Molecular Vision[JOURNAL]

Sun 200 papers

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

Alayed B, Albuainain D, Siddiqui S … +6 more , Li W, Hany U, Anand S, Inglehearn CF, Watson CM, Ali M

Mol Vis · 2025 · PMID 42180433

PURPOSE: A trinucleotide repeat expansion in 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 and... PURPOSE: A trinucleotide repeat expansion in 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 and in a small number of FECD cases, and more controversially, it has been suggested that variants in , , and may also be involved. We screened patients without a 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 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 , , , , and 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 , 2 in , 4 in , and 6 in . No variants were identified in 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 ( = 0.7612 and 0.3275). Only one variant ( 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 repeat expansion in cases over controls but found a paucity of evidence for the involvement of variants in , , , , or in this set of expansion-negative cases, implying knowledge of the causes of FECD remains incomplete.

Molecular Vision[JOURNAL]

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