A 75-years-old male patient presented relapsed heart failure symptoms and was diagnosed with ATTR-CA at the Department of Cardiology, Second Affiliated Hospital of Zhejiang University College of Medicine (SAHZU). Clinical data were obtained from the medical history system at SAHZU from September 2019 to April 2022. We performed genetic sequencing to differentiate hATTR-CA from wtATTR-CA for this patient. When hATTR-CA is confirmed, we recommend genetic screening for the patient’s 46-year-old daughter and 43-year-old daughter because this disease is inherited. Written Informed consent was taken from the patient and relatives before acquiring blood samples and clinical data. In parallel, blood samples and data analysis were conducted at the SAHZU, and biochemical characterization analysis was performed at Chinese Academy of Sciences (CAS) Key Laboratory of Separation Science for Analytical Chemistry. The participants agreed to the publication of clinical data and complementary examinations after the identifiable personal information was removed. This investigation was performed according to the Declaration of Helsinki and with approval by the Institutional Ethics Committee of SAHZU (Ethical Batch Number: 20220273). This was a prospective study involving retrospective clinical data.

Wild-type transthyretin and variants were recombinantly prepared following previous literature. Genes of E. coli transthyretin were codon optimized, synthesized by GenScript, and sub-cloned into pET-29b (+) vectors. WT, L55P, and D39Y plasmids were transformed into BL21 DE3 E. coli cells. Cells were grown to OD600 at 0.6–0.8 before being induced by IPTG (0.5 mM) at 37°C for 4 h. Cultured cells were harvested and resuspended in resuspension buffer (50 mM Tris, 150 mM NaCl, pH = 7.5; 15 mL buffer/L of culture). Cells expressing recombinant proteins were thawed and lysed by sonication at 4°C. Lysed cells were centrifuged for 30 min at 16,000 rpm. Ammonium sulfate (final concentration of 242 g/L) was slowly added to the supernatant with rigorous stirring at 4°C for 20 min. The solution was centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was supplemented with additional ammonium sulfate to a final concentration of 607 g/L with rigorous stirring at 4°C for 20 min. The solution was centrifuged at 12,000 rpm for 15 min at 4°C. The pellet was resuspended in 20 mL of anion exchange buffer A (25 mM Tris, 1 mM EDTA, pH = 8.0) and dialyzed against 4 L of buffer A overnight at 4°C. After dialysis, the sample was filtered and applied to a 50 mL Source 15Q anion exchange column (GE Healthcare) equilibrated with buffer A. TTR was eluted using a linear gradient of NaCl (160 mL; 50–350 mM) followed by a NaCl wash (50 mL; 350 mM). Eluted TTR was purified using a 120 mL Superdex 200 gel filtration column (GE Healthcare) in SEC buffer (10 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, pH = 7.4). The protein containing fractions were identified by SDS-PAGE gel analysis, pooled, and concentrated. No significant impurities were identified and purity was estimated to be 98% based on SDS-PAGE electrophoresis analysis.

To quantify the thermodynamic stability of protein TTR, we denatured D39Y-TTR (3.6 μM) by urea of concentration from 0.5 to 9.0 M in phosphate buffer (10 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, pH = 7.4). After 96 h, the fluorescence of tryptophan was measured from 310 to 410 nm with excitation at 295 nm. The ratio at 355 nm and 335 nm was recorded as a signal of the unfolding of TTR.

Resveratrol increases in its fluorescence quantum yield and displays a blue shift when it selectively binds to the TTR tetramer. We quantified the dissociation of TTR by measuring the concentration of tetrameric TTR to text the equilibrium between TTR tetramers and monomers. 500 μL D39Y TTR (3.6 μM) was incubated with urea (5 M) in phosphate buffer (10 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, pH = 7.4) for 96 h. In order not to disturb the equilibrium of tetramers and monomers, TTR samples were treated with resveratrol (18 μM) just before the fluorescence measurement. Excited at 320 nm, the resveratrol fluorescence spectrum was recorded from 310 to 410 nm. Fluorescence at 394 nm was reported on the concentration of tetrameric D39Y TTR.

Kinetic stability was determined by the rate-limiting dissociation of the tetramer, because monomer unfolding was much faster than tetramer dissociation. To verify this, we incubated TTR (3.6 μM) in urea at the concentration from 3 to 9 M in phosphate buffer (10 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, pH = 7.4, 25°C). Tryptophan fluorescence (I355/335) was recorded at period of time. The kinetics data fit well to a single exponential function: I355/335 = I355/335N + A (1–e-kdiss t); where I355/335N is the native protein fluorescence intensity ratio (355/335 nm), A is the amplitude difference, kdiss is the tetramer dissociation rate constant and t is time in h. The lnkdiss versus urea concentration plot is linear, allowing extrapolation to 0 M urea.

Tafamidis of various concentrations was preincubated with TTR (7.2 μM) in phosphate buffer (10 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, pH = 7.4) at 37°C for 0.5 h. Acidic aggregation buffer (200 mM NaOAc, 100 mM KCl, acidified by AcOH to pH = 4.40) was added to the samples to get a final concentration of TTR of 7.2 μM, and after incubating at 37°C for 72 h, the extent of fibril formation was measured by OD at 330 nm.

Following the protocol reported before, dissociation constants for Tafamidis binding to wild type, L55P and D39Y TTR were determined using a MicroCal PEAQ-ITC (Malvern MAN0572-01-EN-00). A solution of Tafamidis (300 μM in 10 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, 5% DMSO, 5% EtOH, pH = 7.4) was prepared and titrated into an ITC cell containing TTR (25 μM in 10 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, 5% DMSO, 5% EtOH, pH = 7.4). The initial injection of 1 μL was followed by 35 injections of 1 μL each (25°C). Integration of the thermogram after subtraction of blanks yielded a binding isotherm that fit best to a model of one binding site. The data were fit by a non-linear least squares approach with one adjustable parameter, namely, Kd, using the ITC data analysis module in Microcal PEAQ-ITC analysis software.

This protocol was adapted from previous work by the Kelly group. Briefly, UPLC (FLR) Waters Acquity H-Class uplc plus pro and Protein-pakTM Hi RCSQ (5 μm, 4.6 × 100 mm) were from Waters. The A2 molecule was synthesized in the laboratory. The patients’ sera were incubated with A2 for covalent labeling and subjected to UPLC separation and detection of tetramer TTR. Each sample was injected onto a Waters Acquity H-Class UPLC plus pro instrument fitted with a Protein-pakTM Hi RCSQ (5 μm, 4.6 × 100 mm) from Waters. TTR was eluted from the column using the following gradient (Buffer A: 25 mM Tris–HCl, pH = 9.0, 1 mM EDTA; Buffer B: 1 M NaCl, 1 mM EDTA, 25 mM Tris–HCl, pH = 9.0).

The proband presented worsening dyspnea on exertion and lower extremity edema over 1 year. The proband’s ECG revealed an atrial flutter with low voltage in the limb leads, with a QS pattern in lead II, III and aVF. Laboratory investigations showed mild elevated BNP (135.7 pg/ml) and troponin T (0.027 ng/ml). Serum and urine protein electrophoresis with immunofixation and sFLC assay were negative. Echocardiography showed bi-atrial enlargement, symmetrical left ventricular hypertrophy (septal 17.4 mm and posterior wall 16.6 mm), and mild impaired left ventricular systolic function (LVEF 49.2%) (Figure 1A, right side). Speckle tracking echocardiography revealed severely depressed global longitudinal strain with apical sparing (GLS-8.4%) (Figure 1A, left side) which was a hallmark of cardiac amyloidosis. Cardiac enhanced magnetic resonance showed diffuse subendocardial late gadolinium enhancement (Figure 1B). Tc-99m-PYP nuclear scintigraphy showed grade 3 myocardial uptake with increased heart/contralateral lung ratio (HCL 2.38) (Figure 1C). Furthermore, the proband and his two asymptomatic daughters were sequenced and analyzed (Figure 1D). The proband showed a novel homozygous missense variant at base position 175 in exon 2 of the TTR gene (c.175G>T), resulting in an aspartic acid change to tyrosine (p.D39Y, numbered without the first 20 amino acids that serve as signaling peptide and are cleaved during TTR secretion) (Figure 1E). The proband’s eldest daughter was identified as carrying this heterozygous variant of TTR gene c.175G>T. In silico analyses predicted this c.175G>T variant could be pathogenic (SIFT: affect protein function; Polyphen-2: benign; Mutation Taster: polymorphism; M-CAP: damaging).

Point mutants on TTR protein usually affect its thermodynamic and kinetic stability leading to ATTR amyloidosis diseases. In this work, we elucidated the effect of D39Y mutation (Figure 2A) on the thermodynamic stability in three ways and compared it with the parameters of commonly studied mutation L55P and wild type TTR. First, we measured the thermodynamic stability by urea-mediated denaturation (Figure 2B), followed by resveratrol binding measurement (Figure 2C). Normally, the thermodynamic stability was quantified by urea denaturation midpoints (Cm), revealing that D39Y (Cm = 2.8 M) exhibited higher stability than L55P-TTR (Cm = 2.3 M), but lower than that of WT-TTR (Cm = 3.4 M) (Figure 2D). To further validate this result, we performed a thermal shift assay that can quantify the thermodynamic stability of TTR via melting temperature (Tm). TTR (7.2 μM) was heated in acid aggregation buffer (NaOAc 200 mM, KCl 100 mM, acidified by AcOH to pH = 4.4), respectively from 35 to 96°C for 5 min and measured the optical density at 330 nm. The thermal shift assay concluded the same that D39Y (Tm = 341.8 K) is more stable than L55P (Tm = 337.8 K), but less stable than WT (Tm = 351.1 K) and (Figure 2E). Overall, D39Y-TTR is thermodynamically destabilized compared to WT-TTR.

Some TTR mutations destabilize TTR by decreasing the tetramer dissociation kinetic barrier. To verify whether the variant D39Y reduced the kinetic stability of TTR, we quantified it using tetramers dissociation half time (t1/2) measured by urea mediated denaturation (Figures 3A, B). Kinetically, in 6 M urea, D39Y (t1/2 = 5.4 h) was less stable than WT (t1/2 = 17.1 h), but more stable compared with L55P (t1/2 = 0.6 h) (Figure 3C). Under physiological conditions (0 M urea), the conclusion still held true. Extrapolated from the urea mediated tetramer dissociation curve (Figure 3B), D39Y (t1/2 = 9.9 h, 0 M urea) was less stable compared with WT (t1/2 = 42 h), but more stable than L55P (t1/2 = 4.4 h) on kinetic stability under 0 M physiological conditions (Figure 3D).

Transthyretin is usually heterotetramers in patients’ serum due to the subunit exchange effect. To compare the kinetic stability of heterotetramers of variant D39Y and WT, we mixed up D39Y and WT TTR and incubated them in phosphate buffer (10 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, pH = 7.4) at D39Y:WT ratios from 4:0 to 0:4 at 4°C for 48 h for efficient subunit exchange. Then, the subunit exchanged samples were subjected to cross-linking reaction. Through the cross-linking reaction, glutaraldehyde crosslinked two TTR monomers into a dimer, the dimer into a tetramer, and the dimer into a tetramer, as well as octamers and multimers of larger molecular weights. Therefore, we observed these products of higher molecular weights as corresponding bands on this SDS-PAGE gel. Due to the limited resolution of the SDS-PAGE gel and the randomness of crosslinking reaction between TTR multimers, these higher molecular bands representing TTR crosslinked multimers exhibited as a smear bands cluster at the top of the gel. Fortunately, this experiment does not rely on the quantification of these smear bands of higher molecular weights. We monitored the dimeric bands that were well defined and the molecular weight of which was clearly discernible by the gel ladder. SDS-PAGE gel analysis of TTR proteins after cross-linking showed that homotetramers and heterotetramers had the same kinetic stability as the dissociated dimer bands did not change with the increasing ratio of D39Y and WT (Figure 4A). This result may explain the late disease age-of-onset for the D39Y patient reported herein.

On the other hand, besides the deposition of full-length amyloid TTR, fragment 49–127 to trigger TTR aggregation is also a pathway to form amyloid fibrils. To assess whether D39Y is capable of forming digested fragments, D39Y (3.6 μM) was incubated with trypsin (5 μg/mL) at 37°C for 0–345 min. The products were analyzed by SDS-PAGE gel, revealing no digested bands at 9 kDa. This result demonstrates that D39Y is resistant to proteolysis and does not generate amyloid-prone proteolytic fragments (Figure 4B). Overall, these lines of evidence may also explain D39Y late disease age-of-onset.

We next evaluated whether D39Y TTR exhibited a differential response to Tafamidis stabilization compared to WT and L55P-TTR. Using OD300 turbidity assay, we assessed the inhibitory effect of Tafamidis on WT-TTR and mutant TTR amyloid formation under acidic aggregation conditions (200 mM NaOAc, 100 mM KCl, acidified by AcOH to pH = 4.4). D39Y showed a similar response to Tafamidis upon one site occupation at 1:1 stoichiometry (Figures 5A–C). However, upon the increasing concentration of Tafamidis, we did not observe a dose-dependent inhibitory effect after 3.6 μM, indicating its negative cooperativity present in mutant TTR binding to Tafamidis. This result was supported by the ITC experiment as two binding site model was not feasible in D39Y mutant (Figures 5D–F). However, D39Y binds to Tafamidis as tightly as the WT TTR, indicating its potential satisfactory response to Tafamidis treatment.

We next explored the tetramer concentration of TTR in patient’ serum using UPLC method reported by the Kelly group (11) (Figure 6A). Stilbene A2 probe selectively reacts with the Lysine-15 at the weak dimer-dimer interface of TTR tetramer and fluoresces at 430 nm. Due to the stable covalent bonds between A2 and TTR tetramers, we can quantify the folded tetrameric TTR by UPLC (Figure 6A). Based on the above working principle of this assay, we established the linear correlation between the fluorescence intensity of the peak and TTR tetramer concentration spiked in serum for afterward quantification (Figures 6B, C). The results showed that A2 fluorescence intensity increased with the concentration of TTR, giving a wide linear range from 0.5 to 20 μM (Figure 6C).

It has been reported that TTR may partition into folded tetrameric, misfolded oligomeric and amyloid fibril conformations. The decrease in tetramer TTR in blood serum may manifest the disease onset of TTR amyloidosis. Toward this end, we assessed the concentration of TTR tetramer in D39Y symptomatic patient (father), asymptomatic D39Y gene carrier (daughter 1), and wild type non-D39Y gene carrier (daughter 2). Next, we quantified the TTR tetramer concentration in the sera of three subjects of the patient’s family (Figures 6D, E). Intriguingly, the fluorescence intensity of the healthy daughter (WT, 4.8 μM) shows much higher than the symptomatic father (D39Y, 2.5 μM). Whereas the asymptomatic daughter’s TTR tetramer concentration stayed in the middle (D39Y, 3.7 μM) (Figure 6F). In addition, the chromatographic peak shape of D39Y (multiplet peaks) and WT (singlet peak) TTR in this assay reflects its genotypes. These results highlighted that TTR tetramer concentration may serve as a disease onset parameter for D39Y mutation carrier and for the drug interference window time.