Studies of genetic and metabolic disorders, autism and premature aging

遗传和代谢紊乱、自闭症和过早衰老的研究

• 批准号: 8736898
• 负责人: Owen Rennert
• 金额: $ 144.64万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8736898
• 关键词: 3 year old; Adult; Aging; Animal Model; Architecture; Autistic Disorder; Autopsy; Behavior; Biochemical Process; Biological Markers; Brain; Caring; Cell Culture Techniques; Cell model; Cells; Cerebellum; Characteristics; Child; Child health care; Childhood; Clinic; Clinical; Clinical Assessment Tool; Clinical Protocols; Clinical Research; Clinical Skills; Code; Communication; Complex; Congenital Abnormality; DNA biosynthesis; Development; Diagnosis; Dimensions; Disease; Dysmorphology; Early identification; Embryo; Etiology; Europe; Evaluation; Exhibits; Family; Fibroblasts; Fractals; Functional RNA; Functional disorder; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic; Genetic Counseling; Genetic Transcription; Genomic Instability; Genomics; Health Professional; Hereditary Disease; Hereditary Malignant Neoplasm; Heritability; Heterogeneity; Hippocampus (Brain); Human; Human Development; Impairment; In Vitro; Individual; Inherited; Institutes; Investigation; Lymphocyte; Measures; Medical Students; Mental disorders; Messenger RNA; Metabolic; Metabolic Diseases; Methodology; Mission; Modeling; Molecular; Mutation; National Institute of Child Health and Human Development; Nature; Neuronal Differentiation; Neurons; Optics; Other Genetics; Parkinson Disease; Pathogenesis; Pathway Analysis; Pathway interactions; Patient Care; Patient Care Management; Patients; Pattern; Phenotype; Physicians; Physiologic pulse; Play; Pluripotent Stem Cells; Prefrontal Cortex; Premature aging syndrome; Preventive; Proteins; Protocols documentation; Rattus; Research; Research Training; Risk; Role; Services; Signal Transduction; Skin; Socialization; Stimulus; Subgroup; Symptoms; Synaptic Transmission; Syndrome; System; Techniques; Telomere Maintenance; Testing; Tissues; Training; Training Programs; Transcript; Treatment Effectiveness; United States National Institutes of Health; Untranslated RNA; Variant; Werner Syndrome; autism spectrum disorder; base; brain tissue; clinical phenotype; early onset; embryonic stem cell; graduate student; helicase; human WRN protein; human disease; improved; in vitro Model; induced pluripotent stem cell; mRNA Expression; membrane excitation; mutant; neurobehavioral disorder; neurodevelopment; recombinational repair; relating to nervous system; research and development; senescence; skills; telomere; transforming virus; undergraduate student; ward

Clinical Protocol: This protocol has as its objective the training of clinical fellows, graduate students and health professionals to afford them a broader understanding of heritable diseases. Additionally by seeing patients with "undiagnosed" genetic diseases or birth defects it provides clinical cases for the development of new research protocols at NIH. Studies of Pediatric Patients with Metabolic and other Genetic Disorders Under this protocol we provide care for patients with a variety of rare genetic disorders. In addition, we supplement and offer an opportunity for training in clinical genetics, dysmorphology and metabolic genetics in the National Institute of Child Health and Human Development (NICHD) and other Institutes of the National Institutes of Health (NIH), and spearhead the development of new research protocols on particular aspects of diagnosis and care for specific genetic diseases. Evaluations of patients with a broad spectrum of metabolic and genetic conditions are performed, genetic counseling services are offered to patients and their families to assess risk, and give information on preventive measures, and testing options. Disorders that we studied include chromosomal and Mendelian disorders of childhood and/or adult onset, congenital anomalies and/or birth defects, dysmorphic syndromes, familial cancer syndromes, multifactorial disorders, and metabolic abnormalities. If not eligible for another NICHD research protocol (specific for a disease or a treatment), patients with genetic/metabolic-related conditions may be evaluated under the auspices of this protocol to advance the clinical skills of physicians participating in NICHD clinical research and training programs, and to provide stimuli for new clinical research initiatives. The overall purpose of this protocol is to support our Institutes training and research missions by expanding the spectrum of diseases that can be seen in our clinics and wards. We trained IRTAs, undergraduate and graduate students, medical students, residents, and fellows in the care and management of patients with genetic conditions and their families. Autism Research: Autism spectrum disorder, normally exhibits the onset of symptoms before 3 years of age, and is characterized by severe impairment in reciprocal socialization, impairment in communication skills, and repetitive or restrictive behaviors. It is a heterogeneous condition of multiple etiologies; no precise clinical assessment tools currently allow precise definition between the multiple variants, nor are there biological markers to distinguish these variants. A rise in the number of children identified with autism spectrum disorders, from five to 72 cases per 10,000 children in the USA and Europe, and the absence of definitive forms of therapy have resulted in increased public concern (1). Improved strategies for early identification of specific phenotypic characteristics and biological markers (e.g., electrophysiological changes) hopefully might improve the effectiveness of treatment. The invasive nature of collecting primary neuronal tissue from patients might be circumvented through the use of iPSC and their subsequent neuronal differentiation. With the successful reprogramming of human fibroblasts into ES cell‐like state (aka induced pluripotent stem cells, iPSC) by Yamanaka et al in 2007 (2), this methodology has subsequently been successfully employed to derive cultured neural cells from patients with ALS, Parkinson disease, and other disorders (3). These breakthroughs make it possible for us to generate a cell culture model of autism spectrum disorder by application of iPSC reprogramming of human fibroblasts and subsequent neural differentiation. In this study, fibroblast cultures from patients (subject with autism), and non-affected controls have been established; subsequently these cells are reprogrammed into an ES cell-like state (aka induced pluripotent stem cells, iPSC). The reprogrammed cell colonies are cloned, propagated, and induced to differentiate in vitro into neuronal cultures. Based on our underlying assumption that synaptic transmission is aberrant in autism, these patient-specific neuronal cultures will be utilized for neuronal network analysis by using the photoconductive-stimulation system described in Gutierrez et al. Briefly, spontaneous or pulse-stimulated activity of networks is measured by optical techniques, and the structural basis of these patterns will analyzed by fractal dimension analysis. By use of these approaches we have the capacity to characterize the arrangement and complexity of their axonal architecture. This approach has been employed to demonstrate differences in hippocampal cultures of a rat model carrying the neurolignin mutation R471C‐NL3 which has been identified in a subgroup of patients with autism spectrum disorders. This study represents the attempt to evaluate membrane excitation and signal transduction in neural cells derived from patients with autism. The autism spectrum disorders (ASD) have a significant hereditary component, but the implicated genetic loci are heterogeneous and complex. Consequently, there is a gap in understanding how diverse genomic aberrations all result in one clinical ASD phenotype. Gene expression studies from autism brain tissue have demonstrated aberrantly expressed protein-coding genes may converge onto common molecular pathways, potentially reconciling the strong heritability and shared clinical phenotypes with the genomic heterogeneity of the disorder. However, the regulation of gene expression is extremely complex and governed by many mechanisms, including noncoding RNAs. Yet no study in ASD brain tissue has assessed for changes in regulatory long non-coding RNAs (lncRNAs), which represent a large proportion of the human transcriptome, and actively modulate mRNA expression. To assess if aberrant expression of lncRNAs may play a role in the molecular pathogenesis of ASD, we profiled over 33,000 annotated lncRNAs and 30,000 mRNA transcripts from postmortem brain tissue of autistic and control prefrontal cortex and cerebellum by microarray. We detected over 200 differentially expressed lncRNAs in ASD, which were enriched for genomic regions containing genes related to neurodevelopment and psychiatric disease. Additionally, comparison of differences in expression of mRNAs between prefrontal cortex and cerebellum within individual donors showed ASD brains had more transcriptional homogeneity. Moreover, this was also true of the lncRNA transcriptome. Our results suggest that further investigation of lncRNA expression in autistic brain may further elucidate the molecular pathogenesis of this disorder. Premature Aging Syndromes: The diseases of premature aging in human are characterized by the early onset of aging phenotypes that are now known to be caused by mutations of different genes. Werner syndrome (WS) is an adult progeroid syndrome caused by mutations of the RecQ helicase WRN. WRN has been implicated in a variety of biochemical processes including DNA replication, repair, recombination, telomere maintenance and transcription. Loss of the WRN protein results in genomic instability and dysfunctional telomeres. Skin fibroblasts from WS patients demonstrate reduced replication potential and accelerated senescence in culture, possibly due to the dysfunctional telomeres. Previous studies on the pathogenesis of WS were limited to skin fibroblasts or virus-transformed lymphocytes. Secondly, animal models of WRN mutant cannot accurately recapitulate the WS phenotype observed in humans. Reprogramming of WS cells to iPSCs may provide a cell model for the study of the pathogenesis, especially for the differentiation of WS embryonic and adult st

临床方案：该方案的目标是培训临床研究员、研究生和卫生专业人员，使他们对遗传性疾病有更广泛的了解。此外，通过查看患有“未确诊”遗传性疾病或出生缺陷的患者，它为 NIH 制定新的研究方案提供了临床案例。 患有代谢性疾病和其他遗传性疾病的儿科患者的研究 根据该协议，我们为患有各种罕见遗传性疾病的患者提供护理。 此外，我们还为国家儿童健康和人类发展研究所 (NICHD) 以及美国国立卫生研究院 (NIH) 的其他研究所提供临床遗传学、畸形学和代谢遗传学培训的机会，并带头制定针对特定遗传疾病的诊断和护理特定方面的新研究方案。对患有广泛代谢和遗传疾病的患者进行评估，为患者及其家人提供遗传咨询服务以评估风险，并提供有关预防措施和检测选项的信息。我们研究的疾病包括儿童和/或成人发病的染色体和孟德尔疾病、先天性异常和/或出生缺陷、畸形综合征、家族性癌症综合征、多因素疾病和代谢异常。如果不符合另一项 NICHD 研究方案（特定于某种疾病或治疗），患有遗传/代谢相关疾病的患者可以在本方案的支持下进行评估，以提高参与 NICHD 临床研究和培训项目的医生的临床技能，并为新的临床研究举措提供刺激。该协议的总体目的是通过扩大我们诊所和病房中可见的疾病范围来支持我们研究所的培训和研究任务。我们对 IRTA、本科生和研究生、医学生、住院医师和研究员进行了有关遗传性疾病患者及其家人护理和管理的培训。 自闭症研究： 自闭症谱系障碍通常在 3 岁之前出现症状，其特征是严重的相互社交障碍、沟通技巧障碍以及重复或限制性行为。它是多种病因的异质性疾病；目前没有精确的临床评估工具可以精确定义多种变异，也没有生物标记来区分这些变异。在美国和欧洲，患有自闭症谱系障碍的儿童数量从每 10,000 名儿童 5 例增加到 72 例，并且缺乏明确的治疗方法，导致公众日益关注 (1)。早期识别特定表型特征和生物标志物（例如电生理变化）的改进策略有望提高治疗效果。通过使用 iPSC 及其随后的神经元分化，可以避免从患者身上收集原代神经元组织的侵入性。随着 Yamanaka 等人于 2007 年成功将人类成纤维细胞重编程为 ES 细胞样状态（又名诱导多能干细胞，iPSC）(2)，该方法随后已成功用于从 ALS、帕金森病和其他疾病患者中培养神经细胞 (3)。这些突破使我们能够通过应用 iPSC 重编程人类成纤维细胞和随后的神经分化来生成自闭症谱系障碍的细胞培养模型。在这项研究中，建立了来自患者（自闭症受试者）和未受影响对照的成纤维细胞培养物；随后这些细胞被重新编程为 ES 细胞样状态（又名诱导多能干细胞，iPSC）。重新编程的细胞集落被克隆、增殖并诱导在体外分化为神经元培养物。基于我们的基本假设，即自闭症中突触传递是异常的，这些患者特异性神经元培养物将通过使用 Gutierrez 等人中描述的光电导刺激系统用于神经元网络分析。简而言之，通过光学技术测量网络的自发或脉冲刺激活动，并通过分形维数分析来分析这些模式的结构基础。通过使用这些方法，我们有能力表征其轴突结构的排列和复杂性。这种方法已被用来证明携带神经木质素突变 R471C-NL3 的大鼠模型海马培养物的差异，该突变已在自闭症谱系障碍患者亚组中发现。 这项研究代表了评估自闭症患者神经细胞膜兴奋和信号转导的尝试。 自闭症谱系障碍（ASD）具有显着的遗传成分，但所涉及的遗传位点是异质且复杂的。因此，对于不同的基因组畸变如何导致一种临床 ASD 的理解存在差距。 表型。自闭症脑组织的基因表达研究表明，异常表达的蛋白质编码基因可能会汇聚到共同的分子途径上，从而可能协调强遗传性和共同的临床表型 与疾病的基因组异质性。然而，基因表达的调控极其复杂，受多种机制控制，包括非编码RNA。然而，尚无针对自闭症谱系障碍 (ASD) 脑组织的研究评估过 调节性长非编码 RNA (lncRNA) 占人类转录组的很大一部分，并积极调节 mRNA 表达。为了评估 lncRNA 的异常表达是否可能在 ASD 的分子发病机制中发挥作用，我们 通过微阵列分析了来自自闭症患者和对照前额皮质和小脑死后脑组织的超过 33,000 个带注释的 lncRNA 和 30,000 个 mRNA 转录本。我们在 ASD 中检测到了 200 多个差异表达的 lncRNA，这些 lncRNA 富含包含与神经发育和精神疾病相关基因的基因组区域。此外，对个体供体中前额皮质和小脑之间 mRNA 表达差异的比较表明，自闭症谱系障碍 (ASD) 大脑具有更多的转录同质性。此外，lncRNA转录组也是如此。我们的结果表明，进一步研究自闭症大脑中 lncRNA 的表达可能会进一步阐明这种疾病的分子发病机制。 过早衰老综合症： 人类过早衰老疾病的特征是衰老表型提前出现，目前已知这些衰老表型是由不同基因的突变引起的。沃纳综合征 (WS) 是一种由 RecQ 解旋酶 WRN 突变引起的成人早衰综合征。 WRN 参与多种生化过程，包括 DNA 复制、修复、重组、端粒维持和转录。 WRN 蛋白的缺失会导致基因组不稳定和端粒功能障碍。 WS 患者的皮肤成纤维细胞在培养中表现出复制潜力降低并加速衰老，这可能是由于端粒功能失调所致。 先前对WS发病机制的研究仅限于皮肤成纤维细胞或病毒转化的淋巴细胞。其次，WRN 突变体的动物模型无法准确重现在人类中观察到的 WS 表型。将WS细胞重编程为iPSC可能为研究WS的发病机制，特别是WS胚胎和成体的分化提供细胞模型。