Medicine (St Vincent's) - Theses

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    Determining the impact of Recql4 mutations on normal homeostasis, tumour development, and functional genetic interactions
    Castillo Tandazo, Wilson Javier ( 2020)
    Since mutations in the RECQL4 gene were identified as causative of Rothmund-Thomson syndrome (RTS) more than twenty years ago, some inroads have been made in the understanding of this disease and its mutations. It has been discovered that the majority of these mutations are nonsense and frameshift mutations resulting in truncating protein products that delete both the helicase and the C-terminal domain. The deletion of these domains results in a dysfunctional RECQL4 protein and the development of the variable clinical spectrum and the increased predisposition to malignancies, typical of RTS. Several Recql4-mutated mice have been generated as a model for RTS. Although these models have contributed to identify some of the functions of RECQL4, they do not accurately reflect the genetic status of RTS, in which patients generally present with hypomorphic, rather than null alleles. On the other hand, these models have not provided sufficient insight into the specific functions and domains of RECQL4; and the effects of RECQL4 mutations on normal homeostasis, tumour development, and functional genetic interactions. This thesis, through its three related components, aims to address all these gaps by using murine models bearing mutations that inactivate specific functions and domains of RECQL4, and that resemble common mutations seen in humans. In the first component of this thesis, I generated mice carrying an ATP-binding knock-in mutation to assess the physiological requirements and biological functions of the helicase activity, thought to be critical for the overall functions of RECQL4. Through a variety of experiments, I observed that homozygous mice were normal in terms of embryonic development, body weight, haematopoiesis, B and T cell development, and physiological DNA damage repair. Furthermore, to compare the in vivo effects of a helicase-inactive versus truncating mutations, I used conditional deletion models and found that only mice carrying truncating mutations developed bone marrow failure. These findings demonstrated that the ATP-dependent helicase activity of RECQL4 is not essential for its physiological functions, and that truncating mutations are deleterious. For the second component of this thesis, I assessed mice carrying germline truncating RECQL4 mutations to understand the impact of the deletion of the helicase and C-terminal domains on normal homeostasis and tumour development. I found that truncating mutations affected stability and subcellular localisation of RECQL4, which translated to a homozygous embryonic lethality and a haploinsufficient low bone mass phenotype through defects in early osteoblast progenitors. Additionally, I observed that the severity of the defect was related to the degree of the truncation, suggesting that gene dosage is an important determinant of the bone phenotype. However, these mutations were not sufficient to initiate tumorigenesis in mice, even after exposure to irradiation, which pointed to the possibility that mutations in other genes, besides Recql4, might be contributing to this disease. In the third and final component of this thesis, I performed a genome-wide genetic rescue screen to identify genetic interactions with mutant Recql4, an area largely unexplored. Amongst the genes identified, the standout candidate was Klhdc3. In subsequent validation assays, I demonstrated that mutation of Klhdc3 rescued the phenotypes associated with a pathogenic RECQL4 mutation, which is to my knowledge, the first confirmed genetic interaction associated with an improvement of the cellular phenotypes caused by RECQL4 mutations. Collectively, the work presented in this thesis makes an original contribution to knowledge through the finding that the ATP-dependent helicase activity of RECQL4 is dispensable for its physiological functions, the discovery that truncating mutations cause a haploinsufficient low bone mass phenotype and that gene dosage is an unsuspected regulator of bone mass, and the identification of Klhdc3 mutation as being capable of rescuing the proliferation defect caused by a truncating RECQL4 mutation.