A novel phenotype-genotype correlation with an Arg555Trp mutation of TGFBI gene in Thiel-Behnke corneal dystrophy in a Chinese pedigree

Individuals from a four-generation pedigree of Chinese ethnicity with corneal dystrophy (Fig. 1) were recruited from the Eye Center of the Second Affiliated Hospital, Medical College of Zhejiang University, Hangzhou, China. The study adhered to the tenets of the Declaration of Helsinki, and has been aproved by the ethics committee of the 2^nd Affiliated Hospital of Zhejiang University.

We received informed written consent covering DNA extraction and analysis, as well as ocular and ophthalmological examinations from all participating individuals including 150 normal controls. The written consent of the 6-year old patient for participation was obtained by her farther. Full ocular examinations including Snellen visual acuity, slit-lamp examination, applanation tonometry, corneal sensation and anterior segment photography were conducted. In addition, the patients underwent profound ophthalmologic examinations including ultrasound biomicroscopy (UBM) and anterior-segment optical coherence tomography (AS-OCT) to assess depth of the lesions.

Two corneal specimens of the proband were obtained from lamellar keratoplasty and underwent histopathological study. The buttons were processed by standard methods including sectioning of the tissue samples, fixation in 10 % neutral buffered formalin, then stained with hematoxylin and eosin (H&E), Congo red, Periodic acid-Schiff (PAS) and Masson’s trichrome, and analysis by light microscopy to detect the presence of deposits in the cornea.

Genomic DNA was extracted from the peripheral leukocytes of 12 members (three affected and nine unaffected) according to the manufacturer’s (Simgen, Hangzhou, China) standard methods. All 17 exons of TGFBI were amplified by a polymerase chain reaction (PCR) and analyzed with the molecular method described by Zhu et al. previously [8]. PCR products were separated by electrophoresis in a 1.5 % ethidium bromide-stained agarose gel. The bands with the amplified templates were examined, and then, the purified products were sequenced according to protocols accompanying the ABI Big dye Terminator Cycle sequencing kit V 3.1 (ABI Applied Biosystems; Foster City, CA). The nucleotide sequences were analyzed by using Lasergene V. 80 software (DNASTAR, Inc., Madison, WI) and compared manually with the published NCBI TGFBI cDNA sequence (GeneBank accession no. NM. 000358). DNA from 150 normal, unrelated, ethnically Chinese volunteers (75 males and 75 females) was analyzed as the control.

Histopathologic analysis of the two corneal buttons from the proband revealed an irregular, layered structure of corneal epithelium in which eosinophilic deposits were noted predominantly beneath the epithelium and involved the anterior stroma within a small range by hematoxylin and eosin staining (Fig. 3a). The presence of subepithelial congo-red-positive deposits that did not stained with Periodic acid-Schiff and Masson trichrome was also illustrated (Fig. 3b, c, d). UBM and AS-OCT scan of the proband demonstrated markedly increased reflectivity, due to deposits within the lesions and irregular epithelial thickening induced by corneal opacities which project into anterior stroma (Fig. 3e, f).

Directional DNA sequencing revealed that affected members in this pedigree harbored a heterozygous missense mutation (Fig. 4), consisting of a C to T transition at nucleotide position 1663 at the first position of codon 555 in TGFBI exon 12 (c. 1663C > T), resulting in the substitution of a highly conserved Arginine by Tryptophan in the encoded TGFBIp (p. Arg555Trp). With the cosegregation of unaffected individuals and absence of this nucleotide change in 150 normal controls, we strongly assume that this mutation is pathological and exclude the possibility of its being a polymorphic change. The affected members were also heterozygous for three previously described, inconsequential, silent, single-nucleotide polymorphisms (SNPs) in exon 8, 11, 12, which did not affect the protein encoded (c.1142G > A(P.(=)), c.1577 T > C(P.(=)), c.1781C > T(P.(=)). Interestingly, in this pedigree, all patients were heterozygous for another rare variant in exon 6 of the TGFBI gene (p. Arg257Trp), (Fig. 4), which also cosegregated well within this family. We screened 150 controls of exon 6 and found 1 normal control harbored the p. Arg257Trp alteration and, thus, we considered it to be a rare SNP that was not previously reported.