Abstract
DONSON is a novel replisome and replication fork stabilisation factor that is essential for efficient DNA replication and is particularly important during times of increased replicative stress. At stalled replication forks, DONSON is required for promoting the ATR-CHK1 kinase signalling pathway but the precise mechanism is undefined. Biallelic hypomorphic variants in DONSON underlie a spectrum of overlapping, but clinically distinct microcephalic primordial dwarfism disorders that include microcephaly-micromelia syndrome (MIMIS), microcephaly and short stature, with limb abnormalities (MISSLA), and Meier-Gorlin syndrome (MGORS). MIMIS is associated with a more severe loss of function of DONSON protein, but the genotypic-phenotypic association between MISSLA and MGORS pathogenesis is less clear. The role of DONSON in MGORS is further enigmatic as historically, MGORS-associated genes encode proteins involved in early pre-replicative processes, of which DONSON has no known role.
This project utilised in silico tools to identify novel elements in the largely uncharacterised DONSON protein. These included identifying locations of a nuclear localisation signal, CDK phosphorylation sites, and a potential CHK1 binding motif. Structural-based modelling revealed that DONSON is comprised of two protein domains, a disordered N-terminal domain, and a second interfolded domain made up of two globular α/β motifs. Structural similarity suggested that DONSON may function as a scaffold protein important for mediating protein contacts at replication complexes. While patient variants are dispersed throughout DONSON, the core β-sheet of α/β motif 1 reflected a variant hot-spot.
Through our ongoing focus on DONSON, we have been studying a US patient who is compound heterozygous for two novel DONSON variants: an early nonsense variant, c.64C>T (p.R22*), and missense variant c.847G>A (p.V283I), which could not be classified as a causative variant. She had features that generally fit within the DONSON spectrum but don't suggest a clear subtype. Structural modelling revealed that V283 is positioned within the conserved protein core that is also a hot spot for pathogenic variants, with sidechain positioning impacting distances for intraprotein contacts. In vitro analysis confirmed that the p.V283I substitution compromised normal DONSON function by significantly altering DONSON subcellular localisation. From these analyses, the novel variant p.V283I was able to be re-classified as 'Likely Pathogenic' delivering a definitive diagnosis to the patient. These findings further expand the genotypic-phenotypic spectrum of DONSON-associated disorders.
Investigation into the functional consequences of MGORS and MISSLA missense variants on subcellular localisation and protein stability did not reveal clear genotype-phenotype relationships. This work does, however, provide a foundation to explore future research directions such as co-immunoprecipitation experiments.