Accelerating the diagnosis of Chinese cblC type MMA patients by multiplex PCR sequencing method

Ping Zheng; Chaoji Yu; Lina Xie; Xinna Ji; Shuo Feng; Yanyan Gao; Xing Wei; Wenli Wu; Qian Chen

doi:10.1038/s41390-025-03841-4

Accelerating the diagnosis of Chinese cblC type MMA patients by multiplex PCR sequencing method

Pediatr Res. 2025 Jan 15. doi: 10.1038/s41390-025-03841-4. Online ahead of print.

Authors

Ping Zheng¹, Chaoji Yu², Lina Xie¹, Xinna Ji¹, Shuo Feng¹, Yanyan Gao¹, Xing Wei², Wenli Wu², Qian Chen³

Affiliations

¹ Department of Neurology, Children's Hospital Affiliated to Capital Institute of Pediatrics, Beijing, China.
² Beijing Huanuo Aomei Gene Biotech Co. Ltd, Beijing, China.
³ Department of Neurology, Children's Hospital Affiliated to Capital Institute of Pediatrics, Beijing, China. [email protected].

PMID: 39815091
DOI: 10.1038/s41390-025-03841-4

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: 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. 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.