Development of a feasible assay for the detection of GAA mutations in patients with Pompe disease

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.