Padlock Assay in Transthyretin Amyloidosis: A Feasibility Study

Introduction: The ability to distinguish between transcripts that differ by a single nucleotide positions our padlock assay as a highly accurate imaging tool for detecting disorders associated with structural variations in the human genome. In this study, we evaluated the effectiveness of the padlock assay in identifying TTR gene variants in a case of transthyretin amyloidosis (ATTR), a rare multisystemic disease. ATTR may result from autosomal dominant mutations in the TTR gene or occur in a wild-type form. Methods: We applied the padlock assay in combination with rolling circle amplification (RCA) and fluorescence microscopy, using peripheral blood mononuclear cells (PBMCs) as clinical samples. Results: Using the padlock assay, 1) we detected intracellular TTR transcripts in 80% of PBMCs, including a benign variant caused by a single nucleotide substitution in intron 3, 2) we visualized the subcellular localization of both coding and non-coding regions of TTR transcripts, and 3) through dual staining, we simultaneously detected both wild-type and mutated TTR intron 3 in patient- derived cells. Additionally, we found that PBMCs and platelets are immunoreactive to TTR antibodies, suggesting that immunocompetent cells may contribute to the distribution of TTR protein across tissues and organs. Discussion: We demonstrate that the padlock assay can serve as a non-invasive imaging test capable of spatially detecting genomic variants in ATTR. Conclusion: These findings suggest that the padlock assay has potential application in evaluating the efficacy of disease-modifying therapies in extra-hepatic cells. Moreover, this study is the first to highlight PBMCs as a valuable source for advancing our understanding of ATTR pathogenesis and for supporting the development of improved therapeutic approaches.