title: Simple PCR amplification of the entire glucocerebrosidase gene (GBA) coding region for diagnostic sequence analysis.
abstract: Mutations in the human glucocerebrosidase gene (GBA) may lead to Gaucher disease-an autosomal recessive, lysosomal storage disease. In about 15-25% of Caucasian patients with Gaucher disease yet the disease-causing mutations remain to be identified. There exists 16kb downstream from the functional GBA gene (chromosome 1q21) a highly homologous transcribed pseudogene (GBAP) with some sequence differences to GBA. These sequence differences might erroneously imitate a true mutation in the functional gene if an unintentional co-investigation of the pseudogene occurred. We describe a protocol which allows the selective analysis of a PCR-amplified 7.1 kb genomic GBA-fragment encompassing the entire GBA coding region. Direct, nonradioactive double stranded cycle-sequencing procedure of nested PCR fragments from this long range GBA-specific product allowed the sequencing of the coding exons including the flanking splice sites. Several, so far unknown coding mutation were identified in non-Jewish families with Gaucher disease. This protocol allows the rapid detection of new GBA mutations.
authors.affiliation: AG Molekulare Neurobiologie am Institut für Neuropsychopharmakologie, Freie Universität Berlin, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Finckh
authors.forename: P
authors.initials: P
authors.lastname: Seeman
authors.forename: O C
authors.initials: OC
authors.lastname: von Widdern
authors.forename: A
authors.initials: A
authors.lastname: Rolfs
mesh_terms: D002878:Chromosomes, Human, Pair 1; D017931:DNA Primers; D005453:Fluorescence; D005776:Gaucher Disease / Q000175:diagnosis* / Q000235:genetics; D005962:Glucosylceramidase / Q000235:genetics*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011544:Pseudogenes; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D005962:Glucosylceramidase