Most fundus diseases leading to irreversible blindness are associated with genetic variations. Some sequence changes directly cause retinal diseases while others lead toa higher susceptibility to environmental insults common in daily life. Studies of genes related to fundus diseases will lead to a revolutionary change in the prevention and treatment of irreversible blindness.Application of high throughput next-generation sequencing and exome capture techniques will greatly enhance our ability to elucidate genes responsible for fundus diseases. With such technical and analytical advances, we are likely to see continuing and accelerating progress in the genetic study of fundus diseases, particularly in those fields requiring collaborative study of common fundus diseases using large cohorts of samples. The translational clinical application of understanding about these newly identified genes responsible for fundus diseases is also increasing in promise. Thus, strengthening current genetic studies of fundus diseases in both of these areas will make a valuable contribution to the prevention and treatment of blindness in both the near and especially the distant future.

BACKGROUND: 1200009K10 gene is from the lung cDNA pool of adult mouse and announced in January 2002, but its function is still not known.The predicted protein has five domains: Kelch motif, Kelch domain, BTB/POZ domain, BTB domain and β propeller domain, all of which are involved in protein-protein interactions and some enzymatic activities.OBJECTIVE: To explore the correlation between the expression of 1200009K10 gene and retinitis pigmentosa in mice.DESIGN: Observational and comparative trial.SETTING: Institute of Zhongshan Ophthalmic Center of Sun Yat-sen University.MATERIALS: The rd mice, rds mice and passage mice from C3B mice as normal control were purchased from Jackson Laboratory (Bar Harbor,Maine 04609, USA), and raised in second class unit of Experimental Animal Center of Sun Yat-sen University.METHODS: The retinal RNA of mice was extracted and isolated and 1200009K10 gene was amplified by rapid amplification of eDNA ends method (RACE). The gene expressions in the retina of rds, rd and C3B mice were analyzed, respectively by gene-specific real-time quantitative PCR.MAIN OUTCOME MEASURES: The expression contents and patterns of 1200009K10 gene in retina of three kinds of mice.RESULTS: One clone was obtained after RACE of retinal RNA extracted from retina of mice, which had an almost identical sequence with the 1200009K10 gene. Expression of 1200009K10 gene was classified into 6stages: 7, 12, 25, 37, 50 (near sex maturity) and 150 days after born. The expressions of 1200009K10 gene in the retina of rds, rd and C3B mice were low at postnatal day 7 (P7), higher between P7-12, stable between P12-50, and finally increased a little. The expression trend and level of 1200009K10 gene of C3B mice was nearly the same with that of the rds and rd mice, and higher than that of rds and rd mice at P12. Although there were differences in expression levels of 1200009K10 gene among the three kinds of mice, the expression patterns were almost the same.CONCLUSION: 1200009K10 gene may not participate in the development of retinal degeneration.

AIM: To identify the genes that were differentially expressed in the retina of rds mouse during the development of retinitis pigmentosa.METHODS: mRNA differential display method was used to compare and analyze mRNA samples prepared from the retina of rds mouse and normal mouse on postnatal day 25(P25). Differentially expressed fragments were cloned, sequenced and compared with GenBank database by BLASTN. Expression difference was further investigated by gene-specific primer RT-PCR.RESULTS: Obvious difference in gene expression occurred between rds mouse and normal mouse. One fragment, clone No.5, shared 91% homology with rat NADH-cytochrome b5 reductase (b5R) cDNA. Thus, it was identified as mouse b5R cDNA. Gene-specific RT-PCR confirmed that b5R mRNA level was increased in the retina of rds mouse compared with normal mouse on postnatal day 12, 25 and 37, respectively.CONCLUSION: Certain oxidative factors may up-regulate the expression of b5R resulting in large consumption of NADH and production of NAD +, through which apoptotic retinal cell death was enhanced.

Objective To analyze the clinical characteristics and to screen for causative mutations in CRX and GUCY2D genes in children with cone or cone-rod dystrophy. Methods Clinical data and genomic DNA was collected from 18 children with cone or cone-rod dystrophy, aged from 4 months to 8 years. The coding sequence of the cone-rod homeobox (CRX) gene and two exons of the retinal-specific guanylate cyclase GUCY2D gene (exons 2 and 8) were analyzed by using polymerase chain reaction(PCR) and heteroduplex combined with single-strand conformational polymorphism (heteroduplex-SSCP) analysis. Results All of the 18 patients manifested obvious visual impairment. Nystagmus, photophobia and mild ocular fundus changes were found in 13, 8,and 7 cases respectively. Normal fundus was seen in 11 cases. The visual acuity was less than 0.3 in 4 cases and was unable to measure in the other 14 cases because they were too young. Clinical ocular manifestations between cone and cone-rod dystrophy were overlapped. Mutation in the CRX and GUCY2D genes was not detected in the 18 children with cone and cone-rod dystrophy. Conclusion Visual impairment appeared more early and obvious than fundus changes in children with cone or cone-rod dystrophy. Mutation in the CRX gene may not contribute to this series of patients with cone and cone-rod dystrophy.

Objective　To apply two-dimensional (2-D) gel electrophoresis to resolve specially expressed proteins related to retinitis pigmentosa (RP) in the retina of rds mice.Methods　Proteins, prepared from the retinas of rds mice and normal C3B mice at different ages, were separated by using two-dimensional electrophoresis and then analyzed by 2-DE imaging analyzer.Results　2-D gel electrophoresis was established for the retinal proteome analysis. Retinal neuronal tissue was lysed by using chemical lysis solution and ultrasonic. Using carrier ampholyte to set up pH gradient as first dimension and casting vertical 12% SDS-acylamide-bis slab as second dimension, the major retinal proteins showed maps of proteome on 2-D gels clearly. Retinal proteome of rds and C3B mice at 37d has different expressive patterns. Some proteins only expressed in the retina of rds mice while another only in the retina of C3B mice and others had different level between the two kinds of mice.Conclusion　2-D gel electrophoresis is effective to separate specially expressed retinal proteins. The expressed proteins in the rds retina are different in quality and quantity from that in the normal C3B mice.

Objective　To determine the sensitivity and specificity of using the computer-photoscreener and non-cycloplegic retinoscopy in the detection of amblyopiogenic factors in nine to fifty months old infants.Methods　Three hundred children whose ages range from nine to fifty months were screened with the computer-photoscreener and non-cycloplegic retinoscopy. With a masked standardized clinical assessment as the standard, an overall comparison of the results obtained with the two techniques revealed a sensitivity and specificity. Photoscreen images on the computer monitor screen were reviewed and analyzed immediately by two independent observers for indicators of amblyopiogenic risk factors. Simultaneously, the results were compared to the findings of a full ophthalmologic examination.Results　The computer-photoscreener revealed a sensitivity of 94.2% and specificity of 90.1%, and the non-cycloplegic retinocopy revealed a sensitivity of 85.7% and specificity of 81.1% for the detection of amblyopiogenic risk factors, including hyperopia (+2.75 D or more), myopia (-1.50 D or more), astigmatism (2.00 D or more),anisometropia (2.00 D or more), ocular misalignment (5 degrees or more), and media opacity (1.5mm or more). Conclusion　The computer-photoscreener offers an opportunity to identify problems that limit vision, and could provide a feasible and sufficiently reliable screening technique in infants and preschool children who can be screened successfully for amblyopiogenic risk factors.

AIM: To understand the effect of the RB1 gene mutation on the function of pRB (the protein product of the RB1 gene) in the patients with retinoblastoma (RB). METHODS: The genomic DNA from retinoblastoma patients was extracted. After amplification, the promoter and all 27 exons were screened by SSCP-heteroduplex method. The mutation was cloned and identified by sequencing. The effect of the mutation product on the function of pRB was analyzed. RESULTS: One missense mutations of the exon 4 of the RB1 gene was identified in the genomic DNA from RB patients. This mutation was outside the large pocket of the pRB. No mutation of the RB1 gene was found in the genome DNA of the patient's parents. This is the fourth report that there was a genome mutation located outside the large pocket of pRB in the RB patients. CONCLUSION: The amino-terminus of the pRB may be essential for growth suppression.

AIM: To investigate the single nucleotide polymorphisms (SNPs) in the METTL4 gene which was mapped to 18p11.31, and the relationship between the SNPs and high myopia. METHODS: Genomic DNA was collected from 71 control subjects and 177 individuals with high myopia. Among them, there were 59 autosomal dominant high myopia probands (AD group), 46 autosomal recessive probands (AR group) and 72 patients non-transmitted (SF group). The exons of METTL4 gene were analyzed by polymerase chain reaction, heteroduplex-single strand conformation polymorphism (HA-SSCP) and sequencing. RESULTS: There were 2 SNPs of METTL4 gene in high myopia individuals and control subjects: SNP7438A→C, Glu230Asp, which hadn't been reported in GenBank;and SNP131C→A, Gln310Lys. SNP7438A→C genotypes between controls and high myopia groups were not different. SNP131C→A genotypes between controls and AR or SF groups were not different, while SNP131C→A genotypes showed a significant difference between AD group and control subjects. CONCLUSION: In METTL4 gene, SNP7438A→C is not responsible for high myopia. Further studies are needed to confirm whether SNP131C→A is responsible for autosomal dominant high myopia.