Guo Min, Li Fangfang, Ren Lingyan, You Guiqin, Zhang Ying, Liu Juan, Huang Shengwen
School of Medicine, Guizhou University, Guiyang, Guizhou, China.
Department of Medical Genetics, Guizhou Provincial People's Hospital, No. 83, East Zhongshan Road, Nanming District, Guiyang, 550002, Guizhou, China.
Biochem Genet. 2025 Jul 2. doi: 10.1007/s10528-025-11152-w.
The TUBB8 gene is highly conserved in primates, and pathogenic mutations in this gene have been linked to defects in oocyte maturation, leading to infertility in women. This study aimed to identify a mutation in the TUBB8 gene in a family with female infertility and functionally validate the identified mutation to confirm its pathogenicity. Genomic DNA was extracted from the proband's peripheral blood for whole exome sequencing. DNA from the proband's parents was obtained for Sanger sequencing to trace the origin of the proband's mutation. Bioinformatics analysis, conservation analysis, and three-dimensional protein structure prediction were performed on the sequencing results. Wild-type and mutant TUBB8 expression plasmids for the identified mutation sites were constructed and transfected into HEK293T and HeLa cells. Changes in protein structure and gene expression were then assessed. The analysis revealed that the proband carried the TUBB8 mutation c.1039A > G, also present in her father and aunt. This mutation was classified as a Variant of Uncertain Significance (VUS). Protein structure prediction suggested that the TUBB8 p.N347D (c.1039A > G) mutant protein had an additional hydrogen bond compared to the wild-type protein. Still, no significant structural changes were observed in the three-dimensional model. Immunofluorescence staining showed that the TUBB8 c.1039A > G mutation did not disrupt cellular microtubule structure. In vitro assays indicated that the c.1039A > G mutation decreased mRNA and protein expression levels of TUBB8. This study describes a case of female infertility associated with a newly discovered heterozygous c.1039A > G mutation in the TUBB8 gene, which may reduce TUBB8 expression. These findings contribute to the genetic understanding and diagnosis of TUBB8-related diseases.
TUBB8基因在灵长类动物中高度保守，该基因的致病性突变与卵母细胞成熟缺陷有关，导致女性不孕。本研究旨在鉴定一个女性不孕家族中TUBB8基因的突变，并对鉴定出的突变进行功能验证，以确认其致病性。从先证者外周血中提取基因组DNA进行全外显子组测序。获取先证者父母的DNA进行桑格测序，以追踪先证者突变的来源。对测序结果进行生物信息学分析、保守性分析和三维蛋白质结构预测。构建针对鉴定出的突变位点的野生型和突变型TUBB8表达质粒，并转染到HEK293T和HeLa细胞中。然后评估蛋白质结构和基因表达的变化。分析显示，先证者携带TUBB8突变c.1039A＞G，其父亲和姑姑也携带该突变。此突变被分类为意义未明的变异（VUS）。蛋白质结构预测表明，与野生型蛋白质相比，TUBB8 p.N347D（c.1039A＞G）突变蛋白有一个额外的氢键。然而，在三维模型中未观察到明显的结构变化。免疫荧光染色显示，TUBB8 c.1039A＞G突变未破坏细胞微管结构。体外试验表明，c.1039A＞G突变降低了TUBB8的mRNA和蛋白质表达水平。本研究描述了一例与新发现的TUBB8基因杂合c.1039A＞G突变相关的女性不孕病例，该突变可能降低TUBB8表达。这些发现有助于对TUBB8相关疾病的遗传学理解和诊断。
