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.·

Characteristics of corneal dystrophies:a review from clinical,histological and genetic perspectives

Corneal dystrophy is a common type of hereditary corneal diseases. It includes many types, which have varied pathology, histology and clinical manifestations. Recently, the examination techniques of ophthalmology and gene sequencing advance greatly, which do benefit to our understanding of these diseases. However, many aspects remain still unknown. And due to the poor knowledge of these diseases, the results of the treatments are not satisfactory. The purpose of this review was to summarize the clinical, histological and genetic characteristics of different types of corneal dystrophies.

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.

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..

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.

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

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.

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.

Alcohol delamination of the corneal epithelium for recurrent corneal erosion syndrome

AIM： To evaluate the outcomes of alcohol delamination（ALD） of the corneal epithelium for the treatment of recurrent corneal erosion syndrome（RCES） and to implement a standardized treatment protocol for this condition utilizing evidence based practice and the findings of an internal audit. METHODS： A retrospective analysis of 42 eyes of 40 patients diagnosed with RCES who were treated with ALD between January 2006 and March 2016 was conducted. Patients had 20% alcohol applied to the cornea with the use of a well for 40 s. Patients were reviewed one week later in the Outpatient Department. Outcome criteria were established based on standards from other studies in the medical literature. These included, a treatment success rate of at least 72%（defined as complete resolution of symptoms one month after treatment）, a postoperative complication a rate of 〈5%（mainly infective keratitis, and subepithelial haze）, and the absence of any detrimental effect on visual acuity in ≥95% of patients. RESULTS： The mean age at the time of ALD was 41.17±13.44 y. Patients were followed for an average of 12.8±15.65 mo. The majority were female（52.5%, n=21） and the majority of eyes treated with ALD were left eyes（62.9%, n=26）. Trauma was the primary aetiology in our study population. Treatment was successful in 73.8%（n=31） of eyes and in 75%（n=30） of patients. Recurrence occurred in 26.2% of eyes at a mean of 10.41±12.63 mo post treatment. CONCLUSION： ALD is an efficacious and cost-effective primary surgical intervention for RCES.

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.

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.

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.

The role of corneal endothelium in macular corneal dystrophy development and recurrence

Macular corneal dystrophy(MCD)is a progressive,bilateral stromal dystrophic disease that arises from mutations in carbohydrate sulfotransferase 6(CHST6).Corneal transplantation is the ultimate therapeutic solution for MCD patients.Unfortunately,postoperative recurrence remains a significant challenge.We conducted a retrospective review of a clinical cohort comprising 102 MCD patients with 124 eyes that underwent either penetrating keratoplasty(PKP)or deep anterior lamellar keratoplasty(DALK).Our results revealed that the recurrence rate was nearly three times higher in the DALK group(39.13%,9/23 eyes)compared with the PKP group(10.89%,11/101 eyes),suggesting that surgical replacement of the corneal endothelium for treating MCD is advisable to prevent postoperative recurrence.Our experimental data confirmed the robust m RNA and protein expression of CHST6 in human corneal endothelium and the rodent homolog CHST5 in mouse endothelium.Selective knockdown of wild-type Chst5 in mouse corneal endothelium(AC^(siChst5)),but not in the corneal stroma,induced experimental MCD with similar extracellular matrix synthesis impairments and corneal thinning as observed in MCD patients.Mice carrying Chst5 point mutation also recapitulated clinical phenotypes of MCD,along with corneal endothelial abnormalities.Intracameral injection of wild-type Chst5 rescued the corneal impairments in AC^(siChst5)mice and retarded the disease progression in Chst5 mutant mice.Overall,our study provides new mechanistic insights and therapeutic approaches for MCD treatment by highlighting the role of corneal endothelium in MCD development.

Aplication of <i>in Vivo</i>Confocal Microscopy in Ophtalmology—Overview

Confocal microscopy is a method which has been increasingly used over the last decade in the study of the anterior ocular surface. The method allows testing and in vivo high resolution imaging of the structures of the anterior eye segment, at a cellular level, which is close to the histological examination of tissues. The data provided by this method allow for a better understanding of both the functional and pathological processes occurring in the anterior ocular surface not only for scientific purposes but also in clinical practice. The aim of the present work is to summarize the current knowledge and applications of confocal microscopy of the anterior ocular surface.

Genotype-phenotype correlations in Chinese patients with TGFBI gene-linked corneal dystrophy

In this paper,we report the clinical and molecular features of the distinct TGFBI (human transforming growth factor β-induced,OMIM No.601692) gene-linked corneal dystrophy.Altogether,five pedigrees and ten unrelated individuals diagnosed as corneal dystrophy were recruited.Peripheral venous DNA was extracted,and then amplified by polymerase chain reaction (PCR) and scanned for mutation by single-stranded conformation polymorphism (SSCP).Direct DNA sequencing was used to analyze the mutations of the TGFBI gene.In our study,thirty patients from five pedigrees and ten sporadic patients were diagnosed as four TGFBI gene-linked corneal dystrophies of granular corneal dystrophy type I (GGCD I),Avellino corneal dystrophy (ACD),lattice corneal dystrophy type I (LCD I),and lattice corneal dystrophy type ⅢA (LCD IIIA),and in total,seven disease-causing mutations,namely R555W,A546D,A546T,and T538P mutations in exon 12,R124H and R124C mutations in exon 4,and P501T mutation in exon 11,were identified,while four polymorphisms of V327V,L472L,F540F,and 1665-1666insC were screened in exons 8,11,and 12.The study ascertained the tight genotype-phenotype relationship and confirmed the clinical and genetic features of four TGFBI gene-linked corneal dystrophies.

Genetic mutations and molecular mechanisms of Fuchs endothelial corneal dystrophy

Background:Fuchs endothelial corneal dystrophy is a hereditary disease and the most frequent cause of corneal transplantation in the worldwide.Its main clinical signs are an accelerated decrease in the number of endothelial cells,thickening of Descemet’s membrane and formation of guttae in the extracellular matrix.The cornea’s ability to maintain stromal dehydration is impaired,causing painful epithelial bullae and loss of vision at the point when the amount of corneal endothelial cells cannot be compensated.At present,apart from corneal transplantation,there is no other effective treatment that prevents blindness.Main text:In this review,we first summarized the mutations of COL8A2,TCF4,TCF8,SLC4A11 and AGBL1 genes in Fuchs endothelial corneal dystrophy.The molecular mechanisms associated with Fuchs endothelial corneal dystrophy,such as endoplasmic reticulum stress and unfolded protein response pathway,oxidative stress,mitochondrial dysregulation pathway,apoptosis pathway,mitophagy,epithelial-mesenchymal transition pathway,RNA toxicity and repeat-associated non-ATG translation,and other pathogenesis,were then explored.Finally,we discussed several potential treatments related to the pathogenesis of Fuchs endothelial corneal dystrophy,which may be the focus of future research.Conclusions:The pathogenesis of Fuchs endothelial corneal dystrophy is very complicated.Currently,corneal transplantation is an important method in the treatment of Fuchs endothelial corneal dystrophy.It is necessary to continuously explore the pathogenesis of Fuchs endothelial corneal dystrophy and establish the scientific foundations for the development of next-generation corneal therapeutics.

R555W mutation of TGF, βI related to granular corneal dystrophy in Chinese patients

Background Mutations in the transforming growth factor beta I （TGFBI） gene cause several types of autosomal-dominant corneal dystrophies. We investigated the role of this gene in a Chinese family affected by granular corneal dystrophy （GCD）.Methods Family history and phenotypic data were recorded. The diagnosis of GCD was made on the basis of clinical evaluation. The genomic DNA was extracted from peripheral blood leukocytes. All the exons and flanking intron-exon boundary sequences of TGFβ1 were amplified by polymerase chain reaction （PCR） and screened for mutation by direct DNA sequencing.Results A heterozygous C to T transition at nucleotide c.1663 （CGG to TGG R555W） of TGFβ1 gene was present in two affected members but was absent in the rest of the family members. Conclusion A recurrent pathogenic R555W of TGFβ1 gene mutation is identified, which appears to be the predominant mutations causing GCD in different populations.

Analysis of human transforming growth factor β-induced gene mutation in corneal dystrophy

Background Corneal dystrophy is a group of inherited blinding diseases of the cornea. This study was to identify the mutations of the keratoepithelin (KE) gene for proper diagnosis of corneal dystrophy. Methods Three families with corneal dystrophy were analysed. Thirteen individuals at risk for corneal dystrophy in family A, the proband and her son in family B, and the proband in family C were examined after their blood samples were obtained. Mutation screening of human transforming growth factor β-induced gene (BIGH3 gene) was performed. Results Five individuals in family A were found by clinical evaluation to be affected with granular corneal dystrophy and carried the BIGH3 mutation W555R. However, both probands in families B and C, also diagnosed with granular corneal dystrophy, harboured the BIGH3 mutation R124H. Conclusion Molecular genetic analysis can improve accurate diagnosis of corneal dystrophy.