Use of denaturing gradient gel electrophoresis to identify mutant sequences in the β-glucocerebrosidase gene

A marked deficiency of beta -glucocerebrosidase (EC 3.2.1.45), the lysosomal beta -glucocerebrosidase responsible for catalyzing the hydrolysis of the glucosphingolipid glucocerebroside to ceremide and glucose, is the cause of Gaucher disease. The variable onset of Gaucher symptoms causes difficulties in the clinical diagnosis of this disease. Although leukocyte beta -glucocerebrosidase assays are useful for confirming Gaucher disease, the severity of the disease does not always correlate with the level of residual enzyme activity. As a consequence, newer approaches have focused on determining whether the disease severity in Gaucher patients can be associated with specific molecular changes (i.e., mutations) within the beta -glucocerebrosidase gene. When the position of a mutation is not known, techniques like mismatched cleavage of RNA: cDNA hybrids, single-strand conformation polymorphism (SSCP), and denaturing gradient gel electrophoresis (DGGE) can be used to localize mutations to within small regions of a genomic sequence. DGGE is a sensitive, alternative method that also can be used to detect mutant alleles in short DNA fragments. This approach previously has been used to detect mutations in exon 9 of the beta -glucocerebrosidase gene in humans. Extension of this approach allowed analysis of exons 5 through 10 of the beta -glucocerebrosidase gene. This report demonstrates the ability of DGGE to distinguish wildtype, recombinant, and L444P sequences in exon 10, and wildtype and N370S sequences in exon 9. In addition, the DGGE procedure has been used to identify a rare exon 9 mutation in a type 1 patient. The mutation is a G to A change at cDNA base pair 1246, which results in an amino acid coding change from Gly super(377) to Ser super(377) (G377S).