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.     

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 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.     

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.     

Biomarkers in the assessment of therapies for familial amyloidotic polyneuropathy

The identification of specific biomarkers provides opportunities to develop early diagnostic parameters, monitor disease progression, and test drug efficiency in clinical trials. We previously demonstrated that in familial amyloidotic polyneuropathy (FAP) related to the abnormal extracellular tissue deposition of mutant transthyretin (TTR), inflammatory and apoptotic pathways are triggered in the presymptomatic stages of the disease, when nonfibrillar TTR deposits are present. In the present work, to better define biomarkers for future assessment of prophylactic and therapeutic drugs in the treatment of FAP, we extended the search for oxidative stress and apoptotic biomarkers to clinical samples and animal models presenting nonfibrillar and fibrillar TTR. We found that lipid peroxidation measured by hydroxynonenal, oxidative DNA damage measured by 8-hydroxy-2'-deoxyguanosine, and cellular redox homeostasis measured by glutaredoxin 1 were consistently increased in biopsy specimens from FAP patients and in tissues from transgenic mouse models presenting nonfibrillar TTR deposition. Death-receptor Fas, caspase-8, and Bax were also found to be increased, indicative of the involvement of death receptors in the observed apoptosis process. Removal of TTR deposition by an immunization protocol resulted in significant decreases of the selected markers we describe, corroborating the relationship between TTR deposition, oxidative stress, and apoptosis. Taken together, our results provide a robust biomarker profile for initial experimental animal studies and clinical trials to assess the application of the selected markers in therapies aimed at removal and/or inhibition of TTR polymerization.     

A novel transthyretin mutation associated with familial amyloidotic polyneuropathy.

We characterized the mutation associated with familial amyloidotic polyneuropathy in a Japanese patient. Sequence analysis of polymerase chain reaction-amplified exons of the transthyretin gene revealed a novel point mutation resulting in a substitution of arginine for glycine at position 47. The mutation was confirmed using allele-specific olgonucleotide hybridization procedures. This most likely represents a de novo mutation since neither parent carries the mutant allele.     

Decreased thermodynamic stability as a crucial factor for familial amyloidotic polyneuropathy

A single mutation in the wild-type transthyretin (WT TTR) such as V30M causes a familial amyloidotic polyneuropathy disease. Comparison of the three-dimensional crystal structures of WT and V30M does not tell much about the reason. High-pressure NMR revealed that at neutral pH both WT and V30M exist as equilibrium between the native tetramer and the dissociated/unfolded monomer. The native tetramer is highly stable in WT (deltaG(0)=104 kJ/mol at 37 degrees C, pH 7.1), but the stability is significantly reduced in V30M (deltadeltaG(0)=-18 kJ/mol), increasing the fraction of the unfolded monomer by a 1000-fold. Significant reduction of thermodynamic stability of WT TTR by mutation could be a crucial factor for familial amyloidotic polyneuropathy.     

Activation of ERK1/2 MAP kinases in familial amyloidotic polyneuropathy

Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disorder characterized by the extracellular deposition of transthyretin (TTR), especially in the PNS. Given the invasiveness of nerve biopsy, salivary glands (SG) from FAP patients were used previously in microarray analysis; mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) was down-regulated in FAP. Results were validated by RT-PCR and immunohistochemistry both in SG and in nerve biopsies of different stages of disease progression. MKP-3 was also down-regulated in FAP SG biopsies. Given the relationship between MKPs and MAPKs, the latter were investigated. Only extracellular signal-regulated kinases 1/2 (ERK1/2) displayed increased activation in FAP SG and nerves. ERK1/2 kinase (MEK1/2) activation was also up-regulated in FAP nerves. In addition, an FAP transgenic mouse model revealed increased ERK1/2 activation in peripheral nerve affected with TTR deposition when compared to control animals. Cultured rat Schwannoma cell line treatment with TTR aggregates stimulated ERK1/2 activation, which was partially mediated by the receptor for advanced glycation end-products (RAGE). Moreover, caspase-3 activation triggered by TTR aggregates was abrogated by U0126, a MEK1/2 inhibitor, indicating that ERK1/2 activation is essential for TTR aggregates-induced cytotoxicity. Taken together, these data suggest that abnormally sustained activation of ERK in FAP may represent an early signaling cascade leading to neurodegeneration.     

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.     

Identification of amyloid prealbumin variant in familial amyloidotic polyneuropathy (Japanese type)

Structural studies on an amyloid fibril protein of 14 K daltons (AFj(INO] isolated from a Japanese patient who suffered from familial amyloidotic polyneuropathy were carried out to unambiguously identify its difference from normal human serum prealbumin. Sequence analyses performed by comparing peptide maps prepared from cyanogen bromide fragments and tryptic peptides of purified RCM-amyloid protein with those from RCM-prealbumin indicate that only a valine residue at position 30 in prealbumin is replaced by a methionine residue. Furthermore, it was also proved that AFj(INO) consists of four components; the prealbumin variant and its three related proteins, which are derived by successively accumulated deletion of the N-terminal three amino acid residues (Gly1, Pro2 and Thr3) from the prealbumin variant.     

Transthyretin Pro 36 associated with familial amyloidotic polyneuropathy in an Ashkenazic Jewish kindred

Summary Mutations in the serum protein transthyretin (TTR) cause amyloidosis involving the peripheral nerves, heart, and other organs. In Ashkenazic Jews, the only TTR variant described to date has been TTR Ile 33. We have studied DNA from another Ashkenazic Jewish kindred with familial amyloidotic polyneuropathy. Singlestrand conformation polymorphism analysis, DNA sequencing, and restriction analysis indicated that this kindred has the TTR Pro 36 variant, previously described only in a Greek kindred.     

Association of C3 and C4A complement types with familial amyloidotic polyneuropathy

A mutant variant of the serum protein transthyretin (TTR-met30) appears to be a necessary but not sufficient condition for the development of familial amyloidotic polyneuropathy (FAP). We have studied a number of serum protein markers (alpha 1-antitrypsin, properdin factor B, C3, C4A, C4B, haptoglobin, transferrin and group-specific component) in FAP patients and healthy controls in an attempt to identify additional pathogenic factors which may influence the risk for developing FAP in male and female patients as well as the age of onset of the disease. Statistically significant associations were found in the complement systems C3 and C4A. The C3F variant was significantly increased in all FAP patients with a relative risk (RR) of 2.0, more pronounced in female patients (RR = 2.6) and patients with an early onset of the disease (RR = 4.5). In the FAP patients only the variants A3 and A4 were found in the C4A system. C4A3 was found in all patients, which was significantly higher than in the controls. The remaining serum protein systems showed no statistically significant associations with FAP. The results suggest that genetic variants of complement factors C3 and C4A may interact with the mutant TTR-met30 by modifying the expression and onset of FAP.     

Production of recombinant human transthyretin with biological activities toward the understanding of the molecular basis of familial amyloidotic polyneuropathy (FAP).

Transthyretin (TTR) is a plasma protein interacting with thyroxine T4 and retinol binding protein (RBP). Several variants of TTR with single amino acid substitutions have been identified as the major components of the amyloid fibrils of familial amyloidotic polyneuropathy (FAP), a fetal, autosomal dominant genetic disease. The elucidation of the molecular nature of the variants distinct from that of the wild-type TTR is crucial for understanding the amyloidogenesis in FAP, but our understanding is very poor mainly because of the unavailability of pure variant TTRs. In the present study, we used an Escherichia coli OmpA secretion vector (Ghrayeb et al., 1984) and achieved an effective production of the variant TTRs related to FAP including Met-30, Ile-33, Ala-60, Tyr-77, Met-111, and Ile-122 types. The variant TTRs produced in this system were efficiently secreted to the culture media. The chemical analysis showed that the secreted TTR (Met-30 type) has the same N-terminus as the native one. IEF analyses also indicated that the secreted product is properly processed as assessed by its pI. Furthermore, the secreted TTR was shown to have biological activities, namely, the thyroxin binding activity and the ability to associate with retinol binding protein, indicating that the secreted TTR polypeptide is properly folded. The present work also demonstrated that the processing/secretion of the recombinant TTR molecules in E. coli was strongly affected by single amino acid substitutions.     

Transthyretin and familial amyloidotic polyneuropathy. Recent progress in understanding the molecular mechanism of neurodegeneration

Familial amyloidotic polyneuropathy (FAP) is an inherited autosomal dominant disease that is commonly caused by accumulation of deposits of transthyretin (TTR) amyloid around peripheral nerves. The only effective treatment for FAP is liver transplantation. However, recent studies on TTR aggregation provide clues to the mechanism of the molecular pathogenesis of FAP and suggest new avenues for therapeutic intervention. It is increasingly recognized that there are common features of a number of protein-misfolding diseases that can lead to neurodegeneration. As for other amyloidogenic proteins, the most toxic forms of aggregated TTR are likely to be the low-molecular-mass diffusible species, and there is increasing evidence that this toxicity is mediated by disturbances in calcium homeostasis. This article reviews what is already known about the mechanism of TTR aggregation in FAP and describes how recent discoveries in other areas of amyloid research, particularly Alzheimer's disease, provide clues to the molecular pathogenesis of FAP.     

Genetic anticipation in Portuguese kindreds with familial amyloidotic polyneuropathy is unlikely to be caused by triplet repeat expansions

Familial amyloidotic polyneuropathy (FAP) is a lethal autosomal dominant type of amyloidosis resulting from the deposition of transthyretin (ATTR) variants in the peripheral and autonomic nervous systems. ATTR V30M-associated FAP exhibits marked genetic anticipation in some families, with clinical symptoms developing at an earlier age in successive generations. The genetic basis of this phenomenon in FAP is unknown. Anticipation has been associated with the dynamic expansion of trinucleotide repeats in several neurodegenerative disorders, such as Huntington disease, myotonic dystrophy, and fragile X syndrome. We have used the repeat expansion detection (RED) assay to screen affected members of Portuguese FAP kindreds for expansion of any of the ten possible trinucleotide repeats. Nine generational pairs with differences in their age of onset greater than 12 years and a control pair with identical ages of onset were tested. No major differences were found in the lengths of the ten trinucleotide repeats analyzed. The distribution of the maximal repeat sizes was consistent with reported studies in unrelated individuals with no known genetic disease. The present data do not support a role for trinucleotide repeat expansions as the molecular mechanism underlying anticipation in Portuguese FAP. Received: 13 December 1998 / Accepted: 23 March 1999