Editors: Norio Fujiki, M.D. & Darryl R.J. Macer, Ph.D.
Emeritus Professor, Kumamoto University;
Director, Neurological Center, Mitui Ohmuta Hospital, Fukuoka 836, Japan
The rapid advance of molecular biology has led to the development of new tools for the elucidation and diagnosis of human hereditary amyloidosis. The majority of the hereditary amyloidoses are expressed as peripheral neuropathy and for this reason have long been known as familial amyloidotic polyneuropathy (FAP).Within this group of diseases many have been found to be the result of single-base changes in the gene for plasma transthyretin (TTR), with single-amino acid substitutions in the circulating proteins. These variant proteins are the building blocks of the amyloid fibrils that characterize the disease.
Our understanding of TTR related amyloid and hereditary amyloidosis has progressed over the last few years. This report provides a summary of the recent main advances concerning FAP, especially the TTR amyloid syndromes, diagnosis, ethical problems, therapeutic approaches and genetic counseling.
In the past few years, molecular biology has uncovered 41 new TTR genetic variants, and proved the genetic and clinical heterogeneity of TTR associated amyloidosis. The structure of the TTR molecule by X-ray crystallography, the TTR amyloid fibroprotein in patients with familial amyloidotic polyneuropathy (FAP), the clinical syndromes of homozygous TTR mutations, a system for production of variant TTRs by recombinant DNA technology, and transgenic mice, have been studied. PCR technology has made diagnosis possible at the prenatal, presymptomatic and symptomatic levels through RFLP. There are no specific therapies for FAP. Current treatment for FAP is aimed at symptomatic care. Liver transplantation is a new therapeutic approach which only appeared in 1990. A long term follow up will be necessary in order to evaluate the effectiveness of liver transplantation in FAP type I patients. Presymptomatic and prenatal diagnosis have become more and more precise. This, in turn, raises new ethical problems about the identification of individuals doomed to have an untreatable disease, like FAP.
2. Structure and function of transthyretin (TTR) amyloid
Plasma TTR is the product of a single-copy gene on chromosome 18(q11.2-q12.1). The TTR molecule is a stable and symmetrical tetramer compromised of identical 127 amino acid subunits, with a molecular weight of 54,940. The three-dimensional structure of the molecule has been determined by high-resolution X-ray crystallography, and the subunits have extensive beta-sheet structure. TTR has binding protein sites for both thyroxine and plasma retinol-binding protein, and plays a role in plasma transport of both thyroid hormone and retinol (vitamin A). However, to date, no specific role for TTR in the nervous system has become clear. TTR is expressed mainly in the liver, but it is also synthesized in the choroid plexus which is the origin of TTR in the cerebrospinal fluid. TTR mRNA has been detected in the retina and the pancreas. The TTR gene spans approximately 7kb and has 4 exons. No mutations have been described in exon 1, but several mutations have been found in the exon 2, exon 3, and exon 4 codes for amino acid residues (1,2).
3. TTR amyloidosis syndromes and classification of FAP
All forms of hereditary amyloidosis are autosomal dominant. Classification of familial amyloidotic polyneuropathy (FAP) is made according to ethnic origin, differing amyloid proteins and patterns of neuropathy; FAP type I (TTR) -Portuguese, Japanese, and Swedish FAP type II (TTR) -Indiana/Swiss and Maryland /German kindreds; FAP type III (apo A1) -Iowa kindred; and FAP type IV (gelsolin) -Finnish. Subsequently, a number of family lines with FAP have been described. However, these four groups have been found to be separate entities, each distinguished by the unique amyloid fibril subunit protein, as above (3).
In hereditary amyloidosis associated with TTR mutations, peripheral neuropathy is the most common manifestation. Varying degrees of cardiac, renal, gastrointestinal, ocular, and other organ system involvement may occur with each of the genetic mutations. The disease may vary considerably in different family lines with the same mutations. Until July 1993, molecular biology has explored 41 new TTR genetic variants (4). To date, most of the FAP syndromes are related to single-amino acid substitutions in the TTR gene (Table 1).
1: Polyneuropathy starting in the lower extremities.
Met30, Ala30, Leu30 and Ser6 (double mutation), Leu33, Pro36, Gly42, Ala47, Arg47, Ala49, Pro55, Ala60, Lys61, Tyr77, Gln89, Gly42 and Asn90 (double mutation), Gly97, Cys114
2: Polyneuropathy starting in the upper extremities with carpal tunnel syndrome.
Arg50, His58, Arg58, Leu64, Asn70, Ala71, Ser84, Asn84, His114
3: Cardiac amyloidosis with less prominent peripheral neuropathy
Ser24, Tyr45, Ile50, Ala60, Leu68, Val107, Met111, Ile122
4: Euthyroid hyperthyroxinemia
Ser6, Asn90, Arg102, Met119
Met 30 TTR amyloidosis is the most common type of FAP. Since the first description of FAP in Portuguese families by Andrade in 1952(5), similar syndromes have been recognized in a number of family groups in Japan, Sweden, Greece, Cyprus, Majorca, Brazil, Turkey, and the United States.
In Japan, Araki et al. (5) first reported patients with FAP from the Arao district in Kumamoto prefecture in 1968, and Kito reported another large focus of FAP in Ogawa village, Nagano prefecture in 1973. Since then, several other foci of FAP have been reported in Japan.
FAP type I is almost always associated with a peripheral neuropathy, starting in the lower extremities and progressing gradually upward (cephalad). The upper extremities are involved later in the disease, and cranial nerves may also become involved. The initial symptoms of the disease may be bowel dysfunction and/or impotence. The disease is progressive and death follows after about 10-20 years. Varying degrees of renal or cardiac amyloidosis may be the cause of death, but many patients die from malnutrition related to gastrointestinal amyloidosis. The mean age of onset in Japanese patients is 32 years. Biochemical studies have revealed that the amyloid fibril protein of FAP type I consists of a TTR variant caused by a single amino acid substitution, of methionine for valine at position 30 (8).
b) Other mutations of TTR in Japanese amyloidosis patients
Recently, apart from this variant TTR Met 30, there have been new mutations have been reported at position Leu 30, (sporadic, Hamamatsu), Gly 42 (1 kindred, Toyama) , Arg 47 (sporadic, de novo mutation, Hokkaido), Arg 50 (sporadic, Osaka), Arg 58 (1 kindred, Ishikawa) and Cys 114 (1 kindred, Nagasaki).
Leu 30, Gly 42, Arg 47 and Cyst 114 show FAP type I, and Arg 50 and Arg 58 lead to FAP with polyneuropathy starting in the upper extremities, and carpal tunnel syndrome (FAP type II).
4. Diagnosis of FAP
In diagnosing FAP, tissue biopsies of abdominal fat, gastric submucosa, and sural nerve are important to identify the amyloid deposits.
Most of the mutations in the TTR gene result in changes in the restriction enzyme recognition pattern, and therefore direct genomic DNA testing (standard Southern analysis) is possible (9). Recently, with the advent of PCR (polymerase chain reaction) for amplifying genomic DNA, only a minute amount of sample is required and it is no longer necessary to do Southern analysis. When the specific mutation in the TTR gene is known for the member of a family to be tested, the appropriate exon can be amplified from the genomic DNA, and the restriction pattern can be determined using an enzyme specific for the mutation in question. The PCR test can be accomplished in less than a day, after DNA is isolated, and the PCR test can be applied to tissues that have been aldehyde-fixed for many years. The PCR test is now available for diagnosis at prenatal, presymptomatic and symptomatic levels through RFLP (10) (Fig. 1).
Figure 2: Prenatal diagnosis of a German family of FAP. Lanes 1, NsiI digests of the amplified DNAs; lanes 2, the undigested amplified DNAs; lanes 3, Xbal digests of the amplified DNAs. Lane M, size marker (1kb ladder). Sizes are indicated by base pairs.
Recent studies have indicated that several cases within there families showed onset of the disease in the seventh or eighth decade. It has become obvious that many gene carriers may not develop symptoms at all. An example of this is the variant TTR Met-30 that causes the classic FAP found in Japan, Portugal, and Sweden. Although the PCR test is now available for diagnosis at prenatal, presymptomatic, and symptomatic level, this DNA diagnosis raises new ethical problems about the identification of individuals doomed to have an untreatable disease. The problem of presymptomatic diagnosis has become a reality for FAP. We have followed 10 carriers with variant TTR Met-30 gene over the last 3 years and 3 of these 10 have developed amyloid formation which proved by abdominal fat diagnosis. In Japan there have been no examples of requests for prenatal diagnosis.
6. Therapeutic Approach
There is no specific therapy for any form of systemic amyloidosis including the hereditary forms of the disease.
Current treatment for FAP is aimed at symptomatic care; pacemakers for sick sinus syndrome, L-DOPS (L-threo-3,4 dihydroxyphenyl serine) for orthostatic hypotension and general treatment for bowel and renal insufficiency. While none of these measures is specific, significant prolongation of life can be excepted with proper medical care (Table 2).
The Second International Symposium on FAP and Other Transthyretin Related Disorders was held at Skelleftea, Sweden, June 1-3, 1992. On this occasion the new therapeutic approach of liver transplantation was reported from Sweden, Spain, and the United States.
The major site of TTR production is known to be the hepatocyte and epithelial cells of the choroid plexus. However, amyloid deposition has not been found to have occurred in the liver of FAP autopsy cases except for arteries in Glison's sheaths. This is because the variant TTR produced by liver cells is directly secreted into the blood, transported to other tissues, and deposited in situ as a major component of amyloid fibrils. This also explains angiocentric amyloid deposition in various visceral organs and tissues, particular in the cardiovascular system, renal glomeruli, and interfollicular region of the thyroid.
arhythmia pacemaker implantation
orthostatic hypotension L-DOPS
diarrhoea loperamide hydrochloride, L-DOPS
nausea, vomiting domperidone
abdominal pain scopolamine, pentazocine
renal dysfunction hemodialysis
edema albumin, frosemide
vitreous opacity operation
dry mouth salibate
other treatment plasma exchange
The amyloid fibrils contain a variant TTR Met30, over 90% of which is produced in the liver. This is the background for the use of liver transplantation in FAP. The first liver transplantation was performed in a patient with FAP (Met30) in Sweden in April 1990. Since then, 10 patients with FAP have undergone transplantation in Sweden. The biochemical effect of the transplantation has been good to date in all the patients. Analysis for variant TTR with the FD6 antibody and by the RIA method before and after the operation showed a dramatic reduction in TTR Met30. The greatest clinical improvement is seen in the autonomic nerve functions with especially good effects on the intestinal function, resulting in weight gain and normal stools.
In Spain, the first liver transplantation was performed in patient with FAP (TTR non-Met30) in September 1991. After replacement the plasma levels of variant TTR immediately came down to the values found in negative controls. After 6 months the neuropathy had not progressed, constipation had disappeared and weight increased.
In the United States, a FAP patient of Greek lineage received a liver transplantation in Boston in March 1992. After replacement therapy, the gastrointestinal symptoms decreased along with symptoms of numbness.
As far as liver transplantation is concerned, a long term follow up of a large number of patients will be necessary in order to evaluate the efficacy of liver transplantation in FAP type I.
7. Genetic counseling
With the advent of direct DNA tests for FAP, genetic counseling has become more meaningful. As with all autosomal dominant conditions, each child of a gene carrier has a 50% chance of inheriting the variant gene.
Since FAP is usually a late-onset condition,most affected individuals have not themselves benefited from genetic counseling. A few gene carriers have elected not to have children. These are usually individuals who have observed the devastating effects of the disease on their own parents. Another benefit of DNA testing for FAP is prevention of incorrect diagnosis of individuals with varying manifestations of systemic amyloidosis. On carrying out a questionnaire of FAP patients and their families concerning DNA analysis and prenatal diagnosis of FAP we found that about 50% would not request diagnosis. However, in fact only a few had undergone testing for their disease. There is a strong fear of FAP diagnosis.
Our understanding of FAP has progressed in leaps and bounds over the past few years. The identification of the TTR gene has given us substantial new insights into the pathogenesis in FAP. Future studies on TTR in FAP should explore the function that TTR might play in amyloid formation. The new ethical problems arising from precise presymptomatic diagnosis and a therapeutic approach for FAP clearly point the way to a need for future study. We wait for future research and deliberation.
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