Clinical application of NGS-based SNP haplotyping for the preimplantation genetic diagnosis of primary open angle glaucoma.

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. Abbreviations: 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.