Kohji Nishida

Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene

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 phenotypes. The gene responsible for MCD type I has been mapped to chromosome 16q22, and that responsible for MCD type II may involve the same locus. 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.

Characteristics of the human ocular surface epithelium.

An appreciation of the biological characteristics of the human ocular surface epithelium affords us a great insight into the physiology of the human ocular surface in health and disease. Here, we review five important aspects of the human ocular surface epithelium. First, we recognize the discovery of corneal epithelial stem cells, and note how the palisades of Vogt have been suggested as a clinical marker of their presence. Second, we introduce the concept of the gene expression profile of the ocular surface epithelium as arrived at using a new strategy for the systematic analysis of active genes. We also provide a summary of several genes abundantly or uniquely expressed in the human corneal epithelium, namely clusterin, keratin 3, keratin 12, aldehyde dehydrogenase 3 (ALDH3), troponin-I fast-twitch isoform, ssig-h3, cathepsin L2 (cathepsin V), uroplakin Ib, and Ca(2+)-activated chloride channel. Genes related to limbal and conjunctival epithelia are also described. Third, we touch upon the genetic abnormalities thought to be involved with epithelial dysfunction in Meesmanns dystrophy, gelatinous drop-like corneal dystrophy, and the ssig-h3-mutated corneal dystrophies. Fourth, we provide an update regarding the current state of knowledge of the role of cytokines, growth factors and apoptosis in relation to ocular surface homeostasis and tissue reconstruction; the main factors being epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), transforming growth factor-ss (TGF-ss), and some inflammatory cytokines. Fifth, corneal epithelial barrier function and dysfunction as measured by fluorophotometry is remarked upon, with an explanation of the FL-500 fluorophotometer and its ability to detect corneal epithelial dysfunction at a subclinical level. The research described in this review has undoubtedly generated a complete understanding of corneal epithelial pathophysiology-an understanding that, directly or indirectly, has helped advance the development of new therapeutic modalities for ocular surface reconstruction.

Wavefront aberrations measured with Hartmann-Shack sensor in patients with keratoconus.

OBJECTIVEnTo compare the ocular wavefront aberrations of normal and keratoconic eyes and to describe the characteristics of the higher-order aberrations in eyes with keratoconus.nnnDESIGNnProspective case control and observational study.nnnPARTICIPANTSnThirty-five keratoconic eyes and thirty-eight normal controls.nnnMETHODSnHigher-order aberrations in refraction were measured with a wavefront sensor, and those aberrations resulting from the cornea were evaluated by videokeratographic data.nnnMAIN OUTCOME MEASURESnComa-like (S(3 + 5)), spherical-like (S(4 + 6)), and total (S(3 + 4 + 5 + 6)) higher-order aberrations in both refraction and the cornea.nnnRESULTSnThe mean +/- standard deviation of S(3 + 5) (1.88 +/- 1.16), S(4 + 6) (0.70 +/- 0.55), and S(3 + 4 + 5 + 6) (2.03 +/- 1.23) in refraction (6-mm diameter, root mean square, micro m) were significantly higher in the keratoconic eyes than in normal controls (0.26 +/- 0.10, 0.19 +/- 0.10, 0.34 +/- 0.11, respectively; Mann-Whitney U test, P = 0.001). Coma-like aberrations were 2.32 times larger than spherical-like aberrations in keratoconic eyes.nnnCONCLUSIONSnThe increase of ocular higher-order aberrations in keratoconic eyes results from an increase of corneal higher-order aberrations. Coma-like aberrations were dominant compared with spherical-like aberrations in keratoconic eyes. Wavefront sensing will enable us not only to evaluate the quality of vision but also to differentiate keratoconic eyes from normal eyes by analyzing the characteristics of the higher-order aberrations.

Ocular Surface Abnormalities in Aniridia

PURPOSEnThe purpose of this study was to examine ocular surface abnormalities in aniridia.nnnMETHODSnWe prospectively studied the ocular surface in nine consecutive patients (18 eyes) with virtually total aniridia. Of the 18 eyes, two eyes (two patients) were excluded from the study because severe surgical damage was likely to be the main cause of their corneal abnormalities. Six volunteers whose eye examinations yielded normal results served as control subjects. Detailed slit-lamp biomicroscopic examinations were performed to assess corneal findings and confirm the presence or absence of the palisades of Vogt. Goblet cell density on the peripheral corneal epithelium and inferior conjunctival epithelium was calculated via impression cytology and was statistically compared between aniridic eyes and control eyes.nnnRESULTSnAll 16 aniridic eyes had superficial corneal opacification and vascularization of either the peripheral or entire cornea. Palisades of Vogt were completely absent all around the limbal area. Goblet cell density on the peripheral cornea and inferior conjunctiva showed a statistically significant increase in the aniridic eyes (226 +/- 169 cells/mm2 and 406 +/- 203 cells/mm2), as compared with the control eyes (O cells/mm2 and 181 +/- 89 cells/mm2) (P = .0005 and P = .0102).nnnCONCLUSIONSnThese findings suggest that, in the aniridic eye, conjunctival epithelium invades the cornea because of corneal epithelial stem cell deficiency.

Identification of the gene responsible for gelatinous drop-like corneal dystrophy

Gelatinous drop-like corneal dystrophy (GDLD; OMIM 204870) is an autosomal recessive disorder characterized by severe corneal amyloidosis leading to blindness, with an incidence of 1 in 300,000 in Japan. Our previous genetic linkage study localized the gene responsible to a 2.6-cM interval on chromosome 1p (ref. 6). Clinical manifestations, which appear in the first decade of life, include blurred vision, photophobia and foreign-body sensation. By the third decade, raised, yellowish-grey, gelatinous masses severely impair visual acuity, and lamellar keratoplasty is required for most patients. Here we report DNA sequencing, cDNA cloning and mutational analyses of four deleterious mutations (Q118X, 632delA, Q207X and S170X) in M1S1 (formerly TROP2 and GA733-1), encoding a gastrointestinal tumour-associated antigen. The Q118X mutation was the most common alteration in the GDLD patients examined, accounting for 33 of 40 (82.5%) disease alleles in our panel of families. Protein expression anaysis revealed aggregation of the mutated, truncated protein in the perinuclear region, whereas the normal protein was distributed diffusely in the cytoplasm with a homogenous or fine granular pattern. Our successful identification of the gene that is defective in GDLD should facilitate genetic diagnosis and potentially treatment of the disease, and enhance general understanding of the mechanisms of amyloidosis.

Possible role of herpes simplex virus in the origin of Posner-Schlossman syndrome

PURPOSE/METHODSnWe conducted this study to determine if the herpesviruses are possible etiologic agents in Posner-Schlossman syndrome. We aspirated aqueous humor samples from patients during acute attacks of the syndrome. Ten normal aqueous humor specimens from patients undergoing cataract surgery were used as controls. DNA was extracted and subjected to polymerase chain reaction amplification and Southern blot hybridization.nnnRESULTS/CONCLUSIONnAll three specimens were positive for amplified genomic fragments of herpes simplex virus and negative for varicella-zoster virus and cytomegalovirus. Ten normal aqueous specimens were negative for all three. Herpes simplex virus may play a role in the origin of Posner-Schlossman syndrome.

Transforming growth factor-beta 1, -beta 2 and -beta 3 mRNA expression in human cornea.

The expression of TGF-beta 1, TGF-beta 2 and TGF-beta 3 precursors mRNA transcripts in in vivo human corneal cells were studied. Complementary DNA (cDNA) was generated from poly A+RNA extracted from in vivo human corneal epithelial cells, stromal keratocytes and endothelial cells. With the cDNAs as template, polymerase chain reaction (PCR) was carried out using specific primers of TGF-beta 1, TGF-beta 2 and TGF-beta 3 precursors, synthesized by choosing specific nucleotide sequences in the latency-associated peptide region of each precursor. Southern blot analysis of the PCR products was carried out. In corneal epithelial cells, TGF-beta 2 mRNA transcript was strongly expressed; TGF-beta 1 and -beta 3 mRNA transcripts were also expressed; stromal keratocyte samples expressed TGF-beta 1 and -beta 2 mRNA transcripts but not TGF-beta 3 mRNA transcript. Endothelial cells expressed all three transcripts. The present study, together with the authors previous immunohistochemical study demonstrates that both protein and mRNA of TGF-beta 2-LAP are present in human corneal epithelium, indicating that TGF-beta 2 may play a crucial role in corneal epithelial cell layers.

Isolation and Chromosomal Localization of a Cornea-Specific Human Keratin 12 Gene and Detection of Four Mutations in Meesmann Corneal Epithelial Dystrophy

Keratin 12 (K12) is an intermediate-filament protein expressed specifically in corneal epithelium. Recently, we isolated K12 cDNA from a human corneal epithelial cDNA library and determined its full sequence. Herein, we present the exon-intron boundary structure and chromosomal localization of human K12. In addition, we report four K12 mutations in Meesmann corneal epithelial dystrophy (MCD), an autosomal dominant disorder characterized by intraepithelial microcysts and corneal epithelial fragility in which mutations in keratin 3 (K3) and K12 have recently been implicated. In the human K12 gene, we identified seven introns, defining eight individual exons that cover the coding sequence. Together the exons and introns span approximately 6 kb of genomic DNA. Using FISH, we found that the K12 gene mapped to 17q12, where a type I keratin cluster exists. In this study, four new K12 mutations (Arg135Gly, Arg135Ile, Tyr429Asp, and Leu140Arg) were identified in three unrelated MCD pedigrees and in one individual with MCD. All mutations were either in the highly conserved alpha-helix-initiation motif of rod domain 1A or in the alpha-helix-termination motif of rod domain 2B. These sites are essential for keratin filament assembly, suggesting that the mutations described above may be causative for MCD. Of particular interest, one of these mutations (Tyr429Asp), detected in both affected individuals in one of our pedigrees, is the first mutation to be identified within the alpha-helix-termination motif in type I keratin.

Two distinct kerato-epithelin mutations in Reis-Bücklers corneal dystrophy

PURPOSEnTwo patients were diagnosed with Reis-Bücklers corneal dystrophy (RBCD), although the pattern and severity of corneal opacification differed. To see whether there was a genetic basis for these phenotypic variations, we analyzed beta ig-h3, the gene that codes for kerato-epithelin and that contains a mutation (Arg555Gln) that causes RBCD.nnnMETHODSnA 30-year-old man with honeycomb-shaped subepithelial opacities in his central cornea and a 25-year-old man with progressive subepithelial geographic opacities were both considered to have RBCD. We isolated genomic DNA from leukocytes of the two patients and their family members and screened for an Arg555Gln kerato-epithelin mutation. Then we analyzed all exons of the gene using the single-strand conformation polymorphism (SSCP) technique to search for any other kerato-epithelin mutations.nnnRESULTSnThe patient with honeycomb-shaped opacities had an Arg555Gln kerato-epithelin mutation that caused his RBCD, whereas the patient with geographic opacities did not; instead, he had a new kerato-epithelin mutation (Arg124Leu), which cosegregated with his family members.nnnCONCLUSIONSnThe variant of RBCD characterized by honeycomb-shaped opacities is caused by an Arg555Gln kerato-epithelin mutation. On the other hand, a new kerato-epithelin mutation, Arg124Leu, was found to cause the RBCD variant characterized by recurrent epithelial erosions and progressive geographic subepithelial opacification. Codon 124 is a hot spot for kerato-epithelin mutations, where the mutations responsible for three autosomal dominant corneal dystrophies--lattice type I (Arg124Cys), Avellino (Arg124His), and the variant of RBCD with geographic rather than honeycomb opacities (Arg124Leu)--are located.

Detection of herpes simplex virus DNA in human tear film by the polymerase chain reaction.

We investigated the use of the polymerase chain reaction for detecting genomes of herpes simplex virus, varicella-zoster virus, and cytomegalovirus from tear film of patients with clinically diagnosed herpes simplex virus keratitis. Using the polymerase chain reaction with a herpes simplex virus detection sensitivity adjusted to 1.0 plaque-forming units/ml, we detected herpes simplex virus genomic sequences in 12 of 12 epithelial keratitis specimens, two of six stromal keratitis specimens, but in none of 20 normal specimens. Neither varicella-zoster virus nor cytomegalovirus genomic sequences were detected in any sample. These results suggest that polymerase chain reaction quickly performed with reduced sensitivity is useful as a diagnostic tool for confirming clinical observations.