Assessment of pancreas cell type composition is crucial to the understanding of the genesis of diabetes. Current approaches use immunodetection of protein markers, for example insulin as a marker of beta-cells. A major limitation of these methods is that protein content varies in physiological and pathological conditions, complicating the extrapolation to actual cell number. Here we demonstrate the use of cell type-specific DNA methylation markers for determining the fraction of specific cell types in human islet and pancreas specimens. We identified genomic loci that are uniquely demethylated in specific pancreatic cell types and applied targeted PCR to assess the methylation status of these loci in tissue samples, enabling inference of cell type composition. In islet preparations, normalization of insulin secretion to beta-cell DNA revealed similar beta-cell function in pre-T1D, T1D and T2D , which was significantly lower than in non-diabetic donors. In histological pancreas specimens from recent-onset T1D this assay showed beta-cell fraction within the normal range, suggesting a significant contribution of beta-cell dysfunction. In T2D pancreata we observed increased alpha-cell fraction and normal beta-cell fraction. Methylation-based analysis provides an accurate molecular alternative to immune detection of cell types in the human pancreas, with utility in the interpretation of insulin secretion assays and the assessment of pancreas cell composition in health and disease.

中文翻译：

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基于 DNA 甲基化的人类胰腺和胰岛细胞组成评估

胰腺细胞类型组成的评估对于了解糖尿病的起源至关重要。目前的方法使用蛋白质标记物的免疫检测，例如胰岛素作为β细胞的标记物。这些方法的一个主要限制是蛋白质含量在生理和病理条件下变化，使外推到实际细胞数量变得复杂。在这里，我们演示了使用细胞类型特异性 DNA 甲基化标记来确定人类胰岛和胰腺样本中特定细胞类型的比例。我们鉴定了特定胰腺细胞类型中独特去甲基化的基因组位点，并应用靶向 PCR 来评估组织样本中这些位点的甲基化状态，从而能够推断细胞类型组成。在胰岛制剂中，β细胞DNA的胰岛素分泌正常化揭示了T1D前期、T1D和T2D中相似的β细胞功能，其显着低于非糖尿病供体。在新近发病的 T1D 的组织学胰腺标本中，该测定显示 β 细胞分数在正常范围内，表明 β 细胞功能障碍具有重要作用。在 T2D 胰腺中，我们观察到 α 细胞分数增加，而 β 细胞分数正常。基于甲基化的分析为人类胰腺细胞类型的免疫检测提供了一种准确的分子替代方案，可用于解释胰岛素分泌测定以及评估健康和疾病中的胰腺细胞组成。