Assessment of transthyretin tetramer stability in patients with ATTR-CM using a novel probe-based immunoassay
M Nagao, Y K Kobayashi, T I Iino, K N Niwa, H O Obinata, S K Kodera, M N Nakadate, N K Kuwahara, K K Kaneshiro, T I Ishida, A H Harada, H O Otake, M S ShinoharaAbstract
Background
Destabilization of the transthyretin (TTR) tetramer is the rate-limiting step in the development of transthyretin amyloid cardiomyopathy (ATTR-CM) and may serve as a characteristic feature for the diagnosis of ATTR-CM. However, due to the lack of readily available methods, clinical assessment of TTR tetramer stability remains challenging. Tetrameric TTR contains a thyroxine (T4)–binding pocket at the weak dimer–dimer interface. Previous studies have demonstrated that unstable TTR variants exhibit lower T4–binding capacity than stable TTR, indicating that T4 binding can serve as an indicator of TTR tetramer stability. Based on the findings, we designed a small-molecule probe that targets the T4-binding pocket and established a probe-based immunoassay to assess TTR tetramer stability.
Purpose
This study aimed to assess TTR tetramer stability in patients with ATTR-CM using the newly developed assay.
Methods
Serum samples were collected from 20 patients with ATTR-CM (18 wild-type and 2 with the A36D variant) and 27 age- and sex-matched control patients with cardiovascular diseases who had no evidence of cardiac hypertrophy or carpal tunnel syndrome. Tetrameric TTR was captured using the small-molecule probe designed to bind the T4-binding pocket of TTR and detected using an alkaline phosphatase–conjugated anti-TTR antibody. Probe-binding signal was normalized to the total TTR concentration in each sample and used as an index of TTR tetramer stability. The assay was performed on a fully automated immunoassay platform.
Results
Proof-of-concept studies using recombinant TTR proteins showed that probe-binding signals of the thermodynamically and/or kinetically unstable TTR mutants V30M, V122I, and L55P were lower than those of recombinant wild-type TTR and the stable mutant T119M. These findings suggest that the developed assay can provide an index reflecting TTR tetramer stability. In clinical samples, the TTR tetramer stability index was significantly lower in patients with ATTR-CM than those in the control group (Mann–Whitney U test, p < 0.001). Receiver operating characteristic (ROC) analysis yielded an area under the curve (AUC) of 0.819. In contrast, total circulating TTR levels measured using a conventional antibody-based immunoassay did not differ significantly between the ATTR-CM and the control group (Mann–Whitney U test, p = 0.539).
Conclusion
The interaction between TTR and the small-molecule probe targeting the T4-binding pocket was reduced in ATTR-CM, indicating decreased TTR tetramer stability. These results suggest that reduced TTR tetramer stability is a characteristic feature of ATTR-CM and support its potential diagnostic utility in clinical settings.graphical abstract