Tze-Kiong Er1, Chih-Chieh Chen2, Yin-Hsiu Chien3, Wen-Chen Liang4, Tzu-Min Kan5, Yuh-Jyh Jong6. 1. Division of Molecular Diagnostics, Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 100 Shih-Chuan 1st Rd., Kaohsiung 80708, Taiwan; Translational Research Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan. Electronic address: tzekiong@totalbb.net.tw. 2. Institute of Bioinformatics and Systems Biology, College of Biological Science and Technology, National Chiao Tung University, 75 Bo-Ai Street, Hsinchu 30068, Taiwan; Center for Lipid Biosciences, Kaohsiung Medical University Hospital, 100 Shih-Chuan 1st Rd., Kaohsiung 80708, Taiwan; Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, 195, Sec. 4, Chung Hsing Rd., Hsinchu 31040, Taiwan. Electronic address: chieh.bi91g@nctu.edu.tw. 3. Department of Medical Genetics and Pediatrics, National Taiwan University Hospital, Taipei 10041, Taiwan. Electronic address: chienyh@ntu.edu.tw. 4. Center for Lipid Biosciences, Kaohsiung Medical University Hospital, 100 Shih-Chuan 1st Rd., Kaohsiung 80708, Taiwan; Department of Pediatrics, School of Medicine, College of Medicine, Kaohsiung Medical University, 100 Shih-Chuan 1st Rd., Kaohsiung 80708, Taiwan. Electronic address: peggieliang@yahoo.com.tw. 5. Department of Pediatrics, Kaohsiung Medical University Hospital, 100 Shih-Chuan 1st Rd., Kaohsiung 80708, Taiwan. Electronic address: nekokan1983@yahoo.com.tw. 6. Division of Molecular Diagnostics, Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 100 Shih-Chuan 1st Rd., Kaohsiung 80708, Taiwan; Department of Pediatrics, Kaohsiung Medical University Hospital, 100 Shih-Chuan 1st Rd., Kaohsiung 80708, Taiwan; Department of Biological Science and Technology, College of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan; Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. Electronic address: yjjong@gap.kmu.edu.tw.
Abstract
BACKGROUND: Pompe disease is an inherited autosomal recessive deficiency of acid α-glucosidase (GAA) and is due to pathogenic sequence variants in the corresponding GAA gene. While the analysis of enzyme activity remains the diagnostic test of choice for individuals with Pompe disease, mutation analysis remains for establishing a definitive diagnosis. METHODS: High resolution melting (HRM) analysis was performed to screen GAA mutations. Genomic DNA was extracted from peripheral blood samples of the two patients with Pompe disease and 250 normal controls. Exons 2 through 20 of the GAA gene were screened by the HRM analysis. The results were subsequently confirmed by direct sequencing. RESULTS: This assay proved to be feasible in detecting seven known (c.2T>C, c.1726G>A, c.1845G>A, c.1935C>A, c.1958C>A, c.2238G>C, and c.2815_2816del) GAA mutations. Each mutation could be readily and accurately identified in the difference plot curves. We estimated the carrier frequency of the most common mutation, c.1935G>A (p.D645E), in the Taiwanese population to be 0.2%. CONCLUSIONS: In clinical practice, we suggest that HRM analysis is assumed as a fast and reliable method for screening GAA gene mutations especially the most common mutations which are responsible for Pompe disease among the Taiwanese populations.
BACKGROUND: Pompe disease is an inherited autosomal recessive deficiency of acid α-glucosidase (GAA) and is due to pathogenic sequence variants in the corresponding GAA gene. While the analysis of enzyme activity remains the diagnostic test of choice for individuals with Pompe disease, mutation analysis remains for establishing a definitive diagnosis. METHODS: High resolution melting (HRM) analysis was performed to screen GAA mutations. Genomic DNA was extracted from peripheral blood samples of the two patients with Pompe disease and 250 normal controls. Exons 2 through 20 of the GAA gene were screened by the HRM analysis. The results were subsequently confirmed by direct sequencing. RESULTS: This assay proved to be feasible in detecting seven known (c.2T>C, c.1726G>A, c.1845G>A, c.1935C>A, c.1958C>A, c.2238G>C, and c.2815_2816del) GAA mutations. Each mutation could be readily and accurately identified in the difference plot curves. We estimated the carrier frequency of the most common mutation, c.1935G>A (p.D645E), in the Taiwanese population to be 0.2%. CONCLUSIONS: In clinical practice, we suggest that HRM analysis is assumed as a fast and reliable method for screening GAA gene mutations especially the most common mutations which are responsible for Pompe disease among the Taiwanese populations.