Key Information
Abstract
Over 150 transthyretin (TTR) mutations have been identified in hereditary transthyretin (ATTRv) amyloidosis, and new TTR variants have recently emerged. However, the pathogenicity of several new variants remains unclear, making it important to elucidate the differences between amyloidogenic and wild-type TTR. In this study, we report a novel TTR variant (V121A) identified in two unrelated amyloidosis patients aged > 60 years who developed cardiomyopathy. We evaluated the detailed biochemical features of this TTR variant to confirm its amyloidogenicity using plasma samples from these patients and recombinant TTR proteins. While the V121A TTR variant has a similar ability to assemble into a tetramer as wild-type TTR, it aggregates more readily over a wide potential hydrogen range than wild-type TTR. Additionally, the V121A variant is highly prone to dissociation and resistant to binding with known TTR tetramer stabilizers. Clinical and biochemical data suggest that this novel variant is clearly pathogenic, is highly prone to dissociation and aggregation, and is associated with the development of late-onset amyloid cardiomyopathy. Interestingly, amyloid fibril formation due to this variant may not be affected by known TTR stabilizers.
Keywords: amyloid, transthyretin, hereditary transthyretin amyloidosis, ATTR cardiomyopathy
1. Introduction
Transthyretin amyloidosis (ATTR amyloidosis) is a life-threatening disease characterized by the accumulation of amyloid fibrils composed of transthyretin (TTR) [1external link, opens in a new tab,2external link, opens in a new tab]. TTR is a 55 kDa tetramer consisting of four β-sheet-rich subunits, each comprising 127 amino acid residues [3external link, opens in a new tab]. TTR is primarily synthesized in the liver; however, smaller quantities are produced in the choroidal plexus and retina. The TTR tetramer binds and transports thyroxine- and holoretinal-binding proteins. Binding to either ligand slows TTR tetramer dissociation, which is the rate-limiting step of TTR protein aggregation [3external link, opens in a new tab,4external link, opens in a new tab,5external link, opens in a new tab]. Following this rate-limiting tetramer dissociation and partial monomer denaturation, TTR can aggregate into numerous non-native structures, including cross-β-sheet amyloid fibrils, and the soluble non-native TTR structures appear to be the main drivers of degenerative phenotype [6external link, opens in a new tab]. The wild-type TTR (WT-TTR) tetramer is kinetically stable, and it dissociates with a half-life on the order of 1 day in vitro at 25 °C and 2–3 days ex vivo in the blood [7external link, opens in a new tab,8external link, opens in a new tab]. The misfolding of wild-type TTR results in wild-type ATTR amyloidosis (ATTRwt amyloidosis), which presents as an acquired amyloid disease in the elderly [9external link, opens in a new tab].
Hereditary ATTR amyloidosis (ATTRv amyloidosis) associated with variant TTR has historically been an intractable disease. TTR heterotetramers composed of both mutant TTR and WT-TTR caused faster dissociation into monomer subunits and aggregation for amyloid fibril formation. Several therapeutic strategies have been established to slow or halt disease progression in patients with ATTRv amyloidosis. One of the main strategies is the stabilization of the tetramer structure with small-molecule ligands, such as diflunisal [10external link, opens in a new tab], tafamidis [11external link, opens in a new tab], tolcapone [12external link, opens in a new tab], and acoramidis [13external link, opens in a new tab]. These molecules, termed kinetic stabilizers, preferentially bind to the thyroxine-binding sites of TTR. Binding to the tetramer can slow its dissociation into monomers [14external link, opens in a new tab]. In addition to these tetramer stabilizers, TTR silencer drugs, including patisiran [15external link, opens in a new tab], vutrisiran [16external link, opens in a new tab], inotersen [17external link, opens in a new tab], and eplontersen [18external link, opens in a new tab], which reduce the hepatic synthesis of TTR, have also been used in patients with ATTRv amyloidosis.
To date, over 150 TTR variants have been identified in ATTRv amyloidosis. However, the pathogenicity of some of these variants is not fully understood, and it is important to elucidate the differences in amyloidogenicity compared to wild-type TTR. Here, we report the cases of two unrelated patients with late-onset amyloid cardiomyopathy. Both patients carried a novel TTR variant (V121A), and we evaluated the detailed biochemical features of this new TTR variant and compared them to those of wild-type TTR. We also investigated the pharmacological effect of known TTR stabilizers on this TTR variant.