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Novel susceptibility gene SLC23A2 functions via PI3K-AKT-mTOR pathway in etiology of non-syndromic cleft palate

Journal of Human Genetics volume 70, pages 443–452 (2025)Cite this article

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Abstract

The biological interactions between genetic and environmental modifiers play critical roles in the etiology of non-syndromic orofacial cleft (NSOC), but it is rarely studied. This study selected 47 environmental related genes from the metabolic pathways of smoking, drinking, hypoxia, and vitamins (including vitamin A, vitamin B9 (folic acid), vitamin C (ascorbic acid), vitamin D, and vitamin E), and test their associations with NSOC and its subtypes. We found that a novel gene SLC23A2, the vitamin C transporter gene is significantly associated with non-syndromic cleft palate only (NSCPO) (p = 3.25E-07, OR = 8.45, 95%CI:3.73–19.17). SLC23A2 is expressed in the craniofacial region of zebrafish (24hpf to 120hpf), obvious craniofacial abnormalities appeared in zebrafish (48hpf) when knock down the slc23a2 (slc23a2-MO). Knock down SLC23A2 in human embryonic palatal mesenchymal cell line (HEPM) induced decreased intracellular ascorbic acid (AA), increased reactive oxygen species (ROS), inhibited cell proliferation and triggered apoptosis, activated the PI3K-AKT-mTOR signaling pathway and inhibited autophagy; ROS levels and apoptosis ratio significantly decreased when we supplemented AA to HEPM cells with high ROS levels induced by Sin-1 (an exogenous ROS mimic). Knocked down SLC23A2 in HEPM cells or zebrafish, they became more sensitive to Sin-1, and AA supplementation was ineffective. In conclusion, we identified a novel susceptibility gene SLC23A2 for NSCPO, it may function by decreasing AA level, increasing the ROS levels, inducing apoptosis, and inhibiting autophagy through the activation of the PI3K-AKT-mTOR pathway in etiology of cleft palate.

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Fig. 1: Association analysis of SNPs in environmental related genes.
Fig. 2: Expression and functional analysis of slc23a2 in zebrafish.
Fig. 3: Changes of cell biology and ROS in HEPM after SLC23A2 knockdown.
Fig. 4: Effects of SLC23A2/ exogenous ROS/AA interaction on HEPM cellular oxidative stress level and cellular biology.
Fig. 5: Effects of slc23a2/exogenous ROS/AA interaction on oxidative stress and apoptosis in zebrafish.

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Data availability

The data are available from the corresponding author on reasonable request.

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Acknowledgements

The authors thank all the participants who donated samples in this study. All authors gave their final approval and agree to be accountable for all aspects of the work. This project was supported by the National Science Funds of China (No. 82170919 and No. 81600849), Sichuan Province Science and Technology support program (2024NSFSC0649), the Research and Develop Program, West China Hospital of Stomatology Sichuan University (No. RD-03-202301).

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  1. These authors contributed equally: Bin Yin, Mu-Jia Li, Jia-Lin Sun.

Authors and Affiliations

  1. State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Department of cleft lip and palate, West China Hospital of Stomatology, Sichuan University, Chengdu, China

    Bin Yin, Jia-Lin Sun, Yue You, Si-Di Zhang, Qian-Xue Wan, Mei-Lin Yao, Cheng-Wei Yang, Bing Shi & Zhong-Lin Jia

  2. Department of Oral and Maxillofacial Surgery, Affiliated Stomatology Hospital, Medical School of Nanjing University, No. 30 Zhong Yang’s Road, Xuan Wu, Nanjing, 210008, Jiangsu Province, China

    Bin Yin

  3. Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Engineering Research Center of Oral Biomaterials and Devices of Zhejiang Province, Hangzhou, 310000, China

    Mu-Jia Li

  4. SCU-CUHK Joint Laboratory for Reproductive Medicine, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Department of Pediatrics, West China Second University Hospital, Sichuan University, 610041, Chengdu, China

    Hua-Qin Sun & Zi-Yuan Lin

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Contributions

Bin Yin: Data curation, Writing-original draft, Conceptualization, Methodology, Software. Mu-Jia Li: Data curation, Methodology, Software, Writing - Review & Editing. Jia-Lin Sun: Data curation, Methodology, Software, Writing - Review & Editing. Yue You: Visualization, Methodology. Si-Di Zhang: Validation, Writing - Review & Editing. Qian-Xue Wan: Visualization. Mei-Lin Yao: Visualization. Cheng-Wei Yang: Writing - Review & Editing. Hua-Qin Sun: Methodology, Validation. Zi-Yuan Lin: Resources. Bing Shi: Supervision. Zhong-Lin Jia: Conceptualization, Methodology, Validation, Formal analysis, Project administration, Funding acquisition, Writing - Review & Editing.

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Correspondence to Zhong-Lin Jia.

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The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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The study protocol conformed to the STOBE (strengthening the reporting of observational studies in epidemiology) guidelines and was approved by the institutional review board (IRB) of West China Hospital of Stomatology, Sichuan University in 2016 (WCHSIRB-D-2016-012R1). Written informed consent was obtained from recruited individuals of consenting age and from parents on behalf of their participating children.

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Yin, B., Li, MJ., Sun, JL. et al. Novel susceptibility gene SLC23A2 functions via PI3K-AKT-mTOR pathway in etiology of non-syndromic cleft palate. J Hum Genet 70, 443–452 (2025). https://doi.org/10.1038/s10038-025-01352-y

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