Research question

How can the effect of genetic mutations that may cause primary female infertility be evaluated?

Design

Patients and their family members underwent whole-exome sequencing and Sanger sequencing to detect the infertility-causing gene and inheritance pattern. To study the function of mutant proteins in vitro, vectors containing wild-type or mutant TUBB8 cDNA were constructed for transient expression in HeLa cells, and in-vitro transcribed mRNA were used for microinjection in germinal vesicle-stage mouse oocytes. Immunofluorescence staining was used to observe the microtubule structure in HeLa cells or meiotic spindle in mouse oocytes.

Results

A maternally inherited TUBB8 (Tubulin beta 8 class VIII) mutation (NM_177987.2: c. 959G>A: p. R320H) and a previously reported (NM_177987.2: c. 161C>T: p. A54V) recessive mutation from two infertile female patients were identified. The oocytes from the patient carrying p.A54V mutation failed fertilization, whereas oocytes with p.R320H mutation could be fertilized but showed heavy fragmentation during early development. In vitro, functional assays showed that p. A54V mutant disrupted the microtubule structure in HeLa cells (49.3% of transfected cells) and caused large polar body extrusion in mouse oocytes (27.5%), whereas the p.R320H mutant caused a higher abnormal rate (69.7%) in cultured cells and arrested mouse oocytes at meiosis I (38.7%).

Conclusion

Two TUBB8 mutations (p.A54V and p.R320H) were identified and their pathogeny was confirmed by in-vitro functional assays.

中文翻译：

---

TUBB8 的两个突变导致人类卵母细胞和早期胚胎发育停滞

研究问题

如何评估可能导致原发性女性不育的基因突变的影响？

设计

患者及其家属接受了全外显子组测序和 Sanger 测序，以检测导致不孕的基因和遗传模式。为了在体外研究突变蛋白的功能，构建了含有野生型或突变型TUBB8 cDNA的载体，用于在HeLa细胞中瞬时表达，体外转录的mRNA被用于显微注射生泡期小鼠卵母细胞。免疫荧光染色用于观察HeLa细胞中的微管结构或小鼠卵母细胞中的减数分裂纺锤体。

结果

母系遗传的TUBB8（微管蛋白 8 类 VIII）突变（NM_177987.2：c.959G>A：p.R320H）和先前报道的（NM_177987.2：c.161C>T：p.A54V）来自两个隐性突变确定了不孕的女性患者。携带p.A54V突变的患者的卵母细胞受精失败，而携带p.R320H突变的卵母细胞可以受精，但在早期发育过程中表现出严重的碎片化。在体外，功能测定表明 p。A54V 突变体破坏了 HeLa 细胞中的微管结构（占转染细胞的 49.3%）并导致小鼠卵母细胞中的极体大面积挤出（27.5%），而 p.R320H 突变体在培养细胞中导致更高的异常率（69.7%）并停滞减数分裂 I (38.7%) 的小鼠卵母细胞。

结论

鉴定了两个TUBB8突变（p.A54V 和 p.R320H），并通过体外功能测定证实了它们的致病性。