目的:哪些遗传变异可以解释空卵泡综合征(EFS)和透明带异常(ZP)的原因,并影响辅助生殖技术(ART)治疗的成功?
方法:在EFS和ZP异常的先证者中进行全外显子组测序。Sanger测序用于变体验证。使用HEK-293T细胞,通过蛋白质印迹法探索ZP1和ZP2变体对蛋白质表达的影响,通过免疫荧光研究了ZP1变体对蛋白质定位的影响。还分析了蛋白质结构以研究变体的致病性。
结果:ZP1中的纯合无义变体(c.874C>T,在EFS患者中检测到p.Gln292*)。ZP2中的一种新型纯合移码变体(c.836_837delAG,p.Glu279Valfs*6)和ZP3中的一个新型杂合错义变体(c.159G>A,p.Val387Met)在两名ZP形态异常患者中发现,分别。蛋白质印迹和免疫荧光分析显示ZP1变异导致提前终止密码子,导致截短的ZP1蛋白。ZP2变体,它位于N端,引发过早终止蛋白的降解。此外,ZP3变异体患者在卵胞浆内单精子注射治疗后达到临床妊娠.
结论:这些发现扩展了ZP1、ZP2和ZP3的突变谱,为女性不孕症的基因诊断提供了新的证据。建议对ZP基因进行靶向遗传诊断,选择合适的受精方法,提高ART治疗成功率。
Which genetic variants might explain the causes of empty follicle syndrome (EFS) and abnormal zona pellucida (ZP) and affect the success of treatment with assisted reproductive technologies (ART)?
Whole-exome sequencing was performed in probands with EFS and abnormal ZP. Sanger sequencing was used for variant validation. Using HEK-293T cells, the effects of
ZP1 and ZP2 variants on protein expression were explored by western blotting, and the effect of the
ZP1 variant on protein location was investigated via immunofluorescence. The protein structure was also analysed to investigate the pathogenicity of variants.
A homozygous nonsense variant in
ZP1 (c.874C>T, p.Gln292*) was detected in a patient with EFS. A novel homozygous frameshift variant in ZP2 (c.836_837delAG, p.Glu279Valfs*6) and a novel heterozygous missense variant in ZP3 (c.1159G>A, p.Val387Met) were identified in two patients with ZP morphological abnormalities, respectively. Western blotting and immunofluorescence analysis showed that the
ZP1 variant results in a premature stop codon, leading to the truncated
ZP1 protein. The ZP2 variant, which is situated in the N-terminus, triggers the degradation of a premature termination protein. Additionally, the patient with the ZP3 variant achieved clinical pregnancy following intracytoplasmic sperm injection treatment.
These findings expand the mutational spectrum of ZP1, ZP2 and ZP3, and provide new evidence for genetic diagnosis of female infertility. The targeted genetic diagnosis of ZP genes is recommended to choose appropriate fertilization methods and improve success rates of treatment with ART.