Amplification-free long-read sequencing of TCF4 expanded trinucleotide repeats in Fuchs Endothelial Corneal Dystrophy

PLoS One. 2019 Jul 5;14(7):e0219446. doi: 10.1371/journal.pone.0219446. eCollection 2019.


Amplification of a CAG trinucleotide motif (CTG18.1) within the TCF4 gene has been strongly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Nevertheless, a small minority of clinically unaffected elderly patients who have expanded CTG18.1 sequences have been identified. To test the hypothesis that the CAG expansions in these patients are protected from FECD because they have interruptions within the CAG repeats, we utilized a combination of an amplification-free, long-read sequencing method and a new target-enrichment sequence analysis tool developed by Pacific Biosciences to interrogate the sequence structure of expanded repeats. The sequencing was successful in identifying a previously described interruption within an unexpanded allele and provided sequence data on expanded alleles greater than 2000 bases in length. The data revealed considerable heterogeneity in the size distribution of expanded repeats within each patient. Detailed analysis of the long sequence reads did not reveal any instances of interruptions to the expanded CAG repeats, but did reveal novel variants within the AGG repeats that flank the CAG repeats in two of the five samples from clinically unaffected patients with expansions. This first examination of the sequence structure of CAG repeats in CTG18.1 suggests that factors other than interruptions to the repeat structure account for the absence of disease in some elderly patients with repeat expansions in the TCF4 gene.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Computational Biology / methods
  • Fuchs' Endothelial Dystrophy / diagnosis
  • Fuchs' Endothelial Dystrophy / genetics*
  • Gene Amplification*
  • Gene Editing
  • Genetic Association Studies
  • Genetic Predisposition to Disease*
  • Genomics / methods
  • Genotype
  • Humans
  • Phenotype
  • RNA, Guide
  • Transcription Factor 4 / genetics*
  • Trinucleotide Repeat Expansion*
  • Trinucleotide Repeats


  • RNA, Guide
  • TCF4 protein, human
  • Transcription Factor 4

Grant support

Funding for this project was provided by National Eye Institute grants EY21727 (MPF) and EY26490 (MPF), Robert Waller Career Development Award (KHB), and the Mayo Foundation for Medical Education and Research (MPF, KHB, EDW). Pacific Biosciences provided support for employees associated with the company (BB, IJM, JME).