基于相似序列高分辨熔解曲线分析的流产物染色体数目异常快速诊断新技术的研究

Numerical chromosomal abnormalities (NCAs), are responsible for 50%-80% of first trimester miscarriages. The characterization of such chromosomal abnormalities can aid in the identification of the causes underlying a miscarriage and assess the risk of complications in subsequent pregnancies, such as having recurrent miscarriages or viable abnormal offspring. Although there are several cytogenetic or molecular methods such as karyotyping and fluorescence in situ hybridization, can detect NCAs in spontaneous abortions (SA), some limitations remain to be solved in clinical applications. Recently, we have developed a novel molecular method called “high-resolution melting analysis of segmental duplications” (SD-HRM) which can clearly differentiate samples of patients with common aneuploidies from those of unaffected controls, while markedly simplifying assay setup and analysis, and reducing time and costs compared to more conventional methods.The results had published in Clinical Chemistry.In the present study, we will establish a SD-HRM based system for the detection of 13 major types of NCA in SA. Furthermore, we will also evaluate the diagnostic sensitivity and specificity of this system by detecting 1000 SA samples using karyotyping, FISH, array-CGH as control methods.Detection system we established in this study will provide an approach which undoubtedly improve the efficiency and effectiveness of genetic testing for SA, and would also provide experimental and theoretical clues for the understanding of the embryonic development and pregnancy mechanism.

自然流产中50%-80%是因为胚胎染色体数目异常导致胎儿发育障碍所致。流产物的遗传检测，对流产病因机制研究和再发风险评估均具有重要意义。目前以核型分析为主的多种技术，由于检测通量或成本的限制，均难以满足日益增长的临床和科研需求。前期，本课题组发展出一种相似序列高分辨熔解曲线技术，可完成对染色体非整倍体的快速产前诊断，相关成果已发表在《Clinical Chemistry》上。本研究拟以该技术为基础，根据流产物中不同染色体数目异常的发生频率，建立一种简单高效地诊断流产物中13种主要染色体数目异常的分子检测体系。通过对1000例临床流产物样本核型分析、FISH或Array-CGH检测结果的对照分析，评估该体系的检测特异性、灵敏度和重复性，为该方法的推广应用奠定基础，以为临床胚胎发育和妊娠机制研究提供更为有力的技术支撑。

流产物遗传检测对临床明确流产原因、指导下一次妊娠有着重要的指导意义。同时，对其的分析还可为胚胎发育和妊娠流产的病因机制研究提供重要的实验数据。但由于当前各种检测技术的限制，目前流产物遗传检测一直难以成为临床常规，相关报道也仅限于小样本数据，制约了相关研究的进展。 .因此，发展出一种简单、快速、廉价，同时可覆盖绝大多数染色体数目异常的检测技术，对临床流产原因的确认和病理机制研究应具有重要的意义。如必要的话，以该技术作为初期的广度筛检手段，结合Array-CGH的深度分析，应可完成对流产物组织较全面的遗传分析。获得的数据除可指导临床诊治外，还可为胚胎发育和妊娠研究提供实验数据和理论指导。.我们发展了一项基于高分辨熔解曲线（HRM）的技术应用于流产物中染色体数目异常的快速检测，能够准确有效地区分和检测各类染色体数目异常包括多倍体、嵌合体等，获得了96.1%的灵敏度和96.3%的特异性。我们还对该方法进行了成本效益分析。该检测手段如能与Array-CGH或NGS配合使用，有望在操作简便性、性价比、检测通量等多个方面同时满足目前我国临床流产物检测的需求，并使得流产物的大样本遗传检测成为可能，为临床胚胎发育和妊娠机制研究提供更为有力的技术支撑。