Transthyretin Amyloidosis(Adapted from a Review in Amyloid: Int J Exp Clin Invest 3:44-56, 1996)

The transthyretin amyloidoses are the most prevalent type of hereditary systemic amyloidosis. Since the first published recognition of this form of amyloidosis by Andrade in 1952, and the first description of an American kindred by Falls, et al., in 1955, the number of kindreds with transthyretin amyloidosis has steadily increased (Andrade 1952; Falls, et al., 1955; Benson, 2000). While it was expected that variations in clinical presentation (FAP-I, II, III, IV) were the result of heterogeneity in etiology or pathogenesis of the hereditary amyloidosis, it was not until the discovery by Costa, et al., in 1978 showing transthyretin as a constituent of the fibril deposits, that the biochemical basis of these syndromes could be pursued (Costa, et al., 1978). This resulted in the discovery of the first variant form of transthyretin mutation reported in 1983. In 1989 there were approximately 12 known mutations and in 2002 there are at least 90. Over 80 of these mutations are associated with amyloidosis. In addition, there is evidence that normal transthyretin may for amyloid especially in the heart and be the basis for senile cardiac amyloidosis (Westermark, 1990).

The transthyretin amyloidoses were classically associated with peripheral sensorimotor neuropathy. A number of the more recently discovered transthyretin mutations, however, cause little if any clinical neuropathy. Isolated carpal tunnel syndrome, vitreous opacities, and restrictive cardiomyopathy without any clinically significant neuropathy have all been found in association with specific transthyretin mutations.

The transthyretin amyloidoses by definition are all associated with tissue deposits of fibrils having transthyretin as a major protein constituent. While there are a number of other constituents of the amyloid deposits, including proteoglycan, amyloid P component, and various lipoproteins, it is transthyretin that is the essential ingredient in this type of amyloid.Transthyretin

Transthyretin is a normal plasma protein synthesized predominantly by the liver as a single polypeptide chain of 127 amino acids (14,000 daltons) (Kanda, et al., 1974). It folds into a globular pattern with four -peptide strands in each of two planes (Blake, et al., 1974). The protein is secreted into the plasma as a tetramer with four noncovalently bound monomers (Fig.1). Normal plasma concentration is 20-40 mg/dl with significant depression of this level when the liver is participating in the acute phase in response to injury. It would appear that the signals for down regulating production of transthyretin (cytokines such as IL1 and IL6) are the same as those which cause the positive acute phase response of serum amyloid A and C reactive protein (Costa, et al., 1986). The negative acute phase phenomenon of transthyretin is used by clinicians to monitor nutritional status of their patients. Transthyretin appears not to be essential for life since the murine gene knockout model fails to result in any abnormality in fetal development or life-span of the animals which produce no transthyretin (Episkopou, et al., 1993). These animals do have very low plasma levels of vitamin A, but show neither signs of hyperthyroidism nor vitamin A deficiency. Even so, transthyretin is firmly entrenched in the phylogenetic evolution of vertebrate species being present in both birds and reptiles and its primary structure has been stable throughout evolution (Richardson, 1994).

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The gene for transthyretin is a single copy on the long arm of chromosome 18 (Wallace, et al., 1985). It was first localized using mouse/human hybrid cell lines and subsequently more closely localized using in situ hybridization (18 q11.2-q12.1) (Sparkes, et al., 1987). The gene spans approximately 7 kb, has 4 exons each of approximately 200 bases (Fig. 2) (Tsuzuki, et al., 1985; Sasaki, et al.,1985). The first exon codes a 5 region with an 18 amino acid signal peptide and the first three amino acid residues of the mature protein. Exon 2 codes for residues 4-47, exon 3, 47-92, exon 4, 93-127. The gene has 5 regulatory regions located within 200 bp of the initiation codon and further enhancer sequences at least 2,000 bp 5 which may be important for expression at extra-hepatic sites 12. While plasma transthyretin is predominantly synthesized by the adult liver, it is also synthesized by the choroids plexus of the brain and mRNA is also present in the retinal pigment epithelium, pituitary and pancreas19, 20 . Choroid plexus synthesis would appear to be necessary for the thyroid hormone across the basement membrane into the cerebral spinal space. Reason for synthesis is yet unknown.

The metabolism of transthyretin is largely unknown. The binding of RBP to transthyretin saves this small protein (21,000 daltons) from plasma clearance via filtration in the kidney. However, when the complex gives up retinal, RBP dissociates from transthyretin and goes to meet its fate. Transthyretin evidently can recirculate to bind more RBP-vitamin A. Plasma residence time of transthyretin is approximately 20-24 hours, representing a plasma half-life of no more than 15 hours (Benson, et al., 1996). This is really very rapid turnover for a plasma protein, compared to plasma residence time of apolipoprotein AI which is 5 days, and that of albumin which is approximately 27 days (t ½ =19 days).Transthyretin Amyloidosis

There are now over 90 known mutations in transthyretin (Dwulet and Benson, 1983) (Fig. 3). The majority are associated with systemic amyloidosis, a late onset autosomal dominant disease. Some mutations have been found in single families, others in multiple families, and still others show evidence that the same mutation occurred multiple times. This is particularly true for the methionine 30 transthyretin variant which can be present on at least three different haplotypes (Yoshioka, et al., 1989). It has been hypothesized that the presence of the Met30 mutation on several haplotypes is a result of a “hot spot” at this codon with a CpG dinucleotide. There are 13 potential mutations that could be the result of “hot spots” in the transthyretin coding sequence. To date, five have been identified (Gly6Ser, Val30Met, Arg104Cys, Ala109Thr, Thr119Met, Val122Ile). Of these, Gly6Ser, Thr109 and Met119 are not associated with amyloid fibril formation. While the sole individual described with Arg104Cys transthyretin had peripheral neuropathy, nerve biopsy did not show amyloid, and there was not a family history of amyloid. It is possible that this mutation is not associated with amyloidosis. Most variants of transthyretin are not associated with amyloidosis. Most variants of transthyretin are not associated with any postulated “hot spots” in the coding region. The Ser6 variant is the only known polymorphism, prevalence of approximately 12% in the Caucasian population. All the other mutations are present in less than 2% of the population, except in the restricted areas of Northern Sweden where greater than 2% of inhabitants have the Met30 gene and in African Americans, when considered as a group, where approximately 3% have a Val122Ile mutation. One possible explanation of the large number of pathogenic mutations in transthyretin is that the amyloidosis is a delayed onset disease and, therefore, there is a lesseneddegree of selection against perpetuation of a pathogenic mutation. Whether there are many morevariants of transthyretin that are not associated with transthyretin has not been determined, since no large population study has been undertaken to look for nonpathogenic transthyretin mutations. In reality, there may not be many more nonpathogenic mutations to be discovered. The

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argument can be made that practically any structural change in this heavily -pleated sheet protein may result in amyloid fibril formation. The fact that normal transthyretin makes amyloid fibrils argues in favor of this hypothesis.Clinical Features of Transthyretin Amyloidosis

Peripheral neuropathy is the major clinical manifestation of transthyretin amyloidosis (Andrade, 1952). This was the basis for naming the syndrome familial amyloidotic polyneuropathy (FAP). However, it has become obvious that neuropathy does not occur in all patients with transthyretin amyloidosis, and indeed, many of these syndromes have minor degrees of peripheral neuropathy. The classic presentation as described by Andrade in his report of Portuguese families with amyloidosis, is sensory neuropathy starting in the lower extremities. This usually begins as a typical small fiber neuropathy with inability to discriminate temperature, then touch, and is followed by varying degrees of dyesthesias which at times may be relatively debilitating. The signs of neuropathy progress proximally at varying rates. Typically, five years or more may pass before the sensory neuropathy reaches the level of the knees. By this time the neuropathy usually affects the hands. The symptoms in the upper extremities may be complicated by appearance of the carpal tunnel syndrome, a compression neuropathy of the median nerve. While the neuropathy starts in the lower extremities as pure sensory changes, evidence of motor neuropathy follows within a few years. This is often noted as relative loss of extensor function of the toes and foot. A slapping gait ensues and affected subjects are unstable while walking. The sensorimotor neuropathy often progresses over 10, 15, to 20 years with the affected individual progressing from use of ankle braces to canes, to crutches, and then to a wheelchair. Autonomic neuropathy may occur as the first clinical symptom of this disease. Males are often evaluated for other causes of sexual impotence before the diagnosis of amyloidosis is suspected. Urinary retention from bladder dysfunction may be found. Constipation alternating with diarrhea is a common feature as are nausea and vomiting, signs of delayed gastric emptying. Orthostatic hypotension is common and may be incapacitating. This feature is most problematic when individuals also have cardiac amyloid deposition with decreased cardiac filling. Any sudden movement requiring increased cardiac output causes decreased perfusion of the central nervous system. Late in the course, cachexia is common and this may severely complicate the deficiencies caused by neuropathy and cardiomyopathy.

Variations on the theme include the involvement of the vitreous of the eye in a number of the kindreds. Approximately a third of transthyretin mutations are associated with vitreous deposits of amyloid; however, this finding is not uniform within families. In different kindreds, a single mutation may have different presentations. Most notably, Swedish patients with Met30 transthyretin have a high incidence of vitreous opacities with presentation at a fairly advanced age (58 years); whereas Portuguese patients have a lower incidence of vitreous opacities, but have presentation of neuropathy at an early age (mean 32 or 33 years). Some transthyretin variants present as pure cardiomyopathy (e.g. Met111) (Frederikson, et al., 1962). The Indiana/Swiss kindred (Ser84) has 100% incidence of cardiomyopathy (Benson and Dwulet, 1983) and this also appears to be true for the Appalachian kindred (Ala60) (Benson, et al., 1987).

Significant renal amyloidosis is less common than cardiac amyloidosis in most of the kindreds. Recently attention has been directed toward kindreds having transthyretin amyloidosis

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with extensive leptomeningeal amyloid. This is the hallmark of the Ohio kindred with oculoleptomeningeal amyloidosis (Gly30) (Goren, et al., 1980; Peterson, et al., 1997) and a recently reported kindred from Hungary (Gly18) in which the first clinical manifestation is dementia (Vidal, et al.,1996). There are kindreds (e.g. His114) in which the only clinical manifestation is carpal tunnel syndrome (Murakami, et al., 1994). The His69 mutation has been associated with vitreous opacities alone (Zeldenrust, et al., 1994), but in another family causes oculoleptomeningeal amyloidosis. Features of the disease in particular kindreds make familiarity with the different clinical expressions of the various transthyretin variants essential. Following are brief descriptions of reported amyloid syndrome associated with each of the transthyretin mutations (Table 1). References are given for reported mutations so the reader can obtain more detail if needed.Kindreds and Transthyretin Variants

Arginine 10 (C10R): Only one kindred has been described with this variant. The family lives in Eastern Pennsylvania with ancestors from Hungary (Uemichi, et al., 1992). The clinical picture includes restrictive cardiomyopathy, polyneuropathy starting in the lower extremities, gastrointestinal symptoms occurring usually after age 60. Vitreous opacities and carpal tunnel syndrome have also occurred in affected subjects.

Proline 12 (L12P): One patient from the United Kingdom has been described with this mutation (Brett, et al., 1999). Intracerebral hemorrage was associated with extensive meningeal amyloid deposits. This patient also had hepatic amyloid deposits as measured I 123 SAP scintigraphy. The spleen and the kidney were also positive by this scanning technique (Booth, et al., 1996).

Glutamic Acid 18 (D18E): A patient from South America was reported to have typical amyloidotic polyneuropathy (Booth, et al., 1996). This mutation has been found in the United States associated with cardiac amyloidosis.

Glycine 18 (D18G): Members of a Hungarian kindred were reported to present with dementia, spasticity, ataxia and hearing loss. Amyloid deposits in the meningeal vessels and subpial areas were positive for transthyretin by immunohistochemistry. Lesser amounts of amyloid deposition were found in kidney, skin, ovaries, and peripheral nerves and reported as clinically nonsignificant (Garzuly, et al., 1996; Videl , et al., 1996).

Asparagine 18 (D18N): Clinical history not reported (Connors, et al., 2001). Isoleucine 20 (V20I): This mutation has been reported twice; once in a German family in

which the proband was 64 years old and presented with severe cardiac disease (Jenne, et al., 1996). Vascular amyloid was present in the liver, kidney, and rectal biopsy. The second report was of a 50 year old male who presented to the Mayo Clinic with a two year history gastrointestinal discomfort. Again, cardiac amyloidosis was found and an endomyocardial biopsy was positive when stained for transthyretin. It was not mentioned whether the patient was of German extraction.

Asparagine 23 (S23N): A 44 year old man of Portuguese descent presented with severe cardiomyopathy which was ultimately treated by cardiac transplantation. No symptoms of neuropathy were presented (Connors, et al., 1999).

Serine 24 (P24S): This mutation has been found in only one family originally from Kentucky (Uemichi, et al., 1995). Affected subjects have carpal tunnel syndrome,

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gastrointestinal involvement with diarrhea and cardiomyopathy. The syndrome usually starts after age 50 and most patients live to at least age 65 before dying of cardiac complications.

Serine 25 (A25S): This mutation is associated with rapidly progressive neuropathy and cardiomyopathy starting between age 50 and 60 years. In the one family reported it was the result of a de novo mutation. (Yazaki, et al., 2002).

Methionine 28 (V28M): A Portuguese man (62 years of age) had peripheral and autonomic neuropathy without cardio or renal abnormality (De Carvalho, et al., 2000).

Methionine 30 (V30M): The methionine 30 variant of transthyretin is the most common. It has been found in kindreds from many countries including Portugal, Japan, Sweden, England, Brazil, Greece, and the United States. While the original description was a typical sensorimotor neuropathy starting in the lower extremities, practically every variation on the theme has been seen including vitreous opacities alone, and carpal tunnel syndrome (Andrade, 1952). Extensive leptomeningeal amyloid has been reported in some kindreds (Julião, et al., 1974; Ushiyama, et al., 1991).

Alanine 30 (V30A): Only one family of German descent has been described with this mutation (Jones, et. al., 1992).

Leucine 30 (V30L): The transthyretin variant was reported in a Japanese woman in her sixth decade that presented with diarrhea, weight loss and sensory neuropathy in the lower extremities (Murakami, et al., 1992; Nakazato, et al., 1992).

Glycine 30 (V30G): This mutation was first discovered in an American man of French ancestry with vitreous opacities (Herbert, et al., 1993). It has recently been found in the Ohio kindred of a German origin which was reported as oculoleptomeningeal amyloidosis (Goren, et al., 1980; Petersen, et al., 1997). The syndrome as reported had minor systemic deposition of amyloid and no extensive peripheral neuropathy, but was characterized by vitreous opacities and extensive leptomeningeal amyloid deposition. Dementia and ataxia we reported as part of the syndrome.

Isoleucine 33 (F33I): This mutation has been found in one kindred in Israel (Nakazato, et al., 1984). The family had immigrated to Israel from Poland (Gafni,, et al., 1985). Vitreous opacities, peripheral neuropathy, diarrhea, and impotence were features of this disease.

Leucine 33 (F33L): Only one individual has been reported with this mutation (Harding, et al., 1991). He was of Polish and Lithuanian heritage and developed lower limb neuropathy and cardiomyopathy. There was no family history at the time of this report.

Valine 33 (F33V): This mutation was reported from a single individual in the United Kingdom with typical FAP. There was no definite family history (Booth, et al., 1996).

Threonine 34 (R34T): This mutation was described in one family with three affected brothers living in the Puglia area of Italy. The disease presents as polyneuropathy after age 50 with restrictive cardiomyopathy (Patrosso, et al., 1998).

Asparagine 35 (K35N): The one patient reported with this mutation had typical amyloid polyneuropathy. This patient lived in France, but the country of origin could not be determined. (Reilly, et al.,1995).

Proline 36 (A36P): This mutation has been described in two kindreds; one American family of Greek origin and an Ashkenazic Jewish family (Jacobson, et al., 1992; Jones, et al., 1991). Age of onset may be as early as 28. The syndrome includes peripheral neuropathy, autonomic neuropathy, and vitreous opacities.

Alanine 38 (D38A): A 63 year old Japanese woman presented with peripheral neuropathy, atrioventricular heart block, and congestive heart failure (Shimazu, et al., 1998; Kishikawa, et al., 1999).

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Leucine 41 (W41L): A 45 year old woman presented with vitreous amyloid and no systemic symptoms (Yakazi, et al., 2002a).

Glycine 42 (E42G): This mutation has been reported from two areas; one in Japan (Toyama prefecture) with affected subjects having lower limb neuropathy, autonomic neuropathy, cardiomyopathy, and vitreous opacities (Ueno,, et al., 1990; Uemichi, et al., 1992). In addition, an American Caucasian family which also has the Asn 90 mutation was reported to have this disease with cardiomyopathy.

Aspartic Acid 42 (E42D): Amyloid cardiomyopathy was diagnosed in a 62 year old individual without evidence of neuropathy (Dupuy, et al., 1998).

Serine 44 (F44S): An American man of Irish decent presented with peripheral neuropathy at age 26. He subsequently developed severe headaches, autonomic neuropathy, deafness, and cardiomyopathy (Klein, et al., 1998).

Threonine 45 (A45T): This mutation has been reported in an American Family of Irish and Italian descent with affected individuals dying from restrictive cardiomyopathy (Saraiva, et al., 1992). Onset is at approximately age 50 to 60 years of age.

Aspartic Acid 45 (A45D): This mutation associated with peripheral neuropathy and cardiomyopathy has been reported from the United States (Jacobson, et al., 1998). The proband had Asp 45 and Ser6, while the proband’s father who died of amyloidosis had only Asp45.

Serine 45 (A45S): A 73 year old Swedish man had carpal tunnel syndrome and cardiomyopathy without evidence of axonal neuropathy (Janunger, et al., 2000).

Arginine 47 (G47R): This was reported as a de novo mutation in transthyretin in a 38 year old Japanese man (Murakami, et al., 1992). Both parents were negative for the mutation. Autonomic neuropathy started at age 29 with subsequent development of polyneuropathy.

Alanine 47 (G47A): This was first reported in an Italian family with cardiomyopathy and peripheral neuropathy starting in the fifth decade of life (Ferlini, et al., 1994). The mutation has also been discovered in a French patient.

Valine 47 (G47V): This mutation was found in a Sri Lankan kindred with polyneuropathy (Booth, et al., 1993).

Glutamic Acid 47 (G47E): Members of an Italian family had rapidly progressive neuropathy and cardiomyopathy. Death occurred between 35 and 56 years (Pelo, et al., 2002).

Alanine 49 (T49A): Two distinct kindreds have been described with this mutation; one in France, one in Italy, both having cardiomyopathy (Almeida, et al., 1992, Benson II, et al., 1993). The Italian kindred was reported to have vitreous opacities, but not the French kindred. Both have polyneuropathy and carpal tunnel syndrome starting between ages 35 and 40 and subsequent development of cardiomyopathy.

Isoleucine 49 (T49I): A 63 year old Japanese woman presented with painful parathesias in all four extremities. Affected members of kindred had neuropathy and cardiac amyloidosis (Nakamura, et al., 1999).

Arginine 50 (S50R): Members of a Japanese family presented with perpheral neuropathy and autonomic neuropathy in their early 40s (Ueno, et al., 1990). Cardiac amyloid deposits were proven to contain transthyretin amyloid. A French/Italian woman with this mutation was also reported (Reilly, et al., 1995).

Isoleucine 50 (S50I): This mutation was originally reported from Japan in a 56 year old Japanese woman with a seven year history of peripheral neuropathy and autonomic neuropathy (Nishi, et al., 1992, Saeki, et al., 1992). Cardiomyopathy was noted in another affected individual. In a more recent report, affected subjects were as young as 46 and presented with carpal tunnel syndrome and cardiomyopathy, as well as peripheral neuropathy.

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Glycine 51(E51G): This mutation was associated with cardiomyopathy (Jacobson, et al., 1999).

Proline 52 (S52P): This mutation has been reported from the United Kingdom and is associated with peripheral neuropathy, autonomic neuropathy, cardiomyopathy and renal amyloidosis (Booth, et al., 1993).

Glutamic Acid 53 (G53E): Members of a French family had leptomeningeal and cardiac amyloidosis presenting in the 40’s with headaches and subarachnoid hemorrage (Ellie, et al., 2001).

Gylcine 54 (E54G): This mutation was reported in an English kindred. It gave typical amyloid polyneuropathy and vitreous opacities. This kindred was reported to also have Ser6 (Reilly, et al., 1995).

Lysine 54 (E54K): A 32 year old Japanese man presented with neuropathy and cardiomyopathy. He had vitreous opacities (Togashi, et al., 1999).

Proline 55 (L55P): This TTR variant has been reported twice: once in a kindred from West Virginia which was of Dutch and German descent (Jacobson, et al., 1999). It was also reported in a Chinese family in Taiwan (Yamamoto, et al., 1994). In both families the syndrome is characterized by vitreous opacities, peripheral neuropathy, autonomic neuropathy and cardiomyopathy presenting at a very early age. This syndrome has been noted before age 20 and often results in death from restrictive cardiomyopathy with all members of one kindred dead by age 38.

Arginine 55 (L55R): This mutation was reported from Germany and associated with leptomeningeal amyloidosis (Atland, 1999).

Glutamine 55 (L55Q): This mutation was reported from Germany and associated with leptomeningeal amyloidosis (Yazaki, et al., 2002).

Arginine 56 (H56R): This mutation was associated (Jacobson, et al., 1999).Histidine 58 (L58H): This is the mutation of the Maryland/German kindreds originally

reported by Mahloudji, et al, in 1969 (Mahloudji, et al., 1969, Nichols, et al., 1989). It is associated with slowly progressive peripheral neuropathy starting as early as age 40. Death is usually from cardiomyopathy. There are many families with this mutation in the United States and it is presumed that all are descended from German immigrants from Southern Germany in the 1700s. Only one haplotype has been demonstrated for several families. One homozygous individual has been described with a more rapid course of neuropathy and death six years after the presentation at age 46. Recently we have discovered this mutation in a subject in Germany (Goebel, et al., 1997).

Arginine 58 (L58R): This mutation has been reported in a single Japanese family with one individual presenting with peripheral neuropathy, autonomic neuropathy, and carpal tunnel syndrome at age 39 years (Saeki, et al., 1991). Vitreous opacities were also noted.

Lysine 59 (T59K): The mutation was discovered in an Italian kindred with cardiomyopathy as a major feature of the syndrome (Booth, et al., 1991). Patients also showed the features of peripheral neuropathy and autonomic neuropathy. Disease onset was between 49 and 64.

Alanine 60 (T60A): This mutation was discovered in a large kindred from West Virginia which was of Irish descent (Benson, et al., 1987; Wallace, et al., 1986). The syndrome is characterized by cardiomyopathy, although gastrointestinal symptoms are prominent. Peripheral neuropathy is present but of less critical significance. Carpal tunnel syndrome has been reported, vitreous opacities have not been seen. This mutation has been found in many families in the United States, Ireland, England and also in Australia. So far, all have had the same haplotype

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suggesting that the mutation is of common origin (Waits, et al., 1995). Disease presents after age 50 and patients may live into the 8th and 9th decade without serious compromise.

Lysine 61 (E61K): This mutation has been reported from Japan and is associated with peripheral neuropathy (Shiomi, et al., 1993). A patient developed diarrhea at the age of 62 and then sensorimotor neuropathy.

Leucine 64 (F64L): This mutation was reported in an individual with peripheral neuropathy and cardiomyopathy presenting at age 66 (Ii, et al., 1991). He was an American of Italian descent.

Serine 64 (F64S): Members of this Canadian family of Italian origin had severe migraine, periodic obtundation, psychoses, seizures and endocerebral hemorrage. Pathologic examination demonstrated amyloid deposition in leptomeneges around the brain and spinal cord in the retina and in the peripheral nerves (Uemichi, et al., 1999).

Leucine 68 (I68L): This mutation was described in a 61 year old German with dyesthesias, cardiomyopathy and polyneuropathy, but peripheral nervous system amyloid was not proven (Almeida, et al., 1991).

Histidine 69 (Y69H): Symptoms of carpal tunnel syndrome were present in one individual, but amyloid deposition was not proven. One individual died of brain hemorrage at age 62. A family in Texas with origin in New York was recently found to have this variant transthyretin as well as a large family in Canada of Swedish descent (Zeldenrust, et al., 1994).

Aspargine 70 (K70N): A New Jersey family with German ancestry reported with this mutation associated with carpal tunnel syndrome. Disease onset may be as early as age 30 years. Vitreous opacities were also reported (Izumoto, et al., 1992).

Alanine 71 (V71A): This mutation was found in two loci; one family from Northeastern France with carpal tunnel syndrome as early as age 35, followed by peripheral neuropathy and cardiomyopathy; also in subjects from Majorca, Spain. Vitreous opacities were described for both kindreds (Benson II, et al., 1993).

Valine 73 (I73V): Multiple members of a Bangladeshi family had FAP starting at age 50 (Booth, et al., 1998).

Tyrosine 77 (S77Y): The proband of the German family from Illinois with this mutation had typical peripheral neuropathy and diarrhea and died with renal insufficiency. However, many individuals in the family have had cardiomyopathy. This is also the feature of a large family from Texas, and another from Northern France. Two separate haplotypes have been described, suggesting separate mutational events (Wallace, et al., 1988).

Phenylalanine 77 (S77F): This mutation is associated with peripheral and autonomic neuropathy, and cardiomyopathy. Presentation in a French family was between 48 and 68 years of age (Plante-Bordeneuve, et al., 1998).

Phenylalanine 78 (Y78F): This mutation is associated with late-onset carpal tunnel syndrome and dermal amyloid in a French family.

Threonine 81 (A81T): A Caucasian man presented with cardiomyopathy in his late 60’s. He had no neuropathy but had a history of Carpal Tunnel Syndrome (CTS).

Serine 84 (I84S): This is a mutation of the Indiana/Swiss kindred first described by Falls, et. al., in 1955 and Rukavina, et. al., Rukavina, et. al., in 1956 (Falls, et al., 1955; Rukavina, et al., 1956; Dwulet, et al., 1986). The syndrome is characterized by carpal tunnel syndrome as early as age 30, followed by vitreous opacities in the 40s or 50s, then restrictive cardiomyopathy. Essentially 100% of the patients have vitreous opacities and cardiomyopathy. Death is often in the 50s, but may not occur until the 70s. Males seem to have a more rapidly progressive course than females. Subjects with this mutation have very low levels of plasma retinol binding protein

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and this can be used for genetic screening (Benson and Dwulet, et al., 1983). There also is a kindred in Hungary with this mutation whose affected members show the same clinical symptoms (Zólyomi, et al., 1988).

Asparagine 84 (I84N): This mutation has been reported in only one individual of American and Italian descent who presented with vitreous opacities at age 62 (Skinner, et al., 1992). Carpal tunnel syndrome and cardiomyopathy were also reported. This individual also has a low plasma retinol binding protein level.

Threonine 84 (I84T): This mutation was reported in a 60 year old German woman with cardiomyopathy and peripheral neuropathy (Stangou, et al., 1998).

Glutamine 89 (E89Q): This mutation was reported in a Sicilian family with carpal tunnel syndrome, cardiomyopathy and neuropathy presenting in the fifth decade (Almeida, et al., 1992).

Lysine 89 (E89K): This mutation was discovered in an American family with peripheral neuropathy and cardiomyopathy presenting at 55 years (Nakamura, et al., 2000).

Serine 91 (A91S): This mutation was described in a French family with peripheral neuropathy, carpal tunnel syndrome and cardiomyopathy. The proband presented at 72 years of age (Mirashi, et al., 1998; Plante-Bordeneuve, et al., 1998).

Lysine 92 (E92K): A 71 year old Japanese man presented with amyloid cardiomyopathy (Saito, et al., 2001).

Glycine 97 (A97G): This mutation was reported in a Japanese kindred with cardiomyopathy and peripheral neuropathy (Yasuda, et al., 1994). A patient developed sensorimotor neuropathy at the age of 52, but showed well preserved autonomic function and slow progression of the disease.

Serine 97 (A97S): This mutation was found in two Chinese brothers who moved from Taiwan to the United States. Peripheral and autonomic neuropathy plus cardiomyopathy were features of the disease (Lachmann, et al., 2000).

Valine 107 (I117V): This mutation was found in two American men of German descent (Uemichi, et al., 1994). Both presented with carpal tunnel syndrome at age 57 and subsequently developed peripheral neuropathy.

Methionine 107 (I107M): This mutation is associated with peripheral neuropathy and cardiomyopathy (Atland, et al., 1999).

Serine 109 (A109S): A 69 year old Japanese woman had severe amyloid peripheral neuropathy proven by sural nerve biopsy (Date, et al., 1997).

Methionine 111 (L111M): This is the mutation of the Danish kindred reported by Frederiksen, et al., in 1962 (Fredriksen, et al., 1962; Nordlie, et al., 1988; Ranløv et al., 1992). Affected individuals have cardiomyopathy and several studies looking for peripheral neuropathy have failed to reveal any.

Isoleucine 112 (S112I): This mutation was reported from Italy with subjects having peripheral neuropathy and cardiomyopathy (De Lucia, et al., 1993).

Cysteine 114 (Y114C): This mutation was reported in a kindred from Nagasaki with peripheral neuropathy and autonomic neuropathy presenting at age 30 with subsequent development of vitreous opacities (Ueno, et al., 1990). Cardiomyopathy was also a common feature of this disease.

Histidine 114 (Y114H): This mutation was reported in a kindred living in Niigata prefecture of Japan. Patients developed CTS in their 50’s without other manifestations of amyloidosis (Murakami, et al., 1994).

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Serine 116 (Y116S): A 75 year old French man presented with peripheral neuropathy and carpal tunnel syndrome. A sibling and a daughter had the same mutation (Misrahi, et al., 1998).

Serine 120 (A120S): One Afro-Caribbean patient had peripheral and autonomic neuropathy plus cardiomyopathy (Gillmore, et al., 1999).

Isoleucine 122 (V122I): This mutation occurs in individuals presenting after age 60 and was originally thought to represent senile cardiac amyloidosis (Gorevic, et al., 1999). The mutation has been described predominantly in African Americans and it has been now been reported that approximately 3% of African Americans have this mutation. A number of individuals homozygous for the mutation have been described (Jacobson, et al., 1990; Nichols, et al., 1991). Peripheral neuropathy is a minor clinical manifestation of this disease and death is from congestive heart failure.

Valine 122 (122 V) : This is the first deletion mutation transthyretin found to be associated with amyloidosis (Uemichi, et al., 1997). The proband of this kindred was a 64 year old man with approximately two years of impotence before developing peripheral neuropathy. There is also evidence of cardiomyopathy. The proband immigrated to the United States from Ecuador.

Alanine 122 (V122A): A 47 year old American of Welsh and English descent presented with cardiomyopathy (Theberge, et al., 1999).

It should be noted that while some of the hereditary amyloid syndromes have been extensively described (e.g. Met30, Ala60, Ser84), others have been described for only one affected subject or only a few members of one family. The assumption that many of the transthyretin variants cause the disease is by analogy alone. There is still much to be learned about how each of these mutations is related to the clinical disease.

Nonamyloid associated variants of transthyretin include Ser6, His74, Asn90, Ser101, Arg102, His 104, Thr109, Val109, and Met119 (Almeida, et al., 1991; Izumoto, et al., 1993, Uemichi, et al., 1994). A mutation of arginine to cysteine at 104 was found in an individual with symptoms of polyneuropathy, but no definite evidence of amyloid deposition (Torres, et al., 1995). The mutations at 109 (Thr109 and Val109) are associated with euthyroid hyperthyroxenemia (Izumoto, et al., 1993; Moses, et al., 1990). Also, some individuals with Met 119 appear to have increased thyroxine binding. Only the Ser6 mutation can be listed as a polymorphism being present in approximately 12% of the American Caucasian population. As mentioned, the amyloid associated isoleucine 122 allele may be considered a polymorphism in the African American population since approximately 3% of this ethnic group carry this mutation.PathogenesisThe pathogenesis of transthyretin amyloidosis is still not well understood. The extensive ß confirmation of the protein is most certainly important in forming the ß-pleated sheet fibrils (Fig.1). With the discovery of single amino acid substitution variants of transthyretin, it was postulated that perturbations in the structure of the protein in some way might lead to aggregation or polymerization of the subunit protein molecules to form the fibrils. It is not that simple. Tertiary structure analysis of a number of the transthyretin variants has failed to show any unifying theme which predicts the transition of soluble plasma protein to isoluble amyloid fibril (Blake, et al., 1978; Hamilton, et al., 1993; Oatley, et al., 1984). The intramolecular location or type of amino acid substitution in the transthyretin protein also gives little clues to pathogenesis. Mutations from neutral to charged residues, from charged to neutral residues, from hydrophobic to hydrophilic, or hydrophilic to hydrophobic have all been described for this

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protein. In addition, the mutations are distributed over most of the length of the protein molecule. All are due to single base changes causing single amino acid substitutions except for the Val122 which is the result of a loss of codon. If tertiary structure alterations leading to increased aggregation or polymerization are not the basis for fibril formation, the question persists. How do single amino acid substitutions lead to fibril formation? One possibility is the disruption of the stability of the transthyretin tetramer so that altered metabolism and polymerization may occur (Chakrabartty, 2001; Hammarström, et al., 2001). The fact that normal transthyretin can result in amyloid fibril formation indicates that there are factors other than mutation events leading to this pathology. Hypothesized models of amyloid fibril formation have been formulation are not consistent with the tetramer being the basic building block of the fibril forming subunit. Limited studies have shown evidence of significant amounts of proteolysis. Fragments of transthyretin representing amino acid residues 47 to 127, 49 to 127 and 52 to 127 have been demonstrated in extracted fibrils from several tissues, and a relative lack of the amino terminal portion of transthyretin has also been noted. These findings along with the demonstration that synthetic peptides of the carboxyl terminal end of transthyretin can give in vitro formation of fibrils meeting the characteristics of amyloid, suggest that proteolysis may be a significant factor in the formation of transthyretin amyloid deposits (Fig. 4). Accelerated metabolism of variant transthyretin may also be a factor. Plasma transthyretin levels are commonly depressed in individuals with many of the variant forms of amyloidosis. This is found in mutant gene carriers which are not yet affected with systemic amyloid deposition.

Plasma transthyretin is synthesized predominantly by the liver and is the source of substrate for amyloid deposits in the vascular tree, the heart, and the kidney. The liver, however, is rarely involved with significant amyloid deposits nor is the pancreas or spleen. The reason for organ selectivity is not readily apparent. Transthyretin is also synthesized by choroids plexus and amyloid deposition in the leptomeninges is probably the result of this intercranial synthesis. It has not been demonstrated, for certain, that vitreous amyloid is the result of local transthyretin production (Munar-Quéz, et al., 2000). The predilection for the peripheral nerves also is not understood. Amyloid deposits are unusually seen within nerve substance; although they may originally be associated with the vasa nervorum. The involvement of the dorsal root ganglia has not been fully appreciated, but is probably very important in this peripheral neuropathy. An interesting observation is that transgenic mice with the methionine 30 variant of transthyretin, while having amyloid deposits in heart, gastrointestinal tract, kidney, and other organs, have not yet been shown to develop amyloid in peripheral nerves (Yi, et al., 1991).Treatment

The only specific therapy for transthyretin amyloidosis is liver transplantation (Holgren, et al., 1991; 1993; Skinner, et al., 1994). Replacement of the liver results in rapid disappearance of variant transthyretin from the blood. The procedure has increased risk since individuals with cardiomyopathy or severe nutritional abnormalities have greater risk of perioperative death. Most recent reports indicate 78% survival after two years. Partial liver transplant from related living donors has also been reported (Matsunami, et al., 1995). The reports of the appearance of vitreous amyloid deposits after liver transplantation and the association of severe leptomeningeal amyloid deposition with cerebral dysfunction are worrisome (Munar-Quéz, et al., 2000; Dubrey, et al., 1997; Adams, et al., 2000). In addition, progression of cardiomyopathy has been documented in some patients. In general, patients with the methionine 30 and the histidine 58 mutations have fared better than patients with the glycine 42 and arginine 10 mutations who have had disease progression after liver transplantation.

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Nonspecific therapy for patients with transthyretin amyloidosis, including diligent management of heart failure, gastrointestinal dysfunction, and renal dialysis when indicated, can significantly prolong life. The judicious use of cardiac pacing and recognition that any negative inotropic drugs such as antiarrythmics should be avoided if possible in patients with restrictive cardiomyopathy, has added to length of survival. A very significant factor in the treatment of subjects with transthyretin amyloidosis has been genetic screening. This has resulted in the identification of subjects with this key condition so they can receive timely diagnosis and therapy.Diagnosis

As with many rare diseases, diagnosis of transthyretin amyloidosis is a problem, not primarily because of varied expression of clinical disease, but because it is not expected. It is easy to criticize the lack of early consideration of amyloidosis in the medical evaluation of subjects presenting with impotence, chronic diarrhea, carpal tunnel syndrome, angina pectoris, or proteinuria. These are all early signs of amyloidosis, but they are also early signs of much more prevalent diseases. There are, however, combinations or signs and symptoms which should bring the diagnosis of amyloidosis to mind. Peripheral neuropathy or autonomic neuropathy in combination with any of the major systemic conditions (angina, proteinuria, restrictive cardiomyopathy, azotemia) demands consideration of hereditary amyloidosis in the differential diagnosis. Carpal tunnel syndrome or vitreous opacities in a person with sensorimotor neuropathy and any of the cardiac, renal or gastrointestinal signs raises the chance for amyloidosis being the correct diagnosis.

Taking a good family history is most important. The presence of similar syndrome in any relative, no matter how distant, can give reason to suspect hereditary amyloidosis. On the other hand, the lack of the disease in the family history should not deter consideration of hereditary amyloidosis. Most of the transthyretin variants are the result of single nucleotide changes. These are easily detected by SSCP, ASO hybridization, RFLP, or direct DNA sequencing. The transthyretin gene exons are small and easily amplified by PCR (Nichols, et al., 1989).Results can be determined within 48 hours. In addition, hybrid isoelectric focusing of plasma has been used to detect some of the transthyretin variants. Plasma RBP levels can be used to screen individuals in the Ser84 families.

Our current strategy for DNA testing is based upon PCR of genomic DNA. If a particular mutation is known for the family under consideration, previously described RFLP tests are used. This requires amplification of the transthyretin exon having the known mutation and then digestion of the PCR product with the appropriate restrictive enzyme. If the mutation is unknown or a novel mutation is expected, all exons are amplified and then subjected to direct DNA sequencing. If sequencing does not give adequate results, SSCP may localize the mutation to a specific exon. This can then be studied under greater detail (e.g. M13 cloning and sequencing).

DNA testing is a valuable tool in the treatment of individuals and families with this genetic disease. It is also very important for genetic counseling and should be appropriately for the overall medical care of those affected with transthyretin amyloidosis.Summary

A tremendous amount of progress has been made in the study of transthyretin amyloidosis. To date the most visible element of this progress has been the discovery and description of new transthyretin mutations and the disease they cause. This has been essentially descriptive research, but that has not been so bad. The generation of descriptive data has formed the basis for more in-depth research on transthyretin amyloidosis, has resulted in the discovery of

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apolipoprotein AII forms of hereditary amyloidosis and allowed patients afflicted with these diseases to benefit from diagnostic testing, genetic counseling, prenatal testing, and treatment such as liver transplantation. Now we are charged with the task of taking our knowledge base and pursuing mechanistic research into pathogenesis. This hopefully will lead to a means to prevent or cure these diseases.ReferencesAdams D,. Samuel D, Goulon-Goeau C, Nakazato M, Costa PMP, Feray C, Planté V, Ducot B, Ichai P, Lacroix C, Metral S, Bismuth H and Said G (2000). The course and prognostic factors of familial amyloid polyneuropathy after liver transplantation. Brain 123, 1495-1504.

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Figure 1

Figure 2

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Figure 3

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Figure 4

Table 1

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