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. 2013 Nov 14:8:178.
doi: 10.1186/1750-1172-8-178.

Abnormal centrosome and spindle morphology in a patient with autosomal recessive primary microcephaly type 2 due to compound heterozygous WDR62 gene mutation

Heba Gamal Farag, Sebastian Froehler, Konrad Oexle, Ethiraj Ravindran, Detlev Schindler, Timo Staab, Angela Huebner, Nadine Kraemer, Wei Chen  1 , Angela M Kaindl
Affiliations

Abnormal centrosome and spindle morphology in a patient with autosomal recessive primary microcephaly type 2 due to compound heterozygous WDR62 gene mutation

Heba Gamal Farag et al. Orphanet J Rare Dis. .

Abstract

Background: Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disease with severe microcephaly at birth due to a pronounced reduction in brain volume and intellectual disability. Biallelic mutations in the WD repeat-containing protein 62 gene WDR62 are the genetic cause of MCPH2. However, the exact underlying pathomechanism of MCPH2 remains to be clarified.

Methods/results: We characterized the clinical, radiological, and cellular features that add to the human MCPH2 phenotype. Exome sequencing followed by Sanger sequencing in a German family with two affected daughters with primary microcephaly revealed in the index patient the compound heterozygous mutations c.1313G>A (p.R438H) / c.2864-2867delACAG (p.D955Afs*112) of WDR62, the second of which is novel. Radiological examination displayed small frontal lobes, corpus callosum hypoplasia, simplified hippocampal gyration, and cerebellar hypoplasia. We investigated the cellular phenotype in patient-derived lymphoblastoid cells and compared it with that of healthy female controls. WDR62 expression in the patient's immortalized lymphocytes was deranged, and mitotic spindle defects as well as abnormal centrosomal protein localization were apparent.

Conclusion: We propose that a disruption of centrosome integrity and/or spindle organization may play an important role in the development of microcephaly in MCPH2.

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Figures

Figure 1
Figure 1
Phenotype and genotype of index patient. (A) Pedigree. (B) Facial phenotype of the patient. Note the sloping forehead, the convex facial profile, the full lips, and the small chin. The appearance of low-set and posteriorly rotated ears on the lateral picture is partly due to reclination of the head. See Additional file 1: Figure S1 for photo of sibling II:1. (C) Representative electropherogram traces confirm the heterozygous frameshift mutation c.2864-2867delACAG in the index patient and her father and the heterozygous missense mutation c.1313G>A in the index patient and her mother (NM_001083961.1 transcript reference sequence). (D) Sequence alignment of corresponding WDR62 protein regions depict the highly conserved amino acids affected by the maternally inherited missense mutation (p.R438H) and the position of the paternally inherited frameshift mutation (p.D955Afs*112): Human (Homo sapiens) UniProt O43379, Chimpanzee (Pan troglodytes) GenBank JAA38944.1, Rhesus monkey (Macaca mulatta) GenBank AFH29290.1, Cat (Felis catus) GenBank XP_003998018.1, Mouse (Mus musculus) GenBank NP_666298.3, Fruit fly (Drosophila melanogaster) Fly Base ID FBgn0031374. (E) Known WDR62 gene mutations according to HGMD Professional 2012.4 and the present paper. Mutations types are color-coded, i.e. missense in red, frameshift in blue, nonsense in black, and splice site mutations in green. The positions of the mutations detected in the index patient are emphasized through bold letters (c.1313G>A also present in the index patient has been previously reported [4]).
Figure 2
Figure 2
Representation of WDR62 protein in immortalized lymphocytes. (A) Protein extracts were analyzed with an anti-WDR62 antibody that recognizes amino acids 900–950. Beta-actin was used as a control for equal protein loading. Signals were visualized with enhanced chemiluminescence. WDR62 protein levels in immortalized lymphocytes from the patient were significantly lower than in control cells (n = 3, p 0.0104, Student’s t-test). (B) Centrosomal WDR62 levels were below detection levels when assessed through immunocytology in patient cells using the same antibody.
Figure 3
Figure 3
Absence of a prophase-like chromosome phenotype in MCPH2 deficiency. (A) Normal metaphase morphology (arrow) of a cultured lymphoblast in a cell line from the MCPH2 patient after induced mitosic arrest by colcemid. The chromosomes are condensed while the delimitation of the metaphase is irregularly shaped suggesting that there is no more nuclear membrane after its disintegration in prophase. Three other nuclei are stained rather homogeneously, typical of uncondensed chromatin in interphase nuclei (asterisks). (B) An MCPH1 lymphoblastoid cell line treated identically shows nuclei with typical prophase-like chromosome (PLC) morphology. Virtually all of the six nuclei reveal a meandering striped or banded chromatin structure characteristic of beginning chromosome condensation (best seen in the two marked nuclei). Despite prometaphase stage, they present with rounded delimitations (arrows) suggesting that they are bounded by a persisting nuclear membrane, a phenomenon that is designated as premature chromosome condensation (PCC). Giemsa stain.
Figure 4
Figure 4
WDR62 in immortalized lymphocytes and dispersion of centrosomal proteins γ-tubulin and CDK5RAP2 in WDR62 mutant patients cells. Subcellular localization of WDR62 (red) and γ-tubulin (green) throughout the cell cycle in immortalized lymphocytes of (A) controls and (B) the MCPH2 patient. In controls, WDR62 colocalized with γ-tubulin and was present on the centrosome with high levels throughout mitosis until anaphase and telophase, thereafter the signal intensity dropped to interphase levels. Gamma-tubulin immunostaining shows distinct centrosomes in controls whereas in patient cells the γ-tubulin-marked centrosomes appear broad and dispersed. (C) Quantification results of abnormal centrosomes with a dispersed γ-tubulin staining around the centrosome (n = 100 metaphase lymphoblastoid cells, p = 0.0006, Student’s t-test). (D) Abnormal localization of centrosomal protein CDK5RAP2 (red) in WDR62 mutant patient immortalized lymphocytes (see also Figure 5). Cells were stained for WDR62, for the centrosome marker CDK5RAP2, and for α-tubulin as a microtubuli marker. DNA was stained with DAPI (blue).
Figure 5
Figure 5
Spindle defects in WDR62 mutant patient cells. Subcellular localization of CDK5RAP2 and α-tubulin throughout the cell cycle in immortalized lymphocytes of (A) control and (B) MCPH2 patient. While the spindle apparatus has a regular bipolar form of appearance from prometaphase to telophase in controls, patient cells show abnormal spindle formation with an increase of abnormal misdirected spindles and broad, unfocused microtubules poles. CDK5RAP2 signals are weaker in patient’s cells than in controls. Cells were stained for CDK5RAP2 (red), for α-tubulin (green), and for DNA using DAPI (blue). (C) Quantification results of abnormal spindles and spindle pole distance (n = 115 metaphase lymphoblastoid cells, p < 0.0001, Student’s t-test).
Figure 6
Figure 6
Lagging chromosomes in mutant WDR62 lymphocytes. Some chromosomes in patient cells showed lagging defects in metaphase (indicated by arrows).
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