Variability in proliferative and migratory defects in Hirschsprung disease-associated RET pathogenic variants

Area of Science:

• Genetics and Molecular Biology
• Developmental Biology
• Cell Biology

Background:

• Hirschsprung disease (HSCR) is genetically complex, often involving variants in genes crucial for enteric nervous system (ENS) development.
• The receptor tyrosine kinase gene RET is a major contributor to HSCR, but the functional impact of its variants is not fully understood.
• Understanding RET variant function is critical for diagnosing and potentially treating HSCR.

Purpose of the Study:

• To investigate the functional impact of heterozygous pathogenic RET variants on neural crest cell proliferation and migration.
• To assess the pathogenicity of specific missense and nonsense RET variants associated with HSCR.
• To determine the correlation between proliferation defects, migration defects, and HSCR phenotypic severity.

Main Methods:

• Utilized a RET-dependent neural crest-derived cell line.
• Employed cDNA- and CRISPR-based PRIME editing to engineer specific RET variants (five missense, three nonsense).
• Conducted quantitative proliferation and migration assays to analyze cellular phenotypes.

Main Results:

• Three missense and all three nonsense RET variants significantly reduced cell proliferation and migration.
• The missense variant c.2372A>T (p.Tyr791Phe) showed no significant impact, suggesting it may be benign.
• Migration defects did not consistently correlate with proliferation defects, and nonsense variant severity was position-independent.

Conclusions:

• Targeted functional assays are essential for accurate pathogenicity assessment of HSCR-associated RET variants.
• Bioinformatic predictions for HSCR variant pathogenicity could be improved by incorporating functional data.
• The study provides critical insights into the cellular mechanisms underlying RET-dependent HSCR.