Introduction: HLA-G gene is involved in the control of immune response. It plays a primary role on immune tolerance and may participate in controlling autoimmune responses serving as a potential independent susceptibility marker. HLA-G has been isolated in some secretory granules and on the cell surface of primary islet cells induced to secrete insulin. Subsequently, it could be hypothesized that HLA-G methylation at pancreatic islet could sustain T cell activation and onset of diabetes. To the best of our knowledge the methylation of HLA-G gene promoter has not been studied yet in T1D. We aimed to investigate the methylation patterns of HLA-G CpG loci in T1D paediatric population.
Patients and methods: Twenty T1D participants and 20 age-/gender-matched healthy youngsters were enrolled. DNA was extracted from white blood cells, then treated with sodium bisulphate which converts unmethylated cytosines into uracyls, whereas methylated cytosines remain unchanged under the same conditions. DNA was then amplified by PCR using primers:(F)primer:5TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTAGGGAGTTTAGTTTAGGGAT3 and (R)primer:5GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCATAACCACCATCCTTAA3. Amplicons were analyzed by electrophoresis (1% agarose gel stained with ethidium bromide), visualized by ultraviolet trans-illumination, and then Next Generation Sequencing was applied to identify differences in DNA methylation status. The methylation profile was analyzed at 19 CpG sites of the HLA-G gene (1-5446,2-5484,3-5550,4-5568,5-5579, 6-5599, 7-5629, 8-5652, 9-5654, 10-5690, 11-5715, 12-5719, 13-5772, 14-5775, 15-5778, 16-5780, 17-5801, 18-5813,19-5832). Comparisons between groups were performed with students t-test or its non-parametric analogue, Mann Whitney U test, as appropriate.
Results: The results are described in table. HLA-G gene did not exhibit significant differences regarding the methylation status within its promoter sites compared to healthy individuals.
|19 CpGs in HLA-G gene||T1D (n=20)||Controls (n=20)||P|
|Results are expressed as Mean±S.D.|
Conclusions: Despite the described close association of HLA-G with autoimmunity, we failed to find any methylation differences in HLA-G promoter sites between T1D patients and controls.
27 - 29 Sep 2018
European Society for Paediatric Endocrinology