ABSTRACT
BACKGROUND: Haptoglobin is an acute-phase binding protein that scavenges free hemoglobin. The human haptoglobin gene (HP) is polymorphic with two main alleles, haptoglobin allele 1 (Hp1) and haptoglobin allele 2 (Hp2). The smaller Hp1 allele features no duplication and consists of four exons, whereas the larger Hp2 allele, containing a 1.7 kb duplication, consists of six exons, with the fifth and sixth being highly homologous to exons 3 and 4 of Hp1. METHODS: We designed an exonuclease (TaqMan) assay targeting single nucleotide differences between the homologous regions of Hp1 and Hp2. The assay contained one probe specifically binding to a site in intron 4 of Hp2, and another probe binding equally to intron 4 of Hp1 and intron 6 of Hp2. RESULTS: Measurement of post-PCR fluorescence allowed unambiguous discrimination of HP genotypes. Comparison with genotypes obtained by a method based upon allele-specific primers yielded fully corresponding results. CONCLUSIONS: The new HP genotyping method is fast, reliable, does not require real-time instruments and may be especially useful for high-throughput genotyping.
Subject(s)
Exonucleases/metabolism , Genotype , Haptoglobins/genetics , Polymerase Chain Reaction , Alleles , Fluorescence , HumansABSTRACT
OBJECTIVE: Exudative age-related macular degeneration (AMD) is a common cause for a severe central visual loss. The complement system has been implicated in the pathogenesis of drusen. Recently, a complement factor H (CFH) polymorphism, which is characterized by a tyrosine (Y)-to-histidine (H) exchange at position 402 of the CFH gene, has been suggested as a major risk factor for AMD in a North American population. The aim of the present study was to investigate a hypothesized association between the CFH Y402H polymorphism and the presence of exudative AMD in a Central European population of Caucasoid descent as well as to determine the genotype distribution among different types of exudative AMD. DESIGN: Retrospective case-control study. PARTICIPANTS: The study cohort consisted of 179 patients with exudative AMD and 163 controls. METHODS: Determination of genotypes was carried out by allele-specific digestion of polymerase chain reaction products. MAIN OUTCOME MEASURES: Genotypes of CFH Y402H polymorphism. RESULTS: The prevalence of the CFH 402HH genotype was significantly higher in patients with exudative AMD than among controls (35.2% vs. 8.6%; P<0.001). Homozygosity for the CFH Y402H polymorphism was associated with an odds ratio of 5.78 (95% confidence interval, 3.09-10.83) for exudative AMD. Subgroup analysis revealed that the CFH 402HH genotype was significantly more prevalent in eyes with predominantly classic with no occult choroidal neovascularization (CNV) than in those with either retinal angiomatous proliferation, occult with no classic CNV, or predominantly classic with occult CNV. CONCLUSION: Our data suggest that the CFH Y402H polymorphism is a major risk factor for exudative AMD in a Central European population.
