Biochemical analysis of a common human polymorphism associated with age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in developed countries. A large number of human genetic studies have associated a common variant (Y402H) of complement factor H (CFH) with a highly significant increase in AMD risk. CFH is a modular protein with 20 homologous short consensus repeats (SCRs). The Y402H variant is located in SCR7 of both CFH and factor H-like protein 1 (FHL-1), a splice variant of CFH (containing SCR1-7) with unique biochemical properties. Because SCR7 is known to bind to heparin, C-reactive protein (CRP), and M protein from Streptococcus pyogenes, it has been hypothesized that the AMD-associated polymorphism may affect interactions with these CFH ligands. In this study, we tested this hypothesis in the context of full-length CFH (SCR1-20) and FHL-1. We systematically analyzed the interactions of the Y402 and H402 variants of CFH and FHL-1 with heparin, CRP, and several bacterial ligands: M6 protein of Streptococcus pyogenes, PspC of Streptococcus pneumoniea, and BbCRASP-1 of Borrelia burgdorferi. In comparing the Y and H variants of CFH and FHL-1, we found no significant difference in their protein secretion, cofactor activity, or interactions with heparin, BbCRASP-1, or PspC, but a significant difference in binding to CRP and M6 protein. This study reveals the fundamental properties of a common polymorphism of CFH and lays the groundwork for elucidating the role of CFH in AMD pathogenesis.     

Complement factor H allotype 402H is associated with increased C3b opsonization and phagocytosis of Streptococcus pyogenes

The main virulence factor of group A streptococcus (GAS), M protein, binds plasma complement regulators factor H (FH) and FH-like protein 1 (FHL-1) leading to decreased opsonization. The M protein binding site on FH is within domain 7 in which also the age-related macular degeneration (AMD)-associated polymorphism Y402H is located. We studied if FH allotypes 402H and 402Y have different binding affinities to GAS. Plasma-derived FH allotype 402H and its recombinant fragment FH5-7(402H) showed decreased binding to several GAS strains. Growth of GAS in human blood taken from FH(402H) homozygous individuals was decreased when compared with blood taken from FH(402Y) homozygous individuals. The effect of the allotype 402H can be explained by combining the previous M protein mutagenesis data and the recently published crystal structure of FH6-8. In conclusion the data indicate that the AMD-associated allotype 402H leads to diminished binding of FH to GAS and increased opsonophagocytosis of the bacteria in blood. These results suggest that the homozygous presence of the allele 402H could be associated with decreased risk for severe GAS infections offering an explanation for the high frequency of the allele despite its association with visual impairment.     

Y402H polymorphism of complement factor H affects binding affinity to C-reactive protein

Complement factor H (FH) is an important regulator of the alternative complement pathway. The Y402H polymorphism within the seventh short consensus repeat of FH was recently shown to be associated with age-related macular degeneration, the most common cause of irreversible blindness in the Western world. We examined the effects of this polymorphism on various FH functions. FH purified from sera of age-related macular degeneration patients homozygous for the FH(402H) variant showed a significantly reduced binding to C-reactive protein (CRP), an acute phase protein, as compared with FH derived from unaffected controls homozygous for the FH(402Y) variant. Strongly reduced binding to CRP was also observed with a recombinant fragment of FH (short consensus repeat 5-7) containing the same amino acid change. Because the interaction of CRP and FH promotes complement-mediated clearance of cellular debris in a noninflammatory fashion, we propose that the reduced binding of FH(402H) to CRP could lead to an impaired targeting of FH to cellular debris and a reduction in debris clearance and enhanced inflammation along the macular retinal pigmented epithelium-choroid interface in individuals with age-related macular degeneration.     

Complement factor H binds to denatured rather than to native pentameric C-reactive protein

Binding of the complement regulatory protein, factor H, to C-reactive protein has been reported and implicated as the biological basis for association of the H402 polymorphic variant of factor H with macular degeneration. Published studies utilize solid-phase or fluid-phase binding assays to show that the factor H Y402 variant binds C-reactive protein more strongly than H402. Diminished binding of H402 variant to C-reactive protein in retinal drusen is posited to permit increased complement activation, driving inflammation and pathology. We used well validated native human C-reactive protein and pure factor H Y402H variants to test interactions. When factor H variants were incubated with C-reactive protein in the fluid phase at physiological concentrations, no association occurred. When C-reactive protein was immobilized on plastic, either non-specifically by adsorption in the presence of Ca(2+) to maintain its native fold and pentameric subunit assembly or by specific Ca(2+)-dependent binding to immobilized natural ligands, no specific binding of either factor H variant from the fluid phase was observed. In contrast, both factor H variants reproducibly bound to C-reactive protein immobilized in the absence of Ca(2+), conditions that destabilize the native fold and pentameric assembly. Both factor H variants strongly bound C-reactive protein that was denatured by heat treatment before immobilization, confirming interaction with denatured but not native C-reactive protein. We conclude that the reported binding of factor H to C-reactive protein results from denaturation of the C-reactive protein during immobilization. Differential binding to C-reactive protein, thus, does not explain association of the Y402H polymorphism with macular degeneration.     

Molecular Mechanisms of Macular Degeneration Associated with the Complement Factor H Y402H Mutation

A single nucleotide polymorphism, tyrosine at position 402 to histidine (Y402H), within the gene encoding complement factor H (FH) predisposes individuals to acquiring age-related macular degeneration (AMD) after aging. This polymorphism occurs in short consensus repeat (SCR) 7 of FH and results in decreased binding affinity of SCR6-8 for heparin. As FH is responsible for regulating the complement system, decreased affinity for heparin results in decreased regulation on surfaces of self. To understand the involvement of the Y402H polymorphism in AMD, we leverage methods from bioinformatics and computational biophysics to quantify structural and dynamical differences between SCR7 isoforms that contribute to decreased pattern recognition in SCR7^H402. Our data from molecular and Brownian dynamics simulations suggest a revised mechanism for decreased heparin binding. In this model, transient contacts not observed in structures for SCR7 are predicted to occur in molecular dynamics simulations between coevolved residues Y402 and I412, stabilizing SCR7^Y402 in a conformation that promotes association with heparin. H402 in the risk isoform is less likely to form a contact with I412 and samples a larger conformational space than Y402. We observe energy minima for sidechains of Y402 and R404 from SCR7^Y402 that are predicted to associate with heparin at a rate constant faster than energy minima for sidechains of H402 and R404 from SCR7^H402. As both carbohydrate density and degree of sulfation decrease with age in Bruch’s membrane of the macula, the decreased heparin recognition of SCR7^H402 may contribute to the pathogenesis of AMD.     

Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of vision loss. It is associated with development of characteristic plaque-like deposits (soft drusen) in Bruch’s membrane basal to the retinal pigment epithelium (RPE). A sequence variant (Y402H) in short consensus repeat domain 7 (SCR7) of complement factor H (CFH) is associated with risk for “dry” AMD. We asked whether the eye-targeting of this disease might be related to specific interactions of CFH SCR7 with proteins expressed in the aging human RPE/choroid that could contribute to protein deposition in drusen. Yeast 2-hybrid (Y2H) screens of a retinal pigment epithelium/choroid library derived from aged donors using CFH SCR7 baits detected an interaction with EFEMP1/Fibulin 3 (Fib3), which is the locus for an inherited macular degeneration and also accumulates basal to macular RPE in AMD. The CFH/Fib3 interaction was validated by co-immunoprecipitation of native proteins. Quantitative Y2H and ELISA assays with different recombinant protein constructs both demonstrated higher affinity for Fib3 for the disease-related CFH 402H variant. Immuno-labeling revealed colocalization of CFH and Fib3 in globular deposits within cholesterol-rich domains in soft drusen in two AMD donors homozygous for CFH 402H (H/H). This pattern of labeling was quite distinct from those seen in examples of eyes with Y/Y and H/Y genotypes. The CFH 402H/Fib3 interaction could contribute to the development of pathological aggregates in soft drusen in some patients and as such might provide a target for therapeutic intervention in some forms of AMD.     

Association between CFH Y402H Polymorphism and Age Related Macular Degeneration in North Indian Cohort

The purpose of the study was to determine serum complement factor H (CFH) levels in patients of age related macular degeneration (AMD) and examine its association with CFH Y402H polymorphism. 115 AMD patients and 61 normal controls were recruited in this study. The single nucleotide polymorphism was assayed by real time PCR and serum CFH levels were measured by ELISA and standardized to total serum protein. Chi-square test was applied to polymorphism analysis while Mann Whitney U-statistic for CFH-levels. Mendelian randomization approach was used for determining causal relationship. The genotype frequency differed between the AMD patients (TT- 18.3%, TC-41.3% and CC-40.4%) and controls (TT-76.3%, TC-13.6%, and CC-10.1%) (p = 0001). The frequency of alleles was also significantly different when AMD (T-39% and C-61%) was compared to controls (T-83% and C-17%) (p = 0.0001). Level of serum CFH was significantly lower in AMD patients as compared to normal controls (p = 0.001). Our data showed that the CFH Y402H polymorphism is a risk factor for AMD in the North Indian population. Mendelian randomization approach revealed that CFH Y402H polymorphism affects AMD risk through the modification of CFH serum levels.     

Zinc binding to the Tyr402 and His402 allotypes of complement factor H: possible implications for age-related macular degeneration

The Tyr402His polymorphism of complement factor H (FH) with 20 short complement regulator (SCR) domains is associated with age-related macular degeneration (AMD). How FH contributes to disease pathology is not clear. Both FH and high concentrations of zinc are found in drusen deposits, the key feature of AMD. Heterozygous FH is inhibited by zinc, which causes FH to aggregate. Here, zinc binding to homozygous FH was studied. By analytical ultracentrifugation, large amounts of oligomers were observed with both the native Tyr402 and the AMD-risk His402 homozygous allotypes of FH and both the recombinant SCR-6/8 allotypes with Tyr/His402. X-ray scattering also showed that both FH and SCR-6/8 allotypes strongly aggregated at > 10 μM zinc. The SCR-1/5 and SCR-16/20 fragments were less likely to bind zinc. These observations were supported by bioinformatics predictions. Starting from known zinc binding sites in crystal structures, we predicted 202 putative partial surface zinc binding sites in FH, most of which were in SCR-6. Metal site prediction web servers also suggested that SCR-6 and other domains bind zinc. Predicted SCR-6/8 dimer structures showed that zinc binding sites could be formed at the protein-protein interface that would lead to daisy-chained oligomers. It was concluded that zinc binds weakly to FH at multiple surface locations, most probably within the functionally important SCR-6/8 domains, and this explains why zinc inhibits FH activity. Given the high pathophysiological levels of bioavailable zinc present in subretinal deposits, we discuss how zinc binding to FH may contribute to deposit formation and inflammation associated with AMD.     

The Scl1 protein of M6-type group A Streptococcus binds the human complement regulatory protein, factor H, and inhibits the alternative pathway of complement

Non-specific activation of the complement system is regulated by the plasma glycoprotein factor H (FH). Bacteria can avoid complement-mediated opsonization and phagocytosis through acquiring FH to the cell surface. Here, we characterize an interaction between the streptococcal collagen-like protein Scl1.6 of M6-type group A Streptococcus (GAS) and FH. Using affinity chromatography with immobilized recombinant Scl1.6 protein, we co-eluted human plasma proteins with molecular weight of 155 kDa, 43 kDa and 38 kDa. Mass spectrometry identified the 155 kDa band as FH and two other bands as isoforms of the FH-related protein-1. The identities of all three bands were confirmed by Western immunoblotting with specific antibodies. Structure-function relation studies determined that the globular domain of the Scl1.6 variant specifically binds FH while fused to collagenous tails of various lengths. This binding is not restricted to Scl1.6 as the phylogenetically linked Scl1.55 variant also binds FH. Functional analyses demonstrated the cofactor activity of the rScl1.6-bound FH for factor I-mediated cleavage of C3b. Finally, purified FH bound to the Scl1.6 protein present in the cell wall material obtained from M6-type GAS. In conclusion, we have identified a functional interaction between Scl1 and plasma FH, which may contribute to GAS evasion of complement-mediated opsonization and phagocytosis.     

Production of biologically active complement factor H in therapeutically useful quantities

Human complement factor H (FH), an abundant 155-kDa plasma glycoprotein with 40 disulphide bonds, regulates the alternative-pathway complement cascade. Mutations and single nucleotide polymorphisms in the FH gene predispose to development of age-related macular degeneration, atypical haemolytic uraemic syndrome and dense deposit disease. Supplementation with FH variants protective against disease is an enticing therapeutic prospect. Current sources of therapeutic FH are restricted to human blood plasma highlighting a need for recombinant material. Previously FH expression in cultured plant, mammalian or insect cells yielded protein amounts inadequate for full characterisation, and orders of magnitude below therapeutic usefulness. Here, the V62,Y402 variant of FH has been produced recombinantly (rFH) in Pichia pastoris cells. Codon-optimisation proved essential whilst exploitation of the yeast mating α-factor peptide ensured secretion. We thereby produced multiple 10s-of-milligram of rFH. Following endoglycosidase H digestion of N-linked glycans, rFH (with eight residual N-acetylglucosamine moieties) was purified on heparin-affinity resin and anion-exchange chromatography. Full-length rFH was verified by mass spectrometry and Western blot using monoclonal antibodies to the C-terminus. Recombinant FH is a single non-aggregated species (by dynamic light scattering) and fully functional in biochemical and biological assays. An additional version of rFH was produced in which eight N-glycosylation sequons were ablated by Asn-Gln substitutions resulting in a glycan-devoid product. Successful production of rFH in this potentially very highly expressing system makes production of therapeutically useful quantities economically viable. Furthermore, ease of genetic manipulation in P. pastoris would allow production of engineered FH versions with enhanced pharmacokinetic and pharmacodynamic properties.     

Basal laminar drusen caused by compound heterozygous variants in the CFH gene

Age-related macular degeneration (AMD) is a multifactorial disease that is strongly associated with the Tyr402His variant in the complement factor H (CFH) gene. Drusen are hallmark lesions of AMD and consist of focal-inflammatory and/or immune-mediated depositions of extracellular material at the interface of the retinal pigment epithelium (RPE) and the Bruch membrane. We evaluated the role of CFH in 30 probands with early-onset drusen and identified heterozygous nonsense, missense, and splice variants in five families. The affected individuals all carried the Tyr402His AMD risk variant on the other allele. This supports an autosomal-recessive disease model in which individuals who carry a CFH mutation on one allele and the Tyr402His variant on the other allele develop drusen. Our findings strongly suggest that monogenic inheritance of CFH variants can result in basal laminar drusen in young adults, and this can progress to maculopathy and severe vision loss later in life.     

Secreted proteome profiling in human RPE cell cultures derived from donors with age related macular degeneration and age matched healthy donors

Age-related macular degeneration (AMD) is characterized by progressive loss of central vision, which is attributed to abnormal accumulation of macular deposits called "drusen" at the interface between the basal surface of the retinal pigment epithelium (RPE) and Bruch's membrane. In the most severe cases, drusen deposits are accompanied by the growth of new blood vessels that breach the RPE layer and invade photoreceptors. In this study, we hypothesized that RPE secreted proteins are responsible for drusen formation and choroidal neovascularization. We used stable isotope labeling by amino acids in cell culture (SILAC) in combination with LC-MS/MS analysis and ZoomQuant quantification to assess differential protein secretion by RPE cell cultures prepared from human autopsy eyes of AMD donors (diagnosed by histological examinations of the macula and genotyped for the Y402H-complement factor H variant) and age-matched healthy control donors. In general, RPE cells were found to secrete a variety of extracellular matrix proteins, complement factors, and protease inhibitors that have been reported to be major constituents of drusen (hallmark deposits in AMD). Interestingly, RPE cells from AMD donors secreted 2 to 3-fold more galectin 3 binding protein, fibronectin, clusterin, matrix metalloproteinase-2 and pigment epithelium derived factor than RPE cells from age-matched healthy donors. Conversely, secreted protein acidic and rich in cysteine (SPARC) was found to be down regulated by 2-fold in AMD RPE cells versus healthy RPE cells. Ingenuity pathway analysis grouped these differentially secreted proteins into two groups; those involved in tissue development and angiogenesis and those involved in complement regulation and protein aggregation such as clusterin. Overall, these data strongly suggest that RPE cells are involved in the biogenesis of drusen and the pathology of AMD.     

Complement Factor H Binds at Two Independent Sites to C-reactive Protein in Acute Phase Concentrations

Factor H (FH) regulates the activation of C3b in the alternative complement pathway, both in serum and at host cell surfaces. It is composed of 20 short complement regulator (SCR) domains. The Y402H polymorphism in FH is a risk factor for age-related macular degeneration. C-reactive protein (CRP) is an acute phase protein that binds Ca²⁺. We established the FH-CRP interaction using improved analytical ultracentrifugation (AUC), surface plasmon resonance (SPR), and synchrotron x-ray scattering methods. Physiological FH and CRP concentrations were used in 137 mm NaCl and 2 mm Ca²⁺, in which the occurrence of denatured CRP was avoided. In solution, AUC revealed FH-CRP binding. The FH-CRP interaction inhibited the formation of higher FH oligomers, indicating that CRP blocked FH dimerization sites at both SCR-6/8 and SCR-16/20. SPR confirmed the FH-CRP interaction and its NaCl concentration dependence upon using either immobilized FH or CRP. The SCR-1/5 fragment of FH did not bind to CRP. In order of increasing affinity, SCR-16/20, SCR-6/8 (His-402), and SCR-6/8 (Tyr-402) fragments bound to CRP. X-ray scattering showed that FH became more compact when binding to CRP, which is consistent with CRP binding at two different FH sites. We concluded that FH and CRP bind at elevated acute phase concentrations of CRP in physiological buffer. The SCR-16/20 site is novel and indicates the importance of the FH-CRP interaction for both age-related macular degeneration and atypical hemolytic uremic syndrome.     

Group A streptococcal phagocytosis resistance is independent of complement factor H and factor H-like protein 1 binding

Factor H (FH) and factor H-like protein 1 (FHL-1) regulate complement activation through the alternative pathway. Several extracellular bacterial pathogens, prime targets for the complement system, bind FH and FHL-1, thereby acquiring a potential mechanism for minimizing complement deposition on their surface. For group A streptococci (GAS), surface-bound antiphagocytic M proteins mediate the interaction. To study the role of the FH-FHL-1 interaction for complement deposition and opsonophagocytosis of GAS, we first constructed a set of truncated M5 protein variants and expressed them on the surface of a homologous M-negative GAS strain. Binding experiments with the resulting strains demonstrated that the major FH-FHL-1 binding is located in a 42-amino-acid region within the N-terminal third of M5. Measurement of bacteria-bound complement factor C3 after incubation in plasma showed that the presence of this region had little impact upon complement deposition through the alternative pathway. Moreover, streptococci expressing M5 proteins lacking the major FH and FHL-1 binding sequence resisted phagocytosis in human blood as efficiently as bacteria expressing the wild-type protein. Consequently, the data suggest that the binding of the regulators of the alternative pathway is of limited importance for GAS phagocytosis resistance.     

A Newly-Identified Polymorphism in Rhesus Macaque Complement Factor H Modulates Binding Affinity for Meningococcal FHbp

Background

Two meningococcal serogroup B vaccines contain Factor H binding protein (FHbp). Binding of Factor H (FH) to FHbp was thought to be specific for human or chimpanzee FH. However, in a previous study an amino acid polymorphism in rhesus macaque FH domain 6, tyrosine at position 352 (Y352) was associated with high binding to FHbp, whereas histidine at position 352 (H352) was associated with low binding.

Methods and Results

Here we report that a second FH polymorphism at position 360 also affects macaque FH binding. Of 43 macaques, 11 had high FH binding and 32 had low binding. As in our previous study, all 11 animals with high binding had Y352, and 24 with low binding had H352. However the remaining eight with low FH binding had Y352, which was predicted to yield high binding. All eight had S360 instead of P360. Thus, three allelic variants at positions 352 and 360 affect macaque FH binding to FHbp: HP (low), YS (low), and YP (high). We measured binding affinity of each FH sequence type to FHbp by surface plasmon resonance. Two animals with high binding types (YS/YP and HP/YP) had dissociation constants (K _D) of 10.4 and 18.2 nM, respectively, which were similar to human FH (19.8 nM). Two macaques with low binding (HP/HP and HP/YS) had K _D values approximately five-fold higher (100.3 and 99.5 nM, respectively). A third macaque with low binding (YS/YS) had a K _D value too high to be measured.

Conclusions

Macaques have at least three allelic variants encoding FH with different affinities for FHbp (five genotypic combinations of these variants). Since in previous studies binding of FH to FHbp vaccines decreased protective antibody responses, our data will aid in selection of macaques with FH binding that is similar to humans for further investigation of FHbp vaccine immunogenicity.     

Complement factor H variant p.Y402H in pseudoxanthoma elasticum patients

Pseudoxanthoma elasticum (PXE) is a hereditary disorder predominantly affecting the eyes, the skin, and the vascular system. The subretinal neovascularization and retinal hemorrhages leading to the loss of central vision in PXE are similar to the process observed in age-related macular degeneration (AMD). The complement factor H (CFH) variant c.1277T > C (p.Y402H) is a recently discovered risk factor for AMD. The aim of this study was to analyze whether this CFH variant is a secondary genetic risk factor for PXE. Therefore, the genotypes of CFH c.1277T > C (p.Y402H) were determined in 189 German PXE patients and 189 age- and sex-matched controls. The allelic frequencies of the investigated variant did not differ between patients and controls. The frequencies were 33%, 56%, and 11% for wild-type, heterozygous, and homozygous genotypes in the PXE patients and 36%, 51%, and 13% in the control cohort, respectively. Further, no significant associations were identified when allele carriers were analyzed or after adjustment for sex, age, smoking, organ involvement, hypertension, or age at disease onset. No significant genotype-phenotype correlation was detected. In conclusion, our data reliably show that the CFH variant c.1277T > C (p.Y402H) is not a genetic risk factor for PXE.     

Factor H Binds to the Hypervariable Region of Many Streptococcus pyogenes M Proteins but Does Not Promote Phagocytosis Resistance or Acute Virulence

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems.     

Age-related macular degeneration and association of CFH Y402H and LOC387715 A69S polymorphisms in a Turkish population

Age-related macular degeneration (AMD) is a disease with multifactorial etiology characterized by irreversible loss of central visual acuity. The discovery of susceptive single-nucleotide polymorphisms (SNPs) has progressed our understanding of AMD. Complement factor H (CFH) gene Y402H polymorphism and high-temperature requirement A-1 (HTRA1) LOC387715 gene A69S polymorphisms are the most important SNPs reported in the literature. Determination of genetic risk factors and genotype-phenotype relationship in AMD may result in rapid and cost-effective therapeutic applications for young and old population. In this study, we hypothesized a potential association between CFH gene Y402H and HTRA1 LOC387715 gene A69S polymorphism in Turkish AMD patients. In blood samples from a total of 252 individuals, 147 clinically diagnosed as AMD and the others control, polymorphic sites in CFH, Y402H (Tsp509I T/C), and HTRA1, LOC387715 A69S (FnuHI G/T), were determined by polymerase chain reaction-restriction fragment length polymorphism analysis. There was significant difference between CFH genotypes in the AMD group, TT 21.8%, TC 48.3%, and CC 29.9%, and in the control subjects, TT 45% (p=0.003), TC 41% (p=0.0001), and CC 14% (p=0.0001). Further, the A69S polymorphism of LOC387715 was investigated and found to be significantly associated with AMD. LOC387715 genotypes in the AMD group were GG 30.6%, GT 38.1%, and TT 31.3% and in the control subjects were GG 59% (p=0.027), GT 39% (p=0.0001), and TT 2% (p=0.0001), respectively. We also found that Y402H C and A69S T allele were associated with AMD. This is the first study showing that Y402H and LOC387715 are associated with AMD in Turkish population.     

CFH gene variant, Y402H, and smoking, body mass index, environmental associations with advanced age-related macular degeneration

OBJECTIVES: We tested the hypothesis that modifiable lifestyle factors alter the genetic susceptibility associated with a common coding variant in the complement factor H (CFH) gene, Y402H, for the leading cause of blindness among the elderly, age-related macular degeneration (AMD). METHODS: In this case-control association analysis, Caucasian participants in the multicenter Age-Related Eye Disease Study with advanced AMD (n = 574 cases) or no AMD (n = 280 controls) were evaluated. AMD status was determined by grading of fundus photographs. Risk factors including cigarette smoking and body mass index (BMI) were assessed and DNA specimens were genotyped for the variant in the CFH gene. Unconditional logistic regression analyses were performed. Attributable risks and multivariable AMD risk scores were calculated. RESULTS: The number of risk alleles for Y402H was associated with advanced AMD, with odds ratios (OR) of 2.7 (95% confidence interval (CI) 1.8-3.8) for the CT heterozygous genotype and OR 7.4 (4.7-11.8) for the homozygous CC risk genotype, after controlling for demographic and behavioral risk factors. Current cigarette smoking (OR 5.1) and high BMI > or =30 (OR 2.1) were independently related to AMD, controlling for genotype. The association between AMD and BMI varied dependent on genotype (P interaction = 0.006 for the CT vs. TT genotype). The CC genotype plus higher BMI (OR 5.9) or smoking (OR 10.2) conferred the greatest risks. Gene plus environment risk scores provided an area under the receiver operating characteristic (ROC) curve of 0.70-0.75. CONCLUSIONS: Genetic and environmental factors are independently related to advanced AMD, and modifiable factors alter genetic susceptibility. The AMD risk score identifies a highly susceptible population.