Unitat de Biologia Cel·lular i Genètica Mèdica, Facultat de Medicina, Departament de Biologia Cel·lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Congenital Coagulopathies, Blood and Tissue Bank, Barcelona, Spain.
PLoS One. 2018 Oct 17;13(10):e0205692. doi: 10.1371/journal.pone.0205692. eCollection 2018.
In families at risk from monogenic diseases affected offspring, it is fundamental the development of a suitable Double Factor Preimplantation Genetic Testing (DF-PGT) method for both single-gene analysis and chromosome complement screening. Aneuploidy is not only a major issue in advanced-maternal-age patients and balanced translocation carriers, but also the aneuploidy rate is extremely high in patients undergoing in vitro fertilization (IVF), even in young donors. To adequate NGS technology to the DF-PGT strategy four different whole genome amplification systems (Sureplex, MALBAC, and two multiple displacement amplification systems-MDA) were tested using TruSight One panel on cell lines and blastocyst trophectoderm biopsies-TE. Embryo cytogenetic status was analyzed by Nexus software. Sureplex and MALBAC DNA products were considered not suitable for PGT diagnosis due to inconsistent and poor results on Trusight one (TSO) panel. Results obtained with both MDA based methods (GEH-MDA and RG-MDA) were appropriate for direct mutation detection by TSO NGS platform. Nevertheless, RG-MDA amplification products showed better coverage and lower ADO rates than GEH-MDA. The present work also demonstrates that the same TSO sequencing data is suitable not only for the direct mutation detection, but also for the indirect mutation detection by linkage analysis of informative SNPs. The present work also demonstrates that Nexus software is competent for the detection of CNV by using with TSO sequencing data from RG-MDA products, allowing for the whole cytogenetic characterization of the embryos. In conclusion, successfully development of an innovative and promising DF-PGT strategy using TSO-NGS technology in TE biopsies, performed in-house in a single laboratory experience, has been done in the present work. Additional studies should be performed before it could be used as a diagnostic alternative in order to validate this approach for the detection of chromosomal aneuploidies.
在受单基因疾病影响的家族中，对于受影响的后代，开发一种合适的双因素胚胎植入前遗传学检测（DF-PGT）方法，用于单基因分析和染色体互补筛查，这是至关重要的。非整倍体不仅是高龄产妇和平衡易位携带者的主要问题，而且在体外受精（IVF）患者中，非整倍体率极高，即使是年轻供体也是如此。为了使 NGS 技术适应 DF-PGT 策略，使用 TruSight One 面板在细胞系和囊胚滋养外胚层活检-TE 上测试了四种不同的全基因组扩增系统（Sureplex、MALBAC 和两种多重置换扩增系统-MDA）。胚胎细胞遗传学状态通过 Nexus 软件进行分析。Sureplex 和 MALBAC DNA 产物由于在 Trusight one（TSO）面板上的结果不一致和较差，被认为不适合 PGT 诊断。基于两种 MDA 方法（GEH-MDA 和 RG-MDA）的结果适合通过 TSO NGS 平台直接进行突变检测。然而，RG-MDA 扩增产物的覆盖度更好，ADO 率低于 GEH-MDA。本工作还表明，相同的 TSO 测序数据不仅适合直接突变检测，还适合通过有信息 SNP 的连锁分析进行间接突变检测。本工作还表明，Nexus 软件通过使用 RG-MDA 产物的 TSO 测序数据，通过检测 CNV 是胜任的，这允许对胚胎进行全细胞遗传学特征分析。总之，在本工作中，在单个实验室经验中，成功地开发了一种使用 TSO-NGS 技术在 TE 活检中进行的创新且有前途的 DF-PGT 策略。为了验证这种方法在检测染色体非整倍体方面的应用，还需要进行更多的研究，以便将其作为一种诊断替代方法进行验证。
