A mutation in ADIPOR1 causes nonsyndromic autosomal dominant retinitis pigmentosa.

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous disorder characterized by night blindness, visual field constriction, and severely reduced visual acuity. Despite a number of genes being implicated in RP pathogenesis, the genetic etiology of the disease remains unknown in many patients. In this study, our aim was to identify the disease-causing mutation of a large Chinese family with autosomal dominant RP (adRP). Targeted exon capture sequencing was initially performed to screen mutations in known disease-causing genes, followed by exome sequencing. In doing so, a heterozygous mutation in ADIPOR1 (c.929A > G) that results in an amino acid substitution (p.Y310C) was identified to co-segregate with the disease phenotype in this family. Adipor1 is wildly expressed throughout the body, but appears to be enriched in the photoreceptor inner and outer segments. The p.Y310C mutation, predicted to affect the structure and function of the protein, was confirmed to affect protein folding and its subcellular localization in vitro. In addition, knockdown of adipor1 expression in a zebrafish model with morpholino (MO) preferentially reduced the number of rod photoreceptors, with no effect on the number of cones, a phenotype that is characteristic of RP. Furthermore, the knockdown phenotype was partially rescued by injecting wild-type, but not mutant, human ADIPOR1 mRNA. We conclude that ADIPOR1 is a novel adRP-causing gene and plays an important role in rod development and maintenance.

Identification of CYP4V2 mutation in 36 Chinese families with Bietti crystalline corneoretinal dystrophy.

Bietti crystalline corneoretinal dystrophy (BCD) is an inherited eye disease that is most common in the Chinese. It is caused by a mutation in the CYP4V2 gene. In this study, 43 Chinese BCD families were recruited; most patients manifested the characteristic phenotype of BCD, with 2 families initially misdiagnosed with retinitis pigmentosa. Five patients in our cohort presented with BCD and choroidal neovascularization (CNV), and 1 patient presented with typical BCD and abnormality in the terminals of both fingers and toes. A total of 17 pathogenic mutations involving 68 alleles were identified from 36 families using targeted exon sequencing and Sanger sequencing; we achieved a diagnostic rate of approximately 84%. Fifteen families were found to carry homozygous mutations, 17 families carried compound heterozygous mutations, and 4 families carried a single heterozygous mutation. Of the mutations identified, four variants c.802-8_810del17bpinsGC, c.802-8_810del17bpinsGT, c.992A > C (p.H331P), and c.1091-2A > G accounted for 71% of the mutations identified in CYP4V2. These mutations were hotspots in Chinese populations for BCD. Five among them were novel and predicted to be disease-causing, including c.65T > A (p.L22H), c.681_4delTGAG (p.S227Rfs*1), c.802-8_810del17bpinsGT, c.965_7delAAG (p.321delE), and c.994G > A (p.D332N). No apparent correlation between genotype and phenotype was identified. Our findings broaden the spectrum of CYP4V2 mutations that cause BCD and the phenotypic spectrum of the disease in Chinese families. These results will be useful for the genetic diagnosis of BCD, genetic consultation, and gene therapy in the future.

SPATA4 Counteracts Etoposide-Induced Apoptosis via Modulating Bcl-2 Family Proteins in HeLa Cells.

Spermatogenesis associated 4 (SPATA4) is a testis-specific gene first cloned by our laboratory, and plays an important role in maintaining the physiological function of germ cells. Accumulated evidence suggests that SPATA4 might be associated with apoptosis. Here we established HeLa cells that stably expressed SPATA4 to investigate the function of SPATA4 in apoptosis. SPATA4 protected HeLa cells from etoposide-induced apoptosis through the mitochondrial apoptotic pathway, in the way that SPATA4 suppressed decrease of the mitochondrial membrane potential, the release of cytochrome c, and subsequent activation of caspase-9 and -3. We further demonstrated that SPATA4 upregulated anti-apoptotic members of Bcl-2 family proteins, Bcl-2, and downregulated the pro-apoptotic member of Bcl-2 family proteins, Bax. Knockdown of SPATA4 in HeLa/SPATA4 cells could partially rescue expression levels of bcl-2 and bax. In conclusion, SPATA4 protects HeLa cells against etoposide-induced apoptosis through the mitochondrial apoptotic pathway. Our findings provide further evidence that SPATA4 plays a role in regulating apoptosis.

Novel mutations of CRB1 in Chinese families presenting with retinal dystrophies.

PURPOSE: To identify disease-causing mutations in Chinese families who presented with retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). METHODS: The pathogenic variant in a Chinese family with autosomal dominant RP was investigated with a specific hereditary eye disease enrichment panel (HEDEP) based on targeted exome capture technology. The identified variant was confirmed with Sanger sequencing. CRB1 mutations in 67 patients with sporadic retinal dystrophy were examined with Sanger sequencing. RESULTS: Compound heterozygous mutations were identified in a family who had undergone HEDEP analysis. After complete sequence analysis of the CRB1 gene was performed in 67 patients with sporadic retinal dystrophy, other compound heterozygous mutations were detected in three families. The mutations included three novel heterozygous mutations: c.3059delT (p.M1020SfsX1), c.3460T>A (p.C1154S), and c.4207G>C (p.E1403Q). The mutation frequency of CRB1 in this study was 5.9% (8/136). CONCLUSIONS: Our findings broaden the spectrum of CRB1 mutations and the phenotypic spectrum of the disease in Chinese patients. The results from this study show that patients with LCA carry CRB1 null mutations more frequently than patients with RP.

[The anomalies of choroidal and retinal blood flow in retinitis pigmentosa patients].

OBJECTIVE: To study changes of blood flow of ophthalmic artery (OA), central retinal artery (CRA) and posterior ciliary artery (PCA) in patients with primary retinitis pigmentosa. METHODS: Color Doppler flow imaging (CDIF) was used in 184 cases (184 eyes) of RP patient. The peak systolic velocity (PSV), end diastolic velocity (EDV), time-averaged maximum velocity (TAMV), pulsatility index (PI) and resistance index (RI) were measured in OA, CRA and PCA separately. Data was analyzed statistically by One-Sample T Test to compare changes of these parameters between RP and normal group. RESULTS: Concerning RP group, in OA, the PSV,EDV, TAMV[ (33.05 ± 8.62) , (7.74 ± 3.04) , (14.16 ± 4.43) cm/s] vs normal group[ (31.47 ± 9.63) , (7.11 ± 2.34) , (12.44 ± 3.64) cm/s] were increased, the differences were statistically significant (t = -2.481, 2.820, 5.253, P < 0.05). But the PI in OA of RP(1.87 ± 0.73) vs normal group (2.02 ± 0.71) was deceased, the difference was statistically significant (t = -2.794;P < 0.05). Concerning RP group, in CRA, the PSV, EDV, TAMV, PI and RI [(5.30 ± 1.36), (2.11 ± 0.49), (3.01 ± 0.680) cm/s], [ (1.05 ± 0.28) , (0.60 ± 0.18) ] vs normal group [(10.82 ± 2.97) , (3.28 ± 1.11), (5.50 ± 2.06) cm/s], [ (1.48 ± 0.49), (0.71 ± 0.08) ] were decreased, the differences were statistically significant (t = -55.186, -32.015, -49.634, -20.430, -8.704, P < 0.05). Concerning RP group, in PCA, the PSV, EDV, TAMV, PI and RI [(7.60 ± 1.95), (2.59 ± 0.58), (4.07 ± 1.00) cm/s], [ (1.23 ± 0.28), (0.65 ± 0.80) ] vs normal group [(11.61 ± 3.41), (3.34 ± 1.25), (5.83 ± 1.91) cm/s], [ (1.49 ± 0.43), (0.70 ± 0.09)] were decreased, the differences were statistically significant (t = -28.097, -17.541, -23.842, 12.445, -8.720, P < 0.05). CONCLUSIONS: Hemodynamic abnormal changes of OA, RCA and PCA in RP patients possibly suggest the role of blood supply in occurrence or development of RP. Maintaining normal ocular blood flow may be able to offer a new way for prevention and treatment for RP.

[Progress in studies of effects on brain-derived neurotrophic factor in degenerative fundus disease].

Degenerative fundus diseases are a group of outer retinal disease caused by dysfunction of photoreceptors, some of which have the inner layer defection. Brain-derived neurotrophic factor (BDNF) has been shown to play a key role in the neuronal survival, which accomplishes its protection via receptors, including a low affinity p75 and a high affinity tyrosine kinase receptor TrkB, retinal growth cone filopodia and cofactor of Zinc. It is proved that BDNF can induce differentiation of stem cells and retinal progenitor, in case for the effective retinal transplantation. The protection by BDNF has also been observed through the interaction with retinal neurons such as photoreceptors, bipolar cells, horizontal neurons, ganglion cells and dopaminergic cells, as well as other cells of eyes. Furthermore, BDNF has been used to attenuate the injuries of optic nerve and the application of BDNF combined with other neurotrophic factors and drugs also manifests a certain efficiency.

Efficacy and Safety of Antivascular Endothelial Growth Factor (Anti-VEGF) in Treating Neovascular Age-Related Macular Degeneration (AMD): A Systematic Review and Meta-analysis.

This study is aimed at assessing the efficacy and safety of antivascular endothelial growth factor (anti-VEGF) inhibitors in treating age-related macular degeneration (AMD). PubMed, Embase, and Cochrane library were searched. Weighted mean difference (WMD) and relative risk (RR) with 95% confidence interval (CI) were applied to assess outcomes. Eighteen randomized controlled trials involved 8,847 neovascular AMD patients were selected for the meta-analysis. Pegaptanib (WMD: 6.70; P < 0.001) and ranibizumab (WMD: 17.80; P < 0.001) were associated with greater BCVA changes than control after 1 year. Bevacizumab was linked with less changes in central macular thickness after 1 year compared to control (WMD: -38.50; P < 0.001), but more changes compared to ranibizumab (WMD: 10.69; P = 0.024). The incidence of gain of 15 or more letter visual acuity after 1 year was increased when compared with bevacizumab versus control (RR: 7.80; P = 0.001), pegaptanib versus control (RR: 2.83; P = 0.015), and ranibizumab versus control (RR: 3.92; P = 0.003). Moreover, ranibizumab was associated with more BCVA changes and an increased incidence of gain of 15 or more letter visual acuity after 2 years compared with control (RR: 5.77; P < 0.001). This study found that most anti-VEGF inhibitors provided better efficacy than non-anti-VEGF intervention, and the treatment effectiveness among various anti-VEGF agents was equally effective.

Dependable and Efficient Clinical Molecular Diagnosis of Chinese RP Patient with Targeted Exon Sequencing.

Retinitis pigmentosa (RP) is the most common inherited retinal disease. It is a clinically and genetically heterogeneous disorder, which is why it is particularly challenging to diagnose. The aim of this study was to establish a targeted next-generation sequencing (NGS) approach for the comprehensive, rapid, and cost-effective clinical molecular diagnosis of RP. A specific hereditary eye disease enrichment panel (HEDEP) based on exome capture technology was used to collect the protein coding regions of 371 targeted hereditary eye disease genes, followed by high-throughput sequencing on the Illumina HiSeq2000 platform. From a cohort of 34 Chinese RP families, 13 families were successfully diagnosed; thus, the method achieves a diagnostic rate of approximately 40%. Of 16 pathogenic mutations identified, 11 were novel. Our study demonstrates that targeted capture sequencing offers a rapid and effective method for the molecular diagnosis of RP, which helps to provide a more accurate clinical diagnosis and paves the way for genetic counseling, family planning, and future gene-targeted treatment.

Novel mutations of RPGR in Chinese retinitis pigmentosa patients and the genotype-phenotype correlation.

X-linked Retinitis Pigmentosa (XLRP) accounts for 10-20% of all RP cases, and represents the most severe subtype of this disease. Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene are the most common causes of XLRP, accounting for over 70-75% of all XLRP cases. In this work, we analyzed all the exons of RPGR gene with Sanger sequencing in seven Chinese XLRP families, two of these with a provisional diagnosis of adRP but without male-to-male transmission. Three novel deletions (c.2233_34delAG; c.2236_37delGA and c.2403_04delAG) and two known nonsense mutations (c.851C→G and c.2260G→T) were identified in five families. Two novel deletions (c.2233_34delAG and c.2236_37delGA) resulted in the same frame shift (p.E746RfsX22), created similar phenotype in Family 3 and 4. The novel deletion (c.2403_04delAG; p.E802GfsX31) resulted in both XLRP and x-linked cone-rod dystrophy within the male patients of family 5, which suggested the presence of either genetic or environmental modifiers, or both, play a substantial role in disease expression. Genotype-phenotype correlation analysis suggested that (1) both patients and female carriers with mutation in Exon 8 (Family 1) manifest more severe disease than did those with ORF15 mutations (Family 2&3&4); (2) mutation close to downstream of ORF15 (Family 5) demonstrate the early preferential loss of cone function with moderate loss of rod function.

Construction of a eukaryotic expression plasmid for human retina-derived neurotrophin-3.

Neurotrophin-3 (NT-3) can promote the repair of central nervous system and retinal damage. In previous reports, NT-3 has been expressed by viral vectors. However, plasmid vectors have a safer profile compared with viral vectors in clinical studies. This study recombined amplified human retinal NT-3 with a eukaryotic expression plasmid containing green fluorescent protein (GFP) to construct an NT-3 expression plasmid, pEGFP-N1-NT-3. The transfection efficiency 48 hours after pEGFP-N1-NT-3 transfection to 293T cells was 50.06 ± 2.78%. Abundant NT-3-GFP was expressed in 293T cells as observed by fluorescence microscopy, suggesting the construct pEGFP-N1-NT-3 effectively expressed and secreted NT-3-GFP. Secretory vesicles containing NT-3-GFP were observed in a constant location in cells by laser scan confocal microscopy, indicating the expression and secretion processes of NT-3 in eukaryotic cells were in accordance with the physical synthesis processes of secreted proteins. Western blot assay showed that pro-NT-3-GFP had a molecular weight of 56 kDa, further confirming NT-3-GFP expression. At 48 hours after transfection, the concentration of NT-3 in culture medium was 22.3 ng/mL, suggesting NT-3 produced by pEGFP-N1-NT-3 was efficiently secreted. This study constructed a human retinal-derived NT-3 eukaryotic expression plasmid that efficiently expressed and secreted NT-3.

Targeted exome capture and sequencing identifies novel PRPF31 mutations in autosomal dominant retinitis pigmentosa in Chinese families.

OBJECTIVES: To identify disease-causing mutations in two Chinese families with autosomal dominant retinitis pigmentosa (adRP). DESIGN: Prospective analysis. PATIENTS: Two Chinese adRP families underwent genetic diagnosis. A specific hereditary eye disease enrichment panel (HEDEP) based on targeted exome capture technology was used to collect the protein coding regions of targeted 371 hereditary eye disease genes; high throughput sequencing was done with the Illumina HiSeq 2000 platform. The identified variants were confirmed with Sanger sequencing. SETTING: All experiments were performed in a large laboratory specialising in genetic studies in the Department of Ophthalmology, Peking University Third Hospital. RESULTS: Two novel mutations, including one splice site mutation (Int10 c.1074-2 A>T; p.Y359SfsX29) and one insertion (c.824_825insA; p.Y275X) of PRPF31 were identified in the two families. The two mutations segregated with the disease phenotype in their respective families. CONCLUSIONS: Our findings broaden the spectrum of PRPF31 mutations causing adRP and the phenotypic spectrum of the disease in Chinese patients. The HEDEP based on targeted exome capture technology is an efficient method for molecular diagnosis in adRP patients.

Effect of brain-derived neurotrophic factor on c-jun expression in the rd mouse retina.

AIM: To determine the location of c-jun protein, dynamic changes in c-jun mRNA and protein expression, and ultrastructure characteristics in the rd mouse retina, following a single dose of brain-derived neurotrophic factor (BDNF) in a short period of time. METHODS: A single intravitreal injection of BDNF at two dosages (25µg/L or 50µg/L) was given to the right eye of the rd mouse at age 2 and 3 weeks respectively. Two weeks after injection, the location of c-jun protein in the retina was observed by immunofluorescence detection, c-jun mRNA and protein expression in retinas were detected by quantitative real time polymerase chain reaction (RT-PCR) and western immunoblotting analysis, ultrastructure characteristics of retinas were detected by transmission electron microscope (TEM) observation. RESULTS: c-jun protein was expressed in the inner nuclear layer (INL) of retina. BDNF at two dosages (25µg/L and 50µg/L) increased c-jun mRNA expression at PN-4 weeks respectively (P(1)=0.019, P(2)=0.021). 50µg/L BDNF increased c-jun protein expression at PN-4 weeks (P =0.000). The retinal ultrastructure was improved. CONCLUSION: The effects of BDNF exerts on the c-jun expression in the retina are dose-dependent and time-dependent, which may mediate photoreceptor rescue indirectly in the pathological process of retinitis pigmentosa (RP) at early stage.