Haplotype analysis of familial amyloidotic polyneuropathy

Summary Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant genetic disease characterized by systemic accumulation of amyloid fibrils. A major component of FAP anyloid has been identified as variant transthyretin (TTR, also called prealbumin). In particular, a variant with the substitution ³⁰ValMet has been commonly found in FAP of various ethnic groups. To understand the origin and spread of the ValMet mutation, we analyzed DNA polymorphisms associated with the TTR gene in six Japanese FAP families and several Portuguese FAP patients. Three distinct haplotypes associated with the ValMet mutation were identified in Japanese FAP families, one of which was also found in Portuguese patients. On the other hand, it was found that the ValMet mutation can be explained by a C-T transition at the C_pG dinucleotide sequence of a mutation hot spot. Thus, our findings indicate that the ValMet mutation has probably recurred in the human population, to generate FAP families of independent origin.     

Familial amyloidotic polyneuropathy: transthyretin (prealbumin) variants in kindreds of Italian origin

Summary As part of an epidemiological study that aims to characterize chemically the mutation(s) in transthyretin (TTR) related to familial amyloidotic polyneuropathy (FAP) of different ethnic origins, studies were carried out on TTR from two FAP kindreds of Italian origin. Two different criteria were employed in the characterization of TTR from these kindreds: (1) immunoblotting of cyanogen bromide fragments for screening of TTR(Met³⁰) and (2) isoelectric focusing. TTR(Met³⁰) was not detected but other substitutions were demonstrated using isoelectric focusing techniques. One of the variants found is a basic TTR variant. The substitutions occurring in the variant TTRs of these two kindreds are not known and are presently under study.     

Tetramer formation of a variant type human transthyretin (prealbumin) produced by Escherichia coli expression system

A variant of human transthyretin(TTR, prealbumin) with methionine for valine substitution at position 30 is a major component of amyloid fibrils found in patients of familial amyloidotic polyneuropathy(FAP) type I, an autosomal dominant genetic disease. But the molecular nature of the variant TTR has been obscure, because most of plasma TTR from FAP patients is a mixture of variant and wild type TTR and no pure preparation of the variant has been available. For this reason, we constructed a system in which the variant type TTR was efficiently synthesized. In this system, the recombinant variant TTR was first synthesized as a fusion protein with E. coli outer membrane protein A (ompA) signal peptide, processed to eliminate the signal peptide and finally secreted to the culture medium. The final concentration of the recombinant variant TTR in the medium was about 5 mg/l. SDS polyacrylamide gel electrophoresis and gel filtration analysis suggested that the recombinant variant TTR can form tetramer as seen for native one. Purification of the protein was accomplished by only two steps of chromatography.     

Haplotype analysis of common transthyretin mutations

The most frequent transthyretin (TTR) variant associated with hereditary amyloidosis is TTR Met 30, which has its major focus in Portugal, although it also occurs in many other countries. The distribution of the mutation and its occurrence in a CpG dinucleotide lead us to question the origin of the mutation and the possibility of its having originated in Portugal. In order to investigate these questions, we studied the distribution of haplotypes associated with the Met 30 mutation in families from different European countries. All the analysed Portuguese families presented the same haplotype associated with the Met 30 mutation (haplotype I). The same was found for the Swedish and Spanish families studied. However, a distinct haplotype (haplotype III) was found in three families, one Italian, one English and one Turkish. These results suggest that, although the Portuguese Met 30 carriers might have one founder, the mutation probably recurred in populations in Europe in a similar manner to that reported in Japan. In this study, we have also analysed the haplotypes associated with other TTR variants frequent in the Portuguese population.     

Comparative studies of two transthyretin variants with protective effects on familial amyloidotic polyneuropathy: TTR R104H and TTR T119M

Recently, a new nonpathogenic transthyretin (TTR) variant-TTR R104H (TTR H104)-has been described in heterozygotic and compound heterozygotic individuals from a Japanese family with familial amyloidotic polyneuropathy (FAP). The compound heterozygotic individual, a carrier of TTR V30M (TTR M30) and TTR R104H (TTR M30/H104) presented a very mild form of FAP with slow progression of the disease. TTR and retinol binding protein (RBP) levels were found to be increased in serum from TTR H104 carriers. These characteristics are very similar to those found in compound heterozygotic carriers of TTR V30M-T119M (TTR M30/M119). To structurally compare these variants, we performed stability and thyroxine (T(4)) binding studies. TTR M30/H104 showed an increased resistance to dissociation into monomers similar to TTR M30/M119. This suggests that the His104 substitution has the same stabilizing effect on tetrameric TTR as the Met119 substitution. Concerning T(4) binding, TTR H104 presents a T(4) binding affinity lower than that of TTR M119, but still higher than normal TTR. However, TTR from the compound heterozygotic carrier of TTR M30/H104 presented a T(4) binding affinity lower than normal. The results indicate that the His 104 substitution induces structural alterations that increase the stability of the tetramer in compound heterozygotes for TTR M30 despite a lower affinity for T(4) binding. Thus, stability of TTR and binding affinity for T(4) may not be related. More detailed characterization of these variants is needed to clarify the structural alterations responsible for their increased stability.     

Cardiac amyloid in patients with familial amyloid polyneuropathy consists of abundant wild-type transthyretin

Patients with familial amyloid polyneuropathy (FAP) are now cured by liver transplantation, but cardiac amyloidosis would further progress even after liver transplantation in some patients. To clarify the pathological mechanism of the progress of cardiac amyloidosis in FAP, we investigated cardiac tissues obtained from 6 FAP patients with 3 different types of TTR mutations. One of them had undergone liver transplantation and one year later died of cardiac amyloidosis. We determined clinical severity of cardiac involvement of those patients and characterized amyloid fibril proteins depositing in their cardiac muscles by immunohistochemistry, mass spectrometry and isoelectric focusing. All the patients had cardiac dysfunction and increased cardiac weight. Diffuse deposition of TTR-related amyloid was seen in their myocardium on microscopic examination. Amyloid fibrils of the heart were composed of wild-type TTR as well as variant TTR at a ratio of about 1:1 in 5 patients without liver transplantation. In the patient with a transplanted liver, about 80% of the cardiac amyloid consisted of wild-type TTR. Wild-type TTR contributes greatly to the development of amyloid deposition in the heart of FAP patients regardless of the types of TTR mutations.     

Transthyretin stability as a key factor in amyloidogenesis: X-ray analysis at atomic resolution

Transthyretin (TTR) amyloidosis is a conformational disturbance, which, like other amyloidoses, represents a life threat. Here, we report a TTR variant, TTR Thr119Met, that has been shown to have a protective role in the development of clinical symptoms in carriers of TTR Val30Met, one of the most frequent variants among TTR amyloidosis patients. In order to understand this effect, we have determined the structures of the TTR Val30Met/Thr119Met double mutant isolated from the serum of one patient and of both the native and thyroxine complex of TTR Thr119Met. Major conclusions are: (i) new H-bonds within each monomer and monomer-monomer inter-subunit contacts, e.g. Ser117-Ser117 and Met119-Tyr114, increase protein stability, possibly leading to the protective effect of the TTR Val30Met/Thr119Met variant when compared to the single variant TTR Val30Met. (ii) The mutated residue (Met119) extends across the thyroxine binding channel inducing conformational changes that lead to closer contacts between different dimers within the tetramer. The data, at atomic resolution, were essential to detect, for the first time, the subtle changes in the inter-subunit contacts of TTR, and explain the non-amyloidogenic potential of the TTR Thr119Met variant, improving considerably current research on the TTR amyloid fibril formation pathway.     

Study of an anti-human transthyretin immunoadsorbent: Influence of coupling chemistry on binding capacity and ligand leakage

A variant of transthyretin (TTR Val30Met) has been identified as the main protein precursor of the amyloid fibrils deposited in familial amyloidotic polyneuropathy (FAP). Specific removal of TTR in an extracorporeal immunoadsorption procedure is currently under investigation as a possible treatment of FAP. Immunoadsorbents were constructed by immobilizing murine anti-TTR monoclonal antibody 88.6.BA9 onto agarose gel supports via several different coupling chemistries. The influence of coupling conditions such as pH and antibody density, and of perfusion variables, such as antigen concentration and applied flow-rate, on the TTR capture efficiency, was determined. Cyanogen bromide-, carbonyldiimidazole- and aldehyde-activated (ALD) supports conjugated with antibody at optimal pH, provided immunoadsorbents with comparable TTR binding capacities. Regarding stability, leakage was lowest for the ALD based immunoadsorbents, particularly at high pH.     

Characterization of a basic transthyretin variant - TTR Arg 102 - in the German population

A basic transthyretin (TTR) variant, apparently non-pathogenic, has been reported in a German family. Protein analysis of this TTR variant revealed the substitution of arginine for proline at position 102 of the TTR polypeptide chain. This result was confirmed by DNA analysis of PCR amplified DNA.     

Characterization of a basic transthyretin variant--TTR Arg 102--in the German population.

A basic transthyretin (TTR) variant, apparently non-pathogenic, has been reported in a German family. Protein analysis of this TTR variant revealed the substitution of arginine for proline at position 102 of the TTR polypeptide chain. This result was confirmed by DNA analysis of PCR amplified DNA.     

Unusual self-association properties of transthyretin Y114C related to familial amyloidotic polyneuropathy: effects on detection and quantification.

We performed biochemical and immunological examinations of heterozygotic carriers of the transthyretin (TTR) mutant Y114C associated with familial amyloidotic polyneuropathy (FAP). The total serum TTR levels in Y114C TTR carriers were extremely low when analyzed by single radial immunodiffusion (SRID), whereas by indirect enzyme-linked immunosorbent assay (ELISA) procedure, their total TTR concentrations were increased. Recombinant homozygotic Y114C TTR showed no immunoreactivity towards a TTR antibody when analyzed by SRID, whereas by the ELISA procedure presented the same degree of reactivity as that of normal TTR or isolated serum heterozygotic Y114C TTR. These results indicate that immunodifusion based techniques cannot properly determine TTR serum levels in Y114C carriers. Analyses of serum TTR of the Y114C TTR carriers by electrospray ionization mass spectrometry (ESI-MS) with the orifice corn voltage at 60 V revealed a small peak of the free Y114C TTR in addition to large TTR peaks of normal TTR. The levels of the free mutant TTR increased with the orifice corn voltage at 90 V. In contrast, increase in orifice voltage from 60 to 90 V produced a reduction in the level of normal TTR. The results suggest a different pattern of association between monomers in Y114C relative to normal TTR.     

Selective silencing of a mutant transthyretin allele by small interfering RNAs

Familial amyloidotic polyneuropathy (FAP) is a hereditary systemic amyloidosis caused by dominantly acting missense mutations in the gene encoding transthyretin (TTR). The most common mutant TTR is of the Val30Met type, which results from a point mutation. Because the major constituent of amyloid fibrils is mutant TTR, agents that selectively suppress mutant TTR expression could be powerful therapeutic tools. This study has been performed to evaluate the use of small interfering RNAs (siRNAs) for the selective silencing of mutant Val30Met TTR in cell culture systems. We have identified an siRNA that specifically inhibits mutant, but not wild-type, TTR expression even in cells expressing both alleles. Thus, this siRNA-based approach may have potential for the gene therapy of FAP.     

TGGE and HIEF: a comparison of two methods in the detection of carriers of the Z mutation of the alpha-1-antitrypsin gene

Alpha-1-antitrypsin (-1-AT) deficiency can lead to juvenile liver cirrhosis and lung emphysema in adulthood. The deficiency Z allele is caused by a base transition. Temperature gradient gel electrophoresis (TGGE) and hybrid isoelectric focusing (HIEF) were used to detect carriers of the Z mutation of the -1-AT gene. The resulting data were compared. To verify carriers at the sequence level, a manual nonradioactive sequencing strategy was established. Among our sample of carriers of the Z mutation, two were not detected by HIEF that could be identified by TGGE. DNA of all TGGE identified individuals harboring the Z mutation of the -1-AT gene were sequenced nonradioactively. All carriers harbored a G to A transition at position 11.940. This mutation is described to cause the altered protein.     

Proline at position 36: a new transthyretin mutation associated with familial amyloidotic polyneuropathy.

Familial amyloidotic polyneuropathy (FAP) is associated with the deposition of an abnormal transthyretin (TTR) molecule. We have studied DNA from a family of Greek descent with FAP. The proband's TTR gene was asymmetrically amplified by using PCR and then was sequenced directly, to reveal a cytosine-for-guanine substitution in codon 36. This substitution removes a recognition site for endonuclease Fnu4HI. Allele-specific PCR was employed for diagnosis of the mutation. The predicted amino acid change of alanine to proline at position 36 was confirmed by protein sequencing of the proband's plasma TTR.     

Generation of transgenic mice producing a human transthyretin variant: a possible mouse model for familial amyloidotic polyneuropathy

Type I familial amyloidotic polyneuropathy (FAP) results from the systemic deposition of a plasma transthyretin (TTR) variant with a Val----Met change at position 30. In an attempt to establish a model of this disease, we generated transgenic mice producing the variant TTR. A DNA fragment containing the mouse metallothionein-I promoter fused to the structural gene coding for the human TTR variant was microinjected into fertilized mouse eggs. Among 72 mice that developed from these eggs, ten carried the fusion gene and three of these showed significant concentrations of the variant TTR in their serum. These mice may be useful in elucidating the pathogenesis of FAP and in establishing a therapy for this intractable disorder.     

Isolation and characterization of thymosin beta 9 Met from pork spleen

We have identified a new thymosin beta 4-like peptide in pork spleen. The new peptide (12 mg) and thymosin beta 4 (33 mg) were isolated from 230 g of spleen by solid phase extraction, preparative isoelectric focusing, and HPLC. The new peptide was termed thymosin beta 9 Met to indicate its close relationship to thymosin beta 9 from calf. The only difference from thymosin beta 9 is the substitution of leucine by methionine at position 6. This peptide replaces thymosin beta 10 which is the minor thymosin beta 4-like peptide in most mammals, e.g., in man, rat, mouse, cat, and rabbit. The structure was determined by amino acid analysis, tryptic digestion, and carboxypeptidase digestion. Pork spleen contains 192 micrograms of thymosin beta 4 and 117 micrograms of thymosin beta 9 Met per gram of tissue.     

Transthyretin Proteins Regulate Angiogenesis by Conferring Different Molecular Identities to Endothelial Cells

Familial amyloidotic polyneuropathy (FAP) has a high prevalence in Portugal, and the most common form of hereditary amyloidosis is caused by an amyloidogenic variant of transthyretin (TTR) with a substitution of methionine for valine at position 30 (V30M). Until now, the available efficient therapy is liver transplantation, when performed in an early phase of the onset of the disease symptoms. However, transplanted FAP patients have a significantly higher incidence of early hepatic artery thrombosis compared with non-FAP transplanted patients. Because FAP was described as an independent risk factor for early hepatic artery thrombosis, more studies to understand the underlying mechanisms involved in this outcome are of the utmost importance. Knowing that the liver is the major site for TTR production, we investigated the biological effects of TTR proteins in the vasculature and on angiogenesis. In this study, we identified genes differentially expressed in endothelial cells exposed to the WT or V30M tetramer. We found that endothelial cells may acquire different molecular identities when exposed to these proteins, and consequently TTR could regulate angiogenesis. Moreover, we show that V30M decreases endothelial survival by inducing apoptosis, and it inhibits migration. These findings provide new knowledge that may have critical implications in the prevention of early hepatic artery thrombosis in FAP patients after liver transplantation.     

Native state hydrogen exchange study of suppressor and pathogenic variants of transthyretin

Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for numerous familial amyloid diseases, the exact phenotype being dependent on the sequence deposited. Many familial disease variants display decreased stability in vitro, and early onset pathology in vivo. Only subtle structural differences were observed upon crystallographic comparison of the disease-associated variants to the T119M interallelic trans-suppressor. Herein three human TTR single amino acid variant homotetramers including two familial amyloidotic polyneuropathy (FAP) causing variants (V30M and L55P), and a suppressor variant T119M (known to protect V30M carriers from disease by trans-suppression) were investigated in a residue-specific fashion by monitoring (2)H-(1)H exchange employing NMR spectroscopy. The measured protection factors for slowly exchanging amide hydrogen atoms reveal destabilization of the protein core in the FAP variants, the core consisting of strands A, B, E and G and the loop between strands A and B. The same core exhibits much slower exchange in the suppressor variant. Accelerated exchange rates were observed for residues at the subunit interfaces in L55P, but not in the T119M or V30M TTR. The correlation between destabilization of the TTR core strands and the tendency for amyloid formation supports the view that these strands are involved in amyloidogenicity, consistent with previous (2)H-(1)H exchange analysis of the WT-TTR amyloidogenic intermediate.     

Identification of S-sulfonation and S-thiolation of a novel transthyretin Phe33Cys variant from a patient diagnosed with familial transthyretin amyloidosis

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disorder associated with a variant form of the plasma carrier protein transthyretin (TTR). Amyloid fibrils consisting of variant TTR, wild-type TTR, and TTR fragments deposit in tissues and organs. The diagnosis of ATTR relies on the identification of pathologic TTR variants in plasma of symptomatic individuals who have biopsy proven amyloid disease. Previously, we have developed a mass spectrometry-based approach, in combination with direct DNA sequence analysis, to fully identify TTR variants. Our methodology uses immunoprecipitation to isolate TTR from serum, and electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry (MS) peptide mapping to identify TTR variants and posttranslational modifications. Unambiguous identification of the amino acid substitution is performed using tandem MS (MS/MS) analysis and confirmed by direct DNA sequence analysis. The MS and MS/MS analyses also yield information about posttranslational modifications. Using this approach, we have recently identified a novel pathologic TTR variant. This variant has an amino acid substitution (Phe --> Cys) at position 33. In addition, like the Cys10 present in the wild type and in this variant, the Cys33 residue was both S-sulfonated and S-thiolated (conjugated to cysteine, cysteinylglycine, and glutathione). These adducts may play a role in the TTR fibrillogenesis.     

Genetic studies of antithrombin III with IEF and ASO hybridization

Antithrombin III (AT III) was analyzed by two different methods. Isoelectric focusing was used to screen 3 different populations (southwest Germans, Portuguese, Xavante Indians). The same variant was detected both in the German and the Portuguese populations with frequencies of 0.007 and 0.00024, respectively. Further characterization of this variant was performed by allele specific oligonucleotides. By this means, it was possible to identify the variant as AT III Dublin, originally found in 4 Irish families.