A comprehensive framework for the interpretation of TTN missense variants

Area of Science:

• Genetics
• Molecular Biology
• Neurology

Background:

• Missense variants in the TTN gene are common and difficult to interpret, complicating genetic diagnostics for titinopathies.
• While TTN truncating variants are well-established causes of titinopathies, the role of rare missense variants remains unclear.
• Computational and functional tools exist but are underutilized in clinical settings for TTN missense variant assessment.

Purpose of the Study:

• To develop a framework for interpreting rare TTN missense variants in patients with myopathy.
• To integrate clinical, computational, and functional data for improved variant classification.
• To enhance diagnostic accuracy for titinopathies caused by TTN missense variants.

Main Methods:

• Analyzed an international cohort of unsolved myopathy cases with rare TTN missense variants and TTN truncating variants (TTNtv).
• Utilized AlphaMissense for in silico predictions, complemented by minor allele frequency (MAF) and exon usage.
• Performed in vitro functional assays (protein expression, cell imaging) to assess variant effects on protein folding, solubility, and aggregation.

Main Results:

• Identified 30 patients with TTNtv/missense combinations, exhibiting diverse myopathic phenotypes.
• AlphaMissense predictions identified potentially pathogenic missense variants.
• Functional assays revealed three proline substitution variants impairing protein folding and causing aggregation.
• Identified a recurrent TTN missense variant (p.Gln7023Pro) in five families, suggesting a common ancestor and meeting criteria for likely pathogenic classification.

Conclusions:

• A framework integrating clinical, computational, and functional evidence is proposed for TTN missense variant interpretation.
• Multiple lines of evidence are crucial for accurate variant classification due to TTN's complexity.
• Improved diagnostic accuracy requires tailored guidelines and global data sharing for TTN variant interpretation.