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）：此申请地址：广泛的挑战领域：（15）转化科学主题：15-OD（ORDR）-101*预防，早期发现和治疗罕见疾病的试点项目。外显子捕获和大规模测序，用于肾脏综合征中的疾病原因，早期检测和药物发现。 背景：慢性肾脏疾病（CKD）对人类健康造成了最高的通行费之一。它们阴险地导致需要透析或肾脏移植以生存的末期肾脏疾病（ESKD）。在美国，有2000万个人患有CKD，治疗费用> 200亿美元/年。抗类固醇的肾病综合征（SRNS）是一种罕见的疾病，构成了儿童和年轻人中ESKD的第二常见原因。没有可用的治疗方法。我们证明了许多患有SRN的儿科病例是罕见的隐性单基因疾病。所有SRN的病例中有25％是由隐性NPHS2/Podocin突变引起的，并且存在许多其他单基因原因。识别SRN的罕见单基因原因已为儿童和成人的肾病综合征疾病机制提供了基本见解。它允许执行明确的分子遗传诊断，以早期检测SRN（www.enalgenes.org），并分层患者同伴进行治疗试验。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/敲除在动物模型中概括。这些动物模型允许高吞吐药物筛查来定义用于治疗的新分子靶标。 知识差距和新技术：SRN疾病原因的当前知识差距阻碍了这种无法治愈的疾病的诊断，早期发现和面向目标的药物治疗。隐性SRNS引起的基因的异质性及其稀有性对基因鉴定构成了严重的局限性。最近，我们通过开发连续的外显子捕获和大规模测序的纯合性映射（HM）的组合方法来发现稀有隐性单基因原因的基因发现。 可行性和现有资源：我们已经确定了来自全球2,000多家SRN家族的DNA样本和临床数据，并阐明了15％的病例中SRN的分子原因。我们成功地应用于11个新型CKD基因的鉴定。最近，我们证明了HM甚至适用于罕见疾病的单个病例。在30-80％的病例中，它产生了纯合候选区域，其中含有该疾病，导致93％的病例中引起纯合突变。当通过HM隐性PLCE1突变确定为SRN的罕见原因（〜1％）时，我们在斑马鱼中概括了该疾病，从而产生了用于药物筛查的模型。在2,000个SRN家族中的250个（使用250K和1Mill SNP阵列）中，我们在100/250个家庭（40％）中产生了新的纯合候选区域（40％），并证明必须存在20多个SRN的单基因原因。因此，我们将解决以下特定目标：特定目标SA1。通过总基因组纯合绘图来确定罕见疾病抗性肾病综合征（SRN）的新原因，并连续靶向外显子捕获和大规模测序。 SA2。建立新型SRNS基因的斑马鱼模型并测试新的治疗方法。 SA3。快速将基因鉴定的新策略扩展到具有泌尿生殖器表型的其他常染色体隐性疾病。 意义：我们开发的新方法将对SRN和其他罕见疾病产生以下影响：i）基因鉴定将产生对SRNS病因和疾病机制的新见解。 ii）它将允许对这种罕见疾病和病因学分层进行筛查和早期检测。 iii）基因鉴定将产生新的分子靶标，以治疗当前无法治愈的SRN。 iv）斑马鱼中疾病模型的进一步开发用于引起SRNS的基因的疾病模型将使高吞吐量筛查测定法，以便发现新型化合物的药物以治疗SRN。我们已经证明了这种方法的可行性。该项目将通过连续的靶向外显子捕获和大规模测序扩大我们成功的HM的成功初步结果，以扩展到许多罕见疾病的家庭。在此ARRA之后，我们将扩大该项目（使用现有资源），以研究Mott儿童医院及其他地区U的U型小儿疾病。通过大量资金涌入，这些研究将大大受益于SRN和其他罕见疾病的疾病，从而大大受益，通过使用高效但昂贵的外显子捕获和大规模测序的新技术来快速地鉴定疾病，并从中受益。慢性肾脏疾病是人类健康的最高通行费之一。抗类固醇的肾病综合征（SRNS）是一种罕见的疾病，构成了儿童和年轻人中ESKD的第二常见原因。没有可用的治疗方法。所有SRN的病例中有25％是由隐性NPHS2/Podocin突变引起的，我们最近通过遗传学映射证明，必须存在许多其他SRN的单基因原因。识别SRN的罕见单基因原因已为儿童和成人的肾病综合征疾病机制提供了基本见解。最近，我们通过开发连续的外显子捕获和大规模测序的纯合性映射方法来发现罕见隐性单基因原因的基因发现。我们已经确定了来自2,000多个SRNS世界宽的家庭的DNA样品和临床数据，并阐明了15％的病例中SRN的分子原因。我们在这里提议1）通过总基因组纯合绘制映射确定罕见疾病SR​​N的新原因，并连续靶向外显子捕获和大规模测序； 2）建立新型SRN基因和测试新治疗方法的斑马鱼模型； 3）迅速将基因鉴定的新策略扩展到具有泌尿生殖器表型的其他常染色体隐性疾病。