Characterization of Transthyretin Mutation G47V Associated with Hereditary Cardiac Amyloidosis Open Access

Amyloidosis is a multisystem, gain-of-toxicity disease due to the continuous deposition of misfolded and aggregated pathogenic proteins in various tissues [1]. To date, more than 30 different amyloidogenic proteins have been reported to be involved in amyloidosis diseases [2]. One of them is the transthyretin (TTR). The deposition of wild-type (WT)-TTR amyloidosis leads to age-related nonhereditary amyloidosis, which usually develops at an age older than 50 [3]. In contrast, amyloidosis caused by TTR mutations (ATTRv) is a rare inherited and autosomal dominant disease with the deposition of mutant TTR amyloid fibrils in various organs or tissues, including cardiac [4, 5]. Cardiac involvement is one of the most important prognostic factors in patients with generalized amyloidosis [6]. Survival with TTR-related cardiac amyloidosis in untreated patients is measured in years to decades; for example, median survival of ATTR cardiac amyloidosis (ATTR-CA) caused by Val122Ile TTR, found in approximately 3–4% of the US black population, is only 31 months from diagnosis [7, 8]. Various treatment strategies, including small molecule stabilizers (tafamidis, AG10, diflunisal), RNA drugs (patisiran and inotersen), monoclonal antibody (NI-006), etc., are either approved for clinical applications or under clinical trials [5, 9].

TTR, also known as prealbumin, biochemically is a tetrameric protein consisting of 127 amino acids [10], performing functions as transporting thyroid hormones and binding retinol-binding protein [11, 12]. Upon circulation, the structural integrity of TTR is compromised particularly for mutant proteins, leading to tetramer dissociation, protein misfolding, and amyloid fibril deposition in different organs or tissues [8, 13]. The pathogenicity and progressiveness of ATTRv amyloidosis are correlated to the aggregation propensity of mutant proteins into amyloid substances, mainly determined by their thermodynamic and kinetic stabilities [14]. More than 150 mutants of TTR have been reported, resulting in compromised thermodynamic and kinetic stabilities to drastically different extents [15]. The differences in these biochemical and biophysical properties often lead to a different disease age-of-onset, progressiveness, penetration rate, and drug response [4, 14]. Thus, comprehensive studies on these parameters for each mutant may benefit the patients by providing personalized diagnosis, treatment option, and drug response follow-ups.

In this study, we reported an ATTR-CA patient carrying the TTR mutation G47V, also known as p.Gly67Val in the literature, including the 20 amino acid signal peptides in the numbering of residues [16, 17]. Among all clinically reported and biochemically characterized TTR mutations, G47V was rarely reported. Previous reports described the clinical characterization of G47V, but no biochemical characterizations have been performed [18‒27]. Herein, series of biochemical and biophysical experiments were conducted to measure the thermodynamic and kinetic stabilities of the mutant protein. In addition, the response of G47V-TTR to small molecule stabilizers was tested that provided hints for precision medicine in ATTR-CA treatment. Finally, tetrameric TTR concentration in patient’s serum was measured using an ultra-performance liquid chromatography (UPLC) assay, showing significant decrease compared to healthy donors, echoing its compromised stabilities. Together, our work provided the first piece of evidence for the biochemical properties of G47V-TTR mutation, as well as experimental and clinical hints for the pathogenicity of this mutation.

Written informed consent was obtained from the research subject. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by Institutional Review Board (IRB) at the Ethics Committee of the second affiliated hospital of Zhejiang University School of Medicine, approval number [2021LSY0102]. The clinical data including a complete medical history, family history, laboratory tests, 12-lead echocardiogram (ECG), transthoracic echocardiography, cardiac magnetic resonance (CMR), and technetium pyrophosphate (^99mTc-PYP) scintigraphy were systematically reviewed.

Genomic DNA was extracted from peripheral blood lymphocytes by standard procedures using QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany). DNA quality and concentration were examined by NanoDrop2000 (Thermo Fisher Scientific, USA) and were fragmented by Bioruptor Pico to generate paired-end library (200–250 bp). The whole exome was captured using the SureSelect Human All Exon V7 Kit (Agilent Technologies, USA). The final purified DNA libraries were sequenced using the Illumina NovaSeq 6000 platform (Illumina, USA) as PE 150 bp reads, providing an average coverage depth for each sample of at least 100-fold. Quality control was performed before proceeding to the next step.

Adapter and low-quality reads were removed from raw sequencing data using FASTQ preprocessor. High-quality reads were then aligned to the reference sequence (hg19) using Burrows-Wheeler Aligner. The final BAM files were used for variant calling. Single-nucleotide variants, insertions, and deletions were screened using Genome Analysis Toolkit (GATK, https://software.broadinstitute.org/gatk). All single-nucleotide variants and indels were filtered and estimated via multiple databases, including 1000 Genomes (http://www.1000genomes.org/), GenomeAD (http://gnomad.broadinstitute.org/), and ExAC (http://exac.broadinstitute.org/). dbNSFP database (http://database.liulab.science/dbNSFP) was used to predict the effect of missense variants. The Human Gene Mutation Database (HGMD, http://www.hgmd.cf.ac.uk/ac/) and the ClinVar Database (https://www.ncbi.nlm.nih.gov/clinvar/) were used to screen mutations reported in published studies. Pathogenic variants are assessed under the protocol issued by ACMG (http://acmg.cbgc.org.cn). Sanger sequencing was performed bidirectionally for the verification of TTR c.200G>T.

WT-TTR and variants were recombinantly prepared following previous literature [28]. Genes of Escherichia coli TTR were codon optimized, synthesized by GenScript, and sub-cloned into pET-29b ⁽⁺⁾ vectors. WT, L55P, G47V plasmids were transformed into BL21 (DE3) E. coli cells, respectively. Cells were grown to OD₆₀₀ at 0.6–0.8 before 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 365 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.

The thermodynamic stability of the three proteins WT-TTR, L55P-TTR, and G47V-TTR (3.6 μm) was measured in different concentrations of urea between 0.5 and 10 m in phosphate buffer (10 mm sodium phosphate, 100 mm potassium chloride, 1 mm EDTA, pH = 7.4). Excitation at 295 nm was used, and the fluorescence emission intensity of tryptophan from 305 nm to 405 nm was collected. The ratio of 355 nm to 335 nm fluorescence intensity reflected the degree of TTR dissociation.

The monomer unfolding is much faster than dissociation of tetramer, so the rate-limiting of TTR dissociation is the structural changes from quaternary to tertiary, which is measured by tryptophan fluorescence (I₃₅₅/I₃₃₅). The rates of tetramer dissociation were carried out using TTR (3.6 μm) in 3–9 M urea in phosphate buffer (10 mm sodium phosphate, 100 mm KCl, 1 mm EDTA, pH = 7.4, 25°C) as a function of time. The kinetics data fit well to a single exponential function: I_355/335 = I_355/335^N + A (1 – e^{-kdiss t}), where I_355/335^N is the native protein fluorescence intensity ratio (355/335 nm), A is the amplitude difference, k_diss is the tetramer dissociation rate constant, and t is time in hour. The lnk_diss versus urea concentration plot is linear, allowing extrapolation to 0 m urea.

WT-TTR and G47V-TTR were incubated in phosphate buffer (10 mm sodium phosphate, 100 mm KCl, 1 mm EDTA, pH = 7.4) at 4°C for 48 h to enable subunit exchange [29]. In this process, the WT-TTR and G47V-TTR tetramer proteins dissociate into monomers and reassemble. A heterozygous tetramer protein containing five different exchangeable monomer components (4WT-TTR, 3WT•1G47V-TTR, 2WT•2G47V-TTR, 1WT•3G47V-TTL, and 4G47V-TTT) was formed. However, the five components cannot be further separated because they maintain equilibrium in the process of constant subunit exchange. Subsequently, kinetic and thermodynamic stability of the heterozygous protein mixture obtained after subunit exchange was measured, and the effect of the small molecule stabilizer was evaluated.

TTR (7.2 μm) was incubated with different concentrations of tafamidis, diflunisal, and AG10 in phosphate buffer (10 mm sodium phosphate, 100 mm potassium chloride, 1 mm EDTA, pH = 7.4) for 0.5 h at 37°C in advance, and then acidic buffer (NaOAc 200 mm, KCl 100 mm, acidified by AcOH to pH = 4.4) was added in equal volume to allow for amyloid formation. The degree of TTR amyloid formation was measured by OD₃₃₀ after all samples were incubated at 37°C for 72 h.

UPLC (FLR) Waters Acquity H-Class plus pro and Protein-pak™ Hi RCSQ (5 μm, 4.6 × 100 mm) were from Waters. The A2 molecule was synthesized in the laboratory as previously reported. Plasma from six G47V mutation carriers was filtered through 0.22 μm PVDF membrane, followed by 5 μL of each filtrate, 5 μL of PBS was added and mixed well, and 1 μL of A2 (1 mm) was added. After incubation at 25°C for 2 h, the mobile phase was added at a starting ratio of 45 μL, and vortexed and mixed thoroughly, and the supernatant was centrifuged (15,000 g, 4°C, 5 min) for sample analysis. Each sample was loaded in a volume of 20 µL into a Waters Acquity H-Class UPLC plus pro instrument with a Waters Protein-pak™ Hi RCSQ (5 μm, 4.6 × 100 mm). TTR was eluted from the column in a gradient using buffer A (25 mm Tris-HCl, pH = 9.0, 1 mm EDTA) and buffer B (1 m NaCl, 1 mm EDTA, 25 mm Tris-HCl, pH = 9.0) at a flow rate of 0.6 mL/min.

A 52-year-old male was referred to our hospital, complaining of exertional dyspnea and lower limb edema. Four years ago, he suffered from sudden onset syncope and a permanent dual-chamber pacemaker was implanted due to complete heart block in the local hospital. Hypertrophy cardiomyopathy was also documented by the local hospital suggested by the echo test. He denied symptoms of either peripheral neurological disorders or autonomic nervous dysfunction. The proband’s mother also had a history of “cardiac hypertrophy and heart block.” However, she suffered from sudden cardiac death only 1 year after pacemaker implantation at nearly 40 years old (shown in Fig. 1a). Other than his mother, no other first-degree relatives reported history of heart disease. Laboratory testing has shown significantly elevated N-terminal pro b-type natriuretic peptide up to 3,598 pg/mL. The serum monoclonal proteins on immunofixation electrophoresis were negative, and free light chain level/ratio was within normal range.

Electrocardiogram showed pacing rhythm in DDD mode (shown in Fig. 1b). A 24-h ambulatory electrocardiogram indicated sinus rhythm; dual-chamber pacemaker, DDD (atrioventricular sequential pacing) mode, and VAT (atrial synchronous ventricular pacing) mode pacing; frequent atrial and ventricular premature beats. ECG demonstrated severe concentric left ventricular hypertrophy (the interventricular septum and the posterior wall were 23 mm and 20 mm, respectively), bilateral atrial dilatation, granular sparkling of myocardium, thickened atrial-ventricular valves, and minor pericardial effusion. Reduced left ventricular ejection fraction of 28.0% was calculated using Simpson method, and grade 3 diastolic dysfunction was characterized according to Doppler E wave/e’ wave velocities at 12.7 and TAPSE at 13.7 mm (shown in Fig. 2a). 2D longitudinal sparkle tracking showed significantly reduced LV global longitudinal strain at −5.7% and apical sparing pattern based on systolic longitudinal strain apex to base ratio at 3.3. In addition, CMR revealed diffused subendocardial late gadolinium enhancement in the left ventricle, right ventricular wall, left atrial wall, and transmural late gadolinium enhancement at ventricular septal area (shown in Fig. 2b). Native T1 demonstrated abnormally high values (up to 1,192 ms) in basal area rather than apical. ^99mTc-PYP scintigraphy indicated grade 3 myocardial uptake of ^99mTc-PYP and an H/CL ratio of 2.54, strongly suggesting TTR amyloid deposition (shown in Fig. 2c). According to the normal light chain assays and positive bone scintigraphy, clinical diagnosis of ATTR-CA was sufficient even without proceeding to myocardial biopsy.

The whole-exome sequencing of the proband revealed a heterozygous missense variant (G47V) in TTR gene (shown in Fig. 1c) and was validated by Sanger sequencing. This missense variant was not detected in the patient’s first-degree relatives. The glycine encoded by codon 47 of TTR gene was replaced by valine by G47V. The mutation was located in the last nucleotide of exon 2 of TTR gene, at the splicing site of the exon [30, 31]. It was absent in known human genome databases including 1000 Genomes, GenomeAD, and ExAC, predicted to be detrimental by SIFT and MutationTaster, and PolyPhen2. This variant has been reported in the literatures and was characterized as likely pathogenic in the ClinVar database regarding the amyloidosis phenotype. Thus, TTR c.200G>T was regarded as a possible causal variant in our ATTR-CA patient and further functional investigation was warranted.

Characterization of the thermodynamic and kinetic stabilities of G47V-TTR (shown in Fig. 3a) by biochemical experiments may assist to explain the clinical symptoms of G47V patients and foresee the progressiveness. In this study, WT-TTR and L55P-TTR were chosen as controls to analyze the stabilities in parallel together with G47V-TTR. The experimental results showed that the thermodynamic stability of G47V-TTR (C_m = 2.4 m) was significantly lower than that of WT-TTR (C_m = 3.4 m) and comparable to that of L55P-TTR (C_m = 2.3 m), an early age-of-onset mutation (shown in Fig. 3b, e). In addition, the G47V-TTR (t_1/2 = 3.1 h) exhibits poorer kinetic stability than both the WT-TTR (t_1/2 = 42 h) and the L55P-TTR (t_1/2 = 4.4 h), further confirming its compromised stability (Fig. 3c–e). Together, G47V-TTR is severely destabilized in thermodynamic stability and kinetic stability that supports its early disease age-of-onset and pathogenicity to some extent.

Since the patient’s TTR gene is heterozygous, plasma from patients with the G47V mutation expresses both WT-TTR and G47V-TTR. TTR tetramers inherently dissociate into monomers and reassemble into new tetramers with subunit exchanged. Thus, G47V:WT-TTR mixture tetramers of different G47V:WT ratios are theoretically present in human plasma. To this end, we next characterized G47V:WT-TTR heterozygous mixture’s stability parameters. G47V-TTR and WT-TTR were mixed at 4°C for 48 h to allow for efficient subunit exchange. The resulting G47V:WT-TTR heterozygous tetramer protein was immediately measured for the thermodynamic and kinetic stability. The results demonstrated that the thermodynamic stability of the G47V:WT-TTR heterozygous tetramer protein (C_{m G47V:WT} = 2.6 m, shown in Fig. 3b, e) was comparable to that of the pure homozygous G47V-TTR (C_{m G47V} = 2.4 m). However, the kinetic stability (t_{1/2 G47V:WT} = 1.4 h, shown in Fig. 3c–e) was further compromised compared to that of the pure homozygous G47V-TTR (t_{1/2 G47V} = 3.1 h). This result indicates that WT-TTR cannot increase the thermodynamic stability of G47V-TTR but further destabilized the kinetic stability.

Conventionally, the primary treatment for ATTR amyloidosis is liver transplantation. However, the disadvantages of liver transplantation are limited by donor shortage, high cost, and the chronic need for immunosuppression [32‒34]. Small molecule kinetic stabilizers that increase the kinetic energy barrier and slow down tetramer dissociation have been approved for clinical applications or clinical trials, such as tafamidis, diflunisal, and AG10. In this study, three small molecule drugs, tafamidis, diflunisal, and AG10, were selected to analyze their inhibitory effect on G47V-TTR fibril formation in buffer using both of G47V-TTR homozygous proteins and G47V:WT-TTR heterozygous proteins.

Among all kinetic stabilizers, AG10 exhibited the best inhibition to the fibrillation of G47V-TTR homozygous protein, whereas diflunisal showed nearly no effect on amyloid formation (shown in Fig. 4a–c). In addition, consistent with what was observed in other TTR mutations (e.g., E61K-TTR and T96R-TTR) [35, 36], the G47V:WT-TTR heterozygous protein showed much poorer drug response to all three small molecule stabilizers compared to the G47V-TTR homozygous proteins (shown in Fig. 4d–f). This is also consistent with the thermodynamic and kinetic stability data described above. It also illustrates the importance of personalized medicine and rational selection of treatment options.

In the present study, we obtained a whole blood sample from the patient carrying G47V-TTR mutation. TTR tetramer concentration in the patient’s serum was quantified using a fluorescence-based UPLC method reported by the Kelly’s group [29]. The A2 probe can selectively bind to TTR tetramer protein in plasma, chemoselectively and covalently react with Lys-15 at the weak dimer-dimer interface of TTR tetramer, and fluoresce at 430 nm (shown in Fig. 5a). Further separation and quantification of TTR tetramer concentration can be achieved on UPLC coupled with a fluorescence signal detector. To better mimic the patient’s plasma TTR composition, we first obtained a standard correlation curve using G47V:WT-TTR heterozygous tetramer using the above method for later extrapolation (shown in Fig. 5b, c). The normal range of TTR concentrations in human plasma was perfectly included in the linear range (0.5–10 μm).

Using the above method, TTR tetramer plasma concentration in the G47V patient was determined as 0.72 μm (shown in Fig. 5d, e). Further measurement of the total TTR concentration (tetramer and other misfolded and aggregated species) using the current clinical immunoturbidimetry assay was determined as 1.20 μm. This concentration is significantly below the range of TTR total concentrations in normal human plasma (shown in Fig. 5d, e). Overall, 40% (0.48 μm) of the total serum TTR (1.20 μm) from this patient was calculated to be not tetrameric. Together, these results illustrate the severe instability of the G47V mutant and high progressiveness observed in clinics given its high aggregation propensity and strongly destabilized stabilities.

In this current study, a rare heterozygous missense mutation (G47V) in TTR gene was revealed in our proband as the potential causal variant of ATTR-CA. With thorough clinical, laboratory, and imaging investigation, the clinical diagnosis of ATTR-CA was established based on negative pathological light chain and grade 3 cardiac uptake on ^99mTc-PYP scintigraphy, fulfilling the international Task Force criteria for the diagnosis of ATTR-CA [4, 5].

Through a series of biochemical studies, severely compromised kinetic and thermodynamic stability was detected. Also, a distinct response to various small molecule kinetic stabilizers was detected in TTR mutant protein. Our study, for the first time, confirmed the pathogenicity of TTR G47V in causing ATTR-CA. Furthermore, the significant instability of G47V protein was consistent with clinical severity that was observed in our proband, such as early onset and severe clinical symptoms. It thus provided sufficient basis for individualized treatment of the disease.

The mortality of ATTR-CA patients usually is within 2–6 years after diagnosis and carries a very poor prognosis [37]. Therefore, an early diagnosis and treatment of ATTR-CA are crucial for slowing down the deterioration of organ function and prolonging life expectancy. The important characteristic of this case was the early onset of the disease, and in the early stage, arrhythmia was the main phenotype.

Due to the limitations of ultrasound technology and subjective limitations, the proband was misdiagnosed as hypertrophic cardiomyopathy in the local hospital without further appropriate treatment. Reviewing the symptoms of heart failure, family history of cardiomyopathy and arrhythmia, symmetrical hypertrophy of the left ventricle, myocardial granular echo, and signs for involvement of the early-onset cardiac conduction system, we cross-checked the provisional diagnosis of the local hospital. With a detailed patient history and medical examination, aided with diagnostic tests such as two-dimensional spot tracking echocardiography, CMR, ^99mTc-PYP scintigraphy, the results were highly suggestive of ATTR-CA, while excluding other cardiomyopathies or infectious disease. This was confirmed with genetic testing as hereditary cardiac amyloidosis, and a rare TTR gene mutation was found.

At present, only a few incidental TTR mutations have been reported in China, most of which were associated with familial amyloid polyneuropathy, but lacking a comprehensive analysis and diagnosis of ATTR-CA [38‒40]. We found that TTR G47V also showed phenotypic heterogeneity. In Japan, Kensuke Asakura et al. [41] reported the first case of FAP with G47V-TTR, a 71-year-old Japanese lady with peripheral neuropathy, autonomic dysfunction, and refractory diarrhea. A multiethnic Malaysian study reported a pair of siblings with G47V mutations. Both had symptoms of peripheral neuropathy and abnormal ECG, but only one of them had heart failure symptoms [16].

In contrast to the above studies, the G47V TTR case we reported had cardiac involvement but no evidence of extracardiac manifestations (such as polyneuropathy, carpal tunnel syndrome, gastrointestinal dysfunction, etc.). It was also an early-onset type and rapid disease progression. Several reasons may contribute to this heterogeneity, such as multiple epigenetic configurations for TTR gene, additional mutations of unidentified genes, or transcriptional regulation mediated by noncoding RNA [18, 42].

The risk factors of amyloidosis caused by TTR mutants are related to the thermodynamic and kinetic stability of TTR proteins [14]. These stability measurements of the G47V-TTR showed that its stability was significantly lower than the WT-TTR and comparable to the L55P-TTR mutant. These data indicated that the instability of G47V-TTR is one of the key causes of TTR amyloidosis. TTR present in the serum of patients in the form of heterotetramers contains different monomer components due to subunit exchange reaction. Our study also demonstrated that heterozygous tetramer was more unstable than homozygous G47V-TTR. This was consistent with the presence of heterozygous mutation in our patient, suggesting that G47V was a pathogenic mutation in early-onset cardiac amyloidosis in proband.

Multiple studies have reported that the average concentration of serum TTR in healthy adults ranges from 3 to 6 µM [43‒45], but it was noticed to be reduced in patients with ATTR [46, 47]. A small sample prospective cohort study in the USA showed that the decreased serum TTR predicted decreased rate of survival in WT-TTR amyloidosis-CA patients. This could be used as a biomarker to assess disease progression and response to treatment [48]. A Danish cohort study of 16,967 patients showed that the decreased serum TTR concentration correlated with an increased risk of heart failure, ignoring the effects of lower plasma TTR concentrations in patients with malnutrition or chronic inflammation [49]. In addition, patients with higher tetramer TTR levels tend to respond better to treatment [50].

Through A2-UPLC quantitative method, we detected that the concentration of tetramer TTR of proband was significantly lower than normal people, and this also correlated with an early onset of disease, more severe clinical symptoms, and carried a poorer prognosis. However, we cannot completely rule out the interference of other factors that may affect TTR concentration (such as age, sex, medication and nutritional status, etc.) with a single test, and further follow-up is needed to dynamically monitor the change of TTR tetramer concentration.

Emerging medical therapies, including TTR stabilizers and gene-silencing agents, bring hope to ATTR-CA patients [9]. Tafamidis is a selective and effective small molecule stabilizer of TTR tetramer that can be taken orally, and has been approved by the Food and Drug Administration (FDA) for the treatment of FAP and ATTR-CA [51, 52]. AG10 is an effective and highly selective TTR stabilizer, capable of forming hydrogen bonds with 117th Ser residues of each TTR monomer, thereby reducing TTR tetramer dissociation [53]. Judge DP et al. recruited 49 ATTR-CA patients with chronic heart failure, including mutant and WT patients. Compared with placebo group, TTR serum concentration and tetramer TTR ratio in AG10 treatment group were significantly increased and returned to the normal range, indicating that AG10 might be a safe and effective treatment for ATTR-CA [53]. Currently, phase 3 clinical trials of AG10 are still ongoing.

Among the three small molecule stabilizers in this study, AG10 and tafamidis had certain inhibitory effects on G47V-TTR homozygous protein, especially AG10, whereas diflunisal exhibited little effect on the formation of amyloid protein. This is of great significance for the drug optimization strategy for the patient. At present, only tafamidis has been approved for clinical use in China. The patient had been implanted with a permanent dual-chamber pacemaker without receiving guideline-directed medical therapy for the HFrEF. Medication such as oral diuretics was initiated and optimized before proceeding to pacemaker upgrading. Unfortunately, the patient in this study refused TTR stabilizer treatment, rendering it impossible for us to take a deep dive into the true efficacy of small molecule stabilizers.

Our study had a few limitations. First, the mother of the proband, who had strong clinical symptoms suggestive of amyloidosis, had passed away and, therefore, could not be sequenced. This does give strong suspicions about the mutation, but it could not be confirmed. Second, due to logistic limitations, our functional research was limited to biochemical and physiological experiments, and we could not progress to transgenic animal model studies to replicate disease phenotypes. Finally, despite multiple attempts to try the specific drug therapy, the proband and their families did not consent for same. So, the efficacy of TTR stabilizer and dynamic monitoring of TTR concentration of the patient could not be observed.

In this work, we reported a patient presented early onset of clinically typical ATTR-CA due to G47V-TTR mutation and was associated with a high mortality usually within 2–6 years after the diagnosis. We uncovered severely compromised thermodynamic and kinetic stabilities in this mutation supporting its early disease age-of-onset. Meanwhile, most small molecule kinetic stabilizers exhibited poor response to this mutation in vitro. The severe instability of this mutation led to low tetramer TTR concentration in patient’s serum. Our work for the first time not only characterized the biochemical properties of G47V-TTR mutation, but also provided hints for the pathogenicity of this mutation.

This study protocol was reviewed and approved by Institutional Review Board (IRB) at the Ethics Committee of the second affiliated hospital of Zhejiang University School of Medicine, approval number [2021LSY0102]. Written informed consent was obtained from the patient for publication of the details of their medical case and any accompanying images.

The authors have no conflicts of interest to declare.

This work was supported by Zhejiang Provincial Basic Welfare Research Project (LGF22H020004) and National Natural Science Foundation of China (22222410). The funder had no role in the design, data collection, data analysis, and reporting of this study.

Xiaopeng He and Mengdie Wang performed the experimental work, analyzed the data, designed the figures, and wrote the manuscript; Jialu Sun provided experimental support and analyzed the data; Zhengyang Yu and Xinyang Hu analyzed and fitted the data and contributed to manuscript writing; Yu Liu and Xiaoping Lin conceived and designed the experimental work and planned, revised, and contributed to manuscript writing; all authors have read and approved the final manuscript.

Xiaopeng He and Mengdie Wang contributed equally to this work.

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA005185) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human [54, 55].