Background To describe mutations in the transforming growth factor-beta induced (TGFBI) gene in Asian patients with Bowman's membrane as well as stromal corneal dystrophies, and to elucidate their structural implications, using model peptides.
Methods Twenty-two unrelated Asian families were examined clinically including visual acuity testing and ocular examination with slit lamp biomicroscopy. Genomic DNA was extracted and the 17 exons of the TGFBI gene were amplified by PCR and sequenced bi-directionally. Biophysical techniques were used to characterise the wild type and mutant model peptides.
Results Molecular genetic analysis identified a variety of mutations in our patient series including a novel heterozygous C to A transversion mutation in exon 14 (c.1859C→A), resulting in a substitution of a highly conserved alanine residue by aspartic acid (p.A620D). Clinical presentation in the patient with the p.A620D included subepithelial scarring in addition to the linear branching opacities usually seen with lattice dystrophy. Using model peptides, we showed that A620D mutant peptide alters the secondary structure and conformational stability, and increased amyloid formation.
Conclusion A novel mutation (A620D) in transforming growth factor-beta induced protein (TGFβIp) is described, expanding the repertoire of mutations in this protein. Using model peptides, we demonstrated that A→D substitution leads to perturbation of the secondary structure that may be responsible for the amyloid formation in lattice corneal dystrophy.
- lattice corneal dystrophy
- conformational stability
- experimental and 8211 animal models
- treatment lasers
- clinical trial
- experimental and 8211 laboratory
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- lattice corneal dystrophy
- conformational stability
- experimental and 8211 animal models
- treatment lasers
- clinical trial
- experimental and 8211 laboratory
Corneal stromal dystrophies are a heterogeneous group of inherited disorders characterised by age-dependent progressive accumulation of insoluble protein aggregates leading to loss of corneal transparency and vision impairment.1 The opacities occur at various depths in the cornea depending on the nature of mutations. Ever since the discovery of four types of stromal corneal dystrophies due to mutations in the transforming growth factor-beta induced (TGFBI) gene located on chromosome 5q31, several phenotype-specific mutations have been characterised.2–4 The protein product, transforming growth factor-beta induced protein (TGFβIp), is a secreted 68-kDa extracellular matrix protein that is found in multiple cell types throughout the human body.5 It consists of an N-terminal cysteine-rich domain (EMI), four internal repeat domains called fasciclin-like (FAS1) domains, which are very similar to each another, and a C-terminal RGD sequence.6 The first and fourth FAS1 domain of TGFβIp has been identified as mutational hot spots. The fourth FAS1 domain alone accounts for 45 different mutations out of 57 mutations reported to date. Mutations reported include single residue substitution, deletion of one or two contiguous amino acid residues, double mutations, and replacement of two contiguous residues with three.4 The clinical features of the deposition vary from rod-shaped crystalloid structures, amyloid, a combination of both rod-shaped bodies with amyloid, or curly fibres.7
Lattice corneal dystrophy (LCD) is the most common form of corneal stromal dystrophy characterised by deposits of extracellular amyloid within the cornea, and manifest clinically with multiple linear branching or fusiform corneal opacities in the anterior to mid-stromal level.7 LCD may be further classified into four types based on clinical features and depth of the deposits, with LCD types I, IIIA and IV and ‘atypical’ LCD associated with mutations in TGFBI. The exact mechanism(s) responsible for the clinical and histopathological features observed in patients with TGFBI mutations remains unclear.4 7 It has been hypothesised that mutations in the TGFBI gene result in changes to the protein structure or function, which leads to accumulation of insoluble extracellular material in the cornea.3 8 9
Here we report the results of a molecular genetic analysis of 22 unrelated patients who presented to the Singapore National Eye Centre between 1999 and 2008. We also describe the clinical and molecular genetics of a Chinese patient with lattice dystrophy carrying a novel mutation (A620D) in a highly conserved region of the TGFBI gene. To characterise mutant protein behaviour and understand structure–function relationships, we used synthetic model peptides to examine the impact of A620D mutations on secondary structure, stability and amyloid formation. The results indicate that this LCD mutant causes a significant destabilisation of the secondary structure of the protein that may play an important role in the formation of amyloid fibrils and the observed phenotype.
Materials and methods
Patients and clinical diagnosis
Twenty-two people from 22 families who were referred to Singapore National Eye Centre from 1999 to 2008 were examined. Clinical examinations included manifest refraction, visual acuity, contrast sensitivity with glare, corneal sensation, applanation tonometry, pupil reactions, motility, visual fields, corneal pachymetry and dilated fundus examination. Slit lamp examination and photography were performed to assess and document the deposits. Anterior segment optical coherence tomography (ASOCT; Carl Zeiss Meditec, Jena, Germany) was performed to assess depth of the lesions.
Molecular genetic analysis
Genomic DNA was extracted (Nucleon blood extraction kit; Amersham, Buckinghamshire, UK) from leucocytes of the peripheral blood of all participating subjects. All 17 exons of TGFBI gene were amplified by PCR as described previously.10 The PCR products were purified using PCR clean-up kits (Axygen Biosciences, Union City, California, USA) and sequenced using Big Dye Terminator v3.1 chemistry (Applied Biosystems, Foster City, California, USA). Bi-directional sequencing of amplicons was carried out on an ABI prism 3100 genetic analyser (Applied Biosystems). The nucleotide sequences were analysed using the Lasergene V.8.0 software (DNASTAR, Inc., Madison, Wisconsin, USA) and compared with the published TGFBI cDNA sequence (GenBank accession no. NM_000358). Mutation numbering was based on this reference sequence with +1 corresponding to the A of the ATG translation initiation codon.
All the peptides were purchased from EZBiolab Inc. (Indiana, Carmel, USA). The purity of the peptides were confirmed by HPLC and mass spectrometry, and provided by the supplier.
Far ultraviolet circular dichroism spectropolarimetry
Circular dichroism (CD) studies were carried out on a JASCO-810 spectropolarimeter (JASCO, Tokyo, Japan) equipped with a peltier setup. The peptides (0.2 mg/ml) were dissolved in 10 mM phosphate buffer (pH 7.0). The far UV-CD spectra were recorded using a 0.1 cm path length cell under constant nitrogen flush (∼30 l/min) with a step size of 0.1 nm, bandwidth of 2 nm, and an averaging time of 3 s. The final spectrum reported was an average of eight scans. For comparison, the CD intensity is expressed in mean residual weight ellipticity ([θ]mrw). For the guanidine hydrochloride (GdCl) denaturation experiments, the peptides were incubated at various concentration of GdCl (0.25–7 M) in phosphate-buffered saline (PBS) for 16 h and the spectra were recorded from 210 to 260 nm at 25°C. The denaturation data were fitted by non-linear least squares method, which assumes a two state model.11
Thioflavin T assay
Thioflavin T (ThT; 25 μM) was prepared in PBS. About 0.6 mg/ml of the peptide in PBS (pH 7.0) was shaken on a Thermomix shaker at 37°C. At regular time intervals, a 50 μl aliquot of the peptide solution was added to 325 μl of ThT. The steady state fluorescence of the samples were measured on a QuantaMaster spectrofluoromenter (Photon Technology International, Birmingham, New Jersey, USA) using a 10 mm path length quartz cuvette. The excitation and emission wavelengths were set 440 nm and 485 nm, respectively.
Transmission electron microscopy
A 5 μl aliquot of the peptide sample (incubated at 37°C for 48 h) was dropped on a Formvar-coated copper grid (300 mesh; FMB industries Pte Ltd, Singapore) for 5 min and the excess sample was removed by absorbing with the filter paper. A drop of uranyl acetate solution (0.4%) was placed on top of the sample for 5 min. Then the grid was washed twice with water and allowed to dry for 10 min. The grid was viewed and photographed on a JEOL JEM 1010 (JEOL Ltd., Tokyo, Japan) transmission electron microscope.
Mutation analysis and identification of a novel A620D mutation in TGFBI
Table 1 summarises the clinical diagnosis and ethnicity of patients and TGFBI mutations identified in this study. TGFBI gene mutations at the codons R124 and R555 were the most common in our Chinese patients. We observed that these two sites account for ∼78% of mutations in our study. A heterozygous R555Q mutation was detected in one patient initially diagnosed as having Reis–Bucklers dystrophy and led to the re-classification of the diagnosis to Thiel–Behnke dystrophy based on the results of the molecular genetic analysis. We also observed two Chinese patients with LCD exhibiting H626R mutation of the TGFBI gene (figure 1C and D).
A novel mutation in TGFBI gene was also detected in a Chinese patient with LCD. She was first presented with symptoms when she was 22 years old with a history of recurrent corneal erosions. Over the 10 years of follow-up she has had multiple episodes of recurrent corneal erosions treated medically with topical antibiotics, topical ointments such as oral doxycycline, oral TheraTears, and when needed with bandage contact lenses. Her vision has fluctuated depending on the flare-up of erosion and at the time of the study the best spectacle-corrected visual acuity (BSCVA) was 20/40 right eye 20/30 left eye. She has had a documented photographic increase in the amount of subepithelial scarring associated with her lattice dystrophy mainly in the paracentral area. There were multiple radially oriented linear opacities in the anterior to mid-stromal depth of central cornea with subepithelial and anterior stromal scarring bilaterally (figure 1A,B).
During her follow-up, she experienced five episodes of recurrent corneal erosions over 16 months, which was resolved with topical medication, but increased the amount of subepithelial scarring. A novel nucleotide substitution, c.1859C→A, was detected in the heterozygous state in exon 14 of this patient, resulting in a missense mutation, p.Ala620Asp (figure 2A). All coding exons and exon–intron boundaries of TGFBI were screened in this patient. The A620D mutation was the only the pathogenic mutation identified. Two previously reported synonymous mutations, F540F and L472L as well intronic polymorphisms (IVS7+47T→C and IVS12+23G→A) were also detected in this patient. The c.1859C→A sequence variant was not detected in 100 unrelated Chinese control samples. The A620D mutation is located at the C-terminus of the fourth FAS1 domain of TGFβIp, which is highly conserved residue across a number of species (figure 2B). The parents of the proband did not consent to genetic testing but they had no abnormalities on ocular examination.
Design and structural characterisation of TGFβIp model peptides
The x-ray crystal structure of the fourth FAS1 domain of TGFβIp has been reported.12 The tertiary structure is divided into two halves by α-helix and β-sheet structures, that is, the first half of the sequence contains predominantly α-helices whereas the second half consists of β-sheet conformation only (figure 3A). To examine the influence of mutation, we synthesised a 23-residue peptide encompassing the C-terminus of fourth FAS1 domain (underlined in figure 3A) and its A620D variant. Since aspartic acid possesses a low propensity to form a β-sheet structure compared with alanine, we sought to investigate the effect of the substitution on secondary structure and conformational stability.13 For convenience, we describe the 23-residue peptide as TGFβIp611–633wt and mutant peptide as TGFβIp611–633A620D.
Far UV-CD studies indicated that TGFβIp611–633wt exhibited a strong negative minimum in the π-π* region (200 nm) and weak shoulder in the n-π* region (215–225 nm, figure 3B). Since the peptides do not contain aromatic amino acid residues, the shoulder in n-π* region is ascribed to the presence of a β-sheet structure in equilibrium with the unordered conformation. For TGFβIp611–633A620D peptide, the CD intensity at 200 nm is increased, whereas the intensity of the shoulder in the n-π* region decreased, indicating a reduction in β-sheet conformation.14
In order to determine to what extent the mutations influence the conformational stability, we performed GdCl denaturation experiments at 25°C. The CD intensity in the n-π* region decreased upon increasing the concentration of GdCl for both the peptides studied indicating loss of secondary structure (figures 3C,D). The change in CD intensity at 222 nm as a function of GdCl concentration is shown in figure 3E. A smooth co-operative unfolding has been observed for TGFβIp611–633wt with increasing concentration of the denaturant. Analysis of the denaturation curve as described by Pace and Shaw11 allowed the determination of the free energy of unfolding in the absence of denaturant (ΔGH2O), midpoint of the denaturation transition (Cm), and m value, which is a measure of dependence of ΔG on GdCl concentration (table 2). The results indicated that the mutant peptide is substantially destabilised compared with TGFβIp611–633wt.
Amyloid formation observed by ThT fluorescence and electron microscopy
To analyse the effect of A→D substitution on amyloid formation, TGFβIp611–633wt and mutant peptide was probed by ThT assay. Figure 4 shows the time-dependent changes in the ThT fluorescence intensity of the two peptides. Both wild type and mutant peptides showed a progressive increase in fluorescence intensity with time, confirming the formation of amyloid. However, the mutant formed amyloid more rapidly than TGFβIp611–633wt. Transmission electron microscopy of the peptides (figures 5A,B) confirmed the presence of amyloid fibrils in the wild type and mutant peptides, confirming the results of the ThT assay.
In this study, we have expanded the mutation spectrum of TGFBI gene by identifying nine different mutations in Asian patients, including a novel mutation, A620D, identified in a Chinese patient in her early 30s and associated with LCD. A greater amount of subepithelial and anterior stromal scarring was seen in this patient compared with other cases of LCD. The parents of this patient were clinically unaffected, indicating either incomplete penetrance or a de novo mutation event in this patient. We were unable to determine the genetic status of this locus in her parents as they did not consent to genetic testing. Interestingly, incomplete penetrance or variable expressivity was also observed in a Hispanic family in association with mutation M619K, affecting the adjacent residue to A620.15 Although the clinical features associated with M619K mutation were generally age-dependent, there were exceptions as the 55-year-old sibling of the proband demonstrated significantly greater corneal involvement than her 75-year-old mother. It is therefore plausible that one of the parents may have very subtle asymptomatic corneal deposits. Alternatively, the parents could be asymptomatic due to the actions of as yet unknown environmental factors or other modifying genetic factors in his/her genetic background preventing the misfolding/aggregation of the mutant protein. Although not very likely, the A620D mutation could also be a de novo mutation. The H626R mutation, located at the β-strand 9 of the fourth FAS1 domain and found in two of our Chinese patients, presents in diverse ethnic groups and occurs with relatively high frequency among Vietnamese people.16 It is interesting to note that almost all of the mutations in this region are associated with generally a late-onset LCD.4
The mutation of H572R, which is located close to the α-helix six of the fourth FAS1 domain, has been previously reported in a large Thai family with LCD type I.17 This is the second report of this mutation, also occurring in a Thai patient. The L550P mutation, which is located in α-helix 5, observed in the present study in an Indonesian patient has only been previously described in a Mexican family with granular corneal dystrophy (GCD) type II, and this is the first report of this mutation in an Asian patient with the same phenotype.18 A heterozygous R555Q mutation (also located in the α-helix 5) was detected in one patient initially diagnosed as having Reis–Bucklers dystrophy and led to the re-classification of the diagnosis to Thiel–Behnke dystrophy based on the results of the molecular genetic analysis.
Protein misfolding diseases including corneal dystrophies involve conversion of specific peptides or proteins from their soluble functional state to an insoluble and highly organised fibrillar aggregate state.19 Though the fibrillar aggregates are quite similar, proteins associated with these diseases differ considerably in terms of amino acid composition, secondary structure and chain length. For a number of globular proteins (both disease and non-disease associated proteins) an inverse correlation has been observed between conformational stability and tendency to form amyloid fibrils in vitro.20 The extent of unfolding and the fibril morphology depends on the degree of destabilisation. In the present study, we have used model peptides modified with specific mutation (A620D) to understand the extent of destabilisation. Far UV-CD spectra indicated that substitution of alanine to aspartic acid (A620D) decreased the β-sheet structure. These results suggest that A620D, a unique mutation found in the present study, unfolds more readily than the wild type peptide.
Using the equilibrium GdCl denaturation assay, we also found that the mutant denatures at lower guanidine concentration compared with the wild type peptide. Earlier studies with full length TGFβIp showed the formation of amyloid-like fibrils and that this property depends on pH, concentrations of trifluoroethanol and GdCl.21 Both trifluoroethanol and GdCl unfold the recombinant TGFβIp and results in the formation of protofibrils.21 Interestingly, the synthetic wild type peptide used in the present study, TGFβIp611–633wt, showed an identical midpoint of the denaturation (3.5 M GdCl) as reported for the full length TGFβIp (3.55 M GdCl).21 ThT assay and electron microscopy further confirmed that wild type and mutant peptides formed amyloid fibrils, and that the mutant peptide formed more readily than the wild type peptide under physiological conditions. A study by Schmitt-Bernard et al using model peptides of the first FAS1 domain of TGFβIp (L110–E131) showed that R124C, which causes LCD type I, showed an increased amyloid formation compared with the wild type peptides.22 Similarly, Yuan et al have shown that a short peptide in the fourth FAS1 domain (F515–N532) and the L527R variant formed amyloid fibrils, and that the variant has about a 10-fold increased propensity to form amyloid fibrils under physiological conditions.23 These results suggested that model peptides can be used to understand the mechanism of amyloid formation. Using equilibrium GdCl, we showed that significant destabilisation of the secondary structure in the mutant which may play an important role in the formation of amyloid fibrils. These results augment the recent observations that A546T mutant of TGFβIp readily forms amyloid fibrils owing to the destabilisation of the native structure.24 Therefore, we suggest that LCD mutant A620D populates the unfolded states, and contributes to the phenotype.
In conclusion, we report a range of TGFBI gene mutations in an Asian population. We also report a novel mutation in a Chinese patient with LCD that was linked to a mutation resulting in an alanine to aspartate switch at codon 620. This mutation was associated with a greater amount of corneal scarring phenotypically compared with other cases of lattice dystrophies. Using model peptides, we have also identified a previously unreported amyloidogenic region in the fourth FAS1 domain and showed that the mutation destabilises the secondary structure, thereby predisposing to amyloid formation.
We thank Lim Wei Ting for technical assistance. Our work was supported by TCR grant (R626/41/2008, R620/41/2008 and R618/41/2008) from NMRC, Singapore.
Competing interests None to declare.
Patient consent Obtained.
Ethics approval Institutional Review Board, Singapore National Eye Centre, Singapore. All parts of the study were performed according to the tenets of the Declaration of Helsinki.
Provenance and peer review Not commissioned; externally peer reviewed.
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