Objective:To identify and observe the pathogenic genes and clinical phenotypes of a family with a special platelet phenotype, Hermansky-Pudlak syndrome type 6 (HSP6).Methods:A retrospective clinical study. In November 2019, one proband and three family members from six HSP families who visited Henan Eye Hospital were included in the study. The child's medical history and family history were inquired in detail. The proband and all family members underwent best corrected visual acuity (BCVA), fundus color photography, frequency-domain optical coherence tomography, and general physical examination. The proband underwent platelet transmission electron microscopy (PTEM) and colonoscopy. Peripheral venous blood was collected from the proband, her parents and younger brother, and genomic DNA was extracted. Whole exome sequencing (WES) was used to screen pathogenic genes and their loci. Bioinformatics analysis determines the pathogenicity of gene variation sites. Fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to verify the related variations.Results:The proband (Ⅱ-1) was a 7-year-old female. The BCVA in both eyes was 0.1, who exhibited mild horizontal nystagmus and iris depigmentation. Fundus examination revealed obvious depigmentation and an underdeveloped fovea centralis. At the age of 7, the patient underwent colonoscopy due to acute gastrointestinal bleeding. A polyp approximately 5 mm in size was found on the floor of the sigmoid colon, with erosion and mucosal leukoplakia on its surface. PTEM showed that the number of platelet dense granules was normal, but the nuclei were small or exhibited low compactness. The skin on both lower legs showed pigmentation. The clinical phenotypes of the proband’s parents (Ⅰ-1, Ⅰ-2) and younger brother (Ⅱ-2) showed no obvious abnormalities. WES revealed that the proband carried compound heterozygous variants in exon 1 of the HPS6 gene: c.60_64dup (p.L22fs) (M1) and c.1147_1148del (p.L383fs) (M2). The mother carried the M1 variant, while the father and younger brother carried the M2 variant. Bioinformatics analysis predicted that both variants were pathogenic. RT-qPCR results showed that, compared with the relative expression level of HPS6 wt mRNA, the relative expression levels of HPS6 L22fs and HPS6 L383fs mRNA were significantly decreased ( t = 3.549, 4.560; P<0.05). Western blot analysis demonstrated that the HPS6 L383fs protein was truncated, whereas the HPS6 L22fs protein was not detected. Conclusions:This family is a special HPS6 with a normal number of dense platelet granules. The compound heterozygous variations of M1 and M2 in the HPS6 gene are pathogenic genes in this family.

Objective:To identify the pathogenic gene in a family with cone-rod dystrophy (CRD).Methods:A pedigree study was conducted.Clinical data were collected from three generations of six people from a family with CRD who visited Henan Eye Hospital in December 2019, including one patient.After detailed collection of the patient's medical history, the proband and his family members underwent best-corrected visual acuity, slit-lamp microscope+ front-lens examination, optometry, non-mydriatic fundus photography, spectral-domain optical coherence tomography (SD-OCT), and full-field flash electroretinography (ff-ERG). Peripheral venous blood (5 ml) was collected from the proband, his parents and siblings, and the whole genome DNA was extracted.The proband's DNA was sequenced using whole exome sequencing.Hemizygous and potentially pathogenic mutations were verified by Sanger sequencing.Pathogenicity was assessed according to the American College of Medical Genetics and Genomics (ACMG) guidelines.Tools such as SpliceAI and dbscSNV were used to predict the impact of mutations on mRNA splicing.This study strictly followed the Declaration of Helsinki, and the study protocol was approved by the Ethics Committee of Henan Eye Hospital (No.HNEECKY-2019[15]). All subjects and guardians of minor subjects signed informed consent forms.Results:The proband (Ⅲ: 1), a 5-year-old boy, presented with recessive nystagmus in both eyes and a best corrected visual acuity of 0.2.Color vision examination revealed red-green color blindness without night blindness.SD-OCT showed the presence of neuroepithelial structures in both eyes, but the interdigitation zone was blurred in both eyes.ff-ERG showed a slight decrease in rod function and a moderate-severe decrease in cone function in the right eye, and a slight decrease in cone and rod function in the left eye.Gene sequencing results showed that the proband had the hemizygous splice site variant c. 1911-3C>A of the CACNA1F gene on the X chromosome.Sanger sequencing showed that neither his mother nor his younger sister carried the variant, suggesting it was novel.This variant site was not recorded in the normal population database (PM2). Bioinformatics tools SpliceAI and dbscSNV consistently predicted that this variation affects on splicing.According to the ACMG guidelines, this variation is pathogenic. Conclusions:A novel variant c. 1911-3C>A in the CACNA1F gene was found in a family with CRD, and this variant may be a pathogenic variant site in this CRD family.This discovery expands the spectrum of pathogenic variations in CRD.

Objective:To identify the pathogenic gene in a family with cone-rod dystrophy (CRD).Methods:A pedigree study was conducted.Clinical data were collected from three generations of six people from a family with CRD who visited Henan Eye Hospital in December 2019, including one patient.After detailed collection of the patient's medical history, the proband and his family members underwent best-corrected visual acuity, slit-lamp microscope+ front-lens examination, optometry, non-mydriatic fundus photography, spectral-domain optical coherence tomography (SD-OCT), and full-field flash electroretinography (ff-ERG). Peripheral venous blood (5 ml) was collected from the proband, his parents and siblings, and the whole genome DNA was extracted.The proband's DNA was sequenced using whole exome sequencing.Hemizygous and potentially pathogenic mutations were verified by Sanger sequencing.Pathogenicity was assessed according to the American College of Medical Genetics and Genomics (ACMG) guidelines.Tools such as SpliceAI and dbscSNV were used to predict the impact of mutations on mRNA splicing.This study strictly followed the Declaration of Helsinki, and the study protocol was approved by the Ethics Committee of Henan Eye Hospital (No.HNEECKY-2019[15]). All subjects and guardians of minor subjects signed informed consent forms.Results:The proband (Ⅲ: 1), a 5-year-old boy, presented with recessive nystagmus in both eyes and a best corrected visual acuity of 0.2.Color vision examination revealed red-green color blindness without night blindness.SD-OCT showed the presence of neuroepithelial structures in both eyes, but the interdigitation zone was blurred in both eyes.ff-ERG showed a slight decrease in rod function and a moderate-severe decrease in cone function in the right eye, and a slight decrease in cone and rod function in the left eye.Gene sequencing results showed that the proband had the hemizygous splice site variant c. 1911-3C>A of the CACNA1F gene on the X chromosome.Sanger sequencing showed that neither his mother nor his younger sister carried the variant, suggesting it was novel.This variant site was not recorded in the normal population database (PM2). Bioinformatics tools SpliceAI and dbscSNV consistently predicted that this variation affects on splicing.According to the ACMG guidelines, this variation is pathogenic. Conclusions:A novel variant c. 1911-3C>A in the CACNA1F gene was found in a family with CRD, and this variant may be a pathogenic variant site in this CRD family.This discovery expands the spectrum of pathogenic variations in CRD.

Objective:To identify and observe the pathogenic genes and clinical phenotypes of a family with a special platelet phenotype, Hermansky-Pudlak syndrome type 6 (HSP6).Methods:A retrospective clinical study. In November 2019, one proband and three family members from six HSP families who visited Henan Eye Hospital were included in the study. The child's medical history and family history were inquired in detail. The proband and all family members underwent best corrected visual acuity (BCVA), fundus color photography, frequency-domain optical coherence tomography, and general physical examination. The proband underwent platelet transmission electron microscopy (PTEM) and colonoscopy. Peripheral venous blood was collected from the proband, her parents and younger brother, and genomic DNA was extracted. Whole exome sequencing (WES) was used to screen pathogenic genes and their loci. Bioinformatics analysis determines the pathogenicity of gene variation sites. Fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to verify the related variations.Results:The proband (Ⅱ-1) was a 7-year-old female. The BCVA in both eyes was 0.1, who exhibited mild horizontal nystagmus and iris depigmentation. Fundus examination revealed obvious depigmentation and an underdeveloped fovea centralis. At the age of 7, the patient underwent colonoscopy due to acute gastrointestinal bleeding. A polyp approximately 5 mm in size was found on the floor of the sigmoid colon, with erosion and mucosal leukoplakia on its surface. PTEM showed that the number of platelet dense granules was normal, but the nuclei were small or exhibited low compactness. The skin on both lower legs showed pigmentation. The clinical phenotypes of the proband’s parents (Ⅰ-1, Ⅰ-2) and younger brother (Ⅱ-2) showed no obvious abnormalities. WES revealed that the proband carried compound heterozygous variants in exon 1 of the HPS6 gene: c.60_64dup (p.L22fs) (M1) and c.1147_1148del (p.L383fs) (M2). The mother carried the M1 variant, while the father and younger brother carried the M2 variant. Bioinformatics analysis predicted that both variants were pathogenic. RT-qPCR results showed that, compared with the relative expression level of HPS6 wt mRNA, the relative expression levels of HPS6 L22fs and HPS6 L383fs mRNA were significantly decreased ( t = 3.549, 4.560; P<0.05). Western blot analysis demonstrated that the HPS6 L383fs protein was truncated, whereas the HPS6 L22fs protein was not detected. Conclusions:This family is a special HPS6 with a normal number of dense platelet granules. The compound heterozygous variations of M1 and M2 in the HPS6 gene are pathogenic genes in this family.

Objective:To analyze the change of differential genes and signaling pathways in high glucose induced BV2 cells, and to explore the mechanism of transgelin-2 (TAGLN2) regulating cellular inflammatory response and metabolic process.Methods:An experimental study. The cultured BV2 cells were divided into mannitol treatment (Man) group, glucose treatment (Glu) group, overexpression control Glu treatment (Con) group, overexpression TAGLN2 Glu treatment group, silence control Glu treatment (shCon Glu) group, and silence TAGLN2 Glu treatment (shTAGLN2 Glu) group. Cells in the Man group were cultured in modified Eagle high glucose medium (DMEM) containing 25 mmol/L mannitol and 25 mmol/L glucose, cells in other groups (Glu group, Con Glu group, TAGLN2 Glu group, shCon Glu group and shTAGLN2 Glu group) were cultured in DMEM medium containing 50 mmol/L glucose. After 24 hours of cells culture, transcriptome sequencing of cells in each group were performed using high-throughput sequencing technology, and significantly differentially expressed genes (DEG) were screened. |log 2 (fold change)|≥1 and P≤0.05 were adopted as criteria to screen for DEG. Gene Ontology (GO) function enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network analysis were performed. Real-time polymerase chain reaction (RT-PCR) was used to detect the relative expression level of DEG mRNA. The data between groups were compared by independent sample t-test. Results:When compared with Man group, a total of 517 differentially expressed genes were screened in Glu group, which including 277 up-regulated genes and 240 down-regulated genes. KEGG pathway enrichment analysis showed that the up-regulated genes were significantly enriched in immune system processes such as nuclear factor (NF)-κB signal pathway, Jak-signal transducers and activators of transcription (STAT) signal pathway, while down-regulated genes were significantly enriched in glycosaminoglycan degradation and glyceride metabolic pathway. Compared with Con Glu group, a total of 480 DEG were screened in TAGLN2 Glu group, among which 147 up-regulated and 333 down-regulated genes were detected. Up-regulated genes were significantly enriched in the metabolic processes of fatty acid, glyceride and pyruvate, while down-regulated genes were significantly enriched in immune system processes such as NF-κB signal pathway, Jak-STAT signal pathway and tumor necrosis factor (TNF) signal pathway. Compared with shCon Glu group, a total of 582 DEG were screened in shTAGLN2 Glu group, among which 423 up-regulated and 159 down-regulated genes were detected. Up-regulated DEG were significantly enriched in immune system processes such as TNF signal pathway and chemokine signal pathway, while down-regulated DEG were significantly enriched in pattern recognition receptor signal pathway. RT-PCR results showed that the relative expression levels of DEG mRNA Card11 ( t=13.530), Icos ( t=3.482), Chst3 ( t=6.949), Kynu ( t=5.399), interleukin (IL)-1β ( t=2.960), TNF-α ( t=5.800), IL-6 ( t=3.130), interferon-γ ( t=7.690) and IL-17 ( t=6.530) in the TAGLN2 Glu treatment group were decreased significantly compared with Con Glu group, and the difference was statistically significant. Conclusion:TAGLN2 can inhibit glucose induced microglia inflammation by NF-κB and Jak-STAT signaling pathways, Card11, Icos, Chst3 and Kynu play an important role in the anti-inflammatory process of TAGLN2.

Objective:To identify two pathogenic gene mutations in two families with Alstr?m syndrome (ALMS).Methods:A retrospective clinical study. Two patients and five family members from two Han families of ALMS diagnosed at Henan Eye Hospital from August 2020 to December 2021 were enrolled in this study. All participants underwent comprehensive ophthalmic examinations including best corrected visual acuity (BCVA), color test, slit-lamp, fundus biomicroscopy with slit lamp, fundus color photography, optical coherence tomography (OCT) and full-field electroretinography (ff-ERG) after the detailed history of the patient was taken. Five millilitres peripheral venous blood of each subject was collected, and the whole genome DNA was extracted. The pathogenic genes and mutation sites were identified using whole exome sequencing and the identified mutations were verified by Sanger sequencing. Mutation sites were analyzed via bioinformatics softwares.Results:Family one included one victim and two members and family two included one victim and three members. Proband in the first family was a four-year old boy whose chief complaint was poor vision along with photophobia since born, while proband in the second family was a 12-year old girl whose chief complaint was the same. The boy proband could not distinguish color, and both the anterior segment and fundus were normal. Ellipsoid zone of the boy was unclear in both eyes in OCT, and though rod system function decreased mildly-moderately in both eyes, the cone system function decreased severely in ff-ERG. The girl could not distinguish color as well, and the anterior segment was normal, though obvious pigmentary change could be seen in both retinas. The integrity of outer retinal bands was unclear in both eyes in OCT, and both cone and rod systems function decreased severely in both eyes in ff-ERG. Gene tests and bioinformatics analyze showed c.468dupT and c.10819C>T of ALMS1 gene in family one were novel mutations and c.10819C>T in family one and c.10831_10832del in family two were pathogenic mutations. Conclusions:M1, M2 and M3, M4 may be pathogenic gene variants in family 1 and family 2, respectively. The compound heterozygous mutation, c.468dupT and c.10819C>T of ALMS1 gene was a novel mutation.

Objective:To observe the efficacy of pars plana vitrectomy (PPV) in the treatment of different types of chorioretinal coloboma with retinal detachment (RD).Methods:A single-center, retrospective clinical study. From April 2021 to March 2023, 24 eyes of 23 patients who were diagnosed as chorioretinal coloboma with RD in Henan Provincial Eye Hospital were included in this study. There were 11 males with 12 eyes and 12 females with 12 eyes. The mean age was (33.3±13.7) years old. Best corrected visual acuity (BCVA), spectral domain optical coherence tomography were performed. The BCVA examination was performed using a international standard logarithmic visual acuity chart, which was converted into logarithm of the minimum angle of resolution (logMAR) visual acuity during statistics. According to the types of chorioretinal coloboma, the affected eyes were divided into the coloboma involved the optic disc group and the coloboma not involved the optic disc group, with 15 eyes and 9 eyes. According to whether the RD containing the coloboma area, the affected eyes were divided into RD containing the coloboma area group and the RD not containing the coloboma area group, with 15 eyes and 9 eyes. All eyes underwent standard pars plana three-channel 25G PPV, retinal laser photocoagulation combined with silicone oil tamponade. The follow-up time after surgery was (19.5±16.3) months. The last follow-up was the time point for efficacy determination. The retinal reattachment, BCVA recovery and postoperative complications were observed. Paired t-test or t test was performed for comparison of quantitative data. Fisher's exact test was performed for comparison of qualitative data. Results:At the last follow-up, retinal reattachment was achieved in 20 eyes (83.3%, 20/24). The logMAR BCVA of the coloboma involved the optic disc group before and after surgery were 1.85±0.62 and 1.71±0.71, the difference was no significant ( t=0.845 , P=0.412). The logMAR BCVA of the coloboma not involved the optic disc group before and after surgery were 1.75±0.45 and 0.84±0.26, the difference was statistically significant ( t=6.153 , P<0.001). The improvement of BCVA in the coloboma not involved the optic disc group was significantly higher than that in the coloboma involved the optic disc group after surgery, with statistically significant differences ( t=3.024 , P=0.006). There was no significant difference in the retinal reattachment rate between the two groups ( P=0.615). There was no significant difference in the retinal reattachment rate between the RD containing the coloboma area group and the RD not containing the coloboma area group ( P=0.259). Postoperative complications included elevated intraocular pressure in five eyes, cataract progression in ten eyes, recurrent RD in two eyes, bullous keratopathy in one eye and band-shaped keratopathy in one eye. Conclusion:PPV combined with silicone oil tamponade is safe and effective in the treatment of chorioretinal coloboma with RD, the improvement of visual acuity in the coloboma not involved the optic disc group is better than that in the coloboma involved the optic disc group after surgery.

Objective:To identify the pathogenic gene mutations in a Chinese achromatopsia family.Methods:A pedigree investigation was performed.A Chinese Han pedigree from Luoyang city of China was enrolled in Henan Eye Hospital in November 2018.The medical history of the patients was collected.The best corrected visual acuity (BCVA) of the families was examined.The maniafestations of the anterior segment and fundus were obtained via slit lamp biomicroscope and slit lamp lens.The diopter was determined by objective and subjective refraction.Color vision was examined by Farnsworth-Munsell Hue Test.Retinal function was evaluated by international standard electroretinogram (ERG). Retina was observed by color photography, and its structural image was obtained by spectral-domain optical coherence tomography (SD-OCT). The peripheral blood sample was collected from the proband (Ⅲ1) and her younger brother (Ⅲ2) and parents for whole blood DNA extraction, and a whole genome sequencing (WGS) was performed to identify the pathogenic genes and mutation sites, and the sequencing data was compared through disease-related databases such as the Human Genome Databases due to a negative detective result of specific hereditary eye disease enrichment panel based on targeted exome capture technology.Sanger sequencing and bioinformatics analysis was carried out with softwares.The cosegregation analysis was performed.This study protocol was approved by an Ethics Committee of Henan Eye Hospital (No.HNEECKY-2019[15]) and complied with Declaration of Helsinki.Written informed consent was obtained from each subject or the guardian before any medical examination.Results:This family included 2 patients and 8 members with normal phenotypes in 3 generations and showed an autosomal recessive inheritance model.Poor vision and photophobia appeared after birth in both Ⅲ1 and Ⅲ2, and these symptoms did not deteriorate with aging.Pigmentary mottling and atrophic changes could be seen in the retinas of the patients.Reflection bands of external membrane and ellipsoid line in macula of patients were irregular on the OCT image.Color vision examination showed achromatopsia of the patients.ERG indicated that the amplitudes of a-, b-waves of scotopic 0.01, 3.0, 10.0 ERG and oscillatory potentials were slightly reduced, and the amplitudes of a-, b-waves of photopic ERG and wavelets of 30 Hz were seriously reduced in both eyes of Ⅲ1 and Ⅲ2.WGS showed that heterozygous mutations of a novel mutation c. 129+ 1G>A and a known mutation c. 1285dupT of CNGB3 gene in Ⅲ1 and Ⅲ2.The mutations were confirmed by Sanger sequencing.Conclusions:The compound heterozygous mutation in c. 129+ 1G>A/c.1285dupT of CNGB3 gene may be responsible for the achromatopsia pathogenesis in this Chinese Han pedigree.The abnormal phenotype of the patients is the result of both CNGB3 c. 129+ 1G>A and CNGB3 c. 1285dupT mutations simultaneously.

Objective:To identify the pathogenic gene mutations in a family with early onset severe retinal dystrophy (EOSRD).Methods:A retrospective clinical study. One patient and three family members from a Han of EOSRD who were diagnosed at Henan Eye Hospital in August 2018 were included in the study. After the detailed history of the patients was collected, all participants underwent best corrected visual acuity (BCVA), slit-lamp, fundus biomicroscopy with the slit lamp, untra-widefield fundus color photography, spectral-domain optical coherence tomography (SD-OCT) and full-field electroretinography (ff-ERG). The subject’s peripheral venous blood of 5 ml was collected and the whole genome DNA was extracted. A genetic eye disease capture chip containing 441 disease-causing genes was used for targeted capture and enrichment of high-throughput sequencing, and Sanger sequencing was performed for the clear pathogenic mutation sites; the analysis software was used for bioinformatics analysis of the mutation sites.Results:A 6-year-old female proband developed poor night vision in both eyes after 1 year old. The BCVA of both eyes were 0.1. The color of the optic disc was slightly lighter; the diameter of the retinal vessels was slightly reduced, and extensive pigment changes can be seen in the retina outside the vascular arch. SD-OCT examination showed that the outer membrane, ellipsoid zone and chimera zone in the central fovea of both eyes were unclear and intermittent. The visual area outside the fovea was neuroepithelial outer plexiform layer, outer nuclear layer, outer membrane, ellipsoid zone. The chimera zone gradually disappeared, and the thickness of the pigment epithelial layer was not uniform. In ff-ERG examination, the functions of the binocular cone and rod system were severely decreased. The results of genetic testing showed that there were c.921C>A homozygous mutations in the Tubby-like protein (TULP1) gene of the proband, and c.3121C>T and c.3488G>A compound heterozygous mutations in the cyclic nucleotide gated channel beta 1 (CNGB1) gene. Amino acid conservation analysis results showed that the above three mutation sites were highly conserved in multiple species; bioinformatics analysis results showed that TULP1 gene c.921C>A (p.Cys307*) had translation termination in the protein conserved region, CNGB1 gene c.3121C>T (p.Arg1041Trp) and c.3488G>A (p.Gly1163Glu) had amino acid polarity changes in the protein conserved region, which led to major changes in the protein spatial structure.Conclusion:TULP1 gene c.921C>A homozygous mutation, CNGB1 gene c.3121C>T and c.3488G>A compound heterozygous mutation are the mutation sites of this EOSRD family.

Objective:To analyze the early changes of gene expression levels and signaling pathways in 661W cell line under hypoxic conditions and to find potential functional target genes.Methods:The cultured mouse 661W cells were divided into hypoxia treatment group and normoxia control group. Cells in the hypoxia treatment group were cultured in a three-gas incubator with volume fraction of 1% and 5% CO 2 at 37 ℃. Cells in the normoxia control group were cultured in an incubator at 37 ℃ with volume fraction of 5% CO 2. High-throughput sequencing technology was used to sequence the transcriptome of 661W cell treated with hypoxia and normoxia for 4 hours to screen for differentially expressed genes (DEG). Clustering heat map analysis, gene ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network (PPI) analysis were performed. The reverse transcription-polymerase chain reaction (RT-PCR) was used to verify the accuracy of the sequencing results. Results:A total of 506 differentially expressed genes were screened, including 459 up-regulated genes and 47 down-regulated genes. GO functional enrichment analysis showed that the main biological processes of DEG were the cell's response to hypoxia, glycolysis, negative regulation of cell proliferation and apoptosis. hypoxia inducible factor (HIF)-1α pathway, glycolysis, Forkhead box O (FoxO) pathway, Insulin signaling pathway and Adenosine 5′-monophosphate-activated protein kinase (AMPK) pathway were involved in the above process. PPI analysis results showed that hub genes related to hypoxia were Aldoa, Aldoc, Gpi1, Hk2, Hk1, Pfkl, Pfkp, Vhl, Fbxo10 and Fbxo27. The RT-PCR results showed that the relative expression levels of 15 DEG mRNA in the hypoxic treatment group were higher than that of the normoxic control group, and the difference was statistically significant ( P<0.05). The mRNA expression levels of N-myc downstream-regulated gene-1 ( Ndrg1 ), Mt1, and vascular endothelial growth factor A ( VEGFA) were time-dependent on hypoxia. Conclusions:Under hypoxia, DEG is mainly related to glucose metabolism, cell response to hypoxia, regulation of proliferation and apoptosis. HIF-1α pathway, glycolysis, FoxO pathway and AMPK pathway are involved in the early changes of 661W cells under hypoxia. Aldoa, Aldoc, Gpi1, Hk2, Hk1, Pfkl, Pfkp, Vhl, Fbxo10, Fbxo27 may play key roles in the response of 661W cells to hypoxia. Ndrg1, Mt1 and VEGFA could be potential functional target genes for the study of ischemia and hypoxia-related fundus diseases.