基于LR-PCR-SMRT技术体系的常染色体显性多囊肾病基因诊断新方法研究

常染色体显性多囊肾病（ADPKD）是一种危害严重的常见单基因遗传病，基因诊断对该病的防治具有重要的临床价值。现行ADPKD基因诊断方法存在读长短、受GC含量影响大、成本高等局限，亟需发展高效、准确、经济的基因检测方法。前期研究中，我们提出了长片段PCR（LR-PCR）直接联合单分子实时（SMRT）测序的新策略，即LR-PCR-SMRT技术体系，实验结果显示该体系技术路线可行，对假基因干扰严重、GC含量高的ADPKD基因诊断有较其它方法更好的检测效果，且具有快速、经济的优势。本项目拟通过优化LR-PCR-SMRT体系参数设置和检测流程，提高检测灵敏度和特异性，降低检测成本，采用回顾性和前瞻性研究策略评估该方法在临床的应用价值，从而建立基于LR-PCR-SMRT技术体系的ADPKD基因诊断新方法，为发挥其重要的临床应用价值奠定科学依据与实验基础，并为其它疑难单基因遗传病的基因诊断研究提供借鉴。

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, which can cause the life-threatening end-stage renal disease. Genetic diagnosis is very important for the prevention and early medical management of the disease. The present methods developed for genetic diagnosis of ADPKD still have some limitations including short reads length, bias caused by GC content and high cost. Therefore, it is necessary to develop efficient, accurate and economic method for molecular diagnosis of ADPKD. In our preliminary study, we presented a new strategy based on long-range PCR (LR-PCR) combined with direct single molecule real time (SMRT) sequencing, and the results showed that the LR-PCR-SMRT procedure is feasible. This approach is suitable for distinguishing PKD1 from the pseudogenes and detecting GC-rich regions. It also has a fast and economic advantage. In this study, we will systematically optimize the setting of system parameters and the testing processes to improve the sensitivity and specificity of our method and lower testing cost. We will perform validation and clinical application in order to establish a novel method based on the LR-PCR-SMRT strategy for genetic diagnosis of ADPKD. Our study will give the scientific evidence for clinical application of the method, and provide a paradigm for genetic diagnosis of other complicated single-gene disorders.