Cross-Tissue Comparison of Epigenetic Aging Clocks in Humans. Cross-Tissue Comparison of Epigenetic Aging Clocks in Humans

Epigenetic clocks are a common group of tools used to measure biological aging – the progressive deterioration of cells, tissues and organs. Epigenetic clocks have been trained almost exclusively using blood-based tissues but there is growing interest in estimating epigenetic age using less-invasive oral-based tissues (i.e., buccal or saliva) in both research and commercial settings. However, differentiated cell types across body tissues exhibit unique DNA methylation landscapes and age-related alterations to the DNA methylome. Applying epigenetic clocks derived from blood-based tissues to estimate epigenetic age of oral-based tissues may introduce biases. We tested the within-person comparability of common epigenetic clocks across four tissue types: buccal epithelial, saliva, dry blood spots, and peripheral blood mononuclear cells. We tested 163 distinct tissue samples from 47 individuals aged 19-70 years. Overall, there were significant within-person differences in epigenetic clock estimates from oral-based versus blood-based tissues, with average differences of almost 30 years observed in some age clocks. In addition, most epigenetic clock estimates of blood-based tissues exhibited low correlation with estimates from oral-based tissues despite controlling for cellular proportions and other technical factors. Notably, the Skin and Blood clock exhibited the lowest age acceleration values of any clock across all tissue types, indicating its unique ability to accurately estimate chronological age in both oral- and blood-based tissues. Our findings indicate that application of blood-derived epigenetic clocks in oral-based tissues may not yield comparable estimates of epigenetic age, highlighting the need for careful consideration of tissue type when estimating epigenetic age. NOTE: The full study included children and adult samples, however, the current data only includes the adult samples (sample sizes and age range have been adjusted to reflect the adult data only). Overall design: Trained phlebotomists performed antecubital venipuncture to collect 20 mL of whole blood in EDTA tubes. PBMCs were isolated from these whole blood samples through density-gradient centrifugation using Ficoll. Approximately 200 µL of whole blood were applied to a Whatman 903 protein saver card for the dried blood spot (DBS) samples. Participants were also asked to provide 4 mL of saliva across two Oragene tubes (OGR-500, DNA Genotek), which upon completion was mixed with Oragene stabilizing buffer and sealed. Eight buccal samples were collected using Isohelix SK1 swabs to firmly scrape the inside of the cheek per manufacturer’s directions. Collection order for all tissue types was uniform across participants. Participants were asked to refrain from eating or drinking anything other than water for one hour before collection. After collection, tissue samples were stored as follows: PBMCs were stored at -80°C in a solution buffer composed of phosphate buffered saline pH 7.2+EDTA (2mMol) + bovine serum albumin (0.5%) prior to DNA extraction. DBS were stored in sealed Ziploc bags with desiccant packets at room temperature. Buccal swabs were placed in sealed Ziploc bags and stored at -80°C. Saliva samples were aliquoted into 4 cryovials and stored at -80°C.

表观遗传时钟（Epigenetic clocks）是一类常用于评估生物衰老的工具，生物衰老指细胞、组织与器官的进行性退行性衰退。尽管表观遗传时钟几乎完全依托血液来源组织训练而成，但当前研究与商业场景中，利用微创口腔来源组织（即颊部组织或唾液样本）估算表观遗传年龄的需求正日益增长。然而，人体不同组织中的分化细胞类型具有独特的DNA甲基化图谱，且存在与年龄相关的DNA甲基化组改变。直接套用基于血液来源组织训练得到的表观遗传时钟来估算口腔来源组织的表观遗传年龄，可能会引入估算偏差。

本研究针对四种组织类型——颊上皮组织、唾液样本、干血斑以及外周血单个核细胞（peripheral blood mononuclear cells）——测试了常见表观遗传时钟的个体内可比性。研究共纳入47名年龄在19至70岁之间的受试者，获取163份独立组织样本。整体而言，口腔来源组织与血液来源组织的表观遗传时钟估算值存在显著个体内差异，部分时钟的估算差值可达近30年。此外，尽管我们控制了细胞比例与其他技术干扰因素，多数基于血液来源组织的表观遗传时钟估算值与口腔来源组织的估算值相关性仍较低。值得注意的是，皮肤与血液时钟（Skin and Blood clock）在所有组织类型中的年龄加速值均为所有时钟中最低，这表明其能够精准估算口腔及血液来源组织的实际年龄。

本研究结果表明，将血液来源的表观遗传时钟应用于口腔来源组织时，可能无法得到可比的表观遗传年龄估算值，这凸显了在估算表观遗传年龄时需谨慎考量组织类型的必要性。

注：本研究完整数据集包含儿童与成人样本，但本次公开的数据集仅包含成人样本（样本量与年龄范围已调整以仅体现成人数据）。

实验整体设计：经过培训的采血员通过肘静脉穿刺采集20mL全血，置于乙二胺四乙酸（EDTA）抗凝管中。采用Ficoll密度梯度离心法从上述全血样本中分离外周血单个核细胞（PBMCs）。取约200μL全血滴加至Whatman 903蛋白保存卡上，制备干血斑（DBS）样本。受试者还需提供4mL唾液，分装至两支Oragene唾液采集管（OGR-500，DNA Genotek）中，采集完成后与Oragene稳定缓冲液混合并密封。采用Isohelix SK1采样拭子按照制造商说明轻柔刮取颊内侧黏膜，采集8份颊部样本。所有受试者的组织样本采集顺序保持一致。受试者需在采样前1小时禁食禁水（仅可饮用白水）。

采样完成后，组织样本按以下方式保存：外周血单个核细胞在提取DNA前，保存于含pH7.2磷酸盐缓冲液、2mmol/L EDTA及0.5%牛血清白蛋白的缓冲溶液中，置于-80℃冰箱；干血斑样本置于含干燥剂的密封Ziploc保鲜袋中，室温保存；颊部采样拭子放入密封Ziploc保鲜袋中，置于-80℃冰箱；唾液样本分装至4个冻存管中，置于-80℃冰箱保存。