult Hb (HbA, α2β2), due to the switch from y- to β-globin gene expression. In normal cases, HbF constitutes <1% of the total Hb by the end of the 1st year of life. The synthesis of HbF is restricted to a subpopulation of red cells, known as F-cells (FCs), and the HbF levels are directly correlated to the number of FC. DNA mutation may lead to a persistent expression in y-globin gene downregulation. HbF levels, which are regulated by multiple genes with influence of an environmental component, play a dominant role in ameliorating morbidity and mortality of many congenital Hb disorders such as sickle cell disease (SCD). The high concentration of HbF is a well-characterized diagnostic feature and correlates with reducing morbidity and mortality in patients with these blood disorders.
HbF differs from the adult form of the protein in its affinity for oxygen. Production of HbF begins about 2 months into gestation and helps deliver oxygen from the mother's bloodstream to the developing fetus. By about 3–6 months after birth, HbF is almost completely replaced by HbA. The timing explains why sickle cell patients do not experience symptoms of the disease until several months after birth.
The SCD is a disorder that results from the inheritance of two abnormal allelomorphic genes of the β chains of Hb, at least one of which is the sickle gene, in which sickling of red blood cells produces prominent clinical manifestations due to missense mutation of the β-globin chain. Red cell sickling due to HbS is caused by the polymerization of Hb tetramer as a result of replacement of glutamic acid by valine at position 6 of β-globin due to mutant sickle gene, whereas red cell sickling due to hemoglobin C (HbC) is caused due to abnormal Hb, in which substitution of a glutamic acid residue with a lysine residue (E6K substitution) occurred at the 6th position of β-globin chain. The absence of a polar amino acid at this position promotes the noncovalent aggregation of Hb in a low-oxygen environment which distorts red blood cells into a sickle shape and decreases their elasticity. Biochemically, the low-oxygen environment causes the beta chain of neighboring Hb molecules to hook together, becoming rigid and polymerized. These cells fail to return to their normal shape when oxygen is restored and thus fail to deform as they pass through narrow vessels, leading to blockage in the capillaries.
A variety of factors affect the pathophysiology of SCD leading multitude of clinical manifestations including intravascular hemolysis, vascular occlusion, pro-oxidant and pro-inflammatory stress, coagulopathy and altered blood rheology resulting in pain, organ damage, and a low blood count. There is no single-therapeutic modality that serves to abrogate all pathology of SCD but a better understanding of the mechanism of red cells sickling, factors that influence variability of its clinical course, the interactions of the SCDs and as well as their associated complications, had led to a number of clinical interventions including the induction of HbF production.
Elevated levels of HbF have been associated with lessened vaso-occlusive complications and prolonged survival rates of sickle cell disorder owing to its antipolymerization property. HbF reduces HbS concentration in the same red cell, but more importantly, both HbF and its mixed hybrid tetramer cannot enter the deoxy-sickle Hb polymer phase. This modulates the phenotypes of SCD due to variable distribution of HbF in sickle erythrocytes. The blood concentration of HbF, or the number of cells with detectable HbF (FCs), does not measure the amount of HbF/FC. Even patients with high HbF can have severe disease because HbF is unevenly distributed among FCs, and some cells might have insufficient concentrations to inhibit HbS polymerization. With mean HbF levels of 5%, 10%, 20%, and 30%, the distribution of HbF/FC can greatly vary, even if the mean is constant. For example, with 20% HbF, as few as 1% and as many as 24% of cells can have polymer-inhibiting, or protective, levels of HbF of ~ 10 pg; with lower HbF, few or no protected cells can be present. Only when the total HbF concentration is near 30%, is it possible for the number of protected cells to approach 70%. HbF/FC and the proportion of FCs that have enough HbF to thwart HbS polymerization is the most critical predictor of the likelihood of severe SCD. The general objective of this study is to determine the pattern of HbF expression and total Hb concentration by high-performance liquid chromatography (HPLC) and cyanmethemoglobin (HiCN) method, respectively, among sickle cell anemia (SCA) patients attending the University of Abuja Teaching Hospital (UATH), Abuja, Nigeria. Owing to the tests products stability, the high sensitivity, specificity, accuracy, and reliability of these methods (HPLC and HiCN), they were thus employed for the present study. HPLC and HiCN techniques are the internationally recommended methods for determining the percentage fractions of Hb bands and total HbF concentration in humans.
| Materials and Methods|| |
Cases and the controls were recruited from sickle cell day clinic and Outpatient Department of the UATH, Gwagwalada, Federal Capital Territory Abuja, Nigeria. Abuja is the capital city of Nigeria.
The study was a case–control study conducted from February 22 to October 30, 2015. It included sickle cell patients as test cases and nonsickle cell cases as controls who are homozygous HbA.
Ethics statement and informed consent
This study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the ethical research committee of the UATH, Abuja, Nigeria (Ethical approval number: UATH/HREC/PR/2015/09/047). The study was appropriately explained to all participants; thereafter, they individually gave verbal and/or written consent for inclusion before they voluntarily participated in the study. All data were analyzed anonymously throughout the study.
- All consented sickle cell patients attending UATH within the study period
- Individuals between 1 and 15 years of age.
- Patients with any form of illness besides SCD were excluded
- Those currently receiving therapy besides those for SCD were also excluded.
Sample size estimation
A total of 146 cases were consecutively enrolled into this study. Seventy five sickle cell patients of 1–15 years of age constituted the case group while 71 nonsickle cell cases (homozygous for HbA alleles) of 1–15 years of age constituted the control group.
Five milliliters of blood was collected inside the ethylenediaminetetraacetic acid (EDTA) bottle by aseptic venous puncture from sickle cell patients in Abuja, North Central Nigeria, and also 5 ml was collected inside the EDTA bottle from the controls, those determine to be suitable based on the selection criteria, with their consents.
All specimens were analyzed by the Bio-Rad Variant HPLC system with the use of the Variant Beta-Globin Short Program Recorder Pack (Bio-Rad Laboratories, California, USA) as described in the user manual for the assay. After collection, the samples were stored at 2°C –8°C and tested within 3 days of collection. The sample preparation involves dilution of 5 μl of EDTA-anticoagulated blood in 1 ml of hemolysis fluid provided in the kit. Thereafter, the samples were applied in the HPLC instrument. The lysed samples were diluted with the specific hemolyzing/wash buffer and injected into an assay-specific analytic cartridge. The variant dual pumps delivered a programmed buffer gradient of increasing ionic strength to the cartridge, where the Hb fractions were separated based on their ionic interaction with the cartridge material. The separated Hb fractions were passed through a flow cell, where optical absorbance was measured at 415 nm with simultaneous use of secondary wavelength of 690 nm to reduce background noise. The software delivers a printed report showing the chromatogram, with all the Hb fractions eluted. The integrated peaks were assigned to manufacturer-defined “windows” derived from specific retention time (RT). This RT was the time that elapses from the sample injection to the apex of the elution peak, of normal Hb fraction and common variants. The “windows” were established ranges, in which common variants have been observed to elute using the Variant beta-globin short program. The printed chromatogram shows all the Hb fractions eluted, the RTs, the areas of the peaks and the values (%) of different Hb components [Figure 1]a and [Figure 1]b. If a peak elutes at a RT that is not predefined, it is labeled as an unknown. Each analytical cycle, from sampling to printing of results, took about 5 min.
|Figure 1: (a) A chromatogram of a HbAHbA participant. (b) A chromatogram of a HbSHbS participant|
Click here to view
The total Hb concentration was assessed from Becton Dickinson hematology analyzer (New Jersey, USA), and the Hb value was determined based on HiCN (spectrophotometric) method. Owing to the high sensitivity, specificity, accuracy, and reliability of these methods (HPLC and HiCN), they were thus employed for the present study. HPLC and HiCN techniques are the internationally recommended methods for determining the percentage fractions of Hb bands and total Hb concentration in humans. In cynomethemoglobin method, blood sample is diluted in a solution containing potassium cyanide and potassium ferricyanide. The latter converts Hb to methemoglobin which is converted to HiCN by potassium cyanide. The absorbance of the solution is then measured in a spectrophotometer at a wavelength of 540 nm or in a colorimeter using a yellow-green filter. In HiCN method, all forms of Hb except sulfhemoglobin are converted to HiCN, there is no visual error, and as no color matching is required, HiCN solution is stable and its color does not fade with time, so readings may not be taken immediately. Hence, absorbance may be measured soon after dilution. In addition, a reliable and stable reference standard was available from the World Health Organization for direct comparison.
Mean ± standard deviation of mean (SD) was derived; Student's t-test test for unpaired data and correlation analysis was used to compare the variables of HbF, HbA, Hb2 values and Hb concentration of both groups using statistical package for social sciences (SPSS) software version 20 (SPSS Inc., California, USA). A two-sided P < 0.001 at 99% confidence interval is considered statistically significant for the t-test to determine the statistical association between the groups.
| Results|| |
Of 146 cases consecutively recruited in this study, 75 sickle cell patients from 1 to 15 years of age constituted the case group while 71 nonsickle cell cases (homozygous for HbA alleles) from 1 to 15 years of age constituted the control group. The homozygous SS showed HbF levels of 6.5 ± 0.80 (mean ± standard error of mean [SEM]), HbA1 showed mean ± SEM of 2.6 ± 0.30%, and HbA2 showed mean ± SEM of 4.9 ± 0.1%. The controls showed an HbF mean ± SEM of 0.5 ± 0.04%, HbA1 mean ± SEM of 87.3 ± 0.40%, and HbA2 mean ± SEM of 3.2 ± 0.1 [Table 1]. The HbF showed mean ± SEM of 6.5 ± 0.80% and a total Hb concentration mean ± SEM of 6.59 ± 0.16 g/dl, while the controls (HbAHbA) showed HbF mean ± SEM of 0.5 ± 0.04% and total Hb concentration mean ± SEM of 12.32 ± 0.13 g/dl [Table 2].
|Table 1: Fetal hemoglobin concentration and total hemoglobin concentration of sickle cell disease patients and their control counterparts|
Click here to view
|Table 2: Comparison of fetal hemoglobin concentration and total hemoglobin concentration among sickle cell disease patients in the studied population and their control counterparts|
Click here to view
Those who experienced sickle cell crisis <2 times/month were 46, while those who had crisis >2 times in a month were 29 [Table 3]. The HbF mean ± SEM of those who experienced crisis <2 times/month were 6.1 ± 0.98% and a total Hb concentration mean ± SEM of 7.1 ± 0.63 g/dl, while those who experienced sickle cell crisis >2 times in a month shows a mean ± SEM of 7.1 ± 1.39% and a total Hb concentration mean ± SEM of 5.7 ± 0.24 g/dl.
|Table 3: Comparison of fetal hemoglobin and hemoglobin concentration sickle cell disease patients with different durations of vaso.occlusive crisis|
Click here to view
| Discussion|| |
SCA is a monogenic disease with widely heterogeneous phenotypes. Its severity is moderated by high HbF levels. The mechanism responsible for HbF production in adults is not fully comprehended. However, several studies have linked variations in HbF to polymorphism in β-globin cluster. Increased levels of HbF are of no consequence in healthy adults but confer ameliorating effects on the clinical courses of SCA and beta thalassemia.
Of the 75 sickle cell anemic patients enrolled in this study, the mean ± SEM HbF expression was 6.5 ± 0.8%, which is significantly higher than that of controls who constituted by 71 nonsickle cell cases (homozygous for HbA alleles), which showed mean ± SEM HbF genes expression of 0.5 ± 0.04%, this indicates that there is persistence expression of HbF in the study participants after birth. This is in agreement with the work of Boyer et al., who stated that the persistent expression of high levels of HbF after birth helps in ameliorating the rate of frequent sickle cell crisis and subsequently mortality and morbidity of sickle cell patients. It also agrees with the work of Wood et al., who stated that in normal individuals, HbF constitutes <1% of the total Hb as seen in the present study [Table 1] and [Table 2], where the HbF level is 0.5% which is <1% even though the mechanism responsible for the production of HBF in adults are not fully comprehended. However, several studies have linked these variations in HBF to polymorphism in beta-globin cluster.
The findings from this study revealed lower levels of HBA1 among the sickle cell study participants compared to that of controls in the study participants. The sickle cell anemic patients showed mean ± SEM of 2.6 ± 0.3 for HbA1, while that of controls showed mean ± SEM of 87.3 ± 0.4% for HbA1. This is significantly higher than that of sickle cell anemic group. There was also increase in the level of HBA2 among the case group which shows mean ± SEM of 4.9 ± 0.1%, which was also significantly lower than that of nonsickle cell anemic cases.
The total Hb concentration of Group A (sickle cell patients) was significantly lower than that of control group. Group A shows a total Hb concentration of 6.59 g/dl with mean ± SEM of 6.59 ± 0.15652 g/dl, while Group B (non-sickle cell individuals) shows Hb concentration of 12.32 g/dl with mean ± SEM of 12.23 ± 0.12548 g/dl, which was significantly higher than that of sickle cell patients. The relatively low Hb concentration observed among the sickle cell anemic patients invariably impact on their hematocrit levels is an indication of reduced oxygen-carrying capacity and increased propensity for during sickle cell crisis. This may warrant blood transfusion to keep and sustain the affected SCD patients so as to enable sustained energy for the normal function of the body.
The findings from this study showed a positive correlation of Hb concentration with HbS genes expression [Figure 2]. This may be because the lower Hb concentration of HbS correlates with the possibility of anemic condition.
|Figure 2: Correlation between fetal hemoglobin and HbS gene expression among sickle cell disease patient in the studied population. This shows a positive correlation but not significant associated (P = 0.4206 and r = 0.094)|
Click here to view
In regard to the frequency of sickle cell crisis among the patients, those who had sickle cell crisis <2 times/month had lower HbF expression of 6.06 ± 0.98% compared to those who had sickle cell crisis ≥2 times/month, which revealed higher HbF expression of 7.09 ± 1.39. In addition, the Hb concentration of sickle cell patients who experienced sickle cell crisis <2 times/month was significantly higher than those who experienced sickle cell crisis <2 times/month. This implies that the lesser the number of sickle cell crisis, the lower the HbF expression and vice versa. This is because HbF favors relatively higher total Hb concentration, as such the Hb had affinity for oxygen which was needed for steady cardiopulmonary circulation and also for transportation of nutrients to all organs. This may also be due to the fact that after birth, HbF was destroyed early and once destroyed, normal sickle cell activities begin to take place. Hence, the lesser the frequency of crisis, the higher the total Hb concentration, and vice versa. This is because more red blood cells might have been hemolyzed, thereby destroying functional red cells population leading to low Hb and subsequently anemia set in.
There was a positive correlation between HbF and HbS gene expression with no statistical association [Figure 3]. This implies that the higher levels of circulating HbF in a sickle cell patient could lead to low manifestation of sickle cell symptoms because their deoxygenated erythrocytes take a longer period to sickle and will not deform extensively as those of sickle cell trait patients; this agrees with the report by Mabaera et al., who revealed that the HbF did not interact with HbS as such the high level of HbF with the persistent y-globin in the HbF will inhibit the polymerization of HbS because HbF is a powerful modulator of the clinical and hematological features of sickle cell anemia. They attributed the association of the high level of HbF with a reduced rate of acute painful crisis episode.
|Figure 3: Correlation between hemoglobin concentration (mg/dl) and HbS gene expression among sickle cell disease patients in the studied population. This also shows a positive correlation with statistical significant association (P < 0.05 and r = 0.0205)|
Click here to view
| Conclusion|| |
Findings from this study revealed that the lesser episodes of sickle cell crisis, the lower the HbF expression and higher total Hb concentration. Hence, it is recommended that the determination of HbF, HbA1, and HbA2 levels be considered in conjunction with other routine complete blood count, and hematology tests in the diagnosis and clinical management of SCD.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Zago MA, Wood WG, Clegg JB, Weatherall DJ, O'Sullivan M, Gunson H, et al.
Genetic control of F
cells in human adults. Blood 1979;53:977-86.
Boyer SH, Belding TK, Margolet L, Noyes AN. Fetal hemoglobin restriction to a few erythrocytes (F
cells) in normal human adults. Science 1975;188:361-3.
Wood WG, Stamatoyannopoulos G, Lim G, Nute PE. F-cells in the adult: Normal values and levels in individuals with hereditary and acquired elevations of Hb F. Blood 1975;46:671-82.
Miyoshi K, Kaneto Y, Kawai H, Ohchi H, Niki S, Hasegawa K, et al.
X-linked dominant control of F-cells in normal adult life: Characterization of the Swiss type as hereditary persistence of fetal hemoglobin regulated dominantly by gene(s) on X chromosome. Blood 1988;72:1854-60.
Stamatoyannopoulos G. Control of globin gene expression during development and erythroid differentiation. Exp Hematol 2005;33:259-71.
Bank A. Regulation of human fetal hemoglobin: New players, new complexities. Blood 2006;107:435-43.
Powars DR, Weiss JN, Chan LS, Schroeder WA. Is there a threshold level of fetal hemoglobin that ameliorates morbidity in sickle cell anemia? Blood 1984;63:921-6.
Labie D, Elion J. Molecular and pathophysiological basis of hemoglobin disorders. J EMC - Hematologie 2005;2:220-39.
Kohne E. Hemoglobinopathies. Clinical manifestation, diagnosis and treatment. Dtsch Arzteblatt Int 2011;108:534-40.
Mabaera R, West RJ, Conine SJ, Macari ER, Boyd CD, Engman CA, et al.
Acell stress signaling model of fetal hemoglobin induction: What doesn't kill red blood cells may make them stronger. Exp Hematol 2008;36:1057-72.
Akinsheye I, Solovieff N, Ngo D, Malek A, Sebastiani P, Steinberg MH, et al.
Fetal hemoglobin in sickle cell anemia: Molecular characterization of the unusually high fetal hemoglobin phenotype in African Americans. Am J Hematol 2012;87:217-9.
Dover GJ, Boyer SH. Quantitation of hemoglobins within individual red cells: Asynchronous biosynthesis of fetal and adult hemoglobin during erythroid maturation in normal subjects. Blood 1980;56:1082-91.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]