Thiel-Behnke Corneal Dystrophy in a Young Man in Denmark—A Case Report

Background: This case report presents a case of bilateral Thiel-Behnke corneal dystrophy in Denmark. Thiel-Behnke is an autosomal dominant inherited epithelial-stromal TGFBI dystrophy causing visual impairment. Methods and Results: This case study presents a 24-year-old Lithuanian man, with no previous ocular history, who had experienced slowly progressive visual impairment since his childhood. He was examined at the Department of Ophthalmology at Vejle Hospital and Aarhus University Hospital, where he was diagnosed with bilateral Thiel-Behnke corneal dystrophy. Histology confirmed the diagnosis. A lamellar corneal transplantation was performed in the right eye;however, due to epithelial growth under the corneal graft, it was later decided to redo the operation. Following the operations, the patient experienced a visual improvement in best corrected visual acuity (BCVA) from 0.1 (20/25 Snellen equivalent) to 0.3 (20/40 Snellen equivalent) in his right eye. Conclusions: This case of Thiel-Behnke corneal dystrophy is to our knowledge the first reported case in Denmark.

Covering corneal stromal lenticule for macular hole in pathological myopia

AIM:To evaluate the clinical effect of a new surgery technique(covering corneal stromal lenticule,CSL)for macular hole(MH)in pathological myopia.METHODS:This was a prospective non-randomized series case study.Fourteen eyes of 14 patients whose axial length were more than 29 mm and suffered from MH and macular hole retinal detachment(MHRD)were included in this study.All cases were treated with 25-gauge pars plana vitrectomy(PPV)with internal limiting membrane(ILM)peeling,covering CSL and C_(3)F_(8) gas tamponade.These cases were followed for 6mo,and the best-corrected visual acuity(BCVA),healing status of MH,the reattached rate of retinal detachment(RD),and reoperation rate were analyzed.RESULTS:All cases were successfully performed the surgery and the postoperative follow-up was completed.After surgery,MHs were healed in all 14 eyes(100%,14/14)after assessed by optical coherence tomography.The reattachment of retina was achieved in all 6 eyes(100%,6/6)with MHRD.BCVA was improved in 12 eyes(85.71%,12/14),and had no significant change in 2 eyes(14.29%,2/14).The overall mean BCVA was improved from 1.80±0.77 to 0.82±0.46 logMAR(F=10.46,P<0.01).No serious complications occurred in all cases.CONCLUSION:The new surgery technique(covering CSL)has high reattached rate of RD and high healing rate of MH in pathological myopia in the preliminary study.And it can effectively improve the visual function of patients.This new technique offers meaningful new ideas for treating refractory MH in pathological myopia.

Deterioration of Avellino corneal dystrophy in a Chinese family after LASIK

AIM:To reveal the importance of TGFBI gene screening for candidates with a family history of corneal disease or granular opacities in corneal stroma before refractive surgery.METHODS:A 37-year-old male(proband)underwent bilateral laser-assisted in situ keratomileusis(LASIK)in 2002,with right vision decreased significantly in 2006.The proband and other 32 members of the family underwent a detailed ophthalmic examination,including vision acuity,intraocular pressure,slit-lamp photograph,fundus examination,optical coherence tomography(OCT)of cornea,and in vivo confocal microscope(IVCM)and peripheral blood was used for genomic DNA extraction.Seventeen TGFBI gene exons were analyzed via polymerase chain reaction amplification and direct sequencing.RESULTS:Slit-lamp,IVCM,and OCT images showed that a large amount of dense and confluent granular opaque were seen at the interfaces of the flap and remnant stromal bed in right and light degree in left eye.Sanger sequencing showed that there was a 371 G>A mutation(CGC>CAC)in exon 4,which indicated that he harbored a heterozygote R124 H mutation,identifying the diagnosis of Avellino corneal dystrophy(ACD).Among the other 32 family members,6 of them harbored the identical mutation to that in the proband.CONCLUSION:ACD will worsen and recur after LASIK.Preoperative gene-screening for TGFBI mutations is important in diagnosing ACD.

Establishment of an untransfected human corneal stromal cell line and its biocompatibility to acellular porcine corneal stroma

AIM: To establish an untransfected human corneal stromal (HCS) cell line and characterize its biocompatibility to acellular porcine corneal stoma (aPCS). METHODS: Primary culture was initiated with a pure population of HCS cells in DMEM/F12 media (pH 7.2) containing 20% fetal bovine serum and various necessary growth factors. The established cell line was characterized by growth property, chromosome analysis, tumorigenicity assay, expression of marker proteins and functional proteins. Furthermore, the biocompatibility of HCS cells with aPCS was examined through histological and immunocytochemistry analyses and with light, electron microscopies. RESULTS: HCS cells proliferated to confluence 2 weeks later in primary culture and have been subcultured to passage 140 so far. A continuous untransfected HCS cell line with a population doubling time of 41.44 hours at passage 80 has been determined. Results of chromosome analysis, morphology, combined with the results of expression of marker protein and functional proteins suggested that the cells retained HCS cell properties. Furthermore, HCS cells have no tumorigenicity, and with excellent biocompatibility to aPCS. CONCLUSION: An untransfected and non-tumorigenic HCS cell line has been established, and the cells maintained positive expression of marker proteins and functional proteins. The cell line, with excellent biocompatibility to aPCS, might be used for in vitroreconstruction of tissue-engineered HCS.

Chinese family with atypical granular corneal dystrophy type I caused by the typical R555W mutation in TGFBI

·AIM: To investigate the clinical features and genetic defects in four generations of a Chinese family affected with atypical granular corneal dystrophy type I (GCD type I). · METHODS: Family history and clinical data were recorded. Genomic DNA samples were obtained from peripheral blood leukocytes of all participated. Exons of the transforming growth factor-β-induced (TGFBI) gene were directly sequenced after being amplified by polymerase chain reaction (PCR), and multi-point linkage analysis using microsatellite makers flanking the gene was applied to identify the disease-causing mutation. · RESULTS: Clinical features were quite variable in patients, some patients only had opacities in the epithelium, and others revealed multiple bilateral circular, discrete, crumb -like opacities mainly in the epithelium, with several in different depths of corneal stroma, and the performance was different bilaterally, even in the same patient. Directly nucleotide sequencing revealed a heterozygous p.R555W mutation in the coding sequence of the TGFBI gene in all affected individuals of the family, but was not found in all unaffected. The maximum logarithm of odds (LOD) score obtained by multi -point analysis was detected at marker locus D5S393 (LOD = 2.740; α=1.000). ·CONCLUSION: Our case presented with clinical futures and the pathogenic mutations in TGFBI gene, the phenotype of the pedigree was quite different from typical GCD type I, so we suggested that this phenotype was a variant of GCD type I. These findings expand the knowledge about GCD type I, and demonstrate that molecular genetic analysis is important to make an accurate diagnosis of patients with variable corneal dystrophies in clinic.

Local anesthetic lidocaine induces apoptosis in human corneal stromal cells in vitro

AIM:To demonstrate the apoptosis-inducing effect of iidocalne on human corneal stromal(HCS)cells fn vitm,and provide experimental basis for safety anesthetic usage In clinic of ophthalmology.METHODS:In vitro cultured HCS cells were treated with lidocaine at different doses and times,and their morphology was monitored successively with inverted phase contrast microscopy.The membrane permeability of them was detected by acridine orange/ethidium bromide(AO/EB)double staining.The DNA fragmentation of them was examined by agarose gel electrophoresis,and their ultrastructure was observed by transmission electron microscopy(TEM),respectively.RESULTS:Exposure to lidocaine at doses from0.3125g/L to 20g/L induced morphological changes of HCS cells such as cytoplasmic vacuolation,cellular shrinkage,and turning round,and elevated membrane permeability of these cells in AO/EB staining.The change of morphology and membrane permeability was doseand time-dependent,while lidocaine at dose below0.15625g/L could not induce these changes.Furthermore,lidocaine induced DNA fragmentation and ultrastructural changes such as cytoplasmic vacuolation,structural disorganization,chromatin condensation,and apoptotic body appearance of the cells.CONCLUSION:Lidocaine has significant cytotoxicity on human corneal stromal cells in vitro in a dose-and time-dependent manner by inducing apoptosis of these cells.The established experimental model and findingsbased on this model here help provide new insight into the apoptosis-inducing effect of local anesthetics in eye clinic.

Arg124Cys mutation of the TGFBI gene in a Chinese pedigree of Reis-Bücklers corneal dystrophy

AIM: To analyze mutations in transforming growth factor beta-induced (TGFBI) gene in a Chinese pedigree with Reis-Bücklers corneal dystrophy (RBCD,also known as GCD3).METHODS: In a five-generation Chinese family,eight members were identified with RBCD and the rest were unaffected.All members of the family underwent complete ophthalmologic examinations.Exons of TGFBI were amplified by polymerase chain reaction,sequenced,and compared with a reference database.RESULTS: A single heterozygous C>T (R124C) point mutation was found in exon 4 of TGFBI in all the affected members of the pedigree,but not in the unaffected members.CONCLUSION: R124C which was a known mutation for lattice corneal dystrophy type I,segregated with the RBCD in this pedigree.This elucidated the correlation between genotype and phenotype in a Chinese family of RBCD.

Comparing corneal outcome between femtosecond laser-assisted cataract surgery and conventional phaco surgery in Fuchs’endothelial dystrophy patients:a randomized pilot study with 6mo follow up

AIM:To compare the corneal outcome in Fuchs’endothelial dystrophy(FED)patients between femtosecond laser-assisted cataract surgery(FLACS)and conventional phaco surgery(CPS).METHODS:This was a randomized controlled study comparing one eye surgery by FLACS and the contralateral eye operated by CPS(stop and chop technique)in FED patients.Central corneal thickness,corneal light backscatter,corneal densitometry,and central corneal endothelial cell count and hexagonality(noncontact endothelial cell microscope),and corrected distance visual acuity(CDVA)were assessed preoperatively and at day 1,40,and 180 postoperatively.RESULTS:Totally 31 patients(16 women)were included.At day 40 postoperatively,the mean endothelial cell loss(ECL)was 23.67%by FLACS and 17.30%by CPS(P=0.53).At day 180 postoperatively,ECL was 25.58%in FLACS and 21.32%in CPS(P=0.69).Densitometry data in all layers and all annuli from anterior layer to posterior layer in annuli 0-2,2-6,6-10 and 10-12,total densitometry with all layers and all annuli was performed.A significant difference was found in 6-10(posterior layer)at day 1 with-1.42 grayscale units(GSU;95%CI:-2.66 to-0.19,P=0.02).In 10-12(anterior layer,central layer and all layers)at day 40 were significant different with 7.7(95%CI:1.89 to 13.50,P=0.009),3.97(95%CI:0.23 to 7.71,P=0.03),4.73 GSU(95%CI:0.71 to 8.75,P=0.02),respectively.In the remaining parameters we found no difference between the two groups(P>0.05).Three CPS eyes suffered from corneal decompensation.CONCLUSION:There is no significant difference in corneal outcome between FLACS and CPS.Endothelial cell density and pentacam corneal outcome may be inadequate as outcome parameters in FED patients.

Phacoemulsification in a rare case of keratoconus with Fuch's endothelial corneal dystrophy

Dear Sir,Iam Dr.Jaya Kaushik from the Department of Ophthalmology of the Post Graduate Institute of Medical Education and Research,Chandigarh,India.I write to present a case report of phacoemulsification in a rare case of cataract associated with keratoconus and Fuch’s endothelial corneal

A recognition survey of granular corneal dystrophy type 2 genetic detection in China

AIM:To evaluate the feasibility of promoting genetic detection for granular corneal dystrophy type 2(GCD2)by a questionnaire conducted among citizens in five cities in China.METHODS:The data were collected by questionnaire,and analyzed by Chi-square test and one-tailed t test in IBM SPSS statistics.RESULTS:Based on the survey data on the awareness of GCD2 genetic detection in this study and the positive predictive analysis report of the citizens in five cities in China,the vast majority(84.2%)of respondents had never heard of it and did not know that GCD2 patients have been prohibited from performing excimer surgery that can deteriorate GCD2 patients’condition even leading to blindness.Though 3.4%of patients understood GCD2 very much,they have no idea that GCD2 could not be 100%accuracy diagnosed by the conventional inspection methods.CONCLUSION:It is feasible and necessary to use GCD2 genetic detection as an excimer preoperative examination project.In order to promote the development of detection project,a few improvements should be carried out in terms of the promoting efforts,costs,and research progress.

Cytotoxicity of pilocarpine to human corneal stromal cells and its underlying cytotoxic mechanisms

AIM： To examine the cytotoxic effect of pilocarpine, an anti-glaucoma drug, on human corneal stromal（HCS）cells and its underlying cytotoxic mechanisms using an in vitro model of non-transfected HCS cells.· METHODS： After HCS cells were treated with pilocarpine at a concentration from 0.15625 g/L to 20.0 g/L,their morphology and viability were detected by light microscopy and MTT assay. The membrane permeability,DNA fragmentation and ultrastructure were examined by acridine orange（AO）/ethidium bromide（EB） double-staining. DNA electrophoresis and transmission electron microscopy（TEM）, cell cycle, phosphatidylserine（PS）orientation and mitochondrial transmembrane potential（MTP） were assayed by flow cytometry（FCM）. And the activation of caspases was checked by ELISA.· RESULTS： Morphology observations and viability assay showed that pilocarpine at concentrations above0.625 g/L induced dose- and time-dependent morphological abnormality and viability decline of HCS cells. AO/EB double-staining, DNA electrophoresis and TEM noted that pilocarpine at concentrations above 0.625 g/L induced dose- and/or time-dependent membrane permeability elevation, DNA fragmentation, and apoptotic body formation of the cells. Moreover, FCM and ELISA assays revealed that 2.5 g/L pilocarpine also induced S phase arrest, PS externalization, MTP disruption, and caspase-8,-9 and-3 activation of the cells.· CONCLUSION： Pilocarpine at concentrations above0.625 g/L（1/32 of its clinical therapeutic dosage） has a dose- and time-dependent cytotoxicity to HCS cells by inducing apoptosis in these cells, which is most probably regulated by a death receptor-mediated mitochondrion-dependent signaling pathway.

AB027.Varying pattern of proteases secretion in Fuchs corneal endothelial dystrophy

Background:The goal of this project was to analyze the relationship between cell morphology and proteases/proteases inhibitors(PIs)secretion profile in fuchs endothelial corneal dystrophy(FECD)corneal endothelial cells(CECs).Methods:Cell morphology was determined using a circularity index(4π×area/perimeter2)for each CECs population extracted from surgical FECD specimens(N=2)and healthy Eye bank corneas(N=3).CECs were cultured 28 days post-confluency.Supernatant was collected and analysed using Proteome Profiler Array detecting 35 proteases and 32 PIs(R&D Systems).Proteome signal was analyzed using Image Studio Lite and correlated with the population’s circularity index.Results:Calculation of circularity index reported different morphologies among FECD populations(0.59±0.18 and 0.64±0.17)and healthy populations(0.44±0.18,0.66±0.13 and 0.71±0.11).Proteome arrays revealed the presence of 10 proteases(ADAMTS1,Cathepsin A,B,D,and X/Z/P,DPPIV/CD26,MMP-2,3 and 12,uPA/Urokinase)and 10 PIs(Protease Nexin II,Cystatin B and C,EMMPRIN/CD147,Latexin,Lipocalin-1,Serpin E1,TFPI,TFPI-2,TIMP-1,2 and 4).Healthy and FECD specimens showed similar variation patterns according to morphology for secretion of ADAMTS1,MMP-3 and 12.However,opposing patterns between healthy and FECD populations were observed for Cathepsin B and D.Moreover,some proteins did not show variation according to phenotype in healthy CECs,but did in FECD CECs:Cathepsin A,Cystatin C,TFPI-2 and total TIMPs.For the other proteins,secretion did not vary according to morphology or no specific pattern was distinguishable.Conclusions:To conclude,our results suggest that cell phenotype is linked to the secretion of certain proteases/PIs in both groups.However,there seems to be differences in secretion of particular proteases and PIs between FECD and healthy specimens as morphology did not have a similar influence.These differences might initiate an imbalance between proteases and PIs explaining the irregular thickening of the Descemet membrane seen in FECD.

Graft rejection after deep anterior lamellar keratoplasty in fellow eye in macular corneal dystrophy: a case report

Because of the low incidence of immunological rejection,deep anterior lamellar keratoplasty(DALK)is currently the preferred treatment for macular corneal dystrophy(MCD).However,there were few reports about whether the consistent results were obtained when performing DALK in both eyes for MCD,especially when the corneal grafts were taken from different donors.Also,there were few reports about whether the stromal graft rejection occurred typically in both eyes in MCD with DALK.This case may represent the first report of an unusual and misleading manifestation of stromal graft rejection after uneventful DALK with big bubble technique in the fellow eye in MCD.A 32-year-old healthy man with MCD underwent bilateral uneventful DALK with a big bubble technique in the left eye in January and the right eye in July,the corneal grafts were taken from different donors.There was an atypical allograft rejection that occurred in the right eye and none in the left eye;although a timely diagnosis of graft rejection revealed following aqueous determination,it could not be reversed and underwent PK finally.The purpose of this case report is to illustrate the identification of atypical allograft rejection after DALK in the fellow eye,the significance of aqueous detection in the diagnosis of graft rejection,the choosing of grafts,and the timing of bilateral corneal transplantation in patients with MCD.

TGFBI gene mutation analysis in a Chinese pedigree of Avellino corneal dystrophy

AIM: To analyze phenotype and genotype of a Chinese pedigree with Avellino corneal dystrophy (ACD). METHODS: Complete ophthalmic EX aminations were performed on all the family members. Exons of TGFBI were amplified by polymerase chain reaction, sequenced, and compared with a reference database. RESULTS: A single heterozygous G>A(R124H) point mutation was identified in exon 4 of TGFBI in three affected members and two unaffected children who were offsprings of the affected members, but not in the other family members. CONCLUSION: Mutation R124H in TGFBI was identified in this pedigree and appeared to be the disease causing mutation. Atypical phenotype and low penetrance was observed in this pedigree..

Uncovering the profile of mutations of transforming growth factor beta-induced gene in Chinese corneal dystrophy patients

AIM： To uncover the mutations profile of transforming growth factor beta-induced （TGFBI） gene in Chinese corneal dystrophy patients and further investigate the characteristics of genotype-phenotype correlations. METHODS： Forty-two subjects （6 unrelated families including 15 patients and 8 unaffected members, and 19 sporadic patients） of Chinese origin were subjected to phenotypic and genotypic characterization. The corneal phenotypes of patients were documented by slit lamp photography. Mutation screening of the coding regions of TGFBI was performed by direct sequencing. RESULTS： We detected four corneal dystrophy types. The most frequent phenotypes were granular corneal dystrophy （GCD） （including 3 families and 8 sporadic patients） and lattice corneal dystrophy （LCD） （including 2 families and 9 sporadic patients）. The next phenotypes were corneal dystrophy of Bowman layer （CDB） （1 family and 1 sporadic patient） and epithelial basement membrane dystrophy （EBMD） （1 sporadic patient）. Six distinct mutations responsible for TGFBI corneal dystrophies were identified in 30 individuals with corneal dystrophies. Those were, p.R124H mutation in 1 family and 2 sporadic patients with GCD, p.R555W mutation in 2 families and 3 sporadic patients with GCD, p.R124C mutation in 2 families and 7 sporadic patients with LCD, p.A620D mutation in 1 sporadic patient with LCD, p.H626R mutation in 1 sporadic patient with LCD, and p.R555Q in 1 family and 1 sporadic patient with CDB. No mutation was detected in the remaining 3 atypical GCD patients and 1 EBMD patient, CONCLUSION： GCD and LCD are the most frequent phenotypes in Chinese population. R555W was the most common mutation for GCD; R124C was the most common mutation for LCD, Our findings extend the mutational spectrum of TFGBI , and this is the extensively delineated TGFBI mutation profile associated with the various corneal dystrophies in the Chinese population.

Cell viability and extracellular matrix synthesis in a co-culture system of corneal stromal cells and adipose-derived mesenchymal stem cells

AIM：To investigate the impact of adipose-derived mesenchymal stem cells（ADSCs） on cell viability and extracellular matrix（ECM） synthesis of corneal stromal cells（CSCs）. METHODS：ADSCs and CSCs were obtained from the corneas of New Zealand white rabbits and indirectly cocultured in vitro. The proliferative capacity of CSCs in the different groups was assessed by CCK-8 assays. Annexin V-fluorescein isothiocyanate（FITC）/proliferation indices（PI） assays were used to detect the apoptosis of CSCs. The expression levels of matrix metalloproteinase（MMP）, such as MMP1, MMP2, MMP9, and collagens were also evaluated by Western blot. RESULTS：ADSCs significantly promoted proliferation and invasion of CSCs in the indirect co-culture assays. The co-cultural group displayed much higher ability of proliferation, especially under the co-culture conditions of ADSCs for 3d, compared with that CSCs cultured alone. The PI of CSCs in the co-culture system were increased approximately 3-8-fold compared with the control group. A significant change was observed in the proportions of cells at apoptosis（early and late） between the negative control group（6.34% and 2.06%） and the ADCSs-treated group（4.69% and 1.59%）. The expression levels of MMPs were down regulated in the co-culture models. Compared with the control group, the decrease intensities of MMP-1, MMP-2 and MMP-9 in CSCs/ADSCs group were observed, 3.90-fold, 1.09-fold and 3.03-fold, respectively. However, the increase intensities of collagen type（I, II, III, IV, and V） in CSCs were observed in CSCs/ADSCs group, 3.47-fold,4.30-fold, 2.35-fold, 2.55-fold and 2.43-fold, respectively, compared to that in the control group. The expressions of aldehyde dehydrogenase and fibronectin in CSCs were upregulated in the co-culture models.CONCLUSION：ADSCs play a promotive role in CSCs＇ growth and invasion, which may be partially associated with MMPs decrease and collagens increase, resulting in a positive participation in the plasticity and ECM synthesis of CSCs. This provided a new insight into the extensive role of ADSCs in CSCs and a potential molecular target for corneal therapy.

Effects of corneal stromal cell-and bone marrow-derived endothelial progenitor cell-conditioned media on the proliferation of corneal endothelial cells

AIM： To explore the effects of conditioned media on the proliferation of corneal endothelial cells （CECa） and to compare the efficiency of different conditioned media （CM）. METHODS： Rat CECs, corneal stromal cells （CSCs）, bone marrow -derived endothelial progenitor cells （BEPCs）, and bone marrow-derived mesenchymal stem cells （BMSCs） were isolated and cultured in vitra CM was collected from CSCs, BEPCs, and BMSCSo CECs were cultivated in different culture media. Cell morphology was recorded, and gene and protein expression were analyzed.~ RESULTS： After grown in CM for 5d, CECs in each experimental group remained polygonal, in a cobblestone- like monolayer arrangement. Immunocytofluorescence revealed positive expression of Na＋/K＋-ATP, aquaporin 1 （AQP1）, and zonula occludens 1 （ZO-1）. Based on quantitative polymerase chain reaction （qPCR） analysis, Na ＋/K ＋-ATP expression in CSC-CM was notably upregulated by 1.3-fold （＋0.036） （P〈0.05, n=3）. The expression levels of ZO-1, neuron specific enolase （NSE）, Vimentin, paired homebox 6 （PAX6）, and procollagen type VII （COL8A1） were notably upregulated in each experimental group. Each CM had a positive effect on CEC proliferation, and CSC-CM had the strongest effect on proliferation.~ CONCLUSION： CSC-CM, BEPC-CM, and BMSC-CM not only stimulated the proliferation of CECs, but also maintained the characteristic differentiated phenotypes necessary for endothelial functions. CSC-CM had the most notable effect on CEC proliferation. KEYWORDS： conditioned medium; corneal endothelial cell; corneal stromal cell; bone marrow-derived endothelial progenitor cell; proliferation

Two mutations in the transforming growth factor beta-induced gene associated with familial Lattice corneal dystrophy

AIM：To report a phenotypic variant pedigree of lattice corneal dystrophy（LCD）associated with two mutations,R124C and A546 D,in the transforming growth factor betainduced gene（TGFBI）.METHODS：A detailed ocular examination was taken for all participants of a LCD family. Peripheral blood leukocytes from each participant were extracted to obtain the DNA. Polymerase chain reaction（PCR）of all seventeen exons of TGFBI gene was performed. The products were sequenced and analyzed. Histological examination was carried out after a penetrating keratoplasty from the right eye of proband. RESULTS：Genetic analysis showed that the proband and all 6 affected individuals harbored both a heterozygous CGC to TGC mutation at codon 124 and a heterozygous GCC to GAC mutation at codon 546 of TGFBI. None of the 100 control subjects and unaffected family members was positive for these two mutations. Ocular examination displayed multiple refractile lattice-like opacities in anterior stroma of the central cornea and small granular deposits in the peripheral cornea. The deposits were stained positively with Congo red indicating be amyloid in nature and situated mainly in the anterior and middle stroma. CONCLUSION：We observed a novel LCD family which carried two pathogenic mutations（R124C and A546D）in the TGFBI gene. The phenotypic features were apparently different from those associated with corresponding single mutations. The result reveals that although the definite mutation is the most important genetic cause of the disease,some different modifier alleles may influence the phenotype.

Corneal histomorphology and electron microscopic observation of R124L mutated corneal dystrophy in a relapsed pedigree

·AIM:To investigate the histological characteristics and ultrastructure of recurrent Chinese R124 L mutated corneal dystrophy after keratoplasty.·METHODS:The subjects were enrolled from a Chinese family of corneal dystrophy with R124 L heterozygous gene mutation and with a history of consanguineous marriage.Normal corneal samples were used as controls.·RESULTS:In this family,2 patients(3 eyes)underwent penetrating keratoplasty(PKP)and 2 patients(4 eyes)underwent lamellar keratoplasty(LKP).They had recurrence at 33.5±3.0(range 30-36)mo after keratoplasty.Among them,1 patient(1 eye)underwent PKP again and 1 patient(2 eyes)underwent LKP again.In the R124 L mutated recurrent corneal dystrophy,the corneal turbidity was mainly distributed from the upper corneal cortex to the anterior stroma;the corneal epithelium surface was rougher and more uneven;and,the corneal erosions were larger.Hematoxylin-eosin staining showed that the thickness of the corneal epithelium was uneven;the arrangement of the epithelial cells was disordered;and,some corneal epithelial cells were swollen.The results of Congo red staining,Masson’s trichrome staining and Periodic acid-Schiff staining were positive,while that of Alcian blue staining was negative.Under a transmission electron microscope,deposition of high electron density substances between epithelial and basal cells,and,apoptosis of basal cells were observed.Many high electron density depositions were observed in the sub-epithelial and anterior corneal matrix.·CONCLUSION:In the Chinese family of recurrent corneal dystrophy with R124 L gene mutation,the corneal epithelia of the recurrent cases are rougher,and the corneal depositions are extra cellular amyloid fibrin.

TGFBI and CHST6 gene analysis in Chinese stromal corneal dystrophies

AIM:To investigate whether mutations in TGFBI gene or CHST6 gene correlated with stromal corneal dystrophies(CD) in 8 Chinese probands.· METHODS:Eight unrelated patients with stromal corneal dystrophies were recruited in this study;all affected members were assessed by completely ophthalmologic examinations.Genomic DNA was extracted from peripheral leukocytes,17 exons of TGFBI gene and the exon of CHST6 gene were amplified by polymerase chain reaction(PCR),sequenced directly and compared with the reference database.· RESULTS:Three heterozygous mutations in TGFBI gene were identified in six patients:c.370C>T(p.Arg124Cys) was found in exon 4 of TGFBI gene in three members,c.371G>A(p.Arg124His) was found in one patient;c.1663C>T(p.Arg555Trp) was found in exon 12 in other two members.In addition,four polymorphisms with the nucleotide changes rs1442,rs1054124,rs4669,and rs35151677 were found in TGFBI gene.Mutations were not identified in the rest of 2 affected individuals in TGFBI gene or CHST6 gene.· CONCLUSION:Within these patients,R124C,R124H and R555W mutations were co-segregated with the disease phenotypes and were specific mutations for lattice corneal dystrophy type I(LCD I),Avellino corneal dystrophy(ACD,GCDⅡ),granular corneal dystrophy type I(GCD I),respectively.Our study highlights the prevalence of codon 124 and codon 555 mutations in the TGFBI gene among the Chinese stromal corneal dystrophies patients.·