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

描述（由申请人提供）：2006年，在全球范围内，进行了超过80万次体外受精（IVF）周期。在美国进行的150,000个周期中，大约有10,000个涉及植入前遗传学诊断（PGD）。目前的PGD技术不受监管、价格昂贵且极不可靠：筛查疾病相关基因座或非整倍体的错误率约为10%；每次筛选测试的费用超过5000美元；一对夫妇被迫在检测非整倍体（大约40%的试管婴儿胚胎受此困扰）和筛查单细胞上的疾病相关基因座之间做出选择。目前迫切需要一种负担得起的技术，能够可靠地从单细胞中确定遗传数据，以便平行筛选非整倍体、单基因疾病（如囊性纤维化）和对复杂疾病表型的易感性，这些疾病的多种遗传标记通过全基因组关联（WGA）研究已知。体外受精期间的PGD过程包括从早期胚胎的大约8个细胞中提取一个细胞进行分析。由于一个细胞中只有一个DNA拷贝，因此对DNA的直接测量很容易出错，或者很嘈杂。基因安全网络（GSN）开发了一种称为亲代支持tm （PS）的新技术，用于从单个细胞中测定胚胎DNA的数百个位点以及23条染色体的拷贝数，错误率低于0.1%。该专有技术利用母亲和父亲的遗传数据，以及对减数分裂机制的了解和胚胎DNA的噪声测量，以确定亲本染色体的哪些片段有助于配子受精，从而以超过99%的信心重建胚胎DNA。根据我们一期研究的结果，GSN已经与美国5家领先的IVF中心签署了使用GSN诊断服务的意向书。在本提案的目标1中，我们将展示PS技术可靠地重建遗传数据的能力，使用来自出生儿童的分离单细胞的测量遗传数据和父母遗传数据。在目标2中，我们将展示PS技术检测所有23条染色体的非整倍性的能力，也使用分离的单细胞遗传数据，通过一种创新的非整倍性单细胞模型，不需要直接对胚胎进行工作。在目标3中，我们将与斯坦福试管婴儿中心，波士顿试管婴儿中心和亨廷顿生殖中心联合进行临床试验，将目标1和2中的技术应用于试管婴儿中的真实卵裂球，并将我们的预测与出生时测量的事实进行比较。这项研究的目标之一是生成数据，用于获得美国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领域带入可靠的诊断领域，可以在临床决策中进行调节和使用。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。公共卫生相关性：随着将疾病表型与基因型相关联的数据不断增长，问题出现了：如何利用这些知识来改善生活质量和健康？有了这笔拨款，基因安全网络将彻底验证一种在体外受精过程中筛选胚胎的技术，以检测多种疾病相关基因和TM非整倍性。这项技术被称为亲代支持（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之上。与现有技术相比，PS能够：i)以大约低两个数量级的错误率确定疾病相关基因座；Ii)平行测定多个疾病相关基因座；Iii)非整倍体的测定，错误率降低了大约两个数量级；iv)所有染色体的非整倍性以及来自单个细胞的多个疾病相关位点的测定。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，为已签署购买该服务意向书的领先试管婴儿中心提供这项服务，然后向全球试管婴儿社区提供这项服务。这项研究的资金将使我们能够用已知的单细胞真实模型来验证诊断的性能，并通过将这些预测与孩子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，美国和国际上大约有15.2万到65.3万例试管婴儿手术。PGD的年增长率约为33%。这项研究的资金将使我们能够用已知的单细胞真实模型来验证诊断的性能，并通过将这些预测与孩子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领域带入可靠的诊断领域，可以作为体外受精期间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.