Kiyoo Nakayasu

Distribution of Types I, II, III, IV and V Collagen in Normal and Keratoconus Corneas

By using type-specific antibodies to types I, II, III, IV and V collagens, distribution of distinct types of collagen in normal human cornea as well as keratoconus cornea were examined by indirect immunofluorescence microscopy. In normal human cornea, immunohistochemical evidence supported the previous biochemical finding that type I collagen was the major type of collagen in human corneal stroma. No reaction was observed to anti-type II collagen antibody in the whole cornea. Anti-type III collagen antibody reacted with the corneal stroma in a similar fashion as that of anti-type I collagen antibody. Type IV collagen was observed in the basement membrane of the corneal epithelium and in Descemets membrane. Anti-type V collagen antibody also reacted with the corneal stroma diffusely. Bowmans membrane was strongly stained only with he anti-type V collagen antibody. For further details of the distribution of type I, type III and V collagens in human corneal stroma, immunoelectron microscopic study was undertaken. The positive reaction products of anti-type I and anti-type III collagen antibodies were located on the collagen fibrils, while that of anti-type V collagen antibody was either on or close to collagen fibrils. In keratoconus cornea, no difference was observed in terms of the distribution of type I, III and V collagens, while the disruptive and excrescent distribution of type IV collagen was noted in the basement membrane of the corneal epithelium.

A new L527R mutation of the βIGH3 gene in patients with lattice corneal dystrophy with deep stromal opacities

Abstract Mutations in the βIGH3 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 βIGH3 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 βIGH3 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 βIGH3 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 βIGH3 gene on chromosome 5q31 and that lead to corneal dystrophy.

Corneal wound healing following laser in situ keratomileusis (LASIK): a histopathological study in rabbits

AIMS To investigate the histopathological changes of rabbit corneas after laser in situ keratomileusis (LASIK) and to evaluate the corneal wound healing process. METHODS A LASIK was performed on white rabbit eyes. Postoperatively, rabbits were killed on days 1 and 7, and at 1, 3, and 9 months. RESULTS Periodic acid Schiff (PAS) positive material and disorganised collagen fibre were seen along the interface of the corneal flap even 9 months after operation. CONCLUSIONS The wound healing process still continued at 9 months after LASIK indicating that a much longer time than expected was required for corneal wound healing following LASIK.

Leu518Pro mutation of the βig-h3 gene causes lattice corneal dystrophy type I

Abstract PURPOSE: To describe a Japanese family with lattice corneal dystrophy type I, which segregates with a novel mutation, Leu518Pro of the βig-h3 gene. METHODS: DNA was extracted from leukocytes in four members (three affected and one unaffected) of a Japanese family with lattice corneal dystrophy type I. Exon 12 of the βig-h3 gene was amplified and analyzed with a molecular biologic method. Clinical data were also collected. RESULTS: Three generations of this family have been positively diagnosed with lattice corneal dystrophy, indicating autosomal dominant inheritance. We found a heterozygous point mutation that segregates with the disease phenotype. It was a single base-pair transition (CTG to CCG, Leu to Pro). CONCLUSIONS: Although it is extremely rare compared with the Arg124Cys mutation of the βig-h3 gene, Leu518Pro mutation of the βig-h3 also causes lattice corneal dystrophy type I.

Analysis of COL8A2 Gene Mutation in Japanese Patients with Fuchs’ Endothelial Dystrophy and Posterior Polymorphous Dystrophy

PurposeTo 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.MethodsDNA 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.ResultsTwo 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.ConclusionsThe R155Q and T502M mutations of COL8A2 may not be the causative defect in the Japanese FECD and PPMD patients examined in this study. Jpn J Ophthalmol 2004;48:195–198

Corneal dystrophies in Japan

AbstractRecent advances in molecular genetics have increased our understanding of the role of genes. Four autosomal dominant corneal dystrophies (CDs); granular CD (GCD), Avellino CD (ACD), lattice CD (LCD), and Reis-Bücklers CD (RBCD) were mapped to the long arm of chromosome 5 (5q31). These four diseases were shown, in a Caucasian series, to result from different missense mutations in the TGFBI (BIGH3, keratoepithelin) gene. The same mutations were also detected in Japanese patients, from a different ethnic background. Gelatinous drop-like corneal dystrophy (GDLD), on the other hand, which was found in Japanese patients in 1914, is a rare autosomal recessive disorder characterized by corneal amyloidosis. Parents of the patients had a markedly higher frequency of consanguineous marriages than the general population. The gene responsible for GDLD, the membrane component, chromosome 1, surface marker 1 (M1S1) gene was mapped to the short arm of chromosome 1(1p). Four deleterious mutations in this gene were detected in Japanese patients. We review here additional studies on mutations of the TGFBI and M1S1 genes found in Japanese patients. In the TGFBI gene, nine different mutations were detected in Japanese patients with GCD, ACD, LCD, or RBCD. The codons R124 and R555 of the TGFBI gene were hotspots in Japanese patients, of whom many were ACD patients with the R124H mutation. New mutations responsible for LCD were detected in the TGFBI gene of patients with LCD, in addition to the P501T mutation in LCD type IIIA found earlier. These studies showed a clear genotype/phenotype correlation associated with the TGFBI gene. In the M1S1 gene, the Q118X mutation was the most common alteration, and a founder mutation in Japanese GDLD patients, as previously reported. Ninety-two percent of the mutated alleles were the Q118X.

The Ultrastructure of the Lens Capsule Abnormalities in Alport’s Syndrome

The ultrastructure of lens capsule abnormalities in Alports syndrome is reported. An anterior lens capsule from a 29-year-old patient with lenticonus who was affected by Alports syndrome was obtained at phacoemulsification. The histopathological findings showed decreased thickness of the anterior lens capsule and there were many vertical capsular dehiscences localized at the inner part of the lens capsule. Almost every dehiscence was limited to the inner two-thirds of the capsule. One should be cautious in attempting intraocular lens implantation into the lens capsule, since the lens capsule may be fragile in this disease.

Homozygotic patient with βig-h3 gene mutation in granular dystrophy

Purpose This study investigated patients with granular dystrophy and identified a homozygotic patient and his family with a mutation in the βtig-h3 gene. Methods Genomic DNAs were extracted from leukocytes of the peripheral blood of the pro-band, his parents, and his grandmother. All had granular dystrophy. Genomic DNAs from 50 unrelated normal volunteers were used as controls. Exon 4 of βig-h3 gene was amplified and analyzed by direct sequence. Clinical data were collected. Results A single-base-pair transition was detected. This was a substitution of G to A of the second nucleotide position of codon 124 in the βig-h3 gene that led to a replacement of histidine for arginine (Arg124His, CGC->CAC). This mutation was the precise one previously reported for Avellino dystrophy. Although the proband was homozygotic for the mutant alleles, his grandmother, and parents were heterozygotic for these alleles. No sequence modification in the codon 124 from 50 nonaffected control individuals was detected. Clinical findings of the proband were severe. Keratectomies were performed for both his eyes 5 times for a 24-year period. His grandmother and parents showed mild clinical symptoms, had a few annular granules in the subepithelial stroma, and maintained good visual acuities. Conclusion Arg124His mutation of the βig-h3 gene was found in a pedigree with granular dystrophy. This mutation was the precise one previously reported for Avellino dystrophy. This fact shows an existence of Avellino form in Japanese. Homozygotic patient for mutant gene showed severe symptoms and an early onset.

Six different mutations of TGFBI (betaig-h3, keratoepithelin) gene found in Japanese corneal dystrophies.

Purpose. To investigate mutations of the human transforming growth factor beta-induced gene (TGFBI), transforming growth factor–beta-induced gene product (&bgr;ig-h3, keratoepithelin), in Japanese patients with Avellino corneal dystrophy (ACD), lattice corneal dystrophy (LCD), granular corneal dystrophy (GCD), and Reis-Bücklers corneal dystrophy (RBCD). Methods. Genomic DNA was extracted from the peripheral blood of 75 patients and 7 unaffected relatives from 60 families with ACD, 34 patients and 8 unaffected relatives from 21 families with LCD, 4 patients and 4 unaffected relatives from 4 families with GCD, and 4 patients and an unaffected relative from 3 families with RBCD. Fifty normal volunteers served as controls. Exons 4, 11, and 12 of the TGFBI gene were amplified by polymerase chain reaction and were directly sequenced. Results. Six different heterozygous missense mutations were detected in codons R124, L518, L527, and R555 of the TGFBI gene in the 117 patients from 88 families. A R124H mutation was detected in the patients with ACD. A R124C mutation was detected in the patients with LCD type 1 (LCD1), L518P was in atypical LCD1, and L527R in LCD with opacities deep in stroma. A R555W mutation was detected in the patients with GCD. A R555Q mutation was detected in the patients with RBCD. Conclusions. We conclude that codons R124 and R555 of the TGFBI gene are also hot spots in Japanese patients with ACD, LCD, GCD, and RBCD. Many Japanese patients with CD had ACD with R124H mutation. GCD with R555W mutation was rare.

Microarray analysis identified differentially expressed genes in keratocytes from keratoconus patients

Purpose. To identify differentially expressed genes in human keratoconus keratocyte by cDNA microarray. Methods. Normal and keratoconic cornea were cultured for keratocytes. RNA was extracted. cDNA probe labeled with fluorescence dye was made from Poly A+ RNA, hybridized with microarray slide, containing 164 human apoptosis genes. Signal intensity was measured. Expression ratio between keratoconus and normal was determined using ImaGene Ver.3.0. Identified genes were further evaluated by RT-PCR and real-time PCR. Results. Five over-expressed and four under-expressed genes were identified. Of these, differential expression of tumor necrosis factor alpha-induced protein 6 (TNFAIP6), human insulin-like growth factor binding protein 5 (IGFBP5), and IGFBP3 were verified and confirmed by RT-PCR. Real-time PCR showed TNFAIP6 increased by 3.3 folds, while IGFBP5, IGFBP3 decreased by 14 and 11 folds respectively. Conclusions. The identified genes could be important and deserve further investigation. Significant differential expression of TNFAIP6, IGFBP5, and IGFBP3 may indicate an important role of these genes in the mechanism underlying stromal thinning.