Whole-exome sequencing identifies recessive WDR62 mutations in severe brain malformations

Abstract

The development of the human cerebral cortex is an orchestrated process involving the generation of neural progenitors in the periventricular germinal zones, cell proliferation characterized by symmetric and asymmetric mitoses, followed by migration of post-mitotic neurons to their final destinations in six highly ordered, functionally specialized layers. An understanding of the molecular mechanisms guiding these intricate processes is in its infancy, substantially driven by the discovery of rare mutations that cause malformations of cortical development. Mapping of disease loci in putative Mendelian forms of malformations of cortical development has been hindered by marked locus heterogeneity, small kindred sizes and diagnostic classifications that may not reflect molecular pathogenesis. Here we demonstrate the use of whole-exome sequencing to overcome these obstacles by identifying recessive mutations in WD repeat domain 62 (WDR62) as the cause of a wide spectrum of severe cerebral cortical malformations including microcephaly, pachygyria with cortical thickening as well as hypoplasia of the corpus callosum. Some patients with mutations in WDR62 had evidence of additional abnormalities including lissencephaly, schizencephaly, polymicrogyria and, in one instance, cerebellar hypoplasia, all traits traditionally regarded as distinct entities. In mice and humans, WDR62 transcripts and protein are enriched in neural progenitors within the ventricular and subventricular zones. Expression of WDR62 in the neocortex is transient, spanning the period of embryonic neurogenesis. Unlike other known microcephaly genes, WDR62 does not apparently associate with centrosomes and is predominantly nuclear in localization. These findings unify previously disparate aspects of cerebral cortical development and highlight the use of whole-exome sequencing to identify disease loci in settings in which traditional methods have proved challenging.

Fig. 1. Identification of a 4 base pair deletion in the WDR62 gene in a family with microcephaly and pachygria

a-d, Coronal (a) and axial (c) MR images of a control subject as compared to NG 26-1 (b, d) confirms the clinical diagnosis of microcephaly and shows a diffusely thickened cortex, an indistinct gray-white junction, pachgyria and underoperculization. All images are T2 weighted (photographically inverted). Scale bars in centimeters are shown. e, A four base-pair deletion (red box) in the WDR62 is identified through exome sequencing (WT: wild type). f, Sanger sequencing confirms the deleted bases (in green). The altered amino acid sequence (starting at position 1402) leading to a premature stop-codon (X) is shown in red.

Fig. 2. Additional WDR62 mutations

a-f, Pedigree structures along with mutated bases (red) and the corresponding normal alleles (green) are marked on the chromatograms (mutant–left; wild type-right). a, Families NG 26 and 891 harbor the identical 4 base pair deletion, whereas nonsense mutations leading to premature stop codons (X) are observed in NG 30 (b) and NG 294 (d). Missense mutations affecting conserved amino acids are seen in NG 190 (c) and NG 537 (f). In NG 339 (e), a 17 base pair deletion leads to a premature stop codon. (g) The locations of independent mutations are indicated on the genomic organization of WDR62.

Fig. 3. Representative MRI images from patients demonstrating the wide spectrum of findings associated with mutations in WDR62

a, e, i, k, Axial (a), coronal (e), sagittal (k) MRI images and three-dimensional (3D) surface rendering (i) of a control subject are shown. b, Microlissencephalic features with microcephaly, diffusely thickened cortex, loss of gray-white junction and pachygyria. c, Asymmetric microcephalic hemispheres with marked polymicrogyria (arrowheads). d, Significant polymicrogyria (black arrowheads) and open-lip schizencephaly (red arrowhead). f, Unilateral cerebellar hypoplasia (arrowhead). g, Open-lip schizencephaly (red arrowhead) and the polymicrogyric cortex. h, Unilateral brain stem atrophy (arrow). j, 3D surface rendering demonstrating craniofacial dysmorphology. l, Microcephaly, pachygyria and abnormally shaped corpus callosum (arrowheads).

Fig 4. Wdr62 expression in the developing mouse brain

a, Wdr62 expression is enriched in the VZ and SVZ as seen with in-situ hybridization. b, WDR62 protein(red) distribution reveals a similar pattern. c, d, WDR62 (red) localizes to the nuclei and is expressed by neural stem cells and intermediate progenitors, as marked by SOX2 and TBR2 expression (green), respectively. e, Immunofluorescent staining for α-tubulin (cytoplasmic, blue), S0X2 (nuclear, green) and WDR62 (red) in E12.5 cortical neural progenitor cells reveals that the distribution of the WDR62 overlaps with that of SOX2 and is predominantly nuclear. (Nuclear staining by DAPI (blue) in b-d; Right most panels are composite images in b-e).