A duplication on chromosome 16q12 affecting the IRXB gene cluster is associated with autosomal dominant cone dystrophy with early tritanopic color vision defect.

Cone dystrophies are a rare subgroup of inherited retinal dystrophies and hallmarked by color vision defects, low or decreasing visual acuity and central vision loss, nystagmus and photophobia. Applying genome-wide linkage analysis and array comparative genome hybridization, we identified a locus for autosomal dominant cone dystrophy on chromosome 16q12 in four independent multigeneration families. The locus is defined by duplications of variable size with a smallest region of overlap of 608 kb affecting the IRXB gene cluster and encompasses the genes IRX5 and IRX6. IRX5 and IRX6 belong to the Iroquois (Iro) protein family of homeodomain-containing transcription factors involved in patterning and regionalization of embryonic tissue in vertebrates, including the eye and the retina. All patients presented with a unique progressive cone dystrophy phenotype hallmarked by early tritanopic color vision defects. We propose that the disease underlies a misregulation of the IRXB gene cluster on chromosome 16q12 and demonstrate that overexpression of Irx5a and Irx6a, the two orthologous genes in zebrafish, results in visual impairment in 5-day-old zebrafish larvae.

Usher Syndrome and Color Vision.

PURPOSE: The aim of this study is to report on the results of color vision testing in a European cohort of patients with Usher syndrome (USH). We describe the results in relation to Usher type (USH1 and USH2), age and visual acuity. METHODS AND METHODS: The color vision of 220 genetically confirmed adult USH patients, aged 18-70 years, was analyzed with one of three methods: the Farnsworth D-15 Dichotomous test (D-15) along with the Lanthony desaturated 15 Hue tests (D-15d), the Roth 28-Hue test, or the Ishihara 14-plate test. Visual acuity was measured with either the ETDRS or the SNELLEN charts. The Confusion index, the Selectivity index and the Confusion angle were calculated for the panel tests and used for analysis. The numbers of plates that could not be read were analyzed for the Ishihara test. RESULTS: For the panel tests, the degree of color loss (Confusion index) is similar in both subtypes of USH, but the polarization of error scores (Selectivity index) is significantly lower in USH1 than USH2, showing more diffuse errors than those found in USH2. There is no significant correlation between logMAR visual acuity and the Confusion or the Selectivity indices. Additionally, we find a significant correlation between patient age and the degree and the polarity of the loss only in USH2. There was no difference between USH1 and USH2 in the results of the Ishihara test. CONCLUSIONS: The examination of color vision in patients with USH shows a significant difference in the pattern of color vision loss in USH1 and USH2 patients, but not in the severity of the loss. In USH2, we find a correlation between patient age and the degree and the polarity of the loss. These results may be due to differences in the pathogenesis of retinal dystrophy in USH1 and USH2.

Phenotype Variations Caused by Mutations in the RP1L1 Gene in a Large Mainly German Cohort.

Purpose: Mutations in the retinitis pigmentosa-1-like-1 (RP1L1) gene are the major cause of autosomal dominant occult macular dystrophy (OCMD), while recessive mutations have been linked to autosomal recessive retinitis pigmentosa (arRP). We present the clinical phenotype of a large German OCMD cohort, as well as four RP patients. Methods: A total of 42 OCMD patients (27 families) and 4 arRP patients (3 families) with genetically confirmed mutations in RP1L1 were included. Genomic DNA was analyzed by targeted analysis of the c.133C>T;p.R45W mutation for all RP or macular dystrophy-related genes. All patients underwent ophthalmologic examination including psychophysical tests, electrophysiology, fundus autofluorescence (FAF), and spectral domain optical coherence tomography (SD-OCT). Follow-up time was up to 12 years. Results: In 25 OCMD index patients genomic testing revealed the heterozygous mutation c.133C>T;p.R45W in RP1L1; one patient was homozygous for the mutation. Two OCMD patients displayed the variants c.3599G>A;p.G1200D and c.2849G>A;p.R950H, respectively, in a heterozygous state. All OCMD patients showed characteristic clinical findings and typical microstructural photoreceptor changes. Two arRP patients displayed the novel homozygous mutations c.3022C>T;p.Q1008* and c.1107G>A;p.W369*, respectively, while two RP-siblings carried the two heterozygous mutations c.455G>A;p.R152Q and c.5959C>T;p.Q1987*, the first also being novel. All arRP cases were mild with disease onset ≈30 years and preserved ERG-responses. Conclusions: OCMD phenotype showed consistent clinical findings including classical microstructural changes on SD-OCT. An important hallmark of RP1L1-related OCMD is the dominant family history with reduced penetrance. Furthermore, novel mutations in association with arRP were identified, outlining the complexity of the protein.

An innovative strategy for the molecular diagnosis of Usher syndrome identifies causal biallelic mutations in 93% of European patients.

Usher syndrome (USH), the most prevalent cause of hereditary deafness-blindness, is an autosomal recessive and genetically heterogeneous disorder. Three clinical subtypes (USH1-3) are distinguishable based on the severity of the sensorineural hearing impairment, the presence or absence of vestibular dysfunction, and the age of onset of the retinitis pigmentosa. A total of 10 causal genes, 6 for USH1, 3 for USH2, and 1 for USH3, and an USH2 modifier gene, have been identified. A robust molecular diagnosis is required not only to improve genetic counseling, but also to advance gene therapy in USH patients. Here, we present an improved diagnostic strategy that is both cost- and time-effective. It relies on the sequential use of three different techniques to analyze selected genomic regions: targeted exome sequencing, comparative genome hybridization, and quantitative exon amplification. We screened a large cohort of 427 patients (139 USH1, 282 USH2, and six of undefined clinical subtype) from various European medical centers for mutations in all USH genes and the modifier gene. We identified a total of 421 different sequence variants predicted to be pathogenic, about half of which had not been previously reported. Remarkably, we detected large genomic rearrangements, most of which were novel and unique, in 9% of the patients. Thus, our strategy led to the identification of biallelic and monoallelic mutations in 92.7% and 5.8% of the USH patients, respectively. With an overall 98.5% mutation characterization rate, the diagnosis efficiency was substantially improved compared with previously reported methods.

Novel grading system for quantification of cystic macular lesions in Usher syndrome.

BACKGROUND: To evaluate novel grading system used to quantify optical coherence tomography (OCT) scans for cystic macular lesions (CML) in Usher syndrome (USH) patients, focusing on CML associated alterations in MOY7A and USH2A mutations. METHODS: Two readers evaluated 76 patients' (mean age 42 ± 14 years) data prospectively uploaded on Eurush database. OCT was used to obtain high quality cross-sectional images through the fovea. The CML was graded as none, mild, moderate or severe, depending on the following features set: subretinal fluid without clearly detectable CML boundaries; central macular thickness; largest diameter of CML; calculated mean of all detectable CML; total number of detectable CML; retinal layers affected by CML. Intra-and inter-grader reproducibility was evaluated. RESULTS: CML were observed in 37 % of USH eyes, while 45 % were observed in MYO7A and 29 % in USH2A cases. Of those with CML: 52 % had mild, 22 % had moderate and 26 % had severe changes, respectively. CML were found in following retinal layers: 50 % inner nuclear layer, 44 % outer nuclear layer, 6 % retinal ganglion cell layer. For the inter-grader repeatability analysis, agreements rates for CML were 97 % and kappa statistics was 0.91 (95 % CI 0.83-0.99). For the intra-grader analysis, agreement rates for CML were 98 %, while kappa statistics was 0.96 (95 % CI 0.92-0.99). CONCLUSIONS: The novel grading system is a reproducible tool for grading OCT images in USH complicated by CML, and potentially could be used for objective tracking of macular pathology in clinical therapy trials.

A Test of a Model of Glaucomatous Damage of the Macula With High-Density Perimetry: Implications for the Locations of Visual Field Test Points.

PURPOSE: To use high-density perimetry to test a model of local glaucomatous damage to the macula (central visual field [VF]) and to assess the optimal placement of stimuli used to detect this damage. METHODS: Thirty-one eyes of 31 patients showing glaucomatous arcuate damage within the upper hemifield of the central 10° were tested with a customized VF with double the density of the 10-2 (2° grid) test. Individual plots of total deviation (TD) values were generated. A model, which predicts a "vulnerable macular region" (VMR) and a "less vulnerable macular region" (LVMR), was compared with the TD values without (standard model) and with (aligned model) scaling and rotating to align it with the patient's fovea-to-disc axis. Computer simulations assessed alternative VF locations for adding two points to the 6° grid pattern (e.g., 24-2 VF) typically used in the clinic. RESULTS: There were significantly more abnormal points in the VMR than in the LVMR. However, the aligned model did no better than the standard model in describing the data. The optimal locations for adding two points to the 24-2 (6° grid) test were (-1°, 5°) and (1°, 5°), both within the VMR. CONCLUSIONS: The model describes the region of the superior VF vulnerable to arcuate damage. TRANSLATIONAL RELEVANCE: The model can be used to determine the optimal locations for adding test points to the commonly used VF test pattern (24-2). It does not seem necessary to adjust the location of VF test points based upon interindividual differences in the fovea-to-disc axis.

BBS1 mutations in a wide spectrum of phenotypes ranging from nonsyndromic retinitis pigmentosa to Bardet-Biedl syndrome.

OBJECTIVE: To investigate the involvement of the Bardet-Biedl syndrome (BBS) gene BBS1 p.M390R variant in nonsyndromic autosomal recessive retinitis pigmentosa (RP). METHODS: Homozygosity mapping of a patient with isolated RP was followed by BBS1 sequence analysis. We performed restriction fragment length polymorphism analysis of the p.M390R allele in 2007 patients with isolated RP or autosomal recessive RP and in 1824 ethnically matched controls. Patients with 2 BBS1 variants underwent extensive clinical and ophthalmologic assessment. RESULTS: In an RP proband who did not fulfill the clinical criteria for BBS, we identified a large homozygous region encompassing the BBS1 gene, which carried the p.M390R variant. In addition, this variant was detected homozygously in 10 RP patients and 1 control, compound heterozygously in 3 patients, and heterozygously in 5 patients and 6 controls. The 14 patients with 2 BBS1 variants showed the entire clinical spectrum, from nonsyndromic RP to full-blown BBS. In 8 of 14 patients, visual acuity was significantly reduced. In patients with electroretinographic responses, a rod-cone pattern of photoreceptor degeneration was observed. CONCLUSIONS: Variants in BBS1 are significantly associated with nonsyndromic autosomal recessive RP and relatively mild forms of BBS. As exemplified in this study by the identification of a homozygous p.M390R variant in a control individual and in unaffected parents of BBS patients in other studies, cis - or trans -acting modifiers may influence the disease phenotype. CLINICAL RELEVANCE: It is important to monitor patients with an early diagnosis of mild BBS phenotypes for possible life-threatening conditions.

Repeated pupil size measurements in refractive surgery candidates.

PURPOSE: To evaluate the individual variation in pupil diameter measurements in refractive surgery candidates under standardized conditions over 2 testing sessions using a digital infrared pupillometer. SETTING: Department of Ophthalmology, University Medical Center Regensburg, Regensburg, Germany. METHODS: The pupil size in both eyes of consecutive refractive surgery candidates was measured under 2 illumination levels (scotopic at 0.03 lux, low mesopic at 0.82 lux) with a digital infrared pupillometer (Procyon P2000 SA). Before measurement, a standardized dark-adaptation protocol was used. Measurements were taken twice on separate occasions. RESULTS: The study evaluated 318 eyes of 159 patients with a mean age of 36.3 years +/- 10.2 (SD). The mean interval between measurements was 53 +/- 62.8 days. Despite sufficient power (beta < 0.05), there was no statistically significant difference between 2 consecutive measurements under scotopic and low mesopic illumination. CONCLUSION: Using a standardized dark-adaptation protocol, refractive surgery candidates showed no significant variation in scotopic or low mesopic pupil diameter over 2 testing sessions.

Diagnostic compatibility of structural and haemodynamic parameters in open-angle glaucoma patients.

PURPOSE: Current evidence indicates that alteration in ocular blood flow may be relevant in open-angle glaucoma (OAG) patients independent of intraocular pressure (IOP). Presently, the lack of an adequate methodology capable of assessing all vascular beds limits the clinical role of blood flow parameters in glaucoma management. We aimed to compare differences in retinal nerve fibre layer (RNFL) thickness and retrobulbar haemodynamics between OAG patients and healthy age-matched control subjects. METHODS: Sixty eyes of 30 OAG patients and 30 healthy age-matched controls were enrolled into the prospective, randomized study. Retinal nerve fibre layer thickness was analysed by scanning laser polarimetry (SLP). Standard SLP parameters were determined, including: average temporal, superior, nasal, inferior thickness (TSNIT); superior and inferior averages; TSNIT standard deviation (TSNIT-SD), and nerve fibre indicator (NFI). Retrobulbar haemodynamics were assessed using colour Doppler imaging (CDI). Peak systolic velocity (PSV), end-diastolic velocity (EDV), pulsatility index (PI) and resistivity index (RI) in the ophthalmic artery (OA), central retinal artery (CRA) and short posterior ciliary artery (SPCA) were evaluated. RESULTS: The RNFL in OAG patients was statistically significantly thinner compared with that in age-matched controls: the NFI was 24.9 +/- 10.24 in OAG patients and 16.13 +/- 7.95 in healthy controls (p < 0.05). Statistically significant differences were observed: CRA PSV was 20.54 +/- 7.84 cm/second in OAG subjects and 16.5 +/- 6.19 cm/second in healthy controls (p = 0.0038); OA EDV was 8.99 +/- 4.71 cm/second in OAG subjects and 5.93 +/- 3.23 cm/second in healthy controls (p = 0.0048). Correlation analysis of NFI was in positive association with CRA EDV (r = 0.395; p < 0.05) and CRA PI (r = 0.403; p < 0.05) in OAG subjects, but no statistically significant association was seen in healthy controls. CONCLUSIONS: Statistically significant thinning of the RNFL in association with reduced retrobulbar blood flow velocities was observed in OAG patients. Combining ocular structural alterations with ocular circulation assessment may increase our ability to elucidate potential IOP-independent glaucomatous risk factors.

Comparison of intraocular pressure fluctuations measured by goldmann applanation tonometer and pulsatile ocular blood flow analyser.

BACKGROUND: Intraocular pressure (IOP) is the major known risk factor in glaucoma and the primer mover of the functional damage in glaucomatous patients but it is not a unique determinant of glaucomatous damage. Clinical assessment of glaucoma patients may not be a true reflection of overall IOP control. Evaluation of the effect of glaucoma medication is restricted by measurement of IOP as a dynamic physiological parameter. PURPOSE: To compare IOP fluctuations over time using Goldmann applanation tonometry (IOPGAT) and pulsatile ocular blood flow analyzer (IOP-POBFA) under the Dorzolamide/timolol or latanoprost treatment regimes. DESIGN: Prospective 1 year follow-up study. PARTICIPANTS: 30 randomly chosen controlled open angle glaucoma patients (60 eyes): 16 patients (32 eyes) receiving Dorzolamide/timolol fixed combination (D/T) and 14 (28 eyes) latanoprost 0.005% treatment. MAIN OUTCOME MEASURES: Changes in IOP and perfusion pressure dynamics. RESULTS: THERE WAS NO STATISTICALLY SIGNIFICANT DIFFERENCE IN BASELINE IOP PARAMETERS BETWEEN STUDY GROUPS: 15.69 ± 2.02 mmHg with D/T and 16.71 ± 2.84 mmHg with latanoprost (p=0.314). Both treatment regimes were tolerated and patients were adherent to treatment. Determined a strong positive correlation between IOP-GAT and IOP-POBFA; verified over time period under particular treatment regime. After 1 year follow-up D/T and latanoprost results referred to statistically significant tachyphylaxis effect, i.e. IOP-GAT increased in 2.31mmHg with D/T (p=0.007) and 2.72 mmHg (p=0.004) with latanoprost and IOP-POBFA increased in 1.74 mmHg (p=0.026) and 3.13 mmHg (p=0.007) respectively. Multiple regression analysis revealed no important blood flow factors as predictors in the increase of IOP. CONCLUSIONS: Strong positive correlation was revealed between IOP-POBFA and IOP-GAT over a time period. Observed tachyphylaxis effects after 1 year under both treatment regimes should be assessed with respect to patient compliance and persistence to treatment.