The transthyretin (TTR) amyloidoses are a group of degenerative diseases caused by TTR aggregation, requiring rate-limiting tetramer dissociation. Kinetic stabilization of TTR, by preferential binding of a drug to the native tetramer over the dissociative transition state, dramatically slows the progression of familial amyloid polyneuropathy. An established method for quantifying the kinetic stability of recombinant TTR tetramers in buffer is subunit exchange, in which tagged TTR homotetramers are added to untagged homotetramers at equal concentrations to measure the rate at which the subunits exchange. Herein, we report a subunit exchange method for quantifying the kinetic stability of endogenous TTR in human plasma. The subunit exchange reaction is initiated by the addition of a substoichiometric quantity of FLAG-tagged TTR homotetramers to endogenous TTR in plasma. Aliquots of the subunit exchange reaction, taken as a function of time, are then added to an excess of a fluorogenic small molecule, which immediately arrests further subunit exchange. After binding, the small molecule reacts with the TTR tetramers, rendering them fluorescent and detectable in human plasma after subsequent ion exchange chromatography. The ability to report on the extent of TTR kinetic stabilization resulting from treatment with oral tafamidis is important, especially for selection of the appropriate dose for patients carrying rare mutations. This method could also serve as a surrogate biomarker for the prediction of the clinical outcome. Subunit exchange was used to quantify the stabilization of WT TTR from senile systemic amyloidosis patients currently being treated with tafamidis (20 mg orally, once daily). TTR kinetic stability correlated with the tafamidis plasma concentration.

Increasing evidence supports the hypothesis that soluble misfolded protein assemblies contribute to the degeneration of post-mitotic tissue in amyloid diseases. However, there is a dearth of reliable non-antibody based probes for selectively detecting oligomeric aggregate structures circulating in plasma or deposited in tissues, making it difficult to scrutinize this hypothesis in patients. Hence, understanding the structure-proteotoxicity relationships driving amyloid diseases remains challenging, hampering the development of early diagnostic and novel treatment strategies. Here, we report peptide-based probes that selectively label misfolded transthyretin (TTR) oligomers circulating in the plasma of TTR hereditary amyloidosis patients exhibiting a predominant neuropathic phenotype. These probes revealed that there are much fewer misfolded TTR oligomers in healthy controls, in asymptomatic carriers of mutations linked to amyloid polyneuropathy, and in patients with TTR-associated cardiomyopathies. The absence of misfolded TTR oligomers in the plasma of cardiomyopathy patients suggests that the tissue tropism observed in the TTR amyloidoses is structure based. Misfolded oligomers decrease in TTR amyloid polyneuropathy patients treated with disease-modifying therapies (tafamidis or liver transplant-mediated gene therapy). In a subset of TTR amyloid polyneuropathy patients, the probes also detected a circulating TTR fragment that disappeared after tafamidis treatment. Proteomic analysis of the isolated TTR oligomers revealed a specific patient associated-signature comprised of proteins that likely associate with the circulating TTR oligomers. Quantification of plasma oligomer concentrations using peptide probes could become an early diagnostic strategy, a response-to-therapy biomarker, and a useful tool for understanding structure-proteotoxicity relationships in the TTR amyloidoses.

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Conflict of interest: Conflict of interest statement: JWK discovered tafamidis at the Scripps Research Institute; is a shareholder of FoldRx (Pfizer), the companies that developed tafamidis into a drug; and he and ETP receive royalty payments from tafamidis sales. JWK is a paid consultant for the Pfizer Orphan and Rare Diseases organization. Pfizer supported JWK's expenses with travel and accommodation for scientific meetings. TC is a past and current investigator in clinical trials sponsored by FoldRx, Pfizer, Alnylam, Ionis, and Prothena; these trials were paid per protocol to Centro Hospitalar do Porto. TC is a consultant for Pfizer, Alnylam, Ionis, and Prothena. TC has spoken on behalf of Pfizer, Alnylam, Glaxo, and Prothena at scientific meetings and received financial compensation. Pfizer, Alnylam, Ionis, and Biogen supported TC's expenses with travel, accommodation, and registration for scientific meetings.

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Hereditary transthyretin (TTR) amyloidosis associated with the TTRV30M (p.TTRV50M) mutation presents predominantly as an axonal polyneuropathy, with variable involvement of other organs. Serious central nervous system (CNS) and eye manifestations, including stroke, dementia, vitreous opacities and glaucoma, have been reported in untreated V30M TTR amyloidosis patients, and in these patients after treatment with liver transplantation (LT). Distinct therapies for V30M TTR amyloidosis developed during the last decade exhibit promising results in slowing the peripheral and autonomic nervous system pathology. However, the effect of these therapies on the CNS and eye manifestations of V30M TTR amyloidosis is not known. Herein, we show that in a small cohort of patients taking tafamidis orally (20 mg tafamidis meglumine daily) we could detect this small molecule in the cerebrospinal fluid (CSF) and the vitreous body. In the CSF, the ratio of TTR tetramer to tafamidis was ≈2:1, leading to a moderate kinetic stabilization of TTR in the CSF of these patients. Our data suggest that tafamidis can cross the CSF-blood and eye-blood barriers. Future studies comparing CNS and eye manifestations in patients treated with LT, kinetic stabilizers or TTR lowering drugs are essential to understand the clinical effect of our observations.

Ornithine transcarbamylase deficiency (OTCD) is an X-linked inborn defect of metabolism of the urea cycle, which causes hyperamonemia. Mutations of the OTC gene have been recognized as the genetic cause underlying the OTC deficiency. The severity of the disease is associated with the type of mutation, leading either to neonatal onset of hyperammonemia or to a later appearance of the disease. The mutation Thr125Met is associated with neonatal hyperammonemia. Recently, the disease-causing Thr125Met mutation in humans was reported as wild-type neutral allele in chimpanzees. Further analysis confirmed the presence of Met125 fixed in chimpanzees together with Thr135, representing the only two divergent positions between human and chimpanzee OTCs. Thr125 and Thr135 were identified as ancestral mammalian combination, so the Thr135Ala substitution occurred as human-specific event, whereas the substitution of Thr125Met was characteristic of the chimpanzee linage. Only when Met125 emerges in a background with the human-specific Ala135, a highly deleterious effect is observed, suggesting among other hypotheses the existence of a compensatory effect in chimpanzee. To explore this hypothesis, we built an in vitro cell model system to study the effect of the three distinct genetic backgrounds (Ala135-Thr125; Ala135-Met125 and Thr135-Met125) on the OTC protein function. We observed that the human Thr125Met mutant is inactive, whereas the chimp OTC shows an enzymatic activity comparable with the wild-type human OTC. We concluded that the presence of a threonine at position 135 in chimps rescues the deleterious effect of the methionine at position 125, in a mechanism of intra-locus compensation.

The transthyretin (TTR) amyloidoses (ATTR) are progressive, degenerative diseases resulting from dissociation of the TTR tetramer to monomers, which subsequently misfold and aggregate, forming a spectrum of aggregate structures including oligomers and amyloid fibrils. To determine whether circulating nonnative TTR (NNTTR) levels correlate with the clinical status of patients with V30M TTR familial amyloid polyneuropathy (FAP), we quantified plasma NNTTR using a newly developed sandwich enzyme-linked immunosorbent assay. The assay detected significant plasma levels of NNTTR in most presymptomatic V30M TTR carriers and in all FAP patients. NNTTR was not detected in age-matched control plasmas or in subjects with other peripheral neuropathies, suggesting NNTTR can be useful in diagnosing FAP. NNTTR levels were substantially reduced in patients receiving approved FAP disease-modifying therapies (e.g., the TTR stabilizer tafamidis, 20 mg once daily). This NNTTR decrease was seen in both the responders (average reduction 56.4 ± 4.2%; n = 49) and nonresponders (average reduction of 63.3 ± 4.8%; n = 32) at 12 mo posttreatment. Notably, high pretreatment NNTTR levels were associated with a significantly lower likelihood of clinical response to tafamidis. Our data suggest that NNTTR is a disease driver whose reduction is sufficient to ameliorate FAP so long as pretreatment NNTTR levels are below a critical clinical threshold.