CDK6 associates with the centrosome during mitosis and is mutated in a large Pakistani family with primary microcephaly

Abstract

Autosomal recessive primary microcephaly (MCPH) is characterized by reduced head circumference, reduction in the size of the cerebral cortex with otherwise grossly normal brain structure and variable intellectual disability. MCPH is caused by mutations of 11 different genes which code for proteins implicated in cell division and cell cycle regulation. We studied a consanguineous eight-generation family from Pakistan with ten microcephalic children using homozygosity mapping and found a new MCPH locus at HSA 7q21.11-q21.3. Sanger sequencing of the most relevant candidate genes in this region revealed a homozygous single nucleotide substitution c.589G>A in CDK6, which encodes cyclin-dependent kinase 6. The mutation changes a highly conserved alanine at position 197 into threonine (p.Ala197Thr). Post hoc whole-exome sequencing corroborated this mutation's identification as the causal variant. CDK6 is an important protein for the control of the cell cycle and differentiation of various cell types. We show here for the first time that CDK6 associates with the centrosome during mitosis; however, this was not observed in patient fibroblasts. Moreover, the mutant primary fibroblasts exhibited supernumerary centrosomes, disorganized microtubules and mitotic spindles, an increased centrosome nucleus distance, reduced cell proliferation and impaired cell motility and polarity. Upon ectopic expression of the mutant protein and knockdown of CDK6 through shRNA, we noted similar effects. We propose that the identified CDK6 mutation leads to reduced cell proliferation and impairs the correct functioning of the centrosome in microtubule organization and its positioning near the nucleus which are key determinants during neurogenesis.

Exome sequencing and CRISPR/Cas genome editing identify mutations of ZAK as a cause of limb defects in humans and mice

“…Briefly, genomic DNA was enriched for exonic and adjacent splice site sequences with the SeqCap EZ human exome library v2.0 kit, and libraries were run on an Illumina HiSeq 2000 sequencer via a paired-end 100-bp protocol (Hussain et al 2013). For data analysis, the Cologne Center for Genomics (CCG) Varbank pipeline v2.6 and user interface was used (Kawalia et al 2015).…”

What next-generation sequencing (NGS) technology has enabled us to learn about primary autosomal recessive microcephaly (MCPH)

“…CENPJ, STIL and CEP135, others have a nuclear or cytoplasmic localization during interphase and only locate to the spindle poles during mitosis e.g. WDR62, ASPM and CDK6 [18,26,27] and yet others interact with known centrosomal proteins, e.g. CASC5 and PHC1.…”

“…Homozygosity mapping after genome-wide linkage analysis in a consanguineous eight-generation family with seven affected and two unaffected individuals and parents identified a new MCPH locus on chromosome 7q21.11-q21.3 [27]. Despite having head circumferences ranging from -4 to -6 SD below the mean, patients had a relatively mild phenotype with mild intellectual disability.…”

“…Some cells with misshapen nuclei had only a single centrosome, others had supernumerary centrosomes. Patient fibroblasts showed a reduced growth rate when compared to controls and fluorescence-activated cell sorting (FACS) analysis of patient and control primary fibroblasts detected a higher percentage of late-apoptotic patient cells [27].…”

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays

“…12 Germline mutations in CDK genes are exceedingly rare with the exception of an activating germline mutation (encoding p.Arg24Cys) predisposing to malignant melanoma 13,14 in CDK4 (MIM: 123829) and CDK6 mutations that cause autosomal-recessive primary microcephaly 12 (MCPH12 [MIM: 616080]). 15 To the best of our knowledge, no other loss-of-function mutations in any member of the CDK family have been linked to a Mendelian syndrome.…”