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. Female primary infertility was the clinical trait of OOMD2. The corresponding phenotype includes arrest at MI or MII of oocytes, fertilization failure, stagnation of early embryonic development, and failure of embryo implantation [16]. Expression of TUBB8 protein is unique to oocytes and early embryos. Thus, male carriers of the TUBB8 mutation are fertile[9].
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 [17]. In early embryos, this gene occupies almost all of the expressed beta-tubulin. Microtubules are dynamic polymers composed of alpha/beta-tubulin isodimers [18]. 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 [16]. 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[19].
We found a consanguineous mating family in which the proband suffered from primary infertility. After a cycle of ovulation induction treatment, most oocytes of the healthy individual were at the MII stage, while oocytes of the patient were at the GV stage or MI stage. After a period of culture in vitro, the oocytes remained immature. We considered 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) from a family by WES. Because the parents of the proband were cousins and neither of them was affected, we speculated that the inheritance pattern of this mutation was recessive (Fig. 1).
The variant that we found was located in exon 2, and the affected residue(p.A54) was 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 [6]. Thus, the affected residues that we found (p. A54) may influence folding/protein stability. And TUBB8 is an important component of oocyte spindle[9]{, #46}, 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 suggested that heterozygous p.A54V mutations could not affect female fertility. Thus, p.A54V has a haploinsufficiency effect than a dominant-negative effect.
We reviewed variants in TUBB8 that have reported previously (Supplementary Table 1). We found that the inheritance pattern of p.E27_A33del located in exon 2 was recessive[10]. While our newly discovered p.A54V was located in exon 2 and the inheritance pattern was also 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 speculated that the inheritance pattern of this mutation was recessive. Therefore, we selected homozygous mutations from the proband. This helped us to quickly identify this variant of TUBB8. Consanguineous mating families are useful for studying mechanisms of genetic diseases without human intervention.
This study elucidated the cause of oocyte maturation defects. The results confirmed that mutations of TUBB8 contributed genetically to OOMD2 and expanded the mutation spectrum of TUBB8.