Abstract
Background
CblC type methylmalonic aciduria (cblC disease) is the most common inborn error of vitamin B12 metabolism and due to mutations in the MMACHC gene. The earlier the diagnosis, the better the prognosis. Therefore, convenient and inexpensive detection method is needed.
Methods
This study selected mutational hot-spot regions in the MMACHC gene which harbors more than 90% of mutant alleles responsible for cblC disease in China. Subsequently, a hot-spot regions multi-PCR Sanger sequencing method (HsRMSS) was designed. The accuracy and efficiency of HsRMSS was validated using samples from 20 cblC families with known MMACHC gene mutations and 50 healthy volunteers. In addition, patients’ clinical phenotypes and molecular genetic features were analyzed.
Results
A total of 16 different mutations were identified in 20 cblC families. Among them, the most common mutations were c.609 G>A (26/80, 32.5%), c.567dupT (10/80, 12.5%), c.80A>G (8/80, 10.0%), c.658_660delAAG (8/80, 10.0%) and c.394C>T (6/80, 7.5%), which accounted for over 70% of disease alleles. The HsRMSS results were the same as the results using the whole exon sequencing, with a coincidence rate of 100%.
Conclusion
The HsRMSS targeting the mutational hot-spots of MMACHC gene could be a promising tool to accurately and rapidly diagnose cblC disease in China.
Impact
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This study reported the development and validation of a hot-spot regions multi-PCR Sanger sequencing method for targeting hotspots which harbor most of the common MMACHC gene mutations reported in Chinese patients with cblC disease.
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The approach could have a potential clinical application as a rapid diagnosis and screening tool for suspected children with cblC type MMA and population carrier, owing to its high throughput, low cost, and high sensitivity and specificity.
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
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Acknowledgements
We would like to sincerely thank all participants and their families for their cooperation in providing both clinical information and samples for the study.
Funding
The study was supported by the National key Research and Development Program of China (2022YFC2703903) and Rare disease Research Program of E-Town Cooperation & Development Foundation (YCXJ-JZ-2023-017).
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Contributions
Ping Zheng designed the study, performed the experiments, coordinated and supervised data collection, carried out the data analyses and interpretation, and drafted the manuscript. Chaoji Yu helped perform the experiments, Lina Xie and Xinna Ji provide both clinical information and samples, and critically reviewed and revised the manuscript. Shuo Feng and Yanyan Gao collected data and critically reviewed and revised the manuscript. Xing Wei and Wenli Wu provided the experiment guidance and revised the manuscript. Qian Chen conceived and supervised this study, and revised the manuscript. All authors contributed to the article and approved the submitted version.
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The authors declare no competing interests.
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All the samples were collected with informed consent of the individual or his/her parents. The protocols for the study and the written consent were approved by the ethics committee of the Capital Institute of Pediatrics at Beijing, China (Approval ID: SHERLL2023086).
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Zheng, P., Yu, C., Xie, L. et al. Accelerating the diagnosis of Chinese cblC type MMA patients by multiplex PCR sequencing method. Pediatr Res 98, 950–956 (2025). https://doi.org/10.1038/s41390-025-03841-4
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DOI: https://doi.org/10.1038/s41390-025-03841-4
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