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.

Amyloidoses comprise a group of gain-of-toxic function protein misfolding diseases, in which normally soluble proteins in their functional state undergo conformational changes into highly organized and generally intractable thread-like aggregates, termed amyloid fibrils. These structures accumulate predominantly in the extracellular space but growing evidence suggests that amyloids may start to form intracellularly. At least 26 different human proteins, intact or in fragmented form, are known to form amyloid, which is linked with many debilitating neurodegenerative diseases such as Alzheimer’s disease (AD), Creutzfeldt-Jakob disease, and transthyretin (TTR)-related amyloidosis (ATTR). In this work, we focus on ATTR, which is one of the most frequent systemic amyloid diseases. A functional link was established between hereditary ATTR, a severe and fatal disorder and the enhanced propensity of the human plasma protein transthyretin (TTR) to form aggregates, caused by single point mutations in the TTR gene. The disease is heterogeneous and clinical symptoms vary from cardiomyopathy to progressing sensorimotor polyneuropathy depending on TTR variant involved and the amyloid deposition site. Despite the fact that TTR-derived amyloid accumulates in different organs such as heart, kidney, eyes, and predominantly in the peripheral nerves of ATTR patients, the exact mechanism of the disease development is not understood. In contrast to the case of AD, it has been difficult to generate an animal model for ATTR in transgenic mice that would be useful in understanding TTR aggregation processes and the mechanisms of the associated toxicity as these mice did not develop any neuropathic phenotype besides amyloid deposits. Therefore, we created a disease-model in Drosophila due to its huge repertoire of genetic techniques and easy genotype – phenotype translation, as well as its success in modeling human neurodegeneration. We have generated transgenic flies that over-express the clinical amyloidogenic variant TTRL55P, the engineered variant TTR-A (TTRV14N ⁄ V16E), and the wild-type protein. All TTR variants were found in the secreted form in the hemolymph where misfolding occurred and depending on the pool of toxic species, the fate of the fly was decided. Within a few weeks, both mutants (but not the wild-type TTR) demonstrated a time-dependent aggregation of misfolded molecules in vivo. This was associated with neurodegeneration, change in wing posture, attenuation of locomotor activity including compromised flying ability, and shortened life span. In contrast, expression of the wild-type TTR had no discernible effect on either longevity or fly behavior. In this work, we also addressed the correlation between TTR transgene dosage and thus, protein levels, with the severity of the phenotypes observed in TTR-A flies which developed a “dragged wing” phenotype. Remarkably, we established that degenerative changes such as damage to the retina strictly correlated with increased levels of mutated TTR but inversely with behavioral alterations and the dragged wing phenotype. We characterized formation of aggregates in the form of 20 nm spherules and amyloid filaments intracellularly in the thoracic adipose tissue and brain glia (both tissues that do not express the transgene). Moreover, we detected a fraction of neurotoxic TTR-A in the hemolymph of young but not old flies. We proposed that these animals counteract formation and persistence of toxic TTR-A species by removal from the circulation into intracellular compartments of glial and fat body cells and this is part of a mechanism that neutralizes the toxic effects of TTR. We validated the fly model for ATTR by applying a genetic screen during study of modifier genes. We found Serum amyloid P component (a product of the APCS gene) as a potent modifier of TTR amyloid-induced toxicity that was effective in preventing the apoptotic response in cell culture assay and capable of reducing the dragged wings when co-expressed in TTR-A flies. Finally, we optimized this fly model in order to screen for therapeutic compounds effective against ATTR. Feeding assays showed the effectiveness of several compounds among known native-state kinetic stabilizers of TTR against its aggregation. We described several early endpoints in this model, which can be used as a rapid and cost-effective method for optimizing concentrations and pre-screening of drug candidates. As the proof of principle, by feeding flies with increasing doses of diflunisal analogue (an FDA-approved Non-Steroidal Anti-Inflammatory Drug) a dose-dependent reduction of the dragged wings was observed.

Systemic amyloidoses are a group of lethal diseases where proteins aggregate into fibrillar structures, called amyloid fibrils, that deposits throughout the body. Transthyretin (TTR) causes one type of amyloidosis, in which the aggregates mainly infiltrate nervous and cardiac tissue. Almost a hundred different mutations in the TTR gene are known to trigger the disease, but wild-type (wt) TTR is also incorporated into the fibrils, and may alone form amyloid. Patients with the TTRV30M mutation show, for unknown reasons, two clinical phenotypes. Some have an early onset of disease without cardiomyopathy while others have a late onset and cardiomyopathy. It has previously been described that amyloid fibrils formed from TTRV30M can have two different compositions; either with truncated molecules beside full-length TTR (type A) or only-full-length molecules (type B).  In this thesis, the clinical importance of the two types of amyloid fibrils was investigated. We found that the fibril composition types are correlated to the two clinical phenotypes seen among TTRV30M patients, with type A fibrils present in late onset patients and type B fibrils in early onset patients. The only treatment for hereditary TTR amyloidosis has been liver transplantation, whereby the liver producing the mutant TTR is replaced by an organ only producing wt protein. However, in some patients, cardiac symptoms progress post-transplantationally. We demonstrated that the propensity to incorporate wtTTR differs between fibril types and tissue types in TTRV30M patients, with cardiac amyloid of type A having the highest tendency. This offers an explanation to why particularly cardiac amyloidosis develops after transplantation, and suggests which patients that are at risk for such development. By examining patients with other mutations than TTRV30M, we showed that, in contrast to the general belief, a fibril composition with truncated TTR is very common and might even be the general rule. This may explain why TTRV30M patients often have a better outcome after liver transplantation than patients with other mutations. In conclusion, this thesis has contributed with one piece to the puzzle of understanding the differences in clinical phenotype and treatment response between TTR amyloidosis patients, by demonstrating corresponding differences at a molecular level.

The hereditary transthyretin (TTR) amyloidoses are rare, and in severe cases, fatal disorders caused by mutations in the TTR gene. The clinical picture is diverse, involving neuropathies and myopathies, and mainly depends on the causative mutation and the sites and rates of amyloid deposition. The ultimate aim of the field of research presented in this thesis is to prevent TTR amyloid disease. To reach this ambitious goal, a thorough understanding of the normal as well as the pathological properties of the protein is essential. Here, comparisons between TTR from humans and other species may provide valuable information. The three-dimensional structure of TTR from Gilthead sea bream (Sparus aurata) was determined at 1.75 Å resolution by X-ray crystallography, and was found to be structurally similar to human TTR. However, significant differences were observed in the area at and around β-strand D, an area believed to dissociate from the structure prior to amyloid formation, thereby allowing the β-strands A and B to participate in polymerization. During evolution, the preference of TTR for the thyroid hormones, 3,5,3’-triiodo-L-thyronine (T3) and 3,5,3’,5’-tetraiodo-L-thyronine (T4), has shifted. While human TTR has higher affinity for T4, the opposite is true in lower vertebrates, e.g. fish and reptiles, where T3 is the main ligand. We have determined two separate structures of sea bream TTR in complex with T3 and T4, both at 1.9 Å resolution, as well as the complex of human TTR with T3. A significantly wider entrance and narrower thyroid hormone binding channel suggest a structural explanation to the differences in thyroid hormone preference between human and piscine TTR. The Tyr114Cys substitution in TTR is associated with severe systemic amyloidosis. The mutation introduces a second cysteinyl group in the TTR monomer, and has been shown to inhibit the formation of fibril formation in vitro, promoting the formation of disulfide-bonded amorphous aggregates. To deduce the role of intermolecular disulfide bonds in fibril formation we characterized the TTR Cys10Ala/Tyr114Cys double mutant. Our results suggest that an intermolecular disulfide bond at position 114 enhances the exposure of Cys10, which promotes the formation of additional intermolecular disulfide-linked assemblies. Also, we were able to isolate a disulfide-linked dimeric form of this mutant that formed protofibrils in vitro, suggesting the architecture of TTR amyloid may be the result of different underlying structures rather than that of a highly stringent assembly. We have also been able to successfully adapt a method of protein pre-heating to enable crystallization, thereby succeeding in a particularly problematic protein crystallization experiment. By heating the protein solution, we succeeded in separating several forms of protein micro-heterogeneities from the properly folded protein species, thereby allowing the growth of well diffracting crystals. / Ärftlig transthyretinamyloidos är en ovanlig och i allvarliga fall dödlig proteininlagringssjukdom som orsakas av mutationer i genen för transthyretin. Den kliniska bilden är huvudsakligen beroende av den bakomliggande genförändringen samt amyloidlokaliseringen och -depositionshastigheten och omfattar vanligen neuropatier och myopatier av varierande grad. Det slutgiltiga målet med forskningsfältet som presenteras i denna avhandling är att förhindra eller bota transthyretinamyloidos. En förutsättning för att lyckas med detta ambitiösa mål är en ingående förståelse för proteinets grundläggande egenskaper, såväl i normalfallet som i de patologiska processerna, bland annat genom jämförande studier av humant och icke-humant transthyretin (TTR). Den tredimensionella röntgenkristallografiska strukturen av TTR från fisken guldsparid (Sparus aurata) bestämdes till en upplösning på 1,75Å och befanns vara strukturellt snarlik humant TTR. Signifikanta skillnader observerades emellertid i och kring β-sträng D, en region som tros dissociera från huvudstrukturen innan själva bildningen av amyloid. Enligt denna hypotes leder D-strängsdissociationen till exponering av β-strängarna A och B, vilka därmed kan delta i de reaktioner som bildar amyloid. Under evolutionen har bindningspreferenserna för thyroideahormonerna T3 (3,5,3’-trijod-L-thyronin) och T4 (3,5,3’,5’-tetrajod-L-thyronin) hos TTR ändrats. Humant TTR har högre affinitet för T4 än för T3, medan det motsatta förhållandet gäller för lägre vertebrater, t ex fisk, där T3 är den huvudsakliga liganden. Strukturerna bestämdes för guldsparid i komplex med T4 och med T3 till 1,9 Å upplösning, samt för humant TTR i komplex med T3 till 1,7 Å upplösning. Jämförande analyser visade på signifikanta skillnader i thyroideahormonbindningskanalen, vilken var vidare och grundare i fisk än i människa. Dessa strukturella skillnader kan delvis förklara olikheterna i hormonbindning mellan högre och lägre vertebrater. Substitutionen Tyr114Cys i TTR är kopplad till en allvarlig form av systemisk transthyretinamyloidos. Mutationen introducerar en andra cysteinylgrupp i TTR-monomererna, vilket förhindrar fibrillbildning in vitro, men gynnar bildningen av amorfa disulfidbundna aggregat. För att närmare studera betydelsen av disulfidbindningar vid fibrillbildning av detta protein så karakteriserades dubbelmutanten TTR Cys10Ala/Tyr114Cys. Baserat på våra resultat föreslår vi att intermolekylära disulfidbindningar i position 114 ökar exponeringen av Cys10, vilket förstärker tendensen att bilda ytterligare disulfidbundna aggregat. Vi isolerade även en disulfidbunden dimerisk form av dubbelmutanten som kan bilda protofibriller in vitro. Baserat på denna observation föreslår vi att transthyretinamyloids underliggande arkitektur är sammansatt och kan nås genom sammanfogning av olika substrukturer snarare än genom en strikt ordnad uppbyggnad. Vi har också modifierat och anpassat en metod för uppvärmning av proteiner för att möjliggöra kristallisation i ett synnerligen problematiskt proteinkristallisations-experiment. Genom uppvärmning av proteinlösningen lyckades vi separera olika former av mikroheterogeniteter från det rättveckade proteinet, som sedan bildade kristaller av god röntgendiffraktiv kvalitet.

BACKGROUND: Serum amyloid P component (SAP) is a glycoprotein that is universally found associated with different types of amyloid deposits. It has been suggested that it stabilizes amyloid fibrils and therefore protects them from proteolytic degradation. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we show that SAP binds not only to mature amyloid fibrils but also to early aggregates of amyloidogenic mutants of the plasma protein transthyretin (TTR). It does not inhibit fibril formation of TTR mutants, which spontaneously form amyloid in vitro at physiological pH. We found that SAP prevents cell death induced by mutant TTR, while several other molecules that are also known to decorate amyloid fibrils do not have such effect. Using a Drosophila model for TTR-associated amyloidosis, we found a new role for SAP as a protective factor in inhibition of TTR-induced toxicity. Overexpression of mutated TTR leads to a neurological phenotype with changes in wing posture. SAP-transgenic flies were crossed with mutated TTR-expressing flies and the results clearly confirmed a protective effect of SAP on TTR-induced phenotype, with an almost complete reduction in abnormal wing posture. Furthermore, we found in vivo that binding of SAP to mutated TTR counteracts the otherwise detrimental effects of aggregation of amyloidogenic TTR on retinal structure. CONCLUSIONS/SIGNIFICANCE: Together, these two approaches firmly establish the protective effect of SAP on TTR-induced cell death and degenerative phenotypes, and suggest a novel role for SAP through which the toxicity of early amyloidogenic aggregates is attenuated. / <p>Epub 2013 Feb 4.</p>

Immunoglobulin-mediated inhibition of amyloid fibril formation in vivo is a promising strategy for the treatment of protein misfolding diseases such as the amyloidoses. Here we focus on transthyretin amyloidoses, a group of protein conformation diseases caused by the misfolding of the serum protein transthyretin into fibrillar structures that deposit in specific organs and tissues—often with serious pathological consequences. Using a structure-guided immunological approach, we report a novel antibody that selectively recognizes monomeric, misfolded conformations of transthyretin in vitro. Raised to an epitope normally buried in the native form of transthyretin, this antibody was found to suppress transthyretin fibrillogenesis at substoichiometric concentrations in vitro. Overall, the selectivity and inhibitory nature of the antibody signals the potential use of conformation specific antibodies in the diagnosis and treatment of transthyretin amyloidoses, conditions which remain difficult to treat and are widely under/misdiagnosed at the current time.

Immunoglobulin-mediated inhibition of amyloid fibril formation in vivo is a promising strategy for the treatment of protein misfolding diseases such as the amyloidoses. Here we focus on transthyretin amyloidoses, a group of protein conformation diseases caused by the misfolding of the serum protein transthyretin into fibrillar structures that deposit in specific organs and tissues—often with serious pathological consequences. Using a structure-guided immunological approach, we report a novel antibody that selectively recognizes monomeric, misfolded conformations of transthyretin in vitro. Raised to an epitope normally buried in the native form of transthyretin, this antibody was found to suppress transthyretin fibrillogenesis at substoichiometric concentrations in vitro. Overall, the selectivity and inhibitory nature of the antibody signals the potential use of conformation specific antibodies in the diagnosis and treatment of transthyretin amyloidoses, conditions which remain difficult to treat and are widely under/misdiagnosed at the current time.

No description available.

Background Hereditary transthyretin amyloid (ATTRm) amyloidosis is a systemic disease mainly affecting the peripheral nervous system and the heart. The disease is inherited in an autosomal dominant manner with a varying penetrance. It is caused by mutations in the transthyretin (TTR) gene. Today more than 100 disease causing mutations are known. The V30M mutation that is endemic in northern Sweden is the best studied and comprises the majority of the reported disease cases in the world. In ATTRm amyloidosis caused by the V30M mutation two distinct sub populations are seen, one with disease onset early in life and a mainly neuropathic disease and the other with late onset disease and both neuropathic disease and a progressive cardiomyopathy. These phenotypical findings have in Swedish patients been tied to differences in amyloid fibril composition. Generally, patients with early onset disease have amyloid fibrils containing only full length transthyretin (type B) whereas patients with late onset disease have amyloid containing both full length and fragmented transthyretin (type A). Until recently, the only available treatment for the disease has been liver transplantation. Patients with type A fibrils, especially males, have significantly worse survival after liver transplant due to progressive amyloid cardiomyopathy. Furthermore, it appears that type A fibrils may be the most common finding in other mutations. This thesis work aims to in depth investigate the impact amyloid fibril composition has on cardiac manifestations of the disease and on the outcome of available and novel modalities for cardiac amyloid imaging. Methods The four studies included in the thesis were done as part of the on going clinical research at the Swedish centre for transthyretin amyloidosis in Umeå.  Patients in whom amyloid fibril composition had been determined were included. Available echocardiographic data were analysed to find predictors for left ventricular hypertrophy and systolic function as measured by strain analysis in a large cohort of 105 patients (paper I). Serial 12-lead electrocardiograms from 98 patients were gathered and retrospectively interpreted and analysed to investigate the impact of amyloid fibril composition and disease progression on frequency and development of ECG abnormalities (paper IV).  DPD scintigraphy, cardiac biomarkers, clinical data and echocardiograms were analysed in a cohort of 53 consecutive patients. to assess the impact of amyloid fibril composition on the outcome of DPD scintigraphy and its relationship with cardiac hypertrophy. (paper II). To evaluate the usefulness of positron emission tomography (PET) using the amyloid specific tracer PIB, 10 patients, five with each fibril type, were selected and examined. The patients selected had a similar age of onset and similar echocardiographic findings (paper III). Results Paper I: Type A fibrils, male gender and age were independent factors associated with increased LV thickness. The distribution of amyloid fibril composition did not differ between the sexes, but in patients with type A fibrils, females had lower median cardiac wall thickness (p&lt;0.01and better left ventricular septal strain (p=0.04).The gender differences were not apparent in patients with type B fibrils. Paper II: Ninety-seven per cent of patients with type A fibrils had pathological cardiac DPD uptake compared to none of the patients with type B fibrils. Among patients with normal septal thickness, none of 15 patients with type B fibrils had positive scintigraphy compared with 2 out of 2 with type A fibrils (P&lt;0.01) Cardiac biomarkers, demographic data and cardiac biomarkers were significantly different, but could not differentiate between type A and type B fibrils in individual patients. Paper III: All patients had pathological cardiac PIB retention. In patients with type B fibrils the retention was significantly higher (p&lt;0.01) than in patients with type A fibrils. Based on the selection criteria, no significant differences were seen in various echocardiographic measurements. Paper IV: All patients had a high prevalence of AV-blocks, LAH and anterior infarction pattern. Patients with type A fibrils had significantly more electrocardiographic abnormalities compared to those with type B fibrils, both at an early stage of diseases and at later follow up. Conclusion Type A fibrils are associated with more pronounced cardiac involvement, which appear to be more severe in males than in females. In study II we showed that DPD scintigraphy appears to be a very good tool for non-invasive determination of amyloid fibril composition. Papers III and IV show that patients with type B amyloid have cardiac involvement even without echocardiographic or DPD-scintigraphic evidence of amyloid cardiomyopathy and that ECG abnormalities are common irrespectively of amyloid fibril composition, and increase with time for both groups.