To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication, and protect, repair and restart damaged forks. Here we identify DONSON as a novel fork protection factor, and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilises forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATR-dependent signalling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity, and potentiating chromosomal instability. Hypomorphic mutations substantially reduce DONSON protein levels and impair fork stability in patient cells, consistent with defective DNA replication underlying the disease phenotype. In summary, we identify mutations in DONSON as a common cause of microcephalic dwarfism, and establish DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.

Gillespie syndrome (GS) is characterized by bilateral iris hypoplasia, congenital hypotonia, non-progressive ataxia, and progressive cerebellar atrophy. Trio-based exome sequencing identified de novo mutations in ITPR1 in three unrelated individuals with GS recruited to the Deciphering Developmental Disorders study. Whole-exome or targeted sequence analysis identified plausible disease-causing ITPR1 mutations in 10/10 additional GS-affected individuals. These ultra-rare protein-altering variants affected only three residues in ITPR1: Glu2094 missense (one de novo, one co-segregating), Gly2539 missense (five de novo, one inheritance uncertain), and Lys2596 in-frame deletion (four de novo). No clinical or radiological differences were evident between individuals with different mutations. ITPR1 encodes an inositol 1,4,5-triphosphate-responsive calcium channel. The homo-tetrameric structure has been solved by cryoelectron microscopy. Using estimations of the degree of structural change induced by known recessive- and dominant-negative mutations in other disease-associated multimeric channels, we developed a generalizable computational approach to indicate the likely mutational mechanism. This analysis supports a dominant-negative mechanism for GS variants in ITPR1. In GS-derived lymphoblastoid cell lines (LCLs), the proportion of ITPR1-positive cells using immunofluorescence was significantly higher in mutant than control LCLs, consistent with an abnormality of nuclear calcium signaling feedback control. Super-resolution imaging supports the existence of an ITPR1-lined nucleoplasmic reticulum. Mice with Itpr1 heterozygous null mutations showed no major iris defects. Purkinje cells of the cerebellum appear to be the most sensitive to impaired ITPR1 function in humans. Iris hypoplasia is likely to result from either complete loss of ITPR1 activity or structure-specific disruption of multimeric interactions.

Compaction of chromosomes is essential for accurate segregation of the genome during mitosis. In vertebrates, two condensin complexes ensure timely chromosome condensation, sister chromatid disentanglement, and maintenance of mitotic chromosome structure. Here, we report that biallelic mutations in NCAPD2, NCAPH, or NCAPD3, encoding subunits of these complexes, cause microcephaly. In addition, hypomorphic Ncaph2 mice have significantly reduced brain size, with frequent anaphase chromatin bridge formation observed in apical neural progenitors during neurogenesis. Such DNA bridges also arise in condensin-deficient patient cells, where they are the consequence of failed sister chromatid disentanglement during chromosome compaction. This results in chromosome segregation errors, leading to micronucleus formation and increased aneuploidy in daughter cells. These findings establish "condensinopathies" as microcephalic disorders, with decatenation failure as an additional disease mechanism for microcephaly, implicating mitotic chromosome condensation as a key process ensuring mammalian cerebral cortex size.

Preterm infants are at increased risk of alterations in brain structure and connectivity, and subsequent neurocognitive impairment. Breast milk may be more advantageous than formula feed for promoting brain development in infants born at term, but uncertainties remain about its effect on preterm brain development and the optimal nutritional regimen for preterm infants. We test the hypothesis that breast milk exposure is associated with improved markers of brain development and connectivity in preterm infants at term equivalent age. We collected information about neonatal breast milk exposure and brain MRI at term equivalent age from 47 preterm infants (mean postmenstrual age [PMA] 29.43 weeks, range 23.28-33.0). Network-Based Statistics (NBS), Tract-based Spatial Statistics (TBSS) and volumetric analysis were used to investigate the effect of breast milk exposure on white matter water diffusion parameters, tissue volumes, and the structural connectome. Twenty-seven infants received exclusive breast milk feeds for ≥75% of days of in-patient care and this was associated with higher connectivity in the fractional anisotropy (FA)-weighted connectome compared with the group who had < 75% of days receiving exclusive breast milk feeds (NBS, p = 0.04). Within the TBSS white matter skeleton, the group that received ≥75% exclusive breast milk days exhibited higher FA within the corpus callosum, cingulum cingulate gyri, centrum semiovale, corticospinal tracts, arcuate fasciculi and posterior limbs of the internal capsule compared with the low exposure group after adjustment for PMA at birth, PMA at image acquisition, bronchopulmonary dysplasia, and chorioamnionitis (p < 0.05). The effect on structural connectivity and tract water diffusion parameters was greater with ≥90% exposure, suggesting a dose effect. There were no significant groupwise differences in brain volumes. Breast milk feeding in the weeks after preterm birth is associated with improved structural connectivity of developing networks and greater FA in major white matter fasciculi.