To further explore the incidence of RNA foci, we investigated a total of 36 independent CEC lines derived from FECD-affected subjects by FISH (Table S4; Figure 2B). In summary, no foci were detected in 9 CEC lines derived from FECD-affected subjects with CTG18.1 genotype status ranging from 12/12 to 18/31, in addition to a further 4 control lines. Bright nuclear foci were clearly detected in 27 CEC lines derived from individuals with alleles ranging from 25/31 to 12/126. Interestingly, CTG18.1 allele length of 31 repeats appears to represent a critical threshold for foci occurrence in CECs as individuals with genotypes of 18/31 and 25/31 were foci negative and positive, respectively. No samples were available with an expansion in the range between 32 and 52 repeats (Figure 2B; Table S4). On this basis, we classified CEC lines selected for further experimental investigation as non-expanded (NE) if both CTG alleles contained <31 repeats, whereas those with at least one allele ≥53 repeats were considered expanded (Tables S3–S9).