DBS Screening for Glycogen Storage Disease Type 1a: Detection of c.648G>T Mutation in G6PC by Combination of Modified Competitive Oligonucleotide Priming-PCR and Melting Curve Analysis

International Journal of Neonatal Screening

Emma Tabe Eko Niba¹  Yogik Onky Silvana Wijaya¹  Hisahide Nishio¹  Masakazu Shinohara¹  Naoko Taniguchi²  Yasuhiro Takeshima²  Hiroyuki Awano³ 

[1] Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Kobe 650-0017, Japan;Department of Pediatrics, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501, Japan;Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Kobe 650-0017, Japan;

关键词: glycogen storage disease type 1a;    dried blood spot;    allele-specific PCR;    mCOP-PCR;    melting curve;   

DOI  :  10.3390/ijns7040079

来源: DOAJ

【 摘 要 】

Glycogen storage disease type Ia (GSDIa) is an autosomal recessive disorder caused by glucose-6-phosphatase (G6PC) deficiency. GSDIa causes not only life-threatening hypoglycemia in infancy, but also hepatocellular adenoma as a long-term complication. Hepatocellular adenoma may undergo malignant transformation to hepatocellular carcinoma. New treatment approaches are keenly anticipated for the prevention of hepatic tumors. Gene replacement therapy (GRT) is a promising approach, although early treatment in infancy is essential for its safety and efficiency. Thus, GRT requires screening systems for early disease detection. In this study, we developed a screening system for GSDIa using dried blood spots (DBS) on filter paper, which can detect the most common causative mutation in the East-Asian population, c.648G>T in the G6PC gene. Our system consisted of nested PCR analysis with modified competitive oligonucleotide priming (mCOP)-PCR in the second round and melting curve analysis of the amplified products. Here, we tested 54 DBS samples from 50 c.648G (wild type) controls and four c.648T (mutant) patients. This system, using DBS samples, specifically amplified and clearly detected wild-type and mutant alleles from controls and patients, respectively. In conclusion, our system will be applicable to newborn screening for GSDIa in the real world.

【 授权许可】

Unknown