Six Different Mutations of TGFBI (ßig-H3, Keratoepithelin) Gene

Six Different Mutations of TGFBI (␤ig-h3, Keratoepithelin) Gene Found in Japanese Corneal Dystrophies

Keiko Fujiki, Ph.D., Yoshihiro Hotta, M.D., Kiyoo Nakayasu, M.D., Tatsuo Yamaguchi, M.D., Takuji Kato, M.D., Yuko Uesugi, M.D., Nguyen T. Ha, M.D., Shinichiro Endo, M.D., Nobuo Ishida, M.D., Wen-Nan Lu, M.D., and Atsushi Kanai, M.D.

Purpose. To investigate mutations of the human transforming become visually significant in the fourth or fifth decades. Avellino growth factor beta-induced gene (TGFBI), transforming growth corneal dystrophy (ACD) has been described as a combined dys- factor–beta-induced gene product (␤ig-h3, keratoepithelin), in trophy that shows both granular and lattice changes in the same Japanese patients with Avellino corneal dystrophy (ACD), lattice eye.1–3 Each of the three clinically and histopathologically distinct corneal dystrophy (LCD), granular corneal dystrophy (GCD), and phenotypes—lattice type I (LCD1), GCD, and ACD—has been Reis-Bu¨cklers corneal dystrophy (RBCD). Methods. Genomic mapped to chromosome 5q by linkage analysis.4 Reis-Bu¨cklers DNA was extracted from the peripheral blood of 75 patients and 7 corneal dystrophy (RBCD) has been identified and classified as a unaffected relatives from 60 families with ACD, 34 patients and 8 5 unaffected relatives from 21 families with LCD, 4 patients and 4 dystrophy of Bowman’s layer and the superficial stroma. RBCD 6 unaffected relatives from 4 families with GCD, and 4 patients and was also mapped to the same interval on 5q31. an unaffected relative from 3 families with RBCD. Fifty normal The human transforming growth factor beta-induced gene, volunteers served as controls. Exons 4, 11, and 12 of the TGFBI TGFBI (␤ig-h3), was identified by Skonier et al.7 and mapped to gene were amplified by polymerase chain reaction and were di- chromosome 5q31. Keratoepithelin, the product of TGFBI,was rectly sequenced. Results. Six different heterozygous missense shown to be expressed in numerous tissues and displayed a distinct mutations were detected in codons R124, L518, L527, and R555 of expression pattern in the human eye by being transcribed almost the TGFBI gene in the 117 patients from 88 families. A R124H exclusively in the corneal epithelium.8 In 1997, missense muta- mutation was detected in the patients with ACD. A R124C mutations of R555W in one patient with GCD, R555Q in one with tion was detected in the patients with LCD type 1 (LCD1), L518P RBCD, R124C in two with LCD1, and R124H in two with ACD was in atypical LCD1, and L527R in LCD with opacities deep in 9 stroma. A R555W mutation was detected in the patients with were detected in the TGFBI gene by candidate gene approach. GCD. A R555Q mutation was detected in the patients with RBCD. Recently, mutations of the TGFBI gene also have been reported in Conclusions. We conclude that codons R124 and R555 of the Japanese patients.10–18 TGFBI gene are also hot spots in Japanese patients with ACD, Here, we present six different mutations of the TGFBI gene LCD, GCD, and RBCD. Many Japanese patients with CD had found in 117 patients from 88 Japanese families with ACD, LCD, ACD with R124H mutation. GCD with R555W mutation was rare. GCD, or RBCD, including nine previously reported.12,14,17,18 Key Words: TGFBI (␤ig-h3) gene—Keratoepithelin—Avellino corneal dystrophy—Lattice corneal dystrophy—Granular corneal dystrophy—Reis-Bu¨cklers corneal dystrophy. MATERIALS AND METHODS Subjects All of the study was performed according to the tenets of the Granular corneal dystrophy (GCD) and lattice corneal dystrophy Declaration of Helsinki. We randomly collected 75 patients and (LCD) are two of the most common and distinct autosomal dom- 7 unaffected relatives from 60 families with ACD, 4 patients and inant eye diseases. They are characterized by opacities of the cor- 2 unaffected relatives from 4 families with GCD, 34 patients and neal stroma that appear in the first or second decade of life and 8 unaffected relatives from 21 families with LCD, and 4 patients and an unaffected relative from 3 families with RBCD. They had been followed in the Department of Ophthalmology, Juntendo Submitted October 18, 1999. Revision received February 2, 2000. Ac- University, Tokyo; St. Luke’s International Hospital, Tokyo; and cepted February 5, 2000. From the Department of Ophthalmology (K.F., Y.H., K.N., T.K., Y.U., Ishida Eye Hospital, Niigata, Japan. We also analyzed 50 unrelated N.T.H., W-N.L., A.K), Juntendo University School of Medicine, Tokyo; normal volunteers. the Department of Ophthalmology (T.Y.), St. Luke’s International Hospi- tal, Tokyo; and the Ishida Eye Hospital (S.E., N.I), Niigata, Japan. Address correspondence and reprint requests to Dr. K. Fujiki, Depart- DNA Preparation and Analysis ment of Ophthalmology, Jutendo University School of Medicine, 2-1-1 Genomic DNA was extracted from leukocytes of peripheral Hongo Bunkyo-Ku, Tokyo 113-8421, Japan. blood by a standard procedure.19 Each exon of the TGFBI gene

842 TGFBI MUTATIONS IN CORNEAL DYSTROPHIES 843

TABLE 1. Mutations of the TGFBI gene found in the Model 373A (Applied Biosystems) in both sense and antisense present study strands. Nucleotide sequences for coding regions were compared No. of No. of with the nucleotide and deduced amino acid sequence of TGFBI Diseases Mutations families patients Remarks (␤ig-h3) human cDNA published by Skonier et al.7 Avellino CD R124H 60 75 Lattice CD R124C 13 24 Type 1 L518P 1 3 Atypical type 1 RESULTS L527R 7 7 Opacities deep in stroma Granular CD R555W 4 4 Reis-Bu¨cklers CD R555Q 3 4 Table 1 shows six different mutations—R124H, R124C, L518P, Total 88 117 L527R, R555W, and R555Q—found in the TGFBI gene of the 117 patients from 88 families. Figure 1 shows the nucleotide sequences around the mutation in the TGFBI gene and Figure 2 shows pho- was amplified by polymerase chain reaction using each pair of tographs of the corneas of the patients with the mutations. L518P 9 12 primer and slightly modified for exon 12. Polymerase chain is a new mutation found in a family with atypical LCD1.18 reaction products of exon 4, 11, and 12 that include R124, P501, Unaffected family members and normal volunteers showed no L518, L527, and R555 were purified using the High Pure PCR sequence modifications. Sequencing Kit (Boehringer Mannheim, Germany) and were di- rectly sequenced as described previously.20,21 The terminator reaction for direct sequencing was performed using a DNA Sequenc- DISCUSSION ing Kit, Dye Terminator Cycle Sequencing, Ready Reaction (Per- kin Elmer Applied Biosystems, Foster City, CA, U.S.A.). We detected six different mutations of the TGFBI gene in the Sequencing was carried out in an automated DNA Sequencer 117 patients from 88 families with ACD, GCD, LCD, or RBCD.

FIG. 1. Direct sequencing analysis around the mutations in the TGFBI gene of patients with LCD, ACD, GCD, and RBCD. A: Heterozygous R124C (CGC→TGC) mutation at codon 124 in a patient with typical LCD1. B: Heterozygous L518P (CTG→CCG) mutation at codon 518 in a patient with atypical LCD1. C: Heterozygous L527R (CTG→CGG) mutation at codon 527 of a patient with LCD with deposits deep in the stroma. D: Heterozygous R124H (CGC→CAC) mutation at codon 124 of a patient with ACD. E: Heterozygous R555W (CGG→TGG) mutation at codon 555 of a patient with GCD. F: Hetero- zygous R555Q (CGG→CAG) mutation at codon 555 of a patient with RBCD.

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FIG. 2. Photographs of the corneas of patients with mutations in the TGFBI gene. A: LCD1 with heterozygous R124C mutation. B: Atypical LCD1 with heterozygous L518P mutation. C: LCD with opacities deep in stroma with heterozygous L527R mutation. D: ACD with heterozygous R124H mutation. E: Granular CD with heterozygous R555W mutation. F: RBCD with heterozygous R555Q mutation.

Interestingly, many Japanese patients of the ACD with the R124H Recently, N622H and H626R mutations—both in exon 14 of the mutation of the TGFBI gene were detected and only three families TGFBI gene—were shown to cause a form of late onset LCD,23 had GCD with R555W mutation. These results, together with a where the majority of cases showed significant asymmetry. The previous report,15 show that ACD with R124H mutation is com- clinical findings of the patients with these mutations are closely mon and GCD with R555W mutation is rare in the Japanese popu- resembled to the case with L527R mutation. lation. Rosenwasser et al.22 have already pointed out that ACD can L518P was also a new mutation found in a family with LCD1. be found in Italy, Germany, and a wider geographic distribution The clinical findings in the patients with this mutation were than previous proposed. slightly different from those of typical LCD1. The lesion extended With regard to the L527R mutation, although the families were into the deep stroma and the peripheral region is atypical for randomly collected in the present study, LCD with L527R muta- LCD1.18 tion was detected at relatively high frequencies. The patients with Two distinct mutations, R555Q9 and R124L,16 have been de- L527R mutation were not so severely affected, had a late onset, tected in RBCD. In the present study, three families with the and showed significant asymmetry. Deposits showing phenotypic R555Q were detected. variations in size and shape were observed in the deep stromal The TGFBI gene encodes an adhesion molecule characterized layer. The amyloid deposits were found between the deep stromal by a 4-fold internal homologous domain. The keratoepithelin pro- layer and Descemet’s membrane.14 tein is folded into a potential bivalent structure and acts as a bridge

Cornea, Vol. 19, No. 6, 2000 TGFBI MUTATIONS IN CORNEAL DYSTROPHIES 845 between cells expressing the appropriate ligand and has a carboxy- 7. Skonier J, Neubauer M, Madisen L, Bennett K, Plowman GD, Purchio terminal Arg-Gly-Asp (RGD) sequence that can serve as a ligand AF. cDNA cloning and sequence analysis of ␤ig-h3, a novel gene recognition site for several integrins.7 The TGFBI gene displayed induced in a human adenocarcinoma cell line after treatment with transforming growth factor-␤. DNA Cell Biol 1992;11:511–22. a distinct expression pattern in the human eye, being transcribed 8. Escribano J, Hernando N, Ghosh S, Crabb J, Coca-Prados M. cDNA 8 almost exclusively in the corneal epithelium. The true function of from human ocular ciliary epithelium homologous to ␤ig-h3 is pref- the keratoepithelin protein that is the product of the TGFBI gene is erentially expressed as an extracellular protein in the corneal epithe- still unclear. However, it is possible that the tertiary structure of lium. J Cell Physiol 1994;160:511–21. the mutant keratoepithelin is deranged in stroma. The abnormal 9. Munier FL, Korvatska E, Djemaı¨ A, et al. Kerato-epithelin mutations in four 5q31-linked corneal dystrophies. Nat Genet 1997;15:247–51. folding and precipitation of the encoded protein, keratoepithelin, 10. Mashima Y, Imamura Y, Konishi M, et al. Homogeneity of kerato- might lead to the accumulation of amyloid within the corneal epithelin codon 124 mutations in Japanese patients with either of two stroma. Because the disease is a dominant trait, only 50% of this types of corneal stromal dystrophy. Am J Hum Genet 1997;61:1448– protein would be expected to be abnormal. The abnormal protein 50. is deposited largely in the stroma where it causes damage leading 11. Yamamoto S, Okada M, Tsujikawa M, et al. 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