Cost-effective sequence analysis of 113 genes in 1,192 probands with retinitis pigmentosa and Leber congenital amaurosis

Introduction: Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are two groups of inherited retinal diseases (IRDs) where the rod photoreceptors degenerate followed by the cone photoreceptors of the retina. A genetic diagnosis for IRDs is challenging since >280 genes are associated with these conditions. While whole exome sequencing (WES) is commonly used by diagnostic facilities, the costs and required infrastructure prevent its global applicability. Previous studies have shown the cost-effectiveness of sequence analysis using single molecule Molecular Inversion Probes (smMIPs) in a cohort of patients diagnosed with Stargardt disease and other maculopathies. Methods: Here, we introduce a smMIPs panel that targets the exons and splice sites of all currently known genes associated with RP and LCA, the entire RPE65 gene, known causative deep-intronic variants leading to pseudo-exons, and part of the RP17 region associated with autosomal dominant RP, by using a total of 16,812 smMIPs. The RP-LCA smMIPs panel was used to screen 1,192 probands from an international cohort of predominantly RP and LCA cases. Results and discussion: After genetic analysis, a diagnostic yield of 56% was obtained which is on par with results from WES analysis. The effectiveness and the reduced costs compared to WES renders the RP-LCA smMIPs panel a competitive approach to provide IRD patients with a genetic diagnosis, especially in countries with restricted access to genetic testing.This study received funding from Novartis. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. This work was supported by grants from Foundation Fighting Blindness Career Development Award CDGE-0621-0809-RAD (SR), Foundation Fighting Blindness project program award PPA-0123-0841-UCL (SR and SdB), Retinitis Pigmentosa Fighting Blindness, Fight for Sight UK (RP Genome Project GR586), Ghent University Special Research Fund (BOF20/GOA/023) (EDB and BL); EJP RD Solve-RET EJPRD19-234 (EDB, BL, SB, CR, FC, and SR). EDB (1802220N) and BL (1803816N) are FWO Senior Clinical Investigators of the Research Foundation Flanders (FWO). EDB, BL, SB, FC, and SR are members of ERN-EYE (Framework Partnership Agreement No. 739534)

Ocular function and morphology in humans and rabbits exposed to vigabatrin medication

Vigabatrin is an antiepileptic drug that is known to cause visual field defects and retinal dysfunction measured by full-field electroretinography (ff-ERG). Underlying mechanisms have not yet been elucidated. The aims of this thesis were to investigate parameters of vigabatrin treatment; reversibility of visual dysfunction, prevalence of retinal dysfunction in children, role of drug-dose and evaluation of alternative methods for follow-up. A translational approach was applied, investigating issues in an experimental rabbit model and in clinical investigations. Reversibility of reduced retinal function was studied in our rabbit model, showing reduced cone function both during treatment and after withdrawal of medication. No changes in immuno labelling patterns of vimentin, glial fibrillary acidic protein (GFAP), γ-aminobutyric acid (GABA) and GAD (glutamic acid decarboxylase) were found. Re-evaluation of visual field defects and retinal function in patients five years after withdrawal of vigabatrin demonstrated irreversible reduction of the isolated rod and cone function and the combined rod-cone function. Also the 30 Hz flicker implicit time was altered. Indications of progressive decline of the combined response were observed. No changes in visual fields were found. In the rabbit model, dose-related changes of retinal function (rod, combined rod-cone and oscillatory potentials, OPs) and expression of proteins were found. In rod bipolar cells, protein kinase C-α (PKC- α) labelling showed a reduced number of labelled cells and translocation of enzyme activity. Müller cells showed deviant vimentin labelling. In children, vigabatrin medication was associated with reduced rod and cone function. Alterations of cone responses were found in 24% of treated children. Young children were affected more often, showing reduced retinal function in 33% of the subjects. The younger group had received higher daily drug-doses and had started medication at younger age than the older group. No differences were found concerning cumulative drug-doses. Alternative methods for detection of vigabatrin toxicity were tested, indicating that attenuation of the retinal nerve fibre layer (RNFL) can be detected with optical coherence tomography (OCT) in patients with vigabatrin-attributed visual field defects and ff-ERG alterations. Correlations between RNFL thickness and reduced ff-ERG amplitudes were found. Multi focal visual evoked potentials (mfVEP) were not useful

Reduced macular function in ABCA4 carriers.

To study retinal function and morphology in ABCA4 carriers to investigate if ABCA4 carriership is associated with any functional or morphological changes and, if so, to explore whether certain mutations may be associated with particularly severe alterations

Association between genotype and phenotype in families with mutations in the ABCA4 gene.

To investigate the genotype and phenotype in families with adenosine triphosphate-binding cassette, sub-family A, member 4 (ABCA4)-associated retinal degeneration

Full-field ERG as a predictor of the natural course of ABCA4-associated retinal degenerations

Purpose: To assess retinal function in combination with the retinal structure in ABCA4-associated retinal degenerations. Moreover, to evaluate the possibility of predicting the natural course of these disorders. Methods: 34 patients with Stargardt disease or cone rod dystrophy carrying confirmed mutations in ABCA4 were selected from our retinitis pigmentosa (RP) register. Sequence analysis of the entire coding region of the ABCA4 gene was performed. The patients were subdivided into three groups based on their most recent visual fields. Group 1 included ten patients with central scotomas within 10°, group 2 included 19 patients with larger central scotomas of 10-35°, and group 3 included five patients with mere temporal residues. The patients underwent slit-lamp and fundus examinations, visual acuity testing, optical coherence tomography (OCT), fundus photography (color, red-free, and autofluorescence (AF) images), full-field electroretinography (ffERG), and multifocal electroretinography (mERG). FfERG and mERG results were analyzed statistically. Total rod and cone function, as well as macular function, was compared between the three groups and of each group to a normal material. In 23 patients who had undergone ffERG on a previous occasion, the 30 Hz flicker implicit time (IT) from the first visit was also analyzed. Results: The ffERG statistics revealed significant differences between the groups regarding cone and rod function with group 1 showing the highest amplitudes and the shortest ITs while group 3 demonstrated the lowest amplitudes and the most delayed ITs. When compared to controls, group 1 did not show any significant changes while groups 2 and 3 demonstrated reduced amplitudes and delayed 30 Hz ITs. Regarding estimation of the natural course, identical results of the 30 Hz IT were encountered for the groups also at the first visit early in the course of disease. Comparison of the mERGs showed significant differences with group 1 demonstrating the highest amplitudes and group 3 the lowest for all rings but rings 2 and 3 in the right eye for which the amplitudes were the second highest. The mERGs for each group were also compared to controls showing reduced mERG amplitudes for all rings in all groups, except group 1, left eye. OCT showed macular attenuation in all patients. Evaluation of the inner and outer photoreceptor junction (IS/OS) morphology revealed alterations related to macular function measured with mERG in all eyes. Eight patients in group 1 showed foveal IS/OS junction loss, one had foveal IS/OS junction disorganization, and one had IS/OS loss also beyond the fovea. In group 2, one patient had IS/OS junction loss confined to the fovea, and the rest showed total loss of IS/OS junctions. Group 3 was devoid of IS/OS junctions. Concerning the AF images, group 1 showed small areas of absent AF in the macula, peripapillary sparing, and flecks of increased and reduced AF in the posterior pole. In group 2, the central areas of absent AF were larger. Flecks of reduced AF were the most dominant and reached beyond the posterior pole. Seven of 19 patients had peripapillary sparing. In group 3, large confluent areas of reduced AF were found in the posterior pole and beyond with small areas of increased AF in the far periphery. No peripapillary sparing was seen. Conclusions: The current study demonstrates a significant difference in total retinal function, as well as macular function, between patients with ABCA4-associated retinal degeneration and a different degree of visual field defects with gradual deterioration of function along with increased visual field constriction. Likewise, the morphological changes, including the deviant AF pattern and loss of IS/OS junctions, that were related to macular function measured with mERG worsened with the degree of visual field defects. Moreover, in these groups of patients with ABCA4-associated retinal degenerations, full-field cone 30 Hz flicker IT seems to be a predictor of the natural course of the disease also on long-term follow-up

A five-year follow-up of ABCA4 carriers showing deterioration of retinal function and increased structural changes

Purpose: To investigate whether the reduced retinal function and morphological retinal changes previously demonstrated in ABCA4 carriers had remained stationary or had deteriorated over time at 5-year follow-up to further explore if carriers of an autosomal recessive trait also express a weak phenotype, although this is not expected for an autosomal recessive disorder. Methods: Thirteen ABCA4 carriers from a previous study that included parents to patients with well known genetically verified ABCA4-associated retinal degenerations were reexamined 5 years after the initial examination. As novel genes and new variants in already established genes are continuously reported, all subjects underwent renewed genetic testing with a next-generation sequencing (NGS) panel that included 288 genes associated with retinal dystrophies and an analysis of deep intronic mutations and copy number variations in the ABCA4 gene. Moreover, to evaluate any changes in retinal function and/or structure over time, clinical reassessment with Goldmann perimetry, visual acuity testing, fundus photography, fundus autofluorescence (FAF) imaging, optical coherence tomography (OCT), full-field electro-retinography (ffERG), and multifocal ERG (mf ERG) were performed 5 years after the initial investigation. The values of the ffERG parameters were compared between the two time points (the measurements obtained in the initial study versus the measurements at 5-year follow-up) and with the controls. The mf ERG results of the carriers were compared with those of the controls. Results: The renewed genetic testing confirmed the previously established ABCA4 mutations but also revealed the hypomorph ABCA4 variant c.5603A>T in five ABCA4 carriers. In three of them, the variant was found to be associated with known disease-causing alleles that always carry the c.5603A>T in cis. According to recent publications, the subjects could still be considered ABCA4 carriers because both variants are on the same allele. In the remaining two subjects, c.5603A>T could be in trans with the previously known ABCA4 variant, and the subjects were therefore excluded from the study since they could no longer be considered as carriers only. Statistical comparison of ffERG parameters showed significant reduction of the isolated rod,-as well as the combined rod-cone amplitudes over the five years of follow-up, but not compared with the controls. Concerning macular function, mf ERG amplitudes were reduced for all rings in the carriers compared with the controls. Fundus photographs demonstrated morphological changes in 64% of the carriers, and 36% of them had further changes at follow-up. FAF images showed alterations in 55% of the carriers, with increased changes in 36% of them. Abnormalities on OCT were observed in 82% of the carriers, of whom 9% had newly found abnormalities at follow-up. Conclusions: At 5-year follow-up, the ABCA4 carriers, who previously demonstrated reduced macular function, presented with deterioration of general retinal function, including reduced isolated rod and mixed rod-cone ffERG responses combined with a slight increase in morphological changes in some subjects. This indicates that carriership of at least some ABCA4 variants may cause a condition similar to a subgroup of dry age-related macular degeneration (AMD). In the long run, this might be of importance concerning the possibilities to also treat this subgroup of AMD patients with future gene-based and pharmacological drugs targeting ABCA4-associated disorders

Willingness to wind - A panel data study of public policies and municipal factors determining the deployment of wind power in Swedish municipalities

In 2009 the Swedish government adopted a national planning goal of 30 TWh wind power by 2020, implying a large expansion is needed from the current level of 7 TWh. The municipal territorial planning monopoly implies that the municipalities have a high degree of independence in the decision of installing wind power. This has led to large differences between municipalities; some have a large amount of installed wind power while others have none. This thesis uses panel data from the period 2003-2011, with the aim of analyzing the effect of national policy instruments, market conditions and municipal specific factors on the deployment of wind power in Swedish municipalities. We find a significant effect off two policy instruments, the Green Certificate System and the EU´s Emission Trading Scheme. The results show that more wind power is installed in municipalities with a left wing political majority and a successful tourism industry, but appears unrelated to other municipal factors, such as wind capacity. Our findings suggest that social conditions affect the deployment of wind power, not favorable physical conditions

Electrophysiological evaluation of retinal function in children receiving vigabatrin medication

PURPOSE: To evaluate retinal function in children taking vigabatrin and to explore the influence of age and dose parameters on the results of full-field electroretinography (ff-ERG).METHODS: The ff-ERGs from 14 children receiving vigabatrin were compared with ff-ERGs from healthy controls. Treated children were further grouped according to age (pre-school = 12-71 months; older = 72-228 months). Parameters of drug dosage were compared.RESULTS: Treated children showed rod and cone dysfunction reflected by reduced b-wave amplitudes for the isolated rod response, the combined rod-cone response, and the 30-Hz flicker response. The a-wave amplitude and implicit time for the combined rod-cone response, reflecting photoreceptor function, were also altered. Further evaluation of age groups revealed similar findings in the pre-school group but not in the older group. Alterations in ff-ERG were seen in 57% of the treated children. Pre-school children had received significantly higher daily drug doses with start of medication at younger age. No differences were found concerning cumulative doses or duration of medication.CONCLUSION: Alterations in ff-ERG are as frequent in children as in adults and the results indicate that exposure to high daily doses of vigabatrin may be associated with increased risk of retinal dysfunction, including photoreceptor damage, not previously shown in children. Thus, recommendations of careful follow-up for children receiving vigabatrin are supported

Electrophysiological evaluation and 18-month follow-up of two regimens with aflibercept for neovascular age-related macular degeneration

PURPOSE: To compare two aflibercept treatment regimens and the electrophysiological outcome concerning cone and rod function in age-related macular degeneration (nAMD) over 18 months.METHODS: 41 patients with treatment-naïve nAMD were randomized 1:1 to either arm 1 or 2. Arm 1 received three consecutive monthly aflibercept injections, followed by bimonthly treatment until week 52. Thereafter, a treat-and-extend (TAE) regimen was applied. Arm 2 was treated according to a TAE protocol throughout the 18-month follow-up. We assessed visual acuity (VA), central retinal thickness (CRT), injection rate and interval, and evaluated cone and rod function with full-field and multifocal electroretinography (ffERG, mERG).RESULTS: There were no statistically significant differences in mean baseline VA, lesion type, age, gender, or symptom duration between the two arms. During the 18-month follow-up, mean VA improved in arm 1 (n = 19) from 63.5 ± 10.5 to 69.1 ± 9.2 letters; p = 0.098; and in arm 2 (n = 20) from 66.8 ± 13.6 to 73.9 ± 9.0 letters; p = .002. In both arms, mean CRT was significantly reduced; p < 0.000. At month 18, we found no significant difference in the number of injections or injection intervals between groups. Arm 1 had received 11.3 ± 1.7 injections vs. 10.9 ± 2.0 in arm 2. The mean injection interval was 9.2 ± 3.4 weeks vs. 9.5 ± 3.1, with 52% (n = 10) on the maximum 12-week interval in arm 1, and 50% (n = 10) in arm 2. The combined rod-cone a-wave amplitude significantly decreased over time; p = 0.043. The isolated rod b-wave amplitude showed a statistically significant decline; p = 0.026. The overall mERG amplitude and implicit time remained unchanged over time; p = 0.878 vs. p = 0.922. The central ring 1 mERG amplitude improved; p = 0.041, with an unaffected implicit time.CONCLUSIONS: After 18 months, both treatments arms have received a similar number of injections at comparable intervals. Electrophysiological evaluation shows no signs of toxicity concerning cone function. But ffERGs for the combined and isolated rod response have declined, possibly reflecting either toxic effects of the drug to rods or the natural course of the disease itself