Zheng Ping, Yu Chaoji, Xie Lina, Ji Xinna, Feng Shuo, Gao Yanyan, Wei Xing, Wu Wenli, Chen Qian
Department of Neurology, Children's Hospital Affiliated to Capital Institute of Pediatrics, Beijing, China.
Beijing Huanuo Aomei Gene Biotech Co. Ltd, Beijing, China.
Pediatr Res. 2025 Jan 15. doi: 10.1038/s41390-025-03841-4.
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.
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.
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%.
The HsRMSS targeting the mutational hot-spots of MMACHC gene could be a promising tool to accurately and rapidly diagnose cblC disease in China.
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.
CblC型甲基丙二酸血症（cblC病）是维生素B12代谢最常见的先天性缺陷，由MMACHC基因突变引起。诊断越早，预后越好。因此，需要便捷且廉价的检测方法。
本研究选择了MMACHC基因中的突变热点区域，该区域携带了中国超过90%导致cblC病的突变等位基因。随后，设计了一种热点区域多重聚合酶链反应桑格测序方法（HsRMSS）。使用来自20个已知MMACHC基因突变的cblC家族的样本和50名健康志愿者验证了HsRMSS的准确性和效率。此外，分析了患者的临床表型和分子遗传学特征。
在20个cblC家族中总共鉴定出16种不同的突变。其中，最常见的突变是c.609G>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%）和c.394C>T（6/80，7.5%），这些突变占疾病等位基因的70%以上。HsRMSS结果与全外显子测序结果相同，符合率为100%。
针对MMACHC基因突变热点的HsRMSS可能是在中国准确、快速诊断cblC病的一种有前景的工具。
本研究报告了一种针对热点区域的多重聚合酶链反应桑格测序方法的开发和验证，该热点区域包含中国cblC病患者中报告的大多数常见MMACHC基因突变。由于其高通量、低成本以及高灵敏度和特异性，该方法作为疑似cblC型甲基丙二酸血症儿童和人群携带者的快速诊断和筛查工具可能具有潜在的临床应用价值。
