Genome-wide association study of age-related macular degeneration identifies associated variants in the TNXB-FKBPL-NOTCH4 region of chromosome 6p21.3.

Age-related macular degeneration (AMD) is a leading cause of visual loss in Western populations. Susceptibility is influenced by age, environmental and genetic factors. Known genetic risk loci do not account for all the heritability. We therefore carried out a genome-wide association study of AMD in the UK population with 893 cases of advanced AMD and 2199 controls. This showed an association with the well-established AMD risk loci ARMS2 (age-related maculopathy susceptibility 2)-HTRA1 (HtrA serine peptidase 1) (P =2.7 × 10(-72)), CFH (complement factor H) (P =2.3 × 10(-47)), C2 (complement component 2)-CFB (complement factor B) (P =5.2 × 10(-9)), C3 (complement component 3) (P =2.2 × 10(-3)) and CFI (P =3.6 × 10(-3)) and with more recently reported risk loci at VEGFA (P =1.2 × 10(-3)) and LIPC (hepatic lipase) (P =0.04). Using a replication sample of 1411 advanced AMD cases and 1431 examined controls, we confirmed a novel association between AMD and single-nucleotide polymorphisms on chromosome 6p21.3 at TNXB (tenascin XB)-FKBPL (FK506 binding protein like) [rs12153855/rs9391734; discovery P =4.3 × 10(-7), replication P =3.0 × 10(-4), combined P =1.3 × 10(-9), odds ratio (OR) = 1.4, 95% confidence interval (CI) = 1.3-1.6] and the neighbouring gene NOTCH4 (Notch 4) (rs2071277; discovery P =3.2 × 10(-8), replication P =3.8 × 10(-5), combined P =2.0 × 10(-11), OR = 1.3, 95% CI = 1.2-1.4). These associations remained significant in conditional analyses which included the adjacent C2-CFB locus. TNXB, FKBPL and NOTCH4 are all plausible AMD susceptibility genes, but further research will be needed to identify the causal variants and determine whether any of these genes are involved in the pathogenesis of AMD.

The symmetry of phenotype between eyes of patients with early and late bilateral age-related macular degeneration (AMD).

BACKGROUND: Macular degeneration is known to be a bilateral disease. This study set out to determine the symmetry of phenotype between eyes of patients with bilateral early AMD (or drusen) or late-stage AMD. This may be important information when considering the likelihood of anti-VEGF treatment. METHODS: This prospective, observational, cross-sectional study graded the color fundus photographs of both eyes of 1,114 Caucasian patients with either early or late-stage AMD. Patients were recruited from a tertiary referral UK population. The main outcomes were phenotype, comparison of number, type and overall area of drusen in early AMD and symmetry of late AMD. RESULTS: The overall agreement of phenotype in the entire cohort of patients was 53%, kappa statistic (κ)=0.31, (95% CI = 0.27-0.36). Within this group, a total of 271 patients were identified with bilateral soft and hard drusen (early AMD). Symmetry of phenotype within this group was high in terms of total of area of drusen (agreement = 79%, weighted κ = 0.75) and number of drusen. In those with bilateral geographic atrophy (GA), symmetry between area of GA was moderate (agreement 72%, weighted κ = 0.54), and in those with bilateral neovascular disease (choroidal neovascularization or pigment epithelial detachment), symmetry was poor (agreement 45%, weighted κ = 0.16). Out of the entire cohort, 62% (n = 688) had neovascular disease in at least one eye and 37.5% of these had bilateral disease. CONCLUSIONS: The observed symmetry of phenotype between eyes with drusen appears to reduce in GA and neovascular forms of AMD. Overall, 53% of the cohort had symmetrical disease in terms of phenotype, 23% had neovascular disease in both eyes, 9.3% had GA in both eyes, and 39% of patients had neovascular disease in one eye and non-neovascular disease in the other. This may have implications for the potential need for anti-VEGF treatment of AMD in second eye involvement.

Macular dystrophy associated with the A3243G mitochondrial DNA mutation. Distinct retinal and associated features, disease variability, and characterization of asymptomatic family members.

OBJECTIVES: To determine (1) detailed retinal and audiological features of probands harboring the A3243G mitochondrial DNA mutation (m.3243A>G) and their asymptomatic maternal relatives, (2) intrafamilial and interfamilial phenotypic variability, and (3) the presence of other systemic features. METHODS: Seven probands harboring the A3243G mitochondrial DNA mutation and 36 asymptomatic maternal relatives were ascertained. Participants underwent ophthalmologic examination, fundus photography, autofluorescence imaging, and audiological evaluation and completed a questionnaire. Blood samples were taken to test for diabetes, determine renal function, and screen relatives for the A3243G mutation. RESULTS: The A3243G mutation was associated with both intrafamilial and interfamilial variable expressivity regarding retinal appearance, hearing loss, diabetes, and other systemic features. The most common macular appearance in maternal relatives (one-third of those positive for the mutation) was mild abnormalities of the retinal pigment epithelium (more clearly identified using autofluorescence), which may therefore be a useful clinical indicator suggesting positive mutation status. Four probands and 13 mutation-positive relatives were found to have evidence of significant bilateral, cochlear, symmetrical age-adjusted hearing loss, predominantly affecting high frequencies. CONCLUSIONS: Hearing loss and macular disturbance were the most frequent findings in mutation-positive participants, with 95% of mutation-positive relatives having hearing loss. Diabetes was the least frequent finding. Patients with progressive hearing loss may merit ophthalmologic assessment to detect retinal abnormalities consistent with the A3243G mutation. Conversely, patients with macular features in keeping with the A3243G mutation should have audiological testing, even in the absence of diabetes or a positive family history.

Mutations in the gene coding for the pre-mRNA splicing factor, PRPF31, in patients with autosomal dominant retinitis pigmentosa.

PURPOSE: Retinitis pigmentosa is a clinically and genetically heterogeneous disorder. It is characterized by progressive degeneration of the peripheral retina, leading to night blindness and loss of the peripheral visual field. PRPF31 is one of four pre-mRNA splicing factors identified as causing autosomal dominant retinitis pigmentosa, with incomplete penetrance being the unique feature associated with mutations in this gene. The purpose of this study was to identify PRPF31 mutations in a cohort of 118 cases of autosomal dominant retinitis pigmentosa and determine the genotype-phenotype correlation emerging from the spectrum of mutations in this gene. METHODS: Probands with autosomal dominant retinitis pigmentosa underwent ophthalmic evaluation. Blood samples were obtained, genomic DNA was isolated, and PRPF31 exons along with adjacent splice junctions were amplified by PCR and screened by direct sequencing. RESULTS: In the 118 individuals with autosomal dominant retinitis pigmentosa, six mutations were identified, of which four were novel. One previously known splice site mutation was identified in two other apparently unrelated families. CONCLUSIONS: Mutations in PRPF31 causing adRP were present in nearly 5% of a mixed U.K. population. The age of onset and the severity of the disease varied with different mutations. In addition, individuals carrying the same mutation showed a range of phenotypic variation, suggesting the involvement of other modifying genes.

Pseudoxanthoma elasticum with generalized retinal dysfunction, a common finding?

PURPOSE: Pseudoxanthoma elasticum (PXE; [MIM 264800]) is an autosomal recessive systemic disorder characterized by progressive degeneration and calcification of elastic fibers in connective tissue. The phenotype is variable, with cutaneous, vascular, and ophthalmic abnormalities. The disorder is a consequence of mutations in the ABCC6 gene. Visual impairment is mainly due to neovascular complications, and retinal function is usually assumed to be normal. The purpose of this study was the objective assessment of macular and generalized retinal function in unrelated patients with clinical and/or genetic features of PXE. METHODS: Four unrelated patients carrying a clinical diagnosis of PXE presented with unexplained visual loss. After ophthalmic examination, retinal and macular function was assessed by full-field electroretinogram (ERG) and pattern ERG, respectively, according to ISCEV (International Society for Clinical Electrophysiology of Vision) recommendations. Molecular analysis of the ABCC6 gene was performed in three patients by dHPLC (denaturing high-performance liquid chromatography) and direct sequencing. RESULTS: Full-field ERG revealed significant reduction of cone and rod responses in all four patients. Funduscopic appearances varied. Three patients were found to carry ABCC6 mutations. In case 1, a novel nonsense mutation (p.L1474X) was detected in exon 31 paired with a splice-site mutation. Mutation analyses in cases 3 and 4 revealed previously reported ABCC6 mutations. CONCLUSIONS: These findings suggest that retinal dysfunction in PXE may not be uncommon. The mechanism underlying retinal dysfunction is unknown but may result from metabolic disturbance leading to retinal toxicity with a possible role of modifying genetic or environmental factors rather than specific ABCC6 mutations.

Maculopathy due to the R345W substitution in fibulin-3: distinct clinical features, disease variability, and extent of retinal dysfunction.

PURPOSE: To determine (1) clinical features that distinguish maculopathy due to the R345W substitution in fibulin-3 from other forms of inherited or early-onset drusen, (2) the phenotypic variability, and (3) the extent of retinal disease in those with a positive molecular diagnosis. METHODS: Affected individuals underwent ophthalmic examination, digital color fundus photography, fundus autofluorescence (AF) imaging, and psychophysical testing with automated photopic and dark-adapted perimetry and fine matrix mapping. Blood samples were taken for DNA extraction and screening for the R345W mutation in fibulin-3. Patients were subsequently divided into mutation-positive and -negative groups, to compare the identified phenotypic findings in these two sets of subjects. RESULTS: Twenty-nine subjects from 19 families were ascertained with inherited or early-onset drusen. Twenty-four (83%) subjects from 15 families were found to harbor the R345W fibulin-3 mutation. Peripapillary deposition and a radial distribution of macular drusen were consistent, distinguishing signs in the mutation-positive group. Subretinal neovascular membrane (SRNVM) was a rare occurrence, affecting only 1 of 48 eyes, whereas hyperpigmentation and atrophy of the retinal pigment epithelium (RPE) were common in older mutation-positive patients. Increased AF corresponding to the drusen was detected in both the mutation-positive and -negative groups. The phenotype in the group of patients positive for the R345W mutation was extremely variable, with evidence of interocular, intrafamilial, and interfamilial variability in visual loss, natural history, ophthalmoscopic findings, autofluorescence imaging, and psychophysical data. The novel finding of nonpenetrance was observed in a 62-year-old asymptomatic, mutation-positive man. The findings from detailed perimetry performed on a subset of subjects were consistent with the presence of widespread retinal dysfunction not isolated to the macula. CONCLUSIONS: Marked inter- and intrafamilial variation associated with the fibulin-3 R345W mutation in terms of retinal appearance, severity, progression, and nonpenetrance were identified. It was noted that SRNVM is a rare occurrence in R345W fibulin-3 maculopathy. These findings are helpful for advice regarding prognosis and for genetic counseling. The findings established that the presence of peripapillary deposit is highly likely to indicate that a patient carries the R345W mutation.

Identification of novel RPGR ORF15 mutations in X-linked progressive cone-rod dystrophy (XLCORD) families.

PURPOSE: To test the incidence of mutations in RPGR ORF15 in six families with X-linked progressive retinal degeneration (cone-rod dystrophy [XLCORD], macular or cone dystrophy) and to undertake a detailed phenotypic assessment of families in whom ORF15 mutations were identified. METHODS: To amplify and sequence ORF15 in its entirety, a cloning strategy was developed. Families with mutations in ORF15 underwent electrophysiological testing, color vision assessment, color fundus photography, and fundus autofluorescence (AF) imaging. RESULTS: Novel protein truncation mutations were identified in two families. In family A, a 2-bp mutation was identified in ORF15+A1094C G1095T, predicted to result in a truncated protein (E364D/E365X). In family B, a G-to-T transversion (ORF15+1176G>T) resulted in a nonsense mutation (G392X). Characteristics of phenotype in both families included progressive deterioration of central vision and subsequently night vision, mild photophobia, and moderate to high myopia. Ophthalmoscopic abnormalities were generally confined to the macula. A parafoveal ring of increased AF was observed, and electrophysiological evidence of a greater generalized abnormality in cone than rod responses were consistent with a cone-rod dystrophy phenotype. CONCLUSIONS: The cloning strategy for ORF15 facilitated comprehensive sequence analysis in patients. Two families were identified with nonsense mutations, and clinical evaluation revealed them both to have a similar phenotype. The presence of a parafoveal ring of increased AF was an early indicator of affected status in these families. No disease-causing mutations in ORF15 were detected in four other families, suggesting that ORF15 mutations may not be the most common cause of XLCORD.

Autofluorescence imaging of choroidal neovascularization due to age-related macular degeneration.

OBJECTIVE: To describe the autofluorescence (AF) characteristics of choroidal neovascularization (CNV) in patients with age-related macular degeneration. METHODS: Autofluorescence images of 65 consecutive eyes with CNV at various stages of evolution were analyzed. Twenty images were of recent-onset CNV (group 1), 8 were of eyes 1 to 6 months after CNV diagnosis (group 2), and 37 were late-stage CNV (group 3). Autofluorescence images from groups 1 and 2 were compared with fundus fluorescein angiographic images. RESULTS: Group 1 showed areas of hyperfluorescence on fundus fluorescein angiography corresponding to areas of normal AF in 16 of 20 cases, with adjacent areas of increased AF in 13 cases. The main areas of abnormal AF were larger than the main areas of abnormal fluorescence on fundus fluorescein angiography in 18 of the 20 cases. Groups 2 and 3 showed areas of decreased AF corresponding to areas of previous leakage on fundus fluorescein angiography (in group 2) or atrophy. CONCLUSIONS: Preserved AF in group 1 indicates viable retinal pigment epithelium initially, which has implications for visual prognosis. Decreased AF in groups 2 and 3 indicates loss of retinal pigment epithelium and photoreceptors. Autofluorescence imaging may increase our understanding of CNV in age-related macular degeneration.

Complement factor H genetic variant and age-related macular degeneration: effect size, modifiers and relationship to disease subtype.

BACKGROUND: Variation in the complement factor H gene (CFH) is associated with risk of late age-related macular degeneration (AMD). Previous studies have been case-control studies in populations of European ancestry with little differentiation in AMD subtype, and insufficient power to confirm or refute effect modification by smoking. METHODS: To precisely quantify the association of the single nucleotide polymorphism (SNP rs1061170, 'Y402H') with risk of AMD among studies with differing study designs, participant ancestry and AMD grade and to investigate effect modification by smoking, we report two unpublished genetic association studies (n = 2759) combined with data from 24 published studies (26 studies, 26,494 individuals, including 14,174 cases of AMD) of European ancestry, 10 of which provided individual-level data used to test gene-smoking interaction; and 16 published studies from non-European ancestry. RESULTS: In individuals of European ancestry, there was a significant association between Y402H and late-AMD with a per-allele odds ratio (OR) of 2.27 [95% confidence interval (CI) 2.10-2.45; P = 1.1 x 10(-161)]. There was no evidence of effect modification by smoking (P = 0.75). The frequency of Y402H varied by ancestral origin and the association with AMD in non-Europeans was less clear, limited by paucity of studies. CONCLUSION: The Y402H variant confers a 2-fold higher risk of late-AMD per copy in individuals of European descent. This was stable to stratification by study design and AMD classification and not modified by smoking. The lack of association in non-Europeans requires further verification. These findings are of direct relevance for disease prediction. New research is needed to ascertain if differences in circulating levels, expression or activity of factor H protein explain the genetic association.

Mutations in the gene KCNV2 encoding a voltage-gated potassium channel subunit cause "cone dystrophy with supernormal rod electroretinogram" in humans.

"Cone dystrophy with supernormal rod electroretinogram (ERG)" is an autosomal recessive disorder that causes lifelong visual loss combined with a supernormal ERG response to a bright flash of light. We have linked the disorder to a 0.98-cM (1.5-Mb) region on chromosome 9p24, flanked by rs1112534 and rs1074449, using homozygosity mapping in one large consanguineous pedigree. Analysis of one gene within this region, KCNV2, showed a homozygous nonsense mutation. Mutations were also found in 17 alleles of 10 other unrelated families with the same disorder. In situ hybridization demonstrated KCNV2 expression in human rod and cone photoreceptors. The precise function of KCNV2 in human photoreceptors remains to be determined, although this work suggests that mutations might perturb or abrogate I(KX), the potassium current within vertebrate photoreceptor inner segments, which has been shown to set their resting potential and voltage response.