Molecular Vision 2007; 13:1390-1396 <>
Received 21 May 2007 | Accepted 6 August 2007 | Published 10 August 2007

R124C and R555W TGFBI mutations in Spanish families with autosomal-dominant corneal dystrophies

Cristina Blanco-Marchite,1 Francisco Sánchez-Sánchez,2 Enrique López-Sánchez,3 Julio Escribano2

1Servicio de Oftalmología, Complejo Hospitalario Universitario de Albacete (Hospital Perpetuo Socorro), Albacete, Spain, 2Área de Genética, Facultad de Medicina/Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete, Spain, 3Servicio de Oftalmología, Hospital Arnau de Vilanova, Valencia, Spain

Correspondence to: Julio Escribano, Área de Genética, Facultad de Medicina, Avda. de Almansa 14, 02006 Albacete, Spain; Phone: +34 967 599200, ext: 2928; FAX: +34 902 204130; email:


Purpose: Mutations in the transforming growth factor beta I (TGFBI) gene cause several types of autosomal-dominant corneal dystrophies. We investigated the role of this gene in two Spanish families affected by lattice type I or granular type I corneal dystrophies.

Methods: We recruited 13 subjects from two unrelated families diagnosed with autosomal dominant lattice type I or granular type I corneal dystrophies. Corneal phenotypes were assessed by slit lamp examination. Genomic DNA was obtained from blood samples, and exons 4, 11, 12, and 14, which contained mutation hot spots of the TGFBI gene, were screened for mutations by PCR DNA sequencing.

Results: We identified two TGFBI mutations: R124C (exon 4), which segregated with lattice type I corneal dystrophy, and R555W (exon 12), which segregated granular type I corneal dystrophy. Two single-nucleotide polymorphisms were also found, of which H428H was novel and F540F was previously reported.

Conclusions: This is the first report of mutations in the TGFBI gene in Spanish families affected by corneal dystrophy. R124C and R555W TGFBI mutations cause lattice and granular type I corneal dystrophies in the studied families. Our results indicate that the genetic defects underlying corneal dystrophies in Spanish patients are similar to those found in other populations.


The term corneal dystrophy (CD) defines a group of inherited, bilateral, and clinically heterogeneous disorders that impair corneal transparency or refraction. Both lattice type I CD and granular type I CD (also known as Groenouw type I CD) are the most common inherited blinding diseases of the cornea [1]. CDs are genetically heterogeneous, and in recent years, several loci have been linked to the disease with different patterns of inheritance, including autosomal dominant, autosomal recessive, and X-linked recessive [2]. The different types of granular CD and most types of lattice CD are linked to chromosome 5q31. These disorders are transmitted as autosomal-dominant traits and are 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 (BIGH3) [3], consists of 17 exons and is highly expressed in the corneal epithelium and keratocytes [4]. Mapping of TGFBI in the same locus (5q31) as three CDs and the characteristic expression pattern of this gene in the cornea [4], were important clues to elucidate the role of TGFBI mutations in the development of four autosomal dominant CDs [1]. The protein encoded by TGFBI has also been named keratoepithelin because of its presence in the corneal epithelium. Keratoepithelin 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 and to fasciclin-1 of Drosophila [5].

CDs caused by TGFBI mutations are characterized by the formation of deposits of insoluble material in the corneal stroma, which leads to visual impairment. Both granular and lattice CD are divided into different subtypes based on the clinical and histopathological properties of the deposits and on the patients' clinical features. Frequently, these are juvenile-onset diseases that appear during the first or second decade of life. The symptoms manifest as recurrent and extremely painful corneal erosions and visual blurring caused by progressive corneal opacification, which usually leads to corneal transplantation. Lattice type I CD is characterized by a thin, linear network of opacities in the anterior stroma. Recurrent corneal erosions are common, and keratoplasty is frequently required. The mutation R124C in TGFBI is the most prevalent for this type of CD worldwide [6]. Granular CD type I is featured by discrete, rounded, breadcrumb-shaped opacities in the anterior central stroma. As the disease advances, the number and extension of opacities increase and progressively impair vision. This type of CD is frequently associated with the mutation R555W in the TGFBI gene in different ethnic groups [6]. More than 30 TGFBI gene mutations causing lattice and granular CDs have been identified in patients of different ethnic groups [6-8], including Japanese [9,10], Chinese [11,12], Vietnamese [13,14], Korean [15], Indian [16], Mexican [17], North American [18-21], Bulgarian [22], and Ukrainian patients [23]. The role of TGFBI mutations has not been investigated in CDs in the Spanish population to date. In this study, we report for the first time the presence of TGFBI mutations segregating with lattice type I and granular type I CD in Spanish patients.



Eight patients and three unaffected individuals (including partners) from a family (CD1) with lattice type I CD and two patients from another unrelated family (CD2) with granular type I CD, were recruited for this study. All subjects were native Spaniards from the provinces of Albacete (CD1) or Valencia (CD2). A family history of autosomal dominant and bilateral CD was present with documented occurrence over four generations. All patients underwent clinical examination including best-corrected visual acuity measurement (Snellen chart), slit-lamp, and fundus examination.

Blood samples (5 ml) were collected for DNA analysis from all participants in the study. The study protocol was approved by the Ethics Committee for Human Research of the University Hospital of Albacete and adhered to the tenets set forth by the Declaration of Helsinki. Informed consent was obtained from each study subject.

TGFBI mutation screening by PCR DNA sequencing

Genomic DNA was extracted from the peripheral leukocytes with the Perfect gDNA Blood Mini kit (Eppendorf, Madrid, Spain) according to the manufacturer's protocol. TGFBI exons 4, 11, 12, and 14 were screened for mutations because they contained mutation "hot spots." Primers and conditions used to amplify these exons were adapted from those described by Munier and coworkers [8]. PCRs were performed in a 50 μl volume containing 50-100 ng of genomic DNA, 10 pmol of forward and reverse primers, 2 mM MgCl2 for exons 4 and 14, and 2.5 mM MgCl2 for exons 11 and 12. The reactions also contained 100 μM of each dNTP and 1.25 U of Taq DNA polymerase (Biotools, B & M Labs, Madrid, Spain). Dimethylsulphoxide (10% final concentration) was included for the amplification of exons 11 and 12. Thermocycling included an initial denaturation step at 94 °C for 4 min followed by 30 cycles of denaturation, annealing, and extension (Table 1). The extension was performed at 72 °C for 10 s, except for exon 11, which required 60 s. A final cycle was performed at 72 °C for 7 min. Terminator cycle sequencing was carried out using the BigDye® (v3.1) kit (Applied Biosystems, Foster City, CA) and the products of sequencing reactions were analyzed in an automated capillary DNA sequencer (ABI Prism 3130-Avant genetic analyzer, Applied Biosystems).


Phenotype of patients

We analyzed the presence of TGFBI gene mutations in two Spanish families affected by autosomal-dominant CD. Family CD1 was diagnosed with lattice type I CD which affected four generations. Members of this family were from the province of Albacete. The second family (CD2) was affected by granular type I CD and also encompassed four generations from the province of Valencia. The disease was bilateral in all patients. Five (50%) lattice type I CD patients were male and five patients (50%) were female. In the case of granular type I CD, three affected subjects were male and three were female. The age at diagnosis for lattice type I CD patients ranged 3-46 years (mean 17.25 years) and for granular CD patients, the age range was 26-53 years. Two patients (Figure 1A, subjects III:6 and IV:1 from families CD1 and CD2, respectively) were diagnosed during this study. The oldest studied patients in family CD1 were two siblings (Figure 1A, II:1 and II:2), who were 70 and 72 years old at the time of the study. One of them (II:1) had a delayed diagnostic (at 46 years) while the other (II:2) was diagnosed at the age of 20. These two subjects, as well as patient III:2 (age 43 years), underwent several penetrant keratoplasties due to relapses of the CD (Table 2). Small dot opacities were evident on the transplanted corneas of II:1, II:2, and III:2 (see Figure 2A for a representative picture of subject III:2). Particularly the oldest individuals had important visual acuity impairment at the time of the study (Table 2). The other two patients of the third generation (III:4 and III:6) manifested typical lattice deposits of this type of CD, which included opacities forming small nodules, dots, spikes, and numerous refractile branching lattice lines (Figure 2B,C). Patients III:2 and III:4 (43 and 38 years of age, respectively) described repetitive episodes of red eye and pain without traumatism. These patients were treated with occlusion of the painful eye and artificial tears. Keratoplasty relieved the episodes of red eye and pain. Patient III:6 was diagnosed during this study at the age of 27 and complained of reduced visual acuity, which did not improve with optical correction. Under slit lamp examination, her corneas showed lattice deposits without dot-like lesions (Figure 2C). Patients of generation IV (ages at diagnosis ranged 3-5 years) showed a reduced loss of visual acuity (Table 2) and manifested symptoms only after minor ocular traumatisms. Slit lamp examination revealed thin dots mainly in the stroma and subepithelial layers of the central cornea, which were not stained by fluorescein (Figure 2D). The peripheral cornea was free of these deposits. Only one of these young patients (IV:2) had curly line lesions in the form of a snail or comma located in the anterior stroma. These patients occasionally required artificial tears.

Only two members of the family affected by granular type I CD participated in this study. The most severe corneal phenotype was observed in patient III:1 (Table 2). It consisted of white opacities with sharp borders, fine dots, and radial lines in the superficial part of the central corneal stroma and a diffuse anterior stromal haze (Figure 2E). The corneal periphery was free of deposits (Figure 2F). Patient III:1 was 53 years of age at the time of the study and presented a severe visual impairment (Table 2). His son (IV:1) was 26 years old and had a preserved visual acuity (Table 2). However, detailed ocular examination revealed the presence of white opacities and a clear corneal stroma between deposits (Figure 2G,H). These corneal alterations clearly showed that this subject was also affected by granular type I CD. He was diagnosed during the study.

Analysis of TGFBI mutations

In order to determine the genetic defect underlying CD in the two families, we studied by direct PCR sequencing the presence of mutations in four exons of theTGFBI gene, described to contain mutation "hot spots" (exons 4, 11, 12, and 14). Three unaffected individuals were also analyzed as controls. We identified a heterozygous C to T transition at position c.370 (CGC to TGC), located in exon 4, which predicted the substitution of Arg for Cys at codon 124 (Figure 3A) in the eight studied patients from family CD1 (Figure 1). In the two patients recruited from the CD2 family (Figure 1; III:1 and IV:1), we also detected a heterozygous C to T transition at nucleotide c.1663 (CGG to TGG), located in exon 12 (Figure 3B). This nucleotide substitution resulted in the predicted missense mutation Arg555Trp. The two mutations found in this study clearly segregated with the CDs (Figure 1). In addition, we found two heterozygous C to T transitions in members of the CD1 family (Figure 3C,D), at positions c.1446 (H482H; Figure 1, subjects III:1 and IV:1) and c.1620 (F540F; Figure 1, subjects III:2 and III:3) which did not predict any amino acid substitution. Therefore, the two last detected changes were considered polymorphic DNA sequence variations. These polymorphisms mapped to exons 11 (H482H) and 12 (F540F), respectively.


Mutations in TGFBI cause different types of autosomal-dominant CD in different populations worldwide, but until this study, the contribution of this gene to the development of CD in Spanish patients had not been investigated. To our knowledge, this is the first study to analyze the role of the mutations of this gene in CD in families from Spain. We found that mutation Arg124Cys caused lattice type I CD and Arg555Trp caused granular type I CD in the families studied. These mutations arose in two known hot spots in the TGFBI gene, localized at codons Arg124 and Arg555 [7], and their linkage to CDs was first established by Munier et al. [1]. Most individuals with lattice type I CD present mutation Arg124Cys [6]. Mutation Arg555Trp is the most prevalent in granular type I CD in several different ethnic groups [8,16,24]. Our data showed that the spectrum of TGFBI mutations in Spanish patients with autosomal-dominant CD is similar to that found in other populations.

It is likely that the wide distribution of these two mutations in different ethnic groups is the result of independent de novo events [7], even in different ethnic backgrounds, because the two C to T transitions at the arginine codons affected CpG dinucleotides, which are prone to this type of mutation.

Keratoepithelin has four internal repeat domains homologous to one another and also to the Drosophila protein fasciclin-1 [5]. These two mutations are located in solvent-exposed alpha-helical regions of the fasciclin-1 domains 1 (Arg124) and 4 (Arg555). These mutations are predicted to alter either protein solubility or stability rather than protein structure [25], and they likely affect protein-protein interactions directly rather than misfolding [26]. However, it has been suggested that protein misfolding of the rare mutant keratoepithelins is probably involved in the slow accumulation and deposition of amyloid in CD [26]. It is interesting that the cornea displays amyloid, granular, or mixed lesions depending on the presence of Cys, Leu, Ser, or His substituting Arg124, respectively. It has been suggested that this phenotypic variability could be due to the abnormal degradation of the protein [27,28]. It is also interesting that deposits of mutant keratoepithelin are probably present only in the cornea of CD-affected patients, suggesting that specific conditions existing in the cornea may trigger the deposition of abnormal keratoepithelin products [29].

All patients affected by lattice type I CD who were subjected to keratoplasty (III:3, II:2, and II:6) suffered a relapse, which is typical in this disease. However, they did not show the characteristic lattice pattern of lesions of this type of CD; instead they showed round amyloid deposits only in the anterior stroma, which did not affect the deep stroma. This can be due to the quick replacement of the transplanted corneal epithelium of the receptor by epithelial cells, which produced mutant keratoepithelin. In contrast, donor stromal keratocytes survive long enough in the transplanted cornea to prevent the deposition of abnormal keratoepithelin [30].

Of the two single nucleotide polymorphisms detected, F540F was previously reported [12,21,31], while H482H was identified in the Spanish patients for the first time.

We observed the well known evolution of the lattice type I CD over several generations. The younger patients (ages 3-5 years at diagnosis) were mainly asymptomatic. They presented subtle corneal changes on clinical examination and did not manifest the typical lattice deposits. Patients from this family exhibited a clear phenotypic manifestation in the second decade of life.

In contrast with lattice type I CD, the two patients affected by granular type I CD did not complain of corneal erosions. The phenotype in the oldest studied granular type I CD patient was severe. Although this patient presented an important visual impairment, he rejected keratoplasty. His son (IV:1) presented discrete corneal opacities, but the small size of the lesions and the absence of a hazy stroma between deposits helped preserve his visual acuity. This patient was unaware of the disease, which was diagnosed during the course of the study.

Since the mutations at the hot spot codons 124 and 555 can be easily, rapidly, and cost-effectively evaluated by PCR sequencing or by other alternative techniques, identification, particularly of these mutations, will allow Spanish patients to benefit from a timely and accurate molecular diagnosis for CD. Genetic testing of TGFBI mutations in CD patients will also contribute to improve their clinical classification, management and eventual genetic counseling.


We are deeply grateful to the patients and their families for their cooperation in this study. We thank Dr. Juan López-Moya, Chairman of the Servicio de Oftalmología, Complejo Hospitalario Universitario de Albacete for supporting this project. We thank Mrs. Virginia Aznar for her invaluable help in recruiting patients. We also thank Mrs. Carmen Cifuentes for her technical assistance and the nurses of the Servicio de Oftalmología for extracting blood samples. This study was supported in part by research grants PI052494 and 02021-00 from the Fondo de Investigaciones Sanitarias, and Consejería de Sanidad of the Junta de Comunidades de Castilla-La Mancha, respectively. We have no financial or proprietary conflicts relevant to the content of this paper.


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