Technical Brief

A novel diagnostic test detects a low frequency of the hemicentin Gln5345Arg variant among Northern Irish age related macular degeneration patients

Gareth J. McKay, Stephen Clarke, Anne Hughes, Vivienne McConnell, Dennis W. Schultz, Michael Klein, Giuliana Silvestri, David A C Simpson

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Purpose: Age related macular degeneration (AMD) is a common cause of severe vision loss. Identification of genes involved in AMD will facilitate early detection and ultimately help to identify pathways for treatment for this disorder. The A16,263G mutation in the HEMICENTIN-1 gene produces a non-conservative substitution of arginine for glutamine at codon 5345 which has been implicated in familial AMD. The aim of this study is to develop a rapid diagnostic assay for the detection of this mutation and to evaluate its frequency in a sample of AMD patients. Methods: A primer probe set was designed from exon 104 of the HEMICENTIN-1 gene to differentiate between mutant and wild type alleles. A region spanning the mutation was amplified by PCR using a LightCycler (Roche Diagnostic). The mutation was then detected by melt curve analysis of the hybrid formed between the PCR product and a specific fluorescent probe. The frequency of the mutation within the Northern Ireland population was evaluated by assaying 508 affected AMD patients, 25 possibly affected and 163 controls. Results: This assay clearly discriminates between the A16,263G mutant and wild type HEMICENTIN-1 alleles. The wild type sequence has a single base mismatch with the probe which decreases the stability of 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 of the 696 subjects, an affected AMD patient. Conclusions: We describe a rapid assay for the genotyping of the Gln5345Arg mutation using real-time fluorescence PCR to facilitate rapid processing of samples through combined amplification and detection steps. These characteristics are suitable for a clinical setting where high throughput diagnostic procedures are required. The frequency of this mutation within the Northern Ireland population has been estimated at 0.2%, concurring with previous findings that this mutation is a rare variant associated with AMD. A rapid diagnostic assay will facilitate a reliable and convenient evaluation of the frequency of the Gln5345 Arg mutation and its association with AMD within other populations.

Original languageEnglish (US)
Pages (from-to)682-687
Number of pages6
JournalMolecular Vision
Volume10
StatePublished - Sep 24 2004

Fingerprint

Macular Degeneration
Routine Diagnostic Tests
Mutation
Alleles
Northern Ireland
Mutation Rate
Population
Genes
Polymerase Chain Reaction
Glutamine
Fluorescent Dyes
Codon
Arginine
Real-Time Polymerase Chain Reaction
Exons
Fluorescence

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Technical Brief : A novel diagnostic test detects a low frequency of the hemicentin Gln5345Arg variant among Northern Irish age related macular degeneration patients. / McKay, Gareth J.; Clarke, Stephen; Hughes, Anne; McConnell, Vivienne; Schultz, Dennis W.; Klein, Michael; Silvestri, Giuliana; Simpson, David A C.

In: Molecular Vision, Vol. 10, 24.09.2004, p. 682-687.

Research output: Contribution to journalArticle

McKay, Gareth J. ; Clarke, Stephen ; Hughes, Anne ; McConnell, Vivienne ; Schultz, Dennis W. ; Klein, Michael ; Silvestri, Giuliana ; Simpson, David A C. / Technical Brief : A novel diagnostic test detects a low frequency of the hemicentin Gln5345Arg variant among Northern Irish age related macular degeneration patients. In: Molecular Vision. 2004 ; Vol. 10. pp. 682-687.
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abstract = "Purpose: Age related macular degeneration (AMD) is a common cause of severe vision loss. Identification of genes involved in AMD will facilitate early detection and ultimately help to identify pathways for treatment for this disorder. The A16,263G mutation in the HEMICENTIN-1 gene produces a non-conservative substitution of arginine for glutamine at codon 5345 which has been implicated in familial AMD. The aim of this study is to develop a rapid diagnostic assay for the detection of this mutation and to evaluate its frequency in a sample of AMD patients. Methods: A primer probe set was designed from exon 104 of the HEMICENTIN-1 gene to differentiate between mutant and wild type alleles. A region spanning the mutation was amplified by PCR using a LightCycler (Roche Diagnostic). The mutation was then detected by melt curve analysis of the hybrid formed between the PCR product and a specific fluorescent probe. The frequency of the mutation within the Northern Ireland population was evaluated by assaying 508 affected AMD patients, 25 possibly affected and 163 controls. Results: This assay clearly discriminates between the A16,263G mutant and wild type HEMICENTIN-1 alleles. The wild type sequence has a single base mismatch with the probe which decreases the stability of 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 of the 696 subjects, an affected AMD patient. Conclusions: We describe a rapid assay for the genotyping of the Gln5345Arg mutation using real-time fluorescence PCR to facilitate rapid processing of samples through combined amplification and detection steps. These characteristics are suitable for a clinical setting where high throughput diagnostic procedures are required. The frequency of this mutation within the Northern Ireland population has been estimated at 0.2{\%}, concurring with previous findings that this mutation is a rare variant associated with AMD. A rapid diagnostic assay will facilitate a reliable and convenient evaluation of the frequency of the Gln5345 Arg mutation and its association with AMD within other populations.",
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