Transthyretin (TTR)-associated amyloidoses are diseases wherein wild-type or mutant TTR forms amyloid fibrils that infiltrate multiple organs. Wild-type TTR amyloidosis, ATTRwt, is a sporadic disease characterized by deposits that occur mainly in the heart. Alternatively, >100 TTR mutants cause inherited forms, ATTRm, frequently featuring cardiac amyloid deposits. The goals of this research were to create a cell-based model of ATTR amyloidosis, to define the mechanism of cardiac TTR-associated amyloid at the cellular level, and to study several agents that could interrupt the amyloid process. We hypothesized that TTR oligomers were cardiotoxic and played a role in the mechanism of ATTR amyloidosis, and that cytotoxicity could be inhibited by diflunisal, doxycycline, and Kiacta®. Focusing on TTR proteins associated with cardiac amyloidosis (wild-type, L55P, V30A, and V122), we developed a thermal denaturation method for creating TTR oligomers that allowed us to study the direct effect of oligomers on cells. Congo red and thioflavin T analyses confirmed that the oligomers were on pathway to amyloid fibril formation. We tested the effect of TTR oligomers on rat and human cardiac cells by measuring cell viability and stress response (through live protease activity and qPCR). TTR-L55P oligomers elicited a cytotoxic effect; fluorescent microscopy indicated cellular uptake of the oligomers and continued intra-cellular aggregation. Cytotoxicity was blocked when TTR was heated in the presence of doxycycline; the drug appeared to dissociate TTR aggregates or stabilize the monomeric forms. We also investigated retinol-binding protein (RBP), a natural binding partner of TTR. By immuno-histochemistry, RBP was demonstrated in ATTRwt and ATTRm `non-amyloid' transplant heart tissues, localized to areas containing amyloid or in the case of the transplant tissue, regions that appeared to display ischemic damage. Serum RBP levels were significantly different in ATTR vs. age-matched controls (p = 0.03), and in ATTRwt vs. ATTRm (p <0.0001) by ELISA. These data provide evidence that TTR oligomers are cardiotoxic, possibly due to cellular internalization and progressive intracellular aggregation. Furthermore, our results support the use of doxycycline as a therapeutic in ATTR to target these amyloidogenic oligomers, and suggest that RBP may have potential as a disease biomarker.

A common theme in amyloid disorders is the deposition of disease-specific protein aggregates in tissues. Amyloid proteins bind to heparan sulfate (HS), a sulfated glycosaminoglycan, and HS has been found to promote the aggregation process. The present work relates to HS mediated mechanisms of amyloidosis, particularly transthyretin (TTR) amyloidosis, AA-amyloidosis and Alzheimer’s disease (AD). TTR is a transport protein present in the blood and cerebrospinal fluid, which under unclear circumstances can deposit as amyloid in the myocardium of elderly individuals. Examination of cardiac tissue from a 70 year old patient with reported cardiomyopathy reveald co-deposition of TTR amyloid and HS. Studies revealed that HS promotes TTR fibrillization through interaction with a basic motif in the protein. Empolyment of a cell model demonstrated that cell surface HS mediates internalization of TTR, an effect likely facilitated by HS-binding to the basic motif on TTR. Collectively, HS-TTR interactions at the cell surface may have dual outcomes, resulting in either fibrillization or internalization, respectively. During inflammatory conditions, serum amyloid A (SAA), an acute-phase protein associated with the high-density lipoprotein (HDL), can assemble into insoluble amyloid fibrils, causing AA-amyloidosis. We found that HS structures exceeding 12-14 sugar units in length separates SAA from HDL and induces subsequent aggregation of the polypeptide. Our result proposes a novel role for HS in AA-amyloidosis in which a critical length of HS is required for separation of SAA from HDL. Late-onset AD patients show reduced ability to clear cerebral amyloid-β (Aβ) aggregates, a pathological hallmark of the disease. Althought the pathway of Aβ clearance is still unclear, several cell-surface receptors are implicated in Aβ internalization. We found that ApoE facilitated Aβ uptake through interactions with HS-proteoglycans and low-density lipoprotein receptor-related protein 1. The ApoE interaction with Aβ likely promotes Aβ clearance in the brain, but, if unbalanced, may contribute to the pathology of AD.     These findings are in accord with the concept of HS as a promoter of amyloid protein aggregation, but also point to more complex relationship.

Amyloid diseases are a world-wide problem causing great human suffer and large economical costs. Although amyloid deposits, a common denominator in all amyloid disorders, are detrimental to the surrounding tissue, there is a poor correlation between total amyloid burden and clinical symptoms. Soluble oligomers are much more potent to exert a tissue damaging effect.  Alzheimer’s disease (AD) is strongly linked to self-assembly of the amyloid-β (Aβ) peptide. Antibodies selectively targeting cytotoxic Aβ-species are useful both for understanding oligomer formation and for their therapeutic abilities. We hypothesized that the effect of avidity would compensate for a low single site affinity and be enough to selectively target oligomers. To evaluate this hypothesis, we focused on the IgM isotype having ten antigen-binding sites. In accordance with the hypothesis, the IgM isotype effectively bound oligomeric Aβ also in presence of a vast excess of its monomeric counterpart, clearly illustrating the potentiating effect of avidity. As a continuation of this work, we have shown that the avidity effect from a bivalent binding is enough to induce oligomer specificity. This finding facilitates a direct application on the clinically more useful IgG isotype, where the binding properties now can be controlled in detail. The method is general and we have, using this technique, also designed oligomer specific antibodies targeting α-synuclein. Transthyretin (TTR) is an amyloidogenic protein involved in both hereditary and sporadic amyloidosis. The cytotoxicity of TTR is intriguing since studies have shown cytotoxic potential from oligomers, tetramers and even monomers. Elucidation of the molecular properties associated with TTR cytotoxicity is hence of interest. By preventing tetramer dissociation, TTR aggregation and TTR-induced cytotoxicity is abolished. Based on this rationale, a current therapeutic strategy is to stabilize the TTR tetramer with small molecules. The kinetic stability within the spectra of known TTR mutations spans more than three orders of magnitude. However, although the most stable mutants are inert, a poor correlation within the group of cytotoxic variants exists where the cytotoxic effect is not potentiated in proportion to their kinetic stability. Through analysis of a large spectra of TTR variants, our results indicate that TTR induced cytotoxicity requires an intermediate stability of the TTR molecule. The kinetic stability should be low enough to permit tetramer dissociation and the thermodynamic stability high enough to prevent instant aggregation and to allow formation of the cytotoxic fold.

This thesis was made to get a deeper understanding of how chaperones interact with unstable, aggregation prone, misfolded proteins involved in human disease. Over the last two decades, there has been much focus on misfolding diseases within the fields of biochemistry and molecular biotechnology research. It has become obvious that proteins that misfold (as a consequence of a mutation or outer factors), are the cause of many diseases. Molecular chaperones are proteins that have been defined as agents that help other proteins to fold correctly and to prevent aggregation. Their role in the misfolding disease process has been the subject for this thesis. Transthyretin (TTR) is a protein found in human plasma and in cerebrospinal fluid. It works as a transport protein, transporting thyroxin and holo-retinol binding protein. The structure of TTR consists of four identical subunits connected through hydrogen bonds and hydrophobic interactions. Over 100 point mutations in the TTR gene are associated with amyloidosis often involving peripheral neurodegeneration (familial amyloidotic polyneuropathy (FAP)). Amyloidosis represents a group of diseases leading to extra cellular deposition of fibrillar protein known as amyloid. We used human SH-SY5Y neuroblastoma cells as a model for neurodegeneration. Various conformers of TTR were incubated with the cells for different amounts of time. The experiments showed that early prefibrillar oligomers of TTR induced apoptosis when neuroblastoma cells were exposed to these species whereas mature fibrils were not cytotoxic. We also found increased expression of the molecular chaperone BiP in cells challenged with TTR oligomers. Point mutations destabilize TTR and result in monomers that are unstable and prone to aggregate. TTR D18G is naturally occurring and the most destabilized TTR mutant found to date. It leads to central nervous system (CNS) amyloidosis. The CNS phenotype is rare for TTR amyloid disease. Most proteins associated with amyloid disease are secreted proteins and secreted proteins must pass the quality control check within the endoplasmic reticulum (ER). BiP is a Hsp70 molecular chaperone situated in the ER. BiP is one of the most important components of the quality control system in the cell. We have used TTR D18G as a model for understanding how an extremely aggregation prone protein is handled by BiP. We have shown that BiP can selectively capture TTR D18G during co-expression in both E. coli and during over expression in human 293T cells and collects the mutant in oligomeric states. We have also shown that degradation of TTR D18G in human 293T cells occurs slower in presence of BiP, that BiP is present in amyloid deposition in human brain and mitigates cytotoxicity of TTR D18G oligomers. / Denna avhandling handlar om proteiner. Särskilt de som inte fungerar som de ska utan har blivit vad man kallar ”felveckade”. Anledningen till att proteiner veckas fel beror ofta (men inte alltid) på mutationer i arvsmassan. Felveckade proteiner kan leda till sjukdomar hos människor och djur (man brukar tala om amyloidsjukdomar), ofta av neurologisk karaktär. Exempel på amyloidsjukdomar är polyneuropati, där perifera nervsystemet är drabbat, vilket leder till begränsad rörelseförmåga och senare till förlamning; och Alzheimer´s sjukdom, där centrala nervsystemet är drabbat och leder till begränsad tankeförmåga och minnesförluster. Studierna som presenteras i denna avhandling har gått ut på att få en bättre förståelse för hur felveckade proteiner interagerar med det som vi har naturligt i cellerna och som fungerar som skyddande, hjälpande proteiner, så kallade chaperoner. Transtyretin (TTR) är ett protein som cirkulerar i blodet och transporterar tyroxin (som är ett hormon som bland annat har betydelse för ämnesomsättningen) samt retinol-bindande protein (vitamin A). I TTR genen har man funnit över 100 punktmutationer, vilka har kopplats samman med amyloidsjukdomar, bland annat ”Skellefteåsjukan”. Mutationer i TTR genen leder ofta till att proteinet blir instabilt vilket leder till upplösning av TTR tetrameren till monomerer. Dessa monomerer kan därefter sammanfogas på nytt men denna gång på ett sätt som är farligt för organismen. I denna avhandling har fokus legat på en mutation som kallas TTR D18G, vilken har identifierats i olika delar av världen och leder till en dödlig form av amyloidos i centrala nervsystemet. Det chaperon som vi har studerat benämns BiP och är beläget i en cellkomponent som kallas för det endoplasmatiska retiklet (ER). I ER finns cellens kontrollsystem i vilket det ses till att felveckade proteiner inte släpps ut utan istället bryts ned. Denna avhandling har visat att BiP kan fånga upp TTR D18G inuti celler och där samla mutanten i lösliga partiklar som i detta fall är ofarliga för cellen. Avhandligen har också visat att nedbrytningen av TTR D18G sker mycket långsammare när BiP finns i riklig mängd.

Protein stability plays an important role in a wide variety of settings ranging from industrial processes where proteins are used as biocatalysts to medical settings where misfolded proteins are implicated in disease. Understanding protein stability will allow design of improved bioprocess and pharmaceutical formulations as well as aid in the development of therapies for protein-based diseases. The effects of dissolved salts on protein kinetic stability are studied here. We find that ion-solvent interactions, characterized by the Jones-Dole B-viscosity coefficient, are strong indicators of salt effects on protein deactivation. This finding is used to develop a model for predicting protein deactivation in salt solutions in terms of two competing processes. Since protein unfolding and aggregation can lead to a number of protein misfolding diseases, we test the applicability of our model for describing salt effects on transthyretin aggregation. As the factors contributing to protein stability become more understood, the use of enzymes as biocatalyst for industrial process will increase, and the need for enzymes active in a wide range of reaction media will increase. We have developed a process using an enzyme in combination with organic-aqueous tunable solvents (OATS) which allows for monophasic reaction of the enzyme with hypdrophobic substrates. The reaction mixture can be separated into two phases by the addition of carbon dioxide pressure. This separation allows for both convenient recovery of the hydrophobic reaction product from the organic phase as well as recycle of the enzyme in the aqueous phase. Overall reaction conversions of 80% and little enzyme activity loss are observed after six reaction cycles.

In recent years, lower-chlorinated, airborne congeners of polychlorinated biphenyls (PCBs) have evolved as an emerging class of potentially hazardous environmental contaminants. Previous work has demonstrated that sulfation is a major metabolic pathway for these PCBs in vitro and in vivo; however, their metabolic fate and toxicities have not been explored. Hypothyroxinemia is among the most prevalent adverse health effects associated with PCB exposure in human populations and is an assumed cause of a variety of neurodevelopmental effects observed in infants following prenatal PCB exposure. The displacement of L-thyroxine (T4) from binding sites on transthyretin (TTR), a major T4 transport protein and trans-placental carrier of thyroid hormones, is thought to be a significant contributing factor in PCB-induced hypothyroxinemia. Structural similarities between sulfated metabolites of PCBs and T4 led to the central hypothesis that PCB sulfates are bioactive metabolites that exhibit high affinity binding to T4 binding sites on human TTR. An examination of the ability of six lower-chlorinated PCB sulfates to bind to human TTR in vitro, as well as subsequent computational modeling, revealed that these compounds interact with the high-affinity binding site in a non-covalent manner and with affinities comparable to T4. Corroborating evidence for the binding of PCB sulfates stems from their ability to inhibit the formation of TTR amyloid fibrils through stabilization of the protein's native conformation. Fibrillar TTR aggregates are the cause of amyloidoses like senile systemic amyloidosis, familial amyloid polyneuropathy and familial amyloid cardiomyopathy. All PCB sulfates examined were effective inhibitors of TTR fibrillogenesis with equal or higher efficiencies than some of the best previously described inhibitors. In vivo exposure of male Sprague-Dawley rats to a model PCB sulfate, 4-PCB 11 sulfate, resulted in rapid and widespread distribution of the metabolite to various organs, including the brain. Consequently, there is a strong indication for a potential role of PCB sulfates in thyroid disruption and inter-tissue transport of PCBs, and the binding of PCB sulfates to TTR may also provide structural information for improved design of anti-amyloid therapeutics. To date there are no analytical procedures for the quantification of PCB sulfates available, and exposure levels in human populations remain unknown. This study provides, for the first time, evidence that PCB sulfates, if present in human serum samples, are not extracted by current standard protocols for the analysis of PCBs and their metabolites. Consequently, PCB sulfates may have been overlooked in the past decades resulting in potential underestimation of total PCB exposure levels in exposed populations. Based on this finding, an efficient approach for the quantitative extraction of PCB sulfates from a variety of biological samples was developed. This procedure, coupled with quantitative mass spectrometry, has been validated for the future screening of human serum samples, and it was successfully applied to determine the tissue distribution and elimination profile of 4-PCB 11 sulfate in male Sprague-Dawley rats.

This dissertation focuses on the effects of lead (Pb) on the expression of thyroid hormone distributor proteins and how that affects the developing brain in Xenopus laevis tadpoles. Previous work has shown that Pb has the ability to dysregulate thyroid hormone (TH)-signaling in vertebrates and that Pb can impair brain development. This dissertation reports results for a series of Pb-treatment experiments conducted in Xenopus laevis tadpoles. The first primary hypothesis of this dissertation is that Pb impairs TH-dependent mechanisms of brain development. The second primary hypothesis of this dissertation is that Pb-induced impairments of brain development happen via dysregulation of thyroid hormone distributor proteins (THDPs) transthyretin (TTR) and β-trace. Analyses of the effects of Pb on overall body growth showed dose-dependent decreases in body length with increasing concentrations. Evaluation of the effect of Pb on tectal size and cell death in the developing brain yielded bimodal changes that depended upon Pb concentration in both features. Furthermore, Pb impaired TH-induced changes in brain development, including neurogenesis and brain volume. Pb abolished the T4-mediated increase in proliferating cell nuclear antigen (PCNA) expression, while having only marginal effects on neuronal regeneration related protein (NREP) and Krueppel-like factor 9 (klf9). Analyses of the effects of Pb on TTR and β-trace expression yielded results demonstrating a significant decrease in expression of both proteins in response to Pb-treatment. Contrary to prior studies in the literature, I demonstrate here that TTR is present in the brains of Xenopus. While electroporation of TTR morpholino did result in fewer TTR puncta, electroporation with morpholinos for TTR and β-trace knock down did not mimic the effects of Pb on neurogenesis. However, overexpression of these proteins in the choroid plexus (CP) of these animals was sufficient to produce an increase in neurogenesis. Finally, overexpression of these proteins was sufficient to ameliorate the effects of Pb-treatment on neurogenesis. The results affirm both the primary and secondary hypotheses, illustrating that Pb does, indeed, impair TH-mediated mechanisms of brain development and that these impairments are mitigated by dysregulation of TTR and β-trace. / Doctor of Philosophy / This dissertation focuses on the effects of lead (Pb) poisoning on thyroid hormone (TH)-mediated mechanisms of brain development in Xenopus laevis tadpoles. The work detailed here seeks to shine a light on the effects of Pb on brain development and one mechanism by which those effects may be mediated. This dissertation details experiments done in Xenopus laevis tadpoles, which are a prime animal model for studying environmental toxicants, especially those that disrupt TH physiology. This dissertation focuses on two primary hypotheses within a two journal manuscript format. The first primary hypothesis of this dissertation is that Pb impairs TH-dependent mechanisms of brain development. The second primary hypothesis of this dissertation is that Pb-induced impairments of brain development happen via dysregulation of thyroid hormone distributor proteins (THDPs) TTR and β-trace. The results found in this dissertation are consistent with the conclusions that Pb impairs TH-mediated mechanisms of brain development and that those impairments are mitigated by dysregulation of THDPs in the brain and body.

No description available.

Transthyretin (TTR) amyloidosis is a disease that appears in three variants. One variant affects the elderly population with heart failure, the other two variants are hereditary and caused by an amino acid substitution in the gene, resulting in polyneuropathy and/or heart issues depending on the amino acid substitution. However, in all three variants, other organs may also be affected with amyloid deposition in the disease course. Amyloid fibrils of TTR (ATTR) contains a mixture of full-length protein and fragments (50-127). Luminescent conjugated oligothiophenes (LCO’s) are novel amyloid binding probes used to stain amyloid fibrils and these amyloid probes have the feature of characterizing the amyloid structure in terms of fluorescence spectra. Apart from LCO’s, a few other amyloid binding probes are used to stain recombinant amyloid transthyretin and native transthyretin for binding studies. The majority of generated TTR aggregates in vitro did not have the characteristic fluorescence spectra when bound to LCO’s and was observed as a clumped gel-like aggregate. The generation of recombinant TTR fibrils in vitro using the mutant TTR-T49M to obtain an aggregation prone fragment (50-127) after being treated with cyanogen bromide had a low yield of in vivo amyloid-like fibrils, but with characteristic LCO spectra. Carpal tunnel ATTR often precedes ATTR deposition in heart tissue. Amyloid transthyretin in carpal tunnel tissues was stained with LCO’s and used as a reference in the comparison against the in vitro generated recombinant amyloid transthyretin fibrils. This project also includes quantification of amyloid transthyretin in a few selected parts of the carpal tunnel tissue using ImageJ. In the long run this method could help in diagnosing TTR amyloidosis.

Introdução: A amiloidose transtiretina é uma doença autossômica dominante decorrente de uma proteína transtiretina (TTR) variante, que sofre uma mudança conformacional e origina um tetrâmero de TTR instável, passo que é decisivo para o início da formação dos depósitos amilóides em diferentes órgãos e tecidos. Na maioria dos pacientes, o sistema nervoso periférico é o alvo principal, resultando na polineuropatia amiloidótica familiar transtiretina (TTR-FAP), classicamente uma neuropatia sensitivo-motora e autonômica progressiva, evoluindo para o óbito em aproximadamente 10 anos. A mutação de ponto mais frequente no mundo, incluindo o Brasil, é a TTRVal30Met, entretanto mais de 100 mutações de ponto diferentes já foram descritas. Objetivos: descrever a epidemiologia mutacional do gene TTR na polineuropatia amiloidótica familiar e correlacionar estas mutações com seus achados clínicos e eletroneuromiográficos. Métodos: estudo de coorte, descritivo e retrospectivo de um grupo de pacientes brasileiros encaminhados para o serviço de neurogenética do HC da FMRP-USP para investigação de neuropatia periférica, cujo estudo genético identificou uma mutação no gene TTR, com posterior análise transversal dos resultados obtidos entre os subgrupos com as diferentes mutações. Resultados: um total de 128 pacientes tiveram uma mutação de ponto no gene TTR identificada, dos quais 12 (9,4%) pacientes apresentaram uma mutação não TTRVal30Met, incluindo 4 patogênicas (6 pacientes, 4,7%) e 2 não patogênicas (6 pacientes, 4,7%). A mutações não TTRVal30Met patogênicas foram TTRAsp38Tyr (2 pacientes), TTRIle107Val (2 pacientes), TTRVal71Ala (1 paciente) e TTRVal122Ile (1 paciente). Dentre as mutações não patogênicas, foram encontradas TTRGly6Ser (5 pacientes) e TTRThr119Thr (1 paciente). A mutação TTRVal30Met estava presente em 116 (90,6%) pacientes, dos quais 52 possuíam dados clínicos e eletroneuromiográficos completos: 39 (75%) tiveram início precoce e 13 (25%), início tardio. O grupo de início precoce apresentou-se como a forma clássica da PAF-TTR, sem predileção de gênero (homens: 53,8%), manifestação inicial como neuropatia de fibras finas e autonômica (82,1%) e história familiar positiva (90,3%). A ENMG estava normal em 36,7% destes pacientes. O envolvimento cardiovascular foi caracterizado mais frequentemente por alterações da condução cardíaca (84,2%), sendo menos prevalente a cardiomiopatia (11,1%). Por outro lado, o grupo de início tardio mostrou uma predominância do sexo masculino (92,3%), presença de sintomas motores na primeira consulta (38,5%), resultando numa neuropatia sensitivo-motora com acometimento de fibras grossas e história familiar negativa (69,2%). Todos apresentaram neuropatia sensitivo-motora na ENMG. Neste grupo, a cardiomiopatia estava presente em 71,4% dos pacientes. Todos os pacientes, em ambos os grupos, tiveram disautonomia em algum momento do seu seguimento clínico. Conclusões: no nosso estudo aproximadamente 5% dos pacientes com FAP-TTR tinham uma mutação não TTRVal30Met, demonstrando a importância do sequenciamento do gene TTR em pacientes com história clínica sugestiva e screening negativo para a mutação TTR Val30Met. Além disso, os pacientes brasileiros com FAP-TTRVal30Met apresentaram achados clínicos e eletroneuromiográficos similares as populações descritas com esta mutação em outros países. / Background: Transthyretin amyloidosis is an autossomal dominant disease caused by variant transthyretin, that is misfolded, originating a unstable transthyretin tetramer, a rate-limiting step in the formation of the amyloid deposits in different organs and tissues. In most patients, the peripheral nervous system is the main target, leading to transtyretin familial amyloid neuropathy (TTR-FAP), classically characterized as a progressive sensory-motor and autonomic neuropathy, that leads to death in about 10 years. TTRVal30Met is the most frequent point mutation worldwide, including Brazil, but more than 100 different point mutations has been described. Objectives: describe the mutational epidemiology of TTR gene in TTR-FAP and characterize its clinical and electrophysiological findings. Methods: a descriptive and retrospective study of a group of Brazilian patients forwarded to the Neurogenetics or Peripheral Nerve Clinics from FMRP-USP whose etiological investigation identified a mutation in the TTR gene. A cross-sectional analysis evaluating the subgroups with different mutations was also carried on. Results: we identified one hundred and twenty eight patients carrying a TTR point mutation, of whom 12 (9,4%) harbored a non-Val30Met mutation, including 4 pathogenic (6 patients, 4,7%) and 2 non-pathogenic abnormalities (6 patients, 4,7%). The non Val30Met pathogenic mutations were TTRAsp38Tyr (2 patients), TTRIle107Val (2 patients), TTRVal71Ala (1 patient) and TTRVal122Ile (1 patient). Among the non-pathogenic mutations, we found the TTRGly6Ser (5 patients) and the TTRThr119Thr (1 patient). The TTRVal30Met mutation was present in 116 (90,6%) patients, of whom 52 had a complete clinical and neurophysiological data: 39 (75%) with early-onset and 13(25%) with late-onset neuropathies. The early-onset group presented as the classic TTRFAP, with no gender predominance (male: 53,8%), the first manifestations were those of a small fiber sensory and autonomic neuropathy (82,1%) and a highly positive family history (90,3%). EMG was normal in 36,7% of these patients. The cardiovascular involvement was characterized by frequent ECG abnormalities (84,2%), less often associated with cardiomayopathy (11,1%). On the other hand, the late-onset TTRVal30Met showed a male predominance (92,3%), presence of motor complaints in the first evaluation (38,5%) resulting in a sensory-motor polyneuropathy with large fiber involvement and a negative family history (69,2%). All patients presented a sensory and motor neuropathy on EMG examination. In this group, cardiomiopathy was frequently associated with the neuropathy (71,4%). All patients, in both groups, had autonomic symptoms at some point in clinical follow up. Conclusions: In our study almost 5% of the patients with TTR-FAP have a non Val30Met pathogenic mutation, highlighting the importance of sequecing the whole TTR gene in patients with a sugestive clinical history and negative screening for TTRVal30Met mutation. In adition, the Brazilian patients we studied with early and late onset TTR-FAP, present similar findings to TTRVal30Met populations from other countries submitted to similar studies.