Novel Informatics for Highly Reliable Multi-Locus Allele Calling for Embryo Scree

描述(申请人提供)：2006年，全球共进行了超过80万个体外受精(IVF)周期。在美国进行的150,000个周期中，大约有10,000个周期涉及植入前遗传诊断(PGD)。目前的PGD技术不受监管，成本高昂，而且非常不可靠：筛查疾病相关基因座或非整倍体的错误率约为10%；每项筛查测试的费用超过5000美元；一对夫妇被迫在检测非整倍体和在单个细胞上筛查疾病相关基因座之间做出选择。非整倍体大约影响40%的体外受精胚胎。非常需要一种负担得起的技术，能够可靠地确定来自单个细胞的遗传数据，以便同时筛查非整倍体、单基因疾病，如囊性纤维化，以及对复杂疾病表型的易感性，这些疾病的多个遗传标记是通过全基因组关联(WGA)研究已知的。体外受精过程中的PGD过程包括从早期胚胎的大约8个细胞中提取单个细胞进行分析。由于一个细胞只有一个DNA拷贝，直接测量DNA很容易出错，或有噪音。基因安全网络(GSN)开发了一种名为Parental SupportTM(PS)的新技术，用于从单个细胞中确定数百个基因座的胚胎DNA以及23条染色体的拷贝数，误差率低于0.1%。这项专利技术利用母亲和父亲的遗传数据，以及减数分裂机制的知识和胚胎DNA的噪声测量，以确定父母染色体的哪些片段对受精的配子起作用，从而以超过99%的置信度在计算机上重建胚胎DNA。根据我们第一阶段研究的结果，GSN已经与美国5家领先的试管受精中心签署了使用GSN诊断服务的意向书。在这项提案的目标1中，我们将展示PS技术使用从新生儿分离的单个细胞测量的基因数据和父母基因数据可靠地重建基因数据的能力。在目标2中，我们将展示PS技术的能力，也使用分离的单细胞遗传数据，通过创新的单细胞非整倍体模型检测所有23条染色体的非整倍体，而不需要直接对胚胎进行研究。在目标3中，我们将与斯坦福试管受精中心、波士顿试管受精中心和亨廷顿生殖中心一起进行一项临床试验，将目标1和目标2中的技术应用于试管受精环境中的真实卵裂球，并将我们的预测与出生时在孩子身上测量的真实情况进行比较。这项研究的一个目标是产生数据，用于获得食品和药物管理局对这种诊断技术的批准。基因安全网络的PS技术将把PGD的领域带入可靠的诊断领域，可以在临床决策中放心地进行调节和使用。胚胎植入的选择是一项临床决定，对结果有直接和绝对的影响。叙述和与医疗保健公共卫生的相关性：随着将疾病表型与基因相关的数据持续增长，问题出现了：如何利用这些知识来改善生活和健康的质量？有了这笔赠款，基因安全网络将彻底验证一项在体外受精过程中筛选胚胎的技术，以检测多种与疾病相关的基因和TM非整倍体。这项技术被称为父母支持(PS)，它建立在减数分裂的基本原理和最近通过人类基因组计划获得的数据基础上。与现有技术相比，PS能够：i)以大约两个数量级的低错误率来确定疾病相关基因座；ii)并行地确定多个疾病相关基因座；iii)以大约两个数量级的低错误率来确定非整倍体；以及iv)确定所有染色体上的非整倍体以及全部来自单个细胞的多个疾病相关基因座。GSN正在开发增强的报告系统、统计方法和湿实验室基础设施，以向签署了购买服务意向书的领先试管受精中心提供这项服务，然后再提供给全球试管受精社区。这项研究的资金将使我们能够验证在单细胞上使用已知真理模型进行诊断的性能，并通过将这些预测与婴儿出生时测量的基因数据进行比较来验证从单个卵裂球做出的预测。2006年，美国和国际上进行了大约152,000个和653,000个试管受精周期。PGD的年增长率约为33%。这项研究的资金将使我们能够验证在单细胞上使用已知真理模型进行诊断的性能，并通过将这些预测与婴儿出生时测量的基因数据进行比较来验证从单个卵裂球做出的预测。GSN的PS技术将把PGD的领域带入可靠的诊断领域，可以作为体外受精过程中护理标准的一部分进行调节和使用。

DESCRIPTION (provided by applicant): In 2006, across the globe, more than 800,000 in-vitro fertilization (IVF) cycles were run. Of 150,000 cycles run in the US, roughly 10,000 involved pre-implantation genetic diagnosis (PGD). Current PGD techniques are unregulated, expensive and highly unreliable: error rates for screening disease-linked loci or aneuploidy are on the order of 10%; each screening test costs more than $5,000; and a couple is forced to choose between testing aneuploidy, which afflicts roughly 40% of IVF embryos, or screening for disease-linked loci on the single cell. There is a great need for an affordable technology that can reliably determine genetic data from the single cell in order to screen in parallel for aneuploidy, monogenic diseases such as Cystic Fibrosis, and susceptibility to complex disease phenotypes for which the multiple genetic markers are known through whole-genome association (WGA) studies. The process of PGD during IVF involves extracting a single cell from the roughly 8 cells of an early-stage embryo for analysis. Since only a single copy of the DNA is available from one cell, direct measurements of the DNA are highly error-prone, or noisy. Gene Security Network (GSN) has developed a novel technology, termed Parental SupportTM (PS), for determining the embryonic DNA at hundreds of loci together with copy numbers for 23 chromosomes, with error rates below 0.1%, from a single cell. The proprietary technique makes use of genetic data of the mother and the father, together with the knowledge of the mechanism of meiosis and noisy measurements of the embryonic DNA, in order to determine which segments of parent chromosomes contributed to the gametes that fertilized and hence to reconstruct in silico the embryonic DNA with confidence exceeding 99%. Based on the results of our phase I study, GSN has executed letters of intent with the 5 leading IVF centers in the United States to use the GSN diagnostic service. In aim 1 of this proposal, we will demonstrate the ability of the PS technology to reliably reconstruct genetic data using the measured genetic data from isolated single cells from a born child, and parental genetic data. In aim 2 we will demonstrate the ability of the PS technology to detect aneuploidy at all 23 chromosomes, also using isolated single cell genetic data, by means of an innovative single cell model for aneuploidy that does not require direct work on embryos. In Aim 3 we will perform a clinical trial in conjunction with Stanford IVF Center, Boston IVF and Huntington Reproductive Center that applies the techniques from aims 1 and 2 to real blastomeres in the IVF context and compare our predictions with truth measured on the child when born. One goal of the study is to generate data that will be used to obtain approval of this diagnostic technique by the Food and Drug Administration. The PS technology of Gene Security Network will bring the domain of PGD into the realm of reliable diagnostics which can be regulated and used with confidence in clinical decisions. The selection of the embryos to implant is a clinical decision that has direct and absolute impact on outcomes. Narrative and Relevance to Healthcare PUBLIC HEALTH RELEVANCE: As data associating disease phenotypes with genotype continues to grow, the question arises: how can this knowledge be used to improve the quality of life and health? With this grant, Gene Security Network will thoroughly validate a technology for screening embryos during in-vitro fertilization for a multiplicity of disease linked genes and TM aneuploidy. This technology, termed Parental Support (PS) which is built on the fundamental principles of meiosis and data that has recently become available through the human genome project. Compared to existing technologies, PS enables: i) determination of disease linked loci with roughly two orders of magnitude lower error rates; ii) determination of multiple disease-linked loci in parallel; iii) determination of aneuploidy with roughly two orders of magnitude lower error rates; and iv) determination of aneuploidy across all chromosomes together with multiple disease-linked loci all from a single cell. GSN is developing the enhanced reporting system, statistical methods, and wet-lab infrastructure to offer this service to the leading IVF centers who have signed letters of intent to purchase the service, and then to the worldwide IVF community. Funding for this study will enable us to validate the performance of the diagnostic with known truth models on single cells, and to validate predictions made from a single blastomere by comparing those predictions with genetic data measured when a child is born. Roughly 152,000 and 653,000 IVF cycles were performed in 2006 in the US and internationally. The rate of growth of PGD is roughly 33% annually. Funding for this study will enable us to validate the performance of the diagnostic with known truth models on single cells, and to validate predictions made from a single blastomere by comparing those predictions with genetic data measured when a child is born. GSN's PS technology of will bring the domain of PGD into the realm of reliable diagnostics which can be regulated and used as part of the standard of care during in-vitro fertilization.