JAUNDICE NEXT: A diagnostic tool for cholestatic liver disease.

描述（由申请人提供）：胆汁淤积（或病理性黄疸）是儿童肝脏疾病最常见的症状，通常由在胆汁形成和运输中起关键作用的基因突变引起。在成人中，相同基因的突变也可引起慢性胆汁淤积，表现为非特异性门脉纤维化、妊娠肝内胆汁淤积和低磷脂相关性胆石症。利用这些疾病遗传基础的最新知识，我们开发了一种高通量基因芯片，可以识别导致最常见的慢性胆汁淤积遗传性综合征的基因突变。这种芯片现在是一种临床测试，越来越多地被医生用作有症状患者诊断算法的一部分。虽然芯片显著地促进了特定的诊断，但其准确性受到无法可靠地检测插入或缺失的限制，这占致病突变的10-20%，从而降低了芯片的灵敏度。在本申请中，我们建议通过开发下一版本的突变筛选工具来解决这一技术差距，我们将其命名为“JAUNDICENEXT”。该工具定制NextGen测序，准确调查目标基因的核苷酸组成，以诊断致病突变。我们的具体目标是“确定JAUNDICENEXT对目标基因测序的准确性”。为了验证假设1“JAUNDICENEXT对目标基因进行测序，准确度为bb0 99%”，我们将比较JAUNDICENEXT产生的核苷酸序列与标准测序方法产生的序列。为了验证假设2“JAUNDICENEXT识别胆汁淤积症患者的插入和缺失突变”，我们将检查由JAUNDICENEXT产生的序列输出是否检测已知携带这些突变的DNA中的插入、缺失或缺失。总的来说，这些实验将验证JAUNDICENEXT的技术优点，并为我们未来的实验奠定基础，以进一步进行实验室测试，并开发一种自动检测算法，以用于未来的ii期应用。

DESCRIPTION (provided by applicant): Cholestasis (or pathologic jaundice) is the most common sign of liver disease in children and often results from mutations in genes that play a critical role in bile formation and transport. In adults, mutations in the same genes also cause chronic cholestasis that may manifest as non-specific portal fibrosis, intrahepatic cholestasis of pregnancy, and low-phospholipid-associated cholelithiasis. Using recent knowledge of the genetic basis of these disorders, we developed a high-throughput gene chip that identifies mutations in the genes that cause the most common forms of inherited syndromes of chronic cholestasis. The chip is now a clinical test that is increasingly used by physicians as part of diagnostic algorithms in symptomatic patients. Although the chip remarkably facilitates specific diagnoses, its accuracy is limited by the inability to reliably detect insertions or deletions, whih account for 10-20% of the disease-causing mutations, thus decreasing the sensitivity of the chip. In this application, we propose to solve this technological gap by developing the next version of a mutation-screening tool that we are naming "JAUNDICENEXT". This tool customizes NextGen sequencing to accurately survey the nucleotide composition of target genes to diagnose disease-causing mutations. Our Specific Aim is "to determine the accuracy of JAUNDICENEXT to sequence the target genes." To test Hypothesis-1 that "JAUNDICENEXT sequences the target genes at >99% accuracy," we will compare the nucleotide sequence produced by the JaundiceNext with the sequence produced by standard sequencing methodology. To test Hypothesis-2 that "JAUNDICENEXT identifies insertion and deletion mutations in patients with cholestasis," we will examine whether the sequence output produced by JAUNDICENEXT detects insertions, deletions, or indels in DNA known to carry these mutations. Collectively, these experiments will validate the technical merits of JAUNDICENEXT and position us for future experiments to further bench-test the assay and develop an automated detection algorithm to in a future Phase-II application. PUBLIC HEALTH RELEVANCE: Jaundice is a clinical sign that is common to several types of chronic liver diseases in children and adults. In a previous STTR, we developed a gene chip to identify mutations in the genes that cause the most common forms of inherited syndromes of persistent jaundice (also known as cholestasis). The chip is now a clinical test that is increasingly used by physicians as part of diagnostic algorithms in symptomatic patients. Although the chip remarkably facilitates specific diagnoses, its accuracy is limited by the inabiliy to reliably detect insertions or deletions, which account for 10-20% of the disease-causing mutations, thus decreasing the sensitivity of the chip. We propose to solve this technological gap by developing the next version of a mutation-screening tool that we are naming "JAUNDICENEXT". This new tool customizes NextGen, a powerful sequencing technology, to accurately detect the wider spectrum of mutations in four genes simultaneously. These studies will improve the sensitivity and accuracy of a non-invasive diagnostic tool that significantly improves the diagnostic algorithm in patients with liver disease.