Application to transthyretin analysis

Transthyretin (TTR) is the precursor protein of familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis (SSA). TTR-related FAP is a hereditary amyloidosis induced by mutated TTR. The most common type of FAP is FAP amyloidogenic TTR (ATTR) Val30Met: TTR substitution of methionine for valine at position 30 of the TTR sequence. In this type of FAP, amyloid deposition causes organ failure including peripheral neuropathy, autonomic disorders, gastrointestinal symptoms, and cardiac and renal failure. In Japan, it is well known that Kumamoto and Nagano are the two major foci of FAP, but recently, FAP has been found throughout Japan. In addition, various different types of mutations in the TTR gene and phenotypes of FAP have also been documented. Medical care for FAP is becoming more important. Detection of mutant TTR is indispensable to diagnose the disease in order to treat FAP. We have developed a method for detecting mutant TTR in serum and cerebrospinal fluid by means of mass spectrometry to screen for variant TTR in FAP patients. In this article, we reviewed the general characteristics of TTR and clinical features of FAP, and described the identification of mutant TTR using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS). In addition, we analyzed TTR modifications and protein profiles in serum samples of Japanese and Swedish patients using SELDI-TOF-MS to look for a key protein(s) as a trigger of FAP.     

New transthyretin variants SER 91 and SER 116 associated with familial amyloidotic polyneuropathy. Mutations in brief no. 151. Online

Mutations of the transthyretin (TTR) gene are associated with familial amyloidotic polyneuropathy (FAP). Two new mutations were detected in French patients with TTR amyloidosis. The first patient was a 72 year old man who presented with severe and rapidly evolving sensory motor polyneuropathy of the 4 limbs, a bilateral carpal tunnel syndrome and a restrictive cardiomyopathy. His father died after a clinical history suggestive in retrospect of TTR amyloidosis. The second patient was a 75 year old man who presented with axonal sensory neuropathy of the 4 limbs and a bilateral carpal tunnel syndrome. In both cases immunohistochemistry performed on a nerve biopsy reveled TTR positive amyloid. Direct genomic sequencing of the full TTR gene coding region indicated two heterozygous transversions encoding Ser for Ala 91 substitution in the third exon of the gene in patient 1 and Ser for Tyr 116 substitution in the fourth exon of the gene in patient 2. The mutations were confirmed by digesting PCR products with restriction enzymes and were not found in a control population of 100 unrelated individuals. The Ser 116 substitution was also detected in the daughter and the 70 year old sister of the proband. However the absence of symptomatology suggestive of TTR amyloidosis may be related to the late onset of the disease. The clinical immunohistochemical and molecular studies in both patients are highly suggestive of an association between the Ser 91 and Ser 116 TTR variants with amyloidosis.     

Deposition and passage of transthyretin through the blood-nerve barrier in recipients of familial amyloid polyneuropathy livers

Familial amyloid polyneuropathy (FAP) is characterized by deposition of mutated transthyretin (TTR) in the peripheral nervous system. Prior to amyloid fibrils, nonfibrillar TTR aggregates are deposited inducing oxidative stress with increased nitration (3-NT). As the major source of TTR is the liver, liver transplantation (LT) is used to halt FAP. Given the shortage of liver donors, domino LT (DLT) using FAP livers is performed. The correlation between TTR deposition in the skin and nerve was tested in biopsies from normal individuals, asymptomatic carriers (FAP 0) and FAP patients; in FAP 0, nonfibrillar TTR was observed both in the skin and nerve in the same individuals; in patients, amyloid was detected in both tissues. The occurrence of amyloidosis in recipients of FAP livers was evaluated 1-7 years after DLT: TTR deposition occurred in the skin 3 years after transplantation either as amyloid or aggregates; in one of the recipients, fibrillar TTR was present in the epineurium 6 years after DLT. Deposits were scarce and 3-NT immunostaining was irrelevant. Nerve biopsies from DLT recipients had no FAP-related neuropathy. Our findings suggest that TTR amyloid formation occurs faster than predicted and that TTR of liver origin can cross the blood-nerve barrier. Recipients of FAP livers should be under surveillance for TTR deposition and tissue damage.     

Antibody recognition of amyloidogenic transthyretin variants in serum of patients with familial amyloidotic polyneuropathy

Familial amyloidotic polyneuropathy (FAP) is a late-onset inherited disease characterized by the deposition of amyloid fibrils. FAP is associated with mutations on the transthyretin (TTR) gene. A monoclonal antibody, MAb 39-44, reacting with high molecular weight aggregates of TTR but not with tetrameric TTR has recently been generated and characterized. This antibody recognizes a cryptic epitope that is expressed in isolated recombinant amyloidogenic mutants and in ex vivo amyloid. In the present work we show that this amyloid-specific antibody specifically recognizes in a direct enzyme-linked immunoassay (ELISA) plasma TTR from carriers of various mutations associated with FAP, both in asymptomatic individuals and in patients. In contrast, it does not react with plasma TTR from healthy individuals or that from carriers of nonpathogenic mutations. Using the ELISA developed in this study we identified three different TTR mutations in Portuguese patients with neuropathy of unknown cause, later shown to have amyloid tissue deposition. This antibody recognizes conformations that express cryptic epitopes shared by amyloidogenic TTR variants associated with FAP, not present among nonpathogenic TTR molecules. This antibody will contribute to further identify and characterize intermediates of the amyloidogenic cascade. In addition, it will also be useful for screening amyloidogenic TTR mutations in patients with neuropathy of unknown cause, prior to precise molecular diagnosis using protein and/or DNA analysis.     

Transthyretin Ile73Val is associated with familial amyloidotic polyneuropathy in a Bangladeshi family. Mutations in brief no. 158. Online

Amyloidosis is characterised by the extraceullular deposition of certain different proteins in a distinctively abnormal fibrillar conformation. All types of amyloid fibril share remarkably similar structural and biophysical properties despite substantial chemical heterogeneity among their respective precursor proteins. Hereditary amyloidosis associated with genetically determined protein variants is rare, but is extremely important as a model for studying the pathogenesis of amyloidosis generally. We report a novel mutation of the transthyretin (TTR) coding for TTR Ile73Val which is associated with familial amylodotic polyneuropathy (FAP) in a Bangladeshi family. The mutation was detected by direct sequencing of the PCR-amplified TTR exons. It creates an additional Accl restriction exzyme site in exon 3, allowing confirmation of its presence by RFLP. Amyloid detected in sural nerve and colonic biopsies was shown to be composed of TTR by immunohistochemistry. The predominant clinical features were progressive autonomic and sensori-motor peripheral neuropathy, beginning at age 50 years. The proband's father and two siblings had similar illnesses. These findings indicate Val73 is an amyloidogenic variant of TTR.     

Immunohistochemical characterization of amyloid proteins in sural nerves and clinical associations in amyloid neuropathy.

To test whether immunohistochemical characterization of proteins in amyloid deposits in biopsied sural nerves gives reliable and useful diagnostic information using commercially available reagents, biopsy specimens of sural nerves from 38 patients with amyloid neuropathy were studied. Transthyretin (TTR) was detected in the amyloid deposits of 11 nerves, lambda light chains (LC) in 8 nerves, kappa LC in 7 nerves, and both lambda and kappa LC in 3 nerves. In 9 nerves, the amyloid deposits were too small to allow adequate immunohistochemical characterization of amyloid proteins in serial sections. Evidence that immunohistochemical characterization was correct came from: 1) evaluation of kin, 2) search for monoclonal proteins in the plasma, and 3) sequencing of the gene abnormalities in TTR+ cases. In 9 of 11 TTR+ cases, in which DNA could be obtained, sequencing of the gene showed that each of the 9 cases was heterozygous for a gene mutation; 7 had previously described mutations and 2 undescribed mutations. Therefore, in the nine sporadic cases without plasma monoclonal light chains, the immunohistochemical characterization correctly identified the protein in amyloid as transthyretin. Likewise, there was a high concordance between immunoglobulin light chains in plasma and light chains in amyloid in primary amyloidosis. Evaluation of the type, distribution, and severity of the neurologic symptoms and deficits showed: 1) the sensorimotor and autonomic neuropathy of amyloidosis characteristically affects proximal as well as distal limbs, and 2) the type of amyloidosis probably cannot be determined from the characteristics or severity of the neuropathy alone or from the location or size of amyloid deposits in nerve.     

Biochemical effect of liver transplantation in two Swedish patients with familial amyloidotic polyneuropathy (FAP-met30)

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant inherited disorder characterized by progressive peripheral and autonomic neuropathy, associated with neural and systemic amyloid deposits. The amyloid fibrils contain a variant transthyretin (TTR) molecule (TTR met30), over 90% of which is produced in the liver. After liver transplantation in two patients with severe symptomatic FAP, only normal TTR was detectable in circulation. The two patients are being monitored at regular intervals, and, although in one patient there was no evidence of reduction in the quantity of amyloid present at 6 months, there had been no further progression of the neuropathy.     

The transthyretin gene is expressed in human and rodent dorsal root ganglia

The transthyretin (TTR) gene is mainly expressed in the liver and choroid plexus of the brain. Most cases of familial amyloidotic polyneuropathy (FAP) are caused by TTR gene mutations, and characterized by amyloid deposition in the peripheral nervous system. We hypothesized that the TTR gene may be expressed in the peripheral nervous system. We analyzed TTR gene expression in several parts of the human, mouse and rat peripheral nervous systems using RT-PCR. To determine the sites of TTR synthesis in the dorsal root ganglia (DRG), mouse DRG were examined by in situ hybridization, laser capture microdissection and RT-PCR, and immunohistochemistry. TTR mRNA was detected in the DRG and cauda equina of humans and rodents by RT-PCR. TTR mRNA was not detected in the sural nerve, lumbar plexus or sympathetic ganglia in humans, or in the sciatic nerve in rodents. In mouse DRG, TTR mRNA was localized in the peripheral glial cells. No TTR-like immunoreactivity was observed in these tissues except for the perineurium. The TTR gene is probably expressed in the peripheral glial cells of the DRG. TTR synthesis in the DRG may be important for the involvement of the peripheral nervous system in FAP.     

Gene therapy in familial amyloidotic polyneuropathy by single-stranded oligonucleotides (SSOs)

Transthyretin (TTR) related familial amyloidotic polyneuropathy (FAP) is the common form of hereditary generalized amyloidosis and is characterized by the accumulation of amyloid deposits in the peripheral nerves and other organs. Liver transplantation has been utilized as therapy for FAP, because the variant TTR is predominantly synthesized by the liver, but this therapy is associated with several problems. Since we need to develop a new treatment, we examined the feasibility of gene therapy in FAP to prevent the production of variant TTR in the liver by using single-stranded oligonucleotides (SSOs). To examine in vitro and in vivo conversion of the TTR gene by SSOs embedded in atelocollagen, we used HepG2 cells and liver from transgenic mice whose intrinsic wild-type TTR gene was replaced by the mouse TTR V30M gene. The level of gene conversion was determined by real-time RCR combined with mutant-allele-specific amplification. The level of gene conversion was approximately 11% and 9% of the total TTR gene in HepG2 cells and liver from transgenic mice, respectively. Therefore, gene therapy via this method may be a promising alternative to liver transplantation for treatment of FAP.     

A new mutant transthyretin (Arg 10) associated with familial amyloid polyneuropathy.

We report a new kindred with systemic amyloidosis presenting as peripheral neuropathy in the sixth and seventh decades of life. Polymorphism in exon 2 of the transthyretin (TTR) gene was suggested by single strand conformation polymorphism analysis and determined by direct DNA sequencing. We also developed restriction fragment length polymorphism analysis by polymerase chain reaction using a primer with an induced mutation. The point mutation (cytosine for thymine at position 1038 of the TTR gene) is responsible for substitution of arginine for cysteine at position 10 of the TTR molecule. It is hypothesised that the TTR molecules which have no cysteine have a unique structure in heterozygous TTR polymers and are responsible for amyloid fibril formation.     

Diflunisal stabilizes familial amyloid polyneuropathy-associated transthyretin variant tetramers in serum against dissociation required for amyloidogenesis

Transthyretin (TTR) tetramer dissociation, misfolding and misassembly are required for the process of amyloid fibril formation associated with familial amyloid polyneuropathy (FAP). Preferential stabilization of the native TTR tetramer over the dissociative transition state by small molecule binding raises the kinetic barrier of tetramer dissociation, preventing amyloidogenesis. Two NSAIDs, diflunisal and flufenamic acid, and trivalent chromium have this ability. Here, we investigated the feasibility of using these molecules for the treatment of FAP utilizing serum samples from 37 FAP patients with 10 different mutations. We demonstrated that the TTR heterotetramer structures in FAP patients serum are significantly less stable than that in normal subjects, indicating the instability of the variant TTR structure is a fundamental cause of TTR amyloidosis. We also demonstrated that therapeutic serum concentrations of diflunisal (100-200 microM) stabilized serum variant TTR tetramer better than those of flufenamic acid (35-70 microM). Trivalent chromium at levels obtained by oral supplementation did not stabilize TTR in a statistically significant fashion. Importantly, diflunisal increased serum TTR stability in FAP patients beyond the level of normal controls.     

Analyses of amyloid formation mechanism in familial amyloidotic polyneuropathy and therapeutic trial

Familial amyloidotic polyneuropathy (FAP) is characterized by systemic accumulation of amyloid fibrils in the peripheral nerves and other organs. FAP ATTR Val30Met is the most common of the familial forms of amyloidosis. In the Kumamoto district, 5 different points of mutation in transthyretin (TTR) have been discovered. To make a diagnosis of FAP, histochemical analysis using ATTR Val30Met monoclonal antibody and FAP patients' hair, and mass spectrometry which can analyze TTR post-translational modifications in the blood circulation and cerebrospinal fluid. From our examinations, oxidative stress and beta protein metabolism is deeply connected with amyloid formation mechanism. Liver transplantation for FAP is only the therapy to save the life of FAP patients. By 1999, we had 17 FAP patients who underwent liver transplantation. They are all alive and showed some improvement predominantly in autonomic dysfunction after the surgery. Liver transplantation revealed that FAP does not progress if the TTR gene in the liver is normalized, suggesting the therapeutic possibility of gene therapy to the liver in FAP patients.     

Transthyretin-its function and pathogenesis

Transthyretin (TTR) is a transport protein for retinol-binding protein and thyroxin, and works as a rapid turnover protein. Recently, it has been used as a nutrition assessment protein in the assessment of the acute phase nutritional status in various diseases because it contains four tryptophans in the tetramer of the protein and its plasma half life is 1.9 days. However, the wild-type protein and its mutated form become a precursor protein of amyloid fibrils in senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy(FAP), respectively. Recent biochemical and pathological studies revealed that instability of the terameric form of TTR by mutation and post-translational modifications leads to amyloid formation in the tissues of SSA and FAP. In the process of TTR amyloid formation, misfolding of TTR, the trigger of amyloid formation, is also induced. For these amyloid formation mechanisms, Cr3+ administration, BSB(FSB) therapy, gene therapy, and antibody therapy are now on-going therapeutic projects for FAP and SSA.     

Loss of functional albumin triggers acceleration of transthyretin amyloid fibril formation in familial amyloidotic polyneuropathy

Transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP) is characterized by systemic accumulation of amyloid fibrils caused by a point mutation in the TTR gene. Despite the urgent need for alternative therapeutic strategies, the pathogenesis of FAP still remains elusive. In our study reported here, we focused on albumin, the most abundant protein in plasma, and described the role of albumin in the TTR amyloid-formation process. Patients with FAP evidenced significantly decreased serum albumin levels as the disease progressed. Biacore analysis showed that albumin had a binding affinity for TTR and exhibited higher affinity for TTR amyloid than native TTR. Albumin functioning as an antioxidant effectively suppressed TTR amyloid formation. In patients with FAP, albumin was significantly oxidized as the disease progressed. Moreover, loss of functional albumin accelerated TTR deposition in analbuminemic rats possessing a human variant TTR gene. Taken together, these results indicate that albumin may have an inhibitory role in the TTR amyloid-formation process.     

Neurodegeneration in familial amyloid polyneuropathy: from pathology to molecular signaling

Familial amyloid polyneuropathy (FAP) is an autosomal dominant neurodegenerative disorder related to the systemic deposition of mutated transthyretin (TTR) amyloid fibrils, particularly in peripheral nervous system (PNS). TTR fibrils are diffusely distributed in the PNS of FAP patients, involving nerve trunks, plexuses and ganglia. In peripheral nerves, amyloid deposits are prominent in the endoneurium, near blood vessels, Schwann cells and collagen fibrils. Fiber degeneration is axonal, beginning in the unmyelinated and low diameter myelinated fibers. Several hypotheses have been raised to explain axonal and neuronal loss: (i) compression of the nervous tissue by amyloid; however, a cause-effect relationship between amyloid deposition, structural nerve changes and degeneration was never clearly made; (ii) role of nerve ischemia secondary to lesions caused by perivascular amyloid, which is also doubtful as compromised blood flow was never demonstrated; (iii) lesions in the dorsal root ganglia neurons or Schwann cells. Recently, evidence for the presence of toxic non-fibrillar TTR aggregates early in FAP nerves constituted a first step to unravel molecular signaling related to neurodegeneration in FAP. The toxic nature of TTR non-fibrillar aggregates, and not mature TTR fibrils, was evidenced by their ability to induce the expression of oxidative stress and inflammation-related molecules in neuronal cells, driving them into apoptotic pathways. How these TTR aggregates exert their effects is debatable; interaction with cellular receptors, namely, the receptor for advanced glycation endproducts (RAGE), is a probable candidate mechanism. The pathology and the yet unknown molecular signaling mechanisms responsible for neurodegeneration in FAP are discussed.     

Pituicytoma with Gelsolin Amyloid Deposition

Pituicytoma is a rare low-grade (WHO grade I) sellar region glioma. Among sellar tumors, pituitary adenomas, mainly prolactinomas, may show amyloid deposits. Gelsolin is a ubiquitous calcium-dependent protein that regulates actin filament dynamics. Two known gene point mutations result in gelsolin amyloid deposition, a characteristic feature of a rare type of familial amyloid polyneuropathy (FAP), the Finnish-type FAP, or hereditary gelsolin amyloidosis (HGA). HGA is an autosomal-dominant systemic amyloidosis, characterized by slowly progressive neurological deterioration with corneal lattice dystrophy, cranial neuropathy, and cutis laxa. A unique case of pituicytoma with marked gelsolin amyloid deposition in a 67-year-old Chinese woman is described. MRI revealed a 2.6-cm well-circumscribed, uniformly contrast-enhancing solid sellar mass with suprasellar extension. Histologically, the lesion was characterized by solid sheets and fascicles of spindle cells with slightly fibrillary cytoplasm and oval nuclei with pinpoint nucleoli. Surrounding brain parenchyma showed marked reactive piloid gliosis. Remarkably, conspicuous amyloid deposits were identified as pink homogeneous spherules on light microscopy that showed apple-green birefringence on Congo red with polarization. Mass spectrometric-based proteomic analysis identified the amyloid as gelsolin type. Immunohistochemically, diffuse reactivity to S100 protein and TTF1, focal reactivity for GFAP, and no reactivity to EMA, synaptophysin, and chromogranin were observed. HGA-related mutations were not identified in the tumor. No recurrence was noted 14 months after surgery. To the knowledge of the authors, amyloid deposition in pituicytoma or tumor-associated gelsolin amyloidosis has not been previously described. This novel finding expands the spectrum of sellar tumors that may be associated with amyloid deposition.     

Genetic abnormalities and pathogenesis of familial amyloidotic polyneuropathy

Familial amyloidotic polyneuropathy (FAP) is an autosomal inherited disease, characterized by extracellular amyloid deposits and by peripheral neuropathy. Amyloid fibrils derived from most types of FAP consist of variant transthyretin (TTR) with single amino acid substitutions, and methionine 30 TTR is the most common variant TTR. TTR is mainly produced in the liver and the choroid plexus. Biochemical and molecular biological techniques have been revealing the amyloidogenicity of variant TTR in vitro and in vivo using the transgenic mouse as a model. It will be important for the development of effective therapy to find out the factors, other than variant TTR, which affect amyloid deposition and define the tissue specificity of amyloid.     

Energetic characteristics of the new transthyretin variant A25T may explain its atypical central nervous system pathology

Transthyretin (TTR) is a tetrameric protein that must misfold to form amyloid fibrils. Misfolding includes rate-limiting tetramer dissociation, followed by fast tertiary structural changes that enable aggregation. Amyloidogenesis of wild-type (WT) TTR causes a late-onset cardiac disease called senile systemic amyloidosis. The aggregation of one of > 80 TTR variants leads to familial amyloidosis encompassing a collection of disorders characterized by peripheral neuropathy and/or cardiomyopathy. Prominent central nervous system (CNS) impairment is rare in TTR amyloidosis. Herein, we identify a new A25T TTR variant in a Japanese patient who presented with CNS amyloidosis at age 42 and peripheral neuropathy at age 44. The A25T variant is the most destabilized and fastest dissociating TTR tetramer published to date, yet, surprising, disease onset is in the fifth decade. Quantification of A25T TTR in the serum of this heterozygote reveals low levels relative to WT, suggesting that protein concentration influences disease phenotype. Another recently characterized TTR CNS variant (D18G TTR) exhibits strictly analogous characteristics, suggesting that instability coupled with low serum concentrations is the signature of CNS pathology and protects against early-onset systemic amyloidosis. The low A25T serum concentration may be explained either by impaired secretion from the liver or by increased clearance, both scenarios consistent with A25T's low kinetic and thermodynamic stability. Liver transplantation is the only known treatment for familial amyloid polyneuropathy. This is a form of gene therapy that removes the variant protein from serum preventing systemic amyloidosis. Unfortunately, the choroid plexus would have to be resected to remove A25T from the CSF-the source of the CNS TTR amyloid. Herein we demonstrate that small-molecule tetramer stabilizers represent an attractive therapeutic strategy to inhibit A25T misfolding and CNS amyloidosis. Specifically, 2-[(3,5-dichlorophenyl)amino]benzoic acid is an excellent inhibitor of A25T TTR amyloidosis in vitro.     

Evidence of the presence of amyloid substance in the blood of familial amyloidotic polyneuropathy patients with ATTR Val30Met mutation

Transthyretin (TTR) is a major amyloid fibril protein found in patients with familial amyloidotic polynuropathy (FAP) and senile systemic amyloidosis (SSA). Mainly synthesized in the live, TTR is transferred in the form of tetramer bound with thyroxine, retinol-binding protein (RBP) and lipoprotein in the blood. The aim of this study was to demonstrate the presence of amyloid substances in the blood by investigated the hemocoelom amyloid in different tissue sections from autopsies such as brain, kidney, heart and aorta arch tissue. Congo red staining was employed following by application of polarized light examination, to verify the presence of amyloid deposition in the tissues. Immunohistochemical staining was then performed to identify the specific type of amyloid deposition. Matrix-assisted laser desorption-ionization/time of flight mass spectrometry (MALDI-TOF/MS) was also used to analyze TTR mutation in FAP patients. All subjects were FAP ATTR Val30Met patients. In FAP patients, TTR amyloid deposition was found mainly in the tunica intima of the aortic arch. Interestingly, amyloid substance was found in the blood of FAP patient. Our results suggest that amyloid substance was present in the blood of FAP ATTR Val30Met patients.     

Amyloid and nonfibrillar deposits in mice transgenic for wild-type human transthyretin: a possible model for senile systemic amyloidosis

The human serum protein transthyretin (TTR) is highly fibrillogenic in vitro and is the fibril precursor in both autosomal dominant (familial amyloidotic polyneuropathy [FAP] and familial amyloidotic cardiomyopathy [FAC]) and sporadic (senile systemic amyloidosis [SSA]) forms of human cardiac amyloidosis. We have produced mouse strains transgenic for either wild-type or mutant (TTRLeu55Pro) human TTR genes. Eighty-four percent of C57BI/6xDBA/2 mice older than 18 months, transgenic for the wild-type human TTR gene, develop TTR deposits that occur primarily in heart and kidney. In most of the animals, the deposits are nonfibrillar and non-Congophilic, but 20% of animals older than 18 months that bear the transgene have human TTR cardiac amyloid deposits identical to the lesions seen in SSA. Amino terminal amino acid sequence analysis and mass spectrometry of the major component extracted from amyloid and nonamyloid deposits revealed that both were intact human TTR monomers with no evidence of proteolysis or codeposition of murine TTR. This is the first instance in which the proteins from amyloid and nonfibrillar deposits in the same or syngeneic animals have been shown to be identical by sequence analysis. It is also the first time in any form of amyloidosis that nonfibrillar deposits have been shown to systematically occur temporally before the appearance of fibrils derived from the same precursor in the same tissues. These findings suggest, but do not prove, that the nonamyloid deposits represent a precursor of the fibril. The differences in the ultrastructure and binding properties of the deposits, despite the identical sizes and amino terminal amino acid sequences of the TTR and the dissociation of deposition and fibril formation, provide evidence that in vivo factors, perhaps associated with aging, impact on both systemic precursor deposition and amyloid fibril formation.