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