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Objective:To observe clinical phenotypes and analyze the pathogenic genes of Leber congenital amaurosis (LCA).Methods:A retrospective clinical study. From 2019 to 2020, 2 patients diagnosed with LCA by genetic testing in Tianjin Medical University Eye Hospital and their 6 unaffected family members were enrolled in the study. Two patients were from 2 unrelated families, both were probands. The patient's medical history was inquired in detail, slit lamp microscopy, ultra-widefield fundus photography, autofluorescence, and flash visual evoked potential (F-VEP) were performed. Peripheral vein blood (3-5 ml) was collected and genomic DNA was extracted from all study subjects. A total of 381 pathogetic genes associated with inherited retinal diseases, were selected by targeted exome sequencing capture strategy. Sanger sequencing was used to verify suspected pathogenic mutations. Candidate pathogenic mutations were identified after bioinformatics analysis. Sanger sequencing, real-time quantitative polymerase chain reaction and family co-identification were used to confirm the final mutations.Results:Two patients were male, aged 3 and 27 years. One case had vision loss in both eyes, accompanied by nystagmus and acupressure eye sign since childhood. The clinical hallmark of the proband (F1-Ⅱ-3) in F1 includes clearly boundary of optic disc, normal retinal blood vessels and macular fovea. The implied period of the maximum forward wave in both eyes of F-VEP was roughly normal, and its amplitude decreased significantly. The phenotype of the proband (F2-Ⅱ-1) in F2 includes optic nerve head pallor, bone-spicule intraretinal pigmentation, "gold-foil maculopathy" , retina patchy hypo-autofluorescence in both eyes. There was no abnormal phenotype in the eyes of the family members. According to the genetic diagnosis, the proband (F1-Ⅱ-3) carried the GUCY2D gene c.835G>A (p.D279N) (M1) and exon 9-19 deletion (M2) compound heterozygous mutations, in which M1 was derived from healthy mother and M2 was derived from healthy father. The proband (F2-Ⅱ-1) carried CRB1 gene c.1576C>T(R526X) (M3) and c.1522T>C (C508R) (M4) compound heterozygous mutations, in which M3 from the healthy father, M4 from the healthy mother. M2 and M4 were novel mutations. Conclusion:GUCY2D gene mutations lead to LCA1 type in the F1 family, CRB1 gene mutations lead to LCA8 type in the F2 family; there are significant different phenotypes caused by different pathogenic genes.
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Objective:To observe the relationship between the response to anti-vascular endothelial growth factor (VEGF) drug treatment and single nucleotide polymorphism (SNP) genotype in patients with wet age-related macular degeneration (wAMD).Methods:A retrospective clinical study. From August 2019 to September 2020, 103 eyes of 103 wAMD patients diagnosed in Tianjin Medical University Eye Hospital were included in the study. Among them, there were 59 males (57.28%, 59/103) and 44 females (42.72%, 44/103); the average age was 68.74±7.74 years. The standard logarithmic visual acuity chart was used to detect the Best Corrected Visual Acuity of the affected eye and converted to the logarithmic minimum angle of resolution (logMAR) visual acuity during statistics. Optical coherence tomography was used to detect the central retinal thickness (CRT) of the affected eye. At the same time, the patient's high-density lipoprotein cholesterol (HDL-C) was tested. All eyes were treated with intravitreal injection of anti-VEGF drugs once a month for 3 months. Before the initial treatment, peripheral venous blood from the patient were collected. Interleukin-8 ( IL-8), complement C3 gene ( C3), complement factor H ( CFH), liver lipase ( LIPC), cholesterol ester transfer protein ( CETP), ATP binding cassette subfamily a member 1 ( ABCA1), lipoprotein lipase ( LPL), fatty acid desaturation gene cluster ( FADS1) SNP. According to gene frequency, genotypes are divided into wild type and mutant type were detected. Qualitative data such as the frequency difference of the genotype distribution in the clinical phenotype and the Hardy-Weinberg equilibrium of the genotype distribution were compared with the Chi-square test or Fisher's exact test. Results:There were fewer CRT responders in IL-8 rs4073 mutant (TA+AA) patients than wild-type (TT) [odds ratio ( OR)=0.310, 95% confidence interval ( CI) 0.106-0.910, P<0.05). Among them, after the drug stratification test, the proportion of patients with IL-8 rs4073 locus TT genotype in the conbercept treatment group was less CRT non-responders ( OR=0.179, 95% CI=0.034-0.960, P=0.033). Patients with LIPC rs2043085 mutant (CT+TT) with BCVA increased ≥0.2 logMAR are more likely than wild-type (CC) ( OR=3.031, 95% CI 1.036-8.867, P<0.05); HDL-C level was significantly lower Compared with wild type (CC), the difference was statistically significant ( t=2.448, P=0.016). There was no significant difference in logMAR BCVA and CRT between IL-8 rs4073, LIPC rs2043085 mutant and wild-type patients before treatment ( IL-8 rs4073: Z=-0.198, -1.651; P=0.843, 0.099; LIPC rs2043085: Z=-0.532, -0.152; P=0.595, 0.879). C3 rs 225066, CFH rs800292, CETP rs708272, ABCA1 rs1883025, FADS1 rs174547, LPL rs12678919 have no correlation with anti-VEGF drug treatment response. Conclusions:Patients with wAMD are treated with anti-VEGF drugs. Those with IL-8 rs4073 locus A genotype may be less responsive to CRT. LIPC rs2043085 locus T genotypes may be relatively more responsive to BCVA.
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Objective:To observe the MiSeq sequencing analysis results of fulvic acid (FA) intervention in hypoxia-induced human retinal microvascular endothelial cell (hRMEC) gene expression profile.Methods:hRMEC were cultured in vitro and divided into the hypoxia group (hypoxia treatment) and the FA intervention group (FA intervention after hypoxia). The MTT colorimetric method was used to detect the influence of different concentrations and different modes of FA on hRMEC activity. The optimal concentration of FA was chosen. RT-PCR was used to investigated the effect of FA on hypoxia-induced intercellular adhesion molecule-1 (ICAM-1), IL-1β, IL-4, IL-6, IL-6, IL-8, IL-10, MMP-2, TNF-α, TNF-β, other inflammatory factors in hRMEC, and inflammation-related factors mRNA expression. Cells in the hypoxia group and FA intervention group in the logarithmic growth phase were collected. MiSeq sequencing technology was applyed to complete the whole transcriptome sequencing of the two groups of cells, biological data were obtained, and the differentially expressed miRNA were analyzed on this basis. Gene annotation (GO) functionally significant enrichment analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway significant enrichment analysis were used to analyze the functions and signal pathways of differential miRNAs. The expression of inflammatory factors and inflammation-related factors were compared between groups. The expression level of the corresponding miRNA in the cell was regulated by miRNA mimic, and its effect on cell function was observed, so as to judge the effect of the miRNA.Results:Different concentrations and different modes of action of FA had no effect on the cell viability of hRMEC. The mRNA expression of ICAM-1, IL-1β, IL-6 and TNF-β in the hypoxia group hRMEC were significantly up-regulated compared with the normal group, and the difference was statistically significant ( t=3.426, 6.011, 5.282, 6.500; P=0.027, 0.004, 0.006, 0.003); the mRNA expression of ICAM-1, IL-6, TNF-α and TNF-β in the FA intervention group hRMEC was significantly lower than that of the hypoxia group, and the difference was statistically significant ( t=9.961, 3.676, 3.613, 3.387; P=0.001, 0.021, 0.023, 0.028). There were 14 differentially expressed miRNAs between the hypoxia group and the FA intervention group, of which 9 were up-regulated genes and 5 were down-regulated genes. The predicted target genes of 4 differential miRNAs (hsa-miR-1 285-3p, hsa-miR-30d-3p, hsa-miR-3 170, hsa-miR-7 976) were all ICAM-1. The results of significant enrichment analysis of GO function showed that the functions of differential genes were mainly enriched in the process of cell development, cell differentiation and single organism development. Significant enrichment analysis of the KEGG pathway showed that the differential miRNA expression was highly enriched in the proteoglycan pathway and the cytokine-cytokine receptor interaction pathway in cancer, and the arachidonic acid metabolism pathway and the amphetamine pathway were the more obvious differential expressions. Conclusion:FA may affect the expression level of downstream ICAM-1 mRNA by regulating the expression of multiple miRNAs, thereby affecting the inflammatory state of cells after hypoxia-stimulated hRMEC.
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Objective:To observe the effect of pyrimidine bundle-binding protein-associated splicing factors (PSF) on the function of hypoxia-induced human retinal microvascular endothelial cells (hRMECs).Methods:A three-plasmid system was used to construct lentivirus (LV)-PSF. After LV-PSF infected hRMECs in vitro, the infection efficiency was measured by flow cytometry. Real-time quantitative PCR (RT-PCR) was used to detect the expression of PSF mRNA in hRMECs infected with LV-PSF. The experiment was divided into two parts, in vivo and in vitro. In vivo experiments: 20 healthy C57B/L6 mice at the age of postnatal 7 were randomly divided into normal group, oxygen-induced retinopathy (OIR) group, OIR+LV-Vec group, and OIR+LV-PSF group, each group has five mice. Mice in 3 groups were constructed with OIR models except the normal group and the mice in OIR group were not treated. The mice in the OIR + LV-Vec group and the OIR+LV-PSF group were injected with an empty vector (LV-Vec) or LV-PSF in the vitreous cavity, respectively. The effect of LV-PSF on the formation of retinal neovascularization (RNV) was observed then. In vitro experiments: hRMECs were divided into normal group, hypoxia group, vector group, and PSF high expression group. HRMECs in the normal group were cultured in vitro; hRMECs in the hypoxic group were restored to normal culture conditions for 3 h after 3 h of hypoxia stimulation; hRMECs in the vector group and PSF high expression group were infected with LV-Vec and LV-PSF for 48 h, and hRMECs were returned to normal culture conditions for 24 h with hypoxia stimulation for 3 h. The effect of PSF on cell proliferation was observed by MTT colorimetry. Cell scratch test and Transwell migration experiment were used to observe the effect of PSF on cell migration ability under hypoxia stimulation. RT-PCR was used to observe the mRNA expression of HIF-1α, VEGF and PSF in each group of cells.Results:The LV-PSF of stably expressing PSF was successfully constructed. The infection efficiency was 97% determined by flow cytometry. The level of PSF mRNA in hRMECs infected with LV-PSF was significantly increased and detected by RT-PCR. In vivo experiments: The RNV area of the mice in the OIR group and the OIR + LV-Vec group was significantly increased compared to the normal group ( t=18.31, 43.71), and the RNV area of the mice in the OIR + LV-PSF group was smaller than that in the OIR group ( t=11.30) and OIR + The LV-Vec group ( t=15.47), and the differences were statistically significant ( P<0.05). In vitro experiments: MTT colorimetry results showed that the proliferative capacity of hRMECs in the hypoxic group was significantly enhanced compared with the normal group ( t=2.57), and the proliferative capacity of hRMECs in the PSF high expression group was significantly lower than that of the normal, hypoxic, and vector groups ( t=5.26, 5.46, 3.73), the differences were statistically significant ( P<0.05). The results of cell scratch test showed that the hRMECs could be stimulated by the hypoxia stimulation for 3 hours to restore the normal condition for 24 hours or 48 hours ( t=8.35, 13.84; P<0.05). Compared with the vector group, cell migration rate in the PSF-high expression group was not significant ( t=10.99, 18.27, 9.75, 8.93, 26.94, 7.01; P<0.05). Transwell experiments showed that the number of cells stained on the microporous membrane was higher in the normal group and the vector groups, while the number of cells stained in the PSF high expression group was significantly reduced ( t=9.33, 6.15; P<0.05). The results of RT-PCR showed that the mRNA expression of HIF-1α and VEGF in hRMECs in the hypoxic and vector groups increased significantly compared with the normal group ( t=15.23, 21.09; P<0.05), but no change in the mRNA expression of PSF ( t=0.12, 2.15; P>0.05); compared with the hypoxia group and the vector group, the HIF-1α and VEGF mRNA expression in hRMECs in the PSF high expression group were significantly decreased ( t=10.18, 13.10; P<0.05), but the PSF mRNA expression increased ( t=65.00, 85.79; P<0.05). Conclusion:PSF can reduce the RNV area in OIR model mice. PSF may inhibit hypoxia-induced proliferation and migration of hRMECs through the HIF-1α/VEGF signaling pathway.
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Objective To observe the effect of different macular edema on the area of foveal avascular zone (FAZ) and its correlation in eyes with branch retinal vein occlusion (BRVO).Methods A total of 72 patients (75 eyes) diagnosed with BRVO were included in the study.There were 40 patients males (42 eyes) and 32 females (33 eyes),with the mean age of (56.00±9.96) years.All the eyes were examined by BCVA,intraocular pressure,slit lamp microscope combined with preset lens,fundus color photography and optical coherence tomography angiography (OCTA).BRVO patients were divided into two groups according to the degree of macular edema:group M300 that was CRT ≥300 μm (38 patients,39 eyes) and group L300 that was CRT＜300 μmn (34 patients,36 eyes).The macular angiography scan protocol covered a 3 mm × 3 mm area.The parameters of macular were measured by the built-in measurement software of the system:(1) area of FAZ,perimeter ofFAZ (PERIM),avascular index ofFAZ (AI),vascular density within a width of 300 μm around the FAZ region (FD-300);(2) central retinal thickness (CRT);(3) vascular density (VD):the superficial central fovea vascular density (SFVD),the deep central fovea vascular density (DFVD),the superficial hemi-macular vascular density (SHVD),the deep hemi-macular vascular density (DHVD).Spearman test was used to test the correlation between FAZ area and other parameters in each group.Results The FAZ area in group M300 and L300 were 0.388 ± 0.166 mmn2 and 0.596± 0.512 rmm2,respectively.The results of Spearman test showed that the FAZ area of group M300 was positively correlated with PERIM and AI (r=0.932,0.591;P=0.000,0.000),negatively correlated with SFVD,DFVD and SHVD (r=-0.490,-0.429,-0.339;P=0.002,0.006,0.035).But there was no significant negative correlation between FAZ area and FD-300,CRT,DHVD in group M300 (r=-0.129,-0.053,-0.400;P=0.435,0.749,0.395).The FAZ area in group L300 was positively correlated with PERIM and AI (r=0.887,0.633;P=0.000,0.000),negatively correlated with SFVD,DFVD,SHVD and DHVD (r=-0.413,-0.643,-0.630,-0.370,-0.411;P=0.012,0.000,0.000,0.026,0.013).But there was no significant positive correlation between FAZ area and FD-300 in group L300 (r=0.093,P=0.590).Conclusion FAZ area varies with the degree of macular edema.The degree of macular edema is higher,the FAZ area is smaller.FAZ area is positively correlated with PERIM and AI significantly,and negatively correlated with SFVD,DFVD and SHVD.
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Objective To investigate the peripheral vascular findings in eyes with branch retinal vein occlusion (BRVO) and hemi-retinal vein occlusion (HRVO) using ultra-wide field fluorescein angiography (UWFFA),and analyze the influence of relative systemic factors on retinal vascular leakage.Methods A retrospective case-control study was designed.The 153 eyes of 146 patients with BRVO and 40 eyes of 40 patients with HRVO were include in Tianjin Medical University Eye Institute from September 2017 to March 2018.UWFFA was carried out in the patients,and the images were analyzed by Vantage Review software.The eyes were divided into two groups based on the whether the leakage occurred in other quadrant or fellow eye.The eyes with the fluorescence leakage only in the quadrant of affected vessel in late stage of UWFFA were in the RVO1 group,and the eyes with the fluorescence leakage in other quadrants or fellow eye besides affected vessel were in the RVO2 group.Relative past medical histories were recorded,such as hypertension,high cholesterol and diabetes mellitus.The influence of medical histories on vascular leakage in RVO1 group and RVO2 group with different histories was analyzed,respectively,and systemic factors which affected lcakage degree were evaluated.Results In 179 eyes with RVO,fluorescence leakage occurred in late stage of UWFFA besides affected vessel in 25 eyes (14.0％),including 19 eyes in the affected eyes (10.6％) and 6 eyes in fellow eyes (3.4％).Hypertension,high cholesterol and diabetes mellitus were found in 77,28 and 21 patients,respectively.In 77 hypertension patients,66 were in the RVO1 group,and 9 were in the RVO2 group (11.69％),and in 102 non-hypertension patients,86 were in the RVO1 group,and 16 were in the RVO2 group (15.69％),without significant difference was seen in the fluorescence leakage in other quadrants or fellow eye besides affected vessel between hypertension and non-hypertension patients (x2 =0.298,P =0.585).In 28 high cholesterol patients,24 were in the RVO1 group,and 4 were in the RVO2 group (14.29％),and in the 151 patients without high cholesterol,130 were in the RVO1 group,and 21 were in the RVO2 group (13.91％),without significant difference in the fluorescence leakage in other quadrants or fellow eye besides affected vessel between high cholesterol and non-high cholesterol (x2 =0.000,P =1.000).In 21 diabetes mellitus patients,17 patients were in the RVO1 group,and 4 patients were in the RVO2 group (19.05％),and in 158 patients without diabetes mellitus,137 were in the RVO1 group,and 21 were in the RVO2 group (13.29％),without significant difference was seen in the fluorescence leakage in other quadrants or fellow eye besides affected vessel between diabetes mellitus and non-diabetes mellitus (x2 =0.144,P=0.704).Conclusions Unexpected late peripheral retinal leakage can be seen on the UWFFA in the eyes with BRVO and HRVO.Hypertension,high cholesterol and diabetes mellitus are not the main cause of these findings.UWFFA can disclose more peripheral,wider retinal lesions.
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Objective To observe the difference of macular microvascular features in superficial and deep vascular plexi in patients with branch retinal vein occlusion (BRVO).Methods A total of 63 BRVO patients (63 eyes) were enrolled in this study. There were 28 males (28 eyes) and 35 females (35 eyes). The patients aged from 39 to 74 years, with the mean age of (59.76±8.48) years. All eyes were evaluated by optical coherence tomography angiography (OCTA). The macular angiography scan protocol covered a 3 mm×3 mm area. The focus of angiography analysis included superficial vascular plexus and deep vascular plexus. The following vascular morphological parameters were assessed in these two plexi: foveal avascular zone (FAZ) enlargement, capillary non-perfusion (CNP) occurrence, microvascular abnormalities (MA) appearance, and vascular congestion (VC) signs. The FAZ area was measured by the built-in software. The macular microvascular morphology changes in superficial and deep vascular plexi were compared through McNemar test. Results The superficial and deep plexi showed FAZ enlargement in 43 eyes (68.3％) and 50 eyes (79.4％), CNP in 51 eyes (81％) and 50 eyes (79.4％), MA in 62 eyes (98.4％) and 62 eyes (98.4％), VC in 23 eyes (36.5％) and 52 eyes (82.5％), respectively. FAZ area was (0.55±0.37) mm2. There was no difference in CNP (P=1.000) and MA (P=1.000) between superficial and deep plexi. But, there was difference in FAZ enlargement (P=0.039) and VC signs (P<0.001) between superficial and deep plexi.Conclusion Deep vascular plexus showed more FAZ enlargement and VC sign than superficial plexus in BRVO patients.
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Idiopathic parafoveal telangiectasis (IPT) is a retinal vascular disease which is characterized by foveal and parafoveal telangiectasia.The main clinical manifestations are retinal telangiectasis,reduced retinal transparency,retinal venular dilatation,yellow exudation,retinal pigment epithelial lesions,retinal hemorrhage,macular atrophy,macular hole or lamellar hole,subretinal neovascularization and retinal detachment.According to the clinical characteristics and features of fluorescein angiography,IPT can be divided into 3 types and 6 subtypes.Laser photocoagulation,photodynamic therapy,and intravitreal injection of glucocorticoid or anti-vascular endothelial growth factor drugs,can reduce the macular edema and neovascularization.However,due to the unclear etiology of IPT,the existing treatment measures are not specific for its etiology.We need to work hard to understand further the clinical features and pathogenesis of IPT and search the targeted treatments based on its pathogenesis mechanism.
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Objective To observe the optic disc perfusion in anterior ischemic optic neuropathy (AION) patients.Methods Forty eyes of 40 AION patients and 30 eyes of 30 normal subjects were included.The stage of the diseases was defined based on the course of the disease,including acute stage (less than 3 weeks) and recovery stage (more than 3 months).Optic disc blood flow area,outer vascular density and blood flow index were measured by optical coherence tomography angiography in all the subjects.Optic disc perfusion was observed in acute and recovery stage of disease.Results The optic disc blood flow area,outer vascular density and blood flow index were decreased of AION eyes in acute stage compared with the normal subjects,the difference was statistically significant (P＜0.05);while the optic disc blood flow area,outer vascular density and blood flow index of AION eyes in the recovery stage showed no significant difference compared with normal subjects (P＞0.05).Conclusion Disc perfusion is reduced in AION at the acute stage,but recovered at the recovery stage.
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Objective To compare the differences of corneal keratometry and corneal astigmatism measured by Len?star LS900 and KR-1 auto-refractor of age-related cataract. Methods Seventy-six patients with cataract (76 eyes) were in?cluded in this study. Flat keratometry (K1), steep keratometry (K2), mean keratometry (Km) and astigmatism were measured before operation by Lenstar LS900 and KR-1 auto-refractor. The parameters of astigmatism were transformed into J0 and J45 by Fourier vector transform, and which was compared. Results Values of K1, K2, Km, J0 and J45 were (43.960±1.440) D, (44.901±1.319)D, (44.430±1.336)D, 0.043±0.402 and 0.017±0.425 measured by Lenstar LS900, respectively, and which measured by KR-1 auto-refractor were (44.007 ± 1.400)D, (44.859 ± 1.338)D, (44.433 ± 1.330)D,-0.058 ± 0.322 and 0.031 ± 0.419, respectively. There was no statistical difference between these values measured by two instruments ( P>0.05). The Bland-Altman plots showed that two devices had coincident results for corneal parameters. Conclusion Lenstar LS900 and KR-1 auto-refractor can be applied in the measurement of corneal astigmatism of age-related cataract before surgery.