TGFBI Gene Mutations Causing Lattice and Granular Corneal Dystrophies in Indian Patients

Abstract

purpose. To identify mutations in the TGFBI gene in Indian patients with lattice corneal dystrophy (LCD) or granular corneal dystrophy (GCD) and to look for genotype–phenotype correlations.

methods. Thirty-seven unrelated patients were studied, 18 with LCD and 19 with GCD. The diagnosis of LCD or GCD was made on the basis of clinical and/or histopathological evaluation. Exons and flanking intron sequences of the TGFBI gene were amplified by PCR with specific primers. PCR products were screened by the method of single-strand conformation polymorphism followed by sequencing. Mutations were confirmed by screening at least 100 unrelated normal control subjects.

results. Mutations were identified in 14 of 18 patients with LCD and in all 19 patients with GCD. In LCD, three novel heterozygous mutations found were glycine-594-valine (Gly594Val) in 2 of 18 patients, valine-539-aspartic acid (Val539Asp) in 1 patient, and deletion of valine 624, valine 625 (Val624-Val625del) in 1 patient. In addition, mutation of arginine 124-to-cysteine (Arg124Cys) was found in 8 of 18 patients and histidine 626-to-arginine (His626Arg) in 2 of 18 patients. Atypical clinical features for LCD were noted in patients with the Gly594Val and Val624-Val625del mutations. In GCD, 18 patients with GCD type I had a mutation of arginine 555-to-tryptophan (Arg555Trp) and 1 patient with GCD type III (Reis-Bücklers dystrophy), had the Arg124Leu mutation. Seven novel single-nucleotide polymorphisms (SNPs) were also found, of which a change of leucine 269 to phenylalanine (Leu269Phe) was found in 12 of 18 patients with the Arg555Trp mutation.

conclusions. Arg124Cys and Arg555Trp appear to be the predominant mutations causing LCD and GCD, respectively, in the population studied. The novel mutations identified in this study are associated with distinct phenotypes.

Corneal dystrophies involve the formation of corneal opacities that are most often characterized by bilateral, inherited, noninflammatory, and progressive lesions. The opacities are caused by progressive accumulation of deposits in the cornea resulting in loss of transparency and visual impairment. Lattice corneal dystrophy (LCD) and granular corneal dystrophy (GCD) are autosomal dominant dystrophies of the corneal stroma caused by mutations in the TGFBI gene (TGFBI or BIGH3, transforming growth factor-β induced) on chromosome 5 at q31. ^{1 2} The protein product of the TGFBI gene, keratoepithelin, is an extracellular matrix protein expressed in many tissues, as well as in the corneal epithelium. ^{3 4} Its function in the cornea is not understood as yet, although it has been found to affect the adhesion of different types of cells in vitro. ⁵ In the cornea, it presumably contributes to the structure of the extracellular matrix. 

LCD (OMIM 122200; On-line Mendelian Inheritance in Man; http://www.ncbi.nlm.nih.gov/Omim/ provided in the public domain by the National Center for Biotechnology Information [NCBI], Bethesda, MD) is a primary, usually bilateral, corneal amyloidosis characterized by refractile lines that are in the form of a fine, branching network. Histologically, the deposits in LCD stain positively with Congo red and are birefringent under polarized light. LCD has at least four different subtypes (reviewed in Ref. ⁶ ). LCD type I ⁷ is an autosomal dominant, bilaterally symmetrical corneal disorder that is characterized by numerous translucent fine lattice lines that are associated with white dots and faint haze in the superficial and middle layers of the central stroma. LCD type III ⁸ is a late-onset disease of autosomal recessive inheritance that appears with decreased vision in the fifth to seventh decades of life. Asymmetrical findings are common. Lattice lines extend up to the limbus, are thicker, and are more easily seen with direct illumination than those in LCD type I. LCD type IIIA differs from type III in the presence of erosions and an autosomal dominant inheritance pattern. ⁹ Recently, LCD type IV has been described, with deep stromal opacities and late onset of disease. ⁶  

GCD type I (OMIM 121900) is characterized by small, discrete, sharply demarcated grayish white opacities in the anterior central stroma resembling bread crumbs or snowflakes. ¹⁰ Histologically, the corneal deposits stain positively with Masson trichrome and are nonamyloid. ¹¹ As the condition advances, individual lesions increase in size and number and may coalesce, extending into the deeper and more peripheral stroma. GCD type II (Avellino corneal dystrophy, OMIM 607541), shares features of lattice and granular dystrophies and has both granular and amyloid types of deposits. ¹² It is clinically similar to GCD type I. GCD type III is a superficial variant of GCD ¹³ (Reis-Bücklers dystrophy, OMIM 608470) and the deposits are morphologically similar to those in GCD type I, but are present mainly in the Bowman’s layer and beneath the epithelium. 

Phenotype-specific mutations have been characterized in the TGFBI gene. Most patients have mutations at mutational hot spots corresponding to arginine 124 and arginine 555 of the keratoepithelin protein. These include the arginine 124-to-cysteine (Arg124Cys) mutation in LCD type I, arginine 555-to-tryptophan (Arg555Trp) in GCD type I, arginine 124-to-histidine (Arg124His) in Avellino corneal dystrophy, ^{2 14} and arginine124-to-leucine (Arg124Leu) in Reis-Bücklers corneal dystrophy. ¹⁵ In addition to these common mutations, mutational heterogeneity exists, particularly in different forms of lattice dystrophy. ¹⁶  

We screened the TGFBI gene for mutations in Indian patients with LCD and GCD to determine the range of mutations underlying these diseases and to characterize the associated phenotypes. LCD and GCD account for approximately 15% to 25% of all patients with corneal dystrophy requiring corneal grafts in our institution, a tertiary-care referral center in southern India. ¹⁷ No genetic studies have been reported so far on lattice and granular dystrophies in Indians. We report the results of our study on 37 unrelated patients (18 with LCD, 19 with GCD). 

Materials and Methods

The study protocol adhered to the tenets of the Declaration of Helsinki. Corneal dystrophies were diagnosed by clinical and/or histopathological evaluation, and clinical examination was performed by slit lamp biomicroscopy. Histopathological data were available for 11 patients with LCD and 10 patients with GCD who underwent corneal transplantation. For histopathology, corneal sections were processed by standard methods and stained with Congo red or Masson trichrome. The presence of amyloid was confirmed by birefringence under polarized light. Blood samples were collected from patients and available family members after obtaining informed consent and approval of the study by the Institutional Review Board of the L. V. Prasad Eye Institute. Among the patients studied, 7 with LCD and 13 with GCD had a family history of disease, and all patients were bilaterally affected. None of the patients had any systemic manifestations. The control population consisted of 100 unrelated individuals who had no history of the diseases studied. 

Genomic DNA was isolated from blood leukocytes by standard procedures. ¹⁸ Individual exons of the TGFBI gene were amplified with primers designed by us, specific for flanking intron sequences (primer sequences available on request). PCR-amplified products of all 17 exons were screened for sequence changes by the method of single-strand conformation polymorphism (SSCP), as previously described. ¹⁹ Fragments showing altered mobility relative to control subjects were sequenced bidirectionally. Sequencing of purified PCR products was performed (BigDye Terminator Kit on a model 310 Prism sequencer; Applied Biosystems, Inc. [ABI], Foster City, CA). Sequences were compared with the published sequence of TGFBI (GenBank accession no. for genomic sequence, AY149344; mRNA sequence- NM_000358, version NM_000358.1; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). All samples that were negative for mutations on screening by SSCP, were sequenced directly in all exons to identify mutations. At least 100 normal unrelated control individuals as well as family members were screened for identified mutations to confirm pathogenicity. PCR-restriction fragment length polymorphism (RFLP) was used to test for the presence of various mutations in unrelated control subjects and family members. Restriction site changes are detailed in Table 1 . PCR products of normal and mutant DNAs were digested with the relevant restriction enzyme and resolved on polyacrylamide or agarose gels. DNA was visualized by staining with ethidium bromide. 

Results

Details of mutations and phenotypic features of patients are summarized in Table 1 . 

Mutations

Five mutations, of which three are novel, were identified in 14 patients with a diagnosis of LCD. Eight patients had a mutation of arginine 124 to cysteine (Arg124Cys), two patients had a mutation of histidine 626 to arginine (His626Arg), one patient had a mutation of valine 539 to aspartic acid (Val539Asp), two patients had a mutation of glycine 594 to valine (Gly594Val), and one patient had an in-frame deletion of two amino acids, valine 624 and valine 625 (Val624-Val625del). All the mutations detected were heterozygous in probands and were absent in 100 unrelated unaffected individuals. No mutations were identifiable after sequencing of all exons of TGFBI in the remaining four patients. Eighteen patients with a diagnosis of GCD type I had a mutation of arginine 555 to tryptophan (Arg555Trp), and one patient with a diagnosis of GCD type III (or Reis-Bücklers dystrophy) had a mutation of arginine 124 to leucine (Arg124Leu). Pedigrees with segregation of novel mutations in available family members are shown in Figure 1 . 

Clinical and Histopathological Features of Patients

LCD.

Clinical and histopathological features of patients bearing the three novel mutations identified in this study are described in the following sections. 

Val539Asp.

The slit lamp photograph of the cornea of the proband is shown in Figure 2A . The opacities were in the form of lattice lines in the anterior stroma. Cosegregation analysis of the mutation in this pedigree, shown in Figure 1A , revealed that his older son, aged 34 years, affected but asymptomatic, was heterozygous for the mutation. 

Gly594Val.

Two unrelated patients with this mutation had opacities in the posterior stroma on clinical examination. They presented with late-onset disease, in the sixth and seventh decades of life, and thick lattice lines (Figs. 2B 2C 2D)extending into the corneoscleral limbus. No histopathological data were available for these patients. Cosegregation analysis of the available members of the families of the two probands is shown in Figures 1B and 1C . As shown in Figure 1B , two offspring of the proband who were aged 44 and 40 years carried the mutation but showed no signs of disease on slit lamp examination. 

Val624-Val625del.

One patient had this mutation and showed manifestations that are atypical of LCD (Figs. 3A 3B) . He had diffuse corneal opacities with no clear lattice lines. He complained of progressive loss of vision and photophobia during his 20s and at the age of 30 years, underwent corneal grafting. The diagnosis of LCD was based on the histopathology of the corneal button, which showed amyloid deposits in the anterior stroma (Fig. 3C) . The deposits were negative for Masson trichrome. His father and two siblings were reported to be similarly affected in their 20s. The brother of the proband, who was also examined in our institution, had scarring and deposits in the superficial stroma. He was diagnosed to have corneal dystrophy of an unspecified type. After a corneal graft at the age of 30 years, histopathology revealed amyloid deposits in the Bowman’s layer and anterior stroma. The pedigree was analyzed for cosegregation of the mutation with disease, and details are shown in Figure 1D . 

GCD.

Eighteen patients with granular dystrophy had the Arg555Trp mutation and presented with features of type I GCD, with granular opacities, the extent of which ranged from the anterior third to the full thickness of the stroma (summarized in Table 1 ). 

One patient with the Arg124Leu mutation, received a clinical diagnosis of Reis-Bücklers dystrophy (GCD type III) with stromal involvement, based on the presence of multiple opacities in a honeycomb pattern in the subepithelial and superficial stromal layers (Figs. 4A 4B) . Disease had its onset during the second decade. The patient underwent corneal transplantation in both eyes with recurrence of the opacities within a few years of surgery. Histopathological evaluation revealed granular Masson-positive deposits in the stroma. 

Polymorphisms Identified in TGFBI

In addition to the mutations just described, we identified several single nucleotide polymorphisms (SNPs) in the TGFBI gene. Details are shown in Table 2 . Eight exonic polymorphisms, of which four are novel, and four intronic polymorphisms, three novel, were identified. Among the exonic polymorphisms, seven were located at the third base position of the respective codons and did not result in a codon change. 

The remaining exonic polymorphism, located in exon 7, consists of a C→T variation at cDNA position 852, and results in a change of CTT (coding for leucine 269) to TTT (phenylalanine).This change was heterozygous in 12 of 18 patients with GCD type I and in 3 of 100 unrelated normal control subjects. It cosegregated with the Arg555Trp mutation in some affected families and in one family (not included in this series), affected members were homozygous for both the Arg555Trp mutation and the Leu269Phe variant (data not shown), suggesting that the two sequence changes may be in cis. This polymorphism was absent in the patients that we studied who had LCD and Reis-Bücklers dystrophy. 

Discussion

Our study of 37 patients of Indian origin with LCD and GCD, shows that the predominant mutations causing these diseases in the patient cohort screened were Arg124Cys (found in 8/18 patients with LCD) and Arg555Trp (found in 18/19 patients with GCD). Although these two mutations have been found to be the major causes of LCD and GCD respectively in different ethnic groups, ²¹ population-specific variations in the prevalence of different mutations in TGFBI have been documented. For example, GCD type II (Avellino corneal dystrophy) caused by the Arg124His mutation, is the most common type of GCD in Japan ²² and His626Arg may be more frequent than Arg124Cys among Vietnamese patients with LCD. ²³  

We observed a broad correspondence similar to that reported in earlier studies, between mutations at arginine 124 and arginine 555 residues in TGFBI and their associated phenotypes. ^{2 14} In addition, our study demonstrates further mutational heterogeneity in TGFBI and brings to light unusual phenotypes of TGFBI-linked corneal dystrophies (Table 1) . 

The three novel mutations identified in this study were each associated with different phenotypes of LCD. All three mutations involve the fourth fasciclin-like domain in which most pathogenic alterations in TGFBI have been found. The two missense changes at valine 539 and glycine 594, as well as the in-frame deletion at valine residues 624 and 625, involve highly conserved residues, conserved among fasciclin-like domains of several proteins (NCBI Conserved Domain Database; available in the public domain at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd). 

For the Gly594Val mutation, the clinical features in both probands (Table 1)were late onset of disease and the presence of deep stromal opacities that extended up to the limbus. These manifestations are similar to those reported for two other mutations—namely, Leu527Arg ²⁴ and Val631Asp ²¹ —and have been classified as LCD type IV. ⁶ The Gly594Val mutation, to our knowledge, represents the third mutation causing this form of LCD. Cosegregation analysis in the family members revealed that two offspring of one of the patients (Fig. 1B)carried the mutation but did not manifest disease. It is possible that the mutation carriers in this family may manifest disease at a more advanced age or that this mutation has incomplete penetrance. The high degree of conservation of the residue mutated, as well as the absence of the change in 100 unrelated control subjects support the conclusion that it is pathogenic. 

The novel deletion of Val624-Val625 occurred in a patient having diffuse corneal opacities with no clinically evident lattice-type pattern and amyloid deposits in the stroma (Fig. 3) . A nonlattice pattern of corneal opacification has also been observed in association with the Arg124Cys mutation, ²⁵ thus raising the idea that factors such as advanced stage disease, ageing, environmental factors, or modifier genes contribute to the phenotype. In addition, a nonlattice phenotype resulting from a mutation in TGFBI was reported in a family with polymorphic corneal amyloidosis. ²⁶ These data together enlarge the range of phenotypic variability associated with TGFBI gene mutations and suggest that the lattice and nonlattice types of stromal amyloidoses showing autosomal dominant inheritance may be part of a spectrum of phenotypes of the same disease. 

An interesting novel polymorphism identified in this study is a change of leucine269 to phenylalanine (Leu269Phe). Two thirds of the patients with GCD (12/18) with the Arg555Trp mutation were heterozygous for Leu269Phe as were 3% of normal control subjects. Analysis of a larger cohort of families with GCD is necessary to determine whether the Leu269Phe polymorphism is in linkage disequilibrium with the Arg555Trp mutation in this population. To examine the possibility of a common origin of the Arg555Trp allele in patients with both Arg555Trp and Leu269Phe changes, we looked at the haplotypes of the other SNPs that we identified in the TGFBI gene, as well as of flanking microsatellite markers. We found that there was more than one haplotype in this group of 12 patients (data not shown). The small number of patients with GCD studied did not permit a conclusion as to whether there is a significant difference in the frequency of any haplotype between patients and control subjects. 

No mutations were identified in four patients diagnosed with LCD clinically and histopathologically (Table 1) . It is possible that mutations in these cases lie within the introns or promoter of the TGFBI gene. 

The keratoepithelin protein has four internal repeat domains, the FAS1 domains with homology to fasciclin-1, an insect cell-adhesion molecule. ²⁷ Most of the mutations so far reported lie in the fourth fasciclin-like domain. Structural modeling of the fourth FAS1 domain in TGFBI has predicted that the mutations in this domain possibly disrupt the structure by leading to misfolding of the protein. ²⁸ Mutant keratoepithelin especially for the Arg124 mutations, appears to undergo abnormal processing and/or turnover, resulting in the accumulation of mutant protein or fragments thereof. ^{29 30} It may be speculated that the mutants within FAS1 domain 4 also follow a similar route. Leucine 269 is present in the second FAS1 domain of keratoepithelin (NCBI Conserved Domain Database). Because no pathogenic alterations have been found in this region, it is possible that sequence variations, especially replacement of one hydrophobic residue with another, as in the case of Leu269Phe, are tolerated. Knowledge of the functions of the different domains of keratoepithelin may eventually provide insight into the pathogenesis of the different forms of TGFBI-linked corneal dystrophies. 

 Supported by the Hyderabad Eye Research Foundation; and the Department of Biotechnology, Government of India. SVVKC was supported by a fellowship from the Council for Scientific and Industrial Research.

 Submitted for publication April 19, 2004; revised September 2, 2004; accepted September 10, 2004.

 Disclosure: S.V.V.K. Chakravarthi, None; C. Kannabiran, None; M.S. Sridhar, None; G.K. Vemuganti, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Details of TGFBI Gene Mutations and Phenotypes of Probands with LCD or GCD

Probands (n)	Mutation in TGFBI			Restriction Site Change	Clinical Features	Histopathological Features
		Amino Acid	cDNA
8	Arg124Cys	c.417C→T	CpoI−	Anterior-to-mid-stromal lattice lines	Amyloid (4), †
2	His626Arg	c.1924A→G	—	Anterior-to-deep stromal lattice lines	Amyloid (1)
1	Val539Asp*	c.1663T→A	TaaI−	Anterior and mid-stromal lattice lines	Amyloid
2	Gly594Val*	c.1828G→T	HincII+	Posterior stromal, thick lattice lines; late onset	NA
1	Val624, Val625del*	c.1917-22del	AvaII−	Anterior stromal opacities and scarring; no clinically evident lattice lines	Amyloid in anterior stroma
4	None identified	—	—	Anterior to mid-stromal lattice lines	Amyloid
18	Arg555Trp	c.1710C→T	BstXI+	Granular opacities in anterior third to full stroma	Masson +ve (9)
1	Arg124Leu	c.418G→T	CpoI−	Subepithelial and stromal opacities with recurrence after grafting	Masson +ve deposits

 The first column shows the number of patients with each of the mutations. Restriction sites created (+) or destroyed (−) as a result of mutation are shown. NA, data not available.

*  Novel mutations.

†  Number of patients for whom data were available. In those without a number, data were available for all.

Figure 1.

 

Pedigrees of patients having novel mutations in TGFBI, showing segregation of mutations and disease status of individuals examined. (A) Val539Asp, (B) Gly594Val, (C) Gly594Val, and (D) Val624-Val625del. Only parts of the pedigree containing individuals tested are shown. M, mutant allele; +, wild-type allele. Probands are marked by an arrow at the lower left of the symbol. Cosegregation was checked by PCR-RFLP (restriction enzymes used for each mutation are in Table 1 ).

Figure 2.

 

Slit view of patient with mutation Val539Asp showing lattice lines on indirect illumination (A). Diffuse (B) and retroilluminated (C) slit lamp views of patient with Gly594Val showing thick lattice lines with intervening opacity. (D) Slit lamp view of second patient with Gly594Val showing lattice lines.

Figure 3.

 

Diffuse slit lamp view of the patient with deletion of Val624-Val625 (A) showing opacities. Note the associated spheroidal degeneration. Narrow slit view of the cornea in the same patient (B). Congo red stained section (C) when seen under polarized filters showed birefringence in the subepithelial region (arrow). Magnification: × 400.

Figure 4.

 

Diffuse (A) and slit (B) views of proband with Arg124Leu mutation showing multiple opacities in subepithelial and anterior stromal regions.

Table 2.

 

SNPs in the TGFBI Gene

Location	Polymorphism (codon)	Novel/Reported
Exon 6	c.698C→G (Leu217Leu)	Reported (20)
Exon 7	c.852C→T (Leu269Phe)	Novel
Exon 7	c.863C→T (Asn272Asn)	Novel
Exon 8	c.977C→T (His310His)	Novel
Exon 8	c.1028A→G (Val327Val)	Reported (14)
Exon 11	c.1463C→T (Leu472Leu)	Reported (14)
Exon 12	c.1631G→A (Thr528Thr)	Novel
Exon 12	c.1667C→T (Phe540Phe)	Reported (14)
IVS12	g.29683G/A	Reported (14)
IVS13	g.33618A/G	Novel
IVS13	g.33635T/A	Novel
IVS14	g.33836T/C	Novel

 Polymorphisms identified are shown according to position in cDNA or genomic DNA. Codons involved are given in parentheses. In column 3, the numbers indicate the first published report of the polymorphism. IVS, intervening sequence.