Long-read DNA and RNA sequencing to identify disease-causing genetic variation and streamline testing

长读长 DNA 和 RNA 测序可识别致病遗传变异并简化测试

• 批准号: 10704054
• 负责人: Danny Erwin Miller
• 金额: $ 38.88万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-13 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10704054
• 关键词: Adoption; Appointment; Award; Biological Assay; Biological Process; Caring; Clinic; Clinic Visits; Clinical; Collaborations; Communities; Complex; Cost Savings; Critical Illness; DNA Insertion Elements; DNA sequencing; Data; Data Set; Data Sources; Diagnosis; Diagnostic; Diagnostic tests; Disease; Evaluation; Family; Funding; Gene Duplication; Gene Expression; Genetic; Genetic Diseases; Genetic Variation; Genomic Segment; Genomics; Goals; Health system; Healthcare Systems; Individual; Institution; International; Investments; Laboratories; Medical; Medical Genetics; Mendelian disorder; Mentors; Messenger RNA; Methods; Methylation; Nucleotides; Outpatients; Parents; Patients; Pediatric Hospitals; Persons; Postdoctoral Fellow; Precision therapeutics; Protein Isoforms; Research; Research Personnel; Residencies; Sensitivity and Specificity; Spliced Genes; Technology; Testing; Time; Training; Universities; Variant; Vision; Washington; Work; clinical diagnostics; clinical practice; cohort; complex data; exome; exome sequencing; experience; experimental study; genetic disorder diagnosis; genetic testing; genome sequencing; graduate student; human disease; human genome sequencing; imprint; interest; meetings; nanopore; neonate; new technology; novel; novel therapeutics; operation; pediatric department; precision medicine; prediction algorithm; professor; prognostic; rare genetic disorder; research clinical testing; skills; success; transcriptome sequencing; variant detection; whole genome

PROJECT SUMMARY/ABSTRACT Clinical genetic testing, including exome sequencing, has diagnosed many rare and novel genetic disorders. These diagnoses have helped guide treatment, suggest novel or precision therapies, and provide long-term prognostic information for individuals and their families. However, at least 50% of individuals with a suspected genetic disorder remain undiagnosed after a complete clinical evaluation, which can take years to complete. There are two major technical reasons for this low solve rate. First, structural variants such as large deletions, insertions, inversions, and repeat expansions account for a significant fraction of unsolved cases, but they are challenging to analyze using traditional short-read sequencing technologies. Second, many variants lie in recently duplicated genomic regions that are difficult to evaluate using short-read sequencing technologies. Recently, it has become economically feasible to pursue whole-genome long-read sequencing (LRS) of human genomes with sufficient coverage to analyze these complex genomic regions. This presents an opportunity to systematically evaluate the incremental diagnostic rate of LRS over current testing approaches and apply LRS technology to a large cohort of individuals with unsolved genetic disorders. Moreover, LRS is unique in that as a single test it has the potential to replace nearly all existing clinical genetic testing (microarray, exome, methylation studies), making it possible to perform a complete clinical genetic evaluation in days to weeks using a single dataset. The overall goal of this project is to establish a framework for the clinical adoption of long-read DNA and RNA sequencing by systematic evaluation of individuals with suspected or unsolved genetic disorders. To achieve this goal, LRS will be performed on 50 critically ill neonates and their parents who had nondiagnostic trio short-read whole-genome sequencing (WGS) and 25 trios from the same cohort who had diagnostic WGS (Aim 1). This will help establish the incremental diagnostic rate of LRS over short-read WGS. To evaluate the technical advantages of LRS over traditional clinical testing, I will sequence 50 individuals with a suspected genetic disorder who remain unsolved after at least clinical exome sequencing (Aim 2). In up to 20 persons that remain undiagnosed after LRS, long-read RNA sequencing will be used to identify high-priority regions that influence gene expression or splicing for further analysis. I will then evaluate the sensitivity of LRS as a single genetic test by sequencing 50 individuals undergoing a typical outpatient clinical evaluation (Aim 3). Demonstrating that LRS can be used as a single data source will help streamline the current stepwise approach to clinical testing, leading to an increase in the diagnostic rate and an overall cost savings to the health system through reduced testing and clinic visits. Overall, the aims outlined in this proposal will benefit patients and families by increasing the rate of genetic diagnosis, decreasing the time to diagnosis, and identifying novel disease-causing mechanisms, which will lead to a better understanding of the causes of human disease.

项目摘要/摘要 临床基因检测，包括外显子组测序，已诊断出许多罕见和新颖的遗传疾病。 这些诊断有助于指导治疗，提出新颖或精确的疗法，并提供长期 个人及其家人的预后信息。但是，至少有50％的人有可疑的人 经过完整的临床评估后，遗传疾病仍未诊断，这可能需要数年才能完成。 这种低解决率有两个主要的技术原因。首先，结构变体，例如大删除， 插入，反转和重复扩展占未解决案例的很大一部分，但它们是 使用传统的短阅读测序技术进行分析的挑战。其次，许多变体都在 最近重复的基因组区域很难使用短阅读测序技术评估。 最近，追求人类的全基因组长阅读测序（LRS）在经济上变得可行 具有足够覆盖的基因组来分析这些复杂的基因组区域。这是一个机会 系统地评估LRS在当前测试方法上的增量诊断率并应用LRS 对大量未解决遗传疾病的人的技术。而且，LRS是独一无二的 单个测试有可能替代几乎所有现有的临床基因检测（微阵列，外显，甲基化） 研究），使得使用单个单一的几天到几周进行完整的临床遗传评估 数据集。该项目的总体目标是建立一个临床采用长阅读DNA的框架和 通过对具有可疑或未解决遗传疾病的个体的系统评估进行RNA测序。到 实现这一目标，LRS将在50个重病的新生儿及其父母进行非诊断的父母上进行 来自具有诊断WGS的同一队列的三人组短阅读全基因组测序（WGS）和25个三重奏 （目标1）。这将有助于在短读WGS上确定LRS的增量诊断率。评估 LRS比传统临床测试具有技术优势，我将对50个人进行序列，可疑 至少在临床外显子组测序后仍未解决的遗传疾病（AIM 2）。最多20人 在LRS后保持未诊断，将使用长阅读的RNA测序来识别高优先级区域 影响基因表达或剪接以进行进一步分析。然后，我将评估LRS的灵敏度 通过对50个接受典型门诊临床评估的人进行测序（AIM 3）。 证明LR可以用作单个数据源将有助于简化当前的逐步方法 进行临床测试，导致诊断率提高，并节省卫生系统的总体成本 通过减少测试和诊所就诊。总体而言，该提案中概述的目的将使患者受益，并且 通过增加遗传诊断率，减少诊断时间并确定新颖的家族 引起疾病的机制，这将使人们更好地了解人类疾病的原因。

项目成果

Characteristics, Genetic Testing, and Diagnoses of Infants with Neonatal Encephalopathy Not Due to Hypoxic Ischemic Encephalopathy: A Cohort Study.

非缺氧缺血性脑病所致新生儿脑病婴儿的特征、基因检测和诊断：队列研究。

• DOI: 10.1016/j.jpeds.2023.113533
• 发表时间: 2023
• 期刊: The Journal of pediatrics
• 作者: Lenahan,Arthur;Mietzsch,Ulrike;Wood,ThomasR;Callahan,KatharinePress;Weiss,ElliottM;Miller,DannyE;German,Kendell;Natarajan,Niranjana;Puia-Dumitrescu,Mihai;Esposito,Valentine;Kolnik,Sarah;Law,JanessaB
• 通讯作者: Law,JanessaB

Panels, Exomes, Genomes, and More-Finding the Best Path Through the Diagnostic Odyssey.

面板、外显子组、基因组等——通过诊断奥德赛寻找最佳路径。

• DOI: 10.1016/j.pcl.2023.06.001
• 发表时间: 2023
• 期刊: Pediatric clinics of North America
• 影响因子: 2.6
• 作者: Lenahan,ArthurL;Squire,AudreyE;Miller,DannyE
• 通讯作者: Miller,DannyE

Subdural hemorrhage, macrocephaly, rash, and developmental delay in an infant: A pathogenic variant in NLRP3 causes CINCA/NOMID.

婴儿硬膜下出血、大头畸形、皮疹和发育迟缓：NLRP3 的致病性变异导致 CI​​NCA/NOMID。

• DOI: 10.1002/ajmg.a.63366
• 发表时间: 2023
• 期刊: American journal of medical genetics. Part A
• 作者: Koti,AjayS;Lanis,Aviya;Finlayson,Samuel;Canny,Susan;Feldman,ElanaA;Miller,DannyE;Rosenwasser,Natalie;Scott,AbbeyA;Wong,StephenC;Feldman,KennethW
• 通讯作者: Feldman,KennethW

Congenital pleuropulmonary blastoma in a newborn with a variant of uncertain significance in DICER1 evaluated by RNA-sequencing.

• DOI: 10.1186/s40748-023-00148-2
• 发表时间: 2023-03-16
• 期刊: Maternal health, neonatology and perinatology