The Y402H Variant in the Complement Factor H Gene Affects Incidence and Progression of Age-Related Macular Degeneration: Results from Multi-State Models Applied to the Beaver Dam Eye Study

Abstract

Objective

To investigate the impact of age, sex and the Y402H variant in the complement factor H (CFH) gene on incidence, progression and regression of age-related macular degeneration (AMD) and the impact of these factors and AMD on mortality using multi-state models (MSMs).

Methods

Analyses included 4,379 persons aged 43 to 86 years at the time of initial examination. AMD status on a 5-level severity scale was graded from retinal photographs taken at up to 5 study visits between 1988 and 2010. MSMs in continuous time were used to model the effects of age, sex and CFH genotype on incidence, progression and regression of AMD and mortality.

Results

CFH Y402H genotype CC was associated, relative to genotype TT (reported as hazard ratio, 95% confidence interval), with increased incidence of AMD (no to minimally severe early AMD: 1.98, 1.57–2.49), progression of AMD (minimally severe early to moderately severe early AMD: 1.73, 1.29–2.33; moderately severe early to severe early AMD: 1.30, 0.86–1.94; and severe early to late AMD: 1.72, 1.01–2.91), but not with regression of AMD or mortality. Late AMD was associated with increased mortality (1.37, 1.15–1.62) relative to no AMD, but earlier stages of AMD were not.

Conclusions

Using MSMs, we show that the Y402H risk variant elevates lifetime incidence of early AMD and progression of early to late AMD, and that late AMD elevates mortality risk.

Information regarding the development and progression of age-related macular degeneration (AMD) has emerged from clinical and epidemiological studies.^1–11 This has resulted in the development of severity scales ranging from no lesions through the most severe lesions (neovascular AMD and/or geographic atrophy).^6,12–14 Most scales use size, type and area of drusen and retinal pigment epithelium (RPE) pigmentary abnormalities to define intermediate severity levels.

While most epidemiological studies have investigated incidence and progression of AMD, few studies have examined regression and associated protective factors.^2,5,9,11 In the Chesapeake Bay Waterman Study, large soft drusen disappeared in 34% of eyes over a 5-year follow-up.² In the Melton Mowbray study, 20% of soft drusen regressed over a 7-year period.⁹ It is unclear what factors are associated with regression or how AMD transitions through various stages.

Previous studies show that the Y402H polymorphism in the Complement Factor H (CFH) gene on chromosome 1q is strongly associated with AMD,^15–18 suggesting a role for innate immunity and inflammation in AMD pathogenesis. Most studies have examined relationships of the Y402H polymorphism to incidence of early or progression to late AMD but few have considered progression through various stages of AMD or regression of AMD.^19–22

Multi-state models (MSMs) can be used to gain greater insight into this relationship. We use MSMs to model transitions from the state at a study visit to the state at a subsequent visit.^23–27 In practice, disease states are observed at intermittent study visits, and exact transition times are not observed; even when the exact date of death is known, the AMD state at death is unknown.^28–31 Additionally, observations are frequently censored. For example, at the end of follow-up, subjects are known to be alive but their AMD state at that time is unknown. In this paper, we use MSMs to examine the effects of age, sex and the Y402H variant in the CFH gene on incidence, progression and regression of AMD as well as mortality in the population-based Beaver Dam Eye Study (BDES).

METHODS

Population

Methods used to identify the population have been described previously.^32–36 A private census of Beaver Dam, Wisconsin was performed from fall 1987 to spring 1988.³² Of the 5924 persons aged 43 to 84 years, 4926 participated in the baseline examination,³³ 3722 participated in the 5-year follow-up,³⁴ 2962 participated in the 10-year follow-up,³⁵ 2375 participated in the 15-year follow-up,³⁶ and 1913 participated in the 20-year follow-up. Characteristics of the population at each examination and reasons for nonparticipation appear elsewhere.^33–36

Procedures and Definitions

Similar procedures were used at all examinations.^3,5,37–41 Data were collected with Institutional Review Board approval from the University of Wisconsin-Madison, informed consent was obtained from each participant at each examination, and the study adhered to the tenets of the Declaration of Helsinki. Pertinent parts of the examination consisted of taking stereoscopic 30° color fundus photographs centered on the disc (Diabetic Retinopathy Study standard field 1) and macula (Diabetic Retinopathy Study standard field 2) and a nonstereoscopic color fundus photograph temporal to but including the fovea of each eye.

Grading procedures have been described previously.^39–41 In brief, after placing a circular grid on 1 photographic slide of the stereoscopic pair, a preliminary masked grading was done followed by a detailed grading using the Wisconsin Age-Related Maculopathy Grading System; each was performed independently of the fellow eye and the gradings of other observers.^39,40 Next, edits and reviews were performed. Finally, graders made side-by-side comparisons of 15-and 20-year follow-up photographs that showed change in AMD lesions. The senior grader (SMM) and principal investigator (RK) performed a final unmasked review of all 5 visits for progression and regression. Information on gradability has been published elsewhere.^3,5,11,41 AMD status in each eye was classified using the 5-step BDES AMD severity scale (Table 1). Subjects were classified based on the worse eye.

Table 1.

The 5-Level Beaver Dam Eye Study Age-related Macular Degeneration Severity Scale.

Level	Label	Description
1	No AMD	Hard drusen or small soft drusen (<125 μ in diameter only, regardless of area of involvement), and no pigmentary abnormality (increased retinal pigment or RPE depigmentation) present.
2	Minimally severe early AMD	Hard drusen or small soft drusen (<125 μ in diameter, regardless of area of involvement, with any pigmentary abnormality (increased retinal pigment present and/or RPE depigmentation present) OR soft drusen (≥125 μ in diameter) with drusen area <196,350 μ2 (equivalent to a circle with a diameter of 500 μ) and no pigmentary abnormalities.
3	Moderately severe early AMD	Soft drusen (≥125 μ in diameter with drusen area <196,350 μ2 (equivalent to a circle with a diameter of 500 μ) and with any pigmentary abnormality (increased retinal pigment present and/or RPE depigmentation present OR soft drusen (≥125 μ in diameter) with drusen area ≥196,350 μ2 (equivalent to a circle with a diameter of 500 μ) with or without increased retinal pigment but no RPE depigmentation.
4	Severe early AMD	Soft drusen (≥125 μ in diameter) with drusen area ≥196,350 μ2 (equivalent to a circle with a diameter of 500 μ) and RPE depigmentation present, with or without increased retinal pigment.
5	Late AMD	Pure geographic atrophy in the absence of exudative macular degeneration OR exudative macular degeneration with or without geographic atrophy present.

AMD, age-related macular degeneration; RPE, retinal pigment epithelium.

Information on the Y402H polymorphism for CFH, classified as absence (genotype TT), presence of 1 high-risk allele (genotype CT), and presence of 2 high-risk alleles (genotype CC), was available for 4479 participants. Distributions of other characteristics for these subjects did not differ from the rest of the population (data not shown).

Vital status was monitored by reading the obituaries in local newspapers and by making annual telephone contact. Persons not known to have died but could not be contacted had their survival time entered as their last contact date. We ascertained mortality starting after the 1988–1990 baseline examination to the end of the 20-year examination.

Statistical Analysis

Incidence, progression and regression of AMD and mortality were modeled using MSM in continuous time. We identified mutually exclusive and exhaustive states representing the current status of each subject at a given age. Here, subjects were classified as being in 1 of the 5 levels on the BDES AMD severity scale, or death. Figure 1 illustrates the underlying MSM; arrows indicate possible instantaneous transitions. Instantaneous transitions (the next state to which the individual moves and the time of the change) were allowed between adjacent AMD states with one exception; based on clinical observation, regression from late AMD (Level 5) to severe early AMD (Level 4) was not allowed.

Figure 1.

Transition diagram for 5-level age-related macular degeneration scale. Arrows indicate possible instantaneous transitions between states.

Transitions are governed by 12 transition intensities, one for each possible instantaneous transition between states (represented by arrows in Figure 1), which represent the hazard of moving between states. Dependence of transition intensities on age, sex, and CFH Y402H genotype was specified using log-linear regression models. Age was entered as a linear term and updated annually. Sex and CFH Y402H genotype were entered using indicator variables. Covariate effects on transitions within the AMD scale were unconstrained. Covariate effects on transitions to death were constrained to be equal, but intercepts were allowed to vary. Non-linear effects of age and two-way interactions between age, sex and CFH Y402H were evaluated.

The MSM incorporates all available information on the history of disease progression into likelihood calculations. Current AMD state is observed at intermittent study follow-up visits; transition times and numbers of intermediate transitions are unobserved. Death times are available, but AMD state at death is unknown. If subjects are alive at the end of follow-up, final AMD state is unknown. At study visits, the exact AMD state may be unknown. For example, if the right eye has moderately severe early AMD (Level 3) and the left eye is ungradable, the subject’s AMD state could be Level 3, 4 or 5. The timing of follow-up visits is assumed to be uninformative. A sensitivity analysis for which observations from visits outside the scheduled visit window (± 6 months of the anniversary of the baseline visit) were censored was conducted. Results of analyses of the complete data and of this subset were virtually identical, validating the assumption of uninformative visit times (data not shown).

Analyses were conducted in R⁴² using the msm package.⁴³ Covariate effects on transition intensities are summarized as hazard ratios. Using matrix exponentiation, we obtain annual transition matrices for each initial state, age, sex and CFH Y402H genotype. From these transition matrices, we calculate estimated transition probabilities to each AMD state (and death) after 5 years and estimated cumulative incidence of each AMD state (and death) for specified subgroups. Cumulative incidence calculations use annual assessments of AMD status; subjects are assigned to the most severe state observed at or prior to the current age. Confidence intervals (CIs) for these non-linear functions of the transition intensity parameters are obtained from a parametric bootstrap.⁴⁴

RESULTS

Of the 4973 subjects seen at any study visit, 134 were excluded for ungradable AMD status at all visits, and 460 were excluded for missing CFH Y402H genotype; 4379 subjects contributed data from 12,640 BDES follow-up intervals (up to 4 per subject). Table 2 displays characteristics of subjects at the start of each interval by AMD level. Subjects with more severe AMD were older and more likely to be female, to have CFH Y402H genotype CT or CC and to be seen at later visits. Similar relationships were seen among subjects with partial AMD severity information (data not shown).

Table 2.

Characteristics of the Cohort by Current Age-related Macular Degeneration Status (Worse Eye) at the Beginning of the Beaver Dam Eye Study Follow-up Intervals.

Characteristic	No AMD (Level 1)		Minimal Early AMD (Level 2)		Moderate Early AMD (Level 3)		Severe Early AMD (Level 4)		Late AMD (Level 5)
	N=8474		N=1524		N=833		N=250		N=294
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Age (yrs)	63	10	67	10	73	10	76	9	80	8
	N	%	N	%	N	%	N	%	N	%
Sex (male)	3644	43	736	48	357	43	82	33	106	36
Participated in examination
BDES 1	3151	37	530	35	247	30	47	19	75	26
BDES 2	2185	26	444	29	239	29	64	26	67	23
BDES 3	1724	20	316	21	197	24	70	28	70	24
BDES 4	1414	17	234	15	150	18	69	28	82	28
CFH Y402H genotype
TT	3482	41	594	39	287	34	66	26	43	15
CT	3903	46	743	49	404	48	138	55	180	61
CC	1089	13	187	12	142	17	46	18	71	24

AMD, age-related macular degeneration; BDES, Beaver Dam Eye Study; CFH, complement factor H; N, number of subject follow-up intervals; SD, standard deviation.

Table 3 shows observed transitions between consecutive BDES visits. The first column presents transitions for the 8474 BDES visits where subjects had no AMD (Level 1). At their next BDES visit, 71% (n=6050) were still free of AMD, 5% (n=386) progressed to minimally severe early AMD (Level 2), 2% (n=181) progressed to Level 3, 0.2% (n=13) progressed to Level 4, 0.1% (n=8) progressed to late AMD, and 12% (n=1015) died. The remaining subjects (n=821) had missing/partial information. Six percent (n=493) were seen with no information (Levels 1–5), 0.2% (n=17) had minimal early AMD or worse (Levels 2–5), 0.1% (n=10) had moderate early AMD or worse (Levels 3–5), 0.02% (n=2) had severe early AMD or worse (Levels 4–5), and 4% (n=299) were not seen.

Table 3.

Observed State Transitions During Consecutive Visit Intervals.

	AMD Level (Beginning of Interval)
	No AMD (Level 1)		Minimal Early AMD (Level 2)		Moderate Early AMD (Level 3)		Severe Early AMD (Level 4)		Late AMD (Level 5)
	N=8474		N=1524		N=833		N=250		N=294
	N	%	N	%	N	%	N	%	N	%
AMD Level (End of Interval)
1	6050	71	149	10	7	1	0	0
2	386	5	677	44	67	8	7	3
3	181	2	211	14	294	35	9	4
4	13	<1	39	3	115	14	75	30
5	8	<1	13	1	60	6	57	23	157	53
1–5	493	6	60	4	26	3	7	3
2–5	17	<1	28	2	9	1	0	0
3–5	10	<1	16	1	21	2	6	2
4–5	2	<1	1	<1	4	<1	3	1
Dead	1015	12	277	18	203	24	78	31	127	43
Not Seen	299	4	53	4	27	3	8	3	10	3

AMD, age-related macular degeneration; N, number of subject follow-up intervals.

The plurality of subjects maintained the same AMD state at the next visit, but the absolute proportions declined with increasing severity (71%, 44%, 35%, 30%, and 53% for Levels 1–5, respectively). Progression was more common than regression (17% vs. 10% for Level 2; 21% vs. 9% for Level 3; 23% vs. 6% for Level 4). Mortality increased with severity (12%, 18%, 24%, 31%, and 43% for Levels 1–5, respectively). Few subjects were not seen at the next scheduled visit, and the proportion not seen did not vary with AMD severity. Similar relationships were seen for subjects with partial information (data not shown).

Covariate effects on transition intensities

Covariate effects from the MSMs are presented in Table 4. There was no evidence of non-linear age effects (p=0.54 for quadratic terms) or interactions (age and sex: p=0.54; age and CFH Y402H genotype: p=0.84; sex and CFH Y402H genotype: p=0.87).

Table 4.

Estimated Covariate Effects on Transition Intensities or Hazards.

Covariate	Incidence or Progression				Regression			Mortality
	Level 1 to Level 2	Level 2 to Level 3	Level 3 to Level 4	Level 4 to Level 5	Level 2 to Level 1	Level 3 to Level 2	Level 4 to Level 3	Any AMD to Death
	HR (95% CI)	HR (95% CI)	HR (95% CI)	HR (95% CI)	HR (95% CI)	HR (95% CI)	HR (95% CI)	HR (95% CI)
Age (per 5 years)	1.43 (1.37–1.49)	1.31 (1.24–1.39)	1.24 (1.14–1.34)	1.27 (1.14–1.41)	1.04 (0.95–1.13)	0.91 (0.81–1.03)	0.91 (0.68–1.21)	1.66 (1.61–1.70)
Sex (male)	0.96 (0.82–1.13)	0.88 (0.72–1.08)	0.69 (0.52–0.91)	1.17 (0.82–1.68)	0.83 (0.60–1.14)	1.30 (0.83–2.04)	0.46 (0.14–1.50)	1.55 (1.42–1.69)
CFH Y402H genotype
TT	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
CT	1.35 (1.13–1.60)	1.09 (0.87–1.36)	1.44 (1.04–1.98)	1.60 (1.03–2.48)	0.87 (0.62–1.22)	0.49 (0.23–1.04)	0.49 (0.16–1.48)	0.94 (0.85–1.01)
CC	1.98 (1.57–2.49)	1.73 (1.29–2.33)	1.30 (0.86–1.94)	1.72 (1.01–2.91)	0.92 (0.54–1.56)	0.60 (0.37–0.97)	0.41 (0.09–1.83)	0.98 (0.86–1.12)
AMD severity
Level 1								1.00
Level 2								1.04 (0.82–1.32)
Level 3								1.01 (0.79–1.30)
Level 4								1.05 (0.71–1.55)
Level 5								1.37 (1.15–1.62)

AMD, age-related macular degeneration; CI, confidence interval; HR, hazard ratio.

Level 1 = no AMD, Level 2 = minimally severe early AMD, Level 3 = moderately severe early AMD, Level 4 = severe early AMD, Level 5 = late AMD.

Older age was associated with increased AMD incidence and progression and mortality, but not with AMD regression (Table 4). Being male was associated with increased mortality, but not with AMD incidence, progression or regression. CFH Y402H genotypes CC and CT were associated, relative to genotype TT, with increased AMD incidence and progression, but not with regression or mortality. Late AMD was associated with increased mortality relative to no AMD, but earlier stages of AMD were not.

Five-year transition probabilities by age, sex, and CFH Y402H genotype

Five-year transition probabilities by age and CFH Y402H genotype are displayed for women (Figure 2A) and for men (Figure 2B). One can assess relative transitions between states by CFH 402H genotype for initial AMD state and age (comparisons within a single panel) and by age (comparisons between panels within a single column).

Figure 2.

Five year transition probabilities to age-related macular degeneration (AMD) states and death for the specified initial AMD state, age, and complement factor H (CFH) Y402H genotype for women (A) and for men (B).

Effects of the CFH Y402H risk genotype CC on progression on AMD are clear. Relative to individuals with genotype TT, for individuals free of AMD at age 50, the rate of progression to Level 2 at age 55 for those with genotype CC was higher (women: 4.2% vs. 2.2%; men: 4.1% vs. 2.1%). A similar effect of genotype was seen for those free of AMD at age 70 who progressed to Level 2 at age 75 (women: 11.3% vs. 7.1%; men: 11.0% vs. 6.7%). At age 90 years, progression to Level 2 at age 95 was similar regardless of genotype (women: 8.4% vs. 8.2%; men: 6.1% vs. 5.7%), but rates of progression to Level 3, Level 4 and late AMD were higher for those with genotype CC (women: 12.1% vs. 5.9%; 4.2% vs. 1.6%, 2.0% vs. 0.4%, respectively; men: 8.3% vs. 3.6%, 1.8% vs. 0.6%, 1.0% vs. 0.2%, respectively).

For individuals with AMD at Level 2 at age 50, regression to no AMD by age 55 was common (11.7–16.0%) but did not vary with CFH genotype; progression to Level 3 and Level 4 was more common for CC individuals than TT individuals (women: 18.9% vs. 10.6%, 1.9% vs. 0.8%, respectively; men: 17.0% vs. 9.1%, 1.2% vs. 0.5%, respectively). At age 70, regression is less frequent and progression more frequent; CC individuals remain at greater risk of progression. At age 90, regression is infrequent while progression declines due to the competing risk of death; CC individuals remain at greater risk of progression. Similar observations can be made for individuals with Level 3 and Level 4 (Figure 2).

Effects of age on the 5-year transition probabilities are clear. Mortality increases substantially with age. For example, in TT women with no AMD, 5-year mortality rates increase from 0.8% at age 45 to 72.9% at age 95. AMD progression generally increases with age until at least age 80; regression declines with increasing age. For example, in TT women, 5-year rates of progression from Level 3 to Level 4 and late AMD increase from 9.9% and 1.0% at age 55 to peaks of 18.8% and 7.7% at ages 80 and 90 and then declines to 8.2% and 5.9%, respectively, at age 95 while 5-year rates of regression from Level 3 to Level 2 or no AMD decline from 26.5% and 3.2% to 1.2% and 0.2%.

Cumulative incidence of AMD for individuals with no AMD at age 45

Figure 3 shows cumulative incidence of Level 2 or higher, Level 3 or higher, Level 4 or higher and late AMD through age 100 based on annual assessments of AMD state for persons free of AMD at age 45. The upper left panel presents, for TT women, the cumulative incidence of late AMD as the black curve (increasing to 7.8%), the cumulative incidence of Level 4 or higher as the dark gray curve (increasing to 16.5%), the cumulative incidence of Level 3 or higher as the medium gray curve (increasing to 31.1%), and the cumulative incidence of Level 2 or higher as the light gray curve (increasing to 48.3%).

Figure 3.

Cumulative incidence of minimally severe early age related macular degeneration (AMD) (Level 2) or higher (top curve), moderately severe early AMD (Level 3) or higher, severe early AMD (Level 4) or higher, and late AMD (Level 5; bottom curve) through age 100 years for individuals with no AMD (Level 1) at age 45 years by sex and complement factor H (CFH) Y402H genotype (TT, CT, CC). Estimates are based on annual assessments of AMD status.

Cumulative incidence of Level 2 or higher was greater for women and for CC individuals than for men and for TT individuals. For example, cumulative incidence of Level 2 by age 65 was 23.0% (95% CI 19.0–27.6%) for CC women versus 12.4% (10.7–14.6%) for TT women, and 21.8% (18.0–27.2%) for CC men versus 11.7% (9.5–14.4%) for TT men; by age 80, it was 61.2% (54.5–67.1%) versus 39.6% (36.1–43.8%) for women and 54.1% (48.2–61.0%) versus 34.1% (29.5–39.6%) for men; by age 100, it was 67.7% (61.2–72.9%) versus 48.3% (44.4–53.1%) for women and 57.8% (52.1–64.4%) versus 38.6% (33.2–44.1%) for men. Cumulative incidence of late AMD was similarly greater for women and for CC individuals than for men and for TT individuals at all ages.

COMMENT

In a cohort followed over 20 years, we show that, after adjusting for age and sex, the Y402H variant in the CFH gene is associated with incidence of early AMD and progression along the AMD severity scale, up to and including progression to late AMD. We also identify an association between late AMD and mortality. Although other studies have shown associations between incidence of late AMD (and sometimes early AMD) and the CFH Y402H risk allele, our models demonstrate that the effect is monotonic and detectable at all stages of the disease. This provides further evidence of the effects of this risk allele across the continuum of AMD.

The MSM used here is advantageous for modeling staged disease progression in studies like the BDES. The model incorporates all facets of the natural history of AMD as well as death into a single, biologically plausible model rather than modeling aspects of the disease process in isolation. It accommodates the different observation times (including longer times resulting from missing examinations) that are independent of the subjects’ current state. We use a biologically meaningful time scale (subject age) rather than an artificial time scale (time of study). The model also incorporates time-varying covariates by updating covariate values at observation times. Disadvantages of the MSM are the large computation burden involved in model fitting and the sparseness of information regarding some transitions.

Our findings show the profound effect of the competing risk of death on incidence and progression of AMD in persons aged 85 years or older (Figure 2). In our study, late AMD was associated with a 37% increase in overall mortality while earlier stages of AMD and CFH variant were not. This is consistent with some earlier studies that examined associations of AMD and survival.^45–49 Our findings are consistent with the Age-Related Eye Disease Study that showed increased mortality risk in persons with advanced-stage macular degeneration⁴⁶ and with the Copenhagen Eye Study⁴⁷ which showed increased mortality risk in women with late AMD. However, AMD was not associated with mortality in the Rotterdam Eye Study,⁴⁸ the Blue Mountains Eye Study,⁴⁹ or the Beijing Eye Study.⁵⁰ The relationships of AMD and other ocular conditions to mortality adjusting for smoking, hypertension, and other potential confounders will be examined in future work. Better understanding of the role of AMD as an indicator of increased mortality may be especially important for persons with exudative AMD undergoing treatment with intravitreal anti-VEGF agents.⁵¹

Ten percent of eyes at Level 2, 9% of eyes at Level 3 and 7% of eyes at Level 4 regressed between visits (Table 3). Soft drusen and pigmentary abnormalities may regress and disappear, accounting for these findings.^15,16,18,26 Our findings suggest that regression from Levels 3 or 4 to Levels 2 and 3, respectively, are diminished by 40% and 59% in the presence of the high-risk CFH Y402H CC genotype, although only the former was statistically significant. While masked side-by-side comparisons of photographs minimized the effect of media opacity, photographic artifacts, and grader error, it is possible that these factors may explain some apparent regression. Factors leading to disappearance or regression of these lesions remain to be studied.

In conclusion, we demonstrated the advantages of MSMs in assessing the relationships of CFH Y402H, age, and sex with incidence, progression and regression of AMD. This modeling approach should similarly provide greater insight regarding roles of other candidate genes as well as environmental and host factors in AMD.