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Clinical and histopathological features of progressive-type familial amyloidotic polyneuropathy with TTR Lys54
Journal of the Neurological Sciences 276 (2009) 88–94
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Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Clinical and histopathological features of progressive-type familial amyloidotic polyneuropathy with TTR Lys54 Takamura Nagasaka ⁎, Shinji Togashi, Harue Watanabe, Haruyasu Iida, Kaori Nagasaka, Yuki Nakamura, Michiaki Miwa, Fumikazu Kobayashi, Kazumasa Shindo, Zenji Shiozawa Department of Neurology, University of Yamanashi, 1110, Shimokato, Chuou-city, Yamanashi, 409-3898, Japan
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Article history: Received 20 November 2007 Received in revised form 31 August 2008 Accepted 3 September 2008 Available online 18 October 2008 Keywords: Familial amyloidotic polyneuropathy Variant transthyretin Heart failure Autopsy
a b s t r a c t The purpose of this study was to evaluate the clinical and pathological features in patients with progressivetype familial amyloidotic polyneuropathy (FAP) using autopsy and biopsy specimens. A proband is a 33-yearold man with FAP type I who developed motor, sensory and autonomic impairments with neuropathy, heart failure, and anorexia. Genetic findings of transthyretin (TTR) revealed G to A transition in codon 54 causing a rare mutation of TTR Lys54. He died of pneumonia and severe cardiac failure 4 years after onset. Autopsy showed heavy amyloid deposition in the heart, peripheral nerves, thyroid, skin, fat tissue, prostate and testis, moderate in the sympathetic nerve trunk, vagal nerve, celiac plexus, pelvic plexus, bladder, gastrointestinal tract, tongue, pancreas, lung, pituitary, blood vessel, gall bladder, adrenals and muscles, and free in the central nervous system, liver, kidney and spleen. Sural nerve biopsy in a sibling confirmed TTR amyloidosis immunohistochemically. Electronmicroscopic findings of amyloid fibrils were similar to that of FAP Met30. Immunoelectronmicroscopic findings indicated the relationship between amyloid fibrils or non-fibrillar structure and collagen fibers. The distribution of amyloid deposition, heavy in the heart and lacking in the kidney, is a characteristic feature and reflected severity of FAP with TTR Lys54. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Familial amyloidotic polyneuropathy (FAP) is an autosomaldominant inherited disease, characterized by amyloid deposition in various organs. Polyneuropathy, autonomic neuropathy, vitreous opacity, cardiomyopathy and renal failure are the major manifestations of FAP. TTR is a tetrameric serum protein of four identical subunits of 14 kDa synthesized mainly in the liver and the choroid plexus of the brain, acting as a transport protein for thyroxine and retinol, and a cryptic protease for Apolipoprotein AI [1,2] and degraded in the kidney, liver, muscle and skin in the rat. Genetically more than 80 other amino acid substitutions of variant TTR, including cases without pathological findings, have been reported elsewhere [3]. The most common disease associated mutation in inherited amyloidosis is TTR Val122Ile which is responsible for familial amyloidotic cardiomyopathy. Variant of TTR with amino acid substitution of Val30Met (TTRMet30) is the most common in FAP. Genetic differences lead to the heterogeneity of clinical manifestation due to the variant deposition of amyloid fibrils. The distribution of variant TTR is not necessarily in accordance with the region of synthesis and degradation. Several cases of the progressive form with
⁎ Corresponding author. Tel.: +81 55 273 1111; fax: +81 55 273 7108. E-mail addresses: [email protected], [email protected] (T. Nagasaka). 0022-510X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2008.09.011
severe heart failure, including autopsy findings, were reported. Four affected siblings in a Costa Rican family with a severe form of FAP with TTR mutation of Glu54Lys were reported with biopsy findings in various organs (oral mucosa, skin and rectum) [4]. No detailed systemic pathological findings proven by autopsy identified FAP with TTR Glu54Lys. In this report, we present the clinical features and autopsy findings of the progressive form of FAP type 1 in a family with a rare substitution of the variant TTR gene Glu54Lys [5] and compared with the common type of FAP with TTRMet30 and the others. Free of amyloid in the kidney is the major manifestation in autopsy. The severity of this type of FAP owe to heavy amyloid deposition in the heart in addition to periperal nerves. We also present the ultrastructural findings of sural nerve using biopsy specimen and discuss the behavior of amyloid from the morphological views. 2. Material and methods 2.1. Case presentation 2.1.1. Case 1 The patient is a 32-year-old Japanese man who had been complaining of alternate diarrhea and constipation, and vomiting for 3 years following 10 kg weight loss. He had noticed severe paresthesia and weakness of his legs for two years before admission to our hospital at the age of 32 because of gait disturbance. His mother had
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also complained of severe diarrhea, developed ileus, and died at 33 years of age from sudden heart failure. His maternal grandmother died suddenly at the age of 35 of unknown cause. His sister was also affected by FAP (Case 2). On admission, physical examination showed emaciation, vitreous opacity in the right eye, and cardiac enlargement. Neurologic examination showed muscle weakness with atrophy, predominantly affecting the lower extremities. Deep tendon reflexes were all absent. Severe impairment of pain and light touch sensation were present predominantly in the distal part of the four extremities. Autonomic dysfunction included abnormal gastrointestinal motility, neurogenic bladder, postural hypotension and hypohydrosis. ECG showed complete right bundle branch block, and echocardiogram revealed massive thickness of the left ventricular wall (15 mm) and septal wall (21 mm) with high echogenicity, with a 58% decrease of ejection fraction. Myocardial scintigraphy using 123I-meta-iodobenzyl guanidine (MIBG) showed no uptake into the cardiac muscle (Fig. 1a). 99m Tc-(V)-dimercaptosuccinic acid (DMSA) scintigraphy showed uptake into the cardiac muscle, which indicates amyloid deposition (Fig. 1b). Postprandial hypotension lasted 2 or 3 h by ambulatory blood pressure monitoring (ABPM) in the supine position. The 75-g oral glucose tolerance test (75-g OGTT) in the supine position showed excessive secretion of insulin 30 min after glucose administration (Fig. 2a). The postprandial decrease, 25 mmHg, in blood pressure observed at 15 min, recovered to the level at 30 min preadministration and slowly decreased for at least 120 min. The heart rate peaked within 30 min without cathecolamine response (Fig. 2b). Skin sympathetic nerve activity and muscle sympathetic nerve activity were not elicited by microneurography. Decreased coefficient of variation of R–R intervals (1.54%, normal range: 2.5–7.3) in ECG was observed. Nerve conduction studies showed that the velocity and compound muscle action potential amplitude of the right median nerve were 45.7 m/s and 2.4 mV (normal range: 57.7 + −4.9 m/s, 7.0 + −3.0 mV), and 35.7 m/s and 0.02 mV (normal range: 48.5 + −3.6 m/s, 5.8 + −1.9 mV) in the right
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Fig. 2. The 75-g oral glucose tolerance test (75-g OGTT) in Case 1. a: The 75-g OGTT in the supine position showed excessive secretion of insulin 30 min after glucose administration. b: The postprandial decrease, 25 mmHg, in blood pressure observed at 15 min, recovered at the level of 30 min preadministration and slowly decreased for at least 120 min. The heart rate reached a peak within 30 min without cathecolamine response.
posterior tibial nerve. Sensory conduction velocity and sensory action potential amplitude of the right median, ulnar and sural nerves were 33.0 m/s and 16.7 µV (normal range: 58.8 + −5.8 m/s, 38.5 + −15.6 µV), 30.0 m/s and 14.3 µV (normal range: 64.7 + −5.4 m/s, 35.0 + −14.7 µV) and 27.4 m/s and 8.5 µV (normal range: 52.5 + −5.6 m/s, 20.9 + −8.0 µV), respectively. Electromyogram showed a neurogenic pattern in the four extremities. These symptoms and examinations indicated severe polyneuropathy with autonomic failure. Genetic analysis revealed amino acid substitution of Glu 54 for Lys in the transthyretin gene. Sural nerve biopsy was performed. Hepatic transplantation could not be performed because of refusal by his family. Approximately 4 years after the onset of symptoms, he died at the age of 33 years from pneumonia and severe cardiac failure. Autopsy was performed with informed consent from his family.
Fig. 1. Myocardial scintigraphy in Case 1. a: Myocardial scintigraphy using 123I-metaiodobenzyl guanidine (MIBG) show no uptake into the cardiac muscle. b: Technetium 99m Tc-(V)-dimercaptosuccinic acid scintigraphy showed uptake into the cardiac muscle, which indicates amyloid deposition.
2.1.2. Case 2 The patient is a 38-year-old woman, the younger sister of Case 1, who had been complaining of frequent vomiting. She had been diagnosed with schizophrenia at the age of 28. Surgery for vitreous opacity had been performed at the age of 36. [6] At that time, she had noticed severe paresthesia and weakness of her legs. Autonomic dysfunction included abnormal gastrointestinal motility, neurogenic bladder, postural hypotension and hypohydrosis was observed. Echocardiogram revealed massive thickness of the wall. Genetic analysis revealed an amino acid substitution of Glu 54 for Lys in the TTR gene. Nerve conduction studies showed that the motor conduction velocity and compound muscle action potential amplitude of the right median and posterior tibial nerves were 48.4 m/s and 3.8 mV (normal range: according to the indication in Case 1), and 43.8 m/s and 0.56 mV. Sensory conduction velocity and sensory action potential amplitude of the right median, ulnar and sural nerves were 37.8 m/s
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and 14.8 µV, 37.0 m/s and 11.2 µV, and 37.0 m/s and 0.5 µV, respectively. Sural nerve biopsy was performed with consent. 2.2. Histological preparation The sural nerve obtained by biopsy was fixed in 10% formalin and embedded in paraffin. Frozen fixed material was also prepared. Paraffin sections were stained by H–E, Congo red and Direct Fast Scarlet 4BS as an amyloid specific staining. The tissue was also fixed in 2.5% buffered glutaraldehyde, post-fixed in osmium tetroxide and embedded in Epon. Transverse semithin sections were stained with toluidine blue staining. Ultrathin sections were treated for routin electron microscopy. Immunohistological examinations were also performed. For fluorescence microscopy, paraffin and frozen sections were incubated with TTR antibody (DAKO Japan, Kyoto, Japan, 500fold dilution) overnight at 4 °C followed by fluorescein-conjugated secondary antibody for 90 min at room temperature. For immunoelectronmicroscopy, the tissue was fixed with 4% paraformaldehyde and 0.15 M HEPES-KOH buffer (pH 7.4) and embedded in LR White. Ultrathin sections were incubated overnight with TTR antibody followed by protein A-gold probes for 30 min. All tissues removed from the autopsy in Case 1 were fixed in 10% formalin and embedded in paraffin. Paraffin sections were stained by H-E, klüver-barrera and Direct Fast Scarlet 4BS. In the kidney, immunofluorescence staining was performed using TTR antibody for comparing the behavior of staining with Direct Fast Scarlet 4BS. 3. Results 3.1. Sural nerve biopsy Nerve fiber density in Case 2 revealed loss of nerve fibers dominant in unmyelinated and small myelinated fibers (Fig. 3). Severe neuronal loss in myelinated nerves was observed in the sural nerves obtained by biopsy in both cases (Fig. 4a). Amyloid deposition was observed extracellurally in the endoneurium adjacent to the perineurium (Fig. 4b), perineurium (Fig. 4c), and epineurium (Fig. 4e), and in the capillary wall and connective tissue in immunohistochemical staining for TTR. Behavior of staining was coincided between Direct Fast Scarlet 4BS and TTR antibody using paraffin embedded material (Fig. 4c, d). Comparison of the same sections stained with Congo red under ultraviolet light and bright light revealed complete agreement for the pattern of staining (Fig. 4f, g). This pattern was also coincided with the staining with Direct Fast Scarlet 4BS(Fig. 4h). Ultrastructural study revealed amyloid fibril deposition in the subfascicular region. Amyloid deposits were very dense and disorganized, with a hard-edged profile and surrounded by a long process of fibroblasts (Fig. 4i, j). The amyloid
seemed not to invade nerve fiber or Schwann cell for the most part (Fig. 4k), but some of nerve fiber or Schwann cell were seemed to be destructed (Fig. 4l). Ghost structure; covered by amyloid and seemd to be myelinated fiber (Fig. 4m), and slightly shrinked unmyelinated fiber surrounded by amyloid fibrils (Fig. 4n) were scarcely observed. The fibrils were straight, nonbranching tubules with a diameter of 7 nm, and with a periodicity of about 200 nm (Fig. 4o). The size of the amyloid fibers did not differ from other FAP, including TTR Met30. In immunoelectronmicroscopy, the amyloid was positive for TTR. Gold labeling-positive TTR was presented in both pre-fibrillar and fibrillar structures along or intermingled with collagen fibers (Fig. 4p, q). 3.2. Autopsy findings The heart weighed 510 g and the brain weighed 1380 g. Histological findings showed heavy amyloid deposition in the heart, peripheral nerves, thyroid, skin, fat tissue, prostate and testis. The cardiac muscle was atrophic with complemental hypertrophy. Heavy amyloid deposition was detected in the endoneurium of cardiac nerves (Fig. 5a) and in the capillary wall and perimyofibrils (Fig. 5b) in the heart. A little amyloid deposition was detected in the small artery (Fig. 5c). An electron microscopic study showed fine amyloid fibrils in contact with myofibrils. Other organs containing moderate amyloid included the sympathetic nerve trunk, vagal nerve, celiac plexus, pelvic plexus, bladder, gastrointestinal tract, tongue, pancreas (more prominent in the nerves than Langerhans islets) (Fig. 5d), lung, pituitary (anterior lobe) (Fig. 5e), gall bladder, adrenals and muscles. No amyloid was detected in the liver or spleen. In the kidney, no amyloid deposition was observed in Direct Fast Scarlet 4BS staining (Fig. 5f). In TTR immunostaining, no abnormal staining indicating amyloid deposition could be observed (Fig. 5g), while subtle dotted staining was detected only in epithelial cells of proximal tubules in which normal TTR is observed for degradation (Fig. 5h). Deposition limited to blood vessels in the arachnoid, subarachnoid and choroid plexus but lacking in the parenchyma including blood vessels was generally seen in the brain (Fig. 5i). In the spinal cord, loss of anterior horn cells was observed (Fig. 5j). The slender gracile fasciculus was extremely degenerated (Fig. 5k) with no amyloid(Fig. 5l). Comparison of the region of amyloid deposition between FAPMet30 and Lys54 is summarized in Table 1. The major difference between the two types of FAP was renal involvement; free from amyloid deposition in Lys54 and heavy in Met30. 4. Discussion The age at onset (ranging from 20 to 71 with a mean of 40.1 years) and duration of illness (5.1 years) varied considerably in the 107 cases
Fig. 3. The histogram of sural nerve in Case 2. a: Myelinated fiber density is decreased dominant in small fiber. b: Unmyelinated fiber density is decreased and the size of fiber become smaller.
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Fig. 4. Sural nerve obtained by biopsy in Case 2. (a: toluidine blue staining b,c,e: immunofluorescence staining for TTR d, h: Direct Fast Scarlet 4BS staining f, g: Congo red staining i, j, l, m, n: Routine electronmicroscopy k, o, p, q: immunoelectronmicroscopy by immuno-gold labeling for TTR) a: Severe neuronal loss in myelinated and unmyelinated nerves is observed. In TTR immunostaining. b: Amyloid deposition is seen in the endoneurium adjacent to the perineurium. c: Amyloid is detected in the perineurium. d: Neighbouring section of Fig. c. Behavior of staining is coincided between Direct Fast Scarlet 4BS and TTR antibody (Fig. 4c) using paraffin embedded material. e: Amyloid is seen in the epineurial space. f: Amyloid is seen in endoneurium under bright light. g: The same section of Fig. f. Amyloid is clearly seen under ultraviolet light. h: Neighbouring section of Fig. f, g. Behavior of staining is coincided between Congo red and Direct Fast Scarlet 4BS. i: A myelinated fiber and amyloid aggregation (asterisk) are separated by the process of fibroblast (arrow). Bar = 1 µm j: Amyloid aggregation (asterisk) is surrounded by the fibroblast. Bar = 2 µm k: Fibrile aggregation is positive of TTR. Schwann cell is intact near amyloid aggregation. Bar = 0.5 µm l: Schwann cell seems to be destructed near amyloid aggreagation (asterisk). Bar = 2 µm m: Ghost structure (arrow) which is covered by amyloid (asterisk) seems to be myelinated fiber. Bar = 1 µm n: Slightly shrinked unmyelinated nerve is seen surrounded by amyloid fibrils (asterisk). Bar = 1 µm o: The fibrils were straight, nonbranching tubules with a diameter of 7 nm, and with a periodicity of about 200 nm. Bar = 0.5 µm p: Amyloid fibrils intermingled with collagen fibers (asterisk) are seen. Bar = 0.2 µm q: Pre-fibrillar amyloid (arrow head) deposited in collagen fiber (asterisk) is seen. Bar = 0.5 µm.
of FAP Met30 seen in Japan [7]. In our case, initial symptoms and age do not differ from the common form of TTR Met30 FAP [8] and are compatible with early-onset group [9]. Severe cardiac impairment
with amyloid deposition other than neuropathy resulting in rapid progression was a characteristic feature in this patient. Cardiomyopathy is a prominent feature in patients with the Asp45, Ile50, Lys59,
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Fig. 5. Several tissues by autopsy in Case 1. (a, b, c, d, e, f, i, l: Direct Fast Scarlet 4BS staining j, k: klüver-barrera staining g, h: immunofluorescence staining for TTR) a: Amyloid is seen in nerve (arrow) and connective tissue in the space between epicardium and myofibrils(asterisk). Bar = 250 µm b: Amyloid is abundant in the capillary wall (arrow) and perimyofibrils of left ventricle. Bar = 200 µm c: A little deposition of amyloid is detected in the small artery. (asterisk: myofibril) Bar = 500 µm d: Moderate amyloid is observed in pancreas, more prominent in the inner nerve (arrow) than Langerhans islets (asterisk). Bar = 200 µm e: In pituitary, amyloid (arrow) is seen in anterior lobe, not in posterior lobe (margin is indicated by arrowheads). Bar = 200 µm f: Normal renal tissue without amyloid deposition is seen in amyloid staining. Arrow: glomerulus Bar = 150 µm g: TTR immunostaining shows no abnormal staining indicating amyloid deposition other than normal finding in Fig.h. Arrow: glomerulus Bar = 200 µm h: Subtle dotted stainings are seen only in epithelial cells of proximal tubules (arrow) in which normal TTR is observed for degradation. Autofluorescence of blood erythrocytes are seen (arrow head). Bar = 40 µm i: In the brain, amyloid limited to blood vessels (arrow) in the arachnoid and subarachnoid but lacking in the parenchyma (margin is indicated by arrowheads). Bar = 300 µm j: In the spinal cord (C7), loss of the anterior horn cell (arrow) was observed. Bar = 200 µm k: The slender gracile fasciculus in lumbar spinal cord was extremely degenerated. Arrow: posterior median fissure Arrowhead: posterior intermediate fissure Bar = 500 µm l: No amyloid is seen in the slender gracile fasciculus. Bar = 500 µm.
Ala60, Leu68, Ser84 and Gln89 variants among FAP [10]. Three TTR mutations, Thr45, Met111 and Ile122 variants, have also been associated predominantly with cardiac amyloidosis and the absence of polyneuropathy. In our patient, heavy amyloid which was shown in
scintigraphy using DMSA was detected in the endoneurium, perimyofibrils, capillary wall and connective tissue in the heart. Moreover, myocardial scintigraphy using MIBG showed no uptake into the cardiac muscle, which revealed severe dysfunction of sympathetic
T. Nagasaka et al. / Journal of the Neurological Sciences 276 (2009) 88–94 Table 1 Comparison of the region of amyloid deposition between FAPMet30 and FAPLys54 Tissue
Glu54Lys (our case)
Val 30 Met
Heart Lung Liver Kidneys Spleen Pancreas Digestive tract Bladder Adrenals Testis Thyroid gland Prostate Pituitary Skeletal muscle Cerebrum Cerebellum Arachnoid membrane Choroid plexus Spinal cord Peripheral nerves
+++ + − − − ++ ++ ++ ++ +++ +++ +++ ++ ++ − − + − − +
++ − − ++ + ++ ++ +++ + + +++ Unknown + + − − + +++ − ++
The degree of amyloid deposition is classified into the following 4 grades: − negative; + slight; ++ moderate; +++ severe.
nerve activity in the early period [11]. Postprandial hypotension observed in ABPM and 75-g OGTT was due to a lack of compensatory increase of cardiac output and abnormal secretion of insulin with neuronal impairment. In clinical and biochemical evidence of severe amyloidgenicity of TTR Pro55 has been reported [12–16]. An aggressive form of FAP Gly54 with thickened cardiac muscle in echocardiography was reported [17]. Accordingly, increased affinity of amyloid for cardiac muscle may be one of the character of variant TTR with two adjacent amino acid substitutions at position 54 and 55. In the kidneys, a large amount of amyloid was observed in almost all progressive forms of FAP [12], and the common form of FAP Met30. Alternatively, no amyloid deposition other than normal existence in epithelial cells of proximal tubules [18,19] was detected in the kidney in our case. Nearly normal renal function with no proteinuria or creatine retention in four affected siblings with a severe form of FAP with TTR Lys54 in a Costa Rican family was reported [4]. The absence of renal involvement may be a characteristic feature in FAP Lys54 despite its aggressive form with severe cardiac impairment. A combination of severe cardiac involvement and mild deposition in the kidney was also reported in autopsy cases of FAP Ile50 [20] and FAP Gly54 with Ser6 (probably a polymorphism) mutations [21]. The severity might not be necessarily attributed to both the kidney and heart. In the spinal cord, the gracile fasciculus was extremely degenerated without amyloid deposition in our case. A report revealed that minimal to moderate myelinated fiber loss was observed in the posterior columns in early- and late-onset TTRMet30 FAP [22]. So, the change may be caused by progressive ascending degeneration with a ‘dying back’ process, which reflects severe neuropathy including the large fibers. The loss of anterior horn cells observed in our case is thought to have the same significance. Various involvements of the CNS have been reported, including leptomeningeal deposition or severe meningocerebrovascular amyloidosis [23]. In our patient, slight deposition was observed in the blood vessel walls in the leptomeninges, but not in the brain parenchyma including blood vessels. Moreover, in the pituitary, amyloid was not detected in the posterior lobe(neuralgichypophysis). A secretion of variant TTR from the choroid plexus or the blood-brain barrier may play a significant role in the little deposition of amyloid in the brain. In peripheral nerve fibers, small fiber dominant neuropathy shown in Fig. 3, characteristic in FAP, was observed. At autopsy, amyloid was observed in the capillary wall and connective tissue of the endoneurium, epineurium and perineurium of the sympathetic nerve trunk, vagal nerve, celiac
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plexus, pelvic plexus, and the nerve in gastrointestinal tract and pancreas, which were also involved in Met30 FAP. The vagus nerves and celiac ganglion showed extensive endoneurial and intraganglionic deposition of amyloid, as reported previously [24]. 75-g OGTT performed in Case 1 showed excessive secretion of insulin 30 min after glucose administration. The result may reflect abnormal nerve regulation in the pancreatic endocrine system with amyloid deposition, more prominent in the nerves than in Langerhans islets, as reported previously [25]. Endocrinologically, the pituitary [26], thyroid and adrenal glands [27] can be affected in FAP. In our patient, there was no functional abnormality although amyloid deposition was observed in these organs. This may contribute to explaining the lack of correlation between amyloid deposition and the function of the amyloidogenic organs, at least in the endocrine system. Overlook these autopsied pathological findings, each of variant TTR mutations have characteristic organ distribution in amyloid deposition as in Table 1. As for the pathogenesis of peripheral nerve disturbances in FAP, various theories, such as local compression by aggregated amyloid, ischemia, neurotoxicity or apoptosis have been presented [28]. Regarding to the point, we focus on the findings of sural nerve biopsy in Case 2. In electron microscopy, the ultrastructure and the site of deposition of amyloid do not differ from the other types of variant TTR, including Met30. The amyloid seems not to invade nerve fiber or Schwann cell for the most part, but some of nerve fiber or Schwann cell are seemed to be destructed, covered or surrounded by amyloid fibrils. That is, a part of the mechanism of nerve impairment may be in relation to direct invasion of amyloid, although whether the destruction is supported by another factor such as neurotoxicity or apotosis is unclear. The amyloid was located mainly in the endoneurium adjacent to the perineurium, perineurium, and epineurium where the region collagen fiber is abundant. In immunoelectronmicroscopy, gold labeling-positive TTR present along or intermingled with collagen fibers in both pre-fibrillar and fibrillar structures. The findings may suggest that collagen fiber is one of the ground where amyloid fibril or pre-fibrillar structure is fixed or exhibit amyloidgenicity. Recently, evidence that free amyloid or amyloid intermediate has a cytotoxic effect on FAP nerves has been presented [29]. Teng et al. first demonstrated non-fibrillar TTR deposits in mice transgenic for human TTR Leu55Pro [30]. It was proposed that TTR might be deposited in a non-fibrillar form in the nerves prior to major nerve fiber degeneration in the early stages of FAP [31]. A report revealed that beta2-microglobulin amyloid induces the release of MMP-1, leading to uncontrolled collagenolysis followed by the tissue destruction [32]. These observations and our findings may be helpful evidences for the relationship between amyloid and collagen fiber. No precise autopsy findings have described FAP with TTRLys54, a progressive form with a rare substitution of the variant TTR gene. The severity of this patient was reflected in the autopsy findings. References [1] Liz MA, Faro CJ, Saraiva MJ, Sousa MM. Transthyretin, a new cryptic protease. J Biol Chem 2004;279:21431–8. [2] Sousa MM, Berglund L, Saraiva MJ. Transthyretin in high density lipoproteins: association with apolipoprotein A-I. J Lipid Res 2000;41:58–65. [3] http://www.bumc.bu.edu/Dept/Content.aspx?DepartmentID=354&PageID=8850. [4] Busse A, Sánchez MA, Monterroso V, Alvarado MV, León P. A severe form of amyloidotic polyneuropathy in a Costa Rican family with a rare transthyretin mutation (Glu54Lys). Am J Med Genet 2004;128:190–4. [5] Togashi S, Watanabe H, Nagasaka T, Shindo K, Shiozawa Z, Maeda S, et al. An aggressive familial amyloidotic polyneuropathy caused by a new variant transthyretin Lys54. Neurology 1999;53:637–9. [6] Imasawa M, Toda Y, Sakurada Y, Imai M, Iijima H. Vitreous opacities in a case of familial amyloidotic polyneuropathy associated with a transthyretin Lys 54. Acta Ophthalmol Scand 2004;82:635–6. [7] Nakazato M, Shiomi K, Miyazato M, Matsukura S. Type I familial amyloidotic polyneuropathy in Japan. Intern Med 1992;31:1335–8. [8] Araki S, Yi S. Pathology of familial amyloidotic polyneuropathy with TTR Met 30 in kumamoto, Japan. Neuropathology 2000;20:S47–51. [9] Sobue G, Koike H, Misu K, Hattori N, Yamamoto M, Ikeda S, et al. Clinicopathological and genetic features of early- and late-onset FAP type I (FAP ATTR Val30Met) in Japan. Amyloid 2003;10(Suppl1):32–8.
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[21] Reilly MM, Adams D, Booth DR, Davis MB, Said G, Laubriat-Bianchin M, et al. Transthyretin gene analysis in European patients with suspected familial amyloido polyneuropathy. Brain 1995;118:849–56. [22] Koike H, Misu K, Sugiura M, Iijima M, Mori K, Yamamoto M, et al. Pathology of earlyvs late-onset TTR Met30 familial amyloid polyneuropathy. Neurology 2004;63: 129–38. [23] Sakashita N, Ando Y, Jinnouchi K, Yoshimatsu M, Terazaki H, Obayashi K, et al. Familial amyloidotic polyneuropathy(ATTR Val30Met) with widespread cerebral amyloid angiopathy and lethal cerebral hemorrhage. Pathol Int 2001;51:476–80. [24] Ikeda S, Yanagisawa N, Hongo M, Ito N. Vagus nerve and celial ganglion lesions in generalized amyloidosis. J Neurol Sci 1987;79:129–39. [25] Ando Y, Yi S, Nakagawa T, Ikegawa S, Hirota M, Miyazaki A, et al. Disturbed metabolism of glucose and related hormones in familial amyloidotic polyneuropathy : hypersensitivities of the autonomic nervous system and therapeutic prevention. J Auton Nerv Syst 1991;35:63–70. [26] Olofsson B-O, Grankvist K, Olsson T, Boman K, Forsberg K, Lafvas I, et al. Assesment of hypothalamic-pituitary function in patients with familial amyloidotic polyneuropathy. J Intern Med 1991;229:55–9. [27] Olofsson B-O, Grankvist K, Boman K, Forsberg K, Lafvas I, Lithner F. Assesment of thyroid anad adrenal function in patients with familial amyloidotic polyneuropathy. J Intern Med 1989;225:337–41. [28] Said G. Familial amyloid polyneuropathy: mechanisms leading to nerve degeneration. Amyloid. J Protein Folding Disord 2003;10(Suppl 1):7–12. [29] Kelly JW. The alternative conformations of amyloidgenic proteins and their multistep assembly pathways. Curr Opin Struck Biol 1998;8:101–6. [30] Teng M, Yin J, Vidal R, Ghiso J, Kumar A, Rabenou R, et al. Amyloid and nonfibrillar deposits in mice transgenic for wild-type human transthyretin: a possible model for senile systemic amyloidosis. Lab Invest 2001;81:385–96. [31] Sousa MM, Cardoso I, Fernandes R, Guimarães A, Sariva MJ. Deposition of transthyretin in early stages of familial amyloidotic polyneuropathy: evidence for toxicity of non-fibrillar aggregates. Am J Pathol 2001;159:1993–2000. [32] Moe SM, Singh GK, Bailey AM. Beta2-microglobulin induces MMP-1 but not TIMP1 expression in human synovial fibroblasts. Kidney Int 2000;57:2023–34.
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Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Clinical and histopathological features of progressive-type familial amyloidotic polyneuropathy with TTR Lys54 Takamura Nagasaka ⁎, Shinji Togashi, Harue Watanabe, Haruyasu Iida, Kaori Nagasaka, Yuki Nakamura, Michiaki Miwa, Fumikazu Kobayashi, Kazumasa Shindo, Zenji Shiozawa Department of Neurology, University of Yamanashi, 1110, Shimokato, Chuou-city, Yamanashi, 409-3898, Japan
a r t i c l e
i n f o
Article history: Received 20 November 2007 Received in revised form 31 August 2008 Accepted 3 September 2008 Available online 18 October 2008 Keywords: Familial amyloidotic polyneuropathy Variant transthyretin Heart failure Autopsy
a b s t r a c t The purpose of this study was to evaluate the clinical and pathological features in patients with progressivetype familial amyloidotic polyneuropathy (FAP) using autopsy and biopsy specimens. A proband is a 33-yearold man with FAP type I who developed motor, sensory and autonomic impairments with neuropathy, heart failure, and anorexia. Genetic findings of transthyretin (TTR) revealed G to A transition in codon 54 causing a rare mutation of TTR Lys54. He died of pneumonia and severe cardiac failure 4 years after onset. Autopsy showed heavy amyloid deposition in the heart, peripheral nerves, thyroid, skin, fat tissue, prostate and testis, moderate in the sympathetic nerve trunk, vagal nerve, celiac plexus, pelvic plexus, bladder, gastrointestinal tract, tongue, pancreas, lung, pituitary, blood vessel, gall bladder, adrenals and muscles, and free in the central nervous system, liver, kidney and spleen. Sural nerve biopsy in a sibling confirmed TTR amyloidosis immunohistochemically. Electronmicroscopic findings of amyloid fibrils were similar to that of FAP Met30. Immunoelectronmicroscopic findings indicated the relationship between amyloid fibrils or non-fibrillar structure and collagen fibers. The distribution of amyloid deposition, heavy in the heart and lacking in the kidney, is a characteristic feature and reflected severity of FAP with TTR Lys54. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Familial amyloidotic polyneuropathy (FAP) is an autosomaldominant inherited disease, characterized by amyloid deposition in various organs. Polyneuropathy, autonomic neuropathy, vitreous opacity, cardiomyopathy and renal failure are the major manifestations of FAP. TTR is a tetrameric serum protein of four identical subunits of 14 kDa synthesized mainly in the liver and the choroid plexus of the brain, acting as a transport protein for thyroxine and retinol, and a cryptic protease for Apolipoprotein AI [1,2] and degraded in the kidney, liver, muscle and skin in the rat. Genetically more than 80 other amino acid substitutions of variant TTR, including cases without pathological findings, have been reported elsewhere [3]. The most common disease associated mutation in inherited amyloidosis is TTR Val122Ile which is responsible for familial amyloidotic cardiomyopathy. Variant of TTR with amino acid substitution of Val30Met (TTRMet30) is the most common in FAP. Genetic differences lead to the heterogeneity of clinical manifestation due to the variant deposition of amyloid fibrils. The distribution of variant TTR is not necessarily in accordance with the region of synthesis and degradation. Several cases of the progressive form with
⁎ Corresponding author. Tel.: +81 55 273 1111; fax: +81 55 273 7108. E-mail addresses: [email protected], [email protected] (T. Nagasaka). 0022-510X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2008.09.011
severe heart failure, including autopsy findings, were reported. Four affected siblings in a Costa Rican family with a severe form of FAP with TTR mutation of Glu54Lys were reported with biopsy findings in various organs (oral mucosa, skin and rectum) [4]. No detailed systemic pathological findings proven by autopsy identified FAP with TTR Glu54Lys. In this report, we present the clinical features and autopsy findings of the progressive form of FAP type 1 in a family with a rare substitution of the variant TTR gene Glu54Lys [5] and compared with the common type of FAP with TTRMet30 and the others. Free of amyloid in the kidney is the major manifestation in autopsy. The severity of this type of FAP owe to heavy amyloid deposition in the heart in addition to periperal nerves. We also present the ultrastructural findings of sural nerve using biopsy specimen and discuss the behavior of amyloid from the morphological views. 2. Material and methods 2.1. Case presentation 2.1.1. Case 1 The patient is a 32-year-old Japanese man who had been complaining of alternate diarrhea and constipation, and vomiting for 3 years following 10 kg weight loss. He had noticed severe paresthesia and weakness of his legs for two years before admission to our hospital at the age of 32 because of gait disturbance. His mother had
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also complained of severe diarrhea, developed ileus, and died at 33 years of age from sudden heart failure. His maternal grandmother died suddenly at the age of 35 of unknown cause. His sister was also affected by FAP (Case 2). On admission, physical examination showed emaciation, vitreous opacity in the right eye, and cardiac enlargement. Neurologic examination showed muscle weakness with atrophy, predominantly affecting the lower extremities. Deep tendon reflexes were all absent. Severe impairment of pain and light touch sensation were present predominantly in the distal part of the four extremities. Autonomic dysfunction included abnormal gastrointestinal motility, neurogenic bladder, postural hypotension and hypohydrosis. ECG showed complete right bundle branch block, and echocardiogram revealed massive thickness of the left ventricular wall (15 mm) and septal wall (21 mm) with high echogenicity, with a 58% decrease of ejection fraction. Myocardial scintigraphy using 123I-meta-iodobenzyl guanidine (MIBG) showed no uptake into the cardiac muscle (Fig. 1a). 99m Tc-(V)-dimercaptosuccinic acid (DMSA) scintigraphy showed uptake into the cardiac muscle, which indicates amyloid deposition (Fig. 1b). Postprandial hypotension lasted 2 or 3 h by ambulatory blood pressure monitoring (ABPM) in the supine position. The 75-g oral glucose tolerance test (75-g OGTT) in the supine position showed excessive secretion of insulin 30 min after glucose administration (Fig. 2a). The postprandial decrease, 25 mmHg, in blood pressure observed at 15 min, recovered to the level at 30 min preadministration and slowly decreased for at least 120 min. The heart rate peaked within 30 min without cathecolamine response (Fig. 2b). Skin sympathetic nerve activity and muscle sympathetic nerve activity were not elicited by microneurography. Decreased coefficient of variation of R–R intervals (1.54%, normal range: 2.5–7.3) in ECG was observed. Nerve conduction studies showed that the velocity and compound muscle action potential amplitude of the right median nerve were 45.7 m/s and 2.4 mV (normal range: 57.7 + −4.9 m/s, 7.0 + −3.0 mV), and 35.7 m/s and 0.02 mV (normal range: 48.5 + −3.6 m/s, 5.8 + −1.9 mV) in the right
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Fig. 2. The 75-g oral glucose tolerance test (75-g OGTT) in Case 1. a: The 75-g OGTT in the supine position showed excessive secretion of insulin 30 min after glucose administration. b: The postprandial decrease, 25 mmHg, in blood pressure observed at 15 min, recovered at the level of 30 min preadministration and slowly decreased for at least 120 min. The heart rate reached a peak within 30 min without cathecolamine response.
posterior tibial nerve. Sensory conduction velocity and sensory action potential amplitude of the right median, ulnar and sural nerves were 33.0 m/s and 16.7 µV (normal range: 58.8 + −5.8 m/s, 38.5 + −15.6 µV), 30.0 m/s and 14.3 µV (normal range: 64.7 + −5.4 m/s, 35.0 + −14.7 µV) and 27.4 m/s and 8.5 µV (normal range: 52.5 + −5.6 m/s, 20.9 + −8.0 µV), respectively. Electromyogram showed a neurogenic pattern in the four extremities. These symptoms and examinations indicated severe polyneuropathy with autonomic failure. Genetic analysis revealed amino acid substitution of Glu 54 for Lys in the transthyretin gene. Sural nerve biopsy was performed. Hepatic transplantation could not be performed because of refusal by his family. Approximately 4 years after the onset of symptoms, he died at the age of 33 years from pneumonia and severe cardiac failure. Autopsy was performed with informed consent from his family.
Fig. 1. Myocardial scintigraphy in Case 1. a: Myocardial scintigraphy using 123I-metaiodobenzyl guanidine (MIBG) show no uptake into the cardiac muscle. b: Technetium 99m Tc-(V)-dimercaptosuccinic acid scintigraphy showed uptake into the cardiac muscle, which indicates amyloid deposition.
2.1.2. Case 2 The patient is a 38-year-old woman, the younger sister of Case 1, who had been complaining of frequent vomiting. She had been diagnosed with schizophrenia at the age of 28. Surgery for vitreous opacity had been performed at the age of 36. [6] At that time, she had noticed severe paresthesia and weakness of her legs. Autonomic dysfunction included abnormal gastrointestinal motility, neurogenic bladder, postural hypotension and hypohydrosis was observed. Echocardiogram revealed massive thickness of the wall. Genetic analysis revealed an amino acid substitution of Glu 54 for Lys in the TTR gene. Nerve conduction studies showed that the motor conduction velocity and compound muscle action potential amplitude of the right median and posterior tibial nerves were 48.4 m/s and 3.8 mV (normal range: according to the indication in Case 1), and 43.8 m/s and 0.56 mV. Sensory conduction velocity and sensory action potential amplitude of the right median, ulnar and sural nerves were 37.8 m/s
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and 14.8 µV, 37.0 m/s and 11.2 µV, and 37.0 m/s and 0.5 µV, respectively. Sural nerve biopsy was performed with consent. 2.2. Histological preparation The sural nerve obtained by biopsy was fixed in 10% formalin and embedded in paraffin. Frozen fixed material was also prepared. Paraffin sections were stained by H–E, Congo red and Direct Fast Scarlet 4BS as an amyloid specific staining. The tissue was also fixed in 2.5% buffered glutaraldehyde, post-fixed in osmium tetroxide and embedded in Epon. Transverse semithin sections were stained with toluidine blue staining. Ultrathin sections were treated for routin electron microscopy. Immunohistological examinations were also performed. For fluorescence microscopy, paraffin and frozen sections were incubated with TTR antibody (DAKO Japan, Kyoto, Japan, 500fold dilution) overnight at 4 °C followed by fluorescein-conjugated secondary antibody for 90 min at room temperature. For immunoelectronmicroscopy, the tissue was fixed with 4% paraformaldehyde and 0.15 M HEPES-KOH buffer (pH 7.4) and embedded in LR White. Ultrathin sections were incubated overnight with TTR antibody followed by protein A-gold probes for 30 min. All tissues removed from the autopsy in Case 1 were fixed in 10% formalin and embedded in paraffin. Paraffin sections were stained by H-E, klüver-barrera and Direct Fast Scarlet 4BS. In the kidney, immunofluorescence staining was performed using TTR antibody for comparing the behavior of staining with Direct Fast Scarlet 4BS. 3. Results 3.1. Sural nerve biopsy Nerve fiber density in Case 2 revealed loss of nerve fibers dominant in unmyelinated and small myelinated fibers (Fig. 3). Severe neuronal loss in myelinated nerves was observed in the sural nerves obtained by biopsy in both cases (Fig. 4a). Amyloid deposition was observed extracellurally in the endoneurium adjacent to the perineurium (Fig. 4b), perineurium (Fig. 4c), and epineurium (Fig. 4e), and in the capillary wall and connective tissue in immunohistochemical staining for TTR. Behavior of staining was coincided between Direct Fast Scarlet 4BS and TTR antibody using paraffin embedded material (Fig. 4c, d). Comparison of the same sections stained with Congo red under ultraviolet light and bright light revealed complete agreement for the pattern of staining (Fig. 4f, g). This pattern was also coincided with the staining with Direct Fast Scarlet 4BS(Fig. 4h). Ultrastructural study revealed amyloid fibril deposition in the subfascicular region. Amyloid deposits were very dense and disorganized, with a hard-edged profile and surrounded by a long process of fibroblasts (Fig. 4i, j). The amyloid
seemed not to invade nerve fiber or Schwann cell for the most part (Fig. 4k), but some of nerve fiber or Schwann cell were seemed to be destructed (Fig. 4l). Ghost structure; covered by amyloid and seemd to be myelinated fiber (Fig. 4m), and slightly shrinked unmyelinated fiber surrounded by amyloid fibrils (Fig. 4n) were scarcely observed. The fibrils were straight, nonbranching tubules with a diameter of 7 nm, and with a periodicity of about 200 nm (Fig. 4o). The size of the amyloid fibers did not differ from other FAP, including TTR Met30. In immunoelectronmicroscopy, the amyloid was positive for TTR. Gold labeling-positive TTR was presented in both pre-fibrillar and fibrillar structures along or intermingled with collagen fibers (Fig. 4p, q). 3.2. Autopsy findings The heart weighed 510 g and the brain weighed 1380 g. Histological findings showed heavy amyloid deposition in the heart, peripheral nerves, thyroid, skin, fat tissue, prostate and testis. The cardiac muscle was atrophic with complemental hypertrophy. Heavy amyloid deposition was detected in the endoneurium of cardiac nerves (Fig. 5a) and in the capillary wall and perimyofibrils (Fig. 5b) in the heart. A little amyloid deposition was detected in the small artery (Fig. 5c). An electron microscopic study showed fine amyloid fibrils in contact with myofibrils. Other organs containing moderate amyloid included the sympathetic nerve trunk, vagal nerve, celiac plexus, pelvic plexus, bladder, gastrointestinal tract, tongue, pancreas (more prominent in the nerves than Langerhans islets) (Fig. 5d), lung, pituitary (anterior lobe) (Fig. 5e), gall bladder, adrenals and muscles. No amyloid was detected in the liver or spleen. In the kidney, no amyloid deposition was observed in Direct Fast Scarlet 4BS staining (Fig. 5f). In TTR immunostaining, no abnormal staining indicating amyloid deposition could be observed (Fig. 5g), while subtle dotted staining was detected only in epithelial cells of proximal tubules in which normal TTR is observed for degradation (Fig. 5h). Deposition limited to blood vessels in the arachnoid, subarachnoid and choroid plexus but lacking in the parenchyma including blood vessels was generally seen in the brain (Fig. 5i). In the spinal cord, loss of anterior horn cells was observed (Fig. 5j). The slender gracile fasciculus was extremely degenerated (Fig. 5k) with no amyloid(Fig. 5l). Comparison of the region of amyloid deposition between FAPMet30 and Lys54 is summarized in Table 1. The major difference between the two types of FAP was renal involvement; free from amyloid deposition in Lys54 and heavy in Met30. 4. Discussion The age at onset (ranging from 20 to 71 with a mean of 40.1 years) and duration of illness (5.1 years) varied considerably in the 107 cases
Fig. 3. The histogram of sural nerve in Case 2. a: Myelinated fiber density is decreased dominant in small fiber. b: Unmyelinated fiber density is decreased and the size of fiber become smaller.
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Fig. 4. Sural nerve obtained by biopsy in Case 2. (a: toluidine blue staining b,c,e: immunofluorescence staining for TTR d, h: Direct Fast Scarlet 4BS staining f, g: Congo red staining i, j, l, m, n: Routine electronmicroscopy k, o, p, q: immunoelectronmicroscopy by immuno-gold labeling for TTR) a: Severe neuronal loss in myelinated and unmyelinated nerves is observed. In TTR immunostaining. b: Amyloid deposition is seen in the endoneurium adjacent to the perineurium. c: Amyloid is detected in the perineurium. d: Neighbouring section of Fig. c. Behavior of staining is coincided between Direct Fast Scarlet 4BS and TTR antibody (Fig. 4c) using paraffin embedded material. e: Amyloid is seen in the epineurial space. f: Amyloid is seen in endoneurium under bright light. g: The same section of Fig. f. Amyloid is clearly seen under ultraviolet light. h: Neighbouring section of Fig. f, g. Behavior of staining is coincided between Congo red and Direct Fast Scarlet 4BS. i: A myelinated fiber and amyloid aggregation (asterisk) are separated by the process of fibroblast (arrow). Bar = 1 µm j: Amyloid aggregation (asterisk) is surrounded by the fibroblast. Bar = 2 µm k: Fibrile aggregation is positive of TTR. Schwann cell is intact near amyloid aggregation. Bar = 0.5 µm l: Schwann cell seems to be destructed near amyloid aggreagation (asterisk). Bar = 2 µm m: Ghost structure (arrow) which is covered by amyloid (asterisk) seems to be myelinated fiber. Bar = 1 µm n: Slightly shrinked unmyelinated nerve is seen surrounded by amyloid fibrils (asterisk). Bar = 1 µm o: The fibrils were straight, nonbranching tubules with a diameter of 7 nm, and with a periodicity of about 200 nm. Bar = 0.5 µm p: Amyloid fibrils intermingled with collagen fibers (asterisk) are seen. Bar = 0.2 µm q: Pre-fibrillar amyloid (arrow head) deposited in collagen fiber (asterisk) is seen. Bar = 0.5 µm.
of FAP Met30 seen in Japan [7]. In our case, initial symptoms and age do not differ from the common form of TTR Met30 FAP [8] and are compatible with early-onset group [9]. Severe cardiac impairment
with amyloid deposition other than neuropathy resulting in rapid progression was a characteristic feature in this patient. Cardiomyopathy is a prominent feature in patients with the Asp45, Ile50, Lys59,
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Fig. 5. Several tissues by autopsy in Case 1. (a, b, c, d, e, f, i, l: Direct Fast Scarlet 4BS staining j, k: klüver-barrera staining g, h: immunofluorescence staining for TTR) a: Amyloid is seen in nerve (arrow) and connective tissue in the space between epicardium and myofibrils(asterisk). Bar = 250 µm b: Amyloid is abundant in the capillary wall (arrow) and perimyofibrils of left ventricle. Bar = 200 µm c: A little deposition of amyloid is detected in the small artery. (asterisk: myofibril) Bar = 500 µm d: Moderate amyloid is observed in pancreas, more prominent in the inner nerve (arrow) than Langerhans islets (asterisk). Bar = 200 µm e: In pituitary, amyloid (arrow) is seen in anterior lobe, not in posterior lobe (margin is indicated by arrowheads). Bar = 200 µm f: Normal renal tissue without amyloid deposition is seen in amyloid staining. Arrow: glomerulus Bar = 150 µm g: TTR immunostaining shows no abnormal staining indicating amyloid deposition other than normal finding in Fig.h. Arrow: glomerulus Bar = 200 µm h: Subtle dotted stainings are seen only in epithelial cells of proximal tubules (arrow) in which normal TTR is observed for degradation. Autofluorescence of blood erythrocytes are seen (arrow head). Bar = 40 µm i: In the brain, amyloid limited to blood vessels (arrow) in the arachnoid and subarachnoid but lacking in the parenchyma (margin is indicated by arrowheads). Bar = 300 µm j: In the spinal cord (C7), loss of the anterior horn cell (arrow) was observed. Bar = 200 µm k: The slender gracile fasciculus in lumbar spinal cord was extremely degenerated. Arrow: posterior median fissure Arrowhead: posterior intermediate fissure Bar = 500 µm l: No amyloid is seen in the slender gracile fasciculus. Bar = 500 µm.
Ala60, Leu68, Ser84 and Gln89 variants among FAP [10]. Three TTR mutations, Thr45, Met111 and Ile122 variants, have also been associated predominantly with cardiac amyloidosis and the absence of polyneuropathy. In our patient, heavy amyloid which was shown in
scintigraphy using DMSA was detected in the endoneurium, perimyofibrils, capillary wall and connective tissue in the heart. Moreover, myocardial scintigraphy using MIBG showed no uptake into the cardiac muscle, which revealed severe dysfunction of sympathetic
T. Nagasaka et al. / Journal of the Neurological Sciences 276 (2009) 88–94 Table 1 Comparison of the region of amyloid deposition between FAPMet30 and FAPLys54 Tissue
Glu54Lys (our case)
Val 30 Met
Heart Lung Liver Kidneys Spleen Pancreas Digestive tract Bladder Adrenals Testis Thyroid gland Prostate Pituitary Skeletal muscle Cerebrum Cerebellum Arachnoid membrane Choroid plexus Spinal cord Peripheral nerves
+++ + − − − ++ ++ ++ ++ +++ +++ +++ ++ ++ − − + − − +
++ − − ++ + ++ ++ +++ + + +++ Unknown + + − − + +++ − ++
The degree of amyloid deposition is classified into the following 4 grades: − negative; + slight; ++ moderate; +++ severe.
nerve activity in the early period [11]. Postprandial hypotension observed in ABPM and 75-g OGTT was due to a lack of compensatory increase of cardiac output and abnormal secretion of insulin with neuronal impairment. In clinical and biochemical evidence of severe amyloidgenicity of TTR Pro55 has been reported [12–16]. An aggressive form of FAP Gly54 with thickened cardiac muscle in echocardiography was reported [17]. Accordingly, increased affinity of amyloid for cardiac muscle may be one of the character of variant TTR with two adjacent amino acid substitutions at position 54 and 55. In the kidneys, a large amount of amyloid was observed in almost all progressive forms of FAP [12], and the common form of FAP Met30. Alternatively, no amyloid deposition other than normal existence in epithelial cells of proximal tubules [18,19] was detected in the kidney in our case. Nearly normal renal function with no proteinuria or creatine retention in four affected siblings with a severe form of FAP with TTR Lys54 in a Costa Rican family was reported [4]. The absence of renal involvement may be a characteristic feature in FAP Lys54 despite its aggressive form with severe cardiac impairment. A combination of severe cardiac involvement and mild deposition in the kidney was also reported in autopsy cases of FAP Ile50 [20] and FAP Gly54 with Ser6 (probably a polymorphism) mutations [21]. The severity might not be necessarily attributed to both the kidney and heart. In the spinal cord, the gracile fasciculus was extremely degenerated without amyloid deposition in our case. A report revealed that minimal to moderate myelinated fiber loss was observed in the posterior columns in early- and late-onset TTRMet30 FAP [22]. So, the change may be caused by progressive ascending degeneration with a ‘dying back’ process, which reflects severe neuropathy including the large fibers. The loss of anterior horn cells observed in our case is thought to have the same significance. Various involvements of the CNS have been reported, including leptomeningeal deposition or severe meningocerebrovascular amyloidosis [23]. In our patient, slight deposition was observed in the blood vessel walls in the leptomeninges, but not in the brain parenchyma including blood vessels. Moreover, in the pituitary, amyloid was not detected in the posterior lobe(neuralgichypophysis). A secretion of variant TTR from the choroid plexus or the blood-brain barrier may play a significant role in the little deposition of amyloid in the brain. In peripheral nerve fibers, small fiber dominant neuropathy shown in Fig. 3, characteristic in FAP, was observed. At autopsy, amyloid was observed in the capillary wall and connective tissue of the endoneurium, epineurium and perineurium of the sympathetic nerve trunk, vagal nerve, celiac
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plexus, pelvic plexus, and the nerve in gastrointestinal tract and pancreas, which were also involved in Met30 FAP. The vagus nerves and celiac ganglion showed extensive endoneurial and intraganglionic deposition of amyloid, as reported previously [24]. 75-g OGTT performed in Case 1 showed excessive secretion of insulin 30 min after glucose administration. The result may reflect abnormal nerve regulation in the pancreatic endocrine system with amyloid deposition, more prominent in the nerves than in Langerhans islets, as reported previously [25]. Endocrinologically, the pituitary [26], thyroid and adrenal glands [27] can be affected in FAP. In our patient, there was no functional abnormality although amyloid deposition was observed in these organs. This may contribute to explaining the lack of correlation between amyloid deposition and the function of the amyloidogenic organs, at least in the endocrine system. Overlook these autopsied pathological findings, each of variant TTR mutations have characteristic organ distribution in amyloid deposition as in Table 1. As for the pathogenesis of peripheral nerve disturbances in FAP, various theories, such as local compression by aggregated amyloid, ischemia, neurotoxicity or apoptosis have been presented [28]. Regarding to the point, we focus on the findings of sural nerve biopsy in Case 2. In electron microscopy, the ultrastructure and the site of deposition of amyloid do not differ from the other types of variant TTR, including Met30. The amyloid seems not to invade nerve fiber or Schwann cell for the most part, but some of nerve fiber or Schwann cell are seemed to be destructed, covered or surrounded by amyloid fibrils. That is, a part of the mechanism of nerve impairment may be in relation to direct invasion of amyloid, although whether the destruction is supported by another factor such as neurotoxicity or apotosis is unclear. The amyloid was located mainly in the endoneurium adjacent to the perineurium, perineurium, and epineurium where the region collagen fiber is abundant. In immunoelectronmicroscopy, gold labeling-positive TTR present along or intermingled with collagen fibers in both pre-fibrillar and fibrillar structures. The findings may suggest that collagen fiber is one of the ground where amyloid fibril or pre-fibrillar structure is fixed or exhibit amyloidgenicity. Recently, evidence that free amyloid or amyloid intermediate has a cytotoxic effect on FAP nerves has been presented [29]. Teng et al. first demonstrated non-fibrillar TTR deposits in mice transgenic for human TTR Leu55Pro [30]. It was proposed that TTR might be deposited in a non-fibrillar form in the nerves prior to major nerve fiber degeneration in the early stages of FAP [31]. A report revealed that beta2-microglobulin amyloid induces the release of MMP-1, leading to uncontrolled collagenolysis followed by the tissue destruction [32]. These observations and our findings may be helpful evidences for the relationship between amyloid and collagen fiber. No precise autopsy findings have described FAP with TTRLys54, a progressive form with a rare substitution of the variant TTR gene. The severity of this patient was reflected in the autopsy findings. References [1] Liz MA, Faro CJ, Saraiva MJ, Sousa MM. Transthyretin, a new cryptic protease. J Biol Chem 2004;279:21431–8. [2] Sousa MM, Berglund L, Saraiva MJ. Transthyretin in high density lipoproteins: association with apolipoprotein A-I. J Lipid Res 2000;41:58–65. [3] http://www.bumc.bu.edu/Dept/Content.aspx?DepartmentID=354&PageID=8850. [4] Busse A, Sánchez MA, Monterroso V, Alvarado MV, León P. A severe form of amyloidotic polyneuropathy in a Costa Rican family with a rare transthyretin mutation (Glu54Lys). Am J Med Genet 2004;128:190–4. [5] Togashi S, Watanabe H, Nagasaka T, Shindo K, Shiozawa Z, Maeda S, et al. An aggressive familial amyloidotic polyneuropathy caused by a new variant transthyretin Lys54. Neurology 1999;53:637–9. [6] Imasawa M, Toda Y, Sakurada Y, Imai M, Iijima H. Vitreous opacities in a case of familial amyloidotic polyneuropathy associated with a transthyretin Lys 54. Acta Ophthalmol Scand 2004;82:635–6. [7] Nakazato M, Shiomi K, Miyazato M, Matsukura S. Type I familial amyloidotic polyneuropathy in Japan. Intern Med 1992;31:1335–8. [8] Araki S, Yi S. Pathology of familial amyloidotic polyneuropathy with TTR Met 30 in kumamoto, Japan. Neuropathology 2000;20:S47–51. [9] Sobue G, Koike H, Misu K, Hattori N, Yamamoto M, Ikeda S, et al. Clinicopathological and genetic features of early- and late-onset FAP type I (FAP ATTR Val30Met) in Japan. Amyloid 2003;10(Suppl1):32–8.
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