Y669H

Position 669 sits in wolframin's C-terminal lumenal domain. The AlphaFold model places Y669 within 5 Å of GLN668 (2.5 Å), GLY670 (2.5 Å), TRP666 (3.8 Å), THR665 (4.0 Å), ALA671 (4.4 Å), and GLN667 (4.5 Å). The wild-type tyrosine ring makes edge-face π–π stacking with TRP666 and hydrogen-bonding contacts to nearby polar residues through its hydroxyl.

Replacing tyrosine with histidine here preserves more structural character than the cysteine substitution at the same position (Y669C, Atlas card adjacent). The imidazole ring of histidine is aromatic, so some π-stacking interaction with TRP666 is preserved — although the histidine imidazole is smaller, lacks the phenol's hydroxyl arm, and presents a different electrostatic surface. Critically, histidine's pKa is close to physiological pH; the residue can be neutral or positively charged depending on its local environment. The ER lumen is mildly acidic, which favors the protonated, charged form.

This substitution carries a larger DynaMut2 |ΔΔG| (1.20 kcal/mol) than Y669C (0.41 kcal/mol). Two factors explain the difference. First, histidine's larger volume creates steric mismatch where the tyrosine's hydroxyl arm previously projected into the local environment without conflict. Second, the introduced positive charge — when histidine is protonated in the mildly acidic ER lumen — destabilizes the local electrostatic context that the neutral tyrosine occupied. The net effect is a moderate but real destabilization of the local fold, while still leaving the protein fold globally intact.

The pathogenic classification ("Pathogenic" rather than the "Likely Pathogenic" of Y669C) reflects accumulated clinical evidence — multiple submitters, multiple cases — that this specific substitution has reliable functional consequences.