A novel homozygous splicing mutation of CASC5 causes primary microcephaly in a large Pakistani family.

Primary microcephaly is a disorder characterized by a small head and brain associated with impaired cognitive capabilities. Mutations in 13 different genes encoding centrosomal proteins and cell cycle regulators have been reported to cause the disease. CASC5, a gene encoding a protein important for kinetochore formation and proper chromosome segregation during mitosis, has been suggested to be associated with primary microcephaly-4 (MCPH4). This was based on one mutation only and circumstantial functional evidence. By combining homozygosity mapping and whole-exome sequencing in an MCPH family from Pakistan, we identified a second mutation (NM_170589.4;c.6673-19T>A) in CASC5. This mutation induced skipping of exon 25 of CASC5 resulting in a frameshift and the introduction of a premature stop codon (p.Met2225Ilefs*7). The C-terminally truncated protein lacks 118 amino acids that encompass the region responsible for the interaction with the hMIS12 complex, which is essential for proper chromosome alignment and segregation. Furthermore, we showed a down-regulation of CASC5 mRNA and reduction of the amount of CASC5 protein by quantitative RT-PCR and western blot analysis, respectively. As a further sign of functional deficits, we observed dispersed dots of CASC5 immunoreactive material outside the metaphase plate of dividing patient fibroblasts. Normally, CASC5 is a component of the kinetochore of metaphase chromosomes. A higher mitotic index in patient cells indicated a mitotic arrest in the cells carrying the mutation. We also observed lobulated and fragmented nuclei as well as micronuclei in the patient cells. Moreover, we detected an altered DNA damage response with higher levels of γH2AX and 53BP1 in mutant as compared to control fibroblasts. Our findings substantiate the proposed role of CASC5 for primary microcephaly and suggest that it also might be relevant for genome stability.