A novel diagnostic test detects a low frequency of the hemicentinGln5345Arg variant among Northern Irish age related macular degenerationpatients

[摘要] Purpose: Age related macular degeneration (AMD) is a common cause ofsevere vision loss. Identification of genes involved in AMD willfacilitate early detection and ultimately help to identify pathways fortreatment for this disorder. The A16,263G mutation in theHEMICENTIN-1 gene produces a non-conservative substitution ofarginine for glutamine at codon 5345 which has been implicated infamilial AMD. The aim of this study is to develop a rapid diagnosticassay for the detection of this mutation and to evaluate its frequencyin a sample of AMD patients.Methods: A primer probe set was designed from exon 104 of theHEMICENTIN-1 gene to differentiate between mutant and wild typealleles. A region spanning the mutation was amplified by PCR using aLightCycler (Roche Diagnostic). The mutation was then detected by meltcurve analysis of the hybrid formed between the PCR product and aspecific fluorescent probe. The frequency of the mutation within theNorthern Ireland population was evaluated by assaying 508 affected AMDpatients, 25 possibly affected and 163 controls.Results: This assay clearly discriminates between the A16,263Gmutant and wild type HEMICENTIN-1 alleles. The wild type sequencehas a single base mismatch with the probe which decreases the stabilityof the hybrid, resulting in a lower TM (TM=51.27 °C)than that observed for the perfectly matched mutant allele(TM=59.9 °C). The mutant allele was detected in only one ofthe 696 subjects, an affected AMD patient.Conclusions: We describe a rapid assay for the genotyping of theGln5345Arg mutation using real-time fluorescence PCR to facilitate rapidprocessing of samples through combined amplification and detectionsteps. These characteristics are suitable for a clinical setting wherehigh throughput diagnostic procedures are required. The frequency ofthis mutation within the Northern Ireland population has been estimatedat 0.2%, concurring with previous findings that this mutation is a rarevariant associated with AMD. A rapid diagnostic assay will facilitate areliable and convenient evaluation of the frequency of the Gln5345Argmutation and its association with AMD within other populations.

[学科分类] 生物化学/生物物理