Functional analyses of splice site variants in TCF12

Area of Science:

• Genetics
• Molecular Biology

Background:

• Pre-messenger RNA (mRNA) splicing is crucial for protein synthesis; errors can cause human diseases.
• Genetic variants, such as splice donor and acceptor site alterations, can lead to mis-splicing.
• Craniosynostosis, a congenital disorder, can result from aberrant splicing in genes like TCF12.

Purpose of the Study:

• To establish a pipeline for the functional validation of genetic variants suspected of altering pre-mRNA splicing.
• To experimentally classify the pathogenicity of variants in the TCF12 gene associated with craniosynostosis.

Main Methods:

• Identification and revalidation of genetic variants in patients with craniosynostosis.
• In-silico prediction of splicing consequences for identified variants.
• In-vitro functional analysis using a minigene splice assay to assess splicing alterations and transcriptional activity.

Main Results:

• Two novel and one previously described genetic variants in TCF12 were analyzed.
• In-silico predictions suggested altered splicing for all variants.
• In-vitro assays confirmed aberrant splicing for two variants and demonstrated significantly reduced transcriptional activity for all three variants.

Conclusions:

• The combined approach of in-silico prediction and in-vitro functional assays provides a robust method for classifying splice site variants.
• This pipeline enables accurate assessment of variant pathogenicity, even without additional patient RNA.
• The study successfully classified TCF12 variants as likely pathogenic, aiding in the understanding of craniosynostosis.