Genotype-phenotype correlations of low-frequency variants in the complement system in renal disease and age-related macular degeneration.

Genetic alterations in the complement system have been linked to a variety of diseases, including atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G), and age-related macular degeneration (AMD). We performed sequence analysis of the complement genes complement factor H (CFH), complement factor I (CFI), and complement C3 (C3) in 866 aHUS/C3G and 697 AMD patients. In total, we identified 505 low-frequency alleles, representing 121 unique variants, of which 51 are novel. CFH contained the largest number of unique low-frequency variants (n = 64; 53%), followed by C3 (n = 32; 26%) and CFI (n = 25; 21%). A substantial number of variants were found in both patients groups (n = 48; 40%), while 41 (34%) variants were found only in aHUS/C3G and 32 (26%) variants were AMD specific. Genotype-phenotype correlations between the disease groups identified a higher frequency of protein altering alleles in short consensus repeat 20 (SCR20) of factor H (FH), and in the serine protease domain of factor I (FI) in aHUS/C3G patients. In AMD, a higher frequency of protein-altering alleles was observed in SCR3, SCR5, and SCR7 of FH, the SRCR domain of FI, and in the MG3 domain of C3. In conclusion, we observed a substantial overlap of variants between aHUS/C3G and AMD; however, there is a distinct clustering of variants within specific domains.

Further audiovestibular characterization of DFNB77, caused by deleterious variants in LOXHD1, and investigation into the involvement of Fuchs corneal dystrophy.

This study focuses on further characterization of the audiovestibular phenotype and on genotype-phenotype correlations of DFNB77, an autosomal recessive type of hearing impairment (HI). DFNB77 is associated with disease-causing variants in LOXHD1, and is genetically and phenotypically highly heterogeneous. Heterozygous deleterious missense variants in LOXHD1 have been associated with late-onset Fuchs corneal dystrophy (FCD). However, up to now screening for FCD of heterozygous carriers in DFNB77 families has not been reported. This study describes the genotype and audiovestibular phenotype of 9 families with DFNB77. In addition, carriers within the families were screened for FCD. Fifteen pathogenic missense and truncating variants were identified, of which 12 were novel. The hearing phenotype showed high inter- and intrafamilial variation in severity and progression. There was no evidence for involvement of the vestibular system. None of the carriers showed (pre-clinical) symptoms of FCD. Our findings expand the genotypic and phenotypic spectrum of DFNB77, but a clear correlation between the type or location of the variant and the severity or progression of HI could not be established. We hypothesize that environmental factors or genetic modifiers are responsible for phenotypic differences. No association was found between heterozygous LOXHD1 variants and the occurrence of FCD in carriers.

Comparing the Effectiveness of Bevacizumab to Ranibizumab in Patients with Exudative Age-Related Macular Degeneration. The BRAMD Study.

PURPOSE: To compare the effectiveness of bevacizumab and ranibizumab in the treatment of exudative age-related macular degeneration (AMD). DESIGN: Multicentre, randomized, controlled, double-masked clinical trial in 327 patients. The non-inferiority margin was 4 letters. PATIENTS: Patients ≥ 60 years of age with primary or recurrent sub- or juxtafoveal choroidal neovascularization (CNV) secondary to AMD with a total area of CNV < 12 disc areas and a best corrected visual acuity (BCVA) score between 20 and 78 letters on an EDTRS like chart in the study eye. METHODS: Monthly intravitreal injections with 1.25 mg bevacizumab or 0.5 mg ranibizumab were given during one year. Intention to treat with last observation carried forward analysis was performed. MAIN OUTCOME MEASURES: Primary outcome was the change in BCVA in the study eye from baseline to 12 months. RESULTS: The mean gain in BCVA was 5.1 (±14.1) letters in the bevacizumab group (n = 161) and 6.4 (±12.2) letters in the ranibizumab group (n = 166) (p = 0.37). The lower limit of the 95% confidence interval of the difference in BCVA gain was 3.72. The response to bevacizumab was more varied; 24% of patients showed a gain of ≥15 letters, 11% a loss of ≥15 letters and 65% a gain or loss < 15 letters compared to 19%, 5% and 76% respectively for ranibizumab (p = 0.038). No significant differences in absolute CRT and CRT change (p = 0.13) or in the presence of subretinal or intraretinal fluid (p = 0.14 and 0.10, respectively) were observed. However, the presence of any fluid on SD-OCT (subretinal and/or intraretinal) differed significantly (p = 0.020), with definite fluid on SD-OCT in 45% of the patients for bevacizumab versus 31% for ranibizumab. The occurrence of serious adverse events and adverse events was similar, with 34 SAEs and 256 AEs in the bevacizumab group and 37 SAEs and 299 AEs in the ranibizumab group (p = 0.87 and p = 0.48, respectively). CONCLUSIONS: Bevacizumab was not inferior to ranibizumab. The response to bevacizumab was more varied with higher percentages of both gainers and losers and more frequently observed retinal fluid on SD-OCT at 12 months when compared to the ranibizumab group. TRIAL REGISTRATION: Trialregister.nl NTR1704.

Extramacular drusen are highly associated with age-related macular degeneration, but not with CFH and ARMS2 genotypes.

BACKGROUND: To evaluate the association of extramacular drusen (EMD) with age-related macular degeneration (AMD) and with complement factor H (CFH rs1061170) and age-related maculopathy susceptibility 2 (ARMS2 rs10490924) polymorphisms in individuals with and without AMD. METHODS: In this case-control study, AMD staging was performed in 622 individuals. EMD were defined as ≥10 drusen (including ≥1 intermediate drusen) outside the Early Treatment of Diabetic Retinopathy Study Grid within field 2. Genotype associations for CFH and ARMS2 variants were assessed using logistic regression analysis. RESULTS: EMD (n=213) showed a strong association with AMD (OR=3.85; p=1.66×10(-13)). AMD (n=316) was strongly associated with CFH (p=1.78×10(-7)) and ARMS2 genotypes (p=1.67×10(-8)). After adjustment for AMD, age and gender, EMD were neither associated with CFH (p=0.11) nor with ARMS2 (p=0.45) genotypes. In individuals without AMD, the groups with and without EMD showed no differences regarding both genetic variants. CONCLUSIONS: The strong association between drusen within and outside of the macula suggests a common pathogenesis. However, EMD were not AMD-independently associated with CFH or ARMS2 genotypes. Our results indicate that patients without AMD but with EMD can serve as controls in studies evaluating AMD risk factors. Further studies are required to elucidate the aetiology and clinical relevance of EMD.

Comparing the effectiveness and costs of Bevacizumab to Ranibizumab in patients with Diabetic Macular Edema: a randomized clinical trial (the BRDME study).

BACKGROUND: The effectiveness of ranibizumab in the treatment of diabetic macular edema has been proven with large clinical trials. For bevacizumab only two clinical trials have been published and a head-to-head comparison is lacking to date. However, if proved non-inferior to ranibizumab, use of the off-label bevacizumab could reduce costs enormously without a loss in visual acuity. A cost-effectiveness study has been designed to substantiate this hypothesis. AIM: To compare the effectiveness and costs of 1.25 mg of bevacizumab to 0.5 mg ranibizumab given as monthly intravitreal injections during 6 months in patients with diabetic macular edema. It is hypothesized that bevacizumab is non-inferior to ranibizumab regarding its effectiveness. DESIGN: This is a randomized, controlled, double masked, clinical trial in 246 patients in seven academic trial centres in The Netherlands. OUTCOMES: The primary outcome measure is the change in best-corrected visual acuity (BCVA) in the study eye from baseline to month 6. Secondary outcomes are the proportions of patients with a gain or loss of 15 letters or more or a BCVA of 20/40 or more at 6 months, the change in leakage on fluorescein angiography and the change in foveal thickness by optical coherence tomography at 6 months, the number of adverse events in 6 months, and the costs per quality adjusted life-year of the two treatments.

Intravenous diclofenac as prophylactic treatment for verteporfin-associated low back pain.

PURPOSE: The authors report on the therapeutic effect of intravenous diclofenac on verteporfin associated low back pain (LBP), which is the most frequent adverse effect of photodynamic therapy (PDT) for macular degeneration. METHODS: The authors studied 818 patients who received PDT with verteporfin for choroidal neovascularization. Systemic blood pressures were recorded in all study participants half an hour before PDT treatment. All patients who experienced LBP during verteporfin infusion were asked to grade their pain as mild (1), moderate (2), severe (3), or unbearable (4). RESULTS: Thirty-three patients had LBP during their first verteporfin infusion. Of these, 11 subjects (1.34% of all) reported increased pain scores (level 2 to 4) and received intravenous diclofenac ahead of their next PDT. Patients with LBP during verteporfin infusion had significantly higher systolic blood pressures than uncomplicated cases (180 mmHg vs 155 mmHg, p=0.01). Treatment with intravenous diclofenac short before PDT significantly reduced the patients' mean pain score by 1.8 levels (p=0.0001). CONCLUSIONS: In this study, intravenous application of diclofenac short before verteporfin infusion effectively prevented verteporfin associated LBP in patients with systemic hypertension.

Intravitreal triamcinolone for the treatment of refractory macular edema in idiopathic intermediate or posterior uveitis.

PURPOSE: Cystoid macular edema (CME) is the most significant cause of visual loss associated with idiopathic uveitis. The authors report on the use of intravitreal triamcinolone acetonide (IVTA) in a group of patients with macular edema due to idiopathic intermediate and posterior uveitis. METHODS: Retrospective, noncomparative, interventional case series. Thirty-three eyes were included with uveitic CME that was refractory to topical steroids, oral prednisone, or a combination thereof. Previous steroid treatment did not result in elevated intraocular pressure (IOP). The eyes received an intravitreal injection with 10 mg triamcinolone acetonide, after best-corrected visual acuity (BCVA) and fluorescein angiography (FA) were assessed. Ophthalmologic examination including FA was regularly performed during a 1-year follow-up period. RESULTS: Within 12 weeks after injection of IVTA, 50% of the eyes responded with an improvement in vision of more than two lines and 30% of the eyes reached an IOP of > or = 21 mmHg (p<0.01). All eyes with an elevated IOP responded well on topical antiglaucoma medication. After 12 months follow-up 40% of the eyes responded with an improvement in vision of more than two lines and 28% of the affected eyes underwent phacoemulsification during the follow-up. No other complications occurred within a year after the treatment. CONCLUSIONS: In macular edema due to idiopathic intermediate or posterior uveitis IVTA improves the visual acuity within the first 3 months. However, thereafter the visual acuity decreases again. Cataract and elevated IOP are common side effects.

[Fundus autofluorescence in patients with inherited retinal diseases : patterns of fluorescence at two different wavelengths]. / Fundusautofluoreszenz bei erblichen Netzhauterkrankungen : Fluoreszenzmuster in zwei verschiedenen Wellenlängenbereichen.

BACKGROUND: Fundus autofluorescence (FAF) may be excited and measured at different wavelengths. In the present study we compared short wavelength and near-infrared FAF patterns of retinal dystrophies. METHODS: We analysed both eyes of 108 patients with diverse retinal dystrophies. Besides colour fundus photographs, FAF images were obtained with the Heidelberg Retina Angiograph (HRA 2). Excitation wavelengths of 488 nm (blue; filter at 500 nm) and 787 nm (near-infrared; filter at 810 nm) were applied. For improvement of the signal-to-noise ratio a total of nine images were averaged, and the mean images (original grey values, not normalized) were analysed. RESULTS: Useful FAF images of both excitation wavelengths were achieved in all patients. We observed characteristic FAF patterns, which differed between excitation wavelengths depending on the disease. With time, FAF pattern changes and progression could be observed. CONCLUSION: FAF of both wavelengths provided additional information for phenotype description in retinal dystrophies. Other than short wavelength FAF, near-infrared FAF showed different pathological changes, which may be related to changes in RPE melanin. However, any conclusions may be limited by the still incomplete knowledge about the prognostic value of FAF in the diseases studied here.

Octreotide acetate in dominant cystoid macular dystrophy.

PURPOSE: Dominant cystoid macular degeneration (DCMD) is an autosomal dominant trait of cystoid macular edema with poor visual prognosis. Until now, no efficient treatments for DCMD have been reported. The authors evaluated a somatostatin-analogue (octreotide acetate) as treatment for DCMD. METHODS: The authors treated four patients with early DCMD by intramuscular longacting octreotide acetate, 20 mg every 4 weeks for 1 year. In addition to general ophthalmologic examination the authors performed fluorescein angiography (FA) before and after treatment. RESULTS: Seven out of eight eyes showed improvement on FA and stabilization of visual acuity. CONCLUSIONS: Somatostatin-analogues may reduce cystoid edema in DCMD and may thus prevent disease-related visual loss.

Analysis of visual pigment by fundus autofluorescence.

This study investigated changes of short-wavelength fundus autofluorescence (SW-AF) by retinal bleaching effects. All measurements were performed with the Heidelberg Retina Angiograph 2 (HRA 2). Initially, experimental imaging was done on a healthy eye after dark adaptation. Photopigment was bleached within the central 30 degrees of the fundus by HRA 2 excitation light. Then SW-AF imaging of this region was performed, and SW-AF of the surrounding, unbleached 25 degrees fundus region was subsequently studied by a wide-field lens. Next, another 30 degrees SW-AF image of the posterior pole was obtained after complete dark adaptation. Then an extra SW-AF examination was performed with 15 degrees temporal eccentricity, overlapping the original examination area. Finally, a successive image series was carried out on the dark-adapted eye to test for bleaching kinetics. The second and third experiments were also performed on eyes with macular dystrophies. Distinct regions of increased SW-AF were observed after strong illumination with the blue excitation light in all eyes studied. During light adaptation mean gray levels showed a saturation plateau after an initial steep increase. The resulting gray-value maps showed significant differences of pixel intensities between bleached and unbleached parts of the fundus. Two-dimensional density difference maps allowed analysis of visual pigment distribution and density in both healthy eyes and eyes with macular dystrophies. Our observations highlight the viability of objective, non-invasive evaluation of visual pigment in the healthy and diseased human retina by means of confocal fundus autofluorescence.

[The Dutch version of the Radner Reading Chart for assessing vision function]. / De Nederlandse versie van de Radner-leeskaart voor het beoordelen van de functionele visus.

A chart for measuring visual acuity is a better functional test than the routine distance visual acuity testing with single optotypes. The characteristics of a good reading chart are: logarithmically diminishing print size, simultaneous measurement of reading acuity and reading speed, and the calculation of one score for reading acuity corrected for the number of reading errors. The original German-language Radner Reading Chart meets all these requirements, and above all emphasizes the principle of 'sentence optotypes' i.e. highly standardized sentences, because sentence complexity also influences reading performance. Sentence optotypes were created in the Dutch language and tested according to Radner's strict principles. The most equally matched sentence optotypes in terms of reading speed and number of reading errors were selected for the introduction and printing of the first Dutch version of the Radner Reading Chart. The Dutch Radner Reading Chart is precise and practical and therefore useful for research and daily practice.

Ocular syphilis acquired through oral sex in two HIV-infected patients.

Two cases of ocular syphilis are described in HIV-infected individuals after unprotected oral sex. The primary syphilitic lesion remained unnoticed and lues was therefore only diagnosed after visual symptoms developed.

The expanding roles of ABCA4 and CRB1 in inherited blindness.

Mutations in the ABCA4 gene cause Stargardt disease (STGD), most cases with autosomal recessive (ar) cone-rod dystrophy (CRD), and some cases with atypical ar retinitis pigmentosa (arRP). We found compound heterozygous ABCA4 mutations in two unrelated patients with STGD and homozygous splice site mutations in their 2nd and 4th degree cousins with RP. Some ABCA4 mutations display strong founder effects. In Dutch and German STGD patients, the 768G > T mutation is present in 8% and 0.6% of ABCA4 alleles respectively. Vice versa, the complex L541P;A1038V allele is found in 70% of ABCA4 alleles in German STGD patients but absent in Dutch patients. As approximately 70% of ABCA4 mutations are known, a microarray-based analysis of known ABCA4 gene variants allows routine DNA diagnostics in Caucasian patients. Mutations in the CRB1 gene underlie RP12, some cases with classic arRP, 55% of cases with RP and Coats-like exudative vasculopathy, and 13% of patients with Leber congenital amaurosis (LCA), rendering CRB1 a significant cause of autosomal recessive retinal dystrophy. Different combinations of mutations in ABCA4 or CRB1 can be correlated with disease severity, suggesting that small increments of protein activities in patients might have significant therapeutic effects. Mouse and Drosophila studies strongly suggest that both patient groups might benefit from reduced light exposure and therefore should be detected as early as possible using molecular techniques.

Genotyping microarray (gene chip) for the ABCR (ABCA4) gene.

Genetic variation in the ABCR (ABCA4) gene has been associated with five distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), and age-related macular degeneration (AMD). Comparative genetic analyses of ABCR variation and diagnostics have been complicated by substantial allelic heterogeneity and by differences in screening methods. To overcome these limitations, we designed a genotyping microarray (gene chip) for ABCR that includes all approximately 400 disease-associated and other variants currently described, enabling simultaneous detection of all known ABCR variants. The ABCR genotyping microarray (the ABCR400 chip) was constructed by the arrayed primer extension (APEX) technology. Each sequence change in ABCR was included on the chip by synthesis and application of sequence-specific oligonucleotides. We validated the chip by screening 136 confirmed STGD patients and 96 healthy controls, each of whom we had analyzed previously by single strand conformation polymorphism (SSCP) technology and/or heteroduplex analysis. The microarray was >98% effective in determining the existing genetic variation and was comparable to direct sequencing in that it yielded many sequence changes undetected by SSCP. In STGD patient cohorts, the efficiency of the array to detect disease-associated alleles was between 54% and 78%, depending on the ethnic composition and degree of clinical and molecular characterization of a cohort. In addition, chip analysis suggested a high carrier frequency (up to 1:10) of ABCR variants in the general population. The ABCR genotyping microarray is a robust, cost-effective, and comprehensive screening tool for variation in one gene in which mutations are responsible for a substantial fraction of retinal disease. The ABCR chip is a prototype for the next generation of screening and diagnostic tools in ophthalmic genetics, bridging clinical and scientific research.

[From gene to disease: from the ABCA4 gene to Stargardt disease, cone-rod dystrophy and retinitis pigmentosa]. / Van gen naar ziekte; van het ABCA4-gen naar de ziekte van Stargardt, kegel-stavendystrofie en retinitis pigmentosa.

Autosomal recessive Stargardt disease is caused by mutations in the ABCA4 gene. Mutations in ABCA4 are also found in two-thirds of cases with autosomal recessive cone-rod dystrophy, and a small fraction of patients with autosomal recessive retinitis pigmentosa. Patients with autosomal recessive retinitis pigmentosa, the most severe of these three phenotypes, invariably carry ABCA4 inactivating mutations; patients with autosomal recessive cone-rod dystrophy and Stargardt disease carry combinations of mutations that do not completely inactivate the retina specific 'ATP-binding cassette transporter' (ABCR) protein. DNA diagnostics is complicated by the high allelic heterogeneity and the uncertainty as to whether some ABCA4 variants are pathological. Nevertheless, ABCA4 mutation analysis is particularly important for patients with cone-rod dystrophy to confirm the autosomal recessive mode of inheritance.

Neovascular age-related macular degeneration and its relationship to antioxidant intake.

PURPOSE: Experimental and epidemiological studies suggest that low antioxidant intake may be associated with the occurrence of neovascular age-related macular degeneration (AMD). METHODS: We investigated this hypothesis further with a case-control study involving 72 case and 66 control patients attending the Ophthalmology Department of the University Hospital in Nijmegen. Data were collected by interview on antioxidant intake (i.e. in fruit and vegetables), cigarette smoking, sunlight exposure and familial predisposition. Antioxidant intake was calculated according to the method described in the Framingham Eye Study. Logistic regression analysis was used to estimate odds ratios (OR) and 95% confidence intervals (CI). RESULTS: The prevalence rate of AMD in patients with low antioxidant intake and low lutein intake (dichotomized at the median value) was about twice as high as that in patients with high intake: OR = 1.7, 95% CI (0.8-3.7), and OR = 2.4, 95% CI (1.1-5.1). Further specification of intake data into quartiles of antioxidant intake and lutein/zeaxanthine intake showed a clear dose-response relationship. CONCLUSION: The effect of dietary antioxidants upon macular health warrants preventive studies.

Clinical characterization, linkage analysis, and PRPC8 mutation analysis of a family with autosomal dominant retinitis pigmentosa type 13 (RP13).

A Dutch family with autosomal dominant retinitis pigmentosa (adRP) displayed a phenotype characterized by an early age of onset, a diffuse loss of rod and cone sensitivity, and constricted visual fields (type I). One male showed a mild progression of the disease. Linkage analysis showed cosegregation of the genetic defect with markers from chromosome 17p13.1-p13.3, a region overlapping the RP13 locus. The critical interval of the RP locus as defined in this family was flanked by D17S926 and D17S786, with a maximal lod score of 4.2 (theta = 0.00) for marker D17S1529. Soon after the mapping of the underlying defect to the 17p13 region, a missense mutation (6970G>A; R2310K) was identified in exon 42 of the splicing factor gene PRPC8 in one patient of this family. Diagnostic restriction enzyme digestion of exon 42 amplified from genomic DNA of all family members revealed that the R2310K mutation segregated fully with the disease. The type I phenotype observed in this family is similar to that described for three other RP13 families with mutations in PRPC8.

CNGA3 mutations in hereditary cone photoreceptor disorders.

We recently showed that mutations in the CNGA3 gene encoding the alpha-subunit of the cone photoreceptor cGMP-gated channel cause autosomal recessive complete achromatopsia linked to chromosome 2q11. We now report the results of a first comprehensive screening for CNGA3 mutations in a cohort of 258 additional independent families with hereditary cone photoreceptor disorders. CNGA3 mutations were detected not only in patients with the complete form of achromatopsia but also in incomplete achromats with residual cone photoreceptor function and (rarely) in patients with evidence for severe progressive cone dystrophy. In total, mutations were identified in 53 independent families comprising 38 new CNGA3 mutations, in addition to the 8 mutations reported elsewhere. Apparently, both mutant alleles were identified in 47 families, including 16 families with presumed homozygous mutations and 31 families with two heterozygous mutations. Single heterozygous mutations were identified in six additional families. The majority of all known CNGA3 mutations (39/46) are amino acid substitutions compared with only four stop-codon mutations, two 1-bp insertions and one 3-bp in-frame deletion. The missense mutations mostly affect amino acids conserved among the members of the cyclic nucleotide gated (CNG) channel family and cluster at the cytoplasmic face of transmembrane domains (TM) S1 and S2, in TM S4, and in the cGMP-binding domain. Several mutations were identified recurrently (e.g., R277C, R283W, R436W, and F547L). These four mutations account for 41.8% of all detected mutant CNGA3 alleles. Haplotype analysis suggests that the R436W and F547L mutant alleles have multiple origins, whereas we found evidence that the R283W alleles, which are particularly frequent among patients from Scandinavia and northern Italy, have a common origin.