Recurrent de novo variant in NUSAP1 escapes nonsense-mediated decay and leads to syndromic microcephaly

Emuna Paz Ebstein ^2,3 Alisa Mo ¹ Shira Yanovsky-Dagan ² Abbe Lai ⁴ Hagar Mor-Shaked ^2,3 Tal Gilboa ^3,5 Edward Yang ⁶ Diane D. Shao ¹ Christopher A. Walsh ^4,7 Tamar Harel ^2,3

¹Department of Neurology, Boston Children’s Hospital, Harvard Medical School, USA
²Department of Genetics, Hadassah Medical Center, Israel
³Faculty of Medicine, Hebrew University of Jerusalem, Israel
⁴Division of Genetics and Genomics, Department of Pediatrics, Boston Children’s Hospital, USA
⁵Pediatric Neurology Unit, Hadassah Medical Center, Israel
⁶Department of Radiology, Boston Children’s Hospital, USA
⁷Howard Hughes Medical Institute, Boston Children’s Hospital, USA

NUSAP1 encodes Nuclear and Spindle Associated Protein (NUSAP1), a cell cycle dependent protein with key roles in mitotic progression, spindle formation and microtubule stability. Expression and protein abundance of NUSAP1 peak in the G2 and M phases of the cell cycle and sharply decrease at mitotic exit. At mitotic onset, NUSAP1 accumulates at the growing ends of kinetochore microtubules and stabilizes them by negatively regulating the mitotic centromere-associated kinesin (MCAK) microtubule depolymerising activity. Both over- and under-expression of NUSAP1 lead to dysregulation of mitosis and impaired cell proliferation.

We identified two unrelated individuals with an identical recurrent de novo nonsense heterozygous variant (c.1209C>A; p.(Tyr403Ter)) in NUSAP1, which is expected to lead to a premature truncation codon within the last 50 base pairs of the penultimate exon. Both individuals had microcephaly, severe developmental delay, brain abnormalities and seizures. The gene is predicted to be tolerant of heterozygous loss-of-function mutations, and by cDNA sequencing, quantitative RT-PCR and qPCR on cDNA from patient LCLs and fibroblasts, we showed that the mutant transcript escapes nonsense mediated decay (NMD), suggesting that the mechanism is likely dominant-negative or toxic gain of function rather than haploinsufficiency.

Furthermore, single-cell RNA-sequencing of patient post-mortem brain tissue indicated that the NUSAP1 mutant brain contains all main cell lineages, suggesting an underlying defect in neural progenitor cells, consistent with the resultant phenotype of microcephaly. In conclusion, we propose NUSAP1 as a novel disease gene underlying syndromic microcephaly.