# The Relation between Glutathione S Transferase (GSTM1, GSTP1 and GSTT1) Polymorphisms and Clinical Diversity of Sickle Cell Disease among Pediatric Sudanese Patients

Naif Taleb Lahj University Ali, Ozaz Yagoup Mohammed Ahmed, Fayad Osman Mohammed, Huda Mohammed Haroun

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Naif Taleb Lahj University Ali, Ozaz Yagoup Mohammed Ahmed, Fayad Osman Mohammed, Huda Mohammed Haroun. The Relation between Glutathione S Transferase (GSTM1, GSTP1 and GSTT1) Polymorphisms and Clinical Diversity of Sickle Cell Disease among Pediatric Sudanese Patients. Asian Hematology Research Journal, 2021, 4 (2), pp.86-102. 10.9734/ahrj/2021/v4i278. hal-04987221

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# The Relation between Glutathione S Transferase (GSTM1, GSTP1 and GSTT1) Polymorphisms and Clinical Diversity of Sickle Cell Disease among Pediatric Sudanese Patients

Naif Taleb Ali<fnref n="1" />*, Ozaz Yagoup Mohammed Ahmed<fnref n="1" />, Fayad Osman Mohammed<fnref n="2" /> and Huda Mohammed Haroun<fnref n="1" />

## Authors' contributions

This work was carried out in collaboration among all authors. Author NTA designed the study, collect the data, carried out the analysis, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. Authors OYMA and HMH supervised and help in the work and give the information for all sections in Research. Author FOM helped in Molecular analysis. All authors read and approved the final manuscript.

## Article Information

Editor(s):

(1) Dr. Dharmesh Chandra Sharma, G. R. Medical College & J. A. Hospital, India.

Reviewers:

(1) Alice Verticchio, Icahn School of Medicine at Mount Sinai, USA.

(2) Luís Silva Santos, Universidade Católica Portuguesa, Portugal.

Complete Peer review History: http://www.sdiarticle4.com/review-history/66916

## Original Research Article

Received 20 January 2021

Accepted 25 March 2021

Published 14 April 2021

## ABSTRACT

**Background:** Sickle cell disease (SCD) is a highly variable condition, with some patients being asymptomatic and others frequently admitted to hospital. Impairment of the glutathione system due to genetic polymorphisms of glutathione S-transferase (GST) genes is expected to influence on the severity of SCD manifestations.

**Objectives:** This study aimed to investigate the possible association between the presence of GSTM1, GSTT1 and GSTP1 gene polymorphisms and SCD severity, diversity and complications.

**Study Design:** Cross-section hospital based study.

**Place and Duration of Study:** This study carried out in Khartoum town in Jafar Ibn Auf Pediatric Hospital / Khartoum during the period (June 2017 to June 2020).

<footnote n="1">¹Department of Hematology and Immunohematology, Faculty of Medical Laboratory Sciences, Gezira University, Sudan.</footnote>

<footnote n="2">²Institute of Endemic Diseases, University of Khartoum, Khartoum, 11111, Sudan.</footnote>

<footnote>*Corresponding author: E-mail: naiftalebali@gmail.com;</footnote>
**Methodology:** The total subjects of the confirmed diagnosis were 126 patients, 78 (61.9%) are males and 48 (38.1%) are female.

GSTM1 and GSTT1 genotypes were determined by polymerase chain reaction (PCR), GSTP1 genotyping was conducted with a PCR-RFLP, and the data analyzed by SPSS version 23.

**Results:** The GSTM1null genotype was found to be present in male more than female (OR=2.6 and p=0.002) and trend to be protective from development of Dactylitis (OR=0.313 and p=0.006) and reduce risk to develop ACS (OR=0.23 and p=0.002) while this polymorphism increase requirement for blood exchange (OR=1.1 and p=0.044), the GSTT1null genotype found to be present in female more than male (OR=2.6 and p=0.012) and this polymorphism reduce requirements for blood transfusion (OR=0.137 and p< 0.001) and annual hospitalization (OR=0.436 and p=0.029), and reduce risk to development of stroke (OR=0.125 and p=0.008), polymorphism of both GSTM1 and GSTT1 found to be associated with appearance of disease before one year of age (OR=1.43 and p=0.004) and trend to be protective from development of Dactylitis (OR=0.124 and p=0.002), and there are no statistically significant association between GSTP1 gene polymorphism and gender variability and clinical manifestations of SCD.

**Conclusion:** Some GST genes polymorphisms were significantly associated with increased risk and some trend to have protective effect on clinical manifestations of SCD.

Keywords: SCD; GST; GSTM1; GSTT1; GSTP1; polymorphisms; ACS; Sudan.

# 1. INTRODUCTION

Sickle cell diseases (SCD) is a disorder caused by a mutation that results in a single substitution of amino acid valine for glutamic acid in the sixth position on the beta subunit of hemoglobin resulting in abnormal hemoglobin, hemoglobin (Hb) S [1]. In its deoxygenated state, the HbS molecules become polymerized and deform the red blood cells, causing oxidative damage, cellular dehydration, abnormal phospholipid asymmetry, and increased adhesion to vascular endothelium [2]. The multifactorial nature of the SCD involves several changes in erythrocyte sickling, vaso-occlusive episodes, hemolysis, activation of inflammatory mediators, oxidative stress and endothelial dysfunction, which apparently result from HbS instability, generating oxygen radicals [3]. Among the major complications of SCD, stroke, acute chest syndrome, infections, osteoarticular lesions, lower limbs ulcers and priapism are among the most common [4,5]. An altered glutathione (GSH) metabolism in association with increased oxidative stress has been implicated in the pathogenesis of many diseases [6]. Alterations in GSH concentration have been demonstrated in many pathological conditions including SCD [7]. Glutathione S-transferases (GST) are a family of enzymes involved in phase-II detoxification of endogenous and xenobiotic compounds. Polymorphisms in GST genes have been associated with susceptibility to different diseases [8]. The clinical severity and hematological manifestations of sickle cell anemia are varied and are influenced by the

participation of several genes in modulating the phenotype of sickle cell disease; polymorphisms of these genes may be related to the different manifestations between individuals [9].

The study of GSTM1, GSTT1 and GSTP1 gene polymorphisms in Sickle cell disease patients are the first step toward the understanding of the pathophysiology of disease, enabling predictive medicines and providing clinically useful pharmacogenomics, so this study was aimed to investigate the possible association between the presence of GSTM1, GSTT1 and GSTP1 genes polymorphism and SCD severity, diversity and complications.

# 2. MATERIALS AND METHODS

This study is cross-section hospital based study; included 126 pediatric patients with sickle cell disease who were confirmed by hemoglobin electrophoresis, Sickling test and clinical examination. Patients were invited to participate in the study during their regular follow up visits to Jafar Ibn Auf Pediatric Hospital / Khartoum. The study protocol was in accordance with the local hospital research guidelines and informed consent was obtained from patient's legal representatives. The data and blood samples collection were carried out in Khartoum town in Jafar Ibn Auf Pediatric Hospital / Khartoum.

The DNA extraction and storage and molecular biology analysis were carried out in the department of Molecular biology Institute of
Endemic Diseases (IEND) -University of Khartoum.

Clinical data were obtained from medical records and interviews with the patients.

## 2.1 Molecular Analysis

5 ml of blood were obtained from all participants, collected in sterile EDTA tubes, and then stored at -20°C until use.

DNA was extracted from EDTA blood samples by G-spin TM Total DNA extraction kit protocol intron biotechnology: briefly, a total of 200 µl of blood sample was placed in 1.5ml micro centrifuge tube, 20 µl proteinase K and 5 µl of RNase solution were added. The solution was mixed gently by vortex, and then 200 µl of Buffer BL was added into sample and mixed thoroughly and placed at Room temperature for 2 minutes. The lysate was incubated at 56 C for 10 min and briefly centrifuged to remove drops from the inside of the lid. Thereafter, 200 µl of absolute ethanol was added into the lysate and mixed gently by inverting 5-6 times or pipetting. The mixture was applied to the spin column (in a 2 ml collection tube) and centrifuged at 13,000 rpm for 1 min. The filtrate was discarded and the spin column was placed in a new 2ml collection tube then 700 µl of buffer WA was added to the spin column, and centrifuged for 1 min at 13,000 rpm. The flow-through was discarded and 700 µl of buffer WB was added and centrifuged for 1 min at 13,000 rpm. The flow-through was discarded and the column was placed into a new 2 ml collection tube, then again centrifuged for 1 min to dry the membrane. Finally, the spin column was placed into a new 1.5 ml tube, and 40 µl of buffer CE was directly added onto the membrane, and incubated for 1 min at room temperature, DNA was then eluted by centrifugation for 1 min at 13,000 rpm. DNA purity was quantified using a Nano Drop Spectro-photometer (Thermo Scientific 2000) and the DNA integrity was checked using agarose gel electrophoresis.

## 2.2 Genotyping of GSTM1 and GSTT1 Polymorphisms

The primers were synthesized by Sangon and PCR amplifications were carried out using the thermal cycler Applied QIAGEN (Rotor-Gene Q).

For GSTM1 genotype, the following pair of primers was used in the genotyping analysis: Forward primer:

GAACTCCCTGAAAAGCTAAAGC-3, Reverse primer: 5- GTTGGGCTCAAATATACGGTGG -3. PCR was carried out in a total volume of 20 µl. It consists of 2 µl of genomic DNA, 1 µl from each primer, Master mix (Maxime TM premix kit (i-Taq) and 16 µl distilled water. PCR was initiated by denaturation step at 95°C for 2 minutes followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing temperatures ranged between 59°C for 30 second and 72°C for 30 second, and final extension at 72°C for 5 minutes.

For GSTT1 genotype, the following pair of primers was used in the genotyping analysis: Forward primer: TTCCTTACTGGTCCTCACATCTC -3, Reverse primer: 5- TCACCGGATCATGGCCAGCA -3. PCR was carried out in a total volume of 20 µl. It consists of 2 µl of genomic DNA, 1 µl from each primer, Master mix (Maxime TM premix kit (i-Taq) and 16 µl distilled water. PCR was initiated by denaturation step at 95°C for 2 minutes followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing temperatures ranged between 60°C for 30 second and 72°C for 50 second, and final extension at 72°C for 5 minutes. The product obtained from each reaction was subjected to electrophoresis on a 2% agarose gel in an electric field of 10 V/cm, stained with 5 µg/mL ethidium bromide, and visualized and recorded with the aid of a video documentation system (Image Master VDS®, Amersham Pharmacia Biotech). GSTM1 and GSTT1 genotypes were determined by the presence and absence (null) of bands of 219 and 480 bp respectively (Figs. 1 and 2).

## 2.3 For Genotyping of GSTP1 Polymorphism

GSTP1 (Ile105Val) polymorphism was determined with a polymerase chain reaction-restriction fragment length polymorphism assay [PCR-RFLP]. The PCR primers were: 5'-ACCCCAGGGCTCTATGGGAA-3' (F) and 5'-TGAGGGCACAAGAAGCCCCT-3' (R).

PCR was carried out in a total volume of 20µl. It consists 2µl genomic DNA, 1µl each primer, ready to load master mix (Maxime TM premix kit (i-Taq) and 16µl distilled water. PCR condition includes initial denaturation at 95°C for 2 minutes, followed by 30 cycles at 95°C for 30 second, 61.3°C for 30 second, 72°C for 20 second and a last extension at 72°C for 5 minutes. PCR products were analyzed on a 2%
Agarose gel stained with 0.3 µg/mL ethidium bromide, and visualized by gel documentation system (to check the presence of 176 pb of GSTP1) (Fig. 3).

<figure><img src="image_3.png" /><figcaption>Fig. 1. Agarose gel electrophoresis for amplified PCR products of GSTM1 (219bp) fragments</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Fig. 2. Agarose gel electrophoresis for amplified PCR products of GSTT1 (480 bp) fragments</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Fig. 3. Agarose gel electrophoresis for amplified PCR products of GSTP1 (176 bp) fragments</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 4. DNA fragment digestion with Alw261 restriction enzyme</figcaption></figure>

Then the PCR product was digested with the
restriction endonuclease Alw261 (BsmAI)
restriction enzyme {thermoscientific Alw261
(BsmA1) Lot Number 00743699} as follow:

each 7 µl of PCR product, 1 µl from 10X NEB
buffer and 0.5 µl from Alw261 restriction enzyme
were added, then incubated at 37°C for 20 hrs,
followed by incubation at 65°C for 20 minutes to
inhibit the enzyme activity. The products are then resolved on 2% agarose gel electrophoresis containing ethidium bromide, then visualized using UV trans illuminator. The amplified fragment after digestion with Alw261 restriction enzyme, will give rise to: 2 fragments at 176 bp and 85 bp indicating the presence of wild type (Ile/Ile), appearance of 2 fragments at 91 bp and 85bp indicates the presence of homozygous mutant type (Val/Val), while presence of 3 fragments at 176 bp, 91 bp and 85 bp indicates the presence of heterozygous mutant type (Ile/Val). For quality control, genotyping of the samples were repeated blindly and were identical to the initial results (Fig. 4).

Lane DNA ladder: MW 100-1500 bp fragments, lane fragments at 176 bp and 85 bp indicates the presence of wild type (Ile/Ile), lane fragments at 176 bp, 91 bp and 85 bp indicates the presence of heterozygous mutant type (Ile/Val). Lane fragments at 91 bp and 85 bp indicates the presence of homozygous mutant type (Val/Val).

## 2.4 Statistical Analysis

Data were transferred to the Statistical Package for the Social Sciences (SPSS) Software program, version 23 to be statistically analyzed. The obtained data are presented as frequencies, percentage, mean and standard deviation, Descriptive and analytic statistics and crosstabulation were performed. Data were summarized using Chi-square or Fisher exact probability tests. Associations between the GSTM1, GSTT1 and GSTP1 polymorphisms and clinical manifestations of SCD patients were estimated using odd ratio (OR) and 95% confidence intervals (95% CIs). Odd ratio and confidence interval were used to estimate risk of the SCD among the population; the lowest accepted level of significance was 0.05 or less.

## 3. RESULTS

The total subjects of the confirmed diagnosis were 126 patients, 78 (61.9%) are males and 48 (38.1%) are female, the mean age of the study subjects was (8.0) years old; the minimum age was 10 months and the maximum one was 14.5 years.

In the study subjects, 94 (74.6%) were diagnosed with SCD when they were less than one year and 32 (25.4%) were diagnosed when their age one year or more, the frequency of blood transfusion for these patients 82 (65.1%)

were diagnosed with SCD had blood transfusion less than two times per year and 44 (34.9%) had blood transfusion more than two times per year, for the frequency of VOC 50 (39.7%) were diagnosed with SCD had crises less than two times per year and 76 (60.3%) had VOC more than two times per year. Regarding the frequency of annual hospitalization 46 (36.5%) were diagnosed with SCD had less than two times per year and 80 (63.5%) had more than two times per year.

And out of whole subjects 14 (11.1%) were diagnosed with SCD had stroke and 52 (41.3%) had Dactylities, the frequency of ACS 78 (61.9%) were diagnosed with SCD had less than two times per year and 48 (38.1%) had more than two times per year.

And out of whole subjects 8 (6.3%) were diagnosed with SCD had bones problems and 4 (3.2%) had splenomegaly and 2 (1.6%) had blood exchange.

The GSTM1null genotype was found to be present in male more than female (OR=2.6, 95% CI=1.324 – 5.168, p=0.002) and trend to be protective from development of Dactylitis (OR=0.313, 95% CI=0.136 – 0.716, p=0.006) and reduce risk to develop ACS (OR=0.259, 95% CI=0.107 – 0.625, p=0.002) while this polymorphism increased requirements to blood exchange (OR=1.050, 95% CI=0.981 – 1.123, p=0.044).

Also we found, GSTM1null genotype increased risk to stroke but this association not statistically significant (OR=3.33, 95% CI=0.710 – 15.643, p=0.109), and there are no significant association between GSTM1 genotype and time of anemia appearance, frequency of blood transfusion, annual hospitalization, VOC, bone problems and splenomegaly (Table 1).

The GSTT1null genotype found to be present in female more than male (OR=2.6, 95% CI=1.224 – 5.472, p=0.012) and this polymorphism may reduce requirements to blood transfusion (OR=0.137, 95% CI=0.059 – 0.318, p< 0.001) and annual hospitalization (OR=0.436, 95% CI=0.206 – 0.924 p=0.029) and reduce risk to development of stroke (OR=0.125, 95% CI=0.0267 – 0.585, p=0.008). Also this study found GSTT1null genotype increased frequency of VOC, ACS, bone problems and splenomegaly but this association not statistically significant (OR=1.34, 95% CI=0.654 – 2.738, p=0.424),
(OR=1.47, 95% CI=0.713 – 3.044, p=0.294), (OR=2.9, 95% CI=0.562 – 14.96, p=0.203) and (OR=1.1, 95% CI=0.151 – 8.088, p=0.923) respectively. And there are no significant association between GSTT1 null genotype and time of anemia appearance and Dactylities (Table 2).

Polymorphism of both GSTM1 and GSTT1 found to be associated with appearance of disease before one year of age (OR=1.43, 95% CI=1.264

– 1.623, p=0.004) and trend to be protective from development of Dactylitis (OR=0.124, 95% CI=0.027 – 0.563, p=0.002).

Also, there are no significance association between GSTM1/ GSTT1null genotype and gender, frequency of blood transfusion, hospitalization, VOC, stroke, ACS, bone problems, splenomegaly and blood exchange (Table 3).

<table><caption>Table 1. Association between GSTM1 null genotype and clinical manifestations</caption><thead><tr><th rowspan="2">Clinical manifestations</th><th colspan="4">GSTM1</th><th rowspan="2">Confidence interval 95% p. value</th></tr><tr><th>Present</th><th>Null</th><th>Chi-square</th><th>Odd ratio</th></tr></thead><tbody><tr><td><strong>Gender</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male</td><td>44</td><td>34</td><td>9.692</td><td>2.6</td><td>(1.324 – 5.168)</td><td>0.002</td></tr><tr><td>Female</td><td>40</td><td>8</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Anemia appearance</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt; 1 year</td><td>62</td><td>32</td><td>0.084</td><td>0.881</td><td>0.372 – 2.083</td><td>0.772</td></tr><tr><td>≥ 1 year</td><td>22</td><td>10</td><td></td><td></td><td></td><td></td></tr><tr><td>Blood transfusion</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt; 2 per year</td><td>54</td><td>28</td><td>0.070</td><td>0.900</td><td>0.412 – 1.966</td><td>0.792</td></tr><tr><td>≥ 2 per year</td><td>30</td><td>14</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>VOC</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt; 2 per year</td><td>32</td><td>18</td><td>0.265</td><td>0.821</td><td>0.386 – 1.743</td><td>0.607</td></tr><tr><td>≥ 2 per year</td><td>52</td><td>24</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Annual hospitalization</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt; 2 per year</td><td>30</td><td>16</td><td>0.068</td><td>0.903</td><td>0.420 – 1.942</td><td>0.794</td></tr><tr><td>≥ 2 per year</td><td>54</td><td>26</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Stroke</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>12</td><td>2</td><td>2.571</td><td>3.33</td><td>0.710 – 15.643</td><td>0.109</td></tr><tr><td>No</td><td>72</td><td>40</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Dactylities</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>42</td><td>10</td><td>7.924</td><td>0.313</td><td>0.136 – 0.716</td><td>0.006</td></tr><tr><td>No</td><td>42</td><td>32</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>ACS</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt; 2 per year</td><td>44</td><td>34</td><td>9.692</td><td>0.259</td><td>0.107 – 0.625</td><td>0.002</td></tr><tr><td>≥ 2 per year</td><td>40</td><td>8</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Bone problems</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>6</td><td>2</td><td>0.267</td><td>1.54</td><td>0.297 – 7.971</td><td>0.605</td></tr><tr><td>No</td><td>78</td><td>40</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Splenomegaly</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>2</td><td>2</td><td>0.516</td><td>0.488</td><td>0.066 – 3.590</td><td>0.472</td></tr><tr><td>No</td><td>82</td><td>40</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Blood exchange</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>0</td><td>2</td><td>4.065</td><td>1.050</td><td>0.981 – 1.123</td><td>0.044</td></tr><tr><td>No</td><td>84</td><td>40</td><td></td><td></td><td></td><td></td></tr></tbody></table>

ACS= Acute chest syndrome, VOC= Vaso-Occlusive Crisis
<table><caption>Table 2. Association between GSTT1 null genotype and clinical manifestations</caption><thead><tr><th rowspan="2">Clinical manifestations</th><th colspan="5">GSTT1</th></tr><tr><th>Present</th><th>Null</th><th>Chi-square</th><th>Odd ratio</th><th>Confidence interval 95%</th><th>p. value</th></tr></thead><tbody><tr><td><strong>Gender</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male</td><td>44</td><td>34</td><td>6.344</td><td>2.6</td><td>1.224 – 5.472</td><td>0.012</td></tr><tr><td>Female</td><td>16</td><td>32</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Anemia appearance</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt; 1 year</td><td>42</td><td>52</td><td>1.281</td><td>0.628</td><td>0.280 – 1.410</td><td>0.258</td></tr><tr><td>≥ 1 year</td><td>18</td><td>14</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Blood transfusion</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt; 2 per year</td><td>26</td><td>56</td><td>23.84</td><td>0.137</td><td>0.059 – 0.318</td><td>0.000</td></tr><tr><td>≥ 2 per year</td><td>34</td><td>10</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>VOC</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt; 2 per year</td><td>26</td><td>24</td><td>0.638</td><td>1.34</td><td>0.654 – 2.738</td><td>0.424</td></tr><tr><td>≥ 2 per year</td><td>34</td><td>42</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Annual hospitalization</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt; 2 per year</td><td>16</td><td>30</td><td>4.786</td><td>0.436</td><td>0.206 – 0.924</td><td>0.029</td></tr><tr><td>≥ 2 per year</td><td>44</td><td>36</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Stroke</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>12</td><td>2</td><td>9.164</td><td>0.125</td><td>0.0267 – 0.585</td><td>0.008</td></tr><tr><td>No</td><td>48</td><td>64</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Dactylities</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>30</td><td>22</td><td>3.602</td><td>0.500</td><td>0.243 – 1.027</td><td>0.059</td></tr><tr><td>No</td><td>30</td><td>44</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>ACS</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt; 2 per year</td><td>40</td><td>38</td><td>1.101</td><td>1.47</td><td>0.713 – 3.044</td><td>0.294</td></tr><tr><td>≥ 2 per year</td><td>20</td><td>28</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Bone problems</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>2</td><td>6</td><td>1.752</td><td>2.9</td><td>0.562 – 14.96</td><td>0.203</td></tr><tr><td>No</td><td>58</td><td>60</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Splenomegaly</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>2</td><td>2</td><td>0.009</td><td>1.103</td><td>0.151 – 8.088</td><td>0.923</td></tr><tr><td>No</td><td>58</td><td>64</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Blood exchange</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>2</td><td>0</td><td>2.235</td><td>0.967</td><td>0.922 – 1.013</td><td>0.135</td></tr><tr><td>No</td><td>58</td><td>66</td><td></td><td></td><td></td><td></td></tr></tbody></table>

<table><caption>Table 3. Association between GSTM1/GSTT1 null genotype and some clinical manifestations</caption><thead><tr><th rowspan="2">Clinical manifestations</th><th colspan="5">GSTM1/GSTT1</th></tr><tr><th>Present</th><th>Null</th><th>Chi-square</th><th>Odd ratio</th><th>Confidence interval 95%</th><th>p. value</th></tr></thead><tbody><tr><td><strong>Gender</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male</td><td>64</td><td>14</td><td>0.661</td><td>1.5</td><td>0.554 – 4.300</td><td>0.291</td></tr><tr><td>Female</td><td>42</td><td>6</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Anemia appearance</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt; 1 year</td><td>74</td><td>20</td><td>8.093</td><td>1.432</td><td>1.264 – 1.623</td><td>0.004</td></tr><tr><td>≥ 1 year</td><td>32</td><td>0</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Blood transfusion</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt; 2 per year</td><td>66</td><td>16</td><td>2.329</td><td>2.4</td><td>0.757 – 7.764</td><td>0.127</td></tr><tr><td>≥ 2 per year</td><td>40</td><td>4</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>VOC</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt; 2 per year</td><td>40</td><td>10</td><td>1.057</td><td>1.65</td><td>0.631 – 4.311</td><td>0.304</td></tr><tr><td>≥ 2 per year</td><td>66</td><td>10</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Annual hospitalization</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt; 2 per year</td><td>38</td><td>8</td><td>0.125</td><td>1.2</td><td>0.448 – 3.175</td><td>0.724</td></tr><tr><td>≥ 2 per year</td><td>68</td><td>12</td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><thead><tr><th rowspan="2">Clinical manifestations</th><th colspan="5">GSTM1/GSTT1</th></tr><tr><th>Present</th><th>Null</th><th>Chi-square</th><th>Odd ratio</th><th>Confidence interval 95% p. value</th></tr></thead><tbody><tr><td><strong>Stroke</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>14</td><td>0</td><td>2.972</td><td>1.2</td><td>1.070 – 1.241</td><td>0.085</td></tr><tr><td>No</td><td>92</td><td>20</td><td></td><td></td><td></td></tr><tr><td><strong>Dactylities</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>50</td><td>2</td><td>9.591</td><td>0.124</td><td>0.027 – 0.563</td><td>0.002</td></tr><tr><td>No</td><td>56</td><td>18</td><td></td><td></td><td></td></tr><tr><td><strong>ACS</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt; 2 per year</td><td>62</td><td>16</td><td>3.301</td><td>0.352</td><td>0.110 – 1.123</td><td>0.078</td></tr><tr><td>≥ 2 per year</td><td>44</td><td>4</td><td></td><td></td><td></td></tr><tr><td><strong>Bone problems</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>8</td><td>0</td><td>1.612</td><td>0.283</td><td>0.016 – 5.094</td><td>0.283</td></tr><tr><td>No</td><td>98</td><td>20</td><td></td><td></td><td></td></tr><tr><td><strong>Splenomegaly</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>4</td><td>0</td><td>0.779</td><td>0.556</td><td>0.029 – 10.721</td><td>0.697</td></tr><tr><td>No</td><td>102</td><td>20</td><td></td><td></td><td></td></tr><tr><td><strong>Blood exchange</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>2</td><td>0</td><td>0.383</td><td>1.1</td><td>0.993 – 1.046</td><td>0.536</td></tr><tr><td>No</td><td>104</td><td>20</td><td></td><td></td><td></td></tr></tbody></table>

Also the present study found no statistically significance between GSTP1 gene polymorphism and gender variability and clinical manifestations of SCD.

## 4. DISCUSSION

Sickle cell anemia (SCA) is a chronic and progressively debilitating medical condition featuring ongoing hemolytic anemia and recurrent acute vaso-occlusive events [10]. It is characterized by a clinical course highly variable, ranging from death in early childhood [11] to a normal life span with few complications [12].

The complex pathophysiology of SCA which can be affected by a number of modifying factors including haplotype of β-globin gene cluster [13], coinheritance of polymorphisms associated with clinical aspects [14,15] and Hemoglobin fetal (Hb F) levels [16], chronic inflammation and oxidative states [17,18] as well as gender [13].

Human GSTs have been well characterized as ethnic-dependent polymorphism frequencies and largely divergent among populations around the world [19,20].

There are published reports about the association between GSTM1 and GSTT1 and GSTP1 polymorphisms and Sickle cell diseases but to date no study published in Africa except in Egypt, so this study aimed to fill the gap by investigating the possible association between the presence of GSTM1, GSTT1 and GSTP1 genes polymorphisms and SCD severity,

diversity and complications in pediatric Sudanese patients.

In present study, GSTM1null genotype was found to be present in male more than female (OR=2.6 and p=0.002) and this agreed with another study done in Sudan [21], they observed male had GSTM1 null genotype more than female (58.8% and 41.7%) respectively.

Also in this study, the GSTT1null genotype found to be present in female more than male (OR=2.6 and p=0.012), and this agreed with meta-analysis study [22] did report a significantly higher frequency of GSTT1 deletion among healthy Caucasian females, yet was not able to explain it on biological grounds, since GSTT1 gene is not located on the sex chromosome and [23] observed the female (68.6%) to male ratio (31.4%) was high which might explain the higher frequency of GSTT1 deletion among female controls. the difference between males and females may also be related to gender-associated expression of the GST family enzymes [24,25], or the influence of sex hormones, importance of which GST regulation is well established in rodent models [26,27].

Also, the GSTM1null genotype trend to be protective from development of Dactylitis (OR=0.313 and p=0.006) and associated with increase requirement to blood exchange
(OR=1.1 and p=0.044), to date no any published reports agreed or contrast this finding.

Also, GSTM null genotype reduce risk of ACS (OR=0.3 and p=0.002), in contrast to our finding, in Egypt [28], and in Brazil [29], they observed the GSTM1 null genotype was significantly associated with ACS.

We also observed the GSTM1null genotype increased risk to stroke (OR=3.33 and p=0.109), and this association not statistically significant, this agreed with study done in Brazil [29], they observed the patients with GSTM1 null showed a risk 3.9 times higher to develop stroke, Vasculopathy has been implicated in the development of pulmonary hypertension, stroke, leg ulcers and priapism, particularly associated with hemolytic severity.

In this study, there are no significant association between GSTM1 null genotype and the time of disease appearance, annual hospitalization and splenomegaly, to date, no study involving the polymorphism of GSTM1 gene and these clinical manifestations of SCD has been published, and no significant association between GSTM1 null genotype and the frequency of blood transfusion, and this agreed with another studies in Egypt [28,30] and no significant association with VOC, bone problems.

The GSTM1 gene contains four alleles and most widely studied, GSTM1 polymorphism M1*A 0.2 is associated with decreased risk of bladder and breast cancer in Caucasians, M1*B 0.2 with decreased risk of pituitary adenomas; M1*0 0.59 has been shown to increase the risk of lung, colon, bladder, and post-menopausal breast cancer. GSTM1*A has been associated with a decreased risk of bladder cancer and has an allele frequency of 20% [31].

Evolution from the basic identification of polymorphic sites has provided the tools to discover the genetic complexity that affects genotype-phenotype correlation [16].

For GSTT1 null genotype, this study found no significant association with the time of anemia appearance, Dactylities and splenomegaly.

In this study, the GSTT1null genotype associated with decreased requirements to blood transfusion (OR=0.137 and p< 0.001), in contrast to our finding, in Egypt [28], found the GSTT1 null

genotype was associated with significantly increased requirement of blood transfusion, and in the contrast for both, another studies in India [32], observed the requirement of blood transfusion is not dependent on GST deletions, and in Egypt [30] found no significant association between GST genotypes and transfusion frequency.

Also GSTT1 null genotype associated with reduce frequency of annual hospitalization (OR=0.436 and p=0.029), but we don't find any published reports studied this association to confirm or contrast our finding.

And GSTT1 null genotype associated with reduced risk to development of stroke (OR=0.125 and p=0.008) and this agreed with [29] found (OR=0.55 and p=0.45) but no associated significance.

Also, in present study, GSTT1 null genotype increased risk of ACS and bone problems and VOC (OR=1.47 and p=0.294), (OR=1.47 and p=0.294) and (OR=1.34 and p=0.424) respectively, and this association not statistically significant and this agreed with another studies [28,29,33].

The previous reports demonstrated that patients with SCD are subject to increased oxidative stress mainly in ACS [34].

Polymorphism of both GSTM1 and GSTT1 genes found to be associated with appearance of disease before one year of age (OR=1.43 and p=0.004) and trend to be protective from development of Dactylitis (OR=0.124 and p=0.002) but we don't find published reports confirm or contrast this finding.

Also, this study found no statistically significance between GSTM1/ GSTT1null genotype and gender variability, annual hospitalization, splenomegaly and blood exchange, no previous reports confirm or contrast this finding, and no statistically significance with VOC, frequency of blood transfusion, ACS, stroke and bone problems, and this agreed with previous studies [28,29,30,35], except [29], contrast our finding only in ACS.

In this study, there are no statistically significance between GSTP1 gene polymorphism and gender variability and clinical manifestations of SCD, and this agreed with another studies in Egypt [28,30], they found the non-wild-type
GSTP1 polymorphism was not associated with
clinical manifestations of SCD.

In a Brazilian cohort [36] they found no association between GSTM1 and GSTT1 gene polymorphism and oxidative stress parameters in SCD patients, also [37] Pooled analysis of GSTT1 and GSTP1 polymorphisms revealed significantly increased risk of complications in SCD, while GSTM1 null genotypes did not show association with SCD complications. Significant between study heterogeneity ($I^2$ > 50%) was observed for in all three polymorphisms (GSTM1=68.7%), (GSTT1=71.6%), (GSTP1=83%). These contradictory results may be explained by the fact that Silva and colleagues studied the relation between GST gene polymorphisms and biochemical markers of oxidative stress, not the clinical manifestations of SCD. Thus, biochemical markers of oxidative stress may not be an accurate marker for measuring SCD severity. In addition, different ethnicity could explain this contradiction [29].

The difference between the clinical manifestations of this study and previous studies make complexity in detecting the association between these polymorphisms and SCD severity and diversity, this study were the first in the studying of some clinical manifestations to date no study published before such as, the time of disease appearance, frequency of annual hospitalization, Dactylities, splenomegaly and blood exchange in SCD patients.

The unexpected clinical diversity in a monogenic disease such as SCD has led to countless genetic studies and current knowledge has evolved, together with technological development in molecular biology [34].

Moreover, since GST is a multigene family, one or two single polymorphisms and null genotype expression may not be sufficient to alter the overall enzymatic and antioxidant capacity [38].

Previous studies have shown that there is marked geographical and ethnic variation in the distribution of genes for polymorphic GSTs [39].

The protective effects found in this study in some clinical manifestations and differences in the associations or lack of them in the previous studies may be due to gene-gene interactive effects on GSTs and genotype-phenotype correlation, previous studies postulated, there are another mechanism for defense against

oxidative stress, erythrocyte have a self-sustaining activity of antioxidant defense enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR), in addition to low-molecular-weight antioxidants, such as glutathione(GSH) and vitamins E and C [40], RBC superoxide dismutase (SOD) activity has been shown to increase in some SCD studies [41,42]. The primary antioxidant enzymes against superoxide radicals include superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) [43]. [44] Observed higher CAT activity in the plasma of SS children compared to AA controls, and [45] found higher GPX activity in young adults with SCA compared to healthy individuals.

Nur et al. [46] demonstrated an increased GSSG efflux in sickle erythrocytes that can be a protective action, because GSSG is an oxidant itself and its enhanced excretion under oxidative conditions prevents the potentially toxic effects of its intracellular accumulation [47]. But increased GSSG efflux could play an important role in GSH depletion in these cells.

Also, G6PD is an important enzyme related to the antioxidant defense in erythrocytes [36]. Higher activity of this enzyme in patients with sickle cell disease was found than in the control group, previously reported that erythrocytes from patients with sickle cell disease have an increased percentage of reticulocytes, while the activity of G6PD in reticulocytes is normal, but declines exponentially as the red cells age [48].

Also these polymorphisms had protective effect in other diseases; some reports found that, the decreased GST activity served to protect the host erythrocytes against the invading malarial parasite by up-regulating oxidative defense mechanisms [49], homozygous deletion of GSTM1 may interfere with iron chelation therapy and lead to slow unloading of liver iron [50].

GST polymorphisms not only influence susceptibility to disease, but they also appear to influence responsiveness to cancer chemotherapeutic agents [51], protective role for cancer [52], protection from having ALL [53], Diabetic retinopathy in type 1 diabetes [54], Diabetic retinopathy in type 2 diabetes [55,56], Protection against colon cancer [57], protection against hearing impairment in testicular cancer patients [58], protective effect male infertility [59,60]. The GSTT1 null genotype had a
protective effect on the development of schizophrenia and the combination of null genotypes of the GSTT1 and GSTM1 genes was made at a lower risk of schizophrenia [61, 62], protection against coronary artery disease [63].

Due to little papers like this study are published and there are difference in clinical manifestations and population, limited information about this subject has been published, the absence of data obtained on phenotypic effects of GST family genes, the diversity of the clinical manifestations and severity among patients with similar GST polymorphisms may be due to the presence of other modifying genes effects on these genes, Future large studies evaluating GST genes in addition to other antioxidant genes are needed to provide evidence on gene-gene interactive effects on SCD, which makes further functional studies a necessity to determine the exact genotype-phenotype correlation, also any reported differences between studies might be attributed to sampling error.

Most of studies done before agreed with our study in most finding in the same clinical.

## 5. CONCLUSION

In conclusion, this study found, The GSTM1null genotype was found to be present in male more than female and trend to be protective from development of Dactylitis and may reduce risk to develop ACS, while this polymorphism may increase requirements to blood exchange.

The GSTT1null genotype found to be present in female more than male and this polymorphism may reduce requirements to blood transfusion and annual hospitalization and may reduce risk to development of stroke.

Polymorphism of both GSTM1 and GSTT1 found to be associated with appearance of disease before one year of age and trend to be protective from development of Dactylitis.

Also, there are no statistically significance between GSTP1 gene polymorphism and gender variability and clinical manifestations of SCD.

This study were the first in the studying of some clinical manifestations to date no study published before, as time of disease appearance, frequency of annual hospitalization, Dactylities, splenomegaly and blood exchange in SCD

patients and was the first done in Africa except Egypt.

The polymorphism of GSTM1, GSTT1 and GSTP1 genes effect on clinical manifestations diversity of SCD. Future large studies evaluating GST genes are needed in order to minimize severity of their symptoms by using prophylactic antioxidants and other measures that improve their reductive defense mechanisms.

## CONSENT

The study protocol was in accordance with the local hospital research guidelines and informed consent was obtained from patient's legal representatives.

## ETHICAL APPROVAL

Approval was received from ministry of health in Khartoum/Sudan and including the Hospital Ethics commission.

## ACKNOWLEDGEMENTS

The authors would like to thank all the people for their assist and directing in preparation this research, the most thankful for my supervisors Dr. OzazYagoup Mohammed Ahmed and professor/ Huda Mohammed Haroun and Dr. Muzamil Mahdi Abdel Hamid and the patients and controls enrolled in this study

## COMPETING INTERESTS

Authors have declared that no competing interests exist.

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