Investigation of DUF1220 domains in human brain function and disease

总结 DUF 1220蛋白结构域经历了最大的人类谱系特异性拷贝数增加 基因组中任何蛋白质编码区（人类单倍体基因组中约290个拷贝），并主要映射到 1 q21，包含许多DUF 1220拷贝的拷贝数变异重复连锁的区域 自闭症精神分裂症小头症和大头症DUF 1220拷贝数呈现宽高斯分布 在人群中的分布，并且是未经检查的功能等位基因变异的丰富来源。以前我们 已经暗示DUF 1220拷贝数（剂量）在人脑扩增和正常和病理 大脑大小的变化。在过去的一年里，我们已经证明DUF 1220（CON 1亚型）剂量是 与自闭症症状严重程度的增加呈线性相关（现已通过复制研究证实）， 与精神分裂症的严重程度和风险成反比。这些发现是值得注意的，因为它们意味着剂量 同一个蛋白质结构域家族（DUF 1220）内的变异可能与人类大脑进化、自闭症 和精神分裂症，这些过程可能是遗传和机械相关的。 在这些重要发现的基础上，我们将扩大我们对自闭症DUF 1220拷贝数的研究， 精神分裂症的严重程度（目标1）和风险（目标2）和小/大头畸形（目标3）使用的方法，我们有 成功使用（ddPCR，1 q21靶向阵列CGH）以及通过应用新方法， 具有显著改善DUF 1220拷贝数分析的潜力（目标4）。比如我们有 最近优化和验证了我们的序列读取深度方法，用于测量DUF 1220拷贝数， 全基因组序列（WGS）数据高分辨率和精确度。我们将把这种方法应用于WGS 数据来自1）与Stephen谢勒博士合作的自闭症10 K项目，以及2）其他几个 自闭症和精神分裂症的大型WGS数据集。此外，我们还建立了一个 与我们的合作者裴国博士合作，应用Irys，一种自动化的光学映射方法， 长（> 150 kb）单DNA分子的基因组作图，以评估这些DNA分子中DUF 1220拷贝数的变异。 紊乱Irys方法应该得到我们开发的CRISPR-Cas9方法的帮助， DUF 1220-基因组DNA的特异性标记。这些方法，包括我们对长读 测序技术，将通过利用新的、更准确的和无间隙的1 q21组装来促进 我们最近帮助创造的。最后，我们将建立在我们最近的进展，将DUF 1220功能与 通过测试DUF 1220是否促进神经干细胞（H9衍生）增殖来增加神经元数量 以剂量依赖性方式，并确定受逐渐增加的 DUF 1220剂量。

SUMMARY DUF1220 protein domains have undergone the largest human lineage-specific increase in copy number of any protein coding region in the genome (~290 copies in human haploid genome), and map primarily to 1q21, a region where copy number variations containing many DUF1220 copies have been repeatedly linked to autism, schizophrenia, micro- and macrocephaly. DUF1220 copy number exhibits a broad Gaussian distribution in human populations, and is a rich source of unexamined functional allelic variation. Previously we have implicated DUF1220 copy number (dosage) in human brain expansion and normal and pathological changes in brain size. Over the past year we have demonstrated that DUF1220 (CON1 subtype) dosage is linearly associated with increasing symptom severity of autism (now confirmed by a replication study) and also inversely with schizophrenia severity and risk. These findings are remarkable in that they imply that dosage variations within the same protein domain family (DUF1220) may be involved in human brain evolution, autism and schizophrenia, and that these processes may be genetically and mechanistically interrelated. Building on these significant findings, we will expand our study of DUF1220 copy number in autism and schizophrenia severity (Aim 1) and risk (Aim 2) and in micro-/macrocephaly (Aim 3) using methods we have used successfully (ddPCR, 1q21-targeted arrayCGH) as well as through application of novel methods that have the potential to significantly improve DUF1220 copy number analysis (Aim 4). For example, we have recently optimized and validated our sequence read-depth method for measuring DUF1220 copy number at high resolution and precision from whole genome sequence (WGS) data. We will apply this approach to WGS data from 1) the Autism 10K project in collaboration with Dr. Stephen Scherer, and 2) to several additional large WGS datasets that are emerging for autism and schizophrenia. In addition, we have established a subcontract with our collaborator, Dr. Pui Kwok, to apply Irys, an optical mapping method for automated genome mapping of long (>150kb) single DNA molecules, to assess DUF1220 copy number variation in these disorders. The Irys approach should be aided by our development of a CRISPR-Cas9 method which allows DUF1220-specific labeling of genomic DNA. These approaches, including our exploration of long-read sequencing technologies, will be facilitated by utilization of a new, more accurate and gap-free 1q21 assembly we recently helped generate. Finally, we will build on our recent progress linking DUF1220 function to increases in neuron number by testing whether DUF1220 promotes neural stem cell (H9-derived) proliferation in a dosage-dependent manner and identifying cellular pathways affected by progressively increasing DUF1220 dosage.