ABSTRACT

Cystic fibrosis (CF) is a monogenic disease, characterized by massive chronic lung inflammation. The observed variability in clinical phenotypes in monozygotic CF twins is likely associated with the extent of inflammation. This study sought to investigate inflammation-related aberrant DNA methylation in CF twins and to determine to what extent acquired methylation changes may be associated with lung cancer.

Blood-based genome-wide DNA methylation analysis was performed to compare the DNA methylomes of monozygotic twins, from the European CF Twin and Sibling Study with various degrees of disease severity. Putatively inflammation-related and differentially methylated positions were selected from a large lung cancer case-control study and investigated in blood by targeted bisulphite next-generation-sequencing. An inflammation-related locus located in the Plakophilin-3 (PKP3) gene was functionally analysed regarding promoter and enhancer activity in presence and absence of methylation using luciferase reporter assays.

We confirmed in a unique cohort that monozygotic twins, even if clinically discordant, have only minor differences in global DNA methylation patterns and blood cell composition. Further, we determined the most differentially methylated positions, a high proportion of which are blood cell-type-specific, whereas others may be acquired and thus have potential relevance in the context of inflammation as lung cancer risk factors. We identified a sequence in the gene body of PKP3 which is hypermethylated in blood from CF twins with severe phenotype and highly variably methylated in lung cancer patients and controls, independent of known clinical parameters, and showed that this region exhibits methylation-dependent promoter activity in lung epithelial cells.

摘要

囊性纤维化（CF）是一种单基因疾病，以大量慢性肺部炎症为特征。在同卵 CF 双胞胎中观察到的临床表型变异性可能与炎症程度有关。本研究旨在调查 CF 双胞胎中与炎症相关的异常 DNA 甲基化，并确定获得性甲基化变化在何种程度上可能与肺癌相关。

进行了基于血液的全基因组 DNA 甲基化分析，以比较来自欧洲 CF 双胞胎和兄弟姐妹研究的同卵双胞胎的 DNA 甲基化组与不同程度的疾病严重程度。从一项大型肺癌病例对照研究中选择了假定的炎症相关和差异甲基化位置，并通过靶向亚硫酸氢盐下一代测序在血液中进行了研究。使用荧光素酶报告基因分析对位于Plakophilin-3 ( PKP3 ) 基因中的炎症相关基因座在甲基化存在和不存在的情况下的启动子和增强子活性进行了功能分析。

我们在一个独特的队列中证实，同卵双胞胎即使在临床上不一致，在整体 DNA 甲基化模式和血细胞组成方面也只有微小的差异。此外，我们确定了差异最大的甲基化位置，其中很大一部分是血细胞类型特异性的，而其他位置可能是获得性的，因此在炎症背景下作为肺癌危险因素具有潜在的相关性。我们鉴定了PKP3基因体中的一个序列，该序列在来自具有严重表型的 CF 双胞胎的血液中高度甲基化，并且在肺癌患者和对照中高度可变地甲基化，与已知的临床参数无关，并表明该区域在肺上皮细胞。