Rare variants in CFI, C3 and C9 are associated with high risk of advanced age-related macular degeneration

Abstract

To define the role of rare variants in advanced age-related macular degeneration (AMD) risk, we sequenced the exons of 681 genes within all reported AMD loci and related pathways in 2,493 cases and controls. We first tested each gene for increased or decreased burden of rare variants in cases compared to controls. We found that 7.8% of AMD cases compared to 2.3% of controls are carriers of rare missense CFI variants (odds ratio (OR) = 3.6; P = 2 × 10(-8)). There was a predominance of dysfunctional variants in cases compared to controls. We then tested individual variants for association with disease. We observed significant association with rare missense alleles in genes other than CFI. Genotyping in 5,115 independent samples confirmed associations with AMD of an allele in C3 encoding p.Lys155Gln (replication P = 3.5 × 10(-5), OR = 2.8; joint P = 5.2 × 10(-9), OR = 3.8) and an allele in C9 encoding p.Pro167Ser (replication P = 2.4 × 10(-5), OR = 2.2; joint P = 6.5 × 10(-7), OR = 2.2). Finally, we show that the allele of C3 encoding Gln155 results in resistance to proteolytic inactivation by CFH and CFI. These results implicate loss of C3 protein regulation and excessive alternative complement activation in AMD pathogenesis, thus informing both the direction of effect and mechanistic underpinnings of this disorder.

Figure 1. CFI burden of rare coding variants is increased in cases

A. We tested 681 sequenced genes for increased burden of rare (<1%) missense, nonsense, splice, or read-through variants. Here we plot the observed p-value for each gene as a function of expected p-values. We tested enrichment in rare variant carriers in cases (red) separately from enrichment in rare variant carriers in controls (yellow). We indicate the threshold of significance after multiple hypothesis testing (dashed red line, p<3.6×10⁻⁵ = 0.05/(681 genes × 2 tests) ). We observed significant enrichment of rare variants in cases in only one gene, CFI. B. We stratified individuals by their rs4698775 genotype, a nearby associated common variant; the G minor allele has previously been reported to be associated with risk. We observed that rare variants are enriched on all genotypic backgrounds.

Figure 1. CFI burden of rare coding variants is increased in cases

A. We tested 681 sequenced genes for increased burden of rare (<1%) missense, nonsense, splice, or read-through variants. Here we plot the observed p-value for each gene as a function of expected p-values. We tested enrichment in rare variant carriers in cases (red) separately from enrichment in rare variant carriers in controls (yellow). We indicate the threshold of significance after multiple hypothesis testing (dashed red line, p<3.6×10⁻⁵ = 0.05/(681 genes × 2 tests) ). We observed significant enrichment of rare variants in cases in only one gene, CFI. B. We stratified individuals by their rs4698775 genotype, a nearby associated common variant; the G minor allele has previously been reported to be associated with risk. We observed that rare variants are enriched on all genotypic backgrounds.

Figure 2. Burden of CFI rare variants

A. In the top we show the domain structure of the CFI protein, including the factor I membrane attack complex (FIMAC), scavenger receptor cysteine rich (SRCR) domain, the LDL-receptor (LDLR1 and LDLR2) domains, and a peptidase S1 serine protease domain. Below we plot the individual rare variants that we observed in our sequencing experiment in cases (red, upper) and controls (yellow, lower). In our data, we observed no common CFI coding variants with f>0.01. We plot counts of variants along the left y-axes, and the proportions of heterozygote individuals along the right y-axes. We list the actual coding changes conferred by the variants in the middle. Case-only variants are colored red and control-only variants are colored yellow. We put red boxes around variants that are predicted to be loss of function or probably damaging variants. B. Plot of rare variants in CFI categorized with PolyPhen-2 as all variants, “benign”, “possibly damaging”, “probably damaging”, and “loss of function”, with counts in controls (left) compared to counts in cases (right) for each category. Variants that are predicted to have greater liklihood of being damaging are more strongly skewed toward cases (1-tailed p=0.045, logistic regression with permutation of only individuals with a rare variant)

Figure 2. Burden of CFI rare variants

A. In the top we show the domain structure of the CFI protein, including the factor I membrane attack complex (FIMAC), scavenger receptor cysteine rich (SRCR) domain, the LDL-receptor (LDLR1 and LDLR2) domains, and a peptidase S1 serine protease domain. Below we plot the individual rare variants that we observed in our sequencing experiment in cases (red, upper) and controls (yellow, lower). In our data, we observed no common CFI coding variants with f>0.01. We plot counts of variants along the left y-axes, and the proportions of heterozygote individuals along the right y-axes. We list the actual coding changes conferred by the variants in the middle. Case-only variants are colored red and control-only variants are colored yellow. We put red boxes around variants that are predicted to be loss of function or probably damaging variants. B. Plot of rare variants in CFI categorized with PolyPhen-2 as all variants, “benign”, “possibly damaging”, “probably damaging”, and “loss of function”, with counts in controls (left) compared to counts in cases (right) for each category. Variants that are predicted to have greater liklihood of being damaging are more strongly skewed toward cases (1-tailed p=0.045, logistic regression with permutation of only individuals with a rare variant)

Figure 3. The C3 155Q confers resistance to cofactor activity

A. We incubated 155K (WT) and 155Q (variant) C3 proteins at physiologic salt concentration with 20 ng of CFI and 200 ng of CFH at 37°C for 10, 20, and 30 minutes and then stopped the reaction by the addition of 3X reducing sample buffer. Following electrophoresis and transfer to a nitrocellulose membrane, western blots were performed for C3 proteins. This western blot is representative of four similar experiments. We observe that the α-chain is cleaved much less efficiency in 155Q than in 155K. In parallel we see accumulation of the α₄₀ cleavage fragment in proportion to α-chain cleavage. The α₁ cleavage fragment is not visualized since it migrates at the same molecular weight as the β-chain. B. We quantified the reduction of the α-chain by densitometry of four independent experiments; here, we present the mean ±SEM at each time point. We assessed significance with an unpaired t-test.