Wen Xiaojun, Du Jing, Li Zhiming, Liu Nengqing, Huo Junye, Li Jieliang, Ke Wanna, Wu Jiaqi, Fang Xiaowu, Lin Xiufeng
Reproductive Medicine Center, Boai Hospital of Zhongshan Affiliated to Southern Medical University, Zhongshan, China.
The Second School of Clinical Medicine, Southern Medical University, Guangzhou, Guangdong, China.
Front Genet. 2023 Sep 14;14:1169868. doi: 10.3389/fgene.2023.1169868. eCollection 2023.
This study aimed to perform preimplantation genetic testing (PGT) for a female Coffin-Lowry Syndrome (CLS) patient with a mutation (DNM) in RPS6KA3. It was challenging to establish the haplotype in this family because of the lack of information from affected family members. Hence, we explored a new and reliable strategy for the detection of the DNM in PGT, using Oxford Nanopore Technologies (ONT) and the MARSALA platform. We performed whole-exome sequencing (WES) on the proband and confirmed the pathogenic mutation by Sanger sequencing. The proband then underwent PGT to prevent the transmission of the pathogenic mutation to her offspring. We diverged from the conventional methods and used long-read sequencing (LRS) on the ONT platform to directly detect the mutation and nearby SNPs, for construction of the haplotype in the preclinical phase of PGT. In the clinical phase of embryo diagnosis, the MARSALA method was used to detect both the SNP-based haplotype and chromosome copy number variations (CNVs), in each blastocyst. Finally, a normal embryo was selected by comparison to the haplotype of the proband and transferred into the uterus. Sanger sequencing and karyotyping were performed by amniocentesis, at 17 weeks of gestation, to confirm the accuracy of PGT. Using WES, we found the novel, heterozygous, pathogenic c.1496delG (p.Gly499Valfs*25) mutation of RPS6KA3 in the proband. The SNP-based haplotype that was linked to the pathogenic mutation site was successfully established in the proband, without the need for other family members to be tested with ONT. Eight blastocysts were biopsied to perform PGT and were assessed with a haplotype linkage analysis (30 SNP sites selected), to give results that were consistent with direct mutation detection using Sanger sequencing. The results of PGT showed that three of the eight blastocysts were normal, without the DNM. Moreover, the patient had a successful pregnancy, after transfer of a normal blastocyst into the uterus, and delivered a healthy baby. The ONT platform, combined with the MARSALA method, can be used to perform PGT for DNM patients without the need for other samples as a reference.
本研究旨在对一名患有RPS6KA3基因突变（DNM）的女性科芬-洛里综合征（CLS）患者进行植入前基因检测（PGT）。由于缺乏患病家庭成员的信息，在这个家族中确定单倍型具有挑战性。因此，我们探索了一种新的、可靠的策略，利用牛津纳米孔技术（ONT）和MARSALA平台在PGT中检测DNM。我们对先证者进行了全外显子组测序（WES），并通过桑格测序确认了致病突变。然后，先证者接受了PGT，以防止致病突变遗传给她的后代。我们背离了传统方法，在ONT平台上使用长读长测序（LRS）直接检测突变和附近的单核苷酸多态性（SNP），以便在PGT的临床前阶段构建单倍型。在胚胎诊断的临床阶段，使用MARSALA方法检测每个囊胚中基于SNP的单倍型和染色体拷贝数变异（CNV）。最后，通过与先证者的单倍型进行比较，选择了一个正常胚胎并移植到子宫内。在妊娠17周时通过羊膜穿刺术进行桑格测序和核型分析，以确认PGT的准确性。通过WES，我们在先证者中发现了RPS6KA3新的杂合致病突变c.1496delG（p.Gly499Valfs*25）。无需对其他家庭成员进行ONT检测，就成功在先证者中建立了与致病突变位点连锁的基于SNP的单倍型。对8个囊胚进行活检以进行PGT，并通过单倍型连锁分析（选择30个SNP位点）进行评估，结果与使用桑格测序直接检测突变一致。PGT结果显示，8个囊胚中有3个正常，无DNM。此外，在将一个正常囊胚移植到子宫后，患者成功怀孕并生下了一个健康的婴儿。ONT平台与MARSALA方法相结合，可用于对DNM患者进行PGT，而无需其他样本作为参考。
