Changed affinity of apolipoprotein AII to high density lipoprotein (HDL) in patients with familial amyloidotic polyneuropathy (FAP) type I.

Patients with familial amyloidotic polyneuropathy (FAP) showed extremely low plasma apolipoprotein AII (apoAII) levels and apolipoprotein AII/AI (apoAII/AI) ratio while plasma levels of AI, B, CII, CIII, and E were all within normal ranges. To elucidate the reason for these phenomena, we investigated the percent of these proteins contained in high density lipoprotein (HDL) extracted from plasma of FAP patients by ultracentrifugation. The apoAII/AI ratio in extracted HDL was much lower in asymptomatic carriers of FAP as well as FAP patients than that in control subjects. Since a significant amount of apoAII as well as apoAI was recognized in the fraction of the density > 1.21 g/ml in the samples from asymptomatic carriers of FAP and FAP patients, decreased apoAII/AI ratio may result from the increased dissociation of apoAII from HDL during the process of the ultracentrifugation. Electrophoresis of HDL extracted by agarose gel revealed that HDL from asymptomatic carriers of FAP as well as FAP patients increased negative charge, suggesting that nature of HDL itself may change in these subjects. Although urinary excretion of apoAII was increased in carriers of FAP and FAP patients, there was no correlation between plasma and urinary apoAII levels and also no relationship between urinary total protein and apoAII levels. These results suggest that changed affinity of apoAII to HDL may cause the increased secretion of apoAII to the urine and the decreased plasma apoAII level in carriers of FAP and FAP patients.

Down regulation of a harmful variant protein by replacement of its normal protein.

To compensate for the hypoprotein and hypoalbuminemia of familial amyloidotic polyneuropathy (FAP) patients, 800 ml of fresh frozen plasma (FFP) was intravenously administered and change in total and variant transthyretin (TTR) levels were measured in the plasma. After injection of FFP, total plasma TTR levels were elevated and variant TTR levels decreased from 24 to 48 h, accompanied by an elevation of plasma total protein, albumin levels and TTR levels. To elucidate the mechanism of this phenomenon, a large amount of purified normal TTR from normal human plasma was intravenously injected in mice and FAP patients. By intravenous injection of 3 mg of the purified TTR to C57Black6, the expression of TTR mRNA decreased from 6 to 24 h post injection, and gradually increased up to 48 h post injection. After injecting 400 mg of normal TTR in each of 3 FAP patients, total plasma TTR levels were elevated and variant TTR levels decreased significantly from 24 to 48 h. These results suggested that down regulation of the harmful protein by replacement of its normal form of the protein occurred by this method. This phenomenon should be applied as the basis for one of the useful methods for decreasing the harmful proteins in the circulation.

Selective binding to transthyretin and tetramer stabilization in serum from patients with familial amyloidotic polyneuropathy by an iodinated diflunisal derivative.

In familial amyloidotic polyneuropathy, TTR (transthyretin) variants are deposited as amyloid fibrils. It is thought that this process involves TTR tetramer dissociation, which leads to partially unfolded monomers that aggregate and polymerize into amyloid fibrils. This process can be counteracted by stabilization of the tetramer. Several small compounds, such as diclofenac, diflunisal and flufenamic acid, have been reported to bind to TTR in vitro, in the T4 (thyroxine) binding channel that runs through the TTR tetramer, and consequently are considered to stabilize TTR. However, if these agents bind plasma proteins other than TTR, decreased drug availability will occur, compromising their use as therapeutic agents for TTR amyloidosis. In the present work, we compared the action of these compounds and of new derivatives designed to increase both selectivity of binding to TTR and inhibitory potency in relation to TTR amyloid fibril formation. We found two diflunisal derivatives that, in contrast with diclofenac, flufenamic acid and diflunisal, displaced T4 from TTR in plasma preferentially over binding to albumin and thyroxine binding globulin. The same diflunisal derivatives also had a stabilizing effect on TTR tetramers in plasma, as studied by isoelectric focusing of whole plasma under semi-denaturing conditions. In addition, by transmission electron microscopy, we demonstrated that, in contrast with other proposed TTR stabilizers (namely diclofenac, flufenamic acid and diflunisal), one of the diflunisal derivatives tested efficiently inhibited TTR aggregation. Taken together, our ex vivo and in vitro studies present evidence for the selectivity and efficiency of novel diflunisal derivates as TTR stabilizers and as inhibitors of fibril formation.

A trinucleotide deletion in the transthyretin gene (delta V 122) in a kindred with familial amyloidotic polyneuropathy.

A 63-year-old white man of Ecuadorian origin had a subarachnoid hemorrhage at age 57 followed by numbness and paresthesia in his lower extremities. He subsequently developed sexual impotence, alternating constipation and diarrhea, urinary frequency, and difficulty in walking. Rectal biopsy revealed amyloid deposits immunohistochemically reactive with antitransthyretin antisera. Direct DNA sequencing of the transthyretin gene of the patient showed a trinucleotide deletion in exon 4. This deletion resulted in the loss of one of two valines at position 121 or 122. DNA analysis on 11 family members at risk revealed four mutant gene carriers. Plasma transthyretin levels in the mutant gene carriers measured by nephelometry were very low. Peptide sequence analysis revealed that most of plasma transthyretin was normal with only a small amount of variant protein. This is the first report of a DNA deletion in the transthyretin gene. We speculate that the loss of valine in the carboxyl terminal region of the transthyretin monomer alters stability of the tetrameric protein, which leads to rapid clearance from the plasma and amyloid deposition in the tissue.

Familial amyloidotic polyneuropathy type I with extracellular superoxide dismutase mutation: a case report.

We report an autopsy case of familial amyloidotic polyneuropathy (FAP) Type I with mutations in both transthyretin (TTR) and extracellular superoxide dismutase (EC-SOD). This patient started to develop peripheral neuropathy at age 25, followed by cardiac, renal, and autonomic nervous system failure due to massive amyloid deposition. Thirteen years after the initial symptoms, he died of septic shock. Autopsy revealed suppurative peritonitis, multiple abscesses in the bile ducts and urinary tract, and more marked amyloid deposition than commonly seen in FAP. Amyloid deposition occurred in various organs and tissues, especially prominently around blood vessels and in interstitial tissues, and was demonstrated immunohistochemically to be composed of TTR but not amyloid A (AA) and not amyloid L (AL) proteins. The serum EC-SOD content of the patient was 10 fold higher than those seen often in other FAP patients and in healthy controls. Genetic analysis demonstrated the single amino acid substitutions in Val30Met TIR and Arg213Gly EC-SOD. Since these data suggest the dissociation of EC-SOD from the vascular wall, massive amyloid deposition in the present case may be related to increased oxidative stress in loco.

A simple and reliable method of detecting variant transthyretins by multidimensional liquid chromatography coupled to electrospray ionization mass spectrometry.

Transthyretin (TTR) variants cause amyloidosis. A method, originally reported by us, of detecting the variants by high performance liquid chromatography/electrospray ionization mass spectrometry (ESIMS) using materials precipitated with anti-TTR antiserum, has been successfully applied by several institutions. The method is simple and reliable, but some variants may not precipitate with the antiserum or may precipitate in different yields compared to normal TTR. Moreover, unidentified minor peaks were observed, which may have been derived from cross reactive materials. We have now devised a new procedure to overcome these problems. An anion exchange and reversed phase liquid chromatography system and ESI mass spectrometer were connected in a tandem fashion using a 6 port valve and a protein trap cartridge. The profile of ion peaks by the method was the same as that by MS with immunoprecipitates. The minor peaks were proved not to be derived from cross reactive materials, and the molecular species of these peaks were characterized. This method is faster than immunoprecipitation method and using no antibody is a great benefit. The method can be applied widely to the study various proteins, when antibodies are not available.

Familial amyloidotic polyneuropathy type 1 in Brazil is associated with the transthyretin Val30Met variant.

UNLABELLED: Familial amyloidotic polyneuropathy type 1 (FAP1) is an inherited systemic amyloidosis that is secondary to the deposition of transthyretin (TTR) variants in peripheral nerves and in certain visceral organs. More than 50 distinct mutations have already been described in the TTR gene. Yet, the most common mutation found worldwide is a substitution of valine for methionine in position 30 (Val30Met). Currently, the variants of TTR in Brazilian FAP1 patients remain largely unknown and the aim of this study was to analyze the frequency of the TTR Val30Met mutation in such Brazilian subjects. METHODS: Thirty-two FAP1 patients belonging to 24 different families were studied for the presence of Val30Met variant by PCR-RFLP. RESULTS: All Brazilian FAP1 subjects studied were positive for the TTR Val30Met variant. As expected, all of them were heterozygous for the mutation. CONCLUSION: TTR Val30Met mutation was the sole TTR variant found in Brazilian FAP1 patients in this cohort, and it was present even in those subjects without a clear history of Portuguese ancestry.

Usefulness of MALDI/TOF mass spectrometry of immunoprecipitated serum variant transthyretin in the diagnosis of familial amyloid polyneuropathy.

A matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometry (MS) system was used to detect variant transthyretin (TTR) in immunoprecipitated serum TTR molecules obtained from 6 patients with familial amyloid polyneuropathy (FAP) who were already proven not to have ATTR Val30Met. This simple and quick method showed six different patterns of mass spectra of TTR-related immunoprecipitates from these patients, and in each patient the clearly identified characteristic doublet-shaped ion peaks consisted of normal and variant TTR apart from each other peak with a mass difference between them. DNA sequencing confirmed that the patterns of variant TTR corresponded respectively to ATTR Val30Leu, ATTR Phe33Val, ATTR Asp38Ala, ATTR Ser50Arg, ATTR Ala97Gly and ATTR Ala97Ser. ATTR Asp38Ala and ATTR Ala97Ser are previously unknown variants of TTR leading to the development of FAP. ATTR Phe33Val was found in a Chinese FAP patient and ATTR Ala97Ser in a Taiwanese. Serum analysis using immunoprecipitation and MALDI/TOF MS system can provide useful information when investigating FAP patients with diverse types of variant TTR.

Detection of different forms of variant transthyretin (Met30) in cerebrospinal fluid.

To detect the variant transthyretin (TTR; Met30) in cerebrospinal fluid (CSF) of familial amyloidotic polyneuropathy (FAP) patients, we have applied a new method using a centrifugal concentrator device and electrospray ionization mass spectrometry (ESI-MS). Only 100 microl of CSF and 30 microl of the antibody for TTR was needed for the analysis. After preparation of the samples with anti-TTR antibody, they were passed through a 1000 kDa cut-off centrifugal concentrator which retained the antibody. By analyzing the obtained filtrate with ESI-MS, three predominant forms of normal and their variant forms of TTR were detected in CSF samples. TTR (Met30), with a molecular weight 32.0 Da higher than the normal form of TTR, was found in all FAP patients' materials. Although the ratio of the three major peaks of TTR were different in each individual, they were always found in CSF and sera. This method will contribute to make a diagnosis of neurologic disorders having a mutant protein in CSF as well as serum.

Treatment of a Japanese patient with familial amyloidotic polyneuropathy with orthotopic liver transplantation.

A 28-year-old male patient with familial amyloidotic polyneuropathy (FAP) underwent a liver transplantation from a heart-beating cadaveric donor in Sweden. He had suffered from the disease for 2.5 years. It took 5.5 hours to carry out the operation without blood transfusion. After the liver transplantation, serum amyloidgenic variant transthyretin (TTR) levels became extremely low and diarrhea stopped after the 7th day. On day 13, the patient was discharged from the hospital and one month after the transplantation, his general condition remained quite good. This is the first case of a Japanese patient with congenital metabolic disorders as well as FAP to receive a liver transplantation from a heart-beating cadaveric donor.

Liver transplantation for familial amyloid polyneuropathy.

Familiar Amyloid Polyneuropathy (FAP), an autosomal dominant inherited multisystemic disorder was first observed by Corino de Andrade, a Portuguese neurologist, in 1939. This disease of Portuguese origin was probably spread by fishermen, mainly to Sweden and Japan. It is characterized by a progressive peripheral polyneuropathy and autonomic neuropathy (erectile sexual disfunction, gastrointestinal disfunction, bladder dysfunction and cardio vascular disease) and malnutrition. There are neural and systemic amiloid deposits. Type I FAP, of Portuguese origin, is the most common variety. The amyloid protein is the variant transthyretin (TTR) in which methionine (MET) is a substitute for valine in position 30 (TTR MET 30). It is mainly produced by the liver (90%) and, in small amounts, by the choroidal plexus. Symptoms usually start in the 3rd and 4th decade of life and the patients usually die within 10-15 years. From the therapeutic options--plasmapheresis, immunoadsorption and liver transplantation; the latter seems to be the only one, which stops the production of TTR MET 30 in a permanent way, by means of the liver. The lack of any other effective therapy and the success of the first liver transplantation performed in Sweden arouse great hope. So far, around 300 patients have been transplanted all over the world. A hundred and thirty of them were transplanted in Portugal. A Kaplan Meier survival curve of the Portuguese patients shows a survival rate of 78% at 5 years. However, in spite of the progression of the disease being halted, the irreversibility of some neurological lesions seems to persist. This fact raises the problem of the timing of the transplantation. It seems that the patients should be transplanted as soon as the symptoms start, since mortality and severe morbidity seems to mainly involve those in whom symptomatic disease has lasted longer than six years. As the explanted liver is a morphologic normal liver, a sequential (domino) transplant has been carried out in 16 cases so far done--by one of the authors (ALF) on patients with either hepatocellular carcinoma or liver metastatic disease.

[Liver transplantation for the treatment of type I familial amyloidotic polyneuropathy]. / Trasplante hepático para el tratamiento de la polineuropatía amiloidótica familiar tipo I.

The first liver transplantation carried out in Spain for the treatment of type I familial amyloidotic polyneuropathy (FAP I) is presented. The reason for the operation was based on the liver being responsible for the synthesis of abnormal transtirretin (TTR) constituting the peculiar amyloid of the disease. Following transplantation a rapid and noticeable decrease in abnormal TTR was observed and the evolution of the clinical picture after 18 months of surgery is favorable with progressive improvement of the neurologic symptoms and normal function of the graft. These encouraging results coincide with those of the Swedish group of Umea, the pioneer of this procedure.

Serum potassium concentrations after suxamethonium in patients with familial amyloid polyneuropathy type I.

BACKGROUND: Suxamethonium produces an abnormal increase in serum potassium in some neurological diseases and some authors have suggested that it is safer not to use this drug in patients with familial amyloid polyneuropathy (FAP). However, there are no data previously reported to support this hypothesis. The aim of this study was to evaluate the magnitude of the potassium increase produced by suxamethonium in FAP type I. METHOD: Twenty-one FAP Met 30 patients anaesthetised for liver transplantation were studied. Age was 34.9 +/- 6.9 years (mean +/- SD), time elapsed from first symptom 5.5 +/- 3.2 years and weight was 14 +/- 9% below ideal body weight. Anaesthesia was induced with thiopentone and low-dose fentanyl. Samples for blood gas and 5 min after 1 mg/kg of suxamethonium was given for tracheal intubation. RESULTS: Before induction serum potassium levels were 3.8 +/- 0.4 mmol/L. One minute after suxamethonium, values were 3.8 +/- 0.4 mmol/L and 5 min after 4.3 +/- 0.5 mmol/L. The maximal increase observed was 1.6 mmol/L (from 3.4 mmol/L to 5.0 mmol/L). CONCLUSION: The average increase in plasma potassium concentrations observed in FAP patients after suxamethonium was similar to the increase observed in a normal population by others. Our study can exclude the hypothesis that an anomalous increase in potassium would be a typical and frequent response to suxamethonium in FAP met 30 patients. However, we cannot exclude that a dangerous rise in serum potassium may exist in a certain percentage of FAP patients.

Change in variant transthyretin levels in patients with familial amyloidotic polyneuropathy type I following liver transplantation.

Three patients with familial amyloidotic polyneuropathy (FAP) type I underwent liver transplantation from heart-beating cadaveric donors. Since 2 patients underwent blood transfusion during the operation, variant transthyretin (TTR) levels in the plasma did not decrease time dependently. However, in 1 patient without blood transfusion variant TTR levels decreased in a time dependent manner and plasma half life of variant TTR was calculated to be 2.1 days. Total protein, normal, and variant TTR levels in cerebrospinal fluid (CSF) remained unchanged after liver transplantation.

The high frequency of TTR M30 in familial amyloidotic polyneuropathy is not due to a founder effect.

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant disease due to mutations in the transthyretin (TTR) gene. Valine30-->methionine (TTR M30) is by far the most common mutation in patients with FAP. In a sample of 11 North American unrelated patients, we previously found that 6 had TTR M30. By utilizing double PASA, we could perform haplotype analysis despite the absence of DNA samples on relatives. The results indicate that at least four of the six patients with TTR M30 have different haplotypes, an observation that is surprising for North American patients in which the ostensible symptoms generally begin after the reproductive years. It is suggested that the most likely explanation is rapid selection against TTR M30 mutations by one of four possible mechanisms.