Research Article - (2023) Volume 11, Issue 1
Received: 31-Jul-2022, Manuscript No. IPACLR-22-12895; Editor assigned: 03-Aug-2022, Pre QC No. IPACLR-22-12895 (PQ); Reviewed: 18-Aug-2022, QC No. IPACLR-22-12895; Revised: 23-Dec-2022, Manuscript No. IPACLR-22-12895 (R); Published: 02-Jan-2023, DOI: 10.36648/2386-5126.96.36.1990
Background: Gestational diabetes mellitus is the most common metabolic disorder that develops during pregnancy. GDM can cause major health problems for the mother both during pregnancy and after the baby is born. In addition, the metabolism of the mother's kids is abnormal. The study aimed to investigate superoxide dismutase 1 genetic variants in pregnant women diagnosed with gestational diabetes using amplification refractory mutation system analysis.
Results: Our study demonstrated that the frequencies of genotypes in women with GDM were SOD1-AG (90.0%). The study revealed there is a significant difference in the frequency of the genotype of SOD1 between GDM and healthy pregnant women.
Conclusions: To the best of our knowledge, this is the first study to indicate that the genotype of SOD1 (A/G rs2070424) has a significant impact on the development of gestational diabetes mellitus in Egyptian women.
Gestational diabetes; Superoxide dismutase 1 gene; Genetic polymorphism; Oxidative stress
Gestational diabetes; Superoxide dismutase 1 gene; Genetic polymorphism; Oxidative stress
GDM: Gestational Diabetes; SOD1: Superoxide Dismutase 1; ROS: Reactive Oxygen Species; SNPs: Single-Nucleotide Polymorphisms; T-ARMS-PCR: Tetra- Primer Amplification Refractory Mutation System
Gestational Diabetes Mellitus (GDM) is defined as any degree of glucose intolerance that develops or is detected during pregnancy. In the second or third trimester, pregnant women are diagnosed . This condition is associated with adverse pregnancy outcomes, including fetal macrosomia, stillbirth, neonatal metabolic disturbances, and related problems. GDM is not caused by a lack of insulin but by other hormones produced during pregnancy that can make insulin less effective, a condition referred to as insulin resistance .
Oxidative stress is a general term used to describe the steady state of oxidative damage in a cell, tissue, or organ caused by Reactive Oxygen Species (ROS). The majority of ROS is produced endogenously as a byproduct of normal and necessary reactions, such as energy generation from mitochondria. This oxidative stress was found to be greater in women with GDM than in normal pregnant women . A complex and integrated antioxidant system plays a crucial role in protecting cells or tissues from damage as a result of ROS. The expression and activity of antioxidants are changed during oxidative stress .
Human Cu–Zn Superoxide Dismutase (SOD1) gene is located on chromosome 21 (21q22.1), a homo-dimeric metalloprotein consisting of 153 amino acids with the two subunits linked noncovalently . The SOD1 polymorphism (rs2070424) is distinguished by the substitution of Adenine (A) to Guanine (G) at codon 251 in intron 3 of the gene. This polymorphism has been linked to a reduction in antioxidant capacity . Actually, the SOD1 gene encodes an enzyme that catalyzes the dismutation of superoxide into O2 and H2O2 to eliminate free superoxide radicals produced in the body. The delicate balance between the elimination of ROS and limiting oxidative stress is disrupted by genotype related differences in enzyme activity.
The study aimed to evaluate the Single Nucleotide Polymorphisms (SNPs) in SOD1 (A/G rs2070424) and correlate its genotyping to gestational diabetes mellitus. To the best of our knowledge, this is the irst study to report the association of their genotypes with gestational diabetes in Egyptian women.
This case-control study was conducted with 70 women with gestational diabetes and 70 healthy pregnant women. They were recruited from the obstetrics and gynecology department, faculty of medicine, Mansoura university in the period from July 2017 to March 2019. All of the women in the study were at least 18 years old and had been diagnosed at a gestational age of more than 24 weeks.
Pregnant women with type 1 diabetes, previous macrosomia (a baby weighing more than 4000 g at birth), polycystic ovary syndrome, and other serious medical conditions (hypertension, renal disease, moderate to severe anaemia, thyroid disorder) that interfered with maternal and perinatal outcomes were excluded.
Sample collection and DNA extraction
Each patient's fasting blood was drawn and dispensed into an EDTA containing tube. A blood sample was divided into two portions (3 mL and 2 mL). After centrifugation at >2000 g for 10 minutes, the first portion was used to collect plasma. The plasma was extracted and used for biochemical analysis as well as a native-PAGE investigation of SOD activity. The second portion was used to extract DNA. The DNA quantity and quality were measured by reading the absorbance at λ 230 nm and λ 260 nm.
Amplification refractory mutation system (ARMS)- PCR analysis Tri-ARMS-PCR analysis of superoxide dismutase 1 gene (SOD1)
Primers used in this study (F1, F2 and R) are provided in Table 1. In the SOD1 gene (A/G rs2070424).
|Primer||Sequence (5′‐3′)||Fragment size|
|F1||TAGCTTTGTTAGCTATGCCA||A allele: 480 (F1+R)|
|F2||TAGCTTTGT TAGCTATGCCG||G allele: 480 (F2+R)|
|F1=Forward primer 1, F2=Forward primer 2, R=Reverse primer|
Table 1: Primers used in Tri‐ARMS‐PCR of SOD1 gene (A/G rs2070424).
200 ng of genomic DNA, 3 μL of an allele primers (F1 and RP) or G allele primers (F2 and RP) (Table 1) and 8 μL 2 × Taq Master Mix (EmeraldAmp GT PCR Master Mix, Cat. No.RR310A) were used in an 18 μL polymerase chain reaction (PCR). The PCR protocol was as follows: 95°C for 3 min, then 35 cycles at 95°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 30 sec, and a final extension at 72°C for 10 min. The agarose gel electrophoresis (2%) was performed at 125 V and a 100 bp MaestroGen DNA ladder (Cat. No. 02001_500) was used to investigate and confirm the quality of the PCR products (Cleaver Scientific Ltd., UK).
Antioxidant levels were estimated by determining catalase, GR activity and plasma levels of MDA using kit supplied by biodiagnostic (Cairo, Egypt). Plasma levels of catalase, superoxide dismutase and glutathione reductase activity can be determined according to the methods of Fossati, and Ponti, et al. and goldberg and spooner respectively [7-10]. Satoh and Tsikas techniques can be used to detect plasma malondialdehyde and nitric oxide levels respectively .
SOD activity gel method
The SOD activity gel test was developed by a method that is based on the suppression of NBT reduction by SOD. This test is based on the ability of O2•−to interact with NBT and convert the tetrazolium (yellow colour) in the gel to a blue aggregate. SOD active areas generate a clear region (achromatic bands) that competes with NBT for O2•− [12-14].
Statistical analysis was done using the software package, SPSS version 22 and excel. data were expressed as mean ± SD. The significance of the difference between women with gestational diabetes and healthy pregnant women was determined using ANOVA. Take into account that P–values of less than 0.05 were statistically significant. Frequencies of either genotype or allele of SOD1 polymorphism between two groups were analyzed by the Fisher exact test and hardy Weinberg equilibrium.
Plasma catalase, superoxide dismutase and glutathione reductase levels were significantly decreased in women with gestational diabetes when compared with the control group. On the other hand, the levels of malondialdehyde and nitric oxide were significantly higher than normal pregnant women (Table 2).
|Parameters||GDM group (n =70)||Control group (n =70)||OR (95% CI)||P value|
|CAT (U/L) mean ± SD||337.39 ± 133.06||875.54 ± 100.6||0.65 (undefined)||p<0.001*|
|SOD (U/mL) mean ± SD||1.43 ± 0.38||2.32 ± 0.14||undefined||p<0.001*|
|GR (U/L) median (IQR)||6.43 (4.02-10.45)||30.54 (22.51-40.21)||0.54 (0.44-0.65)||p<0.001*|
|MDA (nmol/mL) mean ± SD||13.35 ± 4.30||5.36 ± 2.83||2.15(1.77-2.59)||p<0.001*|
|NO (mg/L) median (IQR)||4.81 (3.53-6.79)||2.04 (1.77-2.78)||undefined||P<0.001*|
Table 2: Comparison of antioxidant enzymes and oxidative stress markers for gestational diabetes mellitus in pregnant women with healthy pregnant women.
Genetic polymorphism and genotype frequencies
Analysis of SOD1 gene: The genetic polymorphism in the SOD1 gene (A251G) was investigated, and the genotypes were shown in Figure 1. The frequencies of the allele and genotype of the SOD1 gene (rs2070424) between women with gestational diabetes and healthy pregnant women were listed in Tables 3 and 4. Genotype frequencies of the rs2070424 A/G polymorphism differed significantly between women with gestational diabetes and control (p=0.025), while allele frequencies did not show any signi icant difference between women with gestational diabetes and control. Moreover, the distribution of genotypes of SOD1 for GDM and control groups was in alignment with the Hardy- Weinberg equilibrium (χ2=44.84, p<0.001; χ2=70, p<0.001), which was analyzed by Fisher’s exact test (Table 5).
|Allele||GDM group||Control group||P value|
|A||71 (50.7%)||70 (50%)||0.905|
|G||69 (49.3%)||70 (50%)|
|The data was expressed as frequency and percentage. GDM: gestational diabetes mellitus.|
Table 3: Allele frequencies of SOD1 gene (rs2070424) in the current study.
|Genotype||GDM group (n=70)||Control group (n=70)||P value|
|A/G||63 (90.0%)||70 (100%)||0.025*|
|The data was expressed as frequency and percentage. GDM: gestational diabetes mellitus.|
Table 4: Genotype frequencies of SOD1 gene (rs2070424) in the current study.
|GDM group (n=70)||Control group (n=70)|
|GDM: Gestational Diabetes Mellitus; χ2: chi-Square Test; HWE: Hardy-Weignberg Equilibrium; *statistically significant if p<0.05.|
Table 5: Fisher exact test for Hardy-Weinberg equilibrium in the current study.
Figure 1: Agarose gel electrophoresis (2%) of PCR product of tri-primer ARMS-PCR for analyzing the rs2070424 polymorphism in the SOD1 gene. Lane M showed DNA marker; lane GD1 showed AA genotype; lane C1, C2, C3, GD3, GD4, GD4, GD5 and GD6 showed AG genotype; lane GD2 showed GG genotype (Where C: control; GD: gestational diabetes).
The major risk of GDM was evaluated by the co-dominant, dominant, recessive, and over dominant models as shown in Table 6. In the co-dominant model (A/A vs. A/G vs. G/G), there was a statistically significant difference between GDM and healthy pregnant women (p=0.025). The dominant model (A/A vs. A/G+G/G) showed a significant risk of GDM when compared with the A/G+G/G genotype (p=0.04). The recessive model (A/A +A/G vs. G/G) did not display any significant risk of GDM compared to the A/A+A/G genotype (p=0.08). Furthermore, the over dominant (A/A+G/G vs. A/G) showed a significant risk of GDM compared to the A/G genotype (p=0.006).
|Model||Genotype||GDM group (n=70)||Control group (n=70)||OR (95% CI)||P value|
|A/G||70 (100%)||63 (90.0%)|
|A/G+G/G||70 (100%)||66 (94.3%)|
|Recessive||A/A+A/G||70 (100%)||67 (95.7%)||undefined||0.08|
|Over dominant||A/A+G/G||0||7 (10%)||undefined||0.006*|
|A/G||70 (100%)||63 (90%)|
|The data was presented as percentage and frequency. GDM: Gestational Diabetes Mellitus; OR: Odds Ratio; 95% CI: 95% Confidence Interval for the difference in means between the two groups; P is significant when <0.005.|
Table 6: Association between genotypes of SOD1 and response status in the current study.
Correlation between antioxidant enzymes, oxidative stress markers and SOD1 (rs2070424) SNP
The association between antioxidant enzymes, oxidative stress markers and the SOD1 gene (rs2070424) SNP in the population under study was presented in Table 7. The study population (GDM and control groups) was divided into three groups according to genotypes in AA, AG, and GG. There wasn’t any signficant difference in the biochemical parameters between the three groups, except that the level of MDA, and nitric oxide showed signi icant differences between AG and GG (p=0.039, p=0.014) respectively.
|Parameters||AA||AG||GG||Test of significance||Significance within groups|
|Catalase mean ± SD||409.63 ± 151.27||331.11 ± 134.65||372.91 ± 28.94||F=0.761 P=0.471||P1=0.598 P2=0.720 P3=0.258|
|Superoxide dismutase mean ± SD||1.41 ± 0.61||1.44 ± 0.38||1.23 ± 0.06||F=0.391 P=0.678||P1=0.383 P2=0.567 P3=0.879|
|Glutathione reductase Median (IQR)||10.05 (2.61-17.48)||6.43 (4.02-10.45)||4.02 (4.01-13.66)||KW P=0.754||P1=0.951 P2=0.721 P3=0.449|
|Malondialdehyde Median (IQR)||11.26 (7.96-17.69)||12.08 (10.49-17.14)||9.66 (7.91-9.71)||KW P=0.109||P1=0.039* P2=0.289 P3=0.578|
|Nitric oxide Median (IQR)||3.79 (3.41-4.40)||5.17 (3.75-7.28)||3.29 (2.82-3.52)||KW P=0.015*||P1=0.014* P2=0.108 P3=0.109|
|F: one way ANOVA test; KW: Kruskal Wallis test; IQR: Interquartile Range; P1: Difference between AG and GG; P2: difference between AA and GG; P3: difference between AA and AG; *statistically significant if p<0.05.|
Table 7: Correlation between antioxidant enzymes, oxidative stress markers and SOD1 (rs2070424) SNP in the current study.
SOD native gel
The native PAGE gel assay facilitated the identification of SOD isoenzymes in normal pregnant women and women with gestational diabetes. Figure 2 showed superoxide dismutase isoenzymes (Mn-SOD and CuZn-SOD) activities in native gel. The stain intensity of the bands in the control lane was higher, whereas the stain intensity of the bands in the gestational diabetes group lanes was lower. Therefore, the activity of SOD isoenzymes was decreased in women with gestational diabetes compared to normal pregnant women.
Figure 2: Superoxide dismutase (SOD) isoenzymes activities in native gel. Lane C1 showed control; lane GD1, GD2, GD3, GD4, GD5, GD6, GD7 and GD8 show gestational diabetes.
Gestational diabetes mellitus is the most common illness among pregnant women all over the world. Insulin resistance distinguishes it as a complicated metabolic condition . The study aimed to find whether there was a link between superoxide dismutase 1 A251G SNP (rs2070424) with the incidence of gestational diabetes in Egyptian women.
The antioxidant defense system includes the enzymes CAT, SOD, and GR. They are in charge of regulating the level of free radicals in cells . In this study, MDA and nitric oxide levels increased, whereas CAT, SOD, and GR activity decreased in women with GDM compared to normal pregnant women.
It was discovered that there was a substantial rise in MDA levels in other studies . MDA levels rise and anti-oxidant enzyme levels fall as pregnancy progresses . The GDM group had higher MDA concentrations throughout the pregnancy, with significant differences in the first and second trimesters . When comparing women with GDM to controls, a significant increase in lipid peroxidation was found .
Previous studies have reported conflicting results related to NO levels in pregnancy. It was found that NO levels dropped during pregnancy, whereas NO levels remained constant throughout the pregnancy in other studies [21-23].
It was discovered that SOD activity was found to be significantly lower in pregnant females with gestational diabetes as compared to normal pregnant females . Furthermore, superoxide dismutase activity is lower in pregnant women than in non-pregnant women in the third trimester of normal pregnancy [25,26]. The reduction in SOD levels was demonstrated by another study .
Another research revealed that CAT activity did not alter throughout pregnancy, despite the fact that CAT activity is greater during pregnancy [28,29]. Reduced CAT activity might be attributed to lower lipid peroxidation rates in the third trimester of pregnancy. In agreement with another study, blood catalase activity in gestational diabetes is decreased . The decrease in CAT as an inducible enzyme may be due to the lower level of H2O2 generated by SOD.
In addition, it was discovered that the GDM group's plasma GSH concentration was lower than that of healthy pregnant women . This is in line with the results of other research. A gradual decrease in the activity of glutathione reductase throughout the three trimesters of pregnancy was observed .
Superoxide dismutase1 is an intracellular enzyme that primarily protects cells against cytosolic generated superoxide. The expression of cytoplasmic SOD1 is stable and its activity is often considered as an internal control for SOD gene expression. SOD1 A251G polymorphisms cause changes in SOD1 enzyme levels or activities, which can lead to decreased protection against oxidative stress . This study aimed to demonstrate that the genotype frequencies of the SOD1 gene (A/G rs2070424) differed significantly between women with gestational diabetes and normal pregnant women.
In the Egyptian population, the CC genotype of SOD1 (rs2234694) SNP was more prone to T2DM . It was revealed that 64.10% of the population has the wild type (AA) SOD1 gene in the south-south pregnant Nigerian population. 10.26% of the women had the homozygous genotype and 25.64% of the population had the heterozygous genotype. In addition, wildtype SOD1 genes were most prevalent in the population of women studied . Data regarding the SOD1 gene polymorphism is available for some populations, including North Indians, Bangladeshis, the Finns, the Romanians, the New Zealanders, and the Czechs, but it is lacking among African populations. There is little information about the frequency or implications of SOD1 .
SOD1 allelic variations (rs17880135) have been linked to the occurrence of diabetic nephropathy . Another study found that significant differences between the allele and genotype frequencies for the SOD1+35A/C polymorphism were observed in type 1 diabetes mellitus as compared to controls . In Mexican women, the number of mutant carriers (GA+GG) of SOD (rs2070424) was substantially greater in the obese population than in the normal weight group .
However, contradictory results indicated that SOD1 (rs2234694) SNP was not related to cardiovascular disease in T2DM patients from North Finland. Similarly, it was found that SOD1+35A/C gene polymorphism may not be associated with the susceptibility to T2DM among the South Indian population . Another study has also reported that the (CC) genotype and C allele were completely absent among the North Indian population and that there was no association between diabetes and SOD1+35A/C gene polymorphism .
The current study found the SOD1 gene was associated with blood glucose, MDA, and nitric oxide. The absence of SOD1 is associated with the development of impaired glucose tolerance that results from impaired cell dysfunction and reduced cell volume. ROS reacts with polyunsaturated fatty acids to induce the release of toxic and reactive aldehyde metabolites such as MDA, one of the end products of lipid peroxidation. The mutant SOD1 may have the capacity to catalyze the production of ROS such as peroxynitrite and also contribute to increased cellular expression of iNOS synthase.
It was concluded that SOD1-AG (90.0%) genotype were shown to be the most common in women with GDM. To the best of our knowledge, this is the first study to show that there is a significant difference in the frequency of the genotype of SOD1 (A/G rs2070424) in the development of gestational diabetes mellitus in Egyptian women.
This study was approved by ethical standards of the institutional research board, faculty of medicine and Mansoura university. The patient provided written informed consent.
The authors declare that they have no competing interests.
MMM contributed to the methodology, analysis, investigation and writing original manuscript. FE contributed to the methodology, acquisition, analysis and investigation. AE contributed to the methodology, analysis and investigation. AAE contributed to interpretation of data, supervision, reviewing and editing of the manuscript. AFA contributed to designing the work, supervision, reviewing and editing of the manuscript. All authors have read and approved the manuscript for publication.
We express gratitude to all of the obstetrics and gynecology department, faculty of medicine, Mansoura University for making it possible to collect maternal blood samples.
Citation: Madkour S, Abd El-Aziz AAF, El-Senduny FF, Elsaid AM, El-Refaey AAA (2023) Gene Polymorphism of Superoxide Dismutase in Gestational Diabetes Mellitus in Pregnant Women. Ann Clin Lab Res Vol:11 No:01