Comprehensive analysis of CRP, CFH Y402H and environmental risk factors on risk of neovascular age-related macular degeneration

Abstract

Purpose: To examine if the gene encoding C-reactive protein (CRP), a biomarker of inflammation, confers risk for neovascular age-related macular degeneration (AMD) in the presence of other modifiers of inflammation, including body mass index (BMI), diabetes, smoking, and complement factor H (CFH) Y402 genotype. Additionally we examined the degree to which CRP common variation was in linkage disequilibrium (LD) within our cohort.

Methods: We ascertained 244 individuals from 104 families where at least one member had neovascular AMD, and a sibling had normal maculae and was past the age of the index patient's diagnosis of neovascular AMD. We employed a direct sequencing approach to analyze the 5'-promoter region as well as the entire coding region and the 3'-untranslated region of the CRP gene. CFH Y402 genotype data was available for all participants. Lifestyle and medical factors were obtained via administration of a standardized questionnaire. The family-based association test, haplotype analysis, McNemar's test, and conditional logistic regression were used to determine significant associations and interactions. Haploview was used to calculate the degree of LD (r2) between all CRP variants identified.

Results: Six single nucleotide polymorphisms (SNPs; rs3091244, rs1417938, rs1800947, rs1130864, rs1205, and rs3093068) comprised one haplotype block of which only rs1130864 and rs1417938 were in high LD (r2=0.94). SNP rs3093068 was in LD but less so with rs3093059 (r2=0.83), which is not part of the haplotype block. Six SNPs made up six different haplotypes with > or = 5% frequency, none of which were significantly associated with AMD risk. No statistically significant association was detected between any of the nine common variants in CRP and neovascular AMD when considering disease status alone or when controlling for smoking exposure, BMI, diabetes, or CFH genotype. Significant interactions were not found between CRP genotypes and any of the risk factors studied. No novel CRP variation was identified.

Conclusions: We provide evidence that if elevated serum/plasma levels of CRP are associated with neovascular AMD, it is likely not due to genetic variation within CRP, but likely due to variations in some other genetic as well as epidemiological factors.

Figure 1

Schematic of the C-reactive protein gene representing the promoter region, the 2 exons, and the 3′ untranslated region. The coding regions of the exons are colored dark. The untranslated regions are depicted in the lighter color. All nine single nucleotide polymorphisms (SNPs) were genotyped by direct sequencing except for rs3093068 which was genotyped using the Sequenom technology. The asterisks represent the 7 SNPs that define common variation in CRP. Please note that 3 of these SNPs are located several base pairs upstream from the ATG start site. Although we sequenced 92.3% of the CRP gene, no novel variation was found.

Figure 2

Linkage Disequilibrium of Single Nucleotide Polymorphisms (SNPs) along the 1q25 region encompassing the CRP gene and illustrating the 1 distinct haplotype block, defined by the confidence intervals, an algorithm proposed by Gabriel et al. [31] using HAPLOVIEW. The linkage disequilibrium (r²) between any two SNPs is listed in the cross cell. Asterisk means the darker the color indicates the higher the linkage disequilibrium between any two SNPs. Please note that SNP rs3091244 consists of three alleles; two minor alleles A and T are combined into one minor allele due to the limitation of the program which only allows for dichotomous SNPs. SNP rs3093068 was genotyped on 205 subjects, whereas the other eight SNPs were genotyped on 244 subjects.