Center for Reproduction and Genetics, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Suzhou, China.
Peking Jabrehoo Med-Tech Co., Ltd, Beijing, China.
Reprod Sci. 2024 Jul;31(7):2123-2134. doi: 10.1007/s43032-024-01470-6. Epub 2024 Feb 12.
It is challenging to distinguish embryos with a balanced translocation karyotype from a normal karyotype by existing conventional genetic testing methods. However, in germ-cell gamete generation, chromosome exchange and separation through cell meiosis form a different proportion of unbalanced gametes. Adverse birth events may occur, such as repeated miscarriages and fetal birth defects. In this study, the exact breakpoints of structural variation (SV) from two balanced translocation carrier families by using Nanopore long reads sequencing technology were obtained, and haplotype analysis and Sanger verified the accuracy of the detection results, confirming the application value of the Nanopore sequencing technology in the detection of balanced translocation before embryo implantation. Nanopore long-read sequencing was performed to find the precise breakpoint of chromosome-balanced translocation carriers. The breakpoints were subsequently verified by designing primers across the breakpoints and Sanger sequencing. Haplotype linkage analysis of SNPs which can be linked by a read block of families around the breakpoint regions was followed. After frozen (-thawed) embryo transfer (FET), prenatal cytogenetic analysis of amniotic fluid cells confirmed the predicted karyotypes from the transferred embryos. The presence of breakpoints was detected in three embryos of patient 1. No breakpoints were detected in either embryo of patient 2. One balanced translocated embryo from patient 1 and one normal euploid embryo from patient 2 were transplanted back into the patients, and amniotic fluid cells were analyzed for the karyotype of fetuses. The results were entirely consistent with the fetal karyotype. And through late follow-up, both patients successfully had a live birth fetus. The breakpoint location of the balanced chromosome translocation can be accurately found by Nanopore sequencing. The haplotype of carriers can be successfully constructed by Nanopore and sanger sequencing confirmed that the results were accurate. This is very advantageous for preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) detection in the families without proband.
利用纳米孔长读测序技术获得 2 个平衡易位携带者家系的结构变异（SV）确切断点，通过设计跨越断点的引物并进行 Sanger 测序对检测结果进行验证，证实了纳米孔测序技术在胚胎植入前平衡易位检测中的应用价值。对染色体平衡易位携带者进行纳米孔长读测序，寻找精确的断点。随后通过设计跨越断点的引物并进行 Sanger 测序来验证断点。对断点周围家系的 SNP 进行单倍型连锁分析，这些 SNP 可以通过读段链接。冷冻（解冻）胚胎移植（FET）后，对羊水细胞进行产前细胞遗传学分析，以确认从移植胚胎中预测的核型。在患者 1 的 3 个胚胎中检测到断点。患者 2 的两个胚胎中均未检测到断点。将患者 1 的一个平衡易位胚胎和患者 2 的一个正常二倍体胚胎移植回患者体内，分析羊水细胞的胎儿核型。结果与胎儿核型完全一致。通过后期随访，两名患者均成功分娩活产胎儿。纳米孔测序可准确找到平衡染色体易位的断点位置。通过纳米孔和 sanger 测序成功构建了携带者的单倍型，证实了结果的准确性。这对于无先证者的染色体结构重排（PGT-SR）检测的胚胎植入前遗传学检测非常有利。
