Mutation detection in glycogen storage disease type II by RT-PCR and automated sequencing

ber of 19 coding exons makes this method quite laboriA new method is described for detection of muta- ous. In this paper we describe a new approach to mutations in the lysosomal a-glucosidase gene (GAA) lead- tion detection in GSDII which is based on reverse ing to Glycogen Storage Disease type II (GSDII). A key transcription of mRNA followed by cDNA amplification feature of the method is isolation and reverse tran- and sequence analysis on an ABI PRISM 377 DNA scription of mRNA followed by PCR amplification of sequencing system using dye-labeled primers in the lysosomal a-glucosidase cDNA with M13-extended cycle sequencing reaction. primers. Dye labeled primers are used for cycle se- The advantages and pitfalls of this new method are quencing and an ABI PRISM 377 DNA sequencing sys- illustrated by the detection of a novel mutation in the tem for analysis. The method is rapid and complemen- splice donor site of exon 16 of the GAA gene of a patient tary to the automated sequencing of all the 19, PCR with infantile GSDII from South Africa (Case D; 6). amplified, coding exons of the GAA gene. The advanThe splice site is lost and a cryptic splice donor site in tages and pitfalls of this new method are discussed in exon 16 is used instead, resulting in partial deletion the light of the results obtained with an infantile of exon 16 sequences and deficiency of lysosomal a