LIVERCHIP - A diagnostic tool for genetic liver diseases

描述(由申请人提供)：已知基因突变可导致急性和慢性肝病，并且越来越多地被认为是表型严重程度的驱动因素。作为一个群体，这些疾病在美国代表着巨大的疾病负担，并构成了对受影响患者的临床评估中的诊断挑战。在第一阶段奖中，我们开发了JAundICENEXT，这是一个测序平台，结合了短程和长程PCR来产生目标基因的扩增产物，生成患者特有的文库，并通过下一代技术进行测序，成功地识别了患有遗传性肝内胆汁淤积症的受试者的突变。随后，我们开始进行实验，进一步提高测序平台的效率和范围，更好地满足遗传性肝病患者的诊断需求。在这项应用的初步研究中，我们进行了原理验证实验，证明了使用简化的两步方案的创新测序平台的可行性和技术优势，该方案将为个别患者编码的特定多路化学与下一代技术的立即测序相结合。在展示了这个较新平台的技术优势后，我们建议通过创建LIVERCHIP来扩大基因覆盖范围，这是一个高通量测序平台，可以筛选12个基因的突变，这些基因表现为病理性黄疸、慢性肝损伤和肝囊性疾病，从而满足更广泛人群的需求。为此，我们提出了三个互补的目标：1)将JAundICENEXT转换为LIVERCHIP作为扩展的诊断工具，2)在慢性遗传性肝病患者中对LIVERCHIP进行台架测试，以及3)开发一种分析工具，优化LIVERCHIP的突变检测。我们的实验策略将从开发和验证TruSeq多路复用化学物质开始，该化学物质能够在两步分析中准确地对12个肝脏基因进行测序。随后将针对每一种目标疾病的一组良好表型患者对该分析进行台架测试。最后，我们将开发一个浏览器，它将减少分析核苷酸读数所需的时间，从而减少诊断时间。我们的最终目标是通过生产一种准确、分析时间短、成本比目前可用的测试低得多的分析方法，积极影响基于肝脏的诊断。这些功能将使最终产品在临床环境中用户友好，并增加其市场适销性。

DESCRIPTION (provided by applicant): Genetic mutations are known to cause acute and chronic liver diseases, and are being increasingly recognized as drivers of phenotype severity. As a group, these diseases represent a substantial disease burden in the United States, and constitute a diagnostic challenge in the clinical evaluation of affected patients. In a Phase I Award, we developed the JAUNDICENEXT, a sequencing platform that combines short- and long-range PCRs to produce amplicons of target genes, the generation of patient-specific libraries, and sequencing by next-generation technologies to successfully identify mutations in subjects with inherited syndromes of intrahepatic cholestasis. Then, we began experiments to further improve the efficacy and scope of the sequencing platform to better meet the diagnostic needs of patients with genetic liver diseases. In preliminary studies for this application, we performed proof-of-principle experiments that demonstrated the feasibility and technical merit of an innovative sequencing platform using a simplified 2- step protocol that combines specific multiplexing chemistry coded for individual patients with an immediate sequencing by next-generation technology. Having demonstrated the technological merit of this newer platform, we propose to expand the gene coverage by creating the LIVERCHIP, a high-throughput sequencing platform that screens for mutations in 12 genes that manifest as pathologic jaundice, chronic liver injury, and cystic diseases of the liver, thus meeting the needs of a broader population. To this end, we propose three complementary aims to: 1) transition the JAUNDICENEXT to the LIVERCHIP as an expanded diagnostic tool, 2) bench-test the LIVERCHIP in patients with chronic inheritable liver diseases, and 3) to develop an analytical tool that optimizes mutation detection by the LIVERCHIP. Our experimental strategy will begin with the development and validation of a TruSeq multiplexing chemistry that enables the sequencing of 12 liver genes accurately in a 2-step assay. This will be followed by bench-testing the assay against a group of well-phenotype patients for each one of the target diseases. Last, we will develop a browser that will decrease the time required for the analysis of the nucleotide readout, thus decreasing the time-to-diagnosis. Our ultimate goal is to positively impact liver-based diagnostics by producing an assay that is accurate, requires short analytical time, and at much lower cost than currently available tests. These features will make the end-product user friendly in the clinical setting and increase its marketability.