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

Hoyeraal-Hreidarsson Syndrome (HHS) is a fatal inherited disorder associated with severe telomere shortening, bone marrow failure, immunodeficiency, cerebellar hypoplasia, microcephaly and other central nervous system defects. Inherited mutations in the helicase regulator of telomere elongation 1 (RTEL1) cause HHS while somatic mutations are associated with brain tumors. RTEL1 is essential in both human and mouse, but its role at telomeres is not yet understood. In attempt to understand its role at telomeres and how mutations in RTEL1 cause a fatal disease, we generated two knock-in mice models, each with a different homozygous missense mutation in a conserved amino acid of RTEL1, using CRISPR/Cas9 technology. The first is an HHS-causing mutation and the second is a variation found in Mus spretus, which has much shorter telomeres than those of M. musculous. Mouse embryonic fibroblasts (MEFs) derived from the mutant mice showed gradual telomere shortening over more than 150 population doublings in culture, suggesting that telomere shortening is not caused by rapid deletion but by a defect in length maintenance. Fluorescence in situ hybridization (FISH) using a telomeric PNA probe to metaphase chromosomes showed various aberrations in the homozygous mutant MEFs, such as telomere loss, heterogeneity, fragility, Robertsonian fusion and chromosome fragmentation. Interestingly, the heterozygous MEFs displayed high frequency of aberrant interstitial telomere insertions, suggesting a dominant effect. These results indicate that missense RTEL1 mutations cause telomere defects, which may affect rapidly-proliferating tissues such as bone marrow, epithelial cells and gonads, but also the developing brain during embryogenesis. The observed genomic instability is likely implicated in cancer. Studying these effects in whole mice will expand our knowledge of RTEL1 and its telomeric role, and elucidate pathways causing HHS, neurodevelopment defects and cancer. Hopefully it will lead to developing novel therapeutic approaches for HHS and other genome instability diseases, aging and cancer.