Functional analysis of a novel missense mutation c.1039A＞G of TUBB8 in infertile women

doi:10.21203/rs.3.rs-6223500/v1

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Functional analysis of a novel missense mutation c.1039A＞G of TUBB8 in infertile women

https://doi.org/10.21203/rs.3.rs-6223500/v1

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published 02 Jul, 2025

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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 gene

c.1039A > G

female infertility

oocyte arrest

The perpetuation of the human species relies on three critical stages: the maturation of sperm and oocytes, the union of sperm and oocytes, and subsequent embryonic development. Deviations in any of these stages can lead to infertility. Oocyte maturation is particularly crucial in female reproduction, as failure in this process results in primary infertility in women^1,2. As of 2024, 28 cases of female infertility due to arrested oocyte maturation have been documented in the literature (data sourced from the PubMed database). Despite advances in assisted reproductive technologies, such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), these treatments have not been effective in addressing this specific issue of arrested oocyte maturation³.

Tubulin β class VIII (TUBB8) is a specialized isoform of β-tubulin unique to primates, playing an essential role in oocyte spindle assembly. It is specifically expressed during oocyte maturation and early embryonic development, and notably, it is absent in mature spermatozoa^4,5. In a landmark study in 2016, Feng et al.⁶ discovered that pathogenic mutations in the TUBB8 gene disrupt the normal assembly of microtubules in oocytes, leading to defects in spindle formation and causing oocyte meiosis to arrest at the MI (metaphase I) stage. Heterozygous mutations in TUBB8 can cause oocyte arrest at the mid-MI stage, either through a dominant-negative effect or haploinsufficiency. Furthermore, compound heterozygote or heterozygote mutations may lead to fertilization failure and early embryonic developmental arrest^7–9. Therefore, screening for pathogenic variants in the TUBB8 gene can offer more precise genetic diagnostics and provide comprehensive genetic counseling for couples facing unexplained infertility.

According to the statistics from the Human Gene Mutation Database (HGMD®; http://www.biobase-international.com/product/hgmd), 157 pathogenic variants of TUBB8 have been identified, associated with five main phenotypes: oocyte maturation arrest, failure of oocyte fertilization, zygotic cleavage failure, early embryonic developmental arrest, and failure of embryo implantation¹⁰. Among these, oocyte maturation arrest is the predominant phenotype. Statistically, oocyte maturation arrest accounts for approximately 30% of all TUBB8 pathogenic mutations related to infertility, making it the most frequent phenotype.

In this study, we present a novel mutation in the TUBB8 gene, which was validated through molecular genetic analysis and in vitro experiments. This discovery expands the genetic landscape of TUBB8 mutations and provides additional evidence for the genetic diagnosis of TUBB8-related infertility. Furthermore, it offers valuable insights and potential assistance for improving reproductive health in women.

1. Subjects

The patient, a 27-year-old woman, had been married for over a year and presented with infertility despite not using contraception. She had a history of Polycystic Ovary Syndrome (PCOS) and a menstrual cycle ranging from 23 to 37 days, with a 3-day menstrual period, light flow, and occasional dysmenorrhea. The patient had undergone ovulation stimulation treatment at the Reproductive Medicine Center of Guizhou Provincial People's Hospital, resulting in dominant follicle maturation and ovulation. However, she remained infertile after regular intercourse. She had not undergone in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and denied consanguinity in her marriage.

The patient's family history revealed four women with a history of infertility: II3, III5, III8, and IV1. In contrast, III3 and III7 had children (see Fig. 1a for details).

In this study, peripheral blood samples were collected from the proband (IV1), her father (III2), mother (III1), and aunt for genetic testing. The remaining family members declined to participate. The study was approved by the Ethics Committee of Guizhou Provincial People's Hospital, and all participants provided written informed consent to participate.

2. Whole Exome Sequencing (WES) and Bioinformatics Analysis

Whole exome sequencing (P039-Exome) was performed on the proband with a target mean depth of 104.99 and target region coverage of 98.79. The sequenced reads were aligned to the UCSC hg19 human reference genome using the BWA algorithm (https://www.sentieon.com/) within Sentieon software (https://www.sentieon.com/). Single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) were identified using Sentieon’s variant calling tool. The detected variants were annotated using ANNOVAR software (http://annovar.openbioinformatics.org/en/latest/), which incorporates data from HGMD® and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar). The pathogenicity of the identified variants was predicted using bioinformatics tools, including SIFT (http://sift.jcvi.org/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), MutationTaster (https://www.mutationtaster.org/), and GERP++ (http://mendel.stanford.edu/SidowLab/downloads/gerp/index.html).

3. Sanger Sequencing

To validate the TUBB8 gene mutation identified by WES, Sanger sequencing was performed on the proband, her parents, and her aunt to confirm the presence of potential pathogenic variations. The forward and reverse primers used for sequencing were 5'-CCGTGGCATCCTGATATTGC-3' and 5'-TGACTGTGGCTGAGCTTACC-3', respectively. PCR amplification products were sequenced using a 3130XL sequencer via Sanger sequencing.

4. Conservation Analysis and Protein Structure Prediction

To assess the conservation of the mutant amino acid sequences, we utilized the MEGA11 software tool (https://www.megasoftware.net/dload_win_beta). The three-dimensional structures of both wild-type (WT) and mutant (MUT) proteins were predicted using Phyre2 software (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgiid=index). Additionally, ChimeraX software (https://www.cgl.ucsf.edu/chimerax/) was used to analyze the structural and physicochemical differences between the wild-type and mutant proteins at the mutation sites.

5. Immunofluorescence

Plasmids TUBB8-WT and TUBB8-A1039G-MUT were constructed using a 3xFlag-pcDNA3.1 vector, expressing the FLAG tag fusion protein. These plasmids were transiently transfected into HeLa cells using Lip3000 (HB-LF3, Hanhen Bio, China). After a 48-hour incubation post-transfection, cells were fixed with 4% formaldehyde at 4°C for 30 minutes, then washed with PBST. Permeabilization was performed using 0.2% Triton X-100 for 20 minutes at room temperature, followed by blocking with 5% BSA for 1–2 hours. Immunostaining was conducted using Mouse anti-FLAG tag primary antibody (1:200 dilution, Abclonal, AE005), 594-conjugated Goat anti-Mouse IgG (H + L) (1:200 dilution, AS054, ABclonal, China), or FITC-conjugated Goat anti-Mouse IgG (H + L) (1:300 dilution, AS001, ABclonal, China). Nuclei were stained with DAPI (10 µg/ml, C0065, Solarbio, China) for 10 minutes. The cellular microtubule structure and protein localization were observed using a confocal microscope (LSM980, ZEISS, Germany), and fluorescence intensity was analyzed using ImageJ software.

6. RT-PCR

Plasmids TUBB8-WT and TUBB8-A1039G-MUT were constructed using the pEGFP-N1 empty plasmid and verified by Western blotting. HEK293T cells or HeLa cells were seeded in six-well plates at a density of 4 × 10^5 cells per well, 24 hours before transfection. The plasmids were transfected into the cells using Lip3000 (HB-LF3, Hanhen Bio, China), and RNA was extracted 36 hours post-transfection. Reverse transcription to cDNA was performed using the RT Master Mix for qPCR II (gDNA digester plus) (HY-K0511A-1, MCE, China), followed by real-time quantitative PCR using the TB Green® Premix Ex Taq™ (Tli RNaseH Plus) (RR420A, TAKARA, Japan) on a real-time PCR instrument (CFX OPUS 96, BIO-RAD, USA). The forward and reverse primers for TUBB8 were: TUBB8-F: 5'-TTCAGGCCAGACAACTTCATCTT-3' and TUBB8-R: 5'-CTCCTTTCTGACAACGTCCATCA-3'. The internal control primers were: ACTB-F: 5'-CCACGAAACTACCTTCAACTCCATC-3' and ACTB-R: 5'-AGTGATCTCCTTCTGCATCCTGTC-3'. The Q-PCR reaction mixture (25 µL) consisted of cDNA (2 µL), forward primer (10 µM, 1 µL), reverse primer (10 µM, 1 µL), 2× TB Ex Taq premix (12.5 µL), and sterile water (8.5 µL). The relative gene expression levels were calculated using the 2^−ΔΔCt method (ΔΔCt = ΔCt(treatment group) - ΔCt(control group), ΔCt = Ct(target gene) - Ct(reference gene)).

7. Western Blot

Plasmids TUBB8-WT and TUBB8-A1039G-MUT were constructed using the pEGFP-N1 empty vector and verified by Western blotting. HEK293T or HeLa cells were seeded in six-well plates at a density of 4 × 10^5 cells per well, 24 hours before transfection. The plasmids were transfected into the cells using Lip3000 (HB-LF3, Hanhen Bio, China). Protein extraction was performed 48 hours post-transfection, and Western blotting was conducted. The primary antibodies used were Mouse anti-GFP tag (AE012, Abclonal, China) and Mouse anti-GAPDH (AC033, Abclonal, China), both diluted to 1:4000 and 1:40,000, respectively. The secondary antibody, Goat anti-Mouse IgG HRP (AS003, Abclonal, China), was diluted to 1:4000. Protein bands were visualized, and signals were analyzed using ImageJ software.

8. Statistical Analysis

Data were analyzed using GraphPad Prism software. Values are presented as the mean ± standard error of the mean (SEM), and statistical significance was determined using the t-test. A P-value of less than 0.05 was considered statistically significant.

1. Mutation Site Analysis of the TUBB8 Gene

Whole exome sequencing (WES) of the proband revealed a c.1039A > G (p.N347D) mutation in the TUBB8 gene. This heterozygous missense mutation has not been previously reported in East Asian populations. According to ACMG guidelines, the variant is classified as "Uncertain Clinical Significance" with supporting evidence of PM2. Sanger sequencing confirmed that the proband's father (III2) and aunt (III5) also carried the TUBB8 c.1039A > G (p.N347D) heterozygous mutation (Fig. 1a,b).

2. Conservation Analysis and Prediction of TUBB8 Protein Structure

Conservation analysis using MEGA11 software showed that the aspartic acid (N) at position 347 of the TUBB8 protein is highly conserved among primates (Fig. 1c). Bioinformatics tools, including SIFT and PolyPhen-2, predicted that the p.N347D mutation is benign. However, MutationTaster predicted it as harmful, and GERP + considered it of unknown significance. To assess the potential impact of the c.1039A > G (p.N347D) mutation on TUBB8 protein structure, we performed structural predictions and analysis using Phyre2 and ChimeraX software based on the Q8WP13.1.A model. The results showed no significant difference in the overall structure between the mutant and wild-type proteins. However, the mutation caused the aspartic acid at position 347 in the mutant protein to be replaced by asparagine, forming a hydrogen bond with lysine at position 350. Despite this change, the overall protein structure remained unaffected (Fig. 1e,f).

3. Effect of the TUBB8 Mutation on Cellular Microtubule Morphology

To investigate the effect of the c.1039A > G (p.N347D) mutation on TUBB8 protein localization and tubulin structure, FLAG-tagged TUBB8-WT (wild-type) and TUBB8-A1039G-MUT (mutant) plasmids were transfected into HeLa cells for immunofluorescence staining. The TUBB8 p.N347D mutant protein was localized in the cytoplasm, with no noticeable change in its distribution compared to the wild-type protein (Fig. 2c). Additionally, we assessed the effect of the p.N347D mutation on the microtubule structure. The results showed that the microtubule architecture was not significantly altered in the mutant cells compared to the wild-type, suggesting that the mutation does not induce functional abnormalities by disrupting the structural integrity of microtubules (Fig. 2c).

4. Expression of Mutant TUBB8 in Cells

To determine whether the c.1039A > G mutation affects the expression of TUBB8, pEGFP-tagged TUBB8-WT, and TUBB8-A1039G-MUT plasmids were transfected into both HEK293T and HeLa cells. Q-PCR and Western blot analyses revealed that both the mRNA and protein levels of the c.1039A > G mutation were significantly lower than those of the wild-type counterpart. This experiment was repeated at least three times, with all results showing statistical significance (P-value < 0.0001) (Fig. 3). Similarly, immunofluorescence staining showed that the mutant protein's expression level was lower than the wild-type protein (P-value < 0.0001), which was consistent with the Western blot results. These findings indicate that the c.1039A > G mutation reduces TUBB8 protein expression, which may impair its function (Fig. 2a,b).

TUBB8 encodes β-tubulin, which forms microtubules in combination with α-tubulin. It is critical in guiding spindle assembly and chromosome migration during meiosis ^11,12. The TUBB8 gene (NM_177987.2) is located on chromosome 10p15.3 and consists of four coding exons, with the c.1039A > G mutation occurring in exon 4, near the 3' end (Fig. 1d). The TUBB8 protein contains several structural domains, including a microtubulin structural domain (aa47–244), a microtubulin C-terminal domain (aa246–383), and a helical domain (aa405–444), among others¹³. To date, 157 mutations in TUBB8 have been identified, including missense mutations (87.9%, 138/157), deletions (7%, 11/157), and duplications (5.1%, 8/157). Missense mutations are the most common, and they primarily impair TUBB8 function by affecting microtubule structure. However, some missense mutations may also impair TUBB8 function by reducing its mRNA and protein expression levels.

The c.1039A > G (p.N347D) mutation identified in this study is located in exon 4, near the C-terminal end of the TUBB8 gene. The proband inherited this mutation from her unaffected father while the proband suffers from infertility. Based on the ACMG guidelines for variant interpretation, and supported by the evidence PM2_Supporting, the c.1039A > G mutation was classified as a variant of uncertain significance (VUS).

Conservation analysis showed that the c.1039A > G (p.N347D) mutation is highly conserved among primates. Protein structure prediction results suggested that the mutation does not significantly alter the overall structure of TUBB8. However, the asparagine at position 347 in the mutant protein formed an additional hydrogen bond with lysine at position 350, which may increase the structural stability of the protein. Previous studies have shown that mutations in TUBB8 can alter its protein structure, potentially disrupting the microtubule structure and affecting cellular function^14,15. Therefore, we conducted immunofluorescence staining to examine the structure of tubulin in both wild-type and mutant TUBB8. The results showed no significant differences between the two groups, indicating that the c.1039A > G (p.N347D) mutation did not affect the protein structure in a way that would lead to dysfunction of the TUBB8 protein.

Subsequently, Q-PCR and Western blot analyses revealed that the TUBB8 c.1039A > G mutation is associated with a significant downregulation of mRNA and protein levels. This is consistent with findings from previous studies, such as the missense mutation c.608A > G (p.D203G) located in exon 4, which also decreases mRNA and protein expression levels¹⁴. These results suggest that the c.1039A > G mutation may reduce TUBB8 gene expression. The impact of missense mutations on gene expression can be multifaceted. Such mutations not only alter the protein sequence, structure, and function but can also interfere with mRNA stability, splice site recognition, transcriptional efficiency, and protein translation efficiency, potentially disrupting protein interactions and recognition^16–18. For instance, the c.936 G > C (p.Q312H) mutation in the LMNA gene was shown to cause splicing errors. Minigene assays and Sanger sequencing confirmed that this mutation induced misrecognition of the 5' splice site at the end of exon 5, leading to decreased mRNA stability and reduced protein levels¹⁹. Similarly, a missense mutation c.5628C > A in the DYSF gene was shown to cause aberrant splicing, resulting in exon 50 skipping and leading to a pathogenic splicing pattern that contributes to disease ²⁰.

The findings from these studies highlight the complex molecular mechanisms by which missense mutations can influence gene expression. To further understand the reduced TUBB8 expression associated with the c.1039A > G mutation, future investigations will focus on splice site alterations, mRNA stability, and transcriptional efficiency.

In conclusion, we have identified a novel TUBB8 c.1039A > G (p.N347D) mutation that likely impacts TUBB8 function by reducing its mRNA and protein expression levels, thereby contributing to female infertility. Our study expands the mutation spectrum and associated phenotypes of TUBB8, providing valuable insights for clinicians in diagnosing and managing TUBB8-related infertility.

Author Contribution: All authors contributed to the conception and design of the study. Min Guo, Fangfang Li, Lingyan Ren, Guiqin You, and Ying Zhang were responsible for material preparation, data collection, and analysis. Min Guo authored the first draft of the manuscript. Dr. Liu Juan and Professor Huang Shengwen reviewed the final draft together, and all authors commented on previous versions of the manuscript. Finally, all authors read and approved the final manuscript.

Conflict of Interest The authors declare no competing interests.

Data Availability Not applicable.Code Availability Not applicable.

Declarations Ethics Approval All experimental procedures involving animals were conducted by the Guizhou Provincial People's Hospital.

Consent to Participate Not applicable.

Consent for Publication Not applicable.

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); the terms of such publishing agreement and applicable law solely govern the author self-archiving of the accepted manuscript version of this article.

Acknowledgment

The authors thank Shengwen Huang, Ph.D and Juan Liu, Ph.D.

Funding information

The research reported is supported by the Guizhou Provincial Genetic Disease Diagnosis and Pathogenesis Research Science and Technology Innovation Talent Team (Guizhou Kehe Platform Talent [2020] 5011; Guizhou Provincial Department of Science and Technology.

The research reported is supported by National Natural Science Foundation of China (NSFC) Project: The Role and Mechanism of the AMPK/TFEB/LAMP1 Axis in Metformin - regulated Autophagy against Ovarian Oxidative Damage (Project Approval No. 82260299).

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No competing interests reported.

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Functional analysis of a novel missense mutation c.1039A＞G of TUBB8 in infertile women

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and methods

1. Subjects

2. Whole Exome Sequencing (WES) and Bioinformatics Analysis

3. Sanger Sequencing

4. Conservation Analysis and Protein Structure Prediction

5. Immunofluorescence

6. RT-PCR

7. Western Blot

8. Statistical Analysis

Results

Discussion

Declarations

References

Additional Declarations

Status:

Journal Publication

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