Uncovering position-specific patterns in codon and codon-pair usage in candidate genes associated with blood coagulation diseases

Current strategies for optimizing gene therapeutics and recombinant protein production typically rely on universal host codon usage indices. However, there is a growing shift toward incorporating gene-specific traits to enhance therapeutic characteristics. In this study, we investigate position-specific variations in codon and adjacent codon-pair usage biases (CPUBs), offering potential for more tailored gene engineering approaches. We focus our analysis on the coding sequences of four coagulation factors: ADAMTS13, von Willebrand factor, factor VIII, and factor IX, which have been used in therapeutic applications. By aligning transcript homologs with human sequences for each gene using Discontiguous Megablast and MACSE, we assess "sequence-position-specific" codon and CPUBs; 157 homologous sequences for <i>ADAMTS13</i>, 148 for <i>F8</i>, 96 for <i>F9</i>, and 202 for <i>VWF</i>. Species with homologs ranged from Primates and Artiodactyla (Even-toed Ungulates) to Testudines. Statistically significant, position-specific positive CPUBs were observed that contrasted with conventional, alignment-specific negative CPUBs. Moreover, we observed that codon and codon-pair usages are highly associated at sequence positions despite little or no association in conventional-position-agnostic analyses. The distinct biases observed at different positions/functionally critical domains in coding sequences highlight the importance of considering position-specific effects in codon optimization strategies.