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脏疾病，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，这些疾病在美国构成了巨大的疾病负担，并对受影响患者的临床评估构成了诊断挑战。在一项I期研究中，我们开发了JAUNDICENEXT，这是一个测序平台，结合了短程和远程pcr来产生目标基因的扩增子，生成患者特异性文库，并通过下一代技术进行测序，成功识别遗传性肝内胆汁淤积综合征患者的突变。然后，我们开始实验，进一步提高测序平台的功效和范围，以更好地满足遗传性肝病患者的诊断需求。在该应用程序的初步研究中，我们进行了原理验证实验，证明了创新测序平台的可行性和技术优点，该平台使用简化的两步方案，将针对个体患者的特定多路复用化学编码与下一代技术的即时测序相结合。在展示了这个新平台的技术优点之后，我们建议通过创建LIVERCHIP来扩大基因覆盖范围，LIVERCHIP是一个高通量测序平台，可以筛选表现为病理性黄疸、慢性肝损伤和肝脏囊性疾病的12种基因突变，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进行实验室测试，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TruSeq多路复用化学的开发和验证开始，该化学能够在两步检测中准确地测序12个肝脏基因。随后将对一组表型良好的患者对每一种目标疾病进行实验测试。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、成本远低于现有检测方法的检测方法，对肝脏诊断产生积极影响。这些功能将使最终产品在临床环境中用户友好，并增加其市场销路。

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.