Comprehensive Assessment of Genetic Variants within TCF4 in Fuchs Corneal Dystrophy

Abstract

Purpose: A genome-wide association study identified the single nucleotide variant (SNV), rs613872, in the transcription factor 4 (TCF4) gene to be strongly associated (p = 6 x 10^-26) with Fuchs endothelial corneal dystrophy (FECD). Subsequently, an unstable expansion of the repeating trinucleotides, TGC, in a non-coding portion of the gene was found to be even more predictive of the disease. We performed comprehensive sequencing of the entire TCF4 gene region in order to identify the best genetic marker for FECD within TCF4 and to identify other novel variants that may be involved in disease pathogenesis.

Methods: Leukocyte DNA was isolated from 78 unrelated subjects with FECD (modified Krachmer grade 2 through 6 in the more affected eye) and 16 unaffected (grade 0) individuals. An Agilent custom capture panel was used to isolate the entire region surrounding the two previously-validated genomic markers of FECD. Sequencing of the captured region, spanning 465 kilobases and including the entire TCF4 coding region, introns and flanking sequence was performed at >200x average coverage using the Illumina HiSeq 2000.

Results: Trinucleotide expansion (>50 TGC repeats) was present in 53 (68%) FECD-affected and 1 (6%) normal subject and absent in the remaining 25 affected and 15 normal subjects. A total of 1651 variants, including 1493 single nucleotide variants (SNVs), were identified in this sample set, of which only 2 previously reported SNVs resided in the coding region of TCF4. Neither of these coding SNVs segregated with disease status. No variant, including TGC expansion, correlated perfectly with disease status. Trinucleotide repeat expansion was a better predictor of disease than any other variant. A total of 366 SNVs found in these samples were previously unreported in either dbSNP or the 1000 genome samples.

Conclusions: Complete sequencing of the genomic region identified by the previous genome-wide association study revealed no single causative variant for FECD. The intronic, trinucleotide repeat expansion within TCF4 was more strongly associated with FECD than any other variant and, therefore, is likely a pathogenic contributor to the disease.

Keywords: 481 cornea: endothelium • 539 genetics