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<p>The <i>TUBB8</i> 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 <i>TUBB8</i> 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 <i>TUBB8</i> 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 <i>TUBB8</i> mutation c.1039A &gt; 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 &gt; 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 <i>TUBB8</i> c.1039A &gt; G mutation did not disrupt cellular microtubule structure. In vitro assays indicated that the c.1039A &gt; G mutation decreased mRNA and protein expression levels of <i>TUBB8</i>. This study describes a case of female infertility associated with a newly discovered heterozygous c.1039A &gt; G mutation in the <i>TUBB8</i> gene, which may reduce <i>TUBB8</i> expression. These findings contribute to the genetic understanding and diagnosis of <i>TUBB8</i>-related diseases.</p>

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Functional Analysis of a Novel Missense Mutation c.1039A > G of TUBB8 in Infertile Women

  • 期刊论文

  • 发表日期：

    2025年7月2日

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  • Min Guo,
  • Fangfang Li,
  • Lingyan Ren,
  • Guiqin You,
  • Ying Zhang,
  • Juan Liu,
  • Shengwen Huang

摘要

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.