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Aims: To describe the clinical phenotype of X linked juvenile retinoschisis in eight Italian families with six different mutations in the XLRS1 gene. Methods: Complete ophthalmic examinations, electroretinography and A [...]

Importance: ABCA4-associated retinopathy is a common inherited retinal disease, and its phenotype spans from late-onset macular dystrophy to extensive cone-rod degeneration. Over 2000 disease-causing variants in the ABCA4 gene have been identified., Objective: To investigate genotype-phenotype correlations in ABCA4-associated retinopathy., Design, Setting, and Participants: This cohort study took place at a single referral center for inherited retinal diseases in Italy. Data were prospectively acquired from January 2015 to June 2022. Patients diagnosed with an inherited retinal disease related to biallelic ABCA4 variants were included for analysis., Exposure: Genotype, classified into 4 groups according to the presence of the (1) p.Gly1961Glu allele, (2) a hypomorphic allele, (3) at least 1 moderate variant (moderate genotypes), or (4) 2 biallelic severe variants (severe genotypes)., Main Outcomes and Measures: Total decreased autofluorescence (TDAF) and definitely decreased autofluorescence (DDAF) areas, inner and outer retinal volumes, and the respective progression rate., Results: A total of 71 patients (median [IQR] age, 34 [22.4-47.2] years; 40 [56%] female) were included in the study, and 54 (76%) were followed up for a median (IQR) of 3.5 (1.6-4.7) years. Compared with moderate genotypes, those with the p.Gly1961Glu allele had smaller TDAF lesions by 61% (95% CI, -78% to -33%; P < .001) and DDAF lesions by 77% (95% CI, -93% to -18%; P = .02), along with slower growth rates for both TDAF (0.05 mm/y; 95% CI, 0.01-0.07; P < .001) and DDAF (0.06 mm/y; 95% CI, 0-0.12; P = .004). Hypomorphic alleles were associated with a thicker inner (+0.19 mm3; 95% CI, +0.02 to +0.36; P = .03) and outer retinal volume (+0.16 mm3; 95% CI, +0.03 to +0.28; P = .01) compared with moderate genotypes as well as a slower TDAF growth rate (0.05 mm/y; 95% CI, 0.01-0.08; P = .007). Severe genotypes had a 7-fold larger TDAF area (95% CI, 3.4-14.7; P < .001) and 11-fold larger DDAF area (95% CI, 2.9-42.1; P < .001) compared with moderate genotypes, along with faster growth rates estimated at 0.16 mm/y for TDAF (95% CI, 0.12-0.20; P < .001) and 0.17 mm/y for DDAF (95% CI, 0.12-0.23; P < .001)., Conclusions and Relevance: In this study of ABCA4-associated retinopathy, a 4-tier classification of genotypes was found to capture substantial variation in disease phenotype severity. These findings could prove beneficial for the prognostication of patients and warrant consideration of genotype in the design of future clinical trials.

Background: Schubert-Bornschein (SB) is the most common type of people with congenital stationary night blindness (CSNB). The aim of the study is to describe the optical coherence tomography (OCT) and optical coherence tomography angiography (OCTA) findings in patients with SB CSNB., Methods: Prospective, observational case series including three patients with genetically confirmed CSNB along with matched controls, who underwent complete ophthalmic examination and multimodal imaging., Results: On SD-OCT, a significant focal outer plexiform layer (OPL) thickening and a corresponding focal outer nuclear layer (ONL) thinning were identified in the macular area (p < 0.001). OCTA analysis overall showed decreased density of macular deep capillary plexus (mDCP) and macular choriocapillaris (mCC) (p = 0.008 and p = 0.033, respectively). DCP vessel density in the area corresponding to OPL thickening was significantly increased compared to the remaining retina (p < 0.001)., Conclusion: SB CSNB is characterized by retinal vascular impairment, as detected on OCTA.

Aim of the Study: To report optical coherence tomography angiography (OCTA) findings in patients affected by CRB1 -associated retinal dystrophies., Method: Patients affected by a genetically confirmed CRB1 -associated retinal dystrophy were prospectively enrolled in an observational study, along with age- and sex-matched healthy volunteers as control subjects. All study and control subjects received a complete ophthalmic examination and multimodal retinal imaging, including OCTA., Result: A total of 12 eyes from 6 patients were included in the study. The mean BCVA of patients was 0.42 ± 0.25 logMAR. Two patients showed large central atrophy, with corresponding definite hypo-autofluorescence on fundus autofluorescence (FAF). Another four patients disclosed different degrees of RPE mottling, with uneven FAF. On OCTA, the macular deep capillary plexus and choriocapillaris had a lower vessel density in eyes affected by CRB1 -associated retinopathy when compared to healthy controls. On the other hand, vessel density at the peripapillary radial capillary plexus, superficial capillary plexus, and deep capillary plexus was significantly altered with respect to control eyes. Statistical analyses disclosed a negative correlation between the deep capillary plexus and both LogMAR best corrected visual acuity and central retinal thickness., Conclusion: Our study reveals that CRB1 -associated retinal dystrophies are characterized by vascular alterations both in the macular and peripapillary region, as assessed by OCTA.

Purpose: To describe the multimodal imaging characteristics of benign foveal depigmentation., Methods: The study was designed as prospective observational case series. Patients with benign foveal depigmentation were prospectively investigated by means of multimodal imaging, including blue-light and near-infrared fundus autofluorescence, optical coherence tomography (OCT), OCT angiography, color testing, microperimetry, and electrophysiology. The main outcome measures were vessel density and retinal pigment epithelium (RPE)/photoreceptor complex OCT reflectivity., Results: Overall, 4 patients were identified, with bilateral and unilateral involvement in 1 case and 3 cases, respectively. Fundus autofluorescence provided variable results, showing more impairment on near-infrared fundus autofluorescence. Structural OCT revealed slight attenuation of the outer retinal bands in the area affected by benign foveal depigmentation, associated with choroidal hypertransmission, whereas enface OCT better delineated the attenuation of the reflectivity signal. The mean reflectivity intensity of RPE/photoreceptor complex was statistically significantly reduced in patients with respect to control subjects in the benign foveal depigmentation area. Optical coherence tomography angiography, color testing, microperimetry, electrooculogram, and electroretinogram findings were normal., Conclusion: Benign foveal depigmentation may represent a focal RPE disease. The limited alterations within the RPE band, as visualized on enface OCT and confirmed on near-infrared fundus autofluorescence, suggest an impairment in melanin production or distribution within the RPE cells.

Background: Gene therapy cannot be yet considered a far perspective, but a tangible therapeutic option in the field of retinal diseases. Although still confined in experimental settings, the preliminary results are promising and provide an overall scenario suggesting that we are not so far from the application of gene therapy in clinical settings. The main aim of this review is to provide a complete and updated overview of the current state of the art and of the future perspectives of gene therapy applied on retinal diseases. Methods: We carefully revised the entire literature to report all the relevant findings related to the experimental procedures and the future scenarios of gene therapy applied in retinal diseases. A clinical background and a detailed description of the genetic features of each retinal disease included are also reported. Results: The current literature strongly support the hope of gene therapy options developed for retinal diseases. Although being considered in advanced stages of investigation for some retinal diseases, such as choroideremia (CHM), retinitis pigmentosa (RP), and Leber's congenital amaurosis (LCA), gene therapy is still quite far from a tangible application in clinical practice for other retinal diseases. Conclusions: Gene therapy is an extremely promising therapeutic tool for retinal diseases. The experimental data reported in this review offer a strong hope that gene therapy will be effectively available in clinical practice in the next years., Competing Interests: FB consultant for Alcon (Fort Worth, Texas, USA), Alimera Sciences (Alpharetta, Georgia, USA), Allergan Inc. (Irvine, California, USA), Farmila-Thea (Clermont-Ferrand, France), Bayer Shering-Pharma (Berlin, Germany), Bausch and Lomb (Rochester, New York, USA), Genentech (San Francisco, California, USA), Hoffmann-La-Roche (Basel, Switzerland), NovagaliPharma (Évry, France), Novartis (Basel, Switzerland), Sanofi-Aventis (Paris, France), Thrombogenics (Heverlee, Belgium), and Zeiss (Dublin, USA). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Amato, Arrigo, Aragona, Manitto, Saladino, Bandello and Battaglia Parodi.)

Competing Interests: The authors report no conflicts of interest.

Our aim is to report a case of asymptomatic retinal arterial macroaneurysm in a 9-year-old female patient. She was referred to our Ophthalmology clinic for a routine ophthalmologic examination and, after the detection of a juxtafoveal saccular vascular enlargement in indirect ophthalmoscopy, underwent a multimodal imaging assessment. Optical coherence tomography-angiography and fluorescein angiography were important to determine the nature of the lesion, identified as a congenital retinal arterial macroaneurysm. Retinal vascular abnormalities represent a rare finding in pediatric patients and must be carefully explored to establish the correct diagnosis. A multimodal imaging approach was very useful to thoroughly reach this target. Vascular abnormalities represent a rare finding in pediatric patients and must be carefully explored to establish the correct diagnosis. A multimodal imaging approach is very useful to study in deep the reported arterial macroaneurysm in a non-invasive way.

Inherited retinal dystrophies (IRD) are a heterogeneous group of rare chronic disorders caused by genetically determined degeneration of photoreceptors and retinal pigment epithelium cells. Ultra-widefield (UWF) imaging is a useful diagnostic tool for evaluating retinal integrity in IRD, including Stargardt disease, retinitis pigmentosa, cone dystrophies, and Best vitelliform dystrophy. Color or pseudocolor and fundus autofluorescence images obtained with UWF provide previously unavailable information on the retinal periphery, which correlates well with visual field measurement or electroretinogram. Despite unavoidable artifacts of the UWF device, the feasibility of investigations in infants and in patients with poor fixation makes UWF imaging a precious resource in the diagnostic armamentarium for IRD.

Purpose: To assess foveal and parafoveal vasculature at the superficial capillary plexus, deep capillary plexus, and choriocapillaris of patients with X-linked retinoschisis by means of optical coherence tomography angiography., Methods: Six patients with X-linked retinoschisis (12 eyes) and seven healthy controls (14 eyes) were recruited and underwent complete ophthalmologic examination, including best-corrected visual acuity, dilated fundoscopy, and 3 × 3-mm optical coherence tomography angiography macular scans (DRI OCT Triton; Topcon Corp). After segmentation and quality review, optical coherence tomography angiography slabs were imported into ImageJ 1.50 (NIH; Bethesda) and digitally binarized. Quantification of vessel density was performed after foveal avascular zone area measurement and exclusion. Patients were additionally divided into "responders" and "nonresponders" to dorzolamide therapy., Results: Foveal avascular zone area resulted markedly enlarged at the deep capillary plexus (P < 0.001), particularly in nonresponders. Moreover, patients disclosed a significant deep capillary plexus rarefaction, when compared with controls (P: 0.04); however, a subanalysis revealed that this damage was limited to the fovea (P: 0.006). Finally, the enlargement of foveal avascular zone area positively correlated with a decline in best-corrected visual acuity (P: 0.01)., Conclusion: Prominent foveal vascular impairment is detectable in the deep capillary plexus of patients with X-linked retinoschisis. Our results correlate with functional outcomes, suggesting a possible vascular role in X-linked retinoschisis clinical manifestations.

Purpose: Choroideremia is a rare degenerative retinal disease that causes incurable blindness. It occurs as a result of the deficiency of the X-linked CHM gene, which encodes the Rab escort protein 1 (REP1). Gene therapy has been developed to treat CHM using adeno-associated viral vectors and is currently undergoing clinical trials. Expression of the CHM gene is ubiquitous throughout the retina, and it is therefore important to identify which retinal layers are affected in the disease process. The purpose of this study was to assess in particular the choriocapillaris using optical coherence tomography angiography because this layer is difficult to see with conventional imaging techniques., Methods: Six men with choroideremia were identified and underwent standardized optical coherence tomography angiography as part of an ethics-approved clinical study and were compared with age-matched control subjects., Results: The choriocapillaris appeared normal in regions where the retinal pigment epithelium remained intact, but it was deficient elsewhere. The outer retinal vasculature showed significant changes peripherally but also some changes centrally. The inner retinal vasculature appeared unaffected by the disease process., Conclusion: Choroideremia is a disease in which the choriocapillaris maintains a normal structure until the loss of the overlying retinal pigment epithelium. The inner retina also appears not to be affected at the vascular level. Although this study is limited by the small number of patients eligible for inclusion in the study, the observations support the concept of targeting gene therapy to the retinal pigment epithelium and outer retina because there is no evidence of independent degeneration of the choriocapillaris.

Purpose: To study multimodal imaging features of combined hamartoma of the retina and retinal pigment epithelium (CHRRPE)., Methods: Six patients (3 males, mean age 11 years) and a healthy age-matched control group made up of 15 healthy subjects (8 males, mean age 12.6 years) were included in the analysis. Complete ophthalmologic examination was performed, including best-corrected visual acuity, anterior and posterior segment slit-lamp evaluation, and tonometry. The multimodal imaging protocol included fundus images, structural optical coherence tomography (OCT), and swept-source OCT angiography (OCTA). The main outcome measures included the qualitative evaluation of both OCT and OCTA features of CHRRPE, retinal and choroidal thickness measurements, and the quantitative analysis of superficial capillary plexus, deep capillary plexus, and choriocapillaris vessel densities., Results: Optical coherence tomography features of CHRRPE were examined extensively. Multiple little hyperreflective triangular outer retinal alterations were found at the CHRRPE edges in all patients; these were dubbed the "shark-teeth" sign. Optical coherence tomography angiography showed rarefaction and morphologic alterations of all retinal plexa. Moreover, quantitative analysis revealed a statistically significant decrease in superficial capillary plexus, deep capillary plexus, and choriocapillaris vessel densities in patients affected by CHRRPE compared with the control group., Conclusion: Optical coherence tomography and OCTA analyses allowed the accurate qualitative and quantitative analyses of CHRRPE features. Further studies are needed to better define OCTA changes of CHRRPE better and to improve our understanding of the possible causes of the shark-teeth sign.

We report the outcome of 3 years of arginine-restricted diet and vitamin B6 supplementation in a boy who presented with gyrate atrophy of the choroid and retina and bilateral cystoid macular edema. The diagnosis of gyrate atrophy was made on the basis of clinical findings and increased plasma ornithine levels. Molecular genetic testing revealed a disease-causing homozygous mutation in the ornithine aminotransferase (OAT) gene. After 3 months of dietary modification and pyridoxine supplementation, visual acuity improved, and optical coherence tomography showed resolution of cystoid macular edema in both eyes. This anatomical and functional improvement was maintained during 3 years of follow-up., (Copyright © 2018 American Association for Pediatric Ophthalmology and Strabismus. Published by Elsevier Inc. All rights reserved.)

In recent years, Next-Generation Sequencing (NGS) opened a new way for the study of pathogenic mechanisms and for molecular diagnosis of inherited disorders. In the present work, we focused our attention on the inherited retinal dystrophies (IRDs), a group of specific disorders of the retina, displaying a very high clinical and genetic heterogeneity, whose genetic diagnosis is not easily feasible. It represents a paradigmatic example for the integration of clinical and molecular examination toward precision medicine. In this paper, we discuss the use of targeted NGS resequencing of selected gene panels in a cohort of patients affected by IRDs. We tested the hypothesis to apply a selective approach based on a careful clinical examination. By this approach we reached a 66% overall detection rate for pathogenic variants, with a 52% diagnostic yield. Reduction of the efforts for validation and classification of variants is a clear advantage for the management of genetic testing in a clinical setting.

Purpose: To describe a peculiar flecked-retina phenotype in a young female affected by early-onset retinal dystrophy due to a heterozygous mutation in the cone-rod transcription factor CRX gene., Case Report: A 5-year-old girl presented with poor vision and nystagmus from the first month after birth. Opththalmologic examination at baseline revealed an altered foveal reflex, epiretinal membrane, and yellow fleck-like retinal deposits in the mid- and extreme periphery bilaterally that disappeared after 3 years of follow-up. Electoretinogram was non-recordable in both rods and cones components bilaterally. Genomic sequencing identified a heterozygous missense mutation -c.425A > G (Tyr142Cys) in CRX., Conclusions: We identified a novel early-onset retinal dystrophy-related heterozygous CRX mutation associated with early and severe rod and cone dysfunction and regressive flecked-retina appearance on ophthalmoscopy.

Background: To correlate patterns in short-wavelength (SW) and near-infrared (NIR) fundus autofluorescence (FAF) with morpho-functional outcomes in eyes affected by Stargardt disease., Methods: Fifty-four eyes of 27 patients were prospectively enrolled. All patients underwent a complete ophthalmologic examination including SW-FAF, NIR-FAF, microperimetry and spectral-domain optical coherence tomography (SD-OCT). The main outcome measures were identification of a correlation between NIR-FAF and SW-FAF patterns within the foveal region and best corrected visual acuity (BCVA) values. Secondary outcome measures were correlation of FAF patterns with SD-OCT findings and retinal sensitivity on microperimetry., Results: Eyes showing a pattern of foveal hyper-FAF on NIR-FAF had a higher BCVA than eyes with a reduced FAF signal (0.44±0.23 LogMAR vs 1.08±0.19, p<0.001). Similarly, mean sensitivity within 2° of the foveal region was significantly better (6.45±2.39 dB) in eyes with hyper-FAF than in eyes with hypo-FAF (0.23±0.45 dB, p<0.001). Moreover, eyes with hyper-FAF on SW-FAF did not present a significant difference in BCVA (0.73±0.31 vs 0.83±0.43, p=0.335) and mean retinal sensitivity (4.34±3.91 dB vs 2.33±2.96, p=0.07) compared with the subgroup with foveal hypo-FAF. The integrity of both the photoreceptor inner/outer segment junction and the photoreceptor outer segment/retinal pigmented epithelium junction was significantly correlated with a preserved BCVA and a foveal hyper-FAF pattern on NIR-FAF., Conclusions: Our data suggest that NIR-FAF patterns correlate with morpho-functional outcomes in eyes affected by Stargardt disease. Longitudinal investigations are warranted to assess more precisely the actual contribution of NIR-FAF in the clinical characterisation of Stargardt disease., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Purpose: To describe the previously unreported concomitance of 2 uncommon ocular conditions: posterior polymorphous corneal dystrophy (PPCD) and large colloid drusen (LCD)., Methods: A 45-year-old woman underwent a complete ophthalmologic examination with slit-lamp biomicroscopy and blue fundus autofluorescence with spectral-domain optical coherence tomography, as well as complete systemic examination and renal function investigation., Results: On slit-lamp biomicroscopy, a corneal lesion located at Descemet membrane was observed in the right eye. The clinical features of deep posterior stromal-endothelial linear bands with vesicles and irregular opacities of posterior corneal surface were consistent with the diagnosis of PPCD. Fundus biomicroscopy and blue fundus autofluorescence showed LCD., Discussions: We report the unusual coexistence of PPCD and LCD in a young, healthy subject. Posterior polymorphous corneal dystrophy and LCD share morphologic similarities and dysfunctions of collagen architecture in the basement membrane layer, which suggests a possible common pathogenic pathway.

Background: To report the morphological macular findings detected by spectral domain optical coherence tomography (SD-OCT) and to determine their prevalence in patients with retinitis pigmentosa (RP)., Methods: SD-OCT scans of 176 eyes from 90 patients affected by RP were reviewed. A careful evaluation was carried out on photoreceptor inner/outer segment (IS/OS) junction, external limiting membrane (ELM), inner limiting membrane thickening (ILMT), epiretinal membranes (ERMs), retinal micropseudocysts (MPCs), cystoid macular edema (CME), macular holes (MHs) and choroidal neovascularization (CNV)., Results: The photoreceptor IS/OS junction was absent in the foveal region of 24 eyes (13.6%) and disrupted in 84 eyes (47.7%). The ELM was absent in 24 eyes (13.6%), whereas the ILMT was found in 118 eyes (67%). The presence of an ERM was detected in 48 eyes (27.3%). Some sort of vitreomacular alteration (ILMT and/or ERM) was identifiable in a total of 94.3% of eyes with RP. The presence of MPCs was detected in 32 eyes (18.2%). An evident CME was found in 22 eyes (12.5%). We also found MHs in 8 eyes (4.5%) and CNV in 3 eyes (1.7%)., Conclusions: Our data indicate that RP is associated with alterations of many retinal layers. In particular, the vitreoretinal interface is affected in 94% of patients, and MPC can be identified in 18% of eyes. SD-OCT may contribute to the understanding of the pathophysiological mechanism involved in RP., (Copyright © 2013 S. Karger AG, Basel.)

Purpose: To report on the phenotype and the genotype of Italian patients carrying BEST1 mutations on both alleles., Methods: Five Italian patients from four independent pedigrees with retinal dystrophy associated with biallelic BEST1 variants were recruited from different parts of Italy. Molecular genetic analysis of the BEST1 gene was performed with direct sequencing techniques. All the subjects included in the study were clinically evaluated with a standard ophthalmologic examination, fundus photography, optical coherence tomography scan, and electrophysiological investigations., Results: Six BEST1 variants were identified. Three, c.1699del (p.Glu557AsnfsX52), c.625delAAC (p.Asn179del), and c.139C>T (p.Arg47Cys), were novel, and three had already been reported in the literature, c.301C>A(p.Pro101Thr), c.934G>A (p.Asp312Asn), and c.638A>G (p.Glu213Gly). Four were missense mutations, and two were deletions. Only one BEST1 mutation was located within one of the four mutational clusters described in typical autosomal dominant Best vitelliform macular dystrophy (BVMD). Four patients showed a BVMD phenotype while one patient presented a clinical picture consistent with autosomal recessive bestrophinopathy (ARB)., Conclusions: Biallelic BEST1 sequence variants can be associated with at least two different phenotypes: BVMD and ARB. The phenotypic result of the molecular changes probably depends on the characteristics and the combination of the different BEST1 mutations, but unknown modifying factors such as other genes or the environment may also play a role.

Background: Recessive Stargardt disease is due to mutation in the retina-specific ABC transporter gene. Established strategies for molecular characterization of this gene include direct detection by a microarray interrogating approximately 500 DNA variations and a scanning denaturing HPLC methodology., Methods: Because 11 mutations were recorded to account for approximately 50% of molecular defects in the Italian population, we evaluated an alternative open microchip-based assay for a fast and simplified level 1 screening for these mutations., Results: This approach allowed the characterization of both mutated alleles in 4% and one mutated allele in 43% of cases when applied to a cohort of 47 Stargardt patients. In the same patients, further investigation by denaturing HPLC for complete characterization identified both mutated allele in 51% and one mutated allele in 19% of cases, allowing the detection of 38 different mutations, five of which had never been described. Notably, new mutations account for a high proportion (13%) of molecular defects in our patient cohort., Conclusion: The findings raises the question about the choice of the optimal diagnostic strategy for complete genotyping of the ABCA4 gene, as new mutations could not be identified by any direct detection technology, irrespective of the total number of variations screened.

Aims: To evaluate the complement factor H (CFH) p.402Y>H polymorphism as a risk factor in age related macular degeneration (AMD) in an Italian population., Methods: 104 unrelated Italian AMD patients and 131 unrelated controls were screened for the CFH polymorphism p.402Y>H (c.1277 T>C), which has been associated with AMD. Retinography was obtained for patients and controls; the AMD diagnosis was confirmed by fluorescein angiograms. The c.1277 T>C polymorphism was genotyped with the TaqMan real time polymerase chain reaction single nucleotide polymorphism assay., Results: The frequency of c.1277C allele was higher in AMD patients than in controls (57.2% v 39.3%; p<0.001). The odds ratio (OR; logistic regression analysis) for AMD was 3.9 (95% confidence interval (CI): 1.9 to 8.2) for CC homozygotes. The CC genotype conferred a higher risk for sporadic (OR 4.6; CI: 2.0 to 10.5) than for familial AMD (OR 2.9; CI: 1.0 to 8.4). Genotypes were not related to either age at AMD diagnosis or to AMD phenotype. However, geographic atrophy and choroidal neovascularisation were more frequent in sporadic than in familial AMD (p = 0.027). Overall, the percentage of population attributable risk for the CC genotype was 28% (95% CI:18% to 33%)., Conclusion: The association between the p.402Y>H (c.1277T>C) polymorphism and AMD applies to the Italian population and the CC genotype is more frequent in sporadic than in familial AMD cases.

Background: Mutations in the retina-specific ABC transporter (ABCA4) gene are associated with different types of macular degeneration, including Stargardt disease, cone-rod dystrophy, Fundus flavimaculatus, Retinitis pigmentosa and probably age-related macular degeneration., Methods: Screening for mutations in the ABCA4 gene was performed using denaturing high-performance liquid chromatography and direct sequencing., Results: We describe the identification of a new de novo 44-bp deletion in an Italian patient affected by cone-rod dystrophy. The mutation, located in intron 48 of the ABCA4 gene, is predicted to cause exon 49 skipping, resulting in loss of the C-terminus of the ABCA4 protein. Interestingly, exon 49 also codes for a highly conserved VFVNFA motif, which has been demonstrated to be essential for the activity of ABCA1, another gene of the ABC transporter family. The presence of CT repeats at the breakpoints might have facilitated the generation of the deletion through a slippage mispairing mechanism., Conclusions: The new 6730-16del44 deletion is the first de novo mutation associated with cone-rod dystrophy and may contribute to a better understanding of the role of ABCA4 mutations in macular dystrophies.

Retinitis pigmentosa is the most common form of retinal degeneration and is heterogeneous both clinically and genetically. The autosomal dominant forms (ADRP) can be caused by mutations in 12 different genes. This report describes the first simultaneous mutation analysis of all the known ADRP genes in the same population, represented by 43 Italian families. This analysis allowed the identification of causative mutations in 12 of the families (28% of the total). Seven different mutations were identified, two of which are novel (458delC and 6901C-->T (P2301S), in the CRX and PRPF8 genes, respectively). Several novel polymorphisms leading to amino acid changes in the FSCN2, NRL, IMPDH1, and RP1 genes were also identified. Analysis of gene prevalences indicates that the relative involvement of the RHO and the RDS genes in the pathogenesis of ADRP is less in Italy than in US and UK populations. As causative mutations were not found in over 70% of the families analysed, this study suggests the presence of further novel genes or sequence elements involved in the pathogenesis of ADRP.

Background: Mutations in the retina-specific ABC transporter (ABCA4) gene have been associated with several forms of macular degenerations. Because the high complexity of the molecular genotype makes scanning of the ABCA4 gene cumbersome, we describe here the first use of denaturing HPLC (DHPLC) to screen for ABCA4 mutations., Methods: Temperature conditions were designed for all 50 exons based on effective separation of 83 samples carrying 86 sequence variations and 19 mutagenized controls. For validation, samples from 23 previously characterized Stargardt patients were subjected to DHPLC profiling. Subsequently, samples from a cohort of 30 patients affected by various forms of macular degeneration were subjected to DHPLC scanning under the same conditions., Results: DHPLC profiling not only identified all 132 sequence alterations previously detected by double-gradient denaturing gradient gel electrophoresis but also identified 5 sequence alterations that this approach had missed. Moreover, DHPLC scanning of an additional panel of 30 previously untested patients led to the identification of 26 different mutations and 29 polymorphisms, accounting for 203 sequence variations on 29 of the 30 patients screened. In total, the DHPLC approach allowed us to identify 16 mutations that had never been reported before., Conclusions: These results provide strong support for the use of DHPLC for molecular characterization of the ABCA4 gene.

By means of computational methods, we identified an uncharacterized human transcript, Chromosome 1 open reading frame 36 (C1orf36), that is expressed in the retina and that maps to 1q32.3. The cDNA contains an open reading frame of 585bp that encodes a 195-aminoacid protein with a predicted mass of 22.7kDa. An alternatively spliced transcript in a retinoblastoma cell line, encoding for a truncated peptide, was also identified. PCR experiments performed using human cDNA from several sources indicate that C1orf36 has a preferential expression in the retina. Accordingly, in situ hybridization experiments, performed using as probe a murine C1orf36 cDNA fragment, detected a hybridization signal on mouse retinal adult sections. The C1orf36 protein shares homology with putative proteins in Mus musculus and Fugu rubripes, suggesting evolutionary conservation of its function. Additional sequence analysis of the C1orf36 gene product predicts its subcellular mitochondrial localization and the presence of both evolutionary conserved phosphorylation sites and regions adopting a coiled-coil conformation. We also defined the genomic structure of the gene. This enabled us to perform a mutational analysis of the C1orf36 coding region of about 300 patients affected by retinitis pigmentosa. No pathological mutations were detected in this analysis.

Objective: Apolipoprotein E (apoE) is an important regulator of cholesterol and lipid transport during compensatory synaptogenesis. Our purpose was to investigate the role of apoE gene polymorphisms in Italian patients with age-related macular degeneration (AMD)., Methods: We used the polymerase chain reaction technique to analyze apoE genotypes in 87 patients with AMD, in 47 age-matched controls and in 1,287 individuals from a general reference population., Results: The frequency of allele epsilon4 carriers was significantly higher (p = 0.002) in the general population than in AMD patients, while the frequency of allele epsilon2 was higher in the patients (p = 0.069) with an increased risk for AMD in the patients versus the population-based controls (odds ratio = 1.7; 95% confidence interval: 1.0-2.9). Allele epsilon4 was associated with a decreased risk for AMD in the patients versus the population-based controls (odds ratio = 0.3; 95% confidence interval: 0.1-0.8)., Clinical Relevance: These data suggest that apoE testing may represent a tool for the evaluation of the relative risk of AMD. Consequently, a preventive strategy can be initiated at an early stage of the disorder., Conclusion: The apoE gene polymorphism showed a significant association with the risk of AMD. The lower frequency of the epsilon4 allele in AMD patients suggests that the apoE gene could play a protective role in the pathogenesis of the disease. In contrast, the epsilon2 allele was found associated with a slightly increased risk of AMD, although we did not find a statistically significant effect., (Copyright 2001 S. Karger AG, Basel)

Mutations in the retina-specific ABC transporter (ABCR) gene are responsible for autosomal recessive Stargardt disease (arSTGD). Mutation detection efficiency in ABCR in arSTGD patients ranges between 30% and 66% in previously published studies, because of high allelic heterogeneity and technical limitations of the employed methods. Conditions were developed to screen the ABCR gene by double-gradient denaturing-gradient gel electrophoresis. The efficacy of this method was evaluated by analysis of DNA samples with previously characterized ABCR mutations. This approach was applied to mutation detection in 44 Italian arSTGD patients corresponding to 36 independent genomes, in order to assess the nature and frequency of the ABCR mutations in this ethnic group. In 34 of 36 (94.4%) STGD patients, 37 sequence changes were identified, including 26 missense, six frameshift, three splicing, and two nonsense variations. Among these, 20 had not been previously described. Several polymorphisms were detected in affected individuals and in matched controls. Our findings extend the spectrum of mutations identified in STGD patients and suggest the existence of a subset of molecular defects specific to the Italian population. The identification of at least two disease-associated mutations in four healthy control individuals indicates a higher than expected carrier frequency of variant ABCR alleles in the general population. Genotype-phenotype analysis in our series showed a possible correlation between the nature and location of some mutations and specific ophthalmoscopic features of STGD disease.

We describe a case of a four year-old boy, with congenital ectopia lentis et pupillae, who developed patchy unilateral depigmentation of the skin, hair and lashes. The association between ectopia lentis et pupillae and transillumination of the iris is well documented in the literature, but it has never been reported with skin hypopigmentation.

Pigmented paravenous retino-choroidal atrophy (P P R C A) is a rare retinal disease characterized by bilateral patches of pigment and areas of chorioretinal atrophy distributed along the veins. The authors present a 21-year-old male with pigmented paravenous retinochoroidal atrophy and unilateral macular dysplasia. To their knowledge, this is the second reported case of macular involvement. They believe that such association is not occasional, but may be suggestive of a variable expressivity of the disease.

The authors report a child with a phenotype typical of a first branchial arch defect. The patient has a balanced translocation involving chromosome 2. They propose a defect that has occurred during the translocation in a gene mapped to chromosome 2 and belonging to the HOXD family. HOX gene defects can perturb the expression of other genes important for head development.

Neuron-specific enolase (NSE) is a unique form of the glycolytic enzyme enolase found exclusively in neurons and neuroendocrine tissues. Immunohistochemical techniques in which antineuron-specific enolase antibodies are used have made it possible to map out derivatives of the neural crest in humans. By using affinity-purified antibodies against NSE, we investigated whether the contribution of the neural crest cells to the development of the anterior ocular structures in the rat is similar to that in man. We found that filtration structures in rats show morphologically striking similarities with the analogous region of the human eye. Hence, the rat eye, with certain reservations, is a suitable model for experimental studies on ocular diseases that are characterized by chamber angle anomalies or congenital glaucoma.

Ocular pharmacokinetics of rufloxacin (MF 934), a new monofluorinated quinolone derivative, has been investigated in rabbits. A long half-life, good g.i. absorption and a higher tissue/plasma concentration than that of other quinolones, are its interesting pharmacokinetic properties. However, there is reason to believe that drug accumulation may occur in deep body compartments. We determined plasma, aqueous, and vitreous concentrations of the drug at 1, 4, 8, and 24h after a single 50 mg/kg i.v. administration of rufloxacin. Our data show that rufloxacin, administered by the i.v. route, rapidly reaches chemotherapeutically useful levels in aqueous and vitreous fluids. Although still present in plasma 8 hours after administration, it proved to be undetectable in ocular fluids, signifying that the depletion of the deep compartments occurs well in advance of the next invasion. Due to its antibacterial effectiveness and pharmacokinetic properties rufloxacin may take a relevant place among the quinolone derivatives in the treatment of ocular infections.