Simple PCR amplification of the entire glucocerebrosidase gene (GBA) coding region for diagnostic sequence analysis

Mutations in the human glucocerebrosidase gene (GBA) may lead to Gaucher di sease-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 G BA gene (chromosome 1q21) a highly homologous transcribed pseudogene (GBAP) with some sequence differences to GBA. These sequence differences might er roneously imitate a true mutation in the functional gene if an unintentiona l co-investigation of the pseudogene occurred. We describe a protocol which allows the selective analysis of a PCR-amplified 7.1 kb genomic GBA-fragme nt 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 incl uding the flanking splice sites. Several, so far unknown coding mutation we re identified in non-Jewish families with Gaucher disease. This protocol al lows the rapid detection of new GBA mutations.