Novel Germline Mutations in FUT7 and EXT1 Linked to Hereditary Multiple Exostoses

Background

Hereditary Multiple Exostoses (HME), also known as Hereditary Multiple Osteochondromas (HMO), is an autosomal dominant skeletal disorder characterized by the development of multiple cartilage-capped bone projections (osteochondromas). These osteochondromas typically occur in the long bones, pelvis, or scapula and can lead to pain, bone deformities, nerve and vascular compression, and short stature. The most severe complication is the potential malignant transformation into chondrosarcoma, which occurs in approximately 3.9% of HME patients. HME is primarily associated with mutations in the EXT1 and EXT2 genes, which encode glycosyltransferases essential for heparan sulfate biosynthesis, a critical component in chondrocyte differentiation, ossification, and apoptosis. However, not all HME cases can be explained by mutations in these genes, suggesting the involvement of other genetic factors.

Research Objectives

The study aimed to identify novel germline mutations associated with HME and to elucidate their functional roles in the pathogenesis of the disease. By using whole-exome sequencing (WES) and functional assays, the researchers sought to uncover new genetic contributors to HME and explore their molecular mechanisms.

Study Overview

The research was conducted by a team from multiple institutions, including the Affiliated Cancer Hospital and Institute of Guangzhou Medical University, Sun Yat-sen University Cancer Center, and the South China University of Technology. The study was published in Oncogene in 2024, with the DOI: 10.1038/s41388-024-03254-3.

Key Findings

1. Identification of Novel Mutations

Through WES analysis of a Chinese family affected by HME, the researchers identified two novel germline mutations: a nonsense mutation in EXT1 (c.204G>A, p.Trp68*) and a missense mutation in FUT7 (c.893T>G, p.Phe298Cys). These mutations co-segregated with the HME phenotype in the family. The EXT1 mutation results in a truncated protein lacking critical functional domains, while the FUT7 mutation affects the cellular localization of the FUT7 protein and its role in cell proliferation.

2. Functional Impact of FUT7 Mutation

Functional assays revealed that the FUT7 mutation (p.Phe298Cys) promotes cell proliferation by inhibiting the p53 signaling pathway. Additionally, the mutation reduces EXT1 expression at both the transcriptional and protein levels. The researchers demonstrated that FUT7 normally enhances EXT1 expression through the IL6/STAT3/Slug axis, but the p.Phe298Cys mutation disrupts this regulation, leading to decreased EXT1 expression and increased protein degradation via ubiquitination.

3. Zebrafish Model Validation

Using a zebrafish model, the researchers showed that simultaneous knockdown of FUT7 and EXT1 orthologues resulted in severe craniofacial cartilage defects, indicating a functional link between these genes in chondrocyte regulation. This finding suggests that FUT7 may act as a “second-hit” mutation, exacerbating the effects of EXT1 mutations in HME pathogenesis.

4. Molecular Mechanism of FUT7-EXT1 Interaction

The study further elucidated the molecular mechanism by which FUT7 regulates EXT1 expression. FUT7 activates the IL6/STAT3/Slug pathway, which in turn enhances EXT1 transcription. The p.Phe298Cys mutation disrupts this pathway, leading to reduced EXT1 expression. Additionally, the mutation promotes the ubiquitination-mediated degradation of the EXT1 protein, further contributing to the HME phenotype.

Conclusions and Significance

This study identified two novel germline mutations in FUT7 and EXT1 that are associated with HME in a Chinese family. The findings highlight the critical interaction between FUT7 and EXT1 in the pathogenesis of HME, particularly the role of FUT7 as a potential “second-hit” mutation that exacerbates the effects of EXT1 mutations. These insights enhance our understanding of the genetic and molecular mechanisms underlying HME and open new avenues for potential therapeutic interventions.

Research Highlights

1. Novel Mutations: The study reports the first germline mutation in FUT7 associated with HME, expanding the genetic landscape of the disease.
2. Functional Mechanism: The research reveals that FUT7 regulates EXT1 expression through the IL6/STAT3/Slug pathway and that the p.Phe298Cys mutation disrupts this regulation, leading to decreased EXT1 expression and increased protein degradation.
3. Zebrafish Model: The use of a zebrafish model demonstrated the functional link between FUT7 and EXT1 in chondrocyte regulation, providing in vivo evidence for their roles in HME pathogenesis.

Future Directions

While this study provides significant insights into the genetic and molecular mechanisms of HME, several questions remain. Further research is needed to explore the precise genetic linkage between FUT7 and EXT1 and their co-segregation with the HME phenotype. Additionally, the regulatory mechanisms of FUT7 on IL6 expression and the exact role of EXT1 in HME pathogenesis warrant further investigation. Other pathways, such as those related to osteoblast differentiation and cartilage development, may also contribute to FUT7-regulated EXT1 expression and should be explored in future studies.

Acknowledgments

The authors thank all participating individuals and their family members for supporting this study. They also acknowledge the staff at the High Throughput Analysis Platform of SYSUCC for data generation and processing. This study was supported by the National Key R&D Program of China, the National Natural Science Foundation, and the Guangzhou Eighth People’s Hospital Research Start-up Funds.

Author Contributions

Jin-Xin Bei supervised the work. Guo-Wang Lin, Xiao-Yu Zuo, Wan Peng, and Gao-Fei Li performed bioinformatics analyses. Wan Peng, Gao-Fei Li, Xi-Xi Cheng, Qiao-Hong Lin, and Shu-Qiang Liu conducted the wet laboratory experiments. Chun-Ling Luo provided suggestions on some wet laboratory experiments. Yi-Yue Zhang supported the zebrafish model. De-Jun Li, Dao-Chao Lin, Jun-Qiang Yin, and Xian-Biao Xie contributed to clinical sample collection. Wan Peng, Gao-Fei Li, and Jin-Xin Bei wrote the manuscript. All authors read and approved the final manuscript.

Competing Interests

The authors declare no competing interests.

Ethics Approval

This study was approved by the Ethics Committee for Clinical Research and Animal Trials of the First Affiliated Hospital of Sun Yat-sen University. Informed consent was obtained from all participants. All work involving zebrafish was reviewed and approved by the Animal Research Advisory Committee of the South China University of Technology.

Additional Information

Supplementary information is available online at https://doi.org/10.1038/s41388-024-03254-3. Correspondence and requests for materials should be addressed to Xian-Biao Xie or Jin-Xin Bei.