Optimization and standardization of telomere length measurement by qPCR for population-based health research

摘要 衰老与生理功能的逐渐下降和主要疾病风险的增加有关。 与衰老有关的疾病。端粒磨损已被认为是细胞机制之一， 伴随衰老和与衰老有关的病理。端粒长度过短导致衰老 并且在病因学上与一系列不同的人类疾病有关。越来越多的生物医学， 流行病学和人口研究已经使用端粒长度作为细胞衰老的生物标志物 这反映了环境暴露和生活经历的累积效应，以及 重大疾病 在用于确定端粒长度的主要方法中，定量PCR（qPCR）仍然是最常用的方法。 适用于数百甚至数千个样本的大规模人群研究，由于其成本低， 易于采用，DNA量要求低，具有高通量的优点。然而，在这方面， 当使用以下方法对相同样品重复qPCR TL测定时，报告了不一致性 不同的分析前和分析条件，强调需要对每种情况进行认真的方法学研究， 这个过程的步骤。样本类型选择、样本采集、储存、处理问题和分析 每个程序都被确定为可能导致测定变异性的重要步骤。系统 迫切需要研究这些步骤中的每一个如何影响测定，以就最佳测定方法达成共识。 qPCR TL分析的实践。 在本提案中，我们计划优化qPCR的预分析和分析条件，重点是 三种标本类型：全血，在基于人群的研究中最常用的TL标本; 干血斑（DBS），一种广泛用于收集新生儿样本的非侵入性收集方法，以及唾液， 另一种非侵入性的家庭收集方法。我们进一步建议创建一套完全特征化的质量 对照（QC）DNA样品和参考标准基因组DNA样品，用作共享资源 用于进行qPCR TL的实验室。最后，我们建议通过验证来增加端粒长度测量， 通过先前的全基因组鉴定与TL相关的单核苷酸多态性（SNP）分析 关联研究（GWAS），并检查是否遗传总和得分（来自所有 SNPs）与儿童组群以及成人组群中的纵向端粒长度变化相关。 我们认识到，样本采集、储存和处理条件以及跨组织比较是 对于qPCR TL至关重要，这些问题由其他U 01提案和U24网络解决。作为投票 作为U24指导委员会的成员，我们将通过分析样品全面参与U24网络 由U24和NIA内部实验室分发，用于委员会指导的方法比较;以及 充分遵守委员会制定的准则和规程。

ABSTRACT Aging is associated with progressive decline of physiological functions and increased risks for major aging-related diseases. Telomere attrition has been proposed as one of the cellular mechanisms that accompany aging and pathologies related to aging. Critically short telomere length (TL) leads to senescence and is etiologically linked to a range of different human diseases. A growing number of biomedical, epidemiological and population research studies have used telomere length as a biomarker of cellular aging that reflects the cumulative effects of environmental exposures and life experiences as well as a risk factor for major diseases. Of the major methods utilized to determine telomere length, quantitative PCR (qPCR) remains the most suitable for large-scale population studies with hundreds or even thousands of samples, due to its low cost, ease of adoption, low DNA quantity requirement and advantages for high throughput. However, inconsistencies have been reported when qPCR TL assays were repeated for the same samples using different pre-analytical and analytical conditions, highlighting the need for careful methodological study of each step of this process. Sample type selection, sample collection, storage, processing issues and assay procedures have each been identified as import steps that may contribute to assay variability. Systematic studies of how each of these steps impacts the assay is urgently needed to reach a consensus on the best practices for qPCR TL analysis. In this proposal, we plan to optimize the pre-analytical and analytical conditions for qPCR focusing on three specimen types: whole blood, the most commonly used specimen for TL in population-based studies; dried blood spots (DBS), a non-invasive collection method widely used to collect neonatal samples, and saliva, another non-invasive home collection method. We further propose to create a set of fully characterized quality control (QC) DNA samples and a reference standard genomic DNA sample, to be used as shared resources for labs that perform qPCR TL. Finally, we propose to augment telomere length measurement by validating assays for single nucleotide polymorphisms (SNP) associated with TL identified through prior genome-wide association studies (GWAS) and examining whether a genetic sum score (from combining the effects of all SNPs) is associated with longitudinal telomere length change in a pediatric cohort as well as an adult cohort. We realize that sample collection, storage and processing conditions, as well as cross-tissue comparisons are critical for qPCR TL and these issues are addressed by other U01 proposals and the U24 network. As a voting member of the steering committee for the U24, we will fully participate in the U24 network by assaying samples distributed by the U24 and the NIA intramural labs for method comparisons as directed by the committee; and by fully adhering to guidelines and protocols established by the committee.