Age-related macular degeneration is the leading cause of late-onset blindness in the industrialized world9
. Common CFH
variants are associated with AMD and meningitis susceptibility. Rare CFH
mutations cause atypical hemolytic uremic syndrome (aHUS), an episodic illness that causes acute renal failure, and membranoproliferative glomerulonephritis type II (MPGNII), a chronic renal disease10,11
. While the possibility that rare CFH
variants confer AMD risk has been raised12
, no advanced AMD risk conferring rare variant has yet been identified.
To identify rare penetrant variants within the CFH
locus, we phased genotypes for 20 common SNPs spanning the CFH/CFHR1-3
region and a common CFHR1-
3 deletion (CFHR1-3
Δ) in 711 advanced AMD cases and 1041 controls (“Boston-phased” dataset)13,14
. Controls included 304 phenotyped controls without evidence of retinal disease and 737 unphenotyped shared controls presumed to not have disease. Almost all these markers are associated with AMD (Supplementary Table 1
). We defined 11 common haplotypes (>0.3% frequency), explaining 97.4% of 3354 chromosomes, and calculated association statistics (, Supplementary Table 2
). Consistent with prior observations1–4
, the most frequent haplotype (H1
) is present in 59% of case chromosomes but only in 37% of control chromosomes; this haplotype exclusively contains the Y402H1–4
risk allele (in LD with rs10801555, r2
=0.99 in 288 genotyped controls). Two haplotypes, H2
, containing a proxy to the rs1410996 CFH
intronic risk SNP5,6
, rs10737680, conferred intermediate risk (OR=0.59 [0.49–0.71] relative to H1
). Excepting H4
, the remaining haplotypes contained none of the two common risk variants, and in aggregate conferred low risk (OR = 0.30 [0.26–0.36] relative to H1
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 (more ...)
Strikingly, 10 of 11 individuals heterozygote for the rare H5 haplotype had AMD. The unaffected individual was a 49 year-old unphenotyped male, unlikely to have advanced disease given his age. This proportion of H5 heterozygote individuals with disease was unexpected since H5 lacked both the Y402H and rs10737680/rs1410996 risk alleles. We speculated that a rare variant on the H5 haplotype might explain the data. However, prior to pursuing a sequencing experiment, we considered that the haplotype association was the consequence of chance or stratification. Therefore we asked whether (1) H5 associated significantly with disease accounting for known AMD-associated common variants; (2) H5 correlated with earlier age of onset; and (3) the H5 association could be explained by case-control stratification or recent ancestry.
We assessed the statistical significance of the association of H5
with advanced AMD by permuting case-control status across all 1,752 subjects. Since we wanted to conservatively assess whether the association was independent of known risk alleles, we were careful to preserve genotypes for four published AMD-associated common markers: Y402H (rs1080155), rs10737680/rs1410996, CFHR1-3
Δ, and rs800292 (I62V). This permutation fixes case-control allele frequencies, association statistics, and odds ratios at those markers, and also constrains association statistics at the other 17 markers due to underlying linkage disequilibrium in region (Supplementary Figure 1
). While common marker association statistics are maintained in these permutations, we rarely observed that ten or more affected heterozygote H5
individuals (p=0.00081, ). Permuting case-control status without preserving marker genotypes or preserving genotypes at only two common markers (Y402H and rs1410996) yield similar results (p=0.00081 and p<0.00001 respectively). Thus the case-association with this haplotype is beyond what might be expected by chance.
The 10 H5 heterozygote individuals were diagnosed earlier than the other 701 cases in the Boston-phased data set (median age 62.5 years versus 71 years, p=0.0023 by 1-tailed rank-sum test), further suggesting that H5 might influence AMD risk.
To assess the possibility that the H5
association might be the consequence of recent relatedness or population stratification, we selected 79,091 independent SNPs from genome-wide data (Methods
). We noted first that there was only modest genome-wide stratification (λgc
. We projectied genotype data into principal components14
and observed that H5
heterozygotes do not cluster discretely (Supplementary Figure 2
) and that H5
heterozygote individuals were not skewed along any of the top 20 components (p
>0.047, Supplementary Table 3
). To confirm that H5
was not the consequence of a recent common ancestry, we estimated the proportion of identity by descent to be <3% between H5
heterozygote pairs; at least ~12.5% would be expected for first cousins or more closely related relatives. Taken together, our data suggest that the observed H5
association is unlikely to be artifactual.
To discover the causal mutation we designed a sequencing experiment. We selected 84 samples representing all 11 haplotypes including homozygote individuals when possible; we included all 10 affected individuals with H5
. We first applied high-throughput sequencing to a 106.7 kb region containing CFH
introns, exons, and promotor. We sequenced a subset of 60 individuals (33 cases and 27 controls), representing each of the 11 haplotypes. These samples included 6 H5
heterozygote individuals. We achieved high coverage with 80% of individuals sequenced at ≥20x for at least 89.3 kb (Supplementary Figure 3
). Genotype calls from sequencing were 98.9% accurate in 19 separately genotyped SNPs. We identified a total of 623 variants; to avoid false positive calls, we focused our analyses on 356 variants seen at least twice (Supplementary Table 4, Supplementary Table 5
). We were confident that H5
alleles, both coding and non-coding, were likely to be among these since two or more H5
heterozygotes achieved >20x coverage in 97.8% of the targetted region. We found only six non-synonymous alleles: (1) rs800292 (I62V); (2) rs1061170 (Y402H); (3) rs1065489 (D936E); (4) rs35274867 (N1050Y); (5) a rare glutamine to histidine variant (Q950H); and (6) a rare exon 22 variant that alters arginine to cysteine, R1210C (Supplementary Figure 4A
). Of all observed variants, only R1210C variant was seen exclusively in all of the 6 sequenced H5
heterozygote individuals. To confirm that R1210C was specific for H5
, we used capillary electrophoresis to sequence exon 22 in all 84 samples (Supplementary Figure 4B
). Only the ten affected individuals with the H5
haplotype had the R1210C mutation (); we concluded that R1210C was specific for H5
. We found no other exon 22 variants in any of these 84 individuals.
Sequencing the H5 haplotype identifies an R1210C mutation
To confirm the R1210C association and to asses its presence on other haplotypic backgrounds, we genotyped 707 out of the 711 cases and 303 out of 304 examined controls from the Boston-phased data set (). We observed 13 R1210C heterozygote individuals. In addition to the 10 H5 heterozygote individuals, we identified three more affected individuals with R1210C, two of whom were H1 homozygote and one that was an H1/H11 heterozygote. We did not find the R1210C mutation on any controls. This suggested that in addition to being present consistently on the H5 haplotype, R1210C was also rarely present on H1. One possibility is that the R1210C mutation initially occurred on the common H1 background, and that the H5 haplotype exists because of a historical recombination with H6. These data suggested that R1210C explained the H5 haplotype association to disease and was associated with advanced AMD (p=0.0094 by 1-tailed Fisher’s exact test, ).
To replicate the R1210C association, we genotyped an independent set of 1,707 cases and 817 controls from Boston and Baltimore sample sets (). We assessed significance with exact statistics since the R1210C heterozygotes were so rare (Methods
). In this independent case-control replication we observed a statistically significant association (20 in cases vs 1 in controls, one-tailed p
=0.0052). Combining our case-control replication together with the Boston-phased discovery data set we observed that R1210C heterozygotes were seen in 33/2414 cases (1.4%) compared to 1/1120 controls (<0.1%), suggesting that this mutation confers a high-degree of risk (one-tailed combined p
) and acts as an almost fully penetrant allele. Since Y402H is an influential risk factor at the population level1–4
, we also assessed significance by stratifying for the Y402H genotype (Methods
). We observed that stratifying on Y402H only increased significance of the R1210C association (one tailed p=9.1×10−7
). We concluded that R1210C confers risk independently of the common Y402H risk allele.
To further replicate the R1210C association in independent first-degree relatives, we identified 11 siblings of 6 cases with R1210C from the Boston-phased and Boston replication. These siblings were either unambiguously affected with advanced AMD (n=9), or had absolutely no clinical evidence of disease (n=2) (, Supplementary Table 6
). We observed that 7 of 9 affected individuals were R1210C heterozygotes and 0 of 2 controls were R1210C heterozygotes (p=0.033, one-tailed binomial). In total, aggregating all of the results from our family and case-control data together, we observed a highly significant association (p
stratifying for Y402H, ). We estimate that this mutation accounts for ~14% increase in disease prevalence among siblings of affected probands.
Since R1210C apparently has a strong genetic effect, we tested whether the mutation also drives an early age of disease onset. We observed that unrelated individuals with the R1210C mutation had significantly earlier disease onset (median of 65 versus 71 years, p=2.3×10−6 by rank-sum test, ). Of the 55 individuals with an age of onset <55 years, 5 had the R1210C mutation (8.3%); no individual with onset after 75 years had the R1210C mutation. The retinal phenotype for individual patients with R1210C typically included numerous small, medium, and large drusen ().
The phenotype of the R1210C mutation
Importantly, in our sequencing experiment we found only one rare intronic allele that might potentially contribute to the phenotype; this was a SNP (at 194905360 (HG18)) in 5 of the 6 H5
heterozygotes (Supplementary Table 4
). However, we consider this allele unlikely to be causal. First, this variant has no evidence of mammalian conservation or regulatory function. Second, applying capillary electrophoresis sequencing to 17 R1210C heterozygote individuals revealed six who individuals lacking the intronic allele, indicating again that the R1210C allele is on multiple haplotypes and that the intronic change is more recent.
The R1210C mutation had been described as an incompletely penetrant allele that causes familial forms of aHUS15–19
, and has also been associated with primary glomerulonephritis20
. This allele has been observed worldwide in multiple aHUS families and in 1–4% of individuals in aHUS cohorts16
. The R1210C mutation has been shown to compromise C-terminal CFH function; mutant CFH protein has normal cofactor activity but exhibits defective binding to C3d, C3b, heparin, and endothelial cells7,8,21
. Moreover, mutant protein product in plasma produces a high-molecular-weight factor H protein, resulting from a covalent interaction with human serum albumin15
We assessed renal function by estimating the glomerular filtration rate from serum creatinine measurements in 17 unrelated R1210C heterozygotes with advanced AMD. None had evidence of clinically significant renal dysfunction (creatinine>1.7 mg/dl, eGFR<30 mL/min), though we noted subclinical renal dysfunction (median eGFR of 62 mL/min). Since AMD patients in general have subclinical renal dysfunction22
, we compared renal function to 17 individuals matched on disease severity, age, and gender but without R1210C. We observed no significant difference (one-tailed p=0.54, Supplementary Figure 5
A previous study proposed that rare CFH
variants might be compound heterozygous alleles acting in trans with common alleles such as Y402H or I62V12
. We found no evidence of this for the R1210C mutation. For the 13 Boston-phased cases with the R1210C (with complete phase information), we observed no excess beyond chance of Y402H risk alleles in trans [61% (=8/13) versus 59% frequency in all cases] or I62V risk [85% (=11/13) versus 87% frequency] in trans.
Our data suggest that compromised CFH function contributes to AMD pathogenesis and facilitates the transition from association to causality that is ubiquitously necessary to advance studies of complex disorders. We do not exclude possible contributions of neighboring CFH-like loci. Nonetheless, abrogated C-terminal activity of this protein represents potentially the first mechanistic clue about pathogenesis, and could inform attempts to understand the action of other cis and trans susceptibility alleles. This highlights the value of finding rare alleles with experimentally tractable biological effect in complex traits.
Our data link two clinically unrelated diseases, AMD and aHUS, with common underlying pathology. We note that AMD has common variants in multiple complement pathway genes (CFI23
, and C34,25
), and that rare predisposing mutations to aHUS have been observed in these same genes26
. Future studies sequencing these genes in large numbers of patients might also reveal rare, high penetrant alleles that could contribute to understanding AMD mechanism.