Investigation of a rare risk variant in complement factor H for age-related macular degeneration in the Amish

Abstract

Purpose : Genetic variants in complement factor H (CFH) are well-known risk factors for age-related macular degeneration (AMD). However, their functional consequences are not fully characterized. We previously identified a missense variant in CFH (rs570523689, CFH P503A) in 19 Amish individuals in Ohio and Indiana. The variant was strongly associated with AMD and computationally predicted to be damaging. In our work, we aimed to elucidate the functional effects of CFH P503A to offer insights into AMD risk and etiology in these carriers.

Methods : We used a customized TaqMan assay for CFH P503A to genotype about 1,300 Amish individuals from Ohio, Indiana, and Pennsylvania. A pedigree for the carriers was constructed using data from the Anabaptist Genealogy Database. To determine if CFH P503A has an effect on CFH transcription, we utilized splicing prediction software and measured CFH transcript levels in 40 blood samples from carriers and related non-carriers. We performed real-time quantitative PCR with 4 different TaqMan assays targeting the 3 protein-coding CFH transcripts and the large retained intron of CFH. P503A falls within the longest coding transcript and the large retained intron of CFH. We quantified expression relative to that observed for ARPE-19 cells in the same assays and compared the means among the sample groups using the Wilcoxon rank sum test and a significance threshold of p<0.05.

Results : We identified 33 additional CFH P503A carriers in the Ohio and Indiana Amish. The 52 carriers are interrelated through a 10-generation pedigree dating back to the 1700s. The variant is located near the end of an exon and is predicted to disrupt an exonic splicing enhancer site, which may alter splicing. The combined relative expression of the three protein-coding transcripts and the relative expression of the large retained intron of CFH were significantly higher in the carriers and non-carriers without AMD (n=14 for each group) compared to the AMD non-carriers (n=5).

Conclusions : We identified 52 CFH P503A carriers in the Ohio and Indiana Amish. We found that there were significant differences in relative CFH expression among some groups in our assays, but carrier status alone could not explain these differences. Functional studies into the effects of this variant on protein folding and binding are underway and may contribute to our knowledge of how this variant contributes to AMD risk.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.