Insulin gene promoter methylation status in Greek children and adolescents with Type&nbsp;1 Diabetes

Mouzaki,Konstantina; Kotanidou,Eleni P.; Fragou,Aikaterini; Kyrgios,Ioannis; Giza,Styliani; Kleisarchaki,Angeliki; Tsinopoulou,Vasiliki Rengina; Serbis,Anastasios; Tzimagiorgis,Georgios; Galli‑Τsinopoulou,Assimina

doi:10.3892/br.2020.1338

Insulin gene promoter methylation status in Greek children and adolescents with Type 1 Diabetes

Authors:
- Konstantina Mouzaki
- Eleni P. Kotanidou
- Aikaterini Fragou
- Ioannis Kyrgios
- Styliani Giza
- Angeliki Kleisarchaki
- Vasiliki Rengina Tsinopoulou
- Anastasios Serbis
- Georgios Tzimagiorgis
- Assimina Galli‑Τsinopoulou
View Affiliations

Affiliations: Second Department of Paediatrics, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, AHEPA General Hospital, 546 36 Thessaloniki, Greece, Laboratory of Biological Chemistry, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 54 124 Thessaloniki, Greece
Published online on: July 31, 2020 https://doi.org/10.3892/br.2020.1338
Article Number: 31

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The insulin (INS) gene is the one of the most important genes involved in the pathogenesis of Type 1 Diabetes (T1D) after the Major Histocompatibility Complex genes. Studies addressing the issue of hyper‑ or hypo‑methylation status of the INS gene promoter have reported inconsistent results. The majority of studies showed hypomethylation; however a few studies have shown hypermethylation at specific cytosine‑guanosine (CpG) sites in the promoter region of the INS gene. The aim of the present study was to analyze the methylation status of the promoter region of the INS gene in Greek children and adolescents with T1D. A total of 20 T1D participants (mean diabetes duration of 6.15±4.12 years) and 20 age‑ and sex‑matched controls were enrolled in the present study. DNA was isolated from whole blood samples, modified using sodium bisulfite and analyzed using PCR and electrophoresis. DNA was then pooled with highly reactive supermagnetic beads at similar molar quantities, submitted for library construction and finally sequenced using next‑generation sequencing. The methylation profile at 10 CpG sites around the transcription start site (TSS) of the INS promoter was analysed and expressed as the mean ± standard deviation. The overall mean methylation in patients with T1D did not differ compared with the healthy controls. There was a statistically significant difference between the two groups in hypermethylation at position ‑345 (P=0.02), while a trend (P=0.06) at position ‑102 was observed. According to the results of the present study, increased methylation in the INS gene promoter at specific CpG sites around the TSS were already present in childhood T1D. These data may possibly serve as a guide towards the identification of a methylation pattern for detection of development of T1D in genetically predisposed children.

View Figures

View References

1	Pugliese A: The insulin gene in type 1 diabetes. IUBMB Life. 57:463–468. 2005.PubMed/NCBI View Article : Google Scholar
2	Steck AK and Rewers MJ: Genetics of type 1 diabetes. Clin Chem. 57:176–185. 2011.PubMed/NCBI View Article : Google Scholar
3	Bansal A and Pinney SE: DNA methylation and its role in the pathogenesis of diabetes. Pediatr Diabetes. 18:167–177. 2017.PubMed/NCBI View Article : Google Scholar
4	Handy DE, Castro R and Loscalzo J: Epigenetic modifications: Basic mechanism and role in cardiovascular disease. Circulation. 17:2145–2156. 2011.PubMed/NCBI View Article : Google Scholar
5	Cano-Rodriguez D and Rots MG: Epigenetic editing: On the verge of reprogramming gene expression at will. Curr Genet Med Rep. 1:170–179. 2016.PubMed/NCBI View Article : Google Scholar
6	Dang MN, Buzzetti R and Pozzilli P: Epigenetics in autoimmune diseases with focus on type 1 diabetes. Diabetes Metab Res Rev. 29:8–18. 2013.PubMed/NCBI View Article : Google Scholar
7	Wang Z, Xie Z, Lu Q, Chang C and Zhou Z: Beyond genetics: What causes type 1 diabetes. Clin Rev Allergy Immunol. 52:273–286. 2017.PubMed/NCBI View Article : Google Scholar
8	Moore LD, Le T and Fan G: DNA methylation and its basic function. Neuropsychopharmacology. 38:23–38. 2013.PubMed/NCBI View Article : Google Scholar
9	Deaton AM and Bird A: CpG islands and the regulation of transcription. Genes Dev. 15:1010–1022. 2011.PubMed/NCBI View Article : Google Scholar
10	Riu J, Deng S, Lebastchi J, Clark PL, Usmani-Brawn S and Herold KC: Methylation of insulin DNA in response to proinflammatory cytokines during the progression of autoimmune diabetes in NOD mice. Diabetologia. 59:1021–1029. 2016.PubMed/NCBI View Article : Google Scholar
11	Zhang K, Lin G, Han Y, Xie J and Li J: Circulating unmethylated insulin DNA as a potential non-invasive biomarker of beta cell death in type 1 diabetes: A review and future prospect. Clin Epigenetics. 26(44)2017.PubMed/NCBI View Article : Google Scholar
12	Husseiny MI, Kaye A, Zebabua E, Kandeel F and Ferreri K: Tissue-specific methylation of human insulin gene and PCR assay for monitoring beta cell death. PLoS One. 9(e94591)2014.PubMed/NCBI View Article : Google Scholar
13	Neiman D, Moss J, Hecht M, Magenheim J, Piyanzin S, Shapiro AM, de Koning EJ, Razin A, Cedar H, Shemer R and Dor Y: Islet cells share promoter hypomethylation independently of expression, but exhibit cell-type-specific methylation in enhancers. Proc Natl Acad Sci USA. 19:13525–13230. 2017.PubMed/NCBI View Article : Google Scholar
14	Fradin D, Le Fur S, Mille C, Naoui N, Groves C, Zelenika D, McCarthy MI, Lathrop M and Bougnères P: Association of the CpG methylation pattern of the proximal insulin gene promoter with type 1 diabetes. PLoS One. 7(336278)2012.PubMed/NCBI View Article : Google Scholar
15	Fisher MM, Watkins RA, Blum J, Evans-Molina C, Chalasani N, DiMeglio LA, Mather KJ, Tersey SA and Mirmira RG: Elevations in circulating methylated and unmethylated preproinsulin DNA in new-onset type 1 diabetes. Diabetes. 64:3867–3872. 2015.PubMed/NCBI View Article : Google Scholar
16	Lehmann-Werman R, Neiman D, Zemmour H, Moss J, Magenheim J, Vaknin-Dembinsky A, Rubertsson S, Nellgård B, Blennow K, Zetterberg H, et al: Identification of tissue-specific cell death using methylation patterns of circulating DNA. Proc Natl Acad Sci USA. 113:E1826–E1834. 2016.PubMed/NCBI View Article : Google Scholar
17	Kuroda A, Rauch TA, Todorov I, Ku HT, Al-Abdullah I, Kandeel F, Mullen Y, Pfeifer GP and Ferreri K: Insulin gene expression is regulated by DNA methylation. PLoS One. 9(e6953)2009.PubMed/NCBI View Article : Google Scholar
18	Herold KC, Usmani-Brown S, Ghazi T, Lebastchi J, Beam CA, Bellin MD, Ledizet M, Sosenko JM, Krischer JP and Palmer JP: Type 1 Diabetes TrialNet Study Group. β cell death and dysfunction during type 1 diabetes development in at-risk individuals. J Clin Invest. 125:1163–1173. 2015.PubMed/NCBI View Article : Google Scholar
19	Mayer-Davis EJ, Kahkoska AR, Jefferies C, Dabelea D, Balde N, Gong CX, Aschner P and Craig ME: ISPAD Clinical practice consensus guidelines 2018: Definition, epidemiology, and classification of diabetes in children and adolescents. Pediatr Diabetes. 19:7–19. 2018.PubMed/NCBI View Article : Google Scholar
20	American Diabetes Association. Classification and diagnosis of diabetes: Standards of medical care in diabetes-2018. Diabetes Care. 41 (Suppl 1):S13–S27. 2018.PubMed/NCBI View Article : Google Scholar
21	Marshall WA and Tanner JM: Variations in the pattern of pubertal changes in boys. Arch Dis Child. 45:13–23. 1970.PubMed/NCBI View Article : Google Scholar
22	Marshall WA and Tanner JM: Variations in pattern of pubertal changes in girls. Arch Dis Child. 44:291–303. 1969.PubMed/NCBI View Article : Google Scholar
23	World Medical Association. World medical association declaration of Helsinki: Ethical principles for medical research involving human subjects. JAMA. 310:2191–2194. 2013.PubMed/NCBI View Article : Google Scholar
24	Li Y and Tollefsbol TO: DNA methylation detection: Bisulfite genomic sequencing analysis. Methods Mol Biol. 791:11–21. 2011.PubMed/NCBI View Article : Google Scholar
25	Scala G, Affinito O, Palumbo D, Florio E, Monticelli A, Miele G, Chiariotti L and Cocozza S: AmpliMethProfiler: A pipeline for the analysis of CpG methylation profiles of targeted deep bisulfite sequenced amplicons. BMC Bioinformatics. 25(484)2016.PubMed/NCBI View Article : Google Scholar
26	Mirmira RG, SimsEK Syed F and Evans-Morina C: Biomarkers of β-cell stress and death in type 1 diabetes. Curr Diab Rep. 16(95)2016.PubMed/NCBI View Article : Google Scholar
27	Husseiny MI, Kuroda A, Kaye AN, Nair I, Kandeel F and Ferreri K: Development of a quantitative methylation-specific polymerase chain reaction method for monitoring beta cell death in type 1 diabetes. PLoS One. 7(e47942)2012.PubMed/NCBI View Article : Google Scholar
28	Fisher MM, Chumbiauca CN, Mather KJ, Mirmira RG and Tersey SA: Detection of islet β-cell death in vivo by multiplex PCR analysis of differentially methylated DNA. Endocrinology. 154:3476–3481. 2013.PubMed/NCBI View Article : Google Scholar
29	Usmani-Brown S, Lebastchi J, Steck AK, Beam S, Herold KC and Ledizet M: Analysis of β-cell death in type 1 diabetes by droplet digital PCR. Endocrinol. 155:3694–3698. 2014.PubMed/NCBI View Article : Google Scholar
30	Akirav EM, Lebastchi J, Galvan EM, Henegariu O, Akirav M, Ablamunits V, Lizardi PM and Herold KC: Detection of β cell death in diabetes using differentially methylated circulating DNA. Proc Natl Acad Sci USA. 108:19018–19023. 2011.PubMed/NCBI View Article : Google Scholar
31	Grada A and Weinbrecht K: Next generation sequencing: Methodology and application. J Invest Dermatol. 133(e11)2013.PubMed/NCBI View Article : Google Scholar
32	Behjati S and Tarpey PS: What is next generation sequencing? Arch Dis Child Educ Pract Ed. 98:236–238. 2013.PubMed/NCBI View Article : Google Scholar
33	Liu L, Li Y, Li S, Hu N, He Y, Pong R, Lin D, Lu L and Law M: Comparison of next generation sequencing systems. J Biomed Biotechnol. 2012(251364)2012.PubMed/NCBI View Article : Google Scholar
34	Buermans HPJ and den Dunnen JT: Next generation sequencing technology: Advantages and applications. Biochim Biophys Acta. 1842:1932–1941. 2014.PubMed/NCBI View Article : Google Scholar