DNA methylation as a determinant of organism size

Microcephalic dwarfism (MD) represents a group of single gene disorders that are characterised by a severe reduction in head and body size. Over 40 genes have been identified as MD genes. A common feature of many of these genes is that they are involved in the cell cycle. Hence, mutations in these genes perturb the cell cycle, which leads to fewer cell cycles and fewer cells. Since cell number is the principal determinant of mammalian size (Conlon & Raff, 1999), this leads to a reduction in organism size. Recently, DNMT3A has been identified as a microcephalic dwarfism gene (Heyn et al., 2019). This discovery of DNMT3A mutations in individuals with microcephalic dwarfism is particularly striking given that haploinsufficiency mutations in DNMT3A cause a reciprocal overgrowth disorder known as Tatton Brown Rahman syndrome (Tatton-Brown et al., 2014). Additionally, DNMT3A is a DNA methyltransferase gene and not a cell cycle gene, which presents the question of how mutations in this DNMT3A act to alter cell number and body size. In order to attain a normal cell number and body size, embryonic stem cells must undergo a sufficient number of cell divisions prior to differentiation. If proliferating, progenitor populations undergo premature terminal differentiation, then adequate expansion of progenitor pools, which ensures a sufficiently large organism is formed, cannot occur. Premature differentiation has been suggested to underlie growth restriction observed in patients with loss-of-function, germline mutations in LARP7 (Dai et al., 2014) and my starting hypothesis was that similar, premature differentiation events were responsible for the cell number and body size reduction caused by gain-of-function mutations in DNMT3A. The objective of my PhD was therefore to gain an understanding of how gain-of-function mutations in DNMT3A act to reduce cell number and body size by characterising the biochemical and phenotypic consequences of DNMT3A mutations, and by using haematopoiesis as a model system to understand the consequences of a Dnmt3a p.W326R mutation (orthologous to the DNMT3A p.W330R variant described in 2 human patients with MD) on stem and progenitor cells. Further objectives were to characterise novel DNMT3A variants identified in human MD patients, and to better understand immunodeficiency-type symptoms which have been identified as a recurrent feature in human MD patients. I found that Dnmt3aW326R/+ haematopoietic stem cells were paradoxically increased in number, yet they were dysfunctional. If such dysfunctional behaviour was to occur in other stem and progenitor cell populations, such as neural stem cells and growth plate stem cells, then stem cell dysfunction and inability to normally self-renew and differentiate could explain the global reduction in body size observed in patients with MD caused by mutations in DNMT3A. Defective formation of B cells was also identified in Dnmt3aW326R/+ mice, which could explain the apparent susceptibility of MD patients with DNMT3A mutations to recurrent infections.