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 Descemet's membrane. Anti-type V collagen antibody also reacted with the corneal stroma diffusely. Bowman’s membrane was strongly stained only with the anti-type V collagen antibody. For further details of the distribution of type I, type III and type V collagens in human cornea! 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.

Recent 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 ReisBücklers 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.

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.

The R155Q and T502M mutations of COL8A2 may not be the causative defect in the Japanese FECD and PPMD patients examined in this study.

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.