Integrative Multi-Omics Characterization and Structural Insights into the Poorly Annotated Integrin ITGA6 X1X2 Isoform in Mammals

Background: Accurate annotation of gene isoforms remains one of the major obstacles in translating genomic data into meaningful biological insight. Laminin-binding integrins, particularly integrin α6 (ITGA6), exemplify this challenge through their complex splicing patterns. The rare ITGA6 X1X2 isoform, generated by the alternative inclusion of exons X1 and X2 within the β-propeller domain, has remained poorly characterized despite decades of integrin research. Methods: We combined comparative genomics across primates with targeted re-alignment to assess exon conservation and annotation fidelity; analyzed RNA-seq for exon-level usage; applied splice-site prediction to evaluate inclusion potential; surveyed cancer mutation resources for exon-specific variants; and used structural/disorder modeling to infer effects on the β-propeller. Results: Exon X2 is conserved at the genomic level but inconsistently annotated, reflecting the limitations of current annotation pipelines rather than genuine evolutionary loss. RNA-seq analyses reveal low but detectable expression of X2, consistent with weak splice site predictions that suggest strict regulatory control and condition-specific expression. Despite its rarity, recurrent mutations in exon X2 are reported in cancer datasets, implying possible roles in disease. Structural modeling further indicates that X2 contributes to a flexible, disordered region within the β-propeller domain, potentially influencing laminin binding or β-subunit dimerization. Conclusions: Altogether, our results suggest that ITGA6 X1X2 could be a rare, tightly regulated isoform with potential functional and pathological relevance.