Conceived and designed the experiments: PF DS SH RW MK. Performed the experiments: PF. Analyzed the data: PF DS SH JO. Contributed reagents/materials/analysis tools: PF. Wrote the paper: PF MK.
The authors have declared that no competing interests exist.
Age-related macular degeneration (AMD), the leading cause of blindness in the Western world, is a complex disease that affects people over 50 years old. The complement factor H (CFH) gene has been repeatedly shown to be a major factor in determining susceptibility to the advanced form of the condition. We aimed to better understand the functional role of this gene in the AMD disease process and assess whether it is associated with earlier forms of the disease.
We genotyped SNPs at the CFH gene locus in three independent populations with AMD: (a) extended families where at least 3 family members had AMD; (b) sporadic cases of advanced AMD and (c) cases from the Age-Related Eye Disease Study (AREDS). We investigated polymorphisms and haplotypes in and around the CFH gene to assess their role in AMD. CFH is associated with early/intermediate and advanced AMD in both familial and sporadic cases. In our populations, the CFH SNP, rs2274700, is most strongly associated with AMD and when incorporated into a haplotype with the Y402H SNP and rs1061147, the strongest association is observed (p<10−9).
Our results, reproduced in three populations that represent the spectrum of AMD cases, provide evidence that the CFH gene is associated with drusen as well as with advanced AMD. We also identified novel susceptibility and protective haplotypes in the AMD populations.
Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world.
Advanced AMD is characterized by poor central vision following the development of: (1) choroidal neovascularization (CNV) associated with leakage, hemorrhage, and subretinal scarring, or (2) geographic atrophy (GA), characterized by the appearance of coalescing foci of retinal pigment epithelial atrophy.
Other genes have been implicated in AMD development, including complement component 2 (CF2) and factor B (BF),
In this article, we present the results of analyses of 3 populations with AMD: extended families genetically enriched for their predisposition to the disease, sporadic cases of advanced AMD, and the Age-Related Eye Disease Study (AREDS) cohort. We investigated associations with polymorphisms and haplotypes in the CFH gene. Furthermore, since our cohorts included individuals with earlier stages of AMD, the potential role of CFH in drusen formation was explored.
The CFH risk allele rs1061170 ‘C’ and AMD was strongly associated with advanced AMD status in each of the three cohorts: (1) a cohort of families with AMD (1253 Caucasian affected and unaffected individuals from 124 extended families (average age 68.2 years; 492 males, 761 females); (2) a sporadic case/control cohort: 211 sporadic cases (age 79 years, range 57–100; 70 male, 141 female; 210 Caucasian, 1 Other racial origin) of advanced AMD and 183 unrelated ophthalmologically evaluated controls (age 74, range 63–92; 81 male, 102 female; 183 Caucasian; 2201 cases and controls from the Age-related Eye Disease Study (AREDS).
Associations with several other SNPs in the CFH gene and advanced AMD status were also surveyed in these populations and significant associations were also noted for SNPs rs529825, rs800292, rs1061147, rs203674 and rs2274700 (
SNP | Location and change | Sample | OR (lower bound–upper bound) | P-value | Fbat p value |
rs529825 | IVS1 | Family Data | |||
Case Control | 2.67 (2.22–3.12) | ||||
AREDS | 2.57 (1.81–3.33) | 0.018 | |||
rs800292 | Exon 2, I62V | Family Data | |||
Case Control | 2.35 (1.95–2.75) | ||||
AREDS | 2.52 (1.75–3.28) | 0.022 | |||
rs3766404 | IVS6 | Family Data | 0.096 | ||
Case Control | 3.82 (3.292–4.354) | ||||
AREDS | 3.77 (2.59–4.95) | 0.030 | |||
rs1061147 | Exon 7, A307A | Family Data | |||
Case Control | 2.53 (2.21–2.85) | ||||
AREDS | 2.05 (1.54–2.56) | ||||
rs1061170 | Exon 9, Y402H | Family Data | 4.36 (3.51–5.21)† | ||
Case Control | 2.40 (2.07–2.73) | ||||
AREDS | 2.04 (1.54–2.54) | ||||
rs203674 | IVS10 | Family Data | |||
Case Control | 2.68 (2.34–3.02) | ||||
AREDS | 2.13 (1.61–2.65) | ||||
rs2274700 | Exon 10, A473A | Family Data | |||
Case Control | 3.07 (2.74–3.41) | ||||
AREDS | 3.32 (2.67–3.97) |
Emboldened numbers indicate statistically significant p-values allowing for multiple testing. Number in italics indicated most significant p-value. For IVS1 and I62V confer decreased odds of developing AMD.
A subtractive approach utilized 27 haplotypes to determine the combination of SNPs with the lowest p-value of association with AMD using FBAT. This best combination comprised the following 3 SNPs: rs1061147, rs1061170, rs2274700 (p<0.0002,
Number of SNPs | SNPs involved in best model | Haplotypes | Frequency | Haplotype p-values | Overall p-value |
7 | rs3766404 rs529825 rs1061147 rs800292 rs1061170 rs203674 rs2274700 | TCACCCC | 0.58 | 0.03 | |
TCCCTAC | 0.12 | 0.90 | |||
TTCTTAT | 0.11 | 0.10 | 0.12 | ||
6 | rs3766404 rs529825 rs1061147 rs800292 rs1061170 rs2274700 | TCACC C | 0.56 | 0.02 | |
TCCCT C | 0.18 | 0.44 | |||
TTCTT T | 0.11 | 0.13 | |||
CCCCT T | 0.06 | 0.09 | 0.02 | ||
5 | rs529825 rs1061147 rs800292 rs1061170 rs2274700 | CACC C | 0.55 | 0.01 | |
CCCT C | 0.18 | 0.31 | |||
TCTT T | 0.11 | 0.18 | |||
CCCT T | 0.10 | 0.02 | 0.006 | ||
4 | rs1061147 rs800292 rs1061170 rs2274700 | ACC C | 0.56 | 0.00 | |
CCT C | 0.18 | 0.31 | |||
CTT T | 0.12 | 0.07 | |||
CCT T | 0.100 | 0.03 | 0.0008 | ||
3 | |||||
C T C | 0.17 | 0.35 | 0.00008 | ||
2 | rs1061147 rs1061170 | A | 0.56 | 0.00008 | |
C T | 0.41 | 0.004 | 0.0001 | ||
1 | rs1061170 | 0.58 | 0.006 | ||
T | 0.42 | 0.006 | 0.006 |
The most strongly associated haplotype (shown in red) is derived from SNPs genotyped in the CFH gene locus using backward selection. Emboldened ‘C’ indicates CFH rs1061170 risk allele. ‘Best’ haplotype shown in italics. Overall p value describes association of the haplotype with AMD independent of alleles
We then investigated whether these haplotypes were associated with AMD development in the AREDS and case-control populations and found similar results (
Case control cohort | |||||||
Haplotype | Hap Score | Pooled Haplotype Frequency | Control Haplotype Frequency | Case Haplotype Frequency | OR (lower bound–upper bound) | Associated P-value | Overall p value |
ACC | −5.85 | 0.47 | 0.36 | 0.57 | 0.43 (0.29–0.64) | <10−9 | <0.00001 |
CTT | −7.83 | 0.33 | 0.48 | 0.20 | 3.88 (2.51–6.01) | <10−14 | |
CTC | −1.07 | 0.20 | 0.16 | 0.23 | 0.62 (0.38–1.03) | 0.28 |
AREDS cohort | |||||||
Haplotype | Hap Score | Pooled Haplotype Frequency | Control Haplotype Frequency | Case Haplotype Frequency | OR (lower bound–upper bound) | Associated P-value | Overall p value |
ACC | −4.28 | 0.44 | 0.35 | 0.58 | 0.40 (0.30–0.52) | <0.0001 | <0.00001 |
CTT | −4.79 | 0.36 | 0.46 | 0.21 | 3.08 (2.30–4.13) | <0.0001 | |
CTC | −0.30 | 0.20 | 0.19 | 0.21 | 0.90 (0.64–1.27) | 0.77 |
The odds ratios are computed by comparing each haplotype against all other haplotypes pooled. Cases defined as AREDS category 4, advanced AMD. Controls defined as AREDS category 1, unaffected. Overall p value describes association of the haplotype with AMD independent of alleles
In our 3 populations, we found that neither individual SNPs nor the 3-SNP haplotype was preferentially associated with either the neovascular or geographic atrophy form of advanced AMD.
AMD category | AREDS cohort | Familial AMD cohort | Case control cohort | ||||||
Number of individuals | ‘C’ allele frequency | P value | Number of individuals | ‘C’ allele frequency | P value | Number of individuals | ‘C’ allele frequency | P value | |
1 | 306 | 0.36 | 97 | 0.47 | 211 | 0.36 | |||
2 | 502 | 0.38 | 0.46 | 55 | 0.49 | 0.84 | |||
3 | 526 | 0.48 | <10−5 | 95 | 0.63 | 0.002 | |||
4 | 578 | 0.61 | <10−22 | 124 | 0.63 | 0.001 | 183 | 0.59 | <10−10 |
P-values calculated for allelic test of association for differences in AMD grade (categories 2, 3 and 4) from category 1 (no AMD)
The highest prevalence of this risk allele is in advanced AMD (category 4) and lowest in the unaffected controls (category 1, no AMD), as illustrated in
In this study, we confirm the strong association between polymorphisms in the complement factor H gene and advanced age-related macular degeneration in 3 independent predominantly Caucasian populations. For the first time, we report this association in families in whom there is increased susceptibility among relatives to develop the condition. The prevalence of the risk allele ‘C’ for the coding SNP rs1061170 (Y402H) present in exon 9 of the gene in cases of advanced AMD is broadly comparable to that previously reported in sporadic case-control populations
We chose the 7 SNPs to be genotyped based upon those previously shown to be associated with AMD at the CFH gene locus: rs529825, rs3766404, rs1061147, rs800292, rs1061170, rs203674 and rs2274700.
There are considerable similarities between the findings in our study of CFH haplotypes and the other three studies previously published on the subject. Hageman and colleagues
Li
Maller and coworkers
In summary, our 3-SNP haplotype is a good ‘fit’ for those previously described. Furthermore, we have been able to confirm its validity in 3 independent well-phenotyped populations. Interestingly, apart from the CFH Y402H SNP, the other two are synonymous coding variants, suggesting that these sequence changes do not combine to effect some reduction in protein function but more likely alter expression levels of the CFH gene and possibly other complement factor-related genes
It is now established that the Y402H CFH change is associated with advanced AMD. Whether this polymorphism also contributes to other earlier AMD phenotypes remains to be fully explored. Only one other report has addressed this issue finding increased frequency of the risk allele in individuals with drusen
We found that the frequency of the risk allele was similar among those with advanced AMD in all three groups (approximately 0.60). Interestingly, this same allele frequency is observed in those individuals with intermediate AMD (drusen) with a strong family history of AMD and higher than in those with the same category of AMD in the AREDS cohort. Our future longitudinal studies will evaluate whether this is because individuals with a family history of AMD, who have both extensive drusen and the CFH risk allele are at very high risk of progression to choroidal neovascularization and/or geographic atrophy.
Previous studies have failed to show that CFH associates more frequently with the presence of either CNV or GA. In our 3 populations, we also found that neither the SNPs in and around the CFH gene nor the 3-SNP haplotype preferentially increased susceptibility to one of these late complications.
In summary, we have examined 3 independent, carefully phenotyped populations with AMD. These populations can be considered to reflect the full diversity of AMD patients: those with familial susceptibility to the condition and 2 case-control populations, one of which was followed for more than 10 years as part of a nationwide clinical trial. We have confirmed the association between the Y402H CFH mutation and advanced AMD finding however the strongest association with another SNP within the gene, rs2274700 in agreement with two other studies
All studies involving human subjects were approved by the Institutional Review Board of Oregon Health & Science University. Research followed the tenets of the Declaration of Helsinki, and written informed consent was obtained from all subjects after explanation of the nature and possible consequences of the study.
Each family comprised at least three examined (and genotyped) affected relatives with AMD. Individuals were identified as follows: category 1, (no AMD) no drusen or drusen less than 63 µm diameter, and no pigment changes in either eye; category 2, mild to moderate drusen consisting of drusen of any size, but <393,744 µm2 in total area within 1500 µm from the fovea, with or without pigment changes; category 3, extensive large drusen (>125 µm in minimum diameter) >393,744 µm2 in area (a minimum of approximately 20 large drusen) within 1500 µm of the fovea, with or without pigment changes and no evidence of advanced AMD; category 4 (advanced AMD), presence in one or both eyes of advanced macular degeneration (choroidal neovascularization or geographic atrophy)
The Age-Related Eye Disease Study was an eleven-center double-masked clinical trial investigating the effects of oral antioxidant and vitamin supplements on disease progression. DNA was extracted from venous blood from 2201 individuals (939 males, 1262 females; 2115 Caucasian, 72 African American, 6 Hispanic, 3 Asian, 5 Other racial origin) which comprised approximately one-half the AREDS participants (obtained from the AREDS Genetic Repository). Participants were aged 67.7 years (range 55–80) years. The AREDS study procedures have been previously reported.
Seven SNPs in and around the CFH gene locus were chosen: rs3766404, rs529825, rs1061147, rs800292, rs1061170, rs203674 and rs2274700. Genotyping was performed using Sanger sequencing at Prevention Genetics (3700 Downwind Drive, Marshfield, Wisconsin, USA) and at the Broad Institute Center for Genotyping and Analysis, Cambridge, MA, USA.
To estimate SNP frequencies and determine if the frequencies differed between affected and unaffected individuals in the pedigrees, the Family Based Association Test (FBAT) was used.
FBAT was used to estimate haplotype frequencies and any that occurred less than 10 times in combined cases and controls were eliminated from the analysis. Since 7 SNPs were measured in this gene, all 7 SNPs (see above genotyping) were used to produce the first haplotype model. Then 1 SNP was removed at a time to create 7 models, each containing 6 SNPs. Of these 7 models, the one with the lowest associated p-value was deemed the best model. Then 1 SNP was removed at a time from this best 6-SNP model to create 6 models, each containing 5 SNPs. The 5-SNP model with the lowest associated p-value was then the best 5-SNP model. This process was continued to find the best 4-SNP, 3-SNP, 2-SNP and 1-SNP models. Overall, we performed 27 tests. All 27 models were evaluated to determine which had the lowest associated p-value. The Bonferroni was used to adjust for multiple testing.
The SNPs from the model with the lowest associated p-value were used to determine if one haplotype effect replicated in 2 additional samples. We used haplo.stat (a program in R) to estimate haplotype frequencies, determine if these differed between cases and controls, determine if individual haplotypes differed significantly between cases and controls and estimate an odds ratio for each individual haplotype.
We thank AREDS participants and investigators and the EMMES Corporation for their work on the AREDS Genetic Repository