Targeted sequencing with single-molecule molecular inversion probes highlights a gap in understanding the cause of Fuchs endothelial corneal dystrophy

Purpose A trinucleotide repeat expansion in TCF4 is thought to cause Fuchs endothelial corneal dystrophy (FECD) in ~70% of European patients. In addition, strong evidence exists for the involvement of rare variants in COL8A2 and SLC4A11 in a small number of FECD cases, and more controversially, it has been suggested that variants in ZEB1, AGBL1, and LOXHD1 may also be involved. We screened patients without a TCF4 repeat expansion for causative variants in the other candidate FECD genes.

Methods Genomic DNA from blood was genotyped for expansion of the CTG18.1 repeat in intron 2 of TCF4 using short-tandem repeat PCR, followed by triplet-repeat primed PCR (STR/TP-PCR). Single-molecule molecular inversion probes (smMIPs) were designed to amplify the coding exons and splice recognition sites of FECD candidate genes COL8A2, SLC4A11, ZEB1, AGBL1, and LOXHD1 using MIPGEN, and the libraries generated were sequenced on a NextSeq 2000. FASTQ-formatted sequence reads were aligned to the human reference genome with MIPVAR, and identified variants were annotated using Annovar. Rare potentially pathogenic variants (minor allele frequency ≤0.01 for assumed dominant inheritance, combined annotation-dependent depletion ≥15) were confirmed by Sanger sequencing and interpreted according to American College of Medical Genetics and Genomics criteria using the Franklin by Genoox interface.

Results Analysis of 114 FECD cases by STR/TP-PCR stratified the patients into FECD expansion-negative cases that had <50 trinucleotide repeats on both alleles at the CTG18.1 locus (n = 33 probands and three additional family members) and FECD expansion-positive (n = 78) cases with at least one allele harboring ≥50 repeats in size. All 36 expansion negative cases were then analyzed by smMIP targeted capture and short-read sequencing of the five other genes implicated in FECD causation. For comparison, two control groups were similarly analyzed: a subset of 29 of the expansion-positive cases whose FECD was assumed to be caused by the repeat expansion and 29 expansion-negative unaffected individuals. Across all groups, 13 variants passed filtration criteria: 1 in COL8A2, 2 in SLC4A11, 4 in AGBL1, and 6 in LOXHD1. No variants were identified in ZEB1. Eight of the variants identified were found in seven FECD expansion-negative cases, five in four FECD expansion-positive cases, and three in two non-FECD controls, with three variants appearing in both expansion-positive and expansion-negative patient groups. Statistical analysis indicated no significant enrichment of variants in expansion-negative cases compared to the other two groups (p = 0.7612 and 0.3275). Only one variant (LOXHD1 NM_144612.7, c.5545G>A, p.(Gly1849Arg)), found in an expansion-negative case, was classified as likely pathogenic, while others were classed as variant of unknown significance (n = 4), likely benign (n = 4), or benign (n = 4).

Conclusions smMIPs were used for targeted screening of candidate genes implicated in FECD and proved a versatile, economic approach for prescreening before whole-exome or genome sequencing. This study confirmed the well-documented enrichment of the TCF4 repeat expansion in cases over controls but found a paucity of evidence for the involvement of variants in COL8A2, SLC4A11, ZEB1, AGBL1, or LOXHD1 in this set of expansion-negative cases, implying knowledge of the causes of FECD remains incomplete.