Non-invasive Aneuploidy Screening of Circulating Fetal Cells for Prenatal Diagnos

描述（由申请人提供）：在怀孕期间，医生和患者希望尽可能多地了解胎儿的健康状况。出于情感和医疗方面的原因，应尽可能早地获取这一信息，并尽可能减少对母亲和婴儿的风险。虽然广泛使用的早期妊娠绒毛膜绒毛取样（CVS）和中期妊娠绒毛膜穿刺术是相对安全的，但这两种方法都不是没有风险的。为了完全避免这些风险，研究人员转向从母体血液中分离循环胎儿有核红细胞（FNRBC）作为胎儿组织的替代性非侵入性来源。尽管开发了FNRBC富集方法， 将它们与随后的非整倍性筛选偶联的成功有限，并且仍必须克服几个挑战，例如测试单个胎儿细胞的24-染色体非整倍性、确认分离细胞的来源（胎儿与母体）以及同时筛选由单核苷酸变体或微插入/缺失引起的疾病的能力。我们创新的Parental SupportTM技术为所有这些挑战提供了解决方案，而开发孕早期非侵入性产前诊断测试是此次拨款申请的最终目标。在第一阶段，我们首先计划优化单细胞裂解和全基因组扩增方案，专门用于抗体染色的FNRBC。单细胞分析的方案优化福尔斯GSN的核心竞争力，因为我们之前已经成功地将创新的单细胞分子核型分析方案商业化，从而能够在24小时内对单个卵裂球进行遗传分析。然后，我们将系统地评估现有FNRBC富集方法的哪种组合提供适合于随后使用预定义的胎儿和成人血液混合物进行基于“父母支持”的遗传分析的最大产量和纯度。第二阶段的主要目标是从预定义的胎儿和成人血液混合物过渡到实际的母体血液样本。我们将首先进行一项试点研究，以确定第一阶段确定的最佳FNRBC分离方法应成为主要方法。使用这种引导方法，我们将进行一项更大的研究，以评估通过Parental SupportTM进行的非整倍体诊断与通过胎盘穿刺或绒毛取样进行的核型分析之间的一致性。如果成功，我们预计这些目标的完成将对产前诊断领域产生重大影响，改善全世界数百万夫妇和儿童的生活，并将非侵入性诊断纳入产前医学的主流。 公共卫生相关性：在缺乏产前诊断的情况下，高达1/50的婴儿有严重的身体或精神障碍，高达1/30的婴儿有某种形式的先天畸形，高达1/200的表型显着的染色体异常，虽然这些异常可以通过诸如胎盘穿刺或绒毛膜绒毛取样等技术进行诊断，这两种方法都增加了对母亲和胎儿造成伤害的风险。我们的创新技术有可能通过简单地分析母亲的血液来评估未出生婴儿的健康状况，从而最大限度地降低手术风险，并将产前筛查扩大到普通人群。

DESCRIPTION (provided by applicant): During the course of a pregnancy, physicians and patients desire as much information as possible regarding the health of the fetus. For both emotional and medical reasons, this information is sought as early in term as possible, and with the fewest possible risks to both mother and child. Although the widely used first trimester chorionic villus sampling (CVS) and second trimester amniocentesis are relatively safe, both procedures are not without negligible risks. In efforts to avoid these risks altogether, researchers have turned toward isolating circulating fetal nucleated red blood cells (FNRBCs) from maternal blood as an alternative, non- invasive source of fetal tissue. Despite the development of FNRBC enrichment methods, there has been limited success with their coupling to subsequent aneuploidy screening and several challenges still must be overcome such as ability to test single fetal cells for 24-chromosome aneuploidy, confirm the isolated cell's origin (fetal versus maternal) and simultaneously screen for diseases caused by single nucleotide variants or micro in/dels. Our innovative Parental SupportTM technology provides a solution to all of these challenges and the development of a first trimester non-invasive prenatal diagnostic test is the ultimate goal of this grant application. In Phase I, we first plan to optimize single cell lysis and whole genome amplification protocols specifically for antibody-stained FNRBCs.. Protocol optimization for single cell analysis falls within the core competencies of GSN as we have previously successfully commercialized an innovative single cell molecular karyotyping protocol to enable genetic analysis of single blastomeres within 24 hours. We will then systematically evaluate which combination of existing FNRBC enrichment methods provides maximum yield and purity suitable for subsequent Parental Support"-based genetic analysis using predefined mixtures of fetal and adult blood. The main objective of Phase II will be to transition from the predefined blood mixtures of fetal and adult blood to actual maternal blood samples. We will first conduct a pilot study to determine which of the best FNRBC isolation method(s) identified in Phase I should become the lead method. Using this lead method, we will then conduct a larger study to evaluate concordance between aneuploidy diagnosis by Parental SupportTM and karyotyping by amniocentesis or chorionic villus sampling. If successful, we expect that the completion of these Aims would have a major impact on the field of prenatal diagnosis, improve the lives of millions of couples and children worldwide, and bring non-invasive diagnosis to the mainstream of prenatal medicine. PUBLIC HEALTH RELEVANCE: In the absence of prenatal diagnosis, up to 1 in 50 babies have serious physical or mental handicaps, up to 1 in 30 babies have some form of congenital malformation, and up to 1 in 200 have a phenotypically significant chromosome abnormality Although these abnormalities can be diagnosed with techniques such as amniocentesis or chorionic villus sampling, both procedures carry an increased risk of harm to both the mother and fetus. Our innovative technology has the potential to evaluate the health of an unborn child by simply analyzing the mother's blood, thereby minimizing the risks of the procedure and expanding prenatal screening to the general population.