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Paul Oeth, Payam Mahboubi, Toni Paladino, Jay Stoerker, Daniel Farkas, Lorah Perlee; Analytical Validation of a Genetic Test for Predicting Risk of Developing Late Stage Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5241.
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Age-related macular degeneration (AMD) is a complex genetic disease involving multiple linked and independent genetic markers demonstrating significant association with the development of late stage advanced disease. Multiple genome-wide association studies (GWAS) have reproduced disease associations with single nucleotide polymorphism and insertion/deletions found on chromosome 1q32 in the complement factor H (CFH) and the CFH-related (CFHR) genes in the regulators of complement activation beta (RCA β) region, chromosome 6p21 in the complement component 2 (C2) and complement factor B genes (CFB), chromosome 19p13 in the complement component 3 gene (C3) and chromosome 10q26 LOC387715 (ARMS2). An assessment of an individual’s genetic profile can be highly informative for better patient management.
A single-multiplexed reaction containing thirteen single-nucleotide polymorphisms (SNPs) directed at targets within RCA β, C2, C3 and ARMS2 was designed for genotyping using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Cross platform concordance studies were conducted with MALDI-TOF MS and compared to bi-directional fluorescent-based dideoxy-termination sequencing and real-time PCR allelic discrimination assays to confirm genotype accuracy. HapMap CEU DNAs with annotated genotypes along with paired buccal and blood genomic DNAs were used to assess and optimize assay performance
The RCA β region within chromosome 1 is structurally complex. We identified multiple mis-typings within the released HapMap genotype database and poor genotype coverage of array-based GWAS genotypes for a highly associated AMD marker. Careful primer design in conjunction with multi-platform cross-validation of all SNPs within the assay confirmed the accuracy of the test for genotyping the RCA β, C2, C3 and ARMS2 SNPs. The 13 SNPs allow for high resolution of extended and recombinant haplotypes within the RCA β region previously shown to be associated with development of late stage AMD.
We have developed a multiplexed, single-reaction genetic laboratory developed test demonstrating robust performance that allows for the detection and resolution of recombinant haplotypes within the RCA β region and accounts for the structural complexity within this region to accurately report genotypes critical to late stage risk prediction.
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