|Molecular Vision 2006;
Received 30 June 2005 | Accepted 20 February 2006 | Published 27 February 2006
Unilateral lattice corneal dystrophy associated with the novel His572del mutation in the TGFBI gene
Anthony J. Aldave, Sylvia A.
Rayner, Brian T. Kim, Apiradi Prechanond, Vivek S. Yellore
Cornea Service, The Jules Stein Eye Institute, University of California Los Angeles Medical Center, Los Angeles, CA
Correspondence to: Anthony J. Aldave, MD, Assistant Professor, The Jules Stein Eye Institute, 100 Stein Plaza, UCLA, Los Angeles, CA, 90095; Phone: (310) 206-7202; FAX: (310) 794-7906; email: email@example.com
Purpose: To report a novel mutation in the TGFBI gene, c.1761_1763del (p.His572del), associated with a unilateral variant of lattice corneal dystrophy (LCD).
Methods: A 63-year-old man presenting with the complaint of decreased vision in one eye was noted to have a unilateral lattice corneal dystrophy. Examination of the patient's wife and two sons, ages 20 and 27 years old, failed to reveal the presence of any corneal opacities. Following the collection of DNA from the patient and his family members, the TGFBI gene was screened for mutations previously associated with lattice corneal dystrophy and any novel coding region changes.
Results: In the affected patient, none of the mutations previously associated with the classic and variant forms of LCD were identified. However, a novel mutation, c.1761_1763del (p.His572del), was identified in exon 13 of TGFBI in the patient and his sons. This mutation was not identified in the patient's wife or in 200 control chromosomes.
Conclusions: The novel TGFBI gene mutation (p.His572del) is associated with a unilateral, late-onset variant of lattice corneal dystrophy. This case highlights the utility of molecular genetic analysis in differentiating corneal dystrophies associated with an atypical phenotype from nondystrophic conditions.
Lattice corneal dystrophy type I (CDL1, LCD; OMIM 122200) has been associated with a large number of missense mutations in the transforming growth factor beta induced (TGFBI) gene. While all corneal dystrophies, including the TGFBI dystrophies, typically present with bilateral, symmetric involvement, unilateral and markedly asymmetric variants of LCD have been reported [1-3]. We report the case of a patient with decreased vision but no history of recurrent corneal erosions who presented with a unilateral, late-onset variant of LCD. Screening of all 17 exons of TGFBI revealed a novel mutation, c.1761_1763del, in exon 13 that resulted in a deletion of the amino acid histidine at codon 572. This case represents the first report of this novel mutation in the TGFBI gene, the second report of a pathogenic mutation in exon 13 of TGFBI, and adds to the number of mutations associated with the late-onset, unilateral LCD phenotype.
Report of a case
A 63-year-old man was referred for evaluation of unilateral LCD in the right eye. He complained of a gradual decline in the vision in the right eye for several years and was noted to have branching stromal opacities in the right cornea three years previously. The patient denied previous contact lens wear, ocular trauma, or a personal or family history of recurrent corneal erosions, and he was unaware of a family history of corneal disease (Figure 1).
Corrected visual acuity measured 20/30 OD and 20/15 OS. The right cornea demonstrated linear, branching, anterior and midstromal opacifications with clear intervening stroma (Figure 2A,B). The left cornea was devoid of any opacities (Figure 3).
Neither the patient's wife nor either of his two sons, ages 20 and 27 years old, demonstrated any corneal deposits. As both parents and one of the patient's three siblings were deceased, and his other two siblings lived in Germany, none were available for examination or DNA analysis.
DNA collection and preparation
After institutional review board approval was granted (UCLA IRB 94-07-243-22B), informed consent was obtained from the patient, his wife, and two sons. Buccal epithelial swabs were collected from each using CytoSoft CP-5B brushes (Medical Packaging Corporation, Camarillo, CA). Genomic DNA was prepared from the buccal epithelial cells using the QIAamp DNA Mini Kit spin protocol (Qiagen, Valencia, CA). DNA collected from 100 unaffected healthy volunteers served as controls.
All 17 exons of the TGFBI gene were amplified from the patient's DNA using custom-designed oligonucleotide primers (Table 1). After identification of the c.1761_1763del in the patient, exon 13 of the TGFBI gene was amplified from his family members' DNA. Each reaction was carried out in a 25 μl mixture containing 12.5 μl of MasterAmp PCR 2X PreMix "D" (100 mM Tris-HCl pH 8.3, 100 mM KCl, 400 μM of each dNTP, proprietary concentrations of MgCl2, and MasterAmp PCR Enhancer; Epicentre, Madison, WI), 0.12 μM of each primer, 1.5 units of RedTaq Genomic DNA polymerase (Sigma-Aldrich Corp., St. Louis, MO), and approximately 100 ng of genomic DNA. Thermal cycling was performed in an iCycler Thermal Cycler (Bio-Rad, Hercules, CA).
Purification of the PCR products was achieved by incubating 15-30 ng DNA with 5 units of Exonuclease I and 0.5 units of Shrimp Alkaline Phosphatase (USB Corp., Cleveland, OH) for 15 min at 37 °C. After inactivation of the nucleases (80 °C for 15 min), sequencing reactions were performed by the addition of 2 μl BigDye Terminator Mix version 3.1 (Applied Biosystems, Foster City, CA), 2 μl of SeqSaver (Sigma-Aldrich) and 0.2 μM primer. Samples were denatured at 96 °C for 2 min, then cycled 25 times at 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min. Unincorporated nucleotides were removed using the CleanSeq reagent and a SPRI plate (Agencourt Bioscience Corporation, Beverly, MA) following the manufacturer's instructions and then analyzed on an ABI-3100 Genetic Analyzer (Applied Biosystems) after resuspension in 0.1 mM EDTA. Nucleotide sequences were compared with the published TGFBI cDNA sequence (GenBank NM_000358).
Polymerase chain reaction restriction fragment length polymorphism
After identification of the c.1761_1763del in the patient, a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay was performed to screen for the deletion in 100 control individuals. This mutation abolishes a site recognized by the restriction enzyme Msi1 (NEB, Beverly, MA), permitting distinction of sequence variants from the wild type. PCR products of the control DNAs were digested with the restriction enzyme and resolved on a 2% agarose gel. The products were then visualized with SYBR Green 1 staining. (Molecular Probes, Eugene, OR)
None of the mutations previously associated with either the classic or variant forms of LCD were identified in the patient. However, a novel deletion, c.1761_1763del, which resulted in an in-frame deletion of a single amino acid (p.His572del), was identified in the heterozygous state in exon 13 (Figure 4). This deletion was not identified in the patient's wife, but was identified in both of the patient's sons. The only other allelic variant noted in the patient was a previously identified polymorphism, Leu217Leu (refSNP ID: rs1442), in exon 6. The c.1761_1763del mutation was not identified in any of 200 control chromosomes.
The identification of the TGFBI gene and characterization of the mutations associated with both the classic and variant forms of LCD have given clinicians the ability to definitively differentiate between the various manifestations of LCD and the myriad presentations of pseudolattice-corneal dystrophy [4-6]. In the case we report, initial screening of the TGFBI gene did not reveal any pathogenic mutations previously associated with classic and variant forms of LCD [7,8]. However, complete screening of all 17 exons of the TGFBI gene revealed a presumed pathogenic mutation in exon 13, resulting in the deletion of histidine at codon 572. The only other mutation previously reported in exon 13 of the TGFBI gene was a single base pair substitution (c.1753T>G) resulting in an amino acid substitution at codon 569 (p.Leu569Arg) that was also associated with lattice corneal dystrophy, albeit the classic CDL1 phenotype . The association of the p.Leu569Arg mutation with the classic LCD type I phenotype, which is typically associated with the p.Arg124Cys mutation [7,8], and the association of the p.His572del mutation with the late onset, unilateral LCD phenotype, which has been previously identified with the Leu527Arg, Asn622His, and His626Arg mutations [1,3], underscore the need to screen the entire coding region prior to excluding a TGFBI corneal dystrophy.
Evidence to support the pathogenicity of the 3 base pair in-frame deletion, c.1761_1763del, is provided by previous descriptions of two other in-frame deletions in the TGFBI gene associated with variants of granular (p.Thr125_Glu126del)  and lattice (p.Val624_Val625del)  corneal dystrophies. In contrast to the novel mutation we report, these deletions involved two adjacent amino acids, and although both were associated with an atypical phenotype, neither was associated with unilateral involvement. However, these reports demonstrate that in-frame deletions in the TGFBI gene, in addition to the more commonly described missense mutations, may be associated with the development of lattice and granular corneal dystrophies. Additionally, the absence of the c.1761_1763del allelic variant in 200 control chromosomes provides further evidence that this mutation is associated with the observed unilateral LCD phenotype. The identification of the same deletion in the patient's unaffected 20- and 27-year-old sons is not surprising given the late onset of the disease phenotype in the patient. Unfortunately, the siblings and parents of the patient were not available for examination to perhaps provide a more complete characterization of the clinical features associated with the c.1761_1763del mutation.
The discovery of this novel mutation, of course, does not explain why the dystrophy that we describe, or any corneal dystrophy for that matter, is associated with unilateral involvement when the epithelial cells and keratocytes of the contralateral cornea harbor the same TGFBI gene mutation. The answer likely resides in the influence of modifier genes and environmental effects. The wide variation in the affected phenotype that has been demonstrated both between families sharing a common mutation in the TGFBI gene and between members of a single family with similar environmental influences suggests a role for both environmental factors and modifier genes [11-14]. Stewart and colleagues have proposed that factors such as the development of recurrent corneal erosions may explain the development of unilateral LCD, as they have noted the development of corneal amyloid deposition shortly after the onset of recurrent corneal erosions or other forms of corneal trauma . Thus, a unilateral corneal abrasion or the development of unilateral or markedly asymmetric corneal erosions may precipitate the development of corneal amyloid deposition, with an uninvolved contralateral eye. The authors have also noted an association between disruption of the corneal epithelium, in association with recurrent corneal erosions and trichiasis, and nondystrophic, unilateral corneal stromal amyloid deposition [4,5]. Others have also noted an association between corneal injury in the form of LASIK surgery and a dramatic acceleration in the number and density of dystrophic corneal deposits, concentrated in the interface between the flap and residual stroma, in a series of patients with combined granular-lattice corneal dystrophy [15,16].
The patient that we report denied a history of recurrent corneal erosions, previous ocular surgery, or ocular trauma. Thus, the explanation for the unilateral nature of the corneal changes remains enigmatic. The identification of modifier genes that may alter the expression of the TGFBI corneal dystrophies would provide not only a greater understanding of the factors that determine age-dependent expression and phenotype variability, but also a target for gene-based therapeutic interventions.
The authors thank the Emily Plumb Estate and Trust for their support.
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