Knock-in Mice Models Carrying Missense Mutations in the DNA Helicase RTEL1

Hoyeraal-Hreidarsson Syndrome (HHS) is a fatal disorder associated with severe telomere shortening and caused by inherited mutations in the helicase RTEL1. RTEL1 is essential in both human and mouse, but its role at telomeres is not yet understood. We generated by CRISPR/Cas9 two knock-in mice models, each with a different homozygous missense mutation in a conserved amino acid of RTEL1. The first is an HHS-causing mutation (M492I) and the second is a variation found in Mus spretus (M492K), which has much shorter telomeres than those of M. musculus. Mouse embryonic fibroblasts (MEFs) derived from the mutant mice showed gradual telomere shortening over 250 population doublings in culture, suggesting that telomere shortening is caused by a defect in length maintenance. FISH using a telomeric PNA probe on metaphase chromosomes showed various aberrations in the mutant MEFs, such as telomere loss, heterogeneity, fragility, Robertsonian fusions, chromosome fragmentations, micronuclei, and aberrant interstitial telomere insertions. The M492K mutation mostly caused telomere shortening but did not significantly affect the mice viability , consistent with its natural occurrence in M. spretus. The M492I mutation compromised the viability and fertility of the mice , consistent with its effect on rapidly proliferating tissues, such as bone marrow and epithelial cells, and its causative role in HHS. Studying these mice models will reveal the telomeric role of RTEL1, how RTEL1 mutations cause a fatal disease, and the implications of short telomeres in cancer and aging.