Reproductive Genetics, Igenomix, Marostica, Italy; Reproductive Genetics, Igenomix Foundation - INCLIVA, Valencia, Spain.
Institute of Genetics and Biophysics, National Research Council, Naples, Italy.
Reprod Biomed Online. 2022 Sep;45(3):508-518. doi: 10.1016/j.rbmo.2022.05.009. Epub 2022 May 21.
Can a methodology be developed for case selection and whole-exome sequencing (WES) analysis of women who are infertile owing to recurrent oocyte maturation defects (OOMD) and/or preimplantation embryo lethality (PREMBL)?
Data were collected from IVF patients attending the Istanbul Memorial Hospital (2015-2021). A statistical methodology to identify infertile endophenotypes (recurrent low oocyte maturation rate, low fertilization rate and preimplantation developmental arrest) was developed using a large IVF dataset (11,221 couples). Twenty-eight infertile women with OOMD/PREMBL were subsequently enrolled for WES on their genomic DNA. Pathogenic variants were prioritized using a custom-made bioinformatic pipeline set to minimize false-positive discoveries through resampling in control cohorts (the Human Genome Diversity Project and 1343 whole-exome sequences from oocyte donors). Individual single-cell RNA sequencing data from 18 human metaphase II (MII) oocytes and antral granulosa cells was used for genome-wide validation. WES and bioinformatics were performed at Igenomix and the National Research Council, Italy.
Variant prioritization analysis identified 265 unique variants in 248 genes (average 22.4 per sample). Of the genes harbouring high-impact variants 78% were expressed by MII oocytes and/or antral granulosa cells, significantly higher than for random sample of controls (odds ratio = 5, Fisher's exact P = 0.0004). Seven of the 28 women (25%) were homozygous carriers of missense pathogenic variants in known candidate genes for OOMD/PREMBL, including PATL2, NLRP5 (n = 2),TLE6, PADI6, TUBB8 and TRIP13. Furthermore, novel gene-disease associations were identified. In fact, one woman with a low oocyte maturation rate was a homozygous carrier of high-impact variants in ENSA, an essential gene for prophase I meiotic transition in mice.
This analytical framework could reveal known and new genes associated with isolated recurrent OOMD/PREMBL, providing essential indications for scaling this strategy to larger studies.
能否针对因卵母细胞成熟缺陷（OOMD）和/或胚胎前植入期死亡（PREMBL）而不孕的女性，开发出一种病例选择和全外显子测序（WES）分析的方法？
数据来自于在伊斯坦布尔纪念医院就诊的 IVF 患者（2015-2021 年）。使用大型 IVF 数据集（11221 对夫妇）开发了一种用于识别不孕表型（反复低卵母细胞成熟率、低受精率和胚胎前植入期发育停滞）的统计方法。随后，招募了 28 名 OOMD/PREMBL 不孕女性进行 WES 检测。通过在对照队列（人类基因组多样性计划和 1343 名卵母细胞供体的全外显子序列）中进行重新取样，使用定制的生物信息学管道对致病性变异进行优先级排序，以尽量减少假阳性发现。从 18 个人中期 II（MII）卵母细胞和窦状颗粒细胞的单个单细胞 RNA 测序数据进行全基因组验证。WES 和生物信息学在 Igenomix 和意大利国家研究委员会进行。
变异优先级分析在 248 个基因中确定了 265 个独特变异（每个样本平均 22.4 个）。在携带高影响变异的基因中，有 78%在 MII 卵母细胞和/或窦状颗粒细胞中表达，显著高于随机对照样本（比值比=5，Fisher 精确检验 P=0.0004）。28 名女性中有 7 名（25%）为已知 OOMD/PREMBL 候选基因的纯合错义致病性变异携带者，包括 PATL2、NLRP5（n=2）、TLE6、PADI6、TUBB8 和 TRIP13。此外，还确定了新的基因-疾病关联。事实上，一名卵母细胞成熟率低的女性是 ENSA 的纯合变异携带者，ENSA 是小鼠减数分裂前期 I 转变所必需的基因。
该分析框架可以揭示与孤立性复发性 OOMD/PREMBL 相关的已知和新基因，为将该策略扩展到更大规模的研究提供了重要依据。
