Genetic studies of Fuchs Endothelial Corneal Dystrophy

Abstract

FECD is a bilateral, mainly inherited corneal endothelial disease. Nearly 75% of European cases are caused by trinucleotide repeat expansions in TCF4, while the cause(s) in the remainder are not fully understood. This thesis presents two screening methods not previously applied to FECD, long-range PCR/long-read nanopore sequencing and single-molecule Molecular Inversion Probes (smMIPs).

Long-range PCR/long-read nanopore sequencing was used to screen FECD patients’ blood genomic DNA for CTG18.1 expansion at the TCF4 locus. 73% (87/119) carried ³50 repeat expansions, compared to 1% (1/83) of controls, confirming previous studies. Compared to the more widely used method of Short Tandem Repeat (STR) PCR and Triplet-repeat Primed (TP) PCR, long-range PCR and long-read nanopore sequencing proved robust, required only nanograms of DNA, and could be scaled up for high throughput and accurate repeat sizing.

Long-range PCR/nanopore sequencing of the wider TCF4 locus identified common haplotypes associated with expansion. Alleles of SNPs rs599550 (G) and rs618869 (T) tagged an 8,296 bp haplotype accounting for 81% of expansion bearing alleles. In contrast, 11 different haplotypes were identified in expansion-negative cases, with C-A haplotypes present in 90% of alleles. This could imply that the expansion arose on a T- G haplotype and alleles bearing the expanded CTG18.1 repeat are founder alleles, or that some unknown sequence feature in the T-G haplotype predisposes to CTG18.1 repeat instability.

smMIPs were used to screen expansion negative FECD cases and two control groups, expansion positive FECD cases and non-disease controls, for variants in five genes implicated in FECD pathogenesis. Variants were found in COL8A2 (c.7G>A, p.Gly3Arg); AGBL1 (c.806C>T, p.Pro269Leu; c.3323+2dup) and LOXHD1 (c.3340G>A, p.Gly1114Arg; c.3269G>A, p.Arg1090Gln; c.4504C>T, p.Arg1502Trp; c.5545G>A, p.Gly1849Arg and c.1570C>T, p.Arg524Cys). None were identified in SLC4A11 and ZEB1. Only one was classified likely pathogenic, and similar variants were found in controls, suggesting variants in these genes are rare as a cause of FECD and knowledge of FECD pathomechanism remains incomplete. The approach used proved robust and cost-effective.

This study reveals new insights into the genetic architecture of FECD and highlights new screening approaches with their advantages and drawbacks.