Structural and thermodynamic characterization of a highly amyloidogenic dimer of transthyretin involved in a severe cardiomyopathy.

Transthyretin (TTR) is an homotetrameric protein involved in the transport of thyroxine. More than 150 different mutations have been described in the TTR gene, several of them associated with familial amyloid cardiomyopathy. Recently, our group described a new variant of TTR in Brazil, namely A39D-TTR, which causes a severe cardiac condition. Position 39 is in the AB loop, a region of the protein that is located within the thyroxine-binding channels and is involved in tetramer formation. In the present study, we solved the structure and characterize the thermodynamic stability of this new variant of TTR using urea and high hydrostatic pressure. Interestingly, during the process of purification, A39D-TTR turned out to be a dimer and not a tetramer, a variation that might be explained by the close contact of the four aspartic acids at position 39, where they face each other inside the thyroxine channel. In the presence of subdenaturing concentrations of urea, bis-ANS binding and dynamic light scattering revealed A39D-TTR in the form of a molten-globule dimer. Co-expression of A39D and WT isoforms in the same bacterial cell did not produce heterodimers or heterotetramers, suggesting that somehow a negative charge at the AB loop precludes tetramer formation. A39D-TTR proved to be highly amyloidogenic, even at mildly acidic pH values where WT-TTR does not aggregate. Interestingly, despite being a dimer, aggregation of A39D-TTR was inhibited by diclofenac, which binds to the thyroxine channel in the tetramer, suggesting the existence of other pockets in A39D-TTR able to accommodate this molecule.

Clinical and Prognostic Implications of Right Ventricular Uptake on Bone Scintigraphy in Transthyretin Amyloid Cardiomyopathy.

BACKGROUND: The extent of myocardial bone tracer uptake with technetium pyrophosphate, hydroxymethylene diphosphonate, and 3,3-diphosphono-1,2-propanodicarboxylate in transthyretin amyloid cardiomyopathy (ATTR-CM) might reflect cardiac amyloid burden and be associated with outcome. METHODS: Consecutive patients with ATTR-CM who underwent diagnostic bone tracer scintigraphy with acquisition of whole-body planar and cardiac single-photon emission computed tomography (SPECT) images from the National Amyloidosis Centre and 4 Italian centers were included. Cardiac uptake was defined according to the Perugini classification: 0=absent cardiac uptake; 1=mild uptake less than bone; 2=moderate uptake equal to bone; and 3=high uptake greater than bone. Extent of right ventricular (RV) uptake was defined as focal (basal segment of the RV free wall only) or diffuse (extending beyond basal segment) on the basis of SPECT imaging. The primary outcome was all-cause mortality. RESULTS: Among 1422 patients with ATTR-CM, RV uptake accompanying left ventricular uptake was identified by SPECT imaging in 100% of cases at diagnosis. Median follow-up in the whole cohort was 34 months (interquartile range, 21 to 50 months), and 494 patients died. By Kaplan-Meier analysis, diffuse RV uptake on SPECT imaging (n=936) was associated with higher all-cause mortality compared with focal (n=486) RV uptake (77.9% versus 22.1%; P<0.001), whereas Perugini grade was not associated with survival (P=0.27 in grade 2 versus grade 3). On multivariable analysis, after adjustment for age at diagnosis (hazard ratio [HR], 1.03 [95% CI, 1.02-1.04]; P<0.001), presence of the p.(V142I) TTR variant (HR, 1.42 [95% CI, 1.20-1.81]; P=0.004), National Amyloidosis Centre stage (each category, P<0.001), stroke volume index (HR, 0.99 [95% CI, 0.97-0.99]; P=0.043), E/e' (HR, 1.02 [95% CI, 1.007-1.03]; P=0.004), right atrial area index (HR, 1.05 [95% CI, 1.02-1.08]; P=0.001), and left ventricular global longitudinal strain (HR, 1.06 [95% CI, 1.03-1.09]; P<0.001), diffuse RV uptake on SPECT imaging (HR, 1.60 [95% CI, 1.26-2.04]; P<0.001) remained an independent predictor of all-cause mortality. The prognostic value of diffuse RV uptake was maintained across each National Amyloidosis Centre stage and in both wild-type and hereditary ATTR-CM (P<0.001 and P=0.02, respectively). CONCLUSIONS: Diffuse RV uptake of bone tracer on SPECT imaging is associated with poor outcomes in patients with ATTR-CM and is an independent prognostic marker at diagnosis.

Transthyretin mutagenesis: impact on amyloidogenesis and disease.

Transthyretin (TTR), a homotetrameric protein found in plasma, cerebrospinal fluid, and the eye, plays a pivotal role in the onset of several amyloid diseases with high morbidity and mortality. Protein aggregation and fibril formation by wild-type TTR and its natural more amyloidogenic variants are hallmarks of ATTRwt and ATTRv amyloidosis, respectively. The formation of soluble amyloid aggregates and the accumulation of insoluble amyloid fibrils and deposits in multiple tissues can lead to organ dysfunction and cell death. The most frequent manifestations of ATTR are polyneuropathies and cardiomyopathies. However, clinical manifestations such as carpal tunnel syndrome, leptomeningeal, and ocular amyloidosis, among several others may also occur. This review provides an up-to-date listing of all single amino-acid mutations in TTR known to date. Of approximately 220 single-point mutations, 93% are considered pathogenic. Aspartic acid is the residue mutated with the highest frequency, whereas tryptophan is highly conserved. "Hot spot" mutation regions are mainly assigned to ß-strands B, C, and D. This manuscript also reviews the protein aggregation models that have been proposed for TTR amyloid fibril formation and the transient conformational states that convert native TTR into aggregation-prone molecular species. Finally, it compiles the various in vitro TTR aggregation protocols currently in use for research and drug development purposes. In short, this article reviews and discusses TTR mutagenesis and amyloidogenesis, and their implications in disease onset.

Silencing of ocular transthyretin, a gene responsible for hereditary transthyretin amyloidosis, by intravitreal injection of an siRNA conjugate into rabbit eyes.

The first small interfering RNA (siRNA) therapeutic received approval for hereditary transthyretin (ATTRv) amyloidosis, and the patients' lifespan extension by specific inhibition of hepatic synthesis of transthyretin (TTR) is expected. However, ocular amyloidosis in these patients has been a crucial issue. This study aims to evaluate the efficacy and safety of intravitreal TTR siRNA conjugate injection into rabbit eyes. Rabbit (r) TTR siRNA is a screened TTR siRNA conjugate from 53 candidates. The intraocular pressure (IOP) immediately after injection was high despite the 65.9 % decrease of aqueous humor TTR protein levels in the rTTR siRNA group compared with those in the Control siRNA group 2 weeks after the 50 µL siRNA injection. The IOP spike was milder after the 30 µL siRNA injection, and aqueous humor TTR levels decreased by ∼50 % in the rTTR siRNA group, which is consistent with the mRNA levels in the retina. The parameters of dark-adapted, light-adapted, and light-adapted 30 Hz electroretinogram and the thickness of each retinal layer in histological analysis demonstrated no significant differences between the groups. In conclusion, we developed TTR siRNA conjugates for rabbit eyes, and the results indicate that intravitreal TTR siRNA conjugate injection could be a therapeutic option for ocular amyloidosis caused by ATTRv amyloidosis.

Hereditary Transthyretin Amyloidosis and the Impact of Classic and New Treatments on Kidney Function: A Review.

Hereditary transthyretin amyloidosis (ATTRv) is a rare, progressive, and life-threatening disease caused by misfolded transthyretin (TTR) proteins that aggregate as abnormal amyloid fibrils and accumulate throughout the body. The kidney is one of the main organs affected in amyloid light chain (AL) amyloidosis and ATTRv amyloidosis. The most common clinical presentation is proteinuria, which consists mainly of albumin; this is the first step in the natural history of ATTRv nephropathy. Not all TTR mutations are equal in terms of ATTRv kidney involvement. Kidney involvement in ATTRv itself is difficult to define, given the numerous associated confounding factors. There are several treatments available to treat ATTRv, including orthotopic liver transplant (OLT), which is the classic treatment for ATTRv. However, we should be careful regarding the use of calcineurin inhibitors in the setting of OLT because these can be nephrotoxic. New treatments for amyloidosis may have an impact on kidney function, including drugs that target specific pathways involved in the disease. Tafamidis and diflunisal, which are TTR stabilizers, patisiran (RNA interference agent), and inotersen (antisense oligonucleotide inhibitor) have been shown to reduce TTR amyloid. Tafamidis and patisiran are medications that have reduced the progression of kidney disease in amyloidosis, but inotersen and diflunisal may damage kidney function.

Skeletal muscle involvement in systemic amyloidosis is often overlooked.

AIM: Systemic amyloidosis is a condition in which misfolded amyloid fibrils are deposited within tissues. Amyloid myopathy is a rare manifestation of systemic amyloidosis. However, whether skeletal muscle involvement is underestimated and whether such deposition guarantees clinical and pathological myopathic features remain to be investigated. METHODS: We retrospectively reviewed patients with systemic amyloidosis, in whom skeletal muscle biopsies were performed at our centre between January 2018 and June 2023. In total, 28 patients with suspected systemic amyloidosis were included. Among these, 21 presented with cardiomyopathy but lacked myopathic symptoms. The clinical and pathological data of these patients were further analysed. The amyloid type was confirmed by immunohistochemistry. RESULTS: Twenty-eight patients with suspected systemic amyloidosis underwent muscle biopsy. Amyloid deposition in the skeletal muscle was confirmed in 24 patients, including 22 with light-chain amyloidosis (AL) and two with transthyretin amyloidosis (ATTR). Among the 24 patients, seven presented with muscle weakness and decreased muscle strength (Group 1, symptomatic myopathy), whereas the remaining 17 exhibited normal muscle strength (Group 2, asymptomatic myopathy). Group 1 included four patients with AL-λ, one with AL-κ and two with ATTR. Group 2 included 15 patients with AL-λ and two patients with AL-κ. In Group 1, six patients exhibited neuropathy, whereas only one patient in Group 2 presented with subclinical neuropathy on nerve conduction studies. Amyloid deposition in the interstitium was the most obvious change, observed in all 24 patients. Neuropathic changes, including denervation atrophy and muscle fibre grouping, were also common. Except for type 2 fibre atrophy, the other myopathic changes were mild and nonspecific. No sarcolemmal disruption was observed. Immunohistochemical analysis revealed marked positivity for MAC and MHC1 expression in the regions with amyloid deposits. Clinicopathological analysis revealed no significant differences in the extent of muscular amyloid deposition between the two groups. Nevertheless, patients in Group 1 displayed more pronounced neurogenic atrophy on skeletal muscle biopsies. CONCLUSIONS: Our study indicates that amyloid deposition in skeletal muscle is commonly observed but rarely causes symptomatic myopathy in systemic amyloidosis.

Survival in a Real-World Cohort of Patients With Transthyretin Amyloid Cardiomyopathy Treated With Tafamidis: An Analysis From the Transthyretin Amyloidosis Outcomes Survey (THAOS).

BACKGROUND: In the pivotal Tafamidis in Transthyretin Cardiomyopathy Clinical Trial (ATTR-ACT), tafamidis significantly reduced mortality rates, leading to its approval in many countries for the treatment of transthyretin amyloid cardiomyopathy (ATTR-CM). Real-world evidence on survival in patients with ATTR-CM following tafamidis treatment has not been extensively reported. METHODS AND RESULTS: The Transthyretin Amyloidosis Outcomes Survey (THAOS) was a longitudinal, observational, phase 4 study of patients with transthyretin amyloidosis and asymptomatic participants carrying pathogenic transthyretin variants. Patients from THAOS with a predominantly cardiac phenotype at enrollment were included, and survival was analyzed according to tafamidis treatment status (treated or untreated). Results are based on the completed THAOS dataset. In tafamidis-treated (n = 587) and tafamidis-untreated (n = 854) patients, respectively, median age at enrollment was 77.7 and 76.4 years, 91.8% and 90.0% were male, and 91.8% and 83.8% had wild-type disease. Survival rates (95% CI) at 30 and 42 months, respectively, were 84.4% (80.5-87.7) and 76.8% (70.9-81.7) in tafamidis-treated patients, and 70.0% (66.4-73.2) and 59.3% (55.2-63.0) in tafamidis-untreated patients. Survival rates in genotype subgroups (wild-type and variant) were similar to those of the overall cohort. Survival rates were better in a contemporary cohort, as reflected by a sensitivity analysis performed in patients enrolled after vs before 2019. No new safety signals were identified. CONCLUSIONS: In this real-world cohort of patients with ATTR-CM, survival rates were higher than in ATTR-ACT and consistent with more recent reports, suggesting early diagnosis and treatment with tafamidis has improved life expectancy in ATTR-CM. These results provide further evidence supporting tafamidis' safety and effectiveness. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00628745.

Temporal Outcomes of Patients Diagnosed With Transthyretin Cardiac Amyloidosis.

BACKGROUND: Transthyretin cardiac amyloidosis (ATTR-CA) is increasingly recognized. Clinical outcomes have evolved over time amid changes in the diagnostic pathway and advances in therapeutics. We sought to evaluate clinical outcomes over time of patients with ATTR-CA with access to disease-modifying therapy. METHODS AND RESULTS: This is a retrospective cohort study of 419 patients diagnosed with ATTR-CA during 2001-2021, comparing clinical characteristics across eras. The primary end point was composite all-cause mortality or orthotopic heart transplantation (OHT). Time-to-event analysis was performed using Cox proportional hazard modeling controlling for differences among cohorts. Patients diagnosed in the more recent years had higher median age (2017-2021, 78 years; 2014-2016, 75 years; 2001-2013, 74 years) and more often had wild-type ATTR (81.9% vs 82.5% vs 56.4%), but less severe phenotypes as evidenced by more individuals with Columbia stage I disease (47.6% vs 35.9% vs 22.4%), owing to lower biomarkers, more patients in New York Heart Association functional classes I and II (68.9% vs 47.6% vs 43.6%), and lower use of loop diuretics (67.0% vs 78.6% vs 89.1%). Over time, patients were treated more frequently with tafamidis (74% vs 37% vs 32%). On multivariable analysis, greater Columbia score (hazard ratio 1.42, 95% confidence interval 1.30-1.54, P < .001) was predictive of death or OHT, whereas tafamidis (hazard ratio 0.31, 95% confidence interval 0.22-0.44, P < .001) was associated with greater survival and freedom from OHT. CONCLUSIONS: Patients recently diagnosed with ATTR-CA have earlier stage disease and substantially lower mortality. Tafamidis is associated with significantly improved survival and freedom from OHT.

Effect of dynamic exclusion and the use of FAIMS, DIA and MALDI-mass spectrometry imaging with ion mobility on amyloid protein identification.

Amyloidosis is a disease characterized by local and systemic extracellular deposition of amyloid protein fibrils where its excessive accumulation in tissues and resistance to degradation can lead to organ failure. Diagnosis is challenging because of approximately 36 different amyloid protein subtypes. Imaging methods like immunohistochemistry and the use of Congo red staining of amyloid proteins for laser capture microdissection combined with liquid chromatography tandem mass spectrometry (LMD/LC-MS/MS) are two diagnostic methods currently used depending on the expertise of the pathology laboratory. Here, we demonstrate a streamlined in situ amyloid peptide spatial mapping by Matrix Assisted Laser Desorption Ionization-Mass Spectrometry Imaging (MALDI-MSI) combined with Trapped Ion Mobility Spectrometry for potential transthyretin (ATTR) amyloidosis subtyping. While we utilized the standard LMD/LC-MS/MS workflow for amyloid subtyping of 31 specimens from different organs, we also evaluated the potential introduction in the MS workflow variations in data acquisition parameters like dynamic exclusion, or testing Data Dependent Acquisition combined with High-Field Asymmetric Waveform Ion Mobility Spectrometry (DDA FAIMS) versus Data Independent Acquisition (DIA) for enhanced amyloid protein identification at shorter acquisition times. We also demonstrate the use of Mascot's Error Tolerant Search and PEAKS de novo sequencing for the sequence variant analysis of amyloidosis specimens.

Sex differences in transthyretin cardiac amyloidosis.

Transthyretin cardiac amyloidosis (ATTR-CA) is a progressive disease characterized by the deposition of abnormal transthyretin protein fibrils in the heart, leading to cardiac dysfunction. Recent evidence suggests that sex differences may play a significant role in various steps of ATTR-CA, including clinical presentation, diagnostic challenges, disease progression, and treatment outcomes. ATTR-CA predominantly affects men, whereas women are older at presentation. Women generally present with a history of heart failure with preserved ejection fraction and/or carpal tunnel syndrome. When indexed, left ventricular (LV) wall thickness is equal, or even increased, than men. Women also have smaller LV cavities, more preserved ejection fractions, and apparently a slightly worse right ventricular and diastolic function. Given the under-representation on women in clinical trials, no data regarding sex influence on the treatment response are currently available. Finally, it seems there are no differences in overall prognosis, even if premenopausal women may have a certain level of myocardial protection. Genetic variations, environmental factors, and hormonal changes are considered as potential contributors to observed disparities. Understanding sex differences in ATTR-CA is vital for accurate diagnosis and management. By considering these differences, clinicians can improve diagnostic accuracy, tailor treatments, and optimize outcomes for both sexes with ATTR-CA.

Molecular mechanisms and emerging therapies in wild-type transthyretin amyloid cardiomyopathy.

Wild-type transthyretin amyloid cardiomyopathy (ATTRwt-CM) is an underrecognized cause of heart failure due to misfolded wild-type transthyretin (TTRwt) myocardial deposition. The development of wild-type TTR amyloid fibrils is a complex pathological process linked to the deterioration of homeostatic mechanisms owing to aging, plausibly implicating multiple molecular mechanisms. The components of amyloid transthyretin often include serum amyloid P, proteoglycans, and clusterin, which may play essential roles in the localization and elimination of amyloid fibrils. Oxidative stress, impaired mitochondrial function, and perturbation of intracellular calcium dynamics induced by TTR contribute to cardiac impairment. Recently, tafamidis has been the only drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of ATTRwt-CM. In addition, small interfering RNAs and antisense oligonucleotides for ATTR-CM are promising therapeutic approaches and are currently in phase III clinical trials. Newly emerging therapies, such as antibodies targeting amyloid, inhibitors of seed formation, and CRISPR‒Cas9 technology, are currently in the early stages of research. The development of novel therapies is based on progress in comprehending the molecular events behind amyloid cardiomyopathy. There is still a need to further advance innovative treatments, providing patients with access to alternative and effective therapies, especially for patients diagnosed at a late stage.

Targeting TNF-α-induced expression of TTR and RAGE in rheumatoid arthritis: Apigenin's mediated therapeutic approach.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory disease induced by TNF-α, which increases fibroblast-like synoviocytes inflammation, resulting in cartilage destruction. The current work sought to comprehend the pathophysiological importance of TNF-α stimulation on differential protein expression and their regulation by apigenin using in-vitro and in-vivo models of RA. METHODS: The human RA synovial fibroblast cells were stimulated with or without TNF-α (10 ng/ml) and treated with 40 µM apigenin. In-silico, in-vitro and in-vivo studies were performed to confirm the pathophysiological significance of apigenin on pro-inflammatory cytokines and on differential expression of TTR and RAGE proteins. RESULTS: TNF-α induced inflammatory response in synoviocytes revealed higher levels of IL-6, IL-1ß, and TNF-α cytokines and upregulated differential expression of TTR and RAGE. In-silico results demonstrated that apigenin has a binding affinity towards TNF-α, indicating its potential effect in the inflammatory process. Both in-vitro and in-vivo results obtained by Western Blot analysis suggested that apigenin reduced the level of p65 (p = 0.005), TTR (p = 0.002), and RAGE (p = 0.020). CONCLUSION: The findings of this study suggested that TNF-α promotes the differential expression of pro-inflammatory cytokines, TTR, and RAGE via NF-kB pathways activation. Anti-inflammatory effect of apigenin impedes TNF-α mediated dysregulation or expression associated with RA pathogenesis.

Establishment of a Comprehensive Cardiac Amyloidosis Center in a Community Hospital Setting.

Cardiac amyloidosis is a great masquerader that often results in misdiagnosis of this condition. Early clinical recognition is crucial for timely therapeutic interventions to improve survival in patients with cardiac amyloidosis. Currently, Food and Drug Administration (FDA)-approved medications work best if started early in the disease. Thus, to increase identification, disease awareness, expertise in diagnostic techniques, and a multidisciplinary team approach is essential. The majority of the patients (~90%) in the United States are treated in community hospitals, thus, it would be helpful for these hospitals to have their own designated, comprehensive cardiac amyloidosis center to provide care to the patients who are widespread in the community. Most of these patients are elderly, and it is difficult for these patients to travel long distances to academic amyloid centers. Our manuscript aims to provide a path to the development of cardiac amyloid centers at community hospitals.

Multi-modality artificial intelligence-based transthyretin amyloid cardiomyopathy detection in patients with severe aortic stenosis.

PURPOSE: Transthyretin amyloid cardiomyopathy (ATTR-CM) is a frequent concomitant condition in patients with severe aortic stenosis (AS), yet it often remains undetected. This study aims to comprehensively evaluate artificial intelligence-based models developed based on preprocedural and routinely collected data to detect ATTR-CM in patients with severe AS planned for transcatheter aortic valve implantation (TAVI). METHODS: In this prospective, single-center study, consecutive patients with AS were screened with [99mTc]-3,3-diphosphono-1,2-propanodicarboxylic acid ([99mTc]-DPD) for the presence of ATTR-CM. Clinical, laboratory, electrocardiogram, echocardiography, invasive measurements, 4-dimensional cardiac CT (4D-CCT) strain data, and CT-radiomic features were used for machine learning modeling of ATTR-CM detection and for outcome prediction. Feature selection and classifier algorithms were applied in single- and multi-modality classification scenarios. We split the dataset into training (70%) and testing (30%) samples. Performance was assessed using various metrics across 100 random seeds. RESULTS: Out of 263 patients with severe AS (57% males, age 83 ± 4.6years) enrolled, ATTR-CM was confirmed in 27 (10.3%). The lowest performances for detection of concomitant ATTR-CM were observed in invasive measurements and ECG data with area under the curve (AUC) < 0.68. Individual clinical, laboratory, interventional imaging, and CT-radiomics-based features showed moderate performances (AUC 0.70-0.76, sensitivity 0.79-0.82, specificity 0.63-0.72), echocardiography demonstrated good performance (AUC 0.79, sensitivity 0.80, specificity 0.78), and 4D-CT-strain showed the highest performance (AUC 0.85, sensitivity 0.90, specificity 0.74). The multi-modality model (AUC 0.84, sensitivity 0.87, specificity 0.76) did not outperform the model performance based on 4D-CT-strain only data (p-value > 0.05). The multi-modality model adequately discriminated low and high-risk individuals for all-cause mortality at a mean follow-up of 13 months. CONCLUSION: Artificial intelligence-based models using collected pre-TAVI evaluation data can effectively detect ATTR-CM in patients with severe AS, offering an alternative diagnostic strategy to scintigraphy and myocardial biopsy.

Preoperative level of serum transthyretin as a novel biomarker predicting survival in resected pancreatic ductal adenocarcinoma with neoadjuvant therapy.

BACKGROUND: Systemic inflammation and altered metabolism are essential hallmarks of cancer. We hypothesized that the rapid turnover protein transthyretin (TTR) (half-life: 2-3 days), compared with the conventional marker albumin (21 days), better reflects the inflammatory/metabolic dynamics of pancreatic ductal adenocarcinoma (PDAC) after neoadjuvant therapy (NAT) and is a useful prognostic marker. METHODS: Serum TTR and albumin levels were measured in 104 consecutive post-NAT PDAC patients before curative resection. The associations of preoperative TTR and albumin levels with overall survival (OS) after pancreatectomy were retrospectively analyzed. RESULTS: The mean (SD) TTR and albumin levels were 21.6 (6.4) mg/dL (normal range: ≥22.0 mg/dL) and 3.9 (0.55) g/dL. A low (<22.0 mg/dL) post-NAT TTR level was associated with an advanced tumor stage and higher CEA and CRP levels. Patients with low TTR levels showed significantly worse OS compared with normal levels (3-year OS 39 % vs. 54 %, P = 0.037), although albumin levels did not. We modified prognostic biomarkers of systemic inflammation/metabolism, such as GPS, PNI, and CONUT scores, using the serum TTR instead of albumin level and successfully showed that modified scores were better associated with OS compared with original scores using serum albumin level. CONCLUSIONS: Our data suggest that the TTR level is a promising prognostic biomarker for PDAC patients after NAT.

Correlation between a commercial electrophysiological test of sudomotor function and intraepidermal nerve fiber density in hereditary transthyretin amyloidosis.

INTRODUCTION/AIMS: In the early stage, hereditary transthyretin (ATTRv) amyloidosis predominantly affects small nerve fibers, resulting in autonomic dysfunction and impaired sensation of pain and temperature. Evaluation of small fiber neuropathy (SFN) is therefore important for early diagnosis and treatment of ATTRv amyloidosis. Herein, we aimed to investigate the accuracy of a quick and non-invasive commercial sudomotor function test (SFT) for the assessment of SFN in ATTRv amyloidosis. METHODS: We performed the SFT in 39 Japanese adults with ATTRv amyloidosis, and we analyzed the correlations between electrochemical skin conductance (ESC) values obtained via the SFT and the parameters of other neuropathy assessment methods. RESULTS: ESC in the feet demonstrated significant, moderate correlations with intraepidermal nerve fiber density (IENFD) results (Spearman's rank correlation coefficient [rs ], 0.58; p < .002) and other neuropathy assessment methods including the sensory nerve action potential amplitude in the nerve conduction studies (rs , 0.52; p < .001), the Neuropathy Impairment Score (rs , -0.45; p < .01), the heat-pain detection threshold (rs , -0.62; p < .0001), and the autonomic section of the Kumamoto ATTRv clinical score (rs , -0.53; p < .0001). DISCUSSION: In this study, we found that ESC values in the feet via the SFT demonstrated significant, moderate correlations with IENFD and other SFN assessment methods in patients with ATTRv amyloidosis, suggesting that the SFT appears to be an appropriate method for assessment of SFN in this disease.

Diagnosis and treatment of hereditary transthyretin amyloidosis with polyneuropathy in the United States: Recommendations from a panel of experts.

Hereditary transthyretin (ATTRv; v for variant) amyloidosis is a rare, multisystem, progressive, and fatal disease in which polyneuropathy is a cardinal manifestation. Due to a lack of United States (US)-specific guidance on ATTRv amyloidosis with polyneuropathy, a panel of US-based expert clinicians convened to address identification, monitoring, and treatment of this disease. ATTRv amyloidosis with polyneuropathy should be suspected in unexplained progressive neuropathy, especially if associated with systemic symptoms or family history. The diagnosis is confirmed through genetic testing, biopsy, or cardiac technetium-based scintigraphy. Treatment should be initiated as soon as possible after diagnosis, with gene-silencing therapeutics recommended as a first-line option. Consensus is lacking on what represents "disease progression" during treatment; however, the aggressive natural history of this disease should be considered when evaluating the effectiveness of any therapy.

Effectiveness of patisiran after switching from tafamidis for the treatment of hereditary transthyretin-mediated amyloidosis with polyneuropathy.

BACKGROUND AND PURPOSE: Hereditary transthyretin-mediated amyloidosis with polyneuropathy (ATTRv-PN [v for variant]) is a rare, progressive disease associated with multisystemic impairments. This study assessed the real-world outcomes of patients with ATTRv-PN who switched from tafamidis to patisiran, as well as the reasons for the treatment switch. METHODS: This was a retrospective chart review study at a large expert referral center. Data were extracted from medical charts of patients with ATTRv-PN who switched from tafamidis to patisiran on or before 30 August 2019. Data elements included demographic and clinical characteristics, rationale for switch, and disease measures evaluated from tafamidis initiation through the 12-month patisiran treatment period. RESULTS: Among the 24 patients with ATTRv-PN included in the study, 50.0% had a V30M variant, and the mean (SD) age was 67.3 (8.0) years. During tafamidis treatment (mean [SD] = 30.1 [17.5] months) before switching to patisiran, patients worsened across multiple polyneuropathy measures, including walking ability, Neuropathy Impairment Score, and autonomic function. Neuropathic disease progression on tafamidis was the principal reason for switching to patisiran. After 12 months on patisiran (mean [SD] = 11.7 [1.4] months), patients experienced attenuated disease progression or improvement in the aforementioned measures of polyneuropathy. CONCLUSIONS: Switching from tafamidis to patisiran attenuated the rate of functional decline, and most patients experienced stabilization or improvement of at least one polyneuropathy measure within 12 months of patisiran treatment. Timely switch from tafamidis to patisiran can be beneficial to avoid rapid disease progression in patients with ATTRv-PN.

Characterization of the G-quadruplexes in the transthyretin gene and its role in silencing transthyretin mRNA transcription.

Transthyretin Amyloidosis arises from the misfolding of monomers or oligomers of the normal transthyretin protein. Our investigation revealed that certain guanine-rich regions within the 5' UTR sequence of the transthyretin gene possess the ability to form G2-quadruplex structures, as determined through analysis with QGRS mapper. We demonstrated that small molecule ligands, including TMPyP4, Braco-19, NMM, and TO, have a significant impact on the stabilization of transthyretin G-quadruplexes. The objective of this study was to confirm the effect of ligands on transthyretin gene transcription through the stabilization of G-quadruplexes. To comprehend the interaction between ligands and transthyretin G-quadruplexes, a range of analytical techniques were employed, includingUV titration, fluorescence titration assays, circular dichroism, quantitative RT-PCR and cytotoxicity tests. The results revealed the presence of four putative G2-quadruplex sequences, which formed stable anti-parallel, parallel, and hybrid G2-quadruplex structures. Notably, Ttrg 3 (5'-GGAAGGAAGGGAGGGAGGG-3') exhibited the highest stability to form G-quadruplex. Furthermore, TmPyP4, Braco-19, NMM and TO were found to stabilize the parallel topology of Ttrg 3. After 48 h of incubation, the RT-PCR experiments revealed a significant reduction in transthyretin mRNA transcription in HepG2 cells when treated with 20 µM TmPyP4 and Braco-19, without inducing apoptosis. Our findings suggested that ligand-mediated stabilization of G-quadruplexes within the 5'-UTR can effectively silence transthyretin expression, highlighting the potential of G-quadruplex as a novel therapeutic target for Transthyretin Amyloidosis. This study might shed valuable lights for the development of innovative therapeutic approach against Transthyretin Amyloidosis.

Quantitative 99mTc-pyrophosphate myocardial uptake: Changes on transthyretin stabilization therapy.

BACKGROUND: Quantitative technetium-99m-pyrophosphate cardiac single-photon emission computed tomography (99mTc-PYP SPECT/CT) is an emerging method for estimating myocardial burden of transthyretin cardiac amyloidosis (ATTR-CA), but its efficacy in monitoring longitudinal changes remains uncertain. We aimed to investigate longitudinal changes in cardiac ATTR amyloid burden following transthyretin stabilization therapy using visual and quantitative 99mTc-PYP SPECT/CT and to relate these with changes in cardiac biomarkers and function. METHODS: This prospective longitudinal cohort study investigated changes in 99mTc-PYP SPECT/CT in 23 participants with ATTR-CA on transthyretin stabilization therapy (median: 2.6 years). Quantitative analysis included left ventricular (LV) standardized uptake values (SUVs) (SUVmax, SUVmean), cardiac amyloid activity (CAA; SUVmean∗LV activity volume), and percent injected dose (%ID) (mean activity concentration∗LV activity volume/injected activity), calculated using a threshold of >1.5 times left atrial blood pool activity concentration on SPECT/CT. Longitudinal changes of paired continuous and ordinal variables were analyzed using Wilcoxon signed-rank test. RESULTS: Following therapy, visual grade decreased significantly (P = 0.003). Several quantitative 99mTc-PYP metrics also decreased significantly: SUVmax (median -0.75, P = 0.011), CAA (median: -406.6, P < 0.001), and %ID (median: -0.45, P < 0.001). Serum transthyretin levels improved (median: +6.5 mg/dL, P = 0.008). Echocardiographic parameters (global longitudinal strain, LV mass index, and LV wall thickness), N-terminal pro-B-type natriuretic peptide, and estimated glomerular filtration rate remained stable. CONCLUSIONS: Favorable changes in 99mTc-PYP myocardial uptake were observed in participants on transthyretin stabilization therapy, whereas echocardiographic parameters and biomarkers remained stable. These results likely signify myocardial ATTR amyloid stabilization rather than amyloid burden regression. Further investigation is needed to understand the implications of these findings.