MUTATION DETECTION IN GLYCOGEN-STORAGE-DISEASE TYPE-II BY RT-PCR AND AUTOMATED SEQUENCING

A new method is described for detection of mutations in the lysosomal alpha-glucosidase gene (GAA) leading to Glycogen Storage Disease type II (GSDII), A key feature of the method is isolation and reverse trans cription of mRNA followed by PCR amplification of lysosomal alpha-gluc osidase cDNA with M13-extended primers. Dye labeled primers are used f or cycle sequencing and an ABI PRISM 377 DNA sequencing system for ana lysis. The method is rapid and complementary to the automated sequenci ng of all the 19, PCR amplified, coding exons of the GAA gene. The adv antages and pitfalls of this new method are discussed in the light of the results obtained with an infantile GSDII patient. A new splice sit e mutation in the GAA gene of this patient was identified, IVS16(+2T - -> C), resulting in the deletion of 16 base pairs of exon 16. (C) 1997 Academic Press.