A rare penetrant mutation in CFH confers high risk of age-related macular degeneration

Abstract

Two common variants in the gene encoding complement factor H (CFH), the Y402H substitution (rs1061170, c.1204C>T)(1-4) and the intronic rs1410996 SNP(5,6), explain 17% of age-related macular degeneration (AMD) liability. However, proof for the involvement of CFH, as opposed to a neighboring transcript, and knowledge of the potential mechanism of susceptibility alleles are lacking. Assuming that rare functional variants might provide mechanistic insights, we used genotype data and high-throughput sequencing to discover a rare, high-risk CFH haplotype with a c.3628C>T mutation that resulted in an R1210C substitution. This allele has been implicated previously in atypical hemolytic uremic syndrome, and it abrogates C-terminal ligand binding(7,8). Genotyping R1210C in 2,423 AMD cases and 1,122 controls demonstrated high penetrance (present in 40 cases versus 1 control, P = 7.0 × 10(-6)) and an association with a 6-year-earlier onset of disease (P = 2.3 × 10(-6)). This result suggests that loss-of-function alleles at CFH are likely to drive AMD risk. This finding represents one of the first instances in which a common complex disease variant has led to the discovery of a rare penetrant mutation.

Figure 1

A. Phasing 21 markers in the CFH/CFHR1/CFHR3 region. Here we present association statistics of 11 haplotypes with frequencies >0.3%, resulting from phasing 20 SNP markers and a CFHR1-3 common copy number polymorphism. We specifically note the CFH rs800292 (I62V) SNP; the CFH Y402H proxy, rs10801555; CFH rs1410996 proxy, rs10737680; and the CFHR1-3 deletion. For most SNPs we list the nucleotide, for the CFHR1-3 deletion the empty circle (◎) indicates the region is deleted while the filled circle (◉) indicates the region is not deleted. To the right of each haplotype, we note the observed frequency in cases and controls. To the far right of each haplotype we note the relative ratio of the odds of disease for each haplotye relative to H1. For the H5 haplotype we note that it has a significantly greater allelic odds ratio than H1. The contrast is more striking when compared to the aggregate odds ratio of haplotypes H4-H11 (red dot to the left of the H5 OR). B. Case-control permutations preserving genotype at four common AMD associated markers. Here we present a histogram of the number of H5 heterozygote individuals that are cases for each of the 100,000 permutations. We place an arrow at 10, the observed number of H5 heterozygote individuals that are cases in the actual data (p=0.00081).

Figure 1

A. Phasing 21 markers in the CFH/CFHR1/CFHR3 region. Here we present association statistics of 11 haplotypes with frequencies >0.3%, resulting from phasing 20 SNP markers and a CFHR1-3 common copy number polymorphism. We specifically note the CFH rs800292 (I62V) SNP; the CFH Y402H proxy, rs10801555; CFH rs1410996 proxy, rs10737680; and the CFHR1-3 deletion. For most SNPs we list the nucleotide, for the CFHR1-3 deletion the empty circle (◎) indicates the region is deleted while the filled circle (◉) indicates the region is not deleted. To the right of each haplotype, we note the observed frequency in cases and controls. To the far right of each haplotype we note the relative ratio of the odds of disease for each haplotye relative to H1. For the H5 haplotype we note that it has a significantly greater allelic odds ratio than H1. The contrast is more striking when compared to the aggregate odds ratio of haplotypes H4-H11 (red dot to the left of the H5 OR). B. Case-control permutations preserving genotype at four common AMD associated markers. Here we present a histogram of the number of H5 heterozygote individuals that are cases for each of the 100,000 permutations. We place an arrow at 10, the observed number of H5 heterozygote individuals that are cases in the actual data (p=0.00081).

Figure 2. Sequencing the H5 haplotype identifies an R1210C mutation

We applied capillary electrophoresis sequencing to 84 individuals representing the common CFH haplotypes depicted in Figure 1. Each circle represents an individual. The position on the grid indicates the two haplotypes for the individual at the CFH locus; individuals along the diagonal are homozygous for a haplotype. Individuals that do not have the R1210C mutation are depicted with an empty circle (◎). Individuals that are heterozygous for the R1210C mutation are indicated with a filled red circle (◉). All 10 individuals with the R1210C mutation are heterozygous for the H5 haplotype, strongly suggesting that the mutation is on that haplotype, and accounts for the increased risk associated with that haplotype.

Figure 3. The phenotype of the R1210C mutation

A. Here we plot a histogram of the age of onset for 23 individuals with the R1210C mutation and also for 1,887 individuals without the R1210C mutation from Boston-phased and Boston-replication with available data. Age of onset is defined as the age when the patient starts showing signs of AMD. While the median age of diagnosis for affected individuals with the R1210C mutation is six years less than those without (65 versus 71 years), the mean age is 8.6 years less (61.9 versus 70.5 years). B. Fundus photograph of the right eye of a subject with the R1210C mutation showing central geographic atrophy (advanced dry age-related macular degeneration) surrounded by numerous large and very large drusen in the posterior pole and along the vascular arcades. Drusen were present nasal and temporal to the macula and in all four quadrants out to the mid-peripheral retina.

Figure 3. The phenotype of the R1210C mutation

A. Here we plot a histogram of the age of onset for 23 individuals with the R1210C mutation and also for 1,887 individuals without the R1210C mutation from Boston-phased and Boston-replication with available data. Age of onset is defined as the age when the patient starts showing signs of AMD. While the median age of diagnosis for affected individuals with the R1210C mutation is six years less than those without (65 versus 71 years), the mean age is 8.6 years less (61.9 versus 70.5 years). B. Fundus photograph of the right eye of a subject with the R1210C mutation showing central geographic atrophy (advanced dry age-related macular degeneration) surrounded by numerous large and very large drusen in the posterior pole and along the vascular arcades. Drusen were present nasal and temporal to the macula and in all four quadrants out to the mid-peripheral retina.