The Brain and Maternal Microchimerism

大脑和母体微嵌合现象

• 批准号: 10610125
• 负责人: J. Lee Nelson
• 金额: $ 23.18万
• 依托单位: FRED HUTCHINSON CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10610125
• 关键词: Brain; Maternal; Microchimerism

PROJECT SUMMARY/ABSTRACT Microchimerism (Mc) refers to harboring a small amount of cells or DNA from a genetically distinct individual. Many years after the physical union of mother and child ends maternal Mc (MMc) is found in her offspring, and Mc of fetal origin in the mother. MMc has been found in children and adults in blood and organs, including heart, liver, spleen, kidney and pancreas. In experimental and human studies Mc appears to be differentiated, creating for example MMc as cardiac myocytes in the heart, islet  cells in the pancreas and hepatocytes in the liver. Despite the importance of the brain to human health and function a fundamental gap of knowledge exists for MMc in the brain. The overall purpose of this proposal is to generate foundational knowledge about MMc cell types, quantities and the MMc transcriptome in human brain. To establish the origin of Mc as specific to the mother requires maternal participation which is generally not available for childhood autopsies. Aim 1 has two parts. Part 1 of Aim 1 will investigate MMc in pediatric patients who have surgical excision for medication refractory epilepsy, for whom mothers are available to participate. HLA and other polymorphism genotyping is done from maternal DNA extracted from a buccal swab sample. After genotyping patients and mothers, each mother-child pair is reviewed to identify a non-transmitted, non-shared polymorphism i.e. unique to the mother. A maternal-specific assay is next selected from a panel of HLA- and other polymorphism-specific quantitative PCR (qPCR) assays we have developed for this purpose. DNA extracted from excised brain tissue is then interrogated for MMc using the selected custom assay for each mother-child pair. A similar approach will be employed to study brain resected from age comparable patients undergoing surgery for gliomas for which maternal participation can be included. Part 2 of the Aim 1 approach will select brain tissues from males to study by fluorescence in situ hybridization (FISH) with X- and Y-chromosome specific probes; this aspect of the Aim 1 approach will permit including brain tissue from children without neurologic disease from autopsy from whom maternal participation is not required. Female cells with two X-chromosome signals, presumed maternal, will be counted with XY male cells enumerated in the same area. Immunofluorescence (IF) will be added to evaluate cell phenotypes. In Aim 2 we will conduct single nuclei RNA sequencing (snRNA-seq) analysis on brain tissue samples. The snRNA-seq studies will comprehensively evaluate what type of cells in the brain are derived from MMc and will assess the MMc transcriptome. The ways in which MMc may affect the brain are multiple and diverse to the potential benefit and/or detriment of a child. In addition to informativeness for epilepsy, if naturally acquired MMc is a basic aspect of biology as we hypothesize, the proposed work will have created a foundation from which diverse disorders of the human brain can be investigated including conditions that are developmental, autoimmune, degenerative, or neoplastic, underscoring significance.

项目概要/摘要 微嵌合现象 (Mc) 是指含有来自遗传上不同的个体的少量细胞或 DNA。 母亲和孩子的身体结合结束多年后，在她的后代中发现了母亲 Mc (MMc)，并且 母亲中胎儿来源的 MMc 已在儿童和成人的血液和器官中发现，包括心脏、 在实验和人类研究中，Mc 似乎是分化的，从而产生了肝脏、脾脏、肾脏和胰腺。 例如，MMc 是心脏中的心肌细胞、胰腺中的胰岛  细胞和肝脏中的肝细胞。 尽管大脑对人类健康和功能很重要，但对于大脑的认识却存在着根本性的差距。 该提案的总体目的是生成有关 MMc 细胞的基础知识。 人类大脑中的类型、数量和 MMc 转录组 确定 Mc 的起源。 母亲需要母亲的参与，而这通常不适用于儿童尸检 目标 1 有两个。 目标 1 的第 1 部分将调查接受手术切除以接受药物治疗的儿科患者的 MMc。 难治性癫痫，母亲可以参加 HLA 和其他多态性基因分型。 在对患者和母亲进行基因分型后，从口腔拭子样本中提取母体 DNA。 对母子对进行审查，以确定非遗传性、非共享的多态性，即母亲独有的多态性。 接下来从一组 HLA 和其他多态性特异性定量中选择母体特异性检测。 我们为此目的开发了 PCR (qPCR) 检测，然后从切除的脑组织中提取 DNA。 使用为每对母子选择的定制检测来询问 MMc。 被用来研究从接受神经胶质瘤手术的年龄相当的患者身上切除的大脑 目标 1 方法的第二部分将选择男性脑组织进行研究。 通过使用 X 和 Y 染色体特异性探针进行荧光原位杂交 (FISH)；Aim 1 的这方面； 该方法将允许纳入尸检中没有神经系统疾病的儿童的脑组织 不需要母体参与，具有两条 X 染色体信号的雌性细胞（假定为母体）将被加入。 将添加免疫荧光 (IF) 来评估同一区域中计数的 XY 雄性细胞。 在目标 2 中，我们将对脑组织进行单核 RNA 测序 (snRNA-seq) 分析。 snRNA-seq研究将全面评估大脑中的细胞类型。 MMc 并将评估 MMc 转录组 MMc 影响大脑的方式是多种的。 除了为癫痫病提供信息（如果自然）之外，其对儿童的潜在益处和/或损害也是多种多样的。 获得 MMc 是我们所追求的生物学的一个基本方面，拟议的工作将为我们奠定基础 从中可以研究人类大脑的各种疾病，包括发育、 自身免疫性、退行性或肿瘤性，强调了重要性。