Data from genetic epidemiological studies suggest that the involvement of genetic variation at the APOE locus in Alzheimer's disease (AD) susceptibility may not be restricted to the well-studied coding polymorphisms, but may also be influenced by polymorphisms in flanking DNA. In particular, we have recently observed an association of the APOC1 HpaI+ allele with AD in groups of people with the "reference" APOE e3/e3 genotype. In this project we will carry out molecular analyses and experiments to test the hypothesis that APOE flanking sequences influence AD susceptibility is that this effect is through the influence of these polymorphisms on allele-specific APOE mRNA expression. First, to test whether AD susceptibility depends on particular combinations of APOE coding and regulatory polymorphisms located in cis on the chromosome, we will develop a method for determining complete haplotypes of polymorphic markers in the APOE promoter, the APOE coding region and the APOC1 promoter and we will generate haplotype data for AD association analysis in the large multi-ethnic population samples discussed in Project 1. Second, using a transfection assay screen, we will test for the existence of functional sequence polymorphisms in the APOE and APOC1 promoters, we will determine the sequence of functional sequence polymorphisms in the APOE and APOC1 promoters, we will determine the sequence of these polymorphisms in the APOE and APOC1 promoters, we will determine the sequence of these polymorphisms and we will apply them as new markers in the haplotype analyses. Third, we will test for functional significance for the individual polymorphisms and haplotypes in vivo by correlating them with allele-specific APOE mRNA levels in human lymphoblastoid cell lines and liver, kidney and brain tissues. Fourth, using apoCI promoter "knockout" mice, we will test a model in which the APOE and APOC1 engage in enhance-competition as an explanation for the observed association of the APOC1 HpaI+ allele with AD. Using transfections and transgenic mice we will also investigate localization of the putative brain-enhancer of APOE and then search for functional polymorphisms in this region. Results of these studies may reveal a basic mechanism for AD which is accessible to pharmacological intervention and may also shed light on the difference sin apparent AD risk who have been observed in different ethnic groups.