Methylation of RUNX3 Promoter 2 in the Whole Blood of Children with Ulcerative Colitis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Patient Recruitment
2.2. Group Characteristics
2.3. Genomic DNA Isolation
2.4. Primer Design
2.5. MSRE-qPCR Conditions
2.6. Assessment of DNA Methylation
2.7. Statistical Analysis
2.8. Ethical Considerations
3. Results
3.1. Demographic Factors
3.2. Nutritional Status
3.3. Inflammatory Markers
3.4. Disease Characteristics
3.5. Applied Therapeutics
4. Discussion
4.1. RUNX3 and Inflammation
4.2. UC in Children and Observations Regarding RUNX3 in This Study
4.3. Limitations
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Taman, H.; Fenton, C.G.; Hensel, I.V.; Anderssen, E.; Florholmen, J.; Paulssen, R.H. Genome-Wide DNA Methylation in Treatment-Naïve Ulcerative Colitis. J. Crohns Colitis 2018, 12, 1338–1347. [Google Scholar] [CrossRef] [PubMed]
- McDermott, E.; Ryan, E.J.; Tosetto, M.; Gibson, D.; Burrage, J.; Keegan, D.; Byrne, K.; Crowe, E.; Sexton, G.; Malone, K.; et al. DNA Methylation Profiling in Inflammatory Bowel Disease Provides New Insights into Disease Pathogenesis. J. Crohns Colitis 2016, 10, 77–86. [Google Scholar] [CrossRef] [PubMed]
- Busch, M.A.; Gröndahl, B.; Knoll, R.L.; Pretsch, L.; Doganci, A.; Hoffmann, I.; Kullmer, U.; Bähner, V.; Zepp, F.; Meyer, C.U.; et al. Patterns of Mucosal Inflammation in Pediatric Inflammatory Bowel Disease: Striking Overexpression of IL-17A in Children with Ulcerative Colitis. Pediatr. Res. 2020, 87, 839–846. [Google Scholar] [CrossRef] [PubMed]
- Tatiya-Aphiradee, N.; Chatuphonprasert, W.; Jarukamjorn, K. Immune Response and Inflammatory Pathway of Ulcerative Colitis. J. Basic Clin. Physiol. Pharmacol. 2018, 30, 1–10. [Google Scholar] [CrossRef]
- Reis, B.S.; Rogoz, A.; Costa-Pinto, F.A.; Taniuchi, I.; Mucida, D. Mutual Expression of the Transcription Factors Runx3 and ThPOK Regulates Intestinal CD4+ T Cell Immunity. Nat. Immunol. 2013, 14, 271–280. [Google Scholar] [CrossRef]
- Maul, J.; Loddenkemper, C.; Mundt, P.; Berg, E.; Giese, T.; Stallmach, A.; Zeitz, M.; Duchmann, R. Peripheral and Intestinal Regulatory CD4+ CD25(High) T Cells in Inflammatory Bowel Disease. Gastroenterology 2005, 128, 1868–1878. [Google Scholar] [CrossRef]
- Djuretic, I.M.; Levanon, D.; Negreanu, V.; Groner, Y.; Rao, A.; Ansel, K.M. Transcription Factors T-Bet and Runx3 Cooperate to Activate Ifng and Silence Il4 in T Helper Type 1 Cells. Nat. Immunol. 2007, 8, 145–153. [Google Scholar] [CrossRef]
- Weersma, R.K.; Zhou, L.; Nolte, I.M.; van der Steege, G.; van Dullemen, H.M.; Oosterom, E.; Bok, L.; Peppelenbosch, M.P.; Faber, K.N.; Kleibeuker, J.H.; et al. Runt-Related Transcription Factor 3 Is Associated with Ulcerative Colitis and Shows Epistasis with Solute Carrier Family 22, Members 4 and 5. Inflamm. Bowel Dis. 2008, 14, 1615–1622. [Google Scholar] [CrossRef]
- Vogiatzi, P.; De Falco, G.; Claudio, P.P.; Giordano, A. How Does the Human RUNX3 Gene Induce Apoptosis in Gastric Cancer? Latest Data, Reflections and Reactions. Cancer Biol. Ther. 2006, 5, 371–374. [Google Scholar] [CrossRef]
- Kurklu, B.; Whitehead, R.; Ong, E.; Minamoto, T.; Fox, J.; Mann, J.; Judd, L.; Giraud, A.; Menheniott, T. Lineage-Specific RUNX3 Hypomethylation Marks the Preneoplastic Immune Component of Gastric Cancer. Oncogene 2015, 34, 2856–2866. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Levanon, D.; Brenner, O.; Otto, F.; Groner, Y. Runx3 Knockouts and Stomach Cancer. EMBO Rep. 2003, 4, 560–564. [Google Scholar] [CrossRef] [PubMed]
- Brenner, O.; Levanon, D.; Negreanu, V.; Golubkov, O.; Fainaru, O.; Woolf, E.; Groner, Y. Loss of Runx3 Function in Leukocytes Is Associated with Spontaneously Developed Colitis and Gastric Mucosal Hyperplasia. Proc. Natl. Acad. Sci. USA 2004, 101, 16016–16021. [Google Scholar] [CrossRef] [PubMed]
- Adams, A.T.; Kennedy, N.A.; Hansen, R.; Ventham, N.T.; O’Leary, K.R.; Drummond, H.E.; Noble, C.L.; El-Omar, E.; Russell, R.K.; Wilson, D.C.; et al. Two-Stage Genome-Wide Methylation Profiling in Childhood-Onset Crohn’s Disease Implicates Epigenetic Alterations at the VMP1/MIR21 and HLA Loci. Inflamm. Bowel Dis. 2014, 20, 1784–1793. [Google Scholar] [CrossRef] [PubMed]
- Kalla, R.; Adams, A.; Nowak, J.; Bergemalm, D.; Vatn, S.; Ventham, N. Analysis of Systemic Epigenetic Alterations in Inflammatory Bowel Disease: Defining Geographical, Genetic, and Immune-Inflammatory Influences on the Circulating Methylome. Available online: https://www.researchsquare.com (accessed on 13 June 2022).
- Krausgruber, T.; Schiering, C.; Adelmann, K.; Harrison, O.J.; Chomka, A.; Pearson, C.; Ahern, P.P.; Shale, M.; Oukka, M.; Powrie, F. T-Bet Is a Key Modulator of IL-23-Driven Pathogenic CD4+ T Cell Responses in the Intestine. Nat. Commun. 2016, 7, 11627. [Google Scholar] [CrossRef] [PubMed]
- Lin, Z.; Hegarty, J.P.; Cappel, J.A.; Yu, W.; Chen, X.; Faber, P.; Wang, Y.; Kelly, A.A.; Poritz, L.S.; Peterson, B.Z.; et al. Identification of Disease-Associated DNA Methylation in Intestinal Tissues from Patients with Inflammatory Bowel Disease. Clin. Genet. 2011, 80, 59–67. [Google Scholar] [CrossRef]
- Garrity-Park, M.M.; Loftus, E.V.; Sandborn, W.J.; Bryant, S.C.; Smyrk, T.C. Methylation Status of Genes in Non-Neoplastic Mucosa from Patients with Ulcerative Colitis-Associated Colorectal Cancer. Am. J. Gastroenterol. 2010, 105, 1610–1619. [Google Scholar] [CrossRef]
- Dybska, E.; Adams, A.T.; Duclaux-Loras, R.; Walkowiak, J.; Nowak, J.K. Waiting in the Wings: RUNX3 Reveals Hidden Depths of Immune Regulation with Potential Implications for Inflammatory Bowel Disease. Scand. J. Immunol. 2021, 93, e13025. [Google Scholar] [CrossRef]
- Kim, T.Y.; Lee, H.J.; Hwang, K.S.; Lee, M.; Kim, J.W.; Bang, Y.-J.; Kang, G.H. Methylation of RUNX3 in Various Types of Human Cancers and Premalignant Stages of Gastric Carcinoma. Lab. Investig. 2004, 84, 479–484. [Google Scholar] [CrossRef]
- Rini, D.; Calabi, F. Identification and Comparative Analysis of a Second Runx3 Promoter. Gene 2001, 273, 13–22. [Google Scholar] [CrossRef]
- Garrity-Park, M.; Loftus, E.V.; Sandborn, W.J.; Smyrk, T.C. Myeloperoxidase Immunohistochemistry as a Measure of Disease Activity in Ulcerative Colitis: Association with Ulcerative Colitis-Colorectal Cancer, Tumor Necrosis Factor Polymorphism and RUNX3 Methylation. Inflamm. Bowel Dis. 2012, 18, 275–283. [Google Scholar] [CrossRef]
- Slattery, M.L.; Lundgreen, A.; Herrick, J.S.; Caan, B.J.; Potter, J.D.; Wolff, R.K. Associations between Genetic Variation in RUNX1, RUNX2, RUNX3, MAPK1 and EIF4E and Risk of Colon and Rectal Cancer: Additional Support for a TGF-β-Signaling Pathway. Carcinogenesis 2011, 32, 318–326. [Google Scholar] [CrossRef] [PubMed]
- Tserel, L.; Kolde, R.; Limbach, M.; Tretyakov, K.; Kasela, S.; Kisand, K.; Saare, M.; Vilo, J.; Metspalu, A.; Milani, L.; et al. Age-Related Profiling of DNA Methylation in CD8+ T Cells Reveals Changes in Immune Response and Transcriptional Regulator Genes. Sci. Rep. 2015, 5, 13107. [Google Scholar] [CrossRef] [PubMed]
- Marttila, S.; Jylhävä, J.; Nevalainen, T.; Nykter, M.; Jylhä, M.; Hervonen, A.; Tserel, L.; Peterson, P.; Hurme, M. Transcriptional Analysis Reveals Gender-Specific Changes in the Aging of the Human Immune System. PLoS ONE 2013, 8, e66229. [Google Scholar] [CrossRef]
- Gasparetto, M.; Payne, F.; Nayak, K.; Kraiczy, J.; Glemas, C.; Philip-McKenzie, Y.; Ross, A.; Edgar, R.D.; Zerbino, D.R.; Salvestrini, C.; et al. Transcription and DNA Methylation Patterns of Blood-Derived CD8+ T Cells Are Associated with Age and Inflammatory Bowel Disease but Do Not Predict Prognosis. Gastroenterology 2021, 160, 232–244.e7. [Google Scholar] [CrossRef]
- Long, M.D.; Crandall, W.V.; Leibowitz, I.H.; Duffy, L.; del Rosario, F.; Kim, S.C.; Integlia, M.J.; Berman, J.; Grunow, J.; Colletti, R.B.; et al. Prevalence and Epidemiology of Overweight and Obesity in Children with Inflammatory Bowel Disease. Inflamm. Bowel Dis. 2011, 17, 2162–2168. [Google Scholar] [CrossRef]
- Pituch-Zdanowska, A.; Banaszkiewicz, A.; Dziekiewicz, M.; Łazowska-Przeorek, I.; Gawrońska, A.; Kowalska-Duplaga, K.; Iwańczak, B.; Klincewicz, B.; Grzybowska-Chlebowczyk, U.; Walkowiak, J.; et al. Overweight and Obesity in Children with Newly Diagnosed Inflammatory Bowel Disease. Adv. Med. Sci. 2016, 61, 28–31. [Google Scholar] [CrossRef]
- Vehmeijer, F.O.L.; Küpers, L.K.; Sharp, G.C.; Salas, L.A.; Lent, S.; Jima, D.D.; Tindula, G.; Reese, S.; Qi, C.; Gruzieva, O.; et al. DNA Methylation and Body Mass Index from Birth to Adolescence: Meta-Analyses of Epigenome-Wide Association Studies. Genome Med. 2020, 12, 105. [Google Scholar] [CrossRef]
- Reed, Z.E.; Suderman, M.J.; Relton, C.L.; Davis, O.S.P.; Hemani, G. The Association of DNA Methylation with Body Mass Index: Distinguishing between Predictors and Biomarkers. Clin. Epigenet. 2020, 12, 50. [Google Scholar] [CrossRef]
- Ishida, N.; Higuchi, T.; Miyazu, T.; Tamura, S.; Tani, S.; Yamade, M.; Iwaizumi, M.; Hamaya, Y.; Osawa, S.; Furuta, T.; et al. C-Reactive Protein Is Superior to Fecal Biomarkers for Evaluating Colon-Wide Active Inflammation in Ulcerative Colitis. Sci. Rep. 2021, 11, 12431. [Google Scholar] [CrossRef]
- Sayar, S.; Kurbuz, K.; Kahraman, R.; Caliskan, Z.; Atalay, R.; Ozturk, O.; Doganay, H.L.; Ozdil, K. A Practical Marker to Determining Acute Severe Ulcerative Colitis: CRP/Albumin Ratio. North. Clin. Istanb. 2019, 7, 49–55. [Google Scholar] [CrossRef]
- Ventham, N.T.; Kennedy, N.A.; Adams, A.T.; Kalla, R.; Heath, S.; O’Leary, K.R.; Drummond, H.; Wilson, D.C.; Gut, I.G.; Nimmo, E.R.; et al. Integrative Epigenome-Wide Analysis Demonstrates That DNA Methylation May Mediate Genetic Risk in Inflammatory Bowel Disease. Nat. Commun. 2016, 7, 13507. [Google Scholar] [CrossRef] [PubMed]
- Yagi, S.; Furukawa, S.; Shiraishi, K.; Hashimoto, Y.; Tange, K.; Mori, K.; Ninomiya, T.; Suzuki, S.; Shibata, N.; Murakami, H.; et al. Effect of Disease Duration on the Association between Serum Albumin and Mucosal Healing in Patients with Ulcerative Colitis. BMJ Open Gastroenterol. 2021, 8, e000662. [Google Scholar] [CrossRef] [PubMed]
- Khan, N.; Patel, D.; Shah, Y.; Trivedi, C.; Yang, Y.-X. Albumin as a Prognostic Marker for Ulcerative Colitis. World J. Gastroenterol. 2017, 23, 8008–8016. [Google Scholar] [CrossRef] [PubMed]
- Vecellio, M.; Roberts, A.R.; Cohen, C.J.; Cortes, A.; Knight, J.C.; Bowness, P.; Wordsworth, B.P. The Genetic Association of RUNX3 with Ankylosing Spondylitis Can Be Explained by Allele-Specific Effects on IRF4 Recruitment That Alter Gene Expression. Ann. Rheum. Dis. 2016, 75, 1534–1540. [Google Scholar] [CrossRef]
- Kalkan, I.H.; Dağli, U.; Oztaş, E.; Tunç, B.; Ulker, A. Comparison of Demographic and Clinical Characteristics of Patients with Early vs. Adult vs. Late Onset Ulcerative Colitis. Eur. J. Intern. Med. 2013, 24, 273–277. [Google Scholar] [CrossRef]
- Nambu, R.; Hagiwara, S.-I.; Kubota, M.; Kagimoto, S. Difference between Early Onset and Late-Onset Pediatric Ulcerative Colitis. Pediatr. Int. Off. J. Jpn. Pediatr. Soc. 2016, 58, 862–866. [Google Scholar] [CrossRef]
- Howell, K.J.; Kraiczy, J.; Nayak, K.M.; Gasparetto, M.; Ross, A.; Lee, C.; Mak, T.N.; Koo, B.-K.; Kumar, N.; Lawley, T.; et al. DNA Methylation and Transcription Patterns in Intestinal Epithelial Cells from Pediatric Patients with Inflammatory Bowel Diseases Differentiate Disease Subtypes and Associate with Outcome. Gastroenterology 2018, 154, 585–598. [Google Scholar] [CrossRef] [Green Version]
Characteristic | Sample | |
---|---|---|
Total no. of patients [n] | 64 | |
Average age [yr] | 14.5 ± 2.8 | |
Average BMI [kg/m2] | 18.0 ± 3.2 | |
Average BMI Z-score | −0.44 ± 1.14 | |
Group | Female | Male |
No. of patients [n (%)] | 33 (51.6%) | 31 (48.4%) |
Average age [yr] | 14.6 ± 2.6 | 14.4 ± 3.1 |
Average weight [kg] | 43.7 ± 18.1 | 44.0 ± 17.8 |
BMI [kg/m2] | 18.0 ± 3.4 | 18.0 ± 2.9 |
Average duration of the disease [yr] | 1.7 ± 2.3 | 2.6 ± 3.3 |
<2.5 mo. since UC diagnosis [n (%)] | 15 (45.5%) | 16 (51.6%) |
≥12 mo. since UC diagnosis [n (%)] | 14 (42.4%) | 13 (41.9%) |
Baseline PUCAI [point] | 39.8 ± 17.3 | 38.7 ± 16.4 |
<10 [n (%)] | 0 (0.0%) | 0 (0.0%) |
10–34 [n (%)] | 12 (36.4%) | 10 (32.3%) |
35–64 [n (%)] | 16 (48.5%) | 14 (45.2%) |
≥65 [n (%)] | 4 (12.1%) | 3 (9.7%) |
Baseline Paris Classification | E3 | E4 |
E1 [n (%)] | 6 (18.2%) | 2 (6.5%) |
E2 [n (%)] | 6 (18.2%) | 6 (19.4%) |
E3 [n (%)] | 6 (18.2%) | 4 (12.9%) |
E4 [n (%)] | 14 (42.4%) | 17 (54.8%) |
S0 [n (%)] | 23 (69.7%) | 27 (87.1%) |
S1 [n (%)] | 9 (27.3%) | 3 (9.7%) |
Baseline CRP [mg/dL] | 6.8 ± 10.7 | 10.9 ± 19.2 |
Baseline albumin [g/dL] | 4.2 ± 0.6 | 4.0 ± 0.7 |
Concomitant pharmacotherapy [n (%)] | 21 (63.6%) | 20 (64.5%) |
Systemic corticosteroids [n (%)] | 19 (90.5%) | 20 (100.0%) |
Immunosuppressants [n (%)] | 15 (71.4%) | 12 (60.0%) |
Biologics [n (%)] | 8 (38.1%) | 4 (20.0%) |
Gene | Primer Sequence 5′ → 3′ | Ta [°C] | MSRE Sites [n]/Amplicon Size [bp] | Reference | |
---|---|---|---|---|---|
MGMT | F | GGTGTGAAAACTTTGAAGGA | 54 | 4/186 | Zymo Research design |
R | CACTATTCAAATTCCAACCC | ||||
RUNX3 | F | CTGAACCTTTTAAGAGAGCC | 54 | 6/264 | Designed using NCBI Primer-BLAST |
R | CAAATGGAATTTACCACCAC |
Medication | Received | RUNX3 P2 Methylation Level [%] | p-Value | ||
---|---|---|---|---|---|
N | Min–Max | Mean ± SD | |||
Corticosteroids | no | 24 | 31.3–72.7 | 55.7 ± 10.7 | 0.4105 |
yes | 39 | 22.0–84.4 | 53.2 ± 15.0 | ||
Immunosuppressants | no | 36 | 26.5–84.3 | 54.6 ± 12.1 | 0.6640 |
yes | 27 | 22.0–84.4 | 53.5 ± 15.2 | ||
Biologics | no | 51 | 26.5–84.4 | 55.9 ± 12.5 | 0.0569 |
yes | 12 | 22.0–74.5 | 46.7 ± 15.3 |
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Dybska, E.; Nowak, J.K.; Banaszkiewicz, A.; Szaflarska-Popławska, A.; Kierkuś, J.; Kwiecień, J.; Grzybowska-Chlebowczyk, U.; Walkowiak, J. Methylation of RUNX3 Promoter 2 in the Whole Blood of Children with Ulcerative Colitis. Genes 2022, 13, 1568. https://doi.org/10.3390/genes13091568
Dybska E, Nowak JK, Banaszkiewicz A, Szaflarska-Popławska A, Kierkuś J, Kwiecień J, Grzybowska-Chlebowczyk U, Walkowiak J. Methylation of RUNX3 Promoter 2 in the Whole Blood of Children with Ulcerative Colitis. Genes. 2022; 13(9):1568. https://doi.org/10.3390/genes13091568
Chicago/Turabian StyleDybska, Emilia, Jan Krzysztof Nowak, Aleksandra Banaszkiewicz, Anna Szaflarska-Popławska, Jarosław Kierkuś, Jarosław Kwiecień, Urszula Grzybowska-Chlebowczyk, and Jarosław Walkowiak. 2022. "Methylation of RUNX3 Promoter 2 in the Whole Blood of Children with Ulcerative Colitis" Genes 13, no. 9: 1568. https://doi.org/10.3390/genes13091568
APA StyleDybska, E., Nowak, J. K., Banaszkiewicz, A., Szaflarska-Popławska, A., Kierkuś, J., Kwiecień, J., Grzybowska-Chlebowczyk, U., & Walkowiak, J. (2022). Methylation of RUNX3 Promoter 2 in the Whole Blood of Children with Ulcerative Colitis. Genes, 13(9), 1568. https://doi.org/10.3390/genes13091568