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

Myoclonic Epilepsy in Gaucher Disease: Genotype-Phenotype Insights from a Rare Patient Subgroup

“…One patient with type 1 Gaucher disease was reported to be homozygous for mutation N188S (52,53). Likewise, mutation G377S, found in two patients in our series, has previously been considered a mild mutation and three Portuguese patients with type 1 disease have been described who are homozygous for G377S (54,55).…”

Gaucher disease: expression and characterization of mild and severe acid β-glucosidase mutations in Portuguese type 1 patients

“…Molecular analysis of the acid -glucosidase gene from 12 unrelated Portuguese type 1 GD patients revealed three novel mutations, and the expression of these, and three other reported, but uncharacterized lesions, 12,28,29 provided insight into the function of these alleles and potential genotype/ phenotype correlations. The three novel mutations were missense mutations: F109V, the sterically non-conservative substitution 63,64 of phenylalanine by valine in a weakly conserved (human vs murine 65 ) region of the acid -glucosidase protein; W184R, the non-conservative substitution of a tryptophan residue by the positively-charged arginine in the middle of 15 (amino acids 176-189) conserved (in fact, identical) residues; and R395P, the non-conservative substitution of arginine by proline, involving the loss of a positive charge and introduction of a rigid side chain at residue 395, which lies in the longest stretch (amino acids 315-406) of conserved (ie identical except for amino acid 376) residues in the human and murine sequences, suggesting an important functional domain.…”

“…DNA isolation, PCR amplification and mutation detection Genomic DNA was isolated from skin fibroblasts or peripheral blood using standard techniques. 30 Initial screening for two common (N370S, L444P) and several rare but nonfamily-specific (84GG, IVS2 + 1 , R463C, R496H, D409H, R120Q, P122S, K157Q, Y212H, F216Y, W312C, G325R, C342G, D409V, P415R, RecTL, and RecNci) mutations 21,[31][32][33][34][35][36][37][38][39][40][41][42][43][44] as well as two mutations found in the Portuguese population, G377S 29 and N396T, 12 was performed by polymerase chain reaction (PCR) amplification and restriction digestion or allele specific oligonucleotide hybridization. 31,45,46 To detect the unknown acid -glucosidase mutations, the complete coding region and adjacent intron/exon boundaries were amplified directly from genomic DNA (or through nested PCR) and subjected to non-radioactive SSCP analysis.…”

“…Complete SSCP analysis of the acid -glucosidase coding region and intron/exon boundaries from non-Jewish Portuguese patients with type 1 GD (Table 1) resulted in the identification of three new (F109V, W184R and R395P) and three rare, but uncharacterized, missense mutations (R359Q, 28 G377S, 29 and N396T 12 ). Table 2 summarizes the nucleotide substitutions and the corresponding changes at the protein level for each mutation.…”

Insulin-Like Growth Factors in Childhood-Onset Gaucher Disease

“…Genomic DNA of all participants was isolated from white blood cells by standard methods (19,20). The mismatched PCR method followed by digestion with XhoI was used to detect the N370S mutation (21); restriction analysis with PvuII was used to detect the G377S mutation (22) (24).…”