Retinoblastoma is the most common intraocular cancer in children. While the primary tumor can often be treated by local or systemic chemotherapy, metastatic dissemination is generally resistant to ...therapy and remains a leading cause of pediatric cancer death in much of the world. In order to identify new therapeutic targets in aggressive tumors, we sequenced RNA transcripts in five snap frozen retinoblastomas which invaded the optic nerve and five which did not. A three-fold increase was noted in mRNA levels of ACVR1C/ALK7, a type I receptor of the TGF-β family, in invasive retinoblastomas, while downregulation of DACT2 and LEFTY2, negative modulators of the ACVR1C signaling, was observed in most invasive tumors. A two- to three-fold increase in ACVR1C mRNA was also found in invasive WERI Rb1 and Y79 cells as compared to non-invasive cells in vitro. Transcripts of ACVR1C receptor and its ligands (Nodal, Activin A/B, and GDF3) were expressed in six retinoblastoma lines, and evidence of downstream SMAD2 signaling was present in all these lines. Pharmacological inhibition of ACVR1C signaling using SB505124, or genetic downregulation of the receptor using shRNA potently suppressed invasion, growth, survival, and reduced the protein levels of the mesenchymal markers ZEB1 and Snail. The inhibitory effects on invasion, growth, and proliferation were recapitulated by knocking down SMAD2, but not SMAD3. Finally, in an orthotopic zebrafish model of retinoblastoma, a 55% decrease in tumor spread was noted (p = 0.0026) when larvae were treated with 3 µM of SB505124, as compared to DMSO. Similarly, knockdown of ACVR1C in injected tumor cells using shRNA also resulted in a 54% reduction in tumor dissemination in the zebrafish eye as compared to scrambled shRNA control (p = 0.0005). Our data support a role for the ACVR1C/SMAD2 pathway in promoting invasion and growth of retinoblastoma.
PurposeThe 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.MethodsExome 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.ResultsA 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.ConclusionsA 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.
Abstract
Ocular pterygium-digital keloid dysplasia (OPDKD) presents in childhood with ingrowth of vascularized connective tissue on the cornea leading to severely reduced vision. Later the patients ...develop keloids on digits but are otherwise healthy. The overgrowth in OPDKD affects body parts that typically have lower temperature than 37°C. We present evidence that OPDKD is associated with a temperature sensitive, activating substitution, p.(Asn666Tyr), in PDGFRB. Phosphorylation levels of PDGFRB and downstream targets were higher in OPDKD fibroblasts at 37°C but were further greatly increased at the average corneal temperature of 32°C. This suggests that the substitution cause significant constitutive autoactivation mainly at lower temperature.
In contrast, a different substitution in the same codon, p.(Asn666Ser), is associated with Penttinen type of premature aging syndrome. This devastating condition is characterized by widespread tissue degeneration, including pronounced chronic ulcers and osteolytic resorption in distal limbs. In Penttinen syndrome fibroblasts, equal and high levels of phosphorylated PDGFRB was present at both 32°C and 37°C. This indicates that this substitution causes severe constitutive autoactivation of PDGFRB regardless of temperature. In line with this, most downstream targets were not affected by lower temperature. However, STAT1, important for tissue wasting, did show further increased phosphorylation at 32°C. Temperature-dependent autoactivation offers an explanation to the strikingly different clinical outcomes of substitutions in the Asn666 codon of PDGFRB.
Using a positional cloning approach supported by comparative genomics, we have identified a previously unreported gene, EYS, at the RP25 locus on chromosome 6q12 commonly mutated in autosomal ...recessive retinitis pigmentosa. Spanning over 2 Mb, this is the largest eye-specific gene identified so far. EYS is independently disrupted in four other mammalian lineages, including that of rodents, but is well conserved from Drosophila to man and is likely to have a role in the modeling of retinal architecture.
Retinal dystrophy (RD) is a heterogeneous group of hereditary diseases caused by loss of photoreceptor function and contributes significantly to the etiology of blindness globally but especially in ...the industrialized world. The extreme locus and allelic heterogeneity of these disorders poses a major diagnostic challenge and often impedes the ability to provide a molecular diagnosis that can inform counseling and gene-specific treatment strategies. In a large cohort of nearly 150 RD families, we used genomic approaches in the form of autozygome-guided mutation analysis and exome sequencing to identify the likely causative genetic lesion in the majority of cases. Additionally, our study revealed six novel candidate disease genes (C21orf2, EMC1, KIAA1549, GPR125, ACBD5, and DTHD1), two of which (ACBD5 and DTHD1) were observed in the context of syndromic forms of RD that are described for the first time.
Our aim is to the report the clinical and histopathological features of benign reactive lymphoid hyperplasia (BRLH) of the conjunctiva in children and the outcomes of treatment.
A retrospective chart ...review was performed for children aged 0-18 years, diagnosed with conjunctival BRLH from January 2000 to December 2013 at two large ophthalmology hospitals in the Middle East. Data were collected on patient demographics, features of the lesions, the site of the lesion, location, adnexal involvement, lymph nodes involvement, local spread, histopathology and molecular genetic studies of the cases (if available), outcomes of treatment and recurrence.
There were 24 patients with lymphoid lesions classified as conjunctival BRLH during the 12-year period evaluated in this study. The mean age at diagnosis was 11.6 years. Twenty-three patients were males (96%). Systemic medical history included three patients with bronchial asthma, one patient with Down's syndrome, one patient with generalised skeletal malformation and one patient with gastritis. The initial uncorrected visual acuity was 20/30 or better in 93.5% of the eyes. At presentation, the tumour was unilateral in 12 cases (50%). The conjunctival mass was located on the bulbar conjunctiva in all cases. The mass was present nasally in 96% of lesions. No cases (that were tested) had an infectious aetiology. PCR demonstrated monoclonality suggestive of lymphoma in two cases; however, this did not alter the final diagnosis as BRLH per histopathological criteria and clinical course, CONCLUSIONS: All investigated cases of paediatric conjunctival BRLH had a benign clinical course with no local or systemic dissemination and a male predominance. Recurrence was rare, and in our cohort, it was not associated with malignant transformation.
Orbital Rhabdomyosarcoma is a highly malignant tumor predominantly affecting children. Our study adds more understanding of this tumor to ophthalmologists from the clinicopathological, radiological ...and genetic aspects.
A retrospective clinicopathological and radiological study of ocular rhabdomyosarcoma with genetic profiling.
All the cases with confirmed tissue diagnosis of orbital rhabdomyosarcoma presenting at a tertiary eye hospital in Riyadh, Saudi Arabia during the period 1985-2015.
Charts and histological slides of 26 patients were reviewed. DNA was extracted from paraffin-embedded biopsies and genotyping was performed to detect chromosomal abnormalities and Copy-number variations regions.
18 males and 8 females were included with a mean age at presentation of 6.9 years (SD of 4.4). Proptosis and globe displacement were the commonest clinical presentations. Embryonal histopathological type was the commonest (73.1%) with superior orbital involvement (p = 0.024). Using magnetic resonance imaging, the embryonal type showed higher Apparent diffusion coefficient (ADC) value compared to the alveolar type (p = 0.98). Genetic profiling showed Copy-number gain in regions spanning PAX3, DDIT3, Gli, Wnt6 genes. DICER1 gene implication was found in 9 sporadic cases.
Rhabdomyosarcoma is rare and occurs with a mean age of 7 years, predominantly among males. The commonest embryonal type is significantly correlated with superior orbital involvement. Radiologically, it shows an ADC of 0.67-0.09 × 10-3mm2/s. (p = 0.98). The gain in PAX3, DDIT3, Gli1, Wnt6 genes is a new finding while the DICER1 gene implication in the absence of familial hereditary carcinoma is another interesting finding.
Le rhabdomyosarcome orbitaire est une tumeur extrêmement maligne qui touche surtout l’enfant. Notre étude vise à aider les ophtalmologistes à mieux connaître cette tumeur du point de vue clinicopathologique, radiologique et génétique.
Étude rétrospective de tous les cas de rhabdomyosarcome orbitaire pour lesquels on dispose d’un diagnostic tissulaire confirmé dans un hôpital ophtalmologique de soins tertiaires entre 1985 et 2015.
Les dossiers et les lames histologiques de 26 patients ont été passés en revue. On a extrait l’ADN des biopsies incluses dans la paraffine pour réaliser un génotypage à la recherche d’anomalies chromosomiques et de régions CNV (copy number variations).
Ainsi, 18 garçons et 8 filles ont été inclus; leur âge moyen lors de la consultation initiale était de 6,9 ans (é.-t.: 4,4). L’exophtalmie et le déplacement du globe oculaire constituaient le tableau clinique le plus fréquent. De même, le type histologique le plus fréquent était embryonnaire (73,1 %), avec une atteinte de la partie supérieure de l’orbite (p = 0,024). Le coefficient de diffusion apparent (ADC, pour apparent diffusion coefficient) du type embryonnaire était plus élevé à l’imagerie par résonance magnétique que celui du type alvéolaire (p = 0,98). Le profilage génétique a révélé une hausse du nombre de copies dans les régions rassemblant les gènes PAX3, DDIT3, Gli et Wnt6. Le gène DICER1 a été mis en cause dans 9 cas sporadiques.
Le rhabdomyosarcome est rare et survient surtout chez les sujets masculins dont l’âge moyen est de 7 ans. Le type embryonnaire le plus courant présente une corrélation significative avec une atteinte de la partie supérieure de l’orbite. Du point de vue radiologique, on note un ADC de 0,67_0,09 x 10-3 mm2/s (p = 0,98). La mise en cause des gènes PAX3, DDIT3, Gli et Wnt6 constitue une découverte intéressante, au même titre que l’implication du gène DICER1 en l’absence de cancer familial héréditaire.
To catalog mutations that underlie retinitis pigmentosa (RP) in Saudi Arabia using a representative sample.
Fifty-two patients with RP were recruited and their homozygosity mapping, with or without ...linkage analysis, was used to suggest the causative genes followed by bidirectional sequencing.
Mutations were identified in 94% of our study cohort, including seven that were novel.
Homozygosity mapping is an extremely robust approach in the study of retinitis pigmentosa in the setting of high rates of consanguinity. BBS3 mutations can rarely present as nonsyndromic RP.
To report novel mutations in two Saudi children with clinical features of Leber congenital amaurosis (LCA) and Alström syndrome.
Case 1 was a child with phenotypic features of LCA including ...oculodigital sign, bilateral enophthalmos, nystagmus, pale disc, and retinal changes. Direct sequencing of the coding sequence of GUCY2D revealed a missense mutation affecting highly conserved position (c. 743C > T; p.S248 L). Case 2 describes a girl with marked nystagmus, photophobia, and retinal changes in both eyes with short and stubby fingers tapering at the distal phalanges. The electroretinograms were nonrecordable in each eye. She had a hearing aid in the left ear, mid-facial hypoplasia, bilateral enophthalmos, and insulin dependent diabetes. Mutation screening of candidates genes revealed a pathogenic mutation in ALMS1 gene (c. 8441C > A, p.S2814). Two novel mutations causing phenotypic LCA and Alström syndrome in Saudi patients from consanguineous families expand the genotypic spectrum of congenital retinal dystrophies.
Bardet-Biedl syndrome (BBS) is a model disease for ciliopathy in humans. The remarkable genetic heterogeneity that characterizes this disease is consistent with accumulating data on the interaction ...between the proteins encoded by the 14 BBS genes identified to date. Previous reports suggested that such interaction may also extend to instances of oligogenic inheritance in the form of triallelism which defies the long held view of BBS as an autosomal recessive disease. In order to investigate the magnitude of triallelism in BBS, we conducted a comprehensive analysis of all 14 BBS genes as well as the CCDC28B-modifier gene in a cohort of 29 BBS families, most of which are multiplex. Two in trans mutations in a BBS gene were identified in each of these families for a total of 20 mutations including 12 that are novel. In no instance did we observe two mutations in unaffected members of a given family, or observe the presence of a third allele that convincingly acted as a modifier of penetrance and supported the triallelic model of BBS. In addition to presenting a comprehensive genotype/phenotype overview of a large set of BBS mutations, including the occurrence of nonsyndromic retinitis pigmentosa in a family with a novel BBS9 mutation, our study argues in favor of straightforward autosomal recessive BBS in most cases.
PDF
