DETERMINING THE PATHOGENICITY OF THE P393H VARIANT OF HUMAN SERPINA1 USING IN SILICO APPROACHES

SERPINA1 codes for the serine protease inhibitor Alpha-1 Antitrypsin (AAT). AAT Deficiency (AATD) is a hereditary condition derived from pathogenic variants of SERPINA1. AATD typically manifest as diseases including chronic obstructive pulmonary disease, emphysema, and chronic liver disease; these conditions present in response to insufficient regulation by AAT and ultimately the disruption of the homeostatic antiprotease-protease balance in the associated organs. Genetic testing has identified the new human variant P393H of SERPINA1 which is currently of uncertain clinical significance. P393H is a nonsynonymous, single-nucleotide variant (c.1175C\u3eA) within the reactive center loop (RCL) which coordinates specificity through the enzymatic cleavage between P1-P1’ residues of AAT. This variant is characterized by the substitution of histidine (basic; positive charge) in place of proline (non-polar) within the RCL which may impact pathogenicity secondary to the amino acid’s physio-chemical difference. To determine the likely clinical significance of the P393H variant we used computational protein modeling, molecular dynamics simulation (MDS), conservation analysis, in silico pathogenicity prediction tools, and phylogenetic modeling. Collectively, sequence alignment and phylogenetic analysis shows that P393 of AAT is completely conserved over a wide array of animal taxa. Additionally, multiple prediction tools whose metrics integrate functional information, structural data, physico-chemical similarity among amino acids, and conservation have confidently predicted this position to be intolerant to variation and pathogenic. MDS trajectories were analyzed using root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) data of wildtype and P393H AAT variants. Collectively P393H exhibited increased structural stability over the time course of the simulation, with a lower mean RMSD compared to wildtype (1.533 vs 1.653), but RMSF data identified widespread flexibility changes which could impact enzymatic function. Most critically, ΔRMSF revealed an increase in rigidity in the P1-P1’ residues (M382-S383) critical for protease-binding specificity. Our results suggest that in vivo experimentation, a logical next step, would demonstrate the inability of P393H to maintain protease-antiprotease homeostasis, thus supporting a finding indicative of pathogenicity