Serpell et. al. (1996), Int. J. Exp. Clin. Invest. 3: 75-85
Familial Amyloidotic Polyneuropathy (FAP) is a category of cognate hereditary diseases. They all involve inhibition of nerve functions due to amyloidosis which in turn leads to several other painful symptoms. They spring from various mutations in the protein transthyretin (TTR) (see table 1 and figure 1 below), and affect about one in every 10,000 to 100,000 people1. These diseases have been classified according to their symptoms since the cause of the disease at the point of categorization was unknown. Hence, there are four different types of FAP2:
Table 1
| Type of FAP | Initial symptoms | Kindred | Main amino acid substitution |
| FAP 1 | Lower limb neuropathy | Portuguese, Japanese and Swedish | Val 30 Met in TTR |
| FAP 2 | Carpal tunnel syndrome, upper limb neuropathy | Indiana (USA) / Swiss
Maryland (USA) / German |
Ile 84 Ser
Leu 58 His in TTR |
| FAP 3 | Neuropathy, lower extremity neuropathy | A family in Iowa, USA | Gly 26 Arg, in Apolipoprotein (AI) |
| FAP 4 | Lattice corneal dystrophy, cranial neuropathy | Finnish | Several substitutions in Gelsolin |
Figure 1
Serpell et. al. (1996), Int. J. Exp. Clin. Invest.
3: 75-85
Familial Amyloidotic Polyneuropathy type 1 (FAP 1) was first diagnosed in 1939 in Portugal. Later on, reports of the disease from Japan and Sweden followed in the middle of the 1960´s. FAP type I is a disease that is prevalent in these countries - Japan, Portugal and Sweden2.
FAP 1 is a very painful disease which, though scarcely known, affects every person in the surroundings of the patient. Even though neither the cause, nor an appropriate cure is discovered as of now, the hope and support of the FAP 1 victims and relatives helps to lead research ahead towards unlocking the mystery behind the disease. Once this key has been found, it is likely to have brought genetic research one step forward in the understanding of genetical mysteries.
The symptoms begin in the lower extremities, where the patient can detect loss of pain and temperature sensation, and eventually problems with walking. As the disease progresses, these symptoms ascend to the upper extremities, and the patient may experience trouble gripping objects and may eventually need the aid of a wheelchair because of paralysis caused by severe sensory motor polyneuropathy. The neuropathy also extends to the autonomous nervous system, particularly in the later stages of the disease, disturbing the gastrointestinal motility causing alternating constipation and diarrhea. Orthostatic hypotension is common, as is cardiac arrhythmia. Vitreous opacities are sometimes present in the eye, impairing vision. In a certain group of FAP 1 patients, vitreous opacities is the initial symptom, and in a few cases, vitreous opacities occurs as a single symptom of FAP3. It also appears as if homozygotes for the TTR met30 mutation in most cases develop vitreous opacities3. Other symptoms of FAP 1 are: sexual impotence, incontinence, severe malabsorption resulting in malnutrition, and polyneuropathy affecting the adrenal glands and kindneys.2, 4, 5
FAP 1 is a steadily progressive disease leading to death as no cure has yet been found. Life expectancy in Sweden is about 9-136 years from the first symptoms of the disease has been detected. Average age of onset of FAP 1 in Sweden is about 57 years, ranging from 26 to 84 years.4
The cause of FAP 1 may aid in explaining why liver transplant could be a useful method of therapy, and will probably open up new dimensions on how this disease could be cured. In this essay the mutation causing FAP 1, the effects of this mutation, the suggestions on how amyloid is formed and the efficiency of liver transplantation as a method of therapy will be further discussed.
Familial Amyloidosis with Polyneuropathy springs from an autosomal dominantly inherited mutation in the protein transthyretin (TTR). The mutation in a gene located in chromosome 18 (q11.2 - q12.1)7, 8 causes the cells to produce defective TTR, in which the original amino acid Valine in position 30 has been replaced by the amino acid Methionin4, 6. Since the mutation is dominantly inherited, one carrier and one non-carrier parent would have offspring where 50% would inherit the mutation, and if one of the parents is homozygous and the other a non-carrier, all children would have mutated TTR, according to the Punnet squares below:
Heterozygous carrier + Non-carrier Homozygous carrier + Non-carrier
In a person who has inherited the mutation from one heterozygous parent, only one of the alleles will produce mutated TTR while the other will produce normal TTR; hence, in a heterozygote, 50% of the plasma TTR will be mutated. On the other hand, a homozygote for the mutation will only produce mutated TTR. However, it appears as if homozygotes are not more severely affected by the disease than heterozygotes for the mutation.3, 9
2.2 TTR structure and function
Transthyretin has binding sites for the hormone thyroxine and for retinol-binding protein (RBP), and helps in transporting thyroxine and retinol (Vitamin A) through the body.7 TTR has a very complex three-dimensional structure which is very important for the functions of the protein; the particular structure of TTR involves a canal in the center where the hormone is situated during transport2. When TTR falls into amyloid, this specific structure is probably ruptured since the amyloid deposits consist of fragments of the original molecule.9 However, it is still unknown if the fragments appear primary or secondary to amyloid formation.9 TTR is mainly synthesized in the liver, but also in the choroid plexus7 and circulates in the blood plasma.5 In blood plasma the TTR is structured as a tetramer, consisting of four monomers bonded together2:
Figure 2
Å. Gustavsson (1994): Transthyretin in senile
systematic
amyloidosis and familial amyloidotic polyneuropathy. Ph D thesis,
Linköping University Medical Dissertations, No. 432
2.3 Chaperonins function in TTR folding
Recent research indicates that there may be molecular "guides"
present
in the human body called chaperonins. These chaperonins bind to a newly
synthesized protein and help the protein through the folding process to
attain the final complicated three-dimensional structure of, for
instance,
the TTR tetramer.1, 10
3.1 Gives TTR affinity for conformational change
It has been found that an amino acid substitution (due to mutation) in the precursor molecule for amyloid (TTR) in FAP "increases the statistical mechanical likelihood of TTR falling into amyloid form by a factor of about 104 to 106" according to Gajdusek8. Hence, it can be deduced that the mutation expressed in TTR gives TTR affinity for conformational change - it makes the precursor molecule TTR more likely to precipitate into amyloid fibrils. How TTR precipitates into amyloid is a subject still under discussion. However, as can be seen above, TTR has a very complicated configuration with extensive -pleated sheet structure. It is easy to imagine how something could go wrong on the way from synthesis to a complete, perfectly folded protein. A mutation in the protein may very well be the catch that makes the protein clump together and form amyloid fibrils instead of readily forming finished TTR. There are several theories on how this occurs. Two of the main theories will be discussed and explained below:
The Kelly theory: According to Kelly, an intermediate in the denaturation (unfolding) / reconstitution (folding) pathway is the amyloidogenic intermediate that leads to amyloid fibril formation. Hence, the mutated TTR causing amyloid deposits can form this amyloidogenic intermediate and self-assemble into amyloid under mildly acidic conditions, whereas "normal" TTR is non-amyloidogenic and conformationally stable. In other words, during the process when the TTR protein is folding to achieve its final structure, or unfolding from final structure to a simple structure, it passes through an intermediate state where it tends to self-assemble, clump together, to form amyloid fibrils.11 Studied in vitro by Kelly show that the TTR tetramer separates into monomers that subsequently unfold partially. These configurationally changed monomers expose certain parts of amino acids, normally buried inside the protein, and these amino acids can now bind to similar acids on another intermediate.1 The mutation somehow destabilizes the TTR which affects its denaturation / reconstruction pathway and is in this way responsible for the amyloid formation. Kelly has also found that the more amyloidogenic intermediate present, the more amyloid will be formed because there is a greater chance of two intermediates "running into each other" and bonding. Thus the more mutated TTR is circulating in a patients body, the greater the chances would be of that TTR forming amyloid.1, 11 See figures 3, 4, 5, and 6:
Figure 3
J. W. Kelly (1996): Alternative conformations of
amyloidogenic
proteins govern their behavior. Current Opinion in Structural
Biology
6: 11-17
Figure 4
Figure 5
J. W. Kelly (1996): Alternative conformations of
amyloidogenic
proteins govern their behavior. Current Opinion in Structural
Biology
6: 11-17
Figure 6
G. Taubes (1996): Misfolding the way to disease. Science
271: 1493-1495
The Gajdusek theory: Gajdusek has found several similarities between FAP, familial Creutzfeldt-Jakob (CJD) disease and the familial form of Gerstmann-Sträussler syndrome (GSS). Mutations in amyloid precursor proteins in all three diseases increases the likelihood of amyloid formation considerably. Furthermore, the mutation in the familial forms of CJD and GSS as well as in FAP appears as an autosomal dominant trait.8 It has also been found that the configurational change of a precursor protein resulting in amyloid may follow the same pattern in FAP, CJD and GSS.8, 9
The amyloid formation process would, according to Gajdusek, occur through nucleation of configurational change of the amyloid precursor protein. Gajdusek explains how protein crystallization can be triggered by pulverized minerals and cause patterns of crystal growth. Any particle of the crystal is in itself able to act as pattern-setting nucleants for more crystal formation. According to Gajdusek, the mutated precursor protein induces a change in structure on the normal precursor protein. Thus the configurationally changed precursor protein originates in a crystallization nucleus and the amyloid starts forming around this nucleus in the same manner as crystals are formed.8 See figures 7 and 8 below:
Figure 7
A computer´s attempt to create an image of the TTR crystal. The
image is based on discoveries made on the amyloid deposits in the
vitreous
body of FAP patients.
E. Lundgren (1994): Portugisiska arvsanlag bakom Skellefteåsjukan? (In Swedish). In "Forskning för livet", Forskningsrådet.
3.2 Chaperonins significant in amyloid formation?
The previously mentioned chaperonins may also have significance in the formation of amyloid. According to Dr. Landry, it is possible that one task of the chaperonins is to help proteins avoid aggregations, such as amyloid, by guiding their folding correctly.10 It has been suggested that amyloid formation could result from chaperonin activity gone awry. 1, 10 Another possibility is that dysfunction of chaperonins is a factor that facilitates the formation of amyloid.
3.3.1 Why does amyloid suddenly appear; random or triggered by factor X?
As previously mentioned there are several theories on how amyloid forms from TTR, which has until that point worked perfectly well in the patient. However, what still remains a mystery, and could be the actual cause of the disease is why amyloid deposits can suddenly be found in the body of an FAP 1 patient.
In Sweden the mean age of onset of FAP 1 (when the symptoms are initially detected) is 57 years, ranging from 26-84 years. Conflicting this fact, in Japan and Portugal where the same mutation as the one in Sweden (TTR met30) causes FAP 1, the mean onset of the symptoms is only 33 years. In Portugal the a range of onset is between 17 and 78 years and in Japan it varies between 18-69 years. Thus, no other country shows such a late onset of the disease as Sweden.2 Moreover, it has been estimated that only about 1 out of 20 carrying the mutation in Sweden actually develops FAP 1.12
If the sudden formation of amyloid in FAP 1 is a random event, the mean age of onset should be approximately the same all over the world, at least for patients carrying the same mutation. However, as shown above, this is not the case. In addition, there is nothing to account for the great divergence of onset ages in Sweden for instance, as some patients detect the first symptoms as late as at the age of 84, while others see the symptoms of amyloid deposits as early as at the age of 26 years. Another dubious factor negating randomness is that only a few people carrying the TTR met30 mutation actually develops FAP 1. What triggers the mutated TTR to suddenly precipitate into amyloid, or what inhibits the expression of the mutation in TTR in certain patients or until a certain age?
A liable answer to why TTR suddenly starts to form amyloid is a unknown factor, "Factor X". This factor would affect TTR in a way that causes it to form amyloid, either by crystallization (Gajdusek) or self-assembling (Kelly). The unknown factor could be compared to the pulverized minerals that would set off the crystallization formation of the precursor proteins mentioned earlier, in a sense it would work as a catalyzer, a starter. On the other hand, it could also be a substance which is present in the body until some point in time. During the time it is present in the body, it inhibits the mutant TTR from precipitating into amyloid, whereas, when this substance would disappear the first symptoms could be perceived.8 Factor X could also be a combination of environmental or genetical factors or events.
Nevertheless, it is important to note that the amyloid formation could be a random event9 and that there is just not enough knowledge, facts and research about FAP 1 to accurately establish this randomness.
4. LIVER TRANSPLANTATION AS A METHOD OF THERAPY
4.1 Reports of liver transplantation
Considering that the liver is the main producer of TTR, a liver transplantation would logically be an efficient method of therapy. The first liver transplantation was performed in Sweden in 1991 on the hypothesis that the mutated TTR is the cause of amyloid formation and that most of the mutated TTR is produced in the liver.13 The aftermath of the initial transplantations showed that the procedure eliminates mutated TTR from the blood plasma, or at least reduces it by more than 90%. It was also found that the progress of the disease was halted and some body functions were regained or improved. Interestingly, it was noted that the total amount of amyloid in the body of two of the patients had decreased after 13 and 24 months respectively.14, 15
In 1993 an international workshop was held on the effects of liver transplantation in FAP, and the survey of the 64 liver transplants that had reportedly been performed in FAP patients indicated that liver transplant was a suitable form of therapy for this disease. Generally, in the patients surviving more than one year, the progression of neurological damage had discontinued, and some neurological functions were restored. It was also mentioned that a slight regression of autonomic symptoms occurred after the transplantation.15 According to Suhr et. al.6, liver transplantation earlier after onset could increase the possibility of reversing the patients symptoms.
4.2 Negative aspects of liver transplantation
Liver transplantation is a major surgical procedure, and the patients have to take immunosuppressives during an extensive period after the operation. The post-operational death rate was 17 percent of the 64 patients. Since some of the FAP patients suffer from severe hypotension they may have problems during or after liver transplantation, the same thing applies to cardiac arrhythmia which is a common symptom of FAP. Some of the transplantation centers in the survey mentioned above routinely placed pacemakers in the patients enclosing the time of operation to prevent heart failure.15
4.3 Factors that may affect outcome of liver transplantation.
Resulting from the fact that liver transplantation is a fairly
exacting
operation, patients in a certain stage of the disease or suffering from
particular symptoms may not benefit from the operation. Therefore,
patients
to receive a liver transplant should be carefully chosen. Certain
factors
have been found to affect the outcome of liver transplantation
decisively,
such as the duration of the disease. A person who has had FAP for a
fairly
long time (nine years or more) seems to do worse than patients who have
only had a short duration of the disease, after receiving a liver
transplant.6
Another unfavorable factor for a liver transplantation is a low Body
Mass
Index (BMI). A BMI indicates the nutritional status of the patient. As
mentioned earlier, hypotension can be a serious preoperative problem.15
Advanced gastrointestinal symptoms may also be a nonbeneficial factor
for
liver tranplantation.15
5.1 Prognostically beneficial factors and Factor X
It has been found that certain symptoms and factors during the progress of the disease are of great significance as to the prognosis of the disease - how ill the patient will become or how long he or she will live. One of these factors is the age at onset; a high age at onset is a prognostically favorable factor. Another factor that affects the survival time is the time of onset of gastrointestinal symptoms and BMI (see paragraph 4), a low BMI is related to a short survival time.4 The fact that an early onset of the disease is a negative prospect favors the "factor X theory" mentioned above; some factor is protecting patients from the disease outbreak, and when this factor is gone, the person gets the disease. A person loosing this protective factor early, will be severely affected by the disease. It could also be viewed as simply an unfortunate event where the person has been exceedingly exposed to "factor X" which sets off the amyloid formation in the patients body and speeds it up to decrease survival time.
It has also been suggested that inheritance of the mutation from the mother gives an earlier age of onset of the disease in the offspring.16 Hence, the earlier the onset of the disease in a patient, the worse the patient will do, and receiving the mutation from the mother gives earlier age of onset. Moreover, it has been found that the mutant gene is inherited three times more often from the mother than from the father.15 This could denote that, though the TTR met30 mutation is autosomally inherited, there is some kind of relationship between the X-chromosome and an aggravation of the disease, as in many other hereditary diseases.
5.2 Content of Amyloid Fibrils
Amyloid consists mainly of mutated TTR, however, it has been found that approximately 20-30% of the amyloid is actually normal TTR.9 Gajdusek stated that: "any particles of the grown crystal are themselves able to act as pattern-setting nucleants for further crystal formation".8 Therefore his theory would be compatible with an amyloid consisting of both normal and mutated TTR . In other words, the crystallization would proceed as a kind of "chain reaction" initiated by mutated TTR, but once it got started could incorporate similar protein, such as normal TTR. This finding is not contradictory to Kelly´s theory either. However Goldberg et. al.1 have stated that the TTR intermediate only associates with itself. Normal TTR and mutated TTR are structurally similar,1, 9 and normal TTR intermediates are also present in the blood, although, in a much lower quantity than the mutated TTR intermediates. Thus, also in Kelly´s theory, the amyloid could consist of both mutant and wild-type TTR.
5.3 Is Liver Transplantation a suitable treatment?
Considering that the amyloid deposits consist of both normal and mutated TTR9, it has been suggested by Thylén that the amyloid fibrils may continue to attract normal TTR after all mutated TTR has been removed from the body through liver transplantation (Thylén, personal communication). Since a minute amount of TTR is also synthesized in the choroid plexus, there will always be some mutated TTR in the blood that could continue to form a small amount of amyloid. Moreover, normal TTR is amyloidotic in senile systematic amyloidosis14 which also suggests that amyloid formation is not necessarily halted after liver transplantation. If this is the case, future follow up on liver transplantations in FAP 1 patients will reveal a slow progress of the disease after the temporary halt the operation seems to cause. Hence, the disease will only have been delayed. This is for the future to unveil. However, it describes the need for long-term follow up, probably for at least 10 years, of the patients with liver transplants (Thylén, personal communication).
Disputing the hypothesis of Thylén are the facts that after liver transplantation, the progress of the disease has been halted,17 and regression of amyloid deposits has even been perceived in some patients.15 The regression of amyloid can be accredited to the immune system - it is feasible that the body has some kind of defense mechanism that can slowly dissolve protein aggregates such as amyloid.
5.4 Other ways of curing FAP 1
If factor X does indeed exist, a plausible method of therapy could be to find this factor and eliminate it. However, searching for factor X could be a tedious job, and there may be much simpler ways to cure the disease, such as trying to find a substance that will inhibit amyloid formation. This could take the form of a drug consisting of a small protein or molecule that binds specifically to the TTR intermediates (Kelly theory), and in that way preventing amyloid formation.1 Such inhibitors have been prepared by Kelly et. al.11 and they appeared to inhibit amyloid formation in vitro. It has recently been found that TTR does not undergo dissociation necessary for fibril formation in the presence of thyroxine.18 Thus, thyroxine seems to work as an inhibitor of amyloid formation. However, since most of the thyroxine is transported through the body by globulin, which has a higher affinity for thyroxine than TTR, thyroxine is not appropriate to use as a drug.18 Therefore, it has been suggested that a thyroxine mimic that will bind specifically to the TTR thyroxine binding site and has higher affinity for TTR than for thyroxine binding globulin, could work as an effective amyloid inihibitor.18
The Gajdusek theory is fairly non-specific regarding how the crystallization process takes place and how it alters the precursor protein in a way that makes it form amyloid. Consequently, it is more difficult to find a simple solution, such as a binding protein, if Gajdusek´s theory is correct for amyloid formation.
Yet another conceivable way of treating FAP 1 would be to artificially create the previously mentioned chaperonins. These chaperonins could be administered as a drug that would help the mutated TTR to fold correctly and thereby avoid amyloid formation.
As observed by Gajdusek, there are numerous similarities between FAP
1, Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler
disease.12
There is also a chance that FAP is also related to a much more common
disease;
Alzheimer´s. Amyloid deposits, amyloid, have been found in the
cerebral
blood vessels of Alzheimer´s patients,19
and it has recently been discovered that this could be the actual cause
of Alzheimer´s rather than a secondary effect of neuronal
breakdown.1
Though amyloid in Alzheimer´s is not precipitated from TTR but
from
a large protein referred to as "amyloid precursor protein" (APP), it
may
form in a similar manner to FAP amyloid.1
The key to unlocking amyloid formation, which controls so many
diseases,
is likely to result in a cure that keeps the amyloid precursor proteins
from forming amyloid. Therefore, the research on this area is of utmost
significance, and cooperation between researchers on Alzheimer´s,
CJD, GSS and FAP could bring us this key.
It is obvious that the formation of amyloid is a very complicated process that may be hard to ever completely elucidate. Even so, since the inhibitors of amyloid formation tested on the TTR intermediate actually worked, the Kelly theory on how amyloid forms seems plausible.
There may be a factor X starting or inhibiting the sudden formation of amyloid. Whether a factor X actually exists or not is still an open issue. However, as pointed out in this essay, it may not be relevant for the research on a cure. Instead, a more feasible cure would be the intermediate inhibitors or the thyroxine mimic mentioned previously. Until such a drug has been produced, liver transplant fills a very important function as a treatment that, at least temporarily, halts the disease.
If the body will be able to break down the amyloid deposited previously to liver tranplantation is yet to be found, although a small regression of amyloid after liver transplantation has been reported.14, 15 It is also possible that the amyloid deposits will continue to grow slowly due to the tendency of amyloid to attract normal TTR, however this is also for the future to unveil. In any case, long-term follow up of the patients that have received a liver transplant will indeed be necessary to establish the success of liver transplantation as a method of therapy. As suggested by Suhr et. al.6, there is a chance that better results will be obtained if the patient receives a liver transplant sooner after onset of the disease. The risks of liver transplantation, despite its apparent improvement of the state of the patient, have to be pointed out.
The similarities of FAP and other amyloid diseases such as
Creutzfeld-Jakob
disease, Gerstmann-Sträussler disease and even Alzheimer´s
have
recently been established, and have lead researchers to believe that a
similar mechanism may cause amyloid formation in all these diseases.
This
indicates a great need for cooperation between researchers of the
different
amyloid diseases. If the need for research on this large area is
officially
acknowledged, researchers may receive increased funds that will
eventually
enable them to find a cure to amyloid diseases, which will in turn mean
the end to a lot of suffering for many people around the world.
I would like to sincerely thank Dr. Christina Thylén for
taking
the time to enhance my understanding of the advanced research done on
this
area. I would also like to thank Dr. Ole Suhr, Dr. Christina
Thylén
and FAMY (the patients´ organization) for helping me find
information.
REFERENCES