Novel mutations and structural deletions in TUBB8: expanding mutational and phenotypic spectrum of patients with arrest in oocyte maturation, fertilization or early embryonic development

Study question Are there any new type of mutations and novel phenotypes in patients with arrest in oocyte maturation, fertilization or early embryonic development having tubulin beta eight class VIII (TUBB8) mutations? Summary answer We identified new types of mutations in TUBB8 associated with maturation, fertilization and developmental arrest. What is known already We previously found heterozygous mutations and a homozygous frameshift/internal seven amino acid deletion in TUBB8 that are responsible for oocyte maturation arrest. Study design, size, duration We recruited 10 new primary infertility patients from 9 families from December 2015 to May 2016, most of which exhibited failures in oocyte maturation. Participants/materials, setting, methods Ten primary infertility patients were recruited from the reproduction centers in local hospitals. Genomic DNA samples from the affected individuals, their family members and healthy controls were extracted from peripheral blood. TUBB8 in the DNA samples were sequenced by Sanger sequencing. TUBB8 sequence was then aligned by CodonCode software to identify rare variants. ExAC database was used to search frequency of corresponding mutations. In silico analysis of mutations was used by Polyphen and PROVEAN. Phenotypes of oocytes and embryos were evaluated by light microscopy, polarization microscopy or immunolabeling. Main results and the role of chance Besides several novel heterozygous missense mutations, we also identified other new types of genetic variants, including homozygous mutations and a de novo compound heterozygous mutation. We also found a patient with a homozygous deletion of the whole TUBB8 gene, which is the first reported case of a large structural variation in this gene. In addition, we found different mutations in TUBB8 that could result in variability in oocyte/embryo phenotypes, including oocyte maturation arrest, first polar body (PB1) oocytes that cannot be fertilized, and PB1 oocytes that can be fertilized but arrest at an early embryonic stage. Limitations, reasons for caution The exact molecular mechanism has not been analyzed and should be further investigated in the future. In addition, immunostaining of more oocytes with mutations and checking spindle status of oocytes with mutations non-invasively by polarization microscopy needs to be done in order to determine exact stage of PB1 oocytes and the functional differences of these mutations. Wider implications of the findings The results not only emphasize the important role of TUBB8 in oocyte maturation, fertilization and early embryonic development but they also provide a basis for determining the genetic variations in TUBB8 as a potential additional criterion for evaluating the quality of patients' functional PB1 oocytes. Study funding/competing interests National Key RD Basic Research Program of China (2015CB943300); National Natural Science Foundation of China (81270747 and 81571501). No competing interests declared.