Long-Range PCR and Nanopore Sequencing Enables High-Throughput Detection of TCF4 Trinucleotide Repeat Expansions in Fuchs Endothelial Corneal Dystrophy.

Introduction: Trinucleotide repeat expansion in CTG18.1, in intron 2 of TCF4 (MIM *602272, #613267), is the main cause of Fuchs endothelial corneal dystrophy (FECD), accounting for around 75% of cases in Caucasians. CTG18.1 repeat expansion has typically been detected in peripheral blood genomic DNA by Southern blotting or short tandem repeat polymerase chain reaction (STR-PCR) combined with triplet-repeat primed PCR (TP-PCR) if needed. However both methods estimate the size of the expanded repeat relative to a size standard, and the former requires microgram amounts of DNA. To support the development of therapies, a high-throughput screening approach for repeat expansions in FECD is required. Here, we present a sensitive assay using long-range PCR and nanopore sequencing of genomic DNA to accurately resolve the CTG18.1 repeat.

Methods: The CTG18.1 locus was analysed in genomic DNA from peripheral blood leukocytes by two different methods, and results were compared. The first approach used STR-PCR and capillary electrophoresis, followed by confirmatory testing of apparent homozygotes by TP-PCR. The second used long-range PCR, library preparation and long-read sequencing on an Oxford Nanopore Technologies MinION, with resolution of repeat length using the STRique algorithm.

Results: CTG18.1 expansion was screened for in 119 patients with FECD and 83 controls, by STR/TP-PCR genotyping and, independently, by long-range PCR/long-read nanopore sequencing. Both methods gave comparable results, but the latter was also able to measure repeat length. A total of 73.1% of FECD cases (87/119) and 1.2% of age-matched controls (1/83) had at least one CTG18.1 expansion that was ≥ 50 repeats. The expanded CTG18.1 allele was inherited across multiple generations in four larger families, in a manner consistent with causing a dominant phenotype, revealing that some younger family members may be at risk. The G allele of SNP rs599550, ~1kb away from the expansion, is linked (in cis) with expanded alleles in 80.8% of FECD alleles with an expansion, compared with 12.5% in FECD alleles in cases without an expansion and 14.6% in Europeans.

Discussion: We demonstrate that long-range PCR and long-read nanopore sequencing is a sensitive method requiring only nanograms of DNA, which can be scaled up for high-throughput detection and accurate sizing of CTG18.1 in peripheral blood DNA. The SNP, rs599550, is in linkage disequilibrium with the expansion and physically closer than rs613872, previously used in FECD association studies, making it better for use in diagnostic or association studies.