Background
Age-related macular degeneration (AMD) is the leading cause of severe vision loss in aging societies [
1‐
3]. An early sign of AMD is the appearance of the so-called drusen, which are yellowish extracellular deposits of protein and lipid material within and beneath the retinal pigment epithelium (RPE). Late-stage AMD can manifest essentially as two distinct forms—geographic atrophy (GA) and neovascular (NV) AMD, with both late-stage forms in a proportion of cases presenting in the same or in different eyes of an individual. GA occurs in up to 50 % of cases and is clinically defined as a distinct area of RPE cell atrophy with slow progression over the years. NV AMD describes the growth of blood vessels beneath and within the retina and is mostly characterized by hemorrhagic detachment of the RPE or the retina and eventually widespread RPE atrophy. Progression to visual loss can be rapid in NV AMD [
4].
Over the past several years, genome-wide association studies and large scale re-sequencing projects have identified a number of single nucleotide variants (SNVs) enriched in complement and complement-related genes that confer a strong risk for AMD [
3,
5‐
8]. Recently, a genome-wide association study conducted by the International AMD Genomics consortium (IAMDGC) identified 52 independent genetic signals at 34 loci to be associated with AMD risk [
9], explaining around half of the genomic heritability of the disease. Six out of those 34 loci harbor one or more complement or complement-related genes.
Assessment of structural variation, particularly in duplicated regions of the genome, and its contribution to disease still remains a challenge for most case-control studies [
10,
11]. Multiallelic loci are especially problematic, due to a lack of suitable tagging variants and a dearth of probes on commercial microarrays in duplication rich regions of the genome [
12]. We and others have previously demonstrated that multiallelic loci can be genotyped economically and accurately using PCR-based quantitative techniques [
13‐
15]. Here, we applied multiplex ligation-dependent probe amplification (MLPA) [
16] to examine the role of the multiallelic complement component 4 (
C4) copy number variations (CNVs) as part of the classical complement pathway in AMD etiology.
In this study, we successfully genotyped CNVs for C4 and its relevant isoforms (C4A, C4B) in 2645 individuals from three large AMD cohorts from Australia and Germany. We identify strong statistical significance for a protective association of C4A copy number and AMD. Stratification of individuals based on age and gender revealed that the protective effect increases with increasing age and is stronger in females. In addition, this association appears to be independent of other, strongly associated AMD variants at the nearby CFB/C2 locus. These data implicate multiallelic CNV of the C4 locus in AMD susceptibility, providing further evidence for the crucial role of complement dysregulation in AMD etiology.
Discussion
Here, we performed a genetic association study of complement
C4CNVs in 1536 AMD cases and 1109 unaffected controls from three independent studies [
32]. This identified a strong protective association with an increase in copy numbers of the
C4A isoform.
C4 is known to play an important role in the activation of the classical and lectin pathways of the complement system [
33,
34]. Although
C4A and
C4B share >99 % sequence identity,
C4A has been suggested to have a primary role in immune complex clearance [
26], greatly supported by the strong association between systemic lupus erythematosus (SLE) and low copy numbers of
C4A [
27,
28]. The involvement of immune complexes have also been reported to contribute to drusen formation [
35], the earliest observable phenotypic changes in AMD etiology. Genomic copy numbers and serum C4A concentration are directly correlated [
24,
36], and increasing plasma concentrations of C4A have been proposed to increase the clearance of immune complexes [
37]. It can be speculated that accumulation of immune complexes are problematic in older individuals with reduced C4A, and as such could partially explain the observed increasing age-dependent association we report in this study.
The
C4 gene is located on chromosome 6p21 in the MHC class III region around 30 kb proximal to the
CFB/
C2 locus known to be strongly associated with AMD [
5,
38]. In SLE, physical proximity between
C4 CNVs and variations at the
CFB/
C2 locus is thought to partially explain the observed association signals [
27]. In contrast, in AMD, significant association of genes flanking the
C4 locus were suspected independently of
CFB/C2 variants [
39,
40]. To further address the latter issue, we investigated the association of
C4A with AMD risk after conditioning for four genetic variants known to carry the main signals at the
CFB/C2 locus [
9]. As a result, after conditioning, effect sizes and association strength of
C4A copy numbers are reduced and no longer statistically significant. The loss of association is mainly driven by rs429608, as C4A copy number is significantly associated with AMD in a model conditioned on the remaining
CFB/C2 variants, namely rs114190211, rs204993, and rs142511358. However, the association of
C4A copy number with AMD in females and in individuals beyond 78 years of age remains statistically significant. Importantly, increased
C4A copy number confers similar risk reduction in females of the age group >78 years in the
C2/CFB conditioned versus the unconditioned model. This independence of association was observed across the three studies included in the analysis.
By analyzing AMD-associated haplotypes, we find
C4A copy number to be correlated to two AMD-associated haplotypes in the
CFB/C2 region (haplotypes H2 and H3). We show that the association of
C4A copy number is independent of the adverse haplotype H2 but not of the protective haplotype H3, the latter tagged by rs429608. Importantly, we observed a strong reduction in the protective effect of H3 in older individuals, in line with previous results [
30,
31]. These findings led us to hypothesize that upon reduced protective impact of the H3 haplotype on older individuals, C4A CNVs significantly influence disease risk in this age group. In contrast, in younger individuals, the protective effect of the H3 haplotype would be stronger and thus would account for most of the protection conferred by the C2/CFB locus in those individuals. With this in mind, our data support the notion that older individuals reveal the CNV at
C4A to be associated with AMD risk independently of known risk variations at the
C2/CFB locus.
So far, AMD pathogenesis has primarily been linked to dysregulation of the alternative pathway of the complement system [
9]; however, expression analysis in cells of the RPE-choroid complex has also identified components and regulatory molecules associated with the classical pathway [
41] and implicated classical complement activation in various retinal degenerations [
42]. Moreover, the presence of immunoglobulin G (IgG) and terminal C5b-9 complement complexes as a component of drusen deposits are indicative of classical pathway activation [
35,
43]. These data, together with our findings in this study, suggest the classical pathway, in addition to the alternative pathway, to play a causal role in pathological events leading to AMD disease.
The mechanisms underlying gender differences in AMD risk are still unknown. We recently reported that genetic variants in the death-associated protein-like 1 (
DAPL1) gene are associated with increased risk for AMD, with risk association significantly higher in females than in males [
17]. In the current study, we have identified another gender-specific locus at
C4A, in this instance with a strong protective effect in females. Taken together, these data provide a reasonable basis for further investigations into the gender bias observed in AMD prevalence.
We considered age to possibly be a confounding factor in this study as previous reports have linked copy number of
C4B and total
C4 with longevity [
44,
45]. However, in this study, we found no correlation between total
C4,
C4A, or
C4B copy number and age when including all individuals in the calculations (
P > 0.05). Additionally, our AMD association analyses were adjusted for age, which should satisfactorily account for confounding effects of longevity.
Conclusions
In conclusion, our results demonstrate that multiallelic CNVs provide another source of genetic variance that need to be considered in complex diseases such as AMD. To date, an 84-kb deletion encompassing the
CFHR3/
CFHR1 genes [
46] and a small complex insertion/deletion polymorphism in the
ARMS2 gene [
47] represent the only CNVs to be reproducibly associated with AMD disease. It is noteworthy that the
CFHR3/
CFHR1 CNV is also associated with SLE, suggesting that these two etiologies may share overlapping disease pathways. While our significance level does not reach current standards of genome-wide significance (~5.00 × 10
−8) for new associated loci, it is questionable whether this threshold is overly conservative for complex CNVs such as
C4, especially since we only investigated three mutually correlated CNVs. In addition, other replicated multiallelic CNV associations did not reach this threshold [
48]. Our finding that increased copy number of
C4A is associated with protection in AMD may have direct implications for therapy, as targeted approaches to molecular constituents of the complement pathway have the potential for early intervention before vision is compromised. This is especially true, in very contrast to most associated single nucleotide variants, as we can directly implicate the relevant gene (
C4A) and the orientation of the protective effect with increased copy numbers and thus with increased C4A protein levels in serum [
49].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
FG, SC, BHFW, and PNB designed the study. FG, SC, ASSK, SJW, and AJR performed the experiments. AWH, BJV, DS, and RHG analyzed the clinical data. FG, SC, BHFW, and PNB wrote the manuscript with input from all authors. All authors read and approved the final manuscript.