Rothmund-Thomson Syndrome-Like RECQL4 Truncating Mutations Cause a Haploinsufficient Low-Bone-Mass Phenotype in Mice

ABSTRACT

Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder characterized by defects in the skeletal system, such as bone hypoplasia, short stature, low bone mass, and an increased incidence of osteosarcoma. RTS type 2 patients have germ line compound biallelic protein-truncating mutations of RECQL4. As existing murine models employ Recql4 null alleles, we have attempted to more accurately model RTS by generating mice with patient-mimicking truncating Recql4 mutations. Truncating mutations impaired the stability and subcellular localization of RECQL4 and resulted in homozygous embryonic lethality and a haploinsufficient low-bone mass phenotype. Combination of a truncating mutation with a conditional Recql4 null allele demonstrated that the skeletal defects were intrinsic to the osteoblast lineage. However, the truncating mutations did not promote tumorigenesis. We utilized murine Recql4 null cells to assess the impact of human RECQL4 mutations using an in vitro complementation assay. While some mutations created unstable protein products, others altered subcellular localization of the protein. Interestingly, the severity of the phenotypes correlated with the extent of protein truncation. Collectively, our results reveal that truncating RECQL4 mutations in mice lead to an osteoporosis-like phenotype through defects in early osteoblast progenitors and identify RECQL4 gene dosage as a novel regulator of bone mass.

KEYWORDS:

• Rothmund-Thomson syndrome
• RECQL4
• RecQ
• osteoporosis
• osteosarcoma
• mouse models
• familial cancer syndrome
• myeloid cells
• osteoblast
• tumor suppressor

SUPPLEMENTAL MATERIAL

Supplemental material is available online only.

ACKNOWLEDGMENTS

We thank R. Brink and the Mouse Engineering Garvan/ABR (MEGA) Facility (Garvan Institute, Sydney, Australia) for the generation of the G522Efs allele, the Australian Phenomics Facility and the Australian National University (Canberra, Australia) for their technical expertise and provision of the R347X allele, S. Galic and L. Murray-Segal (St. Vincent’s Institute) for training on Echo-MRI, T. Enomoto (Musashino University, Tokyo, Japan) for providing human EGFP-RECQL4 WT and K508A constructs, M. Kamps (University of California San Diego, USA) for providing the Hoxb8 vectors used to generate cell lines, D. Thorburn (Murdoch Children’s Research Institute and University of Melbourne, Australia), and J. Heierhorst (St. Vincent’s Institute) for comments on the manuscript.

Author contributions: Conceptualization: Wilson Castillo-Tandazo, Monique F. Smeets, Carl R. Walkley. Funding acquisition: Carl R. Walkley. Investigation: Wilson Castillo-Tandazo, Ann E. Frazier, Monique F. Smeets, Carl R. Walkley. Methodology: Wilson Castillo-Tandazo, Ann E. Frazier, Natalie A. Sims, Monique F. Smeets, Carl R. Walkley. Project administration: Monique F. Smeets, Carl R. Walkley. Supervision: Monique F. Smeets, Carl R. Walkley. Visualization: Wilson Castillo-Tandazo, Monique F. Smeets, Carl R. Walkley. Writing – original draft: Wilson Castillo-Tandazo, Monique F. Smeets, Carl R. Walkley. Writing – review and editing: Wilson Castillo-Tandazo, Ann E. Frazier, Natalie A. Sims, Monique F. Smeets, Carl R. Walkley.

This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Cancer Research under award no. W81XWH-15-1-0315 (to C.R.W.). Funding also came from the National Health and Medical Research Council (NHMRC) Australia project grant (to C.R.W.; APP1102004); a Melbourne Research Scholarship (to W.C.-T.; University of Melbourne); a Victorian Cancer Agency research fellowship (to CRW, MCRF15015); and the Mito Foundation (to A.E.F.). This work was enabled by the Australian Phenomics Network and partly supported by funding from the Australian government’s National Collaborative Research Infrastructure Strategy and the Super Science Initiative through the Education Investment Fund (to the Australian Phenomics Network) and in part supported by the Victorian State Government Operational Infrastructure Support (to St. Vincent’s Institute and the Murdoch Children’s Research Institute).

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense (USA).

We declare no conflicts of interest.