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

Area of Science:

• Genetics
• Developmental Biology
• Molecular Biology

Background:

• Hirschsprung disease (HSCR) has genetic heterogeneity, with RET variants implicated in 12-50% of cases.
• RET is crucial for enteric nervous system (ENS) development, controlling precursor proliferation and migration.
• Functional consequences of RET variants in HSCR remain poorly understood.

Purpose of the Study:

• To investigate the functional impact of specific RET missense and nonsense variants on cell proliferation and migration.
• To assess the pathogenicity of debated RET variants.
• To understand the relationship between proliferation defects, migration loss, and variant position in RET.

Main Methods:

• Utilized a RET-dependent neural crest-derived cell line.
• Employed cDNA-based and CRISPR-based PRIME editing to introduce specific RET variants.
• Conducted quantitative proliferation and migration assays.

Main Results:

• Three missense (E178Q, S922Y, F998L) and all three nonsense variants significantly reduced cell proliferation and migration.
• The Y791F variant, previously debated, showed no significant pathogenicity.
• Migration defects did not consistently correlate with proliferation defects; nonsense variant severity was position-independent.

Conclusions:

• Targeted functional assays are essential for accurate HSCR variant pathogenicity assessment.
• Functional data can refine machine learning predictions for variant classification.
• This study clarifies the functional impact of specific RET variants in HSCR pathogenesis.