Summary
This study presents case-level genetic evidence indicating CK2beta haploinsufficiency as pathogenic, with two patients exhibiting distinct loss-of-function (LoF) mutations: Patient 1 with missense mutation p.Leu39Arg with dysmorphic features but unknown inheritance, and Patient 2 with a frameshift mutation Met132fs showing a significant reduction in CSNK2B mRNA expression and stability. Functional studies support haploinsufficiency, demonstrating reduced protein interaction and enzymatic activity, a significant decrease in mRNA expression and protein stability, and a likely destabilizing effect on protein structure due to altered amino acid spatial orientation. In silico predictions and comparative analysis of multiple mutations suggest a common pathogenetic mechanism involving LoF, reinforced by qRT-PCR and protein expression analysis. These findings underscore CK2beta's critical role in the observed disease phenotypes.
Evidence of Haploinsufficiency
Patient Identifier: Patient 1
Mutation Type: missense mutation
Mutation Details: p.Leu39Arg affecting the KEN box-like motif
Clinical Phenotype: Dysmorphic features including prognathism, pointed chin and abnormalities of the ears
Inheritance Tested: No
Effect on Gene Function: The mutant residue (Arg) introduces a charge in a buried residue, which could result in changes in the amino acid spatial orientation, thereby leading to protein-folding problems.
Direct Quote: "Patient 1 had a missense mutation affecting the very conserved Leucine 39 in the KEN box-like motif (aa 32–40) in the N-terminal segment of the CK2beta (p.Leu39Arg)."
Location in Paper: Discussion
Patient Identifier: Patient 2
Mutation Type: Frameshift
Mutation Details: Met132fs, an 11 bp deletion altering the frame and causing a termination site loss
Clinical Phenotype: Not specified in provided text
Inheritance Tested: Yes
Effect on Gene Function: The CSNK2B mRNA with an 11 bp deletion is very likely unstable, causing a significantly reduced amount of the mRNA
Direct Quote: "The Quantitative Real-time PCR (qRT-PCR) showed no significant difference in CSNK2B expression in patient 1 (Leu39Arg) and a highly significant decrease in CSNK2B mRNA in patient 2 (Met132fs) compared to control mRNA expression...the aberrant mRNA was highly significantly reduced compared to the wild-type CSNK2B transcript...the mutant mRNA had a significantly shortened half-life compared to WT CSNK2B mRNA."
Location in Paper: 3.3. Functional Studies 3.3.1.
Clinical Features
Feature: Dysmorphic features
Frequency: 100%
Direct Quote: "Dysmorphic features were presented in 100% of subjects, characterizing a common facial gestalt"
Location in Paper: Discussion
Feature: Developmental delay and cognitive and language function impairment
Frequency: 100%
Direct Quote: "All subjects (n = 12) showed developmental delay and cognitive and language function impairment, with a variable degree of severity"
Location in Paper: Discussion
Feature: Skeletal abnormalities
Frequency: 87.5%
Direct Quote: "Skeletal abnormalities, mainly of the hands and feet, were identified in 87.5% of the patients"
Location in Paper: Discussion
Feature: Variable teeth hypoplasia, hypodontia or hyperdontia
Frequency: 87.5%
Direct Quote: "the same percentage [87.5%] displayed variable teeth hypoplasia, hypodontia or hyperdontia"
Location in Paper: Discussion
Feature: Cardiac or vascular abnormalities
Frequency: about 86%
Direct Quote: "six out of seven individuals (about 86%) showed cardiac or vascular abnormalities"
Location in Paper: Discussion
Feature: Epilepsy
Frequency: 60%
Direct Quote: "only 60% of the patients presented epilepsy"
Location in Paper: Discussion
Functional Studies
Study Type: In silico prediction
Methodology: In silico tools (PaPI score, Polyphen-2, SIFT, MutationTaster, DANN score), ClustalW alignment of CK2beta orthologues, SWISS-Model for 3D structure prediction, and I-Mutant2.0 and SDM for protein stability upon mutation
Conclusions: The variant is predicted to be deleterious with a CADD Phred score of 29.5. Leucine 39 is evolutionarily conserved. Mutation p.Leu39Arg predicted to introduce a charge that could lead to protein-folding problems and is likely destabilizing with a negative ΔΔG.
Direct Quote: "We evaluated the functional impact of the substitution... The mutant residue (Arg) introduces a charge in a buried residue, which could result in changes in the amino acid spatial orientation, thereby leading to protein-folding problems. A ΔΔG <0.0 corresponds to mutations predicted to be destabilizing."
Location in Paper: Not specified
Study Type: qRT-PCR mRNA expression analysis
Methodology: Expression of CSNK2B mRNA in patients' peripheral blood compared to control, using Quantitative Real-time PCR (qRT-PCR), with and without transcript-specific oligonucleotides.
Conclusions: The mutant CSNK2B mRNA had a significantly reduced expression and stability.
Direct Quote: "Initially, we investigated CSNK2B mRNA expression in the patients’ and control peripheral blood. The Quantitative Real-time PCR (qRT-PCR) showed no significant difference in CSNK2B expression in patient 1 (Leu39Arg) and a highly significant decrease in CSNK2B mRNA in patient 2 (Met132fs) compared to control mRNA expression...the aberrant mRNA was highly significantly reduced compared to the wild-type CSNK2B transcript."
Location in Paper: 3.3.1.
Study Type: Mutant mRNA half-life measurement
Methodology: Measuring the half-life of the wild-type and mutant Met132fs CSNK2B mRNA in transfected cells using actinomycin D.
Conclusions: The mutant mRNA had a significantly shortened half-life compared to the wild-type CSNK2B mRNA.
Direct Quote: "To confirm this, we measured the half-life of the wild-type and mutant Met132fs CSNK2B mRNA in transfected cells. After treatment with actinomycin D to inhibit cellular transcription, we examined the half-life of the CSNK2B mRNA at different time points. The results showed that the mutant mRNA had a significantly shortened half-life compared to WT CSNK2B mRNA."
Location in Paper: 3.3.1.
Study Type: Protein Interaction and Enzymatic Activity Assay
Methodology: HEK293 cells were co-transfected with CSNK2A1 HA-tagged with either wild-type or mutant CSNK2B-Myc constructs, followed by pull-down assays and kinase activity measurements.
Conclusions: The interaction between the catalytic subunit and the Leu39Arg mutant was decreased, likely due to its reduced protein amount. A significant decrease in the kinase activity of the mutant CK2beta/CK2alpha1 complex was observed.
Direct Quote: "Since Leu39Arg CK2beta and CK2alpha1 interaction was still present, but the amount of the complex was impaired, we hypothesized that this could affect the kinase activity of the holoenzyme. The data showed a significant decrease in the kinase activity of the mutant CK2beta/CK2alpha1 complex."
Location in Paper: Section 3.3.5
Study Type: Protein expression level and stability assessment
Methodology: Expression and stability of Pro179Tyrfs*49 were compared to the control
Conclusions: The Pro179Tyrfs*49 was expressed at a level comparable to the control, but it lost its ability to bind the catalytic subunit.
Direct Quote: "Conversely, the Pro179Tyrfs*49 was expressed at a level comparable to the control, according to the crystal structure data of the C-terminal deletion mutant CK2beta1−182, showing an intact global fold of the protein. Nevertheless, it lost its ability to bind the catalytic subunit."
Location in Paper: Not provided in the text
Study Type: Functional analysis of mutant protein
Methodology: Analysis of the mutant protein p.Met132LeufsTer110 with altered C-terminal tail
Conclusions: Suggests a possible correlation between the length and the magnitude of the destabilizing effect on the protein.
Direct Quote: "It is worth noting that the mutant protein p.Met132LeufsTer110 was among these mutants, the one with the longer altered C-terminal tail, which suggests a possible correlation between the length and the magnitude of the destabilizing effect."
Location in Paper: Not provided in the text
Study Type: In silico and functional evidence
Methodology: Comparative analysis of mutations
Conclusions: The mutations behave as LoF, suggestive of haploinsufficiency of CK2beta as a common pathogenetic mechanism in POBINDS.
Direct Quote: "Our data provide in silico and functional evidence indicating that the CSNK2B mutations, p.Leu39Arg and p.Met132LeufsTer110, identified in two POBINDS patients, behave as LoF"
Location in Paper: Conclusions
Study Type: mRNA stability analysis
Methodology: Quantification of CSNK2B transcript by qRT-PCR using RNA extracted from blood of patients and control.
Conclusions: A reduction of about 50% in total CSNK2B mRNA in patient 2 compared to control.
Direct Quote: "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."
Location in Paper: Figure 6
Study Type: protein expression analysis
Methodology: HEK293 cells transfected with wild-type or mutant CSNK2B Myc-tagged constructs; immunoblotting with anti-Myc and anti-Hsp90 antibodies.
Conclusions: A significant reduction in the expression of mutant proteins compared to wild type, and an absent signal for the Met132fs mutant initially with a weak signal appearing after a longer exposure.
Direct Quote: "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 (*)."
Location in Paper: Figure 7