Vol. 5, No. 4, April 2024
E-ISSN: 2723-6692
P-ISSN: 2723-6595
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Jurnal Indonesia Sosial Sains, Vol. 5, No. 4, April 2024 949
Exploring the Relationship Between Sod1, 2 And 3 Gene
Polymorphisms With Post-Covid19 Symptoms
Jihan Samira Thabit, Sisca, Monica Dwi Hartanti, Noviani Prasetyaningsih,
Alvionita Kogoya, Arleen Devita, Isa Bella, Ida Effendi
Universitas Trisakti, Jakarta, Indonesia
Email: [email protected], sisc[email protected].id, [email protected]c.id,
[email protected].id, alvionita0300[email protected]sakti.ac.id,
arleen.devita@trisakti.ac.id, isabella@trisakti.ac.id, idaeffendi@trisakti.ac.id
Correspondence: airajn08@ymail.com
*
KEYWORDS
ABSTRACT
Covid19; Long Covid19; SOD;
Genomic vairation
The coronavirus disease 19 (COVID-19) has become a challenge for
the media world. Even though they have been declared cured, some
Covid-19 survivors still have health complaints. Abnormal
symptoms, signs, or clinical parameters that persist two weeks or
more after the onset of COVID-19 and do not return to their initial
healthy state are potentially considered long-term effects of the
disease. Although such changes are primarily reported in people
with severe and critical illness, lasting effects also occur in
individuals with mild infections that do not require hospitalization.
This study aims to explore the role of antioxidants on the
pathogenesis of Covid19 and its relationship with SOD1, 2 and 3
genomic variations. Getting alternative biomarkers for long covid19
detection. Research Method: The research sample is blood and
questionnaires that will be taken from respondents affected by
Covid19 a maximum of 6 months before data collection is held.
Furthermore, DNA isolation, DNA amplification, cutting with
restriction enzymes, and DNA band documentation with gel
electrophoresis will be carried out. Symptoms related to systemic
are the most common symptoms found in respondents with Long
Covid19. A total of 8 respondents had systemic related symptoms,
namely weakness, lethargy and sweating, while skin-related
complaints were found in three respondents and one respondent
had lung-related complaints. The average SOD activity value of
respondents was 2.19 U/ml. In this study, more symptoms of long
COVID-19 were found associated with systemic and cardiovascular
symptoms. The complaints were not differentiated by the sex of the
respondents. The output draft will be processed immediately after
getting other results.
Attribution-ShareAlike 4.0 International (CC BY-SA 4.0)
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1. Introduction
Coronavirus disease 19 (COVID-19) has become a challenge for the media world. Although
related to the respiratory system, the pathogenesis of this disease is still not fully understood. COVID-
19 was designated a pandemic by the World Health Organization (WHO) in March 2020 because it
has spread worldwide and is considered a disease with a high transmission ability from person to
person. Symptoms of Covid19 include early symptoms of low-grade fever and dry cough, followed by
high fever, fatigue and difficulty breathing with dyspnea, and in more severe cases, hypoxia with
oxygen saturation of less than 85%, multi-organ damage and even death (Xu et al., 2020). Even though
they have been declared cured, some COVID-19 survivors still have health complaints. Abnormal
symptoms, signs, or clinical parameters that persist two weeks or more after the onset of COVID-19
and do not return to their initial healthy condition are potentially considered long-term effects of the
disease (Tenforde et al., 2020). Although such changes are primarily reported in people with severe
and critical illnesses, lasting effects also occur in individuals with mild infections that do not require
hospitalization (Townsend et al., 2021).
Once the virus enters the respiratory tract, the viral replication process begins, and the natural
immune response begins to activate macrophages and dendritic cells via toll-like receptors and NODs
against the production of inflammatory cytokines and reactive oxygen species (ROS) (Uehara et al.,
2015). The spread of the virus in the blood will cause damage to erythrocytes by ROS and other
inflammatory mechanisms (Li et al., 2021), leading to the formation of free hemp and Fe. In addition,
activated macrophages and neutrophils will produce respiratory bursts that will form superoxide
radicals and H2O2, which lead to oxidative stress. Oxidative stress, along with free Fe, will convert
soluble plasma fibrinogen into abnormal fibrin clumps in the form of dense matted deposits (DMD)
that are resistant to degradation by enzymes. This will cause microthrombosis in the vascular system
and pulmonary microcirculation. Cytokine storms arise through the upregulation of cytokine
expression via NF-κB. Furthermore, cytokine storms will induce oxidative stress through
macrophages and neutrophil respiratory bursts and vice versa. This cycle will provoke serious tissue
damage that is not related to viruses. In addition, mitochondria produce ROS, which increases iNOS
expression through NF-κB so that NO is formed which will induce mitochondrial dysfunction and
cause cytopathic hypoxia. The virus will also inhibit Nrf2, which is responsible for increased
antioxidant enzymes, causing oxidative stress. In conclusion, low haemoglobin levels and high lung
exudate protein will cause pulmonary hypoxia, cytopathic hypoxia and endothelial damage and
disseminated coagulation, which will cause multi-organ collapse (Cecchini & Cecchini, 2020).
Reactive Oxygen Species (ROS) can be characterized by several markers, including the enzyme
Superoxide Dismutase (SOD). SOD functions to accelerate the reaction of the superoxide anion (O2)
with itself to form hydrogen peroxide (H2O2) and oxygen (2O2 + 2H → H2O2 + O2) (Wang et al., 2018).
This reaction is important for maintaining redox equilibrium (Sies, 2015). However, excessive ROS
production can exceed the capacity of the antioxidant defence system, causing oxidative stress and
causing cellular damage by oxidized proteins, lipids, DNA/RNA and other macromolecules. There are
three types of SOD; two of them are often found in mitochondria, namely SOD1 and SOD2. SOD 1 can
be found in the intermembrane space, while SOD2 can be found in the mitochondrial matrix (Kim et
al., 2017). SOD3 is often found extracellularly (Kim et al., 2017).
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A relationship between the variation of the third SOD genotype and the risk of occurrence of
several diseases has been found, for example, the CC rs4880 genotype is associated with increased
hepatotoxicity after asparaginase therapy, the CC genotype rs2234694 increases the risk of T2DM,
and the rs1041740 and rs17880487 variants in the SOD1 gene are associated with cardiovascular
mortality and rs7655372 the SOD3 gene is associated with ischemic stroke (Alachkar et al., 2017; Ghattas
& Abo-Elmatty, 2012; Otaki et al., 2016; Yang et al., 2021). However, the relationship between the third
genotypic variant of SOD and post-Covid19 symptoms is still unknown.
The aim of this study was to explore the role of antioxidants in the pathogenesis of COVID-19.
The other objective is to Analyze the SOD1, SOD2 and SOD3 mRNA expression levels of COVID-19
survivors. Analyzing SOD1, SOD2 and SOD3 levels in the plasma of Covid19 survivors, Analyzing the
genotype variation of SOD1, SOD2 and SOD3 of Covid19 survivors, Analyzing the demographic picture
and clinical symptoms of Covid19 survivors, Analyzing the relationship between expression levels
and plasma SOD1, SOD2 and SOD3 levels with the duration of recovery and clinical symptoms of long
covid19, Analyzing the relationship between SOD1, SOD2 and SOD3 genotype variations with the
length of recovery and clinical symptoms of long covid19.
2. Materials and Methods
Research Design
The study will be designed as a case-control study that will test two groups of COVID-19
survivors with and without long-term COVID-19 symptoms.
Research Subjects
This study's research population is composed of COVID-19 survivors in Jakarta. Research
subjects who meet the inclusion and exclusion criteria will be asked to join the study and sign an
informed consent form.
Research Sample
Research samples are divided into 2 types, namely blood and tears. Blood is taken from a 5cc
cubital vein and inserted into an EDTA vacutainer. Cheek mucosal smears will be performed using
sterile cotton swabs that are daubed into the cheeks of research subjects and stored in buffers to
prevent DNA and RNA damage. All blood samples and cheek mucosal smears were transferred to the
laboratory in a styrofoam box filled with ice and stored in a refrigerator at - 80C for further analysis.
Inclusion and exclusion criteria. The inclusion criteria are:
• Willing to sign informed consent.
• Willing to follow research procedures.
The exclusion criteria are:
• Individuals confirmed with COVID-19.
Sample Size
The five groups' gene expression differences were determined using G*Power 3.1.9.4 software.
Based on the calculation of G*Power, which takes into account the effect size of 0.5, the total number
of samples needed is 128 (Figure 1), with a total sample of 64 for each group.
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Figure 1 Calculation of total sample size on G*Power
Research materials and methods
The materials needed in this study are:
1) RNA isolation kit
2) Kit synthesis cDNA
3) The DNA is the.
4) Enzyme restrictions
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5) Conventional PCR mix
6) SYBR Green PCR Master Mix
7) Primer
8) Agarose Powder
9) Buffer TAE 1x
10) DNA Fluorosafe
Research Methods
1) RNA Isolation Research Methods
RNA will be extracted from blood and cervical smear samples in tubes containing RNA later. RNA
extraction and DNase cleaning using RNAqueous-Micro® Kit (Cat# AM1931, Thermo Fisher
Scientific, Waltham, MA, USA) in accordance with the manufacturer's protocol. The RNA
concentration was determined using a nanodrop spectrophotometer based on a wavelength of
260. All samples with an absorption ratio of 2.0 to 260:280 will be used for further analysis.
2) Synthesis Cdna
Complement DNA (cDNA) will be synthesized from 200 ng of DNase-treated RNA. This method
uses 250 ng/ul random hexamers (Sigma, Adelaide, SA, Australia) and 200 U Superscript Reverse
Transcriptase III (Thermo Fisher Scientific, Waltham, MA, USA). To ensure the absence of
genomic contamination, negative controls will be analyzed by adding water that DEPC has added
in place of Superscript Reverse Transcriptase III.
3) Quantitative real-time PCR
For Quantitative real-time PCR (qPCR) analysis, primers will be designed based on publicly
published RNA sequences using Primer3 plus (Rozen and Skaletsky, 2000) and primary Net
software (PREMIERE Biosof). Quantitative real-time PCR analysis will be performed using the
Rotor-Gene 6000 series 1.7 thermal cyclers (Qiagen GmbH) by duplication at 95C for 15 seconds,
then 60C for 60 seconds for 40 cycles. CDNA amplification will be prepared in a 10 ml reaction
containing 2 ml 1:20 cDNA, 5 ml Power SYBR Green PCR Master Mix (Applied Biosystems), 0.2
ml forward and reserve primers for target genes and 2.6 ml water mixed with DEPC. The Ct value
is determined using Rotor-Gene 6000 software (Q series; Qiagen GmbH) at a threshold of 0.05
normalized with fluorescence units. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is
used as a "housekeeping" gene due to its stable expression in human tissues.
4) DNA Isolation
According to the manufacturer's protocol, DNA will be isolated from venous blood and cheek
mucosal smears using Quick-DNA™ Miniprep Plus Kit (Zymo Research, Irvine, CA, USA).
The concentration of DNA was determined using nanophotometers based on 260 wavelengths.
All samples with a ratio of 1.8 to absorbance of 260:280 will be used for further analysis.
5) Target gene amplification
The target gene will be amplified using conventional PCR machines. According to the
manufacturer's protocol, DNA will be mixed with MyTagTM HS Red Mix (Bioline-Meridian
Bioscience Memphis, Tennessee, USA).
6) Incubation with restriction enzymes
The amplification results are incubated at a certain time and temperature with restriction
enzymes (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's
protocol.
7) Gel Elektroforesis
Electrophoresis using 2% agarose gel to see the size of the incubation restriction fragment band
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Analysis Methods
Data In this study, the data obtained on quantitative PCR will be entered into the following
formula:
2 − 𝑎𝑣𝑒∆Ct
Where:
Average ∆Ct = mean of the results of reducing the cycle threshold of the GAPDH gene and the target
gene studied.
The formula compares the expression of the target gene with the expression of the GAPDH
(housekeeping gene) gene, which is constantly expressed in each type of body cell. The final result
will be numerical data describing the target expression. The higher number describes the higher gene
expression, while the lower number also describes the lower gene expression.
All statistical analysis will be performed using Microsoft Office Excel 2010 and GraphPad Prism
Version 6.00 (GraphPad Software Inc.). Results will be presented in mean ± SEM if the data
distribution is normal, and results will be presented in median if the data distribution is abnormal.
All bivariate data will be analyzed using the t-test in the normal distribution; for normal distribution,
data will be analyzed in non-Mann-Whitney statistical tests. The result will be considered significant
at p <0.05 with a 95% confidence interval.
Table 1 Research Achievement Indicators
Aspects
CHECKLIST
Featured Scale
🗸
Research Topics/Themes
🗸
Funding Scheme
RESEARCH IMPLEMENTER
🗸
Source Of Funds
3. Result and Discussion
This study analyzes the demographic characteristics of patients and their relationship with
symptoms of long-term COVID-19 that occur a maximum of 6 months after being declared recovered
from COVID-19. Symptoms will be described based on the organ systems of the human body. There
have been 55 respondents with the same portion for gender. Most respondents had never had
Covid19 before but 10 respondents had had Covid19 before twice. Generally, respondents are self-
isolating at home, and most respondents have no comorbidities for COVID-19 disease and no previous
history of smoking.
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Table 2 Characteristics of research respondents (n = 55)
No.
Information
Frequency (n)
Percentage (%)
1.
Gender
Man
27
49.09
Woman
28
50.91
2.
History of Covid19
Never
24
43.64
Once
21
38.18
Twice
10
18.18
3.
Hospital history
Self-isolation
54
98.18
Treated
1
1.82
4.
Comorbid
None
49
89.09
Diabetes Mellitus
2
3.64
Hypertensive
3
5.45
Heart Disease
0
0
Lung Disease
1
1.82
Other
1
1.82
5.
Habit history
Smoke
16
29.09
Drinking alcohol
0
0
Drugs
0
0
Other
39
70.91
6.
Symptoms of Long COVID-19
Yes
28
51
No
27
49
Of the 55 respondents, there were 27 respondents without long-term covid symptoms and 28
respondents with long-term covid symptoms
Based on the data, it is stated that most patients who experience symptoms of long-term COVID-
19 are women, while male patients have a tendency not to experience symptoms of long-term COVID-
19. Based on the analysis, it was found that around 60% of patients suffering from ling covid were
women. This is in line with a publication from ISOS related to Long Covid which states that people
who have weak immunity to infection, women, and people with severe disease are more likely to
experience Long COVID.
As for the history of COVID-19, patients who experience symptoms of long-term COVID-19 are
mostly patients who have previously experienced positive COVID-19 once. All patients who
experience symptoms are patients undergoing self-isolation. Some patients do not have cormobids.
As many as 3 patients suffered from comorbidity, namely diabetes militias, as many as 1 people and
2 people with hypertension. Most patients do not have the habit of smoking, drinking alcohol or using
drugs (Proskurnina et al., 2020; Uehara et al., 2015). Only 5 patients had a smoking habit. As for
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patients who smoke, both regular cigarettes and vapes. Their smoking habits are intense sometimes,
and they spend as much as 6 cigarettes to 1 pack of cigarettes in one day.
The virus that causes Covid19 is SARS-CoV-2 which has mutated several times. To date, there
have been thousands of variants WHO has recorded in three categories; the dangerous one is the
Variant of Concern (VOC). At the beginning of the COVID-19 pandemic, the alpha and beta variants
had spread throughout the world, but it was indeed the delta variant that had a high morbidity and
mortality rate compared to other variants.
The virus must be able to infect host cells and evade the immune system in order to survive.
Therefore, the virus will continue to mutate. Similar to SARS-CoV2, this virus also continues to mutate,
resulting in many variants, including the omicron variant currently detected in many countries
worldwide.
Symptoms related to systemic are the most common symptoms found in respondents with Long
Covid-19. A total of 8 respondents had systemic-related symptoms, namely weakness, lethargy and
sweating, while skin-related complaints were found in three respondents, and one respondent had
lung-related complaints.
Table 3 Long COVID-19 Complaints Related to Systemic Symptoms
No.
Information
Frequency (n)
1
Systemic Symptoms
Weak and lethargic
6
2
Perspire
Lungs
2
3
Shortness of breath Skin
Hair loss
1
1
Dry scaly skin
1
4
Red stain
Cardiovascular
1
Low blood pressure
2
Heart palpitations
1
Chest pain
1
Low blood pressure is the most common cardiovascular-related symptom, followed by heart
palpitations and chest pain.
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From the results of SOD activity, it was found that the average SOD activity was 2.19 U/ml, with
the highest SOD activity value being 3,164 U/ml while the lowest was 1,062 U/ml.
Table 4 The Value of SOD Activity in Respondents
No. Respond
SOD activity (U/ml)
1
2.570
2
2.088
3
2.317
4
2.852
5
3.164
6
2.852
7
2.570
8
2.341
9
1.786
10
2.570
11
2.088
12
2.317
13
1.882
14
2.088
15
2.317
16
2.317
17
2.707
18
2.852
19
2.440
20
2.852
21
1.529
22
2.199
23
1.451
24
2.199
25
2.088
26
2.570
27
1.982
28
1.882
29
1.882
30
2.852
31
2.088
32
1.529
33
2.088
34
1.882
35
1.378
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36
2.570
37
1.786
38
1.882
39
1.062
40
1.696
Lopez-Leon et al. (2022) showed that there are five most common manifestations: fatigue,
headache, attention disorder, hair loss, and shortness of breath. Other symptoms are related to
breathing, cardiovascular, and neurological organs. Some studies report that complaints are more
common in women, especially fatigue and panting (Townsend et al., 2021; Xiong et al., 2021).
4. Conclusion
In this study, more symptoms of long COVID-19 were found associated with systemic and
cardiovascular symptoms. The complaints were not differentiated by the sex of the respondent.
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