Abstract
Aims: To identify transforming growth factor beta-induced (TGFBI) gene mutations in four Chinese families affected by corneal dystrophies. Methods: In this study, three families (21 patients and 18 normal relatives), respectively, with Reis-Bücklers corneal dystrophy (RBCD), classic lattice corneal dystrophy (LCDI), and variant LCD (LCDI/IIIA) were assessed. All subjects underwent a complete ophthalmological evaluation, including biomicroscopic inspection and dilated fundus examination. As a control, 100 individuals without corneal disease were selected from the general population. Polymerase chain reaction (PCR) and direct sequencing were used to screen for mutations in TGFBI. Results: For the three families, a single heterozygous c.371G>T (R124L) point mutation was found in exon 4 of TGFBI in 14 affected members with RBCD, a single heterozygous c.370C>T (R124C) point mutation was found in exon 4 of TGFBI in four affected members with LCDI, and a single heterozygous c.1877A>G (H626R) point mutation was found in exon 14 of TGFBI in four affected members with LCDI/IIIA. TGFBI gene mutation had not been detected in the unaffected members and 100 normal controls. Conclusions: TGFBI gene mutations were present in all three Chinese families with corneal dystrophy, and our study further verified the relationship between phenotype and genotype of corneal dystrophy.
Introduction
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However, as the disease has a significant genetic heterogeneity, different TGFBI gene mutations can lead to the same phenotype, for example, R124C, V625D, V505D, H626R, and other mutations are associated with LCDI (Tian et al., 2005, 2007; Cho et al., 2012); whereas the same TGFBI mutation in the different ethnic populations may exhibit different clinical phenotypes, for example, R124C mutation is responsible for GCDII, LCDI, and RBCD (Munier et al., 2002; Patel et al., 2010; Yang et al., 2011). The complexity of phenotypic and genetic heterogeneity, therefore, complicates the identification of the causes of the disease. Thus, the challenge to ophthalmologists and researchers is to correctly diagnose and classify CDs, as well as to understand their phenotype-genotype aspects. In this study, we recruited three Chinese families affected by different types of CD and identified the gene mutations responsible for the disease. Our study provided an insight into the mechanisms of the disease.
Materials and Methods
Subjects
As part of a genetic screening program for genetic eye disorders, we collected materials from three families diagnosed with different CDs, including RBCD, LCDI, and variant LCD (LCDI/IIIA) from China. This study was performed in accordance with the Declaration of Helsinki, and written informed consent was signed by all subjects or their parents. The study design was approved by the Institutional Review Board of the Second Affiliated Hospital of Wenzhou Medical University, Zhejiang, China. One hundred unrelated subjects without CD were recruited from the Ophthalmology Clinic of the Second Affiliated Hospital of Wenzhou Medical University as normal controls for the study.
Clinical examination
Snellen best-corrected visual acuity test, slit lamp biomicroscopy, intraocular pressure measurement, and fundus examination were conducted by an experienced ophthalmologist for all subjects. Detailed clinical history such as the age of onset, initial signs and symptoms, progression of disease, and ocular therapeutic procedures were documented.
Molecular analysis
Genomic DNA was extracted from the peripheral blood lymphocytes by using the QIAamp Blood Mini DNA Kit (Qiagen, Santa Clara, CA) according to the manufacturer's instructions. The DNA fragments that encode portions of the TGFBI gene were amplified by polymerase chain reaction (PCR). The sequences of the primers are listed in Table 1. Each PCR was carried out in a 30 μL PCR mixture containing 1 × PCR buffer (GC buffer I was used for several reactions, see Table 1), 30 ng DNA, 1.6 mM MgCl2, 20 mM of each of dNTPs, 1.5 U Taq DNA polymerase, and 0.17 μM of each of the forward and reverse primers. PCR conditions were as follows: 94°C for 3 min; 35 cycles of 94°C for 30 s, 52°C or 55°C or 58°C for 30 s, 72°C for 45 s; and further extension step at 72°C for 5 min, using the Gene Amp PCR System 9700 (PE Applied Biosystems, Foster City, CA). The resulting PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA). Bidirectional sequencing of amplicons was performed using the same PCR forward and reverse primers with the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). The products of the sequencing reaction were analyzed using a fluorescent ABI Prism 3100 DNA sequencer (PerkinElmer Applied Biosystems, Warrington, United Kingdom). Gene mutation was confirmed in all family subjects and unrelated controls.
Results
Reis-Bücklers corneal dystrophy
In this four-generation family, 14 affected members and 18 unaffected members were examined (Fig. 1A). The symptoms of the proband (a 22-year-old male, patient III:1) began with episodes of acute ocular pain, redness, and photophobia at 6 years of age, and progressive vision loss at 9 years of age. Slit lamp examination showed geographic-like opacities at the subepithelial and anterior stroma of the cornea of both eyes (Fig. 2A, B). The clinical symptoms of other affected family members are similar to him. Mutation analysis showed that a heterozygous c.371 G > T mutation (R124L) in exon 4 of TGFBI was identified in affected members, but not in unaffected members or healthy controls (Fig. 1D).
Pedigrees of three Chinese families with corneal dystrophies and sequence chromatograms of their corresponding mutations TGFBI.
Representative corneal phenotypes as shown by slit lamp examination. The proband of RBCD family with R124L mutation had gray-white geographic opacities in the right eye
Classic lattice corneal dystrophy
In this three-generation family, three affected members and four unaffected members were examined (Fig. 1B). The symptoms of the proband (a 37-year-old male, patient II:4) began with episodes of acute ocular pain, redness, and photophobia at 13 years of age. Slit lamp examination revealed the typical lattice lines in the anterior stromal layers (Fig. 2C, D). Patient II:2, the sister of the proband had similar clinical symptoms; patient III:1, the 22-year-old nephew of the proband, presented with no clinical symptoms, but manifested bilateral gray linear opacities at epithelial and subepithelial in the inferior cornea. Mutation analysis showed that a heterozygous c.370 C > T mutation (R124C) in exon 4 of TGFBI was identified in all affected members, but not in unaffected members or healthy controls (Fig. 1E).
Variant lattice corneal dystrophy (LCDI/IIIA)
In this three-generation family, four affected members and three unaffected members were examined (Fig. 1C). The proband (49-year-old female, III:3) had only slow vision deterioration, but had no other ocular problems when growing up and had been diagnosed with CD. Slit lamp examination displayed gray-white thick lattice lines deposit in the deep stromal layer (Fig. 2E). The clinical symptoms of other affected family members are similar to hers. Mutation analysis showed that a heterozygous c.1877 A>G mutation (H626R) in exon 14 of TGFBI was identified in the proband, but not in 100 controls. However, we can not ascertain whether this mutation occurred in unaffected family members because they refused to take part in mutation analysis (Fig. 1F).
Discussion
TGFBI gene (also known as BIGH3), first discovered by Skonier et al. (1992), has been closely involved with the inherited CDs. In this study, we searched for mutations of TGFBI in three unrelated Chinese families affected with RBCD, LCDI, or LCDI/IIIA. Three distinct mutations of TGFBI were identified in all of the families (Fig. 2), and there have been perfect correlations between RBCD and R124L, between LCDI and R124C, and between LCDI/IIIA and H626R.
Of these mutations, RBCD is an autosomal dominant inheritance disease, which has a low incidence in China. In this family with RBCD, there were 14 affected members, including 8 males and 6 females. The ratio of affected to unaffected cases was 14:19 and all affected individuals had an affected parent. Phenotypically, the affected individuals presented with confluent irregular, geographic-like opacities in the subepithelial and anterior stroma of both eyes. To date, TGFBI was the only gene found to be associated with RBCD. In China, four different mutations in the TGFBI gene [p.Arg124Leu (Tian et al., 2005; Liang et al., 2012), p.Arg124Cys (Ma et al., 2010), p.Arg555Gln (Piao et al., 2012), and p.Gly623Asp (Li et al., 2008)] have been detected in pedigrees with RBCD. The p.Arg124Leu mutation was detected in 4 (57.1%) of the seven families, together with our current Chinese family affected with RBCD. It is highly possible that p.Arg124Leu is the most prevalent mutation in Chinese RBCD.
LCD is generally divided into three subtypes. The classification has been made mainly by clinical and pathological findings. LCDI is an autosomal dominantly inherited corneal amyloidosis that is characterized by a network of delicate interdigitating filaments within the corneal stroma. The disease usually begins in the first decade of life with symptoms of recurrent painful epithelial erosions. Lattice lines and diffuse opacification of the central cornea develop gradually after the erosions and amyloid accumulations. Many reports from different ethnic groups have described an R124C mutation of the TGFBI gene as the most common cause of LCDI (Stewart et al., 1999; Kim et al., 2001; Patel et al., 2010). However, Liu et al. (2008) found that clinical features of Chinese patients with the same R124C mutation are quite variable even within the same family. In the current study, the clinical features of affected members in the family with LCDI are similar. The mechanism of the phenotypic variability with the same mutation and even within the same family still remains unknown and needs further study.
The LCDI/IIIA subtype is characterized by bilateral progressive visual impairment and has an intermediate age of disease onset between LCDI and LCDIIIA. The LCDI/IIIA subtype was first described in England (Stewart et al., 1999), and then was named by Chau et al. (2003). In 2010, Yang et al. (2010) found this mutation in Northern Chinese with clinical features as intermediate subtype between LCDI and IIIA; 3 years later, Wang et al. (2013) first reported the TGFBI p.H626R mutation in a Southern Chinese pedigree with LCDI/IIIA. All these findings suggest that TGFBI p.H626R could be a mutation hotspot across different populations for LCDI/IIIA. Similar to previous studies, we also found the p.H626R mutation in the family affected by LCDI/IIIA.
In conclusion, we did not find novel mutation in the three pedigrees with CDs, however, a good phenotype-genotype correlation was observed in all patients with TGFBI-linked CDs. Since the mutation at TGFBI can be easily, rapidly, and cost effectively evaluated by PCR sequencing or by PCR-restriction fragment length polymorphism (RFLP), identification of these mutations will allow Chinese patients to benefit from a timely and accurate molecular diagnosis of CDs.
Footnotes
Acknowledgments
This study was supported by grants from the Wenzhou Science and Technology Bureau (No. Y20130252). The authors thank Yifan Feng for his assistance in statistics in this study and Yanlin Zhang for her helpful comments and suggestions.
Author Disclosure Statement
No competing financial interests exist.