IL-6 Polymorphism as a Predisposing Genetic Factor for Gestational Diabetes or Preeclampsia Development in Pregnancy with Obesity in Relation to VEGF and VEGFF Receptor Gene Expression Modalities
Abstract
1. Introduction
2. Materials and Methods
2.1. Subjects
2.2. Measurements
2.3. Genomic DNA Extraction
2.4. Genetic Analysis Restriction Fragment Length Polymorphism (RFLP)-IL-6 Gene Analysis
- 1.
- The SfaNI polymorphism is due to a replacement of G by C at position 174, and primers were designed to amplify the promoter region of the IL-6 gene. The primers used in the PCR were as follows: forward -5′- TGACTTCAGCTTTACTCTTTGT -3′and reverse -5′- CTGATTGGAAACCTTATTAAG-3′. PCR was performed in 50 μL final volume solution using the Master Mix (Hot Start Promega, Madison, WI, USA). The amplification was conducted by a thermal cycler (96-Well Thermal Cycler, Applied Biosystems, Singapore), as follows: an initial denaturation of 10 min at 94 °C, and a final extension of 10 min at 72 °C. The cycle program consisted of a 1 min denaturation at 94 °C, a 1 min, 35 s annealing at 55 °C, and a 1 min extension at 72 °C. PCR products were digested with SfaNI restriction enzyme (New England BioLabs, Beverly, MA, USA) at 37 °C overnight and electrophoresed on a 2% agarose gel. SfaNI RFLP was detected by ethidium bromide staining. The identified genotypes were named according to the presence or absence of the enzyme restriction sites, so SfaNI (G/G), (G/C), and (C/C) are homozygotes for the presence of the site (140/58 bp), heterozygotes for the presence and absence of the site (198/140/58 bp), and homozygotes for the absence of the site (198 bp), respectively.
- 2.
- NlaIII at restriction enzyme (New England BioLabs, Beverly, MA, USA) at 37 °C for 15 min (1 μL NlaIII, 5 μL buffer 36 μLH2O, and 8 μL PCR product). The enzyme results in the cutting of the 198 bp amplicon into fragments with a size of 122, 45, and 31 bp, which indicates the presence of a wild-type homozygous CC genotype. In addition, two 167 bp and 31 bp fragments indicated the presence of a homozygous GG genotype, and four fragments of 167, 122, 45, and 31 bp indicated the presence of a heterozygous CG genotype.
2.5. RNA Isolation and cDNA Synthesis by Reverse Transcription PCR (RT-PCR)
2.6. Sequence Table
Primer | Primer Sequence (5’-3’) | Annealing Temperature | Product Size (bp) |
β-actin | F: CAAGATCATTGCTCCTCCTG | 60 °C | 90 bp |
β-actin | R: ATCCACATCTGCTGGAAGG | ||
VEGF | F: TGCAGATTATGCGGATCAAACC | 60 °C | 81 bp |
VEGF | R: TGCATTCACATTTGTTGTGCTGTAG | ||
VEGFR1 | F: CAGGCCCAGTTTCTGCCATT | 60 °C | 82 bp |
VEGFR1 | R: TTCCAGCTCAGCGTGGTCGTA |
2.7. Biomedical Ethics Issues
2.8. Statistical Analysis
3. Results
3.1. Association of the -174 G/C Polymorphism of the IL-6 Gene with Patients’ Characteristics
3.2. Association of VEGF and VEGF-R Gene Expression with Patients’ Characteristics
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
References
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Total Sample (N = 36) | Group | p | ||||
---|---|---|---|---|---|---|
Preeclampsia (N = 8; 22.2%) | Diabetes Type 2 (N = 7; 19.4%) | Gestational Diabetes (Diet or Insulin) (N = 11; 30.6%) | Normal (N = 10; 27.8%) | |||
Mean (SD) | Mean (SD) | Mean (SD) | Mean (SD) | Mean (SD) | ||
Age (years) | 32.4 (4.6) | 33.8 (4.2) | 30.9 (4.4) | 33.3 (3.6) | 31.5 (5.9) | 0.537 ‡ |
Weight before pregnancy (kg/m2) | 78.7 (17.5) | 81 (17.5) | 88.6 (18.1) | 78.2 (19.9) | 70.6 (11.5) | 0.209 ‡ |
Weight after pregnancy (kg/m2) | 89.4 (16.7) | 91 (16.9) | 98.7 (17.6) | 88.3 (19.6) | 82.7 (10.2) | 0.277 ‡ |
Weight difference | 10.8 (4.1) | 10 (2.9) | 10.1 (2.6) | 10.5 (4.8) | 12.2 (5.2) | 0.685 ‡ |
BMI before pregnancy (kg/m2) | 29.0 (5.9) | 29.0 (6.7) | 32.9 (6.9) | 28.5 (5.8) | 26.7 (3.9) | 0.204 ‡ |
BMI after pregnancy (kg/m2) | 32.9 (5.5) | 32.6 (6.2) | 36.7 (6.4) | 32.3 (5.5) | 31.3 (3.4) | 0.232 ‡ |
Gestational age (weeks) | 38.2 (0.7) | 37.6 (0.7) | 38.1 (0.7) | 38.4 (0.7) | 38.5 (0.6) | 0.020 ‡ |
n (%) | n | n | n | n | ||
Smoking | 18 (50.0) | 4 | 3 | 5 | 6 | 0.937 + |
Diabetes | ||||||
No | 13 (36.1) | 3 | 0 | 0 | 10 | <0.001 + |
Yes, during pregnancy | 15 (41.7) | 4 | 0 | 11 | 0 | |
Yes, type 2 | 8 (22.2) | 1 | 7 | 0 | 0 | |
Years from type 2 diabetes diagnosis | ||||||
<1 year | 2 (33.3) | 0 | 2 | 0 | 0 | 0.333 + |
1–5 years | 2 (33.3) | 0 | 2 | 0 | 0 | |
5–10 years | 1 (16.7) | 0 | 1 | 0 | 0 | |
>10 years | 1 (16.7) | 1 | 0 | 0 | 0 |
Polymorphism IL-6 -174 G/C | Total Sample (N = 36) | Group | p Fisher’s Exact Test | ||||
---|---|---|---|---|---|---|---|
Pre-ecl Ampsia (N = 8; 22.2%) | Diabetes Type 2 (N = 7; 19.4%) | Gestational Diabetes (Diet or Insulin) (N = 11; 30.6%) | Normal (N = 10; 27.8%) | ||||
N | % | N | N | N | N | ||
CC | 6 | 16.7 | 0 | 1 | 0 | 5 | 0.009 |
GC | 1 | 2.8 | 0 | 0 | 1 | 0 | |
GG | 29 | 80.6 | 8 | 6 | 10 | 5 |
Total Sample (N = 36) | Group | p Kruskal–Wallis Test | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Preecl Ampsia (N = 8; 22.2%) | Diabetes Type 2 (N = 7; 19.4%) | Gestational Diabetes (Diet or Insulin) (N = 11; 30.6%) | Normal (N = 10; 27.8%) | ||||||||
Mean (SD) | Median (IQR) | Mean (SD) | Median (IQR) | Mean (SD) | Median (IQR) | Mean (SD) | Median (IQR) | Mean (SD) | Median (IQR) | ||
VEGF | 10.2 (2.3) | 10.7 (8.7–11.4) | 7.1 (0.3) | 7.2 (7–7.3) | 9.5 (1.2) | 8.8 (8.7–11) | 11.2 (0.3) | 11 (10.9–11.5) | 12.2 (2.2) | 11.2 (10.5–15.1) | <0.001 |
VEGF -R | 12.0 (2.2) | 12.3 (11.3–13.6) | 14.4 (0.4) | 14.3 (14.1–14.9) | 12.6 (1.2) | 13.1 (11.3–13.4) | 11.6 (0.4) | 11.4 (11.3–12.1) | 10.1 (2.7) | 11.2 (6.5–12.3) | <0.001 |
Polymorphism IL-6 -174 G/C | p Mann-Whitney Test | ||||
---|---|---|---|---|---|
CC | GG | ||||
Mean (SD) | Median (IQR) | Mean (SD) | Median (IQR) | ||
VEGF | 13.2 (2.3) | 13.6 (11–15.3) | 9.6 (1.8) | 10.4 (7.6–11) | 0.005 |
VEGF-R | 8.7 (2.6) | 8.3 (6.4–10.5) | 12.7 (1.4) | 12.4 (11.4–14) | 0.002 |
Polymorphism | p Student’s t-Test | ||||
---|---|---|---|---|---|
CC | GG | ||||
Mean | SD | Mean | SD | ||
Age | 31.0 | 5.9 | 32.6 | 4.3 | 0.459 |
Weight before pregnancy | 61.3 | 6.3 | 81.9 | 17.1 | 0.007 |
Weight after pregnancy | 76.1 | 9.6 | 91.7 | 16.8 | 0.035 |
Weight difference | 14.8 | 4.3 | 9.9 | 3.7 | 0.008 |
Body mass index before pregnancy | 22.9 | 0.8 | 30.1 | 5.8 | 0.005 |
Body mass index after pregnancy | 28.4 | 2.2 | 33.7 | 5.6 | 0.029 |
Gestational age in weeks at delivery | 38.7 | 0.6 | 38.1 | 0.7 | 0.046 |
VEGF | VEGF-R | |||
---|---|---|---|---|
rho | p | rho | p | |
Age | −0.04 | 0.823 | 0.06 | 0.729 |
Weight before pregnancy | −0.36 | 0.029 | 0.41 | 0.012 |
Weight after pregnancy | −0.30 | 0.071 | 0.36 | 0.033 |
Weight difference | 0.32 | 0.056 | −0.35 | 0.039 |
Body mass index before pregnancy | −0.25 | 0.136 | 0.31 | 0.070 |
Body mass index after pregnancy | −0.27 | 0.106 | 0.32 | 0.057 |
Gestational age in weeks at delivery | 0.54 | 0.001 | −0.56 | <0.001 |
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Halvatsiotis, P.; Tsokaki, T.; Tsitsis, V.; Palaiodimou, L.; Tsivgoulis, G.; Tsangaris, I.; Panagiotou, M.O.; Houhoula, D. IL-6 Polymorphism as a Predisposing Genetic Factor for Gestational Diabetes or Preeclampsia Development in Pregnancy with Obesity in Relation to VEGF and VEGFF Receptor Gene Expression Modalities. Diagnostics 2024, 14, 1206. https://doi.org/10.3390/diagnostics14111206
Halvatsiotis P, Tsokaki T, Tsitsis V, Palaiodimou L, Tsivgoulis G, Tsangaris I, Panagiotou MO, Houhoula D. IL-6 Polymorphism as a Predisposing Genetic Factor for Gestational Diabetes or Preeclampsia Development in Pregnancy with Obesity in Relation to VEGF and VEGFF Receptor Gene Expression Modalities. Diagnostics. 2024; 14(11):1206. https://doi.org/10.3390/diagnostics14111206
Chicago/Turabian StyleHalvatsiotis, Panagiotis, Theodora Tsokaki, Vasileios Tsitsis, Lina Palaiodimou, Georgios Tsivgoulis, Iraklis Tsangaris, Maria Ourania Panagiotou, and Dimitra Houhoula. 2024. "IL-6 Polymorphism as a Predisposing Genetic Factor for Gestational Diabetes or Preeclampsia Development in Pregnancy with Obesity in Relation to VEGF and VEGFF Receptor Gene Expression Modalities" Diagnostics 14, no. 11: 1206. https://doi.org/10.3390/diagnostics14111206
APA StyleHalvatsiotis, P., Tsokaki, T., Tsitsis, V., Palaiodimou, L., Tsivgoulis, G., Tsangaris, I., Panagiotou, M. O., & Houhoula, D. (2024). IL-6 Polymorphism as a Predisposing Genetic Factor for Gestational Diabetes or Preeclampsia Development in Pregnancy with Obesity in Relation to VEGF and VEGFF Receptor Gene Expression Modalities. Diagnostics, 14(11), 1206. https://doi.org/10.3390/diagnostics14111206