Exon capture and large-scale sequencing for disease-cause identification, early d

外显子捕获和大规模测序用于疾病原因识别、早期诊断

• 批准号: 7936906
• 负责人: FRIEDHELM HILDEBRANDT
• 金额: $ 49.07万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7936906
• 关键词: Address; Adult; Animal Model; Area; Biological Assay; Cells; Child; Childhood; Chromosome Mapping; Chronic Kidney Failure; Classification; Clinical Data; Clinical Trials; DNA; Data; Development; Diagnostic; Dialysis procedure; Disease; Disease model; Early Diagnosis; Early identification; End stage renal failure; Etiology; Exons; Family; Funding; Gene Mutation; Genes; Genitourinary system; Genome; Genotype; Health; Heterogeneity; Human; Immunologics; Individual; Kidney; Kidney Transplantation; Knock-out; Knowledge; Laboratories; Large-Scale Sequencing; Lead; Life; Maps; Modeling; Molecular; Molecular Genetics; Molecular Target; Mus; Mutation; NPHS2 protein; Nephrotic Syndrome; Pathogenesis; Patients; Pediatric Hospitals; Pharmaceutical Preparations; Phenotype; Pilot Projects; Preclinical Drug Evaluation; Prevention; Rare Diseases; Resources; Role; Sampling; Screening procedure; Steroid Resistance; Stratification; Technology; Testing; Therapeutic; Therapy Clinical Trials; Translational Research; Treatment Cost; Variant; Zebrafish; advanced disease; animal model development; base; clinical practice; cohort; drug discovery; drug testing; gene discovery; high throughput screening; improved; insight; new technology; new therapeutic target; novel; novel strategies; podocyte; young adult; zebrafish development

DESCRIPTION (provided by applicant): This application addresses: Broad Challenge Area: (15) Translational Science Topic: 15-OD (ORDR)-101* Pilot projects for prevention, early detection and treatment of rare diseases. Exon capture and large-scale sequencing for disease-cause identification, early detection and drug discovery in nephrotic syndrome. Background: Chronic kidney diseases (CKD) take one of the highest tolls on human health. They insidiously lead to end- stage kidney disease (ESKD) requiring dialysis or kidney transplantation for survival. 20 million individuals in the U.S. suffer from CKD with a treatment cost of >$20 billion/yr. Steroid-resistant nephrotic syndrome (SRNS) is a rare disease that constitutes the second most frequent cause of ESKD in children and young adults. No curative treatment is available. We demonstrated that many pediatric cases with SRNS are rare recessive single-gene disorders. 25% of all SRNS cases are caused by recessive NPHS2/podocin mutations and many additional single-gene causes of SRNS exist. Identification of rare single-gene causes for SRNS has provided fundamental insights into disease mechanisms of nephrotic syndrome in children and adults. It has allowed to perform unequivocal molecular genetic diagnostics for early detection of SRNS (www.renalgenes.org) and to stratify patient cohorts for therapeutic trials. The study of recessive disease mechanisms is particularly powerful, as they can be recapitulated in animal models by gene knockdown/knockout in zebrafish or mice. These animal models allow for high throughput drug screening to define new molecular targets for treatment. Knowledge Gap and New Technology: The current knowledge gap in disease causes of SRNS has hampered diagnostics, early detection and target- oriented drug treatment for this incurable disease. The heterogeneity of recessive SRNS-causing genes, and their rarity, pose severe limitations to gene identification. Recently, we introduced a new technology into gene discovery of rare recessive single-gene causes by developing a combined approach of homozygosity mapping (HM) with consecutive exon capture and large-scale sequencing. Feasibility and Existing Resources: We have ascertained DNA samples and clinical data from over 2,000 families with SRNS world-wide and have clarified the molecular cause of SRNS in 15% of cases. We applied HM successfully to the identification of 11 novel CKD genes. Recently, we demonstrated that HM is broadly applicable even to single cases with rare diseases. In 30-80% of cases it yielded homozygous candidate regions, which contained the disease causing homozygous mutation in 93% of cases. When identifying by HM recessive PLCE1 mutations as a rare cause of SRNS (~1%), we recapitulated the disease in zebrafish, thereby generating a model for drug screening. By total genome HM in 250 of the 2,000 families with SRNS (using 250k and 1Mill SNP arrays) we yielded new homozygous candidate regions in 100/250 families (40%), and demonstrated that more than 20 additional unknown recessive single-gene causes of SRNS must exist. Thus, we will address the following specific aims: Specific Aims SA1. Identify novel causes of the rare disease steroid-resistant nephrotic syndrome (SRNS) by total genome homozygosity mapping, with consecutive targeted exon capture and large-scale sequencing. SA2. Establish zebrafish models of novel SRNS genes and test new treatment approaches. SA3. Rapidly expand the new strategy of gene identification to other autosomal recessive conditions with a urogenital phenotype. Significance: The new approach that we developed will have the following impact for SRNS and other rare diseases: i) Gene identification will generate novel insights into the etiology and disease mechanisms of SRNS. ii) It will allow screening and early detection for this rare disease and etiologic stratification for clinical trials. iii) Gene identification will generate novel molecular targets to treat SRNS, which currently is incurable. iv) Further development of disease models in zebrafish for SRNS-causing genes will enable high throughput screening assays for drug discovery of novel compounds to treat SRNS. We already demonstrated the feasibility of this approach. The project will expand our successful preliminary results of HM with consecutive targeted exon capture and large-scale sequencing to a large number of families with rare diseases. Following this ARRA, we will enlarge the project (with existing resources) to study all recessive pediatric diseases at the U of M Mott Children's Hospital and beyond. These studies, for which all resources and technologies are in place, would greatly benefit from an influx of funds to quickly and significantly advance disease-cause identification in SRNS and other rare diseases by rapidly generating data using highly efficient, but costly, new technology of exon capture and large-scale sequencing, which we have established. Chronic kidney diseases take one of the highest tolls on human health. Steroid-resistant nephrotic syndrome (SRNS) is a rare disease that constitutes the second most frequent cause of ESKD in children and young adults. No curative treatment is available. 25% of all SRNS cases are caused by recessive NPHS2/podocin mutations, and we recently demonstrated, by genetic mapping, that many additional single-gene causes of SRNS must exist. Identification of rare single-gene causes for SRNS has provided fundamental insights into disease mechanisms of nephrotic syndrome in children and adults. Recently, we introduced a new technology into gene discovery of rare recessive single-gene causes by developing a combined approach of homozygosity mapping with consecutive exon capture and large-scale sequencing. We have ascertained DNA samples and clinical data from over 2,000 families with SRNS world- wide and have clarified the molecular cause of SRNS in 15% of cases. We here propose to 1) Identify novel causes of the rare disease SRNS by total genome homozygosity mapping, with consecutive targeted exon capture and large-scale sequencing; 2) Establish zebrafish models of novel SRNS genes and test novel treatment approaches; and 3) Rapidly expand the new strategy of gene identification to other autosomal recessive conditions with a urogenital phenotype.

描述（由申请人提供）：本申请涉及：广泛的挑战领域：(15) 转化科学主题：15-OD (ORDR)-101* 预防、早期检测和治疗罕见疾病的试点项目。外显子捕获和大规模测序，用于肾病综合征的病因识别、早期检测和药物发现。 背景：慢性肾脏疾病（CKD）是对人类健康造成最大损失的疾病之一。它们会不知不觉地导致终末期肾病（ESKD），需要透析或肾移植才能生存。美国有 2000 万人患有 CKD，每年的治疗费用超过 200 亿美元。类固醇抵抗性肾病综合征 (SRNS) 是一种罕见疾病，是儿童和年轻人中 ESKD 的第二常见原因。没有可用的治疗方法。我们证明许多 SRNS 儿科病例是罕见的隐性单基因疾病。所有 SRNS 病例中 25% 是由隐性 NPHS2/podocin 突变引起的，并且存在许多其他 SRNS 的单基因原因。 SRNS 罕见单基因病因的鉴定为了解儿童和成人肾病综合征的疾病机制提供了基础见解。它允许执行明确的分子遗传学诊断以早期检测 SRNS (www.renalgenes.org) 并对治疗试验的患者群体进行分层。对隐性疾病机制的研究特别有力，因为它们可以通过斑马鱼或小鼠的基因敲低/敲除在动物模型中重现。这些动物模型允许进行高通量药物筛选，以确定新的治疗分子靶点。 知识差距和新技术：目前对 SRNS 病因的知识差距阻碍了这种不治之症的诊断、早期检测和靶向药物治疗。隐性 SRN​​S 致病基因的异质性及其稀有性对基因鉴定造成了严重限制。最近，我们通过开发纯合性作图（HM）与连续外显子捕获和大规模测序的组合方法，将新技术引入罕见隐性单基因病因的基因发现中。 可行性和现有资源：我们已经确定了全球 2,000 多个 SRNS 家庭的 DNA 样本和临床数据，并阐明了 15% 病例中 SRNS 的分子病因。我们成功地将 HM 应用于鉴定 11 个新的 CKD 基因。最近，我们证明 HM 甚至广泛适用于罕见疾病的单一病例。在 30-80% 的病例中，它产生了纯合候选区域，其中包含在 93% 的病例中引起纯合突变的疾病。当通过 HM 隐性 PLCE1 突变确定 SRNS 的罕见病因（~1%）时，我们在斑马鱼中重现了该疾病，从而生成了药物筛选模型。通过 2,000 个 SRNS 家族中 250 个家族的总基因组 HM（使用 250k 和 1Mill SNP 阵列），我们在 100/250 个家族 (40%) 中产生了新的纯合候选区域，并证明了 SRNS 必须存在 20 多个额外的未知隐性单基因原因。因此，我们将解决以下具体目标：具体目标 SA1。通过全基因组纯合性作图、连续靶向外显子捕获和大规模测序，确定罕见疾病类固醇抵抗性肾病综合征 (SRNS) 的新病因。 SA2。建立新型 SRNS 基因的斑马鱼模型并测试新的治疗方法。 SA3。迅速将基因鉴定的新策略扩展到具有泌尿生殖表型的其他常染色体隐性遗传疾病。 意义：我们开发的新方法将对 SRNS 和其他罕见疾病产生以下影响：i) 基因鉴定将为 SRNS 的病因学和疾病机制提供新的见解。 ii) 它将允许对这种罕见疾病进行筛查和早期检测，并为临床试验进行病因分层。 iii) 基因鉴定将产生新的分子靶标来治疗目前无法治愈的 SRNS。 iv) 进一步开发斑马鱼引起 SRNS 基因的疾病模型，将使高通量筛选试验成为可能，以发现治疗 SRNS 的新化合物的药物。我们已经证明了这种方法的可行性。该项目将把我们的HM连续靶向外显子捕获和大规模测序的成功初步成果推广到大量罕见病家庭。在此 ARRA 之后，我们将扩大该项目（利用现有资源），以研究密歇根大学莫特儿童医院及其他地区的所有隐性儿科疾病。这些研究的所有资源和技术都已到位，将极大地受益于大量资金的涌入，通过使用我们建立的高效但昂贵的外显子捕获和大规模测序新技术快速生成数据，快速、显着地推进 SRNS 和其他罕见疾病的病因识别。慢性肾脏疾病是对人类健康造成最大损失的疾病之一。类固醇抵抗性肾病综合征 (SRNS) 是一种罕见疾病，是儿童和年轻人中 ESKD 的第二常见原因。没有可用的治疗方法。所有 SRNS 病例中 25% 是由隐性 NPHS2/podocin 突变引起的，我们最近通过基因图谱证明，必定存在许多其他 SRNS 的单基因原因。 SRNS 罕见单基因病因的鉴定为了解儿童和成人肾病综合征的疾病机制提供了基础见解。最近，我们通过开发纯合性作图与连续外显子捕获和大规模测序的组合方法，将新技术引入罕见隐性单基因病因的基因发现中。我们已经确定了全球 2,000 多个 SRNS 家庭的 DNA 样本和临床数据，并阐明了 15% 病例中 SRNS 的分子病因。我们在此建议：1）通过全基因组纯合性作图、连续靶向外显子捕获和大规模测序，确定罕见病 SRNS 的新病因； 2）建立新型SRNS基因的斑马鱼模型并测试新的治疗方法； 3) 迅速将基因鉴定的新策略扩展到具有泌尿生殖表型的其他常染色体隐性遗传疾病。