Recombinant Human Putative Williams-Beuren syndrome chromosomal region 23 protein (WBSCR23)

What is WBSCR23 and how is it related to Williams-Beuren syndrome?

WBSCR23 (Williams-Beuren syndrome chromosomal region 23) is one of approximately 28 genes located in the commonly deleted region of chromosome 7q11.23 that causes Williams-Beuren syndrome. This neurodevelopmental condition results from a hemizygous deletion of about 1.5-1.8 megabases and affects 1 in 7,500-20,000 live births. WBSCR23 is among the genes that show significantly decreased expression in individuals with Williams syndrome, suggesting its potential contribution to the WS phenotype .

What is the expression pattern of WBSCR23 in individuals with Williams syndrome?

Quantitative RT-PCR studies have demonstrated that WBSCR23 expression is significantly decreased in individuals with Williams syndrome compared to normal controls (P<0.0001). This reduced expression is consistent with the hemizygous deletion of the gene in WS patients. In a cohort study of 77 individuals with WS and 48 normal controls, researchers found that WBSCR23 was among nine typically deleted genes showing significantly decreased expression, along with FZD9, WSTF, STX1A, CLDN3, CLDN4, RFC2, CYLN2, and GTF2I .

How might parental origin of the deletion affect WBSCR23 expression?

Research has demonstrated that parental origin of the WS deletion can influence the expression of certain genes in the region, particularly GTF2I, which shows parent-of-origin effects independent of age and gender. While specific data for WBSCR23 parent-of-origin effects were not explicitly mentioned in the available studies, researchers investigating WBSCR23 should consider analyzing their expression data in the context of maternal versus paternal deletions. This approach could reveal potential imprinting or parent-of-origin effects that might contribute to phenotypic variability in Williams syndrome .

What are the potential functional roles of WBSCR23 in neural development?

Given the neurodevelopmental phenotype of Williams syndrome, which includes distinctive cognitive and behavioral characteristics such as relative strengths in language and severe deficits in visuospatial processing, WBSCR23 might play a role in neural development. Researchers should investigate:

How does WBSCR23 interact with other genes in the Williams-Beuren syndrome chromosomal region?

Understanding gene-gene interactions is crucial for elucidating the complex phenotype of Williams syndrome. Studies have indicated that the correlation of expression between GTF2I and other genes in the WS region differs between WS subjects and normal controls, suggesting regulatory relationships among these genes. Similar correlation analyses should be performed to investigate potential interactions between WBSCR23 and other WS region genes. This could be accomplished through:

• Co-expression network analyses

• Protein-protein interaction studies

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify shared regulatory elements

• Genetic manipulation in model systems to identify epistatic relationships

What are the recommended approaches for producing recombinant WBSCR23 protein?

For researchers seeking to produce recombinant WBSCR23 protein for structural or functional studies, the following methodological approach is recommended:

• Expression system selection: Bacterial systems (E. coli) for initial attempts, with mammalian cell lines (HEK293 or CHO cells) as alternatives if proper folding or post-translational modifications are required

• Vector design: Include a 6xHis or GST tag for purification, with a precision protease cleavage site for tag removal

• Expression optimization: Test multiple expression conditions (temperature, IPTG concentration, induction time)

• Purification protocol:

  • Initial capture using affinity chromatography (Ni-NTA or glutathione)

  • Intermediate purification using ion exchange chromatography

  • Final polishing using size exclusion chromatography

• Quality control: Assess purity using SDS-PAGE and western blotting; verify folding using circular dichroism

What methodologies are most effective for studying WBSCR23 expression levels?

Based on successful approaches used in Williams syndrome research, the following methodologies are recommended for WBSCR23 expression analysis:

• Quantitative RT-PCR: Using the comparative Ct method with appropriate endogenous controls such as β-actin (ACTB). This approach has successfully identified differential expression of WBSCR23 and other WS region genes in previous studies .

• RNA-Seq: For genome-wide expression profiling that can place WBSCR23 expression in the broader context of gene expression patterns.

• Normalization and statistical analysis: Multiple regression analysis should be employed to account for factors such as age, gender, and parental origin of deletion when analyzing WBSCR23 expression data .

• Expression protocol recommendations:

  • Extract RNA using standardized protocols that minimize degradation

  • Verify RNA quality using bioanalyzer or gel electrophoresis

  • Use commercial TaqMan gene expression assays for consistent efficiency

  • Include technical replicates to minimize pipetting errors

  • Validate findings using alternative methodologies

How should researchers analyze WBSCR23 expression data in Williams syndrome studies?

For robust analysis of WBSCR23 expression data, researchers should follow these analytical approaches:

• Normalization strategy: Normalize WBSCR23 expression data against stable reference genes (ACTB has been successfully used in previous studies) .

• Statistical analysis:

  • Use Student's t-test to evaluate the significance of differences in expression between WS subjects and controls

  • Employ correlation coefficients to examine relationships between WBSCR23 expression and other genes

  • Apply multiple regression analysis to identify factors influencing gene expression

• Distribution assessment: Evaluate whether expression data follow normal distributions using appropriate statistical software (e.g., JMP 5.0) .

• Expression calculation: Calculate relative gene expression between WS and control populations using the formula 2^(-ΔΔCt), which has proven effective in previous WS gene expression studies .

What factors should be considered when interpreting WBSCR23 functional data?

When interpreting functional data related to WBSCR23, researchers should consider:

• Developmental context: Williams syndrome is a neurodevelopmental disorder, and WBSCR23 may have stage-specific functions during development.

• Tissue specificity: The effects of WBSCR23 deletion may vary across different tissues and cell types.

• Genetic background: Individual genetic variation may influence the phenotypic consequences of WBSCR23 haploinsufficiency.

• Compensatory mechanisms: Consider whether other genes may compensate for reduced WBSCR23 expression.

• Parent-of-origin effects: As demonstrated for other WS region genes, parental origin of deletion may influence WBSCR23 function or expression .

How can CRISPR-Cas9 technology be optimized for WBSCR23 functional studies?

CRISPR-Cas9 technology offers powerful approaches for studying WBSCR23 function:

• Knockout strategies:

  • Design multiple gRNAs targeting different exons of WBSCR23

  • Verify knockout efficiency using both genomic sequencing and expression analysis

  • Create isogenic cell lines differing only in WBSCR23 status

• Knockdown approaches:

  • Use CRISPR interference (CRISPRi) for partial repression to model haploinsufficiency

  • Create stable doxycycline-inducible knockdown systems for temporal control

• Model systems:

  • Neural cell lines for investigating neurodevelopmental aspects

  • iPSC-derived neurons from WS patients and controls

  • Animal models (mouse knockouts) for in vivo studies

• Readout assays:

  • Transcriptome analysis to identify downstream effects

  • Cellular phenotyping (morphology, proliferation, differentiation)

  • Functional assays based on predicted WBSCR23

  • Proteome analysis to identify interacting partners

What are key considerations when designing experiments to study WBSCR23 in the context of Williams syndrome?

When designing experiments to study WBSCR23 in the context of Williams syndrome, researchers should consider:

• Sample selection and characterization:

  • Confirm WS diagnosis using both clinical criteria and molecular testing

  • Determine precise deletion boundaries using microsatellite markers or array-based methods

  • Establish parental origin of deletion using polymorphic markers

  • Match cases and controls for age, gender, and other relevant variables

• Expression analysis approach:

  • Use both relative and absolute quantification methods

  • Consider allele-specific expression analysis to examine potential regulatory mechanisms

  • Analyze expression across multiple tissues when possible

• Functional validation:

  • Complement human studies with model systems

  • Design rescue experiments to confirm specificity of observed phenotypes

  • Consider epistatic interactions with other WS region genes

How does WBSCR23 expression compare to other genes in the Williams syndrome region?

Based on quantitative expression studies, the following patterns have been observed:

This comparative analysis highlights that WBSCR23 follows the pattern of most WS region genes in showing reduced expression proportionate to copy number, unlike a minority of WS region genes that maintain normal expression levels despite deletion .

How might researchers prioritize WBSCR23 for functional studies among Williams syndrome genes?

When prioritizing Williams syndrome genes for functional studies, researchers should consider:

• Expression pattern consistency: WBSCR23 shows consistent decreased expression in WS, making it a good candidate for contributing to the phenotype .

• Evolutionary conservation: Assess the conservation of WBSCR23 across species as an indicator of functional importance.

• Known or predicted function: Evaluate available information about WBSCR23's functional domains and potential roles.

• Expression in relevant tissues: Analyze WBSCR23 expression in tissues relevant to WS features, particularly the brain regions affected in Williams syndrome .

• Interaction potential: Consider WBSCR23's potential interactions with other genes known to contribute to WS features.

What are the most promising approaches for investigating WBSCR23 function in neural development?

Future research on WBSCR23's role in neural development should focus on:

• Single-cell transcriptomics: Apply single-cell RNA-seq to analyze WBSCR23 expression patterns in different neural cell populations during development.

• Brain organoid models: Use iPSC-derived brain organoids from WS patients and controls to investigate WBSCR23's role in three-dimensional neural tissue organization.

• Conditional knockout models: Develop temporally and spatially controlled WBSCR23 knockout models to pinpoint critical developmental windows and brain regions.

• Functional neuroimaging: Correlate WBSCR23 expression levels with neuroimaging findings in WS patients to identify potential structure-function relationships.

• Integrative multi-omics approaches: Combine transcriptomics, proteomics, and epigenomics to build comprehensive models of WBSCR23 function in neural development.

How might targeting WBSCR23 contribute to therapeutic strategies for Williams syndrome?

While therapeutic strategies for Williams syndrome are still in early stages, investigations involving WBSCR23 might contribute through:

• Gene therapy approaches: Develop methods to restore WBSCR23 expression in affected tissues.

• Small molecule screening: Identify compounds that can modulate pathways affected by WBSCR23 haploinsufficiency.

• Biomarker development: Investigate whether WBSCR23 expression levels correlate with specific WS phenotypes, potentially serving as biomarkers for therapeutic response.

• Combinatorial approaches: Determine whether combined restoration of WBSCR23 with other WS region genes produces synergistic therapeutic effects.

• Timing considerations: Establish critical developmental windows during which WBSCR23 restoration would be most beneficial.