Efficacy of Mouthwash Samples for Genotyping the CFH Polymorphism Y402H Associated with Increased Risk of AMD

Abstract

Purpose: : To evaluate mouthwash samples as a source of DNA for rapid detection of the Complement Factor H (CFH) variant Tyr402His associated with increased risk of age–related macular degeneration (AMD).

Methods: : 203 Caucasian subjects with AMD (AREDS categories 3 and 4) and 205 control subjects were recruited over a six–week period. AMD phenotypes were characterized by clinical examination, fundus photography, and fluorescein angiography. Participants provided buccal tissue samples by expectorating after rinsing for 30 seconds with Scope mouthwash. Genomic DNA was prepared from buccal cells using a standard protease–digestion/salting–out/alcohol–precipitation procedure. Exon 9 of the CFH gene was PCR–amplified and genotyped for a single nucleotide polymorphism (T–to–C) by restriction–fragment length analysis and verified by re–sequencing.

Results: : Of 408 mouthwash samples (10–20ml), 377 yielded DNA of ample quality (∼ 20kb) and concentration (50–100µg) for successful genotyping. The frequency of the C allele was 0.55 in AMD cases (188), versus 0.34 in controls (189). Being heterozygous for the genotype (TC) increased the likelihood for AMD 2.1–fold (95% CI 1.3–3.3) in our sample population. Being homozygous for the genotype (CC) increased the likelihood for AMD 6.5–fold (95% CI 3.4–12.5).

Conclusions: : Mouthwash samples provide a convenient source of DNA for genotyping (>90% success rate) with failures largely attributable to the presence of excessive food particles. Frequency of the CFH T–to–C polymorphism (Y402H) in AMD cases and normal controls in this study was similar to that in previous reports.

Keywords: age-related macular degeneration • genetics • mutations