In mice, RTEL1 is essential for survival—it is required during telomere replication, in DNA-damage repair (specifically in homologous recombination), and for efficient elongation of the telomere by telomerase.17 Vannier et al. demonstrated that RTEL1 is necessary to cause T-loop disassembly and that T-loops only form with 3′ single-stranded DNA overhangs.11 It has been suggested that persistent T-loops are inappropriately resolved by the SLX4 complex, which cleaves the T-loop from the telomere. This results in the formation of T-circles and a corresponding loss in telomere length.13,17,18 When RTEL1 is inactivated, T-circle formation increases. To examine this potential mode of telomere shortening in individuals with biallelic RTEL1 mutations, we utilized a T-circle amplification assay.19 The formation of T-circles in genomic DNA from controls and the index cases from DCR families 302 and 303 (for whom there was good-quality genomic DNA) was measured (Figure 2B). Compared to the signal from the telomeric terminal restriction fragment, the relative signal from the T-circles was increased in the index cases (p = 0.0148, unpaired t test, Figure 2C), suggesting that these mutations affect the ability of RTEL1 to correctly process T-loops and thus result in telomere shortening and a relative increase in T-circle formation. By contrast, there was no significant difference in T-circle intensity between five HHS cases with DKC1 (MIM 300126) mutations and the normal controls (p = 0.54, Mann Whitney U, Figure S2). These data suggest that T-circle production is increased in individuals with biallelic RTEL1 mutations, which is consistent with observation in mice and supports a disease-causing mechanism that results in the shortening of telomeres without impacting the function of the telomerase complex.