SNP Technology and Alzheimers disease

One major goal of genetic research is to understand the role of genetic variation. By far the most common type of such variation in humans involves single DNA bases, and is termed single nucleotide polymorphism (SNP). With sufficient technological solutions, one strong belief is that SNPs can enable the mapping of disease genes involved in complex genetic disorders. Alzheimer’s disease (AD) is a complex disorder characterized by progressive cognitive decline and memory impairment. Some individuals acquire this form of dementia before the age of 65 (referred to as early-onset or familial AD) but most often AD occurs late in life. It is in the early-onset form, however, where causative mutations have been found in three different genes; APP, PSEN1, & PSEN2. Other than these, the only additional risk factor identified for AD is the e4 allele of the APOE gene. Together these only account for a fraction of AD, leaving room for studies to identify additional AD susceptibility genes. In the initial investigation of this thesis, polymorphisms in the PSEN1, PSEN2, APOE and VLDL-R genes were tested for association with early-onset Alzheimer’s disease (EOAD). Aside from confirming the well-established APOE-e4 association, an allele of the PSEN2 showed a significant disease association. In an attempt to verify the PSEN2 association and localize the potential pathogenic variant, a series of eight additional SNPs, located throughout the PSEN2 gene, were tested for association with EOAD. None of the tested markers showed significant disease association upon replication in a second set of cases and controls. The remainder of the thesis describes the invention and development of a novel technique for scoring SNPs. The method, called Dynamic Allele Specific Hybridization (DASH), is a great improvement in both throughput and reliability compared to traditional techniques. The crucial step in the procedure is the heating of the DNA duplex (formed from hybridization of the PCR-amplified target with an allele-specific probe) whilst measuring the fluorescence of a double-strand specific intercalating dye. SNP alleles are detected and scored by comparative analysis of the melting profiles. Improvements to the initial DASH format are detailed in the final two papers of this thesis. A novel alternative in fluorescence detection, termed induced fluorescent resonance energy transfer (iFRET), is introduced. IFRET employs energy transfer between a generic intercalating dye and an FRET acceptor attached to the allele-specific probe. This system retains the spectralmultiplex potential offered by traditional FRET systems, while reducing costs and improving fluorescence signal intensities. Aside from detection, DASH was converted from a microtiter-plate format to an array format which greatly improved flexibility and simplified the assay procedure. The complete DASH-2 system is examined in terms of multiplex options, throughput, cost and accuracy. LIST OF PUBLICATIONS This thesis is based on the following articles, which are referred to by their roman numerals: I Brookes AJ, Howell WM, Woodburn K, Johnstone EC, Carothers A. Presenilin-I, presenilin-II, and VLDL-R associations in early onset Alzheimer's disease. Lancet (1997) Aug2;350(9074):336-337 II Howell WM, Brookes AJ. Evaluation of multiple presenilin 2 SNPs for association with early-onset sporadic Alzheimer disease. Am J Med Genet (2002) 111;2:157-163 III Howell WM, Jobs M, Gyllensten M, Brookes AJ. Dynamic allele-specific hybridization. A new method for scoring single nucleotide polymorphisms Nat Biotechnol. (1999) Jan;17(1):87-8. IV Howell WM, Jobs M, Brookes AJ. iFRET: An Improved Fluorescence System for DNA-Melting Analysis Genome Res. (2002) 12:1401-1407. V Jobs M*, Howell WM*, Stromqvist L, Brookes AJ. DASH-2: Flexible, low-cost and high-throughput SNP genotyping by Dynamic Allele Specific Hybridization on membrane arrays. Manuscript. *These authors contributed equally to this work.