Joanna E. Merriam

Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration

Age-related macular degeneration (AMD) is the most common form of irreversible blindness in developed countries. Variants in the factor H gene (CFH, also known as HF1), which encodes a major inhibitor of the alternative complement pathway, are associated with the risk for developing AMD. Here we test the hypothesis that variation in genes encoding other regulatory proteins of the same pathway is associated with AMD. We screened factor B (BF) and complement component 2 (C2) genes, located in the major histocompatibility complex class III region, for genetic variation in two independent cohorts comprising ∼900 individuals with AMD and ∼400 matched controls. Haplotype analyses identify a statistically significant common risk haplotype (H1) and two protective haplotypes. The L9H variant of BF and the E318D variant of C2 (H10), as well as a variant in intron 10 of C2 and the R32Q variant of BF (H7), confer a significantly reduced risk of AMD (odds ratio = 0.45 and 0.36, respectively). Combined analysis of the C2 and BF haplotypes and CFH variants shows that variation in the two loci can predict the clinical outcome in 74% of the affected individuals and 56% of the controls. These data expand and refine our understanding of the genetic risk for AMD.

Genome-wide association study of advanced age-related macular degeneration identifies a role of the hepatic lipase gene (LIPC)

Advanced age-related macular degeneration (AMD) is the leading cause of late onset blindness. We present results of a genome-wide association study of 979 advanced AMD cases and 1,709 controls using the Affymetrix 6.0 platform with replication in seven additional cohorts (totaling 5,789 unrelated cases and 4,234 unrelated controls). We also present a comprehensive analysis of copy-number variations and polymorphisms for AMD. Our discovery data implicated the association between AMD and a variant in the hepatic lipase gene (LIPC) in the high-density lipoprotein cholesterol (HDL) pathway (discovery P = 4.53e-05 for rs493258). Our LIPC association was strongest for a functional promoter variant, rs10468017, (P = 1.34e-08), that influences LIPC expression and serum HDL levels with a protective effect of the minor T allele (HDL increasing) for advanced wet and dry AMD. The association we found with LIPC was corroborated by the Michigan/Penn/Mayo genome-wide association study; the locus near the tissue inhibitor of metalloproteinase 3 was corroborated by our replication cohort for rs9621532 with P = 3.71e-09. We observed weaker associations with other HDL loci (ABCA1, P = 9.73e-04; cholesterylester transfer protein, P = 1.41e-03; FADS1-3, P = 2.69e-02). Based on a lack of consistent association between HDL increasing alleles and AMD risk, the LIPC association may not be the result of an effect on HDL levels, but it could represent a pleiotropic effect of the same functional component. Results implicate different biologic pathways than previously reported and provide new avenues for prevention and treatment of AMD.

Extended haplotypes in the complement factor H (CFH) and CFH‐related (CFHR) family of genes protect against age‐related macular degeneration: Characterization, ethnic distribution and evolutionary implications

Background. Variants in the complement factor H gene (CFH) are associated with age‐related macular degeneration (AMD). CFH and five CFH‐related genes (CFHR1‐5) lie within the regulators of complement activation (RCA) locus on chromosome 1q32. Aims and Methods. In this study, the structural and evolutionary relationships between these genes and AMD was refined using a combined genetic, molecular and immunohistochemical approach. Results. We identify and characterize a large, common deletion that encompasses both the CFHR1 and CFHR3 genes. CFHR1, an abundant serum protein, is absent in subjects homozygous for the deletion. Genotyping analyses of AMD cases and controls from two cohorts demonstrates that deletion homozygotes comprise 1.1% of cases and 5.7% of the controls (chi‐square = 32.8; P = 1.6 E‐09). CFHR1 and CFHR3 transcripts are abundant in liver, but undetectable in the ocular retinal pigmented epithelium/choroid complex. AMD‐associated CFH/CFHR1/CFHR3 haplotypes are widespread in human populations. Conclusion. The absence of CFHR1 and/or CFHR3 may account for the protective effects conferred by some CFH haplotypes. Moreover, the high frequencies of the 402H allele and the delCFHR1/CFHR3 alleles in African populations suggest an ancient origin for these alleles. The considerable diversity accumulated at this locus may be due to selection, which is consistent with an important role for the CFHR genes in innate immunity.

Common Variants near FRK/COL10A1 and VEGFA are Associated with Advanced Age-related Macular Degeneration

Despite significant progress in the identification of genetic loci for age-related macular degeneration (AMD), not all of the heritability has been explained. To identify variants which contribute to the remaining genetic susceptibility, we performed the largest meta-analysis of genome-wide association studies to date for advanced AMD. We imputed 6 036 699 single-nucleotide polymorphisms with the 1000 Genomes Project reference genotypes on 2594 cases and 4134 controls with follow-up replication of top signals in 5640 cases and 52 174 controls. We identified two new common susceptibility alleles, rs1999930 on 6q21-q22.3 near FRK/COL10A1 [odds ratio (OR) 0.87; P = 1.1 × 10−8] and rs4711751 on 6p12 near VEGFA (OR 1.15; P = 8.7 × 10−9). In addition to the two novel loci, 10 previously reported loci in ARMS2/HTRA1 (rs10490924), CFH (rs1061170, and rs1410996), CFB (rs641153), C3 (rs2230199), C2 (rs9332739), CFI (rs10033900), LIPC (rs10468017), TIMP3 (rs9621532) and CETP (rs3764261) were confirmed with genome-wide significant signals in this large study. Loci in the recently reported genes ABCA1 and COL8A1 were also detected with suggestive evidence of association with advanced AMD. The novel variants identified in this study suggest that angiogenesis (VEGFA) and extracellular collagen matrix (FRK/COL10A1) pathways contribute to the development of advanced AMD.

Three Major Loci Involved in Age-Related Macular Degeneration Are Also Associated with Polypoidal Choroidal Vasculopathy

PURPOSE To investigate the frequency of variants in 3 major age-related macular degeneration (AMD)-associated loci in patients of European-American descent with polypoidal choroidal vasculopathy (PCV). DESIGN Cross-sectional, case-control association study. PARTICIPANTS Fifty-five patients with PCV, 368 patients with advanced AMD, and 368 age-matched and ethnically matched unaffected controls of European-American descent. METHODS Association analysis of allele and genotype frequencies, determined by TaqMan assays, was performed for the following haplotype-tagging single nucleotide polymorphisms (htSNPs): risk alleles in the complement factor H (CFH) gene (Y402H and IVS14) in the ARMS2/HTRA1 locus on 10q26 (A69S) and protective alleles in CFH (IVS1 and IVS6) and in the complement factor B/complement component C2 (CFB/C2) locus (IVS10 and H9L). MAIN OUTCOME MEASURES Allele and genotype frequencies of the htSNPs in the CFH, CFB/C2, and ARMS2/HTRA1 loci. RESULTS Four AMD-associated haplotype-tagging alleles (rs547154, rs1061170, rs1410996, rs10490924) in the 3 major loci, CFH, CFB/C2, and ARMS2/HTRA1, also were statistically significantly associated with the PCV phenotype (P<0.05). Three other alleles from the same loci (rs4151667, rs529825, rs3766404) showed a trend toward association (P<0.2) but did not reach statistical significance, possibly because of the combined effects of a relatively small sample size and low minor allele frequency in the screened populations. CONCLUSIONS The PCV phenotype in Caucasian patients is associated with the major alleles/genotypes in the AMD-associated loci, suggesting that PCV and AMD are genetically similar in the tested loci.

Heritability and Genome-Wide Association Study to Assess Genetic Differences between Advanced Age-Related Macular Degeneration Subtypes

PURPOSE To investigate whether the 2 subtypes of advanced age-related macular degeneration (AMD), choroidal neovascularization (CNV), and geographic atrophy (GA) segregate separately in families and to identify which genetic variants are associated with these 2 subtypes. DESIGN Sibling correlation study and genome-wide association study (GWAS). PARTICIPANTS For the sibling correlation study, 209 sibling pairs with advanced AMD were included. For the GWAS, 2594 participants with advanced AMD subtypes and 4134 controls were included. Replication cohorts included 5383 advanced AMD participants and 15 240 controls. METHODS Participants had the AMD grade assigned based on fundus photography, examination, or both. To determine heritability of advanced AMD subtypes, a sibling correlation study was performed. For the GWAS, genome-wide genotyping was conducted and 6 036 699 single nucleotide polymorphisms (SNPs) were imputed. Then, the SNPs were analyzed with a generalized linear model controlling for genotyping platform and genetic ancestry. The most significant associations were evaluated in independent cohorts. MAIN OUTCOME MEASURES Concordance of advanced AMD subtypes in sibling pairs and associations between SNPs with GA and CNV advanced AMD subtypes. RESULTS The difference between the observed and expected proportion of siblings concordant for the same subtype of advanced AMD was different to a statistically significant degree (P = 4.2 × 10(-5)), meaning that in siblings of probands with CNV or GA, the same advanced subtype is more likely to develop. In the analysis comparing participants with CNV to those with GA, a statistically significant association was observed at the ARMS2/HTRA1 locus (rs10490924; odds ratio [OR], 1.47; P = 4.3 × 10(-9)), which was confirmed in the replication samples (OR, 1.38; P = 7.4 × 10(-14) for combined discovery and replication analysis). CONCLUSIONS Whether CNV versus GA develops in a patient with AMD is determined in part by genetic variation. In this large GWAS meta-analysis and replication analysis, the ARMS2/HTRA1 locus confers increased risk for both advanced AMD subtypes, but imparts greater risk for CNV than for GA. This locus explains a small proportion of the excess sibling correlation for advanced AMD subtype. Other loci were detected with suggestive associations that differ for advanced AMD subtypes and deserve follow-up in additional studies.

Mutations in NR2E3 can cause dominant or recessive retinal degenerations in the same family

NR2E3, a photoreceptor‐specific nuclear receptor (PNR), represses cone‐specific genes and activates several rod‐specific genes. In humans, mutations in NR2E3 have been associated with the recessively‐inherited enhanced short‐wavelength sensitive S‐cone syndrome (ESCS) and, recently, with autosomal dominant (ad) retinitis pigmentosa (RP) (adRP). In the present work, we describe two additional families affected by adRP that carry a heterozygous c.166G>A (p.G56R) mutation in the NR2E3 gene. Functional analysis determined the dominant negative activity of the p.G56R mutant protein as the molecular mechanism of adRP. Interestingly, in one pedigree, the most common causal variant for ESCS (p.R311Q) cosegregated with the adRP‐linked p.G56R mutation, and the compound heterozygotes exhibited an ESCS‐like phenotype, which in 1 of the 2 cases was strikingly “milder” than the patients carrying the p.G56R mutation alone. Impaired repression of cone‐specific genes by the corepressors atrophin‐1 (dentatorubral‐pallidoluysian atrophy [DRPLA] gene product) and atrophin‐2 (arginine‐glutamic acid dipeptide repeat [RERE] protein) appeared to be a molecular mechanism mediating the beneficial effect of the p.R311Q mutation. Finally, the functional dominance of the p.R311Q variant to the p.G56R mutation is discussed. Hum Mutat 0,1–10, 2008.

Evaluation of the ARMD1 locus on 1q25-31 in patients with age-related maculopathy: genetic variation in laminin genes and in exon 104 of HEMICENTIN-1.

The age-related maculopathy (ARM) genetics program at Columbia University utilizes comprehensive genetic analysis of candidate genes in large case-control studies to determine genotypes associated with the ARM complex trait. Genes encoding laminins, a class of extracellular matrix proteins, represent attractive candidates for two reasons. First, the presence of laminins in the basal lamina of the retinal pigment epithelium (RPE), Bruchs membrane, and choriocapillaris suggests a possible role in the pathophysiology of ARM. Second, three laminin genes, LAMC1, LAMC2, and LAMB3, are located in the 1q25–31 region, within the previously mapped ARMD1 locus. The entire open reading frame of the three laminin genes was screened for variants by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing in at least 92, and up to 368 ARM patients and matched unaffected controls. Sixty-nine sequence variants were detected in the 69 exons of the LAMC1, LAMC2, and LAMB3 genes. Screening of exon 104 of the recently proposed ARMD1 gene, HEMICENTIN-1, residing in the 1q25–31 locus, did not detect the suggested causal variant, Q5345R, in 632 study subjects. Overall, we did not find statistically significant differences in the frequency of variants between ARM-affected individuals and age-matched controls. Four rare, non-synonymous, variants were detected in single cases of ARM patients. Our data on relatively limited numbers of study subjects do not suggest a significant role for genetic variation in the three laminin genes and in exon 104 of HEMICENTIN-1 in predisposing individuals to ARM. However, as in many instances in similar studies, involvement of rare amino acid-changing variants in a fraction of ARM cannot be ruled out.

Comprehensive analysis of the candidate genes CCL2, CCR2, and TLR4 in age-related macular degeneration.

PURPOSE To determine whether variants in the candidate genes TLR4, CCL2, and CCR2 are associated with age-related macular degeneration (AMD). METHODS This study was performed in two independent Caucasian populations that included 357 cases and 173 controls from the Netherlands and 368 cases and 368 controls from the United States. Exon 4 of the TLR4 gene and the promoter, all exons, and flanking intronic regions of the CCL2 and CCR2 genes were analyzed in the Dutch study and common variants were validated in the U.S. study. Quantitative (q)PCR reactions were performed to evaluate expression of these genes in laser-dissected retinal pigment epithelium from 13 donor AMD and 13 control eyes. RESULTS Analysis of single nucleotide polymorphisms (SNPs) in the TLR4 gene did not show a significant association between D299G or T399I and AMD, nor did haplotypes containing these variants. Univariate analyses of the SNPs in CCL2 and CCR2 did not demonstrate an association with AMD. For CCR2, haplotype frequencies were not significantly different between cases and controls. For CCL2, one haplotype containing the minor allele of C35C was significantly associated with AMD (P = 0.03), but this did not sustain after adjustment for multiple testing (q = 0.30). Expression analysis did not demonstrate altered RNA expression of CCL2 and CCR2 in the retinal pigment epithelium from AMD eyes (for CCL2 P = 0.62; for CCR2 P = 0.97). CONCLUSIONS No evidence was found of an association between TLR4, CCR2, and CCL2 and AMD, which implies that the common genetic variation in these genes does not play a significant role in the etiology of AMD.

Complement Factor H 402H Variant and Reticular Macular Disease

OBJECTIVE To determine the association of high-risk alleles in the complement factor H (CFH; Y402H, rs1061170) and age-related maculopathy susceptibility (ARMS2; A69S, rs10490924) genes with reticular macular disease (RMD), a major clinical subphenotype of age-related macular degeneration (AMD). METHODS Using retinal images from the Columbia Macular Genetics Study, we identified 67 subject individuals with RMD. A comparison group of 64 subjects with AMD without RMD was matched by ethnicity, age, sex, and AMD clinical stage. RESULTS In the RMD group, 53 of 67 subjects (79.1%) were female, the mean age was 83 years, and 47 of 67 (70.1%) had late AMD, with closely matched values in the non-RMD group. The frequencies of the CFH 402H allele were 39.6% in the RMD group (53 of 134 individuals) and 58.6% in the non-RMD group (75 of 128 individuals) (χ(2) = 8.8; P = .003; odds ratio, 0.46 [95% confidence interval, 0.28-0.76]). The corresponding frequencies of the risk allele for ARMS2 were 44.0% (40 of 128 individuals) and 31.3% (40 of 128 individuals), respectively (χ(2) = 4.0; P = .045; odds ratio, 1.73 [95% confidence interval, 1.04-2.90]). Homozygosity for 402H was particularly associated with the absence of RMD, occurring in 8 of 67 subjects (11.9%) with RMD vs 24 of 64 subjects (37.5%) without RMD (P < .001). Retinal macular disease also was associated with hypertension among male patients. CONCLUSIONS The AMD-associated CFH 402H risk variant is significantly associated with the absence of RMD but enhanced risk for RMD is conferred by the ARMS2 69S AMD risk allele. These results are consistent with the hypothesis that 402H may confer a survival benefit against certain infections, some of which may cause RMD. CLINICAL RELEVANCE Reticular macular disease may be genetically distinct from the rest of AMD.