Defects in centrosome, centrosomal-associated and spindle-associated proteins are the most frequent cause of Primary Microcephaly (PM) and Microcephalic Primordial Dwarfism (MPD) syndromes in humans. Mitotic progression and segregation defects, microtubule spindle abnormalities and impaired DNA damage-induced G2-M cell cycle checkpoint proficiency have been documented in cell lines from these patients. This suggests that impaired mitotic entry, progression and exit strongly contribute to PM and MPD. Considering the vast protein networks involved in coordinating this cell cycle stage, the list of potential target genes that could underlie novel developmental disorders is large. One such complex network, with a direct microtubule-mediated physical connection to the centrosome, is the kinetochore. This centromeric-associated structure nucleates microtubule attachments onto mitotic chromosomes. Here, we described novel compound heterozygous variants in CENPE in two siblings who exhibit a profound MPD associated with developmental delay, simplified gyri and other isolated abnormalities. CENPE encodes centromere-associated protein E (CENP-E), a core kinetochore component functioning to mediate chromosome congression initially of misaligned chromosomes and in subsequent spindle microtubule capture during mitosis. Firstly, we present a comprehensive clinical description of these patients. Then, using patient cells we document abnormalities in spindle microtubule organisation, mitotic progression and segregation, before modeling the cellular pathogenicity of these variants in an independent cell system. Our cellular analysis shows that a pathogenic defect in CENP-E, a kinetochore-core protein, largely phenocopies PCNT-mutated Microcephalic Osteodysplastic Primordial Dwarfism type II patient cells. PCNT encodes a centrosome-associated protein. These results highlight a common underlying pathomechanism. Our findings provide the first evidence for a kinetochore-based route to MPD in humans.

Replicating the human genome efficiently and accurately is a daunting challenge involving the duplication of upward of three billion base pairs. At the core of the complex machinery that achieves this task are three members of the B family of DNA polymerases: DNA polymerases a, d, and ε. Collectively these multimeric polymerases ensure DNA replication proceeds at optimal rates approaching 2 3 10 3 nucleotides/min with an error rate of less than one per million nucleotides polymerized. The majority of DNA replication of undamaged DNA is conducted by DNA polymerases d and ε. The DNA polymerase a-primase complex performs limited synthesis to initiate the replication process, along with Okazaki-fragment synthesis on the discontinuous lagging strand. An increasing number of human disorders caused by defects in different components of the DNA-replication apparatus have been described to date. These are clinically diverse and involve a wide range of features, including variable combinations of growth delay, immunodeficiency, endocrine insufficiencies, lipodystrophy, and cancer predisposition. Here, by using various complementary approaches, including classical linkage analysis, targeted next-generation sequencing, and whole-exome sequencing, we describe distinct missense and splice-impacting mutations in POLA1 in five unrelated families presenting with an X-linked syndrome involving intellectual disability, proportionate short stature, microcephaly, and hypogonadism. POLA1 encodes the p180 catalytic subunit of DNA polymerase a-primase. A range of replicative impairments could be demonstrated in lymphoblastoid cell lines derived from affected individuals. Our findings describe the presentation of pathogenic mutations in a catalytic component of a B family DNA polymerase member, DNA polymerase a.

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Wolf-Hirschhorn syndrome (WHS) is a contiguous gene deletion disorder associated with the distal part of the short arm of chromosome 4 (4p16.3). Employing a unique panel of patient-derived cell lines with differing-sized 4p deletions, we provide evidence that haploinsufficiency of SLBP and/or WHSC2 (NELF-A) contributes to several novel cellular phenotypes of WHS, including delayed progression from S-phase into M-phase, reduced DNA replication in asynchronous culture and altered higher order chromatin assembly. The latter is evidenced by reduced histone-chromatin association, elevated levels of soluble chaperone-bound histone H3 and increased sensitivity to micrococcal nuclease digestion in WHS patient-derived cells. We also observed increased camptothecin-induced inhibition of DNA replication and hypersensitivity to killing. Our work provides a novel pathogenomic insight into the aetiology of WHS by describing it, for the first time, as a disorder of impaired chromatin reorganization. Delayed cell-cycle progression and impaired DNA replication likely underlie or contribute to microcephaly, pre- and postnatal growth retardation, which constitute the core clinical features of WHS.

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