Systematic Functional Testing of Rare Variants: Contributions of CFI to Age-Related Macular Degeneration.

Purpose: Genome-wide association (GWAS) and sequencing studies for AMD have highlighted the importance of coding variants at loci that encode components of the complement pathway. However, assessing the contribution of such alleles to AMD, especially when they are rare, remains coarse, in part because of the persistent challenge in establishing their functional relevance. Others and we have shown previously that rare alleles in complement factor I (CFI) can be tested functionally using a surrogate in vivo assay of retinal vascularization in zebrafish embryos. Here, we have implemented and scaled these tools to assess the overall contribution of rare alleles in CFI to AMD.

Methods: We performed targeted sequencing of CFI in 731 AMD patients, followed by replication in a second patient cohort of 511 older healthy individuals. Systematic functional testing of all alleles and post-hoc statistical analysis of functional variants was also performed.

Results: We discovered 20 rare coding nonsynonymous variants, including the previously reported G119R allele. In vivo testing led to the identification of nine variants that alter CFI; six of which are associated with hypoactive complement factor I (FI). Post-hoc analysis in ethnically matched, population controls showed six of these to be present exclusively in cases.

Conclusions: Taken together, our data argue that multiple rare and ultra-rare alleles in CFI contribute to AMD pathogenesis; they improve the precision of the assessment of the contribution of CFI to AMD; and they offer a rational route to establishing both causality and direction of allele effect for genes associated with this disorder.