An increasing number of genes have been linked to Alzheimer disease (AD) over the past decade. Although there is contrary evidence, some recent case-control studies suggested that a C to T polymorphism in exon 2 of the cathepsin D (CATD) gene increases the risk of sporadic AD. The gene encoding cathepsin D is located at the extremity of the short arm of chromosome 11, in the p15 band. The transcribed portion of the gene is about 11,000 bp and it is organized into 9 exons analogous with the human pepsinogen A gene. Cathepsin D is a major intracellular aspartyl protease present in the endosomal-lysosomal system. The C to T polymorphism in exon 2 of the CATD gene is common and results in an amino acid sequence change at residue 224 from Ala to Val. The T allele may be associated with increased protein expression (increased pro-cat D secretion) and altered intracellular maturation. We developed new and practicable methods for detection of the C to T polymorphism in exon 2 of the CATD gene. Using newly designed primers and/or a different restriction endonuclease, we markedly shortened the incubation time and reduced the incubation temperature from 60 oC to 37 oC in conventional PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of DNA extracted from peripheral blood of patients and their controls. Based on PCR-single strand conformation polymorphism (PCR-SSCP) detection, we also developed a semi-automated method. We showed that, under optimized conditions, this method is as reliable as PCR-RFLP and direct DNA sequencing but is simpler and faster to perform. Further, we developed and validated a fully automated, real-time ultrafast PCR method based on the use of the LightCycler instrument for detection of the C to T sequence polymorphism in the CATD gene. With their increased level of automation and speed, the latter two methods would be favorable replacements for PCR-RFLP and direct DNA sequencing for both routine laboratory use and large-scale screening. Regarding the possible role of infectious agents in the development of AD, our collaborative study showed no increased incidence of Herpes simplex type 1 virus (HSV-1) DNA in the brains of patients with AD and apolipoprotein E4 allele, making the participation of HSV-1 in AD unlikely. In other collaborative studies, we continued studying the possible pathogenetic role of apolipoprotein(a) isoforms and alleles in patients with atherosclerotic heart disease and systemic lupus erythematosus.
