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Familial amyloid polyneuropathy
Journal of the Neurological Sciences 284 (2009) 149–154
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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
Familial amyloid polyneuropathy A clinico-pathologic study Gérard Said a,⁎, Violaine Planté-Bordeneuve b a b
Fédération des Maladies Neurologiques-Hôpital de la Salpétrière-Assistance Publique Hôpitaux de Paris, France Service de Neurologie-Hôpital Henri Mondor, Créteil, Assistance Publique Hôpitaux de Paris, France
a r t i c l e
i n f o
Article history: Received 9 February 2009 Received in revised form 23 April 2009 Accepted 1 May 2009 Available online 24 May 2009 Keywords: Peripheral neuropathy Neuropathology Amyloidosis Hereditary neuropathy Liver transplantation
a b s t r a c t In familial amyloid polyneuropathy (FAP), destruction of nerve fibres is related to accumulation of mutated transthyretin (mTTR) derived amyloid deposits (AD) in the endoneurium. Liver transplantation (LT), which removes the main source of mTTR, does not prevent deterioration of the clinical condition in all recipients. Material and methods: We evaluated the distribution of AD in the central and peripheral nervous system in order to better understand the pathophysiology of FAP and the potential role of lesions of nerve blood vessels and of mTTR released by choroid plexuses (CP). Forty nerve biopsy specimens and 3 autopsy cases, including 7 patients who underwent liver transplantation, all from patients with symptomatic FAP and DNA mutation of the TTR gene, were included. Results: Patients were ranged into three categories: Group 1: Patients with early neuropathic manifestations. Five patients carrying an amyloidogenic mutation closely followed to detect early neuropathic manifestations experienced paresthesiae with stockings temperature and pains deficit in two; uncertain changes in the others. Sensory nerve action potentials (SNAPs) were normal. Biopsy was performed to detect morphological changes as an indication for early therapeutic intervention. Only one patient had decreased MF and unmyelinated fiber (UF) density. Group 2: Patients with marked sensory-motor and dysautonomic polyneuropathy (35 patients). Non dissociated sensory loss prevailed in distal limbs, while sensory defect predominated on temperature and pain sensations in proximal limbs and anterior trunk. Weakness and amyotrophy predominated in distal lower limbs. Fiber loss massively predominated on UF. Group 3: Patients at terminal stage of the disease. All 3 patients had been bedbound for months with flaccid quadriplegia and non dissociated sensory deficit affecting all four limbs, up to proximal thighs, arms and anterior trunk along with severe autonomic dysfunction. There was virtually no surviving nerve fibers in distal nerves. Morphological changes: Amyloid predominated around endoneurial capillaries in 37 patients, with occlusion/ destruction of endoneurial capillaries in 15 nerves at late stages of the disease. Post-mortem examination showed amyloid in choroid plexuses and perivascular spaces in the brain and around blood vessels penetrating the endoneurium, following arachnoid and connective tissue septae. Destruction of endoneurial blood vessels is a late event in the natural course of FAP. Morphological findings were similar in patients who underwent liver transplantation and in those who did not. The distribution of amyloid in areas communicating with the subarachnoid space suggests that mutated TTR released in the CSF may move to the endoneurial fluid and accumulate in peripheral nerves, accounting for lack of efficacy of liver transplantation in some individuals. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Familial amyloid polyneuropathy (FAP) is an autosomal dominant polyneuropathy of adult onset which used to lead to death within
⁎ Corresponding author. Fédération des Maladies Neurologiques, Hôpital de la Salpétrière, 47 Boulevard de l'Hôpital, 75651 Paris cedex 13, France. E-mail address: [email protected] (G. Said). 0022-510X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2009.05.001
10 years on average after the first symptoms. Originally described by Andrade in Portugal, FAP was subsequently identified throughout the world [1–4]. In this condition amyloid originates from mutated transthyretin (mTTR) and destruction of nerve fibers is spatially related to its presence in the endoneurium [5–7]. Liver transplantation (LT) which removes the main source of mTTR in patients with TTR-FAP, often has a favourable effect on the course of the disease but does not prevent progression of the neuropathy in all liver recipients [8]. After LT, wildtype TTR secreted by the transplanted liver may aggregate to pre-
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existing amyloid deposits, yet mTTR released by choroid plexuses (CP) can also lead to further alteration of the PNS. In order to learn more on the pathophysiology of FAP, we studied the distribution of amyloid deposits (AD) in 40 nerve biopsy specimens from patients at different stages of the disease, and in three autopsy cases, all with documented TTR gene mutations. 2. Patients and methods
and lead citrate, for electron microscopic examination. Nerve samples were studied on ultrathin sections stained with uranyl acetate and lead citrate under the electron microscope to identify amyloid fibrils when needed, and lesions of unmyelinated fibers. The third fragment was osmicated after fixation, then macerated in 66% glycerin for 48 h before dissection in pure glycerin. Control nerves were obtained from five patients who had a nerve biopsy performed for suspected vasculitis and found to be normal on clinical, electrophysiological and morphological examination.
2.1. Patients 3.1. Morphometric procedures Nerve specimens were sampled by biopsying the superficial peroneal nerve and the adjacent peroneus brevis muscle in patients at different stages of the disease. Clinical manifestations ranged from minor neuropathic manifestations to end-stage quadriplegia with major autonomic disturbances. Nine patients were biopsied before and after liver transplantation to assess the efficacy of this procedure. Only the first biopsy findings were used for quantification of fiber density. All patients have been repeatedly examined by us. Neurological examination included muscle strength and tendon reflex testing and evaluation of light touch, pinprick, vibratory, and temperature sensations at + 4 °C and at +40 °C, and position sense. Cardiovascular autonomic function was assessed by measuring of the variation in blood pressure and pulse rate in the recumbent and standing positions and by recording symptoms of gastroparesis, nausea, vomiting, diarrhea, abnormal constipation, urinary and anal incontinence, impotence. EMG recordings were performed using standard concentric needle electrodes. Motor and sensory nerve conduction studies were performed with surface stimulation and registration electrodes using standard techniques. 2.1.1. TTR gene analysis A blood sample was obtained for DNA analysis from consenting patients. After extraction of genomic DNA from lymphocytes, sequencing of the full coding region of the gene was performed. Permission to use this material was obtained from the local ethic committee. 3. Morphological study Forty patients underwent a nerve biopsy in our center. The superficial peroneal nerve, which was clinically affected in all patients, and the adjacent peroneus brevis muscle were sampled under local anesthesia, after informed consent. The nerve samples were fixed at 4 °C in 3.6% glutaraldehyde, buffered at pH 7.4. One nerve fragment was embedded in paraffin and cut at 5 µm thickness. Serial sections were examined after hematein and eosin staining. Amyloid deposits were characterized by their Congo red affinity and birefringence with thioflavin under polarized microscope and by immunolabeling with polyclonal antibodies to lambda and kappa light chain, and to transthyretin (Dako, Denmark). The second fragment was embedded in epon. Thionin stained 1 µm thick transverse sections were used for morphometry and 0.09 µm thick sections stained with uranyl acetate
The density of myelinated fibers per mm2 was determined on the whole intrafascicular area on enlarged photographs of 1 µm thick sections. The density of the unmyelinated axons were assessed on electron micrographs by measuring at least one third of the intrafascicular area examined at a magnification of l0,000×. Teased nerve fiber study was performed on osmium postfixed nerve specimens. Autopsy was performed within 48 h after death in three patients (patients 41–43). Specimens of the CNS, nerve roots and dorsal root ganglia were carefully removed, embedded in paraffin after formalin fixation. H & E, Congo red staining and immunolabeling with anti-TTR antibody were performed. Multiple level sections of each specimen were studied to clearly delineate the distribution of amyloid deposits. 4. Results 4.1. Clinical data Patients were ranged into three categories: Group 1: patients at an early stage of the disease manifesting sensory symptoms and minor or uncertain sensory changes upon examination; Group 2: patients with confirmed sensory-motor and dysautonomic polyneuropathy; Group 3: patients at end stage of the disease (Tables 1–3). Group 1: (patients 1–5). All patients in this group belonged to an affected family and carried an amyloidogenic mutation. They were periodically assessed for detection of early neuropathic manifestations. These patients were biopsied after complaining of feet numbness for a few months (patients 3, 4, 5) to a year (patients 1, 2). Stocking sensory deficit affected temperature and pain sensations in patients 2 and 5, but remained uncertain in the other three. Muscle strength and tendon reflexes were preserved. Patient 2 complained of impotence, the others mentioned no symptoms of autonomic dysfunction. The sural and superficial peroneal sensory nerve action potentials were within normal range in all. Biopsy was performed to detect morphological changes as an indication for early therapeutic intervention. Group 2: (patients 6–40). In patients with confirmed neuropathy and moderate to severe sensory-motor deficit and autonomic dysfunction, non dissociated sensory loss was present in distal limbs, while sensory defect predominated on temperature and pain sensations in proximal limbs and over the trunk. Muscle weakness predominated in distal lower limbs. Tendon reflexes were abolished in
Table 1 Main pathological findings and TTR gene mutations.
1 2 3 4 5
Gender/age
Family history
Mutation
M F /mm2
U F /mm2
Endoneurial amyloid deposits
Sub-perineurial deposits
Around endoneurial capillaries
Destruction of endoneurial capillaries
Epineurial deposits
M-49 M-27 F-55 F-51 F-37
+ + + + +
Val30Met Val30Met Ser77Phe Ser77Phe Val71Ala
3600 4800 8120 8750 6050
8400 10,600 22,800 23,700 19,800
+ + − − +
− − − − +
+ + − + +
− − − − −
− − − − −
Group 1: patients with minor neuropathic manifestations. All patients in this group were clinically followed because one of their relatives had developed the disease. They carried an amyloidogenic mutation and were periodically examined to detect early neuropathic manifestations.
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Table 2 Main pathological findings and TTR gene mutations. Patient
Gender/age
Family history
Mutation
Myelinated fibers/mm2
Unmyelinated fibers/mm2
Endoneurial amyloid deposits
Sub-perineurial deposits
Around endoneurial capillaries
Destruction of endoneurial capillaries
Epineurial deposits
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
M-65 M-57 M-66 M-66 M-36 F-34 M-46 M-36 H-31 M-72 M-36 M-71 F-43 M-75 M-69 M-71 M-82 M-49 M-39 M-41 F-37 F-60 M-52 M-69 F-58 M-65 F-65 M-68 F-67 F-75 M-57 M-58 M-50 M-73 F-38
− + − − + + + + + − + − + − − − − − + + + + − − − − − − − − − − − − −
Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Ile107Val Ile107Val Ile107Val Ile107Val Ile107Val Val116Ser Ser77Phe Ser77Phe Val28Met Val32Ala Lys35Asn Tyr116Ser Val32Gly
450 2820 660 780 0 220 550 5200 440 3520 0 4200 3650 230 1420 110 880 1450 250 460 4230 3680 0 320 430 210 10 110 1800 6550 4520 10 2200 1620 610
0 180 18 60 0 0 20 1450 0 2300 0 4300 2300 0 30 0 60 2400 60 40 3570 3400 0 0 0 0 0 0 2300 5600 4300 0 60 30 0
− + − − + + + + + + + − + + + − − + + + + + − − − − − − − + − − + − −
− − − − − − + + + − + + − − + + − − + + + + − − − + − − − − − − − − −
− + + + + + + + + + + + + + + + + + + + + + + + + + − + − + + + + + +
− + − + + − + − − − + − + + − − − − − + − − − − + + − − − + + + − + +
− − − − − − − − − − − − − − − − − − − − − − − − − − − + − − − − − + −
Group 2: patients with moderate to severe sensory-motor deficit and autonomic dysfunction.
distal lower limbs in all. Sensory nerve action potentials were markedly decreased or absent in distal lower limbs. Patients 6–9, 11, 13, 18, 19, 21, 23, 27, 34, 35 and 38 subsequently underwent liver transplantation. Patients 11, 12, 14, 16, 25 and 26 were biopsied 2 to 3 years after LT to assess progression of the neuropathy. Group 3: (patient 41–43) were at terminal stage of the disease. All had been bedbound for months with flaccid quadriplegia and non dissociated sensory deficit affecting all four limbs, up to proximal thighs and arms, and anterior trunk. All had severe autonomic dysfunction with episodic diarrhea, vomiting, cardiac enlargement. A cardiac pacemaker had been implanted in all of them. Post mortem examination of the nervous system was performed within 48 h after death. Patient 41 underwent LT 2 years before. 4.2. Pathological data — Tables 1–3 The density of myelinated fibres (MF) varied from 0 to 6550/mm2 (mean: 1596) with marked decrease with deterioration of clinical condition. Normal control values: 8370 ± 706 (SD) MF per mm2. The unmyelinated fibers were proportionally more affected with a density varying from 0 to 23,700 per mm2 of endoneurial area (mean 2799).
Control values of our laboratory: 32,600 ± 3500 (SD). In the patients with early manifestations (Group 1), the mean density of MF per mm2 of endoneurial area was 6264 ± 2174 (SD). Only patients 1 and 2 in this group had decreased myelinated and unmyelinated nerve fibre density; the unmyelinated fibre (UF) density was normal in the others. In the patients with signs and symptoms of sensory-motor and dysautonomic polyneuropathy (Group 2) the mean MF density was 1468 ± 1782 (SD), but the density of unmyelinated fibres was strikingly reduced in all. The mean density of unmyelinated fibers in this group was 984/mm2. In 13 patients there was virtually no unmyelinated fiber left in the nerve specimen. In Group 3 patients, the mean fibre density in distal nerves was reduced to 173 MF/mm2. A varying proportion of MF were demyelinated or at different stages of axonal degeneration, sometimes in contact with endoneurial amyloid deposits. Regenerating fibres were occasionally isolated. Details of teased fiber findings have been reported previously [7,9]. Amyloid was found in 37 out of 40 nerve biopsy specimens, and in the three autopsy cases. Amyloid accumulated around endoneurial capillaries in 37 patients with subsequent occlusion or destruction of capillaries in 15 nerve samples, along with major axon loss in patients
Table 3 Main pathological findings and TTR gene mutations.
41 42 43
Gender/age
Family history
Mutation
M F /mm2
U F/mm2
Endoneurial deposits
Sub-perineurial deposits
Around endoneurial capillaries
Destruction of endoneurial capillaries
Epineurial deposits
M-59 M-68 M-69
+ + +
Val30Met Va30Met Val30Met
80 220 220
0 0 0
− − −
+ − +
+ + −
− − −
− − −
Group 3: post-mortem study of patients at end stage polyneuropathy for months.
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Fig. 1. FAP: endoneurial distribution of amyloid and blood vessel lesions. A: Early stage of FAP: One micron thick section of the superficial peroneal nerve biopsy specimen from patient 35 who carried the Ser77Phe gene mutation. She complained of discomfort in distal lower limbs for a few months and retained 6550 MF/mm2 in the nerve biopsy specimen. The arrow points to amyloid surrounding a normal endoneurial blood vessel. Thionin blue. Bar: 10 µm. B: Same patient as in A. Isolated endoneurial capillary. The arrows point to clumps of amyloid dispersed along the capillary. Bar: 25 µm. C: Patient 24 carrying the Val30Met mutation. Terminal stage of the disease. Only 80 MF/mm2 endoneurial area left. Note the occlusion of the lumen of the vessel surrounded by amyloid (arrow). Thionin blue. Bar: 50 µm. D: Patient 42 carrying the Val32Gly mutation, with a severe polyneuropathy and only 610 MF/mm2, to show destruction of an endoneurial blood vessel surrounded by amyloid (arrow). Bar: 10 µm. Thionin blue. E: Same patient as in D, to show complete destruction of two endoneurial blood vessels (arrows). Bar: 10 µm. Thionin blue. F: Patient 27. Val30Met mutation to illustrate subperineurial massive amyloid deposits (stars), the second common location of amyloid in the endoneurium. Bar: 20 µm. Thionin blue. G: Electron micrograph from patient 5, carrying the Val71Ala mutation, at an early stage of the disease with 6050 MF/ mm2, to show amyloid deposits surrounding an endoneurial blood vessel still permeable to blood (arrow). Uranyl acetate and lead citrate. Bar: 5 µm. H: Electron micrograph from patient 12 who carried the Val30Met mutation, to show destruction of an endoneurial blood vessel by amyloid (stars). Uranyl acetate and lead citrate. Bar: 5 µm.
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with long history of symptomatic neuropathy (Fig. 1A–H). Subperineurial amyloid deposits were present in 7 patients (Fig. 1-F). Amyloid was detected in the epineurium in only two patients: patient 41 who carried the Val116Ser mutation and in patient 33, the Tyr77Phe mutation. The latter had amyloid deposits in muscle vessel walls too. In the three patients (Patients 6, 32 and 36) whose nerve biopsy did not show amyloid deposits other causes of neuropathy were excluded and the diagnosis confirmed by DNA testing, as in all patients of this series. Endoneurial amyloid deposits invaded the vessel wall in some specimens, occluding the lumen and destroying the vessels (Fig. 1C, D, E, H). Destruction of endoneurial capillaries occurs at late stage of progression of amyloid deposit as shown by the conspicuous reduction of the mean fibre density in most patients whose endoneurial capillaries were destroyed (Table 1). Post-mortem examination of the three cases showed amyloid in the choroid plexuses, and around blood vessels that penetrated the CNS, in the Virchow-Robin spaces. The blood vessel walls were not damaged. Distribution of amyloid was similar in patient 41 who underwent LT and in the two patients who did not. Amyloid accumulated in the leptomeninges and was followed down to the capsula of dorsal root ganglia and the perineurium of nerve roots. Amyloid followed blood vessels as they entered dorsal roots perineurium and endoneurium. The cell bodies of many sensory neurons of dorsal root ganglia were degenerating as amyloid accumulated between them (Fig. 2D). At post-mortem examination there was virtually no fibre left in distal nerves.
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5. Comments Our findings illustrate the range of changes observed at different stages of FAP. At the onset of neuropathic symptoms, the MF density remains within normal range, in keeping with clinical and electrophysiological findings, and in spite of the presence of minute amyloid deposits in the endoneurium. Amyloid deposits predominate around endoneurial capillaries, without altering their wall and their lumen. Alteration of UF occurs early, along with loss of pain sensation. As the clinical condition worsens, sensory and autonomic then motor manifestations become prominent along with gradual loss of nerve fibers. Sensory defect progresses following a fiberlength dependent pattern and, in patients with marked sensory loss, the UF have virtually disappeared on EM examination, preceding that of MF. At end stage of the disease the density of myelinated and unmyelinated nerve fibres is close to zero in distal nerves. Nerve fiber lesions and amyloid deposits are often markedly asymmetric between and within nerve fascicles. The asymmetric, patchy, distribution of amyloid illustrated on the teased endoneurial blood vessel, accounts for the negative nerve biopsy findings in some cases, in spite of study of serial sections of the nerve biopsy specimen [9]. We found no difference in the distribution of amyloid deposits between the 7 patients who had LT and those who had not. Furthermore, it is not possible to determine which of these deposits were present before LT. Blood vessel damage is a late event in the course of this devastating neuropathy. Perivascular amyloid deposits occur early, along with
Fig. 2. FAP: epineurial amyloid deposits and postmortem findings. A: One micron thick section of the nerve biopsy specimen form patient 39 who carried the Tyr116Ser mutation, to show amyloid deposit invading the vessel wall of an epineurial blood vessel. Thionin blue. Bar: 20 µm. B: Patient 41 carried the Val30Met mutation. Postmortem examination of a paraffin embedded sample of dorsal root near the sensory ganglion. Immunolabeling with anti-TTR antibody and peroxydase-antiperoxydase technique. The arrows point to amyloid deposits decorated by the antibodies in the perineurium and perineurial septae. The triangles point to amyloid surrounding blood vessels. Bar: 50 µm. C: Patient 43 carried the Val30Met mutation. Postmortem examination. Section of the spinal cord to show antibody labeled deposits around a large (triangles) and smaller blood vessels (arrow) Bar: 50 µm. D: Patient 42 carried the Val30Met mutation. Postmortem examination of a dorsal root ganglion showing degenerating neurons and endocapsular amyloid deposit (stars). Bar: 10 µm. H&E staining.
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fiber degeneration, but destruction of the vessel wall and occlusion of the lumen are seen only in severely affected nerves. A role for nerve ischemia is thus unlikely in the vast majority of cases. However, amyloid deposits in the leptomeninges, and especially around blood vessels penetrating the CNS, can occasionally lead to ischemic or hemorrhagic strokes, dementia, hemiparesis and seizures. Most symptomatic CNS involvements have been reported in non-Val30Met mutations [9–14], although they occur also in the Val30Met mutation [15,16]. The three patients of this series who went to autopsy carried the Val30Met mutation, and had conspicuous, although asymptomatic, leptomeningeal amyloid deposits predominantly around blood vessels. Mutated TTR released in the CSF by choroid plexuses is likely to play a role in CNS damage. Eye complications, the prevalence of which increases with time after LT, can also be related to mTTR released by choroid plexuses [17]. Predominant distribution of amyloid in the subperineurial area and around blood vessels is compatible with its release from the subarachnoid space, which is in continuity with the endoneurial space where amyloid deposits are found in roots and nerves. Communication between the CSF and the endoneurial space is demonstrated by the presence of horseradish peroxidase in epineurial, perineurial and endoneurial spaces after subarachnoid injection [18]. Also, CSF tracers like Evans blue-albumin and lanthanum chloride are rapidly detected in roots, spinal nerves, inside the capsula of dorsal root ganglia after subarachnoid injection [19]. The PNS thus represents a route of resorbtion of the CSF [20]. In roots and nerves there is also a proximo-distal fluid convection in the endoneurial spaces with a proximo-distal gradient of endoneurial fluid pressure which makes the content of endoneurial fluid move distally [21,22]. Substances released in the CSF can thus move slowly towards distal nerves through movements of the endoneurial fluid. Mutated TTR can follow this route and accumulate after LT, increasing peripheral nerve damage, which is the major cause of disability and death in transplanted TTR-FAP patients. In addition, most of the CSF secreted by CP, i.e., roughly 0.5 ml/mn, drained in the blood stream may contribute to increase the amyloid deposits outside the nervous system. Occasional cases of symptomatic neuropathy occurring in receivers of livers from FAP patients [23] illustrate the role of mTTR released by the liver in this condition, but the contribution of choroid plexus is also to consider. After LT accumulation of wild-type TTR released by the normal liver, which predominates in the myocardium, can also contribute to deterioration of the clinical condition after liver transplantation [24]. The need for biochemical or molecular biological means of treating the TTR amyloidoses have been stressed to prevent worsening of CNS manifestations in FAPs due to ongoing synthesis of mutated TTR in patients after liver transplantation [25]. Recent observations of worsening of ocular and meningeal involvement after liver transplantation confirmed this view [26]. Since aggregation of wild-type TTR released by the transplanted liver to pre-existing mutated-TTR amyloid may also contribute to progression of nerve lesions, alternative to liver transplantation with single stranded oligonucleotides, short interference RNA (SiRNA) or antibodies to amyloid fibrils as recently suggested should be considered [27].
6. Conclusion After liver transplantation, choroid plexus is the main source of mutated-TTR. Mutated-TTR released by choroid plexus with the CSF accumulates in the subarachnoid spaces, especially around the blood vessels that penetrate the CNS and the PNS. Sustained release of m-TTR by choroid plexus can contribute to CNS damage and to progression of the neuropathy after liver transplantation. Since the proportion of TTR released by choroid plexuses varies between individuals, the response to LT may vary accordingly.
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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
Familial amyloid polyneuropathy A clinico-pathologic study Gérard Said a,⁎, Violaine Planté-Bordeneuve b a b
Fédération des Maladies Neurologiques-Hôpital de la Salpétrière-Assistance Publique Hôpitaux de Paris, France Service de Neurologie-Hôpital Henri Mondor, Créteil, Assistance Publique Hôpitaux de Paris, France
a r t i c l e
i n f o
Article history: Received 9 February 2009 Received in revised form 23 April 2009 Accepted 1 May 2009 Available online 24 May 2009 Keywords: Peripheral neuropathy Neuropathology Amyloidosis Hereditary neuropathy Liver transplantation
a b s t r a c t In familial amyloid polyneuropathy (FAP), destruction of nerve fibres is related to accumulation of mutated transthyretin (mTTR) derived amyloid deposits (AD) in the endoneurium. Liver transplantation (LT), which removes the main source of mTTR, does not prevent deterioration of the clinical condition in all recipients. Material and methods: We evaluated the distribution of AD in the central and peripheral nervous system in order to better understand the pathophysiology of FAP and the potential role of lesions of nerve blood vessels and of mTTR released by choroid plexuses (CP). Forty nerve biopsy specimens and 3 autopsy cases, including 7 patients who underwent liver transplantation, all from patients with symptomatic FAP and DNA mutation of the TTR gene, were included. Results: Patients were ranged into three categories: Group 1: Patients with early neuropathic manifestations. Five patients carrying an amyloidogenic mutation closely followed to detect early neuropathic manifestations experienced paresthesiae with stockings temperature and pains deficit in two; uncertain changes in the others. Sensory nerve action potentials (SNAPs) were normal. Biopsy was performed to detect morphological changes as an indication for early therapeutic intervention. Only one patient had decreased MF and unmyelinated fiber (UF) density. Group 2: Patients with marked sensory-motor and dysautonomic polyneuropathy (35 patients). Non dissociated sensory loss prevailed in distal limbs, while sensory defect predominated on temperature and pain sensations in proximal limbs and anterior trunk. Weakness and amyotrophy predominated in distal lower limbs. Fiber loss massively predominated on UF. Group 3: Patients at terminal stage of the disease. All 3 patients had been bedbound for months with flaccid quadriplegia and non dissociated sensory deficit affecting all four limbs, up to proximal thighs, arms and anterior trunk along with severe autonomic dysfunction. There was virtually no surviving nerve fibers in distal nerves. Morphological changes: Amyloid predominated around endoneurial capillaries in 37 patients, with occlusion/ destruction of endoneurial capillaries in 15 nerves at late stages of the disease. Post-mortem examination showed amyloid in choroid plexuses and perivascular spaces in the brain and around blood vessels penetrating the endoneurium, following arachnoid and connective tissue septae. Destruction of endoneurial blood vessels is a late event in the natural course of FAP. Morphological findings were similar in patients who underwent liver transplantation and in those who did not. The distribution of amyloid in areas communicating with the subarachnoid space suggests that mutated TTR released in the CSF may move to the endoneurial fluid and accumulate in peripheral nerves, accounting for lack of efficacy of liver transplantation in some individuals. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Familial amyloid polyneuropathy (FAP) is an autosomal dominant polyneuropathy of adult onset which used to lead to death within
⁎ Corresponding author. Fédération des Maladies Neurologiques, Hôpital de la Salpétrière, 47 Boulevard de l'Hôpital, 75651 Paris cedex 13, France. E-mail address: [email protected] (G. Said). 0022-510X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2009.05.001
10 years on average after the first symptoms. Originally described by Andrade in Portugal, FAP was subsequently identified throughout the world [1–4]. In this condition amyloid originates from mutated transthyretin (mTTR) and destruction of nerve fibers is spatially related to its presence in the endoneurium [5–7]. Liver transplantation (LT) which removes the main source of mTTR in patients with TTR-FAP, often has a favourable effect on the course of the disease but does not prevent progression of the neuropathy in all liver recipients [8]. After LT, wildtype TTR secreted by the transplanted liver may aggregate to pre-
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existing amyloid deposits, yet mTTR released by choroid plexuses (CP) can also lead to further alteration of the PNS. In order to learn more on the pathophysiology of FAP, we studied the distribution of amyloid deposits (AD) in 40 nerve biopsy specimens from patients at different stages of the disease, and in three autopsy cases, all with documented TTR gene mutations. 2. Patients and methods
and lead citrate, for electron microscopic examination. Nerve samples were studied on ultrathin sections stained with uranyl acetate and lead citrate under the electron microscope to identify amyloid fibrils when needed, and lesions of unmyelinated fibers. The third fragment was osmicated after fixation, then macerated in 66% glycerin for 48 h before dissection in pure glycerin. Control nerves were obtained from five patients who had a nerve biopsy performed for suspected vasculitis and found to be normal on clinical, electrophysiological and morphological examination.
2.1. Patients 3.1. Morphometric procedures Nerve specimens were sampled by biopsying the superficial peroneal nerve and the adjacent peroneus brevis muscle in patients at different stages of the disease. Clinical manifestations ranged from minor neuropathic manifestations to end-stage quadriplegia with major autonomic disturbances. Nine patients were biopsied before and after liver transplantation to assess the efficacy of this procedure. Only the first biopsy findings were used for quantification of fiber density. All patients have been repeatedly examined by us. Neurological examination included muscle strength and tendon reflex testing and evaluation of light touch, pinprick, vibratory, and temperature sensations at + 4 °C and at +40 °C, and position sense. Cardiovascular autonomic function was assessed by measuring of the variation in blood pressure and pulse rate in the recumbent and standing positions and by recording symptoms of gastroparesis, nausea, vomiting, diarrhea, abnormal constipation, urinary and anal incontinence, impotence. EMG recordings were performed using standard concentric needle electrodes. Motor and sensory nerve conduction studies were performed with surface stimulation and registration electrodes using standard techniques. 2.1.1. TTR gene analysis A blood sample was obtained for DNA analysis from consenting patients. After extraction of genomic DNA from lymphocytes, sequencing of the full coding region of the gene was performed. Permission to use this material was obtained from the local ethic committee. 3. Morphological study Forty patients underwent a nerve biopsy in our center. The superficial peroneal nerve, which was clinically affected in all patients, and the adjacent peroneus brevis muscle were sampled under local anesthesia, after informed consent. The nerve samples were fixed at 4 °C in 3.6% glutaraldehyde, buffered at pH 7.4. One nerve fragment was embedded in paraffin and cut at 5 µm thickness. Serial sections were examined after hematein and eosin staining. Amyloid deposits were characterized by their Congo red affinity and birefringence with thioflavin under polarized microscope and by immunolabeling with polyclonal antibodies to lambda and kappa light chain, and to transthyretin (Dako, Denmark). The second fragment was embedded in epon. Thionin stained 1 µm thick transverse sections were used for morphometry and 0.09 µm thick sections stained with uranyl acetate
The density of myelinated fibers per mm2 was determined on the whole intrafascicular area on enlarged photographs of 1 µm thick sections. The density of the unmyelinated axons were assessed on electron micrographs by measuring at least one third of the intrafascicular area examined at a magnification of l0,000×. Teased nerve fiber study was performed on osmium postfixed nerve specimens. Autopsy was performed within 48 h after death in three patients (patients 41–43). Specimens of the CNS, nerve roots and dorsal root ganglia were carefully removed, embedded in paraffin after formalin fixation. H & E, Congo red staining and immunolabeling with anti-TTR antibody were performed. Multiple level sections of each specimen were studied to clearly delineate the distribution of amyloid deposits. 4. Results 4.1. Clinical data Patients were ranged into three categories: Group 1: patients at an early stage of the disease manifesting sensory symptoms and minor or uncertain sensory changes upon examination; Group 2: patients with confirmed sensory-motor and dysautonomic polyneuropathy; Group 3: patients at end stage of the disease (Tables 1–3). Group 1: (patients 1–5). All patients in this group belonged to an affected family and carried an amyloidogenic mutation. They were periodically assessed for detection of early neuropathic manifestations. These patients were biopsied after complaining of feet numbness for a few months (patients 3, 4, 5) to a year (patients 1, 2). Stocking sensory deficit affected temperature and pain sensations in patients 2 and 5, but remained uncertain in the other three. Muscle strength and tendon reflexes were preserved. Patient 2 complained of impotence, the others mentioned no symptoms of autonomic dysfunction. The sural and superficial peroneal sensory nerve action potentials were within normal range in all. Biopsy was performed to detect morphological changes as an indication for early therapeutic intervention. Group 2: (patients 6–40). In patients with confirmed neuropathy and moderate to severe sensory-motor deficit and autonomic dysfunction, non dissociated sensory loss was present in distal limbs, while sensory defect predominated on temperature and pain sensations in proximal limbs and over the trunk. Muscle weakness predominated in distal lower limbs. Tendon reflexes were abolished in
Table 1 Main pathological findings and TTR gene mutations.
1 2 3 4 5
Gender/age
Family history
Mutation
M F /mm2
U F /mm2
Endoneurial amyloid deposits
Sub-perineurial deposits
Around endoneurial capillaries
Destruction of endoneurial capillaries
Epineurial deposits
M-49 M-27 F-55 F-51 F-37
+ + + + +
Val30Met Val30Met Ser77Phe Ser77Phe Val71Ala
3600 4800 8120 8750 6050
8400 10,600 22,800 23,700 19,800
+ + − − +
− − − − +
+ + − + +
− − − − −
− − − − −
Group 1: patients with minor neuropathic manifestations. All patients in this group were clinically followed because one of their relatives had developed the disease. They carried an amyloidogenic mutation and were periodically examined to detect early neuropathic manifestations.
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Table 2 Main pathological findings and TTR gene mutations. Patient
Gender/age
Family history
Mutation
Myelinated fibers/mm2
Unmyelinated fibers/mm2
Endoneurial amyloid deposits
Sub-perineurial deposits
Around endoneurial capillaries
Destruction of endoneurial capillaries
Epineurial deposits
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
M-65 M-57 M-66 M-66 M-36 F-34 M-46 M-36 H-31 M-72 M-36 M-71 F-43 M-75 M-69 M-71 M-82 M-49 M-39 M-41 F-37 F-60 M-52 M-69 F-58 M-65 F-65 M-68 F-67 F-75 M-57 M-58 M-50 M-73 F-38
− + − − + + + + + − + − + − − − − − + + + + − − − − − − − − − − − − −
Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Val30Met Ile107Val Ile107Val Ile107Val Ile107Val Ile107Val Val116Ser Ser77Phe Ser77Phe Val28Met Val32Ala Lys35Asn Tyr116Ser Val32Gly
450 2820 660 780 0 220 550 5200 440 3520 0 4200 3650 230 1420 110 880 1450 250 460 4230 3680 0 320 430 210 10 110 1800 6550 4520 10 2200 1620 610
0 180 18 60 0 0 20 1450 0 2300 0 4300 2300 0 30 0 60 2400 60 40 3570 3400 0 0 0 0 0 0 2300 5600 4300 0 60 30 0
− + − − + + + + + + + − + + + − − + + + + + − − − − − − − + − − + − −
− − − − − − + + + − + + − − + + − − + + + + − − − + − − − − − − − − −
− + + + + + + + + + + + + + + + + + + + + + + + + + − + − + + + + + +
− + − + + − + − − − + − + + − − − − − + − − − − + + − − − + + + − + +
− − − − − − − − − − − − − − − − − − − − − − − − − − − + − − − − − + −
Group 2: patients with moderate to severe sensory-motor deficit and autonomic dysfunction.
distal lower limbs in all. Sensory nerve action potentials were markedly decreased or absent in distal lower limbs. Patients 6–9, 11, 13, 18, 19, 21, 23, 27, 34, 35 and 38 subsequently underwent liver transplantation. Patients 11, 12, 14, 16, 25 and 26 were biopsied 2 to 3 years after LT to assess progression of the neuropathy. Group 3: (patient 41–43) were at terminal stage of the disease. All had been bedbound for months with flaccid quadriplegia and non dissociated sensory deficit affecting all four limbs, up to proximal thighs and arms, and anterior trunk. All had severe autonomic dysfunction with episodic diarrhea, vomiting, cardiac enlargement. A cardiac pacemaker had been implanted in all of them. Post mortem examination of the nervous system was performed within 48 h after death. Patient 41 underwent LT 2 years before. 4.2. Pathological data — Tables 1–3 The density of myelinated fibres (MF) varied from 0 to 6550/mm2 (mean: 1596) with marked decrease with deterioration of clinical condition. Normal control values: 8370 ± 706 (SD) MF per mm2. The unmyelinated fibers were proportionally more affected with a density varying from 0 to 23,700 per mm2 of endoneurial area (mean 2799).
Control values of our laboratory: 32,600 ± 3500 (SD). In the patients with early manifestations (Group 1), the mean density of MF per mm2 of endoneurial area was 6264 ± 2174 (SD). Only patients 1 and 2 in this group had decreased myelinated and unmyelinated nerve fibre density; the unmyelinated fibre (UF) density was normal in the others. In the patients with signs and symptoms of sensory-motor and dysautonomic polyneuropathy (Group 2) the mean MF density was 1468 ± 1782 (SD), but the density of unmyelinated fibres was strikingly reduced in all. The mean density of unmyelinated fibers in this group was 984/mm2. In 13 patients there was virtually no unmyelinated fiber left in the nerve specimen. In Group 3 patients, the mean fibre density in distal nerves was reduced to 173 MF/mm2. A varying proportion of MF were demyelinated or at different stages of axonal degeneration, sometimes in contact with endoneurial amyloid deposits. Regenerating fibres were occasionally isolated. Details of teased fiber findings have been reported previously [7,9]. Amyloid was found in 37 out of 40 nerve biopsy specimens, and in the three autopsy cases. Amyloid accumulated around endoneurial capillaries in 37 patients with subsequent occlusion or destruction of capillaries in 15 nerve samples, along with major axon loss in patients
Table 3 Main pathological findings and TTR gene mutations.
41 42 43
Gender/age
Family history
Mutation
M F /mm2
U F/mm2
Endoneurial deposits
Sub-perineurial deposits
Around endoneurial capillaries
Destruction of endoneurial capillaries
Epineurial deposits
M-59 M-68 M-69
+ + +
Val30Met Va30Met Val30Met
80 220 220
0 0 0
− − −
+ − +
+ + −
− − −
− − −
Group 3: post-mortem study of patients at end stage polyneuropathy for months.
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Fig. 1. FAP: endoneurial distribution of amyloid and blood vessel lesions. A: Early stage of FAP: One micron thick section of the superficial peroneal nerve biopsy specimen from patient 35 who carried the Ser77Phe gene mutation. She complained of discomfort in distal lower limbs for a few months and retained 6550 MF/mm2 in the nerve biopsy specimen. The arrow points to amyloid surrounding a normal endoneurial blood vessel. Thionin blue. Bar: 10 µm. B: Same patient as in A. Isolated endoneurial capillary. The arrows point to clumps of amyloid dispersed along the capillary. Bar: 25 µm. C: Patient 24 carrying the Val30Met mutation. Terminal stage of the disease. Only 80 MF/mm2 endoneurial area left. Note the occlusion of the lumen of the vessel surrounded by amyloid (arrow). Thionin blue. Bar: 50 µm. D: Patient 42 carrying the Val32Gly mutation, with a severe polyneuropathy and only 610 MF/mm2, to show destruction of an endoneurial blood vessel surrounded by amyloid (arrow). Bar: 10 µm. Thionin blue. E: Same patient as in D, to show complete destruction of two endoneurial blood vessels (arrows). Bar: 10 µm. Thionin blue. F: Patient 27. Val30Met mutation to illustrate subperineurial massive amyloid deposits (stars), the second common location of amyloid in the endoneurium. Bar: 20 µm. Thionin blue. G: Electron micrograph from patient 5, carrying the Val71Ala mutation, at an early stage of the disease with 6050 MF/ mm2, to show amyloid deposits surrounding an endoneurial blood vessel still permeable to blood (arrow). Uranyl acetate and lead citrate. Bar: 5 µm. H: Electron micrograph from patient 12 who carried the Val30Met mutation, to show destruction of an endoneurial blood vessel by amyloid (stars). Uranyl acetate and lead citrate. Bar: 5 µm.
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with long history of symptomatic neuropathy (Fig. 1A–H). Subperineurial amyloid deposits were present in 7 patients (Fig. 1-F). Amyloid was detected in the epineurium in only two patients: patient 41 who carried the Val116Ser mutation and in patient 33, the Tyr77Phe mutation. The latter had amyloid deposits in muscle vessel walls too. In the three patients (Patients 6, 32 and 36) whose nerve biopsy did not show amyloid deposits other causes of neuropathy were excluded and the diagnosis confirmed by DNA testing, as in all patients of this series. Endoneurial amyloid deposits invaded the vessel wall in some specimens, occluding the lumen and destroying the vessels (Fig. 1C, D, E, H). Destruction of endoneurial capillaries occurs at late stage of progression of amyloid deposit as shown by the conspicuous reduction of the mean fibre density in most patients whose endoneurial capillaries were destroyed (Table 1). Post-mortem examination of the three cases showed amyloid in the choroid plexuses, and around blood vessels that penetrated the CNS, in the Virchow-Robin spaces. The blood vessel walls were not damaged. Distribution of amyloid was similar in patient 41 who underwent LT and in the two patients who did not. Amyloid accumulated in the leptomeninges and was followed down to the capsula of dorsal root ganglia and the perineurium of nerve roots. Amyloid followed blood vessels as they entered dorsal roots perineurium and endoneurium. The cell bodies of many sensory neurons of dorsal root ganglia were degenerating as amyloid accumulated between them (Fig. 2D). At post-mortem examination there was virtually no fibre left in distal nerves.
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5. Comments Our findings illustrate the range of changes observed at different stages of FAP. At the onset of neuropathic symptoms, the MF density remains within normal range, in keeping with clinical and electrophysiological findings, and in spite of the presence of minute amyloid deposits in the endoneurium. Amyloid deposits predominate around endoneurial capillaries, without altering their wall and their lumen. Alteration of UF occurs early, along with loss of pain sensation. As the clinical condition worsens, sensory and autonomic then motor manifestations become prominent along with gradual loss of nerve fibers. Sensory defect progresses following a fiberlength dependent pattern and, in patients with marked sensory loss, the UF have virtually disappeared on EM examination, preceding that of MF. At end stage of the disease the density of myelinated and unmyelinated nerve fibres is close to zero in distal nerves. Nerve fiber lesions and amyloid deposits are often markedly asymmetric between and within nerve fascicles. The asymmetric, patchy, distribution of amyloid illustrated on the teased endoneurial blood vessel, accounts for the negative nerve biopsy findings in some cases, in spite of study of serial sections of the nerve biopsy specimen [9]. We found no difference in the distribution of amyloid deposits between the 7 patients who had LT and those who had not. Furthermore, it is not possible to determine which of these deposits were present before LT. Blood vessel damage is a late event in the course of this devastating neuropathy. Perivascular amyloid deposits occur early, along with
Fig. 2. FAP: epineurial amyloid deposits and postmortem findings. A: One micron thick section of the nerve biopsy specimen form patient 39 who carried the Tyr116Ser mutation, to show amyloid deposit invading the vessel wall of an epineurial blood vessel. Thionin blue. Bar: 20 µm. B: Patient 41 carried the Val30Met mutation. Postmortem examination of a paraffin embedded sample of dorsal root near the sensory ganglion. Immunolabeling with anti-TTR antibody and peroxydase-antiperoxydase technique. The arrows point to amyloid deposits decorated by the antibodies in the perineurium and perineurial septae. The triangles point to amyloid surrounding blood vessels. Bar: 50 µm. C: Patient 43 carried the Val30Met mutation. Postmortem examination. Section of the spinal cord to show antibody labeled deposits around a large (triangles) and smaller blood vessels (arrow) Bar: 50 µm. D: Patient 42 carried the Val30Met mutation. Postmortem examination of a dorsal root ganglion showing degenerating neurons and endocapsular amyloid deposit (stars). Bar: 10 µm. H&E staining.
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fiber degeneration, but destruction of the vessel wall and occlusion of the lumen are seen only in severely affected nerves. A role for nerve ischemia is thus unlikely in the vast majority of cases. However, amyloid deposits in the leptomeninges, and especially around blood vessels penetrating the CNS, can occasionally lead to ischemic or hemorrhagic strokes, dementia, hemiparesis and seizures. Most symptomatic CNS involvements have been reported in non-Val30Met mutations [9–14], although they occur also in the Val30Met mutation [15,16]. The three patients of this series who went to autopsy carried the Val30Met mutation, and had conspicuous, although asymptomatic, leptomeningeal amyloid deposits predominantly around blood vessels. Mutated TTR released in the CSF by choroid plexuses is likely to play a role in CNS damage. Eye complications, the prevalence of which increases with time after LT, can also be related to mTTR released by choroid plexuses [17]. Predominant distribution of amyloid in the subperineurial area and around blood vessels is compatible with its release from the subarachnoid space, which is in continuity with the endoneurial space where amyloid deposits are found in roots and nerves. Communication between the CSF and the endoneurial space is demonstrated by the presence of horseradish peroxidase in epineurial, perineurial and endoneurial spaces after subarachnoid injection [18]. Also, CSF tracers like Evans blue-albumin and lanthanum chloride are rapidly detected in roots, spinal nerves, inside the capsula of dorsal root ganglia after subarachnoid injection [19]. The PNS thus represents a route of resorbtion of the CSF [20]. In roots and nerves there is also a proximo-distal fluid convection in the endoneurial spaces with a proximo-distal gradient of endoneurial fluid pressure which makes the content of endoneurial fluid move distally [21,22]. Substances released in the CSF can thus move slowly towards distal nerves through movements of the endoneurial fluid. Mutated TTR can follow this route and accumulate after LT, increasing peripheral nerve damage, which is the major cause of disability and death in transplanted TTR-FAP patients. In addition, most of the CSF secreted by CP, i.e., roughly 0.5 ml/mn, drained in the blood stream may contribute to increase the amyloid deposits outside the nervous system. Occasional cases of symptomatic neuropathy occurring in receivers of livers from FAP patients [23] illustrate the role of mTTR released by the liver in this condition, but the contribution of choroid plexus is also to consider. After LT accumulation of wild-type TTR released by the normal liver, which predominates in the myocardium, can also contribute to deterioration of the clinical condition after liver transplantation [24]. The need for biochemical or molecular biological means of treating the TTR amyloidoses have been stressed to prevent worsening of CNS manifestations in FAPs due to ongoing synthesis of mutated TTR in patients after liver transplantation [25]. Recent observations of worsening of ocular and meningeal involvement after liver transplantation confirmed this view [26]. Since aggregation of wild-type TTR released by the transplanted liver to pre-existing mutated-TTR amyloid may also contribute to progression of nerve lesions, alternative to liver transplantation with single stranded oligonucleotides, short interference RNA (SiRNA) or antibodies to amyloid fibrils as recently suggested should be considered [27].
6. Conclusion After liver transplantation, choroid plexus is the main source of mutated-TTR. Mutated-TTR released by choroid plexus with the CSF accumulates in the subarachnoid spaces, especially around the blood vessels that penetrate the CNS and the PNS. Sustained release of m-TTR by choroid plexus can contribute to CNS damage and to progression of the neuropathy after liver transplantation. Since the proportion of TTR released by choroid plexuses varies between individuals, the response to LT may vary accordingly.
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