Purpose:To identify and characterize a novel gene variant associated with late-onset, non-syndromic retinitis pigmentosa (RP). Methods:Ophthalmological examination included Goldmann perimetry, optical coherence tomography, fundus autofluorescence and full-field electroretinography. Gene variants were identified using whole exome sequencing. Expression of ATRAID in fibroblasts was studied using immunoblot analysis and quantitative PCR. Lysosomes were identified in ATRAID-/- and SLC37A3-/- cells using immunofluorescence analysis. Uptake of fluorescent dextran was measured using immunofluorescence analysis and flow cytometry. Results:We first examined two siblings whose parents were second cousins. Ophthalmological examination revealed symmetrical midperipheral chorioretinal atrophy with bone spicules and vessel attenuation and a corresponding rod-cone dysfunction on full-field electroretinography consistent with RP. Whole exome sequencing identified a novel homozygous variant in the ATRAID gene (NM_001170795, c.120dup, p.Ser41Glufs*13) within a 12.45 Mb region of homozygosity, predicted to result in a premature stop codon. Immunoblot analysis confirmed protein absence, indicating a loss of function variant. Further analysis of individuals with similar clinical features identified this variant in a homozygous state in four additional affected individuals in three non-consanguineous families. ATRAID has been shown to interact with SLC37A3 in lysosomes. Recently, loss of SLC37A3 has been associated with non-syndromic RP with features resembling those seen in loss of ATRAID. Increased numbers of lysosomes and accumulation of fluorescein-labelled dextran were observed both in ATRAID-/- and SLC37A3-/- cells. Conclusions:We propose that biallelic loss of ATRAID causes RP through the disruption of ATRAID and SLC37A3 interaction resulting in impaired endocytic trafficking and lysosomal function.

DSCAM occupies a 1-Mb locus in the original Down syndrome critical region on chromosome 21q22 and encodes a neuronal cell adhesion molecule of importance for brain and eye development. Singleton individuals, both born to first-cousin parents, with intellectual disability and homozygous DSCAM loss-of-function variants were reported in 2017 and in 2021, the latter also presenting with nystagmus and visual impairment. We present a cohort of five individuals, four new, including two sibling pairs with homozygosity or compound heterozygosity for predicted loss-of-function DSCAM variants. We identify a common clinical pattern of moderate to severe neurodevelopmental delay with poor language development, risk of focal seizures with onset in infancy, and nystagmus with poor vision. Electroretinography in two of the affected revealed cone-pathway dysfunction with a b-wave pattern indicating main dysfunction at the level of the cone-associated bipolar cells of the central retina. Our electroclinical findings are in line with previous DSCAM knockout chicken and mice studies that evidenced disturbed horizontal and vertical patterning of the retina. Taken together, we delineate a rare syndromic form of recessive intellectual disability with a distinctive type of visual impairment.

Heterozygous activating variants in platelet-derived growth factor receptor beta (PDGFRβ) are associated with ultra-rare and clinically heterogeneous conditions, including KOGS (Kosaki Overgrowth Syndrome), PS (Penttinen Syndrome) and related conditons. Tyrosine kinase inhibitors (TKIs), which are widely used in hematological and oncological diseases, are now being explored as potential treatments for these conditions. While four published cases have reported encouraging results, there is still insufficient data available to draw definitive conclusions on the benefit-risk ratio. The international consortium Knowing and Treating KOGS/PS was launched to assess the real-life outcomes of such treatments. The consortium presents four new cases and updates four previously published cases of KOGS/PS treated with TKIs from seven countries. A recent publication was also included in the analysis. Individuals received treatment for between three and a half months and eight years (imatinib N = 8/8, dasatinib N = 3/8, sunitinib N = 1/8), with a mean duration of 44.4 months (SD = 29.8). The age at treatment initiation ranged from 6 to 57 years (six patients treated in childhood and two in adulthood). All individuals showed improvement within weeks/months, with minimal side effects. However, efficacy decreased over time in some cases, prompting a switch to a different TKI. These preliminary findings highlight the potential of TKIs for managing KOGS/PS patients. Standardized follow-up protocols and an electronic Case Report Form (eCRF) have been implemented to enhance monitoring and data collection, enabling systematic comparisons between treated and untreated individuals despite the disorders' rarity.

Aims/Purpose: This study evaluates the efficacy of topical dasatinib and axitinib for treating experimentally induced corneal neovascularization (CNV) in a mouse alkali burn model, comparing these treatments to established therapies like dexamethasone and bevacizumab. The study aims to determine which treatment provides superior efficacy in inhibiting CNV in this model.Methods: Thirty‐six C57BL/6; 129Sv mice underwent a standardized alkali burn to induce CNV in both eyes using a paper disc soaked in 1M NaOH. The mice were randomly assigned to one of six treatment groups: saline (0.9% sodium chloride), DMSO (5%), dexamethasone (0.1%), bevacizumab (0.5%), dasatinib (0.5%), or axitinib (0.5%). Treatments were applied topically three times daily. CNV assessments, including corneal neovascularization area (CNA), vessel length index (VLI), and limbus vasculature thickness, were conducted post‐mortem using corneal flat‐mounts stained with CD31 antibody for immunohistochemistry.Results: Dexamethasone proved the most effective in inhibiting alkali burn‐induced CNA (p < 0.0001), with bevacizumab showing comparable efficacy (p < 0.001). Axitinib also effectively reduced CNA (p < 0.001) and VLI (p < 0.01). In contrast, dasatinib did not significantly reduce CNA (p = 0.74) or VLI (p = 0.98).Conclusions: While dexamethasone was most effective, both bevacizumab and axitinib successfully reduced CNA, with axitinib additionally decreasing VLI. Axitinib has a broad‐spectrum inhibition of vascular endothelial growth factor receptors 1, 2, and 3 (VEGFR1, VEGFR2, VEGFR3), and platelet‐derived growth factor receptor β (PDGFRβ). This may offer superior benefits compared to narrower‐spectrum TKIs like dasatinib, which primarily target PDGFRβ and fibroblast growth factor receptor (FGFR). The results highlight the effectiveness of targeting the VEGF‐VEGFR pathway for managing corneal neovascularization, presenting a viable option for patients who are sensitive to steroids.

BACKGROUND:5 years have passed since the formation of the multidisciplinary consortium 'Knowing & Treating Kosaki and Penttinen Syndromes', two ultra-rare degenerative multisystem syndromes caused by heterozygous activating variants in PDGFRB. Neurological, orthopaedic and vascular deterioration can occur. Case reports of patients treated with tyrosine kinase inhibitors (TKIs) suggest that these drugs may be a therapeutic option in the future. The bi-annual remote meetings provide an opportunity to share knowledge on these syndromes. MATERIAL AND METHODS:The consortium has validated the communication process, standardised follow-up guidelines, established a database to improve the natural history of these syndromes and evaluated the real-world safety and efficacy profile of TKIs by comparing treated and untreated patients. The regulatory framework is in place. RESULTS:As of November 2024, 18 teams in 13 countries have joined the consortium. More than 25 patients have been identified worldwide, either published or unpublished; 7 of them were treated with a TKI. The guidelines include retrospective and prospective sections for each organ affected by the disease and are based on literature and expert opinion. They also include recommendations to standardise the assessment of the efficacy and safety of treatments prescribed under compassionate use. CONCLUSION:The consortium welcomes new teams on an ongoing basis. Recommendations are especially useful in such ultra-rare degenerative diseases. The real-life observational study seems to be an appropriate model to improve knowledge, including the assessment of treatment efficacy when the prevalence of the disease does not allow the setting up of clinical trials.

Platelet-derived growth factor receptor-beta (PDGFRβ) is a receptor tyrosine kinase that plays significant roles in cell growth, proliferation, and differentiation. Germline variants of PDGFRB can lead to several different diseases, e.g. infantile myofibromatosis, Kosaki overgrowth syndrome, Penttinen premature aging syndrome, ocular pterygium – digital keloid dysplasia, primary familial brain calcification, and others. Some variants cause the kinase to be constitutively active, even in the absence of ligand, while others lead to inactivation of signaling transduction mechanisms. Constitutive activation of PDGFRβ leads to increased cell growth, proliferation, and differentiation, which can lead to the development of tumors or other abnormal growths. The development of new therapies that target PDGFRβ is an active area of research, primarily in cancer treatment. However, these therapies have the potential to also provide effective treatment options for patients with germline variants of PDGFRB. Here, we provide a summary of recurrent activating germline variants reported in PDGFRB and examine their sensitivity to different tyrosine kinase inhibitors. We show that the respective amino acid substitutions respond differently to treatment with tyrosine kinase inhibitors that correlate with previous in vivo data. Our data may assist healthcare providers when deciding personalized treatment of patients with disorders associated with activating variants in PDGFRB.

Introduction:Biallelic pathogenic variants in the ABCA4 gene are the leading cause of inherited retinal diseases. Over 1,200 pathogenic or likely pathogenic ABCA4 variants have been reported, resulting in a broad clinical spectrum of ABCA4-retinal dystrophies (ABCA4-RD), with Stargardt disease being the most common. Most patients with ABCA4-RD are compound heterozygotes, carrying two pathogenic ABCA4 variants in trans. Case Presentation:We report 2 unrelated patients with early-onset (≤12 years) Stargardt disease, both found to be homozygous for a complex ABCA4 allele containing the hypomorphic c.5882G>A p.(Gly1961Glu) variant and the c.634C>T p.(Arg212Cys) variant. Both patients underwent detailed clinical assessment and genetic screening, including whole exome or genome sequencing. In vitro assays were performed to assess the individual and combined effect of these variants on the ABCA4 protein. The identified ABCA4 variants were expressed in HEK293FT and HeLa cells to assess their protein expression levels and intracellular localization compared to the wild type (WT) ABCA4 protein. Molecular analysis revealed that the Arg212Cys variant and the doubly mutated allele showed similarly reduced protein expression, while Gly1961Glu expressed close to WT level. Both variants, individually and combined, localized to intracellular vesicles similarly to WT ABCA4. Conclusion:This study highlights the genetic complexity of ABCA4-RD and the significance of pathogenic variants in cis. It also emphasizes the challenge of accurately predicting the functional consequences of specific ABCA4 alleles with in vitro assays.

Four different amino acid substitutions have been reported at the p.Asn666 position in platelet-derived growth factor receptor β (PDGFRβ): p.Asn666Lys, p.Asn666Tyr, p.Asn666Ser, and p.Asn666His. All four substitutions result in strikingly different phenotypes, ranging from somatic infantile myofibromatosis in p.Asn666Lys and ocular pterygium-digital keloid dysplasia in p.Asn666Tyr to a severe form of Penttinen syndrome in p.Asn666Ser, while p.Asn666His is associated with a complex phenotype characterized by debilitating hand and foot contractures and facial coarseness. Here, we show that the p.Asn666Lys, p.Asn666Tyr, and p.Asn666His substitutions result in increased total PDGFRβ phosphorylation at 32°C compared to 37°C. All four substitutions exhibit distinct activation patterns of specific PDGFRβ tyrosine residues at both temperatures, indicating a unique activation of each variant. The temperature effect on downstream signaling is present across all substitutions, resulting in substitution-specific downstream signaling at both 37°C and 32°C. This complex interplay of downstream signaling proteins could be important for the clinical manifestations of p.Asn666 PDGFRB variants. Furthermore, variant-specific overactivation of tyrosine residues and downstream signaling at 32°C emphasize the importance of temperature as an environmental factor in the pathogenesis of this diverse group of disorders.

PURPOSE:To evaluate the efficacy of topical dasatinib and axitinib for treating experimentally induced corneal neovascularization (CNV) in a mouse alkali burn model, and to compare these treatments to established therapies like dexamethasone and bevacizumab. METHODS:Thirty-six C57BL/6; 129 Sv mice underwent a standardized alkali burn to induce CNV in both eyes by applying a paper disc soaked in 1M NaOH to the cornea for 20 seconds. The mice were randomly assigned to one of 6 treatment groups: saline (0.9% sodium chloride), DMSO (5%), dexamethasone (0.1%), bevacizumab (0.5%), dasatinib (0.5%), or axitinib (0.5%). Treatments were applied topically 3 times daily. After 2 weeks of treatment, the mice were sacrificed. CNV assessments, including corneal neovascularization area (CNA), vessel length index (VLI), and limbus vasculature thickness, were conducted postmortem using corneal flat-mounts stained with a CD31 antibody for immunohistochemistry. RESULTS:Dexamethasone proved the most effective in inhibiting alkali burn-induced CNA ( P < 0.0001), with bevacizumab showing comparable efficacy ( P < 0.001). Axitinib also effectively reduced CNA ( P < 0.001) and VLI ( P < 0.01). In contrast, dasatinib did not significantly reduce CNA ( P = 0.74) or VLI ( P = 0.98). All eyes in the dexamethasone group developed cataracts compared with 25%-41.7% in the other groups. CONCLUSIONS:Axitinib reduced CNA and VLI, although not as effectively as other established treatment modalities, whereas dasatinib did not demonstrate significant effects.

OBJECTIVE:Kosaki overgrowth syndrome (KOGS) is a rare genetic disorder linked to germline variants in the platelet-derived growth factor receptor beta gene ( PDGFRB ) that is characterized by postnatal overgrowth, hyperelastic skin, lipodystrophy, and craniofacial anomalies. This study aimed to summarize clinical and genetic data from reported KOGS cases and investigate the molecular consequences of two recurrent KOGS-associated PDGFRB variants. METHODS:Clinical and genetic information from 17 previously published KOGS cases was reviewed. Molecular studies were performed using patient-derived fibroblasts and genetically modified cells transduced with the recurrent PDGFRB variants Trp566Arg and Pro584Arg. Downstream signaling and phosphorylation of PDGFRβ tyrosine residues were assessed by immunoblotting and immunocytochemistry. RESULTS:Both variants induced phosphorylation of specific PDGFRβ tyrosine residues and activated downstream signaling pathways in the absence of ligand stimulation, which could contribute to the phenotypic overgrowth observed in KOGS. Immunocytochemistry revealed vesicle-like structures of phosphorylated PDGFRβ (pY740), resembling wild-type cells stimulated with growth factor, thereby supporting the constitutive activation of PDGFRβ in patient fibroblasts. Both variants showed sensitivity to the tyrosine kinase inhibitor imatinib. CONCLUSION:Recurrent PDGFRB variants in KOGS cause ligand-independent activation of PDGFRβ, contributing to the overgrowth phenotype. Imatinib may represent a potential targeted therapeutic option.

Purpose: The purpose of this study was to highlight characteristic clinical and microscopic findings and report the long-term follow-up of pediatric excimer laser-assisted penetrating keratoplasty (excimer-PKP) for congenital stromal corneal dystrophy (CSCD). Methods: A 2-year-old Greek child presented with CSCD at our department. Clinical examination showed bilateral flake-like whitish corneal opacities affecting the entire corneal stroma up to the limbus. Genetic testing identified a mutation of the decorin gene (c.962delA). The variant was not present in the parents and represented a de novo mutation. The uncorrected visual acuity was 20/100 in both eyes. Excimer-PKP (8.0/8.1 mm) was performed on the right eye at the age of 2.5 years and on the left eye at the age of 3 years. Postoperatively, alternating occlusion treatment was performed. Results: The light microscopic examination demonstrated a disorganized extracellular matrix of the corneal stroma characterized by a prominent irregular arrangement of stromal collagen lamellae with large interlamellar clefts containing ground substance, highlighted by periodic acid-Schiff- and Alcian blue-positive reaction detecting acid mucopolysaccharides. Electron microscopy showed disorganization and caliber variation of collagen lamellae and thin filaments within an electron-lucent ground substance. The postoperative course was unremarkable. Both grafts remained completely clear 14 years postoperatively. Corneal tomography showed moderate regular astigmatism with normal corneal thickness. The corrected distance visual acuity was 20/25 in both eyes. Conclusions: Excimer-PKP for CSCD might be associated with excellent long-term results and a good prognosis, particularly when the primary surgery is performed at a very young age. However, this requires close postoperative follow-up examinations by an experienced pediatric ophthalmologist to avoid severe amblyopia.

PURPOSE:The International Committee for the Classification of Corneal Dystrophies (IC3D) was created in 2005 to develop a new classification system integrating current information on phenotype, histopathology, and genetic analysis. This update is the third edition of the IC3D nomenclature.METHODS:Peer-reviewed publications from 2014 to 2023 were evaluated. The new information was used to update the anatomic classification and each of the 22 standardized templates including the level of evidence for being a corneal dystrophy [from category 1 (most evidence) to category 4 (least evidence)].RESULTS:Epithelial recurrent erosion dystrophies now include epithelial recurrent erosion dystrophy, category 1 ( COL17A1 mutations, chromosome 10). Signs and symptoms are similar to Franceschetti corneal dystrophy, dystrophia Smolandiensis, and dystrophia Helsinglandica, category 4. Lisch epithelial corneal dystrophy, previously reported as X-linked, has been discovered to be autosomal dominant ( MCOLN1 mutations, chromosome 19). Classic lattice corneal dystrophy (LCD) results from TGFBI R124C mutation. The LCD variant group has over 80 dystrophies with non-R124C TGFBI mutations, amyloid deposition, and often similar phenotypes to classic LCD. We propose a new nomenclature for specific LCD pathogenic variants by appending the mutation using 1-letter amino acid abbreviations to LCD. Pre-Descemet corneal dystrophies include category 1, autosomal dominant, punctiform and polychromatic pre-Descemet corneal dystrophy (PPPCD) ( PRDX3 mutations, chromosome 10). Typically asymptomatic, it can be distinguished phenotypically from pre-Descemet corneal dystrophy, category 4. We include a corneal dystrophy management table.CONCLUSIONS:The IC3D third edition provides a current summary of corneal dystrophy information. The article is available online at https://corneasociety.org/publications/ic3d .

Rubinstein-Taybi syndrome (RTS) is an archetypical genetic syndrome that is characterised by intellectual disability, well-defined facial features, distal limb anomalies and atypical growth, among numerous other signs and symptoms. It is caused by variants in either of two genes (CREBBP,EP300) which encode for the proteins CBP and p300, which both have a function in transcription regulation and histone acetylation. As a group of international experts and national support groups dedicated to the syndrome, we realised that marked heterogeneity currently exists in clinical and molecular diagnostic approaches and care practices in various parts of the world. Here, we outline a series of recommendations that document the consensus of a group of international experts on clinical diagnostic criteria for types of RTS (RTS1:CREBBP; RTS2:EP300), molecular investigations, long-term management of various particular physical and behavioural issues and care planning. The recommendations as presented here will need to be evaluated for improvements to allow for continued optimisation of diagnostics and care.

Aberrant cholesterol metabolism causes neurological disease and neurodegeneration, and mitochondria have been linked to perturbed cholesterol homeostasis via the study of pathological mutations in the ATAD3 gene cluster. However, whether the cholesterol changes were compensatory or contributory to the disorder was unclear, and the effects on cell membranes and the wider cell were also unknown. Using patient-derived cells, we show that cholesterol perturbation is a conserved feature of pathological ATAD3 variants that is accompanied by an expanded lysosome population containing membrane whorls characteristic of lysosomal storage diseases. Lysosomes are also more numerous in Drosophila neural progenitor cells expressing mutant Atad3, which exhibit abundant membrane-bound cholesterol aggregates, many of which co-localize with lysosomes. By subjecting the Drosophila Atad3 mutant to nutrient restriction and cholesterol supplementation, we show that the mutant displays heightened cholesterol dependence. Collectively, these findings suggest that elevated cholesterol enhances tolerance to pathological ATAD3 variants; however, this comes at the cost of inducing cholesterol aggregation in membranes, which lysosomal clearance only partly mitigates.

Purpose: Biallelic pathogenic variants in the gene encoding the ATP-binding cassette transporter ABCA4 are the leading cause of irreversible vision loss in inherited retinal dystrophies (IRDs). Interpretation of ABCA4 variants is challenging, due to cis-modifying and hypomorphic variants. We have previously detected 10 missense variants of unknown significance (VUS) in patients with suspected ABCA4-retinal dystrophies (ABCA4-RDs) in Norway. In this study, we functionally characterized the VUS to aid interpretation of the variants and to determine if they are associated with the disease. Methods: The ABCA4 VUS were expressed in HEK293T cells and the ABCA4 expression level and ATPase activity were determined and correlated with the patients' phenotype. The functional data further used for reclassification of the VUS following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Of the 10 VUSs, 2 variants, Cys205Phe and Asn415Thr, were categorized as functionally severe. The age at presentation in the 2 patients carrying these variants was divergent and seemed to be driven by the patients' second pathogenic variants Gly1961Glu and c.5461-10T>C, respectively. Three variants, Val643Gly, Pro799Leu, and Val1433Ile were categorized as functionally moderate, and were found in patients with intermediate/late age at presentation. The remaining five variants were categorized as functionally normal/mild. Based on our data, c.614G>T p.(Cys205Phe), c.1244A>C p.(Asn415Thr), and c.2396C>T p.(Pro799Leu) were reclassified to (likely) pathogenic, while 4 of the functionally normal/mild variants could be reclassified to likely benign. Conclusions: Functional analyses of ABCA4 variants are a helpful tool in variant classification and enable us to better predict the disease severity in patients with ABCA4-RDs.

Purpose:The purpose of this study was to identify the genetic cause of aggressive corneal vascularization in otherwise healthy children in one family. Further, to study molecular consequences associated with the identified variant and implications for possible treatment.Methods:Exome sequencing was performed in affected individuals. HeLa cells were transduced with the identified c.1643C>A, p.(Ser548Tyr) variant in the platelet-derived growth factor receptor beta gene (PDGFRB) or wild-type PDGFRB. ELISA and immunoblot analysis were used to detect the phosphorylation levels of PDGFRβ and downstream signaling proteins in untreated and ligand-stimulated cells. Sensitivity to various receptor tyrosine kinase inhibitors (TKIs) was determined.Results:A novel c.1643C>A, p.(Ser548Tyr) PDGFRB variant was found in affected family members. HeLa cells transduced with this variant did not have increased baseline levels of phosphorylated PDGFRβ. However, upon stimulation with ligand, excessive activation of PDGFRβ was observed compared to cells transduced with the wild-type variant. PDGFRβ with the p.(Ser548Tyr) amino acid substitution was successfully inhibited with tyrosine kinase inhibitors (axitinib, dasatinib, imatinib, and sunitinib) in vitro.Conclusions:A novel c.1643C>A, p.(Ser548Tyr) PDGFRB variant was found in family members with isolated corneal vascularization. Cells transduced with the newly identified variant showed increased phosphorylation of PDGFRβ upon ligand stimulation. This suggests that PDGF-PDGFRβ signaling in these patients leads to overactivation of PDGFRβ, which could lead to abnormal wound healing of the cornea. The examined TKIs prevented such overactivation, introducing the possibility for targeted treatment in these patients.

Ocular pterygium–digital keloid dysplasia (OPDKD) is a rare hereditary disease characterized by corneal ingrowth of vascularized conjunctival tissue early in life. Later, patients develop keloids on fingers and toes but are otherwise healthy. In a recently described family with OPDKD, we report the presence of a de novo c.770C > T, p.(Thr257Ile) variant in PELI2 in the affected individual. PELI2 encodes for the E3 ubiquitin ligase Pellino‐2. In transgenic U87MG cells overexpressing Pellino‐2 with the p.(Thr257Ile) amino acid substitution, constitutive activation of the NLRP3 inflammasome was observed. However, the Thr257Ile variant did not affect Pellino‐2 intracellular localization, its binding to known interaction partners, nor its stability. Our findings indicate that constitutive autoactivation of the NLRP3 inflammasome contributes to the development of PELI2‐associated OPDKD.