Copy Number Variation at the CFHR3/CFHR1 Locus Has a Major Impact on AMD Risk That Is Independent of the Effect of the Histidine Risk Allele at CFH Y402H

Abstract

Purpose: : Variation in the number of complement factor H related 3 (R3) and 1 (R1) genes is known to have a major impact on AMD risk, but an accurate measure of the number of copies present in individuals subjects has not been possible. We sought to develop a new assay that would enable accurate determination of the number of copies of each gene that would be sufficiently robust for large-scale studies. The assay was used to determine the impact on AMD risk and if the effect is mediated by competition between CFH and the R3/R1 genes.

Methods: : A multiplex ligation-dependent probe amplification (MLPA) assay was developed, validated, and used to assay the number of R3 and R1 copies in 196 cases with AMD and 204 controls without AMD. Interaction with Y402H in CFH (rs1061170) was studied. Selected deletions were confirmed with quantitative PCR. Logistic regression was used to investigate the association between AMD and the copy number of R3 and R1, Y402H genotypes, and their interactions.

Results: : The MLPA assay distinguished between 0, 1, and 2 deletions of the R3/R1 locus with a single assay run in all subjects, and repeated assay runs were concordant. The distribution of R3/R1 combined deletions is shown in the Table and the frequency was 13%. We found 3 subjects who had a deletion of R3 only, a unique observation that needs molecular confirmation. AMD status was highly associated with the number of R3/R1 deletions (Table) and the number of minor allele of rs1061170 (OR=2.54 with 95% CI: 1.88 to 3.46). There was no interaction between Y402H and the R3/R1 deletion (P=0.53). No effect on AMD subtypes (P=0.40 to 0.49) was observed for either variant.

Conclusions: : A novel assay was developed allowing accurate determination of the number of copies of the R3/R1 locus for the first time. The presence of 2 normal copies of R3/R1 in 76% of subjects increased AMD risk 5.3 fold. The risk conferred by R3/R1 appears to be independent of Y402H genotypes, suggesting that the R3/R1 do not increase AMD risk by competing with CFH at sites of complement activation.

Keywords: age-related macular degeneration • genetics • immunomodulation/immunoregulation