Development and evaluation of multiplex and high-throughput SNP analysis for the ABCA1 gene

The main focus of this work was to develop high-throughput and multiplex assays for the fast and efficient analysis of sequence variations in the gene coding for the ATP-binding cassette transporter 1 (ABCA1), which has recently been identified as the main regulator of plasma HDL cholesterol. Intensive literature and data base research was performed to library all known sequence variations in this gene. Relevant data of subjects with HDL deficiency syndrome were archived, which carry mutations in this gene. These data were stored in a web page archive (www.abca1-mutants.all.at) to provide an information resource for further research in ABCA1. A knowledge-based sequencing approach was performed to identify novel sequence variations in ABCA1. In the promoter region, two novel VNTR polymorphisms at a ZNF202 and a SRY binding-site were identified along with three novel SNPs (G1047C, C1152T and C1440T). While the VNTR polymorphisms might not be of functional relevance, the three SNPs were found in significantly different distribution of genotypes in some cohorts and all three nucleotide exchanges appear to be associated to low HDL. Six novel sequence variations affecting amino acid sequence, including the V771M polymorphism, were found in patients with HDL deficiency syndrome and probands with aberrant HDL-levels. LightCycler and TaqMan assays were developed for the detection of common single nucleotide polymorphisms of potential interest in the ABCA1 gene, including the V771M polymorphism. An automated high-throughput workflow with implemented TaqMan technology was established for fast and efficient genotyping of single nucleotide polymorphisms in large cohorts capable of 40,000 analyses per 24 hours. With the newly established workflow, several polymorphisms were genotyped and evaluated, including the R219K and the V771M variation. The R219K polymorphism was found in significantly increased prevalence in Italian centenarians compared to healthy Italian controls, indicating a protective effect of K219 in aging. This confirms findings from other groups concerning this polymorphism. The novel V771M polymorphism was found with significantly decreased abundance in Hungarian CAD patients compared to healthy Hungarian blood donors, indicating a protective effect of M771 on CAD. All other frequency data showed no significant difference compared to the control groups. A multiplex assay was established for the simultaneous detection of four important polymorphisms in the ABCA1 gene using allele specific primer extension reaction for discrimination of SNP. The method uses zip- and color-coded beads for detection with flow cytometry (Luminex). The new assay provides a fast and cost-efficient method for the determination of single nucleotide polymorphisms with reliability comparable to the well-established TaqMan technology