An Algorithm Measuring Donor Cell-Free DNA in Plasma of Cellular and Solid Organ Transplant Recipients That Does Not Require Donor or Recipient Genotyping.
An Algorithm Measuring Donor Cell-Free DNA in Plasma of Cellular and Solid Organ Transplant Recipients That Does Not Require Donor or Recipient Genotyping.
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DOI：
10.3389/fcvm.2016.00033
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发表时间：
2016
影响因子：
3.6
通讯作者：
Greenway SC
中科院分区：
文献类型：
--
作者：
Gordon PM;Khan A;Sajid U;Chang N;Suresh V;Dimnik L;Lamont RE;Parboosingh JS;Martin SR;Pon RT;Weatherhead J;Wegener S;Isaac D;Greenway SC
游离 DNA (cfDNA) 在临床状况的诊断和监测方面具有巨大潜力。然而，准确、轻松地区分来自两种不同来源（即移植后的供体组织和受体组织）的混合物中 DNA 分子的相对比例具有挑战性。在人类细胞移植中，目前没有可用的方法来检测体内植入，并且基于血液的非侵入性实体器官移植中同种异体移植物排斥测试要么是非特异性的，要么是不存在的。供体 cfDNA 水平升高已被证明与实体器官排斥相关，但复杂的方法限制了这种有前途的生物标志物的实施。我们描述了一种经济高效的方法，使用一组高频单核苷酸多态性、下一代（半导体）测序和一种新颖的混合模型算法来量化受体血浆中的供体 cfDNA。在体外，我们的方法准确、快速地确定了供体：受体 DNA 混合物。对于体内测试，在每天接受六次供体肝细胞输注后，对患有尿素循环障碍的婴儿的供体 cfDNA 进行了连续定量。输注后 24 周检测到从 1 至 2ml 受体血浆中分离出的供体 cfDNA，表明植入。还在接受常规心内膜心肌活检的儿童和成人心脏移植受者中评估了循环供体 cfDNA 的百分比，观察到水平随着时间的推移保持稳定，并且在没有中度或重度细胞排斥的情况下通常测量<1%。与现有用于确定实体器官移植患者中供体 cfDNA 比例的非侵入性方法不同，我们的检测不需要性别不匹配、供体基因分型或全基因组测序，并且可能具有广泛的应用来检测移植后的细胞植入或同种异体移植损伤。
Cell-free DNA (cfDNA) has significant potential in the diagnosis and monitoring of clinical conditions. However, accurately and easily distinguishing the relative proportion of DNA molecules in a mixture derived from two different sources (i.e., donor and recipient tissues after transplantation) is challenging. In human cellular transplantation, there is currently no useable method to detect in vivo engraftment, and blood-based non-invasive tests for allograft rejection in solid organ transplantation are either non-specific or absent. Elevated levels of donor cfDNA have been shown to correlate with solid organ rejection, but complex methodology limits implementation of this promising biomarker. We describe a cost-effective method to quantify donor cfDNA in recipient plasma using a panel of high-frequency single nucleotide polymorphisms, next-generation (semiconductor) sequencing, and a novel mixture model algorithm. In vitro, our method accurately and rapidly determined donor:recipient DNA admixture. For in vivo testing, donor cfDNA was serially quantified in an infant with a urea cycle disorder after receiving six daily infusions of donor liver cells. Donor cfDNA isolated from 1 to 2 ml of recipient plasma was detected as late as 24 weeks after infusion suggesting engraftment. The percentage of circulating donor cfDNA was also assessed in pediatric and adult heart transplant recipients undergoing routine endomyocardial biopsy with levels observed to be stable over time and generally measuring <1% in cases without moderate or severe cellular rejection. Unlike existing non-invasive methods used to define the proportion of donor cfDNA in solid organ transplant patients, our assay does not require sex mismatch, donor genotyping, or whole-genome sequencing and potentially has broad application to detect cellular engraftment or allograft injury after transplantation.