Two mutations in TUBB8 cause developmental arrest in human oocytes and early embryos
Tianqi Cao1, Jing Guo2, Yan Xu2
1MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China; Key Laboratory of Reproductive Medicine of Guangdong Province, the First Affiliated Hospital and School of Life Sciences, Sun Yat-sen University, Guangzhou 510275.
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.
Insights
Genetic mutations in the TUBB8 gene can cause primary female infertility. Functional assays confirmed that p.A54V and p.R320H mutations disrupt microtubule structure, leading to oocyte dysfunction and developmental issues.
Area of Science:
- Genetics and Reproductive Biology
- Molecular and Cellular Biology
Background:
- Primary female infertility affects numerous women globally, with genetic factors playing a significant role.
- Understanding the genetic basis of infertility is crucial for developing diagnostic and therapeutic strategies.
Observation:
- Whole-exome and Sanger sequencing identified two TUBB8 mutations (p.A54V and p.R320H) in infertile patients.
- The p.A54V mutation led to failed fertilization, while p.R320H resulted in fertilization but subsequent oocyte fragmentation.
Findings:
- In vitro assays demonstrated that both p.A54V and p.R320H TUBB8 mutations disrupt microtubule organization in HeLa cells.
- These mutations caused significant meiotic abnormalities in mouse oocytes, including abnormal polar body extrusion and meiotic arrest.
Implications:
- The study confirms TUBB8 as a key gene in female fertility and provides insights into the pathogenic mechanisms of specific mutations.
- These findings can aid in the genetic diagnosis of primary female infertility and inform potential future treatments.
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