Functional constraint, not evolutionary domain expansion, is a stronger correlate of pathogenic variant burden in human RNA-binding proteins.

Whether evolutionary innovation predisposes genes to pathogenic variation is a fundamental question in evolutionary medicine. RBP domain family diversity correlates with neural complexity across metazoans via vertebrate-specific domain expansions. Here, we tested whether such expanded RBP domains harbor elevated pathogenic variant burden relative to evolutionarily conserved domains. We classified 2961 human RBP-encoding genes into expanded (n = 25 genes, 8 Pfam domains; vertebrate/invertebrate gene count ratio ≥ 3.0) and non-expanded control (n = 231 genes, 89 Pfam domains) groups, and integrated ClinVar pathogenic variants with gnomAD v4.1 constraint metrics. Contrary to our hypothesis, expanded domains showed lower median pathogenic variant density than non-expanded domains (P = .140, rank-biserial r = 0.180, 95% CI [-0.089, 0.444]), consistent across sensitivity analyses and count-based models. In multivariable analysis, functional constraint (pLI) was a stronger predictor of variant density than evolutionary expansion status. At the family level, no expanded domain showed significantly elevated burden; instead, the most prominent outliers were conserved non-expanded domains associated with established neurodegenerative diseases (SMN, Nop). The RNaseA family, despite 26-fold vertebrate expansion, harbored ClinVar pathogenic variants in only 1 of 15 members-fewer than expected by chance (binomial P = 0.030). The sole exception, ANG (Angiogenin), concentrates all seven ALS9-associated variants at its catalytic triad and nuclear localization signal, reflecting functional specialization rather than expansion per se. These findings indicate that evolutionary domain expansion does not confer generalized disease vulnerability in RBPs, and that pathogenic variant burden is more closely linked to functional constraint and lineage-specific subfunctionalization than to evolutionary novelty.