No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Mutational Spectrum of Korean Patients with Corneal Dystrophy
Abstract
Corneal dystrophy typically refers to a group of rare hereditary disorders with a heterogeneous genetic background. A comprehensive molecular genetic analysis was performed to characterize the genetic spectrum of corneal dystrophies in Korean patients. Patients with various corneal dystrophies underwent thorough ophthalmic examination, histopathologic examination, and Sanger-sequencing. A total of 120 probands were included, with a mean age of 50 years (SD: 18 years) and 70% were female. A total of 26 mutations in 5 genes (14 clearly pathogenic and 12 likely pathogenic) were identified in 49 probands (41%). Epithelial-stromal TGFBI dystrophies, macular corneal dystrophy and Schnyder corneal dystrophy showed 100% mutation detection rates, while endothelial corneal dystrophies showed lower detection rates of 3%. Twenty six non-duplicate mutations including 8 novel mutations were identified and mutations associated with Schnyder corneal dystrophy were identified genetically for the first time in this population. This study provides a comprehensive characterization of the genetic aberrations in Korean patients and also highlights the diagnostic value of molecular genetic analysis in corneal dystrophies.
... Leu450Trp, NP_005193.1:p.Gln455Lys, and NP_005193.1:p. Gln455Val in the early-onset FECD patients has been confirmed by the results of genetic and molecular studies in native specimens and model systems (13,(38)(39)(40)(41)(42). Although there were early-onset cases with no identified variants in the coding exons and the second intron of the COL8A2 gene, the presence of single-nucleotide variants (SNVs), insertions and deletions, or copy number variations (CNVs) in the non-coding exon and the first intron was not excluded (43). ...
... ZEB1 variants in PPCD subtype 3 were extracted from 23 articles (40, 114-118, 120, 130, 132-136, 139-142, 152, 154, 156, 158-160). Fourteen articles provided information on ZEB1 variants in FECD patients (26,40,49,53,55,56,83,116,119,120,151,153,155,157). LOXHD1 variants in FECD patients were analyzed in six articles (24,28,48,55,83,157). AGBL1 variants in FECD were found in the five articles (25, 28,48,55,86). ...
... We extracted and entered the data on 63 unique ZEB1 variants in PPCD3 cases. In one article among 14 tested probands with PPCD3, no ZEB1 variants were identified (40). Summarized types and pathogenicity statuses of reported variants are presented in Table 1. ...
Introduction
The pathogenic role of variants in TCF4 and COL8A2 in causing Fuchs’ endothelial corneal dystrophy (FECD) is not controversial and has been confirmed by numerous studies. The causal role of other genes, SLC4A11, ZEB1, LOXHD1, and AGBL1, which have been reported to be associated with FECD, is more complicated and less obvious. We performed a systematic review of the variants in the above-mentioned genes in FECD cases, taking into account the currently available population frequency information, transcriptomic data, and the results of functional studies to assess their pathogenicity.
Methods
Search for articles published in 2005–2022 was performed manually between July 2022 and February 2023. We searched for original research articles in peer-reviewed journals, written in English. Variants in the genes of interest identified in patients with FECD were extracted for the analysis. We classified each presented variant by pathogenicity status according to the ACMG criteria implemented in the Varsome tool. Diagnosis, segregation data, presence of affected relatives, functional analysis results, and gene expression in the corneal endothelium were taken into account. Data on the expression of genes of interest in the corneal endothelium were extracted from articles in which transcriptome analysis was performed. The identification of at least one variant in a gene classified as pathogenic or significantly associated with FECD was required to confirm the causal role of the gene in FECD.
Results
The analysis included 34 articles with 102 unique ZEB1 variants, 20 articles with 64 SLC4A11 variants, six articles with 26 LOXHD1 variants, and five articles with four AGBL1 variants. Pathogenic status was confirmed for seven SLC4A11 variants found in FECD. No variants in ZEB1, LOXHD1, and AGBL1 genes were classified as pathogenic for FECD. According to the transcriptome data, AGBL1 and LOXHD1 were not expressed in the corneal endothelium. Functional evidence for the association of LOXHD1, and AGBL1 with FECD was conflicting.
Conclusion
Our analysis confirmed the causal role of SLC4A11 variants in the development of FECD. The causal role of ZEB1, LOXHD1, and AGBL1 variants in FECD has not been confirmed. Further evidence from familial cases and functional analysis is needed to confirm their causal roles in FECD.
... Dotąd opisano 27 niesynonimicznych mutacji punktowych występujących w obrębie genu UBIAD1, które powodują występowanie SCD [4,23,32,34]. Do najczęściej rozpoznawanych mutacji należą p.G177E/R, p.N102S i p.L121F [32]. W polskiej populacji potwierdzono także występowanie mutacji p.D112N (Ołdak i wsp., dane nieopublikowane). ...
... Prawdopodobnie jego umiejscowienie jest związane ze stanem i rodzajem komórek, w których prowadzi się detekcję. Dotychczas białko UBIAD1 zlokalizowano w: [10,16,32,33]; czerwone kółka wskazują miejsca występowania wszystkich opisanych dotychczas mutacji zidentyfikowanych u pacjentów z SCD [4,23,32,34]. CA -przewidywane centrum aktywne białka, CRAC -sekwencja konsensusowa rozpoznawania cholesterolu, CXXC -motyw oksydoreduktazy o sekwencji aa: Cys-Xaa-Xaa-Cys, FARM -pierwszy motyw bogaty w reszty kwasu asparaginowego, HRM -motyw regulowany przez hem, RPWS -motyw sekwencji aa: Arg-Pro-Trp-Ser, SI-IV -konserwowane subdomeny (subdomena I, II, III i IV). Wygenerowano w bezpłatnie dostępnym programie TMRPres2D (RefSeq: NP_037451) [44] Zróżnicowane umiejscowienie białka UBIAD1 może być także związane z funkcją pełnioną przez dany typ komórki. ...
UbiA prenyltransferase domain-containing protein 1 (UBIAD1) is a transmembrane enzyme
that plays an essential physiological role in the human body. The most important functions of
the UBIAD1 protein include (i) the synthesis of endogenous vitamin K, (ii) direct and indirect
participation in the cholesterol synthesis pathway and (iii) synthesis of the non-mitochondrial
ubiquinone Q10 (CoQ10), a cellular antioxidant [27, 28, 30]. UBIAD1 is the only protein
in the human body that is known to convert derivatives of the plant form of vitamin K1 to the
endogenous vitamin K2 (MK-4).
Mutations in the UBIAD1 gene and/or dysfunction of the UBIAD1 protein have severe consequences
on cellular metabolism and are causative of many diseases, including Schnyder corneal
dystrophy (SCD). It is an inherited disease that leads to gradual vision loss by the deposition
of lipids in the cornea. The mechanism of the disease remains largely unknown. Importantly,
dysfunction of UBIAD1 was also found in other diseases, such as (i) Parkinson’s disease, (ii)
cardiovascular diseases and (iii) prostate and bladder cancer.
In this paper we attempt to present a wide characterization of UBIAD1 metabolic functions
at the cellular and tissue level. We also show the mechanisms that may lead to pathological
changes observed in SCD. Based on previous research, we demonstrate the complexity of processes
in which the UBIAD1 protein is involved. The aim of this review is to analyze possible
causes and to present a probable explanation for the occurrence of various diseases related
to the dysfunction of UBIAD1.
... Heterozygous patients are usually diagnosed as teenagers or young adults, although earliest can be at the age of 8 [11]. Severity of heterozygote GCD2 can vary among Mini Review: Granular Corneal Dystrophy type 2 the patients [9,10,[21][22][23][24][25][26][27][28][29][30]. Several severe phenotypic forms were found to be caused by different types of compound heterozygous mutation [31]. ...
Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant corneal stromal dystrophy that is caused by p.Arg124His mutation of transforming growth factor beta induced (TGFBI) gene. It is characterized by well demarcated granular shaped opacities in central anterior stroma and as the disease progresses, extrusion of the deposits results in ocular pain due to corneal epithelial erosion. Also, diffuse corneal haze which appears late, causes decrease in visual acuity. The prevalence of GCD2 is high in East Asia including Korea. Homozygous patients show a severe phenotype from an early age, and the heterozygote phenotype varies among patients, depending on several types of compound heterozygous TGFBI mutations. In the initial stage, conservative treatments such as artificial tears, antibiotic eye drops, and bandage contact lenses are used to treat corneal erosion. Different surgical methods are used depending on the depth and extent of the stromal deposits. Phototherapeutic keratectomy (PTK) removes anterior opacities and is advantageous in terms of its applicability and repeatability. For deeper lesions, deep anterior lamellar keratoplasty can be used as the endothelial layer is not always affected. Recurrence following these treatments are reported within a wide range of rates in different studies due to varying definition of recurrence and follow up period. In patients who have undergone corneal laser vision-correction surgeries such as photorefractive keratectomy, LASEK, or LASIK including SMILE surgery, corneal opacity exacerbates rapidly with severe deterioration of visual acuity. Further investigations on new treatments of GCD2 are necessary.
... Severe phenotypic features due to double mutations in TGFBI have been reported [11][12][13][14][15][16][17][18][19][20][21][22][23] . In the current study, we detected ve novel and two known mutations, occurring as a compound heterozygote in TGFBI along with p.Arg124His, which result in a very severe phenotypic variant of GCD2. ...
We investigated the clinical and genetic features of patients with severe phenotype of granular corneal dystrophy type 2 (GCD2) associated with compound heterozygosity in the transforming growth factor-β-induced ( TGFBI ) gene. Patients with severe GCD2 underwent ophthalmic examination (best-corrected visual acuity test, intraocular pressure measurement, slit-lamp examination, and slit-lamp photograph analysis) and direct Sanger sequencing of whole- TGFBI . The patient’s family was tested to determine the pedigrees. Five novel mutations (p.His174Asp, p.Ile247Asn, p.Tyr88Cys, p.Arg257Pro, and p.Tyr468*) and two known mutations (p.Asn544Ser and p.Arg179*) in TGFBI were identified, along with p.Arg124His, in the patients. Trans-phase of TGFBI second mutations was confirmed by pedigree analysis. Multiple, extensive discoid granular, and increased linear deposits were observed in the probands carrying p.Arg124His and other nonsense mutations. Some patients who had undergone phototherapeutic keratectomy experienced rapid recurrence (p.Ile247Asn and p.Asn544Ser); however, the cornea was well-maintained in a patient who underwent deep anterior lamellar keratoplasty (p.Ile247Asn). Thus, compound heterozygosity of TGFBI is associated with the phenotypic variability of TGFBI corneal dystrophies, suggesting that identifying TGFBI second mutations may be vital in patients with extraordinarily severe phenotypes. Our findings indicate the necessity for a more precise observation of genotype-phenotype correlation and additional care when treating TGFBI corneal dystrophies.
... For example, the prevalence of autosomal recessive dystrophies, such as congenital hereditary endothelial corneal dystrophy, is higher in regions where consanguineous marriages occur more commonly, such as in parts of the Middle East and India [27][28][29][30]. The prevalence of autosomal dominantly inherited corneal dystrophies also varies by region, with the prevalence of Fuchs endothelial corneal dystrophy, posterior polymorphous corneal dystrophy and granular corneal dystrophy type 2 reported to be higher in individuals from Northern Europe, Czech Republic and South Korea, respectively [25,[31][32][33]. However, it is likely that the geographic clustering of reports of MECD is due at least in part to the location of centers with more established research programs producing a higher number of publications. ...
Meesmann epithelial corneal dystrophy (MECD) is a rare dominantly inherited disorder that is characterized by corneal epithelial microcysts and is associated with mutations in the keratin 3 (KRT3) and keratin 12 (KRT12) genes. In this study, we report a novel mutation in the KRT12 gene in a Vietnamese pedigree with MECD. Slit-lamp examination was performed on each of the 7 recruited members of a Vietnamese family to identify characteristic features of MECD. After informed consent was obtained from each individual, genomic DNA was isolated from saliva samples and screening of KRT3and KRT12 genes was performed by Sanger sequencing. The proband, a 31-year-old man, complained of a 1-year history of eye irritation and photophobia. Slit-lamp examination revealed intraepithelial microcysts involving only the corneal periphery in each eye with clear central corneas and no stromal or endothelial involvement. Three family members demonstrated similar intraepithelial microcysts, but with diffuse involvement, extended from limbus to limbus. Sanger sequencing of KRT3 (exon 7) and KRT12 (exons 1 and 6) in the proband revealed a novel heterozygous KRT12 variant (c.1273G>A [p.Glu425Lys]) that was present in the three affected family members but was absent in the three family members with clear corneas. This study is the first report of a Vietnamese family affected with MECD, associated with an atypical peripheral corneal epithelial phenotype in the proband and a novel mutation in KRT12.
... Posterior polymorphous corneal dystrophy (PPCD) is a rare, autosomal dominantly inherited, corneal endothelial dystrophy characterized by an abnormal transition of the corneal endothelium into stratified squamous epithelium and corneal edema [2,3]. PPCD is diagnosed based on ophthalmologic findings, but molecular genetic analysis can differentiate various clinically similar CDs [4,5]. Locus heterogeneity has been identified for PPCD with mutations in the COL8A2 (collagen type VIII alpha 2 chain) gene on chromosome 1p34.3 ...
Corneal dystrophies (CDs) are a diverse group of inherited disorders with a heterogeneous genetic background. Here, we report the identification of a novel ZNF143 heterozygous missense mutation in three individuals of the same family with clinical and pathological features that are consistent with endothelial CD. Ophthalmologic examination revealed diffuse corneal clouding and edema with decreased endothelial cell density. Pathological findings showed increased corneal thickness due to edema of basal epithelial cells and stroma, and abnormal metaplastic endothelium with stratified epithelium-like changes. Patients’ metaplastic corneal endothelial cells expressed predominantly cytokerain 7, cytokeratin 19, and E-cadherin. Although Sanger sequencing did not detect any mutation associated with endothelial CDs, whole exome sequencing identified the ZNF143 c.937G>C p.(Asp313His) mutation as a candidate gene for our patients’ endothelial CD. In-vitro functional studies demonstrated that mutant ZNF143 promoted the mesenchymal-to-epithelial transition; it upregulated the expression of genes associated with epithelialization in human corneal endothelial cells. Additionally, proinflammatory cytokine responsive genes were significantly enriched after mutant ZNF143 transfection, which may contribute to the severe phenotype of the three patients. These findings link a mutation in ZNF143 with endothelial CD for the first time.
... To date, 27 non-synonymous point alterations of the UBIAD1 gene causative of SCD have been described [8][9][10][11]. The most frequent pathogenic variants include p.Asn102Ser, p.Gly177Glu/Arg, and p.Leu121Phe. In 2016, p.Thr103Ile, the first de novo UBIAD1 gene pathogenic variant associated with SCD was identified [9]. ...
Purpose:
Schnyder corneal dystrophy (SCD) is a rare inherited disease that leads to gradual vision loss by the deposition of lipids in the corneal stroma. The aim of this study is to report a novel pathogenic variant in the UBIAD1 gene and present clinical and molecular findings in Polish patients with SCD.
Methods:
Individuals (n = 37) originating from four Polish SCD families were subjected for a complete ophthalmological check-up and genetic testing. Corneal changes were visualized by slit-lamp examination, anterior segment optical coherent tomography (AS-OCT), and in vivo confocal microscopy (IVCM).
Results:
In a proband with primarily mild SCD that progressed rapidly at the end of the fifth decade of life, a novel missense pathogenic variant in UBIAD1 (p.Thr120Arg) was identified. The other studied SCD family represents the second family reported worldwide with the UBIAD1 p.Asp112Asn variant. SCD in the remaining two families resulted from a frequently identified p.Asn102Ser pathogenic variant. All affected subjects presented a crystalline form of SCD. The severity of corneal changes was age-dependent, and their morphology and localization are described in detail.
Conclusion:
The novel p.Thr120Arg is the fourth SCD-causing variant lying within the FARM motif of the UBIAD1 protein, which underlines a high importance of this motif for SCD pathogenesis. The current study provides independent evidence for the pathogenic potential of UBIAD1 p.Asp112Asn and new information useful for clinicians.
... So far, ~50 different mutations causing LCD have been identified in the TGFBI gene (www.hgmd.org). The TGFBI mutation spectrum and their clinical consequences have been investigated in patients with LCDI in different ethnic populations (2,7,(9)(10)(11)(12)(13). For example, the p.Arg124Cys mutation is reported to be associated with the LCDI phenotype in Greek, Japanese, Bangladeshi and Chinese patients (14)(15)(16)(17)(18)(19). ...
Lattice corneal dystrophy type I (LCDI) is associated with a large number of missense mutations in the transforming growth factor β induced (TGFBI) gene. The aim of the present study was to analyze TGFBI mutation in a Chinese family with LCDI, and to describe the clinical features and phenotype-genotype correlation within this family. Three generations of this family with LCDI were enrolled in the current study. Complete ophthalmic examinations were performed on all family members and mutation screenings of the coding regions of TGFBI were analyzed using a direct sequencing method. All family members underwent slit-lamp examination, and two patients and one of normal members in the family were evaluated by laser scanning in vivo confocal microscopy. A single heterozygous c.370C>T (p.R124C) mutation was identified in exon 4 of the TGFBI gene in five affected individuals, but not in the other family members and 400 normal control subjects. The affected members exhibited similar clinical features of LCDI, except that patient III:5 presented with mild symptoms. Confocal microscopy in vivo examination demonstrated that the proband (II:2) and his affected niece (III:4) had disruptions in multiple corneal layers, including the basal epithelial cells, stroma cells and Bowman's membrane. Thus, the R124C mutation in the TGFBI gene was identified in a Chinese family with LCDI. These results characterized the clinical features and revealed a genotype-associated phenotype in this family, which may contribute to understanding the pathogenesis of LCDI.
... In 1997, Munier et al [2] identified TGFBI on chromosome 5q31 and discovered 4 different mutations that associated with 4 inherited corneal dystrophies: R555W resulting in the granular dystrophy (GCD), R124H resulting in the Avellino dystrophy (ACD), R124C resulting in the lattice dystrophy type I (LCD-I), R555Q resulting in the Reis-Bücklers dystrophy (RBCD). Subsequently, several additional mutations of TGFBI throughout the world were found to be responsible for diverse corneal dystrophies and a phenotype-genotype correlation had been established [3][4] . LCD is one of common stromal dystrophy which manifests typically as linear, radially oriented, branching opacities in the anterior stroma [5] . ...
Aim:
To report a phenotypic variant pedigree of lattice corneal dystrophy (LCD) associated with two mutations, R124C and A546D, in the transforming growth factor beta-induced 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.
Introduction:
This study aimed to establish which worldwide population cohorts have a genetic make-up closest to that of a large sample of the Maltese population with regard to corneal dystrophy (CD) genes.
Methods:
Single nucleotide polymorphisms (SNPs) in the Maltese cohort were compared with worldwide cohorts. Fixation index (FST) values were calculated to evaluate population differentiation. The genetic prevalence of CD subtypes in worldwide and Maltese cohorts were calculated, and single nucleotide missense mutations present in the Maltese cohort were evaluated for potential pathogenicity.
Results:
FST values showed that CD-related genes differ substantially among the studied cohorts. FST values for each SNP showed greatest differentiation between the Maltese and African cohorts and least differentiation with the Puerto Rican, Mexican, and Colombian cohorts. One TGFBI casual CD mutation, 502V, which causes a Bowman's layer CD/atypical Thiel-Behnke CD was identified in the Maltese cohort. The KRT3 NC_000012.11:g.53186088G>C mutation was potentially deleterious.
Conclusion:
Identifying populations with least genetic differentiation can facilitate and help guide future diagnostic and treatment strategies for Maltese individuals with CDs in the absence of comparable Maltese data. Analysing the previously unknown CD genetic pool present in a large Maltese cohort adds to the global genetic bank that researchers rely on for medical progress.
We investigated the clinical and genetic features of patients with severe phenotype of granular corneal dystrophy type 2 (GCD2) associated with compound heterozygosity in the transforming growth factor-β-induced ( TGFBI ) gene. Patients with severe GCD2 underwent ophthalmic examination (best-corrected visual acuity test, intraocular pressure measurement, slit-lamp examination, and slit-lamp photograph analysis) and direct Sanger sequencing of whole- TGFBI . The patient’s family was tested to determine the pedigrees. Five novel mutations (p.(His174Asp), p.(Ile247Asn), p.(Tyr88Cys), p.(Arg257Pro), and p.(Tyr468*)) and two known mutations (p.(Asn544Ser) and p.(Arg179*)) in TGFBI were identified, along with p.(Arg124His), in the patients. Trans-phase of TGFBI second mutations was confirmed by pedigree analysis. Multiple, extensive discoid granular, and increased linear deposits were observed in the probands carrying p.(Arg124His) and other nonsense mutations. Some patients who had undergone phototherapeutic keratectomy experienced rapid recurrence (p.(Ile247Asn) and p.(Asn544Ser)); however, the cornea was well-maintained in a patient who underwent deep anterior lamellar keratoplasty (p.(Ile247Asn)). Thus, compound heterozygosity of TGFBI is associated with the phenotypic variability of TGFBI corneal dystrophies, suggesting that identifying TGFBI second mutations may be vital in patients with extraordinarily severe phenotypes. Our findings indicate the necessity for a more precise observation of genotype–phenotype correlation and additional care when treating TGFBI corneal dystrophies.
Transforming growth factor-β-induced protein (TGFBIp), an extracellular matrix protein, is the second most abundant protein in the corneal stroma. In this review, we summarize the current knowledge concerning the expression, molecular structure, binding partners, and functions of human TGFBIp. To date, 74 mutations in the transforming growth factor-β-induced gene (TGFBI) are associated with amyloid and amorphous protein deposition in TGFBI-linked corneal dystrophies. We discuss the current understanding of the biochemical mechanisms of TGFBI-linked corneal dystrophies and propose that mutations leading to granular corneal dystrophy (GCD) decrease the solubility of TGFBIp and affect the interactions between TGFBIp and components of the corneal stroma, whereas mutations associated with lattice corneal dystrophy (LCD) lead to a destabilization of the protein that disrupts proteolytic turnover, especially by the serine protease HtrA1. Future research should focus on TGFBIp function in the cornea, confirmation of the biochemical mechanisms in vivo, and the development of disease models. Future therapies for TGFBI-linked corneal dystrophies might include topical agents that regulate protein aggregation or gene therapy that targets the mutant allele by CRISPR/Cas9 technology.
Human Transforming Growth Factor β‐induced (TGFBI), is a gene responsible for various corneal dystrophies. TGFBI produces a protein called TGFBI which is involved in cell adhesion and serves as a recognition sequence for integrins. An alteration in cell surface interactions could be the underlying cause for the progressive accumulation of extracellular deposits in different layers of the cornea with the resulting changes of refractive index and transparency. To this date 69 different pathogenic or likely pathogenic variants in TGFBI have been identified in heterozygous or homozygous state in various corneal dystrophies, including a novel variant reported here. All disease‐associated variants were inherited as autosomal dominant traits but one; this latter was inherited as an autosomal recessive trait (Afshari et al., 2008). Most corneal dystrophy‐associated variants are located at amino acids Arg124 and Arg555. In order to keep the list of corneal dystrophy‐associated variant current, we generated a locus‐specific database for TGFBI (http://databases.lovd.nl/shared/variants/TGFBI) containing all pathogenic and likely pathogenic variants reported so far. Non disease associated variants are described in specific databases, like gnomAD and ExAC and are not listed here. This article presents the most recent up‐to‐date list of disease‐associated variants.
This article is protected by copyright. All rights reserved.
Fuchs’ endothelial corneal dystrophy (FECD) is a common disease resulting from corneal endothelial cell dysfunction. It is inherited in an autosomal dominant fashion with incomplete penetrance, and with a female bias. Approximately half of cases occur sporadically, and the remainder are familial. Early and late-onset forms of the disease exist. A review of the literature has revealed more than 15 genes harbouring mutations and/or single nucleotide polymorphisms associated with FECD. The proteins encoded by these genes cover a wide range of endothelial function, including transcription regulation, DNA repair, mitochondrial DNA mutations, targeting of proteins to the cell membrane, deglutamylation of proteins, extracellular matrix secretion, formation of cell–cell and cell–extracellular matrix junctions, water pump, and apoptosis. These genetic variations will form the platform for the further understanding of the pathological basis of the disease, and the development of targeted treatments. This review aims to summarise known genetic variations associated with FECD, discuss any known molecular effects of the variations, how these provide opportunities for targeted therapies, and what therapies are currently in development.
A major cause of visual impairment, corneal dystrophies result from accumulation of protein deposits in the cornea. One of the proteins involved is transforming growth factor β-induced protein (TGFBIp), an extracellular matrix component that interacts with integrins but also produces corneal deposits when mutated. Human TGFBIp is a multi-domain 683-residue protein, which contains one CROPT domain and four FAS1 domains. Its structure spans ∼120 Å and reveals that vicinal domains FAS1-1/FAS1-2 and FAS1-3/FAS1-4 tightly interact in an equivalent manner. The FAS1 domains are sandwiches of two orthogonal four-stranded β sheets decorated with two three-helix insertions. The N-terminal FAS1 dimer forms a compact moiety with the structurally novel CROPT domain, which is a five-stranded all-β cysteine-knot solely found in TGFBIp and periostin. The overall TGFBIp architecture discloses regions for integrin binding and that most dystrophic mutations cluster at both molecule ends, within domains FAS1-1 and FAS1-4.
Purpose
To identify the underlying genetic defect in Korean patients with macular corneal dystrophy (MCD).
Methods
Genomic DNA was isolated from peripheral blood leukocytes of seven patients from six unrelated families with MCD (three men and four women). Polymerase chain reaction was performed for coding regions of the carbohydrate sulfotransferase (CHST6), gene followed by bidirectional sequencing. Targeted mutational analysis (exons 4, 11–12, 14) of the transforming growth factor, beta-induced (TGFBI) gene was performed for all patients.
Results
All seven patients were found to have compound heterozygous mutations in the CHST6 gene. In addition to six previously reported mutations, c.95C>A (p.Ser32*), c.521A>G (p.Lys174Arg), c.557C>G (p.Pro186Arg), c.613C>T (p.Arg205Trp), c.820G>A (p.Glu274Lys), and c.1072T>C (p.Tyr358His), three novel mutations were identified in this study, including two missense mutations, c.353C>T (p.Ser118Phe) and c.922C>T (p.His308Tyr), and one frameshift mutation, c.786delC (p.L264Cfs*117). Among the three novel mutations, only the c.353C>T mutation had been reported in the Exon Aggregation Consortium database at an extremely low frequency of 0.00005072. In addition, these three novel mutations were absent from controls in 1,000 genomes, dbSNP, and the TIARA genome database, which is a Korean personal genome database. The most frequent mutation was c.613C>T (p.Arg205Trp), revealed in four unrelated Korean families, which has not previously been reported in other populations. No mutations were detected in the TGFBI gene.
Discussion
This is the first report on genetic analysis of Korean MCD patients. Three novel and six previously reported disease-causing CHST6 mutations were identified, which expands the mutational spectrum of MCD.
A substantial proportion of patients with posterior polymorphous corneal dystrophy (PPCD) lack a molecular diagnosis. We evaluated 14 unrelated probands who had a clinical diagnosis of PPCD who were previously determined to be negative for mutations in ZEB1 by direct sequencing. A combination of techniques was used including whole-exome sequencing (WES), single-nucleotide polymorphism (SNP) array copy number variation (CNV) analysis, quantitative real-time PCR, and long-range PCR. Segregation of potentially pathogenic changes with disease was confirmed, where possible, in family members. A putative run of homozygosity on chromosome 10 was identified by WES in a three-generation PPCD family, suggestive of a heterozygous deletion. SNP array genotyping followed by long-range PCR and direct sequencing to define the breakpoints confirmed the presence of a large deletion that encompassed multiple genes, including ZEB1. Identification of a heterozygous deletion spanning ZEB1 prompted us to further investigate potential CNVs at this locus in the remaining probands, leading to detection of two additional heterozygous ZEB1 gene deletions. This study demonstrates that ZEB1 mutations account for a larger proportion of PPCD than previously estimated, and supports the hypothesis that haploinsufficiency of ZEB1 is the underlying molecular mechanism of disease for PPCD3.European Journal of Human Genetics advance online publication, 28 October 2015; doi:10.1038/ejhg.2015.232.
Mutations in the transforming growth factor β-induced gene (TGFBI) are major causes of genetic corneal dystrophies (CDs), which can be grouped into TGFBI CDs. Although a few studies have reported the clinical and genetic features of Korean patients with TGFBI CD, no data are available regarding the frequency and spectrum of TGFBI mutations in a consecutive series of Korean patients with clinically diagnosed CDs.
Patients with any type of CD, who underwent both ophthalmologic examination and TGFBI gene analysis by Sanger sequencing at a tertiary care hospital in Seoul, Korea from 2006 to 2013, were enrolled in this study.
Among a total of 89 patients, 77 (86.5%) were diagnosed as having clinical TGFBI CD. Seventy-three out of 74 patients (98.6%) with granular CD type 2 (GCD2), had the p.R124H mutation. Of particular note, one patient with rapidly progressive CD had the p.R124H mutation as well as a novel nonsense variant with unknown clinical significance (p.A179*). In three patients with lattice CD type 1 (LCD1), one known mutation (p.R124C) and two novel variants (p.L569Q and p.T621P) in the TGFBI gene were identified.
This study provides epidemiological insight into CDs in a Korean population and reaffirms that GCD2 is the most common TGFBI CD phenotype and that p.R124H is the only mutation identified in patients with GCD2. In addition, we broaden the spectrum of TGFBI mutations by identifying two novel missense variants in patients with LCD1.
An 87-year-old woman visited our clinic for a scheduled cataract surgery. At the time of preoperative evaluation, slit lamp examination showed lattice-shaped and granular deposits with asymmetrical patterns in the stroma of both corneas. Genomic DNA samples of the patient, amplified by polymerase chain reaction, showed a single nucleotide substitution, c. 1580T>G (p.L527R), in the transforming growth factor-β-induced TGFBI gene. We also found two additional SNP mutations, c.1620T>C (p.F540F) and c.1678+23G>A, along with the well-known L527R mutation. This is the first report of lattice corneal dystrophy type IV with an L527R mutation outside of Japan, and could challenge the idea that L527R is caused by a mutation from a single Japanese ancestor.
To report a novel mutation within the CHST6 gene, as well as describe light and electron microscopic features of a case of macular corneal dystrophy. A 59-year old woman with macular corneal dystrophy in both eyes who had decreased visual acuity underwent penetrating keratoplasty. Further studies including light and electron microscopy, as well as DNA analysis were performed. Light microscopy of the cornea revealed glycosaminoglycan deposits in the keratocytes and endothelial cells, as well as extracellularly within the stroma. All samples stained positively with alcian blue, colloidal iron, and periodic acid-Schiff. Electron microscopy showed keratocytes distended by membrane-bound intracytoplasmic vacuoles containing electron-dense fibrillogranular material. These vacuoles were present in the endothelial cells and between stromal lamellae. Some of the vacuoles contained dense osmophilic whorls. A novel homozygous mutation (c.613 C>T [p.Arg205Trp]) was identified within the whole coding region of CHST6. A novel CHST6 mutation was detected in a Korean macular corneal dystrophy patient.
The Total Integrated Archive of short-Read and Array (TIARA; http://tiara.gmi.ac.kr) database stores and integrates human genome data generated from multiple technologies including next-generation sequencing
and high-resolution comparative genomic hybridization array. The TIARA genome browser is a powerful tool for the analysis
of personal genomic information by exploring genomic variants such as SNPs, indels and structural variants simultaneously.
As of September 2012, the TIARA database provides raw data and variant information for 13 sequenced whole genomes, 16 sequenced
transcriptomes and 33 high resolution array assays. Sequencing reads are available at a depth of ∼30× for whole genomes and
50× for transcriptomes. Information on genomic variants includes a total of ∼9.56 million SNPs, 23 025 of which are non-synonymous
SNPs, and ∼1.19 million indels. In this update, by adding high coverage sequencing of additional human individuals, the TIARA
genome database now provides an extensive record of rare variants in humans. Following TIARA’s fundamentally integrative approach,
new transcriptome sequencing data are matched with whole-genome sequencing data in the genome browser. Users can here observe,
for example, the expression levels of human genes with allele-specific quantification. Improvements to the TIARA genome browser
include the intuitive display of new complex and large-scale data sets.
Schnyder corneal dystrophy (SCD) is an autosomal dominant disease characterized by germline variants in UBIAD1 introducing missense alterations leading to deposition of cholesterol in the cornea, progressive opacification, and loss of visual acuity. UBIAD1 was recently shown to synthesize menaquinone-4 (MK-4, vitamin K(2) , but causal mechanisms of SCD are unknown. We report a novel c.864G>A UBIAD1 mutation altering glycine 177 to glutamic acid (p.G177E) in six SCD families, including four families from Finland who share a likely founder mutation. We observed reduced MK-4 synthesis by UBIAD1 altered by SCD mutations p.N102S, p.G177R/E, and p.D112N, and molecular models showed p.G177-mutant UBIAD1 disrupted transmembrane helices and active site residues. We show UBIAD1 interacts with HMGCR and SOAT1, enzymes catalyzing cholesterol synthesis and storage, respectively, using yeast two-hybrid screening and immunoprecipitation. Docking simulations indicate cholesterol binds to UBIAD1 in the substrate binding cleft and binding overlaps with GGPP binding, a MK-4 substrate, suggesting potential competition between these metabolites. Impaired MK-4 synthesis is a biochemical defect identified in SCD suggesting UBIAD1 links vitamin K and cholesterol metabolism through physical contact between enzymes and metabolites. Our data suggests a role for endogenous MK-4 in maintaining cornea health and visual acuity.
Macular corneal dystrophy (MCD; MIM 217800) is an autosomal recessive hereditary disease in which progressive punctate opacities in the cornea result in bilateral loss of vision, eventually necessitating corneal transplantation. MCD is classified into two subtypes, type I and type II, defined by the respective absence and presence of sulphated keratan sulphate in the patient serum, although both types have clinically indistinguishable phenotypes1,
2. The gene responsible for MCD type I has been mapped to chromosome 16q22, and that responsible for MCD type II may involve the same locus3,
4,
5. Here we identify a new carbohydrate sulphotransferase gene (CHST6), encoding an enzyme designated corneal N-acetylglucosamine-6-sulphotransferase (C-GlcNAc6ST), within the critical region of MCD type I. In MCD type I, we identified several mutations that may lead to inactivation of C-GlcNAc6ST within the coding region of CHST6. In MCD type II, we found large deletions and/or replacements caused by homologous recombination in the upstream region of CHST6. In situ hybridization analysis did not detect CHST6 transcripts in corneal epithelium in an MCD type II patient, suggesting that the mutations found in type II lead to loss of cornea-specific expression of CHST6.
To investigate the clinical and genetic features of Korean patients with corneal dystrophies associated with mutations in the human transforming growth factor-β-induced (TGFBI) gene.
In this study, 387 subjects (71 families and 89 individuals - 268 patients having TGFBI corneal dystrophies and 119 normal relatives) were assessed. All subjects underwent a complete ophthalmologic evaluation, including biomicroscopic inspection and dilated fundus examination. As a control, 100 individuals without corneal disease were selected from the general population. The polymerase chain reaction (PCR) and direct sequencing were used to screen for mutations in TGFBI.
All subjects recruited exhibited a range of corneal dystrophies, including Thiel-Behnke corneal dystrophy (TBCD, R555Q; 6 families and 4 individuals), granular corneal dystrophy type 2 (GCD2, R124H; 61 families and 80 individuals), lattice corneal dystrophy (LCD; 4 families and 5 individuals; 7 with type 1 [R124C], and 2 with a variant [L527R, P542R]). The disease showed an autosomal dominant inheritance pattern in all families.
R124H in GCD2 was the most common mutation. GCD1 and Reis-Bucklers corneal dystrophy were not found. In the GCD2 patients there were a large number of laser refractive surgery-induced corneal opacities. A spontaneous R124H mutation was confirmed in an already mutated allele that resulted in a change from a heterozygous into a homozygous form. Also, a novel mutation, P527R, was identified in LCD.
To estimate the prevalence of corneal dystrophies.
Records of almost 8 million enrollees in a national managed-care network throughout the United States who had an eye care visit in 2001 to 2009 were searched for a recording of corneal dystrophy on a claim submitted by an ophthalmologist or optometrist from January 1, 2001, through December 31, 2007.
Unique individuals (n = 27,372) received two or more diagnoses of any type of corneal dystrophy, for an overall corneal dystrophy prevalence rate of 897 per million (10⁶) covered lives. Endothelial and anterior corneal dystrophies accounted for most of the reported dystrophies, and granular corneal dystrophy was the least common, being reported in 167 enrollees. Age, sex, and race variations among the various corneal dystrophies were observed. The mean age of those with macular corneal dystrophy (47.3 years) was 15 years younger than the age of those with endothelial dystrophy (62.9 years), and females were most highly represented (68.5%) among those with lattice corneal dystrophy. Hispanics and blacks were underrepresented relative to enrollees undergoing eye care for reasons other than corneal dystrophy. Keratoplasty was most frequently coded among those with lattice dystrophy.
Although caveats must be considered in using claims data to estimate prevalence in a population, these data provide an indication of corneal dystrophy's prevalence within insured subjects across the United States. Variations in age, sex, and race, within and between the different types of corneal dystrophies, raise questions that warrant further study.
Investigate the genotype-phenotype correlations for five TGFBI (transforming growth factor, beta-induced) mutations including one novel pathogenic variant and one complex allele affecting the fourth FAS1 domain of keratoepithelin, and their potential effects on the protein's structure.
Three unrelated families were clinically diagnosed with lattice corneal dystrophy (CD) and one with an unclassified CD of Bowman's layer. Mutations in the TGFBI gene were detected by direct sequencing, and the functional impact of each variant was predicted using in silico algorithms. Corneal phenotypes, including histological examinations, were compared with the literature data. Furthermore, molecular modeling studies of these mutations were performed.
Two distinct missense mutations affecting the same residue at position 509 of keratoepithelin: p.Leu509Pro (c.1526T>C) and p.Leu509Arg (c.1526T>G) were found to be associated with a lattice-type CD. The novel p.Val613Gly (c.1828T>G) TGFBI mutation was found in a sporadic case of an Algerian individual affected by lattice CD. Finally, the Bowman's layer CD was linked to the association in cis of the p.Met502Val and p.Arg555Gln variants, leading to the reclassification of this CD as atypical Thiel-Behnke CD. Structural modeling of these TGFBI mutations argues in favor of these mutations being responsible for instability and/or incorrect folding of keratoepithelin, predictions that are compatible with the clinical diagnoses.
Description of a novel TGFBI mutation and a complex TGFBI allele further extends the mutational spectrum of TGFBI. Moreover, we show convincing evidence that TGFBI mutations affecting Leu509 are linked to the lattice phenotype in two unrelated French families, contrasting with findings previously reported. The p.Leu509Pro was reported to be associated with both amyloid and non-amyloid aggregates, whereas p.Leu509Arg has been described as being responsible for Epithelial Basement Membrane Dystrophy (EBMD).
Posterior Polymorphous Dystrophy (PPCD) is a genetically heterogeneous corneal dystrophy, with linkage to three different chromosomal loci, with several genes in these loci being implicated. The role of both VSX1 and COL8A2 in PPCD remains controversial but recent work suggests that mutations in the transcription factor gene ZEB1/TCF8 account for disease in up to 30% of subjects, with a significant association with connective tissue abnormalities. This study aimed to determine the phenotype and contribution of ZEB1 mutations in a New Zealand PPCD population
Following informed consent, 11 probands with PPCD underwent extensive clinical characterization; including a questionnaire to determine birth history, general health, and the incidence of connective tissue abnormalities, slit lamp examination, photography and in vivo confocal microscopy. Family members were recruited where available. Biological specimens underwent mutational analysis of all nine coding exons of ZEB1.
ZEB1 mutational analysis identified one mutation in the 11 probands (9.1%), a novel mutation in the initiating methionine of exon 1, c.1A-->G that results in the protein change p.Met1Val, with resultant aberrant initiation of translation. This mutation segregated with disease in the family, and was not present in 100 control chromosomes. No other ZEB1 mutations were observed in this cohort.
Recent studies suggest that ZEB1 mutations may account for PPCD in 18 to 30% of cases, with the majority of the mutations in exons 5 and 7. Clinical and molecular analyses in this New Zealand cohort show a much lower incidence of ZEB1 sequence change, confirming the genetic heterogeneity of PPCD. We also report identification of a novel mutation in the initiating methionine that removes the Kozak sequence, thereby altering the site of initiation translation.
Corneal clarity is maintained by its endothelium, which functions abnormally in the endothelial dystrophies, leading to corneal opacification. This group of conditions includes Fuchs’ endothelial dystrophy of the cornea (FECD), one of the commonest indications for corneal transplantation performed in developed countries, posterior polymorphous dystrophy (PPCD) and the congenital hereditary endothelial dystrophies (CHED). A genome-wide search of a three-generation family with early-onset FECD demonstrated significant linkage with D1S2830 ( Z max = 3.72, &thgr; = 0.0). Refinement of the critical region defined a 6–7 cM interval of chromosome 1p34.3–p32 within which lies the COL8A2 gene. This encodes the 703 amino acid α2 chain of type VIII collagen, a short-chain collagen which is a component of endothelial basement membranes and which represented a strong candidate gene. Analysis of its coding sequence defined a missense mutation (gln455lys) within the triple helical domain of the protein in this family. Mutation analysis in patients with FECD and PPCD demonstrated further missense substitutions in familial and sporadic cases of FECD as well as in a single family with PPCD. This is the first description of the molecular basis of any of the corneal endothelial dystrophies or of mutations in type VIII collagen in association with human disease. This suggests that the underlying pathogenesis of FECD and PPCD may be related to disturbance of the role of type VIII collagen in influencing the terminal differentiation of the neural crest derived corneal endothelial cell.
Harboyan syndrome is a degenerative corneal disorder defined as congenital hereditary endothelial dystrophy (CHED) accompanied by progressive, postlingual sensorineural hearing loss. To date, 24 cases from 11 families of various origin (Asian Indian, South American Indian, Sephardi Jewish, Brazilian Portuguese, Dutch, Gypsy, Moroccan, Dominican) have been reported. More than 50% of the reported cases have been associated with parental consanguinity. The ocular manifestations in Harboyan syndrome include diffuse bilateral corneal edema occurring with severe corneal clouding, blurred vision, visual loss and nystagmus. They are apparent at birth or within the neonatal period and are indistinguishable from those characteristic of the autosomal recessive CHED (CHED2). Hearing deficit in Harboyan is slowly progressive and typically found in patients 10-25 years old. There are no reported cases with prelinglual deafness, however, a significant hearing loss in children as young as 4 years old has been detected by audiometry, suggesting that hearing may be affected earlier, even at birth. Harboyan syndrome is caused by mutations in the SLC4A11 gene located at the CHED2 locus on chromosome 20p13-p12, indicating that CHED2 and Harboyan syndrome are allelic disorders. A total of 62 different SLC4A11 mutations have been reported in 98 families (92 CHED2 and 6 Harboyan). All reported cases have been consistent with autosomal recessive transmission. Diagnosis is based on clinical criteria, detailed ophthalmological assessment and audiometry. A molecular confirmation of the clinical diagnosis is feasible. A variety of genetic, metabolic, developmental and acquired diseases presenting with clouding of the cornea should be considered in the differential diagnosis (Peters anomaly, sclerocornea, limbal dermoids, congenital glaucoma). Audiometry must be performed to differentiate Harboyan syndrome from CHED2. Autosomal recessive types of CHED (CHED2 and Harboyan syndrome) should carefully be distinguished from the less severe autosomal dominant type CHED1. The ocular abnormalities in patients with Harboyan syndrome may be treated with topical hyperosmolar solutions. However, corneal transplantation (penetrating keratoplasty) represents definitive treatment. Corneal transplantation produces a substantial visual gain and has a relatively good surgical prognosis. Audiometric monitoring should be offered to all patients with CHED2. Hearing aids may be necessary in adolescence.
A major interest in human genetics is to determine whether a nonsynonymous single-base nucleotide polymorphism (nsSNP) in a gene affects its protein product and, consequently, impacts the carrier's health. We used the SIFT (Sorting Intolerant From Tolerant) program to predict that 25% of 3084 nsSNPs from dbSNP, a public SNP database, would affect protein function. Some of the nsSNPs predicted to affect function were variants known to be associated with disease. Others were artifacts of SNP discovery. Two reports have indicated that there are thousands of damaging nsSNPs in an individual's human genome; we find the number is likely to be much lower.
Genetic testing for hereditary cancer syndromes contributes to the medical management of patients who may be at increased risk of one or more cancers. BRCA1 and BRCA2 testing for hereditary breast and ovarian cancer is one such widely used test. However, clinical testing methods with high sensitivity for deleterious mutations in these genes also detect many unclassified variants, primarily missense substitutions.
We developed an extension of the Grantham difference, called A-GVGD, to score missense substitutions against the range of variation present at their position in a multiple sequence alignment. Combining two methods, co-occurrence of unclassified variants with clearly deleterious mutations and A-GVGD, we analysed most of the missense substitutions observed in BRCA1.
A-GVGD was able to resolve known neutral and deleterious missense substitutions into distinct sets. Additionally, eight previously unclassified BRCA1 missense substitutions observed in trans with one or more deleterious mutations, and within the cross-species range of variation observed at their position in the protein, are now classified as neutral.
The methods combined here can classify as neutral about 50% of missense substitutions that have been observed with two or more clearly deleterious mutations. Furthermore, odds ratios estimated for sets of substitutions grouped by A-GVGD scores are consistent with the hypothesis that most unclassified substitutions that are within the cross-species range of variation at their position in BRCA1 are also neutral. For most of these, clinical reclassification will require integrated application of other methods such as pooled family histories, segregation analysis, or validated functional assay.
Prediction of the biological effect of missense substitutions has become important because they are often observed in known or candidate disease susceptibility genes. In this paper, we carried out a 3-step analysis of 1514 missense substitutions in the DNA-binding domain (DBD) of TP53, the most frequently mutated gene in human cancers. First, we calculated two types of conservation scores based on a TP53 multiple sequence alignment (MSA) for each substitution: (i) Grantham Variation (GV), which measures the degree of biochemical variation among amino acids found at a given position in the MSA; (ii) Grantham Deviation (GD), which reflects the 'biochemical distance' of the mutant amino acid from the observed amino acid at a particular position (given by GV). Second, we used a method that combines GV and GD scores, Align-GVGD, to predict the transactivation activity of each missense substitution. We compared our predictions against experimentally measured transactivation activity (yeast assays) to evaluate their accuracy. Finally, the prediction results were compared with those obtained by the program Sorting Intolerant from Tolerant (SIFT) and Dayhoff's classification. Our predictions yielded high prediction accuracy for mutants showing a loss of transactivation ( approximately 88% specificity) with lower prediction accuracy for mutants with transactivation similar to that of the wild-type (67.9 to 71.2% sensitivity). Align-GVGD results were comparable to SIFT (88.3 to 90.6% and 67.4 to 70.3% specificity and sensitivity, respectively) and outperformed Dayhoff's classification (80 and 40.9% specificity and sensitivity, respectively). These results further demonstrate the utility of the Align-GVGD method, which was previously applied to BRCA1. Align-GVGD is available online at http://agvgd.iarc.fr.
The lattice corneal dystrophies (LCD) and granular corneal dystrophies (GCD) are autosomal dominant disorders of the corneal stroma. They are bilateral, progressive conditions characterized by the formation of opacities arising due to the deposition of insoluble material in the corneal stroma leading to visual impairment. The LCDs and GCDs are distinguished from each other and are divided into subtypes on the basis of the clinical appearance of the opacities, clinical features of the disease, and on histopathological staining properties of the deposits. The GCDs and most types of LCD arise from mutations in the transforming growth factor beta-induced (TGFBI) gene on chromosome 5q31. Over 30 mutations causing LCD and GCD have been identified so far in the TGFBI. There are two mutation hotspots corresponding to arginine residues at positions 124 and 555 of the transforming growth factor beta induced protein (TGFBIp) and they are the most frequent sites of mutation in various populations. Mutations at either of these two hotspots result in specific types of LCD or GCD. The majority of identified mutations involve residues in the fourth fasciclin-like domain of TGFBIp.
The autosomal recessive form of congenital hereditary endothelial dystrophy (CHED2) is a rare eye disorder caused by mutations in the SLC4A11 gene located at the CHED2 locus on chromosome 20p13-p12. The purpose of this study was to carry out genetic analysis of CHED2 in two Indian families.
Blood samples were collected from individuals for genomic DNA isolation. In order to see if these families had mutations in the SLC4A11 gene, we selected 11 microsatellite markers from the CHED2 candidate region and used them to genotype the families. DNA sequence analysis was used for mutation detection. Allele-specific PCR was used to determine the segregation of mutations in families and also to determine if the mutations were present in 100 ethnically matched normal control chromosomes.
Haplotype analysis suggested linkage of the disorder to the CHED2 locus in both families. DNA sequence analysis showed a novel indel mutation, c.859_862delGAGAinsCCT (E287fsX21) in exon 8 of the SLC4A11 gene in one family. This mutation is predicted to truncate the protein with a lack of all 14 transmembrane domains. DNA sequence analysis of the second family showed a novel in-frame deletion mutation c.2014_2016delTTC or 2017_2019delTTC which will lead to the loss of a phenylalanine residue at position 672 or 673 (F672del or F673del). The mutant protein is expected to lack a conserved phenylalanine residue in transmembrane domain number 8.
This study reports two novel mutations in two CHED2 families and increases the spectrum of mutations in SLC4A11 to a total of 16. PCR-based screening methods were developed for both mutations for rapid screening of individuals.
A summary of normal and abnormal endothelial structure and function is presented. Endothelium originates from neural crest and it elaborates a banded basement membrane in utero. It is involved in mesenchymal dysgenesis of the anterior segment, like the central defect of Peters' anomaly. Cytoplasmic organelles include mitochondria that provide energy for the metabolic pump, rough endoplasmic reticulum that participate in secretion of extracellular matrix, and a terminal web that may participate in cell migration. The endothelium's main function is to control corneal hydration and nutrition with a leaky barrier formed by the apical gap and macula occludens junctions that keep some water out of the stroma but allow nutrients to pass, and with an ATPase-dependent metabolic pump that is located in the lateral plasma membranes. Endothelial wound healing involves flattening and enlargement of cells to maintain an intact monolayer as well as production of abnormal collagenous material posterior to Descemet's membrane. HLA antigens located in the plasma membrane may participate in corneal endothelial graft rejection. Clinical assessment of the endothelium involves three modalities: specular microscopy to study endothelial morphology, fluorophotometry to measure barrier function, and pachymetry to measure corneal thickness.
To update the 2008 International Classification of Corneal Dystrophies (IC3D) incorporating new clinical, histopathologic, and genetic information.
The IC3D reviewed worldwide peer-reviewed articles for new information on corneal dystrophies published between 2008 and 2014. Using this information, corneal dystrophy templates and anatomic classification were updated. New clinical, histopathologic, and confocal photographs were added.
On the basis of revisiting the cellular origin of corneal dystrophy, a modified anatomic classification is proposed consisting of (1) epithelial and subepithelial dystrophies, (2) epithelial-stromal TGFBI dystrophies, (3) stromal dystrophies, and (4) endothelial dystrophies. Most of the dystrophy templates are updated. The entity "Epithelial recurrent erosion dystrophies" actually includes a number of potentially distinct epithelial dystrophies (Franceschetti corneal dystrophy, Dystrophia Smolandiensis, and Dystrophia Helsinglandica) but must be differentiated from dystrophies such as TGFBI-induced dystrophies, which are also often associated with recurrent epithelial erosions. The chromosome locus of Thiel-Behnke corneal dystrophy is only located on 5q31. The entity previously designated as a variant of Thiel-Behnke corneal dystrophy on chromosome 10q24 may represent a novel corneal dystrophy. Congenital hereditary endothelial dystrophy (CHED, formerly CHED2) is most likely only an autosomal recessive disorder. The so-called autosomal dominant inherited CHED (formerly CHED1) is insufficiently distinct to continue to be considered a unique corneal dystrophy. On review of almost all of the published cases, the description appeared most similar to a type of posterior polymorphous corneal dystrophy linked to the same chromosome 20 locus (PPCD1). Confocal microscopy also has emerged as a helpful tool to reveal in vivo features of several corneal dystrophies that previously required histopathologic examination to definitively diagnose.
This revision of the IC3D classification includes an updated anatomic classification of corneal dystrophies more accurately classifying TGFBI dystrophies that affect multiple layers rather than are confined to one corneal layer. Typical histopathologic and confocal images have been added to the corneal dystrophy templates.
Purpose:
The aim of this study was to report a lattice corneal dystrophy (LCD) family with a novel mutation of A620P in the TGFBI gene, its long-term treatment, follow-up data, and related pathologic findings.
Methods:
A total of 28 family members were clinically examined, and blood samples or buccal epithelial cells were taken for DNA analysis. All exons from the entire TGFBI gene coding region were analyzed for mutations in 3 affected members. Exon 14 was amplified in other family members and in 100 normal Korean persons as control. Corneal tissues from 1 affected family member were examined using light and electron microscopy.
Results:
Clinical examination revealed relatively late-onset LCD with asymmetric progression and recurrent corneal erosion. The affected family members have been treated with penetrating keratoplasty, deep lamellar keratoplasty, and phototherapeutic keratectomy for up to 19 years. Screening of the TGFBI gene revealed a novel A620P mutation, which was found in all affected members. The amyloid origin of deposits was confirmed by Congo red and was also partially stained with Masson trichrome. Although there were no electron-dense bodies as in granular dystrophy, transmission electron microscopy demonstrated that the stromal deposits were not homogenous and contained a variety of constituents with different electron densities.
Conclusions:
We present the characteristics and surgical treatment of corneas with a novel A620P mutation in TGFBI showing LCD type IIIA with hyaline component.
The IC3D group, an international group of corneal specialists and ophthalmic pathologists, formed a few years ago to review known corneal dystrophies. Based on personal experience and literature studies the new IC3D classification of corneal dystrophies was published in December 2008 as a freely available supplement to the journal Cornea. The classification suggests new names for some dystrophies, suggests definitions when an entity could be called a dystrophy, and gives examples of typical slit lamp images. It is built in a way to accommodate new and variant dystrophies found in the future. The purpose of this symposium is to draw attention to this new classification with special emphasis on recent variant dystrophies associated with the TBFBI gene. The IC3D group urges authors and reviewers alike to require stringent criteria to be met before publication of a dystrophy as a new entity.
To report a case of a unilateral variant of late-onset lattice corneal dystrophy (LCD) with the Pro501Thr mutation in the TGFBI gene with unilaterality confirmed by confocal microscopy.
Case report. A 58-year-old man presented with visual impairment in his left eye of 20-year duration. Clinical examinations, molecular genetic analysis, and laser confocal microscopic analysis were performed.
The slit-lamp examination showed thick branching lattice lines in the left corneal stroma with radial orientation. The right eye was asymptomatic and appeared normal without deposition or opacification. Laser confocal microscopy showed that highly reflective deposits or lattice-shaped materials in the left cornea were not present in the right cornea, confirming the complete unilaterality of this disease. Molecular genetic analysis confirmed the Pro501Thr mutation in the TGFBI gene previously associated with LCD type IIIA. No other mutations were found in this gene. No mutations in the lactoferrin gene previously associated with secondary amyloidosis were identified.
The authors present a unilateral variant of late-onset LCD with a heterozygous Pro501Thr mutation in the TGFBI gene. No corneal deposits seen in the affected cornea were found in the unaffected cornea even by laser confocal microscopic analysis.
Importance:
Posterior polymorphous corneal dystrophy (PPCD) is a very rare disorder characterized by primary changes of the posterior corneal layers. Sequence variants in 3 genes are associated with the development of PPCD, including ZEB1 that is responsible for PPCD3. Evidence suggests at least 1 more gene remains to be identified.
Objective:
To determine the molecular genetic cause of PPCD3.
Design:
We performed extensive ophthalmological examination, including rotating Scheimpflug imaging technology and specular microscopy, and direct sequencing of the ZEB1 coding region. Comprehensive review of published PPCD3-causing variants was undertaken.
Setting:
Ophthalmology department of a university hospital.
Participants:
Four Czech probands.
Main outcomes and measures:
Results of ophthalmological examination and direct sequencing of the ZEB1 coding region.
Results:
The following 2 novel frameshift mutations within ZEB1 were identified: c.2617dup in exon 8 in a 22-year-old woman, considered to be most likely de novo in origin, and c.698dup in exon 6 in a 20-year-old man. The first patient had mild changes consistent with PPCD and bilateral best-corrected visual acuity of 1.00. The corneal phenotype of the patient in the second case was more severe, with best-corrected visual acuity of 0.40 OD and 0.05 OS. Corneas of both probands were abnormally steep (keratometry readings, flat ≥ 47.4 diopters [D] and steep ≥ 49.2 D) with increased pachymetry values but no pattern indicative of keratoconus. Specular microscopy in both patients revealed reduced endothelial cell density (range, 1055/mm² to 1655/mm²). Both probands had a history of surgery for inguinal hernia; the male patient also reported hydrocele.
Conclusions and relevance:
Nucleotide changes within the coding region of ZEB1 underlie the pathogenesis of PPCD in 4 of 23 Czech probands (17%). The cumulative de novo ZEB1 mutation rate is at least 14%. Possible involvement of ZEB1 sequence variants not readily identified by direct sequencing of coding regions needs to be further investigated. Our findings also have implications for patient counseling.
To describe mutations in the transforming growth factor-beta induced (TGFBI) gene in Asian patients with Bowman's membrane as well as stromal corneal dystrophies, and to elucidate their structural implications, using model peptides.
Twenty-two unrelated Asian families were examined clinically including visual acuity testing and ocular examination with slit lamp biomicroscopy. Genomic DNA was extracted and the 17 exons of the TGFBI gene were amplified by PCR and sequenced bi-directionally. Biophysical techniques were used to characterise the wild type and mutant model peptides.
Molecular genetic analysis identified a variety of mutations in our patient series including a novel heterozygous C to A transversion mutation in exon 14 (c.1859C→A), resulting in a substitution of a highly conserved alanine residue by aspartic acid (p.A620D). Clinical presentation in the patient with the p.A620D included subepithelial scarring in addition to the linear branching opacities usually seen with lattice dystrophy. Using model peptides, we showed that A620D mutant peptide alters the secondary structure and conformational stability, and increased amyloid formation.
A novel mutation (A620D) in transforming growth factor-beta induced protein (TGFβIp) is described, expanding the repertoire of mutations in this protein. Using model peptides, we demonstrated that A→D substitution leads to perturbation of the secondary structure that may be responsible for the amyloid formation in lattice corneal dystrophy.
The International Committee for Classification of Corneal Dystrophies (IC3D) has provided an update of our knowledge on corneal dystrophies. This chapter gives the summary of clinical findings, onset, course, genetics, nosology, light and electron microscopy as well as immunohistochemistry for 25 different entities included as corneal dystrophies in this survey. A category number from 1 through 4 is assigned, reflecting the level of evidence supporting the existence of a given dystrophy.
The recent availability of genetic analyses has demonstrated the shortcomings of the current phenotypic method of corneal dystrophy classification. Abnormalities in different genes can cause a single phenotype, whereas different defects in a single gene can cause different phenotypes. Some disorders termed corneal dystrophies do not appear to have a genetic basis.
The purpose of this study was to develop a new classification system for corneal dystrophies, integrating up-to-date information on phenotypic description, pathologic examination, and genetic analysis.
The International Committee for Classification of Corneal Dystrophies (IC3D) was created to devise a current and accurate nomenclature.
This anatomic classification continues to organize dystrophies according to the level chiefly affected. Each dystrophy has a template summarizing genetic, clinical, and pathologic information. A category number from 1 through 4 is assigned, reflecting the level of evidence supporting the existence of a given dystrophy. The most defined dystrophies belong to category 1 (a well-defined corneal dystrophy in which a gene has been mapped and identified and specific mutations are known) and the least defined belong to category 4 (a suspected dystrophy where the clinical and genetic evidence is not yet convincing). The nomenclature may be updated over time as new information regarding the dystrophies becomes available.
The IC3D Classification of Corneal Dystrophies is a new classification system that incorporates many aspects of the traditional definitions of corneal dystrophies with new genetic, clinical, and pathologic information. Standardized templates provide key information that includes a level of evidence for there being a corneal dystrophy. The system is user-friendly and upgradeable and can be retrieved on the website www.corneasociety.org/ic3d .
Methods for detecting nucleotide substitution rates that are faster or slower than expected under neutral drift are widely used to identify candidate functional elements in genomic sequences. However, most existing methods consider either reductions (conservation) or increases (acceleration) in rate but not both, or assume that selection acts uniformly across the branches of a phylogeny. Here we examine the more general problem of detecting departures from the neutral rate of substitution in either direction, possibly in a clade-specific manner. We consider four statistical, phylogenetic tests for addressing this problem: a likelihood ratio test, a score test, a test based on exact distributions of numbers of substitutions, and the genomic evolutionary rate profiling (GERP) test. All four tests have been implemented in a freely available program called phyloP. Based on extensive simulation experiments, these tests are remarkably similar in statistical power. With 36 mammalian species, they all appear to be capable of fairly good sensitivity with low false-positive rates in detecting strong selection at individual nucleotides, moderate selection in 3-bp elements, and weaker or clade-specific selection in longer elements. By applying phyloP to mammalian multiple alignments from the ENCODE project, we shed light on patterns of conservation/acceleration in known and predicted functional elements, approximate fractions of sites subject to constraint, and differences in clade-specific selection in the primate and glires clades. We also describe new "Conservation" tracks in the UCSC Genome Browser that display both phyloP and phastCons scores for genome-wide alignments of 44 vertebrate species.
The recent availability of genetic analyses has demonstrated the shortcomings of the current phenotypic method of corneal dystrophy classification. Abnormalities in different genes can cause a single phenotype, whereas different defects in a single gene can cause different phenotypes. Some disorders termed corneal dystrophies do not appear to have a genetic basis.
The purpose of this study was to develop a new classification system for corneal dystrophies, integrating up-to-date information on phenotypic description, pathologic examination, and genetic analysis.
The International Committee for Classification of Corneal Dystrophies (IC3D) was created to devise a current and accurate nomenclature.
This anatomic classification continues to organize dystrophies according to the level chiefly affected. Each dystrophy has a template summarizing genetic, clinical, and pathologic information. A category number from 1 through 4 is assigned, reflecting the level of evidence supporting the existence of a given dystrophy. The most defined dystrophies belong to category 1 (a well-defined corneal dystrophy in which a gene has been mapped and identified and specific mutations are known) and the least defined belong to category 4 (a suspected dystrophy where the clinical and genetic evidence is not yet convincing). The nomenclature may be updated over time as new information regarding the dystrophies becomes available.
The IC3D Classification of Corneal Dystrophies is a new classification system that incorporates many aspects of the traditional definitions of corneal dystrophies with new genetic, clinical, and pathologic information. Standardized templates provide key information that includes a level of evidence for there being a corneal dystrophy. The system is user-friendly and upgradeable and can be retrieved on the website www.corneasociety.org/ic3d.
To report the clinical, histopathological and immunohistochemical findings of two novel mutations within the TGFBI gene.
The genotype of 41 affected members of 16 families and nine sporadic cases was investigated by direct sequencing of the TGFBI gene. Clinical, histological and immunohistochemical characteristics of corneal opacification were reported and compared with the coding region changes in the TGFBI gene.
A novel mutation Leu509Pro was detected in one family with a geographic pattern-like clinical phenotype. Histopathologically we found amyloid together with non-amyloid deposits and immunohistochemical staining of Keratoepithelin (KE) KE2 and KE15 antibodies. In two families and one sporadic case the novel mutation Gly623Arg with a late-onset, map-like corneal dystrophy was identified. Here amyloid and immunohistochemical staining of only KE2 antibodies occurred. Further, five already known mutations are reported: Arg124Cys Arg555Trp Arg124His His626Arg, Ala546Asp in 13 families and five sporadic cases of German origin. The underlying gene defect within the TBFBI gene was not identified in any of the four probands with Thiel-Behnke corneal dystrophy.
The two novel mutations within the TGFBI gene add another two phenotypes with atypical immunohistochemical and histopathological features to those so far reported.
A summary of normal and abnormal endothelial structure and function is presented. Endothelium originates from neural crest and it elaborates a banded basement membrane in utero. It is involved in mesenchymal dysgenesis of the anterior segment, like the central defect of Peters' anomaly. Cytoplasmic organelles include mitochondria that provide energy for the metabolic pump, rough endoplasmic reticulum that participate in secretion of extracellular matrix, and a terminal web that may participate in cell migration. The endothelium's main function is to control corneal hydration and nutrition with a leaky barrier formed by the apical gap and macula occludens junctions that keep some water out of the stroma but allow nutrients to pass, and with an ATPase-dependent metabolic pump that is located in the lateral plasma membranes. Endothelial wound healing involves flattening and enlargement of cells to maintain an intact monolayer as well as production of abnormal collagenous material posterior to Descemet's membrane. HLA antigens located in the plasma membrane may participate in corneal endothelial graft rejection. Clinical assessment of the endothelium involves three modalities: specular microscopy to study endothelial morphology, fluorophotometry to measure barrier function, and pachymetry to measure corneal thickness.
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
Mutations in the betaIGH3 gene on chromosome 5q31 cause five distinct autosomal dominant corneal dystrophies: granular Groenouw type I, Reis-Bücklers', lattice type I and IIIA. and Avellino corneal dystrophies. We present here a new mutation of the betaIGH3 gene in patients with late-onset lattice corneal dystrophy manifest as a deep stromal opacity. To test the previously reported R124C, R124H, P501T, R555W, and R555Q mutations of the betaIGH3 gene, 30 patients and 11 normal relatives from 16 independently ascertained families with lattice corneal dystrophy, 49 patients and 12 normal relatives from 40 independently ascertained families with other corneal dystrophies, and 40 unrelated normal volunteers, were analyzed. A L527R (CTG/CGG) mutation of the betaIGH3 gene was found in 6 unrelated patients with lattice corneal dystrophy. A retrospective review of the patients' records showed that the opacities were deep in the stromal layer and of late onset. The mutation was a heterozygous single base-pair transversion from T to G of the second nucleotide position of codon 527. This caused the substitution of arginine for leucine. These six patients did not have mutations in codons 124, 501, or 555. The L527R mutation was not detected in the other corneal dystrophies or 40 normal volunteers. Although phenotypic variations in the size and shape of the deposits were found, all patients with the L527R mutation showed deposits deep in the stromal layer. We conclude that there are now at least six different mutations that have been detected in the betaIGH3 gene on chromosome 5q31 and that lead to corneal dystrophy.
To analyze BIGH3 and M1S1 genes in two Japanese brothers with gelatinous drop-like corneal dystrophy and five unaffected family members.
DNA was extracted, and each part of the two genes was amplified and directly sequenced.
On the BIGH3 gene, a heterozygous P501T mutation was found in the elder brother and three unaffected family members. On the M1S1 gene, both brothers with gelatinous drop-like corneal dystrophy showed a homozygous Q118X mutation, whereas all unaffected members were heterozygous.
The Q118X mutation of M1S1 gene caused gelatinous drop-like corneal dystrophy. Although the P501T of the BIGH3 gene found in this pedigree was precisely the one reported for lattice corneal dystrophy IIIA, no clinical feature was shown, even in the 85-year-old father. This fact shows that the P501T mutation for LCDIIIA has low penetrance.
To evaluate the incidence of BIGH3 gene mutations in 164 unrelated Japanese patients with corneal stromal dystrophies with an autosomal dominant trait.
Data were collected at two major institutions in the eastern and western parts of Japan, where molecular genetic analysis was performed for diagnostic purpose.
The incidence of mutations was ranked as follows: 118 patients (72%), the R124H mutation associated with Avellino corneal dystrophy; 23 patients (14%), the R124C mutation associated with lattice corneal dystrophy type 1; and 10 patients (6%), the P501T mutation associated with lattice corneal dystrophy type 3A.
Avellino corneal dystrophy associated with the R124H mutation is the most common form of corneal stromal dystrophy in Japan. This dystrophy, which is diagnosed histopathologically, has also been called granular corneal dystrophy in Japan. The classification of these diseases according to genetic pathogenesis may be more appropriate than is the use of clinical or histological findings.
To search for novel mutations that cause corneal stromal dystrophies and to confirm or revise the clinical diagnosis of patients with these mutations.
Through review of the records of the Cogan Eye Pathology Laboratory at the Massachusetts Eye and Ear Infirmary, Boston, and of clinical records, we ascertained 14 unrelated patients with the clinical or histopathologic diagnosis of granular (3 cases), Avellino (5 cases), lattice (5 cases), or Reis-Bücklers (1 case) corneal dystrophy.
Clinical records and histopathologic findings of the index patients and their relatives were reviewed. Patients and selected relatives donated a blood sample from which leukocyte DNA was purified and assayed for mutations in the BIGH3 gene and, in 2 patients, the gelsolin gene, using the polymerase chain reaction and direct genomic sequencing.
All index patients with the diagnosis of granular dystrophy or Avellino dystrophy had the missense mutation Arg555Trp or Arg124His, respectively, previously reported in the BIGH3 gene. Of the 5 index patients with a prior diagnosis of lattice dystrophy, 2 had the originally reported lattice mutation (Arg124Cys) in the BIGH3 gene, 1 had a more recently reported missense mutation (His626Arg) in the same gene, 1 had the missense mutation Asp187Asn in the gelsolin gene, and 1 had no detected mutation in either gene. Affected members of the family with Reis-Bücklers dystrophy did not carry the previously reported mutations Arg555Gln or Arg124Leu but instead carried a novel missense mutation Gly623Asp in the BIGH3 gene.
Molecular genetic analysis can improve the accuracy of diagnosis of patients with corneal dystrophies. Two patients with a prior diagnosis of lattice corneal dystrophy had their diagnosis changed to gelsolin-related amyloidosis (1 case) or secondary, nonhereditary localized amyloidosis (1 case). A novel mutation in the BIGH3 gene that causes Reis-Bücklers dystrophy was uncovered through this analysis, and another recently reported novel mutation was encountered. These findings serve to expand our knowledge of the spectrum of pathogenic mutations in BIGH3.
Single nucleotide polymorphisms (SNPs) constitute the bulk of human genetic variation, occurring with an average density of approximately 1/1000 nucleotides of a genotype. SNPs are either neutral allelic variants or are under selection of various strengths, and the impact of SNPs on fitness remains unknown. Identification of SNPs affecting human phenotype, especially leading to risks of complex disorders, is one of the key problems of medical genetics. SNPs in protein-coding regions that cause amino acid variants (non-synonymous cSNPs) are most likely to affect phenotypes. We have developed a straightforward and reliable method based on physical and comparative considerations that estimates the impact of an amino acid replacement on the three-dimensional structure and function of the protein. We estimate that approximately 20% of common human non-synonymous SNPs damage the protein. The average minor allele frequency of such SNPs in our data set was two times lower than that of benign non-synonymous SNPs. The average human genotype carries approximately 10(3) damaging non-synonymous SNPs that together cause a substantial reduction in fitness.
Lattice corneal dystrophies (LCDs) are caused by mutations of the transforming growth factor beta-induced gene (TGFBI, formerly betaig-h3). LCD type IIIA (LCDIIIA) has been reported mostly from Japan. In this study, we demonstrate allelic homogeneity for Japanese patients with LCDIIIA, using intragenic polymorphic markers. When exon 11 of TGFBI was analyzed, all 18 patients examined were found to be heterozygous for both a P501T mutation and an IVS10-3C --> T variation. On the other hand, none of 54 normal Japanese control subjects had the P501T, and 5 of the controls were heterozygous for IVS10-3C --> T. Haplotype analysis of the patients revealed that both P501T and IVS10-3C --> T were located on the same chromosome, and a significant linkage disequilibrium (P < 0.001, Fisher's exact probability test) was observed between LCDIIIA (P501T) and IVS10-3C --> T. When exon 8 of the gene was analyzed, all these patients possessed the "G allele" of a 1028G/A polymorphism. A significant linkage disequilibrium (P < 0.003; chi-square test) was also observed between P501T and the G allele in the patients. These results suggest that allelic homogeneity seen in Japanese patients with LCDIIIA may result from a single founder mutation.
To determine whether Japanese patients with Fuchs' endothelial corneal dystrophy (FECD) and posterior polymorphous dystrophy (PPMD) carry mutations in the COL8A2 gene, and to investigate the possible pathogenicity of the COL8A2 gene in these corneal dystrophies.
DNA analysis of the COL8A2 gene was performed in 15 unrelated Japanese patients with FECD, and 5 patients with PPMD using polymerase chain reaction and direct sequencing. Mutation screenings were also performed in 36 unrelated normal volunteers as controls, as well as slit-lamp and specular microscopy.
Two types of heterozygous missense mutations of the COL8A2 gene (R155Q and T502M) in 5 of 15 FECD probands (R155Q, 3/30 chromosomes, 10.0%; T502M, 3/30 chromosomes, 10.0%) were found. No mutation was detected in the coding region of the COL8A2 gene in the remaining 10 patients with FECD nor in any of the 5 patients with PPMD. These two mutations were also found in normal Japanese volunteers (R155Q, 5/72 chromosomes, 6.9%; T502M, 11/70 chromosomes, 15.7%). The chromosomal frequency of the two mutations was not significant between the patients and normal controls.
The R155Q and T502M mutations of COL8A2 may not be the causative defect in the Japanese FECD and PPMD patients examined in this study.
Posterior polymorphous corneal dystrophy (PPCD, also known as PPMD) is a rare disease involving metaplasia and overgrowth of corneal endothelial cells. In patients with PPCD, these cells manifest in an epithelial morphology and gene expression pattern, produce an aberrant basement membrane, and, sometimes, spread over the iris and nearby structures in a way that increases the risk for glaucoma. We previously mapped PPCD to a region (PPCD3) on chromosome 10 containing the gene that encodes the two-handed zinc-finger homeodomain transcription factor TCF8. Here, we report a heterozygous frameshift mutation in TCF8 that segregates with PPCD in the family used to map PPCD3 and four different heterozygous nonsense and frameshift mutations in TCF8 in four other PPCD probands. Family reports of inguinal hernia, hydrocele, and possible bone anomalies in affected individuals suggest that individuals with TCF8 mutations should be examined for nonocular anomalies. We detect transcripts of all three identified PPCD genes (VSX1, COL8A2, and TCF8) in the cornea. We show presence of a complex (core plus secondary) binding site for TCF8 in the promoter of Alport syndrome gene COL4A3, which encodes collagen type IV alpha 3, and we present immunohistochemical evidence of ectopic expression of COL4A3 in corneal endothelium of the proband of the original PPCD3 family. Identification of TCF8 as the PPCD3 gene provides a valuable tool for the study of critical gene regulation events in PPCD pathology and suggests a possible role for TCF8 mutations in altered structure and function of cells lining body cavities other than the anterior chamber of the eye. Thus, this study has identified TCF8 as the gene responsible for approximately half of the cases of PPCD, has implicated TCF8 mutations in developmental abnormalities outside the eye, and has presented the TCF8 regulatory target, COL4A3, as a key, shared molecular component of two different diseases, PPCD and Alport syndrome.
To characterize histologically Descemet's membrane in an early-onset Fuchs corneal dystrophy (FCD) COL8A2 mutant and compare these findings with corneas from late-onset FCD and normal corneas.
A corneal explant from a patient with the L450W COL8A2 mutation and others with late-onset disease were studied with antibodies to collagens IV, VIIIA1, VIIIA2, fibronectin, and laminin. Transmission electron microscopy was performed on a portion of the explant. Control explants included eye bank corneas without known disease and surgical explants from unrelated conditions.
In normal corneas, a regular array of colocalized COL8A1 and COL8A2 was observed in the anterior half of Descemet's membrane. In the L450W mutant, Descemet's membrane was several times thicker than normal and traversed by refractile strands and blebs that stained intensely for COL8A2, a feature also observed in late-onset FCD. Both the alpha1 and alpha2 subtypes of collagen VIII were observed at high levels along the anterior edge of Descemet's, another abnormal feature also found in late-onset FCD. Ultrastructure of the L450W cornea revealed a well-formed anterior banded layer more than three times thicker than normal. An unusual, thin internal layer was rich in patches of wide-spaced collagen. The layer is a distinctive pathologic structure that is associated with FCD and is characterized by approximately 120 nm periodicity and the presence of collagen VIII. Depositions of collagen IV, fibronectin, and laminin were also greatly increased in the of posterior Descemet's membrane, yet another general feature shared between early- and late-onset disease.
Early-onset COL8A2 L450W disease involves massive accumulation and abnormal assembly of collagen VIII within Descemet's membrane, a process that is presumed to begin during fetal development. Both early- and late-onset subtypes of FCD appear to be the result of abnormal basement membrane assembly rather than a primary defect in endothelial metabolism.
To present the results of a national survey on bullous keratopathy (BK) in Japan.
A cross-sectional national survey was conducted for 963 eyes with BK seen between 1999 and 2001 by members of the Japan Cornea Society. Demographic characteristics, type of surgery, complications, and postoperative outcome were analyzed.
BK accounted for 24.2% (963 eyes) of total keratoplasties performed during the period. Graft clarity was maintained in 77.4% of cases, and immunologic rejection and elevated intraocular pressure was noted in 10.8% and 15.3%, respectively. Cataract surgery was the most common cause of BK (n = 428, 44.4%), and phacoemulsification and aspiration were performed in approximately 40% of cases. BK secondary to laser iridotomy (LI) was the second most common cause of BK (n = 225, 23.4%). LI was performed as a prophylactic measure in approximately one half of these cases. BK developed with a mean duration of 6.8 years after LI. Fuchs dystrophy was the cause of BK in 18 eyes (1.9%).
The causes of BK in Japan are considerably different from those in other Western countries. LI-related BKs showed a remarkably high number, whereas Fuchs dystrophy was observed only rarely.
Mutations in the two-handed zinc-finger homeodomain transcription factor gene (TCF8) have been associated with posterior polymorphous corneal dystrophy (PPCD) and extraocular developmental abnormalities. We performed screening of TCF8 in 32 affected, unrelated probands, affected and unaffected family members of probands identified with a TCF8 mutation, and in 100 control individuals. Eight different pathogenic mutations were identified in eight probands: four frameshift (c.953_954insA, c.1506dupA, c.1592delA, and c.3012_3013delAG); three nonsense (Gln12X, Gln214X, Arg325X); and one missense (Met1Arg). Screening of TCF8 in affected and unaffected family members in six families demonstrated that each identified mutation segregated with the disease phenotype in each family; two probands did not have additional family members available for analysis. None of the eight TCF8 mutations was identified in 200 control chromosomes. The prevalence of hernias of the abdominal region in affected individuals with PPCD associated with TCF8 mutations was significantly higher than the prevalence in both individuals with PPCD not associated with a TCF8 mutation and in unaffected individuals. Therefore, PPCD is associated with TCF8 mutations in one quarter of affected families in this study, or about one third of all PPCD families that have been screened thus far. In these families, the presence of apparently causative TCF8 mutations is associated with abdominal and inguinal hernias.
To identify the genetic basis of Schnyder crystalline corneal dystrophy (SCCD) through screening positional candidate genes and UBIAD1, in which mutations have been associated with SCCD, in affected families.
The coding region of each of the 16 positional candidate genes for which mutation screening has not been previously reported was screened with polymerase chain reaction (PCR) amplification and automated sequencing in four affected individuals from two families with SCCD. In addition, the coding region of UBIAD1, located just outside of the originally described SCCD candidate interval on chromosome 1p36, was directly sequenced in affected and unaffected individuals from three families with SCCD.
Eighteen novel and 15 previously reported sequence variants were identified in 10 of the 16 positional candidate genes. Only two of the sequence variants segregated with the affected phenotype in either of the families screened. Both were novel single nucleotide polymorphisms (SNPs) predicted to result in synonymous amino acid substitutions in different predicted genes. However, one of these SNPs was also identified in control individuals, and the other SNP was not predicted to alter splicing. Screening of UBIAD1 revealed a different missense mutation in each of the three unrelated probands that was screened: p.Asn102Ser, p.Arg119Gly, and p.Leu121Val. Screening of the affected and unaffected relatives of the probands in whom the p.Asn102Ser and p.Leu121Val mutations were identified demonstrated that each mutation segregated with the affected phenotype. None of the three missense mutations was identified in 110 control individuals.
No presumed pathogenic coding region mutations were identified in the genes mapped to the candidate region for SCCD. However, missense mutations in UBIAD1, located just outside of the originally described SCCD fine mapped region, were identified in each of the three families with SCCD, confirming that mutations in UBIAD1 are associated with SCCD.