Chen Songchang, Shi Weihui, Qian Yeqing, Wang Liya, Zhang Junyu, Li Shuyuan, Chen Yiyao, Chang Chunxin, Fei Hongjun, Zhang Lanlan, Huang Hefeng, Xu Chenming
The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Shanghai Key Laboratory of Embryo Original Diseases, Shanghai, China.
Front Genet. 2020 Nov 4;11:550507. doi: 10.3389/fgene.2020.550507. eCollection 2020.
X-linked lymphoproliferative disease (XLP) is a rare primary immunodeficiency disorder. We performed experiments based on two strategies of preimplantation genetic testing (PGT) for a family with XLP caused by a mutation in (c.191G > A).
First, a single-cell polymerase chain reaction (PCR) protocol was established using single lymphocytes. A nested PCR experiment was performed with direct sequencing after whole genome amplification of single cells to assess the accuracy of the genetic diagnosis. Embryos obtained after intracytoplasmic sperm injection were biopsied on day 3 and detected using the established single-cell PCR protocol. In the second PGT cycle, targeted next generation sequencing (NGS) was performed and the single nucleotide polymorphism (SNP) markers flanking were selected to determine the disease-carrying haplotype phase in each embryo.
In the first PGT cycle, six embryos were biopsied. Discounting an embryo from a single failed PCR experiment, five embryos were identified, including three unaffected and two hemizygous. After PCR, one normal embryo was transferred when it was developing into an early blastocyst. Although the ultrasound images indicated a viable singleton pregnancy, the implantation was on the cesarean scar. Therefore, an artificial abortion was performed. In the haplotyping cycle, six embryos were identified to have inherited a haplotype without pathogenic mutations. After the embryo implantation process failed twice, a successful singleton pregnancy was established, and subsequently, a healthy female child was born.
Targeted NGS with haplotyping analysis circumvents the laborious process of multiplex PCR and is more likely to ensure diagnostic accuracy. However, when a genetic recombination occurs close to the site of mutation, confirmed identification using selected SNP markers can be challenging.
X连锁淋巴增生性疾病（XLP）是一种罕见的原发性免疫缺陷病。我们针对一个因（c.191G > A）突变导致XLP的家庭，基于两种植入前基因检测（PGT）策略进行了实验。
首先，使用单个淋巴细胞建立单细胞聚合酶链反应（PCR）方案。对单细胞进行全基因组扩增后，进行巢式PCR实验并直接测序，以评估基因诊断的准确性。在卵胞浆内单精子注射后获得的胚胎于第3天进行活检，并使用已建立的单细胞PCR方案进行检测。在第二个PGT周期中，进行靶向新一代测序（NGS），并选择位于侧翼的单核苷酸多态性（SNP）标记来确定每个胚胎中携带疾病的单倍型阶段。
在第一个PGT周期中，对6个胚胎进行了活检。排除一次PCR实验失败的单个胚胎后，鉴定出5个胚胎，包括3个未受影响的和2个半合子的。PCR后，当一个正常胚胎发育为早期囊胚时进行了移植。尽管超声图像显示为单胎存活妊娠，但着床在剖宫产瘢痕处。因此，进行了人工流产。在单倍型分析周期中，鉴定出6个胚胎继承了无致病突变的单倍型。在胚胎移植过程两次失败后，成功建立了单胎妊娠，随后，一名健康女婴出生。
具有单倍型分析的靶向NGS避免了多重PCR的繁琐过程，更有可能确保诊断准确性。然而，当基因重组发生在靠近突变位点时，使用选定的SNP标记进行确认鉴定可能具有挑战性。
