Jan E.E. Keunen

The spectrum of ocular phenotypes caused by mutations in the BEST1 gene

Bestrophin-1 is an integral membrane protein, encoded by the BEST1 gene, which is located in the basolateral membrane of the retinal pigment epithelium. The bestrophin-1 protein forms a Ca(2+) activated Cl(-) channel and is involved in the regulation of voltage-dependent Ca(2+) channels. In addition, bestrophin-1 appears to play a role in ocular development. Over 120 different human BEST1 mutations have been described to date, associated with a broad range of ocular phenotypes. The purpose of this review is to describe this spectrum of phenotypes, which includes Best vitelliform macular dystrophy and adult-onset foveomacular vitelliform dystrophy, autosomal dominant vitreoretinochoroidopathy, the MRCS (microcornea, rod-cone dystrophy, cataract, posterior staphyloma) syndrome, and autosomal recessive bestrophinopathy. The genotype-phenotype correlations that are observed in association with BEST1 mutations are discussed. In addition, in vitro studies and animal models that clarify the pathophysiological mechanisms are reviewed.

The spectrum of retinal dystrophies caused by mutations in the peripherin/RDS gene

Peripherin/rds is an integral membrane glycoprotein, mainly located in the rod and cone outer segments. The relevance of this protein to photoreceptor outer segment morphology was first demonstrated in retinal degeneration slow (rds) mice. Thus far, over 90 human peripherin/RDS gene mutations have been identified. These mutations have been associated with a variety of retinal dystrophies, in which there is a remarkable inter- and intrafamilial variation of the retinal phenotype. In this paper, we discuss the characteristics of the peripherin/RDS gene and its protein product. An overview is presented of the broad spectrum of clinical phenotypes caused by human peripherin/RDS gene mutations, ranging from various macular dystrophies to widespread forms of retinal dystrophy such as retinitis pigmentosa. Finally, we review the proposed genotype-phenotype correlation and the pathophysiologic mechanisms underlying this group of retinal dystrophies.

Cumulative Effect of Risk Alleles in CFH, ARMS2, and VEGFA on the Response to Ranibizumab Treatment in Age-Related Macular Degeneration

PURPOSE Intravitreal ranibizumab injections currently are the standard treatment for neovascular age-related macular degeneration (AMD). However, a broad range of response rates have been observed, the reasons for which are poorly understood. This pharmacogenetic study evaluated the impact of high-risk alleles in CFH, ARMS2, VEGFA, vascular endothelial growth factor (VEGF) receptor KDR, and genes involved in angiogenesis (LRP5, FZD4) on the response to ranibizumab treatment and on the age of treatment onset. In contrast to previous studies, the data were stratified according to the number of high-risk alleles to enable the study of the combined effects of these genotypes on the treatment response. DESIGN Case series study. PARTICIPANTS A cohort of 420 eyes of 397 neovascular AMD patients. METHODS The change in visual acuity (VA) between baseline and after 3 ranibizumab injections was calculated. Genotyping of single nucleotide polymorphisms in the CFH, ARMS2, VEGFA, KDR, LPR5, and FZD4 genes was performed. Associations were assessed using linear mixed models. MAIN OUTCOME MEASURES The VA change after 3 ranibizumab injections and the age of neovascular disease onset. RESULTS After ranibizumab treatment, AMD patients without risk alleles in the CFH and ARMS2 genes (4.8%) demonstrated a mean VA improvement of 10 Early Treatment Diabetic Retinopathy Study (ETDRS) letters, whereas no VA improvement was observed in AMD patients with 4 CFH and ARMS2 risk alleles (6.9%; P = 0.014). Patients with 4 high-risk alleles in CFH and ARMS2 were 5.2 years younger than patients with 1 or 2 risk alleles, respectively (63.5%; P<0.0001). The mean age at which the first ranibizumab treatment was carried out among AMD patients with all 6 risk alleles in CFH, ARMS2, and VEGFA was 65.9 years (2%) versus 75.3 years in patients with 0 or 1 high-risk allele (8.8%; P = 0.001). After ranibizumab treatment, patients with 6 high-risk alleles demonstrated a mean VA loss of 10 ETDRS letters (P<0.0001). CONCLUSIONS This study evaluated the largest pharmacogenetic AMD cohort reported to date. A cumulative effect of high-risk alleles in CFH, ARMS2, and VEGFA seems to be associated with a younger age of onset in combination with poor response rates to ranibizumab treatment.

The spectrum of phenotypes caused by variants in the CFH gene.

Complement factor H (CFH) is a complement inhibitor, which is present as a soluble protein and attached to cell surfaces throughout the human body. As such, CFH is a key player in complement homeostasis, inhibiting excessive activation of the complement cascade, with an emphasis on the alternative pathway. The significance of CFH is demonstrated by the broad range of phenotypes associated with specific CFH gene variants. This phenotypic spectrum includes renal phenotypes, such as membranoproliferative glomerulonephritis and atypical hemolytic uremic syndrome, as well as ocular phenotypes, such as basal laminar drusen and age-related macular degeneration. In addition, several overlapping phenotypes have been described in association with CFH gene variants. The phenotypic outcome of these CFH variants depends on their differential impact on plasma- and surface-bound CFH function. Consequently, distinct genotype-phenotype correlations may be observed.

Fundus autofluorescence imaging of retinal dystrophies

Fundus autofluorescence (FAF) is a non-invasive imaging technique that enables the visualization of lipofuscin changes in the retinal pigment epithelium. This study aims to illustrate the spectrum of FAF changes in a variety of retinal dystrophies. For this purpose, we examined patients with retinal dystrophies such as Stargardt disease, Best vitelliform macular dystrophy, and retinal dystrophies associated with mutations in the peripherin/RDS gene. All retinal dystrophies were confirmed by molecular genetic analysis. A broad range of characteristic FAF patterns was observed. Our results indicate that FAF imaging constitutes a useful additive tool in the diagnosis and follow-up of various retinal dystrophies.

Autosomal Recessive Bestrophinopathy: Differential Diagnosis and Treatment Options

OBJECTIVE To describe the clinical and genetic characteristics of patients with autosomal recessive bestrophinopathy (ARB). DESIGN Retrospective case series. PARTICIPANTS Ten patients with ARB from 7 different families. METHODS All patients underwent a complete ophthalmic examination, including dilated fundus examination, fundus photography, and fluorescein angiography (FA). In all probands, fundus autofluorescence (FAF) imaging, spectral-domain optical coherence tomography (OCT), full-field electroretinography (ERG), electro-oculography (EOG), and Goldmann perimetry were performed. In selected patients, multifocal ERG was performed. Blood samples were obtained to analyze the BEST1 gene for biallelic mutations that confirmed the diagnosis of ARB. MAIN OUTCOME MEASURES Age at onset; visual acuity; fundus appearance; characteristics on FA, FAF, OCT, full-field ERG, and EOG; BEST1 gene mutations; and genotype-phenotype correlation. RESULTS The age at onset varied widely, from 2 to 54 years. A spectrum of fundus abnormalities was observed, such as multifocal yellowish subretinal deposits, subretinal fibrous scars, and cystoid intraretinal fluid collections in the macula. All ARB patients were hyperopic, and some had shallow anterior chamber angles that predisposed them to angle-closure glaucoma. The EOG results were abnormal in all patients. The full-field ERG results were abnormal in 8 ARB patients, whereas 2 patients demonstrated normal cone and rod responses on full-field ERG. Nine ARB patients carried biallelic mutations in the BEST1 gene, and in 1 patient with a characteristic ARB phenotype, only 1 mutation could be identified. Seven different mutations were detected, including 4 novel mutations. CONCLUSIONS Autosomal recessive bestrophinopathy is a recognizable phenotype caused by autosomal recessively inherited mutations in the BEST1 gene. A differential diagnosis with other conditions can be made on the basis of marked autofluorescence changes in combination with an absent light rise on the EOG that outweighs the full-field ERG abnormalities, which point to the BEST1-related hereditary nature of the disease. A number of currently available therapeutic options should be considered in ARB, a disease that seems to be a suitable candidate for future gene therapy.

Chronic Central Serous Chorioretinopathy Is Associated with Genetic Variants Implicated in Age-Related Macular Degeneration

PURPOSE In this study, single nucleotide polymorphisms (SNPs) at 19 loci, previously associated with age-related macular degeneration (AMD), were systematically tested for association in patients with chronic central serous chorioretinopathy (cCSC). In addition, we evaluated the effect of detailed phenotyping on these genetic associations. DESIGN Case-control study. PARTICIPANTS We included 292 cCSC patients, 1147 AMD patients, and 1311 control individuals. METHODS We genotyped SNPs at 19 AMD-associated loci and 6 additional SNPs at the complement factor H (CFH) locus. Phenotyping of all patients was based on fundoscopy, spectral-domain optical coherence tomography, fluorescein angiography (FA), and indocyanine green angiography. MAIN OUTCOME MEASURES We measured the allele frequencies of 25 AMD-associated SNPs and CFH haplotype frequencies in patients with cCSC and the effect of phenotypic subdivision of cCSC on genetic associations. RESULTS One SNP in ARMS2 (rs10490924) was significant after Bonferroni correction (Punadjusted=0.002; odds ratio [OR]=0.64). The SNPs at 3 other AMD loci (CFH, TNFRSF10A, ADAMTS9) showed a trend toward association with typical cCSC. Further analysis of the CFH locus identified 2 SNPs that significantly conferred increased risk for cCSC and 1 that was protective. The CFH-H3 haplotype was also found to be protective (P=0.01; OR=0.54). Using multimodal imaging, 197 patients were classified as having typical cCSC, 52 patients had unilateral abnormalities on FA that were otherwise typical of cCSC, and 43 patients had a clinical picture that could be compatible with cCSC, but with features that could also indicate other macular diseases. Significant differences of the minor allele frequencies of the tested SNPs were observed between these 3 phenotypic subgroups. CONCLUSIONS Chronic CSC is associated with genetic variants in ARMS2 and CFH, indicating a genetic and pathophysiologic overlap between cCSC and AMD. Intriguingly, alleles in ARMS2 and CFH that confer risk of AMD may be protective for cCSC, and alleles in CFH that are protective for AMD confer risk for cCSC. Significant differences in allele frequencies were found among the phenotypic subgroups for several SNPs, illustrating the importance of correct clinical classification.

Long-term outcomes of eye-conserving treatment with Ruthenium106 brachytherapy for choroidal melanoma ☆

PURPOSE To evaluate long-term outcomes of eye-conserving treatment using Ruthenium-106 plaque brachytherapy with or without transpupillary thermotherapy (TTT) for small to intermediate size choroidal melanomas. METHODS Outcomes of 425 consecutive patients were analysed. The median basal tumour diameter was 10.9 mm (range 4.8-15.9 mm), and the median apical height 4.2 mm (range 1.2-9.3 mm). Brachytherapy doses ranged from 400 to 600Gy with TTT (86%), or from 600 to 800Gy without TTT (14%), specified at the scleral surface. Kaplan-Meier survival curves, log-rank tests and Cox regression analysis were used for analysis. RESULTS Median follow-up was 50 months. Five-year actuarial local control was 96%. Five-year overall and metastases-free survival rates were 79.6% and 76.5%. Prognostic factors for metastasis-free survival were peripheral location (p=0.02) and smaller basal diameter (p<0.001). No dose effect relationships were found. Radiation side effects were frequent, with 2- and 5-year rates free of radiation complications of 60% and 35%. Five-year enucleation rate was 4.4% (10 for local recurrence, 7 for complications). Cosmetic and functional (visual acuity >0.10) eye preservation rates were 96% and 52% at 5 years. CONCLUSIONS Ruthenium-106 brachytherapy for choroidal melanoma provides excellent rates of local control and eye preservation.

CLRN1 Mutations Cause Nonsyndromic Retinitis Pigmentosa

OBJECTIVE To describe the mutations in the CLRN1 gene in patients from 2 consanguineous Pakistani families diagnosed with autosomal recessive retinitis pigmentosa (arRP). DESIGN Case-series study. PARTICIPANTS Affected and unaffected individuals of 2 consanguineous Pakistani families and 90 unaffected controls from the same population. Informed consent was obtained from participants and the protocol was approved by a local institutional review board. METHODS Patients of 2 consanguineous families were genotyped with single-nucleotide polymorphism microarrays for genome-wide linkage analysis. The search for potential candidate genes within the 8-Mb overlapping homozygous region in these families revealed the presence of CLRN1, a gene previously known to cause Ushers syndrome type III (USH3), which was analyzed by direct sequence analysis. The clinical diagnosis was based on the presence of night blindness, fundoscopic findings, and electroretinography (ERG) results. Additionally, pure tone audiometry was performed to rule out Ushers syndrome. MAIN OUTCOME MEASURES Fundoscopy, single-nucleotide polymorphism microarray, DNA sequence analysis, ERG, and audiometry. RESULTS Sequencing of CLRN1 revealed novel missense mutations (p.Pro31Leu and p.Leu154Trp) segregating in 2 families. Analysis of fundus photographs indicated attenuation of the retinal vessels, and bone spicule pigmentation in the periphery of the retina. The ERG responses were indicative of a rod-cone pattern of the disease. Audiometric assessment revealed no hearing impairment, thereby excluding Ushers syndrome. Subcellular localization studies demonstrated the retention of the mutant proteins in the endoplasmic reticulum, whereas the wild-type protein was mainly present at the cell membrane. CONCLUSIONS The RP-associated mutations p.Pro31Leu and p.Leu154Trp may represent hypomorphic mutations, because the substituted amino acids located in the transmembrane domains remain polar, whereas more severe changes have been detected in patients with USH3. These data indicate that mutations in CLRN1 are associated not only with USH3, but also with nonsyndromic arRP.

Association of whirlin with Cav1.3 (alpha1D) channels in photoreceptors, defining a novel member of the usher protein network.

PURPOSE Usher syndrome is the most common form of hereditary deaf-blindness. It is both clinically and genetically heterogeneous. The USH2D protein whirlin interacts via its PDZ domains with other Usher-associated proteins containing a C-terminal type I PDZ-binding motif. These proteins co-localize with whirlin at the region of the connecting cilium and at the synapse of photoreceptor cells. This study was undertaken to identify novel, Usher syndrome-associated, interacting partners of whirlin and thereby obtain more insights into the function of whirlin. METHODS The database of ciliary proteins was searched for proteins that are present in both the retina and inner ear and contain a PDZ-binding motif. Interactions with whirlin were evaluated by yeast two-hybrid analyses and validated by glutathione S-transferase pull-down assays, co-immunoprecipitation, and co-localization in the retina with immunofluorescence and immunoelectron microscopy. RESULTS The L-type calcium channel subunit Ca(v)1.3 (alpha(1D)) specifically interacts with whirlin. In adult photoreceptors, Ca(v)1.3 (alpha(1D)) and whirlin co-localize in the region of the connecting cilium and at the synapse. During murine embryonic development, the expression patterns of the Whrn and Cacna1d genes show significant overlap and include expression in the eye, the inner ear, and the central nervous system. CONCLUSIONS The findings indicate that Ca(v)1.3 (alpha(1D)) is connected to the Usher protein network. This conclusion leads to the hypothesis that, in the retina, whirlin scaffolds Ca(v)1.3 (alpha(1D)) and therefore contributes to the organization of calcium channels in the photoreceptor cells, where both proteins may be involved in membrane fusions.