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Original article

R555W mutation ofTGFβIrelated to granular corneal dystrophy in Chinese patients

HU, Lei; XU, Fei; MA, Wei-jian; ZHANG, Hua; SUI, Rui-fang

Author Information
doi: 10.3760/cma.j.issn.0366-6999.2009.22.003


Granular corneal dystrophy (GCD, MIM121900; Online Mendelian Inheritance in Man; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD, USA) is characterized by small, discrete, sharply demarcated grayish white opacities in the anterior central stroma resembling bread crumbs or snowflakes. The histological findings are hyaline degeneration with absence of acid mucopoly-saccharide depositions. As the disease advances, the number and extension of opacities increase and progressively impair vision. GCD is linked to chromosome 5q31. The disorder is transmitted as autosomal-dominant traits and is caused by mutations in the transforming growth factor beta I (TGFBI) gene.1 This gene, also known as transforming growth factor-beta-inducible gene-h3, consists of 17 exons and is highly expressed in the corneal epithelium and keratocytes.2

In the current study, we examined the TGFBI gene for mutations in a Chinese family with a clinical diagnosis of GCD.



A single two-generation family of three study participants, of whom two were known to be affected, was studied (Figure 1). The proband was a 45-year-old woman, who complained of progressive visual loss and was referred for evaluation. Her only daughter also suffered from blurred vision. No medical history of the proband's biological parents were collected since she was an adopted child. All patients underwent clinical examination including visual acuity measurement, slit-lamp, and fundus examination. All the investigations followed the tenets of the Declaration of Helsinki, and informed consent was obtained from the subjects after an explanation of the study's purpose.

Figure 1.
Figure 1.:
Two-generation family. Squares: males. Circles: females. Filled circles: affected family members. Arrow: proband.

Mutation screening

Genomic DNA was extracted from the peripheral white blood cells of all the participants. Amplication of all the coding regions of TGFBI and exon-intron boundaries was achieved using 17 pairs of primers.3 Fifty microlitres polymerase chain reaction (PCR) reactions were set up using 2 μl of genomic DNA (20 ng/μl), 4 μl (10 pmol/μl) of forward and reverse primers, 25 μl TransGenePfu taq mix (TransGene, China) and 19 μl ddH2O. PCR reactions were carried out on a dry block thermal cycler (Eppendorf, Germany). Reaction mixtures were denatured at 95°C for 5 minutes, then 34 cycles at 94°C for 30 seconds, X°C for 30 seconds (where X is the optimized annealing temperature for each primer pair) and 72°C for 45 seconds. Final extension was performed at 72°C for 10 minutes. After purification, amplicons were sequenced using both forward and reverse primers on an ABI 377 Genetic Analyzer (ABI, USA). Sequencing results from affected and unaffected individuals as well as TGFBI consensus sequences from the NCBI Human Genome Database were imported into the Chromas program and then aligned to identify variations.


Clinical findings

The proband presented a severe visual impairment with an best corrected visual acuity 0.05 OS, 0.03 OD. Slit lamp examination revealed multiple superficial grayish white granular opacities in the whole cornea (Figure 2A). Her daughter was 21 years old and had a preserved visual acuity of 0.5 OS and 0.3 OD. However, detailed ocular examination revealed the presence of white opacities in the central cornea and clear corneal stroma between deposits could be observed. The peripheral cornea was free of these deposits (Figure 2B). These corneal alterations clearly showed that this subject was also affected by GCD.

Figure 2.
Figure 2.:
Corneal photograph of the proband (A) shows multiple superficial grayish white granular opacities in the central and peripheral cornea; corneal photograph of her daughter (B) shows grayish white granular opacities mainly in the central part of the cornea.

Mutation screening

In the two patients recruited from the family, we detected a heterozygous C to T transition at nucleotide c.1663 (CGG to TGG), located in exon 12. This nucleotide substitution resulted in the amino acid change from arginine to tryptophan at codon555 (R555W, Figure 3). This mutation was not found among unaffected family member. Generally speaking, the existence of a single nucleotide change may be due to two reasons: 1) pathogenic point mutation; 2) single nucleotide polymorphism (SNP). Usually, we need to screen 50 normal controls (100 chromesomes) in order to rule out the possibility of a single nucleotide change is an unknown SNP. However, this mutation has been reported both in the Chinese and western populations, so we do not need to screen the normal controls.

Figure 3.
Figure 3.:
Partial nucleotide sequences of exon 12 of the TGFBI gene in affected and unaffected family members. A: Sequence in affected person, showing the presence of heterozygous C to T transition. B: Unaffected family members lack these nucleotide changes.


Generally, there are two clinical variants of GCD, the classic and the superficial forms. Superficial variant of GCD, the severe form, begins early in childhood with frequent recurrent erosive attacks and rapid visual loss. The classic type is a milder, late-onset form with few corneal erosion and is characterized by multiple snowflakes-like opacities that lead to visual impairment only in adulthood. However, besides these two well-characterized subtypes of granular dystrophy, other rare clinical variants exist.4 The patients we investigated from this Chinese family presented with classical form of GCD. Slow progress was observed in this family as the proband presented both severe visual impairment and worse cornea pathology, but her daughter had a preserved visual acuity as well as milder cornea lesion.

Mutations in TGFBI cause different types of autosomal-dominant corneal dystrophy in different populations worldwide, including GCD, Reis-Bucklers' (CDRB), lattice type I and IIIA (CDL I/CDL IIIA), and Avellino (CDA) corneal dystrophies. Up to date, seven mutations of TGFBI related to GCD have been reported. Among them, R555W is the most frequent mutation among different ethical groups, which usually related to classical GCD.5-8 It is likely that the wide distribution of this mutation in different ethnic groups is the result of independent events,9 even in different ethnic backgrounds, because the C to T transitions at the Arginine codon affected CpG, which are prone to this type of mutation. However, the another six mutations may related to other non-classical types.4,10-14

The protein encoded by TGFBI has also been named keratoepithelin because of its presence in the corneal epithelium. Keratoepithlin is an extracellular molecule involved in cell adhesion.1 It is composed of 682 amino acids and has four internal repeat domains homologous to one another. The mutation identified in our study is located in solvent-exposed alpha-helical regions of the fasciclin-4 domains. This mutation is predicted to alter either protein solubility or stability rather than protein structure,15 and they may be involved in corneal specific protein-protein interactions directly rather than misfolding in the endoplasmic reticulum.16 It is interesting that deposits of mutant keratoepithelin are probably present only in the cornea of affected patients, suggesting that specific conditions existing in the cornea may trigger the deposition of abnormal keratoepithelin products.17

Until relatively recently, penetrating keratoplasty has been the traditional method for treating GCD. The graft can remain free of recurrence for at least 30 months. However, the opacities may recur in the grafts within a year, usually superficial to the donor tissue.18 Laser in situ keratomileusis (LASIK) has been shown to aggravate corneal deposits in corneal dystrophy hence should be avoided. Phototherapeutic keratectomy (PTK) has shown its usefulness in clearing opacities with visual improvement and prevents painful erosion, resulting in delay or postponement of corneal grafting in some corneal dystrophies. Mitomycin-C may be used topically in conjunction with PTK to reduce the recurrence of the opacities. Topical use of antibody to TGF-beta can also be considered to suppress recurrence of corneal opacities after PTK or lamellar keratectomy.19 Since accumulation of mutant proteins leads to corneal opacities and poor epithelial adhesion to the corneal stroma, it is feasible of using shRNAs as effective “molecular silencers” to suppress the expression of keratoepithelin.20

Over the past few years, significant advances have been made in the genetics of corneal dystrophies. Based on this knowledge, diagnosis of corneal dystrophies, which generally has been made by clinical findings, is now beginning to depend on genetic findings. Mutations at the hot spot can be easily, rapidly evaluated by sequencing. Gene test will allow patients to benefit from a timely and accurate molecular diagnosis for GCD, as well as improving classification, management and eventual genetic counseling.


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granular corneal dystrophy; TGFβI gene; mutation screening

© 2009 Chinese Medical Association