Identification of a prealbumin variant in the serum of a Japanese patient with familial amyloidotic polyneuropathy

Serum prealbumin isolated from a Japanese patient with familial amyloidotic polyneuropathy (FAP) has been found to consist of a mixture of normal prealbumin and a prealbumin variant which contains a methionine for valine substitution at position 30. The prealbumin variant in the serum is identical to the prealbumin variant derived from amyloid fibrils of a Japanese FAP patient. FAP likely results from the deposition of abnormal serum prealbumin in various organs as amyloid fibrils.     

Radioimmunoassay for detecting abnormal prealbumin in the serum for diagnosis of familial amyloidotic polyneuropathy (Japanese type)

In the serum of a Japanese patient with familial amyloidotic polyneuropathy (FAP), we demonstrated the presence of a prealbumin variant having a single amino acid substitution of a methionine residue for a valine at position 30. We have developed a highly sensitive and specific method for quantitative analysis of the prealbumin variant in the sera of FAP patients by using radioimmunoassay for a nonapeptide corresponding to subsequence [22-30] of the prealbumin variant. This peptide is produced from the prealbumin variant by cyanogen bromide cleavage followed by tryptic digestion. The serum concentration of the prealbumin variant in five Japanese FAP patients ranges from 4.0 mg/dl to 7.8 mg/dl, which is 100 times or even higher than normal controls. This method should be helpful for an early diagnosis of this hereditary disease.     

Mass spectrometric detection of the plasma prealbumin (transthyretin) variant associated with familial amyloidotic polyneuropathy

A plasma prealbumin variant with a methionine-for-valine substitution at position 30 is closely associated with familial amyloidotic polyneuropathy (FAP) type I. Secondary ion mass spectrometry of the tryptic digest of a carrier's prealbumin could easily detect an abnormal peptide containing the substitution besides the normal peptide. This is a sensitive and reliable method for the diagnosis of FAP.     

Role of variant prealbumin in the pathogenesis of familial amyloidotic polyneuropathy: fate of normal and variant prealbumin in the circulation

According to recent studies on protein chemistry and genetic engineering, replacement of the Val30 residue of prealbumin by methionine is believed to play a critical role in the formation of amyloid deposit and the pathogenesis of familial amyloidotic polyneuropathy (FAP). However, only limited information is available concerning the behavior of prealbumin in the circulation. To obtain the molecular insight into the mechanism of amyloid deposition, it is indispensable to know the fates of normal and variant prealbumin in vivo. Thus, the fates of prealbumin samples from normal and FAP patients were studied in normal rats as well as in animals that were challenged with acute inflammation induced by turpentine. The effect of in vitro photooxidation of prealbumin samples on their behavior was also examined in vivo. Kinetic analysis revealed no appreciable difference between prealbumin samples from normal and FAP patients. These results suggest that factors other than the rate of transfer of the variant form prealbumin from plasma to an extravascular compartment may play a critical role in the pathogenesis of amyloid deposition in FAP patients.     

Revised analysis of amino acid replacement in a prealbumin variant (SKO-III) associated with familial amyloidotic polyneuropathy of Jewish origin

Amyloid fibril protein (SKO-III) of 14K daltons associated with familial amyloidotic polyneuropathy of Jewish type was identified by Pras et al. as a prealbumin variant with a single amino acid substitution of a glycine for a threonine at position 49, mainly based on data obtained by automated sequence analyses. Structural re-investigation of SKO-III was performed by comparing tryptic peptide maps of SKO-III and normal human prealbumin. The present analysis reveals that the reported replacement at position 49 is not present in the molecule of SKO-III. SKO-III should be revised to be a prealbumin variant with one amino acid substitution of an isoleucine for a phenylalanine at position 33.     

The prealbumin nature of the amyloid protein in familial amyloid polyneuropathy (FAP)-Swedish variety

Familial amyloid polyneuropathy (FAP) is a dominant hereditary type of amyloidosis affecting kinships originating in many countries. We have isolated a 15,000 dalton protein from the amyloid laden tissue of a patient of Swedish origin with familial amyloid polyneuropathy. By N-terminal sequence analysis it is homologous to the normal plasma protein, prealbumin. An antiserum prepared to the isolated protein confirms this by reacting identically with the amyloid protein and prealbumin. The normal plasma protein, prealbumin, is linked to a disease syndrome for the first time.     

Identification of a novel transthyretin variant (Val30----Leu) associated with familial amyloidotic polyneuropathy.

A novel variant transthyretin which contains a leucine-for-valine substitution at position 30 was isolated and identified in the serum of a patient with familial amyloidotic polyneuropathy (FAP). The amino acid substitution was proven to result from a guanine-to-cytosine change at the first base of codon 30 located in exon 2 in the mutated transthyretin gene by restriction fragment length analysis on the amplified transthyretin gene using Cfr13 I. The study indicates that the point mutation of the transthyretin gene is a cause of the disorder.     

A variant prealbumin-related low molecular weight amyloid fibril protein in familial amyloid polyneuropathy of Japanese origin

Amyloid fibril protein with a molecular weight of 8K daltons, in addition to one of 16K daltons, has been isolated and characterized from an autopsy sample of a patient with familial amyloid polyneuropathy in a Japanese family from Ogawa Village, Nagano Prefecture. The component was shown to react with an antiserum to normal plasma prealbumin, as did the other. Following the purification by reverse phase liquid chromatography, it was digested with trypsin and the peptides, after the purification by HPLC were sequenced. The data showed that the component was a distinct fragment whose sequence was identical with that of the residues from Gly-6 to Tyr-78 of the prealbumin, except that it had a methionine for a valine at position 25. This corresponded with the position 30 where a valine residues has been reported for the sequence of the normal plasma prealbumin.     

Isolation and characterization of cardiac amyloid in familial amyloid polyneuropathy type IV (Finnish): relation of the amyloid protein to variant gelsolin

Amyloid subunit protein was isolated from familial amyloid polyneuropathy type IV (Finnish type) cardiac tissue and purified to homogeneity. N-terminal amino acid sequence analysis shows that the amyloid protein is a fragment of the inner region of human gelsolin. When compared with the predicted sequence of human plasma gelsolin, the amyloid protein contains an asparagine-for-aspartic acid substitution at position 15 corresponding to residue 187 of the secreted protein. Antibodies raised against the amyloidogenic region of gelsolin specifically stained the amyloid deposited in tissues in familial amyloidosis type IV. The results show that the subunit amyloid protein in familial amyloid polyneuropathy type IV represents a unique type of amyloid derived from a variant (Asn-187) gelsolin molecule by limited proteolysis.     

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.     

Comparison of amyloid deposition in two lines of transgenic mouse that model familial amyloidotic polyneuropathy,type I

We previously produced a transgenic mouse line designated MT-hMet30 by introducing the human mutant transthyretin (TTR) gene carrying the mouse metallothionein promoter, and showed that the presence of human variant TTR is sufficient for amyloid deposition in various tissues of these transgenic mice. However, the expression pattern of human mutant transthyretin gene in the mouse was different from that in man. To analyse pathologic processes, it is essential to establish a transgenic mouse line in which the developmental and tissue- specific expression of the human mutant TTR gene is the same as in man. Thus, we produced two additional transgenic mouse lines carrying the human mutant TTR gene containing either 0.6 kb (0.6- hMet30) or 6.0 kb (6.0-hMet30) of the upstream region. The expression levels of 6.0-hMet30 gene in the liver and serum were the same as in man and about 10 times higher than those of 0.6- hMet30 gene. In both lines amyloid deposition was observed in similar tissues to human patients except for the peripheral and autonomic nervous tissues. The amyloid deposition started earlier and was more extensive in 6.0-hMet30 than 0.6-hMet30 mice, suggesting that the serum levels of human mutant TTR are correlated with the occurrence and degree of amyloid deposition, to some extent. Neither amyloid deposition nor degenerative changes were observed in the peripheral and autonomic nervous systems despite the transgene expression in the choroid plexus of the 6.0-hMet30 mice. In the 6.0-hMet30 mice, amyloid deposition started at 9 months of age, although the serum level of human mutant TTR reached the adult level at 1 month. These results suggest that intrinsic environmental factors other than the mutant gene are involved in the late-onset deposition of amyloid fibrils. Transgenic mice described here should be useful for analysing such factors     

Molecular genetics of familial amyloidotic polyneuropathy

We established a diagnosis of familial amyloidotic polyneuropathy (FAP) based on DNA and demonstrated a direct link between prealbumin gene mutation and FAP. The individuals with FAP, so far examined, were heterozygous for the prealbumin gene, carrying one normal and one mutant gene. To investigate the molecular pathogenesis of FAP, we isolated a normal prealbumin gene (7 kb in length) and also a mutant prealbumin gene associated with FAP. We compared their nucleotide sequences and found that they matched except for a single-base substitution present in the second exon. In an effort to construct mouse model systems for the FAP, we developed strains of transgenic mice carrying human mutant prealbumin genes.     

Genetic basis for familial amyloidotic polyneuropathy

Familial amyloidotic polyneuropathy (FAP) is an inherited systemic amyloidosis, characterized by the extracellular deposition of fibrillar amyloid protein, i.e. a variant type of prealbumin, and by prominent peripheral nerve involvement. We recently established the basis of FAP, using a cloned human prealbumin cDNA, restriction endonuclease(s) and Southern blot procedures. This approach clearly revealed a direct link between mutation in the prealbumin gene and FAP; individuals with FAP are heterozygous for the prealbumin gene, carrying one normal and one mutant gene. Molecular analysis of the prealbumin gene yielded pertinent data on the genetic basis for FAP.     

A mutation in apolipoprotein A-I in the Iowa type of familial amyloidotic polyneuropathy

Immunoblotting of isoelectric focusing gels of plasma and direct genomic DNA sequencing have been used to characterize a mutation in apolipoprotein A-I associated with the familial amyloidotic polyneuropathy originally described by Van Allen in an Iowa kindred. An arginine for glycine substitution in apolipoprotein A-I identified in the proband's amyloid fibrils was determined to be the result of a mutation of guanine to cytosine in the apolipoprotein A-I gene at the position corresponding to the first base of codon 26. Direct sequencing of genomic DNA of three affected individuals who died in the 1960s confirmed the inheritance of the disorder. Immunoblot analysis detected the variant apolipoprotein A-I in the proband's plasma and in several at-risk members of the kindred. In addition, allele-specific amplification by the polymerase chain reaction was used to detect carriers of the variant gene.     

A new mutation causing familial amyloidotic polyneuropathy

The DNA from an individual with familial amyloidotic polyneuropathy was examined. It did not possess any of the mutations which have previously been associated with familial amyloidotic polyneuropathy. However, a novel 7.0 kb Sph I restriction fragment was discovered, and the mutation creating it was localized to exon 3 of the transthyretin gene. This mutation was inherited from a parent, and may result in an amino acid substitution for glu89, his90 or ala91. The patient's transthyretin has a lower pI than normal transthyretin.     

Identification of a new transthyretin variant (Ile49) in familial amyloidotic polyneuropathy using electrospray ionization mass spectrometry and nonisotopic RNase cleavage assay.

Mutation of the transthyretin (TTR) plasma protein and gene in a Japanese patient with amyloid polyneuropathy was investigated by electrospray ionization mass spectrometry (ESI-MS) and nonisotopic RNase cleavage assay (NIRCA), respectively. ESI-MS analysis showed normal TTR peaks and additionally a variant TTR with 12-dalton-higher molecular weight than normal TTR. NIRCA suggested that the mutation existed near either the 5' or 3' end of exon 3. Direct DNA sequencing revealed both a normal ACC (threonine) and a variant ATC (isoleucine) at codon 49, which was located near the 5' end of exon 3. The molecular weight shift of this mutation was 12 D, consistent with the result of ESI-MS.     

The genetic aspect of the familial amyloidotic polyneuropathy

Conclusions and Summary The authors begin with a historical review of the first genetic investigations concerning familial amyloidotic polyneuropathy in the North of Portugal by C. Andrade. Then they review the studies made by other authors about this curious hereditary disease.A dominant mode of transmission was found in all investigations and the area of Povoa de Varzim could be confirmed as the focus where the mutation took place for the first time. Later on the affection spread throughout the northern part of Portugal before finally migrating also to the American continent.The early inquiries of Andrade in 1951–52 were concerned with a still fairly limited number of cases (74 cases). A systematic genetic and statistical study became, however, necessary for the whole material collected over almost 30 years by C. Andrade et al. The individuals examined now amount to 623, 249 of whom are affected (153 males and 96 females, distributed over 148 sibships).After applying various methods of correction (maximum-likelihood-method and Weinberg's method) and while making a distinction between simple selection (k=0) and complete selection (k=1), the authors come to the following evaluations: for K=0, the average of individuals affected (corrected) per sibship is of 21.26±1.88% (method of maximum likelihood scores); for k=1, this value is of 30.84±2.26%. When considering separately within the sibships the male and female patients, the following figures are obtained: for k=0, respectively 13.06±1.33% for males and 8.20±0,98% for females; for k=1, 18,95±1.68% for males and 11.89±1.29% for females.The percentages obtained with Weinberg's method are more or less the same (21.26±1.75% and 31.39±2.50% for k=0 and k=1, respectively). Since the material was collected during a systematic survey of all families affected, the figures computed for k=1 (complete selection) must be considered correct (30.8% by the maximum-likelihood-method and 31.4% by Weinberg's method).In respect of the sex ratio in the families studied, a predilection for the male sex was revealed when all families with at least one member affected are taken into account. However, when considering the families with at least two members affected, the sex ratio corresponds to 1:1.As to morbidity risk, our values are clearly below those of Becker (1964): whereas the figures found by Becker range between 0.32 and 0.50, ours lie between 0.26 and 0.31. The discrepancy must be attributed to the fact that our material which was numerically more important than Becker's provided for a more equitable evaluation of the sex ratio and excluded the possibility of a sex controlled gene.According to our investigations, there is therefore no difference between the sexes as to the degree of penetrance of the affection. The discordance between the sexes is practically restricted to the age of manifestation of the disease, 33 years on an average for males and 44 years for females. The assumption must consequently be made that this one difference is sufficient to guarantee transmission of amyloidotic polyneuropathy through the generations, in spite of counterselection that operates to the detriment of the responsible gene.This work has been supported by the U.S. Public Health Service (Research Grant A. M. 05728), the Calouste Gulbenkian Foundation and the Portuguese Ministry of Health.