Direct measurement of the male germline mutation rate using sequential sperm samples

项目总结/摘要 人类大多数新的基因突变出现在男性生殖系中。虽然大多数这样的新生突变是 良性的，它们共同推动人类进化，并对巨大的疾病负担负责- 导致自闭症、智力残疾和各种发育障碍等疾病。 但值得注意的是，突变在男性生殖细胞中积累的速度， 人类遗传学的基本过程，从未被直接测量过。间接估计数 生殖系突变率是基于家庭研究，这些研究确定了儿童中的新生突变， 在父母的血液中检测不到这些研究表明，大约80%的新生突变， 在男性生殖细胞中出现，此外，父亲通过精子传递的突变数量 随着父亲年龄的增长。然而，这些基于“家庭”的测量受到 因此，他们只能估计一个个体的平均突变率。 大量的个人。他们也对不能受精或存活的精子突变视而不见 怀孕。因此，依赖于以家庭为基础的测量排除了生殖系如何量化 在单个个体的水平上，突变随着父亲年龄的增长而积累， 突变率在人群中各不相同，发现遗传和环境因素， 控制突变率我们假设直接测序单个个体的连续精子样本 将定义个体生殖系突变率及其个体间变异性。在这个项目中，我们将 开发一种新的方法，首次直接量化男性生殖系突变率， 个体的水平及其个体间的变异性。目标1将开发一种新的DNA测序方法 技术（HiDEF-seq），用于以显著低于 目前的方法。然后，Aim 2将使用HiDEF-seq以一种新的方法测量生殖系突变率 我们将从同一个人那里收集间隔超过10年的精子样本， 拥有世界上最大的两家精子银行我们还将开发数据收集和分析方法， 用于未来更大规模的研究，以确定测量的突变率和遗传因素之间的关系 (e.g., DNA修复/复制基因的变异）、健康史、环境暴露、生育能力和精子 质量.这项工作将提供一个新的，可扩展的平台，直接测量男性生殖细胞突变率 并且，在未来更大的群体中，为了发现其遗传和环境修饰剂， 人类遗传学对这些问题的深入研究将有助于深入了解基因突变， 衰老、健康和疾病。这项工作也将提供一种方法，发展生殖细胞突变率作为一个 衰老、后代疾病风险和生育力的潜在新生物标志物。

PROJECT SUMMARY/ABSTRACT Most new genetic mutations in humans arise in the male germline. While most such de novo mutations are benign, collectively they drive human evolution and are responsible for an immense burden of disease— contributing to disorders such as autism, intellectual disability, and a wide range of developmental disorders. But remarkably, the rate at which mutations accumulate in the male germline, one of the most fundamental processes in human genetics, has never been directly measured. Indirect estimates of germline mutation rates have been based on family studies that identify de novo mutations in children that are not detected in their parents’ blood. These studies have shown that approximately 80% of de novo mutations arise in the male germline, and moreover, that the number of mutations transmitted by fathers via their sperm increases with paternal age. However, these ”family”-based measurements are limited by the number of children borne by any single individual, so they are only able to estimate the average mutation rate across a large number of individuals. They are also blind to mutations in sperm incapable of fertilization or viable pregnancies. Reliance on family-based measurements has therefore precluded quantification of how germline mutations accumulate with paternal age at the level of single individuals, the degree to which germline mutation rates vary across the population, and discovery of genetic and environmental factors that modify and control mutation rates. We hypothesize that directly sequencing sequential sperm samples of single individuals will define individual germline mutation rates and their inter-individual variability. In this project, we will develop a novel methodology to directly quantify, for the first time, the male germline mutation rate at the level of individuals and its variability among individuals. Aim 1 will develop a novel DNA sequencing technology (HiDEF-seq) for ultra-high fidelity detection of mosaic mutations at significantly lower cost than current methods. Aim 2 will then use HiDEF-seq to measure germline mutation rates in a novel approach whereby we will obtain sperm samples collected > 10 years apart from the same individuals in a collaboration with two of the largest sperm banks in the world. We will also develop data collection and analytic methods for use in future larger studies to define the relationships between measured mutation rates and genetic factors (e.g., variants in DNA repair/replication genes), health history, environmental exposures, fertility, and sperm quality. This work will provide a novel, scalable platform to directly measure the male germline mutation rate and, in future larger cohorts, to discover its genetic and environmental modifiers, all fundamental unknowns in human genetics. Answering these questions will provide key insights into the relationships between mutation, aging, health, and disease. This work will also provide an approach for developing germline mutation rate as a potential novel biomarker for aging, offspring disease risk, and fertility.