Molecular Genetic Analysis of Autosomal Recessive Primary Microcephaly in Pakistani Kindreds

Autosomal recessive primary microcephaly (MCPH) is a rare genetic disorder in which the afflicted individuals have head circumference more than 3 SDs below the age- and sex-related mean. The reduced head circumference is due to a small but architecturally normal cerebral cortex. MCPH is characterized by a pronounced heterogeneity with seven loci, designated MCPH1-7, have already been identified. The underlying genetic defects were found in the following seven genes, MCPH1, WDR62, CDK5RAP2, CEP152, ASPM, CENPJ, and STIL/SIL. The incidence of this disorder is highest in Pakistan (Woods et al., 2005). Here, I ascertained thirty families with MCPH from various regions of Pakistan. Homozygosity mapping revealed linkage in 19 families to the MCPH5 locus, in 2 to MCPH2, in 2 to MCPH4, in 1 to MCPH1, in 1 to MCPH6, and in 5 families linkage to all known MCPH loci was excluded. Families linked to the MCPH1, MCPH2, MCPH5, and MCPH6 loci were also subjected to direct genomic sequencing of the corresponding genes, i.e. MCPH1, WDR62, ASPM, and CENPJ, respectively. This revealed one, two, and nine novel mutations in MCPH1, WDR62, and ASPM, respectively. Genome-wide linkage analysis in the 5 families previously excluded to be linked to any of the known loci resulted in 5 different new gene loci, MCPH8-MCPH12, situated on different chromosomes. For two of the five new loci, namely MCPH8 on chromosome 7q21-q22 (LOD score 10.47) and MCPH9 on chromosome 4p14-4q12 (LOD score 2.53), the causative genes could be identified. Positional candidate gene sequencing revealed mutations in CDK6 (c.589G>A, p.A197T) at the MCPH8 locus and in CEP135 (c.970delC, p.Gln324Serfs*2) at the MCPH9 locus as the most likely pathogenic variants. These variants were not found in 768 chromosomes from healthy Pakistani controls. These two novel MCPH proteins cyclin-dependent kinase 6 (CDK6) and a centrosomal protein of 135kDa (CEP135) presented as transient or permanent components of the centrosome. Cdk6 and Cep135 showed a high expression level in the developing neuroepithelium of the mouse cerebral cortex of E11.5 and E15.5 embryos. In human cell lines, the localization of CDK6 at the spindle pole was observed. Primary fibroblasts of the patient with the CDK6 mutation failed to grow normally and showed an aberrant nuclear shape as well as centrosome-nucleus distance. CDK6 suppression by shRNA mimicked the defects in cell proliferation, nuclear shape, and microtubule organization. Likewise, overexpression of mutant CDK6 resulted in the production of multiple centrosomes and disorganised microtubules. Primary fibroblasts of the patient with the CEP135 mutation showed multiple and fragmented centrosomes, a disorganised microtubule system, misshapen and fragmented nuclei, and sometimes a complete loss of centrosomes. Altered levels of wild-type and mutant CEP135 protein by overexpression caused disorganization of microtubules, while overexpression of mutant CEP135 showed also multiple centrosomes observed before in the patient’s primary fibroblasts. Based on the data on CDK6, I propose that mutation p.A197T may lead to a reduced cell proliferation and may also affect the correct functioning of the centrosome in microtubule organisation and its positioning near the nucleus. The abnormal centrosome number associated with mutant CEP135 strengthens its role in centriole biogenesis, whereas a disorganisation of the microtubule network points to its role at the centrosome as a microtubule organising center. The data obtained lend further support to the hypothesis that the exquisite control of the cleavage furrow orientation in mammalian neural precursor cell mitosis, controlled in great part by the centrosomes and spindle poles, is critical in the etiology of MCPH (Fish et al., 2006, Thornton and Woods, 2009).