Genotypic, functional, and phenotypic characterization in CTNNB1 neurodevelopmental syndrome

Abstract

CTNNB1 neurodevelopmental syndrome is a rare disorder caused by de novo heterozygous variants in the CTNNB1 gene encoding β-catenin. This study aimed to characterize genetic variants in individuals with CTNNB1 neurodevelopmental syndrome, systematically assess the spectrum of clinical phenotypes using standardized measures, and explore potential genotype-phenotype correlations. In this cross-sectional cohort study, individuals diagnosed with CTNNB1 neurodevelopmental syndrome underwent structured interviews using standardized scales to evaluate motor skills, speech, communication, feeding abilities, visual function, neurodevelopment, and psychopathology. Genetic variants were analyzed, and, in a subset of cases, the impact of β-catenin variants on the Wnt/β-catenin signaling pathway was assessed. Across the 127 included participants (mean age, 70 months; range, 7-242 months) from 20 countries, we identified 88 different variants of the CTNNB1 gene, 87 of which were predicted to lead to loss of CTNNB1 function. Functional assays demonstrated reduced Wnt signaling activity, including 11 variants that also exhibited a dominant-negative effect. One missense variant demonstrated a gain-of-function effect. Dominant-negative variants were not clearly associated with a distinct phenotype; however, those with missense variants presented a milder phenotype, including earlier achievement of independent walking, fewer motor impairments, better conceptual and social skills, improved communication, and fewer feeding difficulties. This study describes the genetic, functional, and phenotypic characteristics in individuals with CTNNB1 neurodevelopmental syndrome. Further investigation into the genotypic and phenotypic characteristics of this syndrome and their interrelationships is essential to deepen our understanding of the disorder and inform the development of targeted therapies.

Keywords: CTNNB1 neurodevelopmental syndrome; Wnt signaling pathway; autism; developmental delay; genotype; genotype-phenotype correlations; microcephaly; phenotype; β-catenin.

Figure 1

Alternative splicing in CTNNB1 (A) Genomic co-ordinates GRCh38/hg38 are shown at the top for the region around exons 2 and 3. Nucleotide resolution of the median read counts of RNA sequencing gene expression data from 176 postmortem human prefrontal cortices are shown organized by developmental stage; data are auto-scaled and include zero. A representative subset of protein-coding transcripts from GENCODEv46 are shown, the alternative splicing event of an additional 132 bp exon (chr3:41,224,190-41,224,321, hg38) is highlighted in light red. Species conservation across 100 vertebrates is represented at the bottom, including PhyloP scores (top) and homology across species (bottom). (B) Corresponding data for an intron with variable inclusion in the 3′ UTR. Images were generated from UCSC genome browser.

Figure 2

Neurodevelopmental disorder variants in CTNNB1 Variants reported by clinical genetics reports are annotated against the MANE Select transcript (ENST00000349496.11), the 125 variants are listed in Table S2. Predicted functional impact is indicated by shape and color (see legend). A blue bar indicates the region (p.32–45) frequently associated with somatic GoF mutations in cancer. For stop-gain single nucleotide variants, the predicted mutability based on trinucleotide genomic sequence is shown under the variants; predicted highly mutable stop gain sites are labeled. Below, protein domains, regions, and structure from UniProt and AlphaMissense are shown. At the bottom, the exon number, CDS exon number, and regions predicted to escape nonsense-mediated decay (NMD) are shown with protein residue number. Nine variants disrupt the canonical splice site, and all of these are predicted to impact splicing (SpliceAI score ≥0.8). Two additional variants had high SpliceAI scores, suggesting they act as cryptic splice sites: the missense variant p.E692D (SpliceAI donor loss score 0.94, donor gain score 0.78) and the synonymous variant p.V561= (SpliceAI donor loss score 0.41, donor gain score 0.76).

Figure 3

TOP/FOP Flash luciferase assay-based functional assessment of mutant β-catenin on Wnt transcriptional activity Dual luciferase activity assay results from SK-N-MC cells transfected with CTNNB1^wt or CTNNB1^mutant. Each bar indicates the relative luciferase activity for each construct following normalization to CTNNB1^wt group. Results are from three independent experiments. Error bars denote SEM. ∗∗∗∗p < 0.0001.

Figure 4

Study of dominant-negative effect of β-catenin variants on Wnt transcriptional activity using TOP/FOP Flash luciferase assay Dual luciferase activity assay results from SK-N-MC cells co-transfected with CTNNB1^wt and empty vector (pcDNA3.1(+)) or CTNNB1^mutant. Each bar indicates the relative luciferase activity for each construct following normalization to the CTNNB1^wt (WT) + empty vector group. Variants marked in red are those for which a significant dominant-negative effect was observed. Results are from three to six independent experiments. Error bars denote SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 5

Prevalence of dysmorphic features in participants with CTNNB1 neurodevelopmental syndrome Percentage with 95% CIs.

Figure 6

Neurological status of CTNNB1 participants as reported by parents Percentage with 95% CIs.

Figure 7

Developmental milestones of CTNNB1 neurodevelopmental syndrome participants aged 24 months or older (N = 111) Percentages represent the proportion of participants achieving a particular milestone. Boxplots represent the median and IQR. Dots represent outliers.

Figure 8

Kaplan-Meier curves showing the probability of independent walking by age with 95% CIs

Figure 9

Scores on functional assessment scales by individuals in relation to their variant type (missense variants, nonsense variants, frameshift variants, whole gene deletions and (canonical) splice variants) Level 1, milder phenotype; level 5, more severe phenotype.

Figure 10

Adaptive skills, age at independent walking, and psychopathological symptoms for individuals with a confirmed GoF (n = 2; green), LoF (n = 24; red), or other variants (n = 99); please see the Table S2 for the list of specific variants included in these groups Median standardized z-scores for each domain are shown (range for z-scores, −3.00 to 3.00) with higher scores indicating better functioning. CBCL, Child Behavior Check List.