Trinucleotide Repeat Expansion in the TCF4 Gene in Fuchs' Endothelial Corneal Dystrophy in Japanese

Abstract

Purpose: The purpose of this study was to evaluate the association between the intronic expansion of a trinucleotide repeat (TNR) in the TCF4 gene and Fuchs' endothelial corneal dystrophy (FECD) in a Japanese population.

Methods: Forty-seven Japanese FECD patients and 96 age-matched controls were recruited. FECD patients and controls were examined by slit-lamp and noncontact specular microscopy. The repeat length was determined by direct sequencing and short tandem repeat assay of PCR-amplified DNA and Southern blotting of unamplified DNA.

Results: A TNR expansion, defined as >50 CTG repeats in the TCF4 gene was identified in 12 of 47 FECD cases (26%) and 0 of 96 controls (0%; P < 0.001). Sensitivity and specificity in this study were 26% and 100%, respectively. The clinical characteristics of FECD patients with TNR expansion were not distinct from those without TNR expansion.

Conclusions: These findings show for the first time in a Japanese population the association of the TNR expansion in TCF4 with FECD. In contrast to Caucasian cohorts in whom the TNR expansion is present in most patients with FECD, a CTG expansion is present in a minority of Japanese subjects, indicating other genetic variants as common causes of phenotypically identical disease in this population.

Fuchs' endothelial corneal dystrophy (FECD) is a bilateral, inherited degenerative disease of the corneal endothelium. This nonreplicating tissue layer is essential for maintaining corneal clarity through an active ionic pump function and barrier function. Mild FECD is asymptomatic, but advanced stages result in a functional impairment of the endothelial layer with eventual corneal edema, vision loss, and pain. Corneal transplantation is the only therapeutic option for treating FECD,¹ and it is the most frequent indication for corneal transplantation, accounting for more than 60% of endothelial keratoplasty procedures performed in the United States.² 

FECD is an autosomal dominant disease with wide variation in penetrance and expression.³ A rare form of early onset FECD is associated with a mutation in COL8A2.^4,5 The common form of late-onset FECD has been linked to chromosomes 13,^6,7 18,⁸ 5,⁹ and 9.¹⁰ In addition, missense mutations in the zinc finger E-box binding homeobox 1 (ZEB1) gene^10,11 and the solute carrier family member 4A11 (SLC4A11) gene^12,13 were suggested to be causes of late-onset FECD, although those mutations were detected in a small proportion of the FECD population. 

A genome-wide association study by Baratz et al.¹⁴ identified an association between late-onset FECD and the intronic single nucleotide polymorphism rs613872 in the transcription factor 4 (TCF4) gene on chromosome 18, and both association and linkage analyses performed by subsequent investigators replicated this association.^15–18 Another study reported that FECD in families mapped to the FCD2 locus on chromosome 18 did not cosegregate with rs613872, suggesting that FCD2 was independent of this minor allele.^15,16 In 2012, Wieben et al.¹⁹ revealed that the expansion of a CTG trinucleotide repeat (TNR) in a different intron of TCF4 was commonly detected in FECD and that the sensitivity and specificity of >50 repeats identifying FECD were 79% and 96%, respectively. In addition, subsequent investigators recently replicated the TNR expansion in other FECD cohorts.^20–22 

Here we present additional evidence for the association between the intronic CTG expansion in TCF4 with late-onset FECD in Japanese. We demonstrated that a repeat length >50 repeats was identified in 12 of 47 FECD cases (26%) and 0 of 96 controls (0%), with a sensitivity and specificity of 26% and 100%, respectively. We also showed that the clinical phenotype and severity of disease status of FECD patients with TNR expansion were not different from those without expansion, suggesting that genetic abnormalities responsible for non−expansion-associated disease cause an indistinguishable phenotype. 

Materials and Methods

Ethical Statements

This study was approved by the Institutional Review Boards of Kyoto Prefectural University of Medicine and the Mayo Clinic and was conducted in accordance with the ethical principles of the Declaration of Helsinki. Written informed consent was obtained from all FECD and normal participants. 

FECD Patients and Control Subjects

Forty-seven FECD and 96 healthy Japanese subjects were recruited between September 2010 and April 2014 at the University Hospital of Kyoto Prefectural University of Medicine in order to provide peripheral blood samples. An investigator who was masked to both the blood sampling and the experiments assigned an anonymous code to each blood sample. 

All participants were examined by slit-lamp biomicroscopy (Pentacam; Oculus Optikgeräte GmbH, Wetzlar, Germany) and noncontact specular microscopy (EM3000; Tomey Corporation, Nagoya, Japan; if images were obtainable). FECD cases were graded based on a scale proposed by Krachmer et al.²³ and had a minimum grade of 1, which represented 12 or more central nonconfluent guttae. Control subjects were selected based on (1) age > 60 years old, (2) absence of guttae observed by slit-lamp microscopy and noncontact specular microscopy, and (3) no other corneal disease. 

Measurement of TCF4 Triplet Repeat Length

Genomic DNA was isolated from 350 μL of peripheral blood by BioRobot EZ1 (Qiagen, Valencia, CA, USA) using the EZ1 DNA blood 350 μL mini-kit (Qiagen), according to the manufacturer's instructions. The amount and quality of each isolated DNA sample was analyzed by UV spectrophotometer (NanoDrop; NanoDrop Technologies, DE, USA) and stored at −80°C until use. In order to measure the length of the CTG repeat in the third intron of the TCF4 gene, we first performed a short tandem repeat assay as described previously.¹⁹ Briefly, PCR of the repeat region was performed using one fluorescent-labeled primer. Following amplification, the size of the product was determined by capillary electrophoresis (3730xl model DNA analyzer; Applied Biosystems, Grand Island, NY, USA). 

For samples demonstrating a single signal from the short tandem repeat assay, we performed Southern blotting using unamplified DNA as previously described¹⁹ to confirm whether each subject possessed either two short alleles of the same repeat length or had an additional allele with a CTG expansion too large to detect by the PCR-based short tandem repeat assay. 

Statistical Analysis

In order to examine the possible confounding effects of sex and age of the subjects, we assessed the correlations between the case and control samples by means of Student's t-test or chi-square test (Table 1). Fisher's exact test was used for analyzing the difference in the frequency of patients and controls in the three groups divided by the length of TNR (>50, 40–50, and <40 repeats) (Table 2). We also applied the Wilcoxon rank sum test and Spearman's rank correlation coefficient to determine whether there were significant correlations between the clinical parameters and the TNR length. Results are expressed as means ± SD. 

Table 1

 

Sample Information

 

Table 2

 

TGC Repeat Length in TCF4 Genes of FECD Patients and Normal Controls

 

Results

Clinical Manifestations of FECD in Japanese Patients

Demographics of the subjects participating in this study are shown in Table 1. No differences were observed in male-to-female ratios or subject age between the FECD and control groups. All control subjects exhibited a normal morphological corneal endothelium without guttae (Fig. 1A). In contrast, all FECD patients exhibited guttae formation associated with decreased corneal endothelial cell density. Coincident with FECD in Caucasians, guttae formation and morphological abnormalities of the endothelium were more evident in the center than in the periphery of corneas, and edema started from the center in early FECD cases (Figs. 1B, 1C). 

Figure 1

 

Clinical manifestations of FECD in Japanese patients. (A) Noncontact specular microscopy image of normal subject. (B) Noncontact specular microscopy images of an FECD patient with 30 CTG repeats. Images were obtained from the corneal center and mid-periphery. (C) Representative slit-lamp photograph from an FECD patient with 70 CTG repeats.

CTG Repeat Expansion of TCF4 in Japanese Subjects

The lengths of CTG TNRs for 47 FECD cases and 96 controls are listed in Table 2. Among FECD participants, 12 of 47 cases (26%) possessed >50 CTG repeats (Table 2); of note, one FECD patient had >100 repeats, and Southern blotting confirmed an expansion of ∼2600 repeats. Similar to findings in a previous study,¹⁹ the distribution of repeat length among affected subjects was bimodal; no subjects had between 31 and 59 repeats (Fig. 2). No control subject possessed more than 41 repeats. The sensitivity and specificity values of an expanded repeat length to determine an FECD case in our subjects were 26% and 100%, respectively; however, the threshold value of an “expanded repeat” could not be determined due to the absence of cases or controls with repeat lengths between 42 and 59. 

Figure 2

 

Frequency histogram of the CTG repeat length. CTG repeat length of the longest allele in all samples of both FECD cases (black bars) and normal control (white bars) samples. Note that one FECD patient had more than 100 repeats.

Correlation Between Clinical Parameters and Repeat Length

We assessed the correlation between the TNR length and clinical parameters in the case subjects. As a result, there was no significant correlation between the length of TNR and visual acuity (P = 0.989) or central corneal thickness (P = 0.124). Additionally, there were no differences in these parameters between the FECD groups, based on the presence or absence of TNR expansion (Figs. 3A–D). Notably, the mean ages of the two groups were similar (TNR+ mean age = 68.3 ± 9.3 years; TNR− mean age = 68.5 ± 13.8 years.) Our subjective evaluation of corneal appearance by slit-lamp biomicroscopy and noncontact specular microscopy was unable to distinguish between cases with and without TNR expansion (Figs. 4A–F). 

Figure 3

 

Correlation between the TGC repeat length and visual acuity; central corneal thickness in the case subjects. (A, B) Graph shows the correlation between TGC repeat length and visual acuity. (C, D) Graph shows the correlation between TGC repeat length and central corneal thickness.

Figure 4

 

Noncontact specular microscopy images and slit-lamp photographs of FECD patients. (A, B) A 56-year-old woman with 25 CTG repeats. (C, D) A 55-year-old man with 100 CTG repeats. (E, F) A 68-year-old woman with ∼2600 CTG repeats.

Discussion

FECD is the most common inherited corneal dystrophy, and as many as 5% of the population over 40 years of age in the United States may exhibit guttae.²⁴ Accordingly, visual loss due to FECD is the most frequent indication for corneal transplantation in the United States² but less commonly so in Asian countries such as Japan. Data comprising the prevalence of FECD in Japan is not well elucidated, but a national survey described FECD as an indication for keratoplasty in 1.9% of cases of bullous keratopathy between 1999 and 2001.²⁵ Similarly, FECD accounted for only 16.6% of penetrating keratoplasty in India.²⁶ 

Evidence is accumulating that variation in TCF4 and particularly the intronic CTG repeat expansion^19–22,27 contributes to FECD. The apparent differences in prevalence of FECD between Caucasian and Asian populations led us to investigate whether Japanese FECD patients share the intronic TNR expansion in TCF4. In this study, we showed that 12 of 47 FECD patients (sensitivity = 26%) harbored a heterozygous CTG expansion of >50 repeats in comparison to no control patients (specificity = 100%). A homozygous expansion was not found in any participant. The prevalence of repeat expansion in the current study population is lower than the 79% reported in the initial report in a Caucasian population¹⁹ and an independent U.S. cohort reported by Mootha et al.²⁰ confirming repeat expansion in 88 of 120 cases (73%) and in 5 of 70 control subjects (7%). The results of this current study are more comparable to the prevalence in an Indian population in which 14 of 44 subjects (34%) were found to have repeat expansion.²¹ A study describing ethnic Chinese in Singapore found a prevalence of repeat expansion in 25 of 57 FECD subjects (44%), but the definition of CTG expansion was >40 repeats.²² The lack of CTG expansion in any of our control subjects is also in contrast to these prior studies in which a small proportion of normal subjects harbored repeat expansion. The U.S. cohorts reported TCF4 repeat expansion in 3 of 63 subjects (5%)¹⁹ and 7 of 100 subjects (7%),²⁰ respectively. Repeat expansion was found in 5 of 97 normal subjects (5%) in an Indian study by Nanda et al.²¹ and in 2 of 121 controls (2%) in a study of ethnic Chinese by Xing et al.²² One consideration in comparing studies is the threshold defined for repeat expansion. Nanda et al.²¹ used a threshold of 50 repeats, whereas the other studies considered CTG expansion as >40 repeats. However, in any population, CTG repeat sizes between 40 and 50 are uncommon in either control or FECD participants. In the original U.S. study, 1 control subject and 1 FECD subject had repeat lengths in this range.¹⁹ Of course, the functional significance of these intermediate repeat lengths or the threshold size for a pathogenic expansion is currently unknown. 

The TCF4 gene (also known as ITF2 and SEF2, and not to be confused with T-cell factor 4, which is now known as TCF7L2) encodes the E2-2 protein that is a member of the helix-loop-helix (bHLH) family of transcription factors and plays an important role in a variety of developmental processes.²⁸ TCF4 was shown to be one of the first genes associated with schizophrenia in a large-scale genetic association study.²⁹ In addition, TCF4 is associated with primary sclerosing cholangitis and Pitt-Hopkins syndrome, a form of severe developmental retardation.²⁸ Except for Pitt-Hopkins syndrome, which results from a haploinsufficiency of TCF4, the involvement of TCF4 in the pathogenesis of other diseases has not been elucidated.³⁰ 

Du et al.³¹ recently reported that transcription of CTG repeats resulted in nuclear foci containing condensed poly(CUG)_n RNA and muscleblind-like1 (MBNL1) protein, an mRNA-splicing factor, in corneal endothelial cells from FECD patients with repeat expansions but not in an FECD patient without repeat expansion, and these results have been replicated by Mootha et al.³² Loss of MBNL1 activity due to sequestration in these toxic RNA foci has been shown to be a pathogenic event in myotonic dystrophy type 1, leading to a pattern of missplicing of MBNL1-regulated mRNAs that was also duplicated in FECD endothelium.³¹ Previous work has also demonstrated that MBNL1 plays a role in epithelial-to-mesenchymal transition (EMT),^33,34 which has been proposed as an important event in the pathogenesis of FECD.^14,31,35 Thus, TNR expansion in TCF4 could play a role in the development of FECD cases in a subset of the Japanese population through several possible mechanisms, including RNA toxicity. However, other genetic and/or environmental factors must be responsible for most cases in Japan. We recently reported that a Rho kinase inhibitor eye drop is effective for Japanese FECD patients.^36,37 Two FECD patients who were successfully treated with Rho kinase inhibitor were included in the current study; 1 patient had a TNR expansion and 1 patient had no TNR expansion. This very preliminary finding that Rho kinase inhibition may be useful regardless of the TCF4 TNR status warrants more detailed investigation but also motivates us to expand our clinical studies of Rho kinase inhibitors to Caucasian populations. 

We also showed that clinical manifestations of FECD, such as guttae, corneal thickness, and visual acuity, were indistinguishable between patients who carry a CTG repeat expansion and those who lack the expansion. Our data are consistent with the knowledge that FECD is a genetically heterogeneous disease and suggest that one or more additional undiscovered variants may be common causes of FECD in Japan. 

Acknowledgments

The authors thank all the patients and volunteers who enrolled in the study. 

Supported by an unrestricted grant from Research to Prevent Blindness, Inc., to the Mayo Clinic Department of Ophthalmology, by a Health Labour Sciences research grant (KS and TK), and by Research Funding for Longevity Sciences from National Center for Geriatrics and Gerontology (KS, TK, and UM). 

Disclosure: M. Nakano, None; N. Okumura, None; H. Nakagawa, None; N. Koizumi, None; Y. Ikeda, None; M Ueno, None; K. Yoshii, None; H. Adachi, None; R.A. Aleff, None; M.L. Butz, None; W.E. Highsmith, None; K. Tashiro, None; E.D. Wieben, None; S. Kinoshita, None; K.H. Baratz, None