An increasing number of gene polymorphisms have been linked to Alzheimer disease over the past decade. Real-time polymerase chain reaction (PCR) followed by melting curve analysis using hybridization probes is an important tool in routine diagnosis of these polymorphisms. Previously, we developed a LightCycler method for the study of a possible relationship between polymorphism of the cathepsin D gene and Alzheimer disease. Cathepsin D is a major intracellular protease present in the endosomal-lysosomal system. Its activity is limited to acidic pH with a strong preference for cleavage near hydrophobic amino acids. A common polymorphism (C to T) in exon 2 (a coding sequence) of the cathepsin D gene changes the amino acid sequence in the enzyme product at residue 224 from alanine to valine. The T allele may be associated with increased protein expression (increased pro-cathepsin D secretion) and altered intracellular maturation and has been suggested to be a marker for Alzheimer disease. During the study of more than 500 subjects, we identified a case with a variant pattern of the cathepsin D gene-LightCycler melting profile preventing allelic discrimination. As a consequence, in subjects with unusual melting curve profile DNA sequencing or restriction fragment-length polymorphism (RFLP) analysis is necessary to unequivocally assign the correct cathepsin D genotype. In a collaborative study, we studied the stability of cerebrospinal fluid beta-amyloid(1-42) and tau levels (two ?established Alzheimer markers?) by apolipoprotein E genotype in patients with Alzheimer disease.
