It is well established for a wide range of repeat expansion disorders that disease onset and incidence of RNA foci manifest only above a critical level of nucleotide repeats.47 A threshold for CTG18.1 repeat length and FECD association is yet to be fully defined. Performing FISH with 36 distinct CEC lines derived from FECD-affected subjects has enabled us to identify the threshold for the number of repeats required to produce nuclear RNA foci in our model (Figure 2B; Table S4). Nine CEC lines with CTG18.1 genotypes ranging from 12/12 to 18/31 were found to lack RNA foci. A further 27 lines with CTG18.1 genotypes ranging from 25/31 to 12/126 were all found to exhibit punctate nuclear RNA foci. These data allow us to correlate CTG18.1 genotype status with CUG RNA foci incidence and indicate that a repeat size of more than 31 trinucleotide repeats is sufficient to drive the accumulation of stable CUG RNA foci in primary CECs (Figure 2B; Table S4). This identified threshold also correlates notably with the bi-nominal distribution of CTG18.1 repeat length observed in our FECD cohort (Figure 1A), which is likely attributed to the instability of the repeat above approximately 30 copies. Interestingly, RNAs containing more than 30 CUG repeats have recently been demonstrated to undergo phase separation to form nuclear foci.47 The threshold for this repeat length-dependent process (30 CUG repeats) is remarkably similar to what we have observed with respect to CTG18.1-related foci occurrence in CECs (Figure 2B). These phase separation data further support the use of agents that disrupt RNA-RNA base-pairing, such as ASOs, as viable treatment options for RNA foci-induced cellular toxicity.47 Taken together, these data suggest that a CTG18.1 length ≥32 should, in future, be considered as FECD risk associated. We repeated the tests for association with FECD defining the expanded repeats as ≥32, instead of the previously used more conservative threshold of ≥50. The association model became more significant (p = 3.79 × 10−74), although the disease risk conferred by this locus was lower (OR = 34.14; 95% CI: 23.35–49.91). Future analysis of CECs from individuals affected by FECD with repeat lengths in the unidentified range of 31–53 could further refine this important threshold.