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Objective@#To identify choroideremia and retinitis pigmentosa(RP)using next-generation sequencing(NGS)technology.@*Methods@#A cross-sectional study was adopted.The participants were two pedigrees that was suspected of RP in previous hospital treatments in He Eye Specialist Hospital between January 2017 and December 2018.The relevant medical and family history were collected, and the family tree was plotted.The visual acuity, intraocular pressure, fundus photography, electroretinogram(ERG), B-mode ultrasond, visual field, color vision, optical coherence tomography(OCT), and slit lamp assessment were performed on all subjects.Target region sequencing and Sanger sequencing were performed, deletion insertion of large fragments was verified by real-time quantitative PCR.This study followed the Helsinki Declaration and was approved by the Ethics Committee of Shenyang He Eye Hospital(NO. IRB[2017]k002.01)@*Results@#Two known pathogenic mutations were identified in CHM gene: EX9 DEL and c. 715C>T.Furthermore, the clinical diagnosis of choroideremia was confirmed.The Family 1 proband Ⅱ1 with the EX9 DEL hemizygous mutation had a special clinical phenotype, "Star" small piece intact choroid was visible in the macular fovea of both eyes.The patients in pedigree 2 carried the pathogenic mutation c. 715C>T, the proband Ⅲ1 carried a hemizygous mutation and the reddish island area in the fovea macula was seen in both eyes.The mother Ⅱ2 and grandmother I2 of proband carried heterozygous mutations, the degree of fundus atrophy was lighter than that of the proband Ⅲ1, the fundus had a mottled appearance, the optic disc boundary was clear, the blood vessel thickness was moderate, and the exposed choroid was visible in the fundus atrophy area.@*Conclusions@#This study is the first to identify two known pathogenic mutations in CHM gene, EX9 DEL and c. 715C>T in Asian Manchu and Han populations.The clinical phenotypes of female carriers with the c. 715C>t pathogenic mutation are different from those of male patients.The NGS technology may become a powerful tool to distinguish choroideremia from RP.
ABSTRACT
Objective To identify choroideremia and retinitis pigmentosa ( RP ) using next-generation sequencing( NGS) technology. Methods A cross-sectional study was adopted. The participants were two pedigrees that was suspected of RP in previous hospital treatments in He Eye Specialist Hospital between January 2017 and December 2018. The relevant medical and family history were collected,and the family tree was plotted. The visual acuity, intraocular pressure, fundus photography, electroretinogram ( ERG ) , B-mode ultrasond, visual field, color vision,optical coherence tomography(OCT),and slit lamp assessment were performed on all subjects. Target region sequencing and Sanger sequencing were performed, deletion insertion of large fragments was verified by real-time quantitative PCR. This study followed the Helsinki Declaration and was approved by the Ethics Committee of Shenyang He Eye Hospital(NO. IRB[2017]k002. 01) Results Two known pathogenic mutations were identified in CHM gene:EX9 DEL and c. 715C>T. Furthermore, the clinical diagnosis of choroideremia was confirmed. The Family 1 probandⅡ1 with the EX9 DEL hemizygous mutation had a special clinical phenotype,"Star" small piece intact choroid was visible in the macular fovea of both eyes. The patients in pedigree 2 carried the pathogenic mutation c. 715C>T,the probandⅢ1 carried a hemizygous mutation and the reddish island area in the fovea macula was seen in both eyes. The motherⅡ2 and grandmother I2 of proband carried heterozygous mutations,the degree of fundus atrophy was lighter than that of the proband Ⅲ1, the fundus had a mottled appearance, the optic disc boundary was clear, the blood vessel thickness was moderate,and the exposed choroid was visible in the fundus atrophy area. Conclusions This study is the first to identify two known pathogenic mutations in CHM gene,EX9 DEL and c. 715C>T in Asian Manchu and Han populations. The clinical phenotypes of female carriers with the c. 715C>t pathogenic mutation are different from those of male patients. The NGS technology may become a powerful tool to distinguish choroideremia from RP.
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Objective To investigate the promoting repair effects of chitosan/gelatin electrospinning membrane implantation on conjunctival injury.Methods Chitosan/gelatin electrospinning membrane was prepared using electrospinning technique,and the general characteristics of the membrane was examined under the scanning electron microscope.Conjunctival injury models were established in the right eyes of 30 New Zealand white rabbits by surgical resection of 6 mm×8 mm bulbar conjunctiva,bums of weak acid at injury site and then randomized into model group,human amniotic membrane (AM) implanted group and electrospinning membrane implanted group.AM or electrospinning membrane was interlayerly implanted between conjunctiva and sclera during operation according to grouping.The inflammatory response and histopathological change at injury site were examined by slit lamp microscope and optical microscope in 4,8,14 and 28 days after surgery.The use and care of the animals followed by code of ethics for animal experiments at the animal experiment center of He University.Results Chitosan/gelatin electrospinning membrane presented with a crosslinked network structure like AM,with the nano-microstructure of 0.1-0.5 μm fibre.No obvious inflammatory symptom was seen in the experimental eyes of various groups in 4 days after operation.In 8 days after operation,conjunctival edema and erosion were found in the model group,and slight conjunctival congestion was exhibited in the eyes of AM implanted group and electrospinning membrane implanted group.In 14 to 28 days after operation,conjunctival edema and new blood vessels were seen in the model group,and the conjunctivas were well recovered in the AM implanted group and electrospinning membrane implanted group.Histopathological examination showed dissociation,attaching,thickening and epithelization of injuried conjunctivas gradually in the model group from 4 to 28 days after operation.In the AM implanted group,attaching of AM was in 4 days,melting of AM was in 8 days,epithelization of injuried conjunctivas was in 14 days and renovation of conjunctivas was in 24 days after operation,and a rapid repairing procedure was found in the electrospinning membrane implanted group under the optical microscope.Conclusions Compared with the self-repairing process of conjunctival injury,implantion of chitosan/gelatin electrospinning membrane or AM can speed up the renovation of injuried conjunctivas,and the effects of chitosan/gelatin electrospinning membrane are better than those of AM in promoting conjunctiva repair and inhibiting scarring.
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Background The deposition of advanced glycation end products (AGEs) in lens is the risk factor of diabetic complications.Researches revealed that AGEs has autofluorescence.Crystallin is a longevity protein.AGEs accumulation is probably associated with diabetic retinopathy (DR).Objective This study was to evaluate the association of AGEs autofluorescence intensity with diabetes and with DR.Methods A cross-sectional study was carried out under the approval of Ethic Committee of Shenyang He Eye Hospital and informed consent of each patient.One hundred eyes of 100 patients with age-related cataract aged 50-70 years were included in He Eye Hospital from September to December 2015.The patients were divided into non-diabetes group (40 patients) and diabetes group (60 patients),and then the patients in diabetes group were subdivided into non-DR (NDR) group,non-proliferating DR (NPDR) group and proliferating DR (PDR) group according to the DR grading criteria,20 patients for each.Glycosylated henoglobin A1c (HbA1c) and fasting plasma glucose (FPG) were detected for each subject,and the lens autofluorescence was assayed with lens fluorescence biomicroscope (Clearpath DS120).The association of lens autofluorescence intensity with serum HbA1c level or DR severity was analyzed.Results The age and diabetes course were matched among the non-diabetes group,NDR group,NPDR group and PDR group (F=2.587,2.899,both at P＞0.05),and the FBS and HbA1c level were evidently higher in the NDR group,NPDR group and PDR group than those in the non-diabetes group (all at P＜0.01).The autofluorescence intensity of lens was (0.159±0.032),(0.256±0.024),(0.319 ±0.013) and (0.394±0.035) cd in the non-diabetes group,NDR group,NPDR group and PDR group,respectively,showing a significant difference among the groups (F =90.265,P =0.000).The autofluorescence intensity of lens in the NDR group,NPDR group and PDR group was significantly increased in comparison with the non-diabetes group and the autofluorescence intensity of lens was gradually increased with the severity of DR (all at P＜0.01).A positive linear correlation was found between autofluorescence intensity of lens and serum HbA1 c level in diabetes patients (r =0.654,P ＜ 0.05).Conclusions The autofluorescence intensity of AGEs in lens appears to be associated with the severity of DR and HbA1 c.The autofluorescence intensity of AGEs in the lens of diabetes patient is probably one of the evaluation indexes of early stage of DR.
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Background The generation of advanced glycosylation end products (AGEs) is one of the most important characteristics in diabetes mellitus (DM).Its role in diabetic eye disease is still unclear.Objective This study was to investigate the changes of AGE levels in human cataractous lens in diabetic patients and further explore the relationship of lens AGEs with early diabetic retinopathy (DR).Methods This study protocol was approved by Ethic Committee of He Eye Hospital.A cases controlled clinical study was carried out.Forty human nuclei were obtained from 40 cataract patients during the phacoemulcification in He Eye Hospital from August to December 2014,with the nuclear hardness of Ⅲ-Ⅳ grades (Emery grading).The eyes were divided into non-DM (NDM) group and DM group,and the DM group was further divided into non-DR (NDR) group,non-proliferative DR (NPDR) group and proliferative DR (PDR) group.Lens AGE level was quantitatively detected by ELISA.Results The content of pentosidine was (31.90±5.00),(35.83±4.22) and (38.59±5.11)pg/ml,and that of carboxymethyl lysine (CML) was (18.82±3.84),(19.65±2.12) and (23.77±4.73) μg/L,and that of imidazolone was (13.12±2.71),(14.48±2.01) and (15.37 ±2.74) pg/ml in the NDR group,NPDR group and PDR group,respectively,which was significantly higher than (27.69 ± 1.53) pg/ml,(11.36 ±1.47) μg/L and (10.21 ± 0.54) pg/ml in the NDM group (all at P＜0.01).The contents of pentosidine and CML in the lens were significantly higher in the PDR group than those in the NDR group (all at P＜0.05);while there was no significant differences in the pentosidine content in the lens among the NDR group,NPDR group and PDR group (all at P＞0.05).The positive correlations were found between CML content and pentosidine content (r =0.623,P =0.000),between CML content and imidazolone content (r =0.717,P =0.000) as well as between pentosidine content and imidazolone content (r=0.669,P =0.000).Conclusions The levels of pentosidine,CML and imidazolone in cataractous lens are obviously elevated in the DM patients.The levels of pentosidine and CML in the lens are gradually raised as the aggravation of DR.AGEs might be a potential maker for the assessment of diabetes.
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Background Scarring of filter bleb is a main cause of failure after trabeculectomy.Administration of anti-proliferation dugs during filtering surgery can maintain the opening of filtering pathway,but some serious complications occure after the drug use.Researches showed that polylactic acid caprolactone (PLCA) drug-loaded electrospinning film can release drug slowly,but its application in glaucoma is seldom.Objective The aim of this study was to evaluate the anti-scarring effects of non-penetrating trabeculectomy combined with sclera interlayer implantation of triamcinolone acetonide (TA)/PLCA drug-loaded electrospinning film in rabbit.Methods TA/PLCA drug-loaded electospinning film was prepared by electrospinning and its surface ultrastructure was observed under the scanning electron microscope.The drug release properties were detected by high performance liquid chromatograph.Ocular hypertensive models were established in New Zealand white rabbits by injecting carbomer into the anterior chamber of the right eyes,and the 40 models were randomized into 5 groups,8 eyes for each group.TA/PLCA drugloaded electrospun membrane,PLCA electrospun membrane or amniotic membrane was implanted beneath the scleral flap during trabeculectomy respectively in the TA/PLCA group,PLCA group or amniotic group,and 40 mg/ml TA was subconjunctivally injected in the TA group.Only trabeculectomy was performed in the simple trabeculectomy group.Intraocular pressure (IOP) was measured,and the shape of filtering bleb was examined under the slit lamp 1 week,2,4,8 and 12 weeks after surgery.The section of filtering bleb was prepared 12 weeks after surgery for the histopathological examination.The use and management of experimental animals was in line with animal ethics.Results The similar three-dimensional reticular structure was seen between TA/PLCA and electrospun membrane with the fiber diameter 0.5-1.5 μm.TA was released stably for 14 days.All the filtering blebs were disappeared in the simple trabeculectomy group during 8-week duration after operation.At 12 weeks after operation,the functional bleb was found in all the 8 eyes of the TA/PLCA group,5 eyes of PLCA group,4 eyes of the amniotic group and 4 eyes of the TA group.The IOP was significantly different among the groups,with the lowest IOP in the TA/PLCA group and the highest IOP in the simple trabeculectomy group (all at P =0.000).Histopathological examination showed that the filtering pathway remained opening in all 8 eyes with the epithelization of bleb surface in the TA/PLCA group,and interspaces of the filtering pathway was explayed in the PLCA group,amniotic group and TA group,while fibrosis of filtering pathway was seen in the simple trabeculectomy group at 12 weeks after surgery.Conclusions TA/PLCA drugloading electrospun membranes presents with nanoscale microstructure and good drug-release properties in vitro.Implantaion of TA/PLCA beneath the scleral flap during trabeculectomy can inhibit the fibrosis of filtering pathway.