重复序列分子标记的筛选鉴定及其在染色体疾病诊断中的应用

Chromosome aneuploidy abnormality is the common cause of abortion and fetal death, therefore, should be to diagnosis early. In the rapid prenatal diagnosis, the aneuploidies of chromosomes 21, 18, 13, X and Y are mainly tested. STR-based quantitative fluorescent PCR (STR-QF-PCR) and multiplex ligation-dependent probe amplification (MLPA) are the most common methods for rapid diagnosis. However, there existed some defects or deficiencies in the detections of rapid prenatal diagnosis. In order to make up the shortages in the existing detection methods, the project intends to use a two copies of segmental duplication (SD) as a new molecule marker to conduct development and application, and plans to adopt the SD molecule to establish a single-tube and quadruple color fluorescence dual relative quantitative PCR (SD-qPCR) detection system which can realize simultaneous detection of four chromosomes by a single-tube to replace the existing rapid prenatal diagnosis. In the rapid detection of apoblema, it is necessary to detect the variations of all 23 pairs of human chromosomes. There is no a simple and stable detection method, this project intends to develop a series of new SD molecular markers, and combined with capillary electrophoresis, the project intends to adopt the segmental duplication quantitative fluorescent PCR (SD-QF-PCR) to simultaneously detect all chromosome numbers of people by a single tube. Thereby, the development of new molecular markers of SD and the establishment of the methodology, provides a new theoretical foundation and diagnostics method for the diagnosis chromosome aneuploidy.

染色体非整倍体异常是孕妇流产及胎儿死亡的常见原因，因此，应及早诊断。在产前快速诊断中，主要检测21号，18号，13号，X和Y染色体的数目异常，定量荧光PCR(STR-QF-PCR)和多重连接探针扩增(MLPA)是最常用的快速诊断方法，但其均需要PCR后处理，检测相对繁琐且价格昂贵，为了建立一种快速而简便的检测方法，本项目拟开发一种新的重复序列分子标记，并将其与qPCR技术相结合，从而建立一种单管四色荧光双重相对定量PCR（SD-qPCR）的检测体系，可实现单管同时检测4条染色体数目；在流产物非整倍体的检测中，需要检测人所有23对染色体的异常，本项目拟将开发的重复序列分子标记与毛细管电泳技术相结合，从而建立一种单管同时检测人所有23对染色体数目的重复序列定量荧光PCR（SD-QF-PCR）。新的重复序列分子标记的开发和方法学的建立将为染色体非整倍体的诊断提供一种新的理论基础和诊断学方法。

本课题开发了一系列的重复序列分子标记，并将其与qPCR技术和QF-PCR技术相结合，从而开发了可以快速诊断13，18，21，X 和Y染色体的SD-qPCR检测技术，以及可以快速检测人所有染色体的SD-QF-PCR技术。SD-qPCR技术可用于产前染色体常见数目异常的检测，而SD-QF-PCR技术可用于人所有染色体数目异常的检测。SD-qPCR和SD-QF-PCR检测体系和检测技术的成功建立和开发，帮助2095例流产患者进行了染色体非整倍体方面的检测，并帮助700余名患者找到了流产方面的遗传学病因。同时，通过分析检测结果与患者表型，还帮助医生和患者明确很多染色体异常与患者表型的相关性。