122 results on '"McLenachan S"'
Search Results
2. Measurement of changes in uterine and fibroid volume during treatment of heavy menstrual bleeding (HMB)
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Yin, K, primary, Whitaker, L, additional, Hojo, E, additional, McLenachan, S, additional, Walker, J, additional, McKillop, G, additional, Stubbs, C, additional, Priest, L, additional, Cruz, M, additional, Roberts, N, additional, and Critchley, H, additional
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- 2023
- Full Text
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3. Characterising splicing defects of ABCA4 variants within exons 13–50 in patient-derived fibroblasts
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Huang, D., Thompson, J.A., Chen, S-C, Adams, A., Pitout, I., Lima, A., Zhang, D., Jeffery, R.C.H., Attia, M.S., McLaren, T.L., Lamey, T.M., De Roach, J.N., McLenachan, S., Aung-Htut, M.T., Fletcher, S., Wilton, S.D., Chen, F.K., Huang, D., Thompson, J.A., Chen, S-C, Adams, A., Pitout, I., Lima, A., Zhang, D., Jeffery, R.C.H., Attia, M.S., McLaren, T.L., Lamey, T.M., De Roach, J.N., McLenachan, S., Aung-Htut, M.T., Fletcher, S., Wilton, S.D., and Chen, F.K.
- Abstract
The ATP-binding cassette subfamily A member 4 gene (ABCA4)-associated retinopathy, Stargardt disease, is the most common monogenic inherited retinal disease. Given the pathogenicity of numerous ABCA4 variants is yet to be examined and a significant proportion (more than 15%) of ABCA4 variants are categorized as splice variants in silico, we therefore established a fibroblast-based splice assay to analyze ABCA4 variants in an Australian Stargardt disease cohort and characterize the pathogenic mechanisms of ABCA4 variants. A cohort of 67 patients clinically diagnosed with Stargardt disease was recruited. Genomic DNA was analysed using a commercial panel for ABCA4 variant detection and the consequences of ABCA4 variants were predicted in silico. Dermal fibroblasts were propagated from skin biopsies, total RNA was extracted and the ABCA4 transcript was amplified by RT-PCR. Our analysis identified a total of 67 unique alleles carrying 74 unique variants. The most prevalent splice-affecting complex allele c.[5461-10T > C; 5603A > T] was carried by 10% of patients in a compound heterozygous state. ABCA4 transcripts from exon 13 to exon 50 were readily detected in fibroblasts. In this region, aberrant splicing was evident in 10 out of 57 variant transcripts (18%), carried by 19 patients (28%). Patient-derived fibroblasts provide a feasible platform for identification of ABCA4 splice variants located within exons 13–50. Experimental evidence of aberrant splicing contributes to the pathogenic classification for ABCA4 variants. Moreover, identification of variants that affect splicing processes provides opportunities for intervention, in particular antisense oligonucleotide-mediated splice correction.
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- 2022
4. Pathogenesis and Treatment of Usher Syndrome Type IIA
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Zaw, K., Carvalho, L.S., Aung-Htut, M.T., Fletcher, S., Wilton, S.D., Chen, F.K., McLenachan, S., Zaw, K., Carvalho, L.S., Aung-Htut, M.T., Fletcher, S., Wilton, S.D., Chen, F.K., and McLenachan, S.
- Abstract
Usher syndrome (USH) is the most common form of deaf-blindness, with an estimated prevalence of 4.4 to 16.6 per 100,000 people worldwide. The most common form of USH is type IIA (USH2A), which is caused by homozygous or compound heterozygous mutations in the USH2A gene and accounts for around half of all USH cases. USH2A patients show moderate to severe hearing loss from birth, with diagnosis of retinitis pigmentosa in the second decade of life and variable vestibular involvement. Although hearing aids or cochlear implants can provide some mitigation of hearing deficits, there are currently no treatments aimed at preventing or restoring vision loss in USH2A patients. In this review, we first provide an overview of the molecular biology of the USH2A gene and its protein isoforms, which include a transmembrane protein (TM usherin) and an extracellular protein (EC usherin). The role of these proteins in the inner ear and retina and their impact on the pathogenesis of USH2A is discussed. We review animal cell-derived and patient cell-derived models currently used in USH2A research and conclude with an overview of potential treatment strategies currently in preclinical development and clinical trials.
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- 2022
5. Growth hormone promotes proliferation of adult neurosphere cultures
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McLenachan, S., Lum, M.-G., Waters, M.J., and Turnley, A.M.
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- 2009
- Full Text
- View/download PDF
6. Inhibition of neurosphere proliferation by IFNγ but not IFNβ is coupled to neuronal differentiation
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Lum, M., Croze, E., Wagner, C., McLenachan, S., Mitrovic, B., and Turnley, A.M.
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- 2009
- Full Text
- View/download PDF
7. Determinants of disease penetrance in PRPF31-associated retinopathy
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McLenachan, S., Zhang, D., Grainok, J., Zhang, X., Huang, Z., Chen, S-C, Zaw, K., Lima, A., Jennings, L., Roshandel, D., Moon, S.Y., Heath Jeffery, R.C., Attia, M.S., Thompson, J.A., Lamey, T.M., McLaren, T.L., De Roach, J., Fletcher, S., Chen, F.K., McLenachan, S., Zhang, D., Grainok, J., Zhang, X., Huang, Z., Chen, S-C, Zaw, K., Lima, A., Jennings, L., Roshandel, D., Moon, S.Y., Heath Jeffery, R.C., Attia, M.S., Thompson, J.A., Lamey, T.M., McLaren, T.L., De Roach, J., Fletcher, S., and Chen, F.K.
- Abstract
Retinitis pigmentosa 11 (RP11) is caused by dominant mutations in PRPF31, however a significant proportion of mutation carriers do not develop retinopathy. Here, we investigated the relationship between CNOT3 polymorphism, MSR1 repeat copy number and disease penetrance in RP11 patients and non-penetrant carriers (NPCs). We further characterized PRPF31 and CNOT3 expression in fibroblasts from eight RP11 patients and one NPC from a family carrying the c.1205C>T variant. Retinal organoids (ROs) and retinal pigment epithelium (RPE) were differentiated from induced pluripotent stem cells derived from RP11 patients, an NPC and a control subject. All RP11 patients were homozygous for the 3-copy MSR1 repeat in the PRPF31 promoter, while 3/5 NPCs carried a 4-copy MSR1 repeat. The CNOT3 rs4806718 genotype did not correlate with disease penetrance. PRFP31 expression declined with age in adult cadaveric retina. PRPF31 and CNOT3 expression was reduced in RP11 fibroblasts, RO and RPE compared with controls. Both RP11 and NPC RPE displayed shortened primary cilia compared with controls, however a subpopulation of cells with normal cilia lengths was present in NPC RPE monolayers. Our results indicate that RP11 non-penetrance is associated with the inheritance of a 4-copy MSR1 repeat, but not with CNOT3 polymorphisms.
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- 2021
8. Using induced pluripotent stem cell-derived retinal pigment epithelial cells to model splicing defects of ABCA4 c.5461-10T > C detected in an Australian Stargardt disease cohort
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Huang, D., Thompson, J.A., McLenachan, S., Chen, S-C, Zhang, D., Heath Jeffery, R.C., Attia, M., McLaren, T.L., Lamey, T.M., De Roach, J.N., Aung-Htut, M.T., Adams, A., Fletcher, S., Wilton, S., Chen, F.K., Huang, D., Thompson, J.A., McLenachan, S., Chen, S-C, Zhang, D., Heath Jeffery, R.C., Attia, M., McLaren, T.L., Lamey, T.M., De Roach, J.N., Aung-Htut, M.T., Adams, A., Fletcher, S., Wilton, S., and Chen, F.K.
- Abstract
Purpose : Stargardt disease (STGD1, OMIM: 248200) is mainly caused by missense, frameshifting or nonsense mutations in the ATP-binding cassette transporter gene, ABCA4. However, sequence variants that alter splicing are also pathogenic. Herein, we describe an in vitro investigation of aberrant splicing in ABCA4 variants detected in a STGD1 cohort using patient-derived fibroblast-based assay. In addition, retinal pigment epithelium (RPE) cells differentiated from patient-derived induced pluripotent stem cells (iPSC) were used to further validate such splicing errors. Methods : A cohort of 68 patients clinically diagnosed with STGD1 were recruited in this study. Genomic DNA obtained from recruited STGD1 patients was analysed by a commercial Stargardt/Macular dystrophy screening panel, targeting all exons of ABCA4 and flanking intronic regions, as well as already-known deep-intronic variants of ABCA4. Fibroblasts were propagated from 68 patients, total RNA was extracted and ABCA4 transcript structure was analysed by RT-PCR. The iPSCs reprogrammed from 2 patients carrying heterozygous c.[5461-10T>C;5603A>T] alleles were differentiated into RPE cells and the ABCA4 transcripts re-examined by RT-PCR. Results : A total of 73 unique ABCA4 alleles were identified. Biallelic ABCA4 variants were detected in 66 patients (66/68, 97.06%) and 2 patients (2/68, 2.94%) had a single ABCA4 variant detected. Only exons 13-50 of ABCA4 could be readily amplified from fibroblast RNA. In this region, 9 out of 55 (16.36%) variants, carried by 19 patients (28%), resulted in aberrant splicing. The most prevalent splice variant, c.5461-10T>C, is complexed with c.5603A>T and carried heterozygously by 7 patients (10%). This variant results in mature ABCA4 mRNA transcripts missing exon 39, or exons 39 and 40. The splicing defect was also evident in patient-derived iPSC-RPE cells. Conclusions : Patient-derived fibroblasts are useful for identifying ABCA4 splicing variants affecting exons 13-50. The
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- 2021
9. Stargardt disease and progress in therapeutic strategies
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Huang, D., Heath Jeffery, R.C., Aung-Htut, M.T., McLenachan, S., Fletcher, S., Wilton, S.D., Chen, F.K., Huang, D., Heath Jeffery, R.C., Aung-Htut, M.T., McLenachan, S., Fletcher, S., Wilton, S.D., and Chen, F.K.
- Abstract
Background: Stargardt disease (STGD1) is an autosomal recessive retinal dystrophy due to mutations in ABCA4, characterized by subretinal deposition of lipofuscin-like substances and bilateral centrifugal vision loss. Despite the tremendous progress made in the understanding of STGD1, there are no approved treatments to date. This review examines the challenges in the development of an effective STGD1 therapy. Materials and Methods: A literature review was performed through to June 2021 summarizing the spectrum of retinal phenotypes in STGD1, the molecular biology of ABCA4 protein, the in vivo and in vitro models used to investigate the mechanisms of ABCA4 mutations and current clinical trials.Results: STGD1 phenotypic variability remains an challenge for clinical trial design and patient selection. Pre-clinical development of therapeutic options has been limited by the lack of animal models reflecting the diverse phenotypic spectrum of STDG1. Patient-derived cell lines have facilitated the characterization of splice mutations but the clinical presentation is not always predicted by the effect of specific mutations on retinoid metabolism in cellular models. Current therapies primarily aim to delay vision loss whilst strategies to restore vision are less well developed. Conclusions: STGD1 therapy development can be accelerated by a deeper understanding of genotype-phenotype correlations.
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- 2021
10. Generation of an induced pluripotent stem cell line from a patient with Stargardt disease caused by biallelic c.[5461–10T>C;5603A>T];[6077T>C] mutations in the ABCA4 gene
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Huang, D., Zhang, D., Chen, S-C, Thandar Aung-Htut, M., Lamey, T.M., Thompson, J.A., McLaren, T.L., De Roach, J.N., Fletcher, S., Wilton, S.D., McLenachan, S., Chen, F.K., Huang, D., Zhang, D., Chen, S-C, Thandar Aung-Htut, M., Lamey, T.M., Thompson, J.A., McLaren, T.L., De Roach, J.N., Fletcher, S., Wilton, S.D., McLenachan, S., and Chen, F.K.
- Abstract
Mutations in ABCA4 gene are causative for autosomal recessive Stargardt disease (STGD1), the most common inherited retinal dystrophy. Here, we report the generation of an induced pluripotent stem cell (iPSC) line from a STGD1 patient carrying biallelic c.[5461–10T>C;5603A>T];[6077T>C] mutations in the ABCA4 gene. Episomes carrying OCT4, SOX2, KLF4, L-MYC, LIN28 and mp53DD were employed for the reprogramming of patient-derived fibroblasts. This iPSC line expressed comparable pluripotency markers as in a commercially available human iPSC line, displayed normal karyotype and potential for trilineage differentiation, and were negative for both reprogramming episomes and mycoplasma test.
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- 2021
11. Generation of two induced pluripotent stem cell lines from a patient with Stargardt disease caused by compound heterozygous mutations in the ABCA4 gene
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Huang, D., Zhang, D., Chen, S-C, Aung-Htut, M.T., Lamey, T.M., Thompson, J.A., McLaren, T.L., De Roach, J.N., Fletcher, S., Wilton, S.D., Chen, F.K., McLenachan, S., Huang, D., Zhang, D., Chen, S-C, Aung-Htut, M.T., Lamey, T.M., Thompson, J.A., McLaren, T.L., De Roach, J.N., Fletcher, S., Wilton, S.D., Chen, F.K., and McLenachan, S.
- Abstract
Stargardt disease (STGD1) is the most common inherited retinal dystrophy and ABCA4 c.546-–10 T>C is the most commonly reported splice mutation. Here, we generated and characterized two induced pluripotent stem cell (iPSC) lines from a STGD1 patient with compound heterozygous mutations in ABCA4 (c.[5461-10 T > C;5603A > T];[4163 T > C;455G > A]). Episomal vectors containing OCT4, SOX2, KLF4, L-MYC, LIN28 and mp53DD were employed to conduct the reprogramming of patient-derived fibroblasts. Both lines had a normal karyotype, displayed iPSC morphology, expressed pluripotency markers and showed trilineage differentiation potential. These lines can provide a powerful platform for further investigating the pathophysiological consequences of mutations in ABCA4.
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- 2021
12. Generation of three induced pluripotent stem cell lines from a patient with Usher syndrome caused by biallelic c.949C > A and c.1256G > T mutations in the USH2A gene
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Zaw, K., Wong, E.Y.M., Zhang, X., Zhang, D., Chen, S-C, Thompson, J.A., Lamey, T., McLaren, T., De Roach, J.N., Wilton, S.D., Fletcher, S., Mitrpant, C., Atlas, M.D., Chen, F.K., McLenachan, S., Zaw, K., Wong, E.Y.M., Zhang, X., Zhang, D., Chen, S-C, Thompson, J.A., Lamey, T., McLaren, T., De Roach, J.N., Wilton, S.D., Fletcher, S., Mitrpant, C., Atlas, M.D., Chen, F.K., and McLenachan, S.
- Abstract
Mutations in the USH2A gene are the most common cause of Usher syndrome and autosomal recessive non-syndromic retinitis pigmentosa. Here, we describe the generation of three induced pluripotent stem cell lines from dermal fibroblasts derived from a patient carrying biallelic c.949C > A and c.1256G > T variants in the USH2A gene, using episomal reprogramming plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28, mir302/367 and shRNA targeting TP53. All three lines expressed pluripotency markers, displayed unaltered karyotypes as well as trilineage differentiation potential, and were negative for reprogramming episomes and mycoplasma.
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- 2021
13. Short-Term Parafoveal Cone Loss Despite Preserved Ellipsoid Zone in Rod Cone Dystrophy
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Roshandel, D, Jeffery, RCH, Charng, J, Sampson, DM, McLenachan, S, Mackey, DA, Chen, FK, Roshandel, D, Jeffery, RCH, Charng, J, Sampson, DM, McLenachan, S, Mackey, DA, and Chen, FK
- Abstract
PURPOSE: Rod-cone dystrophy (RCD) is characterized by centripetal loss of rod followed by cone photoreceptors. In this prospective, observational cohort, we used flood-illumination adaptive optics (AO) imaging to investigate parafoveal cone loss in regions with preserved ellipsoid zone (EZ) in patients with RCD. METHODS: Eight patients with RCD and 10 age-matched healthy controls underwent spectral-domain optical coherence tomography and AO imaging. The RCD cohort underwent a follow-up examination after 6 months. Cone density (CD) and intercone distance (ICD) measurements were performed at 2° temporal from the fovea. Baseline CD and ICD values were compared between the control and patient groups, and longitudinal changes were calculated in the patient group. Residual EZ span in patients was measured in horizontal foveal B-scans. RESULTS: Between the control and patient groups, there was no significant difference in the baseline CD (2094 vs. 1750 cells/deg2, respectively; P = 0.09) and ICD (1.46 vs. 1.62 arcmin, respectively; P = 0.08). Mean CD declined by 198 cells/deg2 (-11.3%; P < 0.01), and mean ICD increased by 0.09 arcmin (+5.6%; P = 0.01) at the 6-month follow-up in the patient group. Mean baseline and follow-up residual EZ spans in the six patients with EZ defect were 3189 µm and 3065 µm, respectively (-3.9%; P = 0.08). CONCLUSIONS: AO imaging detected significant parafoveal cone loss over 6-month follow-up even in regions with preserved EZ. Further studies to refine AO imaging protocol and validate cone metrics as a structural endpoint in early RCD are warranted. TRANSLATIONAL RELEVANCE: CD and ICD may change prior to EZ span shortening in RCD.
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- 2021
14. Clinical Evidence for the Importance of the Wild-Type PRPF31 Allele in the Phenotypic Expression of RP11
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Roshandel, D, Thompson, JA, Jeffery, RCH, Zhang, D, Lamey, TM, McLaren, TL, De Roach, JN, McLenachan, S, Mackey, DA, Chen, FK, Roshandel, D, Thompson, JA, Jeffery, RCH, Zhang, D, Lamey, TM, McLaren, TL, De Roach, JN, McLenachan, S, Mackey, DA, and Chen, FK
- Abstract
PRPF31-associated retinopathy (RP11) is a common form of autosomal dominant retinitis pigmentosa (adRP) that exhibits wide variation in phenotype ranging from non-penetrance to early-onset RP. Herein, we report inter-familial and intra-familial variation in the natural history of RP11 using multimodal imaging and microperimetry. Patients were recruited prospectively. The age of symptom onset, best-corrected visual acuity, microperimetry mean sensitivity (MS), residual ellipsoid zone span and hyperautofluorescent ring area were recorded. Genotyping was performed using targeted next-generation and Sanger sequencing and copy number variant analysis. PRPF31 mutations were found in 14 individuals from seven unrelated families. Four disease patterns were observed: (A) childhood onset with rapid progression (N = 4), (B) adult-onset with rapid progression (N = 4), (C) adult-onset with slow progression (N = 4) and (D) non-penetrance (N = 2). Four different patterns were observed in a family harbouring c.267del; patterns B, C and D were observed in a family with c.772_773delins16 and patterns A, B and C were observed in 3 unrelated individuals with large deletions. Our findings suggest that the RP11 phenotype may be related to the wild-type PRPF31 allele rather than the type of mutation. Further studies that correlate in vitro wild-type PRPF31 allele expression level with the disease patterns are required to investigate this association.
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- 2021
15. Generation of two induced pluripotent stem cell lines from a patient with recessive inherited retinal disease caused by compound heterozygous mutations in SNRNP200
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Zhang, D., McLenachan, S., Chen, S.-C., Zaw, K., Alziyadat, Y., Zhang, X., Lamey, T.M., Thompson, J.A., McLaren, T.L., Mellough, C., De Roach, J.N., Chen, F.K., Zhang, D., McLenachan, S., Chen, S.-C., Zaw, K., Alziyadat, Y., Zhang, X., Lamey, T.M., Thompson, J.A., McLaren, T.L., Mellough, C., De Roach, J.N., and Chen, F.K.
- Abstract
The human induced pluripotent stem cell (iPSC) lines LEIi015-A and LEIi015-B were derived from a patient with inherited retinal disease caused by compound heterozygous mutations in the SNRNP200 gene (c.[1792C>T];[3341T>C]). Dermal fibroblasts were transfected with episomal plasmids carrying transgenes encoding OCT4, SOX2, KLF4, L-MYC, LIN28, mir302/367 microRNA and shRNA for P53. The clonal iPSC lines LEIi015-A and LEIi015-B expressed iPSC markers, were free from genomic alterations and demonstrated trilineage differentiation potential.
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- 2021
16. Gene replacement therapy restores RCBTB1 expression and cilium length in patient‐derived retinal pigment epithelium
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Huang, Z., Zhang, D., Chen, S‐C, Jennings, L., Carvalho, L.S., Fletcher, S., Chen, F.K., McLenachan, S., Huang, Z., Zhang, D., Chen, S‐C, Jennings, L., Carvalho, L.S., Fletcher, S., Chen, F.K., and McLenachan, S.
- Abstract
Biallelic mutations in the RCBTB1 gene cause retinal dystrophy. Here, we characterized the effects of RCBTB1 gene deficiency in retinal pigment epithelial (RPE) cells derived from a patient with RCBTB1-associated retinopathy and restored RCBTB1 expression in these cells using adeno-associated viral (AAV) vectors. Induced pluripotent stem cells derived from a patient with compound heterozygous RCBTB1 mutations (c.170delG and c.707delA) and healthy control subjects were differentiated into RPE cells. RPE cells were treated with AAV vectors carrying a RCBTB1 transgene. Patient-derived RPE cells showed reduced expression of RCBTB1. Expression of NFE2L2 showed a non-significant reduction in patient RPE cells compared with controls, while expression of its target genes (RXRA, IDH1 and SLC25A25) was significantly reduced. Trans-epithelial electrical resistance, surface microvillus densities and primary cilium lengths were reduced in patient-derived RPE cells, compared with controls. Treatment of patient RPE with AAV vectors significantly increased RCBTB1, NFE2L2 and RXRA expression and cilium lengths. Our study provides the first report examining the phenotype of RPE cells derived from a patient with RCBTB1-associated retinopathy. Furthermore, treatment of patient-derived RPE with AAV-RCBTB1 vectors corrected deficits in gene expression and RPE ultrastructure, supporting the use of gene replacement therapy for treating this inherited retinal disease.
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- 2021
17. Exploring microperimetry and autofluorescence endpoints for monitoring disease progression in PRPF31-associated retinopathy
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Roshandel, D., Thompson, J.A., Charng, J., Zhang, D., Chelva, E., Arunachalam, S., Attia, M.S., Lamey, T.M., McLaren, T.L., De Roach, J.N., Mackey, D.A., Wilton, S.D., Fletcher, S., McLenachan, S., Chen, F.K., Roshandel, D., Thompson, J.A., Charng, J., Zhang, D., Chelva, E., Arunachalam, S., Attia, M.S., Lamey, T.M., McLaren, T.L., De Roach, J.N., Mackey, D.A., Wilton, S.D., Fletcher, S., McLenachan, S., and Chen, F.K.
- Abstract
Background Mutations in the splicing factor pre-messenger RNA processing factor 31 (PRPF31) gene cause autosomal dominant retinitis pigmentosa 11 (RP11) through a haplo-insufficiency mechanism. We describe the phenotype and progression of microperimetry and autofluorescence endpoints in an Indigenous Australian RP11 family. Patients and Methods Ophthalmic examination, optical coherence tomography, fundus autofluorescence and microperimetry were performed at baseline and every 6–12 months. Baseline and annual change in best-corrected visual acuity (BCVA), microperimetry mean sensitivity (MS) and number of scotoma loci, residual ellipsoid zone (EZ) span and hyperautofluorescent ring (HAR) area were reported. Next-generation and Sanger sequencing were performed in available members. Results 12 affected members from three generations were examined. Mean (SD, range) age at onset of symptoms was 11 (4.5, 4–19) years. MS declined steadily from the third decade and EZ span and HAR area declined rapidly during the second decade. Serial microperimetry showed negligible change in MS over 2–3 years. However, mean EZ span, near-infrared and short-wavelength HAR area reduction was 203 (6.4%) µm/year, 1.8 (8.7%) mm2/year and 1.1 (8.6%) mm2/year, respectively. Genetic testing was performed on 11 affected and 10 asymptomatic members and PRPF31 c.1205 C > A (p.Ser402Ter) mutation was detected in all affected and two asymptomatic members (non-penetrant carriers). Conclusions Our findings suggest that in the studied cohort, the optimal window for therapeutic intervention is the second decade of life and residual EZ span and HAR area can be considered as efficacy outcome measures. Further studies on larger samples with different PRPF31 mutations and longer follow-up duration are recommended.
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- 2020
18. Phenotype–genotype correlations in a pseudodominant Stargardt disease pedigree due to a novel ABCA4 deletion–insertion variant causing a splicing defect
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Huang, D., Thompson, J.A., Charng, J., Chelva, E., McLenachan, S., Chen, S‐C, Zhang, D., McLaren, T.L., Lamey, T.M., Constable, I.J., De Roach, J.N., Aung‐Htut, M.T., Adams, A.M., Fletcher, S., Wilton, S.D., Chen, F.K., Huang, D., Thompson, J.A., Charng, J., Chelva, E., McLenachan, S., Chen, S‐C, Zhang, D., McLaren, T.L., Lamey, T.M., Constable, I.J., De Roach, J.N., Aung‐Htut, M.T., Adams, A.M., Fletcher, S., Wilton, S.D., and Chen, F.K.
- Abstract
Background Deletion–insertion (delins) variants in the retina‐specific ATP‐binding cassette transporter gene, subfamily A, member 4 (ABCA4) accounts for <1% in Stargardt disease. The consequences of these delins variants on splicing cannot be predicted with certainty without supporting in vitro data. Methods Candidate ABCA4 variants were revealed by genetic and segregation analysis of a family with pseudodominant Stargardt disease using a commercial panel and Sanger sequencing. RNA extracted from patient‐derived fibroblasts was analyzed by RT‐PCR to evaluate splicing behavior of the ABCA4 variants. Results Affected members carrying the novel c.6031_6044delinsAGTATTTAACCAATATTT variant in exon 44 presented with contrasting phenotypes; from early‐onset cone‐rod dystrophy to late‐onset macular dystrophy. This variant resulted in a 56‐nucleotide deletion in the mutant allele by activation of a cryptic splice acceptor site which disrupts the reading frame and results in a premature termination codon (p.Ile2003LeufsTer41). If translated, the crucial functional domains near the C‐terminus would be truncated from the ABCA4 protein. Conclusion This work demonstrates the intrafamilial phenotypic variability in a pseudodominant Stargardt disease pedigree and the use of patient‐derived fibroblasts to evaluate the effect of a novel ABCA4 delins variant on splicing to complement in silico pathogenicity assessment.
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- 2020
19. Utilising patient-specific retinal organoids to investigate the role of SNRNP200 variants of unknown significance in severe early onset retinitis pigmentosa
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Mellough, C.M., Ackerman, M., Thompson, J.A., De Roach, J., McLaren, T., Lamey, T., Akkari, A., Ram, R., Leary, S., Chopra, A., Chen, S.C., Zhang, D., McLenachan, S., Chen, F.K., Mellough, C.M., Ackerman, M., Thompson, J.A., De Roach, J., McLaren, T., Lamey, T., Akkari, A., Ram, R., Leary, S., Chopra, A., Chen, S.C., Zhang, D., McLenachan, S., and Chen, F.K.
- Abstract
Purpose : The consequence of variants in many genes implicated in retinitis pigmentosa (RP) remain unknown. This applies to SNRNP200, which encodes for a component of the spliceosome complex. To investigate the effect of SNRNP200 variants on the human retina, we generated retinal organoids (ROs) from a patient with severe early-onset RP found to harbour compound heterozygous missense variants in SNRNP200 that segregated with the disease. Methods : Computer-assisted pathogenicity programs and other tools were used to predict the pathogenicity of candidate variants detected by a 537-gene next-generation sequencing panel (MVL, Oregon, US). Induced pluripotent stem cells (iPSCs) derived from patient and control dermal fibroblasts were differentiated into ROs and maintained for up to 350 days. Western Blotting and immunohistochemical (IHC) analysis was performed using antibodies directed against SNRNP200 and retinal-specific proteins. Gene expression was analysed by qRT-PCR and retinal ultrastructure examined by transmission electron microscopy. Single cell sorting of control and patient iPSCs and ROs was performed to capture individual cells which were then subjected to RNA-Seq and bioinformatic analysis. Results : A patient-specific in vitro model of RP was generated. The emergence of retinal-specific phenotypes in ROs was confirmed by IHC and qRT-PCR. Several morphological differences in patient ROs were identified including (1) reduced neuroepithelial integrity, (2) a high number of vacuole-like inclusions within photoreceptor soma, and (3) abnormal mitochondrial morphology at later stages of differentiation. Single cell transcriptomic analysis revealed significantly significant differential gene expression between control and patient cells. Notably, multiple genes associated with immunologic signatures were upregulated, and genes associated with cell coenzyme biosynthesis, an important process for normal vision, were differentially expressed. Conclusions : Using a h
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- 2019
20. Optical coherence tomography derived macular volume loss over 5 years in Stargardt disease
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Chen, F.K., Arunachalam, S., Vallis, N., Huang, D., Chen, Y., Thompson, J.A., McLaren, T., Lamey, T., De Roach, J., McLenachan, S., Chen, F.K., Arunachalam, S., Vallis, N., Huang, D., Chen, Y., Thompson, J.A., McLaren, T., Lamey, T., De Roach, J., and McLenachan, S.
- Abstract
Purpose : To examine the variability in the rate of macular volume loss (MVL) in Stargardt disease derived from spectral domain-optical coherence tomography (SD-OCT). Methods : Subjects with clinical Stargardt disease and biphasic ABCA4 mutations were eligible. Assessments included ETDRS visual acuity, fundus examination, widefield fundus autofluorescence (FAF, Optos) and SD-OCT (30°×25°, 61 slices with 119µm separation, Heidelberg Spectralis OCT) imaging. FAF pattern was divided into types A and B, defined by lesions confined to within or extended beyond the central 50°, respectively. Manual centration of the ETDRS grid and correction of segmentation errors in the inner limiting and Bruch’s membranes were performed. Patients with choroidal neovascular membrane, severe epiretinal membrane and decentred scans leading to image truncation within the ETDRS grid boundary were excluded. Annualised MVL was calculated by linear regression after confirming the rate of decline was constant. Inter-ocular (OD-OS) and inter-individual variabilities (OD only) in baseline total macular volume (TMV) and annualised MVL were explored. Results : Forty eyes of 20 patients (mean age 39 years, mean symptom duration 13 years) were recruited and followed for a mean (SD) of 5.0 (1.0) years (range: 3.5-6.6 years). Five patients had late-onset disease (defined as symptom onset >40yrs) and 8 had a Type A FAF pattern. Mean (SD, range) number of OCT scanning sessions was 7.3 (2.1, 4-12) during follow up. Inter-ocular differences and agreements (bias, SD, 95% CI) in TMV (mm3) and MVL (mm3/year) were +0.03, 0.31, -0.12 to +0.18 and +0.02, 0.10, -0.03 to +0.06, respectively. Baseline TMV (OD) was negatively correlated with duration of symptoms (Pearson correlation, r = -0.76, p < 0.001) and associated with FAF pattern (7.55 vs 6.01 mm3, A vs B, p = 0.01). In contrast, there was no significant relationship between MVL (OD) and duration of symptoms or FAF patterns. A trend for lower rates of MVL in t
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- 2019
21. Generation of two induced pluripotent stem cell lines from a patient with dominant PRPF31 mutation and a related non-penetrant carrier
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McLenachan, S., Zhang, D., Zhang, X., Chen, S-C, Lamey, T., Thompson, J.A., McLaren, T., De Roach, J.N., Fletcher, S., Chen, F.K., McLenachan, S., Zhang, D., Zhang, X., Chen, S-C, Lamey, T., Thompson, J.A., McLaren, T., De Roach, J.N., Fletcher, S., and Chen, F.K.
- Abstract
We report the generation of the human iPSC line LEIi008-A from a patient with retinitis pigmentosa-11 caused by a dominant nonsense mutation in the PRPF31 gene (NM_015629.3:c.1205C > A p.(Ser402Ter)). A second line, LEIi009-A, was generated from a related non-penetrant carrier of the same mutation with no retinal disease. Reprogramming of patient dermal fibroblasts using episomal plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, shRNA for p53 and mir302/367 microRNA generated cell lines displaying pluripotent stem cell marker expression, a normal karyotype and the capability to differentiate into the three germ layer lineages.
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- 2018
22. Inherited Retinal Disease Therapies Targeting Precursor Messenger Ribonucleic Acid.
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Huang, D, Fletcher, S, Wilton, SD, Palmer, N, McLenachan, S, Mackey, DA, Chen, FK, Huang, D, Fletcher, S, Wilton, SD, Palmer, N, McLenachan, S, Mackey, DA, and Chen, FK
- Abstract
Inherited retinal diseases are an extremely diverse group of genetically and phenotypically heterogeneous conditions characterized by variable maturation of retinal development, impairment of photoreceptor cell function and gradual loss of photoreceptor cells and vision. Significant progress has been made over the last two decades in identifying the many genes implicated in inherited retinal diseases and developing novel therapies to address the underlying genetic defects. Approximately one-quarter of exonic mutations related to human inherited diseases are likely to induce aberrant splicing products, providing opportunities for the development of novel therapeutics that target splicing processes. The feasibility of antisense oligomer mediated splice intervention to treat inherited diseases has been demonstrated in vitro, in vivo and in clinical trials. In this review, we will discuss therapeutic approaches to treat inherited retinal disease, including strategies to correct splicing and modify exon selection at the level of pre-mRNA. The challenges of clinical translation of this class of emerging therapeutics will also be discussed.
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- 2017
23. Inherited retinal disease therapies targeting precursor messenger ribonucleic acid
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Huang, D., Fletcher, S., Wilton, S., Palmer, N., McLenachan, S., Mackey, D., Chen, F., Huang, D., Fletcher, S., Wilton, S., Palmer, N., McLenachan, S., Mackey, D., and Chen, F.
- Abstract
Inherited retinal diseases are an extremely diverse group of genetically and phenotypically heterogeneous conditions characterized by variable maturation of retinal development, impairment of photoreceptor cell function and gradual loss of photoreceptor cells and vision. Significant progress has been made over the last two decades in identifying the many genes implicated in inherited retinal diseases and developing novel therapies to address the underlying genetic defects. Approximately one-quarter of exonic mutations related to human inherited diseases are likely to induce aberrant splicing products, providing opportunities for the development of novel therapeutics that target splicing processes. The feasibility of antisense oligomer mediated splice intervention to treat inherited diseases has been demonstrated in vitro, in vivo and in clinical trials. In this review, we will discuss therapeutic approaches to treat inherited retinal disease, including strategies to correct splicing and modify exon selection at the level of pre-mRNA. The challenges of clinical translation of this class of emerging therapeutics will also be discussed.
- Published
- 2017
24. Prospects for clinical use of reprogrammed cells for autologous treatment of macular degeneration
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Alvarez Palomo, A.B., McLenachan, S., Chen, F.K., Da Cruz, L., Dilley, R.J., Requena, J., Lucas, M., Lucas, A., Drukker, M., Edel, M.J., Alvarez Palomo, A.B., McLenachan, S., Chen, F.K., Da Cruz, L., Dilley, R.J., Requena, J., Lucas, M., Lucas, A., Drukker, M., and Edel, M.J.
- Abstract
Since the discovery of induced pluripotent stem cells (iPSC) in 2006, the symptoms of many human diseases have been reversed in animal models with iPSC therapy, setting the stage for future clinical development. From the animal data it is clear that iPSC are rapidly becoming the lead cell type for cell replacement therapy and for the newly developing field of iPSC-derived body organ transplantation. The first human pathology that might be treated in the near future with iPSC is age-related macular degeneration (AMD), which has recently passed the criteria set down by regulators for phase I clinical trials with allogeneic human embryonic stem cell-derived cell transplantation in humans. Given that iPSC are currently in clinical trial in Japan (RIKEN) to treat AMD, the establishment of a set of international criteria to make clinical-grade iPSC and their differentiated progeny is the next step in order to prepare for future autologous cell therapy clinical trials. Armed with clinical-grade iPSC, we can then specifically test for their threat of cancer, for proper and efficient differentiation to the correct cell type to treat human disease and then to determine their immunogenicity. Such a rigorous approach sets a far more relevant paradigm for their intended future use than non-clinical-grade iPSC. This review focuses on the latest developments regarding the first possible use of iPSC-derived retinal pigment epithelial cells in treating human disease, covers data gathered on animal models to date and methods to make clinical-grade iPSC, suggests techniques to ensure quality control and discusses possible clinical immune responses.
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- 2015
25. The Power and the Promise of Cell Reprogramming: Personalized Autologous Body Organ and Cell Transplantation
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Palomo, A., Lucas, M., Dilley, R., McLenachan, S., Chen, F., Requena, J., Sal, M., Lucas, A., Alvarez, I., Jaraquemada, D., Edel, M., Palomo, A., Lucas, M., Dilley, R., McLenachan, S., Chen, F., Requena, J., Sal, M., Lucas, A., Alvarez, I., Jaraquemada, D., and Edel, M.
- Abstract
Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) or direct reprogramming to desired cell types are powerful and new in vitro methods for the study of human disease, cell replacement therapy, and drug development. Both methods to reprogram cells are unconstrained by the ethical and social questions raised by embryonic stem cells. iPSC technology promises to enable personalized autologous cell therapy and has the potential to revolutionize cell replacement therapy and regenerative medicine. Potential applications of iPSC technology are rapidly increasing in ambition from discrete cell replacement applications to the iPSC assisted bioengineering of body organs for personalized autologous body organ transplant. Recent work has demonstrated that the generation of organs from iPSCs is a future possibility. The development of embryonic-like organ structures bioengineered from iPSCs has been achieved, such as an early brain structure (cerebral organoids), bone, optic vesicle-like structures (eye), cardiac muscle tissue (heart), primitive pancreas islet cells, a tooth-like structure (teeth), and functional liver buds (liver). Thus, iPSC technology offers, in the future, the powerful and unique possibility to make body organs for transplantation removing the need for organ donation and immune suppressing drugs. Whilst it is clear that iPSCs are rapidly becoming the lead cell type for research into cell replacement therapy and body organ transplantation strategies in humans, it is not known whether (1) such transplants will stimulate host immune responses; and (2) whether this technology will be capable of the bioengineering of a complete and fully functional human organ. This review will not focus on reprogramming to iPSCs, of which a plethora of reviews can be found, but instead focus on the latest developments in direct reprogramming of cells, the bioengineering of body organs from iPSCs, and an analysis of the immune response induced by iPSC-derived ce
- Published
- 2014
26. Application of the TLR9 ligand CpG DNA to the injured corneal epithelium induces intraocular inflammation
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[Unknown], Chinnery, H.R., McLenachan, S., Degli-Esposti, M., Forrester, J.V., Pearlman, E., McMenamin, P.G., [Unknown], Chinnery, H.R., McLenachan, S., Degli-Esposti, M., Forrester, J.V., Pearlman, E., and McMenamin, P.G.
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- 2010
27. Growth hormone promotes proliferation of adult neurosphere cultures
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McLenachan, S, Lum, M-G, Waters, MJ, Turnley, AM, McLenachan, S, Lum, M-G, Waters, MJ, and Turnley, AM
- Abstract
OBJECTIVES: Growth hormone (GH) and its receptor (GHR) are widely expressed in the CNS. During development, GH signaling regulates both proliferation of neural progenitor cells as well as their differentiation into neurons and glia. Here we have examined the effect of GH signaling on adult subventricular zone derived neural progenitor cells cultured as neurospheres. DESIGN: GH was added to adult wild-type (WT) neurosphere cultures and neurosphere growth measured using the MTT cell proliferation assay. To examine the influence of endogenous GH production on neural progenitors, neurospheres derived from GH receptor knockout (GHRKO) mice were examined by measuring neurosphere sizes and Ki67 and TUNEL immunoreactivity. In addition, neurosphere growth curves were compared following long term culture. Finally, the differentiation of WT vs. GHRKO neurospheres was compared using immunocytochemistry for betaIII-tubulin and GFAP. RESULTS: While GH alone was insufficient to support neurosphere formation, it enhanced neurosphere growth by 20% in the presence of epidermal growth factor and fibroblast growth factor-2. Compared to wildtype neurospheres, GHRKO neurospheres were smaller, contained fewer proliferating cells and exhibited reduced self-renewal in long term culture. Addition of GH increased STAT5 phosphorylation levels in neurosphere cells. Upon differentiation, GHRKO neurospheres showed accelerated neurogenesis, although over time similar numbers of betaIII-tubulin positive neurons were generated by cells of both genotypes. CONCLUSIONS: GH functions as an autocrine mitogen in adult neurosphere cultures and promotes proliferation of neural progenitor cells as well as self-renewal of neurosphere cultures. In addition, signaling through the GHR appeared to delay neuronal differentiation in adult neurospheres.
- Published
- 2009
28. Generation of two induced pluripotent stem cell lines from an Usher syndrome type 1B patient with the homozygous c.496del MYO7A variant.
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Wong EYM, Khoh XE, Chen SC, Lye J, Leith FK, Zhang D, Lamey TM, Thompson JA, McLaren TL, Atlas MD, Chen FK, and McLenachan S
- Subjects
- Humans, Cell Line, Cell Differentiation, Male, Fibroblasts metabolism, Induced Pluripotent Stem Cells metabolism, Usher Syndromes genetics, Usher Syndromes pathology, Kruppel-Like Factor 4, Myosin VIIa, Homozygote
- Abstract
Usher syndrome (USH) is the most common cause of inherited deaf-blindness. Here, we produced the LEIi020-A and LEIi020-B induced pluripotent stem cell (iPSC) lines from dermal fibroblasts derived from a patient with USH1B caused by inheritance of homozygous c.496del variants in MYO7A using episomal plasmids encoding OCT4, SOX2, KLF4, L-MYC, LIN28, mir302/367 microRNA and shRNA for TP53. Both iPSC lines expressed pluripotency markers, demonstrated trilineage differentiation potential and displayed a 46,XY karyotype. These cell lines represent a valuable resource for the production of retinal and otic tissues to support research into the pathogenesis and treatment of USH1B., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
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- 2024
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29. Establishment of an induced pluripotent stem cell line LEIi019-A from an early-onset retinal dystrophy patient with the autosomal dominant OTX2 c.259G>A variant.
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Zhang D, Jennings L, Chen SC, Zaw K, Lamey TM, Thompson JA, McLaren TL, Chen FK, and McLenachan S
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- Humans, Cell Line, Cell Differentiation, Male, Mutation, Induced Pluripotent Stem Cells metabolism, Otx Transcription Factors genetics, Otx Transcription Factors metabolism, Kruppel-Like Factor 4, Retinal Dystrophies genetics, Retinal Dystrophies pathology
- Abstract
The human induced pluripotent stem cell (iPSC) line LEIi019-A was generated from a patient with early-onset pattern dystrophy caused by a heterozygous mutation NM_001270525.1:c.259G>A (p.Glu87Lys) in OTX2. Patient-derived dermal fibroblasts were reprogrammed using episomal plasmids containing reprogramming factors OCT4, SOX2, KLF4, MYCL, LIN28, TP53 shRNA and miR-302/367. The iPSC line expressed pluripotency markers, displayed a normal 46,XY karyotype and demonstrated the ability to differentiate into the three primary germ layers, retinal organoids and retinal pigment epithelial cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2024
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30. Polymer-Based Nanoparticles with Probucol and Lithocholic Acid: A Novel Therapeutic Approach for Oxidative Stress-Induced Retinopathies.
- Author
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Wagle SR, Kovacevic B, Ionescu CM, Foster T, Lim P, Brunet A, McLenachan S, Carvalho L, Mikov M, Mooranian A, and Al-Salami H
- Subjects
- Animals, Cell Line, NF-E2-Related Factor 2 metabolism, Cell Survival drug effects, Mice, Heme Oxygenase-1 metabolism, Humans, Probucol pharmacology, Probucol administration & dosage, Probucol chemistry, Oxidative Stress drug effects, Nanoparticles chemistry, Reactive Oxygen Species metabolism, Lithocholic Acid chemistry, Lithocholic Acid pharmacology, Polymers chemistry, Antioxidants pharmacology, Antioxidants chemistry
- Abstract
Oxidative stress is pivotal in retinal disease progression, causing dysfunction in various retinal components. An effective antioxidant, such as probucol (PB), is vital to counteract oxidative stress and emerges as a potential candidate for treating retinal degeneration. However, the challenges associated with delivering lipophilic drugs such as PB to the posterior segment of the eye, specifically targeting photoreceptor cells, necessitate innovative solutions. This study uses formulation-based spray dry encapsulation technology to develop polymer-based PB-lithocholic acid (LCA) nanoparticles and assesses their efficacy in the 661W photoreceptor-like cell line. Incorporating LCA enhances nanoparticles' biological efficacy without compromising PB stability. In vitro studies demonstrate that PB-LCA nanoparticles prevent reactive oxygen species (ROS)-induced oxidative stress by improving cellular viability through the nuclear erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. These findings propose PB-LCA nanoparticles as a promising therapeutic strategy for oxidative stress-induced retinopathies.
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- 2024
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31. Cytokine Levels in Experimental Branch Retinal Vein Occlusion Treated With Either Bevacizumab or Triamcinolone Acetonide.
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McAllister IL, Vijayasekaran S, McLenachan S, Bhikoo R, Chen FK, Zhang D, Kanagalingam E, and Yu DY
- Subjects
- Animals, Swine, Vascular Endothelial Growth Factor A metabolism, Vascular Endothelial Growth Factor A genetics, RNA, Messenger metabolism, RNA, Messenger genetics, Glucocorticoids pharmacology, Glucocorticoids therapeutic use, Gene Expression Regulation drug effects, Glial Fibrillary Acidic Protein metabolism, Glial Fibrillary Acidic Protein genetics, Potassium Channels, Inwardly Rectifying, Bevacizumab pharmacology, Bevacizumab therapeutic use, Triamcinolone Acetonide pharmacology, Triamcinolone Acetonide administration & dosage, Triamcinolone Acetonide therapeutic use, Retinal Vein Occlusion drug therapy, Retinal Vein Occlusion metabolism, Disease Models, Animal, Angiogenesis Inhibitors pharmacology, Angiogenesis Inhibitors therapeutic use, Angiogenesis Inhibitors administration & dosage, Cytokines metabolism, Cytokines genetics, Intravitreal Injections
- Abstract
Purpose: To compare gene expression changes following branch retinal vein occlusion (BRVO) in the pig with and without bevacizumab (BEV) and triamcinolone acetonide (TA)., Methods: Photothrombotic BRVOs were created in both eyes of four groups of nine pigs (2, 6, 10, and 20 days). In each group, six pigs received intravitreal injections of BEV in one eye and TA in the fellow eye, with three pigs serving as untreated BRVO controls. Three untreated pigs served as healthy controls. Expression of mRNA of vascular endothelial growth factor (VEGF), glial fibrillary acidic protein (GFAP), dystrophin (DMD), potassium inwardly rectifying channel subfamily J member 10 protein (Kir4.1, KCNJ10), aquaporin-4 (AQP4), stromal cell-derived factor-1α (CXCL12), interleukin-6 (IL6), interleukin-8 (IL8), monocyte chemoattractant protein-1 (CCL2), intercellular adhesion molecule 1 (ICAM1), and heat shock factor 1 (HSF1) were analyzed by quantitative reverse-transcription polymerase chain reaction. Retinal VEGF protein levels were characterized by immunohistochemistry., Results: In untreated eyes, BRVO significantly increased expression of GFAP, IL8, CCL2, ICAM1, HSF1, and AQP4. Expression of VEGF, KCNJ10, and CXCL12 was significantly reduced by 6 days post-BRVO, with expression recovering to healthy control levels by day 20. Treatment with BEV or TA significantly increased VEGF, DMD, and IL6 expression compared with untreated BRVO eyes and suppressed BRVO-induced CCL2 and AQP4 upregulation, as well as recovery of KCNJ10 expression, at 10 to 20 days post-BRVO., Conclusions: Inflammation and cellular osmohomeostasis rather than VEGF suppression appear to play important roles in BRVO-induced retinal neurodegeneration, enhanced in both BEV- and TA-treated retinas., Translational Relevance: Inner retinal neurodegeneration seen in this acute model of BRVO appears to be mediated by inflammation and alterations in osmohomeostasis rather than VEGF inhibition, which may have implications for more specific treatment modalities in the acute phase of BRVO.
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- 2024
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32. Retinal Dystrophies Associated With Peripherin-2: Genetic Spectrum and Novel Clinical Observations in 241 Patients.
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Heath Jeffery RC, Thompson JA, Lo J, Chelva ES, Armstrong S, Pulido JS, Procopio R, Vincent AL, Bianco L, Battaglia Parodi M, Ziccardi L, Antonelli G, Barbano L, Marques JP, Geada S, Carvalho AL, Tang WC, Chan CM, Boon CJF, Hensman J, Chen TC, Lin CY, Chen PL, Vincent A, Tumber A, Heon E, Grigg JR, Jamieson RV, Cornish EE, Nash BM, Borooah S, Ayton LN, Britten-Jones AC, Edwards TL, Ruddle JB, Sharma A, Porter RG, Lamey TM, McLaren TL, McLenachan S, Roshandel D, and Chen FK
- Subjects
- Humans, Middle Aged, Adult, Male, Female, Adolescent, Aged, Child, Young Adult, Child, Preschool, Tomography, Optical Coherence, Mutation, Fluorescein Angiography, Genetic Association Studies, Retrospective Studies, DNA Mutational Analysis, DNA genetics, Pedigree, Peripherins genetics, Retinal Dystrophies genetics, Retinal Dystrophies physiopathology, Retinal Dystrophies diagnosis, Visual Acuity physiology, Electroretinography, Phenotype
- Abstract
Purpose: To describe the clinical, electrophysiological and genetic spectrum of inherited retinal diseases associated with variants in the PRPH2 gene., Methods: A total of 241 patients from 168 families across 15 sites in 9 countries with pathogenic or likely pathogenic variants in PRPH2 were included. Records were reviewed for age at symptom onset, visual acuity, full-field ERG, fundus colour photography, fundus autofluorescence (FAF), and SD-OCT. Images were graded into six phenotypes. Statistical analyses were performed to determine genotype-phenotype correlations., Results: The median age at symptom onset was 40 years (range, 4-78 years). FAF phenotypes included normal (5%), butterfly pattern dystrophy, or vitelliform macular dystrophy (11%), central areolar choroidal dystrophy (28%), pseudo-Stargardt pattern dystrophy (41%), and retinitis pigmentosa (25%). Symptom onset was earlier in retinitis pigmentosa as compared with pseudo-Stargardt pattern dystrophy (34 vs 44 years; P = 0.004). The median visual acuity was 0.18 logMAR (interquartile range, 0-0.54 logMAR) and 0.18 logMAR (interquartile range 0-0.42 logMAR) in the right and left eyes, respectively. ERG showed a significantly reduced amplitude across all components (P < 0.001) and a peak time delay in the light-adapted 30-Hz flicker and single-flash b-wave (P < 0.001). Twenty-two variants were novel. The central areolar choroidal dystrophy phenotype was associated with 13 missense variants. The remaining variants showed marked phenotypic variability., Conclusions: We described six distinct FAF phenotypes associated with variants in the PRPH2 gene. One FAF phenotype may have multiple ERG phenotypes, demonstrating a discordance between structure and function. Given the vast spectrum of PRPH2 disease our findings are useful for future clinical trials.
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- 2024
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33. Rapid Variant Pathogenicity Analysis by CRISPR Activation of CRB1 Gene Expression in Patient-Derived Fibroblasts.
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Moon SY, Zhang D, Chen SC, Lamey TM, Thompson JA, McLaren TL, Chen FK, and McLenachan S
- Subjects
- Humans, Reactive Oxygen Species metabolism, Virulence, Gene Editing, Gene Expression, Eye Proteins genetics, Eye Proteins metabolism, Membrane Proteins genetics, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
- Abstract
Inherited retinal diseases (IRDs) are a heterogeneous group of blinding genetic disorders caused by pathogenic variants in genes expressed in the retina. In this study, we sought to develop a method for rapid evaluation of IRD gene variant pathogenicity by inducing expression of retinal genes in patient-derived fibroblasts using CRISPR-activation (CRISPRa). We demonstrate CRISPRa of CRB1 expression in fibroblasts derived from patients with retinitis pigmentosa, enabling investigation of pathogenic mechanisms associated with specific variants. We show the CRB1 c.4005 + 1G>A variant caused exon 11 skipping in CRISPR-activated fibroblasts and retinal organoids (ROs) derived from the same RP12 patient. The c.652 + 5G>C variant was shown to enhance exon 2 skipping in CRISPR-activated fibroblasts and differentially affected CRB1 isoform expression in fibroblasts and ROs. Our study demonstrates an accessible platform for transcript screening of IRD gene variants in patient-derived fibroblasts, which can potentially be applied for rapid pathogenicity assessments of any gene variant.
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- 2024
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34. A Precision Therapy Approach for Retinitis Pigmentosa 11 Using Splice-Switching Antisense Oligonucleotides to Restore the Open Reading Frame of PRPF31.
- Author
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Grainok J, Pitout IL, Chen FK, McLenachan S, Heath Jeffery RC, Mitrpant C, and Fletcher S
- Subjects
- Humans, Open Reading Frames, Mutation, Codon, Nonsense, Eye Proteins genetics, Eye Proteins metabolism, Pedigree, RNA Precursors genetics, Oligonucleotides, Antisense genetics, Oligonucleotides, Antisense therapeutic use, Retinitis Pigmentosa
- Abstract
Retinitis pigmentosa 11 is an untreatable, dominantly inherited retinal disease caused by heterozygous mutations in pre-mRNA processing factor 31 PRPF31 . The expression level of PRPF31 is linked to incomplete penetrance in affected families; mutation carriers with higher PRPF31 expression can remain asymptomatic. The current study explores an antisense oligonucleotide exon skipping strategy to treat RP11 caused by truncating mutations within PRPF31 exon 12 since it does not appear to encode any domains essential for PRPF31 protein function. Cells derived from a patient carrying a PRPF31 1205C>A nonsense mutation were investigated; PRPF31 transcripts encoded by the 1205C>A allele were undetectable due to nonsense-mediated mRNA decay, resulting in a 46% reduction in PRPF31 mRNA, relative to healthy donor cells. Antisense oligonucleotide-induced skipping of exon 12 rescued the open reading frame with consequent 1.7-fold PRPF31 mRNA upregulation in the RP11 patient fibroblasts. The level of PRPF31 upregulation met the predicted therapeutic threshold of expression inferred in a non-penetrant carrier family member harbouring the same mutation. This study demonstrated increased PRPF31 expression and retention of the nuclear translocation capability for the induced PRPF31 isoform. Future studies should evaluate the function of the induced PRPF31 protein on pre-mRNA splicing in retinal cells to validate the therapeutic approach for amenable RP11-causing mutations.
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- 2024
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35. Recent Therapeutic Progress and Future Perspectives for the Treatment of Hearing Loss.
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Lye J, Delaney DS, Leith FK, Sardesai VS, McLenachan S, Chen FK, Atlas MD, and Wong EYM
- Abstract
Up to 1.5 billion people worldwide suffer from various forms of hearing loss, with an additional 1.1 billion people at risk from various insults such as increased consumption of recreational noise-emitting devices and ageing. The most common type of hearing impairment is sensorineural hearing loss caused by the degeneration or malfunction of cochlear hair cells or spiral ganglion nerves in the inner ear. There is currently no cure for hearing loss. However, emerging frontier technologies such as gene, drug or cell-based therapies offer hope for an effective cure. In this review, we discuss the current therapeutic progress for the treatment of hearing loss. We describe and evaluate the major therapeutic approaches being applied to hearing loss and summarize the key trials and studies.
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- 2023
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36. Incidence and Mortality of Conjunctival Melanoma in Australia (1982 to 2014).
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Beasley AB, Preen DB, McLenachan S, Gray ES, and Chen FK
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- Male, Female, Humans, Incidence, Australia epidemiology, Databases, Factual, Melanoma epidemiology, Conjunctival Neoplasms epidemiology
- Abstract
Purpose: The purpose of this study was to estimate the incidence and mortality of conjunctival melanoma in Australia from 1982 to 2014., Methods: De-identified unit data for all cases of ocular melanoma were extracted from the Australian Cancer Database from 1982 to 2014. Conjunctival melanoma cases were extracted, and the incidence and mortality were analyzed. Incidence rates were age-standardized against the 2001 Australian Standard Population. Mortality was assessed using log-rank and Cox regression., Results: From 1982 to 2014, there were 299 cases of conjunctival melanoma. The age-standardized incidence rate was 0.48 (95% confidence interval [CI] = 0.41 to 0.54) per million per year. Women (0.52, 95% CI = 0.42 to 0.62) had a higher incidence than men (0.42, 95% CI = 0.33 to 0.51). The incidence of conjunctival melanoma increased in men (+1.46%) and significantly women (+1.41%, P = 0.023) over the study period. The mean 5-, 10-, and 15-year disease-specific survival were 90%, 82%, and 80%, respectively, during the 33-year interval. Comparisons of survival among age, sex, and state revealed no significant differences when tested using log-rank or Cox regression., Conclusions: In conclusion, we found an increase in the rate of conjunctival melanoma diagnoses in Australia from 1982 to 2014. Over the same period, disease survival remained unchanged at a mean of 90%.
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- 2023
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37. In vivo retinal imaging is associated with cognitive decline, blood-brain barrier disruption and neuroinflammation in type 2 diabetic mice.
- Author
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Majimbi M, McLenachan S, Nesbit M, Chen FK, Lam V, Mamo J, and Takechi R
- Subjects
- Mice, Animals, Neuroinflammatory Diseases, Blood-Brain Barrier metabolism, Retina, Inflammation diagnostic imaging, Inflammation metabolism, Diabetes Mellitus, Type 2 complications, Diabetes Mellitus, Type 2 diagnostic imaging, Diabetes Mellitus, Type 2 metabolism, Diabetes Mellitus, Experimental complications, Diabetes Mellitus, Experimental diagnostic imaging, Diabetes Mellitus, Experimental metabolism, Diabetic Retinopathy metabolism, Cognitive Dysfunction diagnostic imaging, Cognitive Dysfunction etiology, Cognitive Dysfunction metabolism
- Abstract
Introduction: Type 2 diabetes (T2D) is associated with chronic inflammation and neurovascular changes that lead to functional impairment and atrophy in neural-derived tissue. A reduction in retinal thickness is an early indicator of diabetic retinopathy (DR), with progressive loss of neuroglia corresponding to DR severity. The brain undergoes similar pathophysiological events as the retina, which contribute to T2D-related cognitive decline., Methods: This study explored the relationship between retinal thinning and cognitive decline in the LepR db/db model of T2D. Diabetic db/db and non-diabetic db/+ mice aged 14 and 28 weeks underwent cognitive testing in short and long-term memory domains and in vivo retinal imaging using optical coherence tomography (OCT), followed by plasma metabolic measures and ex vivo quantification of neuroinflammation, oxidative stress and microvascular leakage., Results: At 28 weeks, mice exhibited retinal thinning in the ganglion cell complex and inner nuclear layer, concomitant with diabetic insulin resistance, memory deficits, increased expression of inflammation markers and cerebrovascular leakage. Interestingly, alterations in retinal thickness at both experimental timepoints were correlated with cognitive decline and elevated immune response in the brain and retina., Discussion: These results suggest that changes in retinal thickness quantified with in vivo OCT imaging may be an indicator of diabetic cognitive dysfunction and neuroinflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Majimbi, McLenachan, Nesbit, Chen, Lam, Mamo and Takechi.)
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- 2023
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38. Genetic predisposition to ocular surface disorders and opportunities for gene-based therapies.
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Roshandel D, Semnani F, Rayati Damavandi A, Masoudi A, Baradaran-Rafii A, Watson SL, Morgan WH, and McLenachan S
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- Humans, Genetic Predisposition to Disease, Cornea, Corneal Diseases genetics, Corneal Diseases therapy, Aniridia complications
- Abstract
The ocular surface, comprised of the corneal and conjunctival epithelium, innervation system, immune components, and tear-film apparatus, plays a key role in ocular integrity as well as comfort and vision. Gene defects may result in congenital ocular or systemic disorders with prominent ocular surface involvement. Examples include epithelial corneal dystrophies, aniridia, ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome, xeroderma pigmentosum (XP), and hereditary sensory and autonomic neuropathy. In addition, genetic factors may interact with environmental risk factors in the development of several multifactorial ocular surface disorders (OSDs) such as autoimmune disorders, allergies, neoplasms, and dry eye disease. Advanced gene-based technologies have already been introduced in disease modelling and proof-of-concept gene therapies for monogenic OSDs. For instance, patient-derived induced pluripotent stem cells have been used for modelling aniridia-associated keratopathy (AAK), XP, and EEC syndrome. Moreover, CRISPR/Cas9 genome editing has been used for disease modelling and/or gene therapy for AAK and Meesmann's epithelial corneal dystrophy. A better understanding of the role of genetic factors in OSDs may be helpful in designing personalized disease models and treatment approaches. Gene-based approaches in monogenic OSDs and genetic predisposition to multifactorial OSDs such as immune-mediated disorders and neoplasms with known or possible genetic risk factors has been seldom reviewed. In this narrative review, we discuss the role of genetic factors in monogenic and multifactorial OSDs and potential opportunities for gene therapy., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2023
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39. Mitochondrial Dysfunction and Impaired Antioxidant Responses in Retinal Pigment Epithelial Cells Derived from a Patient with RCBTB1 -Associated Retinopathy.
- Author
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Huang Z, Zhang D, Chen SC, Huang D, Mackey D, Chen FK, and McLenachan S
- Subjects
- Humans, Reactive Oxygen Species metabolism, Epithelial Cells metabolism, Mitochondria metabolism, Retinal Pigments metabolism, Guanine Nucleotide Exchange Factors metabolism, Antioxidants metabolism, Retinal Diseases metabolism
- Abstract
Mutations in the RCBTB1 gene cause inherited retinal disease; however, the pathogenic mechanisms associated with RCBTB1 deficiency remain poorly understood. Here, we investigated the effect of RCBTB1 deficiency on mitochondria and oxidative stress responses in induced pluripotent stem cell (iPSC)-derived retinal pigment epithelial (RPE) cells from control subjects and a patient with RCBTB1 -associated retinopathy. Oxidative stress was induced with tert-butyl hydroperoxide (tBHP). RPE cells were characterized by immunostaining, transmission electron microscopy (TEM), CellROX assay, MitoTracker assay, quantitative PCR and immunoprecipitation assay. Patient-derived RPE cells displayed abnormal mitochondrial ultrastructure and reduced MitoTracker fluorescence compared with controls. Patient RPE cells displayed increased levels of reactive oxygen species (ROS) and were more sensitive to tBHP-induced ROS generation than control RPE. Control RPE upregulated RCBTB1 and NFE2L2 expression in response to tBHP treatment; however, this response was highly attenuated in patient RPE. RCBTB1 was co-immunoprecipitated from control RPE protein lysates by antibodies for either UBE2E3 or CUL3. Together, these results demonstrate that RCBTB1 deficiency in patient-derived RPE cells is associated with mitochondrial damage, increased oxidative stress and an attenuated oxidative stress response.
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- 2023
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40. Serum miRNA modulations indicate changes in retinal morphology.
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Aggio-Bruce R, Schumann U, Cioanca AV, Chen FK, McLenachan S, Heath Jeffery RC, Das S, and Natoli R
- Abstract
Background: Age-related macular degeneration (AMD) is the leading cause of vision loss in the developed world and the detection of its onset and progression are based on retinal morphological assessments. MicroRNA (miRNA) have been explored extensively as biomarkers for a range of neurological diseases including AMD, however differences in experimental design and the complexity of human biology have resulted in little overlap between studies. Using preclinical animal models and clinical samples, this study employs a novel approach to determine a serum signature of AMD progression., Methods: Serum miRNAs were extracted from mice exposed to photo-oxidative damage (PD; 0, 1, 3 and 5 days), and clinical samples from patients diagnosed with reticular pseudodrusen or atrophic AMD. The expression of ~800 miRNAs was measured using OpenArray™, and differential abundance from controls was determined using the HTqPCR R package followed by pathway analysis with DAVID. MiRNA expression changes were compared against quantifiable retinal histological indicators. Finally, the overlap of miRNA changes observed in the mouse model and human patient samples was investigated., Results: Differential miRNA abundance was identified at all PD time-points and in clinical samples. Importantly, these were associated with inflammatory pathways and histological changes in the retina. Further, we were able to align findings in the mouse serum to those of clinical patients., Conclusion: In conclusion, serum miRNAs are a valid tool as diagnostics for the early detection of retinal degeneration, as they reflect key changes in retinal health. The combination of pre-clinical animal models and human patient samples led to the identification of a preliminary serum miRNA signature for AMD. This study is an important platform for the future development of a diagnostic serum miRNA panel for the early detection of retinal degeneration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Aggio-Bruce, Schumann, Cioanca, Chen, McLenachan, Heath Jeffery, Das and Natoli.)
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- 2023
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41. Incidence and mortality of uveal melanoma in Australia (1982-2014).
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Beasley AB, Preen DB, McLenachan S, Gray ES, and Chen FK
- Subjects
- Humans, Middle Aged, Incidence, Australia epidemiology, Melanoma pathology, Uveal Neoplasms pathology
- Abstract
Aims: We aimed to estimate the incidence and mortality of uveal melanoma (UM) in Australia from 1982 to 2014., Methods: Deidentified unit data for all cases of ocular melanoma were extracted from the Australian Cancer Database from 1 January 1982 to 31 December 2014. UM cases were extracted and trends in incidence and disease-specific mortality were calculated. Incidence rates were age-standardised against the 2001 Australian Standard Population. Mortality was assessed using Cox regression., Results: From 1982 to 2014, there were 5087 cases of ocular melanoma in Australia, of which 4617 were classified as UM. The average age-standardised incidence rate of UM was 7.6 (95% CI 7.3 to 7.9) per million. There was an increase (p=0.0502) in the incidence of UM from 1982 to 1993 with an annual percent change (APC) of +2.5%, followed by a significant decrease in the incidence of UM from 1993 to 2014 (APC -1.2%). The average 5-year survival from 1982 to 2011 did not significantly change from an average of 81%, with an average APC (AAPC) of +0.1%. A multivariate Cox regression revealed that residence in Western Australia (p=0.001) or Tasmania (p=0.05), age ≥60 years (p<0.001) and histological classification as mixed (p<0.001) or epithelioid cells (p<0.001) were significantly associated with reduced survival., Conclusion: In conclusion, we found that the incidence of UM peaked in the 1990s. Although treatment for primary UM has improved in the last 30 years, overall survival did not change significantly in the last 30 years., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)
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- 2023
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42. Knockout of AMD-associated gene POLDIP2 reduces mitochondrial superoxide in human retinal pigment epithelial cells.
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Nguyen T, Urrutia-Cabrera D, Wang L, Lees JG, Wang JH, Hung SSC, Hewitt AW, Edwards TL, McLenachan S, Chen FK, Lim SY, Luu CD, Guymer R, and Wong RCB
- Subjects
- Humans, Oxidative Stress genetics, Retinal Pigment Epithelium pathology, Epithelial Cells metabolism, Retinal Pigments metabolism, Nuclear Proteins metabolism, Superoxides metabolism, Macular Degeneration genetics, Macular Degeneration pathology
- Abstract
Genetic and epidemiologic studies have significantly advanced our understanding of the genetic factors contributing to age-related macular degeneration (AMD). In particular, recent expression quantitative trait loci (eQTL) studies have highlighted POLDIP2 as a significant gene that confers risk of developing AMD. However, the role of POLDIP2 in retinal cells such as retinal pigment epithelium (RPE) and how it contributes to AMD pathology are unknown. Here we report the generation of a stable human RPE cell line ARPE-19 with POLDIP2 knockout using CRISPR/Cas, providing an in vitro model to investigate the functions of POLDIP2 . We conducted functional studies on the POLDIP2 knockout cell line and showed that it retained normal levels of cell proliferation, cell viability, phagocytosis and autophagy. Also, we performed RNA sequencing to profile the transcriptome of POLDIP2 knockout cells. Our results highlighted significant changes in genes involved in immune response, complement activation, oxidative damage and vascular development. We showed that loss of POLDIP2 caused a reduction in mitochondrial superoxide levels, which is consistent with the upregulation of the mitochondrial superoxide dismutase SOD2 . In conclusion, this study demonstrates a novel link between POLDIP2 and SOD2 in ARPE-19, which supports a potential role of POLDIP2 in regulating oxidative stress in AMD pathology.
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- 2023
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43. Inner Retinal Changes in Acute Experimental BRVO Treated With Bevacizumab or Triamcinolone Acetonide.
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McAllister IL, Vijayasekaran S, Bhikoo R, Chen FK, Zhang D, Kanagalingam E, McLenachan S, and Yu DY
- Subjects
- Swine, Animals, Bevacizumab pharmacology, Bevacizumab therapeutic use, Vascular Endothelial Growth Factor A, Retina metabolism, Triamcinolone Acetonide pharmacology, Triamcinolone Acetonide therapeutic use, Retinal Vein Occlusion drug therapy
- Abstract
Purpose: Apoptosis is a key process in neural degeneration associated with retinal vascular diseases. Vascular endothelial growth factor (VEGF) antagonists, including bevacizumab, are used to treat macular edema in these diseases. As VEGF has a critical role in the preservation of retinal neuronal cells, this study investigates the effects of bevacizumab on neural damage in a pig model of branch retinal vein occlusion (BRVO) and compares it with triamcinolone acetonide (TA) which is reported to possess neuroprotective properties., Methods: Thirty-six pigs had a photothrombotic BRVO in both eyes. Six pigs were injected with bevacizumab in one eye and TA in the fellow eye, then they were sacrificed, the eyes enucleated, and retinas processed at 2, 6, 10, and 20 days, respectively, together with three pigs (six eyes) BRVO only and three normal pigs (six eyes). Neuronal degeneration (apoptosis) and associated inner retinal changes were determined by terminal deoxyynuclotidyl transferase dUTP nick-end labeling (TUNEL), histology, and immunohistochemistry for macrophages., Results: TUNEL labeling showed significantly higher apoptosis rates in the ganglion cell layer (GCL) and the inner nuclear layer (INL) in the bevacizumab-treated compared with the TA-treated retinas at 2, 10, and 20 day time points after occlusion (P < 0.05). Pyknotic cells were significantly higher in the GCL in bevacizumab-treated eyes at 6, 10, and 20 days and in the INL at 2 days compared to TA-treated retinas (P < 0.05). Macrophage infiltration was seen at all time points in both untreated and treated retinas with an absence of significance between bevacizumab- and TA-treated retinas (P > 0.05)., Conclusions: Neurodegeneration in the BRVO acute phase is exacerbated by current standard treatments for BRVO. These results may have implications for the timing and treatment type., Translational Relevance: In the acute phase of BRVO, VEGF suppression with bevacizumab and to a lesser extent with triamcinolone exacerbates apoptosis in the inner retinal layers, which has implications for both the timing and choice of treatment.
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- 2023
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44. Microperimetry and Adaptive Optics Imaging Reveal Localized Functional and Structural Changes in Asymptomatic RPGR Mutation Carriers.
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Roshandel D, Lamey TM, Charng J, Heath Jeffery RC, McLaren TL, Thompson JA, De Roach JN, McLenachan S, Mackey DA, and Chen FK
- Subjects
- Humans, Female, Cross-Sectional Studies, Case-Control Studies, Mutation, Eye Proteins genetics, Visual Field Tests, Tomography, Optical Coherence methods
- Abstract
Purpose: Female carriers of RPGR mutations demonstrate no significant retinal dysfunction or structural change despite a characteristic tapetal-like reflex. In this study, we examined localized changes of pointwise sensitivity (PWS) and cone density (CD) using microperimetry (MP) and adaptive optics (AO) imaging in female carriers of RPGR mutations., Methods: In this cross-sectional case-control study, MP (MAIA, 10-2 test grid) and AO imaging (rtx1) were performed in female carriers of RPGR mutations and unrelated age-matched healthy controls. PWS at 68 loci located 1 degree to 9 degrees away from the preferred retinal locus and CD at 12 loci located 1 degree to 3 degrees away from the foveal center were measured. Severity of defect was defined by standard deviation (SD) from age-matched healthy control means: normal (<1 SD from normal average), moderate defect (1-2 SD from normal average), and severe defect (>2 SD from normal average)., Results: Twelve patients from seven unrelated families were enrolled. Seven patients were asymptomatic, 5 of whom had visual acuity 20/20 or better in both eyes. PWS and CD were available in 12 and 8 patients, respectively. Severe PWS and CD defect in at least 1 test location was observed in 10 of 12 patients and 7 of 8 patients, respectively. Among the five asymptomatic patients who had normal visual acuity, severe PWS and CD defects were observed in three of five and four of five patients, respectively., Conclusions: MP and AO imaging revealed early functional and structural changes in asymptomatic RPGR mutation carriers and should be considered in clinical assessment of these patients.
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- 2023
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45. Characterising splicing defects of ABCA4 variants within exons 13-50 in patient-derived fibroblasts.
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Huang D, Thompson JA, Chen SC, Adams A, Pitout I, Lima A, Zhang D, Jeffery RCH, Attia MS, McLaren TL, Lamey TM, De Roach JN, McLenachan S, Aung-Htut MT, Fletcher S, Wilton SD, and Chen FK
- Subjects
- Humans, Stargardt Disease genetics, Introns genetics, Australia, Exons genetics, Mutation, Fibroblasts, Pedigree, ATP-Binding Cassette Transporters genetics, Retinal Diseases genetics
- Abstract
The ATP-binding cassette subfamily A member 4 gene (ABCA4)-associated retinopathy, Stargardt disease, is the most common monogenic inherited retinal disease. Given the pathogenicity of numerous ABCA4 variants is yet to be examined and a significant proportion (more than 15%) of ABCA4 variants are categorized as splice variants in silico, we therefore established a fibroblast-based splice assay to analyze ABCA4 variants in an Australian Stargardt disease cohort and characterize the pathogenic mechanisms of ABCA4 variants. A cohort of 67 patients clinically diagnosed with Stargardt disease was recruited. Genomic DNA was analysed using a commercial panel for ABCA4 variant detection and the consequences of ABCA4 variants were predicted in silico. Dermal fibroblasts were propagated from skin biopsies, total RNA was extracted and the ABCA4 transcript was amplified by RT-PCR. Our analysis identified a total of 67 unique alleles carrying 74 unique variants. The most prevalent splice-affecting complex allele c.[5461-10T>C; 5603A>T] was carried by 10% of patients in a compound heterozygous state. ABCA4 transcripts from exon 13 to exon 50 were readily detected in fibroblasts. In this region, aberrant splicing was evident in 10 out of 57 variant transcripts (18%), carried by 19 patients (28%). Patient-derived fibroblasts provide a feasible platform for identification of ABCA4 splice variants located within exons 13-50. Experimental evidence of aberrant splicing contributes to the pathogenic classification for ABCA4 variants. Moreover, identification of variants that affect splicing processes provides opportunities for intervention, in particular antisense oligonucleotide-mediated splice correction., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2022
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46. Pathogenesis and Treatment of Usher Syndrome Type IIA.
- Author
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Zaw K, Carvalho LS, Aung-Htut MT, Fletcher S, Wilton SD, Chen FK, and McLenachan S
- Subjects
- Animals, Humans, Mutation, Retina, Retinitis Pigmentosa genetics, Usher Syndromes diagnosis, Usher Syndromes genetics, Usher Syndromes therapy
- Abstract
Usher syndrome (USH) is the most common form of deaf-blindness, with an estimated prevalence of 4.4 to 16.6 per 100,000 people worldwide. The most common form of USH is type IIA (USH2A), which is caused by homozygous or compound heterozygous mutations in the USH2A gene and accounts for around half of all USH cases. USH2A patients show moderate to severe hearing loss from birth, with diagnosis of retinitis pigmentosa in the second decade of life and variable vestibular involvement. Although hearing aids or cochlear implants can provide some mitigation of hearing deficits, there are currently no treatments aimed at preventing or restoring vision loss in USH2A patients. In this review, we first provide an overview of the molecular biology of the USH2A gene and its protein isoforms, which include a transmembrane protein (TM usherin) and an extracellular protein (EC usherin). The role of these proteins in the inner ear and retina and their impact on the pathogenesis of USH2A is discussed. We review animal cell-derived and patient cell-derived models currently used in USH2A research and conclude with an overview of potential treatment strategies currently in preclinical development and clinical trials., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2022 Asia-Pacific Academy of Ophthalmology. Published by Wolters Kluwer Health, Inc. on behalf of the Asia-Pacific Academy of Ophthalmology.)
- Published
- 2022
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47. Anti-retinal IgG antibodies in patients with early and advanced type 2 macular telangiectasia.
- Author
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McLenachan S, Balaratnasingam C, Heath Jeffery RC, Chen SC, Zhang D, Chan G, Dolz-Marco R, Bacci T, Lo J, Wiffen S, Yannuzzi LA, and Chen FK
- Subjects
- Adult, Female, Humans, Immunoglobulin G, Retina pathology, Tomography, Optical Coherence methods, Diabetic Retinopathy pathology, Macular Degeneration pathology, Retinal Telangiectasis diagnosis, Retinal Telangiectasis pathology
- Abstract
Type 2 idiopathic macular telangiectasia (MacTel-2) is a progressive adult-onset macular disease associated with bilateral perifoveal vascular changes, Muller cell degeneration and increased blood-retinal barrier permeability. The pathophysiological mechanisms of MacTel-2 remain unclear, however it was previously reported that anti-retinal antibodies in MacTel-2 patients are a significant feature of the disease. In this study, we aimed to compare the prevalence of anti-retinal antibodies in patients MacTel-2, healthy controls and patients with other retinal diseases. MacTel-2 patients diagnosed with multimodal imaging were enrolled and their disease severities were graded using spectral-domain optical coherence tomography. For comparison, patients with age-related macular degeneration (AMD), inherited retinal diseases (IRDs) or no retinal disease (healthy controls) were recruited as controls. Blood serum samples were screened for immunoglobulin G anti-retinal antibodies by western blotting, followed by densitometry analysis. Odds ratios (OR) with 95% confidence intervals (CI) were calculated and p < 0.05 considered statistically significant. Overall, anti-retinal antibody-positive cases were older (64 ± 15 vs 53 ± 17 years, p < 0.001) and females were more likely to develop anti-retinal antibodies (OR: 2.41, CI: 1.12-5.18). The frequency of anti-retinal antibody detection in MacTel-2 patients (n = 42, 36%) was not significantly different from healthy controls (n = 52, 25%) or IRD patients (n = 18, 25%) and the majority of MacTel-2 patients had no anti-retinal antibodies. In contrast, the frequency of anti-retinal antibody detection was significantly higher in patients with AMD (n = 15, 73%, p < 0.001). The lack of a greater anti-retinal antibody frequency or specificity in the MacTel-2 cohort suggests that antibody mediated immunological mechanisms may play a less significant role in MacTel-2 disease pathogenesis., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2022
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48. Stargardt disease and progress in therapeutic strategies.
- Author
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Huang D, Heath Jeffery RC, Aung-Htut MT, McLenachan S, Fletcher S, Wilton SD, and Chen FK
- Subjects
- Animals, Humans, Lipofuscin metabolism, Mutation, Phenotype, Retina metabolism, Stargardt Disease, ATP-Binding Cassette Transporters genetics, Retinal Dystrophies
- Abstract
Background: Stargardt disease (STGD1) is an autosomal recessive retinal dystrophy due to mutations in ABCA4, characterized by subretinal deposition of lipofuscin-like substances and bilateral centrifugal vision loss. Despite the tremendous progress made in the understanding of STGD1, there are no approved treatments to date. This review examines the challenges in the development of an effective STGD1 therapy., Materials and Methods: A literature review was performed through to June 2021 summarizing the spectrum of retinal phenotypes in STGD1, the molecular biology of ABCA4 protein, the in vivo and in vitro models used to investigate the mechanisms of ABCA4 mutations and current clinical trials., Results: STGD1 phenotypic variability remains an challenge for clinical trial design and patient selection. Pre-clinical development of therapeutic options has been limited by the lack of animal models reflecting the diverse phenotypic spectrum of STDG1. Patient-derived cell lines have facilitated the characterization of splice mutations but the clinical presentation is not always predicted by the effect of specific mutations on retinoid metabolism in cellular models. Current therapies primarily aim to delay vision loss whilst strategies to restore vision are less well developed., Conclusions: STGD1 therapy development can be accelerated by a deeper understanding of genotype-phenotype correlations.
- Published
- 2022
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49. Short-Term Parafoveal Cone Loss Despite Preserved Ellipsoid Zone in Rod Cone Dystrophy.
- Author
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Roshandel D, Heath Jeffery RC, Charng J, Sampson DM, McLenachan S, Mackey DA, and Chen FK
- Subjects
- Fovea Centralis diagnostic imaging, Humans, Prospective Studies, Retinal Cone Photoreceptor Cells, Visual Acuity, Cone-Rod Dystrophies
- Abstract
Purpose: Rod-cone dystrophy (RCD) is characterized by centripetal loss of rod followed by cone photoreceptors. In this prospective, observational cohort, we used flood-illumination adaptive optics (AO) imaging to investigate parafoveal cone loss in regions with preserved ellipsoid zone (EZ) in patients with RCD., Methods: Eight patients with RCD and 10 age-matched healthy controls underwent spectral-domain optical coherence tomography and AO imaging. The RCD cohort underwent a follow-up examination after 6 months. Cone density (CD) and intercone distance (ICD) measurements were performed at 2° temporal from the fovea. Baseline CD and ICD values were compared between the control and patient groups, and longitudinal changes were calculated in the patient group. Residual EZ span in patients was measured in horizontal foveal B-scans., Results: Between the control and patient groups, there was no significant difference in the baseline CD (2094 vs. 1750 cells/deg2, respectively; P = 0.09) and ICD (1.46 vs. 1.62 arcmin, respectively; P = 0.08). Mean CD declined by 198 cells/deg2 (-11.3%; P < 0.01), and mean ICD increased by 0.09 arcmin (+5.6%; P = 0.01) at the 6-month follow-up in the patient group. Mean baseline and follow-up residual EZ spans in the six patients with EZ defect were 3189 µm and 3065 µm, respectively (-3.9%; P = 0.08)., Conclusions: AO imaging detected significant parafoveal cone loss over 6-month follow-up even in regions with preserved EZ. Further studies to refine AO imaging protocol and validate cone metrics as a structural endpoint in early RCD are warranted., Translational Relevance: CD and ICD may change prior to EZ span shortening in RCD.
- Published
- 2021
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50. Gene replacement therapy restores RCBTB1 expression and cilium length in patient-derived retinal pigment epithelium.
- Author
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Huang Z, Zhang D, Chen SC, Jennings L, Carvalho LS, Fletcher S, Chen FK, and McLenachan S
- Subjects
- Cell Differentiation, Cells, Cultured, Cilia ultrastructure, Dependovirus genetics, Genetic Vectors genetics, Humans, Induced Pluripotent Stem Cells cytology, Induced Pluripotent Stem Cells metabolism, Retinal Dystrophies genetics, Retinal Dystrophies therapy, Retinal Pigment Epithelium ultrastructure, Transduction, Genetic, Cilia metabolism, Gene Expression, Genetic Therapy methods, Guanine Nucleotide Exchange Factors genetics, Retinal Pigment Epithelium metabolism, Transgenes
- Abstract
Biallelic mutations in the RCBTB1 gene cause retinal dystrophy. Here, we characterized the effects of RCBTB1 gene deficiency in retinal pigment epithelial (RPE) cells derived from a patient with RCBTB1-associated retinopathy and restored RCBTB1 expression in these cells using adeno-associated viral (AAV) vectors. Induced pluripotent stem cells derived from a patient with compound heterozygous RCBTB1 mutations (c.170delG and c.707delA) and healthy control subjects were differentiated into RPE cells. RPE cells were treated with AAV vectors carrying a RCBTB1 transgene. Patient-derived RPE cells showed reduced expression of RCBTB1. Expression of NFE2L2 showed a non-significant reduction in patient RPE cells compared with controls, while expression of its target genes (RXRA, IDH1 and SLC25A25) was significantly reduced. Trans-epithelial electrical resistance, surface microvillus densities and primary cilium lengths were reduced in patient-derived RPE cells, compared with controls. Treatment of patient RPE with AAV vectors significantly increased RCBTB1, NFE2L2 and RXRA expression and cilium lengths. Our study provides the first report examining the phenotype of RPE cells derived from a patient with RCBTB1-associated retinopathy. Furthermore, treatment of patient-derived RPE with AAV-RCBTB1 vectors corrected deficits in gene expression and RPE ultrastructure, supporting the use of gene replacement therapy for treating this inherited retinal disease., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)
- Published
- 2021
- Full Text
- View/download PDF
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