title: Identification of a missense mutation in one allele of a patient with Pompe disease, and use of endonuclease digestion of PCR-amplified RNA to demonstrate lack of mRNA expression from the second allele.
abstract: Infantile-onset glycogen storage disease type II, or Pompe disease, results from a genetic deficiency of the lysosomal enzyme acid alpha glucosidase (GAA). Sequencing of the cDNA from a cell line (GM 244) derived from a patient with Pompe disease demonstrated a T953-to-C transition that predicted a methionine-to-threonine substitution at codon 318. The basepair substitution resulted in loss of restriction-endonuclease sites for NcoI and StyI. Analysis of genomic DNA revealed both a normal and an abnormal NcoI fragment, indicating that the patient was a genetic compound. NcoI and StyI digestion of cDNA, amplified by PCR from reverse-transcribed RNA, demonstrated that greater than 95% of the GAA mRNA in GM 244 was derived from the allele carrying the missense mutation. The missense mutation was uncommon, since it was not detected in 37 additional GAA-deficient chromosomes, as determined by digestion of genomic DNA with NcoI and hybridization. The amino acid substitution predicts a new potential site for N-linked glycosylation, as well as major changes in secondary structure of the protein. We could confirm that the mutation was responsible for the enzyme deficiency by demonstrating that a hybrid minigene containing the mutation did not express GAA enzyme activity after transient gene expression. We have therefore now provided the first identification of a single-basepair missense mutation in a patient with Pompe disease and furthermore have demonstrated that the patient is a genetic compound with the second allele barely expressing mRNA.
authors.affiliation: Division of Medical Genetics, New York University Medical Center, NY 10016.
authors.forename: N
authors.initials: N
authors.lastname: Zhong
authors.forename: F
authors.initials: F
authors.lastname: Martiniuk
authors.forename: S
authors.initials: S
authors.lastname: Tzall
authors.forename: R
authors.initials: R
authors.lastname: Hirschhorn
mesh_terms: D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular; D004720:Endonucleases / Q000378:metabolism; D005260:Female; D006009:Glycogen Storage Disease Type II / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008715:Methionine / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D013912:Threonine / Q000235:genetics; D044465:White People; D000520:alpha-Glucosidases / Q000172:deficiency / Q000235:genetics*
chemical_list: D013912:Threonine; D008715:Methionine; D004720:Endonucleases; D000520:alpha-Glucosidases