Subject(s)
Aspartylglucosylaminase/analysis , Mucolipidoses/diagnosis , Aspartylglucosylaminase/blood , Aspartylglucosylaminase/urine , Biomarkers/analysis , Biomarkers/blood , Biomarkers/urine , Cell Line , Child , Clinical Enzyme Tests , Fibroblasts/enzymology , Humans , Skin/enzymology , Skin/pathologyABSTRACT
Aspartylglycosaminuria (AGU) is a recessively inherited lysosomal storage disease that occurs with much higher frequency in Finland than elsewhere. AGU is caused by a deficiency in glycosylasparaginase (GA), which results in the accumulation of glycoasparagines in lysosomes. In the Finnish population, a single nucleotide change in the gene encoding GA is responsible for the disease. We have used the oligonucleotide ligation assay (OLA) to detect the mutation in polymerase chain reaction (PCR)-amplified DNA samples from normal, carrier, and affected individuals. Screening for AGU among 415 random Finnish DNA samples with PCR/OLA revealed five carriers of the mutant allele and demonstrated the potential of the method for use in carrier screening. PCR/OLA provides a rapid, reliable, nonisotopic method to detect the mutation responsible for AGU that can readily be applied to large population screening.
Subject(s)
Acetylglucosamine/analogs & derivatives , DNA Ligases/metabolism , Lysosomal Storage Diseases/genetics , Mutation , Oligonucleotides/metabolism , Acetylglucosamine/urine , Alleles , Aspartylglucosaminuria , Base Sequence , Finland , Heterozygote , Homozygote , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Templates, GeneticABSTRACT
Recent experimental work on the mechanism of action of glycosylasparaginase suggests that the enzyme specifically reacts toward the L-asparagine or L-aspartic acid moiety of its substrates. Based on this, a new sensitive assay for glycosylasparaginase activity has been developed using L-aspartic acid beta-(7-amido-4-methylcoumarin) as substrate. Release of 7-amino-4-methylcoumarin was determined fluorometrically. At pH 7.5, Km = 93 microM, and as little as 1 ng of glycosylasparaginase could be detected with the assay. Hydrolysis of the substrate was inhibited by diazo-oxonorvaline, a specific inhibitor of glycosylasparaginase. In biological samples, the fluorometric assay is 40-100 times more sensitive than other published methods for glycosylasparaginase. This new assay enables a rapid enzymatic diagnosis of aspartylglycosaminuria--a genetic deficiency of glycosylasparaginase activity--with leukocyte and fibroblast samples.
