Haploinsufficiency as a Foreground Pathomechanism of Poirer-Bienvenu Syndrome and Novel Insights Underlying the Phenotypic Continuum of CSNK2B-Associated Disorders

Abstract

CSNK2B encodes for the regulatory subunit of the casein kinase II, a serine/threonine kinase that is highly expressed in the brain and implicated in development, neuritogenesis, synaptic transmission and plasticity. De novo variants in this gene have been identified as the cause of the Poirier-Bienvenu Neurodevelopmental Syndrome (POBINDS) characterized by seizures and variably impaired intellectual development. More than sixty mutations have been described so far. However, data clarifying their functional impact and the possible pathomechanism are still scarce. Recently, a subset of CSNK2B missense variants affecting the Asp32 in the KEN box-like domain were proposed as the cause of a new intellectual disability-craniodigital syndrome (IDCS). In this study, we combined predictive functional and structural analysis and in vitro experiments to investigate the effect of two CSNK2B mutations, p.Leu39Arg and p.Met132LeufsTer110, identified by WES in two children with POBINDS. Our data prove that loss of the CK2beta protein, due to the instability of mutant CSNK2B mRNA and protein, resulting in a reduced amount of CK2 complex and affecting its kinase activity, may underlie the POBINDS phenotype. In addition, the deep reverse phenotyping of the patient carrying p.Leu39Arg, with an analysis of the available literature for individuals with either POBINDS or IDCS and a mutation in the KEN box-like motif, might suggest the existence of a continuous spectrum of CSNK2B-associated phenotypes rather than a sharp distinction between them.

Keywords: CK2beta haploinsufficiency; IDCS; KEN like-box; POBINDS; WES; mRNA and protein instability; phenotypic continuum.

Figure 1

(A,B) Pedigrees of the two families included in the study. Square, male; circle, female; filled, affected; unfilled, unaffected; arrows indicate index patients. (C) Dysmorphic craniofacial features (front and side views) of patient 1 at 27 months (left panel), 5 years and 7 months (middle panels) and 11 years (right panels). (D) Dysmorphic craniofacial features (front and side views) presented by patient 2 at 8 months (left panel) and 26 months of age (right panels).

Figure 2

Skeletal anomalies in patient 1. (A) Picture and radiograph of the hands. (B) Picture and radiograph of the feet. (C) Orthopantomography of the teeth. L (left), R (right).

Figure 3

(A) Schematic representation of CSNK2B gene. The pathogenic variants identified in this study and their position are shown. Lines and boxes indicate introns and exons, respectively. Exons and introns are numbered with respect to the exon upstream of the first coding exon. The coding regions are indicated by filled boxes, whereas the non-translated sequences are depicted by open boxes. (B) Schematic illustration of the CK2beta protein. The position of the mutations is shown. Important regions in the CK2beta protein are indicated by filled boxes: KEN box-like (green) and destruction domain (orange) represent putative degradation motifs; the acidic groove (grey) is involved in the modulation of the catalytic activity; the zinc-finger motif (yellow) mediates the CK2beta dimerization; the region involved in the CK2alpha binding (blue). (C) Sanger chromatograms from patients (1–2) and their parents. Mutated nucleotides identified in patient 1 (CSNK2B, c.116T > G) and in patient 2 (CSNK2B, c.384_394delAGGTGAAGCCA) are indicated by arrows. (D) ClustalW multiple-sequence alignment showing conservation of Leu39 of CK2beta across species which is highlighted by the blue box. Asterisk (*) indicates conserved amino acid and colon (:) for conservative changes.

Figure 4

(A) CSNK2B c.384_394delAGGTGAAGCCA, identified in patient 2, is predicted to result in a longer protein (p.Met132LeufsTer110), with 240 amino acids instead of 215, and with a different C-terminal amino acid sequence (shown in red) compared to the wild-type CK2beta. * indicates the translation termination codon. (B) Details of the nucleotide sequences (black letters) deleted in patient 2 (current study)and in patient 19 [12] (violet and blue box, respectively). The corresponding amino acid sequences are shown and the underlined regions indicate different amino acid sequence in the mutant proteins compare to the wild type (CK2B).

Figure 5

(A) Predicted secondary structure of the wild-type CK2beta and (B) of the predicted mutant elongated protein (p.Met132LeufsTer110) using PSIPRED Protein Analysis Workbench. The mutant is composed of new α-helices (pink) and strands (yellow); compare to the wild type in the predicted C-terminal tail of the mutant CK2beta protein (p.Met132LeufsTer110).

Figure 6

CSNK2B frameshift mutation reduces its mRNA stability. Quantification of CSNK2B transcript by qRT-PCR was performed using RNA extracted from peripheral blood of control (Cntrl) and patients (1 and 2) using primers for CSNK2B (pairs 1, 2 and 3) and transcript-specific oligonucleotides able to amplify either wild-type (primers wt 1, wt 2) or mutant CSNK2B mRNA (primers Leu39Arg and Met132fs). (A) Histogram showing no differences in the expression of total CSNK2B mRNA in patient 1 (NM_001320.7: c.116T>G, p.Leu39Arg) as compared to control. (B) Histogram showing a reduction of about 50% in total CSNK2B mRNA in patient 2 (NM_001320.7: c.384_394del11, p.Met132fs) compared to control. Each bar represents the mean ± SD of three independent experiments. ** p < 0.01 calculated by Student’s t-test (two-tailed, homoscedastic). (C) Allele-specific qRT-PCR in patient 1 shows a similar expression of wild-type and mutant transcripts (primers wt 1 and primers Leu39Arg, respectively). (D) Allele-specific qRT-PCR in patient 2 shows a significant reduction in the mutant compared to wild-type transcript (primers wt 2 and primers Met132fs, respectively). Each bar represents the mean ± SD of three independent experiments. ** p < 0.01 calculated by Student’s t-test (two-tailed, homoscedastic); Actin served as an internal control. (E) Sanger chromatograms of RT-PCR products using RNA extracted from patient 2. Upper panel: chromatogram showing the presence of both wild-type and mutant CSNK2B transcripts. Middle panel: sequence chromatogram of the wild-type and lower panel: mutant CSNK2B transcripts after cloning of single transcript in the pJet1.2/Blunt vector. The arrows indicate the position of the mutation. The peaks of the 11 nucleotides present in the wild-type transcript and deleted in the mutant transcript in patient 2 are highlighted with an open box. (F–G) HEK293 cells were either transfected with wild-type or mutant CSNK2B Myc-tagged constructs. RT-PCR assays, using primer pair 1, were performed to investigate the half-life of WT and frameshift mutant CSNK2B mRNAs after Actinomycin D treatment at different time points. Each bar represents the mean ± SD of three independent experiments, * p < 0.05; ** p < 0.01; *** p < 0.001 calculated by Student’s t-test (two-tailed, homoscedastic).

Figure 7

CSNK2B mutations impair the amount of CK2beta (CK2B). (A) HEK293 cells were transfected with wild-type or mutant CSNK2B Myc-tagged constructs. Whole-cell lysates were subjected to SDS-PAGE and immunoblotting with anti-Myc and anti-Hsp90 antibodies. A representative Western blot shows a reduced amount of mutant Leu39Arg protein compared to the wild type and an absent signal for the Met132fs mutant. After a longer exposure, a weak signal appears for the Met132fs mutant (*). (B) Histogram showing a significant reduction in the expression of mutant proteins compared to wild type. Each bar represents the mean ± SD of three independent experiments. ** p < 0.01; *** p < 0.001 calculated by Student’s t-test (two-tailed, homoscedastic). The CK2beta protein level was normalized to Hsp90 expression used as a loading control.

Figure 8

The mutant CK2beta(CK2B) protein amount is regulated by proteasome-mediated degradation. (A) HEK293 cells were either transfected with wild-type or mutant CSNK2B Myc-tagged constructs and then treated or not treated with MG132 or NH₄Cl for 4 h. A representative Western blot shows that MG132 effectively blocks the proteolysis of mutant CK2beta proteins. (B) Histogram showing a significant increment in the expression of both mutant proteins upon MG132 treatment compared to untreated or treated with NH₄Cl. ** p < 0.01 calculated by Student’s t-test (two-tailed, homoscedastic). The CK2beta protein level was normalized to Hsp90 expression used as a loading control.

Figure 9

CK2beta cellular localization is not impaired by Leu39Arg mutation. (A) Epi-fluorescence microscopy showing the cytosolic localization of CK2beta wild type (WT) and mutant (Leu39Arg) in HeLa cells. The red signals correspond to CK2beta staining, and the blue corresponds to nuclear DAPI staining. Scale bars = 10 μM. (B) Nuclear and cytosolic fractions from HeLa cells transfected with wild-type CK2beta or mutants (Leu39Arg and Met132fs) were run on SDS-PAGE. The gel was blotted and probed with an anti-Myc antibody. The blot was probed with anti-β-Actin and anti-Sp1 antibodies as loading controls for cytosolic and nuclear fractions, respectively.

Figure 10

Impact of CSNK2B mutations on CK2 subunits’ interaction and kinase activity. (A) HEK293 cells were transiently co-transfected with CSNK2A1-HA and the Myc-tagged CSNK2B wild type and mutants. Protein complexes were immunoprecipitated using an anti-HA antibody. Proteins were subjected to SDS–PAGE gel and immunoblotted with an anti-Myc antibody and an anti-HA antibody. Total cell lysates from co-transfected and non-transfected cells (mock) were probed with anti-Myc, anti-HA and anti-Hsp90 as a loading control. (B) Measure of the kinase activity of wild type (WT) and mutant (Leu39Arg) CK2beta proteins. CSNK2B wild-type and mutant constructs were overexpressed with the catalytic subunit CSNK2A1-HA, followed by immunoprecipitation with anti-Myc antibody and luminescent ADP-Glo kinase assay with (+) and without (−) CK2 specific substrate. (C) Histogram showing a significant decrease in the kinase activity of the mutant complex compared to the wild type. Experiments were repeated three times with independently prepared cell extracts. ** p < 0.01 calculated by Student’s t-test (two-tailed, homoscedastic).

Figure 11

(A) Recurrent or unique missense mutations reported so far (HGMD database and current study) affecting the KEN-box-like domain (aa 32-40) of the CK2beta protein (NP_001311.3). The p.Leu39Arg identified in patient 1 is indicated in bold. (B) MetaDome web server for the CSNK2B gene. The tolerance landscape (upper panel) indicated the region that is intolerant to missense variation. Enlargement of the KEN box-like motif located between protein positions 32 and 40. The mutations identified in patients with IDCS or POBINDS are indicated in green. The analysis shows that these mutations are located in a region of high intolerance. The non-synonymous over-synonymous ratio, or dn/ds score, is used to quantify genetic tolerance [42].