Development of a feasible assay for the detection of GAA mutations in patients with Pompe disease.
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.
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.
Tze-Kiong
TK
Er
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.
Chih-Chieh
CC
Chen
Department of Medical Genetics and Pediatrics, National Taiwan University Hospital, Taipei 10041, Taiwan. Electronic address: chienyh@ntu.edu.tw.
Yin-Hsiu
YH
Chien
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.
Wen-Chen
WC
Liang
Department of Pediatrics, Kaohsiung Medical University Hospital, 100 Shih-Chuan 1st Rd., Kaohsiung 80708, Taiwan. Electronic address: nekokan1983@yahoo.com.tw.
Tzu-Min
TM
Kan
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.
Yuh-Jyh
YJ
Jong
D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D017124:Conserved Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D005240:Feasibility Studies; D005260:Female; D005787:Gene Frequency; D006009:Glycogen Storage Disease Type II / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D008297:Male; D009691:Nucleic Acid Denaturation; D060888:Real-Time Polymerase Chain Reaction; D044366:Transition Temperature; D000520:alpha-Glucosidases / Q000235:genetics*
D000520:alpha-Glucosidases
Acid α-glucosidase (GAA) gene; DBS; DHPLC; GAA; HRM; High resolution melting; Mutation analysis; PCR; Pompe disease; WCN; acid α-glucosidase; denaturing high performance liquid chromatography; dried blood spot; high resolution melting; polymerase chain reaction; weighted contact number