Variants in CUL4B are associated with cerebral malformations

Abstract

Variants in cullin 4B (CUL4B) are a known cause of syndromic X-linked intellectual disability. Here, we describe an additional 25 patients from 11 families with variants in CUL4B. We identified nine different novel variants in these families and confirmed the pathogenicity of all nontruncating variants. Neuroimaging data, available for 15 patients, showed the presence of cerebral malformations in ten patients. The cerebral anomalies comprised malformations of cortical development (MCD), ventriculomegaly, and diminished white matter volume. The phenotypic heterogeneity of the cerebral malformations might result from the involvement of CUL-4B in various cellular pathways essential for normal brain development. Accordingly, we show that CUL-4B interacts with WDR62, a protein in which variants were previously identified in patients with microcephaly and a wide range of MCD. This interaction might contribute to the development of cerebral malformations in patients with variants in CUL4B.

Keywords: CUL4B; WDR62; cortical dysplasia; hydrocephalus; intellectual disability; mutation.

Figure 1

Representative MRI and computed tomography (CT) images from patients with CUL4B variants. A and B: Family 10—individual II-1 (2.5 years): MRI T1 axial (A) and sagittal (B) sections show severe ventricular enlargement (colpocephaly), arched and thin corpus callosum, and a slightly thick and undersulcated cortex (arrows). C and D: Family 10—individual II-2 (6 months, before placement of VP-shunt): MRI T2 axial (C) and CT scan sagittal (D) sections show severe ventricular enlargement, arched and thin corpus callosum, and a very simplified gyral pattern (arrows). E: Family 1—individual IV-3 (6 weeks): CT scan axial section shows severe ventricular enlargement and bilateral perisylvian PMG (arrows). F and G: Family 11—individual II-2 (Day 13): T2 axial section (F) and parasagittal section through left perisylvian area (G) show bilateral perisylvian PMG (arrows). H and I: Family 2—individual III-1 (2 years): T1 axial (H), and sagittal (I) sections show a mild predominantly posterior ventriculomegaly and a thin corpus callosum.

Figure 2

Images of patients with CUL4B variants. Recurrent facial dysmorphisms include high/prominent forehead, malformed, and abnormally positioned ears, hyperplastic supraorbital ridges/deep-set eyes with narrow palpebral fissures, low nasal bridge with a rounded nasal tip, prominent lower lip, and prognathia. Extremities show small hands/feet with brachydactyly, clinodactyly, syndactyly 2–3 of toes, and sandal gap. For description of dysmorphisms, see Table 2.

Figure 3

Overview of CUL4B variants identified in this study. A: Schematic overview of the CUL4B gene including the 22 exons and known domains. The positions of all previously described variants [Tarpey et al., 2007; Zou et al., 2007; Badura-Stronka et al., 2010; Isidor et al., 2010; Whibley et al., 2010; Ravn et al., 2012; Londin et al., 2014] and novel variants are depicted. Recurrent variants are depicted in bold; novel variants from this study are marked with a hash (#). Nuclear localization signal (NLS) is shown in red, cullin domain in blue, and neddylation domain (Nedd8) in green. B: Pedigrees of families with CUL4B variant and results from segregation studies. Mutant alleles are represented by a plus (+) and WT alleles by a minus (−). Arrows indicate probands. Below each pedigree, Sanger sequencing chromatograms of the proband (top) and a control (bottom) are depicted. C: RT-PCR analysis of CUL4B splice site variant of family 5. Alternative splicing occurs between exon 20 and 21. Two alternative splice donor sites within exon 20 are used, which result in premature termination codons at amino acid position 806 (splice variant 1; SV1) and 811 (SV2), respectively. P, patient; C, control. D: RT-PCR analysis of CUL4B splice site variant of family 7. The variant leads to skipping of exon 15, which results in a 111 base pair shorter transcript as confirmed by agarose gel electrophoresis and subsequent Sanger sequencing. L, DNA size ladder; P, patient; C, control.

Figure 4

Effect of the novel amino acid altering variants p.(Leu785del), p.(Ala621dup), and p.(Pro50Leu) on the function of CUL-4B (A and B) and interaction of normal CUL-4B with WDR62 (C). A: Western blot showing the WDR5, LIS-1, and WDR62 protein levels in HEK293T cells expressing WT, p.(Pro50Leu), p.(Ala621dup), and p.(Leu785del) FLAG-tagged CUL-4B (left panel). Levels of WDR5 in all three mutants are increased compared to WDR5 levels in WT. Levels of LIS-1 and WDR62 are normal. Tubulin is shown as control. B: Cycloheximide chase experiments in HEK293T cells show that novel amino acid altering variants p.(Ala621dup) and p.(Leu785del) result in an unstable protein as compared to WT, whereas CUL-4B with the p.(Pro50Leu) change is not degraded faster. Tubulin is shown as a control. C: IP experiments in HEK293T cells with antibodies against CUL-4A (upper panel) and CUL-4B (lower panel) with subsequent immunoblot of the precipitates show that WDR62 binds to CUL-4B, but not CUL-4A. DDB1 is shown as a control.