Rare <i><scp>CFH</scp></i> mutations and early‐onset age‐related macular degeneration (2015)

Factor H (FH), one of the most abundant proteins in human blood is the key inhibitory regulator of the complement system. The complement factor H (CFH) gene which encodes it is highly polymorphic within and between populations: haplotypes carrying functional variants, including deletion of CFHR3-CFHR1 (CNP147) and rs1061170 (Y402H), alter risk of age-related macular degeneration (AMD) and autoimmune disease (Bradley et al. 2011). The common form of FH protein is composed of 20 repetitive short consensus repeat (SCR) units which are folded to form a protein with several active sites. Rare mutations in the FH N-terminal SCRs have recently been reported to cause early-onset familial AMD in two families (Yu et al. 2014). We conducted a pooled next-generation sequencing (NGS) experiment using fragmented and indexed DNA from 35 AMD cases. All cases and controls for this study were recruited and described previously (Hughes et al. 2007). DNA was retrieved from the Anonymised DNA, RNA and Serum Bank in the Centre for Public Health at Queen's University Belfast, under ethical approval of the Office for Research Ethics Committees Northern Ireland (reference 11/NI/0139). Patients were of European descent and had neovascular AMD in at least one eye, but no details about the clinical course or treatment were available. A total of 5 Mb, including CFH, was targeted using a Nimblegen SeqCap EZ library and sequenced on a HiSeq 2000 (Illumina). Paired-end reads (100 bp) were aligned to hg19 human reference sequence with the Burroughs Wheeler Aligner 0.5.9 aln algorithm. Sorting and indexing was performed with SAMtools 0.1.14. Genome Analysis Toolkit was used to recalibrate, realign and call polymorphisms, and sequence was viewed in Integrative Genomics Viewer. We identified a novel missense mutation, CFH P139A, for which the G allele contributed 13 of 1061 reads from the pooled DNA (Fig. 1A) and verified it in a single contributing individual by Sanger sequencing (Fig. 1B). It was not found in a further 181 AMD cases or 151 elderly controls. The individual had neovascular AMD with onset at the age of 55 years and carried an otherwise low-risk genetic background (two protective copies of CFH rs800292 [62I] and heterozygous for the common ARMS2 rs10490924 risk allele). Only two patients in our database of 303 cases had a disease onset at a younger age than this individual. The P139A mutation is located near the C-terminal end of SCR2 and changes a highly conserved amino acid. It is predicted to be damaging by both SIFT and PolyPhen-2. Modelling suggests that the SCR2-SCR3 junction is crucial for the docking of complement factor I (FI) into the groove formed between FH SCRs 1 to 3 and C3b (Roversi et al. 2011). Disruption of the interaction between FH and FI is expected to inhibit decay of C3b and therefore to result in a greater level of alternative pathway activation. We cannot conduct protein studies as our DNA collection is anonymized, and we have no serum from this individual. Evidence about the effects of other mutations and polymorphisms in this region suggests that a functional effect through disruption of interaction with FI is likely: the protective allele of rs800292 (62I in SCR1) results in enhanced FI cofactor activity and reduced risk of AMD. A mutation of a conserved amino acid in SCR1, G69E, has also been reported in association with AMD (Raychaudhuri et al. 2011). A mutation of CFH (P503H) which is strongly associated with AMD in Amish people alters the equivalent amino acid in SCR8 as that which P139A alters in SCR2 (Hoffman et al. 2014). It is possible that rare mutations of CFH SCRs 1-4 are relatively common in people with early-onset or severe AMD. We would expect to find clustering of AMD cases in families that carry these mutations. The increasing use of massively parallel sequencing in other AMD research DNA collections will show whether this is the case. Developing real-world applications for advancing the understanding of inherited diseases is a challenge, but eye diseases continue to be prime targets for ‘personalized medicine’ based on the best knowledge of disease processes and individual risk.