Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by reduced head circumference, a reduction in the size of the cerebral cortex and a mild to moderate mental retardation. The MCPH-associated genes described to date have been implicated in cell division and cell cycle regulation and many of them have been localized to the centrosome. We carry out searches to discover further MCPH-associated genes and study the function of the proteins.
MCPH is a neurodevelopmental disorder characterized by reduced head circumference, reduction in the size of the cerebral cortex and mild to moderate intellectual disability. It is a heterogeneous group of disorders caused by mutations of many different genes. MCPH-associated proteins are ubiquitously expressed and the majority of them is associated with the centrosome at least during part of the cell cycle. They have functions in mitotic spindle assembly and spindle structure, centrosome and centriole formation and function, chromosome condensation, DNA repair and DNA damage response.
We are performing homozygosity mapping and exome sequencing in families affected with primary microcephaly as well as of syndromic forms of microcephaly and have identified several novel loci. By elucidating the function of the corresponding proteins and the consequences of the specific mutations resulting in brain-related disorders, we may get new insight into the molecular mechanisms underlying human brain development.
Filippi syndrome is a rare, presumed autosomal recessive disorder characterized by microcephaly, pre- and post-natal growth failure, syndactyly, and distinctive facial features, including a broad nasal bridge and underdeveloped alae nasi. Some affected individuals have intellectual disability, seizures, undescended testicles in males, as well as teeth and hair abnormalities.
Through homozygosity mapping and whole-exome sequencing in a family from Sardinia with two affected children we identified a homozygous frameshift mutation, c.571dupA (p.Ile191Asnfs*6), in CKAP2L encoding the protein cytoskeleton-associated protein 2-like. This protein plays a pivotal role in cell division of neural progenitors. Sanger sequencing of CKAP2L in a further eight unrelated individuals with clinical features consistent with Fillippi syndrome, revealed biallelic mutations in four cases. In mutant lymphoblastoid cell lines no CKAP2L was detectable at the spindle poles of dividing LCLs.
Furthermore, we observed an increase of the number of disorganized spindle microtubules and defects in chromosome segregation. We conclude that loss-of-function mutations of CKAP2L are a major cause of Filippi syndrome (Hussain et al., 2014).
Centrioles are essential for ciliogenesis. However, mutations in centriole biogenesis genes have been reported in primary microcephaly and Seckel syndrome, disorders that do not show clinical features of ciliopathies. We identified mutations in the master regulator of centriole duplication, the PLK4 kinase, and its substrate TUBGCP6 in patients with microcephalic primordial dwarfism and additional congenital anomalies including retinopathy, extending the human phenotype spectrum associated with centriole dysfunction.
Furthermore, we observed that different levels of impaired Plk4 activity result in growth and cilia phenoptyes, providing a mechanism by which microcephaly disorders can occur with or without ciliopathic features. Investigation of centriole biogenesis in patient-derived fibroblast during mitosis revealed reduced centriole number and an increased number of monopolar spindles (Martin et al., 2014).
Mutation in CASC5 is the cause of primary microcephaly-4 (MCPH4). The gene encodes a protein important for kinetochore formation and proper chromosome segregation during mitosis. We identified a second mutation (NM_170589.4;c.6673-19T>A) of CASC5 in a Pakistani MCP family. Post-hoc patient cDNA analysis revealed the skipping of exon 25 of CASC5 causing a frameshift and introducing a premature stop codon (p.Met2225Ilefs*7) which resulted in a C-terminally truncated protein lacking 118 amino acids that encompass the region responsible for the interaction with the hMIS12 complex.
This complex is essential for proper chromosome alignment and segregation. Quantitative RT-PCR showed a down-regulation of CASC5 mutant mRNA. We observed CASC5 is a component of the kinetochore of metaphase chromosomes. In patient fibroblasts we observed dispersed dots of CASC5 outside the metaphase plate of dividing cells. In addition, nuclear morphology was altered as well as the DNA damage response (Szczepanski et al., 2016).
We recruited 35 MCPH families in Pakistan and characterized them genetically. Using homozygosity mapping complemented with whole-exome sequencing (WES) or gene panel sequencing (GPS), we identified several novel and known mutations in previously reported MCPH-associated genes. Linkage to known MCPH loci was found in 31 families, including 27 linked to MCPH5, 2 to MCPH1, 1 to MCPH2 and 1 to the MCPH3 locus, whereas 4 families were not linked to any of the known loci. In total we identified 12 novel mutations, 9 in ASPM, two in MCPH1 and one in CDK5RAP2 (Figure 1).
We also identified a few known mutations, three in ASPM and one in WDR62. The latter was initially deemed to be a missense mutation but we demonstrate here that it affects splicing. As many as 17 of the MCPH5-linked families were found to carry the previously reported ASPM founder mutation p.Trp1326*. Interestingly, the two novel MCPH1 mutations are homozygous microdeletions of 164,250 kb and 577,594 kb, respectively, for which we mapped the exact breakpoints.
Our investigations will result in the discovery of new genes and mechanisms resulting in MCPH. Further, we expect a considerable expansion of our knowledge about the mutational spectra of MCPH-associated genes and also the incidence of particular mutations in different ethnicities.
Szczepanski, S. et al. (2016). A novel homozygous splicing mutation of CASC5 causes primary microcephaly in a large Pakistani family. Hum Genet. 135, 157-170.
Hussain, M.S. et al. (2014). Mutations in CKAP2L, the Human Ortholog of the Mouse Radmis Gene, Cause Filippi Syndrome. Am. J. Hum. Genet. 95, 622-632.
Martin, C-A. et al. (2014). Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy. Nat. Genet. 46, 1283-1292.
Hussain, M.S. et al. (2013). CDK6 associates with the centrosome during mitosis and is mutated in a large Pakistani family with primary microcephaly. Hum. Mol. Genet. 22, 5199-5214.
Hussain, M.S. et al. (2012). A Truncating Mutation of CEP135 is associated with primary microcephaly and disturbed centrosomal function. Am. J. Hum. Genet. 90, 871-878.
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Muhammad Sajid Hussain (Research Associate)
Ilyas Ahmad (PostDoc)
Emrah Kaygusuz (doctoral student)
Salem Alawbathani (doctoral student)
Kathrin Schrage (doctoral student)
Rolf Müller (technician)