Isolation of a cDNA for human acid alpha-glucosidase and detection of genetic heterogeneity for mRNA in three alpha-glucosidase-deficient patients.

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Abstract
Lysosomal acid .alpha.-glucosidase (EC 3.2.1.3) hydrolyzes 1,4-linked .alpha.-D-glucose polymers present in glycogen. Genetic deficiency of acid .alpha.-glucosidase results in glycogen-storage disease type II, encompassing a spectrum of disorders of varying severity. To study the molecular basis for this heterogeneity, we sought to clone the coding sequence for human acid .alpha.-glucosidase. We screened 106 recombinant phage from a human liver cDNA expression library with an affinity-purified polyclonal antibody to human acid .alpha.-glucosidase. When we retested positive phage for reactivity to monoclonal antibodies, we identified a single phage, containing a 2-kilobase (kb) cDNA insert, that reacted with both polyclonal and monoclonal antibodies. The 2-kb cDNA hybridized to a 20-kb EcoRI fragment of human genomic DNA. This 20-kb EcoRI fragment was present only in DNA from somatic cell hybrids that retained the human chromosome 17 segment q21-q23, which contains the gene for human acid .alpha.-glucosidase. The cDNA also hybridized to a 3.4-kb mRNA, consistent with the size (.apprxeq. 105 kDa) of the acid .alpha.-glucosidase protein. Finally, in one of the two infantile-onset acid .alpha.-glucosidase-deficient cell lines tested, the 3.4-kb mRNA was not detectable, whereas in an adult-onset cell line, an mRNA of reduced size and amount was found. Examination of DNA digested with restriction enzymes did not reveal any major deletions in the genomic DNA of the patients.