a Assisted Reproduction Centre , Northwest Women's and Children's Hospital , Xi'an , China.
b Prenatal Diagnosis Centre , Northwest Women's and Children's Hospital , Xi'an , China.
Syst Biol Reprod Med. 2019 Jun;65(3):258-263. doi: 10.1080/19396368.2019.1590479. Epub 2019 Apr 12.
The current study describes a successful case of preimplantation genetic diagnosis (PGD) of primary open angle glaucoma (POAG) and verifies the efficiency of next-generation sequencing (NGS)-based haplotyping for PGD of POAG. In this study, we applied NGS as part of PGD to effectively detect POAG prior to embryo implantation and avoid the prospect of pregnancy termination in event of vertical inheritance of POAG. We used the technique of multiple annealing and looping based amplification cycles (MALBAC) to conduct whole genome amplification (WGA) and to reduce the allele dropout (ADO). We also employed Sanger sequencing to directly detect the mutation c.1109 C > T in MYOC and NGS-based single nucleotide polymorphism (SNP) haplotyping to distinguish the chromosomes that carried the mutation. Copy number variation (CNV) analysis was carried out to determine the copy number of embryos' chromosomes. Of the 4 blastocysts obtained in this study, only 2 (sample 5 and 7) could be successfully amplified by WGA. CNV results indicated that chromosomes of both these samples were balanced (46, XN). Sanger sequencing and NGS-based SNP haplotyping confirmed that sample 7 carried the mutation c.1109 C > T in MYOC, while sample 5 did not. Moreover, no ADO was observed. Thus, blastocyst 5 was transferred into the uterus of the patient, and a healthy baby without the MYOC mutation c. 1109C>T was born 39 weeks after transplantation. Our study suggests that NGS-based SNP haplotyping is an effective technique for the PGD of POAG. PGD: preimplantation genetic diagnosis; POAG: primary open angle glaucoma; NGS: next-generation sequencing; MALBAC: multiple annealing and looping based amplification cycles chemistry; WGA: whole genome amplification; ADO: allele dropout; SNP: single nucleotide polymorphism; CNV: copy number variation; MYOC: Myocilin; OPTN: Optineurin; WDR36: WD repeat domain 36; CYP1B1: Cytochrome P450 1 B Chain; ICSI: intracytoplasmic sperm injection; TFNA: testicular fine-needle aspiration; TE: trophectoderm; PCR: polymerase chain reaction.
本研究描述了一例原发性开角型青光眼（POAG）的植入前遗传学诊断（PGD）成功案例，并验证了基于下一代测序（NGS）的单体型分析在 POAG 的 PGD 中的效率。在这项研究中，我们应用 NGS 作为 PGD 的一部分，在胚胎植入前有效检测 POAG，并避免在 POAG 垂直遗传的情况下终止妊娠。我们使用基于多次退火和环扩增循环（MALBAC）的技术进行全基因组扩增（WGA），并减少等位基因缺失（ADO）。我们还采用 Sanger 测序直接检测 MYOC 中的突变 c.1109C>T，以及基于 NGS 的单核苷酸多态性（SNP）单体型分析来区分携带突变的染色体。拷贝数变异（CNV）分析用于确定胚胎染色体的拷贝数。在本研究中获得的 4 个囊胚中，只有 2 个（样本 5 和 7）可以通过 WGA 成功扩增。CNV 结果表明，这两个样本的染色体均平衡（46，XN）。Sanger 测序和基于 NGS 的 SNP 单体型分析证实，样本 7 携带 MYOC 中的突变 c.1109C>T，而样本 5 没有。此外，没有观察到 ADO。因此，将囊胚 5 移植到患者的子宫内，在移植后 39 周出生了一个没有 MYOC 突变 c.1109C>T 的健康婴儿。我们的研究表明，基于 NGS 的 SNP 单体型分析是 POAG 的 PGD 的有效技术。PGD：植入前遗传学诊断；POAG：原发性开角型青光眼；NGS：下一代测序；MALBAC：基于多次退火和环扩增循环化学的扩增；WGA：全基因组扩增；ADO：等位基因缺失；SNP：单核苷酸多态性；CNV：拷贝数变异；MYOC：Myocilin；OPTN：Optineurin；WDR36：WD 重复结构域 36；CYP1B1：细胞色素 P450 1 B 链；ICSI：胞浆内精子注射；TFNA：睾丸细针抽吸术；TE：滋养外胚层；PCR：聚合酶链反应。
