Purpose: To quantify retinal function longitudinally and crosssectionally in p atients with autosomal-recessive early-onset severe retinal dystrophy (EOSRD) associated with RPE65 mutations. Subjects and methods: The o...Purpose: To quantify retinal function longitudinally and crosssectionally in p atients with autosomal-recessive early-onset severe retinal dystrophy (EOSRD) associated with RPE65 mutations. Subjects and methods: The ocular phenotype was characterized in four children from three families up to the second decade of li fe, and in three siblings from one family aged 43-54 years carrying compound he terozygous or homozygous mutations in RPE65. Standard clinical examination inclu ded colour vision testing, fundus photography and Goldmann visual fields (GVF). Full-field ERGs (in all) and multifocal ERGs (in two patients) were also record ed. Visual performance and fundus appearance were compared to literature data. R esults: In childhood, visual acuity (VA) ranged from 0.1 to 0.3, and GVF for tar get V4 was well preserved. VA and GVF were measurable in only one of the three a dult siblings. Nystagmus was present in two of four children and two of three ad ults. Photophobia was absent in childhood and developed in adulthood. Funduscopi c changes were discrete during the first decade of life in three of four childre n; one patient had clear macular changes already at age 5 years. All three adult siblings had distinct retinal changes including the macula. Bone spicules were not a feature. Residual colour vision was present in all patients with measurabl e VA. Rod ERGs were absent at any age; cone ERGs were detectable in early childh ood. To date, VA data have been reported in 51 patients, visual fields in 29 pat ients, and a detailed fundus description in 34 patients. For all three parameters, data were compara ble to the results in our patient cohort. Conclusion: In childhood, patients wit h RPE65 mutations have better visual functions than typically seen in Leber cong enital amaurosis. The phenotype shows a common progressive pattern with intrafam ilial and interfamilial variation. The data suggest a preserved retinal morpholo gy at young ages, arguing for vision-restoring gene therapy trials in childhood .展开更多
Background: In a recent report we described RPE65, a protein originally characterized in retinal pigment epithelium, to be expressed in normal human epidermis. RPE65 is suspected to be involved in cellular uptake of r...Background: In a recent report we described RPE65, a protein originally characterized in retinal pigment epithelium, to be expressed in normal human epidermis. RPE65 is suspected to be involved in cellular uptake of retinol which is transported in the bloodstream complexed with plasma retinol-binding protein. Objectives: To evaluate protein and mRNA expression of RPE65 in actinic keratosis (AK), squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) compared with normal skin. Methods: RPE65 mRNA expression in skin tumours relative to normal skin of the respective donor was studied by real-time polymerase chain reaction in AK (n=15), invasive SCC (n=30) and BCC (n=18). A peptide-specific anti-RPE65 a ntibody was used for immunohistochemical staining of formalin-fixed and paraffi n-embedded tissue sections of the respective tumours. Results: RPE65 mRNA expre ssion was reduced in AK. A highly significant reduction of RPE65 mRNA was observ ed in invasive SCC relative to normal skin of the respective donors. Immunohisto chemistry revealed a continuous staining of basal and suprabasal keratinocytes i n normal human epidermis. RPE65 in AK shown by immunohistochemical staining was reduced and quite irregular, whereas invasive SCC revealed no staining of tumour cells with the anti-RPE65 antibody. RPE65 mRNA values were elevated, whereas i mmunohistochemical staining for RPE65 proteinwas heterogeneous in BCC. Conclusio ns: These results suggest progressive downregulation of RPE65 from AK to invasive SCC.展开更多
Purpose Fundus autofluorescence is due to accumulation of lipofuscin in the retinal pigment ep ithelium(RPE)resulting from incomplete digestion of N-retinylidene-phosphatidyl-ethanolamine from shed photorecep tor oute...Purpose Fundus autofluorescence is due to accumulation of lipofuscin in the retinal pigment ep ithelium(RPE)resulting from incomplete digestion of N-retinylidene-phosphatidyl-ethanolamine from shed photorecep tor outer segment discs.Alteration in autofluorescence reflects changes in lipofuscin content of the RPE.Mutations on both alleles of RPE65result in absent or largely decrease d formation of rhodopsin,due to a defect in alltrans retinol is omerization in the RPE.Autofluorescence could therefore b e altered.This study was conducted to evaluate fundus autofl uorescence in patients with early-onset severe retinal dystrophy(EOSRD,or ear-ly-onset rod-cone dystrophy)associated with mutations on both alleles of RPE65.Design Case se ries.Participants and controls Ten 10-to 55-year-old p atients with EOSRD and compound heterozygous or homozy gous mutations in RPE65.For comparison,6heterozygous parents and 2patients with other forms of EOSRD we re examined.Methods Participants underwent,in addition to standard clinical and electrophysiological examination,autofluores-cence imaging using a confocal scanning laser ophthalmo-scope.Three of the patients were als o examined by optical coherence tomography(OCT)to evaluate the status of retinal degeneration.Mutations in7patients have been reported previously;the other pati ents were investigated by polymerase chain reaction-single-strand conformation poly-morphism and direct sequencing for mutations in RPE65and lecithin retinol acyltransfera se(LRAT).Main outcome measures Fundus autofluorescence a nd OCT.Results Ab-sent or minimal autofluorescence wa s found in all patients with compound heterozygous or homozygous RPE65muta-tions.Autofluorescence was normal in the heterozygous parents.Autofluorescence was present in 2children with EOSRD not associated with mutations in RPE65or LRAT,another gene involved in retinol recycling.Optical coher-ence tomography in younger patients revealed an intraretinal appearance similar to that of their h ealthy,heterozygous parents.Conclusions Lack of autofl uorescence in patients with EOSRD associated with mutation s in RPE65is in ac-cordance with the biochemical defect and can be used as a clinical marker of this genotype.Optical coherence tomog-raphy results in younger patients wo uld indicate still viable photoreceptors despite the absence of autofluorescence.展开更多
Background： Leber congenital anaaurosis （LCA） is a visual disease which is caused by RPE65 mutations and results in retinal degeneration and severe vision loss in early infancy. According to previous researches, mu...Background： Leber congenital anaaurosis （LCA） is a visual disease which is caused by RPE65 mutations and results in retinal degeneration and severe vision loss in early infancy. According to previous researches, mutations of the RPE65 gene account for 16% of all cases of LCA. This study aimed to identify RPE65 gene mutations in Chinese patients with LCA. Methods： We recruited 52 sporadic patients from Peking University Third Hospital in 2016 and applied Sanger sequencing to identil＇y variants among exons responsible for the disease. The genomic DNAs from blood leukocytes of these patients were isolated, and tile entire coding region of the RPE65 gene was amplified by polymerase chain reaction. We then deterrnined the sequence of RPE65 using ABI 3100 Genetic Analyzer. Results： Our study identified that only 1 out of the 52 patients with LCA carried the previously unreported homozygosis missense mutation c1174A〉C （T392P） of the RPE65 gene. However, the mutation was associated with the disease phenotype and not detected in 100 normal controls. Conclusions： Though we identified a novel missense mutation in the RPE65 gene that causes LCA, our result indicates that RPE65 mutations may not play a major role in the LCA patients in China since only 1 out of the 52 patients carried mutation in the RPE65 gene.展开更多
Leber congenital amaurosis(LCA)is a severe,genetically heterogeneous recessive eye disease in which∼35%of genemutations are in-frame nonsensemutations coding for loss-of-function premature termination codons(PTCs)inm...Leber congenital amaurosis(LCA)is a severe,genetically heterogeneous recessive eye disease in which∼35%of genemutations are in-frame nonsensemutations coding for loss-of-function premature termination codons(PTCs)inmRNA.Nonsense suppression therapy allows read-through of PTCs leading to production of full-length protein.A limitation of nonsense suppression is that nonsense-mediated decay(NMD)degrades PTC-containing RNA transcripts.The purpose of this study was to determine whether inhibition of NMD could improve nonsense suppression efficacy in vivo.Using a high-throughput approach in the recessive cep290 zebrafish model of LCA(cep290;Q1223X),we first tested the NMD inhibitor Amlexanox in combination with the nonsense suppression drug Ataluren.We observed reduced retinal cell death and improved visual function.With these positive data,we next investigated whether this strategy was also applicable across species in two mammalianmodels:Rd12(rpe65;R44X)and Rd3(rd3;R107X)mouse models of LCA.In the Rd12 model,cell death was reduced,RPE65 protein was produced,and in vivo visual function testing was improved.We establish for the first time that the mechanism of action of Amlexanox in Rd12 retina was through reduced UPF1 phosphorylation.In the Rd3 model,however,no beneficial effect was observed with Ataluren alone or in combination with Amlexanox.This variation in response establishes that some forms of nonsensemutation LCA can be targeted by RNA therapies,but that this needs to be verified for each genotype.The implementation of precision medicine by identifying better responders to specific drugs is essential for development of validated retinal therapies.展开更多
文摘Purpose: To quantify retinal function longitudinally and crosssectionally in p atients with autosomal-recessive early-onset severe retinal dystrophy (EOSRD) associated with RPE65 mutations. Subjects and methods: The ocular phenotype was characterized in four children from three families up to the second decade of li fe, and in three siblings from one family aged 43-54 years carrying compound he terozygous or homozygous mutations in RPE65. Standard clinical examination inclu ded colour vision testing, fundus photography and Goldmann visual fields (GVF). Full-field ERGs (in all) and multifocal ERGs (in two patients) were also record ed. Visual performance and fundus appearance were compared to literature data. R esults: In childhood, visual acuity (VA) ranged from 0.1 to 0.3, and GVF for tar get V4 was well preserved. VA and GVF were measurable in only one of the three a dult siblings. Nystagmus was present in two of four children and two of three ad ults. Photophobia was absent in childhood and developed in adulthood. Funduscopi c changes were discrete during the first decade of life in three of four childre n; one patient had clear macular changes already at age 5 years. All three adult siblings had distinct retinal changes including the macula. Bone spicules were not a feature. Residual colour vision was present in all patients with measurabl e VA. Rod ERGs were absent at any age; cone ERGs were detectable in early childh ood. To date, VA data have been reported in 51 patients, visual fields in 29 pat ients, and a detailed fundus description in 34 patients. For all three parameters, data were compara ble to the results in our patient cohort. Conclusion: In childhood, patients wit h RPE65 mutations have better visual functions than typically seen in Leber cong enital amaurosis. The phenotype shows a common progressive pattern with intrafam ilial and interfamilial variation. The data suggest a preserved retinal morpholo gy at young ages, arguing for vision-restoring gene therapy trials in childhood .
文摘Background: In a recent report we described RPE65, a protein originally characterized in retinal pigment epithelium, to be expressed in normal human epidermis. RPE65 is suspected to be involved in cellular uptake of retinol which is transported in the bloodstream complexed with plasma retinol-binding protein. Objectives: To evaluate protein and mRNA expression of RPE65 in actinic keratosis (AK), squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) compared with normal skin. Methods: RPE65 mRNA expression in skin tumours relative to normal skin of the respective donor was studied by real-time polymerase chain reaction in AK (n=15), invasive SCC (n=30) and BCC (n=18). A peptide-specific anti-RPE65 a ntibody was used for immunohistochemical staining of formalin-fixed and paraffi n-embedded tissue sections of the respective tumours. Results: RPE65 mRNA expre ssion was reduced in AK. A highly significant reduction of RPE65 mRNA was observ ed in invasive SCC relative to normal skin of the respective donors. Immunohisto chemistry revealed a continuous staining of basal and suprabasal keratinocytes i n normal human epidermis. RPE65 in AK shown by immunohistochemical staining was reduced and quite irregular, whereas invasive SCC revealed no staining of tumour cells with the anti-RPE65 antibody. RPE65 mRNA values were elevated, whereas i mmunohistochemical staining for RPE65 proteinwas heterogeneous in BCC. Conclusio ns: These results suggest progressive downregulation of RPE65 from AK to invasive SCC.
文摘Purpose Fundus autofluorescence is due to accumulation of lipofuscin in the retinal pigment ep ithelium(RPE)resulting from incomplete digestion of N-retinylidene-phosphatidyl-ethanolamine from shed photorecep tor outer segment discs.Alteration in autofluorescence reflects changes in lipofuscin content of the RPE.Mutations on both alleles of RPE65result in absent or largely decrease d formation of rhodopsin,due to a defect in alltrans retinol is omerization in the RPE.Autofluorescence could therefore b e altered.This study was conducted to evaluate fundus autofl uorescence in patients with early-onset severe retinal dystrophy(EOSRD,or ear-ly-onset rod-cone dystrophy)associated with mutations on both alleles of RPE65.Design Case se ries.Participants and controls Ten 10-to 55-year-old p atients with EOSRD and compound heterozygous or homozy gous mutations in RPE65.For comparison,6heterozygous parents and 2patients with other forms of EOSRD we re examined.Methods Participants underwent,in addition to standard clinical and electrophysiological examination,autofluores-cence imaging using a confocal scanning laser ophthalmo-scope.Three of the patients were als o examined by optical coherence tomography(OCT)to evaluate the status of retinal degeneration.Mutations in7patients have been reported previously;the other pati ents were investigated by polymerase chain reaction-single-strand conformation poly-morphism and direct sequencing for mutations in RPE65and lecithin retinol acyltransfera se(LRAT).Main outcome measures Fundus autofluorescence a nd OCT.Results Ab-sent or minimal autofluorescence wa s found in all patients with compound heterozygous or homozygous RPE65muta-tions.Autofluorescence was normal in the heterozygous parents.Autofluorescence was present in 2children with EOSRD not associated with mutations in RPE65or LRAT,another gene involved in retinol recycling.Optical coher-ence tomography in younger patients revealed an intraretinal appearance similar to that of their h ealthy,heterozygous parents.Conclusions Lack of autofl uorescence in patients with EOSRD associated with mutation s in RPE65is in ac-cordance with the biochemical defect and can be used as a clinical marker of this genotype.Optical coherence tomog-raphy results in younger patients wo uld indicate still viable photoreceptors despite the absence of autofluorescence.
基金This study was supported by grants from the Science and Technology Commission of Beijing Municipality Fund Project （No. Z171100000417039） and National Natural Science Foundation of China （No. 81300789）.
文摘Background： Leber congenital anaaurosis （LCA） is a visual disease which is caused by RPE65 mutations and results in retinal degeneration and severe vision loss in early infancy. According to previous researches, mutations of the RPE65 gene account for 16% of all cases of LCA. This study aimed to identify RPE65 gene mutations in Chinese patients with LCA. Methods： We recruited 52 sporadic patients from Peking University Third Hospital in 2016 and applied Sanger sequencing to identil＇y variants among exons responsible for the disease. The genomic DNAs from blood leukocytes of these patients were isolated, and tile entire coding region of the RPE65 gene was amplified by polymerase chain reaction. We then deterrnined the sequence of RPE65 using ABI 3100 Genetic Analyzer. Results： Our study identified that only 1 out of the 52 patients with LCA carried the previously unreported homozygosis missense mutation c1174A〉C （T392P） of the RPE65 gene. However, the mutation was associated with the disease phenotype and not detected in 100 normal controls. Conclusions： Though we identified a novel missense mutation in the RPE65 gene that causes LCA, our result indicates that RPE65 mutations may not play a major role in the LCA patients in China since only 1 out of the 52 patients carried mutation in the RPE65 gene.
基金This work was supported by Fighting Blindness Canada(grant No.20R23299).
文摘Leber congenital amaurosis(LCA)is a severe,genetically heterogeneous recessive eye disease in which∼35%of genemutations are in-frame nonsensemutations coding for loss-of-function premature termination codons(PTCs)inmRNA.Nonsense suppression therapy allows read-through of PTCs leading to production of full-length protein.A limitation of nonsense suppression is that nonsense-mediated decay(NMD)degrades PTC-containing RNA transcripts.The purpose of this study was to determine whether inhibition of NMD could improve nonsense suppression efficacy in vivo.Using a high-throughput approach in the recessive cep290 zebrafish model of LCA(cep290;Q1223X),we first tested the NMD inhibitor Amlexanox in combination with the nonsense suppression drug Ataluren.We observed reduced retinal cell death and improved visual function.With these positive data,we next investigated whether this strategy was also applicable across species in two mammalianmodels:Rd12(rpe65;R44X)and Rd3(rd3;R107X)mouse models of LCA.In the Rd12 model,cell death was reduced,RPE65 protein was produced,and in vivo visual function testing was improved.We establish for the first time that the mechanism of action of Amlexanox in Rd12 retina was through reduced UPF1 phosphorylation.In the Rd3 model,however,no beneficial effect was observed with Ataluren alone or in combination with Amlexanox.This variation in response establishes that some forms of nonsensemutation LCA can be targeted by RNA therapies,but that this needs to be verified for each genotype.The implementation of precision medicine by identifying better responders to specific drugs is essential for development of validated retinal therapies.
