Familial Transthyretin Amyloidosis Research Articles

Clinical applications of gene therapy for rare diseases: A review.

Rare diseases collectively exact a high toll on society due to their sheer number and overall prevalence. Their heterogeneity, diversity, and nature pose daunting clinical challenges for both management and treatment. In this review, we discuss recent advances in clinical applications of gene therapy for rare diseases, focusing on a variety of viral and non-viral strategies. The use of adeno-associated virus (AAV) vectors is discussed in the context of Luxturna, licenced for the treatment of RPE65 deficiency in the retinal epithelium. Imlygic, a herpes virus vector licenced for the treatment of refractory metastatic melanoma, will be an example of oncolytic vectors developed against rare cancers. Yescarta and Kymriah will showcase the use of retrovirus and lentivirus vectors in the autologous ex vivo production of chimeric antigen receptor T cells (CAR-T), licenced for the treatment of refractory leukaemias and lymphomas. Similar retroviral and lentiviral technology can be applied to autologous haematopoietic stem cells, exemplified by Strimvelis and Zynteglo, licenced treatments for adenosine deaminase-severe combined immunodeficiency (ADA-SCID) and β-thalassaemia respectively. Antisense oligonucleotide technologies will be highlighted through Onpattro and Tegsedi, RNA interference drugs licenced for familial transthyretin (TTR) amyloidosis, and Spinraza, a splice-switching treatment for spinal muscular atrophy (SMA). An initial comparison of the effectiveness of AAV and oligonucleotide therapies in SMA is possible with Zolgensma, an AAV serotype 9 vector, and Spinraza. Through these examples of marketed gene therapies and gene cell therapies, we will discuss the expanding applications of such novel technologies to previously intractable rare diseases.

Determinants of ventilatory inefficiency in transthyretin cardiac amyloidosis: The role of excessive ventilatory drive.

Background and objective: Along with impaired aerobic capacity, increased slope of the relationship between ventilation (VE) and pulmonary CO2 output (VCO2), i.e., VE-VCO2 slope is a common finding in patients with cardiac amyloidosis (CA), which suggests ventilatory inefficiency. Little is known about mechanisms leading to ventilatory inefficiency in CA patients. The purpose of this investigation was to examine the factors that underlie the abnormal ventilatory efficiency in transthyretin hereditary CA patients, such as excessive ventilatory drive, inability of pulmonary blood flow to increase adequately during exercise and excessive sympathetic stimulation, which are known mechanisms of VE-VCO2 slope increase. Methods: In this single-center retrospective observational study, consecutive patients (n = 41) with known familial transthyretin amyloidosis p.Val142Ile mutation carriers with confirmed cardiac phenotype were included. Results: Compared with CA patients without ventilatory inefficiency (VE-VCO2 slope < 36), patients with ventilatory inefficiency (VE-VCO2 slope ≥ 36) had increased inter-ventricular septum thickness, lower VO2 peak along with hyperventilation, and prolonged post-exercise heart rate recovery. By multivariate analysis, only excess of minute-ventilation at anaerobic threshold (β = 0.127; p = 0.011) remained an independent predictor of ventilatory inefficiency. Conclusion: Our data suggest that high ventilatory stimulation during exercise leading to hyperventilation is the main determinant of ventilatory inefficiency in hereditary transthyretin cardiac amyloidosis patients. This novel finding helps to better understand the mechanism of exercise intolerance in these patients where physiological limitation may be related to both heart dysfunction and abnormal pulmonary response.

OCULAR MANIFESTATIONS OF ASP38ALA AND THR59LYS FAMILIAL TRANSTHYRETIN AMYLOIDOSIS.

To describe the ophthalmic manifestations of familial transthyretin amyloidosis (FTA) mutations, including Asp38Ala and Thr59Lys, which have not been previously reported to have ocular involvement. This is an observational case series of prospectively collected data of 16 patients with FTA who were taking tafamidis for mild peripheral neuropathy and underwent a comprehensive ophthalmic examination at a single tertiary center, between January 2013 and March 2020. The ocular involvement of each FTA mutation type and the specific manifestations were the main outcome measures. Six of 16 patients with FTA manifested ocular involvement. Ocular involvement was noted in two of three patients with Glu89Lys mutations having retinal deposits, retinal hemorrhages, and corneal opacity. Three of nine patients with Asp38Ala mutations and one of two patients with Thr59Lys mutations showed ocular involvement that had not been previously described. The ophthalmic findings included glaucoma, anterior lens capsule opacity, vitreous opacity, and retinal deposits. The decrease in vascular flow due to perivascular cuffing of the amyloid deposits was detected by optical coherence tomography angiography. The current study newly described that two transthyretin mutation types of FTA, Asp38Ala and Thr59Lys, may manifest with ocular findings such as anterior lens capsule opacity and retinal deposits.

Thermodynamic Stability and Aggregation Kinetics of EF Helix and EF Loop Variants of Transthyretin.

Misfolding and aggregation of transthyretin (TTR) are linked to amyloid disease. Amyloidosis occurs when the TTR homotetramer dissociates into aggregation-prone monomers that self-assemble into amyloid. In familial transthyretin amyloidosis, hereditary amino acid substitutions destabilize TTR and promote aggregation. In this work, we used 19F nuclear magnetic resonance (NMR) to determine the effect of mutations in the EF helix (Y78F, K80D, K80E, and A81T) and EF loop (G83R and I84S) on the aggregation kinetics and stability of the TTR tetramer and monomer. The EF region acts as a scaffold that stabilizes interactions in both the strong and weak dimer interfaces of the tetramer and is the site of a cluster of pathogenic mutations. K80D and K80E are non-natural mutants that destabilize the EF helix and yield an equilibrium mixture of tetramer and monomer at neutral pH, providing a unique opportunity to determine the thermodynamic parameters for tetramer assembly under nondenaturing conditions. Of the pathogenic mutants studied, only A81T formed appreciable monomer at neutral pH. Real-time 19F NMR measurements showed that the pathogenic Y78F mutation accelerates aggregation by destabilizing both the tetrameric and monomeric species. The pathogenic mutations A81T, G83R, and I84S destabilize the monomer and increase its aggregation rate by disrupting a Schellman helix C-capping motif. These studies provide new insights into the mechanism by which relatively subtle mutations that affect tetramer or monomer stability promote entry of TTR into the dissociation-aggregation pathway.

S2418 Gifting Your Entire Liver to Another: A Unique Case of Domino Liver Transplantation

INTRODUCTION: Organ transplants are a novel treatment that can significantly improve the health of patients. Domino liver transplants (DLT) offer a way to increase the supply of livers that are in dire shortage. DLTs are unique because they provide an opportunity to use the original liver of liver transplant recipients as a graft for another patient. DLT is performed with patients with disorders such as amyloidosis, a condition where abnormal protein aggregates are produced and deposited in a variety of organs. It allows for the transplantation of a complete liver, that is anatomically and functionally normal besides the production of amyloid. Familial transthyretin amyloidosis (TTR) is a form of amyloidosis where transthyretin protein variants build-up commonly leading to liver and cardiac dysfunction. We present a case where a patient with TTR underwent a liver transplant and the explanted liver was donated to another patient with primary biliary cirrhosis, an autoimmune liver disease that damages intrahepatic bile ducts. CASE DESCRIPTION/METHODS: A 53-year-old female with a TTR type familial amyloidosis (transthyretin serine-84 mutation) undergoes a liver transplant. She has lost a father and 2 siblings to heart disease secondary to amyloidosis. She also had cardiac involvement and a previous carpal tunnel release with a biopsy positive for amyloid. Her original liver is used as the donor liver for a 74-year-old male patient with primary biliary cirrhosis. The recipient had previously developed esophageal varices and an increase in ascites. After the transplant, the donor experienced complications including graft-versus-host disease (GVHD), pain, nausea, vomiting, and diarrhea. GVHD was localized to a rash to her trunk and resolved with treatment. The recipient of the DLT liver had initial edema but is doing well now. Both patients are still doing well 2 years post-surgery. DISCUSSION: DLT can offer a solution for patients in need of a liver, but factors such as age must be considered. A liver transplant is curative of TTR and the removed liver can be considered for DLT for recipients who are not expected to outlive the symptoms of TTR. The transplant is curative because the liver is the source of the abnormal transthyretin protein, so both the donor and recipient should be monitored closely. We present a case of successful DLT, the donor also underwent a successful liver transplant that was curative for TTR. The recipient was an older patient who is not likely to develop symptoms of TTR.

Stroke risk and atrial mechanical dysfunction in cardiac amyloidosis

Amyloidosis is associated with poor prognosis, and patients with cardiac involvement have especially poor outcomes. Cardiac amyloidosis leads to higher rates of atrial arrhythmia and an increased risk of intracardiac thrombus formation. However, atrial mechanical dysfunction due to protein deposition in amyloidosis may lead to thrombus formation in the absence of atrial arrhythmia. We present a 42‐year‐old male patient with familial transthyretin amyloidosis who suffered an embolic stroke that originated from a left atrial appendage thrombus in the absence of any documented atrial fibrillation. This case highlights atrial mechanical dysfunction in patients with cardiac amyloidosis and the need to better stratify thrombotic risk in this population with integration of echocardiographic parameters and transesophageal echocardiography.

Fatal TTR amyloidosis with neuropathy from domino liver p.Val71Ala transplant.

Familial transthyretin (TTR) amyloidosis (FTA) is a serious autosomal dominant neuropathy caused by TTR germline mutations that lead to death on average 10 years from symptom onset. The mutant tetramer protein is produced predominantly in the liver, and liver transplantation is commonly performed to reduce the production of the mutant protein.1 The otherwise well-working explanted TTR liver can be transplanted into recipients who require liver transplantation but are unable to receive a liver in a timely fashion, the so-called domino liver transplant (DLTX). Recently, oligonucleotide drugs that reduce circulating TTR have been approved by the Food and Drug Administration (FDA), and these drugs use either antisense (inotersen) or RNA interference (patisiran), whereas earlier tetramer-stabilizing drugs (tafamidis and diflunisal) were less efficacious.2 Herein, we describe a domino liver recipient who died of respiratory failure from transplanted p.Val71Ala amyloidosis.

Progressive Cardiomyopathy in TTR Amyloidosis after Liver Transplant

Introduction Familial transthyretin (TTR) amyloidosis is a rare disorder characterized primarily by progressive neuropathy and cardiomyopathy. The source of amyloid protein is the liver, thus early liver transplantation can significantly slow disease progression. Unfortunately, for some patients without significant cardiomyopathy at the time of transplant, cardiac disease can subsequently progress due to enhanced deposition of wild-type amyloid protein. Promising new medical therapies may soon replace liver transplant as the gold standard treatment. Case report A 67 year-old male with bilateral carpal tunnel syndrome presented with hematuria and was found to have bladder masses that stained positive for amyloidosis. A work-up for systemic amyloidosis included a normal bone marrow biopsy and an echocardiogram with LVH in the absence of increased voltage on ECG. Two years later he had a cardiac arrest in the setting of sustained VT. A left heart catheterization revealed normal coronary arteries. His endomyocardial biopsy Congo red stain was positive. He was diagnosed with a previously undiscovered mutation of familial TTR amyloidosis (Glu61Gly) and five years later underwent liver transplantation. He was NYHA Class I and did well for ten years before his functional capacity gradually declined. Serial echocardiograms showed a slow progression of LV wall thickness from 1.3 to 1.7 cm and a decline in LVEF from 50% to 30%. In addition, the defibrillation threshold of his ICD progressively increased. He underwent heart transplantation due to end stage amyloid cardiomyopathy. Postoperatively he developed ulcerative esophagitis and ultimately expired due to fungal pericarditis. Discussion Although liver transplantation can slow the progression of TTR amyloidosis and is an appropriate treatment for select individuals, new alternative medical therapies may replace early transplantation. Diflunisal, an anti-inflammatory drug, stabilizes the TTR tetramer in vitro and in a randomized trial slowed progression of neuropathy in familial TTR. Combined doxycycline and tauroursodeoxycholic acid disrupt TTR amyloid fibrils and appear to slow disease progression. Tafamadis, a small molecule that inhibits the dissociation of TTR tetramers has been shown in phase 2 and 3 trials to slow neuropathy progression and decrease mortality and heart failure hospitalizations. Patisiran is a small interfering RNA that blocks TTR synthesis and has demonstrated stabilization of neuropathy. Inotersen is an antisense oligonucleotide that has also shown promise in familial TTR patients with polyneuropathy. Most recently, an investigational monoclonal antibody has been designed to target and remove amyloid deposits. Conclusion Familial TTR amyloidosis is characterized by progressive neuropathy and cardiomyopathy for which patients may undergo liver transplant, though cardiac disease can subsequently progress. Promising new medical therapies may soon replace liver transplant as the gold standard.

A selection of advances in neuromuscular medicine

A selection of advances in neuromuscular medicine Abstract. Significant developments in the realm of neuromuscular medicine have occurred in both non-invasive diagnostics as well as treatments. Whole body muscle MRI can detect disease specific patterns and lead to the implementation of direct molecular genetic diagnostics. Bedside neuromuscular ultrasound can assist in diagnosing inflammatory neuropathies even in some cases enabling the omission of nerve biopsy and lumbar puncture. Specific antibodies aid in the classification and management of acquired autoimmune diseases. First effective genetic therapies for monogenetic neuromuscular illnesses are now available for spinal muscular atrophy and familial transthyretin amyloidosis. This overview aims to highlight select new diagnostic and therapeutic achievements in the realm of neuromuscular medicine.

Angiographic Signatures of the Predominant Form of Familial Transthyretin Amyloidosis (Val30Met Mutation)

To describe abnormalities in choroidal and retinal vasculature associated with Val30Met familial transthyretin amyloidosis (V30M-FTA) using fluorescein and indocyanine green (ICG) angiography. Prospective, cross-sectional study. This study was conducted at the French National Reference Center for FTA. We included 18 consecutive genetically confirmed V30M-FTA patients (36 eyes) who underwent complete neurologic examination, including staging with polyneuropathy disability (PND) score, and complete ophthalmic evaluation, including staging of intraocular amyloid deposits and fluorescein and ICG angiograms (ICG-A). The grading of choroidal and retinal angiopathy, and their association with neurologic functional impairment, were the main outcome measures. Eleven men and 7 women, mean age 61.6 ± 12.1 years, were included. Retinal amyloid angiopathy (RAA) was detected in 24 eyes (92%) of 13 patients, with microaneurysms, retinal hemorrhages, and retinal ischemia of variable extent. Three patients (5 eyes) had neovascular glaucoma and 2 (2 eyes) had preretinal neovascularization. ICG-A indicated choroidal amyloid angiopathy (CAA) in all patients, with 3 distinct patterns-diffuse (9/18 patients), focal (5/18 patients), or punctiform (4/18 patients)-based on the extent of late hypercyanescence along the choroidal arteries. PND scores were significantly higher in patients with diffuse CAA (firework pattern) compared to those with limited CAA (focal and punctiform patterns) (2.89 vs 1.78, P= .045). RAA is a frequent and severe complication of V30M-FTA that may lead to anterior and posterior segment neovascularization. CAA was detected in all patients, with a late hypercyanescent delineation of the choroidal arterial vasculature, which was more extensive with increased disease severity.

Increasing the Efficiency of Amino Acids Detection By Electrochemical Methods on Amorphous Carbon Nitride a-CNx Electrodes

The naphthalene-2,3-dicarboxyaldehyde (NDA) label is widely used for the derivatization of amino acids (AAs) to form N-2-substituted-1-cyanobenz-[f]-isoindole derivatives (CBI) which are both fluorescent and electroactive. [1],[2] Optimal conditions for labeling were previously obtained when [NDA]tot/[CN-] ratio =1 and [NDA]tot /[amino acid or peptide]=40 with a buffer pH of 10. [3],[4] The detection step was achieved by using a carbon reference material (glassy carbon electrode ) exhibiting an obvious oxidation peak for CBI-AA observed at 0.5V/SCE [1]. These experiments confirmed that NDA can by used as a derivative agent for non electroactive AA, allowing for simultaneous electrochemical and fluorescence detections. In this work, amorphous carbon nitride a-CN0.26 thin films were elaborated on transparent and conductive glass/indium-tin oxide (ITO) wafers to improve the electroanalytical detection of AAs. The results obtained on as-grown a-CN0.26 were compared to the glassy carbon (GC). It was shown that a soft anodic pre-treatment protocol on glass/ITO/a-CN0.26 electrode in KCl solution drastically improved the CBI-AA oxidation peak performances. The oxidation peak potential for all CBI derivatives varied in the same range that those on GC and pre-treated glass/ITO/a-CN0.26, while no discrimination could be obtained for as-grown glass/ITO/a-CN0.26. For almost all the tested CBI derivatives, the electrochemical properties determined by electrochemical impedance spectroscopy (R ct, charge transfert resistance) and by differential pulse voltammetry (peak areas, full-widths at peak mid-height, peak current density and their SD values) were improved on pre-treated a-CN0.26 in comparison with GC. [1] M. Faure, S. Korchane, I. Le Potier, A. Pallandre, C. Deslouis, A.-M. Haghiri-Gosnet, J. Gamby, Investigating of labeling and detection of transthyretin synthetic peptide derivatized with naphthalene-2,3-dicarboxaldehyde, Talanta, 116 (2013) 8-13. [2] M. Faure, A. Pallandre, S. Chebil, I. Le Potier, M. Taverna, B. Tribollet, C. Deslouis, A.-M. Haghiri-Gosnet, J. Gamby, Improved electrochemical detection of a transthyretin synthetic peptide in the nanomolar range with a two-electrode system integrated in a glass/PDMS microchip, Lab Chip, 14 (2014) 2800-2805. [3] S. Korchane, A. Pallandre, C. Przybylski, C. Poas, F. Gonnet, M. Taverna, R. Daniel, I. Le Potier, Derivatization strategies for CE-LIF analysis of biomarkers: Toward a clinical diagnostic of familial transthyretin amyloidosis, Electrophoresis, 35 (2014) 1050-1059. [4] M. Faure, I. Le Potier, A. Pallandre, S. Chebil, A.-M. Haghiri-Gosnet, C. Deslouis, E. Maisonhaute, J. Gamby, Determination of the isomeric forms proportion of fluorogenic naphthalene-2,3-dicarboxaldehyde in a binary mixture of water:methanol using electrochemical methods, Talanta, 148 (2016) 494-501.

Cardiac denervation evidenced by MIBG occurs earlier than amyloid deposits detection by diphosphonate scintigraphy in TTR mutation carriers.

Cardiac involvement in familial transthyretin (TTR) amyloidosis is of major prognostic value, and the development of early-diagnostic tools that could trigger the use of new disease-modifying treatments is crucial. The aim of our study was to compare the respective contributions of 99mTc-diphosphonate scintigraphy (DPD, detecting amyloid deposits) and 123I-MIBG (MIBG, assessing cardiac sympathetic denervation) in patients with genetically proven TTR mutation referred for the assessment of cardiac involvement. We prospectively studied 75 consecutive patients (classified as symptomatic or asymptomatic carriers), using clinical evaluation, biomarkers (troponin and BNP), echocardiography, and nuclear imaging. Patients were classified as having normal heart-to-mediastinum (HMR) MIBG uptake ratio 4h after injection (defined by HM4 ≥ 1.85) or abnormal HM4 < 1.85, and positive DPD uptake (grade ≥ 1 of Perugini classification) or negative DPD uptake. Among 75 patients, 49 (65%) presented with scintigraphic sympathetic cardiac denervation and 29 (39%) with myocardial diphosphonate uptake. When MIBG was normal, DPD was negative except for two patients. Age was an independent predictor of abnormal scintigraphic result of both MIBG and DPD (HR 1.08 and 1.15 respectively), whereas echocardiographic-derived indicators of increased left ventricular filling pressure (E/e' ratio) was an independent predictor of abnormal MIBG (HR 1.33) and global longitudinal strain of positive DPD (HR 1.45). In asymptomatic patients (n = 31), MIBG was abnormal in 48% (n = 15) among whom 50% had a normal DPD; all those with a normal MIBG (n = 16) had a normal DPD. In TTR mutation carriers, cardiac sympathetic denervation evidenced by decreased MIBG uptake is detected earlier than amyloid burden evidenced by DPD. These results raise the possibility of a diagnostic role for MIBG scintigraphy at an early stage of cardiac involvement in TTR-mutated carriers, in addition to its well-established prognostic value.

On-a-chip tryptic digestion of transthyretin: a step toward an integrated microfluidic system for the follow-up of familial transthyretin amyloidosis.

A microfluidic microreactor for trypsin mediated transthyretin (TTR) digestion has been developed as a step towards the elaboration of a fully integrated microdevice for the detection of a rare and disabling disease, the familial transthyretin amyloidosis (ATTR) which is related to specific TTR mutations. Therefore, an enzymatic microreactor coupled to an analytical step able to monitor the mutation of TTR on specific peptide fragments would allow an accurate monitoring of the treatment efficiency of ATTR. In this study, two types of immobilized trypsin microreactors have been investigated: a new miniaturized, microfluidic fluidized bed packed with trypsin functionalized magnetic particles (MPs), and a thiol-ene (TE) monolith-based chip. Their performances were first demonstrated with N-benzoyl-dl-arginine-4-nitroanilide hydrochloride BApNA, a low molecular weight substrate. High reaction yields (75.2%) have been reached within 0.6 min for the TE-based trypsin microreactor, while a lower yield (12.4%) was obtained for the micro-fluidized bed within a similar residence time. Transposition of the optimized conditions, developed with BApNA, to TTR digestion in the TE-based trypsin microreactor was successfully performed. We demonstrated that the TE-chip can achieve an efficient and reproducible digestion of TTR. This has been assessed by MS detection. In addition, TTR hydrolysis led to the production of a fragment of interest allowing the therapeutic follow-up of more than twenty possible ATTR mutations. High sequence coverage (90%), similar to those obtained with free trypsin, was achieved in a short time (2.4 min). Repeated experiments showed good reproducibility (RSD = 6.8%). These promising results open up the route for an innovative treatment follow-up dedicated to ATTR.

Abnormal Coronary Flow Velocity Reserve and Decreased Myocardial Contractile Reserve Are Main Factors in Relation to Physical Exercise Capacity in Cardiac Amyloidosis

The aim of the present study was to evaluate the clinical importance of echocardiographic coronary flow velocity reserve (CFVR), resting and exercise left ventricular global longitudinal strain (LVGLS), and myocardial work efficiency (MWE) in patients with cardiac amyloidosis (CA). The study population comprised 69 subjects: group A, 27 patients with CA confirmed by endomyocardial biopsy (CA positive); group B, 42 healthy control subjects. The amyloid phenotype in group A was as follows: patients with wild-type transthyretin-related amyloidosis (n=10), carriers of the Danish familial transthyretin amyloidosis mutation with cardiac involvement (n=5), and patients with amyloid light chain amyloidosis with cardiac involvement (n=12). All subjects underwent comprehensive echocardiographic evaluation during rest and during symptom-limited, semisupine exercise testing. Furthermore, CFVR was assessed using Doppler echocardiography. Patients with CA had significantly lower CFVR (1.7±0.6 vs 3.9±0.8, P<.0001), MWE (1.9±1.0 vs 3.0±0.7, P<.0001), and LVGLS magnitude (11% [10%-14%] vs 20% [18%-21%], P<.0001) than control subjects. Patients with CA showed severely reduced deformation and efficiency reserve compared with control subjects (ΔLVGLS 0.9±2.8% vs 5.6±2.3%, P<.0001; ΔMWE 2.5±2.8 vs 8.8±2.6, P<.0001). In patients with CA, a strong relation was seen between physical capacity by the metabolic equivalent of tasks test and CFVR (r=0.55, P<.01), peak exercise LVGLS (r=0.64, P<.0001), and peak exercise MWE (r=0.60, P<.01). Patients with CA had a profound lack of CFVR and longitudinal myocardial deformation reserve compared with healthy control subjects. Both parameters were significantly associated with exercise capacity and may prove useful for evaluating cardiac performance in patients with CA.

Inotropic myocardial reserve deficiency is the predominant feature of exercise haemodynamics in cardiac amyloidosis.

This study aimed to characterize invasive haemodynamics during exercise in subjects with cardiac amyloidosis (CA). The study population numbered 44 subjects. Group A (CA-positive, n = 24) comprised wild-type transthyretin patients (n = 10), familial transthyretin amyloidosis mutation carriers (ATTRm) with cardiac involvement (n = 5), and light-chain amyloidosis patients with cardiac involvement (n = 9). Group B (CA-negative, n = 20) comprised four healthy ATTRm subjects without cardiac involvement documented by 11 C-PIB positron emission tomography and 16 healthy controls. All subjects underwent a symptom-limited, semi-supine exercise test with expired gas analysis and simultaneous right heart catheterization. CA patients had lower peak oxygen consumption [15 ± 6 mL/min/kg bodyweight (bwt) vs. 33 ± 7 mL/min/kg bwt; P < 0.0001] than controls. Myocardial reserve during exercise was significantly reduced in CA patients as reflected in a small increase in stroke volume index (SVI) and cardiac index (CI) compared with controls [ΔSVI: 4 mL/m2 (range: -1 to 8) vs. 14 mL/m2 (range: 5-25); P < 0.0001; ΔCI: 2 ± 2 L/min vs. 7 ± 2 L/min; P < 0.0001]. During exercise, CA patients had significantly higher left and right ventricular filling pressures than controls. Furthermore, CA patients had severely impaired pulmonary arterial compliance (PAC) compared with controls [2.9 mL/mmHg (range: 2.1-4.5) vs. 7.5 mL/mmHg (range: 5.7-10.4); P < 0.0001]. Cardiac amyloid deposits are associated with severely reduced inotropic myocardial reserve and increased left and right ventricular filling pressures during exercise. Furthermore, CA subjects have severely reduced PAC, which may contribute to right heart failure and reduced exercise capacity.

Outcomes of Patients With Familial Transthyretin Amyloidosis After Liver Transplantation.

Familial transthyretin amyloidosis is a disease caused by misfolded transthyretin aggregates that can impair multiple organ systems. Liver transplantation is the first-line treatment for familial transthyretin amyloidosis. Our objective is to study outcomes and survival among patients with familial transthyretin amyloidosis after transplantation. All patients undergoing orthotopic liver transplant for familial transthyretin amyloidosis at Mayo Clinic between 1997 and 2012 were reviewed. Baseline clinical characteristics, organs transplanted, and posttransplant clinical course were assessed. Of the 40 patients, 7 patients had the V30M mutation and 33 had other mutations. Nineteen patients received liver only, 19 liver and heart, and 2 combined liver, heart, and kidney transplants. The 5-year overall survival was 85% for those receiving multiple organ transplant and 52% for those receiving liver transplant only ( P = .057). There was no difference in overall survival based on mutation (V30M vs other mutations), but survival was confounded by varied disease involvement and organs transplanted. Those who had early death (≤24 months from liver transplant) had a higher incidence of baseline peripheral neuropathy, autonomic neuropathy, lower modified BMI, and higher alkaline phosphatase. Outcomes of orthotopic liver transplant in familial transthyretin amyloidosis are variable due to heterogeneity in mutations and patient status at the time of transplant. Familial transthyretin amyloidosis can progress, despite liver transplantation. Patients receiving combined liver, heart/kidney transplant demonstrated improved survival compared to liver transplant alone.

Outcome in patients treated with isolated liver transplantation for familial transthyretin amyloidosis to prevent cardiomyopathy.

Familial transthyretin (TTR) amyloidosis is caused by different TTR mutations resulting in different clinical phenotypes of the disease. The Leu111Met mutation causes severe restrictive cardiomyopathy. Liver transplantation (LTx) is an established treatment option for patients with TTR amyloidosis; however, information on outcome after isolated LTx in patients with Leu111Met mutation amyloidosis is limited. Between 2005 and 2012, six patients with TTR Leu111Met amyloidosis underwent isolated orthotopic LTx. None suffered from neuropathy. Prior to LTx, patients presented with echocardiographic manifestations of early cardiac amyloid involvement and in five endomyocardial biopsy was positive for TTR amyloid. Median age at LTx was 45.5 yr (range 39-54), and four were male (67%). All patients were alive at a median follow-up of 56.6 months (range 18-104). No surgical complications occurred. Two patients (33%) underwent cardiac transplantation during follow-up due to progressive cardiomyopathy. The remaining four patients experienced no echocardiographic or clinical deterioration of cardiac function following LTx. Isolated LTx appears to be a valuable treatment option for patients with familial TTR amyloidosis due to Leu111Met mutation. Appropriate timing of LTx is of utmost importance to avoid development of severe amyloid cardiomyopathy and the need for combined heart and liver transplantation.

Derivatization strategies for CE‐LIF analysis of biomarkers: Toward a clinical diagnostic of familial transthyretin amyloidosis

We report three derivatization strategies for CE analysis with LIF detection (CE-LIF) of two synthetic peptides mimicking the wild and mutated fragments of interest for the diagnosis of familial transthyretin amyloidosis. The precapillary derivatization of the peptides with three optical tags, 5-carboxytetramethylrhodamin succinimidyl ester (TAMRA-SE), naphtalene-2,3-dicarboxyaldehyde (NDA), and 3-(2-furoyl)quinoline-2-carboxyaldehyde (FQ) has been investigated by CE-LIF detection and MS. Results provide evidence that high reaction yields have been reached whereas the multitagging phenomenon has occurred for both NDA and TAMRA-SE labeling procedures. The derivatization and electrokinetic separation of a mixture of the two peptides of interest for the pathology diagnosis (22-aa peptides that differ only from one amino acid) were achieved using both approaches. The highest resolution with a value of 2.5 was obtained with TAMRA-SE labeled derivatives whereas NDA gave the best detection sensitivity (LOD of 2.5 μM). The validation of the developed methods showed a good linearity (R ≥ 0.997) between the peak area of the labeled derivatives and the peptide concentration for both NDA and FQ labeling procedures. The intraday RSDs of A and the migration times were less than 3.8 and 2.2%, respectively.

Multisystemic Disease Modeling of Liver-Derived Protein Folding Disorders Using Induced Pluripotent Stem Cells (iPSCs).

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant protein-folding disorder caused by over 100 distinct mutations in the transthyretin (TTR) gene. In ATTR, protein secreted from the liver aggregates and forms fibrils in target organs, chiefly the heart and peripheral nervous system, highlighting the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Here, we describe detailed methodologies for the directed differentiation of protein folding disease-specific iPSCs into hepatocytes that produce mutant protein, and neural-lineage cells often targeted in disease. Methodologies are also described for the construction of multisystem models and drug screening using iPSCs.

Improved electrochemical detection of a transthyretin synthetic peptide in the nanomolar range with a two-electrode system integrated in a glass/PDMS microchip.

An alternative to a three-electrode set-up for electrochemical detection and analysis in microfluidic chips is described here. The design of the electrochemical sensor consists of the surface of the glass substrate covered with a PDMS block which bears the microfluidic channels. A band microelectrode which acts as a working electrode surrounded by a large counter electrode is obtained at the micrometric level to propose a simple and efficient sensing area for on-a-chip analysis. The counter-electrode with a surface area about 22-fold greater than the working-microelectrode can also be considered as a pseudo reference since its current density is low and thus limits the potential variations around the rest potential. To this purpose, the [Fe(III)(CN)₆]³⁻/[Fe(II)(CN)₆]⁴⁻ redox couple was used in order to set a reference potential at 0 V since both electrodes were platinum. The electrochemical microchip performance was characterized using differential pulse voltammetric (DPV) detection and quantification of the optically multi-labelled transthyretin synthetic peptide mimicking a tryptic fragment of interest for the diagnosis of familial transthyretin amyloidosis (ATTR). The limit of detection of the peptide by the working microelectrode was 25 nM, a value 100-fold lower than the one reported with conventional capillary electrophoresis coupled with laser-induced fluorescence under the same analytical conditions.