Index of ophthalmology: Volume 1: Issue 1
1Postdoctoral Research Station of Basic Medicine, the Third Xiangya Hospital, Central South University, Changsha, China.
2Center for Experimental Medicine, the Third Xiangya Hospital, Central South University, Changsha, China.
3Department of Ophthalmology, the Third Xiangya Hospital, Central South University, Changsha, China.
#The first two authors contributed equally to this work.
Hao Deng, PhD
Professor of Center for Experimental Medicine
The Third Xiangya Hospital, Central South University
138 Tongzipo Road, Changsha 410013, China
Background/Aims: Corneal dystrophies (CDs) belong to a group of hereditary heterogeneous corneal diseases which result in visual impairment due to the progressive accumulation of deposits in different corneal layers. So far, mutations in several genes have been responsible for various CDs. The purpose of this study is to identify gene mutations in a three-generation Hui-Chinese family associated with Granular corneal dystrophy type I (GCD1). Methods: A three-generation Hui-Chinese pedigree with GCD1 was recruited for this study. Slit-lamp biomicroscopy, optical coherence tomography (OCT) and confocal microscopy were performed to determine the clinical features of available members. Whole exome sequencing was performed on two patients to screen for potential diseasing-causing variants in the family. Sanger sequencing was used to test the variant in the family members.
Results: Clinical Examinations demonstrated bilaterally abundant multiple grayish white opacities in the basal epithelial and superficial stroma layers of corneas of the two patients. Genetic analysis revealed that a heterozygous c.1663C>T (p.Arg555Trp) mutation in the TGFBI gene was shared by the two patients and it co-segregated with this disease in the family. Conclusions: The results suggested that the heterozygous c.1663C>T mutation (p.Arg555Trp) of the TGFBI gene was responsible for GCD1 in the Hui-Chinese family, which should be of great help in genetic counseling for this family.
Key words: Corneal dystrophy, Granular corneal dystrophy type I, Whole exome sequencing, TGFBI, missense mutation
Corneal dystrophies (CDs) belong to a group of hereditary and non-inflammatory corneal disorders that give rise to corneal transparency loss and visual impairment due to the progressive accumulation of extracellular amyloid and non-amyloid deposits in different corneal layers [1-3]. Symptoms typically initiate in the first or second decade of life, and slowly progress throughout life [1, 4]. Based on clinical features, pathologic exams and genetic data, CDs were sub-categorized as: epithelial and sub-epithelial dystrophies, epithelial-stromal TGFBI dystrophies, stromal dystrophies, and endothelial dystrophies [5, 6]. Though a few are inherited as autosomal recessive (AR) forms, the majorities are inherited as autosomal dominant (AD) forms with a high degree of penetrance [4, 7]. Mutations in the transforming growth factor beta induced gene (TGFBI, OMIM 601692), the solute carrier family 4, member 11 gene
(SLC4A11, OMIM 610206), the collagen, type VIII, alpha-2 gene (COL8A2, OMIM 120252), the keratin 3 gene (KRT3, OMIM 148043), the keratin 12 gene (KRT12,OMIM 601687), the chromosome 1, surface marker 1 gene (M1S1, OMIM 137290), and the carbohydrate sulfotransferase 6 gene (CHST6, OMIM 605294) have been reported as being responsible for various CDs, in which the TGFBI-CDs are the mostcommon [1, 8-12]. CDs are highly heterogeneous disorders, both clinically andgenetically . A certain subtype could result from different genetic defects, and adefinite gene could also cause different subtypes [5, 13]. The human TGFBI gene, expression of which was induced by transforming growth factor-beta (TGF-β), encodes a TGF-β induced protein (TGFBIp) which locates in an extracellular matrix . Although it is believed to be involved in many cell processes including cell proliferation, differentiation, migration, adhesion, angiogenesis and apoptosis, its function has not yet been completely understood . TGFBI gene mutations are primarily involved in AD inherited CDs characterized by the progressive accumulation of extracellular insoluble protein deposits within corneal tissue, which can present as amyloid, non-amyloid (granular), or both [15, 16]. Depending upon deposition features and locations in the corneal layers, disease-causing mutations identified in the TGFBI gene have been involved in various phenotypes, including Thiel-Behnke corneal dystrophy (TBCD, OMIM 602082), Reis-Bucklers corneal dystrophy (RBCD, OMIM 608470), Groenouw type I granular cornea dystrophy (CDGG1, also known as GCD1, OMIM 121900), Avellino corneal dystrophy (ACD, OMIM 607541), lattice corneal dystrophy type I and IIIA (LCD1, OMIM 122200 and LCD3A, OMIM 608471), and epithelial basement membrane corneal dystrophy (EBMD, OMIM 121820) [2, 4, 17]. Currently, TGFBI-CDs present as dominantly inherited monogenic with a high penetrance of 60-90% . To our knowledge, at least 63 TGFBI gene mutations have been described as being involved in different subtype of CDs . This study identified a TGFBI gene heterozygous c.1663C>T mutation (p.Arg555Trp) in a three-generation Hui-Chinese family with CD.
2. Materials and Methods
2.1 Subjects and clinical evaluations
Our study recruited a three-generation Hui-Chinese pedigree with CD (Figure 1 A). Two experienced ophthalmologists performed ophthalmologic examinations on available members. The exams included best corrected visual acuity (BCVA) assessed by the Snellen visual chart, slit-lamp biomicroscopy, optical coherence tomography (OCT) and confocal microscopy. All patients were diagnosed at the Third Xiangya Hospital of Central South University, Changsha, Hunan, China. This study followed the tenets of the Declaration of Helsinki and the guidance of the Ethical Committee of the Third Xiangya Hospital of Central South University. After written informed consent was obtained, peripheral blood samples were collected from four family members (I:1, I:2, II:1 and III:1). Genomic DNA (gDNA) was extracted from peripheral blood leukocytes using a phenol-chloroform extraction procedure [19, 20].
2.2 Whole exome sequencing and data analysis
Whole exome sequencing (WES) was performed by a commercial service from BGI-Shenzhen (Shenzhen, China) as previously described . In brief, gDNA samples of patients (I:2 and II:1, Figure 1A) were randomly fragmented into 150 bp -250 bp by Covaris. DNA fragments were repaired with an „A‟ base at the 3′-end of each strand and were then ligated with adapters. Ligation-mediated polymerase chain reaction (PCR) was used to amplify size-selected DNA fragments. PCR products were further purified and enriched with an exome array. High-throughput sequencing was then performed for each captured exome library on BGISEQ-500 platforms according to the manufacturer‟s instructions. After variants called by BGISEQ-500 Basecalling Software, clean reads per sample were mapped to the human reference genome(GRCh37/hg19) via the Burrows-Wheeler Aligner (BWA, v0.7.15). Picard tools (v2.5.0) and the Genome Analysis Toolkit (GATK) were used to mark and remove duplicate reads, respectively. All single nucleotide polymorphisms (SNPs) and insertions/deletions (Indels) were called by the HaplotypeCaller of GATK (v3.3.0). All variants screened were further filtered using the dbSNP v141, the 1000 Genomes Project, and the NHLBI-ESP6500 with MAF ≥ 0.1%. Annotations for variants were performed by the SnpEff tool
(http://snpeff.sourceforge.net/SnpEff_manual.html).Variant (s) shared by two patients (I:2 and II:1) and occurring in CD-causing genes were considered preliminary as candidate variant (s). Prediction for protein functions caused by candidate variant (s) (non-synonymous variants, splicing mutations and frameshift Indels) was performed on Polymorphism Phenotyping v2 (PolyPhen-2), Sorting Intolerant from Tolerant (SIFT), and MutationTaster software. According to the American College of Medical Genetics and Genomics (ACMG) recommendations for interpretative categories of variants in Mendelian disorders, candidate variant (s) was further classified as: „pathogenic‟, „likely pathogenic‟, „uncertain significance‟„likely benign‟, and „benign‟ .
2.3 Variant validation
Potential CD-related pathogenic variant (s) from the WES was further confirmed in other family members using Sanger sequencing and evaluated to determine whether it co-segregated with the disease phenotype in the family. Primers for PCR
amplification were designed by Primer3 software (http://primer3.ut.ee/) based on human reference genome (GRCh37/hg19) and synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) as follows: 5‟-GACTGACGGAGACCCTCAAC-3‟ (forward) and 5‟-GATGTGCCAACTGTTTGCTG-3‟ (reverse). PCR products were sequenced according to manufacturer‟s instructions on an ABI 3500 sequencer (Applied Biosystems, CA, USA). Variant was compared with those of the 528 Chinese controls of our in-house exome databases and 1,943 Chinese controls without CDs from in-house exome databases of BGI-Shenzhen.
3.1 Subjects and clinical assessment
The transmission forms of this family were consistent with AD inheritance (Figure 1A). Patient I:2 was a 61-year-old woman who complained of both poor vision and bilateral hyperdacryosis for about 20 years. Her vision was 20/667 OD and 20/1000 OS. Patient II:1 was a 41-year-old woman. Her vision was 20/33 OD and 20/40 OS.
Patient III:1 was a 19-year-old man. His vision was 20/33 OD and 20/50 OS.
Slit-lamp examinations revealed that the unaffected family member (I:1) had the normal corneas (Figure 2A), but the patients had bilaterally abundant multiple crumb-shaped and round grayish white opacities in the central corneas (Figure 2B-2C). OCT scan of patient II:1 and III:1 demonstrated markedly increased reflectivity due to deposits within the cornea (Figure 3A). In vivo laser scanning confocal microscopy revealed many abnormal hyper-reflective dots with sharp shapes primarily existed in the basal epithelial (Figure 3B) and superficial stroma layers
(Figure 3C) in the cornea of patients II:1 and III:1.
3.2 Whole exome sequencing and variant validation
WES of patient I:2 and patient II:1 generated 26,402 Mb and 22,397.76 Mb raw bases of sequence with an average sequencing depth of target regions 230.75×and 202.01×, respectively. A total of 108,186 and 107,154 SNPs, and 19,029 and 18,426 indels were separately obtained from patients I:2 and II:1. After filtering databases and functional analysis, only a heterozygous c.1663C>T mutation (p.Arg555Trp) in the TGFBI gene, shared by these two patients and reported previously for GCD1 ,was proposed as the potential pathogenic mutation in this family. This mutation was absent in the unaffected family member (I:1) and the 2,471 Chinese controls from the in-house databases. Sanger sequencing tested the mutation in all patients and its co-segregation with GCD1 in the family. The p.Arg555Trp mutation of TGFBIp was predicted to be probably damaging, damaging and disease-causing by PolyPhen-2, SIFT and MutationTaster, respectively. According to the ACMG guidelines for variants interpretation, the heterozygous mutation was categorized as „pathogenic‟. Overall, the genetic and clinical data supported a diagnosis of granular corneal dystrophy 1 (GCD1) in this family.
GCD1, also termed as classic granular CD or Groenouw CD, is one of the most common phenotypes of the TGFBI gene associated with CDs in China, which has an AD trait [13, 24]. It is featured by the progressive accumulation of white or gray white granules in the corneal stroma, with onset usually in the first or second decade of life [7, 25]. In addition to superficial stroma, granule deposits also appear between the basal epithelium cell layer and the Bowman layer with various shapes including drop-, crumb-, and ring-shaped . Histopathologically, GCD1 is featured by eosinophilic and rod-shaped hyaline deposits in the cornea, which can be stained bright red by Masson’s trichrome . In this study, clinical examinations revealed abundant crumb-shaped and round grayish white opacities in the subepithelial and anterior stroma layers of the cornea in patients of this family. In previous reports, mutations in the TGFBI gene including p.Val113Ile, p.Asp123His, p.Arg124Ser, p.Ser516Arg, p.Arg555Trp, and p.Leu559Val had been described as being involved in GCD1 development [24, 26-29]. In this study, a heterozygous missense mutation (c.1663C>T, p.Arg555Trp) in the TFGBI gene was identified in a three-generation Hui-Chinese family which displayed autosomal dominant inheritance. The mutation was predicted to be probably damaging, damaging and disease-causing by PolyPhen-2, SIFT and MutationTaster, respectively. It was also categorized as „pathogenic‟ according to the ACMG guidelines for variants interpretation. These indicated it was importance for protein structure or function. Clinical manifestations of patients are typical GCD1, including non-amyloid, granular stromal deposits, and are consistent with TGFBIp.Arg555Trp-GCD1 reported previously . To our knowledge, this is the first report of the p.Arg555Trp mutation in the TGFBIp in Hui-Chinese, though the mutation has been described in various populations worldwide . The discovery of the TGFBI gene c.1663C>T mutation (p.Arg555Trp) in several distinct ethnic groups with many families existing in a certain population suggests that both hotspot mutation and founder effect should be considered. The TGFBI gene, located at chromosome 5q31, contains 17 exons and encodes a 683 amino-acid extracellular matrix protein (TGFBIp) with a molecular weight of 68-kDa [25, 31]. TGFBIp contains an N-terminal cysteine-rich EMILIN-like (EMI) domain, four consecutive and highly homologous fascilin 1 (FAS1) domains, and a C-terminal arginine-glycine-aspartate acid (RGD) motif [18, 32]. In human corneas, TGFBIp primarily expresses in the epithelium, Bowman‟s membrane, stroma and endothelial cell layers, which suggests that it plays crucial roles in corneal damage repair and extracellular matrix maintenance [13, 32]. High levels of TGFBIp expression were associated with postnatal cornea maturation during the early stage of life .However, the progressive accumulation of insoluble deposits of mutant proteins in the cornea is involved in TGFBI-associated CDs, such as GCD1 . Although the precise pathogenesis of TGFBI gene mutations resulting in CDs remains to be illustrated, abnormal folding and location alteration of mutant proteins has been proposed as the central mechanism for TGFBI-related CDs . It appears that mutations are also likely to change protein degradation pathways and impact structure and stability of aggregated proteins . However, pathogenic mutations in the TGFBI gene were reported to have no effect on proteins secretion, indicating that mutant proteins escaped from protein secretion regulation system monitoring . Although most of the TGFBI gene mutations related to CDs are heterozygous, several patients with homozygous mutations have severe phenotypes, indicating potential toxic functions with a dose-response effect .
The Arg555 is located in the fourth FAS1 core domain of the TGFBIp, which is very susceptible to proteolysis . The TGFBIp with the p.Arg555Trp mutation accumulated as crystalloid deposits in GCD1 patient cornea, indicating that the mutation disrupted normal proteolytic degradation by reducing electrostatic repulsion levels . Compared to the wild type protein, the p.Arg555Trp mutant protein is more stable under physiological pH . The p.Arg555Trp mutation of TGFBIp was also reported to promote human corneal epithelial cells apoptosis by activating the α3β1 integrin-related pathway, indicating that it was likely to affect TGFBIp-α3β1 integrin interactions . Therefore, the p.Arg555Trp mutation of TGFBIp seems to result in solubility decrease and stability increase rather than structure alteration, aswell as impacting interactions with other proteins rather than misfolding. There are no effective approaches to prevent or cure TGFBI-related CDs. Although corneal transplantation has been the recommended treatment, a major limitation is the recurrence of post-transplantation corneal deposits . Thus, developing a therapeutic strategy that focuses on preventing mutant TGFBIp deposition by reducing its expression and/or increasing its degradation is likely to be achieved in treatment of TGFBI-related CDs. Recently, gene therapy has developed new insights into the treatment of several genetic diseases . In vitro, decreasing mutant TGFBIp expression with an allele-specific nature siRNA and correcting mutant DNA in TGFBIp mutant cells with site-specific genome editing technologies seem to provide promising approaches for TGFBI-linked CDs [37, 38]. Taken together, this study demonstrates that a heterozygous c.1663C>T mutation (p.Arg555Trp) of the TGFBI gene is responsible for GCD1 in a Hui-Chinese family, which will be of great help in genetic counseling for this family. Generation of animal models expressing the p.Arg555Trp mutant TGFBIp is likely to reveal the pathogenic mechanisms of GCD1 and shed new light on development of experimental therapy for this disorder.
Conflicts of Interest
These authors confirm that there are no conflicts of interest.
The authors thank all the subjects and investigators for their contributions to this research. This study was supported by grants from National Key Research and Development Program of China [2016YFC1306604], National Natural Science Foundation of China [81670216, 81800219 and 81873686], Natural Science Foundation of Hunan Province [2016JJ2166 and 2018JJ2556], Grant for the Foster Key Subject of the Third Xiangya Hospital Clinical Laboratory Diagnostics (H.D.), the New Xiangya Talent Project of the Third Xiangya Hospital of Central South University , Scientific Research Project of Health and Family Planning Commission of Hunan Province [B20180729], National-level College Students’ Innovative Training Plan Program , China.
 T. Zhang, N. Yan, W. Yu et al., “Molecular genetics of Chinese families with TGFBI corneal dystrophies,” Mol Vis, vol. 17, pp. 380-387, 2011.
 J.S. Song, D.H. Lim, E.S. Chung et al., “Mutation analysis of the TGFBI gene in consecutive Korean patients with corneal dystrophies,” Ann Lab Med, vol. 35, no. 3, pp. 336-340, 2015.
 M. Bustamante, A. Tasinato, F. Maurer et al., “Overexpression of a mutant form of TGFBI/BIGH3 induces retinal degeneration in transgenic mice,” Mol Vis, vol. 14, pp.1129-1137, 2008.
 X.D. Hao, Y.Y. Zhang, P. Chen et al., “Uncovering the profile of mutations of transforming growth factor beta-induced gene in Chinese corneal dystrophy patients,” Int J Ophthalmol, vol. 9, no. 2, pp.198-203, 2016.
 M. Sacchetti, I. Macchi, A. Tiezzi et al., “Pathophysiology of Corneal Dystrophies: From Cellular Genetic Alteration to Clinical Findings,” J Cell Physiol, vol. 231, no. 2, pp. 261-269, 2016.
 J.S. Weiss, H.U. Møller, A.J. Aldave et al., “IC3D classification of corneal dystrophies-edition 2,”Cornea, vol. 34, no. 2, pp. 117-159, 2015.
 S.J. Zhao, Y.N. Zhu, X.C. Shentu et al., “Chinese family with atypical granular corneal dystrophy type I caused by the typical R555W mutation in TGFBI,” Int J Ophthalmol, vol. 6, no. 4, pp. 458-462, 2013.
 E.N. Vithana, P. Morgan, P. Sundaresan et al., “Mutations in sodium-borate cotransporter SLC4A11 cause recessive congenital hereditary endothelial dystrophy (CHED2),” Nat Genet, vol. 38, no. 7, pp. 755-757, 2006.
 S. Biswas, F.L. Munier, J. Yardley et al., “Missense mutations in COL8A2, the gene encoding the alpha 2 chain of type VIII collagen, cause two forms of corneal endothelial dystrophy,” Hum Mol Genet, vol. 10, no. 21, pp. 2415-2423, 2001.
 Y.T. Chen, S.H. Tseng, S.C. Chao, “Novel mutations in the helix termination motif of keratin 3 and keratin 12 in 2 Taiwanese families with Meesmann corneal dystrophy,” Cornea, vol. 24, no. 8, pp. 928-932, 2005.
 Z. Ren, P.Y. Lin, G.K. Klintworth et al., “Allelic and locus heterogeneity in autosomal recessive gelatinous drop-like corneal dystrophy,” Hum Genet, vol. 110, no. 6, pp. 568-577, 2002.
 A.J. Aldave, V.S. Yellore, E.J. Thonar et al., “Novel mutations in the carbohydrate sulfotransferas gene (CHST6) in American patients with macular corneal dystrophy,” Am J Ophthalmol, vol. 137,no. 3, pp. 465-473, 2004.
 K.E. Han, S.I. Choi, T.I. Kim et al., “Pathogenesis and treatments of TGFBI corneal dystrophies,”Prog Retin Eye Res, vol. 50, pp. 67-88, 2016.
 H. Ge, P. Tian, L. Guan et al., “A C-terminal fragment BIGH3 protein with an RGDRGD motif inhibits corneal neovascularization in vitro and in vivo,” Exp Eye Res, vol. 112, pp. 10-20, 2013
 J.Y. Niu, J. Liu, L. Liu et al., “Construction of eukaryotic plasmid expressing human TGFBI and its influence on human corneal epithelial cells,” Int J Ophthalmol, vol. 5, no. 1, pp. 38-44, 2012.
 Y. Long, Y.S. Gu, W. Han et al., “Genotype-phenotype correlations in Chinese patients with TGFBI gene-linked corneal dystrophy,” J Zhejiang Univ Sci B, vol. 12, no. 4, pp. 287-292, 2011.
 J. Yang, X. Han, D. Huang et al., “Analysis of TGFBI gene mutations in Chinese patients with corneal dystrophies and review of the literature,” Mol Vis, vol. 16, pp. 1186-1193, 2010.
 D.G. Courtney, E.T. Poulsen, S. Kennedy et al., “Protein Composition of TGFBI-R124C- and TGFBI-R555W-Associated Aggregates Suggests Multiple Mechanisms Leading to Lattice and Granular Corneal Dystrophy,” Invest Ophthalmol Vis Sci, vol. 56, no. 8, pp. 4653-4661, 2015.
 L. Yuan, X. Deng, Z. Song et al., “Genetic analysis of the RAB39B gene in Chinese Han patients with Parkinson’s disease,” Neurobiol Aging, vol. 36, no. 10, pp. 2907.e11-2907.e12, 2015.
 W. Zheng, H. Chen, X. Deng et al., “Identification of a Novel Mutation in the Titin Gene in a Chinese Family with Limb-Girdle Muscular Dystrophy 2J,” Mol Neurobiol, vol. 53, no. 8, pp. 5097-5102, 2016.
 H. Deng, Q. Lu, H. Xu et al., “Identification of a Novel Missense FBN2 Mutation in a Chinese Family with Congenital Contractural Arachnodactyly Using Exome Sequencing,” Plos One, vol. 11, no. 5, Article ID e0155908, 2016.
 S. Richards, N. Aziz, S. Bale et al., “Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology,” Genet Med, vol. 17, no. 5, pp. 405-424, 2015.
 Y.C. Hou, I.J. Wang, C.H. Hsiao et al., “Phenotype-genotype correlations in patients with TGFBI-linked corneal dystrophies in Taiwan,” Mol Vis, vol. 18, pp. 362-371, 2012.
 J.C. Zenteno, A. Ramirez-Miranda, C. Santacruz-Valdes et al., “Expanding the mutational spectrum in TGFBI-linked corneal dystrophies: Identification of a novel and unusual mutation (Val113Ile) in a family with granular dystrophy,” Mol Vis, vol. 12, pp. 331-335, 2006.
 Y.P. Han, A.J. Sim, S.C. Vora et al., “A unique TGFBI protein in granular corneal dystrophy types 1 and 2,” Curr Eye Res, vol. 37, no. 11, pp. 990-996, 2012.
 N.T. Ha, l.X. Cung, H.M. Chau et al., “A novel mutation of the TGFBI gene found in a Vietnamese family with atypical granular corneal dystrophy,” Jpn J Ophthalmol, vol. 47, no. 3, pp. 246-248, 2003.
 F.L. Munier, B.E. Frueh, P. Othenin-Girard et al., “BIGH3 mutation spectrum in corneal dystrophies,” Invest Ophthalmol Vis Sci, vol. 43, no. 4, pp. 949-954, 2002.
 P. Paliwal, A. Sharma, R. Tandon et al., “TGFBI mutation screening and genotype-phenotype correlation in north Indian patients with corneal dystrophies,” Mol Vis, vol. 16, pp. 1429-1438, 2010.
 S.V. Chakravarthi, C. Kannabiran, M.S. Sridhar et al., “TGFBI gene mutations causing lattice and granular corneal dystrophies in Indian patients,” Invest Ophthalmol Vis Sci, vol. 46, no. 1, pp.121-125, 2005.
 R. Lakshminarayanan, S.S. Chaurasia, V. Anandalakshmi et al., “Clinical and genetic aspects of the TGFBI-associated corneal dystrophies,” Ocul Surf, vol. 12, no.4, pp. 234-251, 2014.
 C. Gruenauer-Kloevekorn, I. Clausen, E. Weidle et al., “TGFBI (BIGH3) gene mutations in German families: two novel mutations associated with unique clinical and histopathological findings,” Br J Ophthalmol, vol. 93, no. 7, pp. 932-937, 2009.
 R. Lakshminarayanan, S.S. Chaurasia, E. Murugan et al., “Biochemical properties and aggregation propensity of transforming growth factor-induced protein (TGFBIp) and the amyloid forming mutants,” Ocul Surf, vol. 13, no. 1, pp. 9-25, 2015.
 B.Y. Kim, J.A. Olzmann, S.I. Choi et al., “Corneal dystrophy-associated R124H mutation disrupts TGFBI interaction with Periostin and causes mislocalization to the lysosome,” J Biol Chem, vol. 284, no. 29, pp. 19580-19591, 2009.
 J. Underhaug, H. Koldso, K. Runager et al., “Mutation in transforming growth factor beta induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization,” Biochim Biophys Acta, vol. 1834, no. 12, pp. 2812-2822, 2013.
 M. Elavazhagan, R. Lakshminarayanan, L. Zhou et al., “Expression, purification and characterization of fourth FAS1 domain of TGF beta Ip-associated corneal dystrophic mutants,” Protein Expr Purif, vol. 84, no. 1, pp. 108-115, 2012.
 S. Morand, V. Buchillier, F. Maurer et al., “Induction of apoptosis in human corneal and HeLa cells by mutated BIGH3,” Invest Ophthalmol Vis Sci, vol. 44, no. 7, pp. 2973-2979, 2003.
 D.G. Courtney, S.D. Atkinson, J.E. Moore et al., “Development of allele specific gene silencing siRNAs for TGFBI Arg124Cys in Lattice Corneal Dystrophy Type I,” Invest Ophthalmol Vis Sci, vol. 55, no. 2, pp. 977-985, 2014.
 Y. Taketani, K. Kitamoto, T. Sakisaka et al., “Repair of the TGFBI gene in human corneal keratocytes derived from a granular corneal dystrophy patient via CRISPR/Cas9-induced homology-directed repair,” Sci Rep, vol. 7, no. 1, p. 16713, 2017.
Figure 1. Pedigree of the Hui-Chinese family with GCD1 and sequencing analysis of TGFBI c.1663C>T variant. (A) Pedigree of the GCD1 family, N: allele with
wild-type, M: allele with c.1663C>T. Squares and circles represent males and females, respectively. Solid symbols indicate patients, open symbols indicate unaffected individuals. (B) Patient II:1 with the heterozygous TGFBI c.1663C>T variant. (C) Unaffected family member (I:1) with the TGFBI c.1663C. GCD1, granular corneal dystrophy 1; TGFBI, the transforming growth factor beta induced gene.
Figure 2. Slit-lamp examinations of the Hui-Chinese family members. (A) The unaffected family member (I:1) showed bilateral normal cornea. (B-C) The patients II:1 (B) and III:1(C) showed bilateral abundant multiple crumb-shaped and round grayish white opacities in their central cornea, indicating a GCDI phenotype in the family.
Figure 3. Optical coherence tomography and in vivo laser scanning confocal
microscopy images of patients in the Hui-Chinese family. (A) Optical coherence tomography scan of the patients (II:1 and III:1) demonstrated markedly increased reflectivity due to deposits within the superficial cornea. (B-C) In vivo laser scanning confocal microscopy images showed many abnormal hyper-reflective regions with irregular shapes existed in the basal epithelial cells layer (B) and the superficial stroma layer (C) of cornea in the patients (II:1 and III:1).
Patient II:1 Patient III:1
Patient II:1 Patient III:1
Patient II:1 Patient III:1
Review Status: Pending