OOMDs can be classified into six subtypes, and different pathogenic genes lead to different genetic and clinical characteristics of different subtypes.[15, 7, 9, 10, 4, 8, 5] Mutation of the TUBB8 gene leads to OOMD2. Inheritance of OOMD2 can be either autosomal dominant or autosomal recessive. The clinical trait of OOMD2 is female primary infertility. The corresponding phenotype includes not only arrest at MI or MII of oocytes, fertilization failure, and stagnation of early embryonic development, but also failure of embryo implantation. Expression of TUBB8 protein is unique to oocytes and early embryos. Therefore, male carriers of the TUBB8 mutation are fertile.
TUBB8 is one of the microtubulin family genes. There are nine types of beta-tubulin in mammals. Beta-tubulin can be distinguished mainly by a change in the C-terminal domain affecting specific cell functions . In early embryos, this gene occupies almost all of the expressed beta-tubulin. Microtubules are dynamic polymers composed of alpha/beta-tubulin isodimers . TUBB8 protein has two domains, including a GTPase domain and C-terminal domain.
In previous studies, researchers have found several inheritance patterns of TUBB8 mutations, including heterozygous mutations [6, 16, 9, 10, 19–21], homozygous mutations [6, 10, 16, 22], compound heterozygous mutations [16, 21], and homozygous deletions . These mutations affect folding and assembly of alpha/beta-tubulin isodimers. This process changes the dynamics of microtubules in vivo, and results in disastrous spindle assembly defects and arrest of oocyte maturation in human oocytes. Some TUBB8 variants of dominant inheritance have significant negative effects, which interfere with microtubule behavior and meiotic spindle assembly of oocytes, leading to arrest of oocyte maturation and female infertility.
We found an inbred family in whom the proband suffered from primary infertility (Fig. 1). After a cycle of ovulation induction treatment, most oocytes of the healthy individual were at the MII stage (Fig. 4), while oocytes of the patient were at the GV stage or MI stage (Fig. 3). After a period of culture in vitro, the oocytes remained immature (Table 2). We consider that there might be some genetic factors leading to arrest of oocyte development.
We identified a TUBB8 variant (TUBB8: NM_177987: exon 2: c.C161T: p.A54V) (Table 3) from a family (Fig. 1) by WES. Because the parents of the proband were cousins and neither of them was affected, we speculate that the inheritance pattern of this mutation is recessive (Fig. 1).
The variant that we found was located in exon 2, and the affected residue(p.A54) is located in the GTPase domain(Fig. 2a). And we referred to a previous study of missense mutations (p.P70L and p.C12Y) located in β-tubulin subunit in the GTPase domain of which their inheritance patterns were also recessive, the two affected residues may influenced folding or protein stability . So the affected residues that we found (p. A54) may influence folding/protein stability. And TUBB8 is an important component of oocyte spindle, so the homozygote of the variant that we found might affect spindle assembly, which will result in arrest of oocyte maturation. Heterozygous missense mutations cause arrest of oocyte maturation through dominant-negative effects. In this study, however, the patient with homozygous p.A54V TUBB8 mutations suffered from OOMD2, while her parents with the heterozygous p.A54V missense mutations were fertile. This finding suggests that heterozygous p.A54V mutations do not affect female fertility. Therefore, p.A54V has a haploinsufficiency effect than a dominant-negative effect.
We reviewed variants in TUBB8 that have reported previously (Supplementary Table 1). We attempted to investigate the effects of the location of variants on inheritance patterns. We found that the location of variants in TUBB8 hardly affected inheritance patterns (Supplementary Table 1). However, we found that the inheritance pattern of p.E27_A33del located in exon 2 is recessive. Our newly discovered p.A54V was located in exon 2 and the inheritance pattern was also recessive. However, the number of samples in our study is small. Therefore, we cannot be sure that the inheritance pattern of variants in exon 2 is recessive.
The discovery of this variant started with investigation of the pedigree of OOMD (Fig. 1). Because the proband’s parents were cousins and neither of them was affected, we speculate that the inheritance pattern of this mutation is recessive. Therefore, we selected homozygous mutations in the proband. This helped us to quickly identify this variant of TUBB8. Inbred families are useful for studying mechanisms of genetic diseases without human intervention.
Our study clarified why oocytes maturation arrested. Our findings indicated that mutations of TUBB8 contributed genetically to OOMD2. Our results expanded the mutation spectrum of TUBB8 causing OOMD2.