Identification and rescue of a novel TUBB8 mutation that causes the first mitotic division defects and infertility

• Published in: Journal of assisted reproduction and genetics Vol. 37; no. 11; pp. 2713 - 2722
• Main Authors: Jia, Yanping, Li, Kunming, Zheng, Caihong, Tang, Yuanyuan, Bai, Dandan, Yin, Jiqing, Chi, Fengli, Zhang, Yalin, Li, Yanhe, Tu, Zhifen, Wang, Yu, Pan, Jiaping, Liang, Shanshan, Guo, Yi, Ruan, Jingling, Kong, Pengcheng, Wu, Bi, Hu, Ye, Wang, Hong, Liu, Wenqiang, Teng, Xiaoming, Gao, Shaorong
• Format: Journal Article
• Language: English
• Published: New York Springer US 01-11-2020; Springer Nature B.V
• Subjects: Animals; Cell Division - genetics; Developmental stages; Embryo Biology; Embryo, Mammalian; Embryos; Female; Fertilization; Gynecology; Human Genetics; Humans; Immunofluorescence; Infertility; Infertility, Female - genetics; Infertility, Female - pathology; Medicine; Medicine & Public Health; Meiosis; Metaphase; Mice; Microinjection; Mitosis; Mitosis - genetics; Mutants; Mutation; Mutation - genetics; Oocytes; Oogenesis; Oogenesis - genetics; Phenotypes; Plasmids; Point mutation; Reproductive Medicine; Sequence analysis; Transcription; Tubulin; Tubulin - genetics; Rescue; Embryo development arrest; Mutation; Infertility; TUBB8
• ISSN: 1058-0468; 1573-7330; 1573-7330
• DOI: 10.1007/s10815-020-01945-w

Purpose
Tubulin beta eight class VIII (TUBB8) is essential for oogenesis, fertilization, and pre-implantation embryo development in human. Although
TUBB8
mutations were recently discovered in meiosis-arrested oocytes of infertile females, there is no effective therapy for this gene mutation caused infertility. Our study aims to further reveal the infertility-causing gene mutations in the patient’s family and to explore whether the infertility could be rescued by optimizing the conditions of embryo culture and finally achieve the purpose of making the patient pregnant.
Methods
Whole-exome sequence analysis and Sanger sequencing were performed on patients’ family members to screen and identify candidate mutant genes. Construction of plasmids, in vitro transcription, microinjection of disease-causing gene cRNA, and immunofluorescence staining were used to recapitulate the infertility phenotype observed in patients and to understand the pathogenic principles. Simultaneously, overexpression of mutant and wild-type cRNA of the candidate gene in mouse oocytes at either germinal vesicle (GV) or metaphase II (MII) stage was performed in the rescue experiment.
Results
We first identified a novel heritable
TUBB8
mutation (c.1041C>A: p.N347K) in the coding region which specifically affects the first mitosis and causes the developmental arrest of early embryos in a three-generation family. We further demonstrated that
TUBB8
mutation could lead to abnormal spindle assemble. And moreover, additional expression of wild-type
TUBB8
cRNA in the mouse oocytes in which the mutant
TUBB8
were expressed can successfully rescue the developmental defects of resulting embryo and produce full-term offspring.
Conclusions
Our study not only defines a novel mutation of
TUBB8
causing the early cleavage arrest of embryos, but also provides an important basis for treating such female infertility in the future.