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.

Background: Hereditary transthyretin amyloidosis is an autosomal dominant disease with a reduced penetrance. The most common mutation in Sweden is the V30M mutation in the transthyretin gene. Clustering areas of the disease can be found in Northern Sweden, Portugal, Brazil and Japan, although sporadic cases exist worldwide. Despite being caused by the same mutation, there are large differences in onset, penetrance and symptoms of the disease. Swedish V30M patients typically have a later onset with a lower penetrance compared to those from the clustering Portuguese V30M areas. The reasons for these differences have not been fully understood. The aim of this thesis is to study mechanisms that may influence onset and symptoms and investigate why patients carrying the same mutation have different phenotypes. Methods: Genealogy studies were performed on all known V30M carriers in Sweden using standard genealogy methods. DNA samples from patients, asymptomatic carriers and controls from different countries were collected and the transthyretin gene was sequenced. Liver biopsies from patients were used for allele specific expression analysis and a cell assay was used for miRNA analysis with the mutated allele. Gene expression analysis was performed on biopsies from liver and fat from patients and controls. Results and conclusions: Genealogic analysis of all known Swedish V30M carriers managed to link together 73% of the Swedish ATTR V30M population to six different ancestors from the 17th and 18th century, thus dating the Swedish V30M mutation to be more than 400 years old. A founder effect was also visible in descendants to one of the ancestors, producing a later age at onset. Sequencing of the transthyretin gene revealed a SNP in the 3’ UTR of all Swedish V30M carriers that was not found in any of the Japanese or French V30M carriers. The SNP was present on the Swedish transthyretin haplotype and defined the Swedish V30M population as separate from others. However, the SNP itself had no effect upon phenotype or onset of disease. Gene expression analysis of liver and fat tissue revealed a change in genetic profile of the patients’ livers, in contrast to the unchanged profile of the fat tissue. A changed genetic profile of the liver could explain why domino liver recipients develop the disease much earlier than expected.

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.

Background Transthyretin amyloid (ATTR) amyloidosis is a systemic disorder caused by amyloid deposits formed by misfolded transthyretin (TTR) monomers. Two main forms exist – wild-type and hereditary ATTR amyloidosis, the latter associated with TTR gene mutations. Wild-type ATTR amyloidosis has a late onset and primarily cardiac manifestations, whereas hereditary ATTR amyloidosis is a rare autosomal dominant condition with a considerable phenotypic diversity. Both disorders are present all over the world, but endemic areas of the hereditary form are found in Sweden, Portugal, Brazil and Japan. Gastrointestinal (GI) complications are common in hereditary ATTR amyloidosis and play an important role in the patients’ morbidity and mortality. Malfunction of the autonomic and enteric nervous systems has been proposed to contribute to the GI disturbances, but the underlying mechanisms have not been fully elucidated. The aims of this thesis were to assess the prevalence of GI disturbances for different subtypes of ATTR amyloidosis, to further explore the mechanisms behind these disturbances, and to evaluate the outcome of the patients’ GI function after liver transplantation, which currently is the standard treatment for hereditary ATTR amyloidosis. Methods The Transthyretin Amyloidosis Outcomes Survey (THAOS) is the first global, multicenter, longitudinal, observational survey that collects data on patients with ATTR amyloidosis. THAOS enrollment data were used to assess the prevalence of GI symptoms and to evaluate their impact on nutritional status (mBMI) and health-related quality of life (EQ-5D Index Score). Data from routine investigations of heart-rate variability and cardio-vascular response to tilt tests were utilized to evaluate the impact of autonomic neuropathy on the scintigraphically measured gastric emptying half-times in Swedish patients with hereditary ATTR amyloidosis. Gastric wall autopsy specimens from Japanese patients with hereditary ATTR amyloidosis and Japanese non-amyloidosis controls were analyzed with immunohistochemistry and computerized image analysis to assess the densities of interstitial cells of Cajal (ICC) and nervous tissue. Data from gastric emptying scintigraphies and validated questionnaires were used to evaluate the outcome of Swedish patients’ GI function after liver transplantation for hereditary ATTR amyloidosis. Results Sixty-three percent of the patients with TTR mutations and 15 % of those with wild-type ATTR amyloidosis reported GI symptoms at enrollment into THAOS. Subsequent analyses focused on patients with TTR mutations and, among them, unintentional weight loss was the most frequent symptom (32 %) followed by early satiety (26 %). Early-onset patients (<50 years of age) reported GI symptoms more frequently than late-onset cases (70 % vs. 50 %, p <0.01), and GI symptoms were more common in patients with the V30M mutation than in those with non-V30M mutations (69 % vs. 56 %, p <0.01). Both upper and lower GI symptoms were significant negative predictors of nutritional status and health-related quality of life (p <0.01 for both). Weak but significant correlations were found between gastric emptying half-times and the function of both the sympathetic (rs = -0.4, p <0.01) and parasympathetic (rs = -0.3, p <0.01) nervous systems. The densities of c-Kit-immunoreactive ICC were significantly lower in the circular (median density 0.0 vs. 2.6, p <0.01) and longitudinal (median density 0.0 vs. 1.8, p <0.01) muscle layers of the gastric wall in patients compared to controls. Yet, no significant differences in protein gene product 9.5-immunoreactive nervous cells were found between patients and controls either in the circular (median density 3.0 vs. 6.8, p = 0.17) or longitudinal (median density 1.4 vs. 2.5, p = 0.10) muscle layers. Lastly, the patients’ GI symptoms scores had increased slightly from before liver transplantation to the follow-ups performed in median two and nine years after transplantation (median score 7 vs. 10 vs. 13, p <0.01). However, their gastric emptying half-times (median half-time 137 vs. 132 vs. 125 min, p = 0.52) and nutritional statuses (median mBMI 975 vs. 991 vs. 973, p = 0.75) were maintained at follow-ups in median two and five years after transplantation. Conclusion GI disturbances are common in hereditary ATTR amyloidosis and have a negative impact on the patients’ nutritional status and health-related quality of life. Fortunately, a liver transplantation appears to halt the progressive GI involvement of the disease, although the patients’ GI symptoms tend to increase after transplantation. An autonomic neuropathy and a depletion of gastrointestinal ICC seem to contribute to the GI disturbances, but additional factors must be involved.