Loss-of-function variants in UBAP1L cause autosomal recessive retinal degeneration.

PURPOSE: Inherited retinal diseases (IRDs) are a group of monogenic conditions that can lead to progressive blindness. Their missing heritability is still considerable, due in part to the presence of disease genes that await molecular identification. The purpose of this work was to identify novel genetic associations with IRDs. METHODS: Patients underwent a comprehensive ophthalmological evaluation using standard-of-care tests, such as detailed retinal imaging (macular optical coherence tomography and short-wavelength fundus autofluorescence) and electrophysiological testing. Exome and genome sequencing, as well as computer-assisted data analysis were used for genotyping and detection of DNA variants. A minigene-driven splicing assay was performed to validate the deleterious effects of 1 of such variants. RESULTS: We identified 8 unrelated families from Hungary, the United States, Israel, and The Netherlands with members presenting with a form of autosomal recessive and nonsyndromic retinal degeneration, predominantly described as rod-cone dystrophy but also including cases of cone/cone-rod dystrophy. Age of disease onset was very variable, with some patients experiencing first symptoms during their fourth decade of life or later. Myopia greater than 5 diopters was present in 5 of 7 cases with available refractive data, and retinal detachment was reported in 2 cases. All ascertained patients carried biallelic loss-of-function variants in UBAP1L (HGNC: 40028), a gene with unknown function and with homologies to UBAP1, encoding a protein involved in ubiquitin metabolism. One of these pathogenic variants, the intronic NM_001163692.2:c.910-7G>A substitution, was identified in 5 unrelated families. Minigene-driven splicing assays in HEK293T cells confirmed that this DNA change is responsible for the creation of a new acceptor splice site, resulting in aberrant splicing. CONCLUSION: We identified UBAP1L as a novel IRD gene. Although its function is currently unknown, UBAP1L is almost exclusively expressed in photoreceptors and the retinal pigment epithelium, hence possibly explaining the link between pathogenic variants in this gene and an ocular phenotype.

Comprehensive variant spectrum of the CNGA3 gene in patients affected by achromatopsia.

Achromatopsia (ACHM) is a congenital cone photoreceptor disorder characterized by impaired color discrimination, low visual acuity, photosensitivity, and nystagmus. To date, six genes have been associated with ACHM (CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, and ATF6), the majority of these being implicated in the cone phototransduction cascade. CNGA3 encodes the CNGA3 subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors and is one of the major disease-associated genes for ACHM. Herein, we provide a comprehensive overview of the CNGA3 variant spectrum in a cohort of 1060 genetically confirmed ACHM patients, 385 (36.3%) of these carrying "likely disease-causing" variants in CNGA3. Compiling our own genetic data with those reported in the literature and in public databases, we further extend the CNGA3 variant spectrum to a total of 316 variants, 244 of which we interpreted as "likely disease-causing" according to ACMG/AMP criteria. We report 48 novel "likely disease-causing" variants, 24 of which are missense substitutions underlining the predominant role of this mutation class in the CNGA3 variant spectrum. In addition, we provide extensive in silico analyses and summarize reported functional data of previously analyzed missense, nonsense and splicing variants to further advance the pathogenicity assessment of the identified variants.

Deep-intronic variants in CNGB3 cause achromatopsia by pseudoexon activation.

Our comprehensive cohort of 1100 unrelated achromatopsia (ACHM) patients comprises a considerable number of cases (~5%) harboring only a single pathogenic variant in the major ACHM gene CNGB3. We sequenced the entire CNGB3 locus in 33 of these patients to find a second variant which eventually explained the patients' phenotype. Forty-seven intronic CNGB3 variants were identified in 28 subjects after a filtering step based on frequency and the exclusion of variants found in cis with pathogenic alleles. In a second step, in silico prediction tools were used to filter out those variants with little odds of being deleterious. This left three variants that were analyzed using heterologous splicing assays. Variant c.1663-1205G>A, found in 14 subjects, and variant c.1663-2137C>T, found in two subjects, were indeed shown to exert a splicing defect by causing pseudoexon insertion into the transcript. Subsequent screening of further unsolved CNGB3 subjects identified four additional cases harboring the c.1663-1205G>A variant which makes it the eighth most frequent CNGB3 variant in our cohort. Compound heterozygosity could be validated in ten cases. Our study demonstrates that whole gene sequencing can be a powerful approach to identify the second pathogenic allele in patients apparently harboring only one disease-causing variant.

Ultrastructure of neurovascular changes in human diabetic retinopathy.

The previous concept regarding diabetic retinopathy assigned a primary role to hyperglycemia-induced microvascular alterations, while neuronal and glial abnormalities were considered to be secondary to either ischemia or exudation. The aim of this study was to reveal the potential role of neuronal and glial cells in initial and advanced alterations of the retinopathy in human type 2 diabetes. Electron microscopy and histochemical studies were performed on 38 surgically removed human eyes (28 obtained from diabetic patients and 10 from non-diabetic patients). Morphometric analysis of basement membrane material and lipids was performed. An accumulation of metabolic by-products was found in the capillary wall with aging: this aspect was significantly more pronounced in diabetics. Müller glial cells were found to contribute to alterations of the capillary wall and to occlusion, as well as to the development of proliferative retinopathy and cystoid degeneration of the retina. Our results showed morphological evidence regarding the role of neuronal and glial cells in the pathology of diabetic retinopathy, prior and in addition to microangiopathy. These morphological findings support a neurovascular pathogenesis at the origin of diabetic retinopathy, thus the current treatment approach should be completed by neuroprotective measures.

Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing.

Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes.

Leber congenital amaurosis: first genotyped Hungarian patients and report of 2 novel mutations in the CRB1 and CEP290 genes.

PURPOSE: To introduce the first Hungarian patients with genetically defined Leber congenital amaurosis (LCA) and to report 2 novel mutations. METHODS: Seven otherwise healthy patients (4-29 years, 5 male and 2 female) who had an onset of severe visual impairment before age 2 years were investigated. The diagnosis was established in all individuals by medical history, funduscopy, and full-field electroretinogram (ERG). Ocular examination included visual acuity testing, digital fundus photography, and in 6 patients retinal imaging with optical coherence tomography (OCT). Arrayed primer extension microarray screening was performed in all probands. In 2 patients, further Sanger sequencing and targeted next-generation sequencing revealed the second disease allele. RESULTS: A cone-rod type LCA was revealed in 4 patients and a rod-cone type disease in 3 patients. Five patients presented with maculopathy. Optical coherence tomography (OCT) imaging showed diffuse retinal thickening in 3 probands with severe macular atrophy in one. Full-field ERGs were undetectable or residual in all patients. Genetic screening revealed AIPL1, CRB1, and CEP290 gene-related pathology in 6 patients; in 1 proband, no mutation was found. Three homozygous and 3 compound heterozygous mutations were identified. Two novel variants were detected: c.2536G>T (p.G846X) in the CRB1 gene and c.4929delA (p.Lys1643fsX2) in the CEP290 gene. CONCLUSIONS: Genetic subtypes identified are among the most common ones in LCA; the phenotypes are consistent with those reported previously. Both novel mutations are predicted to result in a premature translation termination. The phenotype related to the novel CRB1 mutation results in severe atrophic maculopathy.

Large deletions of the KCNV2 gene are common in patients with cone dystrophy with supernormal rod response.

Cone dystrophy with supernormal rod response (CDSRR) is considered to be a very rare autosomal recessive retinal disorder. CDSRR is associated with mutations in KCNV2, a gene that encodes a modulatory subunit (Kv8.2) of a voltage-gated potassium channel. In this study, we found that KCNV2 mutations are present in a substantial fraction (2.2-4.3%) of a sample of 367 independent patients with a variety of initial clinical diagnoses of cone malfunction, indicating that CDSRR is underdiagnosed and more common than previously thought. In total, we identified 20 different KCNV2 mutations; 15 of them are novel. A new finding of this study is the substantial proportion of large deletions at the KCNV2 locus that accounts for 15.5% of the mutant alleles in our sample. We determined the breakpoints and size of all five different deletions, which ranged between 10.9 and 236.8 kb. Two deletions encompass the entire KCNV2 gene and one also includes the adjacent VLDLR gene. Furthermore, we investigated N-terminal amino acid substitution mutations for its effect on interaction with Kv2.1 using yeast two-hybrid technology. We found that these mutations dramatically reduce or abolish this interaction suggesting a lack of assembly of heteromeric Kv channels as one underlying pathomechanism of CDSRR.

Comparison of pattern VEP results acquired using CRT and TFT stimulators in the clinical practice.

There are several electrophysiological systems available commercially. Usually, control groups are required to compare their results, due to the differences between display types. Our aim was to examine the differences between CRT and LCD/TFT stimulators used in pattern VEP responses performed according to the ISCEV standards. We also aimed to check different contrast values toward thresholds. In order to obtain more precise results, we intended to measure the intensity and temporal response characteristics of the monitors with photometric methods. To record VEP signals, a Roland RetiPort electrophysiological system was used. The pattern VEP tests were carried out according to ISCEV protocols on a CRT and a TFT monitor consecutively. Achromatic checkerboard pattern was used at three different contrast levels (maximal, 75, 25%) using 1° and 15' check sizes. Both CRT and TFT displays were luminance and contrast matched, according to the gamma functions based on measurements at several DAC values. Monitor-specific luminance parameters were measured by means of spectroradiometric instruments. Temporal differences between the displays' electronic and radiometric signals were measured with a device specifically built for the purpose. We tested six healthy control subjects with visual acuity of at least 20/20. The tests were performed on each subject three times on different days. We found significant temporal differences between the CRT and the LCD monitors at all contrast levels and spatial frequencies. In average, the latency times were 9.0 ms (±3.3 ms) longer with the TFT stimulator. This value is in accordance with the average of the measured TFT input-output temporal difference values (10.1 ± 2.2 ms). According to our findings, measuring the temporal parameters of the TFT monitor with an adequately calibrated measurement setup and correcting the VEP data with the resulting values, the VEP signals obtained with different display types can be transformed to be comparable.

Toothguide Trainer tests with color vision deficiency simulation monitor.

OBJECTIVES: The aim of this study was to evaluate whether simulated severe red and green color vision deficiency (CVD) influenced color matching results and to investigate whether training with Toothguide Trainer (TT) computer program enabled better color matching results. METHODS: A total of 31 color normal dental students participated in the study. Every participant had to pass the Ishihara Test. Participants with a red/green color vision deficiency were excluded. A lecture on tooth color matching was given, and individual training with TT was performed. To measure the individual tooth color matching results in normal and color deficient display modes, the TT final exam was displayed on a calibrated monitor that served as a hardware-based method of simulating protanopy and deuteranopy. Data from the TT final exams were collected in normal and in severe red and green CVD-simulating monitor display modes. Color difference values for each participant in each display mode were computed (∑ΔE(ab)(*)), and the respective means and standard deviations were calculated. The Student's t-test was used in statistical evaluation. RESULTS: Participants made larger ΔE(ab)(*) errors in severe color vision deficient display modes than in the normal monitor mode. TT tests showed significant (p<0.05) difference in the tooth color matching results of severe green color vision deficiency simulation mode compared to normal vision mode. Students' shade matching results were significantly better after training (p=0.009). CONCLUSIONS: Computer-simulated severe color vision deficiency mode resulted in significantly worse color matching quality compared to normal color vision mode. Toothguide Trainer computer program improved color matching results.

Role of multifocal electroretinography in the diagnosis of idiopathic macular hole.

PURPOSE: To analyze the preoperative results of multifocal electroretinography (mfERG) in the fellow eyes of patients with idiopathic unilateral macular hole and to evaluate the usefulness of this method in predicting the likelihood of macular hole formation in the fellow eye. METHODS: Over a period of 5 years, 80 eyes of 40 patients (mean age, 64.9 years) with unilateral idiopathic macular hole were examined. The diagnosis of idiopathic macular hole was confirmed by optical coherence tomography (OCT). The fellow eyes were intact in all cases. All patients underwent vitreoretinal surgery. Before the surgery, both eyes of the patients were examined by mfERG. During the follow-up period, the 40 fellow eyes were also observed by OCT, and the changes in the vitreofoveal attachment were investigated. The preoperative response densities and ring ratios of mfERG were analyzed in both eyes, and discriminant analysis was used to calculate the best separator function. RESULTS: Preoperative mfERGs demonstrated significantly lower mean response densities in the central area of the 40 eyes with macular hole than in the fellow eyes. During the follow-up period, macular hole was diagnosed in 13 fellow eyes by OCT. The preoperative values of the mfERGs in these eyes were significantly lower than in the other 27 cases. The mfERG ring ratios were significantly lower in the fellow eyes in which macular holes developed than in those that remained intact. CONCLUSIONS: The analysis of ERG in the fellow eyes of patients with macular hole seems clinically useful. The lower amplitude may forecast the propensity for subsequent development of a macular hole. Patients with low central ERG amplitude and lower ring ratios in the healthy fellow eyes should have stricter follow-up.

Truncation of retinoschisin protein associated with a novel splice site mutation in the RS1 gene.

PURPOSE: To present the ocular findings of a Hungarian family with X-linked juvenile retinoschisis (XLRS) and to reveal a novel putative splice mutation leading to serious truncation of retinoschisin (RS1) protein. Our genetic results were compared to a mouse model of XLRS. METHODS: Complete ophthalmic examinations were performed on five members (two male patients, two female carriers, and one healthy fraternal male twin) of the family. The examinations included optical coherence tomography (OCT) and full-field and multifocal electroretinography (mfERG). OCT and ERG results were compared to the normative database of our laboratory. All exons and the flanking intronic regions of the RS1 gene were amplified by polymerase chain reaction and directly sequenced in all family members and in 50 male controls. RESULTS: Typical microcystic foveal changes were found on fundoscopy and OCT in two male patients. Large foveal and smaller perifoveal cysts were detected by OCT in the inner nuclear layer and another deeper retinal cleavage in the photoreceptor layer. The standard combined b-wave amplitudes and b/a amplitude ratios of full-field ERGs of the male patients were decreased compared with controls, but the typical "negative-type" ERG was not observed. The amplitudes of mfERGs were reduced in all rings but mainly in the central part of the examined retina. Implicit times were delayed across almost the whole testing field. Female carriers and the healthy fraternal twin brother were without any symptoms and had normal clinical examination results, but the implicit times of female carriers were delayed in all rings. DNA sequence analyses revealed a novel putative splice mutation (c.78+1G>C) in the splice donor site of intron 2 in RS1 of two male patients and two female carriers. Mutations were absent in the 50 control samples. CONCLUSIONS: Male patients exhibited typical bilateral foveal retinoschisis in two retinal layers and characteristic ERG changes. The inheritance of the novel putative splice mutation (c.78+1G>C) followed the classic inheritance of an X-linked recessive disease in two male patients and two female obligate carriers. There are two possible ways the c.78+1G>C splice site mutation may lead to frameshift and introduce a premature termination codon at the beginning of exon 3: after activation of the next cryptic splice site by a 10 bp insertion or after exon skipping by a 26 bp deletion. The splice site mutation in the second intron of RS1 identified in these XLRS patients is practically identical to the N-ethyl-N-nitrosourea (ENU) induced splice site mutation in the mouse model of XLRS described by the Tennessee Mouse Genome Consortium. The genetic findings of the mutant mouse model confirm and support our human results.

A comparison of macular structure imaged by optical coherence tomography in preterm and full-term children.

PURPOSE: Macular anatomic abnormalities were examined by optical coherence tomography (OCT) imaging in premature children and compared with those of full-term children. METHODS: In a prospective case-control study, premature patients 7 to 14 years of age were divided into three groups (group I, laser-treated retinopathy of prematurity [ROP]; group II, spontaneously regressed ROP; group III, no ROP), and age-matched children (group IV). All the eligible 74 eyes had normal-appearing posterior pole, myopia < or =3 D, and best corrected visual acuity 1.0. When both eyes of a subject were eligible for the study, one eye was randomly selected (10 eyes of 10 children in each group). Retinal thicknesses of the macula measured by OCT3 were compared. The correlation between central foveal thickness and prematurity (gestational age at birth < or = 30 weeks; birth weight < or = 1250 g) or ROP was determined. RESULTS: The mean foveal and central retinal thicknesses decreased significantly in group I (laser-treated ROP) and group IV (term birth). Significant differences in central retinal thickness were found between the premature groups and full-term children (Mann-Whitney U test). The cutoff point of central retinal thickness, determined by receiver operating characteristic curve was 209 microm. The general estimating equation model statistics found a significant effect of ROP severity (P = 0.003), P value for the category of prematurity was 0.063. CONCLUSIONS: The central retinal thickness was significantly higher in the preterm groups than in the full-term group. This subtle macular modification may be related mainly to ROP. Prematurity had only a marginally significant role.

Optical coherence tomography of the macula in congenital achromatopsia.

PURPOSE: It is known that symptoms of congenital achromatopsia are caused by the lack of functioning cones, but there are very few published data on histologic changes in the retina in these cases. This study was conducted to examine in vivo the anatomic structure of the retina of patients with achromatopsia. METHODS: Fifteen eyes of eight patients with congenital achromatopsia and 18 eyes of nine control subjects were examined by optical coherence tomography. Radial 6-mm scans were taken of the macula. The thickness of the neuroretina was measured both automatically and manually. Measurements were taken at the foveola and at distances of 1.5 and 3 mm. Total macular volume and the retinal thickness in the nine ETDRS regions were also calculated. RESULTS: In patients with achromatopsia, statistically significant reductions were found in total macular volume and in the thickness of the central retina. Remarkable differences were found between the results obtained from different methods of measuring retinal thickness. Automated methods underestimated retinal thickness compared with manual measurements. CONCLUSIONS: The structure of the macula in achromats differs from that in normal subjects. A possible reason for the structural alteration is the qualitative and/or quantitative disorder of the cone photoreceptors, as the morphologic change is most expressed in the foveola. The automated methods are not always suitable for measuring retinal thickness in the foveola. The structural changes seen in the central retina of the patients could provide useful information for future gene therapy.

Clinical and genetic features of Hungarian achromatopsia patients.

PURPOSE: To describe the clinical features and molecular genetic findings in a collection of Hungarian achromatopsia patients. METHODS: Twelve patients with congenital achromatopsia from nine Hungarian families were analyzed in this study. The patients underwent standard ophthalmological examination including detailed full-field electroretinography and color vision testing. In two patients, dark adaptation and spectral luminosity tests were also performed. PCR/RFLP analysis and DNA sequencing was applied for mutation screening of CNGA3 and CNGB3. Heterologous minigene expression was used to evaluate transcript splicing of a new intronic mutation in CNGB3. RESULTS: Mutations in CNGA3 were present in four families and mutations in CNGB3 in the remaining five families, including mutations known from Western European patient samples and two new CNGB3 mutations: c.112C>T/Gln38X and c.1663-5T>G. Heterologous expression in COS7 cells shows that the latter induces a splicing defect through the activation of a cryptic splice site 4 bases upstream of the genuine splice site. The patients presented with a clinical picture typical for congenital achromatopsia and there was no significant difference in the phenotype of subjects with either CNGA3 or CNGB3 mutations based on standard ophthalmological examination. However, we assume residual cone function in a subject homozygous for the Phe547Leu mutation in CNGA3 based on prior detailed psychophysical testing (i.e., dark adaptation and spectral luminosity). CONCLUSIONS: Mutations in CNGA3 and CNGB3 account for achromatopsia in Hungarian patients including known mutations and a few new CNGB3 mutations. While standard ophthalmological examination revealed a phenotype of complete achromatopsia, we show that thorough psychophysical testing can help to identify subjects with some minute cone function.

CNGB3 mutations account for 50% of all cases with autosomal recessive achromatopsia.

Achromatopsia is a congenital, autosomal recessively inherited disorder characterized by a lack of color discrimination, low visual acuity (<0.2), photophobia, and nystagmus. Mutations in the genes for CNGA3, CNGB3, and GNAT2 have been associated with this disorder. Here, we analyzed the spectrum and prevalence of CNGB3 gene mutations in a cohort of 341 independent patients with achromatopsia. In 163 patients, CNGB3 mutations could be identified. A total of 105 achromats carried apparent homozygous mutations, 44 were compound (double) heterozygotes, and 14 patients had only a single mutant allele. The derived CNGB3 mutation spectrum comprises 28 different mutations including 12 nonsense mutations, eight insertions and/or deletions, five putative splice site mutations, and three missense mutations. Thus, the majority of mutations in the CNGB3 gene result in significantly altered and/or truncated polypeptides. Several mutations were found recurrently, in particular a 1 bp deletion, c.1148delC, which accounts for over 70% of all CNGB3 mutant alleles. In conclusion, mutations in the CNGB3 gene are responsible for approximately 50% of all patients with achromatopsia. This indicates that the CNGB3/ACHM3 locus on chromosome 8q21 is the major locus for achromatopsia in patients of European origin or descent.