Novel parent-of-origin specific differentially methylated loci on chromosome 16

To identify parent-of-origin biased DNA methylation, we performed high-resolution bisulfite sequencing of chromosome 16 on peripheral blood and cultured skin fibroblasts from individuals with maternal and paternal UPD(16) as well as lung tissue from patients with 16q24.1 ACDMPV-causative deletions and a normal control. We identified 22 differentially methylated regions (DMRs) with ≥5 consecutive CpG methylation sites and varying tissue-specificity, including the known DMRs associated with the established imprinted gene ZNF597 and DMRs supporting maternal methylation of PRR25, thought to be paternally expressed in lymphoblastoid cells. Lastly, we found evidence of paternal methylation on 16q24.1 near LINC01082 mapping to the FOXF1 enhancer. Using high-resolution bisulfite sequencing to evaluate DNA methylation across chromosome 16, we found evidence of novel candidate imprinted loci on chromosome 16 that would not be evident in array-based assays and could contribute to the birth defects observed in association with UPD(16)mat and in ACDMPV. Comparison of DNA methylation profiles between individuals with UPD(16)mat (n=2), UPD(16)pat (n=2), and normal controls in blood, juxtaposed with UPD(16)mat from fibroblast cultures (n=1), and lung tissue samples from ACDMPV individuals with maternal deletions (n=2) and normal control (n=1).

为鉴定亲本偏好性DNA甲基化（DNA methylation），我们对携带母源、父源单亲二体性16号染色体（uniparental disomy(16), UPD(16)）的个体的外周血、培养皮肤成纤维细胞，以及携带16q24.1区域致肺泡毛细血管发育不良伴肺静脉错位（Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins, ACDMPV）致病性缺失的患者的肺组织与正常对照样本，开展了16号染色体（chromosome 16）的高分辨率亚硫酸氢盐测序（bisulfite sequencing）。我们共鉴定出22个差异甲基化区域（differentially methylated regions, DMRs），这些区域包含≥5个连续的CpG甲基化位点，且具有不同程度的组织特异性；其中包括与已验证印迹基因（imprinted gene）ZNF597相关的已知DMRs，以及支持PRR25母源甲基化的新DMRs——此前学界认为PRR25在淋巴母细胞中呈父源表达。最后，我们在16q24.1区域靠近LINC01082的位置发现了父源甲基化的证据，该位点定位于FOXF1增强子（FOXF1 enhancer）。通过对16号染色体全区域开展高分辨率亚硫酸氢盐测序以评估DNA甲基化水平，我们发现了16号染色体上的新型候选印迹基因座（imprinted locus），这类位点无法通过基于芯片的检测（array-based assay）被检出，且可能与母源UPD(16)相关的出生缺陷以及ACDMPV的发病机制相关。本研究对以下样本的DNA甲基化谱进行了比较：2例母源UPD(16)个体、2例父源UPD(16)个体的外周血样本，1例培养成纤维细胞来源的母源UPD(16)样本，以及2例携带16q24.1缺失的ACDMPV患者肺组织样本与1例正常对照肺组织样本。